Modulatory effects of TGF-ß1 and BMP6 on thecal angiogenesis and steroidogenesis in the bovine ovary.

Angiogenesis plays an integral role in follicular and luteal development and is positively regulated by several intra-ovarian factors including vascular endothelial growth factor A (VEGFA) and fibroblast growth factor 2 (FGF2). Various transforming growth factor-ß (TGF-ß) superfamily members function as intra-ovarian regulators of follicle and luteal function, but their potential roles in modulating ovarian angiogenesis have received little attention. In this study, we used a bovine theca interna culture model (exhibiting characteristics of luteinization) to examine the effects of TGF-ß1 and bone morphogenetic protein 6 (BMP6) on angiogenesis and steroidogenesis. VEGFA/FGF2 treatment promoted endothelial cell network formation but had little or no effect on progesterone and androstenedione secretion or expression of key steroidogenesis-related genes. TGF-ß1 suppressed basal and VEGFA/FGF2-induced endothelial cell network formation and progesterone secretion, effects that were reversed by an activin receptor-like kinase 5 (ALK5) inhibitor (SB-431542). The ALK5 inhibitor alone raised androstenedione secretion and expression of several transcripts including CYP17A1. BMP6 also suppressed endothelial cell network formation under VEGFA/FGF2-stimulated conditions and inhibited progesterone secretion and expression of several steroidogenesis-related genes under basal and VEGFA/FGF2-stimulated conditions. These effects were reversed by an ALK1/2 inhibitor (K02288). Moreover, the ALK1/2 inhibitor alone augmented endothelial network formation, progesterone secretion, androstenedione secretion and expression of several steroidogenesis-related genes. The results indicate dual suppressive actions of both TGF-ß1 and BMP6 on follicular angiogenesis and steroidogenesis. Further experiments are needed to unravel the complex interactions between TGF-ß superfamily signalling and other regulatory factors controlling ovarian angiogenesis and steroidogenesis.

Circulating insulin-like factor 3 (INSL3) in healthy and infertile women.

STUDY QUESTION: How does insulin-like factor 3 (INSL3) concentration in blood vary across the menstrual cycle in women? SUMMARY ANSWER: INSL3 is secreted by the theca interna cells of growing antral follicles and is phasic in its expression. WHAT IS KNOWN ALREADY: The relaxin-like hormone INSL3 is known to be expressed in follicles of several mammal species, and was recently shown in cows to be specifically secreted into the bloodstream by growing antral follicles, corresponding to follicular waves. In males INSL3 is known to be acutely independent of the hormones of the hypothalamic-pituitary-gonadal axis, suggesting that in women INSL3 might be a novel biomarker for antral follicle recruitment and development. STUDY DESIGN, SIZE, DURATION: Two cohorts of women were studied. First, 18 healthy women of reproductive age were followed longitudinally for one and a half cycles, with blood sampling and hormone measurement every 2-3 days. A second cohort comprised a cross-sectional study of 909 women attending an infertility clinic, with a single blood sample taken at entry, together with other clinical and hormonal parameters. PARTICIPANTS/MATERIALS, SETTING, METHODS: Blood samples from both retrospective cohorts were analyzed for INSL3 using a highly sensitive time-resolved fluorescent immunoassay, and data were analyzed in comparison with other clinical and hormonal parameters. MAIN RESULT AND THE ROLE OF CHANCE: For young healthy women of reproductive age, we showed a phasic expression of INSL3 corresponding to antral follicle growth in both the follicular and luteal phases of the cycle, which was significantly (P < 0.05) elevated compared with that during menses. For women attending an infertility clinic, those with diagnosed polycystic ovarian syndrome indicated significantly (P < 0.0005) greater circulating INSL3 levels and those with low ovarian reserve showed significantly (P < 0.002) decreased INSL3 values. LIMITATIONS, REASONS FOR CAUTION: These were retrospective studies and the results were obtained from natural cycles only, with their inherent variability. WIDER IMPLICATIONS OF THE FINDINGS: We show for the first time that INSL3 in women does vary across the menstrual cycle, and appears to reflect the number of growing antral follicles recruited within both follicular and luteal phases. STUDY FUNDING/COMPETING INTEREST(S): The present retrospective study was largely supported by departmental funds. There were no competing interests.

The effect of exogenous gonadotropins on ovarian function in goats actively immunized against inhibin.

The aim of this investigation was to compare the ovarian response to superovulatory treatments in does before and after inhibin immunization, with a view to optimizing the superovulatory potential of the caprine ovary. To avoid interference by the ovarian cycle, the experiment was conducted out-of-season. At the onset of the experiment 48 does were subjected to treatment with an sc implant of the progestogen norgestomet, combined with a gonadotropin; eight does each received a single injection of 1200 IU eCG, 400 IU eCG or 2 mL physiological saline (control) or six injections (at 12 h intervals) constituting 16 or 5.4 AU pFSH. The does were mated and subjected to embryo collection 6 to 7 d later. Throughout the experiment ovarian function (by ultrasonography) and plasma levels of inhibin antibodies and progesterone were monitored. Of 40 does treated during the first part of the experiment, 48% showed estrus. The ovarian response in does treated with a high or low dose of eCG or a low dose of pFSH was barely in excess of the ovarian response in the saline-treated controls, whereas a superovulatory dose of pFSH (16 AU) gave a satisfactory response of, on average, 14.5 ovulations (yielding 8.8 flushed ova and embryos). Immediately after the does had been subjected to embryo collection they were actively immunized against inhibin by administering two injections of a recombinant α-subunit of ovine inhibin at four week intervals. All immunized does produced antibodies with the maximal titer reached two weeks after the second injection. Groups of immunized does were subjected to the same gonadotropin treatments as before (avoiding allocation of individuals to the same treatments). This time all does showed estrous symptoms. The ovulatory response to the various treatments, including the saline controls, was virtually identical, the overall average being 21.8 follicles and 9.1 ovulations. The average embryo yield per doe was 5.7. The results imply that inhibin acted as the key factor in determining the ovulatory response since no impact of any of the supplementary gonadotropins was noted in inhibin-immunized does. This finding gives rise to the notion that inhibin antibodies may act primarily by an intraovarian paracrine action rather than by reducing the suppressive action of inhibin on pituitary FSH release. Further, these findings confirm earlier reports that eCG is less suitable than FSH for inducing superovulation in goats, and indicate that active immunization against inhibin may be considered a viable alternative to using exogenous gonadotropin for inducing superovulation in goats.

Variation in the ovarian reserve is linked to alterations in intrafollicular estradiol production and ovarian biomarkers of follicular differentiation and oocyte quality in cattle.

The mechanisms whereby the high variation in numbers of morphologically healthy oocytes and follicles in ovaries (ovarian reserve) may have an impact onovarian function, oocyte quality, and fertility are poorly understood. The objective was to determine whether previously validated biomarkers for follicular differentiation and function, as well as oocyte quality differed between cattle with low versus a high antral follicle count (AFC). Ovaries were removed (n = 5 per group) near the beginning of the nonovulatory follicular wave, before follicles could be identified via ultrasonography as being dominant, from heifers with high versus a low AFC. The F1, F2, and F3 follicles were dissected and diameters determined. Follicular fluid and thecal, granulosal, and cumulus cells and the oocyte were isolated and subjected to biomarker analyses. Although the size and numerous biomarkers of differentiation, such as mRNAs for the gonadotropin receptors, were similar, intrafollicular concentrations of estradiol and the abundance of mRNAs for CYP19A1 in granulosal cells and ESR1, ESR2, and CTSB in cumulus cells were greater, whereas mRNAs for AMH in granulosal cells and TBC1D1 in thecal cells were lower for animals with low versus a high AFC during follicle waves. Hence, variation in the ovarian reserve may have an impact on follicular function and oocyte quality via alterations in intrafollicular estradiol production and expression of key genes involved in follicle-stimulating hormone action (AMH) and estradiol (CYP19A1) production by granulosal cells, function and survival of thecal cells (TBC1D1), responsiveness of cumulus cells to estradiol (ESR1, ESR2), and cumulus cell determinants of oocyte quality (CTSB).

The long-term effect of active immunization against inhibin in goats.

This experiment addresses the long-term effect of active immunization of goats against a recombinant ovine inhibin alpha subunit (roIHN-alpha). In late anestrus 100microg of roINH-alpha was administered to 40 pluriparous Boer goat does, followed, 4 weeks later, by a booster injection. Weekly blood samples were drawn to monitor the inhibin binding capacity with the aid of a radio-tracer binding assay. From the onset until 48h after the end of each estrus, follicular development and ovulation rate were monitored at 24h intervals by transrectal ultrasonography. Beginning in August and continuing into January, does were mated at every other estrus, and submitted to transcervical embryo collection. Seven months after the first immunization, the does were mated again and permitted to carry to term. All immunized does produced inhibin antibodies, an elevated titre being first detected 2 weeks after primary immunization. Maximum titres were reached after 6 weeks, i.e. 2 weeks after the booster injection. Thereafter, in the course of the following 32 weeks, the titre subsided gradually. The does started cycling by mid-August. At that stage the average number of follicles more than 4mm in diameter, ovulations and total embryos and ova recovered were 14.7 (+/-2.3), 5.3 (+/-0.7) and 4.4 (+/-1.0), respectively. A steady decline followed and in January the corresponding means were: 5.2 (+/-0.6) follicles, 3.1 (+/-0.6) ovulations and 1.2 (+/-0.4) embryos and ova recovered. When mated toward the end of the breeding season, 85% of the does became pregnant to the first mating and 73% went to term. Healthy kids were born, the average litter size being 2.2 (+/-0.1). In conclusion, immunization of goats against a recombinant inhibin alpha-subunit proved to be a practicable means of producing embryos for transfer purposes. After about half a year, when the inhibin antibody titre has subsided, it is possible to return the does to the breeding flock without risking complications with normal breeding activity.

Developmental programming: deficits in reproductive hormone dynamics and ovulatory outcomes in prenatal, testosterone-treated sheep.

Prenatal testosterone excess leads to neuroendocrine, ovarian, and metabolic disruptions, culminating in reproductive phenotypes mimicking that of women with polycystic ovary syndrome (PCOS). The objective of this study was to determine the consequences of prenatal testosterone treatment on periovulatory hormonal dynamics and ovulatory outcomes. To generate prenatal testosterone-treated females, pregnant sheep were injected intramuscularly (days 30-90 of gestation, term=147 days) with 100 mg of testosterone-propionate in cottonseed oil semi-weekly. Female offspring born to untreated control females and prenatal testosterone-treated females were then studied during their first two breeding seasons. Sheep were given two injections of prostaglandin F2alpha 11 days apart, and blood samples were collected at 2-h intervals for 120 h, 10-min intervals for 8 h during the luteal phase (first breeding season only), and daily for an additional 15 days to characterize changes in reproductive hormonal dynamics. During the first breeding season, prenatal testosterone-treated females manifested disruptions in the timing and magnitude of primary gonadotropin surges, luteal defects, and reduced responsiveness to progesterone negative feedback. Disruptions in the periovulatory sequence of events during the second breeding season included: 1) delayed but increased preovulatory estradiol rise, 2) delayed and severely reduced primary gonadotropin surge in prenatal testosterone-treated females having an LH surge, 3) tendency for an amplified secondary FSH surge and a shift in the relative balance of FSH regulatory proteins, and 4) luteal responses that ranged from normal to anovulatory. These outcomes are likely to be of relevance to developmental origin of infertility disorders and suggest that differences in fetal exposure or fetal susceptibility to testosterone may account for the variability in reproductive phenotypes.

Seasonal variations in the developmental competence of bovine oocytes matured in vitro.

Ovaries were collected over a period of two years from heifers slaughtered at under 30 months of age and used to harvest 1757 oocytes. After in vitro maturation, fertilisation and culture, the proportions of oocytes and cleaved embryos that developed to blastocysts were significantly higher (P<0.01) in the autumn, from September to November, than in the spring, from March to May. In contrast, embryo development, as assessed by oocytes that developed to eight or more cells and blastocysts, was lowest (P<0.01) in the spring. These results were consistent during the two-year study, indicating a seasonal fluctuation in oocyte competence.

Differential expression of mRNAs encoding the putative inhibin co-receptor (betaglycan) and activin type-I and type-II receptors in preovulatory and prehierarchical follicles of the laying hen ovary.

Ovarian follicle development is primarily regulated by an interplay between the pituitary gonadotrophins, LH and FSH, and ovary-derived steroids. Increasing evidence implicates regulatory roles of transforming growth factor-beta (TGFbeta) superfamily members, including inhibins and activins. The aim of this study was to identify the expression of mRNAs encoding key receptors of the inhibin/activin system in ovarian follicles ranging from 4 mm in diameter to the dominant F1 follicle (approximately 40 mm). Ovaries were collected (n = 16) from mid-sequence hens maintained on a long-day photoschedule (16 h of light:8 h of darkness). All follicles removed were dissected into individual granulosa and thecal layers. RNA was extracted and cDNA synthesized. Real-time quantitative PCR was used to quantify the expression of mRNA encoding betaglycan, activin receptor (ActR) subtypes (type-I, -IIA and -IIB) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH); receptor expression data were normalized to GAPDH expression. Detectable levels of ActRI, -IIA and -IIB and the inhibin co-receptor (betaglycan) expression were found in all granulosa and thecal layers analysed. Granulosa ActRI mRNA peaked (P < 0.05) in 8-9.9 mm follicles, whereas ActRIIA rose significantly from 6-7.9 mm to 8-9.9 mm, before falling to F3/2; levels then rose sharply (3-fold) to F1 levels. Granulosa betaglycan mRNA expression rose 3-fold from 4-5.9 mm to 8-9.9 mm, before falling 4-fold to F3/2; levels then rose sharply (4-fold) to F1 levels. ActRIIB levels did not vary significantly during follicular development. Thecal ActRI mRNA expression was similar from 4-7.9 mm then decreased significantly to a nadir at the F4 position, before increasing 2-fold to the F1 (P < 0.05). Although thecal ActRIIB and -IIA expression did not vary significantly from 4 mm to F3, ActRIIB expression increased significantly (2-fold) from F3 to F1 and ActRIIA increased 2-fold from F2 to F1 (P < 0.05). Thecal betaglycan fell to a nadir at F6 after follicle selection; levels then increased significantly to F2, before falling approximately 50% in the F1. In all follicles studied expression of betaglycan and ActRI (granulosa: r = 0.65, P < 0.001, n = 144/group; theca: r = 0.49, P < 0.001, n = 144/group) was well correlated. No significant correlations were identified between betaglycan and ActRIIA or -IIB. Considering all follicles analysed, granulosa mRNA expression of betaglycan, ActRI, ActRIIA and ActRIIB were all significantly lower than in corresponding thecal tissue (betaglycan, 11.4-fold; ActRIIB, 5.1-fold; ActRI, 3.8-fold; ActRIIA, 2.8-fold). The co-localization of type-I and -II activin receptors and betaglycan on granulosa and thecal cells are consistent with a local auto/paracrine role of inhibins and activins in modulating ovarian follicle development, selection and progression in the domestic fowl.

Variation in pituitary expression of mRNAs encoding the putative inhibin co-receptor (betaglycan) and type-I and type-II activin receptors during the chicken ovulatory cycle.

Secretion of LH and FSH from the anterior pituitary is regulated primarily by hypothalamic GnRH and ovarian steroid hormones. More recent evidence indicates regulatory roles for certain members of the transforming growth factor beta (TGFbeta) superfamily including inhibin and activin. The aim of this study was to identify expression of mRNAs encoding key receptors and ligands of the inhibin/activin system in the hen pituitary gland and to monitor their expression throughout the 24-25-h ovulatory cycle. Hens maintained on long days (16 h light/8 h dark) were killed 20, 12, 6 and 2 h before predicted ovulation of a midsequence egg (n = 8 per group). Anterior pituitary glands were removed, RNA extracted and cDNA synthesized. Plasma concentrations of LH, FSH, progesterone and inhibin A were measured. Real-time quantitative PCR was used to quantify pituitary expression of mRNAs encoding betaglycan, activin receptor (ActR) subtypes (type I, IIA), GnRH receptor (GnRH-R), LH beta subunit, FSH beta subunit and GAPDH. Levels of mRNA for inhibin/activin betaA and betaB subunits, inhibin alpha subunit, follistatin and ActRIIB mRNA in pituitary were undetectable by quantitative PCR (<2 amol/reaction). Significant changes in expression (P<0.05) of ActRIIA and betaglycan mRNA were found, both peaking 6 h before ovulation just prior to the preovulatory LH surge and reaching a nadir 2 h before ovulation, just after the LH surge. There were no significant changes in expression of ActRI mRNA throughout the cycle although values were correlated with mRNA levels for both ActRIIA (r = 0.77; P<0.001) and beta-glycan (r = 0.45; P<0.01). Expression of GnRH-R mRNA was lowest 20 h before ovulation and highest (P<0.05) 6 h before ovulation; values were weakly correlated with betaglycan (r = 0.33; P = 0.06) and ActRIIA (r = 0.34; P = 0.06) mRNA levels. Expression of mRNAs encoding LH beta and FSH beta subunit were both lowest (P<0.05) after the LH surge, 2 h before ovulation. These results are consistent with an endocrine, but not a local intrapituitary, role of inhibin-related proteins in modulating gonadotroph function during the ovulatory cycle of the hen, potentially through interaction with betaglycan and ActRIIA. In contrast to mammals, intrapituitary expression of inhibin/activin subunits and follistatin appears to be extremely low or absent in the domestic fowl.

The reproductive response of rams to thyroidectomy: mediation by impaired inhibin feedback rather than a change in LH pulsatility.

A series of experiments was conducted to examine the mechanism by which removal of the thyroid glands in seasonally suppressed rams brings about rapid testicular growth. In the first experiment, thyroidectomy at the nadir of the testicular cycle (late winter) initiated testis growth without any detectable change in the extent of spermatogenesis compared with sham-operated controls. The serum concentration of FSH, but not LH, was also markedly increased by thyroidectomy. In the second experiment, serum FSH concentration was again increased by thyroidectomy in late winter but there was no effect of thyroidectomy on LH concentration, LH pulses (measured in frequent blood samples) or testosterone concentration. Furthermore, there was no evidence of a change in central dopaminergic inhibition of GnRH, as measured by the pulsatile LH response to an i.m. injection of the dopaminergic D(2) agonist bromocriptine or antagonist sulpiride. The rapid increase in FSH concentration occurred despite a markedly increased serum inhibin A concentration in thyroidectomized rams. Therefore, the efficacy of inhibin feedback was examined by testing the FSH-suppressive effect of an inhibin preparation (5 ml charcoal-stripped bovine follicular fluid i.v.) in long-term thyroidectomized and thyroid intact castrated rams. Bovine follicular fluid suppressed FSH concentrations in control rams as expected but in marked contrast, was completely without effect in thyroidectomized animals. In castrated rams, the FSH concentration was only marginally increased by thyroidectomy, indicating that there is a major component of the mediation of the effects of thyroidectomy that is testicular in origin. It was concluded that a reduction in the ability of endogenous inhibin to inhibit FSH release at the pituitary, rather than a hypothalamic mechanism, is the primary cause of the stimulation of testis growth by thyroidectomy.

Ovarian follicle development in the laying hen is accompanied by divergent changes in inhibin A, inhibin B, activin A and follistatin production in granulosa and theca layers.

To study the potential involvement of inhibin A (inhA), inhibin B (inhB), activin A (actA) and follistatin (FS) in the recruitment of follicles into the preovulatory hierarchy, growing follicles (ranging from 1 mm to the largest designated F1) and the three most recent postovulatory follicles (POFs) were recovered from laying hens (n=11). With the exception of <4 mm follicles and POFs, follicle walls were dissected into separate granulosa (G) and theca (T) layers before extraction. Contents of inhA, inhB, actA and FS in tissue extracts were assayed using specific two-site ELISAs and results are expressed per mg DNA. InhB content of both G and T followed a similar developmental pattern, although the content was >4-fold higher in G than in T at all stages. InhB content was very low in follicles <4 mm but increased ~50-fold (P<0.0001) to peak in 7-9 mm follicles, before falling steadily as follicles entered and moved up the follicular hierarchy (40-fold; 8 mm vs F2). In stark contrast, inhA remained very low in prehierarchical follicles (< or =9 mm) but then increased progressively as follicles moved up the preovulatory hierarchy to peak in F1 (approximately 100-fold increase; P<0.0001); In F1 >97% of inhA was confined to the G layer whereas in 5-9 mm follicles inhA was only detected in the T layer. Both inhA and inhB contents of POFs were significantly reduced compared with F1. Follicular actA was mainly confined to the T layer although detectable levels were present in G from 9 mm; actA was low between 1 and 9 mm but increased sharply as follicles entered the preovulatory hierarchy (approximately 6-fold higher in F4; P<0.0001); levels then fell approximately 2-fold as the follicle progressed to F1. Like actA, FS predominated in the T although significant amounts were also present in the G of prehierarchical follicles (4-9 mm), in contrast to actA, which was absent from the G. The FS content of T rose approximately 3-fold from 6 mm to a plateau which was sustained until F1. In contrast, the FS content of G was greatest in prehierarchical follicles and fell approximately 4-fold in F4-F1 follicles. ActA and FS contents of POFs were reduced compared with F1. In vitro studies on follicle wall explants confirmed the striking divergence in the secretion of inhA and inhB during follicle development. These findings of marked stage-dependent differences in the expression of inhA, inhB, actA and FS proteins imply a significant functional role for these peptides in the recruitment and ordered progression of follicles within the avian ovary.

Immunohistochemical localization of inhibin/activin alpha, betaA and betaB subunits and follistatin in bovine oocytes during in vitro maturation and fertilization.

The aim of this study was to evaluate the distribution of inhibin/activin alpha, beta(A) and beta(B) subunits and follistatin in immature oocytes and in matured oocytes before and after IVF. Denuded oocytes were submitted to a whole-mount immunofluorescence procedure. Specimens were imaged and fluorescent intensities quantified by scanning laser confocal microscopy. Immunoreactivity for inhibin alpha subunit (both alpha(C) and pro-alpha regions), abundant in the ooplasm of immature oocytes, decreased after maturation (a 68% and 88% decrease, respectively; P < 0.001), but increased after IVF by 2- and 5.7-fold, respectively (P < 0.01). Intense staining for beta(A) was detected in immature oocytes (predominantly in the outer ooplasm and zona pellucida) but after maturation and fertilization it was localized mainly in the zona pellucida, perivitelline space and oolemma. Immunoreactivity for beta(A) in the ooplasm decreased by 58% after maturation (P < 0.001) but increased again by 75% after fertilization (P < 0.01). Immunoreactivity for beta(B) was localized mainly in the zona pellucida and did not change after maturation. However, immunoreactivity for beta(B) was not detected in the zona pellucida after fertilization, but remained unchanged in unfertilized oocytes. Immunoreactivity for follistatin was detected in the ooplasm and zona pellucida of immature oocytes but decreased progressively in the ooplasm after maturation (a 63% decrease; P < 0.001) and did not change after IVF. Examination of partially denuded cumulus-oocyte complexes confirmed abundant expression of alpha(C), pro-alpha, beta(A) and follistatin immunoreactivity in cumulus cells, whereas beta(B) subunit staining was weak or absent in cumulus cells, but intense in the zona pellucida. In conclusion, the present study shows that qualitative and quantitative changes in the distribution of inhibin/activin subunits and follistatin accompany oocyte maturation and fertilization. The possibility, indicated by these observations, that activin A and activin B may play distinct roles in bovine oocyte maturation and fertilization warrants further study.

Differential effects of activin A on basal and gonadotrophin-induced secretion of inhibin A and progesterone by granulosa cells from preovulatory (F1-F3) chicken follicles.

Previous work has shown that activin A is expressed selectively within the theca rather than the granulosa layer of preovulatory chicken follicles. In the present study, this finding was verified and the potential paracrine actions of activin A on basal and gonadotrophin-induced secretion of inhibin A, inhibin B and progesterone by granulosa cells from the three largest preovulatory follicles (F1-F3) were investigated. Treatment with activin A (0, 0.25, 2.5 and 25 ng ml(-1)) alone increased inhibin A secretion markedly in a follicle- and time-dependent manner, with the greatest response (up to 15-fold increase; P < 0.0001) in F1 follicles after 3 days of treatment. In contrast, activin A alone had no effect on progesterone output at any time. Cells from F3 follicles were more responsive to FSH than were F1 cells in terms of both inhibin A (P < 0.02) and progesterone (P < 0.01) secretion. Furthermore, activin A greatly enhanced FSH-induced secretion of both inhibin A (up to tenfold; P < 0.0001) and progesterone (up to sixfold; P < 0.0001). In terms of LH-induced inhibin A and progesterone secretion, cells from F1, F2 and F3 follicles showed similar responses. Co-treatment with activin A enhanced LH-induced secretion of inhibin A markedly (up to ninefold; P < 0.0001) but had only a marginal effect on LH-induced progesterone secretion (up to twofold; P < 0.001). The presence of activin receptor subtypes IA, IB, IIA and IIB in cultured granulosa cells from F1, F2 and F3 follicles was demonstrated using immunocytochemistry. These findings support the hypothesis that activin A secreted by the theca layers of avian preovulatory follicles exerts a local paracrine action on granulosa cells to modulate 'basal' inhibin A secretion and to upregulate gonadotrophin-induced secretion of both inhibin A and progesterone. However, the extent to which this local role of activin A contributes to the generation of the preovulatory LH-progesterone surge remains to be established.

Follicle wave growth in cattle.

Ovarian follicle growth in cattle culminates in the selection of a single dominant follicle which attains the ability for final maturation and ovulation once or twice during the luteal phase and at the end of the oestrous cycle, as well as during other reproductive states. This review will describe in detail the first follicle wave of the cycle leading to selection of the first wave dominant follicle, indicating the specific gonadotrophin dependencies of cohort and dominant follicles, and relating follicle fate to steroidogenesis. As a differential gonadotrophin response of growing antral follicles during the follies-stimulating hormone (FSH) decline may determine which follicle becomes selected, first wave follicles are also characterized in relation to intrafollicular growth factors, which may modify the gonadotrophin response, such as inhibins and members of the insulin-like growth factor (IGF) family. Subsequently, the follicular control of the transient FSH rise and decline so crucial to dominant follicle selection will be discussed. It is concluded that successful hormonal manipulation of follicle wave growth and dominant follicle selection will depend on our detailed understanding of the gonadotrophin requirements of differentiating wave follicles.

Day 5 inhibin B levels in a treatment cycle are predictive of IVF outcome.

BACKGROUND: Day 5 serum inhibin B during IVF treatment has been investigated as a predictor of outcome. METHODS: A total of 54 women (< or = 39 years, normal menses and endocrine profiles) were treated with urinary gonadotrophins or recombinant FSH following pituitary down-regulation. Serum day 3 FSH in a preceding cycle was <8.5 IU/l. Plasma inhibin B, inhibin A and estradiol were determined after 4 days of gonadotrophin administration (day 5). RESULTS: Day 5 inhibin B was the most highly correlated with the number of mature follicles (>14 mm), oocytes retrieved and fertilized. Receiver operating characteristic analysis gave high accuracy for day 5 inhibin B in predicting ovarian response and indicated that a threshold of 400 pg/ml may be helpful in the decision as to whether to continue treatment. Women with <400 pg/ml (n = 16) had lower numbers of follicles, mature follicles, oocytes retrieved, fertilized and cleaved compared with those >400 pg/ml (n = 36) and this threshold gave a positive likelihood ratio of 30, 92.9% sensitivity, 95.0% specificity and 86.7% positive predictive value to detect poor ovarian response. Day 5 inhibin B was the best predictor of pregnancy (no live births and four cycles cancelled, low inhibin group; nine live births and no cancelled cycles, high inhibin group). CONCLUSIONS: Normogonadotrophic, normogonadal women with day 5 inhibin B <400 pg/ml in down-regulated cycles have a poor response to ovarian stimulation and are less likely to conceive compared with women with higher day 5 inhibin B.

Effects of ovarian follicle ablation on FSH, oestradiol and inhibin A concentrations and growth of other follicles in sheep.

The aim of this study was to examine the effect of removal of the largest follicle or all visible follicles during the first follicle wave on subsequent follicular growth, steroid, inhibin A and gonadotrophin secretion in sheep. On day 4.5 of a synchronized oestrous cycle, ewes (n = 18) were assigned to one of three groups which underwent either no treatment (control), ablation of the largest follicle (largest follicle aspirated and cauterized via laparotomy) or ablation of all follicles (all visible follicles ablated). Between day 0 and day 10 of the oestrous cycle, blood samples were collected every 8 h and ovaries were examined daily using transrectal ultrasonography. The lifespan of the second largest follicle (number of days > 3 mm in diameter) was longer (6.7 +/- 0.9 days; P < 0.05) and the maximum diameter tended to be greater (4.8 +/- 0.3 mm; P = 0.07) in ewes in which the largest follicle was ablated than in the control ewes (3.8 +/- 0.4 days; 4.2 +/- 0.3 mm). There was no difference in the day of emergence of the second follicular wave between groups (day 6.9 +/- 0.4). However, the peak of the transient increase in FSH concentrations after ablation was earlier (day 5.67 +/- 0.15; P < 0.05) in ewes in which all follicles were ablated than in control ewes (day 6.72 +/- 0.36); the timing in ewes that had only the largest follicle ablated was intermediate (day 6.11 +/- 0.28). Serum inhibin A concentrations were about three-fold lower (P < 0.05) in both follicle ablation groups than in the control group. The numbers of follicles 2-3 mm in diameter during the first 3 days of the second follicular wave were greater in 'ablated ewes' (both groups had 2.6 +/- 0.2 follicles day-1) than in control ewes (1.7 +/- 0.3 follicles day-1). It is concluded that: (i) transient increases in FSH concentrations precede the emergence of follicle waves; (ii) ablation of all follicles on day 4.5 after oestrus advanced the timing of the next peak in FSH concentrations and the numbers of small follicles associated with the development of the second follicular wave; and (iii) ablation of the largest follicle resulted in an increase in the lifespan of the second largest follicle, indicating a regulatory role of large dominant follicles over smaller subordinate follicles.

Modulation of sex hormone secretion in cows by acute infection with bovine viral diarrhoea virus.

Bovine viral diarrhoea virus (BVDV) is a major pathogen of cattle and is responsible for considerable reproductive loss. In this study, the in vivo responses in six multiparous cows were investigated after a non-cytopathogenic BVDV challenge (strain Pe 515; 5 x 10(6) tissue culture infective dose 50) given 9 days before a synchronized ovulation. Six similar cows challenged with non-infectious culture medium served as controls. The experimental noncytopathogenic BVDV infection was followed by a viraemia and leucopenia at days 5-9 after challenge, but no other clinical signs of infection were detected. However, the BVDV infection altered endocrine function. Mean LH pulse frequency immediately before CIDR withdrawal was lower (P < or = 0.05) in the BVDV-infected (2.17 +/- 0.34 pulses per 8 h) compared with the sham-infected (4.83 +/- 1.04 pulses per 8 h) animals. At day 3 after CIDR withdrawal, plasma oestradiol concentrations remained high (P < 0.05) in the infected cows (2.19 +/- 0.51 pg ml(-1)) compared with the sham-infected controls (0.72 +/- 0.29 pg ml(-1)). However, there was no difference in the peak oestradiol concentration (BVDV: 2.31 +/- 0.29 versus sham: 2.34 +/- 0.41 pg ml(-1)). In addition, non-cytopathogenic BVDV significantly (P < 0.05) increased the duration of the interval between ovulation and onset of the postovulatory progesterone increase (values 1.0 ng ml(-1)) (BVDV: 3.0 +/- 0.26 versus sham: 4.0 +/- 0.26 days). The viral infection also significantly (P < 0.01) decreased mean plasma progesterone concentrations between day 3 and day 11 after ovulation (BVDV: 2.59 +/- 0.32 versus sham: 4.13 +/- 0.27 ng ml(-1)). These data show that non-cytopathogenic BVDV viraemias during the follicular phase can modulate the secretion of gonadotrophins and sex steroids, in particular progesterone, during a synchronized oestrous cycle. Therefore, viraemias during the follicular phase may reduce the fertility of cattle by disrupting the capacity of the ovulatory follicle to form a competent corpus luteum, thereby compromising early embryo development and maternal recognition of pregnancy.

Modulatory effects of gonadotrophins and insulin-like growth factor on the secretion of inhibin A and progesterone by granulosa cells from chicken preovulatory (F1-F3) follicles.

The aim of this study was to compare the actions and interactions of gonadotrophins (LH and FSH) and an analogue of insulin-like growth factor I (LR3-IGF-I) on the secretion of inhibin A, inhibin B and progesterone by cultured chicken granulosa cells derived from the three largest (F1--F3) follicles of the preovulatory hierarchy. Treatment with LH or FSH promoted marked dose-(P < 0.0001) and time- (P < 0.0001) dependent increases in both inhibin A and progesterone secretion, with the magnitude of response (< 15-fold compared with basal) increasing over time in culture. Concentrations of inhibin B were below the detection limit in all samples. Initially, F1 cells were more LH-responsive than were F3 cells in terms of progesterone secretion (P < 0.02) but this difference between follicles decreased over time in culture. In contrast, LH-induced inhibin A secretion tended to be highest from F3 cells, although this was not significant. Cells from F3 follicles were consistently more FSH-responsive than F1 cells in terms of both progesterone (P < 0.01) and inhibin A (P < 0.02) secretion. Initially, F1 cells were more responsive to LR3-IGF-I than were F3 cells in terms of progesterone secretion (P < 0.001) but were less responsive in terms of inhibin A secretion (P < 0.001). Again, these inter-follicle differences decreased over time in culture (not significant on day 3 of treatment). Co-treatment experiments showed that LR3-IGF-I enhanced both LH- and FSH-induced secretion of inhibin A and progesterone in a time- (P < 0.001) and follicle- (P < 0.001) dependent way. Initially, F1 cells showed highest LR3-IGF-I enhancement of LH-induced inhibin A and progesterone secretion; in contrast, F3 cells showed the highest LR3- IGF-1 enhancement of FSH-induced inhibin A and progesterone secretion. These inter-follicle differences persisted over time in the case of FSH-induced hormone responses but not in the case of LH-induced responses, even though the relative degree of LR3-IGF-I enhancement increased markedly over time. Collectively, these data support a positive role for IGF-I, presumably of thecal origin, as an amplifier of gonadotrophin action on granulosa cell inhibin A and progesterone production by preovulatory chicken follicles.

Inter-relationships among gonadotrophins, reproductive steroids and inhibin in freemartin ewes.

Freemartins are sterile XX/XY chimaeras that occur as a result of placental fusion between male and female fetuses during early pregnancy. Freemartins occur predominantly in cattle, although the prevalence of ovine freemartinism is increasing. In this study, the reproductive endocrinology of ovine freemartins was compared with that of normal sheep. Freemartins had significantly (P < 0.001) higher basal concentrations of LH and FSH than did normal ewes or rams, although the response of LH to GnRH (10 microg) was similar in freemartins, ewes and rams. Resting concentrations of oestradiol were similar in freemartins and ewes and were increased in both after eCG administration. Testosterone concentrations were higher in freemartins than in ewes, but were unresponsive to GnRH or eCG. Administration of 62.5 mg progesterone or 25 lg oestradiol twice a day for 3 days suppressed LH concentrations to baseline values in freemartins, ewes and rams. In ewes, 500 microg oestradiol administered twice a day caused preovulatory surges in LH concentrations, but suppressed LH in freemartins to baseline values. Thus, LH secretion can potentially be regulated in freemartins by gonadal steroids. FSH concentrations in freemartins were not suppressed by doses of inhibin that were effective in ewes and rams. Therefore, freemartins behave in part like castrated animals, as they have high basal concentrations of LH and FSH, which can be stimulated by GnRH and suppressed by gonadal steroids. Conversely, inhibin does not suppress FSH concentrations in freemartins, and freemartins have circulating concentrations of steroids intermediate between those of castrated and normal animals.

Interactions between follicle-stimulating hormone and growth factors in modulating secretion of steroids and inhibin-related peptides by nonluteinized bovine granulosa cells.

The aim was to investigate potential interactions between FSH and intraovarian growth factors in modulating secretion of inhibin A (inh A), activin A (act A), follistatin (FS), estradiol (E2), and progesterone (P4) by bovine granulosa cells cultured under conditions in which a nonluteinized FSH-responsive phenotype is maintained. Cells from 4- to 6-mm follicles were cultured in serum-free medium containing insulin (10 ng/ml) and androstenedione (10(-7) M), and effects of ovine FSH (0.037-3 ng/ml) were tested alone and in combination with insulin-like growth factors (IGF) (LR3 IGF-I analogue; 2-50 ng/ml) and epidermal growth factor (EGF; 0.1-10 ng/ml). Medium was changed every 48 h and cultures ended after 144 h, when cell number was determined. Between 48-96 h and 96-144 h, FSH promoted (P < 0.0001) increases in output of inh A (6-fold), act A (15-fold), FS (6-fold), and E2 (18-fold), with maximal responses (in parentheses) elicited by 0.33 ng/ml FSH during the final period. Higher FSH doses (1 and 3 ng/ml) gave reduced responses for each of the above hormones, whereas P(4) output was maximal (3-fold) at these doses. FSH promoted a slight increase in cell number ( approximately 1.7-fold; P < 0.001). LR3 IGF-I alone markedly increased (P < 0.0001) output of inh A (8-fold), act A (41-fold), FS (12-fold), and E2 (18-fold); this was accompanied by modest increases (P < 0.01) in P4 output ( approximately 2.5-fold) and cell number ( approximately 2-fold). Whereas FSH enhanced inh A, act A, FS, and E2 secretion evoked by lower doses of LR3 IGF-I, it suppressed (P < 0.001) the response to the highest dose. EGF alone promoted a 1.7-fold increase in cell number (P < 0.001) without affecting hormone release; however, it abolished (P < 0.001) FSH-induced secretion of inh A, act A, FS, and E2. Both FSH alone and LR3 IGF-I alone dose-dependently increased the act A:FS ratio ( approximately 3-fold; P < 0.005) and act A:inh A ratio (3-fold to 6-fold; P < 0.001), suggesting that both factors selectively raise activin "tone" and that this could be a key requirement for FSH and IGF-induction of follicular E2 production. This hypothesis was reinforced by the finding that addition of FS, to reduce the act A:FS ratio and sequester secreted activin, markedly suppressed (P < 0.001) FSH (3-fold)-, and LR3 IGF-I (2-fold)-induced E2 output.