Ann E. Drummond

The role of steroids in follicular growth

The steroidogenic pathway within the ovary gives rise to progestins, androgens and oestrogens, all of which act via specific nuclear receptors to regulate reproductive function and maintain fertility. The role of progestins in follicular growth and development is limited, its action confined largely to ovulation, although direct effects on granulosa cell function have been reported. Consistent with these findings, progesterone receptor knockout mice are infertile because they cannot ovulate. Androgens have been shown to promote early follicular growth, but also to impede follicular development by stimulating atresia and apoptosis. The inability of androgens to transduce a signal in mice lacking androgen receptors culminates in reduced fertility. Oestrogens are known to exert effects on granulosa cell growth and differentiation in association with gonadotrophins. Studies with oestrogen receptor knockouts and oestrogen depleted mice have shown us that oestrogen is essential for folliculogenesis beyond the antral stage and is necessary to maintain the female phenotype of ovarian somatic cells. In summary, the action of steroids within the ovary is based on the developmental status of the follicle. In the absence of any single sex steroid, ovarian function and subsequently fertility, are compromised.

Recruitment and development of the follicle; the roles of the transforming growth factor-β 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 activins 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.

The ovarian phenotype of the aromatase knockout (ArKO) mouse

Targeted disruption of exon 9 of the cyp19 gene gives rise to a non-functional aromatase enzyme incapable of converting androgens to oestrogens. The aromatase knockout (ArKO) mouse is, thus, characterised by a dysfunctional pituitary-gonadal axis, which manifests in non-detectable levels of oestrogen in serum. These mice also exhibit elevated levels of circulating gonadotrophins (luteinising hormone (LH) and follicle stimulating hormone (FSH)) and testosterone. The ArKO mouse is infertile due to folliculogenic disruption and a failure to ovulate. The age-dependent ovarian phenotype revealed a block in follicular development at the antral stage and a complete absence of corpora lutea. By 21-23 weeks of age haemorrhagic cystic follicles were present and by 1 year there were abnormal follicles, an absence of secondary and antral follicles and atretic primary follicles. Interstitial tissue remodelling was extensive and exemplified by an increase in collagen deposition and an influx of macrophages, coincident with the loss of follicles. In mice, maintained on a soy-free and, thus, phytoestrogen-free diet, the ovarian phenotype was accelerated and exacerbated. In conclusion, the ovarian phenotype of the ArKO mouse can be attributed to the altered hormonal environment brought about by the absence of aromatase and the failure of androgens to be converted to oestrogens in the presence of elevated gonadotropins.

Ovarian estrogen receptor α and β mRNA expression: impact of development and estrogen

Abstract We tested the hypothesis that ERα and ERβ mRNAs in the rat ovary are regulated during the post-natal period and in immature rats in response to estrogen treatment. Total ovarian ERβ mRNA was more abundant than ERα mRNA and expression of ERβ increased between post-natal days 4 and 12, coinciding with advancing folliculogenesis and an increase in granulosa cell numbers. In contrast, ERα mRNA levels remained relatively constant during this period. In situ hybridisation studies localised both ERα and ERβ to granulosa cells of growing follicles, in 25 day old ovaries, although not all granulosa cells in a follicle or all follicles expressed the ERs. Diethylstilboestrol (DES) administered in vivo to 21 day old rats, for up to 4 days, did not significantly alter the expression of either ER as determined by RT-PCR, despite a 5.5-fold increase in granulosa cell number in these ovaries. In situ hybridisation studies established that DES-treatment down-regulated granulosa cell ER mRNAs. RT-PCR analyses on isolated granulosa cells confirmed that ERα was significantly down-regulated by DES. The predominance of ERβ over ERα in the ovary and the regulation of ERβ mRNA expression during ovarian development, is consistent with an important biological role for ERβ in granulosa cell proliferation and differentiation.

Expression and Localization of Activin Receptors, Smads, and βglycan to the Postnatal Rat Ovary

Despite understanding the molecular basis of activin/TGF beta and bone morphogenetic protein (BMP) signaling, this study is the first to characterize multiple, sequential elements of these pathways in the ovary concurrently. The expression of activin/BMP receptor, Smad, and beta glycan mRNAs by postnatal rat ovaries were investigated by real-time PCR. Activin/BMP receptors (ActRIA, ActRIB, ActRIIA, and ActRIIB), beta glycan, and Smad 1-8 mRNAs were expressed by the ovary. Activin receptor and Smad 1, 2, 4, 5, and 7 mRNAs declined up to 4-fold between postnatal d 4-8, coinciding with secondary follicle formation. The emergence of antral follicles (postnatal d 12) saw ActRIA, ActRIIB, and Smad 2 mRNA expression return to d 4 levels, whereas ActRIB, ActRIIA, and Smads 1, 4, 5, and 7 remained at lower levels. beta glycan mRNA levels increased 2-fold between d 8 and 12, suggesting expression by the developing theca. Smad 3, 6, and 8 mRNAs were unchanged. Activin receptor and Smad proteins were present in oocytes at all stages of follicular development; granulosa cells of primary-antral follicles, and theca cells. beta glycan protein was present in oocytes, granulosa cells, and theca cells at all stages of folliculogenesis. The colocalization of receptors and Smads supports the notion that activin/TGF beta and BMP signaling pathways are functional in the cellular compartments of the follicle.

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.

Ovarian steroid receptors and their role in ovarian function

The steroidogenic pathway within the ovary gives rise to progestins, androgens and oestrogens, all of which act via specific nuclear receptors to regulate reproductive function and maintain fertility. The precise role of oestrogen in the ovary remains to be elucidated, hence the data presented here which arises from studies designed to resolve this issue. Oestrogens signal via two receptor subtypes ERalpha and ERbeta, both of which are present in the ovary. ERbeta, the most abundant mRNA, is primarily expressed by GC where it transduces signals from ovarian-derived and exogenous oestrogens. Specific roles for each of the ERs in the ovary have yet to be established, despite ER knockout studies indicating both are required for normal function. The ArKO mouse is a model of oestrogen insufficiency. These mice are infertile as a result of arrested folliculogenesis (at the antral stage) and a failure to ovulate. Trans/re-differentiation of somatic cells in the ovary gives rise to Sertoli cell-like and Leydig cell-like cells within abnormal follicular structures. Disruption to the balance of sex steroids in the ovary is likely to facilitate this phenotype. Future studies will focus on the regulation of somatic cell differentiation, assigning roles to individual ERs and establishing definitive targets of oestrogen action in the ovary.

The importance of ERβ signalling in the ovary

This review examines the evidence for a central role of oestrogen receptor beta (ERbeta or ESR2 as listed in the MGI Database) in folliculogenesis and hence reproductive biology. Knockout mouse models have been a valuable resource in this respect. The ERbeta-null mouse exhibits a granulosa cell phenotype associated with the partial arrest of folliculogenesis and ovulatory dysfunction. Phyto-oestrogens such as genistein, which preferentially activate ERbeta, have been shown to alleviate the ovarian phenotype of the oestrogen-depleted aromatase knockout mouse. In normal adult mice, genistein has been shown to cause reproductive defectives following neonatal administration. Studies of ovarian cancer have also informed the literature. A decline in ERbeta levels in epithelial ovarian cancers has been hypothesised to be associated with severity of disease and prognosis. Whereas the abundant expression of ERbeta in granulosa cell tumours (GCT) of the ovary and evidence that ERbeta signalling is transrepressed by the nuclear factor-kappaB pathway in GCT cell lines suggest a pathogenetic role for ERbeta in GCT. In recent years, studies into the impact of environmental oestrogens (either in the form of pesticides or plastics) on reproductive function have shown that ERbeta-selective toxins cause reproductive dysfunction and impair fertility. It remains to be established as to what genes are regulated by ERbeta in the ovary. Finally, ERbeta has been shown to be regulated by gonadotrophins, the pituitary hormones mediating ovarian function.

TGFβ signalling in the development of ovarian function

Ovarian development begins back in the embryo with the formation of primordial germ cells and their subsequent migration and colonisation of the genital ridges. Once the ovary has been defined structurally, the primordial germ cells transform into oocytes and become housed in structures called follicles (in this case, primordial follicles), a procedure that, in most mammals, occurs either shortly before or during the first few days after birth. The growth and differentiation of follicles from the primordial population is termed folliculogenesis. Primordial follicles give rise to primary follicles that transform into preantral follicles, then antral follicles (secondary follicles) and, finally (preovulatory) Graafian follicles (tertiary follicles) in a co-ordinated series of transitions regulated by hormones and local intraovarian factors. Members of the transforming growth factor-β (TGFβ) superfamily have been shown to play important roles in this developmental process starting with the specification of primordial germ cells by the bone morphogenetic proteins through to the recruitment of primordial follicles by anti-Mullerian hormone and, potentially, growth and differentiation factor-9 (GDF9) and, finally, their transformation into preantral and antral follicles in response to activin and TGF-β. Developmental and mutant mouse models have been used to show the importance of this family of growth factors in establishing the first wave of folliculogenesis.

The roles of activins, inhibins and estrogen in early committed follicles.

The hypothesis that activin and inhibin are autocrine/paracrine mediators of ovarian folliculogenesis has a solid basis. In mouse and rat models, granulosa cells (GC) of committed follicles express mRNA and protein for the activin/inhibin subunits and mRNA for the activin receptors (type I and II). Dimeric inhibin-A and -B are produced by postnatal ovarian cell dispersates and (GC) in culture. Similar levels of inhibin-A and -B are produced by postnatal ovarian cells, but thereafter as the ovary develops, inhibin-A becomes the predominant form. Activin was more effective than transforming growth factor-beta (TGF-beta) in enhancing follicle stimulating hormone (FSH)-stimulated inhibin production by ovarian cells. Evidence for a local regulatory role of estrogen in the ovary is also accumulating. Murine models of estrogen receptor (ERalpha or ERbeta) disruption produce mice with abnormal ovarian phenotypes. Female mice, which lack the capacity to produce estrogen (ArKO mice), have arrested folliculogenesis, no corpora lutea, elevated levels of luteinising hormone (LH), FSH and testosterone and are infertile. These data are consistent with autocrine/paracrine actions of activin in the early growth of committed follicles and estrogen in follicular maturation.