Secretion of inhibin A, inhibin B and inhibin pro-alphaC during the oestrous cycle of the golden hamster (Mesocricetus auratus).

Plasma concentrations of inhibin pro-alphaC, inhibin A and inhibin B were determined by enzyme-linked immunosorbent assay at 6 h intervals throughout the 4-day oestrous cycle of the golden hamster. Plasma concentrations of follicle-stimulating hormone (FSH) and oestradiol-17beta were also measured by radioimmunoassay during the oestrous cycle. Plasma concentrations of inhibin A increased from the early morning of day 1 (day 1=day of ovulation) and reached plateau levels at 0500 h on day 2. An abrupt increase in plasma concentrations of inhibin A was found at 1700 h on day 4, when the preovulatory FSH surge was observed. An increase in plasma concentrations of inhibin B occurred on day 1 and reached plateau levels at 1700 h on day 1. The levels remained elevated until 0500 h on day 4 and declined gradually by 2300 h on day 4. Plasma concentrations of inhibin pro-alphaC gradually increased with some fluctuation from day 1 to 1700 h on day 4 and then declined. Significant negative relationships were noted between plasma FSH and both dimeric forms of inhibin from day 1 to day 3. Significant positive relationships were found between plasma oestradiol-17beta and inhibin A or inhibin pro-alphaC throughout the oestrous cycle. In contrast, no significant relationship was found between plasma oestradiol-17beta and inhibin B. These findings suggest that both dimeric forms of inhibin play a role in the regulation of FSH secretion during follicular development. These findings also suggest that inhibin pro-alphaC could be secreted primarily by large follicles, and early atretic follicles could also be responsible for inhibin pro-alphaC secretion. On the other hand, the secretory pattern of dimeric inhibins might shift from inhibin B to inhibin A with follicular development.     

Temporal and hormonal regulation of inhibin protein and subunit mRNA expression by post-natal and immature rat ovaries

The contribution of specific follicle populations to dimeric inhibin production and inhibin subunit mRNA expression by the rat ovary has been investigated in two model systems, granulosa cells isolated from 25-day-old diethylstilboestrol (DES)-treated rats and post-natal rat ovaries, dispersed in culture or whole ovaries, using specific two-site immunoassays and 'real time' PCR. Media from FSH-stimulated granulosa cell cultures fractionated by gel filtration and RP-high performance liquid chromatography revealed two predominant peaks of alpha subunit activity which were attributed to alpha subunit and 31 k dimeric inhibin-A. The corresponding inhibin-B levels were low. FSH stimulation did not alter the ratio of inhibin-A:alpha subunit produced by granulosa cells. All three inhibin subunit mRNAs were expressed by granulosa cells, with eight-fold more alpha subunit mRNA relative to either of the beta subunits. Administration of DES to immature rats prior to the isolation of granulosa cells from the ovary led to beta(A) and beta(B) mRNA expression being down-regulated in the absence of any significant change in alpha subunit expression by the granulosa cells. Inhibin-A, -B and -alpha subunit were produced by basal and stimulated cultures of ovarian cells prepared from 4-, 8- and 12-day-old rats, indicating that primary, preantral and antral follicles contribute to total inhibin production. Consistent with these results, follicles within these ovaries expressed all three inhibin subunit mRNAs, with maximal expression observed in the ovaries of 8-day-old rats. The appearance of antral follicles in the ovary at day 12 led to a decline in the mRNA levels of each of the subunits but was most evident for the beta subunits. There was a profound influence of secondary preantral follicles on dimeric inhibin-A production, with FSH stimulation increasing inhibin-A relative to alpha subunit levels in cultures of ovarian cells prepared from 8-day-old rats. Thus, preantral follicles exposed to FSH contribute significantly to beta(A) subunit production by the ovary. In contrast, primary and preantral follicles did not produce inhibin-B in response to FSH stimulation. Transforming growth factor-beta (TGF-beta) enhanced, in a time-dependent manner, the production of the inhibin forms by ovarian cells in culture, although inhibin-B production was not responsive until day 8. The simultaneous treatment of ovarian cell cultures with FSH and TGF-beta elicited the greatest increases in production of all the inhibin forms. In summary, ovaries of 4-, 8- and 12-day-old rats expressed inhibin subunit mRNAs and produced dimeric inhibin-A and -B and free alpha subunit. Preantral follicles (day-8 ovarian cell cultures) were particularly sensitive to stimulation by FSH and TGF-beta and had a substantial capacity for inhibin production. The production of oestrogen by follicles may be instrumental in regulating inhibin production given that beta subunit mRNA expression was down-regulated by DES. The mechanisms by which inhibin-A and inhibin-B are individually regulated are likely to be similar during the post-natal period, when folliculogenesis is being established, and diverge thereafter, when inhibin-A becomes the predominant form in the fully differentiated ovary.     

Effects of photoperiod and temperature on the binding of follicle-stimulating hormone (FSH) to testicular preparations and plasma FSH concentration in the Djungarian hamster, Phodopus sungorus

The effects of artificial photoperiod and temperature on testicular FSH binding and plasma FSH levels were studied in adult male Djungarian hamsters. In Exp I, hamsters were transferred to long day (LD) photoperiods (16-h light, 8-h dark) after 8 weeks of adaptation in short day (SD) photoperiods (8-h light, 16-h dark), but the ambient temperature was maintained at 25 C throughout the experiments. A marked increase in the total FSH binding per two testes occurred between 10 and 47 days after transfer to LD, a change that was accompanied by testicular growth. Binding of FSH per testicular weight decreased during the same period. Scatchard plot analyses of the parameters indicated that LD decreased both the concentration of FSH binding sites and the equilibrium dissociation constant (Kd). Plasma FSH levels increased between 10 and 47 days after transfer to LD. In contrast, when hamsters reared under LD for 12 weeks were transferred to SD (Exp II), FSH binding per unit weight basis increased 19 weeks after transfer to SD, but the total binding per two testes decreased markedly. In Exp III, sexually mature male hamsters were subjected to different ambient temperature and photoperiods. There were no significant effects of different ambient temperatures on the testicular weight and testicular FSH binding in animals exposed for 8 weeks to either LD or SD. However, plasma FSH levels of hamsters maintained at 25 C under LD was significantly higher than FSH levels at 7 C under LD. A similar effect of temperature on plasma FSH levels was observed in hamsters under SD. The present study indicates that photoperiod is a more important environmental factor than temperature for the regulation of FSH receptor in the Djungarian hamster.     

Decreased inhibin gene expression in preovulatory follicles requires primary gonadotropin surges

We have examined the role of the primary gonadotropin surges in regulating inhibin alpha- and beta A-subunit mRNA levels in rat ovarian follicles. Inhibin subunit mRNA levels decline dramatically on the evening of proestrus in follicles of the ovulatory pool. Because this decline is temporally associated with primary gonadotropin surges, we investigated the contribution of LH and FSH to this process. The primary gonadotropin surges were blocked by injection of a GnRH antagonist (WY45760) at 1200 h on proestrus. This resulted in sustained elevation of inhibin mRNA levels through 0700 h of the subsequent day, a time when inhibin mRNA levels would normally be very low. Replacement of either exogenous LH or FSH in ovulatory doses to an antagonist-treated animal at 1530 h on proestrus resulted in a decrease in inhibin mRNA levels by 4-5 h postreplacement. We conclude that LH and FSH act via a common mechanism to repress inhibin mRNA levels in stimulated preovulatory follicles.     

Photoperiod-dependent regulation of inhibin in Siberian hamsters: II. Regulation of inhibin production and secretion by pregnant mare serum gonadotropin

Inhibin production differs in ovaries of Siberian hamsters (Phodopus sungorus) exposed to long days (LD) or short days (SD). We believe that seasonal differences in serum follicle-stimulating hormone contribute to this difference. However, given the profound photoperiodic differences in follicle maturation, serum gonadotropins alone may not account for all of the observed differences in inhibin processing. To test this hypothesis, we challenged LD and SD female hamsters with exogenous gonadotropins. While both groups responded with increased inhibin expression, the effects were muted in ovaries of SD females and there was no evidence of ovulation in these animals. These data indicate that the ovaries of SD females are not immediately equipped to respond to gonadotropin stimulation. More generally, these data suggest that photoperiodic history affects ovarian inhibin production and secretion in response to gonadotropins.     

Enhanced serum oestrogen levels and highly steroidogenic,luteinized atretic follicles in the ovaries of the Djungarian hamster (Phodopus sungorus) kept under a short photoperiod from birth

OBJECTIVE: In contrast to the elaborate information available on the effects of the photoperiod on the testes of hamsters, little is known about the influence on their ovaries. This study aimed to describe the ovarian follicular development and steroid hormone production in Djungarian hamsters kept from birth under a short daylight regime. DESIGN AND METHODS: Female Djungarian hamsters (Phodopus sungorus) were kept under two different light regimes: (i) 16 h light:8 h darkness (long daylight; LD) and (ii) 4 h light:20 h darkness (short daylight; SD). They were killed at 28, 56 and 80 days after birth; blood and ovaries were collected. Ovaries were either fixed in Bouin's solution or frozen. Fixed material was dehydrated, embedded in paraffin, serially sectioned at 5 microm and stained with haematoxylin and eosin, whereafter all healthy and atretic follicles were classified and counted. 3beta-Hydroxysteroid dehydrogenase (3beta-HSD) was histochemically demonstrated in 10 microm sections of frozen ovaries. Serum oestradiol-17beta and progesterone levels were determined by RIA. RESULTS: The numbers of healthy preantral and antral follicles were higher in LD than in SD hamsters. Antral follicles did not significantly differ in number during development in LD hamsters, but they were completely absent from 80-day-old SD animals. In LD animals the number of apoptotic preantral follicles dramatically increased with age. In SD animals the numbers of apoptotic antral follicles strongly decreased with age, whereas numerous non-apoptotic follicles with luteinized granulosa cells and a degenerated oocyte appeared, and in increasing numbers with age. During development, moderate 3beta-HSD activity was present in interstitial cells, theca cells of healthy follicles, and in both theca and granulosa cells of degenerating follicles. Strong enzyme activity was found in the hypertrophied granulosa cells of luteinized atretic follicles. Mean serum progesterone values varied from 2 to 6 nmol/l and were not different in LD and SD hamsters. Mean serum oestradiol levels varied from 132 to 542 and 325 to 2353 pmol/l in LD and SD hamsters respectively. The highest oestradiol levels were found in SD animals at day 28 of development. CONCLUSIONS: Folliculogenesis was dramatically disturbed in Djungarian hamsters raised under a short photoperiod. These animals developed high serum oestradiol levels and numerous luteinized atretic follicles with highly steroidogenic granulosa cells, which appear to be the source of the increased serum oestradiol levels.     

Unilateral ovariectomy increases inhibin messenger ribonucleic acid levels in newly recruited follicles

We have used the unilaterally ovariectomized (ULO) rat as an experimental model to assess transient relationships between follicular recruitment and inhibin mRNA levels in the ovary. Animals were divided into three experimental groups (ULO, sham-operated, and intact) and killed 0, 9, or 24 h postsurgery. Inhibin alpha- and beta A mRNA were measured within individual ovarian follicles by in situ hybridization. Serum FSH concentrations in ULO animals increased significantly at 9 h and returned to presurgery levels by 24 h. There was no effect of surgery on serum LH concentrations. In the ULO animals, compensatory follicular recruitment was observed in the remaining ovary at 24 h as a significant increase in the number of follicles with mean diameter greater than 350 micron. Two distinct populations of healthy maturing follicles, based on diameter and hybridization intensity, were observed in ovaries from 24 h post-ULO rats. The first class, NRF-1 (newly recruited follicle-1), were those recruited 48 h previously by the secondary FSH surge of the preceding cycle. These large follicles (greater than 500 micron mean diameter) hybridized to both inhibin probes. The second class of follicles (NRF-2), which represent those newly recruited by the ULO-induced increase in serum FSH, were smaller in size than NRF-1 (350-500 micron mean diameter). The intensity of hybridization of both inhibin probes to NRF-1 was higher than that to NRF-2 in the majority of follicles. Each NRF-1 contained higher levels of both alpha- and beta A-inhibin mRNA than nonrecruited follicles (150-350 micron diameter). These data suggest that subtle changes in serum FSH concentrations, which are sufficient to induce follicular recruitment into a size class capable of ovulating within a given cycle, play the additional role of enhancing inhibin mRNA levels in developing follicles.     

An essential role for ovarian inhibin in pineal gland-mediated anestrus in Syrian hamsters

ABSTRACT: When transferred from long photoperiod (LP) to short photoperiod (SP), female Syrian hamsters exhibit depressions of follicle stimulating hormone (FSH) and follicular development, cessation of ovulation, and marked ovarian interstitial tissue hyperplasia. Pinealectomy prevents these effects of SP. The object of this study was to determine the role of inhibin in the regulation of FSH during SP-induced anestrus. Adult LSH/SsLak hamsters maintained in LP (LD 14: 10 hr) were transferred to SP (LD 8: 16 hr) on the day of estrus, and groups of animals killed at either 16.00 hr on proestrus or 08.00 hr on estrus during each of five consecutive 4-day estrus cycles after transfer. Groups of females that became anestrus in SP were killed either at 08.00 or 16.00 hr, 12 days after the last observed estrus discharge. Compared to LP controls, serum FSH levels on estrus increased significantly ( P <0.01) during the first two cycles in SP before declining to concentrations that were significantly lower than control values ( P <0.01). Serum inhibin levels increased significantly by the third, fourth, and five days of estrus in SP. Regression analysis revealed a significant inverse correlation between serum inhibin and FSH levels on estrus during SP exposure (F=0.021) but not on proestrus. Relative levels of inhibin a- and_βA-subunit mRNAs were lower in ovaries from SP proestrus and anestrus females killed at 16.00 hr as compared to those from proestrus LP controls; they were elevated in ovaries from SP estrus and anestrus females killed at 08.00 hr compared to those from estrus LP controls. The absence of antral follicles on estrus in the last cycles of SP and anestrus suggests that the increase in circulating inhibin and inhibin mRNAs may be derived from hyperplastic interstitium. These observations suggest that inhibin may play an essential role in suppressing FSH secretion during pineal gland-mediated anestrus in Syrian hamsters.     

The effect of testosterone, dihydrotestosterone and oestradiol on the re-initiation of spermatogenesis in the adult photoinhibited Djungarian hamster

The roles of testosterone (T) and its metabolites on hamster spermatogenesis are poorly defined. This study assessed the effects of T, dihydrotestosterone (DHT) and oestradiol (E) on the re-initiation of spermatogenesis in the adult Djungarian hamster. Hamsters raised under long photoperiods (LD, 16 h light:8 h darkness) were exposed to short photoperiods (SD, 8 h light:16 h darkness) for 11 weeks to suppress gonadotrophins. Groups of eight animals then received T, DHT and E for 5 weeks. Cell numbers were determined using the optical disector (sic). The number of Sertoli cells was suppressed in SD controls to 48% (P < 0.001) of LD control and restored either fully or partially by exogenous DHTand E (2.6- and 1.8-fold above SD levels) respectively, corresponding with a twofold elevation of serum FSH. The number of germ cells in SD animals was reduced (all P < 0.001) to levels reported. The number of type A spermatogonia increased in line with the rise in Sertoli cell number, by 2.6-fold (P < 0.01) and 1.8-fold (NS) above SD controls after DHT and E treatments respectively. DHT increased the number of type B spermatogonia/preleptotene spermatocytes, leptotene/zygotene and pachytene spermatocytes by 3.5-, 5.7- and 21-fold above SD (all P < 0.01) respectively, compared with a 2.2-fold (P < 0.01), 2.4-fold (not significant, NS) and 6-fold (NS) in E-treated animals respectively. Exogenous T had little effect on cell numbers or serum FSH compared with SD controls. Spermatids were rarely observed after steroid treatment. We believe this study suggests that steroids can regulate the re-initiation of early spermatogenic cells via a mechanism which includes FSH.     

Activation of the bone morphogenetic protein signaling pathway induces inhibin beta(B)-subunit mRNA and secreted inhibin B levels in cultured human granulosa-luteal cells

During the human menstrual cycle the circulating levels of inhibin B, a dimer of inhibin alpha- and beta(B)-subunits, fluctuate in a fashion distinct from that of inhibin A, the alpha-beta(A)-subunit dimer. This suggests that human inhibin subunits are each regulated in a distinct manner in human ovarian granulosa cells by endocrine and local factors. We have previously shown using cultures of human granulosa-luteal (hGL) cells that gonadotropins stimulate the steady state mRNA levels of inhibin alpha- and beta(A)-subunits, but not those of the beta(B)-subunit, which, on the other hand, are up-regulated by, for instance, activin and TGF beta. We recently identified the TGF beta gene family member bone morphogenetic protein-3 (BMP-3) as a granulosa cell-derived growth factor, but whether BMP-3 or other structurally related BMPs regulate human granulosa cell inhibin production is not known. We show here that hGL cells express mRNAs for distinct serine/threonine kinase receptors (BMP-RIA and BMP-RII) and Smad signaling proteins (Smad1, Smad4, and Smad5) involved in the mediation of cellular effects of BMPs. Subsequently, we determined in hGL cell cultures the effects of distinct members of the BMP family previously found to be expressed in mammalian ovaries. Recombinant BMP-2 induces potently in a time- and concentration-dependent manner the expression of the inhibin beta(B)-subunit mRNAs in hGL cells without affecting the levels of alpha- or beta(A)-subunit mRNAs. BMP-6 has a similar, but weaker, effect than BMP-2, whereas BMP-3 and its close homolog, BMP-3b (also known as growth differentiation factor-10) had no effect on inhibin subunit mRNA expression. hCG treatment of hGL cells was previously shown to abolish the stimulatory effect of activin on beta(B)-subunit mRNA levels, and here hCG is also shown to suppress the effect of BMP-2. Furthermore, BMP-2 stimulates hGL cell secreted dimeric inhibin B levels in a concentration-dependent manner. Depending on the experiment, maximal increases in inhibin B levels of 6- to 28-fold above basal levels were detected during a 72-h culture period. We conclude that activation of the BMP-signaling pathway in hGL cells stimulates inhibin beta(B)-subunit mRNA levels and leads at the protein level to a dramatic stimulation of secreted inhibin B dimers. Our results are consistent with the suggestion that in addition to the distinct activin- and TGF beta-activated signaling pathways, the BMP-activated pathway is likely to be implicated in the complex regulation of inhibins in the human ovary.     

Possible role of luteinizing hormone and follicle-stimulating hormone in modulating inhibin secretion and expression during the estrous cycle of the rat

In the female rat, plasma immunoreactive inhibin alpha (irl alpha) levels show marked changes during proestrus and estrus. We investigated the modulating effect of LH and FSH on these changes by injecting the GnRH antagonist DNal-DCpa-DPal-Dpr-(Ac)Dal-Leu-Arg-Pro-Asn-NH2, with or without exogenous LH replacement. Administration of the antagonist at noon on proestrus abolished the primary (proestrus) LH and FSH surge and markedly reduced the secondary (estrus) FSH surge. This treatment also reduced the release of irl alpha normally measured during proestrus afternoon, and partially prevented the decrease in irI alpha secretion on proestrus evening. Exogenous LH injected at 1545 h on proestrus had no measurable effect on irI alpha or FSH levels in control rats; however, in antagonist-treated animals, it restored the secondary FSH surge to control values while augmenting the late proestrus fall in irI alpha. This suggests that the decrease in inhibin secretion measured after exogenous LH treatment represents the mechanism through which LH induced the secondary FSH surge in antagonist-blocked rats. We also used in situ hybridization techniques to examine the changes in the expression of inhibin subunits in the ovary at 0200 h on estrus. The antagonist reduced expression of the alpha-, beta A-, and beta B-subunits in all follicle and tissue types, with the exception of the granulosa cells of large tertiary (possibly preovulatory) follicles where the signal appeared greatly enhanced. These changes were reversed by LH. The alteration in inhibin subunit messages caused by blockade of the primary gonadotropin surge suggests the presence of a cross-regulation between LH and inhibin/activin secretion, so that a decline in circulating LH levels might stimulate inhibin/activin secretion in the granulosa cells of preovulatory follicles, while reducing the production of these proteins in less mature follicles and in other ovarian cell types.     

Serum levels of gonadotrophins in hamsters exposed to short photoperiods: effects of adrenalectomy and ovariectomy

Female hamsters rendered acyclic by exposure to short photoperiods (10 h light: 14 h darkness) showed diurnal surges of both LH and FSH which persisted after removal of the ovaries and adrenal glands. The reduced increase in gonadotrophin secretion after ovariectomy, typical of hamsters exposed to short days, was also not contingent upon the presence of the adrenal glands. The results demonstrate that 'supersensitivity' of the neuroendocrine axis to feedback of ovarian or adrenal steroids cannot fully account for either the daily surges of gonadotrophins or the diminished response of LH and FSH to ovariectomy in hamsters maintained on short days. It is suggested that these changes result instead from other pineal-mediated actions of short photoperiods upon the brain.     

Pachytene spermatocytes in co-culture inhibit rat Sertoli cell synthesis of inhibin beta B-subunit and inhibin B but not the inhibin alpha-subunit

This study investigates the effects of spermatogenic germ cells on inhibin alpha-subunit and beta B-subunit expression, and inhibin alpha-subunit and inhibin B production by rat Sertoli cells in vitro. Sertoli cells isolated from 19-day-old rats were cultured for 48 h at 32 degrees C, in the presence or absence of FSH (2.3-2350 mIU/ml), and in the presence of pachytene spermatocytes, round spermatids or cytoplasts of elongated spermatids purified from adult rat testis by elutriation and density gradient separation. Sertoli cell secretion of inhibin alpha-subunit and inhibin B, as measured by immunoassay, was dose-dependently stimulated by FSH (maximal stimulation 13- and 2-fold, respectively). Round spermatids or cytoplasts co-cultured with Sertoli cells had no effect on basal or FSH-induced secretion of inhibin alpha-subunit or inhibin B. When Sertoli cells were co-cultured with pachytene spermatocytes, inhibin alpha-subunit secretion was unaltered, while inhibin B secretion was suppressed in a cell concentration-dependent manner to reach a maximal suppression of 45% compared with Sertoli cells alone (P<0.01). A similar suppression in inhibin B was still observed (64% of Sertoli cells alone) when the pachytene spermatocytes were separated from Sertoli cells by a 0.45 microm pore membrane barrier in bicameral chambers. Pachytene spermatocytes also suppressed FSH-induced inhibin B levels in Sertoli cell co-cultures and this suppression was attributed to a decrease in basal inhibin B production rather than a change in FSH responsiveness. Quantitation of Sertoli cell inhibin alpha- and beta B-subunit mRNA by quantitative (real-time) PCR demonstrated that pachytene spermatocytes did not alter Sertoli cell alpha-subunit mRNA expression, but significantly (P<0.01) suppressed basal and FSH-induced beta B-subunit mRNA expression to a similar degree to that seen with inhibin B protein levels. It is concluded that pachytene spermatocytes in vitro suppress Sertoli cell inhibin B secretion via factor-mediated suppression of inhibin beta B-subunit expression. These findings support the hypothesis that specific germ cell types can influence inhibin B secretion by the testis independent of FSH regulation.     

Differential expression of matrix metalloproteinases during stimulated ovarian recrudescence in Siberian hamsters (Phodopus sungorus)

The matrix metalloproteinases (MMPs) are a family of extracellular matrix-cleaving enzymes involved in ovarian remodeling. In many non-tropical species, including Siberian hamsters, ovarian remodeling is necessary for the functional changes associated with seasonal reproduction. We evaluated MMPs and their endogenous inhibitors (TIMPs), during photoperiod-induced ovarian recrudescence in Siberian hamsters. Hamsters were transferred from long day (LD; 16:8) to short day (SD; 8:16) photoperiods for 14weeks, and then returned to LD for 0, 1, 2, 4, or 8weeks for collection of ovaries and plasma. Post-transfer (PT) LD exposure increased body and ovarian mass. Number of corpora lutea and antral, but not preantral follicles increased in PT groups. Plasma estradiol concentrations were lower in PT weeks 0-4, and returned to LD levels at PT week 8. No change was observed in relative MMP/TIMP mRNA levels at PT week 0 (SD week 14) as compared to LD. Photostimulation increased MMP-2 mRNA at PT week 8 as compared to PT weeks 0-1. MMP-14 mRNA expression peaked at PT weeks 1-2 as compared to LD levels, while MMP-13 expression was low during this time. TIMP-1 mRNA peaked at PT week 8 as compared to PT weeks 0-4. No changes were noted in MMP-9 and TIMP-2 mRNA expression. In general, MMP/TIMP protein immunodetection followed the same patterns with most staining occurring in granulosa cells of follicles and corpora lutea. Our data suggest that mRNA and protein for several members of the MMP/TIMP families are expressed in Siberian hamster ovaries during recrudescence. Because of the variation observed in expression patterns, MMPs and TIMPs may be differentially involved with photostimulated return to ovarian function.     

Relationship between serum inhibin A and B and ovarian follicle development after a daily fixed dose administration of recombinant follicle-stimulating hormone

The aim of this study was to investigate the relationship of serum inhibin A and inhibin B to ovarian follicular development in women undergoing pituitary down-regulation and ovarian stimulation with a fixed daily dose of recombinant human FSH in an in vitro fertilization program. Thirty-eight patients were treated randomly with either 100 or 200 IU/day recombinant human FSH (Puregon) for a period of 9-14 days. Serum FSH, inhibin A, inhibin B, 17beta-estradiol, and follicular size and number were determined before FSH treatment and every second day from days 4-6 throughout FSH treatment. Serum FSH increased in a dose-related manner to reach a maximum by days 4-6 and remained unchanged over the duration of treatment. Serum inhibin A and 17beta-estradiol also increased with increasing FSH dose and continued to rise throughout the FSH treatment period. By contrast, serum inhibin B was increased by days 4-6 at both doses of FSH to reach a maximum by days 7-8, remaining unchanged thereafter. Serum inhibin B and, to a lesser extent, inhibin A correlated significantly with the number of oocytes retrieved even when assessed early (days 4-6) in the treatment period (inhibin B vs. number of oocytes: r = 0.89; P < 0.001; inhibin A vs. number of oocytes: r = 0.61; P < 0.05). Serum inhibin A, inhibin B, and 17beta-estradiol were weakly correlated with the number of follicles less than 11 mm when assessed on a daily basis; stronger correlations were observed with the greater than 11-mm follicles during the late stages of treatment. It is concluded that serum inhibin B levels determined during the early stages (e.g. days 4-6) of fixed dose FSH treatment provide an early indicator of the number of recruited follicles that are destined to form mature oocytes. In this context, serum inhibin B may be of predictive value in monitoring ovarian hyperstimulation treatment for in vitro fertilization.     

Secretory pattern of inhibin A, inhibin B and inhibin pro-alpha C during induced follicular atresia and subsequent follicular development in the golden hamster (Mesocricetus auratus)

The changes in plasma concentrations of inhibins A, B and pro-alpha C were determined in the cyclic golden hamster during follicular atresia induced with antiserum against luteinizing hormone releasing hormone (LHRH-AS) at 1100 h on day 4 (day 1=day of ovulation). Follicular status in the ovary was also studied by determining the number of follicles ovulating in response to human chorionic gonadotrophin (hCG) injection. The time-courses of changes in plasma concentrations of inhibins A, B and pro-alpha C were different from each other during induced follicular atresia and subsequent follicular development. Plasma concentrations of inhibin A decreased to 58.6% of initial values by 24 h after LHRH-AS treatment, and then remained relatively low until at least 60 h later. Plasma concentrations of inhibin B decreased to 64.2% of the initial values by 18 h after LHRH-AS treatment and remained at basal values for 36 h, but increased abruptly to greater than initial values at 42 h after the treatment. Plasma concentrations of inhibin pro-alpha C increased at 6 and 12 h, decreased suddenly to 21.9% of the initial values by 24 h after LHRH-AS treatment, and then gradually increased until 60 h after LHRH-AS. The number of follicles responding to hCG decreased gradually between 0 and 30 h after LHRH-AS, when no ovulations were observed, and then gradually increased until 60 h. The changes in follicular ovulatory responses to hCG correlated with the plasma profile of inhibin A throughout the experiment. These results suggest that inhibin A is mainly secreted by large antral follicles. In contrast, during the subsequent follicular development, the plasma concentration of inhibin B increased earlier than that of inhibin A. These results suggest that inhibin B is secreted by small and large antral follicles. Plasma concentrations of inhibin pro-alpha C were high at a time when plasma concentrations of oestradiol-17 beta had already decreased, indicating that inhibin pro-alpha C is secreted not only from healthy follicles but also from early atretic antral follicles.     

Expression of ovarian inhibin during pregnancy in the rat

We have examined the expression of the rat inhibin genes in the maternal ovary during pregnancy. RNA blot analysis indicates that the inhibin-alpha chain mRNA is expressed in the ovary throughout gestation at levels comparable to those observed in cycling rats. In situ hybridization shows that the inhibin-alpha and -beta A mRNAs are produced in the granulosa cells of developing antral follicles; little or no hybridization to functional corpora lutea is observed. Early in pregnancy, a large number of follicles hybridize to both alpha- and beta A-inhibin cDNA probes. Many of these follicles undergo atresia during the first half of pregnancy, and the number of inhibin-expressing follicles reaches a nadir on day 15. This is followed by an increase in inhibin-producing follicles, which peaks just before parturition. The increase in inhibin-expressing follicles observed in late pregnancy corresponds to a small rise in serum inhibin levels, as measured using an alpha chain-specific RIA. After the first postpartum ovulation, few hybridizing follicles are observed. Ovariectomy in either early (day 6) or mid (day 15) pregnancy results in a significant fall in serum inhibin levels and a robust increase in serum FSH levels 9 h after surgery. These results suggest that inhibin is produced by the maternal ovary during pregnancy, that its synthesis is modulated during late gestation, and that inhibin may play a role in regulating FSH secretion during pregnancy.     

Relationship between inhibin A and B, estradiol and follicle growth dynamics during ovarian stimulation in normo-ovulatory women

OBJECTIVE: To investigate the relationship between serum concentrations of inhibin A, inhibin B and estradiol (E(2)) and the number of developing follicles during the administration of exogenous follicle-stimulating hormone (FSH) in various regimens in normo-ovulatory volunteers and to evaluate if inhibins act as suitable markers for the number of developing follicles during ovarian stimulation. DESIGN AND METHODS: Serial hormone determinations and assessment of follicle numbers were carried out during unstimulated cycles and during various interventions with exogenous FSH. Subjects were randomized for FSH administration into the following groups: a single high dose (375 IU) during the early follicular phase (group A), 5 consecutive low doses (75 IU/day) starting in the mid follicular phase (group B) or daily low doses (75 IU/day) during the early to late follicular phase (starting on cycle days 3, 5 or 7; groups C, D and E respectively). RESULTS: Extending the FSH window increases the number of small antral follicles and hence inhibin B serum concentrations. If such an intervention results in multi-follicular growth, mid follicular phase inhibin B (P = 0.001) as well as late follicular phase inhibin B and inhibin A levels are significantly (P < 0.05 and P < 0.01 respectively) increased compared with mono-follicular cycles or the natural cycle. Although mid follicular inhibin B levels correlated well with the number of small antral (P < 0.05) and pre-ovulatory (P < 0.001) follicles in the late follicular phase, mid follicular inhibin A and estradiol serum concentrations only correlated with the number of pre-ovulatory follicles (P < 0.001 and P < 0.01 respectively). CONCLUSIONS: The present data extend our understanding of the relationship between follicle dynamics, serum inhibins and FSH during ovarian hyperstimulation. However, although mid follicular inhibin B does correlate with the number of developing follicles, it does not facilitate the identification of women at risk for multiple follicle development.     

Differential regulation of inhibin B and inhibin a by follicle-stimulating hormone and local growth factors in human granulosa cells from small antral follicles

Serum inhibin B rises across the luteal-follicular transition, whereas inhibin A does not increase until the late follicular phase of the menstrual cycle. To test the hypothesis that inhibin B is secreted from preantral and small antral follicles and that FSH and local growth factors differentially regulate inhibin B and inhibin A from these developing follicles, human ovaries were obtained after oophorectomy. Basal secretion of inhibin B and inhibin A was examined in intact preantral follicles in culture (n = 6). Basal secretion and regulation of inhibin B and inhibin A secretion by gonadotropins, androstenedione, activin A, insulin, and IGF-I were examined in cultured granulosa cells from small antral follicles (n = 21). Inhibin B secretion from preantral follicle cultures was detectable at baseline (range, 17-96 pg/mL), whereas inhibin A was not detectable. In contrast, both inhibin B and inhibin A were detectable in granulosa cell cultures from small antral follicles. In granulosa cells from small antral follicles, FSH (30 ng/mL) stimulated inhibin A 3-fold (10.5 +/- 2.2 to 32.5 +/- 8.3 IU/mL; P < 0.001), but not inhibin B secretion (1730 +/- 354 to 2314 +/- 532 pg/mL; P = NS). Likewise, cAMP (1 mmol/L) stimulated inhibin A 4-fold (16.6 +/- 4.3 to 62.5 +/- 21.9 IU/mL; P < 0.002), but not inhibin B secretion (2327 +/- 546 to 1877 +/- 377 pg/mL; P = NS). hCG (30 ng/mL) did not stimulate inhibin A or inhibin B. Androstenedione (10(-)(7) mol/L), activin (30 ng/mL), insulin (30 ng/mL), and insulin-like growth factor I (IGF-I; 100 ng/mL) alone did not stimulate inhibin A or inhibin B secretion. Further, FSH-stimulated inhibin A secretion was not augmented by androstenedione, activin, insulin, or IGF-I. In contrast, the combination of IGF-I and FSH was the only treatment that stimulated inhibin B secretion (1742 +/- 380 to 2881 +/- 731 pg/mL; P < 0.03). However, FSH in combination with IGF-I resulted in greater stimulation of inhibin A (340%) than inhibin B (65%). These findings demonstrate that inhibin B is secreted from developing preantral and small antral follicles, but is not directly stimulated by FSH. However, the combination of FSH and IGF-I enhanced inhibin B secretion. In contrast, inhibin A is not secreted from preantral follicles, but in small antral follicles FSH and cAMP stimulate inhibin A secretion. Further, FSH in combination with IGF-I results in a greater degree of stimulation of inhibin A than of inhibin B. These findings suggest that FSH and IGF-I differentially regulate inhibin A and inhibin B secretion. However, additional growth factors or increasing granulosa cell number may contribute to the preferential serum inhibin B increase across the luteal-follicular transition in the menstrual cycle.     

Changes in dimeric inhibin A and B during normal early puberty in boys and girls

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.