help button home button Endocrine Society JCEM JCEM Call for Nominations for EIC
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Journal of Clinical Endocrinology & Metabolism, doi:10.1210/jc.2006-1697
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
92/3/1034    most recent
Author Manuscript (PDF)
Right arrow Submit a related Letter to the Editor
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Rice, S.
Right arrow Articles by Mason, H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Rice, S.
Right arrow Articles by Mason, H.
Related Collections
Right arrow Female Endocrinology
The Journal of Clinical Endocrinology & Metabolism Vol. 92, No. 3 1034-1040
Copyright © 2007 by The Endocrine Society

Stage-Specific Expression of Androgen Receptor, Follicle-Stimulating Hormone Receptor, and Anti-Müllerian Hormone Type II Receptor in Single, Isolated, Human Preantral Follicles: Relevance to Polycystic Ovaries

Suman Rice, Kamal Ojha, Saffron Whitehead and Helen Mason

Basic Medical Sciences (S.R., S.W., H.M.), Clinical Development Sciences (S.R., H.M.), and Department of Obstetrics and Gynaecology (K.O.), St. George’s, University of London, London SW17 0RE, United Kingdom

Address all correspondence and requests for reprints to: Suman Rice, Basic Medical Sciences, Jenner Wing, St. George’s, University of London, Cranmer Terrace, London SW17 0RE, United Kingdom. E-mail: srice{at}sgul.ac.uk.


   Abstract
Top
 Abstract
Introduction
 Patients and Methods
 Results
 Discussion
 References
 
Context: Recent evidence indicates that the increase in follicle numbers seen in polycystic ovary syndrome occurs early in folliculogenesis, with androgens being a likely causative candidate. In primates and sheep, androgen excess in utero results in ovarian changes similar to those in polycystic ovary syndrome. There is also increasing interest in the role of anti-Müllerian hormone (AMH) in early folliculogenesis because AMH knockout mice have an early depletion of their stock of primordial follicles. Initiation and early folliculogenesis may therefore be under negative control by AMH and positive control by androgens.

Objective: Because AMH signals exclusively through its type II receptor (AMHRII), the aim of this study was to determine and colocalize the stage-specific expression of AMHRII, androgen receptor (AR), and FSH receptor (FSHR) mRNA in individual, well-characterized preantral follicles.

Method: Follicles were isolated from human ovarian cortex obtained from either oophorectomies or cortical biopsies at cesarean section. Expression of AR, FSHR, and AMHRII mRNA was determined using a nested RT-PCR protocol.

Results: AR mRNA was not detected in any primordial follicles but was from the transitional stage onward. The number of AR-positive follicles increased at each progressive growth stage. The expression of AR preceded that of FSHR, and only a small percentage of primary follicles expressed FSHR. AMHRII expression was rarely detected.

Conclusions: This is the first study to identify the expression of AR in human transitional follicles. Results suggest a role for androgens in promoting early follicle growth and challenging the hypothesis that AMH exerts a direct, inhibitory effect on follicles at this stage.


   Introduction
Top
 Abstract
 Introduction
Patients and Methods
 Results
 Discussion
 References
 
IN THE NORMAL ovary, folliculogenesis begins with recruitment of a cohort of primordial follicles into the growing phase in which the surrounding single layer of flattened granulosa cells (GCs) of the primordial follicle become cuboidal, forming the primary follicle. Growth then proceeds with expansion of the oocyte, an increase in numbers of GCs, and formation of the theca, eventually resulting in ovulation of the dominant follicle with atresia being the fate of others in the cohort (1).

It has become apparent from several studies that this process is abnormal in the polycystic ovary (PCO). The main feature of this abnormality is that the proportion of primary (early growing) follicles is much higher in women with PCOs, compared with those with normal ovaries (2, 3, 4). The cause of this change is not clear. Webber et al. (3), however, found a reciprocal decrease in the proportion of primordial follicles, but this was not supported by the other two studies.

The factors responsible for these changes are unknown. There is considerable evidence from animal research that exposure to high levels of androgens may cause the increase in primary follicles seen in PCO, by either increasing recruitment of primordial follicles and/or decreasing atresia of larger follicles (reviewed in Ref. 5). Prenatally androgenized rhesus monkeys have multiple follicles of 1 mm diameter (5, 6), and similarly treated sheep also have enlarged ovaries with multifollicular morphology (7). Androgen treatment of adult monkeys increased the total number of preantral follicles (8). The cellular actions of androgen require binding and activation of its ligand-specific nuclear receptor [androgen receptor (AR)] to regulate transcriptional events. Studies in rats, marmoset monkeys, and humans have shown that the expression of AR is most abundant in GCs of pre- and early antral ovarian follicles and decreases as the follicle develops (9, 10, 11, 12).

In addition to their effects on growth, androgens also enhance FSH-mediated differentiation of GCs as indicated by an increase in estradiol and progesterone production (reviewed in Ref. 13). AR mRNA levels were positively correlated with FSH receptor (FSHR) mRNA levels in GCs from normal cyclic, androgen and FSH-treated primates: testosterone increased FSHR mRNA levels in follicles at all stages of development, whereas FSH increased AR in primary follicles (14). Thus, there appears to be some interdependence between androgens and FSH in early follicular development.

Initiation of follicle growth is under the influence of not only activating factors but also inhibitors. One such factor is anti-Müllerian hormone (AMH), also known as Müllerian-inhibiting substance or factor. This hormone classically is responsible for regression of the Müllerian ducts during differentiation of the male reproductive tract (15) and may also function as an inhibitor of follicle development. Female Amh null mice were depleted of their stock of primordial follicles much earlier than wild-type control, and this was due to increased recruitment into the growing pool (16). Further studies, in which neonatal mice ovaries were cultured with AMH, resulted in an inhibition of the initiation of primordial follicle growth with a decrease in the proportion of growing follicles (17). Interestingly PCOs had significantly fewer primordial and transitional follicles staining for AMH protein, compared with those from normal ovaries (18). It was proposed that this reduced local exposure to AMH resulted in a higher proportion of primordial follicles initiating growth, thus leading to the higher proportion of primary follicles and lower number of primordials previously reported (3, 18). AMH is a member of the TGF-ß family and shares the general characteristics of its signaling pathway. It binds specifically to an AMH type II receptor (AMHRII) which is a membrane bound serine/threonine kinase receptor, and is unable to signal through other type II receptors like bone morphogenetic protein type II receptor or activin type IIB receptor (19). The pattern of expression of the AMH receptor in the ovary should therefore give us an indication of the size of follicle at which AMH is able to exert its effects.

Although both androgens and AMH have been implicated in the disordered early folliculogenesis seen in PCO, few data exist on the expression of the receptors for these ligands in human preantral follicles. The aim of this study was to determine the stage-specific onset of gene transcription for AR and AMHRII and to correlate this to FSHR in single, well-characterized preantral follicles isolated from human ovarian cortex.


   Patients and Methods
Top
 Abstract
 Introduction
 Patients and Methods
Results
 Discussion
 References
 
Tissue samples and follicle isolation

Ovarian cortex was obtained (St. George’s Hospital, London, UK) by ovarian cortical biopsy with ethical approval and informed consent from women undergoing elective cesarean sections (n = 4) or total abdominal hysterectomy (TAH)/bilateral salpingo-oophorectomy (BSO) (n = 4) for a variety of benign gynecological conditions. The age of the women ranged from 28 to 43 yr for the TAH/BSO group and 30 to 40 yr for the C-section group. Patient details are listed in Table 1Go.


View this table:
[in this window]
[in a new window]

  		TABLE 1. Clinical details of patients involved in the study along with number of follicles retrieved and used in this study from their ovarian tissue samples

 
Follicle isolation was performed according to a well-established protocol with some modifications (20, 21, 22). Briefly, follicles were manually isolated from the tissue after a 30- to 40-min enzymatic digestion at 37 C with gentle shaking in Krebs-Ringer bicarbonate buffer containing 15 mM HEPES, 180 U/ml deoxyribonuclease I, and 1240 U/ml collagenase (all chemicals from Sigma, Poole, Dorset, UK) to soften the ovarian matrix. Enzymatics activity was stopped by the addition of 10 ml ice-cold M199 containing 4 mg/ml BSA (Sigma) before centrifuging the mixture at 50 g for 3 min at 4 C. After resuspension of the pellet in 20 ml M199+BSA (4 mg/ml), follicles were mechanically dissected free of the partially digested ovarian stroma using fine acupuncture needles under an inverted microscope with Hoffman optics (Leica, Milton Keynes, Buckinghamshire, UK). Individual follicles were transferred to fresh drops of media using pulled pipettes and photographed using a digital camera (DXM1200) attached to an inverted microscope (Eclipse TE300) with phase contrast and Hoffmann optics (both from Nikon, Surrey, UK). The stage of development and health of each follicle was ascertained using imaging capture analysis software ACT (Nikon U.K. Ltd.).

Follicle stages were classified using the following criteria: primordial (one layer of flattened GCs), transitional (one layer of mixed flattened and cuboidal GCs), primary (one layer of fully expanded, cuboidal GCs), primary-secondary transitional (intermediate between one and two layers of GCs), secondary (two distinct layers of expanded, GCs) and multilaminar (multiple layers of GCs without an antrum) (1, 23) (Fig. 1Go). For this study the primary-secondary transitional stage was classified as secondary.


Figure 1
View larger version (147K):
[in this window]
[in a new window]

  		FIG. 1. Representative photographs of preantral follicles (a), primordial follicles with one layer of flattened GCs (b), transitional follicle with one layer of mixed cuboidal and flattened GCs (c), and primary follicle with one layer of fully expanded, cuboidal GCs (d) secondary follicles with two layers of GCs. Scale bar, 10 µm.

 
RNA extraction and reverse transcription (RT)

Individual follicles were washed in PBS/BSA to remove any adherent stromal cells and then placed by direct observation into RNase-free sterile 0.5 ml Eppendorf tubes (Ambion, Applied Biosystems, Warrington, UK) containing 10 µl of lysis buffer [0.5% IGEPAL CA-630, 10 mM Tris (pH 8.0), 10 mM NaCl, and 3 mM MgCl2 (all from Sigma)] (24). The lysed follicles were then snap frozen in liquid nitrogen and stored at –80 C before RT and nested PCR.

The lysates were defrosted on ice and spun at maximum speed in a microcentrifuge at 4 C for 1 min. Usually the total lysate (10 µl) from each follicle was used for RT. As a result, it was not possible to include RT(–) controls (i.e. samples with no reverse transcriptase enzyme) for all follicles. Each follicular lysate was reverse transcribed in a 20 µl volume with 1 µl 10 mM deoxynucleotide triphosphate mix, 2 µl 0.1 M dithiothreitol, 4 µl 5x first-strand buffer, 1 µl of 200 U Moloney murine leukemia virus reverse transcriptase and 0.5 µg oligo (dT)12–18 primer (all from Invitrogen, Paisley, Scotland, UK) before incubation at 37 C for 1 h. The reaction was terminated by heating tubes at 95 C for 5 min before immediately placing on ice. cDNA was stored at –20 C until further use.

RNA was also extracted using TRIZOL reagent from various tissues, e.g. ovarian cortex and stroma (OV), theca from various-sized follicles, and granulosa-lutein cells, for use as positive control (PCR+) cDNA. The extracted RNA was DNase treated with deoxyribonuclease I (amplification grade) (Invitrogen) to eliminate contaminating DNA, assessed for quality and quantity using the bioanalyzer (Agilent Technologies UK Ltd., Wokingham, Berkshire, UK) and 1 µg of RNA reverse transcribed as detailed above.

Nested PCR

All the primers were designed so that their products spanned introns so as to discriminate genomic DNA from cDNA. Primer sequences and annealing temperatures are listed in Table 2Go. A human ß-actin control was run for each sample. Only those follicles positive for ß-actin mRNA expression were used to investigate mRNA expression of the other genes. Negative controls were performed using no cDNA (PCR–), lysis buffer alone that had been reverse transcribed, and samples prepared without reverse transcriptase enzyme (RT–). Positive controls used were cDNA from ovarian cortex/stroma for AR, granulosa-luteal cells for FSHR and theca for AMHRII.


View this table:
[in this window]
[in a new window]

  		TABLE 2. Nested primer sequences and annealing temperature (Ta) for AR, FSHR, and AMHRII

 
First-stage PCR contained 2 µl cDNA, 5 µl 10x PCR buffer (200 mM Tris-HCl, 200 mM KCl), 1.5 µl of 50 mM MgCl2, 1 µl of 10 mM deoxynucleotide triphosphate mix, 1–1.5 U Platinum Taq DNA polymerase, and 1 µl of each specific 40 µM forward and reverse outer primers, made up to 50 µl with DNase/RNase-free water (all from Invitrogen). Second-stage (nested PCR) assays were made up as above except that between 3 and 4 µl of first-stage (outer) product was used as the template with appropriate forward and reverse inner primers.

After initial denaturation at 94 C for 5 min on a Px2 thermal cycler (Thermo Electron Corp., Waltham, MA), the following cycling conditions were used: denaturation at 94 C for 1 min, annealing at specific temperature for each primer pair (Table 2Go) for 1 min, extension at 72 C for 1 min (1.30 min for ß-actin, 2 min for FSHR), with a final extension at 72 C of 10 min (6 min for ß-actin). The first-stage (outer) reactions were run at 25 cycles, and the second-stage (inner) at 35 cycles, except for FSHR, which were run at 30 cycles each. PCR products were viewed after electrophoresis on 2% (wt/vol) agarose gel (Invitrogen) stained with ethidium bromide (Sigma) and photographed under UV light. RT-PCR products were verified by sequencing (Advanced Biotechnology Centre, Imperial College, London, UK).

Primer sensitivity

The sensitivity of the nested PCR protocol and primer pairs for ß-actin, AR, FSHR, and AMHRII was demonstrated by 10-fold serial dilution of cDNA from each positive control tissue. RNA from the tissue was extracted, assessed, and reverse transcribed as detailed above. The cDNA obtained was measured on a NanoDrop spectrophotometer (NanoDrop Technologies, Wilmington, DE) and then diluted down from nanogram per microliter amounts to attogram per microliter. Where possible, cDNA levels of the follicles were also assessed using the NanoDrop spectrophotometer.


   Results
Top
 Abstract
 Introduction
 Patients and Methods
 Results
Discussion
 References
 
Tissue was collected from four patients having TAH/BSO (OV) and four patients having elective C-sections (CB). A total of 148 follicles were retrieved and 72 were apportioned to this study. Of these 57 were classifiable and usable. The remaining 14 follicles could not be classified due to atresia or clustering or being solely oocytes. The 58 follicles used constituted 19 primordial, 12 transitional, 15 primary, and 11 secondary. The numbers of follicles at each stage were evenly distributed from either source of tissue apart from the primordial follicles of which two were from OV and the remaining 15 from CB (Table 3Go).


View this table:
[in this window]
[in a new window]

  		TABLE 3. Total number of individual follicles isolated from ovarian cortical tissue obtained from women undergoing either CB or TAH/BSO (OV) (left column) and number of follicles expressing mRNA expression for ß-actin, AR, FSHR, and AMHRII at different follicle stages

 
Fifty-two of the 57 follicles (91%) were positive for ß-actin expression with numbers being approximately even from the two sources (Table 3Go). Of these 52, 27% (14) were AR+ve, 6% (3) FSHR+ve, and 6% (3) AMHRII+ve. AR expression was not detected in any primordial follicles, but as folliculogenesis progressed, the percentage of AR+ve follicles at each stage increased (linear correlation coefficient = 0.995) from 18% of transitional follicles to 43% of primaries and 60% of secondary follicles. Only 3 of 14 primary follicles were FSHR+ve. The three AMHRII+ve follicles consisted of one primordial, one transitional, and one secondary follicle (Table 3Go and Fig. 2Go). AR mRNA expression colocalized with FSHR in two primary follicles (Fig. 3AGo). AMHRII mRNA expression did not colocalize with FSHR but did with AR in one secondary follicle (Fig. 3BGo).


Figure 2
View larger version (12K):
[in this window]
[in a new window]

  		FIG. 2. Histogram showing distribution of follicles positive for ß-actin, AR, FSHR, and AMHRII mRNA expression during different stages of follicle growth.

 

Figure 3
View larger version (82K):
[in this window]
[in a new window]

  		FIG. 3. Top left panel, Representative gels of PCR products for ß-actin, AR, and FSHR on a range of follicles from the same ovarian cortical tissue. All the follicles were negative for AMHRII (data not shown). Lanes: L, 100-bp ladder; 1, antral follicle; 2, secondary follicle; 3, secondary follicle; 4, primordial follicle; 5, mixed group of primary and primordial follicles; 6, secondary follicle; 7, primary follicle; 8, secondary follicle; 9, secondary follicle; 10, primary follicle; 11, primordial follicle; 12, primary follicle; 13–16, groups of unclassified follicles; 17, positive control cDNA; 18, PCR negative (no cDNA template). Top right panel, Picture of the primary follicle (lane 10) in which AR expression colocalized with FSHR. Bottom left panel, Representative gels of PCR products for ß-actin, AR, FSHR, and AMHRII on four follicles from the same cortical biopsy. Lanes: L, 100-bp ladder; 1, primary follicle; 2, transitional follicle; 3, secondary follicle; 4, primary follicle; 5, positive control cDNA; 6, PCR-negative control (no cDNA template). Bottom right panel, Picture of the secondary follicle (lane 3), which is positive for both AR and AMHRII expression but not FSHR.

 
Primer sensitivity was assessed by using 10-fold serial dilutions of a fixed amount of cDNA. Using the nested PCR assay, the detection limit was 500 pg/µl for FSHR and AMHRII, 22 pg/µl for AR, and 15 fg/µl for ß-actin. The amount of cDNA at each follicle stage was: primordial 1169.8 ± 53.7 ng/µl (n = 11); transitional 1378.8 ± 91.9 ng/µl (n = 10); primary 1333.2 ± 57.5 ng/µl (n = 11); and secondary 1417.3 ± 90.9 ng/µl (n = 9). All results expressed as mean ± SEM.


   Discussion
Top
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
References
 
We have shown for the first time that the cognate receptor through which all androgens act is present from the earliest stage at which follicles enter the growing pool, i.e. the transitional stage. The number of follicles positive for AR mRNA increased linearly with growth. Early follicles acquired AR before FSHR and the finding that a greater number of preantral follicles (27%) expressed mRNA for AR than FSHR or AMHRII (6% each) further highlights the importance of androgens in early folliculogenesis. Importantly, we found that mRNA for AMHRII was rarely expressed in preantral follicles from normal ovaries. This challenges the hypothesis that AMH exerts a direct, inhibitory effect on follicles at this stage, although absence of mRNA does not prove absence of protein.

Likewise, whereas mRNA expression is not indicative of a functionally active protein, our findings do allow for the possibility that androgens exert an effect from the earliest growing phase onwards. AR protein and mRNA have been located in preantral follicles of primate ovary by immunohistochemistry (10) and in situ hybridization, respectively (25). In human ovaries AR was immunohistochemically localized to preantral, antral follicles, theca, and stroma (11, 26, 27). However, the stage of preantral follicle development could not be specified due to the inherent insensitivity of these techniques. The unique advantage of the technique used here is that it enabled us to study individual carefully characterized follicles. When this was coupled to the sensitivity of nested RT-PCR, it allowed for detection of expression even in the smallest single primordial follicle.

This approach has been widely used and validated in the diagnosis of preimplantation genetic defects in single, blastomere biopsies from human preimplantation embryos in which mRNA levels are probably considerably lower than even the smallest single primordial follicle, which consists of an oocyte surrounded by at least five to six GCs (28). This is demonstrated by the fact that the nested primers were able to detect down to picogram per microliter amounts of input cDNA, which is more than adequately sensitive because the amount of cDNA in the preantral follicles ranged from 881 to 1885.35 ng/µl (average of 1324.8 ng/µl). Moreover the robustness of the technique is supported by the fact that 91% of follicles isolated had intact mRNA as indicated by PCR for ß-actin. Whereas the absolute levels of ß-actin mRNA transcripts will be higher than that of the receptor transcripts, it provides us with a measure of the efficiency of the RT reaction and also an indication that if a receptor transcript is not detected, then this is not due to failure of RT.

In primate follicles AR mRNA appeared in the GCs coincidentally with mature theca interna cells and was positively correlated with cell proliferation and negatively correlated with apoptosis (25). This suggested that thecal androgens may drive primate follicle growth. The authors proposed that androgens were stimulating initiation of follicle growth by one of two mechanisms: either directly via AR on the primordial follicle or indirectly via other growth factors. The latter was proposed because AR expression was also seen in theca-interstitium and stroma, a finding also supported by other studies (25, 26, 27). We also found substantial and consistent mRNA expression of AR in human ovarian stroma and cortex (results not shown). To date, there were no reports of AR mRNA or protein expression in primordial follicles of primates or humans, but this could have been due to lack of sensitivity of the techniques used. By using isolated, individual follicles, we have demonstrated that AR mRNA was not present in any of the 17 human primordial follicles studied and appeared only from the transitional stage onward. It would seem therefore that androgens are not able to exert a direct effect on initiation of primordial follicle growth, but once the follicles have initiated growth, androgens would be increasingly able to exert an effect. This was illustrated by the fact that lamb ovarian cortex cultured on the chorioallantoic membrane of chick eggs in the presence of testosterone showed a selective increase in primary follicle numbers, compared with those cultured without testosterone (29). Conversely, mature AR knockout mice had no primary-secondary follicles, compared with wild type (30). Culturing follicles (100–200 µm) mechanically isolated from immature mouse ovaries, with a range of androgens increased their diameter in a dose-dependent manner, an effect not seen with either estradiol or estrone (31).

It has been suggested that androgens interact with FSH to promote follicular development, even in the gonadotropin-independent phase of folliculogenesis, and significant colocalization between mRNA for AR and FSHR has been seen in healthy, growing antral follicles from primate ovaries (14). Moreover, FSH-treated animals showed a marked induction in AR mRNA expression in primary follicles but not larger follicles, suggesting a potential physiological mechanism whereby FSH may promote early follicular development (14). Likewise, androgen treatment caused an increase in FSHR mRNA expression at all follicle stages (14).

We found FSHR mRNA expression only at the primary stage and at similar levels to those found by Oktay et al. (32) (21 vs. 33%). We did not find FSHR mRNA expression in any of the postprimary stage follicles, which is not surprising because Oktay et al. found that expression at the two-layer stage was very variable, with only a third of follicles positive for FSHR mRNA. This heterogeneity in expression was not related to the amount of cDNA present in the follicles because there was no correlation between measurements of cDNA levels and the presence/absence of receptor transcripts in follicles (data not shown). In addition, this is not a reflection of the fact that some tissue was obtained from women in late pregnancy because Oktay et al. (32) also used this source of tissue, and there is no evidence to suggest that preantral follicle growth is altered during pregnancy (1, 33). There was colocalization of AR and FSHR mRNA in two of three primary follicles, but it was clear that AR mRNA expression preceded that of FSHR and overall more follicles were positive for AR than FSHR (15 vs. three), again highlighting the functional importance of androgens in the preantral follicle.

The finding that there is heterogeneity of expression of these important receptors within individual small follicles is a significant observation as it suggests that all follicles are not created equal and selection maybe occurring even at this early stage. For example, it may be envisaged that follicles lacking FSHR would be unable to progress, but because preantral folliculogenesis is largely considered to be FSH independent, a more likely explanation is that follicles acquire their FSHR progressively, but the driving force may be the acquisition of AR.

It has been hypothesized from the Amh null mice studies (16) and in vitro culture (17) that AMH functions as a general inhibitor of primordial follicle recruitment. AMH exerts all its biological effects through AMHRII, as shown in AMHRII knockout male mouse in which there was no Müllerian duct regression (34). This is further emphasized by the fact that Müllerian ducts persist in those patients who have mutations in the AMHRII gene (35, 36). It was surprising therefore that only one of 17 primordial follicles in our study was positive for AMHRII mRNA. In fact, overall, only three of 52 preantral follicles were AMHRII mRNA positive, the remaining two follicles being a transitional and secondary follicle. In the adult rat ovary, AMHRII mRNA was found in preantral and small antral follicles but not in primordial follicles using in situ hybridization (37). With regard to human follicles, no studies have been performed on the pattern of AMHRII expression. In rats, mice, or humans, however, AMH protein identified by in situ hybridization or immunohistochemistry was not found in resting follicles but only early growing follicles (17, 37, 38).

To explain the increased number of primary follicles in the PCOs, compared with normal ovaries (2, 3), it has been proposed that reduced local exposure to AMH in the PCOs would result in a higher proportion of primordial follicles initiating growth, compared with normal (18). This is supported by the observation that the percentage of primordial and transitional follicles staining for AMH protein was less in tissue from women with anovulatory PCOs, compared with those with ovulatory PCOs or normal ovaries, despite the observations that women with PCO syndrome have significantly higher levels of AMH in both serum and follicular fluid (39, 40). The fact that nearly all of the isolated primordial follicles in this study did not express mRNA for the AMHRII now casts some doubt on this model. However because the presence/absence of a functional AMHRII protein was not determined, this model cannot be wholly disproved.

In conclusion, we have shown that mRNA for AR is present from the transitional stage onward. Our data indicate that preantral follicles acquire AR before FSHR with the number of AR-positive follicles increasing as growth progresses, further supporting the importance of androgens in early folliculogenesis. Furthermore, it is doubtful that AMH exerts a direct inhibition on the primordial pool of follicles or early preantral growth in the normal human ovary because the majority of follicles do not express mRNA for the AMHRII.


   Acknowledgments
 
We acknowledge and thank the surgeons and midwives in the Department of Obstetrics and Gynaecology (St. George’s Hospital, London) for their help in the collection of ovarian cortical biopsies and thank the patients who consented to donate tissue for this study.


   Footnotes
 
This work was supported by a Charitable Trust of St. George’s Hospital grant (to H.M. and S.R.) and a Wellcome Trust grant (to H.M., S.W., and S.R.).

Disclosure Statement: The authors have nothing to disclose.

First Published Online December 19, 2006

Abbreviations: AMH, Anti-Müllerian hormone; AMHRII, AMH type II receptor; AR, androgen receptor; BSO, bilateral salpingo-oophorectomy; CB, elective C-section; FSHR, FSH receptor; GC, granulosa cell; OV, ovarian cortex and stroma; PCO, polycystic ovary; RT, reverse transcription; TAH, total abdominal hysterectomy.

Received August 7, 2006.

Accepted December 13, 2006.


   References
Top
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
 References
 

  1. Gougeon A 1996 Regulation of ovarian follicular development in primates: facts and hypotheses. Endocr Rev 17:121–155[CrossRef][Medline]
  2. Maciel GAF, Baracat EC, Benda JA, Markaham SM, Hensinger K, Chang JR, Erickson GF 2004 Stockpiling of transitional and classic primary follicles in ovaries of women with polycystic ovary syndrome. J Clin Endocrinol Metab 89:5321–5327[Abstract/Free Full Text]
  3. Webber LJ, Stubbs S, Stark J, Trew GH, Margara R, Hardy K, Franks S 2003 Formation and early development of follicles in the polycystic ovary. Lancet 362:1017–1021[CrossRef][Medline]
  4. Hughesdon PE 1982 Morphology and morphogenesis of the Stein-Leventhal ovary and of so-called "hyperthecosis." Obstet Gynecol Surv 37:59–77[Medline]
  5. Abbott DH, Barnett DK, Bruns CM, Dumesic DA 2005 Androgen excess fetal programming of female reproduction: a developmental aetiology for polycystic ovary syndrome? Hum Reprod Update 11:357–374[Abstract/Free Full Text]
  6. Abbott DH, Dumesic DA, Eisner JR, Colman RJ, Kemnitz JW 1999 Insights into the development of PCOS from studies in the prenatally androgenized female rhesus monkey. Trends Endocrinol Metab 9:62–67[Medline]
  7. West C, Foster DL, Evans NP, Robinson J, Padmanabhan V 2001 Intra-follicular activin availability is altered in prenatally androgenized lambs. Mol Cell Endocrinol 185:51–59[CrossRef][Medline]
  8. Vendola KA, Zhou J, Adesanya OO, Weil SJ, Bondy CA 1998 Androgens stimulate early stages of follicular growth in the primate ovary. J Clin Invest 101:2622–2629[Medline]
  9. Tetsuka M, Whitelaw PF, Bremner WJ, Millar MR, Smyth CD, Hillier SG 1995 Developmental regulation of androgen receptor in rat ovary. J Endocrinol 145:535–543[Abstract]
  10. Hillier SG, Tetsuka M, Fraser HM 1997 Location and developmental regulation of androgen receptor in primate ovary. Hum Reprod 12:107–111[Abstract]
  11. Saunders PT, Millar MR, Williams K, Macpherson S, Harkiss D, Anderson RA, Orr B, Groome NP, Scobie G, Fraser HM 2000 Differential expression of estrogen receptor-{alpha} and -ß and androgen receptor in the ovaries of marmosets and humans. Biol Reprod 63:1098–1105[Abstract/Free Full Text]
  12. Szoltys M, Slomczynska M, Duda M, Sakiewicz A, Otak A 2005 Distribution of androgen receptor in rat ovarian follicles undergoing atresia at the beginning of pregnancy. Acta Histochem 107:357–364[CrossRef][Medline]
  13. Tetsuka M, Hillier SG 1997 Differential regulation of aromatase and androgen receptor in granulosa cells. J Steroid Biochem Mol Biol 61:233–239[CrossRef][Medline]
  14. Weil S, Vendola K, Zhou J, Bondy CA 1999 Androgen and follicle-stimulating hormone interactions in primate ovarian follicle development. J Clin Endocrinol Metab 84:2951–2956[Abstract/Free Full Text]
  15. Jost A 1947 Reserches sur la differenciation sexuelle de l’embryon de lapin. Arch Anat Microsc Morphol Exp 36:271–315
  16. Durlinger AL, Kramer P, Karels B, de Jong FH, Uilenbroek JT, Grootegoed JA, Themmen AP 1999 Control of primordial follicle recruitment by anti-Mullerian hormone in the mouse ovary. Endocrinology 140:5789–5796[Abstract/Free Full Text]
  17. Durlinger AL, Gruijters MJ, Kramer P, Karels B, Ingraham HA, Nachtigal MW, Uilenbroek JT, Grootegoed JA, Themmen AP 2002 Anti-Mullerian hormone inhibits initiation of primordial follicle growth in the mouse ovary. Endocrinology 143:1076–1084[Abstract/Free Full Text]
  18. Stubbs SA, Hardy K, Da Silva-Buttkus P, Stark J, Webber LJ, Flanagan AM, Themmen AP, Visser JA, Groome NP, Franks S 2005 Anti-Mullerian hormone protein expression is reduced during the initial stages of follicle development in human polycystic ovaries. J Clin Endocrinol Metab 90:5536–5543[Abstract/Free Full Text]
  19. Josso N, di Clemente N 2003 Transduction pathway of anti-Müllerian hormone, a sex-specific member of the TGF-ß family. Trends Endocrinol Metab 14:91–97[CrossRef][Medline]
  20. Roy SK, Treacy MD 1993 Isolation and long-term culture of human preantral follicles. Fert Steril 59:783–790[Medline]
  21. Roy SK, Greenwald GS 1996 Methods of separation and in vitro culture of pre-antral follicles from mammalian ovaries. Hum Reprod Update 2:236–245[Abstract/Free Full Text]
  22. Oktay K, Nugent D, Newton H, Salha O, Chatterjee P, Gosden RG 1997 Isolation and characterization of primordial follicles from fresh and cryopreserved human ovarian tissue. Fertil Steril 67:481–486[CrossRef][Medline]
  23. Picton HM 2001 Activation of follicle development: the primordial follicle. Theriogenology 55:1193–1210[CrossRef][Medline]
  24. Gilliland G, Perrin S, Bunn HF 1990 Competitive PCR for quantification of mRNA. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ, eds. PCR protocols: a guide to methods and applications. London: Academic Press, Ltd.
  25. Weil SJ, Vendola K, Zhou J, Adesanya OO, Wang J, Okafor J, Bondy CA 1998 Androgen receptor gene expression in the primate ovary: cellular localization, regulation, and functional correlations. J Clin Endocrinol Metab 83:2479–2485[Abstract/Free Full Text]
  26. Chadha S, Pache TD, Huikeshoven JM, Brinkmann AO, van der Kwast TH 1994 Androgen receptor expression in human ovarian and uterine tissue of long-term androgen-treated transsexual women. Hum Pathol 25:1198–1204[CrossRef][Medline]
  27. Takayama K, Fukaya T, Sasano H, Funayama Y, Suzuki T, Takaya R, Wada Y, Yajima A 1996 Immunohistochemical study of steroidogenesis and cell proliferation in polycystic ovarian syndrome. Hum Reprod 11:1387–1392[Abstract/Free Full Text]
  28. Sermon K 2002 Current concepts in preimplantation genetic diagnosis (PGD): a molecular biologist’s view. Hum Reprod Update 8:11–20[Abstract/Free Full Text]
  29. Qureshi AI, Bano G, Whitehead SA, Nussey SS, Mason HD, Androgens increase primary follicle numbers and reduce atresia in ovarian tissue: a new model for the polycytic ovary? Program of the 87th Annual Meeting of The Endocrine Society, San Diego, CA, 2005, p 114 (Abstract OR28-1)
  30. Shiina H, Matsumoto T, Sato T, Igarashi K, Miyamoto J, Takemasa S, Sakari M, Takada I, Nakamura T, Metzger D, Chambon P, Kanno J, Yoshikawa H, Kato S 2006 Premature ovarian failure in androgen receptor-deficient mice. Proc Natl Acad Sci USA 103:224–229[Abstract/Free Full Text]
  31. Wang H, Andoh K, Hagiwara H, Xiaowei L, Kikuchi N, Abe Y, Yamada K, Fatima R, Mizunuma H 2001 Effect of adrenal and ovarian androgens on type 4 follicles unresponsive to FSH in immature mice. Endocrinology 142:4930–4936[Abstract/Free Full Text]
  32. Oktay K, Briggs D, Gosden RG 1997 Ontogeny of follicle-stimulating hormone receptor gene expression in isolated human ovarian follicles. J Clin Endocrinol Metab 82:3748–3751[Abstract/Free Full Text]
  33. Amorim CA, Lucci CM, Rodrigues AP, Carvalho FC, Figueiredo JR, Rondina D, Cecchi R, Giorgetti A, Martini A, Goncalves PB 2000 Quantitative and qualitative analysis of the effectiveness of a mechanical method for the isolation of preantral follicles from ovine ovaries. Theriogenology 53:1251–1262[CrossRef][Medline]
  34. Mishina Y, Rey R, Finegold MJ, Matzuk MM, Josso N, Cate RL, Behringer RR 1996 Genetic analysis of the Mullerian-inhibiting substance signal transduction pathway in mammalian sexual differentiation. Genes Dev 10:2577–2587[Abstract/Free Full Text]
  35. Imbeaud S, Belville C, Messika-Zeitoun L, Rey R, di Clemente N, Josso N, Picard JY 1996 A 27 base-pair deletion of the anti-Mullerian type II receptor gene is the most common cause of the persistent Mullerian duct syndrome. Hum Mol Genet 5:1269–1277[Abstract/Free Full Text]
  36. Josso N, Belville C, di Clemente N, Picard JY 2005 AMH and AMH receptor defects in persistent Mullerian duct syndrome. Hum Reprod Update 11:351–356[Abstract/Free Full Text]
  37. Baarends WM, Uilenbroek JT, Kramer P, Hoogerbrugge JW, van Leeuwen EC, Themmen AP, Grootegoed JA 1995 Anti-Mullerian hormone and anti-Mullerian hormone type II receptor messenger ribonucleic acid expression in rat ovaries during postnatal development, the estrous cycle, and gonadotropin-induced follicle growth. Endocrinology 136:4951–4962[Abstract]
  38. Weenen C, Laven JS, Von Bergh AR, Cranfield M, Groome NP, Visser JA, Kramer P, Fauser BC, Themmen AP 2004 Anti-Mullerian hormone expression pattern in the human ovary: potential implications for initial and cyclic follicle recruitment. Mol Hum Reprod 10:77–83[Abstract/Free Full Text]
  39. Cook CL, Siow Y, Brenner AG, Fallat ME 2002 Relationship between serum Mullerian-inhibiting substance and other reproductive hormones in untreated women with polycystic ovary syndrome and normal women. Fertil Steril 77:141–146[CrossRef][Medline]
  40. Pigny P, Merlen E, Robert Y, Cortet-Rudelli C, Decanter C, Jonard S, Dewailly D 2003 Elevated serum level of anti-Mullerian hormone in patients with polycystic ovary syndrome: relationship to the ovarian follicle excess and to the follicular arrest. J Clin Endocrinol Metab 88:5957–5962[Abstract/Free Full Text]
  41. Spanos S, Rice S, Karagiannis P, Taylor D, Becker DL, Winston RML, Hardy K 2002 Caspase activity and expression of cell death genes during development of human preimplantation embryos. Reproduction 124:353–363[Abstract]
  42. Apparao KB, Lovely LP, Gui Y, Lininger RA, Lessey BA 2002 Elevated endometrial androgen receptor expression in women with polycystic ovary syndrome. Biol Reprod 66:297–304[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Hum Reprod UpdateHome page
S. Franks, J. Stark, and K. Hardy
Follicle dynamics and anovulation in polycystic ovary syndrome
Hum. Reprod. Update, May 22, 2008; (2008) dmn015v1.
[Abstract] [Full Text] [PDF]

Home page
 J. Clin. Endocrinol. Metab.Home page
D. S. Wachs, M. S. Coffler, P. J. Malcom, S. Shimasaki, and R. J. Chang
Increased Androgen Response to Follicle-Stimulating Hormone Administration in Women with Polycystic Ovary Syndrome
J. Clin. Endocrinol. Metab., May 1, 2008; 93(5): 1827 - 1833.
[Abstract] [Full Text] [PDF]

Home page
 Hum ReprodHome page
S. Rice, K. Ojha, and H. Mason
Human ovarian biopsies as a viable source of pre-antral follicles
Hum. Reprod., March 1, 2008; 23(3): 600 - 605.
[Abstract] [Full Text] [PDF]

Home page
 Hum ReprodHome page
K. R. Hansen, N. S. Knowlton, A. C. Thyer, J. S. Charleston, M. R. Soules, and N. A. Klein
A new model of reproductive aging: the decline in ovarian non-growing follicle number from birth to menopause
Hum. Reprod., March 1, 2008; 23(3): 699 - 708.
[Abstract] [Full Text] [PDF]

Home page
 Hum ReprodHome page
S. M. Nelson, R. W. Yates, and R. Fleming
Serum anti-Mullerian hormone and FSH: prediction of live birth and extremes of response in stimulated cycles implications for individualization of therapy
Hum. Reprod., September 1, 2007; 22(9): 2414 - 2421.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
92/3/1034    most recent
Author Manuscript (PDF)
Right arrow Submit a related Letter to the Editor
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Rice, S.
Right arrow Articles by Mason, H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Rice, S.
Right arrow Articles by Mason, H.
Related Collections
Right arrow Female Endocrinology

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals