Accepted Manuscript

Photoperiodic regulation of melatonin membrane receptor (MT1R) expressionand steroidogenesis in testis of adult golden hamster, Mesocricetus auratus

Arun Mukherjee, Chandana Haldar

PII: S1011-1344(14)00273-5DOI: http://dx.doi.org/10.1016/j.jphotobiol.2014.08.022Reference: JPB 9825

To appear in: Journal of Photochemistry and Photobiology B: Bi-ology

Received Date: 1 July 2014Revised Date: 27 August 2014Accepted Date: 28 August 2014

Please cite this article as: A. Mukherjee, C. Haldar, Photoperiodic regulation of melatonin membrane receptor(MT1R) expression and steroidogenesis in testis of adult golden hamster, Mesocricetus auratus, Journal ofPhotochemistry and Photobiology B: Biology (2014), doi: http://dx.doi.org/10.1016/j.jphotobiol.2014.08.022

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Photoperiodic regulation of melatonin membrane receptor (MT1R) expression and

steroidogenesis in testis of adult golden hamster, Mesocricetus auratus.

Arun Mukherjee and Chandana Haldar*

Pineal Research Lab., Department of Zoology, Banaras Hindu University, Varanasi-221005,

India.

E-mail ID of the authors:

Arun Mukherjee: arunmukherjee16@gmail.com,

Chandana Haldar: chaldar2001@yahoo.com,

*Address for correspondence:

Prof. Chandana Haldar,

Pineal Research Lab.

Department of Zoology,

Banaras Hindu University,

Varanasi-221005, India

Ph: 91-542-6702535 Ext. 209; M. 91-9415222261

Fax: 91-542-2368174

E-mail: chaldar2001@yahoo.com

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Abstract

Photoperiodic modulation of melatonin membrane receptor (MT1R) expression in testis has never

been reported for any seasonal breeder. Thus, the aim of the present study was to investigate the

expression dynamics of MT1R in testis and its interaction with testicular steroidogenesis in a long-

day breeder, Mesocricetus auratus. Hamsters were exposed to different photoperiodic conditions

i.e. critical- (CP; 12.5L:11.5D); short-day- (SD; 8L:16D) and long-day- (LD; 16L:8D) for 10 weeks

wherein testicular steroidogenesis, local melatonin synthesis and the expression of MT1R were

analyzed. SD induced melatonin suppressed testicular steroidogenesis as evident from regressed

testicular histoarchitecture, decreased expression of AR, StAR, LH-R, P450SCC and enzyme

activities of 3β- and 17β-HSD. Differential photoperiodic regulation of MT1R expression in testis

suggests its involvement in photoperiodic signal transduction for seasonal adjustment of

reproduction. Increased S-NAT (Serotonin N-acetyl transferase) activity and local testicular

melatonin under SD condition suggest an inhibitory effect of the local melatonergic system on

testicular steroidogenesis. Completely opposite responses were recorded for all the parameters

analyzed when hamsters were exposed to CP or LD conditions. In conclusion, we may suggest that

photoperiod via regulating circulatory and local melatonin level as well as MT1R expression in

testes fine tunes the steroidogenesis and thereby, the reproductive status of male golden hamster.

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1. Introduction

Melatonin, the principal indoleamine secreted from the pineal gland of vertebrates,

synchronizes the seasonal and circadian physiological functions, including reproduction [1,

2]. It has been established that melatonin regulates reproduction in phylogenetically distant

seasonal breeders in a pro- or anti-gonadotropic manner [3, 4] depending on the species.

Being exposed to different photoperiodic and climatic conditions throughout the year,

seasonal breeders breed at a particular time of the year to avoid infant mortality arising due to

adverse climatic conditions. Herein, melatonin acts as a principal transducer of the

photoperiodic stimuli and provides the animal with the ‘time-of-year signal’, restricting the

delivery of the offspring to the most favourable period of the year [5], which helps in

minimizing the maternal cost to benefit ratio and maximizes the chances of offspring survival

[6]. Melatonin modulates seasonality in reproductive status by influencing the hypothalamo-

hypophysial-gonadal (H-P-G) axis [7]. Photoperiodically modulated peripheral melatonin via

MT1R influences the GnRH neurons in the hypothalamus and pars tuberalis of the pituitary,

thereby inhibiting the secretion of the gonadotropin releasing hormone (GnRH) from the

hypothalamus [8] and the gonadotropins (LH and FSH) from the gonadotrophs of the anterior

pituitary [9]. Till date, two high affinity G-protein coupled melatonin membrane receptors

(MT1R and MT2R) have been cloned and characterized and are reported to be expressed

ubiquitously in mammalian tissues [10, 11]. Despite the presence of both the receptor

subtypes in the testis [12], the major points of cross-talk of the melatonergic system with the

reproductive system are mainly mediated via the MT1R subtype [13, 14]. The presence of

two nonsense mutations in the coding frame of the MT2R subtype renders it inactive in the

transduction of photoperiodic stimuli in hamster [15,16] to an extent that transduction of

photoperiodic stimuli occurs principally via the MT1R [13]. The sexual cycle of the golden

hamster, M. auratus, depends on the environmental photoperiod, wherein, the durations of

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the dark and the light periods affect its sexual activity [17]. Being a long-day breeder, it

remains sexually active throughout the year when exposed to ≥ 12.5 hours of light (critical

photoperiod) under laboratory conditions [18]. The presence of MT1R has been reported in

rat Leydig cells [19] and it has been suggested that melatonin may have a local inhibitory

action on the testicular steroidogenic pathway [4]. Previous studies established that, exposure

to inhibitory short photoperiods [4] or exogenous administration of melatonin [20] inhibits

testicular testosterone production. With the aforementioned reports, it is imperative that

photoperiodic stimulus is an important modulator of the testicular steroidogenic pathway.

Since, melatonin is the principal transducer of such stimuli it can be presumed that the local

melatonergic system may have substantial cross-talk with the testicular steroidogenic

pathway, via the MT1R. Based on the above hypothesis, we attempted to explicate the

photoperiodic regulation of the expression of MT1R and its interaction with testicular

steroidogenic pathways in adult male golden hamsters, M auratus.

2. Materials and methods

All the experiments were conducted in accordance with Institutional practice and within the

framework of experimental animals (Scientific Procedure) Act 2007, of the Committee for

the Purpose of Supervision and Control on Experiments on Animals (CPSCEA), Government

of India, on animal welfare.

2.1.Animal procurement and maintenance

Golden hamsters were procured from Central Drug Research Institute (CDRI), Lucknow,

India and colonies were developed and maintained in the departmental animal house facility.

Hamsters were kept under constant temperature (25±2°C) and light/dark cycle (Critical

photoperiod; 12.5 h light, 11.5 h dark; i.e. lights on at 07:00 a.m. and lights off at 07:30 p.m.).

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Animals were maintained in polypropylene cages of equal sizes and provided with

commercial rodent pellet and tap - water ad libitum.

2.2.Photoperiodic treatments

Adult male golden hamsters (average weight 125 g, 90-100 days old) were randomly selected

and divided into three experimental groups (N=5/group) and exposed to different

photoperiodic regimes for 10 weeks:

Group I: Critical photoperiod (CP; 12.5 Light: 11.5 Dark),

Group II: Short-day photoperiod (SD; 8Light: 16Dark) and

Group III: Long day photoperiod (LD; 16Light: 8Dark)

Hamsters were exposed to photostimulatory long day condition and photoinhibitory short day

condition for 10 weeks to achieve maximum testicular growth and regression respectively.

The additional group of hamsters exposed to the critical photoperiod, served as control.

2.3.Tissue collection and processing

At the end of the treatment, hamsters were weighed and sacrificed under deep ether

anaesthesia. Right and left testes were immediately removed, blotted dry and weighed. Testes

were then either quickly fixed in 10% neutral formalin for histological and

immunohistochemical analyses or kept at -80°C for biochemical estimations and western blot

analyses.

2.4.Histology

For histological studies, testes were cut into two halves through its short axis and immersed

in 10% neutral formalin for 24 hours and then cut into small slices of 1-2 mm thickness and

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kept in fresh 10% neutral formalin for 24 hours. After fixation, testes slices were washed

under running tap-water to remove extra fixative and sequentially dehydrated. After clearing

in xylene, tissues were embedded in paraffin and cut into 5µm sections. The sections were

stretched on clean glass slides pre-coated with 1% gelatine. Deparaffinised sections were

stained using hematoxylin and eosin stain. Testicular morphology was observed under

research microscope (Nikon, E 200, Japan) in randomly selected sections. Seminiferous

tubular diameter and area were measured from 100 round seminiferous tubules from the

observed cross-sections.

2.5.Immunohistochemical localization

For immunohistochemical localization, stretched sections on glass slides were sequentially

rehydrated. Endogenous peroxidase activity was blocked by 0.1 % H2O2 in methanol for 30

min. at room temperature (RT). Sections were washed thrice with phosphate buffered saline

(PBS; 0.1 M NaH2PO4, Na2HPO4, NaCl; pH 7.4) and incubated with horse blocking serum

(1:100 in PBS; PK-6200, Vector laboratories, Burlinghame, CA) for 2 hours. Then sections

were incubated with primary antibodies against AR (AR, N-20, sc-816, Lot # E0412, Santa

Cruz Biotech, USA, dilution 1:50) and MT1 (MT1R, R-18, sc- 13186, Lot # L1812, Santa

Cruz Biotech, USA, dilution 1:100) overnight at 40C. Sections were washed thrice with PBS

and incubated with biotinylated secondary antibody (Vectastain ABC Universal kit; PK-

6200, Vector laboratories, Burlinghame, CA, dilution 1:50). Sections were then washed with

PBS and a pre-formed ABC reagent was conjugated to the free biotin of the secondary

antibody. The antigens were visualized using the 0.03% peroxidase substrate 3,3-

diaminobenzidine (DAB, Sigma chemicals, St. Louis, USA) in 0.01M Tris-HCl (pH 7.6) and

0.1% H2O2 and counterstained with Ehrlich’s hematoxylin. Counterstaining with hematoxylin

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was avoided in case of nuclear antigen (i.e. AR). Sections were dehydrated, mounted with

DPX and observed under research microscope (Nikon, E 200, Japan).

2.6.Western blot analyses

For western blot analyses, testes were homogenized and lysed in RIPA buffer containing

aprotinin, sodium orthovanadate and phenyl methylsulphonyl fluoride (PMSF) protease

inhibitor cocktail and was centrifuged at 12,000×g for 30 min, at 4ºC. The protein content of

the lysates was estimated [21]. The aliquots containing 100 µg protein was resolved on 12%

SDS-polyacrylamide gel (PAGE) for MT1R, P450SCC and StAR, , proteins and on 10% SDS-

PAGE for AR and LH-R, followed by electrotransfer (Biometra, Germany) on nitrocellulose

membrane (Boiscience, Keene, NH, USA) for 1 hour. Nitrocellulose membranes were

blocked for 60 min in Tris-buffered saline (TBS; Tris-HCl 50 mM, pH 7.5, NaCl 150 mM)

containing 5% fat free dry milk and were incubated with primary antibodies against AR (AR,

N-20, sc-816, Lot # E0412, Santa Cruz Biotech, USA, dilution 1:250); StAR (StAR, Rabbit

anti-mouse, dilution 1: 250); P450SCC (CYP11A1, H-165, sc-292456, Lot # J2511, Santa

Cruz Biotech, USA, dilution 1:250) and LH-R (LH-R, Rabbit anti-human, dilution 1:100),

MT1 (MT1R, R-18, sc- 13186, Lot # L1812, Santa Cruz Biotech, USA, dilution 1:200).

Antibodies against StAR and LH-receptor were kind gifts from D.M. Stocco (Texas Tech

University, Health Sciences Center, Lubbock, TX, USA) and Craig S. Atwood (William S.

Middleton Memorial Veterans Hospital, Madison, WI, USA) respectively. After overnight

incubation at 4°C, the membranes were washed thrice with TBS. The immunodetection was

carried out using horseradish peroxidase (HRP) conjugated secondary antibody (Goat anti-

rabbit, 1:1000; Donkey anti-goat, 1: 1000). Finally, the blots were washed thrice with TBS

and developed with Super Signal West Pico Chemiluminescent substrate (34080, Thermo

Scientific, USA). Further, the nitrocellulose membranes were then stripped with stripping

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buffer (10% sodium azide) and were immunostained with β-actin antibodies at 1:5,000

dilution (A-2228; Sigma–Aldrich, USA) as internal loading control. Immunodetection of β-

actin was performed with HRP conjugated anti-mouse IgG (1:10,000). Band intensities were

quantified by densitometry using the Image-J analysis software. Values were expressed as the

ratio of the density of the specific signal to that of β-actin.

2.7.Testicular enzyme assay

To observe the effect of different photoperiodic conditions on the steroidogenic pathway,

testicular Δ5 3β-Hydroxysteroid Dehydrogenase (3β-HSD) and 17β-Hydroxysteroid

Dehydrogenase (17β-HSD) activity were measured according to the method of Talalay

(1962) and Jarabek et al. (1962) [22, 23], respectively with minor modifications. Briefly,

testes were homogenized in 20% (v/v) spectroscopic grade glycerol (Sisco Research

Laboratories Pvt. Ltd, India) containing 5 mM potassium phosphate and 1 mM EDTA at a

tissue concentration of 100 mg/ml to prepare 10% (w/v) homogenate and was centrifuged at

12,000 g for 30 min, at 4ºC. The supernatant (approx. 950 µl) was used for enzyme assay.

3β-HSD was measured by mixing 250 µl of the supernatant with 250 µl of 100 µM sodium

pyrophosphate buffer, (pH 8.9), 10µl ethanol containing 30 µg DHEA (Sigma, St. Louis,

USA) and 240 µl of BSA (Sigma, St. Louis, USA) at a concentration of 0.25 mg/ml. Enzyme

activity was measured after addition of 50 µl of 0.5 µM NAD to the mixture in a

spectrophotometer at 340 nm against a blank (without NAD). One unit of enzyme activity is

the amount causing a change in absorbance of 0.001/min at 340 nm. 17β-HSD was measured

by mixing 250 µl of the supernatant with 250 µl of 440 µM sodium pyrophosphate buffer

(pH 10.2), 10 µl ethanol containing 0.3 µM testosterone (Sigma, St. Louis, USA) and 240 µl

of BSA at a concentration of 0.25 mg/ml. Enzyme activity was measured after addition of 50

µl of 0.5 µM NAD to the mixture in a spectrophotometer at 340 nm against a blank (without

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NAD). One unit of enzyme activity is the amount causing a change in absorbance of

0.001/min at 340 nm.

2.8.Plasma testosterone assay

The level of testosterone in serum was measured by using a highly sensitive and specific

commercial ELISA kit (DiaMetra, Italy-DKO002) as per the manufacturer’s instructions. All

samples were quantified in a single assay. The sensitivity of kit was 0.07ng/ml and the intra

and inter-assay variations were < 5.8% and < 10.5% respectively-as per the manufacturer.

2.9.Plasma melatonin assay

The assay was performed according to the manufacturer's protocol provided with the kit

(Uscn Life Science Inc. USA). The sensitivity of the kit was 4.68 pg/ml––as per the

manufacturer. All samples were quantified in a single assay. The intra and inter- assay

variation was <10% and <12% respectively and the recovery percentage was between 90 and

115.

2.10. Testicular S-NAT activity assay

Testicular S-NAT activity was measured following the radioisotopic method of Chae et al.,

(1999) [24]. Briefly, testes were dissected out under red light and quickly frozen in liquid

nitrogen. Testis was homogenized in 0.2M phosphate buffer pH 7.4 at a tissue concentration

of 100 mg/ml to prepare 10% (w/v) homogenate and was centrifuged at 12,000 g for 30 min,

at 4ºC. 10µl of homogenates were mixed in a reaction mixture containing 6 mM tryptamine

hydrochloride, 0.5mM Acetyl-coA and 3H Acetyl-coA (0.2µCi/assay). The reaction mixture

was incubated at 370C for 20 minutes and the reaction was stopped by addition of 180µl

0.2M phosphate buffer. The mixtures were then transferred to scintillation vials containing

3ml of scintillation fluid (PPO and POPOP in toluene). After 24 hours of incubation at room

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temperature in dark, the radioactivity was recorded in a beta scintillation counter (Beckman

Coulter, USA). The activity of the enzyme was expressed as nmoles of N-acetyl tryptamine

formed /min/gm tissue weight.

2.11. Testicular melatonin concentration

Testicular melatonin concentration was determined with an ELISA kit (IBL, Hamburg,

Germany). Testes were homogenized in PBS with 0.1% ethanol in a sonicator at a tissue

concentration of 100 mg/ml to prepare 10% (w/v) homogenate and centrifuged at 3000 g for

10 min. Standards, controls and testicular homogenates were extracted using C18 extraction

columns (IBL, Hamburg, Germany) according to manufacturer’s protocol. The extract was

evaporated to dryness in an evaporator centrifuge (Speed-Vac®, SC110, Savant Instruments,

INC). The dried extracts were used for ELISA. Signals were analyzed at 405 nm using a

microplate reader (Multiskan Ex, Thermo Electron Corporation). Melatonin concentration in

testis was expressed as femtomole (fmol)/gm tissue weight.

2.12. Statistical analyses

Statistical analysis of the data was performed using SPSS 17.0 (SPSS Corp., USA) with one-

way ANOVA followed by Tukey’s multiple range tests for multiple comparisons. The

differences were considered statistically significant when p < 0.05.

3. Results:

3.1.Effect of different photoperiodic regimes on body weight and relative testes

weight

Variations were observed in body weight and relative testes weight in the hamsters exposed

to different photoperiodic conditions. A significant (p<0.01) increase in body weight (Table

1) with a significant (p<0.01) decrease in relative testes weight (Table 1) was recorded in

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hamsters exposed to SD as compared to CP, whereas a significant (p<0.01) decrease in body

weight with a concomitant increase in paired testes weight was observed under LD condition

when compared with SD exposed hamsters.

3.2.Effect of different photoperiod on morphometry and testicular histoarchitecture

Morphometric analyses of seminiferous tubules revealed a significant (p<0.01) decrease in

seminiferous tubular diameter (Table 2) and seminiferous tubular area (Table 2) under SD

condition when compared with CP, whereas significant (p<0.01) increase in diameter and

area of seminiferous tubules was noted in LD group as compared to CP and SD exposed

hamsters. Marked differences in testicular histoarchitecture were noted in hamsters exposed

to SD and LD photoperiodic conditions. However, no such alterations were observed when

comparison was made between CP and LD group (Data not shown). Under LD condition,

normal histoarchitecture of the testis with sequential arrangement of germ cells at different

stages of development and lumen filled with sperm was observed (Figure 1A). Histologically,

the testis of SD exposed hamsters showed signs of atrophy with vacuolization and presence

of large number of giant cells in the seminiferous tubules with the lumen being completely

devoid of sperm (Figure 1B).

3.3. Immunohistochemical localization of MT1R and AR in testis

The product of immune reactions for MT1R was observed in the interstitial compartment, in

general and specifically on the Leydig cells and peritubular cells. Weak immunoreactivity for

MT1R was observed in testis under LD condition (Figure 1C) as compared to strong

immunoreactivity under SD condition (Figure 1D). Strong nuclear immunoreactivity for AR

was observed in the Leydig cells and Sertoli cells of hamsters exposed to LD (Figure 1E) as

compared to weak nuclear immunoreactivity for AR in the Leydig cells under SD condition

(Figure 1F).

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3.4.Effect of different photoperiod on testicular steroidogenesis

Photoperiodic modulation of testicular steroidogenesis was assessed through western blot

analyses of key regulators i.e. LH-R (Lutinizing hormone receptor), StAR (Steroidogenic

acute regulatory protein), P450SCC (P450 Side-chain cleavage) enzyme, AR and estimations of

activity for the key enzymes i.e. 3β-HSD and 17β- HSD of steroidogenesis. Western blot

analyses revealed a significant (p<0.01) decrease in the expression of AR (Figure 2A), StAR

(Figure 2A), P450SCC (Figure 2B) and LH-R (Figure 2B) in hamsters exposed to SD

condition as compared to CP and LD hamsters. However, a significant (p<0.01) increase in

the expression of all the key regulators of steroidogenesis was noted in hamsters exposed to

LD condition when compared to CP and SD groups. Estimations of enzyme activities for 3β-

HSD and 17β- HSD followed the same pattern with a significantly (p<0.01) decreased

enzyme activity under SD condition and vice-versa (Table 3). Significant (p<0.01) decrease

in plasma level of testosterone was noted in SD exposed hamsters as compared to CP and LD

groups (Figure 2A). Taken together, an inhibitory action of SD exposure and a stimulatory

action of LD exposure on testicular steroidogenesis might be suggested.

3.5.Effect of different photoperiod on plasma melatonin level and testicular MT1R

expression

Marked alterations in plasma levels of melatonin and testicular MT1R expression was

observed in hamsters exposed to different photoperiodic conditions. A significant (p<0.01)

elevation in the plasma level of melatonin was observed under SD condition as compared to

CP and LD exposed hamsters (Figure 3A). Western blot analysis for MT1R expression in

testis showed a significant (p<0.01) increase under SD condition as compared to CP and LD

hamsters with a significantly (p<0.01) decreased expression under LD exposure when

compared with SD and CP exposed hamsters (Figure 3A).

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3.6.Effect of different photoperiod on testicular melatonin synthesis

Photoperiodic variation in testicular melatonin production was assessed through measurement

of enzyme activity for the S-NAT, the rate-limiting enzyme for melatonin biosynthesis, and

testicular melatonin level. A significant (p<0.01) increase in S-NAT enzyme activity (Figure

3B) and testicular melatonin concentration (Figure 3B) was recorded in hamsters exposed to

SD condition as compared to CP and LD groups. LD exposure significantly (p<0.01)

abolished the S-NAT enzyme activity and testicular melatonin concentration when compared

with CP and SD hamsters.

4. Discussion:

The essential role played by melatonin in regulation of seasonal reproductive rhythms has

been known for quite some time [25, 26]. The golden hamster, M. auratus is a long-day

seasonal breeder with its neuroendocrine axis being highly sensitive to the changing

photoperiod [25]. It is an excellent animal model for the study of photoperiodic modulation

of reproduction [27] and immunity [28]. Testis is the prime source of circulatory level of

testosterone and the unique site for spermatogenesis in mammals. Hence, detailed

understanding of the photoperiodic modulation of testicular steroidogenesis is of great

significance. The question of whether photoperiodic stimuli influence testicular steroidogenic

pathways via modulation of the melatonin-induced MT1R has never been explored.

In the present study, a significant increase in body mass was noted in hamsters exposed to SD

condition along with a concomitant decrease in paired testes weight. The increased body

weight in the hamsters is due to the increased carcass lipid content, adapted as a means of

successful hibernation strategy [29]. Further, the decreased testes weight in hamsters exposed

to SD photoperiod may be attributed to the inhibitory effect of SD on testicular growth which

thereby minimizes reproductive efforts under photoinhibitory SD condition [1]. The decrease

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in testicular weight and in the diameter and area of seminiferous tubules was accompanied by

the presence of giant cells and intra-epithelial vacuolization in the seminiferous tubules with

lumen being completely devoid of spermatozoa as evidenced from the histological analyses

of testis under SD photoperiod. Our findings receive substantial support from previous

studies [30] suggesting that the testicular regression is due to androgen deficiency under SD

condition. Further, the presence of intraepithelial vacuoles and giant cells in the tubules can

be regarded as a hallmark of decreased plasma levels of testosterone as observed during the

normal process of ageing [31] or during exposure to immunosupessive drugs [32]. The

significant decrease in the level of plasma testosterone under SD condition complies with the

observed histological alterations in the seminiferous tubules. Therefore, it can be suggested

that a trade-off relationship exists between testosterone and melatonin to provide the best

possible way for the seasonal adjustment of reproduction [33].

The present study provides evidence for the photoperiodic variation in MT1R expression in

testis, being higher during the SD condition and lower during LD condition in golden

hamster, M. auratus. Till now, reports suggesting the photoperiodic regulation of MT1R

expression in testis are unavailable for any seasonal mammal, in general, and specially for

golden hamster. Melatonin is classically known to inhibit testicular androgen biosynthesis by

acting at multiple levels of the H-P-G axis through MT1R [7]. In the present study, an

elevated level of peripheral melatonin was observed under SD condition, implying that the

increased melatonin titre inhibits testicular steroidogenesis by up-regulating MT1R in the

Leydig cells.

Further, immunohistochemical (IHC) studies revealed the presence of MT1R in the

interstitial Leydig cells with strong immunoreactivity under SD condition and weak

immunoreactivity under LD condition suggesting a direct action of photoperiodically

15

modulated peripheral melatonin on Leydig cell steroidogenesis. To confirm our IHC data, we

quantitatively assessed the expression of MT1R through western blot analyses. Our

immunoblot analyses revealed highest expression of MT1R under SD condition and lowest

under the LD condition. The high circulatory level of melatonin paralleled the increased

expression of MT1R under SD condition whereas decreased expression of MT1R was

accompanied by low circulatory level of melatonin. Our results are in agreement with earlier

reports demonstrating increased expression of MT1R coincident with high circulatory level

of melatonin [34]. Analyses of AR expression showed an opposite pattern as compared to

MT1R expression i.e. minimal expression of AR under SD exposure and vice versa. Thus,

decreased AR expression in testis parallels the decreased plasma level of testosterone. Our

results mimic earlier reports in the bank vole [35] implying that the stabilization and function

of AR protein is dependent on prolonged receptor occupancy with androgen [36]. Thus,

under LD condition the decreased level of melatonin and MT1R expression coincide with the

increased level of testosterone and AR expression and vice versa suggesting an antagonistic

relationship between melatonin and testosterone and their receptors.

To further elucidate the mechanism of decreased androgen biosynthesis under SD exposure,

we delved into the testicular steroid biosynthetic pathway. Although many enzymes are

involved, it is believed that the rate-limiting step in the steroidogenesis is the movement of

cholesterol across the mitochondrial membrane so that it can be acted upon by the

cytochrome P450 side chain cleavage enzyme (P450SCC) [37]. In mammals, the movement has

been shown to be mediated by the steroidogenic acute regulatory protein (StAR) [38]. StAR

transcripts are rapidly synthesized in response to luteinizing hormone and cAMP [39]. A

significant decrease in StAR, P450SCC and LH-R expression with a simultaneous decrease in

3β- and 17β-HSD enzyme activity was observed under SD condition as compared to LD and

CP exposed hamsters, proposing an inhibitory action of SD condition on testicular

16

steroidogenic machinery. The increased levels of StAR P450SCC and LH-R expression and

enzyme activities of 3β- and 17β-HSD under LD condition concord with the elevated plasma

level of testosterone and are supported by earlier studies in golden hamster [4]. LH is known

to up-regulate the expression of StAR and P450SCC in a cAMP mediated pathway and thereby

stimulate the synthesis of testosterone [37, 40]. On the other hand, binding of melatonin to

MT1R decreases the cAMP level [41] and thus may down-regulate LH-R signaling in Leydig

cells and decreases the expression of steroidogenic genes. The decreased expression LH-R is

concurrent to the decreased expression of StAR, P450SCC and plasma level of testosterone

and is in accordance to the previous reports [4]. Further, evidences suggest local synthesis of

melatonin in rat testis [42]. To check, whether melatonin is being produced in hamster testis,

we measured the enzyme activity for S-NAT-rate limiting enzyme in melatonin synthesis.

Our observations confirmed testicular synthesis of melatonin and also in the variation of

testicular melatonin concentration in a photoperiod dependent manner. Therefore, an elevated

S-NAT enzyme activity and testicular melatonin level under SD condition suggested a local

inhibitory action of melatonin on testicular steroidogenesis.

Conclusion:

The present study demonstrates that the changing photoperiod causes alterations not only in

the peripheral levels of melatonin and androgen but also in the expression pattern of MT1R

and AR in the testis of a long-day breeder, M. auratus. Photoperiodic modulation of plasma

level of melatonin and expression of MT1R demonstrate a reciprocal relationship with the

plasma level of testosterone and expression of AR implying a trade-off relationship between

melatonin and testosterone in the seasonal regulation of testicular steroidogenesis. Further,

alterations in the S-NAT activity and local melatonin level in hamster testis suggest a direct

effect of photoperiodically modulated peripheral and local melatonin levels on testicular

17

steroidogenesis establishing a local microcircuit of this indoleamine in the regulation of

testicular androgen biosynthesis. In the light of the aforementioned, it can be concluded that

seasonality in reproductive status in golden hamster might be modulated by photoperiodic

moderation of the local melatonergic system in the testis.

Declaration of Interest

Authors would like to acknowledge the Council of Scientific and Industrial Research, New

Delhi, government of India (grant scheme number: 37/1462/11/EMR-II) for providing

financial support and Senior Research Fellowship (SRF) to Mr. Arun Mukherjee. Generous

gift of antibodies against LH-receptor and StAR from Dr. Craig S. Atwood (William S.

Middleton Memorial Veterans Hospital, Madison, WI, USA) and Dr. D.M. Stocco (Texas

Tech University, Health Sciences Center, Lubbock, TX, USA) respectively is gratefully

acknowledged. Instrument gift from the Alexander von Humboldt Foundation, Bonn,

Germany to Professor C. Haldar is gratefully acknowledged. The authors report no conflicts

of interest either financial or personal.

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Figure legends

Figure 1

Effect of different photoperiodic conditions (SD and LD) on histoarchitecture and

immunohistochemical localization of MT1R and AR in testes of golden hamster,

Mesocricetus auratus. SD = short-day photoperiod and LD = long-day photoperiod. A)

Showing normal histo-architecture of seminiferous tubule with compact germinal epithelium,

sequentially arranged germ cells at different stages of differentiation with the lumen packed

with spermatozoa under LD condition. B) Showing histological alterations in the

seminiferous tubule under SD condition with signs of testicular atrophy by the presence of

vacuoles and large number of giant cells in the seminiferous tubules with the lumen

completely devoid of sperm. C) Showing weak immunoreactivity for MT1 on the Leydig

cells under LD condition. D) Showing strong immunoreactivity for MT1 on Leydig cells

23

(LC) and peritubular cells (PC) under SD condition. E) Showing strong nuclear

immunoreactivity for AR in the Leydig cells (arrow) and Sertoli cells (dashed arrow) under

LD condition. F) Showing weak nuclear immunoreactivity for AR in the Leydig cells under

SD condition. Scale bar= 50μM.

Figure 2

Effect of different photoperiodic conditions (CP, SD and LD) on testicular steroidogenesis in

golden hamster, Mesocricetus auratus. CP = critical photoperiod, SD = short-day photoperiod

and LD = long-day photoperiod. A) The histogram represents densitometric analyses of AR

and StAR expression in testes. Line diagram on secondary axis shows plasma level of

testosterone. B) The histogram represents densitometric analyses of P450SCC and LH-R

expression in testes. β-actin expression was used as loading control. Values are expressed as

mean ± SEM, N = 5 for each group. Significance of difference; *p < 0.01, CP vs. all

experimental groups and #p < 0.01, SD vs. LD.

Figure 3

Effect of different photoperiodic conditions (CP, SD and LD) on melatonin concentration

(peripheral and local) and MT1R expression in testes of golden hamster, Mesocricetus

auratus. CP = critical photoperiod, SD = short-day photoperiod and LD = long-day

photoperiod. A) The histogram represents plasma level of melatonin and line diagram on

secondary axis shows densitometric analyses of MT1R expression in testes. β-actin

expression was used as loading control. B) The histogram represents testicular S-NAT

enzyme activity and line diagram on secondary axis shows melatonin concentration in testes.

Values are expressed as mean ± SEM, N = 5 for each group. Significance of difference; *p <

0.01, CP vs. all experimental groups and #p < 0.01, SD vs. LD.

24

Table 1

Effect of different photoperiodic conditions (CP, SD and LD) on body weight and relative

testes weight of golden hamster, Mesocricetus auratus. CP = critical photoperiod, SD =

short-day photoperiod and LD = long-day photoperiod. Values are expressed as mean ±SEM.

N = 5 for each group. Significance of difference; *p < 0.01, CP vs. all experimental groups

and #p < 0.01, SD vs. LD.

Table 2

Effect of different photoperiodic conditions (CP, SD and LD) on diameter and area of

semniferous tubule of golden hamster, Mesocricetus auratus. CP = critical photoperiod, SD =

short-day photoperiod and LD = long-day photoperiod. Values are expressed as mean ±SEM.

N = 5 for each group. Significance of difference; *p < 0.01, CP vs. all experimental groups

and #p < 0.01, SD vs. LD.

Table 3

Effect of different photoperiodic conditions (CP, SD and LD) on 3β-HSD and 17β-HSD

enzyme activity in testes of golden hamster, Mesocricetus auratus. CP = critical photoperiod,

SD = short-day photoperiod and LD = long-day photoperiod. Values are expressed as mean

±SEM. N = 5 for each group. Significance of difference; *p < 0.01, CP vs. all experimental

groups and #p < 0.01, SD vs. LD.

Giant Cell

Vacuole

Lumen devoid of

spermatozoa

B

50 µm

Lumen packed with

spermatozoa

50 µm 50 µm

50 µm

Figure 1

LC

C

LC

PC

D

LC

50 µm

50 µm 50 µm

F

LC

LC

LC

A

0

0.5

1

1.5

2

0

20

40

60

80

100

120

CP SD LD

Pla

sm

a te

sto

ste

ron

e (

ng/m

l)

Rela

tive

ba

nd

inte

nsity

AR

STAR

Testosterone

0

10

20

30

40

50

60

70

80

CP SD LD

Rela

tive

ba

nd

inte

nsity

P450SCC

LHR

P450SCC (49 KDa)

LH-receptor (85 KDa)

β-actin (42 KDa)

AR (110 KDa)

StAR (30 KDa)

CP SD LD

β-actin (42 KDa)

Figure 2

A B

*

* #

*

*

* #

* #

* *

* #

* #

CP SD LD

0

5

10

15

20

25

0

20

40

60

CP SD LD

Me

lato

nin

co

nce

ntr

ation

in te

stis

(fm

ol/gm

tis

su

e)

nm

ole

s n

-ace

tyl tr

yp

tam

ine

fo

rme

d/m

in/g

m tis

su

e

S-NAT

Melatonin

0

15

30

45

0

5

10

15

20

25

30

CP SD LD

Re

lative

ba

nd

in

ten

sity

Pla

sm

a m

ela

ton

in (

pg/m

l)

Melatonin

MT1R

β-actin (42 KDa)

CP SD LD

MT1 (~37 KDa)

Figure 3

A

B

*

* #

*

# *

*

* #

*

* #

Groups CP SD LD

Body weight (g)

135.33±4.62

170.34±5.33 *

123.33±4.72 *#

Relative testes weight (g/100g body wt.) 1.665±0.15

0.4095±0.083 *

1.852±0.108 *#

Table 1

Effect of different photoperiodic conditions (CP, SD and LD) on body weight and relative testes weight of

golden hamster, Mesocricetus auratus. CP = critical photoperiod, SD = short-day photoperiod and LD =

long-day photoperiod. Values are expressed as mean ±SEM. N = 5 for each group.

* Significance of difference; p < 0.01, CP vs. all experimental groups. # Significance of difference; p < 0.01, SD vs. LD.

Table 2

Effect of different photoperiodic conditions (CP, SD and LD) on diameter and area of semniferous tubule

of golden hamster, Mesocricetus auratus. CP = critical photoperiod, SD = short-day photoperiod and LD =

long-day photoperiod. Values are expressed as mean ±SEM. N = 5 for each group.

Groups CP SD LD

Seminiferous tubular diameter (µm)

179..43±8.42

89.22±6.34 *

239.75±8.88 *#

Seminiferous tubular area (µm2×103) 51.34±2.08

26.73±0.88 *

64.66±1.45 *#

* Significance of difference; p < 0.01, CP vs. all experimental groups. # Significance of difference; p < 0.01, SD vs. LD.

Groups YH AH OH

3β-HSD activity (U/mg tissue/hr.) 30.63±1.11

16.54±0.50 *

35.70±0.82 *#

17β-HSD activity (U/mg tissue/hr.) 24.59±0.58

11.09±0.48 *

36.44±1.45 *#

Table 3

Effect of different photoperiodic conditions (CP, SD and LD) on 3β-HSD and 17β-HSD enzyme activity in

testes of golden hamster, Mesocricetus auratus. CP = critical photoperiod, SD = short-day photoperiod

and LD = long-day photoperiod. Values are expressed as mean ±SEM. N = 5 for each group.

* Significance of difference; p < 0.01, CP vs. all experimental groups. # Significance of difference; p < 0.01, SD vs. LD.

Highlights

Present study established photoperiodic modulation of MT1R expression in testes for

the first time.

Presence of a local melatonergic system and its photoperiodic modulation is being

proposed.

Photoperiodically modulated peripheral and local melatonin might regulate testicular

steroidogenesis via MT1R.

Photoperiod via melatonin modulate testicular steroidogenesis to restrict male fertility

to a particular time of year.