Sex-dimorphic effects of progesterone and its reduced metabolites on gene expression of myelin...

RESEARCHREPORT

Sex-dimorphic effects of progesterone and its reducedmetabolites on gene expression of myelin proteins by rat

Schwann cells

Valerio Magnaghi1, Sergio Veiga2, Marinella Ballabio1, Lucas C. Gonzalez3, Luis M.Garcia-Segura2, and Roberto C. Melcangi1

1Department of Endocrinology and Center of Excellence of Neurodegenerative Diseases, University of Milan, Milan, Italy;2Instituto Cajal, C.S.I.C., Madrid; and 3Dipartimiento de Biologıa Funcional y Ciencias de la Salud, University of Vigo, Vigo, Spain

Abstract Data obtained in our and other laboratories have indicated that progesterone

(P) and its derivatives, dihydroprogesterone (DHP) and tetrahydroprogesterone (THP),

stimulate the expression of two myelin proteins of the peripheral nervous system (PNS)

[i.e., glycoprotein zero (P0) and peripheral myelin protein 22 (PMP22)]. We have now

considered the effects of P and its derivatives on these and other myelin proteins [i.e.,

myelin-associated glycoprotein (MAG) and myelin and lymphocyte protein (MAL)] in sex-

specific cultures of rat Schwann cells. Gene expression of myelin proteins was assessed

by RNase protection assay. Treatment with P or DHP induced a stimulatory effect on P0

mRNA levels in male but not in female Schwann cells. In contrast, treatment with THP

increased gene expression of P0 exclusively in female Schwann cells. A similar sex-

difference was also evident for other myelin proteins. Indeed, PMP22 expression was

stimulated by treatment with P in male cultures, whereas THP induced an increase of

mRNA levels in female cultures. Moreover, MAG was stimulated by THP treatment in

male cultures only, whereas MAL expression was unaffected by neuroactive steroid

treatment in both male and female cultures. In conclusion, the present observations

indicate that the effects of neuroactive steroids on myelin proteins are sexually dimorphic.

This finding might represent an important background for sex-specific therapies of

acquired and inherited peripheral neuropathies.

Key words: gene expression, myelin proteins, neuroactive steroids, sex difference, sex-

specific Schwann cells

IntroductionIt is now clear that the peripheral nervous system

(PNS), and particularly its glia compartment (i.e.,

Schwann cells), represents a target for neuroactive

steroids. Indeed, in vivo and in vitro observations

have indicated that neuroactive steroids, locally pro-

duced or coming from the blood stream, are able to

affect several parameters of the PNS, such as

Schwann cell proliferation, formation of myelin mem-

branes, expression of myelin proteins, and transcrip-

tion factors involved in the myelination process (for

Address correspondence to: Roberto Cosimo Melcangi, Departmentof Endocrinology and Center of Excellence of NeurodegenerativeDiseases, University of Milan, Via G. Balzaretti 9, 20133 Milan,Italy. Tel: þ39-02-5031-8238; Fax: þ39-02-5031-8204; E-mail: [email protected]

Valerio Magnaghi, Sergio Veiga are co-authors who participatedequally in this work.

Journal of the Peripheral Nervous System 11:111–118 (2006)

� 2006 Peripheral Nerve Society 111 Blackwell Publishing

review, see Melcangi et al., 2005). For instance, in

cultures of rat Schwann cells, progesterone (P) and/or

its metabolites dihydroprogesterone (DHP) and tetra-

hydroprogesterone (THP) stimulate the messenger

levels of two important myelin proteins of the PNS,

such as glycoprotein zero (P0) and the peripheral mye-

lin protein 22 (PMP22) (Melcangi et al., 1998; 1999;

Magnaghi et al., 2001). These effects are mediated by

interactions of these steroids with the classical proges-

terone receptor (PR) and with the GABA-A receptor,

both of them expressed by Schwann cells (Melcangi

et al., 1999; Magnaghi et al., 2001).

These effects are particularly intriguing because

P0 and PMP22 exert a crucial physiological role in the

maintenance of the multilamellar structure of the PNS

myelin (for review, see Melcangi et al., 2000b).

Moreover, alterations of their expression are asso-

ciated with inherited [e.g., Charcot-Marie-Tooth type

1A, 1B, and 2 (CMT1A, CMT1B, and CMT2),

Dejerine-Sottas syndrome, hereditary neuropathy

with liability to pressure palsies, congenital hypomyeli-

nating neuropathy] (Martini et al., 1995; Shy et al.,

1997; Suter and Scherer, 2003) and acquired peripheral

neuropathies, such as those occurring after peripheral

trauma (Melcangi et al., 2000a) or during the aging

process (Melcangi et al., 1998; 1999; 2000b).

On the basis of the effects exerted by neuroactive

steroids on P0 and PMP22, the treatment with these

molecules, or with synthetic ligands of classical or non-

classical steroid receptors, has been proposed as a

new therapeutic approach for the treatment of periph-

eral neuropathies (Melcangi et al., 2003; 2005). Indeed,

we have demonstrated that P and its derivatives sti-

mulate the low expression of P0 and PMP22 occurring

in the sciatic nerve of aged male rats and that these

effects are associated with a reduction of myelin

abnormalities and to a remyelination of small fibers

(Azcoitia et al., 2003; Melcangi et al., 2003). In addition,

as demonstrated by Sereda and co-workers in a model

of CMT1A (i.e., PMP22 transgenic rats), the treatment

with an antagonist of PR is able to reduce the over-

expression of PMP22 and improve the CMT pheno-

type (Sereda et al., 2003).

However, it is important to highlight that, in addition

to P0 and PMP22, other proteins expressed by

Schwann cells, such as myelin-associated glycoprotein

(MAG) (Garbay et al., 2000; Schachner and Bartsch,

2000) and myelin and lymphocyte protein (MAL)

(Schaeren-Wiemers et al., 1995; Frank et al., 1998;

Frank, 2000; Erne et al., 2002), are involved in peripheral

myelination. Indeed, MAG knockout mice exhibit subtle

structural abnormalities in the periaxonal region of the

myelin sheaths (Li et al., 1994; Montag et al., 1994).

Both MAG and MAL have not been so far consid-

ered as possible targets for the action of neuroactive

steroids. Moreover, another important aspect that has

been so far poorly taken into consideration is whether

the effects of neuroactive steroids on Schwann cells

might be different depending on animal sex. Thus, only

one report has analyzed this aspect observing that P is

more effective in stimulating Schwann cell proliferation

in females than in males (Svenningsen and Kanje,

1999), but analysis on myelin proteins is still missing.

To this end, utilizing RNase protection assay

(RPA), which is a profitable method that allows the

simultaneous analysis of several mRNA transcripts,

we have here evaluated the effect of P and its deriva-

tives, DHP and THP, on the expression of P0, PMP22,

MAG, and MAL proteins in sex-specific cultures of rat

Schwann cells.

Materials and MethodsSex-specific Schwann cell cultures

The minimal number of animals was utilized in

accordance with institutional guidelines of the

National Institute of Health (NIH No. 80–23) approved

by the European Communities Council (Directive 86/

609/EEC).

Male and female pups were distinguished by mea-

suring anogenital distance. Male and female Schwann

cell cultures were prepared with the method described

by Brockes et al. (1980) with minor modifications. In

particular, we have introduced a passage with a stream

of cold buffer to block fibroblast proliferation. As sug-

gested by Jirsova et al. (1997), this useful method

allows the detachment and separation of floating

superficial Schwann cells from a carpet of fibroblasts.

Briefly, sciatic nerves from male or female 3-day-old

Sprague-Dawley rats were harvested, digested for

60 min with 1% collagenase (Sigma) and 0.25% tryp-

sin (Sigma), and gently triturated through a 21G nee-

dle. The digested samples were filtered through a

30 mm nylon membrane (Falcon) to eliminate debris

and centrifuged for 10 min at 80 g. The pellets were

resuspended in 10% DMEM-FCS (Invitrogen) and pla-

ted onto 3.5 Petri dishes for 24 h. The medium was

then changed and 10 mM Ara-C (Sigma) was added for

48 h to block fibroblast growth. Then, the cultures

were washed three times with 10% DMEM-FCS to

remove Ara-C and gently treated with a stream of

cold (4�C) 10% DMEM-FCS. The remaining cells,

mainly Schwann cells, were plated in growing medium

made with 10% DMEM-FCS with 10 mM forskolin

(Sigma) and 200 mg/mL bovine pituitary extract

(Invitrogen). In about 10 days, the cells became con-

fluent, and the final stage of purification was carried

out. Cells were then trypsinized, resuspended in

DMEM with mouse anti-rat Thy1.1 (CD90 Serotec)

Magnaghi et al. Journal of the Peripheral Nervous System 11:111–118 (2006)

112

diluted 1 : 100 and incubated for 30 min at 37�C. The

resulting cell suspensions were further incubated for

20 min at 37�C after the addition of 500 mL baby rabbit

complement (Cedarlane). The purification step was

repeated twice. The final cell suspension was then

pelleted, resuspended in 10% DMEM-FCS and plated

onto poly-L-lysine (Sigma)-coated dishes. Cells were

grown in the presence of 2 mM forskolin and 200 mg/

mL BPE. At the third passage in vitro, Schwann cell

cultures characterized by retrotranscriptase polymerase

chain reaction (RT)-PCR, and immunofluorescence

procedure, respectively, for SRY gene, PR, and P0

(see below) were then steroid treated for mRNA

analysis by RPA.

Characterization of Schwann cell cultures

Immunocytochemistry for the specific marker P0

was performed to characterize the sex-specific

Schwann cell cultures. Cultures were fixed for 20 min

at room temperature in 4% paraformaldehyde (Sigma)

in phosphate-buffered saline (PBS). Immunolabeling

was performed using an anti-P0 polyclonal antibody

(raised in rabbit by Sigma-Genosys; diluted 1 : 500).

Cells were washed for 40 min in PBS containing

0.25% bovine serum albumin (BSA) (Sigma) and

0.1% Triton X-100. Cells were then incubated over-

night at 4�C with the primary antibody in the same

PBS–BSA–Triton buffer. The following day, cells were

washed two times and incubated for 2 h at room

temperature with an anti-rabbit secondary antibody

conjugated to fluorescein isothiocyanate (Alexa 488

1 : 800). After washing in buffer, cell cultures were

stained with Dapi (Sigma) to label cell nuclei and

mounted. Omission of the primary antibody was used

as a control for specificity. Images were captured in a

Zeiss microscope and processed with Adobe

Photoshop 5.0.2.

PCR and RT-PCR analyses

Schwann cells were cultured from sex-identified

animals using differences in anogenital distance for

identification. In addition, as a further control to verify

the male or female origin of Schwann cell cultures, a

PCR for the male-specific SRY gene, located on the Y

chromosome, was performed according to An et al.

(1997). Total genomic DNA was extracted at 55�Covernight in Tris, pH 8.3, with 0.45% NP40, 0.45%

Tween 20, and 0.25 mg/mL proteinase K, precipitated

with isopropanol and resuspended in sterile water. A

PCR reaction for 35 cycles (95�C 1 min, 42�C 1 min,

and 72�C 1 min) was performed using the follow-

ing primers: 50-TAC-AGCCTGAGGACATATTA-30 and

50-GCACTTTAACCCTTCGATGA-30. The amplified pro-

ducts were identified by ethidium bromide staining

after separation on 2% agarose gels. Analysis of the

PR was done by RT-PCR as previously described

(Magnaghi et al., 1999).

Steroid treatments

Before treatment with steroids, Schwann cells

were differentiated with 4 mM forskolin for 2 days

and then maintained for 24 h in serum-free condition.

The steroids (P, DHP, and THP; Sigma) were dissolved

in ethanol and added at a final concentration of 10 nM.

Cells were harvested 2 h after the addition of steroids.

Control cultures received an equal volume of ethanol.

cRNA probes

The specific pCR�II-TOP0� (Invitrogen) plasmids

contain, respectively, the following inserts: a 387 bp

for P0, 414 bp for PMP22, a 554 bp for MAL, a 601 bp

for MAG and 290 bp in the case of 18s. The cRNA

antisense probes were generated by in vitro transcrip-

tion of different pCR�II-TOP0� specific plasmids in the

presence of [32P]-CTP (Amersham) as labeled nucleo-

tide. All the cRNA probes were obtained with specific

activity >108 cpm/mg

RNase protection assay

Total RNA was isolated by phenol–chloroform

extraction according to the method of Chomczynski

and Sacchi (1987). Samples of total RNA (10–12 mg)

were utilized in the RPA, as previously described

(Magnaghi et al., 2000). Briefly, after ethanol precipita-

tion samples were dissolved in 20 mL of hybridization

solution [80% formamide, 40 mM piperazine-N,

N�bis (2N-ethanesulfonic acid) (PIPES) pH 6.4,

400 mM sodium acetate pH 6.4, and 1 mM ethylene-

diaminetetraacetic acid (EDTA)] containing

150,000 cpm of each 32P-labeled cRNA probes and

50,000 cpm of 32P-labeled cRNA 18s probe. After

being heated at 85�C for 10 min, the probes were

allowed to hybridize the endogenous RNAs at 45�Covernight. Subsequently, the samples were diluted

with 200 mL of RNase digestion buffer (300 mM NaCl,

10 mM Tris-HCl pH 7.4, 5 mM EDTA pH 7.4) containing

a 1 : 400 dilution of an RNase cocktail (1 mg/mL RNase

A and 20 U/mL RNase T1) and incubated for 30 min at

30�C. Then, 10 mg of proteinase K and sodium dodecyl

sulfate (10 mL of 20% stock solution) were added to

the samples, and the mixture was incubated at 37�Cfor 15 min. At the end of the incubation, the samples

were phenol–chloroform extracted and ethanol preci-

pitated. The pellet was dried and resuspended in


113

loading buffer (80% formamide, 0.1% xylene cyanol,

0.1% brophenol blue, 2 mM EDTA) boiled at 95�C for

5 min and separated on a 5% polyacrylamide gel,

under denaturing conditions (7 M urea). The protected

fragments were visualized by autoradiography, and

their sizes were determined by the use of 32P-end-

labeled (T4 polynucleotide kinase) MspI-digested

pBR322 fragments.

RNA calculation

The levels of mRNA for different myelin proteins

P0, PMP22, MAG, and MAL mRNAs and 18s rRNA

were calculated by measuring the peak densitometric

area of the autoradiography analyzed with a Kodak

Snap Scanner. To ensure that the autoradiographic

bands were in the linear range of intensity, we used

different exposure times. The mean value of the con-

trol (ethanol treated) within a single experiment was

set to 100, and all the other values were expressed as

percent vs. the levels detected in controls. Values of

controls from different experiments were within 10%

of each other.

Statistical analysis

Statistical evaluation of the changes in mRNA

expression was performed using a one-way analysis

of variance (ANOVA). Significant changes were deter-

mined by Tukey’s test for multiple comparisons.

ResultsCharacterization of sex-specific cultures of ratSchwann cells

The purity of each sex-specific cell culture was

tested by immunofluorescence analysis with the anti-

P0 antibody. Both male (Fig. 1A) and female (Fig. 1B)

Schwann cell cultures showed a purity of more than

98%. Moreover, because Schwann cell cultures were

obtained by simply separating the rats on the basis of

their anogenital distance, to confirm their sex-specificity,

a DNA PCR analysis using specific primers directed to

recognize the SRY gene, which is a gene located on

the chromosome Y and represents a specific marker

for the male gender (An et al., 1997), has been per-

formed. As shown in Fig. 2, a band of about 300 bp

corresponding to the SRY gene is present in the DNA

extracted from the male rat Schwann cell cultures

while it is absent in the female cultures. Furthermore,

we have also evaluated by RT-PCR whether, as in the

case of non-sex-specific rat Schwann cells (Magnaghi

et al., 1999), PR was expressed in male or female

Schwann cell cultures. As indicated by the presence

of a band of 325 bp, which is characteristic of the

transcript coding for the rat PR, both male and female

cultures express this steroid receptor (Fig. 3).

Set-up of the RNase protection assay for theexpression analysis of the myelin proteins

The RPA method allows analysis of the relative

expression levels of different transcripts simulta-

neously. In particular, because PMP22 is regulated by

two alternative promoters, located immediately

upstream of two alternative 50-non-coding exons

(exons 1A and 1B) (Suter et al., 1994), the PMP22

pan-probe was designed to detect, in the same

protected fragment, both the exon 1A- and the exon

1B-containing transcripts. Moreover, because two

isoforms of MAG (large and small that are generated

by alternative splicing and that differ only by the car-

boxy-terminal portion of their respective cytoplasmic

domains) have been described (Tropak et al., 1988),

also the MAG pan-probe was designed to detect, in

the same protected fragment, both large and small

transcripts. Furthermore, the method was designed

to obtain the transcripts in order, from the bottom to

the top of the gel, according to the increasing level of

expression. Therefore, at the bottom is present the

mRNA coding for the housekeeping gene 18s rRNA.

As shown in a representative RPA analysis (Fig. 4),

from the bottom to the top, the order of the transcripts

was 18s, P0, PMP22, MAL, and MAG. Samples carry-

ing only tRNA were used as negative controls for the

specificity of the RPA (Fig. 4). In this case, the nega-

tive-control sample demonstrates the assay specificity

because no protected fragments were observed for

either of the specific probes utilized. Moreover, as

shown in Fig. 4, the transcripts for these myelin pro-

teins are constitutively expressed in both male and

female Schwann cells.

Effects of P, DHP, andTHP on gene expression ofP0, PMP22, MAL, and MAG in sex-specificSchwann cell culture

As shown in Fig. 5 (panel A), P, DHP and THP

were all able to significantly stimulate the mRNA levels

of P0 normalized by 18s RNA, but the effect was

different depending on the sex of the cultured cells.

Namely, P and its direct metabolite, DHP, were effec-

tive in stimulating P0 mRNA levels only in cultures of

Schwann cells obtained from male rats, whereas THP

evoked a similar effect only in cells of female origin.

However, it is also important to highlight that the

extent of stimulation was different. Indeed, although

P and DHP resulted in a 100–125% increase in P0

mRNA levels in male cultures, the increase exerted


114

by THP on P0 mRNA levels in female cultures was only

of about 30% of control values.

A very similar situation was observed for PMP22

(Fig. 5, panel B). In cultures of male rat Schwann cells,

the mRNA levels of PMP22 were stimulated only by

treatment with P, whereas in cultures of female origin,

only THP was effective. However, in this case, the extent

of P and THP stimulation was similar in both sexes. Also,

MAG mRNA levels were increased by the treatment with

THP, and interestingly, this effect was only observed in

cultures of male origin (Fig. 5, panel C). Treatments with

P or DHP were ineffective in cultures of both male and

female Schwann cells. Finally, P and its metabolites,

DHP and THP, did not affect the mRNA levels of MAL

in male or female cultures (Fig. 5, panel D).

DiscussionOur previous observations have indicated that neu-

roactive steroids, such as P and/or its derivatives, DHP

and THP, are able to stimulate the gene expression of

two important myelin proteins (P0 and PMP22)

(Melcangi et al., 1998; 1999; 2005; Magnaghi et al.,

2001). In particular, we have demonstrated that, in

cultures of rat Schwann cells, all these neuroactive

steroids are able to stimulate P0, whereas only THP

stimulates the expression of PMP22 (Melcangi et al.,

1999; Magnaghi et al., 2001). Data reported here indi-

cate that these effects are different depending on the

sex of the rat Schwann cell cultures. Thus, P and DHP

are able to stimulate P0 mRNA levels in cultures

obtained from male rats, whereas THP significantly

stimulates the mRNA levels of this myelin protein in

Schwann cell cultures of female origin.

A difference also appears in the case of PMP22.

We presently report that the effect of THP is only

evident in cultures of female origin. Moreover, in

male cultures, P is able to exert a stimulatory effect

A B

Figure 1. Immunostaining of sex-specific rat Schwann cell cultures with anti-P0 polyclonal antibody (raised in rabbit by Sigma-Genosys; diluted 1 : 500). Both male (panel A) and female (panel B) cultures are immunoreactive for this myelin protein (greensignal). Nuclei in blue were stained with Dapi. Scale bar 30 mM.

MK

Mal

e

Fem

ale

Schwann cells

SRY

Figure 2. Polymerase chain reaction analysis of genomicDNA reveals that SRY, a specific gene localized on the chro-mosome Y, is only present in male rat Schwann cells. MK:markers of molecular weight.

PR

Schwann cells

MK

Mal

e

Fem

ale

Figure 3. Representative retrotranscriptase polymerasechain reaction experiments showing the presence of proges-terone receptor mRNA in cultures of male and female ratSchwann cells. MK: markers of molecular weight.


115

on PMP22 mRNA levels. This finding is very interest-

ing because previous observations obtained by others

using a different experimental set up (i.e., Schwann

cells transiently transfected with a reporter construct

in which the expression of the luciferase is controlled

by the promoters of PMP22) have indicated that P is

able to stimulate the gene expression of PMP22 acting

on promoter 1 but not on promoter 2 of the corre-

sponding gene (Desarnaud et al., 1998).

On the basis of previous results obtained in non-

sex-specific cultures, it was proposed that the effects

of P and its derivatives on P0 gene expression are

mediated by an activation of PR, whereas those on

PMP22 need the GABA-A receptor (Melcangi et al.,

1999; Magnaghi et al., 2001). Data presented here

suggest that sex is another variable. Thus, although

male Schwann cells respond in terms of P0 and

PMP22 to neuroactive steroids which directly bind to

PR (i.e., P or DHP), female Schwann cells, even if as

demonstrated here express PR, preferentially utilize a

neuroactive steroid like THP, which is a potent ligand

of GABA-A receptor (Melcangi et al., 2001).

The present observations also extend our knowl-

edge on the effects exerted by neuroactive steroids on

other myelin proteins of the PNS. We demonstrate

here that MAG is a target for these molecules, and

also in this case, a sex difference exists. Indeed, MAG

expression was stimulated in male Schwann cells only

and exclusively by THP. On the contrary, MAL seems

to be unaffected by P and its derivatives both in male

and female Schwann cell cultures.

Altogether these observations indicate for the first

time that, in Schwann cells, the stimulatory effects of

neuroactive steroids on myelin proteins is sexually

dimorphic. Similarly, a sex-dependent effect of ster-

oids on the PNS also occurs with other parameters.

For instance, when the effects of P and estrogens

have been analyzed on the proliferation of Schwann

cells in cultures of segments of the rat sciatic nerve

obtained from both sexes, it has been observed that

these neuroactive steroids are able to enhance thymi-

dine incorporation into Schwann cells in a gender-

specific manner. Indeed, estrogens are only effective

on Schwann cell proliferation in segments from male

rats, whereas P increases Schwann cell proliferation

only in segments obtained from female rats

(Svenningsen and Kanje, 1999). Moreover, in rat

pudendal nerve, a sex difference in terms of number,

caliber, and density of myelinated and unmyelinated

axons, and Schwann cells has been observed (Moore

and White, 1996). Furthermore, experimental models

of acquired peripheral neuropathy, such as sural nerve

crush injury, have shown gender-related differences on

regeneration of nociceptive axons and recovery of

nociception (Kovacic et al., 2004). Finally, also in

other experimental models of acquired peripheral neu-

ropathy, such as chronic constriction injury of sciatic

nerve (Tall et al., 2001), treatment with cancer che-

motherapeutic agents (Joseph and Levine, 2003a) or

with streptozotocin (i.e., diabetic peripheral neuropathy)

(Joseph and Levine, 2003b), nociceptive function differs

as a function of gender and gonadal hormone status.

In conclusion, our findings indicate that the effects

of neuroactive steroids on myelin proteins are different

in Schwann cells from male and female animals. This

sexually dimorphic effect of neuroactive steroids on

Schwann cells is functionally relevant because myelin

proteins are essential to maintain myelin stability, and

changes in their expression are associated with differ-

ent pathological alterations of myelin (Martini et al.,

Male

Female

MAG

MAL

PMP22

P0

18 s

Prob

es

tRN

A

Schw

ann

cells

Figure 4. Representative RNase protection assay showingthat mRNA levels of P0, PMP22, MAL, and MAG are expressedboth in male and female rat Schwann cell cultures.


116

1995; Shy et al., 1997; Melcangi et al., 1998; 1999;

2000a; 2000b; Suter and Scherer, 2003). Furthermore,

neuroactive steroids have been recently proposed as a

new possible therapeutic tool for acquired and inher-

ited peripheral neuropathy. Consequently, the finding

that the effects of neuroactive steroids on myelin pro-

teins are sexually dimorphic might represent a back-

ground for future research aimed to a sex-specific

targeting of the therapy.

AcknowledgementsThe financial support of European Community – RTD

program QLK6-CT-2000-00179, HPMF-CT-2001-01144,

and FIRB 2001(RBAU01kje4_001) are gratefully acknowl-

edged. We thank Miss Elena Crespi for technical

assistance.

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(4)(4)

(4) (4)(4)

(5)

(6)

(6)

MA

L m

RN

A/1

8s r

RN

A(%

of

cont

rols

)

D

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(7)

(10)(7) (10) (8)

*

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G m

RN

A/1

8s r

RN

A(%

of

cont

rols

)

C

*

(11)

(8)(10)

(12)

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(12) (8) (11)

(12)*

P0 m

RN

A/1

8s r

RN

A(%

of

cont

rols

)

A

Male

C DHP THP

Female

B

(11)

*(10)

(10) (10)

(12)(8) (11)

(12)

***

0

50

100

150

200

250

300

0

50

100

150

200

250

300

0

50

100

150

200

250

300

0

50

100

150

200

250

300

PMP2

2 m

RN

A/1

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A(%

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)

P C DHP THPP

Male

C DHP THP

Female

P C DHP THPP

Male

C DHP THP

Female

P C DHP THPP

Male

C DHP THP

Female

P C DHP THPP

Figure 5. P0 (panel A), PMP22 (panel B), MAG (panel C), and MAL (panel D) gene expression in male and female rat Schwanncell cultures 2 h after treatment with progesterone (P), dihydroprogesterone (DHP), or tetrahydroprogesterone (THP) 10 nM ofconcentration. (C) Schwann cells treated with the vehicle only. Quantitative data (after normalization with 18s RNA) obtainedfrom steroid-treated cultures were expressed as percent vs. the levels detected in control cultures (C). The columns representthe means � SEM of the determinations performed (numbers in parentheses). *p < 0.05; **p < 0.01.


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Sex-dimorphic effects of progesterone and its reduced metabolites on gene expression of myelin...

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