Molecular basis of bone morphogenetic protein-4 inhibitory action on progesterone secretion by ovine granulosa cells.

We have recently reported that bone morphogenetic protein-4 (BMP-4) can inhibit progesterone production by ovine granulosa cells (GCs). Here, we have investigated the underlying mechanisms of this effect in basal as well as in FSH-induced conditions. We have confirmed that treatment with BMP-4 decreased basal GC progesterone secretion and totally abolished FSH-stimulating action. This inhibitory action was associated with a decrease in the expression of cAMP-regulated genes, steroidogenic acute regulatory protein (StAR) and P450 side-chain cleavage (P450 scc) at mRNA and protein levels. However, BMP-4 did not alter basal cAMP production by GCs. In contrast, BMP-4 decreased by half the FSH-induced cAMP production and strongly inhibited cAMP-induced progesterone production. Thus, the inhibitory effect of BMP-4 was exerted both upstream and downstream of cAMP signalling. We next examined the downstream effect, focusing on cAMP-dependent transcription factors, steroidogenic factor-1 (SF-1) and CREB, through the BMP factor signalling intermediary, Smad1. As expected, BMP-4 induced phosphorylation and transcriptional activity of Smad1 in ovine GCs. BMP-4-activated Smad1 did not affect CREB activity but inhibited the transcriptional activity of SF-1 on the canonical SF-1 responsive element. Interestingly, this transcriptional inhibitory mechanism occurred on transfected StAR and P450 scc promoter. Based on these results, we propose that SF-1 is a key target in the inhibitory mechanism exerted by BMP-4 on progesterone synthesis by ovine GCs in culture. Because SF-1 plays an essential role in the differentiation of GCs, our findings could have new implications in understanding the role of BMP family members in the control of ovarian folliculogenesis.

The Booroola mutation in sheep is associated with an alteration of the bone morphogenetic protein receptor-IB functionality.

The hyperprolificacy phenotype of Booroola ewes is due to the presence of the FecB(B) allele at the FecB locus, recently identified as a single amino acid substitution (Q249R) in the bone morphogenetic protein (BMP) type-IB receptor (BMPR1B), and is associated with a more precocious differentiation of ovarian granulosa cells (GCs). To evaluate the consequences of the Booroola mutation on BMPR1B functions, the action of ligands of the transforming growth factor-beta (TGFbeta)/BMP family that act through (growth and differentiation factor-5, BMP-4) or independently of (activin A, TGFbeta-1) BMPR1B were studied on primary cultures of GCs from homozygous FecB(+) and FecB(B) ewes. All the tested TGFbeta/BMP family ligands inhibited progesterone secretion by FecB(+) GCs. Those inhibitory effects were lower for GCs from preovulatory (5-7 mm diameter) than from small antral follicles (1-3 mm diameter). The presence of the Booroola mutation was associated with a 3- to 4-fold (P<0.001) decreased responsiveness of GCs from FecB(B) compared with FecB(+) small follicles to the action of BMPR1B ligands. In contrast, TGFbeta-1 and activin A had similar inhibitory effects on progesterone secretion by GCs from FecB(+) and FecB(B) small follicles. No difference between genotypes was observed with GCs from preovulatory follicles. In transfection experiments with HEK-293 cells, co-expression of FecB(+) BMPR1B and BMPR2 resulted in a 2.6-fold (P<0.01) induction of the activity of a BMP-specific luciferase reporter construct by BMP-4. Interestingly, no response to BMP-4 was observed when cells were transfected with the FecB(B) form of the BMPR1B receptor. Overall, these data strongly suggest that the Q249R mutation is associated with a specific alteration of BMPR1B signaling in hyperprolific Booroola ewes.

Differences in splicing of mRNA encoding LH receptor in theca cells according to breeding season in ewes.

Splice variants of mRNA encoding the LH receptor (LHR) during follicular development were characterized in cyclic and non-cyclic ewes. Granulosa and theca cells were collected from individual follicles. After amplification by RT-PCR of a region situated between exon 9 and exon 11 of the LHR gene, three distinct bands, LHR1 (full length), LHR2 (deletion of exon 10), LHR3 (deletion of 262 bp in exon 11), were observed in the granulosa and theca cells of ovine antral follicles of various sizes (2.5-6.0 mm). Expression of LHR mRNA in theca cells varied with the annual cycle of reproduction (P < 0.001), and was highly expressed in all classes of follicle collected from anoestrous ewes (1.3 +/- 0.1, n = 8 in small follicles; 1.8 +/- 0.2, n = 8 in medium follicles; 1.7 +/- 0.3, n = 4 in large follicles; arbitrary units) compared with follicles collected from oestrous ewes (0.19 +/- 0.06, n = 8 in small follicles; 0.2 +/- 0.04, n = 9 in medium follicles; 0.18 +/- 0.04, n = 5 in large follicles). During the breeding season, no differences in the relative expression of the different splice variants were observed according to follicle size. In contrast, during anoestrus, LHR3 mRNA was significantly more abundant in large (6.0-6.5 mm) and medium (4.0-5.5 mm) than it was in small (2.5-3.5 mm) follicles. These results indicate that RNA alternative splicing plays a role in the seasonal and physiological control of LH receptor expression in theca cells.

Laminin-alpha6beta1 integrin interaction enhances survival and proliferation and modulates steroidogenesis of ovine granulosa cells.

This study aimed to determine the physiological role of laminin (LN) and its receptor, alpha(6)beta(1) integrin, in controlling the functions of granulosa cells (GC) during follicular development in sheep ovary. Immunohistochemistry experiments showed the presence of increasing levels of LN (P<0.0001), and high levels of mature alpha(6)beta(1) integrin in GC layers of healthy antral follicles during the follicular and the preovulatory phases of the estrous cycle. In vitro, the addition of a function-blocking antibody raised against alpha(6) subunit (anti-alpha(6) IgG) to the medium of ovine GC cultured on LN impaired cell spreading (P<0.0001), decreased the proliferation rate (P<0.05) and increased the apoptosis rate (P<0.05). Furthermore, addition of anti-alpha(6) IgG enhanced estradiol (E2) secretion by GC in the presence or absence of follicle-stimulating hormone (FSH), luteinizing hormone or insulin-like growth factor-I in culture medium (P<0.0001), and inhibited progesterone (P4) secretion in basal conditions or in the presence of low (0.5 ng/ml) FSH concentrations only (P<0.0001). The anti-alpha(6) IgG effect was specific to an interaction of LN with alpha(6)beta(1) integrin since it was ineffective on GC cultured on heat-denatured LN, RGD (arginine-glycine-aspartic acid) peptides and non-coated substratum. Hence, this study established that alpha(6)beta(1) integrin 1) was expressed in GC of antral follicles, 2) mediated the actions of LN on survival, proliferation and steroidogenesis of GC, and 3) was able to dramatically modulate P4 and E2 secretion by GC in vitro. It is suggested that during the follicular and the preovulatory phases of the estrous cycle, the increasing levels of LN in GC of large antral follicles might support their final development to ovulation.

Extracellular matrix regulates ovine granulosa cell survival, proliferation and steroidogenesis: relationships between cell shape and function.

The extracellular matrix (ECM), constituting the follicular basal lamina and present also between follicular cells and in the follicular fluid, is believed to regulate granulosa cell (GC) function during follicular development. Ovine GCs isolated from small (1-3 mm in diameter) or large (4-7 mm in diameter) antral follicles were cultured on various pure ECM components (type I collagen, fibronectin, laminin), synthetic substrata enhancing (RGD peptides) or impairing (poly 2-hydroxyethylmethacrylate (poly-hema)) cell adhesion, or in the presence of heparin. The effects of these factors, used alone or in combination with IGF-I and/or FSH, were evaluated in terms of GC spread, survival, proliferation and steroidogenesis. When grown on type I collagen (CI) gel, poly-hema or heparin, GCs from both large and small follicles exhibited a round shape and a low proliferation rate. Compared with non-coated plastic substratum as a control, these ECM or synthetic compounds enhanced estradiol secretion and reduced progesterone secretion by large-follicle GCs. In contrast, GCs from both large and small follicles spread extensively on CI coating, fibronectin, laminin and RGD peptides. Fibronectin and laminin dramatically increased the proliferation rate and enhanced survival of GCs from both origins. Moreover, fibronectin, laminin and RGD peptides reduced estradiol secretion by large-follicle GCs. Unexpectedly, CI coating increased estradiol secretion and reduced progesterone secretion by large-follicle GCs, suggesting that type I collagen was able to maintain estradiol secretion independently of GC shape. Finally, GC responsiveness to IGF-I and FSH, in terms of proliferation and steroidogenesis, was generally maintained when cells were grown on ECM components, RGD peptides and in the presence of heparin. However, when large-follicle GCs were grown as non-adherent clusters (as observed on poly-hema) basal and IGF-I- and/or FSH-stimulated progesterone secretions were totally abolished. Overall, this study shows that GC shape, survival, proliferation and steroidogenesis can be modulated in vitro by pure ECM components in a specific and coordinated manner. It is suggested that, in vivo, fibronectin and laminin would sustain follicular development by enhancing the survival and proliferation of GCs, whereas type I collagen might participate in the maintenance of estradiol secretion in large antral follicles.

Mutation in bone morphogenetic protein receptor-IB is associated with increased ovulation rate in Booroola Mérino ewes.

Ewes from the Booroola strain of Australian Mérino sheep are characterized by high ovulation rate and litter size. This phenotype is due to the action of the FecB(B) allele of a major gene named FecB, as determined by statistical analysis of phenotypic data. By genetic analysis of 31 informative half-sib families from heterozygous sires, we showed that the FecB locus is situated in the region of ovine chromosome 6 corresponding to the human chromosome 4q22-23 that contains the bone morphogenetic protein receptor IB (BMPR-IB) gene encoding a member of the transforming growth factor-beta (TGF-beta) receptor family. A nonconservative substitution (Q249R) in the BMPR-IB coding sequence was found to be associated fully with the hyperprolificacy phenotype of Booroola ewes. In vitro, ovarian granulosa cells from FecB(B)/FecB(B) ewes were less responsive than granulosa cells from FecB(+)/FecB(+) ewes to the inhibitory effect on steroidogenesis of GDF-5 and BMP-4, natural ligands of BMPR-IB. It is suggested that in FecB(B)/FecB(B) ewes, BMPR-IB would be inactivated partially, leading to an advanced differentiation of granulosa cells and an advanced maturation of ovulatory follicles.

Consequences of the presence of the Booroola F gene on the intraovarian insulin-like growth factor system and terminal follicular maturation in Mérinos d'Arles ewes.

In sheep, the presence of the Booroola F gene has several important consequences for ovarian function. This study investigated the consequences of the presence of the F gene for the insulin-like growth factor (IGF) system in the ewe ovary. Studies were undertaken in ovaries from F+ and ++ Mérinos d'Arles ewes to determine 1) the levels of type I IGF receptors and IGF binding proteins (IGFBPs) in follicular cells by quantitative autoradiography of [(125)]-IGF-I binding sites on ovarian sections; 2) the pattern of intrafollicular IGFBPs, by Western-ligand blotting on follicular fluids; and 3) the effects of IGF-I and FSH on proliferation and differentiation of granulosa cells in vitro, assessed by progesterone secretion and cytochrome P450 side-chain cleavage (P450(scc)) expression. The amounts of type I IGF receptors were similar in F+ and ++ follicular cells; however, at the same follicular size, F+ healthy follicles contained lower concentrations of IGFBPs smaller than 40 kDa (particularly IGFBP-2) than ++ healthy follicles. In vitro, in basal conditions as well as in IGF-I- or FSH-stimulated conditions (or both), granulosa cells from F+ follicles had a lower proliferative activity, secreted higher amounts of progesterone, and expressed higher levels of P450(scc) than granulosa cells from ++ follicles of the same size. When F+ and ++ preovulatory follicles were compared at the end of the follicular phase, IGFBPs <40 kDa concentrations were slightly higher, and responsiveness of granulosa cells to FSH in vitro was lower in F+ than in ++ follicles, suggesting that terminal maturation of F+ follicles, although precocious, was less complete than it was in ++ follicles. The early decrease in intrafollicular IGFBPs <40 kDa concentrations observed in F+ antral follicles, which likely leads to an early increase in IGF bioavailability, may at least partly account for the increased ovulation rate that characterizes F-carrier ewes.

Fraction of proliferating cells in granulosa during terminal follicular development in high and low prolific sheep breeds.

During terminal development of antral follicles, granulosa cells progressively lose their proliferative activity. Romanov (ovulation rate = 3) and Ile-de-France (ovulation rate = 1) breeds of sheep were compared for fractions of proliferating granulosa cells, determined by in vitro continuous [3H] thymidine labelling. In both breeds, the fraction of proliferating cells decreased with increasing follicular size according to a sigmoid-shaped curve. After linearization, the slope of the regression line was higher (in absolute value) in Romanov, compared to Ile-de-France ewes (p = 0.02). In vivo FSH treatment led to a decrease in the slope of the regression line in Romanov ewes only (p = 0.03). These results suggest that during terminal follicular development (1) the rate of cell cycle exit is higher in granulosa cells of Romanov, compared to lie-de-France follicles, and (2) Romanov granulosa cells are more responsive to exogenous FSH in term of proliferation. These mechanisms may underlie differential dynamics of follicular development in poly- and mono-ovulating breeds of sheep.

Anastomosis of the ovarian vein to the hepatic portal vein in sheep induces ovarian hyperstimulation associated with increased LH pulsatility, but only in the absence of the contralateral ovary.

In this study, two experiments were performed, the first of which examined the ovarian response in ewes that were subject to unilateral ovariectomy (ULO) at different intervals (0-14 days) after surgical anastomosis (AN) of the ovarian vein to the mesenteric vein (n=7 ewes), or sham operation (SO; n=4 ewes). Hypertrophy and development of multiple follicular and luteal structures on AN ovaries were observed after ULO, while SO ovaries remained of normal size and appearance after ULO. The second experiment involving 11 ewes (five AN; six SO) aimed to clarify the mechanism by which AN following ULO-induced ovarian hypertrophy and increased follicle development. The results confirmed that there were more large (>5 mm) follicles on AN compared with SO ovaries; however, their rate of atresia was similar. Oestradiol and progesterone concentrations in follicular fluid of class 1 follicles (5-9 mm) were higher in AN ovaries than those in control follicles of the same size collected in the late follicular phase of an induced oestrous cycle. In AN ewes, intrafollicular progesterone concentrations increased while follicular aromatase activity and intrafollicular oestradiol, inhibin A, follistatin and activin A concentrations all decreased as follicle size increased. Oestradiol and progesterone concentrations were substantially higher in ovarian venous blood than in hepatic venous blood, both in AN and SO ewes, whereas inhibin A levels were not significantly modified by passage through the liver in either group. Mean plasma LH concentration, and LH pulse frequency and amplitude increased markedly after AN but were not affected by SO. Plasma FSH showed only a small transient increase after AN, presumably due to the maintenance of inhibin feedback. Injection of prostaglandin F(2)(alpha) 4 days later did not further modify LH or FSH secretion in either group. Full ovariectomy (FO) 9-14 days after AN or SO increased LH secretion markedly in SO ewes but to a lesser degree in AN ewes; FO induced a large and rapid increase in FSH levels in both groups. In conclusion, AN of the ovary to the liver via the mesenteric vein provides a useful model for studying the feedback between the ovary and the hypothalamo-pituitary system and the mechanisms controlling follicle development. The present results indicate that the pattern of LH secretion is an important factor controlling the terminal phase of follicle development in the ewe.

The xenotransplantation of goat and human hematopoietic cells to sheep fetuses.

Hematopoietic xenografts were carried out in three experiments using goat fetal liver (44-48 days, experiments I and II) or purified human CD 34+ cells (experiment III) as the donor cells. Recipients were sheep fetuses at 41-47 days of gestation. Goat fetal liver cells were either injected without any pretreatment or stimulated by preincubation in a culturing in goat phytohemagglutinin-stimulated lymphocyte supernatant. Human CD 34+ myeloid progenitor cells were purified from bone marrow by minimacs immunomagnetic purification and cultured in medium supplemented with stem cell factor, IL3, and IL6. Goat-sheep chimerism was assessed by flow cytometry analysis (FCA) of peripheral blood and bone marrow cells using a mouse anti-goat CD 45 monoclonal antibody and by karyotype analysis of peripheral blood from goat/sheep chimeras. Human cell engraftment was assessed by polymerase chain reaction amplification of the human DAX1 gene in blood and bone marrow DNA from sheep which had received human cells. In the three experiments, a mean of 76% (26 of 34) of injected fetuses were born alive without any clinical evidence of graft-versus-host disease. Three lambs were found to be goat/sheep chimeric after flow cytometry analysis (peripheral blood and bone marrow) and karyotype (peripheral blood) analysis. Both tissues continued to express goat cells at 6 or 12 months (last assessment) depending on the experiment. No human chimerism was detected using polymerase chain reaction amplification in peripheral blood and bone marrow of any of the six sheep grafted with human cells. These data and those also obtained on other species (human, pig/sheep) show that it is possible to carry out hematopoietic xenografts using the sheep fetus as recipient provided both donor and recipient fetal cells are processed during the period of tolerance to foreign antigens.

Comparative expression of luteinizing hormone and follicle-stimulating hormone receptors in ovarian follicles from high and low prolific sheep breeds.

Expression of gonadotropin receptors and granulosa cell sensitivity to gonadotropin hormones by small (1-3 mm) and large (3.5-7 mm) follicles were compared in Romanov (ROM, ovulation rate = 3) and Ile-de-France (IF, ovulation rate = 1) ewes in the early and late follicular phase. In healthy follicles, LH receptor levels in granulosa cells increased with increasing follicular size (p < 0. 001) while FSH receptor levels decreased (p < 0.05). In granulosa cells of large follicles, LH receptor (LHR) mRNA levels were greater in the late than in the early follicular phase (p < 0.001, p < 0.05, for ROM and IF, respectively). In the early follicular phase, LHR levels in granulosa (p < 0.001) and theca cells (p < 0.05) of small follicles were greater in ROM than in IF ewes. FSH receptor mRNA levels in granulosa cells of small and large ROM follicles were greater than in the corresponding IF follicles (p < 0.05). Finally, a greater responsiveness (increase in cAMP secretion) to both FSH and hCG was observed by granulosa cells collected during the early follicular phase from ROM vs. IF ewes. Data provide evidence that the greater ovulation rate in the ROM as compared to the IF breed is associated with a greater gonadotropin responsiveness during the early follicular phase.

[Mechanism, regulation, and manipulations of follicular atresia]. / Mécanismes, régulations et manipulations de l'atrésie folliculaire.

In ovaries of mammals, an intense loss of germinal cells occurs by follicular atresia throughout the life. In atretic antral follicles, granulosa cells stop proliferating and become apoptotic. Main effectors of apoptosis are caspases which are activated by two ways in granulosa cells, the one involving Fas/TNF-alpha receptor, the other involving factors of the bel-2 family. Atresia is triggered when some essential factors supporting follicular development are lacking. Particularly, terminal follicular development is strictly dependent upon gonadotropin (FSH, then LH in the final preovulatory stage) supply, but factors acting in a paracrine way (growth factors, cytokines, steroids, constituents of extracellular matrix) play also important roles in amplifying gonadotropin action in follicular cells. Some pathological situations such as premature ovarian failure would result from accelerated follicular atresia, triggered by interactions between follicular cells and cells of the immune system. Current methods to control atresia consist in administrating exogenous gonadotropins, or indirectly increasing endogenous gonadotropins, or increasing follicular cell responsiveness to gonadotropins.

Expression of insulin-like growth factor binding protein-5 by ovine granulosa cells is regulated by cell density and programmed cell death in vitro.

In vivo, in the sheep ovary, the expression of insulin-like growth factor binding protein (IGFBP)-2 and particularly IGFBP-5 has been shown to increase dramatically in apoptotic granulosa cells from atretic follicles. The aim of this work was to study the relationship between apoptosis induced by serum starvation in vitro and expression of IGFBP-2 and -5 by ovine granulosa cells. For this purpose, granulosa cells from follicles 1-3 mm in diameter were cultured in the presence of serum for 2 days, then cultured in the presence or absence of serum for 24, 48, or 72 hr. At the end of the culture, cells were counted, cell viability was assessed by studying DNA fragmentation, and IGFBPs expression was studied by quantitative autoradiography, Western-ligand blotting, immunoblotting, and quantitative in situ hybridization. In vitro, IGFBP-2 and particularly IGFBP-5 were the main IGFBPs secreted by ovine granulosa cells. Serum starvation provoked (i) apoptosis of granulosa cells within 48 hr, (ii) a marked decrease in cell density, and (iii) a marked increase in the amount of IGFBP-5 associated with cell membranes and with the walls of culture wells, but no change in culture medium. The increase in the amount of cell- and wall-associated IGFBP-5 after serum starvation was essentially due to the consecutive decrease in cell density rather than to an increase in cell apoptosis. Indeed, irrespective of the presence or absence of serum, the amount of IGFBP-5 associated to cell membranes was inversely correlated to cell density. In contrast, the amount of IGFBP-5 present in culture medium was positively correlated to cell density. Furthermore, expression of IGFBP-5 mRNA was shown to increase with both cell density and cell death. Indeed, the expression of IGFBP-5 mRNA dramatically increased with cell density, irrespective of the presence or absence of serum, but at a similar cell density, expression was higher in serum-free than in serum conditions. Overall, these results indicate that, in vitro, the localization of IGFBP-5 on ovine granulosa cell membranes and in culture medium, respectively, was mainly dependent on cell density, whereas expression of IGFBP-5 mRNA was related to both cell density and cell death. These data suggest that IGFBP-5 is involved in both growth arrest and apoptosis of granulosa cells in the sheep.

Immunization of sheep against GnRH early in life: effects on gonadotropins, follicular growth and responsiveness of granulosa cells to FSH and IGF-I in two breeds of sheep with different prolificacy (Romanov and Ile-de-France).

The profile Romanov (R, ovulation rate = 3) and non-prolific Ile-de-France (IF, ovulation rate = 1) breeds were compared for their ovarian sensitivity to gonadotropins and IGF-I before puberty. For this purpose, the effects of in vivo immunization against GnRH on populations of ovarian follicles and in vitro sensitivity of granulosa cells to FSH and IGF-I were studied in prepuberal lambs from both breeds. Seventeen prepuberal lambs of each breed were actively immunized against GnRH between 3 wk and 6 mo of age. Relative to untreated lambs, FSH levels at 4, 5, and 6 mo of age were (respectively) 41%, 25% and 29% for IF, and 43%, 24%, and 36% for R lambs. In a first experiment, histological analysis of ovaries was performed. Immunization treatment decreased the number of small (100-390 microns in diameter) and large size follicles (< 1500 microns) in both breeds at 6 mo of age. In both breeds, gonadotropin (FSH-LH-hCG) treatment increased the number of large size follicles (< 1500 microns in diameter) and induced the formation of preovulatory follicles in immunized as well as untreated lambs. The ovulation rate was less in immunized animals, but it was not different between breeds. In a second experiment, the effects of FSH and IGF-I were studied on granulosa cells from follicles between 1000 and 2000 microns in diameter. In both breeds, IGF-I increased granulosa cell proliferation, but enhanced progesterone secretion was observed only in R lambs after FSH and IGF-I stimulation. Granulosa cell response to FSH treatment was lost by immunization, whereas response to IGF-I remained unchanged in both breeds. These results indicate that long-term immunization of prepuberal lambs against GnRH reduced systemic concentrations of FSH, follicular development, and response to gonadotropins in vivo, similarly in the prolific R and the non-prolific IF breed. However, granulosa cells from R lambs had higher steroidogenic capacities and were more responsive to FSH. In addition, these results suggest that IGF-I could play an important role in regulating growth of small follicles both in immunized and non-immunized lambs.

Anti-Müllerian hormone (AMH) secretion in prepubertal and adult rams.

The aim of the present analysis was to determine whether anti-Müllerian hormone concentrations in prepubertal plasma or adult rete testis fluid are related to the number or function of Sertoli cells in rams or to the presence of the FecB Booroola gene. Twenty rams from two Booroola crosses, differing in their testicular masses were analysed; in each cross, half of the animals were heterozygous carriers of the FecB gene. The data from rams, during prepuberty and at adulthood during the non-sexual season, were analysed by two-way ANOVA and residual correlations. In 4-week-old intact male lambs, the mean anti-Müllerian hormone plasma concentration was 15 ng ml-1, irrespective of cross, genotype or eCG stimulation; it was significantly negatively correlated with FSH (r = -0.51; P = 0.02; n = 19). In adults, anti-Müllerian hormone was not detectable in plasma and was 0.5 ng ml-1 in rete testis fluid, irrespective of cross or genotype. The total number of Sertoli cells per testis was not related to anti-Müllerian hormone concentration in lamb prepubertal plasma or in adult rete testis fluid. The concentration of anti-Müllerian hormone in adult rete testis fluid was significantly and negatively correlated with the daily production of leptotene primary spermatocytes per testis (r = -0.56; P = 0.02; n = 16). The mean oestrogen concentration in the adult testicular vein was 2 pg ml-1 and was correlated negatively with the rete testis fluid concentration of anti-Müllerian hormone (r = -0.60; P = 0.02; n = 15) and correlated positively with the daily production of leptotene primary spermatocytes per testis (r = 0.53; P < 0.05; n = 19). In conclusion, anti-Müllerian hormone secretion was not correlated with the total numbers of Sertoli cells per testis and cannot be used as a predictor of the number of Sertoli cells. Anti-Müllerian hormone secretions were not affected by the presence of FecB gene. However, anti-Müllerian hormone secretion could be considered to be inversely related to the daily production of primary spermatocytes by the testis.

Chronology of events accompanying follicular atresia in hypophysectomized ewes. Changes in levels of steroidogenic enzymes, connexin 43, insulin-like growth factor II/mannose 6 phosphate receptor, extracellular matrix components, and matrix metalloproteinases.

The chronology of changes in levels of some proteins known to be altered during atresia of ovarian follicles was studied in ewes hypophysectomized at the end of the follicular phase of the estrous cycle. This study was performed by quantitative immunohistochemistry and zymography on large antral follicles (diameter > 3.5 mm) normally destined to ovulate, recovered 24, 36, or 72 h after pituitary ablation. The process of atresia was followed by comparing healthy follicles from intact ewes, with early atretic follicles recovered 24 h after hypophysectomy, clearly atretic follicles recovered 36 h after hypophysectomy, and late atretic follicles recovered 72 h after hypophysectomy. The earliest events of atresia induced by hypophysectomy were detected in theca and consisted of a strong decrease in levels of fibronectin (p < 0.001) and insulin-like growth factor II/mannose 6 phosphate (IGF-II/M6P) receptors (p < 0.05), occurring within the first 24 h following pituitary ablation. In intact animals, similar changes were observed in theca of early atretic follicles, suggesting that these changes may be important events involved in the onset of follicular atresia. In parallel, intrafollicular levels of matrix metalloproteinase-2 and -9 were shown to increase as early as 24 h after hypophysectomy, and a further increase was observed until 72 h after hypophysectomy. These early events were followed by the disappearance of P450 aromatase in granulosa cells 36 h after hypophysectomy (p < 0.05), and a progressive decrease in levels of P450 17alpha-hydroxylase in the theca interna and of gap junction protein connexin-43 in granulosa cells; these markers were still detectable in late atretic follicles 72 h after hypophysectomy. The increase in levels of fibronectin, type IV collagen, laminin, and IGF-II/M6P receptors within granulosa cell layers (p < 0.05) was significant only in late atretic stages, suggesting that these changes may be consequences rather than causes of atresia.

Ram lambs need FSH for normal testicular growth, Sertoli cell numbers and onset of spermatogenesis.

The effect of FSH on the development of the testis in the ram lamb was examined in two experiments where lambs were passively immunized against ovine beta-FSH from birth until 100 or 160 d. In both experiments, immunization resulted in a slower testicular growth relative to that of controls. This effect became apparent at around the start of the period of rapid testicular growth (60-70 d of age) and resulted in testicular weights at the end of treatment ranging from 37 to 51% of those of control groups. Within the testis, this was reflected in shorter seminiferous tubules (48-64% of controls) and in lower numbers of Sertoli cells per testis (57-82%). In the rams immunized until 160 d of age, spermatogenesis had begun and immunization against FSH provoked a lower production of germinal cells which was not solely due to the lower number of Sertoli cells but also due to fewer germinal cells being supported by each Sertoli cell. However, the numbers of A0 spermatogonia per testis and the daily production of the A1 spermatogonia were unaffected by immunization, but the production of the B2 spermatogonia and, as a consequence, of leptotene and pachytene spermatocytes and of round spermatids were all markedly lower (43-47% of controls). These effects were not due to any decreases in the secretion of LH or testosterone as seen in the blood levels of these two hormones. These results show that, in the ram lamb, FSH is essential for normal testicular development and for the establishment of a normal population of Sertoli cells. They also confirm that, once spermatogenesis is established, FSH is necessary for a normal production of germinal cells, with one or more of the divisions between the A1 and B2 spermatogonia being sensitive to suppression of FSH.

Estrogen receptor protein and mRNA expression in the ovary of sheep.

Using immunohistochemistry and in situ hybridization, we attempted to identify the estrogen receptor (ER) protein and messenger RNA (mRNA) in sheep ovaries during the follicular phase of the estrous cycle. Monoclonal anti-ER antibodies H222 and 1D5 were used for localizing estrogen receptor on ovarian cryo-sections. Labeling for ER was found over the nuclei of surface epithelium, interstitial tissue, and granulosa cells of small as well as large ovarian follicles. In the preantral and small antral follicles, intense nuclear ER labeling was observed in mural granulosa cells and particularly in cumulus/granulosa cells surrounding the oocyte. In the large healthy looking follicles, greater diversity in labeling for ER was observed, which is characterized by mixed populations of granulosa cells expressing positive and more or less negative nuclear labeling. Such a pattern of labeling was particularly evident in follicles showing the signs of atresia. Generally, more intense nuclear staining was localized in granulosa cells proximal to basal membrane. In situ hybridization studies revealed the presence of ER mRNA in ovarian tissue. Autoradiographic visualization localized ER mRNA expression over the granulosa cells of healthy follicles of all sizes. Level of hybridization signal was comparable in mural and cumulus granulosa cells. In atretic follicles, the level of hybridization signal in granulosa cells was comparable to that of healthy follicles. A relatively weaker level of labeling was observed in granulosa cells dispersed in follicular antrum in follicles with advanced atretic lesions. Theca cells expressed a lower level of labeling than granulosa cells. Specificity of labeling for both ER protein and mRNA in ovary was proved by parallel probing the ovine uterus. Ovine ER recognition by both H222 and 1D5 antibodies was also proved by immunoblotting. These studies demonstrate the presence of the estrogen receptor and its messenger RNA in the sheep ovary and suggest an autocrine/paracrine role of estradiol and its receptor in the regulation of ovarian follicle development in sheep.

Changes in extracellular matrix components and steroidogenic enzymes during growth and atresia of antral ovarian follicles in the sheep.

To investigate the involvement of extracellular matrix (ECM) in folliculogenesis in the sheep, parallel changes in ECM components and key steroidogenic enzymes were studied by quantitative immunohistochemistry and immunoblotting during follicular growth and atresia. Growth of ovarian follicles from 1 to 5 mm in diameter was characterized by a progressive increase in P450 cholesterol sidechain cleavage levels in both thecal (p < 0.001) and granulosa cells (p < 0.001), an increase in P450 aromatase levels in granulosa cells of follicles larger than 3.5 mm (p < 0.001), and an increase in levels of P450 17 alpha-hydroxylase C17,20 lyase (P450(17 alpha)) in the theca interna. In addition, during follicular growth, a change in localization of cells expressing P450(17 alpha) within the theca interna was observed, positive cells being sparse within the theca interna of small follicles and specifically located close to the basal laminae in large follicles. In parallel, follicular growth was associated with an increase in levels of type I collagen in granulosa cell layers (p < 0.01) and an increase in levels of fibronectin (p < 0.05), particularly the specific ED-A alternatively spliced variant of fibronectin, in the theca externa. Follicular atresia was characterized by a loss of P450 aromatase in granulosa cells (p < 0.001) and a decrease in levels of P450(17 alpha) in the theca interna (p < 0.05). Simultaneously, levels of fibronectin (p < 0.05), particularly the ED-A variant of fibronectin, decreased in the theca externa of atretic follicles. Within the wall of granulosa cells, levels of fibronectin (p < 0.05), laminin, type IV collagen, and heparan sulfate proteoglycans strongly increased during follicular atresia. Overall, these results show that follicular growth and atresia were associated with distinct changes in levels of ECM components, suggesting that ECM components may play a role in the regulation of proliferation, differentiation, and apoptosis of follicular cells.

Proteolytic activity degrading insulin-like growth factor-binding protein-2, -3, -4, and -5 in healthy growing and atretic follicles in the pig ovary.

In the pig, ovarian follicular growth is characterized by an increase in intrafollicular levels of insulin-like growth factor-binding protein (IGFBP)-3 and a decrease in the levels of IGFBPs < 40 kDa (IGFBP-2, -4 and, to a lesser extent, a 30-kDa IGFBP likely corresponding to IGFBP-5). In contrast, atresia is primarily associated with a strong increase in intrafollicular levels of IGFBP-2 and -4, with intrafollicular levels of IGFBP-3 and -5 varying slightly or not at all. The purpose of the present study was to determine whether intrafollicular proteases are involved in such changes. Porcine follicular development was synchronized with a progestin, and individual follicles were isolated 12 h and 96 h after progestin withdrawal. Follicular fluid from follicles of various sizes and qualities was collected and incubated alone or with a source of exogenous bovine IGFBP-2 or human IGFBP-3, -4, or -5 for 20 h at 37 degrees C. Samples were then analyzed by Western ligand blotting and by immunoblotting using specific antisera. Porcine follicular fluid from various classes of follicles contained proteolytic activity degrading IGFBP-2, -4, and -5. In contrast, intrafollicular IGFBP-3 proteolytic activity was very low or nondetectable. In preovulatory follicles, degradation of IGFBPs < 40 kDa was 1) accompanied by the generation of small proteolytic fragments visualized by immunoblotting, 2) strongly inhibited by EDTA and 1,10-phenanthroline, and 3) dependent on the presence of zinc and calcium chloride. PMSF (1 mM, serine protease inhibitor) inhibited degradation of IGFBP-2 and to a lesser extent IGFBP-4, but not IGFBP-5. Other serine and cysteine protease inhibitors as well as TIMP-2 and BB-2116 (natural tissue inhibitor-2 and synthetic inhibitor of matrix metalloproteinases [MMPs], respectively) were ineffective. Gelatin-substrate zymography revealed the presence of two major intrafollicular gelatinase MMPs at 60 kDa and 76-85 kDa (likely MMPs 2 and 9, respectively), the levels of which decreased (76-85 kDa) or strongly increased (60 kDa) during follicular atresia. Follicular growth at diameters between 2 and 6-7 mm was characterized by a dramatic increase in proteolytic activity degrading IGFBP-2, -5 and, to a lesser extent, IGFBP-4. Atresia, in contrast, was associated with a marked decrease in proteolytic activity degrading IGFBP-2, -4, and -5. These results suggest that 1) changes in proteolytic activity of intrafollicular IGFBPs < 40 kDa are at least partly responsible for the changes in intrafollicular IGFBP levels during follicular growth and atresia in the pig and 2) calcium- and zinc-dependent metalloprotease(s) as well as serine protease(s) are involved in degradation of intrafollicular IGFBPs < 40 kDa.