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The annual photoperiod cycle provides the critical environmental cue synchronizing rhythms of life in seasonal habitats. In 1936, Bünning proposed a circadian-based coincidence timer for photoperiodic synchronization in plants. Formal studies support the universality of this so-called coincidence timer, but we lack understanding of the mechanisms involved. Here we show in mammals that long photoperiods induce the circadian transcription factor BMAL2, in the pars tuberalis of the pituitary, and triggers summer biology through the eyes absent/thyrotrophin (EYA3/TSH) pathway. Conversely, long-duration melatonin signals on short photoperiods induce circadian repressors including DEC1, suppressing BMAL2 and the EYA3/TSH pathway, triggering winter biology. These actions are associated with progressive genome-wide changes in chromatin state, elaborating the effect of the circadian coincidence timer. Hence, circadian clock-pituitary epigenetic pathway interactions form the basis of the mammalian coincidence timer mechanism. Our results constitute a blueprint for circadian-based seasonal timekeeping in vertebrates., “Life in a seasonal environment requires appropriate timing of physiological changes to survive, but how the circadian clockwork times these changes remains unclear. Here the authors show that the circadian clock genes BMAL2 and DEC1, in concert with epigenetic pathways in the pituitary, have a central role in seasonal timekeeping in mammals.”

Adult reproductive ability is to a large extent determined by the appropriate development of the reproductive axis during fetal life. Studies have investigated the role of the fetal hypothalamus in the ontogeny and regulation of pituitary gonadal function during fetal development in sheep. Using immunocytochemistry, we examined the ontogeny of gonadotroph development in the pituitary of female sheep fetuses. At day 70 of gestation (term = 145 days), only immunopositive LH beta cells were present. The number and intensity of staining of these LH beta cells had increased by day 100 but had declined again by day 130. Immunopositive alpha-subunit and FSH beta cells appeared at day 100 of gestation and had further increased in number and staining intensity by day 130 of gestation. Treatment of fetuses with the GnRH agonist buserelin resulted in desensitization of the fetal pituitary gonadotrophs, inhibition of pituitary LH beta and FSH beta mRNA expression and a reduction in the number of immunopositive gonadotrophin-containing cells. Pulsatile GnRH treatment resulted in pituitary-gonadal activation and an increase in LH, FSH and testosterone secretion in males. Thus, the synthesis and secretion of the gonadotrophins during fetal development is critically dependent on the secretion of GnRH from the fetal hypothalamus. Inhibition of fetal gonadotrophins by buserelin treatment from day 70 of gestation resulted in a 40% reduction in the size of the fetal testis at birth, and there were no effects on the fetal ovaries. This reduction in testis size was due to a 45% reduction in the number of Sertoli cells. However, when buserelin was given between day 70 and day 110 of gestation, there were no effects on testis size or morphological development of the testis, suggesting that gonadotrophins regulate testicular development during a 'critical window' late in gestation. Taken together, these studies provide convincing evidence that GnRH plays a central role in the ontogeny and regulation of pituitary-gonadal function during fetal life.

In the sheep both FSH and LH are necessary for development of large antral follicles. The secretion of FSH is controlled by the negative feedback effect of two ovarian hormones, oestradiol and inhibin, acting at the level of the anterior pituitary. Both are derived from the granulosa cells of large antral follicles which are present in sheep ovaries throughout the oestrous cycle. FSH stimulates growth and mitosis and so the fully differentiated granulosa cells of the large preovulatory follicles acquire receptors for LH, have maximal aromatase activity and produce large amounts of inhibin. The number of these large antral follicles (oestrogenic) which have the potential for ovulation corresponds to the ovulation rate specific for each particular breed of sheep. Over 90% of the oestradiol secreted by the ovaries is derived from these chosen follicles. In contrast, inhibin (and androstenedione) is also secreted by large antral follicles which have lost or not yet acquired maximal aromatase activity. The secretion of oestradiol by the preovulatory follicle(s) is dependent on the supply of androgen precursors produced by the theca which is stimulated by LH. When the concentration of progesterone falls at the end of the luteal phase the increased secretion of LH stimulates the progressive increase in secretion of oestradiol which occurs during the follicular phase. At this stage of the cycle there is a modest inconsistent rise in inhibin, the secretion of which is not stimulated by LH but is related to the increase in the number of large antral follicles. It is suggested that inhibin with its long half-life sets the overall level of negative feedback while oestradiol is responsible for the day-to-day fluctuations in the concentration of FSH which determines the number of ovulatory follicles. This dual control of FSH is adapted to monitor both the total number of large antral follicles in the ovaries (inhibin) and the number which are selected for ovulation (oestradiol).

Preovulatory follicle growth in the ewe is dependent on FSH although no precise relationship appears to exist between plasma concentrations of FSH and the number of preovulatory follicles which develop or ovulation rate. This may be related to a hitherto unrecognized influence of pulsatile LH on the growth of large follicles. Preovulatory follicle growth is dependent on the presence of basal amounts of LH, but pulsatile LH, while being essential to supply an increase in androgen substrate to the granulosa cells of the follicle, may also play a role in reducing the responsiveness of many large follicles to FSH, in particular during the preovulatory phase when plasma concentrations of FSH are reduced. Thus selection of the preovulatory follicle(s) may involve a previously unrecognized interaction between FSH and pulsatile LH secretion in which pulses of LH act in a negative rather than positive manner.

The Booroola phenotype is associated with a point mutation in the kinase domain of the bone morphogenetic protein receptor 1 B (BMPR1 B), and is characterized by 'precocious' differentiation of ovarian follicles, leading to the production of large numbers of ovulatory follicles that are smaller in diameter than wild-type follicles. These smaller follicles attain differentiation markers, such as expression of mRNA for P450 aromatase and inhibin-betaA subunit, granulosa cell LH receptors and aromatase activity, earlier than follicles from wild-type ewes. However, the preovulatory follicles from mutant ewes collectively secrete similar quantities of oestradiol, androstenedione and inhibin A in exactly the same pattern as wild-type ewes, which result in similar concentrations of FSH. The available evidence strongly indicates that the Booroola mutation exerts its action at the ovary rather than by altering gonadotrophin secretion. The bone morphogenetic protein (BMP) receptors and putative ligands are ubiquitously expressed within the ovary and BMPs seem to be involved in the paracrine regulation of FSH action. Thus, if the mutation is causing a reduction in BMPR1 B signalling, it may act on an inhibitor of follicle differentiation. Further research in this area will concentrate on the elucidation of the natural ligands for BMPR1 B at different stages of follicle development and examine the effect of BMPR1 B mutation on the downstream signalling cascade.

Seasonal mammals integrate changes in the duration of nocturnal melatonin secretion to drive annual physiologic cycles. Melatonin receptors within the proximal pituitary region, the pars tuberalis (PT), are essential in regulating seasonal neuroendocrine responses. In the ovine PT, melatonin is known to influence acute changes in transcriptional dynamics coupled to the onset (dusk) and offset (dawn) of melatonin secretion, leading to a potential interval-timing mechanism capable of decoding changes in day length (photoperiod). Melatonin offset at dawn is linked to cAMP accumulation, which directly induces transcription of the clock gene Per1. The rise of melatonin at dusk induces a separate and distinct cohort, including the clock-regulated genes Cry1 and Nampt, but little is known of the up-stream mechanisms involved. Here, we used next-generation sequencing of the ovine PT transcriptome at melatonin onset and identified Npas4 as a rapidly induced basic helix-loop-helix Per-Arnt-Sim domain transcription factor. In vivo we show nuclear localization of NPAS4 protein in presumptive melatonin target cells of the PT (α-glycoprotein hormone-expressing cells), whereas in situ hybridization studies identified acute and transient expression in the PT of Npas4 in response to melatonin. In vitro, NPAS4 forms functional dimers with basic helix loop helix-PAS domain cofactors aryl hydrocarbon receptor nuclear translocator (ARNT), ARNT2, and ARNTL, transactivating both Cry1 and Nampt ovine promoter reporters. Using a combination of 5′-deletions and site-directed mutagenesis, we show NPAS4-ARNT transactivation to be codependent upon two conserved central midline elements within the Cry1 promoter. Our data thus reveal NPAS4 as a candidate immediate early-response gene in the ovine PT, driving molecular responses to melatonin.

Prenatal androgenization induces a polycystic ovary syndrome-like phenotype in adult female offspring, which is associated with alterations that can be detected in the fetal ovary, suggesting gestational origins of this condition. We therefore investigated whether increased prenatal androgen exposure also altered testicular development using ovine animal models. Biweekly maternal testosterone propionate (TP; 100 mg) from day 62 to day 70/day 90 of gestation altered male developmental trajectory. In male fetuses serum LH was decreased (P < .01), and testicular STAR, CYP11, and CYP17 abundance were reduced. Coincident with this, basal testicular T synthesis was decreased in vitro (P < .001). Leydig cell distribution was severely perturbed in all testes prenatally exposed to TP (P < .001). To examine the contribution of estrogens, fetuses were injected with TP (20 mg), the potent estrogen agonist, diethylstilbestrol (DES; 20 mg), or vehicle control at day 62 and day 82 and assessed at day 90. The effects of fetal (direct) TP treatment, but not DES, paralleled maternal (indirect) TP exposure, supporting a direct androgen effect. Cessation of maternal androgenization at day 102 returned Leydig cell distribution to normal but increased basal T output, at day 112, demonstrating Leydig cell developmental plasticity. Earlier maternal androgen exposure from day 30 similarly influenced Leydig cell development at day 90 but additionally affected the expression of Sertoli and germ cell markers. We show in this study that increased prenatal androgen exposure alters development and function of Leydig cells at a time when androgen production is paramount for male development. This supports the concept that gestational antecedents associated with polycystic ovary syndrome may have effects on the male fetus.

The beta-subunits of luteinizing hormone (LH beta) and follicle-stimulating hormone (FSH beta) are differentially expressed, and this may contribute to the unique expression and storage patterns of LH and FSH. Therefore, to determine if the in vivo expression profile of FSH beta could be altered to that of LH beta, a truncated ovine FSH beta (oFSH beta) gene, which would encode a mRNA lacking the putative destabilizing 3' untranslated region, was fused downstream of the ovine LH beta (oLH beta) promoter and expressed in transgenic mice. In two independent lines, line 16 and 17, we measured oFSH beta, mouse LH beta (mLHbeta) and mouse FSH beta (mFSH beta) mRNA levels: (i) after castration in males; (ii) after administering inhibin to ovariectomized mice; and (iii) during the oestrous cycle. In each experiment, the expression profile of oFSH beta mRNA mimicked mLH beta and not mFSH beta mRNA. In addition, after actinomycin D treatment of pituitary cultures, while mFSH beta mRNA did decay, there was no measurable decay of the oFSH beta mRNA transcript. These differences increased total FSH beta steady-state mRNA expression levels in male transgenics. However, there was no detectable increase in pituitary FSH by either radioimmunoassay or western blotting analysis of pituitary extracts. Subsequent analysis revealed that pituitary FSH beta in line 16 was heavily glycosylated; in contrast, pituitary FSH beta in line 17 was largely unmodified. These differences in post-translational modification of the beta-subunit, and the lack of intracellular storage, contributed to increased plasma FSH levels and ovulation rate in line 16, but not line 17. In conclusion, the expression profile of oFSH beta mRNA was manipulated to mimic mLH beta mRNA and this increased FSH beta mRNA expression levels, but did not increase storage of FSH. This suggests that, regardless of the levels of synthesis, post-translational sorting preferentially promotes FSH secretion from the pituitary.

The current study investigated hormonal and ovarian changes during physiological reproductive aging in Sarda ewes. In a first experiment, follicular and corpus luteum dynamics were compared during an induced oestrus cycle in aged (12–14 years) and young adult ewes (4–5 years). Oestrus cycle characteristics did not differ between the two experimental groups. However, follicular function during the follicular phase showed significant alterations in aged ewes, as determined by a lack of dominance effect and by lower mean values of circulating oestradiol (E2) and inhibin levels, compared with young adult ewes. In a second experiment, differences in follicle growth, hormonal milieu and oocyte quality in response to exogenous FSH administration were assessed in aged and adult ewes. No differences were recorded in ovarian response to FSH treatment between young adult and aged ewes, as evaluated by ultrasonographic data and circulating concentrations of LH, E2 and inhibin-A. Although the total number of recovered oocytes was similar in the two age groups, the number of good quality oocytes selected for IVM was significantly lower in aged ewes compared with adult ones. Thereafter, no differences were recorded in cleavage rates, total blastocyst output, embryo developmental kinetic and quality between aged and adult groups. In conclusion, this study demonstrated that reproductive aging in sheep is associated with impaired follicle functionality and an increase in the proportion of oocytes showing morphological abnormalities. However interestingly, oocyte developmental competence in vitro and embryo cryotolerance were not affected by the aging process, when only good quality oocytes were chosen.

Activin and inhibin are important local modulators of theca cell steroidogenesis in the ovary. Using a serum-free primary theca cell culture system, this study investigated the effects of inhibin on theca cell androgen production and expression of steroidogenic enzymes. Androstenedione secretion from theca cells cultured in media containing activin, inhibin and follistatin was assessed by RIA over 144 h. Activin (1–100 ng/ml) suppressed androstenedione production. Inhibin (1–100 ng/ml) blocked the suppressive effects of added activin, but increased androstenedione production when added alone, suggesting it was blocking endogenous activin produced by theca cells. Addition of SB-431542 (activin receptor inhibitor) and follistatin (500 ng/ml) increased androstenedione production, supporting this concept. Infection of theca cells with adenoviruses expressing inhibitory Smad6 or 7 increased androstenedione secretion, confirming that the suppressive effects of activin required activation of the Smad2/3 pathway. Activin decreased the expression levels of steroidogenic acute regulatory protein (STAR), whereas STAR expression was increased by inhibin and SB-431542, alone and in combination. CYP11A was unaffected. The expression of CYP17 encoding 17α-hydroxylase was unaffected by activin but increased by inhibin and SB-431542, and when added in combination the effect was further enhanced. The expression of 3β-hydroxysteroid dehydrogenase (3β-HSD) was significantly decreased by activin, while inhibin alone and in combination with SB-431542 both potently increased the expression of 3β-HSD. In conclusion, activin suppressed theca cell androstenedione production by decreasing the expression of STAR and 3β-HSD. Inhibin and other blockers of activin action reversed this effect, supporting the concept that endogenous thecal activin modulates androgen production in theca cells.

This review examines the importance of the epithelial origin of granulosa cells and their possible contribution to the development of ovarian cancers in three animal models. We hypothesise that undifferentiated granulosa cells, devoid of their germ cell regulator, retain their embryonic plasticity and may give rise to ovarian cancers of epithelial origin. Dazl-KO and FancD2-KO mice and BMP15-KO sheep are animal models in which germ cells or oocytes are lost at specific stages of follicular formation or growth, leaving behind clusters of residual, but healthy somatic cells. A common feature in Dazl- and Fancd2-KO animals following germ cell/oocyte loss is the presence of sex cords and intraovarian epithelial ducts or tubules. In Dazl-KO mice, cord/tubule-like structures, OSE invaginations and clusters of steroidogenic cells became increasingly prominent with age, but these abnormal structures remained benign. In Fancd2-KO mice, the formation of sex-cords and intraovarian tubules lead to the formation of tumours with multiple phenotypes including luteomas, papillary cysts and malignant carcinomas (e.g. adenocarcinomas). In BMP15-KO sheep, oocytes die as follicles start to grow, leaving ‘nodules’ containing granulosa cells with a capacity to respond to follicle stimulating hormone and synthesize inhibin. Thereafter, these nodules coalesced and a range of benign solid or semi-solid tumour phenotypes developed. We conclude that premature loss of oocytes, but not granulosa cells, leads to tumour formation with multiple phenotypes. Moreover, the severity of tumour development is linked to both the speci-ficity of the mutation and the timing of oocyte loss relative to that of follicular formation.

Inhibitor of differentiation (Id) proteins act during embryogenesis and development to repress gene transcription required for lineage commitment, while promoting cell growth. Growth factors belonging to the TGF beta superfamily of signaling molecules, notably the bone morphogenetic proteins (BMPs) and activin, can regulate Id expression in these tissues. Id expression and function in adult physiology is less well determined, and we hypothesized a role for Id proteins in the adult mammalian ovary. Immunohistochemistry for Id1, Id2, Id3, and Id4 in the sheep ovary revealed consistent expression in granulosa and thecal cells of ovarian follicles throughout development. In atretic follicles, Id proteins were selectively down-regulated in thecal cells (P < 0.0001). Additionally, Id1 was universally up-regulated in the cumulus cells adjacent to the oocyte. Immunohistochemistry for phospho (p)-smad 1/5/8 signaling components (stimulated by BMPs) showed a punctate pattern of expression whereas p-smad 2/3 (stimulated by activin) was ubiquitously expressed in follicles. Neither pathway, however, displayed differential staining in line with Id1 cumulus-specific expression, suggesting a more complex relationship between Id1 expression and TGF beta signaling in these cells. Nevertheless, in vitro, stimulation of ovine granulosa cells with BMP6 or activin A led to a respective increase and decrease in Id1 (P < 0.0001), Id2 (P < 0.0001), Id3 (P < 0.0001), and Id4 (P < 0.05) transcripts, and Id1 gene expression was further manipulated by the oocyte-secreted factors BMP15 and growth differentiation factor 9 (P < 0.001). These data confirm that TGF beta signaling can regulate Id gene expression in the sheep ovarian follicle and suggest a functional role for the Id family in the mammalian ovary. (Endocrinology 151: 1247-1256, 2010)

The transition of the dominant follicle into the corpus luteum is of fundamental reproductive importance. Luteinisation involves disparate changes in the gene expression of follicular granulosa cells that differentiate into the granulosa-lutein cells of the corpus luteum after the gonadotrophin surge. We have shown that activin and human chorionic gonadotropin (hCG) have opposing effects during luteolysis. Therefore, we hypothesised that activin A was an inhibitor of luteinisation that was blocked during the pre–ovulatory gonadotrophin surge. Ovarian tissue and cells were collected from women with regular cycles having hysterectomy and women undergoing oocyte retrieval for assisted conception. Genes that changes during luteinisation were investigated in primary cultures of luteinised granulosa cells exposed to activin A and hCG in vitro. hCG promotes a luteinised granulosa cell phenotype, while activin A promotes a more follicular phenotype in luteinised cells by upregulating granulosa cells markers such as FSHR, HSD11B2 and downregulating LHCGR. In addition, activin A blocked hCG upregulation of STAR, HSD3B1 and HSD11B1 and downregulation of oestrogen receptor α. Activin A antagonised hCG effects in a dose-dependent manner and could block the hCG-stimulated molecular inhibitors of activin action (inhibin α-subunit, follistatin and TGFBR3). These studies show that hCG and activin A have opposing effects on luteinised granulosa cells and some effects of activin are seen only in the presence of hCG. While hCG can inhibit activin action in granulosa cells to facilitate luteinisation, activin A can promote an unluteinised phenotype in luteinised granulosa cells. This confirms the importance of adequate activin withdrawal during luteinisation in women.

International audience; We have shown previously that, in sheep primary pituitary cells, bone morphogenetic proteins (BMP)-4 inhibits FSH beta mRNA expression and FSH release. In contrast, in mouse L beta T2 gonadotrophs, others have shown a stimulatory effect of BMPs on basal or activin-stimulated FSH beta promoter-driven transcription. As a species comparison with our previous results, we used L beta T2 cells to investigate the effects of BMP-4 on gonadotrophin mRNA and secretion modulated by activin and GnRH. BMP-4 alone had no effect on FSH production, but enhanced the activin+GnRH-induced stimulation of FSH beta mRNA and FSH secretion, without any effect on follistatin mRNA. BMP-4 reduced LH beta nnRNA up-regulation in response to GnRH ( activin) and decreased GnRH receptor expression, which would favour FSH, rather than LH, synthesis and secretion. In contrast to sheep pituitary gonadotrophs, which express only BNIP receptor types IA (BMPRIA) and II (BMPRII), L beta T2 cells also express BMPRIB. Smad1/5 phosphorylation induced by BMP-4, indicating activation of BMP signalling, was the same whether BMP-4 was used alone or combined with activin +/- GnRH. We hypothesized that activin and/or GnRH pathways may be modulated by BMP-4, but neither the activin-stimulated phosphorylation of Smad2/3 nor the GnRH-induced ERK1/2 or cAMP response element-binding phosphorylation were modified. However, the GnRH-induced activation of p38 MAPK was decreased by BMP-4. This was associated with increased FSH beta mRNA levels and FSH secretion, but decreased LH beta mRNA levels. These results confirm 1. BMPs as important modulators of activin and/or GnRH-stimulated gonadotrophin synthesis and release and 2. important species differences in these effects, which could relate to differences in BMP receptor expression in gonadotrophs.

Bone morphogenetic proteins (BMPs) have been shown to influence the regulation of FSH synthesis and secretion at the level of the pituitary. Primary pituitary cells were harvested and cultured from Booroola ewes homozygous for a mutation in activin receptor-like kinase 6 (ALK6) also known as BMP receptor IB (BMPRIB), and from wild-type (WT) ewes to determine if the mutation caused alterations in FSH secretion in vitro. The cells were collected 24 h following induction of luteolysis and cultured for 72 h prior to being challenged for 24 h with BMP2, BMP4, BMP6, growth and differentiation factor-9 (GDF9), transforming growth factor-β 1, activin-A and GnRH. The levels of FSH and LH were measured by RIA and then compared with the untreated controls. Primary pituitary cell cultures from Booroola ewes secreted less FSH than WT cells in the presence of BMP2, BMP4 and BMP6. These BMPs did not affect the FSH stores within the cells, or the levels of LH released. GDF9 appeared to act in a BMP-like manner by suppressing FSH secretion. The ALK6 receptor however, was not found to co-localise with gonadotroph cells in either Booroola or WT pituitary tissues. These findings imply that the increased sensitivity of Booroola cells to BMP2, BMP4, BMP6 and GDF9 cannot be due to the direct action of the ALK6 mutant Booroola receptor in the cells that synthesise FSH.

Summary Persistent free-running circannual (approximately year-long) rhythms have evolved in animals to regulate hormone cycles, drive metabolic rhythms (including hibernation), and time annual reproduction. Recent studies have defined the photoperiodic input to this rhythm, wherein melatonin acts on thyrotroph cells of the pituitary pars tuberalis (PT), leading to seasonal changes in the control of thyroid hormone metabolism in the hypothalamus. However, seasonal rhythms persist in constant conditions in many species in the absence of a changing photoperiod signal, leading to the generation of circannual cycles. It is not known which cells, tissues, and pathways generate these remarkable long-term rhythmic processes. We show that individual PT thyrotrophs can be in one of two binary states reflecting either a long (EYA3+) or short (CHGA+) photoperiod, with the relative proportion in each state defining the phase of the circannual cycle. We also show that a morphogenic cycle driven by the PT leads to extensive re-modeling of the PT and hypothalamus over the circannual cycle. We propose that the PT may employ a recapitulated developmental pathway to drive changes in morphology of tissues and cells. Our data are consistent with the hypothesis that the circannual timer may reside within the PT thyrotroph and is encoded by a binary switch timing mechanism, which may regulate the generation of circannual neuroendocrine rhythms, leading to dynamic re-modeling of the hypothalamic interface. In summary, the PT-ventral hypothalamus now appears to be a prime structure involved in long-term rhythm generation., Graphical Abstract, Highlights • A circannual timer may reside in the pituitary pars tuberalis thyrotroph • This is defined by a digital switching mechanism controlling EYA3 expression • The circannual clockwork drives a morphogenic cycle in the PT and hypothalamus • This involves recapitulation of a developmental program, Circannual rhythms have evolved to regulate and time annual changes in physiology. Wood et al. report that the pars tuberalis generates the circannual rhythm in mammals through the digital switching of EYA3 expression. A recapitulated developmental pathway is used by the circannual clock to drive a morphogenic cycle in the PT and hypothalamus.

cAMP response-element binding (CREB) transcription factors transduce cell survival responses to peptide hormones and growth factors in normal tissues and mutant CREB proteins are implicated in tumorigenesis. Ovarian cancer most frequently arises from the ovarian surface epithelium (OSE), possibly due to repeat inflammation-associated injury-repair episodes that promote neoplasia. We asked if post-receptor signalling involving the CREB family of proteins plays a role in OSE cell survival. In an ovine ovulation model, abundant expression of phospho-CREB/activating transcription factor (ATF) protein was detected immunohistochemically, strongly localised to OSE cells in the proximity of pre-ovulatory follicles. Treatment of primary sheep OSE cell cultures with LH stimulated cAMP accumulation and reduced apoptosis (caspase 3/7 activity) in response to serum withdrawal. When OSE cells were infected with an adenovirus containing a CRE-luciferase construct, exposure to LH and FSH induced CRE-directed transcription. Finally, when a non-phosphorylatable mutant of CREB (Ad CREBS133A) was adenovirally expressed, apoptosis measured by activation of caspases was increased several fold relative to that caused by transfection with wild-type CREB (Ad CREBWT) or lacZ (Ad lacZ). To test the potential clinical relevance of these findings, we expressed mutant CREB protein in normal human OSE cells from four women and a series of cell lines derived from human ovarian cancers. Infection with Ad CREBS133A markedly increased apoptosis in normal human OSE but had no detectable effect on apoptosis in any of the cancer cell lines. We conclude that CREB/ATF signalling is important for the maintenance of OSE cell survival in vitro and is altered in human cell lines derived from ovarian cancers.

We report the development and validation of a highly specific heterologous radioimmunoassay (RIA) to measure meerkat prolactin (PRL) by using rabbit antiserum to human prolactin and canine [125I]iodo-PRL. Dilutions of meerkat pituitary standard and plasma gave parallel inhibition curves in the assay. Gel filtration of meerkat pituitary extracts and canine [125I]iodo-PRL run separately on a Sephadex G-100 generated identical peaks of activity, and Western blot analysis of meerkat pituitary extract with the human prolactin antiserum used in the RIA gave a molecular weight similar to canine prolactin (21kDa). We carried out a biological validation of the prolactin assay by administering three different doses each of sulpiride and cabergoline to adult male meerkats. Increasing doses of sulpiride and cabergoline caused substantial increases and decreases, respectively, in the plasma prolactin of the study animals as expected. Activation of the stress response in meerkats by capture and ketamine hydrochloride anesthesia caused short-term but significant increases in prolactin levels in individuals bled repeatedly. The RIA developed and described here was able to determine plasma concentrations of prolactin in all animals sampled. We conclude, however, that it will be important in all future studies to confine blood sampling times to 4-7 min after capture/administration of anesthesia to avoid the confounding effects of the stress response on prolactin levels.

Background Analyses of the follicular reserve and activity of the ovary are central to our understanding of the regulation of follicular development. We have carried out a prospective analysis of endocrine and biophysical assessments under three differing basal conditions: the early follicular and mid-luteal phases, and following GnRH analogue down-regulation. Methods Hormonal analyses were carried out before and after a single dose of FSH on spontaneously ovulating women (n = 58). Ovarian volume and antral follicle count (AFC) were also determined. Results Inhibin B and estradiol concentrations were increased by FSH under all three conditions, and inhibin A in the follicular phase and after down-regulation. Basal hormone concentrations, except inhibin A and B after down-regulation, did not generally correlate with AFC. A close relationship between inhibin B and AFC was evident at all stages after FSH administration (r = 0.70-0.77). AFC and inhibin B after FSH stimulation were well correlated with the number of oocytes recovered after superovulation. Multivariate analysis demonstrated that inhibin B after FSH administration in the down-regulated state showed the closest correlation with oocyte number. In the more clinically useful early follicular and luteal phases, basal FSH was the most significant contributor to the number of oocytes, with a significant contribution from luteal phase AFC. Conclusions These data extend our understanding of the relationships between follicular number, follicular functional activity, and the recruitable follicular population. Down-regulation and subsequent FSH stimulation was required to clearly demonstrate the close relationship between inhibin B and the ovarian reserve. Without such complex manipulation, early follicular phase FSH (supplemented by AFC in the relatively hypogonadotrophic luteal phase) remains of greater value in predicting the ovarian reserve than the currently known direct products of the ovary.

Intracellular associations indicate that granins may play a role in the regulatory mechanisms involved in differential secretion of gonadotrophins. The effect of GnRH on mRNA expression, storage and secretory patterns of granins and gonadotrophins was investigated in male mice. GnRH antiserum (G/A) was injected into mice in the treatment group (n = 15) at 12 h intervals for 2 days and a subset (n = 9) was killed. Buserelin (G/A + B) was administered to the remaining mice (n = 6), which were killed 2 h later; control mice (n = 6) were killed at the onset of the study. LHb mRNA content was lower in G/A and G/A + B mice compared with controls, whereas plasma LH concentrations were higher in G/A + B mice. FSHbeta mRNA content did not change, whereas plasma FSH concentrations were lower in G/A mice compared with controls, and higher in G/A + B mice compared with both G/A and control mice. Secretogranin II (SgII) and CgA mRNA contents were not different between experimental groups. There were more granules per gonadotroph in G/A mice, and considerably fewer after Buserelin treatment. Immunogold labelling of gonadotrophs revealed the presence of LH(+ve)/SgII(+ve) and LH(+ve)/SgII(-ve) granules, and negligible numbers of LH(-ve)/SgII(+ve) granules. Both the numbers of LH(+ve)/SgII(+ve) granules and overall granule antigenicity for SgII were higher in G/A mice compared with controls and G/A + B mice. In contrast, there were fewer LH(+ve)/SgII(-ve) granules per gonadotroph in G/A mice compared with controls. In conclusion, absence of GnRH input to the pituitary gland resulted in preferential storage of SgII and subsequently increased intragranular co-aggregation with LH. Administration of Buserelin to G/A mice resulted in the apparent release of LH(+ve)/SgII(+ve) granules that was reflected by an increase in plasma LH concentrations, indicating that these granules were in the regulated secretory pathway. In contrast, secretion of LH(+ve)/SgII(-ve) granules did not appear to be influenced by the actions of Buserelin and, therefore, may have been destined for constitutive release, possibly to maintain basal plasma LH concentrations.

Previous studies have shown that changes in the plasma concentrations of immunoreactive inhibin measured by radioimmunoassay occur in parallel with growth and regression of the testes during a reproductive cycle in adult Soay rams induced by exposure to an artificial lighting regimen of alternating 16 week periods of long days and short days. With the development of new two-site ELISAs for sheep inhibin A and inhibin B, we have re-examined the relationship between FSH and dimeric, biologically active inhibin in the reproductive cycle in adult Soay rams. No signal was generated by sheep testicular extract, ram or ewe plasma, or sheep ovarian follicular fluid in the inhibin B ELISA. In contrast, ram plasma contained significant activity in the inhibin A ELISA, which diluted in parallel to the inhibin A standard, and was abolished by preincubation of ram plasma with monoclonal antibodies specific for the betaA, but not the betaB, subunit. These results indicate that the ram is the first adult male mammalian species identified to date in which the testes produce and secrete dimeric inhibin A and not inhibin B. Northern blot analysis and immunocytochemistry confirmed the presence of alpha, betaA and betaB inhibin/activin subunit mRNA and protein in the testes of adult rams. Changes in plasma inhibin A concentrations occurred in parallel with the growth and regression of the testes during the long day: short day: long day lighting regimen in adult Soay rams, confirming our previous observations with immunoreactive inhibin. During the growth phase of the testes in the first 8 weeks of exposure to short days there was a positive correlation between plasma FSH and inhibin A concentrations, indicating that during this phase the secretion of inhibin A is stimulated by FSH and that inhibin A did not act as a negative feedback hormone on FSH secretion. From week 8.5 to week 16.0 of exposure to short days, there was a negative correlation between FSH and testosterone concentrations, but not inhibin, indicating that when inhibin concentrations are high, testosterone acts as the negative regulator of FSH secretion. Thus, in intact adult rams, when the testes are fully active it appears that inhibin A may sensitize the pituitary to the negative feedback effects of testosterone, at which time they act synergistically to maintain plasma concentrations of FSH.

Inhibins and activins have roles in the regulation of cell proliferation and differentiation in a variety of tissues. This study investigated the distribution of the three inhibin/activin subunits (alpha, betaA and betaB) and their receptors in the human testis between week 13 and week 19 of gestation using RT-PCR and immunohistochemistry. mRNA for all three subunits and for the activin type II receptors ActRIIA and ActRIIB was detected at all stages of gestation examined. Sertoli cells showed intense immunostaining for the alpha subunit and some staining for the betaB subunit, whereas only the betaB subunit was detected in gonocytes. No betaA subunit staining was detected within the tubules. All three subunits were localized to interstitial Leydig cells. Cells of the rete testis and the epididymal epithelium also showed immunostaining for betaB; however, staining for the other subunits was weak or absent. Peritubular cells showed intense immunostaining for the beta-glycan inhibin receptor, which was also localized to interstitial cells, but was not detected within the tubular compartment, rete testis or epididymal epithelium. ActRIIA was detected in gonocytes and in interstitial cells; ActRIIB was distributed widely. These data indicate that fetal Leydig and Sertoli cells have the potential to produce both activins and inhibins, whereas gonocytes may produce only activin B. The distribution of activin and inhibin receptors implies that the intratubular compartment and developing duct system are sites of action of activin B but not inhibin at this stage of development, whereas both activins and inhibins may be involved in the development and function of the peritubular and interstitial cells.

The bone morphogenetic proteins (BMPs) have been implicated in the paracrine regulation of ovarian follicular development. In this study, we investigated the expression of the BMP receptors (BMPRs) in sheep ovaries by immunohistochemistry and the effect of BMP2, a natural ligand for these receptors, on granulosa cells cultured in vitro. Ovaries from cyclic ewes were fixed, embedded in paraffin wax and cut into sections. The sections were rehydrated, submitted to microwave antigen retrieval and treated with polyclonal antibodies against BMPR1A, BMPR1B and BMPR2. Strong immunostaining for all three receptors was observed in the granulosa cell layer of follicles from the primary to late antral stages of development. Staining was also present in the oocyte, corpus luteum, ovarian surface epithelium and, to a lesser extent, the theca layer of antral follicles. For functional studies, granulosa cells were obtained from immature follicles 1-3 mm in diameter. The cells were cultured for 6 days in serum-free medium containing 1 ng oFSH-20 ml(-1) in the presence of 0, 3, 10 or 30 ng ml(-1) human recombinant BMP2. The medium was replaced every 2 days and oestradiol and inhibin A concentrations were measured in the spent medium. In the absence of BMP2, oestradiol and inhibin A production increased as the granulosa cells differentiated in vitro. The addition of the highest dose of BMP2 enhanced oestradiol production (P < 0.05) without affecting the proliferation of the cells. It is concluded that BMP receptors are present in sheep ovaries and that BMPs may have a role in the differentiation of granulosa cells by enhancing the action of FSH.

The biosynthesis of oestrogens from androgens is catalysed by the aromatase complex, an essential component of which is the aromatase cytochrome P450 (P450 arom) protein. Expression of a functional P450 arom is essential for normal fertility in males and females and the sequence of the protein is highly conserved. We have raised a new monoclonal antibody against a conserved peptide and validated it on fixed tissue sections of the rat, common marmoset (Callthrix jacchus) and human. The monoclonal antibody was used successfully for Western analysis and specifically reacted with a 55 kDa protein in microsomal extracts. On sections of ovaries in all three species, expression in follicles was specific to the mural granulosa cells of antral follicles and was present in corpora lutea. In the human and marmoset, staining of luteal cells was markedly heterogeneous and did not appear to vary consistently with the stage of the cycle. The intensity of immunostaining was elevated in corpora lutea from pregnant rats and following human chorionic gonadotropin rescue in the human. In the testis, the highest levels of expression were observed in the Leydig cells within the interstitium. In adult rat and marmoset, and possibly also in the human, some P450 arom was associated with the cytoplasm surrounding elongate spermatids but other germ cells were immunonegative. In conclusion, a new monoclonal antibody specific for P450 arom recognises the protein in rodent, primate and human. Its ability to work on fixed tissue sections will facilitate identification of individual cells expressing P450 arom within complex tissues.

Several aspects of equine ovarian physiology are unique among domestic species. Moreover, follicular growth patterns are very similar between horses and humans. This study aimed to characterize, for the first time, global gene expression profiles associated with growth and preovulatory (PO) maturation of equine dominant follicles. Granulosa cells (GCs) and theca interna cells (TCs) were harvested from follicles (n = 5) at different stages of an ovulatory wave in mares corresponding to early dominance (ED; diameter ≥22 mm), late dominance (LD; ≥33 mm) and PO stage (34 h after administration of crude equine gonadotropins at LD stage), and separately analyzed on a horse gene expression microarray, followed by validation using quantitative PCR and immunoblotting/immunohistochemistry. Numbers of differentially expressed transcripts (DETs; ≥2-fold; P < 0.05) during the ED-LD and LD-PO transitions were 546 and 2419 in GCs and 5 and 582 in TCs. The most prominent change in GCs was the down-regulation of transcripts associated with cell division during both ED-LD and LD-PO. In addition, DET sets during LD-PO in GCs were enriched for genes involved in cell communication/adhesion, antioxidation/detoxification, immunity/inflammation, and cholesterol biosynthesis. In contrast, the largest change in TCs during the LD-PO transition was an up-regulation of genes involved in immune activation, with other DET sets mapping to GPCR/cAMP signaling, lipid/amino acid metabolism, and cell proliferation/survival and differentiation. In conclusion, distinct expression profiles were identified between growing and PO follicles and, particularly, between GCs and TCs within each stage. Several DETs were identified that have not been associated with follicle development in other species.

Current therapies for pituitary tumours are moderately successful in many cases but still suffer from significant limitations, with relatively poor long-term rates of endocrine cure from surgery, and long-term hypopituitarism after radiotherapy. Even in the case of the most readily treatable tumours, prolactinomas, medical therapy with dopamine agonists is limited by lack of response or side-effects in up to 10% of patients. This has led to increasing interest in the application of our knowledge of pituitary cell and molecular biology to evaluate the potential of gene therapy. Various vectors are available to facilitate gene delivery, and recombinant adenoviruses have been studied in detail because of their ability to transduce the postmitotic, nondividing cells of the pituitary gland. Various studies with reporter genes such as beta-galactosidase have demonstrated high efficiency and long lasting expression of adenoviral transgenes in cultured pituitary cells in vitro. The feasibility of high level transgene expression has also been shown in vivo, but so far this requires stereotaxic intrapituitary injection to achieve adequate transduction. Ablation of pituitary cells has been demonstrated in cultured cell lines and in subcutaneous tumours in nude mice, though alternative animal models will be required to evaluate efficacy in more slowly proliferating tumours as found in man. Inflammatory responses have been documented in the pituitary gland as in other tissues, and this will require the evaluation of modified vectors to avoid significant adverse effects before human applications can be considered. In summary, gene therapy for pituitary disease is likely to be feasible in the future, but will require careful and extensive evaluation of efficacy and safety, using a variety of possible methods of gene delivery.

Twenty-five normal ovulatory women underwent three-dimensional transvaginal ultrasonography and blood sampling before and after oral glucose tolerance testing to compare ovarian morphology and circulating hormone levels in the early follicular phase as predictors of the number of oocytes retrieved after gonadotropin stimulation for in vitro fertilization. Serum levels of gonadotropins, inhibins, testosterone, dehydroepiandrosterone sulfate, and estradiol as well as summed ovarian volume were unrelated to oocyte number. Antral follicle number and serum androstenedione level, however, positively correlated, whereas postoral glucose tolerance test (post-OGTT) insulin release negatively correlated, with total and mature oocyte numbers. Adjusting for age and body mass index by regression analysis, the serum androstenedione level significantly predicted mature, but not total, oocyte number. The relationships of antral follicle number and post-OGTT insulin release to total oocyte number were additive; each was significant after controlling for the other. In contrast, antral follicle number significantly correlated with mature oocyte number after controlling for post-OGTT insulin release, whereas post-OGTT insulin release was unrelated to mature oocyte number after controlling for antral follicle number. Therefore, early follicular phase antral follicle number positively correlates with total and mature oocyte numbers after gonadotropin stimulation for in vitro fertilization and is linked to androgen and insulin actions in predicting ovarian follicle recruitment by gonadotropins.

A thorough understanding of the factors that regulate the secretion of FSH is critical for the development of efficient methods for fertility regulation. The purpose of this review is to evaluate what is currently known about the existence of FSH-releasing factor(s). It considers the obstacles encountered in understanding the control of FSH secretion, current knowledge of the nature of FSH secretion and the mechanisms involved in controlling FSH secretion, the arguments that have been posed against the need for an FSH-releasing factor and finally the evidence available to date supporting the existence of FSH-releasing factor(s).

Within 2 days of birth, the mouse ovary is mainly composed of oocytes surrounded by a few pregranulosa cells forming primordial follicles that remain quiescent until they are recruited by intraovarian or other unknown factors to initiate growth of the oocyte and proliferation of the attendant granulosa cells. However, the role of the oocyte in this early development and organization of the follicle is poorly understood. The Dazl knockout (2/2) mouse in which there is total ablation of oocytes in fetal life has allowed us to address this issue. Ovaries from 2/2 females lack any follicular structure and have no cells positive for either Mullerian inhibiting factor or sulfated glycoprotein-1, indicating a lack of small follicles or corpora lutea. However, by immunocytochemistry, there are cells positive for 3bhydroxysteroid dehydrogenase, 17a-hydroxylase, and aromatase, indicating the presence of steroidogenically active cells capable of producing estrogen. This was confirmed by the presence of hypertrophied uterine endometrium expressing both estrogen receptor a (ERa) and ERb together with normal levels of plasma estradiol. In addition, these steroidogenically active cells contain ERb, inhibin a, and bBsubunits, and 2/2 mice have low measurable plasma inhibin A and B levels. The ovarian steroids and inhibins had no significant effect on either plasma or pituitary gonadotropin levels, with significantly (P , 0.01) lower LH and FSH in intact 1/1 and 1/2 females. However, significantly (P , 0.05) increased plasma inhibin B together with significantly (P , 0.05) lower FSH were observed in the 1/2 females. In conclusion, our data showed that despite oocyte loss in fetal life, the adult ovaries contained steroidogenically active cells capable of producing estradiol and inhibin. Furthermore, in the 1/2 mice, the enhanced plasma inhibin B implies a role for Dazl protein within the oocyte either from more small follicles or increased inhibin B production from each follicle. (Endocrinology 141: 4284 ‐ 4294, 2000)

An interaction between gonadotroph and lactotroph cells of the pituitary gland has long been recognized in several species. The current study was conducted to investigate whether an association between gonadotrophs and lactotrophs occurs in mares and whether prolactin receptors are expressed within the pituitary gland of this species. The effects of both reproductive state and season on these variables were examined in pituitary glands obtained from sexually active mares in July (breeding season), sexually active mares in November (non-breeding season) and anoestrous mares in November. Pituitaries were dissected out immediately after death and immunofluorescent staining was carried out on 6 micrometer sections using specific antibodies to the LHbeta subunit, FSHbeta subunit, prolactin and prolactin receptor. Gonadotrophs were observed in both the pars distalis and pars tuberalis; although they appeared mostly as isolated cells, small groups of gonadotrophs were also identified in the pars distalis. In contrast, lactotrophs were observed only as clusters of cells exclusively in the pars distalis of sexually active and anoestrous mares in November and in most of the sexually active mares in July. A specific gonadotroph-lactotroph association was identified only between large isolated gonadotrophs and lactotroph clusters. Double immunofluorescent staining for FSHbeta and prolactin revealed a similar gonadotroph-lactotroph association to the one detected for LH gonadotrophs. No statistical difference in the gonadotroph:lactotroph ratio was observed as a result of changes in reproductive status or season. However, a tendency for a simultaneous decrease in the number of gonadotrophs and an increase in the number of lactotrophs was detected in anoestrous animals. Prolactin receptor immunoreactivity was found in the pars distalis, but not in the pars tuberalis, of sexually active (July and November) and anoestrous animals for both long and short forms of the receptor. No prolactin receptor co-localization for either form of the receptor was observed in LH or FSH gonadotrophs in either of the reproductive states examined during both summer and winter seasons. Furthermore, no significant difference was apparent in the proportion of cells expressing prolactin receptors between mares of different reproductive state or season. The specific anatomical association between gonadotroph and lactotroph cells and the expression of prolactin receptors in the equine pituitary gland indicate a potential role of prolactin in the regulation of gonadotrophin secretion. However, the absence of evidence for co-localization of prolactin receptors in LH or FSH cells does not support the hypothesis of a direct effect of prolactin on the gonadotroph as reported in a short day breeder. The results raise the possibility that, in horses, an intermediate regulatory cell may mediate the action of prolactin on gonadotroph function.

The purpose of this study was to document the morphological changes in the seminiferous epithelium that underlie the compensatory testicular hypertrophy observed in response to unilateral orchidectomy (UO) in the adult rhesus monkey and to describe the concomitant response in the endocrine feedback loops controlling testicular function in this species. Adult male monkeys were implanted with indwelling venous catheters; seven animals were then subjected to UO (data are presented from six) and three to sham UO. Profiles of circulating concentrations of FSH, LH, testosterone (T), inhibin B, and pro-alpha-C were monitored in 12-h series of sequential blood samples collected before, on the day of UO (day 0), and on days 1, 2, 4, 8, 16, 32, and 42 or 43 after UO. In the UO monkeys, the remaining testis was taken on day 44. Sertoli and germ cells in the removed and remaining testes were counted and expressed either as number per testis or, in the case of the differentiated spermatogonia (B1, B2, B3, and B4), as number per cross-section of the seminiferous tubule. UO was associated with a marked increase in the number of all germ cells more mature than undifferentiated spermatogonia (Ap) in the remaining testis. Sertoli cell number, on the other hand, did not change, and it is therefore reasonable to propose that the primary locus of the spermatogenic compensation was the differentiated spermatogonia. The additional finding that the relationship between the number of Sertoli cells and total germ cells in the remaining testis became robust (r = 0.92; P 0.05 for the removed testis) indicated that in the monkey, spermatogenesis does not normally operate at its ceiling. The increased drive to the seminiferous tubule of the remaining testis is hypothesized to be mediated by the sustained increase in FSH secretion that was observed after UO, although a role for increased testicular T production cannot be excluded. The stimulus for increased FSH secretion was presumably provided by the abrupt, 50% decline in circulating inhibin B levels. Interestingly, inhibin B secretion by the remaining testis was not dramatically affected by UO, and therefore, the deficit in circulating levels of this hormone and thus the error signal to FSH secretion were maintained for the duration of the experiment. In contrast, the changes in circulating LH and T concentrations were only transient, and within 48 h of UO, these hormonal parameters had returned to control values. The mechanisms by which the remaining testis rapidly acquires the capacity to double T production in the face of an unchanging LH drive remains to be determined. The foregoing body of evidence suggests that sperm output by the monkey testis is regulated by the circulating concentration of FSH and that in physiological situations, FSH secretion is insufficient to stimulate spermatogenesis to its ceiling. The results also indicate that FSH secretion is controlled by a feedback system in which the feedforward arm (FSH-inhibin B) is less robust than the feedback loop (inhibin B-FSH). Thus, a decrease in the inhibin B feedback signal results in a sustained increase in FSH secretion that drives the testes toward their spermatogenic ceiling, which is presumably set by Sertoli cell number.

The objective of this study was to assess the effect of ovine follicular fluid (FF) treatment (with or without FSH replacement) during the late follicular phase on plasma concentrations of gonadotrophins and the development of the ovulatory follicle. Ovarian steroid secretion and expression of mRNA encoding inhibin alpha and beta A, beta B subunits, P450 aromatase and P450 17 alpha-hydroxylase were used as endpoints. After induction of luteolysis by injection of 100 micrograms cloprostenol on days 10-12, Scottish Blackface ewes were allocated to one of three groups: (1) control (n = 7): no further treatment; (2) FF (n = 9): subcutaneous injections of 3 ml steroid-free ovine follicular fluid at 9 h intervals, 18 and 27 h after cloprostenol injection; (3) FF + FSH (n = 8): injections of follicular fluid as above plus subcutaneous injections of 0.36 iu ovine FSH at 6 h intervals, 18, 24, and 30 h after cloprostenol injection. Jugular venous blood samples were obtained via indwelling cannulae at 6 h intervals from 0 to 36 h after cloprostenol injection, and at 10 min intervals from 12 to 18 h (control phase) and from 30 to 36 h after cloprostenol injection (treatment phase). At laparotomy, 36 h after cloprostenol injection, ovarian venous blood was collected and ovaries were removed and processed for in situ hybridization. Plasma concentrations of FSH, luteinizing hormone (LH) and oestradiol were determined by radioimmunoassay. Follicular fluid treatment resulted in a decrease (P0.001) in FSH concentrations associated with an acute decrease in ovarian steroid secretion (P0.01) and a specific depression in P450 aromatase, (P0.001), inhibin-activin beta B subunit (P0.05) and thecal LH receptor (P0.001) expression. Follicular fluid treatment had no effect on inhibin-activin alpha and beta A, subunit or P450 17 alpha-hydroxylase expression. FSH co-treatment with follicular fluid restored circulating FSH concentrations to normal values and reversed some of the effects of follicular fluid (androstenedione, testosterone and progesterone secretion, and inhibin beta B and thecal LH receptor expression) but not oestradiol secretion or P450 aromatase expression. It was concluded that the actions of follicular fluid are mediated via both central effects on pituitary FSH secretion and by direct ovarian effects on granulosa cell aromatase activity. The results indicate that follicular fluid contains a factor that inhibits aromatase activity of granulosa cells directly and may play a role in the selection of the dominant follicle.

We investigated the concentrations of serum inhibin B and the pro-alphaC-containing inhibins (pro-alphaC inhibin) and their relations to serum FSH levels in 38 healthy boys during their progression through puberty. Furthermore, we studied the effect of recombinant human FSH (rhFSH) on pro-alphaC inhibin production in three prepubertal gonadotropin-deficient boys. The serum inhibin B level increased between Tanner stages G1 and G2 (p0.02), simultaneously with the serum LH and testosterone concentrations. In advanced puberty, inhibin B changed less, and at stage G4, correlated negatively with serum FSH level (r = -0.57, p0.001, n = 37). The serum pro-alphaC inhibin level changed differently and increased also in advanced puberty. In prepubertal healthy subjects, the serum pro-alphaC inhibin and FSH levels correlated positively (r = 0.50, p = 0.051, n = 16), and during the rhFSH treatment of gonadotropin-deficient boys, serum pro-alphaC inhibin levels increased. These findings suggest that an increased production of inhibin B is an early event in puberty and that inhibin B can inhibit pituitary FSH secretion in the course of sexual maturation. Furthermore, they suggest that during puberty free inhibin (pro)-alpha-subunits, detected by the pro-alphaC inhibin assay, are present in large amounts and that FSH stimulates their production by prepubertal testes.

Luteolysis is associated with tissue remodeling probably involving the matrix metalloproteinases (MMPs) and their specific tissue inhibitors (TIMPs). This study investigated the expression and localization of the major MMPs and TIMPs in the human corpus luteum throughout the luteal phase and after luteal rescue with hCG. Corpora lutea (n = 9) were collected at hysterectomy and were dated by serial urinary LH estimation. In addition, corpora lutea (n = 3) were collected from women who had received daily doubling doses of hCG to mimic the hormonal changes of early pregnancy. MMP-1, MMP-2, MMP-9, TIMP-1, TIMP-2, and TIMP-3 were investigated by zymography, reverse zymography, Northern blotting, and in situ hybridization. There was no change in the expression of MMP-1, TIMP-1, and TIMP-2 throughout the luteal phase or after luteal rescue. Little TIMP-3 could be detected in the corpus luteum. MMP-9 activity peaked in the early and late luteal phase. The expression and activity of MMP-2 were maximal in the late luteal phase. Exposure to hCG during luteal rescue in vivo was associated with a reduction (P < 0.05) in the expression and activity of MMP-2. Messenger ribonucleic acids (mRNAs) for MMP-1, MMP-2, and TIMP-2 were localized to the connective tissue stroma and the thecal-lutein cells of the corpus luteum. In contrast, TIMP-1 mRNA was localized to the granulosa-lutein cells, and MMP-9 mRNA was expressed in scattered cells within the steroidogenic and nonsteroidogenic cell layers. In conclusion, during maternal recognition of pregnancy, hCG prevents the normal increase in MMP-2 in the late luteal phase. MMPs can function in an environment containing large amounts of TIMP-1, as they have a different cellular localization.

Fractionation of bovine corpus luteum (CL) homogenates on continuous sucrose density gradients with and without preincubation with H-3-progesterone demonstrated high levels of tracer binding and high content of endogenous progesterone associated with particulate membrane fractions. Analysis of gradient fractions for a range of luteal plasma membrane and intracellular organelle marker enzyme activities indicated that endogenous progesterone content and H-3-progesterone-binding activity were associated with fractions enriched in luteal plasma membrane markers. This was confirmed by pretreatment of homogenates with the saponin, digitonin, prior to fractionation. Digitonin perturbed the buoyant density of luteal surface membrane markers and H-3-progesterone binding to a similar extent, but did not perturb the buoyant densities of other intracellular markers to the same degree. Interestingly, digitonin pretreatment also increased the proportion of progesterone tracer that entered the gradients. We consistently failed to demonstrate significant binding of H-3-progesterone to membrane fractions incubated with progesterone tracer in vitro. However, when digitonin was included in the in vitro binding assay, we observed a dramatic, dose-dependent stimulation of H-3-progesterone binding by digitonin. Other radiolabeled steroids tested (H-3-cortisol, H-3-testosterone) bound poorly in the presence or absence of digitonin. H-3-Progesterone binding in the presence of optimal digitonin concentrations increased linearly with increasing luteal membrane concentration; was dependent on the pH, duration, and temperature of incubation; and low levels of progesterone (68 nM) competed for tracer binding. A range of other steroids tested (androgens, estrogens, corticosteroids, steroid precursors) competed at higher concentrations (10- to 100-fold) or did not compete at all for H-3-progesterone binding. There was no correlation between the hydrophobicity of various steroids and their ability to compete for binding. Moreover, a number of agonists and antagonists specific for the genomic progesterone receptor, agonists of peripheral benzodiazepine receptors, and inhibitors of a range of steroidogenic enzymes did not compete for H-3-progesterone binding. Western blots confirmed that detergent-solubilized progesterone-binding sites could be resolved from cytochrome P450 side-chain cleavage and 3 beta-hydroxysteroid dehydrogenase. Moreover, extraction of bound steroid from the binding and HPLC demonstrated identity to progesterone, suggesting that no metabolism of the progesterone tracer had occurred during incubation. Progesterone binding to membranes of large luteal cells was higher compared with binding to small luteal cells, and levels were similar in membranes prepared from CL at all stages of the luteal phase. We suggest that bovine luteal progesterone-binding sites may play a role either in sequestration of newly synthesized progesterone or in the mediation of autocrine and/or paracrine actions of progesterone in the CL.

The purpose of this study was to determine the relative concentrations of inhibin A and B in peripheral serum of the adult male rhesus monkey and to examine the testicular contribution to these circulating forms of inhibin. In addition, inhibin B concentrations were also determined in peripheral sera of neonatal and juvenile males and in spermatic vein blood of adults. Immunoradiometric assays specific for the measurement of inhibin A and B were used. These assays also provided an opportunity to reexamine the physiological significance of a replacement infusion of recombinant human (rh)-inhibin A previously employed to study the role of this hormone in regulating FSH secretion in the monkey. In intact adults, the mean (± se) serum concentration of inhibin B was 1008 ± 184 pg/mL. In contrast, circulating inhibin A concentrations were very low (

To investigate the mechanism controlling the fall in maternal pituitary responsiveness to GnRH, LH synthesis and pituitary GnRH receptor content during pregnancy, maternal pituitaries were collected from sheep on days 35, 45, 60, 90, 110, 125 and 135 of pregnancy. Circulating steroids and gonadotrophins were determined in blood samples collected from these ewes immediately before death. Pituitary blocks from each ewe were perifused with either medium alone (control) or medium supplemented with oestradiol, oestradiol plus progesterone or oestradiol plus RU486, for 150 min before administration of two 15 s GnRH pulses 90 min apart. The amounts of mRNA encoding LH beta and GnRH receptor were determined in pituitary tissue fragments snap-frozen in liquid N2 at the time of collection from the ewes. While basal LH secretion fell during pregnancy, pituitary responsiveness to GnRH remained high (up to seven times basal LH concentrations). After day 90, the first GnRH pulse elicited LH peaks equivalent to the LH peaks produced by the second GnRH pulse. Therefore, GnRH self-priming was not evident possibly because the pituitaries were constantly primed by increased concentrations of maternal oestradiol. Around day 90, circulating concentrations of progesterone rose from 7.8 +/- 1.5 to 12.2 +/- 3.8 ng ml-1. Up to day 60, oestradiol in the perifusion buffer had stimulatory effects on LH secretion although this was reduced by RU486. By day 125, the content of mRNA encoding LH beta had declined during pregnancy to 7% of the content on day 35, although the content of mRNA encoding GnRH receptor remained unchanged. From these data, there appears to be a transitional period at around day 90 of gestation when pituitary sensitivity to steroids in vitro is lost together with detectable GnRH self-priming. In conclusion, the marked decline in pituitary amounts of mRNA encoding LH beta, but not in GnRH responsiveness or expression of GnRH receptor, after day 45 of pregnancy suggests that the principal effect of pregnancy on gonadotroph function is mediated via a mechanism other than reduced pituitary amounts of GnRH receptors. Two possible mechanisms are (1) a reduction in GnRH output leading to lowered LH synthesis, or (2) the presence of an inhibitory factor with a short half-life in the maternal circulation.

OBJECTIVE: Although recently developed specific and sensitive assays of bioactive dimeric inhibin A and B have given new insights into the pituitary-gonadal axis in adult men and during the adult female menstrual cycle, there have been no reports on circulating inhibin A and B during normal human puberty. The aim of this study was to assess the relationship of dimeric inhibin A and B to pubertal stage, FSH and testosterone or oestradiol in late prepuberty and in early puberty. STUDY DESIGN AND SUBJECTS: Serial samples were collected during a prospective longitudinal trial of GH treatment in short normal children. Seven boys were studied from late prepuberty to genital stage 3, and six pre-menarche girls from late prepuberty to breast stage 4. MEASUREMENTS: Dimeric inhibin A (girls only) and inhibin B (boys and girls) were measured by highly specific and sensitive two-site ELISAs, FSH by IRMA, testosterone and oestradiol by RIA. RESULTS: In boys, inhibin B increased progressively from pubertal stages 1 to 3 (ANOVA P < 0.0001) and correlated strongly with mean testicular volume (r = 0.72, P = 0.0005). Prepubertal boys showed a positive correlation between inhibin B and FSH (r = 0.65, P = 0.056), whereas pubertal boys gave a strong negative correlation (r = 0.75, P = 0.012). In both prepubertal and pubertal boys positive correlations were observed between inhibin B (y) and testosterone (x) (r = 0.81, P = 0.008 and r = 0.62, P = 0.054 respectively), but the slope of the regression line between the two was much steeper before than after the onset of clinical puberty. In girls, both inhibin A and B increased through pubertal stages 1-4 (ANOVA P = 0.01 and P = 0.047 respectively). Both showed strong positive correlations with oestradiol (r = 0.80 and 0.79, P = 0.001) and with FSH (r = 0.83, P = 0.0004 and r = 0.80, P = 0.001). Inhibin A and B were also strongly correlated with each other (r = 0.92, P = 0.0001). CONCLUSIONS: In boys, testicular production of inhibin B increases as puberty progresses. Our results show for the first time that the initiation of puberty is accompanied by a dramatic switch from a positive to a negative relation between inhibin B and FSH as inhibin B begins to exert the expected negative feedback on FSH. The results in girls suggest that, prior to menarche, the ovarian follicles produce inhibin A and B in strict proportion, and in progressively greater amounts as puberty proceeds. Measurement of dimeric inhibin A and B may provide a sensitive new tool for determining gonadal maturity in late prepuberty and early puberty.

Using an ovine model of polycystic ovary syndrome (PCOS), (pregnant ewes injected with testosterone propionate (TP) (100 mg twice weekly) from day (d)62 to d102 of d147 gestation (maternal injection – MI-TP)), we previously reported female offspring with normal glucose tolerance but hyperinsulinemia. We therefore examined insulin signalling and pancreatic morphology in these offspring using quantitative (Q) RT-PCR and western blotting. In addition the fetal pancreatic responses to MI-TP, and androgenic and estrogenic contributions to such responses (direct fetal injection (FI) of TP (20 mg) or diethylstilbestrol (DES) (20 mg) at d62 and d82 gestation) were assessed at d90 gestation. Fetal plasma was assayed for insulin, testosterone and estradiol, pancreatic tissue was cultured, and expression of key b-cell developmental genes was assessed by QRT-PCR. In female d62MI-TP offspring insulin signalling was unaltered but there was a pancreatic phenotype with increased numbers of b-cells (P,0.05). The fetal pancreas expressed androgen receptors in islets and genes involved in b-cell development and function (PDX1, IGF1R, INSR and INS) were up-regulated in female fetuses after d62MI-TP treatment (P,0.05–0.01). In addition the d62MI-TP pancreas showed increased insulin secretion under euglycaemic conditions (P,0.05) in vitro. The same effects were not seen in the male fetal pancreas or when MI-TP was started at d30, before the male programming window. As d62MI-TP increased both fetal plasma testosterone (P,0.05) and estradiol concentrations (P,0.05) we assessed the relative contribution of androgens and estrogens. FI-TP (commencing d62) (not FIDES treatment) caused elevated basal insulin secretion in vitro and the genes altered by d62MI-TP treatment were similarly altered by FI-TP but not FI-DES. In conclusion, androgen over-exposure alters fetal pancreatic development and b-cell numbers in offspring. These data suggest that that there may be a primary pancreatic phenotype in models of PCOS, and that there may be a distinct male and female pancreas.

Luteal progesterone production is dependent on luteinizing hormone (LH) from the pituitary gland. Despite continuing LH secretion, the human corpus luteum undergoes functional luteolysis unless it is 'rescued' by human chorionic gonadotrophin (HCG), produced by the implanting blastocyst. As LH and HCG act through a common receptor, this study sought to determine the expression of the LH/ HCG receptor in the corpus luteum during maternal recognition of pregnancy. Corpora lutea were collected at hysterectomy from women in the normal luteal phase and after luteal 'rescue' with exogenous HCG. In each case the corpus luteum was classified according to the date of the LH surge measured in daily urine samples. The expression of the LH receptor was investigated by Northern blotting, in-situ hybridization and in-situ ligand binding. LH receptor mRNA and ligand binding activity were detected in corpora lutea from all stages of the luteal phase. LH receptor expression and binding were maintained during maternal recognition of pregnancy in the presence of exponentially increasing HCG concentrations. These data show that the LH receptor is maintained throughout the functional life-span of the human corpus luteum and is not down-regulated during maternal recognition of pregnancy.

In breast-feeding women prolactin released in response to suckling is essential for the maintenance of lactation. This physiological hyperprolactinaemia is also associated with lactational infertility. However, it is not clear whether there is any direct relationship between changes in prolactin per se and the duration of infertility. To address this question, our study determined the pattern of prolactin secretion in relation to suckling and the return of ovarian activity in the same cohort of breast-feeding women. Blood samples were withdrawn at 10 min intervals for 24 h from 09:00 to 09:00 h at either 4 (n = 9) or 8 weeks (n = 11) post-partum when the women had completely suppressed ovarian activity, at the time of the introduction of supplements to the baby (n = 17), a time associated with reduction of suckling activity, at first menses while still breast-feeding (n = 13) and in the follicular phase (n = 9) of the first menstrual cycle after weaning. During sampling, mothers and babies continued their normal pattern of suckling activity. The pattern of prolactin release was very variable at each stage of lactation, depending on the pattern of suckling. Frequent suckling was associated with elevated prolactin concentrations during the 24 h period throughout lactation. When suckling was less frequent, prolactin concentrations fell to baseline values between breast-feeds, but prolactin was released in response to all suckling episodes. An increase in prolactin concentrations at night, independent of suckling, was only evident once breast-feeding had ceased. The prolactin response to suckling declined significantly only after the return of menses at 33.6 +/- 3.5 weeks post-partum. There was no relationship between the duration of amenorrhoea and the plasma concentrations of prolactin over 24 h, or day or night separately, throughout lactation. However, there was a strong correlation (r = 0.843; P < 0.01) between the timing of the introduction of dietary supplements to the baby and the duration of amenorrhoea. These results suggest that there may be no precise link between the release of prolactin during lactation and the duration of lactational infertility in breast-feeding women.

The interaction of gonadotrophin-releasing hormone (GnRH) with its receptor is of crucial importance to gonadotroph function, although until recently, little was known of the structure of the GnRH receptor (GnRH-R). This report has addressed the steps leading to the cloning of the sheep GnRH-R cDNA and the endocrine mechanisms associated with the regulation of GnRH-R mRNA expression. Differences between species and between in vivo and in vitro studies are apparent. The expression of GnRH-R mRNA was shown to increase prior to the gonadotrophin surge and decrease thereafter; these changes were correlated with GnRH binding in the pituitary and follicle oestradiol production. In the early follicular phase of the sheep oestrous cycle, oestradiol appears to be the primary regulator of GnRH-R mRNA, whereas in the periovulatory period, the action of GnRH itself is more important.

The object of the study was to investigate the effect on gonadotrophin secretion of a small increase in oestradiol concentration. A total of 13 fully breast-feeding women (12 weeks post-partum) underwent serial blood sampling at 10 min intervals for 12 h on 2 different days; day 1 untreated and day 5 after 3 days of treatment with transcutaneous oestradiol (100 micrograms/day). On both days bolus gonadotrophin-releasing hormone (GnRH; 10 micrograms i.v.) was given after a 10 h baseline period. In six of the subjects, a naloxone infusion was administered during the second study day. Application of transdermal oestradiol raised the oestradiol concentration within the normal follicular phase range. The mean luteinizing hormone (LH) concentration on day 5 was found to be significantly lower than that on day 1 (P < 0.05). The LH response to GnRH was, however, significantly higher on day 5 than day 1 (P < 0.001). The mean follicle stimulating hormone (FSH) concentration on day 5 was also significantly lower than that on day 1 (P < 0.01), while the peak concentration after GnRH was unchanged. When the opioid antagonist naloxone was infused after oestradiol treatment, the subjects with low pre-study oestradiol concentrations exhibited no effect on LH concentration, while in the subjects with higher oestradiol concentrations the LH concentration was increased. It was concluded that the administration of small doses of oestradiol caused a significant fall in gonadotrophin concentration in breast-feeding women.(ABSTRACT TRUNCATED AT 250 WORDS)

In order to investigate the ontogeny of gonadal inhibin production in the male fetal sheep, testes were collected from male fetuses at days 70, 100, 130 and 140 of gestation (term=145 days). The expression and localization of inhibin α- and inhibin βA-subunit mRNA and protein were evaluated using in situ hybridization and immunocytochemistry. The expression of inhibin α-subunit mRNA was localized within the seminiferous cords of the developing fetal testis and progressively increased with gestational age. Immunostaining corresponding to immunoreactive inhibin α-subunit was detected in Sertoli cells within the seminiferous cords at days 100, 130 and 140 of gestation. In addition, immunostaining was detectable in a small proportion of Leydig cells. No expression of inhibin βA-subunit mRNA or immunoreactivity was detected in any testicular tissue at any stage of gestation. These data show that the Sertoli cells of the developing fetal sheep testis have the capacity to produce inhibin α-subunit by day 100 of gestation and that production increases during late gestation. Journal of Endocrinology (1995) 145, 35–42

In adult sheep, inhibin expression in developing follicles appears to be associated with antrum formation. Our objective was to investigate using in situ hybridization and immunohistochemistry whether antral follicles present before birth in the sheep expressed mRNA or peptide for inhibin alpha- and beta A-subunits. At days 70 and 100 when only primordial and primary follicles were present, there was no detectable mRNA or peptide for either inhibin subunit. By days 130 and 140 (term = 145 days), many secondary follicles were present, a proportion of which (approximately 50%) expressed detectable levels of alpha-subunit mRNA but not peptide. A number of antral follicles were present by this stage, all of which expressed alpha-subunit mRNA and peptide. Expression of beta A-subunit mRNA and peptide was undetectable at all stages of gestation. Our results indicates that even in non-ovulatory follicles present before birth, expression of inhibin, at least the alpha-subunit, is developmentally linked with antrum formation.

The endocrine system in higher mammals represents one of the most complex and fundamental systems that regulates nearly all of an organism's biological functions. This system is composed of multiple organs, tissues, hormones, and receptor modalities. Its ability to regulate critical functions such as reproduction, development, metabolism, stress responses, blood pressure, wakefulness, and digestion places it as one of the most important regulators of life-long physiology. Therefore, at any one point in time the physiological status of the majority of organs in the body is a function of the activity of the whole endocrine system. However, while appreciating the role of the endocrine system in such “frozen” points in time is important, the temporal variation in endocrine function across one's lifespan is of crucial interest to researchers investigating age-related disorders. The importance of gerontological research is becoming more and more evident, given the ever-increasing proportion of aged people in Western countries.

One of the hallmarks of polycystic ovary syndrome (PCOS) is increased ovarian androgen secretion that contributes to the ovarian, hormonal, and metabolic features of this condition. Thecal cells from women with PCOS have an enhanced capacity for androgen synthesis. To investigate whether this propensity is a potential cause, rather than a consequence, of PCOS, we used an ovine prenatal androgenization model of PCOS and assessed ewes at 11 months of age. Pregnant Scottish Greyface ewes were administered 100 mg testosterone propionate (TP) or vehicle control twice weekly from d 62 to 102 of gestation, and female offspring (TP = 9, control = 5) were studied. Prenatal TP exposure did not alter ovarian morphology or cyclicity, or plasma androgen, estrogen, and gonadotropin concentrations, at this stage. However, follicle function was reprogrammed in vivo with increased proportions of estrogenic follicles (P < 0.05) in the TP-exposed cohort. Furthermore, in vitro the thecal cells of follicles (>4 mm) secreted more LH-stimulated androstenedione after prenatal androgenization (P < 0.05), associated with increased basal expression of thecal StAR (P < 0.01), CYP11A (P < 0.05), HSD3B1 (P < 0.01), CYP17 (P < 0.05), and LHR (P < 0.05). This provides the first evidence of increased thecal androgenic capacity in the absence of a PCOS phenotype, suggesting a thecal defect induced during fetal life.

Inhibin and activins play major roles as paracrine and autocrine signaling molecules in reproduction and development where the main emphasis has been placed in developing potential diagnostic applications. While a role for activin assays in diagnostics has so far been unfounded, ELISAs specific for the biologically active inhibin A and B dimers, and for free inhibin alpha subunits, alone or in combination have found some specific diagnostic applications. Addition of inhibin A to the triple test for Down syndrome in the second trimester of pregnancy, measurement of total inhibin as a marker of certain forms of ovarian cancer in specific circumstances, and inhibin B for male fertility are useful diagnostics. A review of the evidence so far suggests that other applications for inhibin and activin assays have yet to be confirmed, or translated into reliable tools for clinical practice.

While the germ cell-specific RNA binding protein, DAZL, is essential for oocytes to survive meiotic arrest, DAZL heterozygous (het) mice have an increased ovulation rate that is associated with elevated inhibin B and decreased plasma follicle-stimulating hormone (FSH). The relationship between decreased oocyte DAZL expression and enhanced follicular development in het mice was investigated using in vitro follicle cultures and in vivo modulation of endogenous FSH, by treating mice with inhibin and exogenous FSH. In vitro, follicles from het mice are more sensitive to FSH than those of wild-type (wt) mice and can grow in FSH concentrations that are deleterious to wildtype follicles. In vivo, despite no differences between genotypes in follicle population profiles, analysis of granulosa cell areas in antral follicles identified a significantly greater number of antral follicles with increased granulosa cell area in het ovaries. Modulation of FSH in vivo, using decreasing doses of FSH or ovine follicular fluid as a source of inhibin, confirmed the increased responsiveness of het antral follicles to FSH. Significantly more follicles expressing aromatase protein confirmed the earlier maturation of granulosa cells in het mice. In conclusion, it is suggested that DAZL expression represses specific unknown genes that regulate the response of granulosa cells to FSH. If this repression is reduced, as in DAZL het mice, then follicles can grow to the late follicular stage despite declining levels of circulating FSH, thus leading to more follicles ovulating and increased litter size. FSH, FSH receptor, follicular development, granulosa cells, inhibin, ovulation rate

Metformin is an insulin sensitizer molecule used for the treatment of infertility in women with polycystic ovary syndrome and insulin resistance. It modulates the reproductive axis, affecting the release of gonadotropin-releasing hormone (GnRH) and luteinizing hormone (LH). However, metformin's mechanism of action in pituitary gonadotropin-secreting cells remains unclear. Adenosine 5' monophosphate-activated protein kinase (PRKA) is involved in metformin action in various cell types. Here, we investigated the effects of metformin on gonadotropin secretion in response to activin and GnRH in primary rat pituitary cells (PRP), and studied PRKA in rat pituitary. In PRP, metformin (10 mM) reduced LH and follicle-stimulating hormone (FSH) secretion induced by GnRH (10(-8) M, 3 h), FSH secretion, and mRNA FSHbeta subunit expression induced by activin (10(-8) M, 12 or 24 h). The different subunits of PRKA are expressed in pituitary. In particular, PRKAA1 is detected mainly in gonadotrophs and thyrotrophs, is less abundant in lactotrophs and somatotrophs, and is undetectable in corticotrophs. In PRP, metformin increased phosphorylation of both PRKA and acetyl-CoA carboxylase. Metformin decreased activin-induced SMAD2 phosphorylation and GnRH-induced mitogen-activated protein kinase (MAPK) 3/1 (ERK1/2) phosphorylation. The PRKA inhibitor compound C abolished the effects of metformin on gonadotropin release induced by GnRH and on FSH secretion and Fshb mRNA induced by activin. The adenovirus-mediated production of dominant negative PRKA abolished the effects of metformin on the FSHbeta subunit mRNA and SMAD2 phosphorylation induced by activin and on the MAPK3/1 phosphorylation induced by GnRH. Thus, in rat pituitary cells, metformin decreases gonadotropin secretion and MAPK3/1 phosphorylation induced by GnRH and FSH release, FSHbeta subunit expression, and SMAD2 phosphorylation induced by activin through PRKA activation.