Gonadal development and transcript profiling of steroidogenic enzymes in response to...

Elsevier Editorial System(tm) for The Journal of Steroid Biochemistry and Molecular Biology Manuscript Draft Manuscript Number SBMB-D-13-00199R1 Title Gonadal development and transcript profiling of steroidogenic enzymes in response to 17α-methyltestosterone in the rare minnow Gobiocypris rarus Article Type Full Length Article Keywords steroidogenic genes 17α-methyltestosterone 17α-ethinylestradiol histology Gobiocypris rarus Corresponding Author Dr Zaizhao Wang Corresponding Authors Institution Northwest AampF University First Author Shaozhen Liu Order of Authors Shaozhen Liu Lihong Wang Fang Qin Yao Zheng Meng Li Yingying Zhang Cong Yuan Zaizhao Wang Abstract It is well known that natural and anthropogenic chemicals interfere with the hormonal system of vertebrate and invertebrate organisms How these chemicals regulate gonadal steroidogenesis remains to be determined The main objective of this study was to evaluate the effects of 17α-methyltestosterone (MT) a synthetic model androgen on gene expression profiles of six key steroidogenic genes in adult rare minnow The full-length cDNA encoding 11β-hydroxysteroid dehydrogenase-2 (11β-HSD2) was firstly isolated and characterized by RT-PCR and RACE methods The gonadal transcript changes of StAR cyp11a1 3β-HSD cyp17a1 11β-HSD2 and cyp19a1a in 6-month adult G rarus exposed to MT and 17α-ethinylestradiol (EE2) for 7 14 and 21 days were detected by qRT-PCR To make an effort to connect the transcriptional changes of steroidogenic enzymes with effects on higher levels of biological organization and on VTG one remarkable sensitive target of steroids body and gonad weights histology of gonads and hepatic vtg mRNA level were measured MT caused varying degree of abnormalities in ovaries and testes in a time- and concentration-dependent manner The hepatic vtg mRNA level was highly inhibited in the females and slightly induced by MT Transcripts of several steroidogenic genes including StAR cyp17a1 and cyp11a1 showed high responsiveness to MT exposure in G rarus The gene expression profiles of these steroidogenic genes in MT-treated groups were much distinct with the EE2-treated group Suggested Reviewers Paola Irato paolairatounipdit Montserrat Soleacute msoleicmcsices

Response to Reviewers

Dear editor

Thank you very much for your letter and the comments from the reviewers about our paper

submitted to Steroid Biochemistry amp Molecular Biology (Ms Ref No SBMB-D-13-00199)

We have checked and revised the manuscript carefully according to the comments We submit here

the revised manuscript as well as the responses to reviews

If you need any other information please contact me immediately by email My email account is

zzwangnwsuafeducn and Tel is +86-29-87092139 and Fax is +86-29-87092164

Sincerely yours

Zaizhao Wang

Response to reviewer

Dear reviewer

Thanks a lot for your comments on our manuscript According to those helpful comments we have

revised this manuscript carefully point-by-point The comments need to be explained are listed as

following

1 General comment Despite the amount of results in the current paper the research

hypothesis on the effects of the synthetic androgen 17-MT in terms of histological and

steroidogenic gene expressions has been largely demonstrated in many fish species So in

the introductory section of the present study the authors should better justify the reasons

for testing low levels of MT and EE as a further confirmation of the known responses to

Detailed Response to Reviewers

MT exposure in rare minnow in the ecological context of their region (case study)

Response Thank you very much for your suggestion In the present study we detected the

effect of MT (25 50 and 100 ngL) and EE2 (25 ngL) on rare minnow EE2 exposure group as a

control group appeared in the present study The aim of present study was to evaluate the effects

of MT at low concentrations (25-100ngL) on gene expression profiles of six key steroidogenic

genes There was no manuscript study the effects of MT at low concentrations on six key

steroidogenic genes simultaneously Our purpose was to study the mechanism of MT in gonads of

fish And someone else in our research group studied the effects of MT in brain or liver of rare

minnow

According your suggestion we plan to test the effect of MT with environment concentration

on Chinese freshwater cyprinid rare minnow

2 Introduction and discussion should be updated There are at least five references between

2010-2012 while there is rich and timely information on the MT and EE response in

various fish species

Response Thank you very much for your suggestion We indeed neglected the time of

references in the present study According your suggestion we carefully consulted a lot of

literature about MT and EE2 Meanwhile the introduction and discussion in the present study

have been updated

3 Pg2 L39-39 The hepatic vtg mRNA level was highly inhibited in the females and slightly

induced by MT What does mean slightly induced while only inhibition or suppression

by MT was observed for females May be this is stated for males but also a decrease was

observed for 21-d exposure

Response Thanks for your suggestion This error should be attributed to our careless We

have replaced the sentence with ldquoThe hepatic vtg mRNA level was highly inhibited in

females and slightly altered in males by MTrdquo (Pg2 L36 ) in the revised manuscript

4 Pg5-L107 The choice of testing low levels of MT (25-100ngL) and EE (25ngL) should be

justified in regard to the levels in the environment

Response Thanks for your suggestion The answer of this question is similar to the

question one Our choice of testing low levels of MT (25-100ngL and EE2 (25ngL) is

referred to previous study in zebrafish Error Reference source not found In the present

study other than detected the effect of MT and EE2 on vtg and steroidogenic enzymes we

attempted to make certain the metabolic pathway of steroid influenced by MT So we chose

these testing levels We will choose testing level in regard to that of environment in next

study

5 Pg7-L134-135 Half of the exposure solution was changed every day On what basis water

was changed every day while the frac12 time for the targeted hormonal compounds are longer

Response Thanks for your suggestion In the present study half of the exposure solution

was changed every day The main reason is fish excrement and remnant foods should be

cleared out timely When we cleared out the excrement and remnant foods part of exposure

solution would be taken away from the tanks To add water and hormonal compounds

expediently half of the exposure solution was changed every day

The frac12 time for the targeted hormonal compounds are longer than one day (Barel-Cohen

et al 2006 Homklin et al 2011)

6 P7-L137 All exposure experiments were conducted in triplicate separate tanks at the same

time How water quality was managed to avoid any false related among triplicates of the

same treatment or among treatments

Response Thanks for your suggestion In the present study the tap water in big tanks

were dechlorinated before used in exposure tests Water quality was often managed by

measuring physicochemical parameters such as temperature pH hardness dissolved oxygen

All the water in experiment from above-mentioned big tanks So the water quality should be

the same in our study

In the present study measurements of temperature and pH in exposure tanks were done

everyday to determine the quality of water further

7 Pg7-L146 pg10-L210 Through materials and methods the number of sampled fish and

related analyses is not well described in a triplicate tank 10 fish were chosen for histology

+ the number of fish sampled for RNA isolation and RT is not specified + 6 fish were

sampled for VTG and gonadal transcripts A line time with steps and types of samples is

recommended for more explanation of the sampling protocol

Response Thanks for your suggestion The number of fish sampled for RNA isolation and

RT is not specified in para 24 because in this section we aimed to introduce the sampling

protocol In this study the fish sampled for RNA isolation and RT consist of two independent

sections fish sampled for investigating tissue distribution of 11β-HSD2 (10 male fish and 10

female fish which were not exposure to MT and EE2) and for detecting mRNA expressions

of hepatic vtg and gonadal steroidogenic genes following MT and EE2 exposure (18 male fish

and 18 female fish control MT or EE2 groups) Thus the number of fish sampled for RNA

isolation and RT was divided into two sections and shown in para 28 (pg10 line 204-205)

and 29 (pg10 line 210-211) respectively

The line time with steps and types of samples as follows

a Mar 2012 Ten male and 10 female adult fish were sampled for investigating tissue

distribution of 11β-HSD2

b May 2012 For detecting mRNA expressions of hepatic vtg and gonadal steroidogenic

genes following MT and EE2 exposure we sampled 18 fish (6 per group in triplicate) in each

group (control EE2 and MT groups in males and females) in which 12 gonads of 10 fish

were chosen for histological examination

8 Pg10-L214 What was the statistical unit and model How the time effect was included in

the model

Response Thanks for your suggestion In the present study the statistical unit is Ct value

of each detected gene (StAR cyp11a1 cyp17a1 cyp19a1a 3β-HSD and 11β-HSD) Statistical

differences were first tested with two-way ANOVA with treatment and time as two factors

Treatment was further analyzed with one-way ANOVA followed by LSD multiple comparison

(Pg7-L194-197)

As there is no way to rule out the effects of growth from day 7 to day 21 on the mRNA

expression of the detected genes the data of two-way ANOVA were not put in our manuscript We

added the sentence bdquoIn the female and male fish for gonadal StAR cyp17a1 11β-HSD2 and

cyp19a1a the interaction of treatment and time was statistically significant (P lt 005)‟ in the

revised manuscript (Pg18-L297-298) The two-way ANOVA analyzed results are as follows

Table 1 Transcriptional profiles of ovarian steroidogenic enzyme genes of two-way ANOVA

analysis results in female G ruras exposed to control MT and EE2 a

a Gene expression is expressed as the mean plusmn SD followed two-way ANOVA analysis P-values in

bold show significant difference (P lt 005)


analysis results in male G ruras exposed to control MT and EE2 a

a Gene expression is expressed as mean plusmn SD followed two-way ANOVA analysis P-values in


9 Table 1 only GSI values are of importance other data should be deleted

Response Thanks for your suggestion We have deleted other data as your suggestion in the

revised manuscript Revised table is showed below

Table 3 Gonadosomatic index (GSI) in G rarus following EE2 and MT exposure a

Exposure time

(days)

Exposure

concentrations

GSI ()

Female Male

7

control 938 plusmn 005 283 plusmn 001

25 ngL EE2 366 plusmn 002darr 230 plusmn 001

25 ngL MT 580 plusmn 004 273 plusmn 001

50 ngL MT 550 plusmn 003darr 299 plusmn 002


14




50 ngL MT 778 plusmn 006 428 plusmn 001uarr


21


25 ngL EE2 636 plusmn 003 141 plusmn 001darr



100 ngL MT 547 plusmn 004 414 plusmn004

a Data are expressed as mean plusmn SD Asterisks indicate significant difference from the control

groups ( P lt 005) The arrows bdquouarr‟ or bdquodarr‟ indicate the significant up-regulation or

down-regulation of the morphological data Absence of the arrows bdquouarr‟ or bdquodarr‟ indicates no

significant difference between exposure groups and control group

10 Pg12-L245-247 The number of spz decreased in the testes as exposure time went on

from 7 to 21 days The authors should precise if such time effect was significant and how such

decrease was related to the MT concentration

Response Thanks very much We have read references about analysis of gonads histological

results (Zha et al 2007 Kang et al 2008) After our close analysis we think that the

developmental stages of the germ cells of the gonads in our histological pictures are hardly

distinguishable So the diverse germ cells can‟t be counted and not statistical analysis can‟t be

conducted So we have changed the sentence ldquoIn MT exposures at 25 50 and 100 ngL the tissue

vacuolation increased and the number of spermatozoa decreased in the testes as exposure time

went on from 7 to 21 daysrdquo into ldquoIn MT exposures at 25 50 and 100 ngL for 7 to 21 days

macroscopic alterations (compared to controls) that the tissue vacuolation increased and the

number of spermatozoa decreased were found in the testesrdquo (Pg11 line 226-229)

11 The statistical conclusions concerning the time effect in relation to MT concentrations are

not clearly specified in the text so the authors should specify such conclusions for each studied

variable adding f and p values

Response Thanks for your suggestion In the present study we discussed the different

concentration of MT on rare minnow gonads steroidogenic genes and hepatic vtg According your

suggestion we have added the f and p values in the line 277 281 284 and 319 (Pg15-L277-278

Pg15-L281-283 Pg15-L284-285 Pg17-L319-321)

12 Pg12-L248 Features in para 32 have been described in the methodology section and this

section may not be considered as a result This is the same for the stability of reference genes in

para 33

Response Thanks for your suggestion We found the corresponding contents that ldquoThe

full-length cDNA of 11β-HSD2 (GeneBank ID KC454276) was obtained by RT-PCR and RACE

methods (Fig S1)rdquo in para 32 and ldquoThe geNorm Comparative Delta CT method BestKeeper

and NormFinder analysis results are shown in Table S2 β-actin showed most stable among the

four candidate reference genes by four methods of geNorm Delt CT analysis BestKeeper and

NormFinderrdquo in para 33 According your suggestion we have recomposed these sentences in the

revised manuscript (Pg 14-L261 Pg 14-L266)

References

M Li L Wang H Wang H Liang Y Zheng F Qin S LiuY Zhang Z Wang Molecular

cloning and characterization of amh dax1 and cyp19a1a genes and their response to

17α-methyltestosterone in Pengze crucian carp Comp Biochem Physiol Part C 157 (2013)

372ndash381

S Oumlrn H Holbech TH Madsen L Norrgren GI Petersen Gonad development and vitellogenin

production in zebrafish (Danio rerio) exposed to ethinylestradiol and methyltestosterone

Aquatic Toxicology 65 (2003) 397-411

Y Zheng L Wang M Li H Liang F Qin S Liu H Wang T Wu Y Zhang Z Wang

Molecular characterization of five steroid receptors from pengze crucian carp and their

expression profiles of juveniles in response to 17α-ethinylestradiol and 17α-methyltestosterone

General and comparative endocrinology 191 (2013) 113-122

IG Kang H Yokota Y Oshimaa Y Tsuruda Y Shimasaki T Honjo The effects of

methyltestosterone on the sexual development and reproduction of adult medaka (Oryzias

latipes) Aquat Toxicol 87 (2008) 37ndash46

J Zha Z Wang N Wang C Ingersoll Histological alternation and vitellogenin induction in

adult rare minnow (Gobiocypris rarus) after exposure to ethynylestradiol and nonylphenol

Chemosphere 66 (2007) 488ndash495

S Homklin SK Ong T Limpiyakorn Biotransformation of 17a-methyltestosterone in sediment

under different electron acceptor conditions Chemosphere 82 (2011) 1401ndash1407

K Barel-Cohen LS Shore M Shemesh A Wenzel J Mueller N Kronfeld-Schor Monitoring

of natural and synthetic hormones in a polluted river Journal of Environmental Management

78 (2006) 16ndash23

Highlights

The full-length cDNA of 11β-HSD2 was isolated and characterized successfully

Histological data demonstrated exposure of MT impaired gonadal development

MT disturbed mRNA expressions of hepatic vtg and gonadal steroidogenic genes

Highlights (for review)

1

Gonadal development and transcript profiling of steroidogenic enzymes in response to 1

17α-methyltestosterone in the rare minnow Gobiocypris rarus 2

Shaozhen Liua Lihong Wang

b Fang Qin

a Yao Zheng

a Meng Li

a Yingying Zhang

a Cong Yuan

a 3

Zaizhao Wanga

4

5

a College of Animal Science and Technology Northwest AampF University Shaanxi Key Laboratory 6

of Molecular Biology for Agriculture Yangling Shaanxi 712100 China 7

b The Hospital of Northwest AampF University Yanglin Shaanxi 712100 China 8

9

10

11

Corresponding Author 12

Zaizhao Wang PhD 13

College of Animal Science and Technology 14

Northwest AampF University 15

22 Xinong Road 16

Yangling Shaanxi 712100 China 17

Tel +86-29-87092139 18

Fax +86-29-87092164 19

E-mail zzwangnwsuafeducn 20

21

22

ManuscriptClick here to view linked References

2

Abstract 23

It is well known that natural and anthropogenic chemicals interfere with the hormonal system 24

of vertebrate and invertebrate organisms How these chemicals regulate gonadal steroidogenesis 25

remains to be determined The main objective of this study was to evaluate the effects of 26

17α-methyltestosterone (MT) a synthetic model androgen on gene expression profiles of six key 27

steroidogenic genes in adult rare minnow The full-length cDNA encoding 11β-hydroxysteroid 28

dehydrogenase-2 (11β-HSD2) was firstly isolated and characterized by RT-PCR and RACE 29

methods The gonadal transcript changes of StAR cyp11a1 3β-HSD cyp17a1 11β-HSD2 and 30

cyp19a1a in 6-month adult G rarus exposed to MT and 17α-ethinylestradiol (EE2) for 7 14 and 31

21 days were detected by qRT-PCR To make an effort to connect the transcriptional changes of 32

steroidogenic enzymes with effects on higher levels of biological organization and on VTG one 33

remarkable sensitive target of steroids body and gonad weights histology of gonads and hepatic 34

vtg mRNA level were measured MT caused varying degree of abnormalities in ovaries and testes 35

The hepatic vtg mRNA level was highly inhibited in females and slightly altered in males by MT 36

Transcripts of several steroidogenic genes including StAR cyp17a1 and cyp11a1 showed high 37

responsiveness to MT exposure in G rarus The gene expression profiles of these steroidogenic 38

genes in MT-treated groups were much distinct with the EE2-treated group 39

Keywords steroidogenic genes 17α-methyltestosterone 17α-ethinylestradiol histology 40

Gobiocypris rarus 41

42

43

44

3

1 Introduction 45

It is well known that a wide range of natural and anthropogenic chemicals interfere with the 46

hormonal system of vertebrate and invertebrate organisms [1 2] The field studies showed 47

physiologic and developmental abnormalities in wild fish such as increased vitellogenin (VTG) 48

concentrations and gonadal intersexuality of male fish in polluted aquatic environment [3-5] 49

Anthropogenic chemicals have several distinct effects including estrogenic androgenic 50

anti-estrogenic and anti-androgenic effects [6-8] Natural and man-made androgenic chemicals 51

are attracting more attention increasingly 52

The undetermined androgenic substances from water collected downstream of a kraft mill of 53

Fen Holloway River is associated with masculinization of female mosquitofish (Gambusia affinis 54

holbrooki) [9] The 17α-methyltestosterone (MT) is widely applied in aquaculture to control 55

sexual differentiation and induce sex-reversal of genetic females to phenotypic males [10-12] 56

MT was detected (133 ngL) in samples of effluent from a chemical factory [13] Concentrations 57

of 61 ngL testosterone and 45 ngL androstenedione were reported in final effluent of 58

municipal wastewater treatment plant [14] The MT could be detected in levels ranging from 59

41ndash70 ngL in waste water obtained from the Beijing area [15] MT has been recommended as a 60

reference chemical in tests for evaluating chemicals for endocrine disruption [16] In the present 61

study we chose the synthetic androgen MT as a test chemical 62

In the laboratory experiments MT demonstrates both androgenic and estrogenic effects in 63

several model fish MT exposure caused masculinization of female adult fathead minnow 64

(Pimephales promelas) exhibited development of nuptial tubercle which is a secondary sex 65

characteristic structure normally found in sexually mature males [17 18] In adult male medaka 66

4

(Oryzias latipes) both full life-cycle MT exposure at 2775 ngL and 3-week MT exposure at 380 67

ngL induced the development of testis-ova within the testicular tissues [19 20] Several studies 68

reported the VTG response in fish exposed to MT In both fathead minnow and medaka MT 69

exposure induced significant increase of VTG in adult males [17ndash19] In goldfish and fathead 70

minnow MT can induce VTG synthesis in their juveniles [21 22] However MT also resulted in 71

inhibition of VTG synthesis in female medaka and had no effect on the VTG level in the males 72

[20] 73

Xenoandrogens might affect the enzymes involved in steroidogenesis The changes of 74

steroidogenic enzyme activities resulted from xenoandrogens will most likely elicit alteration of 75

endogenous steriod hormone levels Several studies showed changes in estrogen and androgen 76

concentrations in fish exposed to MT MT can modulate 17β-estradiol (E2) and testosterone (T) 77

levels in a concentration- and time-dependent manner in cultured previtellogenic oocytes of 78

Atlantic cod (Gadus morhua) in vitro [23] The levels of 11-ketotestosterone (11-KT) and T 79

decreased in a concentration-dependent manner in male zebrafish exposed to MT at 45 to 623 80

ngL for 7 days [24] 81

Steroidogenesis involves a variety of enzymes and potential biochemical pathways Among 82

these biochemical pathways the regulatory step in acute steroid production is the transfer of 83

cholesterol across the mitochondrial membrane by steroidogenic acute regulatory protein (StAR) 84

and the subsequent conversion to pregnenolone by Cytochrome P450-mediated side-chain 85

cleavage enzyme (P450scc cyp11a1) [25ndash27] Cytochrome P450 aromatase (cyp19a1) is another 86

key steroidogenic enzyme responsible for the conversion of a range of androgens into oestrogen 87

[28] It has been demonstrated that teleost fish have two distinct isoforms of cyp19a1 genes 88

5

cyp19a1a and cyp19a1b Cyp19a1a is predominantly expressed in the ovary and involved in 89

sexual differentiation and ovarian development whereas cyp19a1b mainly expressed in the brain 90

[29] The 3β-hydroxysteroid dehydrogenase (3β-HSD) is a key enzyme catalyzing the synthesis of 91

the potent steroid hormones progesterone 17α-hydroxyprogesterone androstenedione and 92

testosterone from their much less hormonally active precursors pregnenolone 93

17α-hydroxypregnenolone dehydroepiandrosterone respectively [30 31] Hydroxylase and lyase 94

activity of cytochrome 17α-hydroxylase 17 20-lyase 1 (CYP17A1) are required for the synthesis 95

of testosterone [32] The functions of 11β-hydroxysteroid dehydrogenase 2 (11β-HSD2) is to 96

convert 11β-hydroxytestosterone to 11-ketotestosterone (11-KT) the main androgen found in the 97

majority of fish species [33 34] 98

The present study was aimed to evaluate the effects of MT at low concentrations (25-100ngL) 99

on gene expression profiles of six key steroidogenic genes In addition we investigated the effects 100

of MT on the transcriptional level of hepatic VTG and gonadal development Rare minnow 101

Gobiocypris rarus was used as model fish in the present study G rarus is a Chinese freshwater 102

cyprinid endemic to the upstream of Yangze River Sichuan Province China It is an appropriate 103

ecological test organism for the assessment of endocrine disrupting chemicals due to its small size 104

(30-80 mm in total length) ease of culture short life cycle (maturation within six to eight weeks) 105

and high sensitivity to aquatic pollutants [35] We deduce that MT exposure will produce 106

differential gene expression profiles of these steroidogenic genes and subsequently change the 107

levels of endogenous steroid hormones with significant effects on gonad development in Chinarsquos 108

indigenous model fish G rarus 109

2 Materials and methods 110

6

21 Animals and chemicals 111

Fertilized eggs were obtained from the female fish spawned in our laboratory After hatching 112

the larvae were fed Artemia nauplii (lt 24 h after hatching) twice a day After 30 days post 113

fertilization (dpf) fish were fed chironomid larvae once a day The adult fish were raised in 125 L 114

glass tanks with dechlorinated tap water at 25 plusmn 2 degC The photoperiod was 14 h 10 h lightdark 115

EE2 (purity gt 98) and MT (purity gt 990) were purchased from Sigma Chemicals Inc (St 116

Louis MO USA) MT and EE2 were dissolved in ethanol (AR analytical reagent) 117

22 MT and EE2 exposure 118

Adult rare minnow at 184 dpf (113 plusmn 033 g in weight 48 plusmn 05 cm in total length) were 119

exposed to MT and EE2 or solvent control (0001 ethylalcohol vv) in 45 liters glass tanks (36 120

liter water per tank) for 7 14 and 21 days (approximately 1 gram fish per liter water) The nominal 121

MT concentrations were 25 50 and 100 ngL As a potent synthetic estrogen EE2 served as a 122

reference estrogen control in the present exposure experiment The nominal EE2 concentration 123

was 25 ngL [35] Half of the exposure solution was changed every day Following exposure the 124

fish were immediately dissected and gonad tissues were frozen in liquid nitrogen and kept 125

individually in frozen tubes at -80 oC until use All exposure experiments were conducted in 126

triplicate in separate tanks at the same time 127

23 Morphometry and gonadal histological examination 128

The body weight and body length of fish were firstly measured Following sampling the 129

gonad tissues were weighed And the gonadosomatic index was calculated using the formula of 130

GSI = 100 times gonad weightbody weight [17] These data are expressed as mean plusmn SD and 131

statistical differences were tested by Analysis of Variance (ANOVA) and least significant 132

7

difference (LSD) Then the gonads were immersed in paraformaldehyde (4) solution over 24 133

hours at 4 degC and prepared for histological examination We chose 10 fish in each group (control 134

EE2 and MT groups) Then formalin-fixed gonads were dehydrated and processed for paraffin 135

wax embedding Serial longitudinal sections with thickness of 7 μm were cut with a rotary 136

microtome (Leica RM2235 Leica Microsystems) and stained using hematoxylin-eosin (HE) 137

Microscopic examination was carried out using an Olympus CHC binocular microscope and 138

photography was done with a Motic Digital Microscope 139

24 RNA isolation and reverse transcription (RT) 140

The tissues of gonad brain liver muscle eye intestines and gill were sampled from adult 141

male and female G rarus to assay the tissue distribution of 11β-HSD2 mRNA Tissue samples for 142

cDNA cloning and quantitative real-time PCR (qRT-PCR) were homogenized in TRIZOL reagent 143

(Invitrogen) and the total RNAs were extracted as described previously [36] cDNAs were 144

synthesized from total RNA with M-MLV reverse transcriptase (Invitrogen) and oligo (dT)18 145

primer in 20 μL final volume 146

25 Complementary DNA cloning of 11β-HSD2 147

The full-length cDNA sequence of G rarus 11β-HSD2 gene was generated in three 148

consecutive steps using reverse transcription polymerase chain reaction (RT-PCR) and rapid 149

amplification of cDNA ends (RACE) strategies [37] 11β-HSD2 was isolated from the testis The 150

cDNA fragment of 11β-HSD2 was obtained by PCR using primers designed from the consensus 151

sequences of teleosts (Table S1) The methods of TA cloning and RACE were described in the 152

previous study [38] 153

26 Sequence analysis 154

8

The full-length cDNA of 11β-HSD2 was assembled by alignment of the internal core fragment 155

5rsquo- and 3rsquo-RACE fragment using SeqMan program of Lasergene software (DNASTAR Inc) 156

Amino acid multiple alignments were performed using the Megalign program of Lasergene 157

software To establish phylogenetic tree for the G rarus 11β-HSD2 we aligned diverse vertebrate 158

11β-HSD2 gene at the level of amino acid sequences by Clustal X (183) sequences alignment 159

program respectively [39] The neighbor-joining algorithms method of Mega 40 program 160

(Molecular Evolutionary Genetic Analysis) was used to construct phylogenetic tree [4041] 161

Bootstrap analyses were conducted using 1000 replicates 162

27 Reference gene Screening 163

To obtain a reliable reference gene for the normalization of qRT-PCR data four candidate 164

reference genes were selected to validate their expression stability These genes are β-actin 165

elongation factor 1-alpha (ef1a) glyceraldehyde-3-phosphate dehydrogenase (gapdh) and tubulin 166

alpha 1 (tuba1) qRT-PCR was performed using total RNA from the samples for mRNA tissue 167

distribution and exposure (including MT EE2 and control groups) qRT-PCR was performed by 168

CFX96 real-time PCR detection systems (Bio-Rad) thermocycler and SYBR Premix ExTaq II kit 169

(TaKaRa) The detailed procedures of qRT-PCR were the same as described previously [42] The 170

qRT-PCR primers are listed in Table S1 qRT-PCR efficiency (E) was calculated from the given 171

slops in CFX Manager software by a 10-fold diluted cDNA sample series with five dilution points 172

measured in triplicate E was determined by the equation E=10 (minus1slope)

[43] The programs of 173

geNorm [44] Comparative Delta CT method [45] BestKeeper [46] and NormFinder [47] were 174

used to calculate gene expression stability (M) value of reference genes 175

(httpwwwleonxiecomreferencegenephp) The M value is defined as the average pairwise 176

9

variation of a certain gene with the remaining genes tested The lowest M value indicates the most 177

stable reference gene whereas the highest M value indicates the least stable reference gene 178

28 Tissue distribution of 11β-HSD2 179

The distribution of 11β-HSD2 transcript was determined in the tissues of gonad brain liver 180

muscle eye intestine and gill of adult fish by qRT-PCR The qRT-PCR primers are listed in Table 181

S1 To verify the specificity of the primers they were tested in normal PCR amplification and the 182

PCR products were visualized on a 15 agarose gel before qRT-PCR qRT-PCR data were 183

obtained as Ct The amounts of 11β-HSD2 mRNA were normalized to the most reliable reference 184

gene The relative change in mRNA expression levels of the four genes in different tissues were 185

calculated by 2minusΔΔCt

method [48] The analysis was performed on tissues of 10 males and 10 186

females All data are expressed as mean plusmn SD 187

29 Detecting mRNA expressions of hepatic vtg and gonadal steroidogenic genes following MT 188

and EE2 exposure 189

The gene expression patterns of hepatic vtg and gonadal StAR cyp11a1 3β-HSD cyp17a1 190

11β-HSD2 and cyp19a1a were detected for the fish exposed to MT and EE2 by qRT-PCR Each 191

transcript was analyzed on 18 individuals (6 per group in triplicate) The relative changes of 192

mRNA levels of these genes after MT and EE2 exposure were calculated using 2minusΔΔCt

method with 193

the formula F=2minusΔΔCt

ΔΔCt=(Ct target gene - Ct reference gene)MT or EE2-(Ct target gene - Ct reference gene)control 194

[48] Data are expressed as the fold change compared to control groups Statistical differences 195

were first tested with two-way ANOVA with treatment and time as two factors Treatment was 196

further analyzed with one-way ANOVA followed by LSD multiple comparison 197

3 Results 198

10

31 GSI and gonadal histology 199

In the present study the data of GSI were shown in Table 1 In the female fish exposure for 7 200

days MT at 50 and 100 ngL significantly decreased the GSI compared to the control groups 201

Meanwhile EE2 exposure for 7 and 14 days significantly reduced the GSI compared to the control 202

groups in female fish Following exposure for 21 days EE2 significantly reduced GSI compared 203

to the control groups respectively in the male fish 204

The histological results of EE2 and MT exposure showed inhibition of oogenesis and 205

spermatogenesis (Fig 1 and 2) The ovaries of female fish exposed to 25 ngL EE2 for 7 days 206

contained a high proportion of perinucleolar oocytes (Poc the most immature oocyte) a small 207

number of cortical alveolus stage oocytes (Coc the less mature oocyte) and no vitellogenic 208

oocyte (Voc the most mature oocyte) (Fig 1B1) The prolonged exposure time of 14 and 21 days 209

elicited the most immature oocyte Poc as the only component of the ovaries (Fig 1B2 1B3) In 210

the MT exposures at 25 and 50 ngL the number of immature oocytes (Coc and Poc) increased 211

however the number of mature oocyte (Voc) decreased as treatment time went on from 7 to 21 212

days (Fig 1Cs and 1Ds) MT exposure at 100 ngL for 7 days strongly inhibited the oocyte 213

development with the most immature oocyte Poc as the sole component in the ovaries (Fig 1E1) 214

However as exposure time prolonged (14 and 21 days) Poc and Coc were observed in the ovaries 215

(Fig 1E2 and 1E3) 216

In the male G rarus no abnormal testicular development was found in the control groups (Fig 217

2A1 2A2 and 2A3) In the male fish of EE2 groups more interstitial tissue was observed in testes 218

compared to control groups (Fig 2B1 and B2) EE2 exposure for 14 days more serious 219

vacuolation and extremely abnormal connective tissue were detected with few spermatozoa or 220

11

spermatocytes observed in the testes (Fig 2B2) Meanwhile abnormal connective tissues with few 221

spermatozoa were present in all three EE2 exposure groups In MT exposures at 25 50 and 100 222

ngL for 7 to 21 days macroscopic alterations (compared to controls) that the tissue vacuolation 223

increased and the number of spermatozoa decreased were found in the testes (Fig 2C1 C2 and 224

C3) 225

32 Molecular characterization of 11β-HSD2 226

The full-length cDNA of 11β-HSD2 (GeneBank ID KC454276) is 1965 bp including 1242 bp 227

ORF encoding a putative protein of 413 amino acids with a theoretical pI of 794 and a calculated 228

molecular weight of 460 kDa a 238 bp 5rsquo-UTR and a 485 bp 3rsquo-UTR (Fig S2) Polyadenylation 229

signal (AATAAA) was found in the 3rsquo-UTR of 11β-HSD2 (Fig S2) 230

33 Homology and phylogenetic analysis of putative amino acid sequences for 11β-HSD2 231

The amino acid sequence of G rarus 11β-HSD2 shows the highest homology with zebrafish 232

and the conserved regions are NAD-binding domain 11β-HSD2 Conserved sequence and 233

Catalytic-site (Fig S3) To better understand the position of G rarus 11β-HSD2 in the 234

evolutionary history of the respective protein phylogenetic tree was constructed from the amino 235

acid alignments for 11β-HSD2 of teleosts and mammals using the neighbor-joining method G 236

rarus 11β-HSD2 was more close to their counterparts of zebrafish than that of other teleosts (Fig 237

S4) 238

34 Expression stability analysis of reference genes 239

The analysis of reference gene results is shown in Table S2 β-actin showed most stable 240

among the four candidate reference genes The recommended comprehensive ranking indicated 241

β-actin as the most stable reference gene Therefore β-actin was selected as the reference gene in 242

12

the present study 243


The tissue distribution of 11β-HSD2 mRNA is shown in Fig 3 11β-HSD2 was predominantly 245

expressed in liver The transcript of 11β-HSD2 in the testis was 4173-fold higher than that in the 246

ovary In females 11β-HSD2 transcript in the liver was 68154-fold higher than that in the ovary 247

However in males the 11β-HSD2 transcript in the liver was 201 times of that in testis The 248

transcript of 11β-HSD2 in the male brain was 165-fold higher than those in the female brain 249

36 Expression profiles of hepatic vtg in adult G rarus under MT and EE2 exposures 250

The expression of hepatic vtg in adult female and male G rarus was detected in the present 251

study (Table 2) In the female fish transcript of hepatic vtg significantly increased for 929 252

3119 and 740 fold (P=001 P=002 and P=005 respectively) following 7- 14- and 21-day EE2 253

exposure compared to the control respectively Seven-day exposure of MT at 50 ngL caused 254

extremely significant decrease of the hepatic vtg transcript in the female fish compared to control 255

groups (16937 fold P=004) The 14-day MT treatments at 25 to 100 ngL significantly 256

suppressed expression of hepatic vtg for 1552 to 369 fold in the female fish As time went on (7 257

14 and 21 days) MT treatment at 100 ngL significantly decreased the expression of hepatic vtg 258

for 1137 3161 and 78350 fold (P=005 P=005 and P=005 respectively) in the female fish 259

In the male fish EE2 exposure for 7 to 21 days caused significant increase of hepatic vtg 260

transcript with huge magnitude orders (760 230 and 106 thousand-fold Plt001 P=002 and 261

P=005 respectively) In the male fish the vtg expression was significantly stimulated for 39 to 262

22 fold by 7- and 14-day MT exposures at 50 and 100 ngL compared to control However 21-day 263

MT exposures at 25 and 50 ngL caused 1911- and 44-fold (P=005 and P=005) significant 264

13

decrease of vtg transcript in male fish respectively 265

37 Expression profiles of steroidogenic genes in adult G rarus under MT and EE2 exposure 266

In the female and male fish for gonadal StAR cyp17a1 11β-HSD2 and cyp19a1a the 267

interaction of treatment and time was statistically significant (P lt 005) The fold changes of gene 268

expression for gonadal StAR cyp11a1 3β-HSD cyp17a1 11β-HSD2 and cyp19a1a under MT and 269

EE2 exposure compared to controls are shown in Table 3 and 4 In the female G rarus EE2 270

exposure at 25 ngL for 7 14 and 21 days caused extremely significant decrease of the 271

steroidogenic enzyme genes except for 3β-HSD and 11β-HSD2 In the female G rarus the effects 272

of MT on 3β-HSD and 11β-HSD2 were weaker than that on the other 4 genes (StAR cyp11a1 273

cyp17a1 and cyp19a1a) Seven-day MT exposure at 25 ngL caused 222-fold significant decrease 274

of transcript for ovarian cyp11a1 and respective 184- 157-fold increase of ovarian cyp17a1 and 275

11β-HSD2 compared to controls (Table 3) Seven-day exposure to MT at 50 ngL caused 276

extremely significant decrease the ovarian transcript of StAR (824-fold) while significant increase 277

the ovarian transcript of cyp17a1 compared to controls (653-fold Table 3) Seven-day MT 278

exposure at 100 ngL significantly down-regulated expressions of ovarian StAR cyp11a1 3β-HSD 279

and cyp17a1 (84 to 5731 fold) Fourteen-day exposure to MT at 25 and 50 ngL significantly 280

down-regulated the ovarian transcripts of cyp11a1 and cyp17a1 compared to controls (367 to 281

1372 fold respectively Table 3) MT exposure at 50 ngL for 14 days significantly up-regulated 282

expression of ovarian cyp19a1a (603-fold Table 3) MT exposure at 100 ngL for 14 days 283

significantly down-regulated expression of all six ovarian steroidogenic enzyme genes compared 284

to controls (gt 232 fold Table 3) MT exposure at 25 ngL for 21 days significantly increased the 285

expression of StAR and cyp19a1a (323- and 247-fold respectively) However 21-day MT 286

14

exposure at 25 ngL significantly decreased the expression of cyp17a1 compared to controls 287

(396-fold Table 3) MT exposure at 50 ngL for 21 days extremely significantly stimulated the 288

expression of StAR cyp17a1 11β-HSD2 and cyp19a1a for 422 163 888 and 286 times in the 289

female fish respectively MT exposure at 100 ngL for 21 days significantly up-regulated the 290

expression of ovarian StAR and cyp19a1a (497 and 225 fold Table 3) The expression of 291

cyp17a1 was extremely significantly down-regulated for 1061 (Plt001) 6075 (Plt001) and 194 292

(Plt001) times following 7- 14- and 21-day exposure to MT at 100 ngL (Table 3) 293

In male G rarus EE2 exposure at 25 ngL in three durations caused generally significant 294

decrease of all transcripts of steroidogenic enzyme genes except for cyp19a1a compared to 295

controls (Table 4) Seven-day exposure to MT at 25 ngL significantly down-regulated the 296

expression of testicular StAR cyp11a1 and 11β-HSD2 for 53 159 and 359 fold respectively 297

(Table 4) MT exposure at 50 ngL for 7 days significantly suppressed the testicular transcripts of 298

StAR and 11β-HSD2 (239 and 180 fold Table 4) Seven-day exposure to MT at 100 ngL caused 299

extremely significant decrease of testicular transcripts for StAR cyp11a1 3β-HSD cyp17a1 and 300

11β-HSD2 (237 to 2117 fold Table 4) Fourteen-day MT treatment at 25 ngL significantly 301

down-regulated the expression of 3β-HSD for 244 times MT exposure at 50 ngL for 14 days 302

resulted in 5091-fold extremely significant increase of testicular cyp17a1 transcript (Table 4) MT 303

exposure at 100 ngL for 14 days significantly up-regulated expression of cyp17a1 and 11β-HSD2 304

for 7782 and 220 fold respectively In 21-day exposure duration MT at 25 ngL extremely 305

significantly up-regulated the expression of 11β-HSD2 (239 fold) MT at 50 ngL resulted in 168- 306

and 208-fold significant increase of testicular 11β-HSD2 and cyp17a1 transcripts (Table 4) MT at 307

100 ngL resulted in extremely significant decrease of testicular StAR cyp11a1 and cyp17a1 308

15

transcript (532 to 3425 fold Table 4) MT at 100 ngL exposure for 7 14 and 21 days the mRNA 309

expression of gene StAR were all decreased (873 236 and 532 fold Plt001 P=005 and Plt001 310

respectively) 311

4 Discussion 312

In the present study gonadal development gonadosomatic index (GSI) and vtg mRNA were 313

employed as biological endpoints for detecting the MT effects EE2 treatment at 25 ngL caused 314

degeneration of ovaries and testes reduced GSI of female and male fish which is in line with the 315

findings of experiment on rare minnow [35] EE2 treatment at relatively low concentrations (1-25 316

ngL) induced testes-ova in adult rare minnow [35] However in the present study no testis-ova 317

were observed in EE2-treated fish The previous study demonstrated that the Betta splendens had 318

smaller gonads after EE2 (100 ngL) exposure for 4 weeks compared to the control [49] Similarly 319

in our study EE2 had inhibited the rare minnow gonads The discrepancy of exposure duration 320

andor developmental status of testis of the subject male fish in the two experiments could be the 321

cause of the difference MT treatment caused reduced GSI in 7-day exposure and degeneration of 322

oocytes in the ovaries of the female fish in three exposure durations (7 14 and 21 days) at three 323

concentrations (25 50 and 100 ngL) which is consistent with the results of experiments on 324

MT-treated female fathead minnow and medaka [20 50] MT exposure at 50 and 100 ngL also 325

resulted in histological abnormalities in testes of the adult male fish However MT at 01-50 μgL 326

had no effect on the testicular structure and amount of sperm in fathead minnow [50] In 327

MT-treated male medaka the testes in the 225-188 ngL groups had no histological aberrations 328

and only 380 ngL MT caused the occurance of testis-ova [20] Therefore in the present study the 329

testis of rare minnow showed higher responsiveness to MT compared to fathead minnow and 330

16

medaka The mechanism by which MT induced these phenomena in female and male rare minnow 331

is undetermined Further investigations are needed to address the mechanism underlying abnormal 332

gonadal development in rare minnow exposed to MT 333

It is well established that EE2 is a potent inducer of VTG in fish acting via the hepatic ERs 334

So EE2 was chosen as a positive estrogenic control in the experiment The response of vtg mRNA 335

to estrogenic chemicals is more sensitive than the response of VTG protein For example the 336

induction of vtg mRNA expression was two-order magnitude greater than that of plasma VTG 337

protein level in the male fathead minnow exposed to EE2 at 10 ngL [51] So in the present study 338

vtg mRNA levels were detected in liver where VTG is synthesized As expected EE2 induced 339

hepatic mRNA expression of vtg gene in both male and female rare minnow and the response in 340

treated male fish was much more dramatic than the EE2-treated females which is consistent with 341

the observations for fathead minnow in both field and laboratory EE2 exposures [51 52] 342

In the male fish MT exposure for 7 and 14 days induced the transcription of hepatic vtg gene 343

albeit not significantly in some cases Interestingly the 21-day MT exposure inhibited mRNA 344

expression of hepatic vtg in male fish The present findings suggest that androgens may involve in 345

the regulation of VTG synthesis Several previous studies have been demonstrated the ability of 346

MT to influence VTG synthesis Seven-day exposure of MT at measured concentration of 45 ngL 347

increased the VTG concentration in whole-body homogenates of adult male fish [24] Furthermore 348

in full life cycle test of MT exposure on Japanese medaka the hepatic VTG level was increased in 349

F1 generation male at 60 days posthatch exposed to 998 ngL MT [19] In fathead minnow MT at 350

higher concentrations (02-200 microgL) also induced VTG synthesis in the male fish [1718] The 351

cause of induced VTG synthesis for MT probably is that MT can be aromatized into 352

17

methylestradiol (ME2) and ME2 with estrogenic effect subsequently up-regulate vtg expression 353

via the hepatic estrogen receptor [18] 354

MT exposures at 25-100 ngL in three durations nearly all significantly suppressed expression 355

of hepatic vtg gene in female rare minnow The response of vtg transcript upon MT in female fish 356

is in line with several previous studies In full life cycle of MT exposure in Japanese medaka the 357

hepatic VTG level was significantly decreased in F1 generation female at 60 days posthatch 358

exposed to MT at 035-998 ngL [19] Significant decrease of hepatic vtg mRNA showed in the 359

hermaphrodite fish (Kryptolebias marmoratus) treated with MT [53] However 21-day MT 360

exposure at high concentrations (02 and 2 mgL) induced plasma VTG in adult female fathead 361

minnow [17] We speculate that there are species and sexual differences in VTGvtg mRNA 362

response to MT for fish 363

In the present study we analyzed the transcriptional responses of main steroidogenic genes to 364

MT and EE2 Our investigations demonstrated that the treatment of the potent estrogen EE2 365

(25ngL) for 7 to 21 days pronouncedly disturbed mRNA expressions of the main steroidogenic 366

genes in gonads of both male and female fish In testis almost the three durations of EE2 exposure 367

(7 14 and 21 day) markedly suppressed the expressions of StAR cyp11a1 cyp17a1 and 11β-HSD2 368

but did not affect mRNA levels of 3β-HSD and cyp19a1a EE2 exposure of the three durations 369

caused much greater inhibition of ovarian StAR cyp11a1 and cyp17a1 than those in testis The 370

EE2 exposures resulted in significant decrease of cyp19a1a mRNA and no effect on the transcript 371

of 11β-HSD2 in ovary Similar to the situation of 3β-HSD in testis the ovarian 3β-HSD transcript 372

was almost not affected by EE2 The results in the present study are consistent with the inhibitory 373

effects of EE2 on cyp17 and 11β-HSD in male fathead minnow [51] In testis of rainbow trout 374

18

(Oncorhynchus mykiss) EE2 also inhibited the expression of four genes coding for enzymes 375

involved in androgen production including cyp11a1 cyp17 3β-HSD and 11β-HSD2 [54] In the 376

zebrafish A clear down-regulation of StAR mRNA in gonads were found in the EE2 groups [55] 377

The transcripts of ovarian StAR cyp11a1 cyp17a1 and cyp19a1a were strongly inhibited by 25 378

ngL EE2 for 7 14 and 21 days All these genes code for important enzymes for estrogen 379

production in female fish For example StAR and CYP11A1 are the key proteins involved in the 380

early steroidogenic pathway The CYP19A1 is a crucial steroidogenic enzyme catalyzing the final 381

step in the conversion of androgens to estrogens in fish [29] The marked decrease of mRNA 382

levels for these key steroidogenic genes in response to EE2 implies the reduced estrogen in ovary 383

of female fish which is in line with the significant decrease of plasma E2 in zebrafish exposed to 384

EE2 at 15-100 ngL for 2 and 7 days [56] The concurrent transcriptional responses of several 385

steroidogenic genes in testis and ovary could result from the inhibition of a common 386

transcriptional factor such as the SF-1 gene In fathead minnow EE2 (10ngL) exposure for 21 387

days significantly inhibited mRNA expression of SF-1 in both male and female fish [51] In the 388

present study the strong inhibition of steroidogenic enzymes at transcriptional level in the female 389

exposed to EE2 suggests that it is the exogenous EE2 but not the endogenous E2 that significantly 390

induces the expression of hepatic vtg 391

In the present study several genes of steroidogenic enzymes in ovary of female fish were 392

inhibited by MT Cyp17a1 mRNA expression was strongly repressed by 7- 14- and 21-day MT 393

treatment at 25 to 100 ngL MT exposures at 50 ngL for 7 day and at 100 ngL for both 7 and 14 394

days significantly inhibited StAR mRNA level MT treatment for duration of 7 days at 25 and 100 395

ngL and 14 days at 25 to 100 ngL caused marked decrease of cyp11a1 transcript 3β-HSD was 396

19

strongly repressed by MT (100 ngL) for 7 and 14 days The down-regulation of genes encoding 397

steroid enzymes by androgens has already been reported in fish species including rainbow trout 398

[57 58] and the Nile tilapia [59] In mammal the mRNA expression of StAR cyp11a1 cyp17a1 399

and 3β-HSD was also inhibited by androgens [60 61] A few regulatory factors have been studied 400

on this down-regulation of androgens SF-1 is a transcription factor which has been shown to 401

stimulate the mRNA expression of StAR cyp11a1 cyp17a1 and 3β-HSD in mammal [62 63] In 402

female rainbow trout three-month treatment of another androgen 11β-hydroxyandrostenedione 403

(11βOHΔ4) down-regulated sf-1 expression in ovary [57] So SF-1 may be one of regulators 404

involved in the transcriptional inhibition of steroidogenic enzyme genes The prolonged MT 405

treatment (21 days) at 25-100 ngL resulted in significant increase of transcripts for StAR and 406

cyp19a1a which both code for enzymes involving in key steps of steroid production in ovary The 407

up-regulation of the two key steroidogenic genes which imply the increase of the enzymes could 408

counteract the continuing complete block of ovarian steroidogenesis Our previous study found 409

100 ngL MT significantly increased the mRNA expression of cyp19a1a in Pengze crucian carp 410

[64] In addition the up-regulation of cyp19a1a upon MT treatment for 21 days could be 411

conducive to the enzymatic conversion of MT to 17α-methylestradiol (ME2) by CYP19A1 412

aromatase In female fathead minnow ME2 was detected in plasma samples following the 7-day 413

MT exposure which confirms the conversion of MT to ME2 [18] 414

Our experiment demonstrates that MT treatment of male rare minnow resulted in transcriptional 415

down-regulation of testicular steroidogenic enzymes genes in the short duration (7 days) and as 416

the treatment time prolonged some genes had a decelerating trend of down-regulation or reversed 417

to up-regulation For example 7-day MT exposure at 25-100 ngL all strongly inhibited StAR 418

20

expression in testis (239- to 873-fold P lt 001) whereas only the highest concentration (100 419

ngL) of MT caused significant decrease of StAR transcript in the longer exposure (236-fold P = 420

005 and 532-fold P lt 001 in 14- and 21-day exposure respectively) For cyp17a1 gene 7-day 421

MT treatment at 100 ngL markedly suppressed (2117-fold P lt 001) its expression however 422

14-day MT exposures at 50 and 100 ngL strongly up-regulated its expression (5091- and 423

7782-fold P lt 001) Similar to cyp17a1 gene the 11β-HSD2 expression also had a reversed 424

regulation with the down-regulation upon MT exposure of the short duration (7 days) at 25-100 425

ngL (180- to 1182-fold P le 001) and up-regulation upon MT exposure for longer duration 426

(220-fold for 14-day exposure at 100 ngL 239- and 168-fold for 21-day exposure at 25-50 ngL 427

P le 001) The data of the present study is in line with the regulatory effects of another androgen 428

11βOHΔ4 on the transcripts of several steroidogenic enzymes in male rainbow trout [54] The 429

11βOHΔ4 treatment for 4 to 10 days caused significant decrease of mRNA levels for cyp11a1 430

3β-HSD and 11β-HSD2 in male rainbow trout For cyp17a1 gene it expression was 431

down-regulated by short term of exposure (4-10 days) of 11βOHΔ4 and up-regulated by long term 432

of treatment (16 days) on the contrary The transcriptional modulation of steroidogenic enzymes in 433

response to MT could be triggered by factors in HPG-axis In Leydig cells of male rat treated with 434

testosterone for 2 months the transcriptional down-regulation of steroidogenic enzymes (StAR 435

cyp11 and cyp17) coupled with significantly decreased LH levels in circulation suggest that MT 436

could regulate androgen production through LH-LHR-cAMP signaling [65] In the present study 437

MT at 25 to 100 ngL barely had effects on mRNA level of cyp19a1a which is consistent with 438

previous findings in male zebrafish [24] 439

In summary MT treatment induced abnormalities of ovaries and testes in adult rare minnow 440

21

which can not be distinguished with the observation in the fish treated with EE2 The hepatic vtg 441

as a well-known sensitive biomarker was barely weakly stimulated in male fish and markedly 442

inhibited in female upon MT exposure which hardly leads to conclusion that MT has estrogenic 443

effects in adult rare minnow as in the MT exposure tests on other teleosts such as fathead minnow 444

The transcripts of several steroidogenic enzymes genes such as StAR cyp17a1 and cyp11a1 445

showed high responsiveness to MT exposure in rare minnow Moreover the MT- and EE2-treated 446

fish demonstrated distinct gene expression profiling for these steroidogenic enzymes genes 447

suggesting that the similar phenotypic effects in the higher levels of biological organization for 448

MT and EE2 could result from distinct molecular mechanisms The mechanisms underlying the 449

adverse effects of exogenous androgens can not be determined by the studies merely limited to the 450

gene expression of steroidogenic enzymes and future investigations needs to be performed in wide 451

spectrum of systems such as transcription regulation nuclear receptor signaling and 452

LH-LHR-cAMP signaling 453

Acknowledgments 454

This study was supported by grants from the National Natural Science Foundation of China 455

(31270547) the Natural Science Foundation of Shaanxi Province China (2011JM3009) and the 456

Fundamental Research Funds for the Central Universities in Northwest AampF University 457

(QN2011062) 458

Appendix A Supplementary data 459

Supplementary data associated with this article can be found in the online version 460

461

462

22

Reference 463

[1] P Matthiesen P Gibbs Critical appraisal of the evidence for tributyltin-mediated endocrine 464

disruption in mollusks Environ Tox Chem 17 (1997) 37ndash43 465

[2] M Tillmann U Schulte-Oehlmann M Duft B Markert J Oehlmann Effects of endocrine 466

disruptors on prosobranch snails (Mollusca Gastropoda) in the laboratory Part III 467

Cyproterone acetate and vinclozolin as antiandrogens Ecotoxicology 10 (2001) 373ndash388 468

[3] S Hashimoto H Bessho A Hara M Nakamura T Iguchi K Fujita Elevated serum 469

vitellogenin levels and gonadal abnormalities in wild male flounder (Pleuronectes yokohamae) 470

from Tokyo Bay Japan Mar Environ Res 49 (2000) 37ndash53 471

[4] R Van Aerle M Nolan S Jobling LB Christiansen JP Sumpter CR Tyler Sexual 472

disruption in a second species of wild cyprinid fish (the gudgeon Gobio gobio) in united 473

kingdom freshwaters Environ Toxicol Chem 20 (2001) 2841ndash2847 474

[5] JH Writer LB Barber GK Brown HE Taylor RL Kiesling ML Ferrey ND Jahns 475

SE Bartell HL Schoenfuss Anthropogenic tracers endocrine disrupting chemicals and 476

endocrine disruption in Minnesota lakes Sci Total Environ 409 (2010) 100ndash111 477

[6] JS Fisher Environmental anti-androgens and male reproductive health focus on phthalates 478

and testicular dysgenesis syndrome Reproduction 127 (2004) 305ndash315 479

[7] JP Sumpter Endocrine disrupters in the aquatic environment an overview Acta Hydrochim 480

Hydrobiol 33 (2005) 9ndash16 481

23

[8] AM Al-Ansari SK Atkinson JR Doyle VL Trudeau JM Blais Dynamics of uptake and 482

elimination of 17α-ethinylestradiol in male goldfish (Carassius auratus) Aquat Toxicol 483

132ndash133 (2013) 134ndash140 484

[9] LG Parks CR Lambright EF Orland LJ Guillette Jr GT Ankley LE Gray Jr 485

Masculinization of female mosquito fish in Kraft mill effluent-contaminated Fenholloway 486

River water is associated with androgen receptor agonist activity Toxicol Sci 62 (2001) 487

257ndash267 488

[10] K Cailleaud H Budzinski S Lardy S Augagneur S Barka S Souissi J Forget-Leray 489

Uptake and elimination and effect of estrogen-like contaminants in estuarine copepods an 490

experimental study Environ Sci Pollut Res 18 (2011) 226ndash236 491

[11] T Haugen E Andersson B Norberg GL Taranger The production of hermaphrodites of 492

Atlantic cod (Gadus morhua) by masculinization with orally administered 493

17α-methyltestosterone and subsequent production of all-female cod populations Aquaculture 494

311 (2011) 248-254 495

[12] El-Greisy ZA and AE El-Gamal Monosex production of tilapia Oreochromis niloticus 496

using different doses of 17α-methyltestosterone with respect to the degree of sex stability after 497

one year of treatment Egyptian Journal of Aquatic Research 38 (2012) 59-66 498

[13] BMG Blankvoort RJT Rodenburg AJ Murk JH Koeman R Schilt JMMJG Aarts 499

Androgenic activity in surface water samples detected using the AR-LUX assay indication for 500

mixture effects Environ Toxicol Pharmacol 19 (2005) 263ndash272 501

[14] EP Kolodziej JL Gray DL Sedlak Quantification of steroid hormones with pheronmonal 502

24

properties in municipal wastewater effluent Environ Toxicol Chem 22 (2003) 2622ndash2629 503

[15] L Sun Y Liu X Chu J-M Lin Trace Analysis of Fifteen Androgens in Environmental 504

Waters by LC-ESI-MS-MS Combined with Solid-Phase Disk Extraction Cleanup 505

Chromatographia 9-10 (2010) 867ndash873 506

[16] Organization for Economic Cooperation and Development Final report from the OECD 507

expert consultation meeting London UK 28ndash29th October 1998 Report 9906 Environmental 508

Health and Safety Division Paris (1999) France 509

[17] GT Ankley KM Jensen MD Kahl JJ Korte EA Makynen Description and evaluation 510

of a short-term reproductive test with the fathead minnow (Pimephales promelas) Environ 511

Toxicol Chem 20 (2001) 1276ndash1290 512

[18] MW Hornung KM Jensen JJ Korte MD Kahl ED Durhan JS Denny TR Henry 513

GT Ankley Mechanistic basis for estrogenic effects in fathead minnow (Pimephales 514

promelas) following exposure to the androgen MT conversion of 17α-methyltestosterone to 515

17α-methylestradiol Aquat Toxicol 66 (2004) 15ndash23 516

[19] M Seki H Yokota H Matsubara M Maeda H Tadokoro K Kobayashi Fish full 517

life-cycle testing for androgen methyltestosterone on medaka (Oryzias latipes) Environ 518


[20] IG Kang H Yokota Y Oshimaa Y Tsuruda Y Shimasaki T Honjo The effects of 520

methyltestosterone on the sexual development and reproduction of adult medaka (Oryzias 521

latipes) Aquat Toxicol 87 (2008) 37ndash46 522

[21] SH Hori T Kodama K Tanahashi Induction of vitellogenin synthesis in goldfish by 523

massive doses of androgens Gen Comp Endocrinol 37 (1979) 306ndash320 524

25

[22] M Zerulla R Laumlnge T Steger-Hartmann G Panter T Hutchinson DR Dietrich 525

Morphological sex reversal upon short-term exposure to endocrine modulators in juvenile 526

fathead minnow (Pimephales promelas) Toxicol Lett 131 (2002) 51ndash63 527

[23] TM Kortner A Arukwe Effects of 17α-methyltestosterone exposure on steroidogenesis and 528

cyclin-B mRNA expression in previtellogenic oocytes of Atlantic cod (Gadus morhua) Comp 529

Biochem Physiol Part C 146 (2007) 569ndash580 530

[24] L Andersen R Goto-Kazeto JM Trant JP Nash B Korsgaard P Bjerregaard Short-term 531

exposure to low concentrations of the synthetic androgen methyltestosterone affects 532

vitellogenin and steroid levels in adult male zebrafish (Danio rerio) Aquat Toxicol 76 (2006) 533

343ndash352 534

[25] BJ Clark DM Stocco StAR-A tissue specific acute mediator of steroidogenesis Trends 535

Endocrin Met 7 (1996) 227ndash233 536

[26] DM Stocco X Wang Y Jo PR Manna Multiple signaling pathways regulating 537

steroidogenesis and steroidogenic acute regulatory protein expression more complicated than 538

we thought Mol Endocrinol 19 (2005) 2647ndash2659 539

[27] SY Skolness EJ Durhan N Garcia-Reyero KM Jensen MD Kahl EA Makynen D 540

Martinovic-Weigelt E Perkins DL Villeneuve GT Ankley Effects of a short-term 541

exposure to the fungicide prochloraz on endocrine function and gene expression in female 542

fathead minnows (Pimephales promelas) Aquat Toxicol 103 (2011) 170ndash178 543

[28] ER Simpson MS Mahendroo GD Means MW Kilgore MM Hinshelwood S 544

Graham-Lorence B Amarneh Y Ito CR Fisher MD Michael CR Mendelson SE 545

26

Bulun Aromatase cytochrome P450 the enzyme responsible for estrogen biosynthesis Endocr 546

Rev 15 (1994) 342ndash355 547

[29] GV Callard AV Tchoudakova M Kishida E Wood Differential tissue distribution 548

developmental programming estrogen regulation and promoter characteristics of cyp19 genes 549

in teleost fish J Steroid Biochem Mol Biol 1ndash5 (2001) 305ndash314 550

[30] A Arukwe Steroidogenic acute regulatory (StAR) protein and cholesterol side-chain 551

cleavage (P450scc)-regulated steroidogenesis as an organ-specific molecular and cellular 552

target for endocrine disrupting chemicals in fish Cell Biol Toxicol 24 (2008) 527ndash540 553

[31] N Sakai M Tanaka M Takahashi S Fukada JI Mason Y Nagahama Ovarian 3 554

beta-hydroxysteroid dehydrogenasedelta 5-4-isomerase of rainbow trout its cDNA cloning 555

and properties of the enzyme expressed in a mammalian cell FEBS Lett 350 (1994) 309-313 556

[32] RS Kumar S Ijiri JM Trant Changes in the expression of genes encoding steroidogenic 557

enzymes in the channel catfish (Ictalurus punctatus) ovary throughout a reproductive cycle 558

Biol Reprod 63 (2000) 1676ndash1682 559

[33] JQ Jiang DS Wang B Senthilkumaran T Kobayashi HK Kobayashi A Yamaguchi W 560

Ge G Young Y Nagahama Isolation characterization and expression of 11β-hydroxysteroid 561

dehydrogenase type 2 cDNAs from the testes of Japanese eel (Anguilla japonica) and Nile 562

tilapia (Oreochromis niloticus) J Mol Endocrinol 31 (2003) 305ndash315 563

[34] ME Baker 11β-Hydroxysteroid dehydrogenase-type 2 evolved from an ancestral 564

17β-Hydroxysteroid dehydrogenase-type 2 Biochem Biophys Res Commun (2010) 565

215ndash220 566

[35] J Zha Z Wang N Wang C Ingersoll Histological alternation and vitellogenin induction in 567

27

adult rare minnow (Gobiocypris rarus) after exposure to ethynylestradiol and nonylphenol 568

Chemosphere 66 (2007) 488ndash495 569

[36] H Wang J Wang T Wu F Qin X Hu L Wang Z Wang Molecular characterization of 570

estrogen receptor genes in Gobiocypris rarus and their expression upon endocrine disrupting 571

chemicals exposure in juveniles AquatToxicol 101 (2011) 276-287 572

[37] MA Frohman MK Dush GR Martin Rapid production of full-length cDNAs from rare 573

transcripts Amplification using a single gene-specific oligonucleotide primer Proc Natl 574

Acad Sci USA 89 (1988) 8998ndash9002 575

[38] T Wu H Wang F Qin SZ Liu M Li P Xu Z Wang Expression of zona pellucida B 576

proteins in juvenile rare minnow (Gobiocypris rarus) exposed to 17α-ethinylestradiol 577

4-nonylphenol and bisphenol A Comp Biochem Physiol C 155 (2012) 259ndash268 578

[39] JD Thompson TJ Gibson F Plewniak F Jeanmougin DG Higgins The ClustalX 579

Windows interface flexible strategies for multiple sequence alignment aided by quality 580

analysis tools Nucleic Acids Res 25 (1997) 4876ndash4882 581

[40] N Saitou M Nei The Neighbor-Joining method-a new method for reconstructing 582

phylogenetic trees Mol Biol Evol 4 (1987) 406ndash425 583

[41] K Tamura J Dudley M Nei S Kumar MEGA4 Molecular Evolutionary Genetics 584

Analysis (MEGA) software version 40 Mol Biol Evol 24 (2007) 1596ndash1599 585

[42] SZ Liu F Qin HP Wang TT Wu YY Zhang Y Zheng M Li ZZ Wang Effects of 586

17α-ethinylestradiol and bisphenol A on steroidogenic messenger ribonucleic acid levels in the 587

gonads of the rare minnow Gobiocypris rarus Aquat Toxicol 10 (2012) 19ndash27 588

[43] R Rasmussen Quantification on the Light Cycler In Meuer S Wittwer C Nakagawara K 589

28

(Eds) Rapid cycle real-time PCR Methods and Applications Springer Press Heidelberg 590

2001 pp 21ndash34 591

[44] J Vandesompele K De Preter F Pattyn B Poppe N Van Roy A De Paepe F Speleman 592

Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of 593

multiple internal control genes Genome Biol 3 (2002) RESEARCH0034 594

[45] N Silver S Best J Jiang SL Thein Selection of housekeeping genes for gene expression 595

studies in human reticulocytes using real-time PCR BMC mol biol 7 (2006) 33 596

[46] MW Pfaffl A Tichopad C Prgomet TP Neuvians Determination of stable housekeeping 597

genes differentially regulated target genes and sample integrity BestKeeper-Excel-based tool 598

using pair-wise correlations Biotechnol Lett 26 (2004) 509ndash515 599

[47] CL Andersen JL Jensen TF Orntoft Normalization of real-time quantitative reverse 600

transcription-PCR data A model-based variance estimation approach to identify genes suited 601

for normalization applied to bladder and colon cancer data sets Cancer Res 64 (2004) 602

5245ndash5250 603

[48] KJ Livak TD Schmittgen Analysis of relative gene expression data using real-time 604

quantitative PCR and the 2(-Delta Delta C(T)) method Methods 25 (2001) 402ndash408 605

[49] TM Montgomery AC Brown HK Gendelman M Ota ED Clotfelter Exposure to 606

17a-Ethinylestradiol Decreases Motility and ATP in Sperm of Male Fighting Fish Betta 607

splendens Environ Toxicol (2012) httpdxdoiorg101002tox21752 Advance online 608

publication 609

[50] S Pawlowski A Sauer JA Shears CR Tyler T Braunbeck Androgenic and estrogenic 610

effects of the synthetic androgen 17α-methyltestosterone on sexual development and 611

29

reproductive performance in the fathead minnow (Pimephales promelas) determined using the 612

gonadal recrudescence assay Aquat Toxicol 68 (2004) 277ndash291 613

[51] AL Filby KL Thorpe G Maack CR Tyler Gene expression profiles revealing the 614

mechanisms of anti-androgen- and estrogen-induced feminization in fish Aquat Toxicol 81 615

(2007) 219ndash231 616

[52] KA Kidd PJ Blanchfield KH Mills VP Palace RE Evans JM Lazorchak RW Flick 617

Collapse of a fish population after exposure to a synthetic estrogen Proc Natl Acad Sci U S 618

A 104 (2007) 8897ndash8901 619

[53] C-B Park K Soyano S Kiros T Kitamura M Minamiyama YSuzuki Transient effects of 620

methyltestosterone injection on different reproductive parameters of the hermaphrodite fish 621

Kryptolebias marmoratus Ecotoxicology 22 (2013) 1145ndash1154 622

[54] D Baron A Fostier B Breton Y Guiguen Androgen and estrogen treatments alter steady 623

state messengers RNA (mRNA) levels of testicular steroidogenic enzymes in the rainbow trout 624

Oncorhynchus mykiss Mol Reprod Dev 71 (2005) 471ndash479 625

[55] R Urbatzka E Rocha B Reis C Cruzeiro RAF Monteiro MJ Rocha Effects of 626

ethinylestradiol and of an environmentally relevant mixture of xenoestrogens on steroidogenic 627

gene expression and specific transcription factors in zebrafish Environ Pollut 164 (2012) 628

28ndash35 629

[56] JL Hoffmann SP Torontali RG Thomason DM Lee JL Brill BB Price GJ Carr DJ 630

Versteeg Hepatic gene expression profiling using Genechips in zebrafish exposed to 631

17α-ethynylestradiol Aquat Toxicol 79 (2006) 233ndash246 632

[57] M Govoroun OM Mcmeel H DrsquoCotta MJ Ricordel T Smith A Fostier Y Guiguen 633

30

Steroid enzyme gene expressions during natural and androgen-induced gonadal differentiation 634

in the rainbow trout Oncorhynchus mykiss J Exp Zool 290 (2001) 558-566 635

[58] D Baron R Houlgatte A Fostier Y Guiguen Expression profiling of candidate genes 636

during ovary-to-testis trans-differentiation in rainbow trout masculinized by androgens Gen 637

Comp Endocrinol 156 (2008) 369ndash378 638

[59] RK Bhandari M Nakamura T Kobayashi Y Nagahama Suppression of steroidogenic 639

enzyme expression during androgen-induced sex reversal in Nile tilapia (Oreochromis 640

niloticus) Gen Comp Endocrinol 145 (2006) 20ndash24 641

[60] M Burgos-Trinidad GL Youngblood MR Maroto A Scheller DM Robins AH Payne 642

Repression of cAMP-induced expression of the mouse P450 17 alpha-hydroxylaseC17-20 643

lyase gene (cyp17) by androgens Mol Endocrinol 11 (1997) 87ndash96 644

[61] Q Zhou JE Shima R Nie PJ Friel MD Griswold Androgen-regulated transcripts in the 645

neonatal mouse testis as determined through microarray analysis Biol Reprod 72 (2005) 646

1010ndash1019 647

[62] KL Parker BP Schimmer Genes essential for early events in gonadal development Ann 648

Med 34 (2002) 171ndash178 649

[63] LJ Luense A Veiga-Lopez V Padmanabhan Lane K Christenson Developmental 650

Programming Gestational Testosterone Treatment Alters Fetal Ovarian Gene Expression 651

Endocrinology December 12 (2011 ) 4974ndash4983 652

[64] M Li L Wang H Wang H Liang Y Zheng F Qin S LiuY Zhang Z Wang Molecular 653

cloning and characterization of amh dax1 and cyp19a1a genes and their response to 654

17α-methyltestosterone in Pengze crucian carp Comp Biochem Physiol Part C 157 (2013) 655

31

372ndash381 656

[65] TS Kostic NJ Stojkov MM Bjelic AI Mihajlovic MM Janjic SA Andric 657

Pharmacological doses of testosterone upregulated androgen receptor and 658

3-beta-hydroxysteroid dehydrogenasedelta-5-delta-4 isomerase and impaired leydig cells 659

steroidogenesis in adult rats Toxicol Sci 121 (2011) 397ndash407 660

661

662

663

664

665

666

667

668

669

670

671

672

673

674

675

676

677

32

Tables and figure legends 678

Table 1 Gonadosomatic index (GSI) in G rarus following EE2 and MT exposure a 679

680

a Data are expressed as mean plusmn SD Asterisks indicate significant difference from the control 681

groups ( P lt 005 P lt 001) The arrows lsquouarrrsquo or lsquodarrrsquo indicate the significant up-regulation or 682

down-regulation of the GSI Absence of the arrows lsquouarrrsquo or lsquodarrrsquo indicates no significant difference 683

between exposure groups and control group 684

685

Table 2 Expression profiles of hepatic vtg in adult G rarus after MT and EE2 exposure a 686

687

33

a The number indicates fold induction or repression (-) from control groups of hepatic vtg in 688

female and male G ruras P-values in bold show significant difference compared to control 689

groups (P lt 005) 690

691

Table 3 Transcriptional profiles of ovarian steroidogenic enzyme genes in female G ruras 692

exposed to MT and EE2 a 693

694

a Gene expression is expressed as the fold change compared to control groups The number 695

indicates fold induction or repression (-) from control groups of the six genes (StAR cyp11a1 696

3β-HSD cyp17a1 11β-HSD2 and cyp19a1a) in female G ruras P-values in bold show 697

significant difference compared to control groups (P lt 005) 698

699

Table 4 Transcriptional profiles of testicular steroidogenic enzyme genes in male G ruras 700


702

34



3β-HSD cyp17a1 11β-HSD2 and cyp19a1a) in male G ruras P-values in bold show significant 705

difference compared to control groups (P lt 005) 706

707

Fig 1 Photomicrographs of transverse ovary sections of adult G rarus unexposed and exposed to 708

EE2 and MT using hematoxylin and eosin stain are shown Scale bars 150 μm A1-A3 the ovary 709

of control female fish (7 14 and 21 days) B1-B3 the ovary of EE2 (25 ngL) exposure for 7 14 710

and 21 days C1-C3 the ovary of MT (25 ngL) exposure for 7 14 and 21 days D1-D3 the ovary 711

of MT (50 ngL) exposure for 7 14 and 21 days E1-E3 the ovary of MT (100 ngL) exposure for 712

7 14 and 21 days Voc vitellogenic oocyte Coc cortical alveolus stage Poc perinucleolar oocyte 713

Do degenerating oocyte 714

715

Fig 2 Photomicrographs of transverse testis sections of adult G rarus unexposed and exposed to 716

EE2 and MT using hematoxylin and eosin stain are shown Scale bars 50 μm A1-A3 the testis of 717

control group (7 14 and 21 days) B1 and B2 the testis of EE2 exposure for 7 14 days B3 the 718

testis of MT at 50 ngL for 21 days C1 and C2 the testis of MT at 50 ngL exposure for 7 14 719

days C3 the testis of MT at 100 ngL exposure for 21 days SZ spermatozoa S spermatocyte 720

AC abnormal connective V vacuolation 721

722

Fig 3 The mRNA tissue distribution of 11β-HSD2 was in G rarus different tissues (brain eye 723

gill intestine liver muscle and gonad) The results were represented the mean plusmn SD of 10 male 724

35

and 10 female fish 725

726

727

728

729

730

731

732

733

734

735

736

737

738

739

740

741

742

743

744

745

746

36

Fig 1 747

748

749

750

751

752

753

754

755

756

757

758

759

760

761

762

37

Fig 2 763

764

765

766

767

768

769

770

771

772

773

774

775

38

Fig 3 776

777

778

779

780

781

782

783

784

785

786

787

788

789

790

791

792

39

Supplement tables and figure legends 793

Table S1 Primers used for fragments cloning RACE and qRT-PCR of StAR cyp11a1 3β-HSD 794

cyp17a1 11β-HSD2 and cyp19a1a 795

796

797

Table S2 Ranking output of the four reference genes according to their expression stability by 798

geNorm Comparative Delta CT method BestKeeper and NormFinder programs in G rarus 799

800

801

40

Fig S1 The cloning strategy for the cDNA of 11β-HSD2 gene 802

803

Fig S2 Nucleotide sequence and deduced amino acid sequence of G rarus 11β-HSD2 Boxes 804

indicated initiator codon and stop codon Polyadenylation signal shaded of light gray 805

806

Fig S3 Comparision of 11β-HSD2 amino acid sequences among vertebrates GenBank IDs of the 807

sequences used for comparision of amino acid residues are shown as follows NP_997885 808

(zebrafish D rerio) ADI60062 (North African catfish C gariepinus) NP_001117690 (rainbow 809

trout O mykiss) BAF35260 (Japanese eel A japonica) NP_001086062 (African clawed frog X 810

laevis) AAH36780 (human H sapiens) and CAA62219 (house mouse M musculus) The 811

conserved regions of 11β-HSD2 are in boxes (I NAD-binding domain II Conserved sequence III 812

Catalytic-site) The identical highly conserved and less conserved amino acid residues were 813

indicated by () () and () respectively with the aid of an internet tool at 814

httpwwwebiacukToolsmsa muscle 815

816

Fig S4 Phylogenetic tree for G rarus 11β-HSD2 using the putative amino acid sequences The 817

phylogenetic tree were constructed using Mega 40 program [41] with neighbor-joining method 818

and bootstrap re-sampling (1000 replications) The numbers in this tree indicate the bootstrap 819

values () GenBank accession numbers of the 11β-HSD2 used in the alignment are NP_997885 820

(zebrafish D rerio) ADI60062 (North African catfish Clarias gariepinus) NP_001117690 821

(rainbow trout O mykiss) BAF35260 (Japanese eel Anguilla japonica) NP_001086062 (African 822

clawed frog Xenopus laevis) AAH36780 (human Homo sapiens) and CAA62219 (house mouse 823

41

Mus musculus) 824

825

826

827

828

829

830

831

832

833

834

835

836

837

838

839

840

841

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843

844

845

42

Fig S1 846

847

848

849

850

851

852

853

854

855

856

857

858

859

860

861

862

863

864

43

Fig S2 865

866

867

868

869

870

44

Fig S3 871

872

873

874

875

876

877

878

879

880

881

882

883

884

885

45

Fig S4 886

887

Response to Reviewers

Dear editor

Thank you very much for your letter and the comments from the reviewers about our paper

submitted to Steroid Biochemistry amp Molecular Biology (Ms Ref No SBMB-D-13-00199)

We have checked and revised the manuscript carefully according to the comments We submit here

the revised manuscript as well as the responses to reviews

If you need any other information please contact me immediately by email My email account is

zzwangnwsuafeducn and Tel is +86-29-87092139 and Fax is +86-29-87092164

Sincerely yours

Zaizhao Wang

Response to reviewer

Dear reviewer

Thanks a lot for your comments on our manuscript According to those helpful comments we have

revised this manuscript carefully point-by-point The comments need to be explained are listed as

following

1 General comment Despite the amount of results in the current paper the research

hypothesis on the effects of the synthetic androgen 17-MT in terms of histological and

steroidogenic gene expressions has been largely demonstrated in many fish species So in

the introductory section of the present study the authors should better justify the reasons

for testing low levels of MT and EE as a further confirmation of the known responses to

Detailed Response to Reviewers









minnow









have been updated
















study










































the model





(Pg7-L194-197)


















Exposure time

(days)

Exposure

concentrations

GSI ()

Female Male

7






14






21




























Pg15-L281-283 Pg15-L284-285 Pg17-L319-321)



para 33








References




372ndash381


















78 (2006) 16ndash23

Highlights





1




b Fang Qin

a Yao Zheng

a Meng Li

a Yingying Zhang

a Cong Yuan

a 3

Zaizhao Wanga

4

5




9

10

11


Zaizhao Wang PhD 13



22 Xinong Road 16


Tel +86-29-87092139 18

Fax +86-29-87092164 19


21

22


2

Abstract 23



















42

43

44

3

1 Introduction 45






















4







[20] 73
















5























6























7























8
























9



















method with 193






3 Results 198

10


















(Fig 1E2 and 1E3) 216





11





C3) 225













S4) 238





12























13























14























15



respectively) 311

4 Discussion 312



















16























17























18























19























20























21














Acknowledgments 454




(QN2011062) 458



461

462

22

Reference 463



















23



132ndash133 (2013) 134ndash140 484




257ndash267 488







311 (2011) 248-254 495








24























25










343ndash352 534












26


Rev 15 (1994) 342ndash355 547



















215ndash220 566


27























28


2001 pp 21ndash34 591












5245ndash5250 603






publication 609



29





(2007) 219ndash231 616



A 104 (2007) 8897ndash8901 619










28ndash35 629





30














1010ndash1019 647


Med 34 (2002) 171ndash178 649







31

372ndash381 656





661

662

663

664

665

666

667

668

669

670

671

672

673

674

675

676

677

32



680





685


687

33




691



694





699



702

34





707








715







722



35


726

727

728

729

730

731

732

733

734

735

736

737

738

739

740

741

742

743

744

745

746

36

Fig 1 747

748

749

750

751

752

753

754

755

756

757

758

759

760

761

762

37

Fig 2 763

764

765

766

767

768

769

770

771

772

773

774

775

38

Fig 3 776

777

778

779

780

781

782

783

784

785

786

787

788

789

790

791

792

39




796

797



800

801

40


803



806










816








41

Mus musculus) 824

825

826

827

828

829

830

831

832

833

834

835

836

837

838

839

840

841

842

843

844

845

42

Fig S1 846

847

848

849

850

851

852

853

854

855

856

857

858

859

860

861

862

863

864

43

Fig S2 865

866

867

868

869

870

44

Fig S3 871

872

873

874

875

876

877

878

879

880

881

882

883

884

885

45

Fig S4 886

887









minnow









have been updated
















study










































the model





(Pg7-L194-197)


















Exposure time

(days)

Exposure

concentrations

GSI ()

Female Male

7






14






21




























Pg15-L281-283 Pg15-L284-285 Pg17-L319-321)



para 33








References




372ndash381


















78 (2006) 16ndash23

Highlights





1




b Fang Qin

a Yao Zheng

a Meng Li

a Yingying Zhang

a Cong Yuan

a 3

Zaizhao Wanga

4

5




9

10

11


Zaizhao Wang PhD 13



22 Xinong Road 16


Tel +86-29-87092139 18

Fax +86-29-87092164 19


21

22


2

Abstract 23



















42

43

44

3

1 Introduction 45






















4







[20] 73
















5























6























7























8
























9



















method with 193






3 Results 198

10


















(Fig 1E2 and 1E3) 216





11





C3) 225













S4) 238





12























13























14























15



respectively) 311

4 Discussion 312



















16























17























18























19























20























21














Acknowledgments 454




(QN2011062) 458



461

462

22

Reference 463



















23



132ndash133 (2013) 134ndash140 484




257ndash267 488







311 (2011) 248-254 495








24























25










343ndash352 534












26


Rev 15 (1994) 342ndash355 547



















215ndash220 566


27























28


2001 pp 21ndash34 591












5245ndash5250 603






publication 609



29





(2007) 219ndash231 616



A 104 (2007) 8897ndash8901 619










28ndash35 629





30














1010ndash1019 647


Med 34 (2002) 171ndash178 649







31

372ndash381 656





661

662

663

664

665

666

667

668

669

670

671

672

673

674

675

676

677

32



680





685


687

33




691



694





699



702

34





707








715







722



35


726

727

728

729

730

731

732

733

734

735

736

737

738

739

740

741

742

743

744

745

746

36

Fig 1 747

748

749

750

751

752

753

754

755

756

757

758

759

760

761

762

37

Fig 2 763

764

765

766

767

768

769

770

771

772

773

774

775

38

Fig 3 776

777

778

779

780

781

782

783

784

785

786

787

788

789

790

791

792

39




796

797



800

801

40


803



806










816








41

Mus musculus) 824

825

826

827

828

829

830

831

832

833

834

835

836

837

838

839

840

841

842

843

844

845

42

Fig S1 846

847

848

849

850

851

852

853

854

855

856

857

858

859

860

861

862

863

864

43

Fig S2 865

866

867

868

869

870

44

Fig S3 871

872

873

874

875

876

877

878

879

880

881

882

883

884

885

45

Fig S4 886

887














study










































the model





(Pg7-L194-197)


















Exposure time

(days)

Exposure

concentrations

GSI ()

Female Male

7






14






21




























Pg15-L281-283 Pg15-L284-285 Pg17-L319-321)



para 33








References




372ndash381


















78 (2006) 16ndash23

Highlights





1




b Fang Qin

a Yao Zheng

a Meng Li

a Yingying Zhang

a Cong Yuan

a 3

Zaizhao Wanga

4

5




9

10

11


Zaizhao Wang PhD 13



22 Xinong Road 16


Tel +86-29-87092139 18

Fax +86-29-87092164 19


21

22


2

Abstract 23



















42

43

44

3

1 Introduction 45






















4







[20] 73
















5























6























7























8
























9



















method with 193






3 Results 198

10


















(Fig 1E2 and 1E3) 216





11





C3) 225













S4) 238





12























13























14























15



respectively) 311

4 Discussion 312



















16























17























18























19























20























21














Acknowledgments 454




(QN2011062) 458



461

462

22

Reference 463



















23



132ndash133 (2013) 134ndash140 484




257ndash267 488







311 (2011) 248-254 495








24























25










343ndash352 534












26


Rev 15 (1994) 342ndash355 547



















215ndash220 566


27























28


2001 pp 21ndash34 591












5245ndash5250 603






publication 609



29





(2007) 219ndash231 616



A 104 (2007) 8897ndash8901 619










28ndash35 629





30














1010ndash1019 647


Med 34 (2002) 171ndash178 649







31

372ndash381 656





661

662

663

664

665

666

667

668

669

670

671

672

673

674

675

676

677

32



680





685


687

33




691



694





699



702

34





707








715







722



35


726

727

728

729

730

731

732

733

734

735

736

737

738

739

740

741

742

743

744

745

746

36

Fig 1 747

748

749

750

751

752

753

754

755

756

757

758

759

760

761

762

37

Fig 2 763

764

765

766

767

768

769

770

771

772

773

774

775

38

Fig 3 776

777

778

779

780

781

782

783

784

785

786

787

788

789

790

791

792

39




796

797



800

801

40


803



806










816








41

Mus musculus) 824

825

826

827

828

829

830

831

832

833

834

835

836

837

838

839

840

841

842

843

844

845

42

Fig S1 846

847

848

849

850

851

852

853

854

855

856

857

858

859

860

861

862

863

864

43

Fig S2 865

866

867

868

869

870

44

Fig S3 871

872

873

874

875

876

877

878

879

880

881

882

883

884

885

45

Fig S4 886

887




































the model





(Pg7-L194-197)


















Exposure time

(days)

Exposure

concentrations

GSI ()

Female Male

7






14






21




























Pg15-L281-283 Pg15-L284-285 Pg17-L319-321)



para 33








References




372ndash381


















78 (2006) 16ndash23

Highlights





1




b Fang Qin

a Yao Zheng

a Meng Li

a Yingying Zhang

a Cong Yuan

a 3

Zaizhao Wanga

4

5




9

10

11


Zaizhao Wang PhD 13



22 Xinong Road 16


Tel +86-29-87092139 18

Fax +86-29-87092164 19


21

22


2

Abstract 23



















42

43

44

3

1 Introduction 45






















4







[20] 73
















5























6























7























8
























9



















method with 193






3 Results 198

10


















(Fig 1E2 and 1E3) 216





11





C3) 225













S4) 238





12























13























14























15



respectively) 311

4 Discussion 312



















16























17























18























19























20























21














Acknowledgments 454




(QN2011062) 458



461

462

22

Reference 463



















23



132ndash133 (2013) 134ndash140 484




257ndash267 488







311 (2011) 248-254 495








24























25










343ndash352 534












26


Rev 15 (1994) 342ndash355 547



















215ndash220 566


27























28


2001 pp 21ndash34 591












5245ndash5250 603






publication 609



29





(2007) 219ndash231 616



A 104 (2007) 8897ndash8901 619










28ndash35 629





30














1010ndash1019 647


Med 34 (2002) 171ndash178 649







31

372ndash381 656





661

662

663

664

665

666

667

668

669

670

671

672

673

674

675

676

677

32



680





685


687

33




691



694





699



702

34





707








715







722



35


726

727

728

729

730

731

732

733

734

735

736

737

738

739

740

741

742

743

744

745

746

36

Fig 1 747

748

749

750

751

752

753

754

755

756

757

758

759

760

761

762

37

Fig 2 763

764

765

766

767

768

769

770

771

772

773

774

775

38

Fig 3 776

777

778

779

780

781

782

783

784

785

786

787

788

789

790

791

792

39




796

797



800

801

40


803



806










816








41

Mus musculus) 824

825

826

827

828

829

830

831

832

833

834

835

836

837

838

839

840

841

842

843

844

845

42

Fig S1 846

847

848

849

850

851

852

853

854

855

856

857

858

859

860

861

862

863

864

43

Fig S2 865

866

867

868

869

870

44

Fig S3 871

872

873

874

875

876

877

878

879

880

881

882

883

884

885

45

Fig S4 886

887


















Exposure time

(days)

Exposure

concentrations

GSI ()

Female Male

7






14






21




























Pg15-L281-283 Pg15-L284-285 Pg17-L319-321)



para 33








References




372ndash381


















78 (2006) 16ndash23

Highlights





1




b Fang Qin

a Yao Zheng

a Meng Li

a Yingying Zhang

a Cong Yuan

a 3

Zaizhao Wanga

4

5




9

10

11


Zaizhao Wang PhD 13



22 Xinong Road 16


Tel +86-29-87092139 18

Fax +86-29-87092164 19


21

22


2

Abstract 23



















42

43

44

3

1 Introduction 45






















4







[20] 73
















5























6























7























8
























9



















method with 193






3 Results 198

10


















(Fig 1E2 and 1E3) 216





11





C3) 225













S4) 238





12























13























14























15



respectively) 311

4 Discussion 312



















16























17























18























19























20























21














Acknowledgments 454




(QN2011062) 458



461

462

22

Reference 463



















23



132ndash133 (2013) 134ndash140 484




257ndash267 488







311 (2011) 248-254 495








24























25










343ndash352 534












26


Rev 15 (1994) 342ndash355 547



















215ndash220 566


27























28


2001 pp 21ndash34 591












5245ndash5250 603






publication 609



29





(2007) 219ndash231 616



A 104 (2007) 8897ndash8901 619










28ndash35 629





30














1010ndash1019 647


Med 34 (2002) 171ndash178 649







31

372ndash381 656





661

662

663

664

665

666

667

668

669

670

671

672

673

674

675

676

677

32



680





685


687

33




691



694





699



702

34





707








715







722



35


726

727

728

729

730

731

732

733

734

735

736

737

738

739

740

741

742

743

744

745

746

36

Fig 1 747

748

749

750

751

752

753

754

755

756

757

758

759

760

761

762

37

Fig 2 763

764

765

766

767

768

769

770

771

772

773

774

775

38

Fig 3 776

777

778

779

780

781

782

783

784

785

786

787

788

789

790

791

792

39




796

797



800

801

40


803



806










816








41

Mus musculus) 824

825

826

827

828

829

830

831

832

833

834

835

836

837

838

839

840

841

842

843

844

845

42

Fig S1 846

847

848

849

850

851

852

853

854

855

856

857

858

859

860

861

862

863

864

43

Fig S2 865

866

867

868

869

870

44

Fig S3 871

872

873

874

875

876

877

878

879

880

881

882

883

884

885

45

Fig S4 886

887
















Pg15-L281-283 Pg15-L284-285 Pg17-L319-321)



para 33








References




372ndash381


















78 (2006) 16ndash23

Highlights





1




b Fang Qin

a Yao Zheng

a Meng Li

a Yingying Zhang

a Cong Yuan

a 3

Zaizhao Wanga

4

5




9

10

11


Zaizhao Wang PhD 13



22 Xinong Road 16


Tel +86-29-87092139 18

Fax +86-29-87092164 19


21

22


2

Abstract 23



















42

43

44

3

1 Introduction 45






















4







[20] 73
















5























6























7























8
























9



















method with 193






3 Results 198

10


















(Fig 1E2 and 1E3) 216





11





C3) 225













S4) 238





12























13























14























15



respectively) 311

4 Discussion 312



















16























17























18























19























20























21














Acknowledgments 454




(QN2011062) 458



461

462

22

Reference 463



















23



132ndash133 (2013) 134ndash140 484




257ndash267 488







311 (2011) 248-254 495








24























25










343ndash352 534












26


Rev 15 (1994) 342ndash355 547



















215ndash220 566


27























28


2001 pp 21ndash34 591












5245ndash5250 603






publication 609



29





(2007) 219ndash231 616



A 104 (2007) 8897ndash8901 619










28ndash35 629





30














1010ndash1019 647


Med 34 (2002) 171ndash178 649







31

372ndash381 656





661

662

663

664

665

666

667

668

669

670

671

672

673

674

675

676

677

32



680





685


687

33




691



694





699



702

34





707








715







722



35


726

727

728

729

730

731

732

733

734

735

736

737

738

739

740

741

742

743

744

745

746

36

Fig 1 747

748

749

750

751

752

753

754

755

756

757

758

759

760

761

762

37

Fig 2 763

764

765

766

767

768

769

770

771

772

773

774

775

38

Fig 3 776

777

778

779

780

781

782

783

784

785

786

787

788

789

790

791

792

39




796

797



800

801

40


803



806










816








41

Mus musculus) 824

825

826

827

828

829

830

831

832

833

834

835

836

837

838

839

840

841

842

843

844

845

42

Fig S1 846

847

848

849

850

851

852

853

854

855

856

857

858

859

860

861

862

863

864

43

Fig S2 865

866

867

868

869

870

44

Fig S3 871

872

873

874

875

876

877

878

879

880

881

882

883

884

885

45

Fig S4 886

887







References




372ndash381


















78 (2006) 16ndash23

Highlights





1




b Fang Qin

a Yao Zheng

a Meng Li

a Yingying Zhang

a Cong Yuan

a 3

Zaizhao Wanga

4

5




9

10

11


Zaizhao Wang PhD 13



22 Xinong Road 16


Tel +86-29-87092139 18

Fax +86-29-87092164 19


21

22


2

Abstract 23



















42

43

44

3

1 Introduction 45






















4







[20] 73
















5























6























7























8
























9



















method with 193






3 Results 198

10


















(Fig 1E2 and 1E3) 216





11





C3) 225













S4) 238





12























13























14























15



respectively) 311

4 Discussion 312



















16























17























18























19























20























21














Acknowledgments 454




(QN2011062) 458



461

462

22

Reference 463



















23



132ndash133 (2013) 134ndash140 484




257ndash267 488







311 (2011) 248-254 495








24























25










343ndash352 534












26


Rev 15 (1994) 342ndash355 547



















215ndash220 566


27























28


2001 pp 21ndash34 591












5245ndash5250 603






publication 609



29





(2007) 219ndash231 616



A 104 (2007) 8897ndash8901 619










28ndash35 629





30














1010ndash1019 647


Med 34 (2002) 171ndash178 649







31

372ndash381 656





661

662

663

664

665

666

667

668

669

670

671

672

673

674

675

676

677

32



680





685


687

33




691



694





699



702

34





707








715







722



35


726

727

728

729

730

731

732

733

734

735

736

737

738

739

740

741

742

743

744

745

746

36

Fig 1 747

748

749

750

751

752

753

754

755

756

757

758

759

760

761

762

37

Fig 2 763

764

765

766

767

768

769

770

771

772

773

774

775

38

Fig 3 776

777

778

779

780

781

782

783

784

785

786

787

788

789

790

791

792

39




796

797



800

801

40


803



806










816








41

Mus musculus) 824

825

826

827

828

829

830

831

832

833

834

835

836

837

838

839

840

841

842

843

844

845

42

Fig S1 846

847

848

849

850

851

852

853

854

855

856

857

858

859

860

861

862

863

864

43

Fig S2 865

866

867

868

869

870

44

Fig S3 871

872

873

874

875

876

877

878

879

880

881

882

883

884

885

45

Fig S4 886

887


78 (2006) 16ndash23

Highlights





1




b Fang Qin

a Yao Zheng

a Meng Li

a Yingying Zhang

a Cong Yuan

a 3

Zaizhao Wanga

4

5




9

10

11


Zaizhao Wang PhD 13



22 Xinong Road 16


Tel +86-29-87092139 18

Fax +86-29-87092164 19


21

22


2

Abstract 23



















42

43

44

3

1 Introduction 45






















4







[20] 73
















5























6























7























8
























9



















method with 193






3 Results 198

10


















(Fig 1E2 and 1E3) 216





11





C3) 225













S4) 238





12























13























14























15



respectively) 311

4 Discussion 312



















16























17























18























19























20























21














Acknowledgments 454




(QN2011062) 458



461

462

22

Reference 463



















23



132ndash133 (2013) 134ndash140 484




257ndash267 488







311 (2011) 248-254 495








24























25










343ndash352 534












26


Rev 15 (1994) 342ndash355 547



















215ndash220 566


27























28


2001 pp 21ndash34 591












5245ndash5250 603






publication 609



29





(2007) 219ndash231 616



A 104 (2007) 8897ndash8901 619










28ndash35 629





30














1010ndash1019 647


Med 34 (2002) 171ndash178 649







31

372ndash381 656





661

662

663

664

665

666

667

668

669

670

671

672

673

674

675

676

677

32



680





685


687

33




691



694





699



702

34





707








715







722



35


726

727

728

729

730

731

732

733

734

735

736

737

738

739

740

741

742

743

744

745

746

36

Fig 1 747

748

749

750

751

752

753

754

755

756

757

758

759

760

761

762

37

Fig 2 763

764

765

766

767

768

769

770

771

772

773

774

775

38

Fig 3 776

777

778

779

780

781

782

783

784

785

786

787

788

789

790

791

792

39




796

797



800

801

40


803



806










816








41

Mus musculus) 824

825

826

827

828

829

830

831

832

833

834

835

836

837

838

839

840

841

842

843

844

845

42

Fig S1 846

847

848

849

850

851

852

853

854

855

856

857

858

859

860

861

862

863

864

43

Fig S2 865

866

867

868

869

870

44

Fig S3 871

872

873

874

875

876

877

878

879

880

881

882

883

884

885

45

Fig S4 886

887

Highlights





1




b Fang Qin

a Yao Zheng

a Meng Li

a Yingying Zhang

a Cong Yuan

a 3

Zaizhao Wanga

4

5




9

10

11


Zaizhao Wang PhD 13



22 Xinong Road 16


Tel +86-29-87092139 18

Fax +86-29-87092164 19


21

22


2

Abstract 23



















42

43

44

3

1 Introduction 45






















4







[20] 73
















5























6























7























8
























9



















method with 193






3 Results 198

10


















(Fig 1E2 and 1E3) 216





11





C3) 225













S4) 238





12























13























14























15



respectively) 311

4 Discussion 312



















16























17























18























19























20























21














Acknowledgments 454




(QN2011062) 458



461

462

22

Reference 463



















23



132ndash133 (2013) 134ndash140 484




257ndash267 488







311 (2011) 248-254 495








24























25










343ndash352 534












26


Rev 15 (1994) 342ndash355 547



















215ndash220 566


27























28


2001 pp 21ndash34 591












5245ndash5250 603






publication 609



29





(2007) 219ndash231 616



A 104 (2007) 8897ndash8901 619










28ndash35 629





30














1010ndash1019 647


Med 34 (2002) 171ndash178 649







31

372ndash381 656





661

662

663

664

665

666

667

668

669

670

671

672

673

674

675

676

677

32



680





685


687

33




691



694





699



702

34





707








715







722



35


726

727

728

729

730

731

732

733

734

735

736

737

738

739

740

741

742

743

744

745

746

36

Fig 1 747

748

749

750

751

752

753

754

755

756

757

758

759

760

761

762

37

Fig 2 763

764

765

766

767

768

769

770

771

772

773

774

775

38

Fig 3 776

777

778

779

780

781

782

783

784

785

786

787

788

789

790

791

792

39




796

797



800

801

40


803



806










816








41

Mus musculus) 824

825

826

827

828

829

830

831

832

833

834

835

836

837

838

839

840

841

842

843

844

845

42

Fig S1 846

847

848

849

850

851

852

853

854

855

856

857

858

859

860

861

862

863

864

43

Fig S2 865

866

867

868

869

870

44

Fig S3 871

872

873

874

875

876

877

878

879

880

881

882

883

884

885

45

Fig S4 886

887

1




b Fang Qin

a Yao Zheng

a Meng Li

a Yingying Zhang

a Cong Yuan

a 3

Zaizhao Wanga

4

5




9

10

11


Zaizhao Wang PhD 13



22 Xinong Road 16


Tel +86-29-87092139 18

Fax +86-29-87092164 19


21

22


2

Abstract 23



















42

43

44

3

1 Introduction 45






















4







[20] 73
















5























6























7























8
























9



















method with 193






3 Results 198

10


















(Fig 1E2 and 1E3) 216





11





C3) 225













S4) 238





12























13























14























15



respectively) 311

4 Discussion 312



















16























17























18























19























20























21














Acknowledgments 454




(QN2011062) 458



461

462

22

Reference 463



















23



132ndash133 (2013) 134ndash140 484




257ndash267 488







311 (2011) 248-254 495








24























25










343ndash352 534












26


Rev 15 (1994) 342ndash355 547



















215ndash220 566


27























28


2001 pp 21ndash34 591












5245ndash5250 603






publication 609



29





(2007) 219ndash231 616



A 104 (2007) 8897ndash8901 619










28ndash35 629





30














1010ndash1019 647


Med 34 (2002) 171ndash178 649







31

372ndash381 656





661

662

663

664

665

666

667

668

669

670

671

672

673

674

675

676

677

32



680





685


687

33




691



694





699



702

34





707








715







722



35


726

727

728

729

730

731

732

733

734

735

736

737

738

739

740

741

742

743

744

745

746

36

Fig 1 747

748

749

750

751

752

753

754

755

756

757

758

759

760

761

762

37

Fig 2 763

764

765

766

767

768

769

770

771

772

773

774

775

38

Fig 3 776

777

778

779

780

781

782

783

784

785

786

787

788

789

790

791

792

39




796

797



800

801

40


803



806










816








41

Mus musculus) 824

825

826

827

828

829

830

831

832

833

834

835

836

837

838

839

840

841

842

843

844

845

42

Fig S1 846

847

848

849

850

851

852

853

854

855

856

857

858

859

860

861

862

863

864

43

Fig S2 865

866

867

868

869

870

44

Fig S3 871

872

873

874

875

876

877

878

879

880

881

882

883

884

885

45

Fig S4 886

887

2

Abstract 23



















42

43

44

3

1 Introduction 45






















4







[20] 73
















5























6























7























8
























9



















method with 193






3 Results 198

10


















(Fig 1E2 and 1E3) 216





11





C3) 225













S4) 238





12























13























14























15



respectively) 311

4 Discussion 312



















16























17























18























19























20























21














Acknowledgments 454




(QN2011062) 458



461

462

22

Reference 463



















23



132ndash133 (2013) 134ndash140 484




257ndash267 488







311 (2011) 248-254 495








24























25










343ndash352 534












26


Rev 15 (1994) 342ndash355 547



















215ndash220 566


27























28


2001 pp 21ndash34 591












5245ndash5250 603






publication 609



29





(2007) 219ndash231 616



A 104 (2007) 8897ndash8901 619










28ndash35 629





30














1010ndash1019 647


Med 34 (2002) 171ndash178 649







31

372ndash381 656





661

662

663

664

665

666

667

668

669

670

671

672

673

674

675

676

677

32



680





685


687

33




691



694





699



702

34





707








715







722



35


726

727

728

729

730

731

732

733

734

735

736

737

738

739

740

741

742

743

744

745

746

36

Fig 1 747

748

749

750

751

752

753

754

755

756

757

758

759

760

761

762

37

Fig 2 763

764

765

766

767

768

769

770

771

772

773

774

775

38

Fig 3 776

777

778

779

780

781

782

783

784

785

786

787

788

789

790

791

792

39




796

797



800

801

40


803



806










816








41

Mus musculus) 824

825

826

827

828

829

830

831

832

833

834

835

836

837

838

839

840

841

842

843

844

845

42

Fig S1 846

847

848

849

850

851

852

853

854

855

856

857

858

859

860

861

862

863

864

43

Fig S2 865

866

867

868

869

870

44

Fig S3 871

872

873

874

875

876

877

878

879

880

881

882

883

884

885

45

Fig S4 886

887

3

1 Introduction 45






















4







[20] 73
















5























6























7























8
























9



















method with 193






3 Results 198

10


















(Fig 1E2 and 1E3) 216





11





C3) 225













S4) 238





12























13























14























15



respectively) 311

4 Discussion 312



















16























17























18























19























20























21














Acknowledgments 454




(QN2011062) 458



461

462

22

Reference 463



















23



132ndash133 (2013) 134ndash140 484




257ndash267 488







311 (2011) 248-254 495








24























25










343ndash352 534












26


Rev 15 (1994) 342ndash355 547



















215ndash220 566


27























28


2001 pp 21ndash34 591












5245ndash5250 603






publication 609



29





(2007) 219ndash231 616



A 104 (2007) 8897ndash8901 619










28ndash35 629





30














1010ndash1019 647


Med 34 (2002) 171ndash178 649







31

372ndash381 656





661

662

663

664

665

666

667

668

669

670

671

672

673

674

675

676

677

32



680





685


687

33




691



694





699



702

34





707








715







722



35


726

727

728

729

730

731

732

733

734

735

736

737

738

739

740

741

742

743

744

745

746

36

Fig 1 747

748

749

750

751

752

753

754

755

756

757

758

759

760

761

762

37

Fig 2 763

764

765

766

767

768

769

770

771

772

773

774

775

38

Fig 3 776

777

778

779

780

781

782

783

784

785

786

787

788

789

790

791

792

39




796

797



800

801

40


803



806










816








41

Mus musculus) 824

825

826

827

828

829

830

831

832

833

834

835

836

837

838

839

840

841

842

843

844

845

42

Fig S1 846

847

848

849

850

851

852

853

854

855

856

857

858

859

860

861

862

863

864

43

Fig S2 865

866

867

868

869

870

44

Fig S3 871

872

873

874

875

876

877

878

879

880

881

882

883

884

885

45

Fig S4 886

887

4







[20] 73
















5























6























7























8
























9



















method with 193






3 Results 198

10


















(Fig 1E2 and 1E3) 216





11





C3) 225













S4) 238





12























13























14























15



respectively) 311

4 Discussion 312



















16























17























18























19























20























21














Acknowledgments 454




(QN2011062) 458



461

462

22

Reference 463



















23



132ndash133 (2013) 134ndash140 484




257ndash267 488







311 (2011) 248-254 495








24























25










343ndash352 534












26


Rev 15 (1994) 342ndash355 547



















215ndash220 566


27























28


2001 pp 21ndash34 591












5245ndash5250 603






publication 609



29





(2007) 219ndash231 616



A 104 (2007) 8897ndash8901 619










28ndash35 629





30














1010ndash1019 647


Med 34 (2002) 171ndash178 649







31

372ndash381 656





661

662

663

664

665

666

667

668

669

670

671

672

673

674

675

676

677

32



680





685


687

33




691



694





699



702

34





707








715







722



35


726

727

728

729

730

731

732

733

734

735

736

737

738

739

740

741

742

743

744

745

746

36

Fig 1 747

748

749

750

751

752

753

754

755

756

757

758

759

760

761

762

37

Fig 2 763

764

765

766

767

768

769

770

771

772

773

774

775

38

Fig 3 776

777

778

779

780

781

782

783

784

785

786

787

788

789

790

791

792

39




796

797



800

801

40


803



806










816








41

Mus musculus) 824

825

826

827

828

829

830

831

832

833

834

835

836

837

838

839

840

841

842

843

844

845

42

Fig S1 846

847

848

849

850

851

852

853

854

855

856

857

858

859

860

861

862

863

864

43

Fig S2 865

866

867

868

869

870

44

Fig S3 871

872

873

874

875

876

877

878

879

880

881

882

883

884

885

45

Fig S4 886

887

5























6























7























8
























9



















method with 193






3 Results 198

10


















(Fig 1E2 and 1E3) 216





11





C3) 225













S4) 238





12























13























14























15



respectively) 311

4 Discussion 312



















16























17























18























19























20























21














Acknowledgments 454




(QN2011062) 458



461

462

22

Reference 463



















23



132ndash133 (2013) 134ndash140 484




257ndash267 488







311 (2011) 248-254 495








24























25










343ndash352 534












26


Rev 15 (1994) 342ndash355 547



















215ndash220 566


27























28


2001 pp 21ndash34 591












5245ndash5250 603






publication 609



29





(2007) 219ndash231 616



A 104 (2007) 8897ndash8901 619










28ndash35 629





30














1010ndash1019 647


Med 34 (2002) 171ndash178 649







31

372ndash381 656





661

662

663

664

665

666

667

668

669

670

671

672

673

674

675

676

677

32



680





685


687

33




691



694





699



702

34





707








715







722



35


726

727

728

729

730

731

732

733

734

735

736

737

738

739

740

741

742

743

744

745

746

36

Fig 1 747

748

749

750

751

752

753

754

755

756

757

758

759

760

761

762

37

Fig 2 763

764

765

766

767

768

769

770

771

772

773

774

775

38

Fig 3 776

777

778

779

780

781

782

783

784

785

786

787

788

789

790

791

792

39




796

797



800

801

40


803



806










816








41

Mus musculus) 824

825

826

827

828

829

830

831

832

833

834

835

836

837

838

839

840

841

842

843

844

845

42

Fig S1 846

847

848

849

850

851

852

853

854

855

856

857

858

859

860

861

862

863

864

43

Fig S2 865

866

867

868

869

870

44

Fig S3 871

872

873

874

875

876

877

878

879

880

881

882

883

884

885

45

Fig S4 886

887

6























7























8
























9



















method with 193






3 Results 198

10


















(Fig 1E2 and 1E3) 216





11





C3) 225













S4) 238





12























13























14























15



respectively) 311

4 Discussion 312



















16























17























18























19























20























21














Acknowledgments 454




(QN2011062) 458



461

462

22

Reference 463



















23



132ndash133 (2013) 134ndash140 484




257ndash267 488







311 (2011) 248-254 495








24























25










343ndash352 534












26


Rev 15 (1994) 342ndash355 547



















215ndash220 566


27























28


2001 pp 21ndash34 591












5245ndash5250 603






publication 609



29





(2007) 219ndash231 616



A 104 (2007) 8897ndash8901 619










28ndash35 629





30














1010ndash1019 647


Med 34 (2002) 171ndash178 649







31

372ndash381 656





661

662

663

664

665

666

667

668

669

670

671

672

673

674

675

676

677

32



680





685


687

33




691



694





699



702

34





707








715







722



35


726

727

728

729

730

731

732

733

734

735

736

737

738

739

740

741

742

743

744

745

746

36

Fig 1 747

748

749

750

751

752

753

754

755

756

757

758

759

760

761

762

37

Fig 2 763

764

765

766

767

768

769

770

771

772

773

774

775

38

Fig 3 776

777

778

779

780

781

782

783

784

785

786

787

788

789

790

791

792

39




796

797



800

801

40


803



806










816








41

Mus musculus) 824

825

826

827

828

829

830

831

832

833

834

835

836

837

838

839

840

841

842

843

844

845

42

Fig S1 846

847

848

849

850

851

852

853

854

855

856

857

858

859

860

861

862

863

864

43

Fig S2 865

866

867

868

869

870

44

Fig S3 871

872

873

874

875

876

877

878

879

880

881

882

883

884

885

45

Fig S4 886

887

7























8
























9



















method with 193






3 Results 198

10


















(Fig 1E2 and 1E3) 216





11





C3) 225













S4) 238





12























13























14























15



respectively) 311

4 Discussion 312



















16























17























18























19























20























21














Acknowledgments 454




(QN2011062) 458



461

462

22

Reference 463



















23



132ndash133 (2013) 134ndash140 484




257ndash267 488







311 (2011) 248-254 495








24























25










343ndash352 534












26


Rev 15 (1994) 342ndash355 547



















215ndash220 566


27























28


2001 pp 21ndash34 591












5245ndash5250 603






publication 609



29





(2007) 219ndash231 616



A 104 (2007) 8897ndash8901 619










28ndash35 629





30














1010ndash1019 647


Med 34 (2002) 171ndash178 649







31

372ndash381 656





661

662

663

664

665

666

667

668

669

670

671

672

673

674

675

676

677

32



680





685


687

33




691



694





699



702

34





707








715







722



35


726

727

728

729

730

731

732

733

734

735

736

737

738

739

740

741

742

743

744

745

746

36

Fig 1 747

748

749

750

751

752

753

754

755

756

757

758

759

760

761

762

37

Fig 2 763

764

765

766

767

768

769

770

771

772

773

774

775

38

Fig 3 776

777

778

779

780

781

782

783

784

785

786

787

788

789

790

791

792

39




796

797



800

801

40


803



806










816








41

Mus musculus) 824

825

826

827

828

829

830

831

832

833

834

835

836

837

838

839

840

841

842

843

844

845

42

Fig S1 846

847

848

849

850

851

852

853

854

855

856

857

858

859

860

861

862

863

864

43

Fig S2 865

866

867

868

869

870

44

Fig S3 871

872

873

874

875

876

877

878

879

880

881

882

883

884

885

45

Fig S4 886

887

8
























9



















method with 193






3 Results 198

10


















(Fig 1E2 and 1E3) 216





11





C3) 225













S4) 238





12























13























14























15



respectively) 311

4 Discussion 312



















16























17























18























19























20























21














Acknowledgments 454




(QN2011062) 458



461

462

22

Reference 463



















23



132ndash133 (2013) 134ndash140 484




257ndash267 488







311 (2011) 248-254 495








24























25










343ndash352 534












26


Rev 15 (1994) 342ndash355 547



















215ndash220 566


27























28


2001 pp 21ndash34 591












5245ndash5250 603






publication 609



29





(2007) 219ndash231 616



A 104 (2007) 8897ndash8901 619










28ndash35 629





30














1010ndash1019 647


Med 34 (2002) 171ndash178 649







31

372ndash381 656





661

662

663

664

665

666

667

668

669

670

671

672

673

674

675

676

677

32



680





685


687

33




691



694





699



702

34





707








715







722



35


726

727

728

729

730

731

732

733

734

735

736

737

738

739

740

741

742

743

744

745

746

36

Fig 1 747

748

749

750

751

752

753

754

755

756

757

758

759

760

761

762

37

Fig 2 763

764

765

766

767

768

769

770

771

772

773

774

775

38

Fig 3 776

777

778

779

780

781

782

783

784

785

786

787

788

789

790

791

792

39




796

797



800

801

40


803



806










816








41

Mus musculus) 824

825

826

827

828

829

830

831

832

833

834

835

836

837

838

839

840

841

842

843

844

845

42

Fig S1 846

847

848

849

850

851

852

853

854

855

856

857

858

859

860

861

862

863

864

43

Fig S2 865

866

867

868

869

870

44

Fig S3 871

872

873

874

875

876

877

878

879

880

881

882

883

884

885

45

Fig S4 886

887

9



















method with 193






3 Results 198

10


















(Fig 1E2 and 1E3) 216





11





C3) 225













S4) 238





12























13























14























15



respectively) 311

4 Discussion 312



















16























17























18























19























20























21














Acknowledgments 454




(QN2011062) 458



461

462

22

Reference 463



















23



132ndash133 (2013) 134ndash140 484




257ndash267 488







311 (2011) 248-254 495








24























25










343ndash352 534












26


Rev 15 (1994) 342ndash355 547



















215ndash220 566


27























28


2001 pp 21ndash34 591












5245ndash5250 603






publication 609



29





(2007) 219ndash231 616



A 104 (2007) 8897ndash8901 619










28ndash35 629





30














1010ndash1019 647


Med 34 (2002) 171ndash178 649







31

372ndash381 656





661

662

663

664

665

666

667

668

669

670

671

672

673

674

675

676

677

32



680





685


687

33




691



694





699



702

34





707








715







722



35


726

727

728

729

730

731

732

733

734

735

736

737

738

739

740

741

742

743

744

745

746

36

Fig 1 747

748

749

750

751

752

753

754

755

756

757

758

759

760

761

762

37

Fig 2 763

764

765

766

767

768

769

770

771

772

773

774

775

38

Fig 3 776

777

778

779

780

781

782

783

784

785

786

787

788

789

790

791

792

39




796

797



800

801

40


803



806










816








41

Mus musculus) 824

825

826

827

828

829

830

831

832

833

834

835

836

837

838

839

840

841

842

843

844

845

42

Fig S1 846

847

848

849

850

851

852

853

854

855

856

857

858

859

860

861

862

863

864

43

Fig S2 865

866

867

868

869

870

44

Fig S3 871

872

873

874

875

876

877

878

879

880

881

882

883

884

885

45

Fig S4 886

887

10


















(Fig 1E2 and 1E3) 216





11





C3) 225













S4) 238





12























13























14























15



respectively) 311

4 Discussion 312



















16























17























18























19























20























21














Acknowledgments 454




(QN2011062) 458



461

462

22

Reference 463



















23



132ndash133 (2013) 134ndash140 484




257ndash267 488







311 (2011) 248-254 495








24























25










343ndash352 534












26


Rev 15 (1994) 342ndash355 547



















215ndash220 566


27























28


2001 pp 21ndash34 591












5245ndash5250 603






publication 609



29





(2007) 219ndash231 616



A 104 (2007) 8897ndash8901 619










28ndash35 629





30














1010ndash1019 647


Med 34 (2002) 171ndash178 649







31

372ndash381 656





661

662

663

664

665

666

667

668

669

670

671

672

673

674

675

676

677

32



680





685


687

33




691



694





699



702

34





707








715







722



35


726

727

728

729

730

731

732

733

734

735

736

737

738

739

740

741

742

743

744

745

746

36

Fig 1 747

748

749

750

751

752

753

754

755

756

757

758

759

760

761

762

37

Fig 2 763

764

765

766

767

768

769

770

771

772

773

774

775

38

Fig 3 776

777

778

779

780

781

782

783

784

785

786

787

788

789

790

791

792

39




796

797



800

801

40


803



806










816








41

Mus musculus) 824

825

826

827

828

829

830

831

832

833

834

835

836

837

838

839

840

841

842

843

844

845

42

Fig S1 846

847

848

849

850

851

852

853

854

855

856

857

858

859

860

861

862

863

864

43

Fig S2 865

866

867

868

869

870

44

Fig S3 871

872

873

874

875

876

877

878

879

880

881

882

883

884

885

45

Fig S4 886

887

11





C3) 225













S4) 238





12























13























14























15



respectively) 311

4 Discussion 312



















16























17























18























19























20























21














Acknowledgments 454




(QN2011062) 458



461

462

22

Reference 463



















23



132ndash133 (2013) 134ndash140 484




257ndash267 488







311 (2011) 248-254 495








24























25










343ndash352 534












26


Rev 15 (1994) 342ndash355 547



















215ndash220 566


27























28


2001 pp 21ndash34 591












5245ndash5250 603






publication 609



29





(2007) 219ndash231 616



A 104 (2007) 8897ndash8901 619










28ndash35 629





30














1010ndash1019 647


Med 34 (2002) 171ndash178 649







31

372ndash381 656





661

662

663

664

665

666

667

668

669

670

671

672

673

674

675

676

677

32



680





685


687

33




691



694





699



702

34





707








715







722



35


726

727

728

729

730

731

732

733

734

735

736

737

738

739

740

741

742

743

744

745

746

36

Fig 1 747

748

749

750

751

752

753

754

755

756

757

758

759

760

761

762

37

Fig 2 763

764

765

766

767

768

769

770

771

772

773

774

775

38

Fig 3 776

777

778

779

780

781

782

783

784

785

786

787

788

789

790

791

792

39




796

797



800

801

40


803



806










816








41

Mus musculus) 824

825

826

827

828

829

830

831

832

833

834

835

836

837

838

839

840

841

842

843

844

845

42

Fig S1 846

847

848

849

850

851

852

853

854

855

856

857

858

859

860

861

862

863

864

43

Fig S2 865

866

867

868

869

870

44

Fig S3 871

872

873

874

875

876

877

878

879

880

881

882

883

884

885

45

Fig S4 886

887

12























13























14























15



respectively) 311

4 Discussion 312



















16























17























18























19























20























21














Acknowledgments 454




(QN2011062) 458



461

462

22

Reference 463



















23



132ndash133 (2013) 134ndash140 484




257ndash267 488







311 (2011) 248-254 495








24























25










343ndash352 534












26


Rev 15 (1994) 342ndash355 547



















215ndash220 566


27























28


2001 pp 21ndash34 591












5245ndash5250 603






publication 609



29





(2007) 219ndash231 616



A 104 (2007) 8897ndash8901 619










28ndash35 629





30














1010ndash1019 647


Med 34 (2002) 171ndash178 649







31

372ndash381 656





661

662

663

664

665

666

667

668

669

670

671

672

673

674

675

676

677

32



680





685


687

33




691



694





699



702

34





707








715







722



35


726

727

728

729

730

731

732

733

734

735

736

737

738

739

740

741

742

743

744

745

746

36

Fig 1 747

748

749

750

751

752

753

754

755

756

757

758

759

760

761

762

37

Fig 2 763

764

765

766

767

768

769

770

771

772

773

774

775

38

Fig 3 776

777

778

779

780

781

782

783

784

785

786

787

788

789

790

791

792

39




796

797



800

801

40


803



806










816








41

Mus musculus) 824

825

826

827

828

829

830

831

832

833

834

835

836

837

838

839

840

841

842

843

844

845

42

Fig S1 846

847

848

849

850

851

852

853

854

855

856

857

858

859

860

861

862

863

864

43

Fig S2 865

866

867

868

869

870

44

Fig S3 871

872

873

874

875

876

877

878

879

880

881

882

883

884

885

45

Fig S4 886

887

13























14























15



respectively) 311

4 Discussion 312



















16























17























18























19























20























21














Acknowledgments 454




(QN2011062) 458



461

462

22

Reference 463



















23



132ndash133 (2013) 134ndash140 484




257ndash267 488







311 (2011) 248-254 495








24























25










343ndash352 534












26


Rev 15 (1994) 342ndash355 547



















215ndash220 566


27























28


2001 pp 21ndash34 591












5245ndash5250 603






publication 609



29





(2007) 219ndash231 616



A 104 (2007) 8897ndash8901 619










28ndash35 629





30














1010ndash1019 647


Med 34 (2002) 171ndash178 649







31

372ndash381 656





661

662

663

664

665

666

667

668

669

670

671

672

673

674

675

676

677

32



680





685


687

33




691



694





699



702

34





707








715







722



35


726

727

728

729

730

731

732

733

734

735

736

737

738

739

740

741

742

743

744

745

746

36

Fig 1 747

748

749

750

751

752

753

754

755

756

757

758

759

760

761

762

37

Fig 2 763

764

765

766

767

768

769

770

771

772

773

774

775

38

Fig 3 776

777

778

779

780

781

782

783

784

785

786

787

788

789

790

791

792

39




796

797



800

801

40


803



806










816








41

Mus musculus) 824

825

826

827

828

829

830

831

832

833

834

835

836

837

838

839

840

841

842

843

844

845

42

Fig S1 846

847

848

849

850

851

852

853

854

855

856

857

858

859

860

861

862

863

864

43

Fig S2 865

866

867

868

869

870

44

Fig S3 871

872

873

874

875

876

877

878

879

880

881

882

883

884

885

45

Fig S4 886

887

14























15



respectively) 311

4 Discussion 312



















16























17























18























19























20























21














Acknowledgments 454




(QN2011062) 458



461

462

22

Reference 463



















23



132ndash133 (2013) 134ndash140 484




257ndash267 488







311 (2011) 248-254 495








24























25










343ndash352 534












26


Rev 15 (1994) 342ndash355 547



















215ndash220 566


27























28


2001 pp 21ndash34 591












5245ndash5250 603






publication 609



29





(2007) 219ndash231 616



A 104 (2007) 8897ndash8901 619










28ndash35 629





30














1010ndash1019 647


Med 34 (2002) 171ndash178 649







31

372ndash381 656





661

662

663

664

665

666

667

668

669

670

671

672

673

674

675

676

677

32



680





685


687

33




691



694





699



702

34





707








715







722



35


726

727

728

729

730

731

732

733

734

735

736

737

738

739

740

741

742

743

744

745

746

36

Fig 1 747

748

749

750

751

752

753

754

755

756

757

758

759

760

761

762

37

Fig 2 763

764

765

766

767

768

769

770

771

772

773

774

775

38

Fig 3 776

777

778

779

780

781

782

783

784

785

786

787

788

789

790

791

792

39




796

797



800

801

40


803



806










816








41

Mus musculus) 824

825

826

827

828

829

830

831

832

833

834

835

836

837

838

839

840

841

842

843

844

845

42

Fig S1 846

847

848

849

850

851

852

853

854

855

856

857

858

859

860

861

862

863

864

43

Fig S2 865

866

867

868

869

870

44

Fig S3 871

872

873

874

875

876

877

878

879

880

881

882

883

884

885

45

Fig S4 886

887

15



respectively) 311

4 Discussion 312



















16























17























18























19























20























21














Acknowledgments 454




(QN2011062) 458



461

462

22

Reference 463



















23



132ndash133 (2013) 134ndash140 484




257ndash267 488







311 (2011) 248-254 495








24























25










343ndash352 534












26


Rev 15 (1994) 342ndash355 547



















215ndash220 566


27























28


2001 pp 21ndash34 591












5245ndash5250 603






publication 609



29





(2007) 219ndash231 616



A 104 (2007) 8897ndash8901 619










28ndash35 629





30














1010ndash1019 647


Med 34 (2002) 171ndash178 649







31

372ndash381 656





661

662

663

664

665

666

667

668

669

670

671

672

673

674

675

676

677

32



680





685


687

33




691



694





699



702

34





707








715







722



35


726

727

728

729

730

731

732

733

734

735

736

737

738

739

740

741

742

743

744

745

746

36

Fig 1 747

748

749

750

751

752

753

754

755

756

757

758

759

760

761

762

37

Fig 2 763

764

765

766

767

768

769

770

771

772

773

774

775

38

Fig 3 776

777

778

779

780

781

782

783

784

785

786

787

788

789

790

791

792

39




796

797



800

801

40


803



806










816








41

Mus musculus) 824

825

826

827

828

829

830

831

832

833

834

835

836

837

838

839

840

841

842

843

844

845

42

Fig S1 846

847

848

849

850

851

852

853

854

855

856

857

858

859

860

861

862

863

864

43

Fig S2 865

866

867

868

869

870

44

Fig S3 871

872

873

874

875

876

877

878

879

880

881

882

883

884

885

45

Fig S4 886

887

16























17























18























19























20























21














Acknowledgments 454




(QN2011062) 458



461

462

22

Reference 463



















23



132ndash133 (2013) 134ndash140 484




257ndash267 488







311 (2011) 248-254 495








24























25










343ndash352 534












26


Rev 15 (1994) 342ndash355 547



















215ndash220 566


27























28


2001 pp 21ndash34 591












5245ndash5250 603






publication 609



29





(2007) 219ndash231 616



A 104 (2007) 8897ndash8901 619










28ndash35 629





30














1010ndash1019 647


Med 34 (2002) 171ndash178 649







31

372ndash381 656





661

662

663

664

665

666

667

668

669

670

671

672

673

674

675

676

677

32



680





685


687

33




691



694





699



702

34





707








715







722



35


726

727

728

729

730

731

732

733

734

735

736

737

738

739

740

741

742

743

744

745

746

36

Fig 1 747

748

749

750

751

752

753

754

755

756

757

758

759

760

761

762

37

Fig 2 763

764

765

766

767

768

769

770

771

772

773

774

775

38

Fig 3 776

777

778

779

780

781

782

783

784

785

786

787

788

789

790

791

792

39




796

797



800

801

40


803



806










816








41

Mus musculus) 824

825

826

827

828

829

830

831

832

833

834

835

836

837

838

839

840

841

842

843

844

845

42

Fig S1 846

847

848

849

850

851

852

853

854

855

856

857

858

859

860

861

862

863

864

43

Fig S2 865

866

867

868

869

870

44

Fig S3 871

872

873

874

875

876

877

878

879

880

881

882

883

884

885

45

Fig S4 886

887

17























18























19























20























21














Acknowledgments 454




(QN2011062) 458



461

462

22

Reference 463



















23



132ndash133 (2013) 134ndash140 484




257ndash267 488







311 (2011) 248-254 495








24























25










343ndash352 534












26


Rev 15 (1994) 342ndash355 547



















215ndash220 566


27























28


2001 pp 21ndash34 591












5245ndash5250 603






publication 609



29





(2007) 219ndash231 616



A 104 (2007) 8897ndash8901 619










28ndash35 629





30














1010ndash1019 647


Med 34 (2002) 171ndash178 649







31

372ndash381 656





661

662

663

664

665

666

667

668

669

670

671

672

673

674

675

676

677

32



680





685


687

33




691



694





699



702

34





707








715







722



35


726

727

728

729

730

731

732

733

734

735

736

737

738

739

740

741

742

743

744

745

746

36

Fig 1 747

748

749

750

751

752

753

754

755

756

757

758

759

760

761

762

37

Fig 2 763

764

765

766

767

768

769

770

771

772

773

774

775

38

Fig 3 776

777

778

779

780

781

782

783

784

785

786

787

788

789

790

791

792

39




796

797



800

801

40


803



806










816








41

Mus musculus) 824

825

826

827

828

829

830

831

832

833

834

835

836

837

838

839

840

841

842

843

844

845

42

Fig S1 846

847

848

849

850

851

852

853

854

855

856

857

858

859

860

861

862

863

864

43

Fig S2 865

866

867

868

869

870

44

Fig S3 871

872

873

874

875

876

877

878

879

880

881

882

883

884

885

45

Fig S4 886

887

18























19























20























21














Acknowledgments 454




(QN2011062) 458



461

462

22

Reference 463



















23



132ndash133 (2013) 134ndash140 484




257ndash267 488







311 (2011) 248-254 495








24























25










343ndash352 534












26


Rev 15 (1994) 342ndash355 547



















215ndash220 566


27























28


2001 pp 21ndash34 591












5245ndash5250 603






publication 609



29





(2007) 219ndash231 616



A 104 (2007) 8897ndash8901 619










28ndash35 629





30














1010ndash1019 647


Med 34 (2002) 171ndash178 649







31

372ndash381 656





661

662

663

664

665

666

667

668

669

670

671

672

673

674

675

676

677

32



680





685


687

33




691



694





699



702

34





707








715







722



35


726

727

728

729

730

731

732

733

734

735

736

737

738

739

740

741

742

743

744

745

746

36

Fig 1 747

748

749

750

751

752

753

754

755

756

757

758

759

760

761

762

37

Fig 2 763

764

765

766

767

768

769

770

771

772

773

774

775

38

Fig 3 776

777

778

779

780

781

782

783

784

785

786

787

788

789

790

791

792

39




796

797



800

801

40


803



806










816








41

Mus musculus) 824

825

826

827

828

829

830

831

832

833

834

835

836

837

838

839

840

841

842

843

844

845

42

Fig S1 846

847

848

849

850

851

852

853

854

855

856

857

858

859

860

861

862

863

864

43

Fig S2 865

866

867

868

869

870

44

Fig S3 871

872

873

874

875

876

877

878

879

880

881

882

883

884

885

45

Fig S4 886

887

19























20























21














Acknowledgments 454




(QN2011062) 458



461

462

22

Reference 463



















23



132ndash133 (2013) 134ndash140 484




257ndash267 488







311 (2011) 248-254 495








24























25










343ndash352 534












26


Rev 15 (1994) 342ndash355 547



















215ndash220 566


27























28


2001 pp 21ndash34 591












5245ndash5250 603






publication 609



29





(2007) 219ndash231 616



A 104 (2007) 8897ndash8901 619










28ndash35 629





30














1010ndash1019 647


Med 34 (2002) 171ndash178 649







31

372ndash381 656





661

662

663

664

665

666

667

668

669

670

671

672

673

674

675

676

677

32



680





685


687

33




691



694





699



702

34





707








715







722



35


726

727

728

729

730

731

732

733

734

735

736

737

738

739

740

741

742

743

744

745

746

36

Fig 1 747

748

749

750

751

752

753

754

755

756

757

758

759

760

761

762

37

Fig 2 763

764

765

766

767

768

769

770

771

772

773

774

775

38

Fig 3 776

777

778

779

780

781

782

783

784

785

786

787

788

789

790

791

792

39




796

797



800

801

40


803



806










816








41

Mus musculus) 824

825

826

827

828

829

830

831

832

833

834

835

836

837

838

839

840

841

842

843

844

845

42

Fig S1 846

847

848

849

850

851

852

853

854

855

856

857

858

859

860

861

862

863

864

43

Fig S2 865

866

867

868

869

870

44

Fig S3 871

872

873

874

875

876

877

878

879

880

881

882

883

884

885

45

Fig S4 886

887

20























21














Acknowledgments 454




(QN2011062) 458



461

462

22

Reference 463



















23



132ndash133 (2013) 134ndash140 484




257ndash267 488







311 (2011) 248-254 495








24























25










343ndash352 534












26


Rev 15 (1994) 342ndash355 547



















215ndash220 566


27























28


2001 pp 21ndash34 591












5245ndash5250 603






publication 609



29





(2007) 219ndash231 616



A 104 (2007) 8897ndash8901 619










28ndash35 629





30














1010ndash1019 647


Med 34 (2002) 171ndash178 649







31

372ndash381 656





661

662

663

664

665

666

667

668

669

670

671

672

673

674

675

676

677

32



680





685


687

33




691



694





699



702

34





707








715







722



35


726

727

728

729

730

731

732

733

734

735

736

737

738

739

740

741

742

743

744

745

746

36

Fig 1 747

748

749

750

751

752

753

754

755

756

757

758

759

760

761

762

37

Fig 2 763

764

765

766

767

768

769

770

771

772

773

774

775

38

Fig 3 776

777

778

779

780

781

782

783

784

785

786

787

788

789

790

791

792

39




796

797



800

801

40


803



806










816








41

Mus musculus) 824

825

826

827

828

829

830

831

832

833

834

835

836

837

838

839

840

841

842

843

844

845

42

Fig S1 846

847

848

849

850

851

852

853

854

855

856

857

858

859

860

861

862

863

864

43

Fig S2 865

866

867

868

869

870

44

Fig S3 871

872

873

874

875

876

877

878

879

880

881

882

883

884

885

45

Fig S4 886

887

21














Acknowledgments 454




(QN2011062) 458



461

462

22

Reference 463



















23



132ndash133 (2013) 134ndash140 484




257ndash267 488







311 (2011) 248-254 495








24























25










343ndash352 534












26


Rev 15 (1994) 342ndash355 547



















215ndash220 566


27























28


2001 pp 21ndash34 591












5245ndash5250 603






publication 609



29





(2007) 219ndash231 616



A 104 (2007) 8897ndash8901 619










28ndash35 629





30














1010ndash1019 647


Med 34 (2002) 171ndash178 649







31

372ndash381 656





661

662

663

664

665

666

667

668

669

670

671

672

673

674

675

676

677

32



680





685


687

33




691



694





699



702

34





707








715







722



35


726

727

728

729

730

731

732

733

734

735

736

737

738

739

740

741

742

743

744

745

746

36

Fig 1 747

748

749

750

751

752

753

754

755

756

757

758

759

760

761

762

37

Fig 2 763

764

765

766

767

768

769

770

771

772

773

774

775

38

Fig 3 776

777

778

779

780

781

782

783

784

785

786

787

788

789

790

791

792

39




796

797



800

801

40


803



806










816








41

Mus musculus) 824

825

826

827

828

829

830

831

832

833

834

835

836

837

838

839

840

841

842

843

844

845

42

Fig S1 846

847

848

849

850

851

852

853

854

855

856

857

858

859

860

861

862

863

864

43

Fig S2 865

866

867

868

869

870

44

Fig S3 871

872

873

874

875

876

877

878

879

880

881

882

883

884

885

45

Fig S4 886

887

22

Reference 463



















23



132ndash133 (2013) 134ndash140 484




257ndash267 488







311 (2011) 248-254 495








24























25










343ndash352 534












26


Rev 15 (1994) 342ndash355 547



















215ndash220 566


27























28


2001 pp 21ndash34 591












5245ndash5250 603






publication 609



29





(2007) 219ndash231 616



A 104 (2007) 8897ndash8901 619










28ndash35 629





30














1010ndash1019 647


Med 34 (2002) 171ndash178 649







31

372ndash381 656





661

662

663

664

665

666

667

668

669

670

671

672

673

674

675

676

677

32



680





685


687

33




691



694





699



702

34





707








715







722



35


726

727

728

729

730

731

732

733

734

735

736

737

738

739

740

741

742

743

744

745

746

36

Fig 1 747

748

749

750

751

752

753

754

755

756

757

758

759

760

761

762

37

Fig 2 763

764

765

766

767

768

769

770

771

772

773

774

775

38

Fig 3 776

777

778

779

780

781

782

783

784

785

786

787

788

789

790

791

792

39




796

797



800

801

40


803



806










816








41

Mus musculus) 824

825

826

827

828

829

830

831

832

833

834

835

836

837

838

839

840

841

842

843

844

845

42

Fig S1 846

847

848

849

850

851

852

853

854

855

856

857

858

859

860

861

862

863

864

43

Fig S2 865

866

867

868

869

870

44

Fig S3 871

872

873

874

875

876

877

878

879

880

881

882

883

884

885

45

Fig S4 886

887

23



132ndash133 (2013) 134ndash140 484




257ndash267 488







311 (2011) 248-254 495








24























25










343ndash352 534












26


Rev 15 (1994) 342ndash355 547



















215ndash220 566


27























28


2001 pp 21ndash34 591












5245ndash5250 603






publication 609



29





(2007) 219ndash231 616



A 104 (2007) 8897ndash8901 619










28ndash35 629





30














1010ndash1019 647


Med 34 (2002) 171ndash178 649







31

372ndash381 656





661

662

663

664

665

666

667

668

669

670

671

672

673

674

675

676

677

32



680





685


687

33




691



694





699



702

34





707








715







722



35


726

727

728

729

730

731

732

733

734

735

736

737

738

739

740

741

742

743

744

745

746

36

Fig 1 747

748

749

750

751

752

753

754

755

756

757

758

759

760

761

762

37

Fig 2 763

764

765

766

767

768

769

770

771

772

773

774

775

38

Fig 3 776

777

778

779

780

781

782

783

784

785

786

787

788

789

790

791

792

39




796

797



800

801

40


803



806










816








41

Mus musculus) 824

825

826

827

828

829

830

831

832

833

834

835

836

837

838

839

840

841

842

843

844

845

42

Fig S1 846

847

848

849

850

851

852

853

854

855

856

857

858

859

860

861

862

863

864

43

Fig S2 865

866

867

868

869

870

44

Fig S3 871

872

873

874

875

876

877

878

879

880

881

882

883

884

885

45

Fig S4 886

887

24























25










343ndash352 534












26


Rev 15 (1994) 342ndash355 547



















215ndash220 566


27























28


2001 pp 21ndash34 591












5245ndash5250 603






publication 609



29





(2007) 219ndash231 616



A 104 (2007) 8897ndash8901 619










28ndash35 629





30














1010ndash1019 647


Med 34 (2002) 171ndash178 649







31

372ndash381 656





661

662

663

664

665

666

667

668

669

670

671

672

673

674

675

676

677

32



680





685


687

33




691



694





699



702

34





707








715







722



35


726

727

728

729

730

731

732

733

734

735

736

737

738

739

740

741

742

743

744

745

746

36

Fig 1 747

748

749

750

751

752

753

754

755

756

757

758

759

760

761

762

37

Fig 2 763

764

765

766

767

768

769

770

771

772

773

774

775

38

Fig 3 776

777

778

779

780

781

782

783

784

785

786

787

788

789

790

791

792

39




796

797



800

801

40


803



806










816








41

Mus musculus) 824

825

826

827

828

829

830

831

832

833

834

835

836

837

838

839

840

841

842

843

844

845

42

Fig S1 846

847

848

849

850

851

852

853

854

855

856

857

858

859

860

861

862

863

864

43

Fig S2 865

866

867

868

869

870

44

Fig S3 871

872

873

874

875

876

877

878

879

880

881

882

883

884

885

45

Fig S4 886

887

25










343ndash352 534












26


Rev 15 (1994) 342ndash355 547



















215ndash220 566


27























28


2001 pp 21ndash34 591












5245ndash5250 603






publication 609



29





(2007) 219ndash231 616



A 104 (2007) 8897ndash8901 619










28ndash35 629





30














1010ndash1019 647


Med 34 (2002) 171ndash178 649







31

372ndash381 656





661

662

663

664

665

666

667

668

669

670

671

672

673

674

675

676

677

32



680





685


687

33




691



694





699



702

34





707








715







722



35


726

727

728

729

730

731

732

733

734

735

736

737

738

739

740

741

742

743

744

745

746

36

Fig 1 747

748

749

750

751

752

753

754

755

756

757

758

759

760

761

762

37

Fig 2 763

764

765

766

767

768

769

770

771

772

773

774

775

38

Fig 3 776

777

778

779

780

781

782

783

784

785

786

787

788

789

790

791

792

39




796

797



800

801

40


803



806










816








41

Mus musculus) 824

825

826

827

828

829

830

831

832

833

834

835

836

837

838

839

840

841

842

843

844

845

42

Fig S1 846

847

848

849

850

851

852

853

854

855

856

857

858

859

860

861

862

863

864

43

Fig S2 865

866

867

868

869

870

44

Fig S3 871

872

873

874

875

876

877

878

879

880

881

882

883

884

885

45

Fig S4 886

887

26


Rev 15 (1994) 342ndash355 547



















215ndash220 566


27























28


2001 pp 21ndash34 591












5245ndash5250 603






publication 609



29





(2007) 219ndash231 616



A 104 (2007) 8897ndash8901 619










28ndash35 629





30














1010ndash1019 647


Med 34 (2002) 171ndash178 649







31

372ndash381 656





661

662

663

664

665

666

667

668

669

670

671

672

673

674

675

676

677

32



680





685


687

33




691



694





699



702

34





707








715







722



35


726

727

728

729

730

731

732

733

734

735

736

737

738

739

740

741

742

743

744

745

746

36

Fig 1 747

748

749

750

751

752

753

754

755

756

757

758

759

760

761

762

37

Fig 2 763

764

765

766

767

768

769

770

771

772

773

774

775

38

Fig 3 776

777

778

779

780

781

782

783

784

785

786

787

788

789

790

791

792

39




796

797



800

801

40


803



806










816








41

Mus musculus) 824

825

826

827

828

829

830

831

832

833

834

835

836

837

838

839

840

841

842

843

844

845

42

Fig S1 846

847

848

849

850

851

852

853

854

855

856

857

858

859

860

861

862

863

864

43

Fig S2 865

866

867

868

869

870

44

Fig S3 871

872

873

874

875

876

877

878

879

880

881

882

883

884

885

45

Fig S4 886

887

27























28


2001 pp 21ndash34 591












5245ndash5250 603






publication 609



29





(2007) 219ndash231 616



A 104 (2007) 8897ndash8901 619










28ndash35 629





30














1010ndash1019 647


Med 34 (2002) 171ndash178 649







31

372ndash381 656





661

662

663

664

665

666

667

668

669

670

671

672

673

674

675

676

677

32



680





685


687

33




691



694





699



702

34





707








715







722



35


726

727

728

729

730

731

732

733

734

735

736

737

738

739

740

741

742

743

744

745

746

36

Fig 1 747

748

749

750

751

752

753

754

755

756

757

758

759

760

761

762

37

Fig 2 763

764

765

766

767

768

769

770

771

772

773

774

775

38

Fig 3 776

777

778

779

780

781

782

783

784

785

786

787

788

789

790

791

792

39




796

797



800

801

40


803



806










816








41

Mus musculus) 824

825

826

827

828

829

830

831

832

833

834

835

836

837

838

839

840

841

842

843

844

845

42

Fig S1 846

847

848

849

850

851

852

853

854

855

856

857

858

859

860

861

862

863

864

43

Fig S2 865

866

867

868

869

870

44

Fig S3 871

872

873

874

875

876

877

878

879

880

881

882

883

884

885

45

Fig S4 886

887

28


2001 pp 21ndash34 591












5245ndash5250 603






publication 609



29





(2007) 219ndash231 616



A 104 (2007) 8897ndash8901 619










28ndash35 629





30














1010ndash1019 647


Med 34 (2002) 171ndash178 649







31

372ndash381 656





661

662

663

664

665

666

667

668

669

670

671

672

673

674

675

676

677

32



680





685


687

33




691



694





699



702

34





707








715







722



35


726

727

728

729

730

731

732

733

734

735

736

737

738

739

740

741

742

743

744

745

746

36

Fig 1 747

748

749

750

751

752

753

754

755

756

757

758

759

760

761

762

37

Fig 2 763

764

765

766

767

768

769

770

771

772

773

774

775

38

Fig 3 776

777

778

779

780

781

782

783

784

785

786

787

788

789

790

791

792

39




796

797



800

801

40


803



806










816








41

Mus musculus) 824

825

826

827

828

829

830

831

832

833

834

835

836

837

838

839

840

841

842

843

844

845

42

Fig S1 846

847

848

849

850

851

852

853

854

855

856

857

858

859

860

861

862

863

864

43

Fig S2 865

866

867

868

869

870

44

Fig S3 871

872

873

874

875

876

877

878

879

880

881

882

883

884

885

45

Fig S4 886

887

29





(2007) 219ndash231 616



A 104 (2007) 8897ndash8901 619










28ndash35 629





30














1010ndash1019 647


Med 34 (2002) 171ndash178 649







31

372ndash381 656





661

662

663

664

665

666

667

668

669

670

671

672

673

674

675

676

677

32



680





685


687

33




691



694





699



702

34





707








715







722



35


726

727

728

729

730

731

732

733

734

735

736

737

738

739

740

741

742

743

744

745

746

36

Fig 1 747

748

749

750

751

752

753

754

755

756

757

758

759

760

761

762

37

Fig 2 763

764

765

766

767

768

769

770

771

772

773

774

775

38

Fig 3 776

777

778

779

780

781

782

783

784

785

786

787

788

789

790

791

792

39




796

797



800

801

40


803



806










816








41

Mus musculus) 824

825

826

827

828

829

830

831

832

833

834

835

836

837

838

839

840

841

842

843

844

845

42

Fig S1 846

847

848

849

850

851

852

853

854

855

856

857

858

859

860

861

862

863

864

43

Fig S2 865

866

867

868

869

870

44

Fig S3 871

872

873

874

875

876

877

878

879

880

881

882

883

884

885

45

Fig S4 886

887

30














1010ndash1019 647


Med 34 (2002) 171ndash178 649







31

372ndash381 656





661

662

663

664

665

666

667

668

669

670

671

672

673

674

675

676

677

32



680





685


687

33




691



694





699



702

34





707








715







722



35


726

727

728

729

730

731

732

733

734

735

736

737

738

739

740

741

742

743

744

745

746

36

Fig 1 747

748

749

750

751

752

753

754

755

756

757

758

759

760

761

762

37

Fig 2 763

764

765

766

767

768

769

770

771

772

773

774

775

38

Fig 3 776

777

778

779

780

781

782

783

784

785

786

787

788

789

790

791

792

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Gonadal development and transcript profiling of steroidogenic enzymes in response to...

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Transcript of Gonadal development and transcript profiling of steroidogenic enzymes in response to...