Cisplatin- and cyclophosphamide-induced primordial follicle depletion is caused by direct damage to oocytes.

It is well established that DNA-damaging chemotherapies can cause infertility and ovarian endocrine failure by depleting the ovarian reserve of primordial follicles. Currently, no effective pharmacological therapies exist for the preservation of long-term fertility and ovarian function in female cancer patients, due to a limited understanding of the mechanisms of chemotherapy-induced follicle depletion. This study investigated the cellular targets, molecular mechanisms, and temporal course of ovarian reserve depletion following treatment with commonly used chemotherapeutic drugs. Adult female C57BL/6 mice were injected i.p. with saline, cisplatin (5mg/kg), or cyclophosphamide (300mg/kg); ovaries were harvested after 8 or 24 hours. Follicle counts showed depletion of all follicular stages 24 hours after administration of cisplatin or cyclophosphamide. Eight hours post-treatment, H2A histone family member X (γH2AX) immunofluorescence showed DNA double-stranded breaks at all follicular stages, including within primordial follicle oocytes. This staining was resolving by 24 hours, indicating that primordial follicle oocytes begin to undergo either apoptosis or repair in this timeframe. γH2AX-positive follicles were further examined to identify the specific cell types damaged. In primordial, transitional, and primary follicles, only oocytes sustained DNA damage, whereas in secondary and antral follicles, only somatic cells were affected. TUNEL staining confirmed that apoptosis occurs in these targeted cell types. Whilst multi-drug and multi-dose regimens were not examined, this study conclusively shows that cyclophosphamide and cisplatin cause direct damage to primordial follicle oocytes, which then undergo apoptosis. Therefore, future pharmacological strategies to prevent chemotherapy-induced infertility in females must specifically prevent primordial follicle oocyte death.

The primordial follicle reserve is not renewed after chemical or γ-irradiation mediated depletion.

Reports indicate that germ-line stem cells present in adult mice can rapidly generate new oocytes and contribute to the primordial follicle reserve following conditions of ovotoxic stress. We further investigated the hypothesis that adult mice have the capacity to generate new oocytes by monitoring primordial follicle numbers throughout postnatal life and following depletion of the primordial follicle reserve by exposure to doxorubicin (DXR), trichostatin A (TSA), or whole-body γ-irradiation. We show that primordial follicle number remains stable in adult C57BL/6 mice between the ages of 25 and 100 days. However, within 2 days of treatment with DXR or TSA, primordial follicle numbers had declined to 65 and 51% respectively (P<0.05-0.01 when compared to untreated controls), with no restoration of follicle numbers evident after 7 days for either treatment. Furthermore, ovaries from mice subjected to sterilizing doses of γ-irradiation (0.45 or 4.5 Gy) revealed complete ablation of all primordial follicles 5 days after treatment, with no indication of follicular renewal. We conclude that neo-folliculogenesis does not occur following chemical or γ-irradiation mediated depletion of the primordial follicle reserve.

Estrogen signaling in the regulation of female reproductive functions.

Estrogens influence fertility and infertility in animals. This chapter reviews the use of estrogen as a contraceptive through the regulation of its production and action. It is concluded that the use of specific agonists and antagonists of estrogen action that avoid the global and unwanted side effects of estrogen offers new potential methods of contraception.

A randomized controlled trial of treatment options for troublesome uterine bleeding in Implanon users.

BACKGROUND: Pilot data have indicated that both doxycycline alone and mifepristone combined with ethinyl estradiol (EE) are effective in stopping episodes of bleeding in Implanon users with troublesome bleeding. We compared four treatments against a placebo in Implanon users and tested whether repeated treatment improved subsequent bleeding patterns. METHOD: Implanon users aged 18-45 years were randomized to treatment with (i) mifepristone 25 mg given twice on day 1 followed by 4 days of EE 20 microg; (ii) doxycycline 100 mg twice daily for 5 days; (iii) mifepristone 25 mg given twice on day 1 plus doxycycline 100 mg twice daily for 5 days; (iv) doxycycline 100 mg twice daily with EE 20 microg daily; and (v) placebo twice daily for 5 days. The primary end-point was the number of days of bleeding/spotting immediately following initiation of the first 5-day course of each therapy, compared with placebo. RESULTS: There were 204 women assigned to treatment. Mifepristone in combination with either EE or doxycycline was significantly more effective in stopping an episode of bleeding (mean 4.0 days (CI 3.5-4.6) and 4.4 days (CI 3.8-5.2), respectively) than doxycycline alone or in combination with EE, or placebo (6.4 days (CI 4.4-9.2), 6.4 days (CI 4.8-8.6) and 6.4 days (CL 5.1-8.0), respectively). CONCLUSION: Mifepristone combined with either EE or doxycycline was significantly more effective than placebo in terminating an episode of bleeding in Implanon users. However there was no improvement in subsequent bleeding patterns. TRIAL REGISTRATION NUMBER: ACTR # 012605000206628.

Human embryo: a biological definition.

This paper defines a human embryo from a biological standpoint that takes into account emerging technologies in reproductive science. The paper does not consider legal, moral, religious or social views. As the definition of a human embryo must reflect the multifactorial processes of development, an approach has been adopted which combines recognition of observed events with potential for further development. This acknowledges that fertilization and development are not static processes, and as such embryo status can only be defined by observation of specific markers. The following biological definition of 'human embryo' is proposed. A human embryo is a discrete entity that has arisen from either: the first mitotic division when fertilization of a human oocyte by a human sperm is complete or any other process that initiates organized development of a biological entity with a human nuclear genome or altered human nuclear genome that has the potential to develop up to, or beyond, the stage at which the primitive streak appears, and has not yet reached 8 weeks of development since the first mitotic division.

Quantification of healthy follicles in the neonatal and adult mouse ovary: evidence for maintenance of primordial follicle supply.

Proliferation and partial meiotic maturation of germ cells in fetal ovaries is believed to establish a finite, non-renewable pool of primordial follicles at birth. The supply of primordial follicles in postnatal life should be depleted during folliculogenesis, either undergoing atresia or surviving to ovulation. Recent studies of mouse ovaries propose that intra- and extraovarian germline stem cells replenish oocytes and form new primordial follicles. We quantified all healthy follicles in C57BL/6 mouse ovaries from day 1 to 200 using unbiased stereological methods, immunolabelling of oocyte meiosis (germ cell nuclear antigen (GCNA)) and ovarian cell proliferation (proliferating cell nuclear antigen (PCNA)) and electronmicroscopy. Day 1 ovaries contained 7924+/-1564 (s.e.m.) oocytes or primordial follicles, declining on day 7 to 1987+/-203, with 200-800 oocytes ejected from individual ovaries on that day and day 12. Discarded oocytes and those subjacent to the surface epithelium were GCNA-positive indicating their incomplete meiotic maturation. From day 7 to 100 mean numbers of primordial follicles per ovary were not significantly depleted but declined at 200 days to 254+/-71. Mean numbers of all healthy follicles per ovary were not significantly different from day 7 to 100 (range 2332+/-349-3007+/-322). Primordial follicle oocytes were PCNA-negative. Occasional unidentified cells were PCNA-positive with mitotic figures observed in the cortex of day 1 and 12 ovaries. Although we found no evidence for ovarian germline stem cells, our data support the hypothesis of postnatal follicle renewal in postnatal and adult ovaries of C57BL/6 mice.

A pilot study to assess the effect of three short-term treatments on frequent and/or prolonged bleeding compared to placebo in women using Implanon.

BACKGROUND: The major side-effect of progestogen-only contraception is disruption of menstrual bleeding patterns, which can lead to a high incidence of early discontinuation. The aim of this study was to compare three treatments with placebo on the duration and recurrence of frequent and/or prolonged bleeding in Implanon users. METHOD: Women between the ages of 18 and 45 years, who had used Implanon for > or =3 months and were experiencing prolonged or frequent bleeding patterns, were recruited at four Australian sites. Subjects were randomized to treatment using computer-generated random number table if they met the World Health Organization criteria for prolonged and/or frequent bleeding in the previous 90 days [Belsey, E.M., Pinol, A.P.Y. and Taskforce on Long-Acting Systemic Agents for Fertility Regulation, World Health Organization (1997) Contraception 55,57-65]. Treatments were: (1) mifepristone 25 mg given twice on day 1 followed by 4 days of twice daily placebo; (2) mifepristone 25 mg given twice on day 1 followed by 4 days of ethinyl estradiol (EE) 20 microg in the morning and placebo at night; (3) doxycycline 100 mg twice daily for 5 days; and (4) placebo twice daily for 5 days. Analysis was by intention to treat. The primary endpoint was the number of days of bleeding and spotting immediately following initiation of the 5 day course of each active therapy compared with placebo. RESULTS: A total of 179 women was assigned to treatment. Both mifepristone in combination with EE and doxycycline alone were significantly more effective in stopping an episode of bleeding {mean 4. 3 days [confidence interval (CI) 3.5-5.2], and 4.8 days (CI 3.9-5.8) respectively} than mifepristone alone or placebo [5.9 days (CI 4.8-7.2) and 7.5 days (CI 6.1-9.1) respectively]. No effect on subsequent bleeding patterns was observed in any treatment group. CONCLUSION: Both mifepristone plus EE and doxycycline alone were significantly more effective than placebo in terminating an episode of bleeding in women with prolonged and/or frequent bleeding using Implanon. We believe that the observed reduction in the number of bleeding days by almost 50% compared to placebo in both the mifepristone combination group and the doxycycline group demonstrates a clinically significant improvement in bleeding patterns and that further trials are needed to compare different combinations of therapy as well as multiple dosing regimens in order to establish which is the most effective treatment option. The effect of repeat administration or combinations of these preparations on long-term bleeding patterns requires further investigation.

HtrA3, a serine protease possessing an IGF-binding domain, is selectively expressed at the maternal-fetal interface during placentation in the mouse.

Hemochorial placentation involves highly regulated interactions between fetal- and maternal-derived cells. HtrA3, a novel serine protease containing an insulin-like growth factor (IGF) binding domain, was previously shown to increase during early pregnancy in the mouse uterus, being dramatically upregulated post-implantation. The present study examined the regulation of HtrA3 gene in the mouse uterus from post-implantation to late gestation. Both mRNA and protein of HtrA3 were localized specifically in the maternal decidua. In contrast, HtrA3 expression was below detection in trophoblasts, including the giant cells that are in direct contact with the decidua. This pattern persisted from the early stages of placentation to near term. The level of decidual HtrA3 mRNA and its protein gradually decreased as the placenta matured. In the decidua, only the maternal decidual cells, but not blood vessels or uterine NK cells that are present in large numbers, were positive for HtrA3. The specific localization of a protease possessing an IGF-binding domain at the maternal-fetal interface suggests that HtrA3 plays a critical role in mediating maternal decidual remodelling and maintenance, likely in association with the IGF system, in placental development and function.

Estrogen actions in the ovary revisited.

Estrogens are synonymous with fertility and infertility in mammals. Our knowledge of the biological actions of estrogens, however, is incomplete. Three recent developments have thrown new light on the actions of estrogens in mammalian reproduction that will lead to a greater understanding of their functions. They are (a) the identification of a second estrogen receptor, called ERbeta, (b) the identification of ligand-specific ER coactivators and (c) mouse models with targeted disruption of the genes encoding both ER and the aromatase enzyme. These models provide for the first time animals which are either unable to respond to endogenous or exogenous estrogens (ER 'knockouts'), or can respond to exogenous estrogen but do not make endogenous estrogen (aromatase 'knockout' or ArKO). Furthermore, the ArKO mouse has provided a model to study the effects on the ovary of exogenous estrogens of plant and synthetic origin that are of clinical relevance. The data show that estrogens are essential for fertility but not for survival after birth or for the formation of the reproductive tract. This commentary focuses on the roles of estrogen in folliculogenesis and in the maintenance of the ovarian somatic cell phenotype in the mouse. We also hypothesize that the ERalpha and ERbeta may subserve the proliferative and differentiative actions of estrogen, respectively, within a follicle. In summary, estrogen is obligatory for normal folliculogenesis beyond the antral stage and for the maintenance of the female phenotype of the somatic cells within the ovaries. This clearly demonstrates a major role for sex steroids in somatic cell differentiation in the gonads of eutherian mammals and challenges the central paradigm that the ovary is the default gonad, arising due to the absence of testicular defining signals. Evidence is also provided for the plasticity of the adult female gonad. Understanding the mechanisms of estrogen actions will provide an insight into the regulation of reproductive disorders afflicting women today, notably ovarian dysfunction and the menopause.

A potential molecular mechanism for regulating pre-mRNA splicing of implantation-related genes through unique uterine expression of splicing factor SC35 in women and rhesus monkeys.

Splicing factor SC35 is an essential component of the spliceosome, the cellular apparatus that removes introns from pre-mRNA to provide alternatively spliced isoforms. Many proteins associated with development of uterine receptivity and embryo implantation are present as isoforms, the tissue-specific expression of which may be regulated through alternative splicing. SC35 was identified as being increased at implantation sites during early pregnancy in mice. However, the present study has demonstrated that SC35 is present in human and rhesus monkey endometrium, that the protein is increased during the secretory phase of the oestrous cycle compared with the proliferative phase in both these primates and that it is present in a distinct pattern within the nucleus of both epithelial and stromal cells, as well as in cells of the vasculature. Both the intensity of immunoreactive protein and the proportion of cells that stain for SC35 alter with the phase of the oestrous cycle. A very precise expression pattern of SC35 (both protein and mRNA) was seen during early placentation in rhesus monkeys. At implantation sites between day 24 and day 35 of early pregnancy, SC35 was expressed strongly in cytotrophoblasts within the trophoblastic shell, in syncytiotrophoblast at the periphery of the cell column and in both cytotrophoblast and syncytiotrophoblast in the floating villi. In the adjacent maternal decidua, expression of SC35 was weak. These results indicate a role for SC35 in preparation of a receptive uterus, in the provision of secreted proteins to support blastocyst development and in trophoblast invasion.

Recruitment and development of the follicle; the roles of the transforming growth factor-beta superfamily.

Peripheral endocrine hormones and local paracrine and autocrine factors contribute, in a coordinated fashion, to the processes of recruitment, development or atresia, selection and ovulation of follicles. Among the local ovarian factors, there is growing evidence from genetic and experimental data that many members of the transforming growth factor (TGFbeta) superfamily have a biological role to play in folliculogenesis. These members include activin, inhibin, TGFbeta, BMP, GDF9 and perhaps MIS. In this review, we discuss the potential roles of the TGFbeta superfamily members, in particular activin, during folliculogenesis. Since the actions of these factors are determined by ligand availability, receptor expression and modulation of their signal transduction pathways, we also collate information on the expression of their signalling components in the follicle. We conclude that the TGFbeta superfamily signalling pathways, in particular activin's pathway, reside in the ovary. Furthermore, follistatin and beta-glycan-components of the accessory binding protein system that modifies activin action-are also present in follicles. In the post-natal rat ovary, the changes in receptor/Smad expression coincide with granulosa cell proliferation and antrum formation. We hypothesise that these pathway components are expressed in a temporal and cell-specific manner to meet the changing demands of cells during follicular development. The analysis of the components of the signal transduction pathways of the TGFbeta family members in populations of defined follicles and the identification of activated pathways in individually stimulated follicles should help clarify the roles of the TGFbeta members in folliculogenesis.

Genes involved in implantation.

The endometrium is normally a hostile environment for an embryo, except for a short phase in each reproductive cycle known as the 'window of receptivity'. The precise molecular events involved in this transformation are not well understood. Application of state-of-the-art techniques of the 1990s has identified some of the genes involved, which are reviewed here. Mice with a null mutation in either the gene for leukemia inhibitory factor or the interleukin-11 receptor alpha chain are infertile, owing in both cases to a failure of embryo implantation. Both of these genes are expressed in the human endometrium with patterns suggesting a role in human fertility. The technique of RNA differential display has been applied to a comparison of the expression of genes at implantation sites v. inter-implantation sites in the mouse uterus on the first day of implantation, and has defined additional genes whose products may be important for this process. Among these are the calcium-binding protein D9K, the monoclonal non-specific suppressor factor beta, and the splicing factor SC35. The major challenge is to determine whether manipulation of such genes can increase or decrease endometrial receptivity in humans.

Inhibin binding sites and proteins in pituitary, gonadal, adrenal and bone cells.

Activin signals via complexes of type I (50-55 kDa) and II (70-75 kDa) activin receptors, but the mechanism of inhibin action is unclear. Proposed models range from an anti-activin action at the type II activin receptor to independent actions involving putative inhibin receptors. Two membrane-embedded proteoglycans, betaglycan and p120, have recently been implicated in inhibin binding, but neither appears to be a signalling receptor. The present studies on primary cultures of rat pituitary and adrenal cells, and several murine and human cell lines were undertaken to characterise inhibin binding to its physiological targets. High affinity binding of inhibin to the primary cultures and several of the cell lines, like that previously described for ovine pituitary cells, was saturable and reversible. Scatchard analysis revealed two classes of binding sites (K(d) of 40-400 and 500-5000 pM, respectively). Affinity labelling identified [125I]inhibin binding proteins with apparent molecular weights of 41, 74, 114 and >170 kDa in all cell types that displayed high affinity, high capacity binding of inhibin. Additional labelling of a 124 kDa species was evident in gonadal TM3 and TM4 cell lines. In several cases, activin (> or =20 nM) competed poorly or not at all for binding to these proteins. The 74, 114 and >170 kDa inhibin binding proteins in TM3 and TM4 cells were immunoprecipitated by an anti-betaglycan antiserum. These three proteins correspond in size to the activin receptor type II and the core protein and glycosylated forms of betaglycan, respectively, that have been proposed to mediate anti-activin actions of inhibin, but the identity of the 74 kDa species is yet to be confirmed. Studies of [125I]inhibin binding kinetics and competition for affinity labelling of individual binding proteins in several cell lines suggest these three species and the 41 and 124 kDa proteins form a high affinity inhibin binding complex. In summary, common patterns of inhibin binding and affinity labelling were observed in inhibin target cells. Novel inhibin binding proteins of around 41 and 124 kDa were implicated in the high affinity binding of inhibin to cells from several sources.

Production and actions of inhibin and activin during folliculogenesis in the rat.

Evidence to enhance the premise that inhibin and activin are local regulators of ovarian folliculogenesis is presented in this review. Granulosa cells (GC) have been identified as the source of inhibin/activin in the ovary on the basis of mRNA and protein localisation and the measurement of the inhibin forms in GC conditioned media. Expression of the subunit mRNAs changed with follicular development, being maximal in the ovaries of 8-day-old rats, where secondary follicles predominate. The expression of beta subunit mRNAs by GC isolated from diethylstilboestrol (DES)-treated immature rats, was reduced in the absence of any change in alpha subunit mRNA expression. Dimeric inhibin-A, -B and free alpha subunit were produced by ovarian cell cultures prepared from 4- to 12-day-old rats. Inhibin-A production by these cultures was responsive to FSH and TGF-beta, with preantral follicles of day 8 ovaries exerting effects so profound that the inhibin A/alpha subunit ratio increased, most likely due to a stimulation of beta(A) subunit production. In contrast, inhibin-B was not stimulated by TGF-beta until day 8 and FSH until day 12. Fractionation of GC conditioned media revealed a prominence of free alpha subunit and inhibin-A, but little inhibin-B, suggesting that inhibin-B production declines with follicular development. Activin receptor types I and II, Smads 1-8 and betaglycan (beta-glycan) mRNAs were present in the rat ovary and showed distinct patterns of expression between postnatal days 4 and 12. Oocytes and GC localised activin receptor, Smad and beta-glycan proteins, with beta-glycan also present in theca cells (TC). These data indicate that activin/TGF-beta signalling machinery and factors which influence these pathways, are present in the postnatal rat ovary. Our hypothesis that inhibin and activin play important and changing autocrine/paracrine roles in the growth and differentiation of follicles, including the oocyte, has been supported by these studies.

Kallikrein gene expression in the gonadotrophin-stimulated rat ovary.

The kallikreins (KLKs) are a highly conserved multi-gene family of serine proteases that are expressed in a wide variety of tissues and act on a diverse range of substrates. KLK-like enzyme activity has variously been reported to increase or decrease during the period leading up to ovulation in the equine chorionic gonadotrophin (eCG)primed, human chorionic gonadotrophin (hCG)-stimulated immature rat ovary. These earlier studies, which used biochemical assays to detect enzyme activity, lacked the specificity and sensitivity necessary to characterise conclusively the activity of the individual KLK gene family members. In this study, we have used a gene-specific RT-PCR/Southern hybridisation strategy to delineate the expression patterns of six of the individual KLK genes expressed in the rat ovary (rKLK1-3 and rKLK7-9). We have identified three broad patterns of expression in the eCG/hCG-stimulated ovary in which there is either a post-eCG increase/pre-ovulatory decrease in rKLK expression (rKLK1, rKLK3), a peri-ovulatory decrease in expression (rKLK2, rKLK8) or a relatively unchanged pattern of expression (rKLK7, rKLK9). In addition to clarifying the earlier biochemical studies, these findings support a differential role for the individual KLKs in the ovulatory process.

Roles of activin and its signal transduction mechanisms in reproductive tissues.

Activins were identified initially as gonadal proteins having a stimulating effect on FSH production by the pituitary gland. Strong evidence has accumulated that activins are important regulating factors for many reproductive processes. Activin may have paracrine or autocrine roles rather than solely an endocrine action on FSH secretion. Activins together with their signalling molecules must be shown to be produced locally in a particular tissue to provide support for their paracrine or autocrine action in that tissue. The discovery of the activin receptors, the intracellular signalling mediators (Smads) and some transcription co-factors involved in activin responses has helped to unravel the activin-transforming growth factor beta signalling mechanism. However, few reports have clearly demonstrated the presence of all of the activin signalling molecules in reproductive tissues, despite the important roles of activin in these tissues. Several activin receptor types and Smad molecules have been identified, indicating either a redundancy in signalling molecules or different signalling pathways. At present, it is not clear which particular subset of these signalling molecules is important in reproductive processes. The aim of this review is to collate the information available on activin actions, as well as on the signalling molecules, to understand how activins may transduce their paracrine or autocrine signals in reproductive tissues.

Effect of immunization against the alpha N (alphaN) and alpha C (alphaC) peptides of the alpha43 subunit of inhibin on antral follicular growth and atresia and the patterns of gonadotrophin secretion in ewes.

The aims of this study were to investigate the role of inhibin in the distribution of healthy and atretic antral follicles and the secretion patterns of gonadotrophins. Ewes were actively immunized against either alphaN or alphaC of the inhibin alpha subunit with a primary injection and three booster injections. The control ewes received adjuvant only. The ovaries were removed either before or at 24 h after hCG administration in a synchronized follicular phase 48 h after removal of intravaginal progesterone pessaries. Morphological observations were made on every fifth section of the complete ovary (one per ewe) stained with haematoxylin and eosin. The mean number of corpora lutea observed per ewe with corpora lutea was not significantly different in ewes immunized against alphaN (2.4; alphaN-immunized ewes) or alphaC (2.6; alphaC-immunized ewes), and control (2.4) ewes, although some corpora lutea appeared cystic in the immunized ovaries. Compared with luteal phase concentrations, mean basal FSH concentrations in the early follicular phase were significantly increased in the alphaC-immunized ewes, similar in alphaN-immunized ewes and reduced in control ewes. No differences were observed in any of the LH parameters. Before hCG treatment, healthy antral follicles > 1 mm in diameter were not observed in any of the 52 follicles in the aC-immunized ewes and were observed in one of 37 follicles from alphaN-immunized ewes compared with 19 of 28 follicles in control ewes (P < 0.0001). For healthy antral follicles < 1 mm in diameter, there were 72 of 85 follicles in the alphaC-immunized ewes, 79 of 81 follicles in the alphaN-immunized ewes and 81 of 82 follicles in the control ewes. Similar results were obtained in healthy antral follicles < 1 mm in diameter at 24 h after hCG administration. In contrast to the control ewes, no healthy preovulatory follicles (> 6 mm in diameter) were observed in alphaN- and alphaC-immunized ewes either before or 24 h after hCG administration. Two newly formed corpora lutea from alphaC-immunized ovaries contained retained oocytes compared with none in control and alphaN-immunized ovaries. In conclusion, immunization against alphaN and alphaC may result in disruption of the normal processes of antral follicular growth and maturation independent of the concentrations of FSH and LH.

Identification of specific inhibin A-binding proteins on mouse Leydig (TM3) and sertoli (TM4) cell lines.

The binding of human inhibin A to cell surface binding proteins of mouse Leydig (TM3) and Sertoli (TM4) cell lines was investigated. Scatchard analysis identified two classes of inhibin A-binding sites on TM3 (K(d(1)) = 85 pM and 4,160 sites/cell; K(d(2)) = 520 pM and 12,500 sites/cell) and TM4 (K(d(1)) = 61 pM and 2,620 sites/cell; K(d(2)) = 520 pM and 10,400 sites/cell) cells. Compared with inhibin A, inhibin B only partially competed [(125)I]inhibin A binding (6-8%), whereas activin A competed weakly (<0.01%). Chemical cross-linking of [(125)I]inhibin A to both cell lines identified five [(125)I]inhibin A binding complexes with apparent molecular masses of 70, 95, 145, 155, and more than 200 kDa. Inhibin A displacement of [(125)I]inhibin A from each of these cross-linked species (ED(50) = 60-110 pM) closely resembled displacement from intact TM3 (ED(50) = 97 +/- 32 pM) and TM4 (ED(50) = 75 +/- 28 pM) cells, suggesting that all of these proteins are involved in the high affinity inhibin A binding complex. Immunoprecipitation of iodinated inhibin A complexed to TM3 and TM4 cells with an antibody against human betaglycan identified protein complexes of more than 200, 145, and 95 kDa. It is concluded that the high affinity binding complex for inhibin A found in these cell lines consists of betaglycan and several proteins of unknown identity and may represent the putative inhibin receptor complex.

The road to ovulation: the role of oestrogens.

Oestrogens have been known for many years to have a direct influence on folliculogenesis. Oestradiol-17beta (E2) and its analogues have both proliferative and differentiative effects on somatic cells of follicles. Nevertheless, definitive proof of an obligatory role for oestrogen in folliculogenesis and elucidation of the mechanisms subserving its different actions in follicular cells remains elusive. Several recent developments permit a re-examination of the roles and actions of E2 in the follicle. They are: (i) the discovery of a second form of the oestrogen receptor, ERbeta; (ii) the advent of genetically modified mice with deletions in the ERalpha (alphaERKO) ERbeta (BERKO) and the double ER deletions (alphabetaERKO); and (iii) a mouse model of oestrogen deficiency (ArKO) by targeted disruption of the cyp 19 gene encoding the aromatase enzyme. Recent information derived from these models is reviewed to re-assess the roles and actions of oestrogens in follicular dynamics and the phenotypic differentiation of ovarian somatic cells in the ovary. The data demonstrate that oestrogen is obligatory for normal folliculogenesis and that the phenotype of the ovarian somatic cells depends on the steroid milieu. The ArKO mouse provides a model to test the roles of the respective ERs in proliferation and differentiation using specific agonists and antagonists, and to study regulation of the differentiation of ovarian and testicular somatic cells.