Regulation of folliculogenesis and the determination of ovulation rate in ruminants.

The paper presents an update of our 1993 model of ovarian follicular development in ruminants, based on knowledge gained from the past 15 years of research. The model addresses the sequence of events from follicular formation in fetal life, through the successive waves of follicular growth and atresia, culminating with the emergence of ovulatory follicles during reproductive cycles. The original concept of five developmental classes of follicles, defined primarily by their responses to gonadotrophins, is retained: primordial, committed, gonadotrophin-responsive, gonadotrophin-dependent and ovulatory follicles. The updated model has more extensive integration of the morphological, molecular and cellular events during folliculogenesis with systemic events in the whole animal. It also incorporates knowledge on factors that influence oocyte quality and the critical roles of the oocyte in regulating follicular development and ovulation rate. The original hypothetical mechanisms determining ovulation rate are retained but with some refinements; the enhanced viability of gonadotrophin-dependent follicles and increases in the number of gonadotrophin-responsive follicles by increases in the throughput of follicles to this stage of growth. Finally, we reexamine how these two mechanisms, which are thought not to be mutually exclusive, appear to account for most of the known genetic and environmental effects on ovulation rate.

Factors affecting oocyte quality: who is driving the follicle?

Mammalian ovaries contain a large stock of oocytes enclosed in primordial follicles. Ovarian cyclic activity induces some of these follicles to initiate growth towards a possible ovulation. However, most of these follicles terminate their growth at any moment and degenerate through atresia. In growing follicles, only a subset of oocytes are capable to support meiosis, fertilization and early embryo development to the blastocyst stage, as shown through embryo in vitro production experiments. This proportion of competent oocytes is increasing along with follicular size. Growing lines of evidence suggest that oocyte competence relies on the storage of gene products (messenger RNA or protein) that will be determinant to support early stages of embryo development, before full activation of embryonic genome. Given these facts, the question is: are these gene products stored in oocytes during late folliculogenesis, allowing an increasing proportion of them to become competent? Alternatively, these transcripts may be stored during early folliculogenesis as the oocyte grows and displays high transcription activity. Several arguments support this latter hypothesis and are discussed in this review: (i) many attempts at prolonged culture of oocytes from antral follicles have failed to increase developmental competence, suggesting that developmental competence may be acquired before antral formation; (ii) the recent discovery of oocyte secreted factors and of their ability to regulate many parameters of surrounding somatic cells, possibly influencing the fate of follicles between ovulation or atresia, suggests a central role of oocyte quality in the success of folliculogenesis. Finally, in addition to their role in interfollicular regulation of ovulation rate, late folliculogenesis regulation and atresia could also be seen as a selective process aimed at the elimination through follicular atresia of oocytes that did not succeed to store proper gene products set during their growth.

Expression and biological effects of bone morphogenetic protein-15 in the hen ovary.

The bone morphogenetic protein 15 (Bmp15) and growth differentiation factor 9 (Gdf9) genes are two members of the transforming growth factor-beta superfamily. In mammals, these genes are known to be specifically expressed in oocytes and to be essential for female fertility. However, potential ovarian roles of BMPs remain unexplored in birds. The aim of the present work was to study for the first time the expression of Bmp15 in the hen ovary, to compare its expression pattern with that of Gdf9, and then to investigate the effects of BMP15 on granulosa cell (GC) proliferation and steroidogenesis. We found that chicken Bmp15 and Gdf9 genes were preferentially expressed in the ovary. We showed using in situ hybridization that Bmp15 and Gdf9 mRNAs were specifically localized in oocytes of all ovarian follicles examined. We also demonstrated using real-time quantitative RT-PCR that Bmp15 and Gdf9 expression was maintained during hierarchical follicular maturation in the gerrminal disc region and then progressively declined after ovulation. BMP15 was able to activate Smad1 (mothers against decapentaplegichomolog1) signaling pathway in hen GCs. Moreover, we showed a strong inhibitory effect of BMP15 on gonadotropin-induced progesterone production in hen GCs. This inhibitory effect was associated with a decrease in steroidogenic acute regulatory protein (STAR) level. Taken together, our results suggest that BMP15 may have a key role in the female fertility of birds.

Regulatory elements controlling pituitary-specific expression of the human prolactin gene.

We have performed transfection and DNase I footprinting experiments to investigate pituitary-specific expression of the human prolactin (hPRL) gene. When fused to the chloramphenicol acetyltransferase (CAT) reporter gene, 5,000 base pairs of the 5'-flanking sequences of the hPRL gene were able to drive high cat gene expression in prolactin-expressing GH3B6 cells specifically. Deletion analysis indicated that this pituitary-specific expression was controlled by three main positive regulatory regions. The first was located just upstream from the TATA box between coordinates -40 and -250 (proximal region). We have previously shown that three motifs of this region bind the pituitary-specific Pit-1 factor. The second positive region was located in the vicinity of coordinates -1300 to -1750 (distal region). DNase I footprinting assays revealed that eight DNA motifs of this distal region bound protein Pit-1 and that two other motifs were recognized by ubiquitous factors, one of which seems to belong to the AP-1 (jun) family. The third positive region was located further upstream, between -3500 and -5000 (superdistal region). This region appears to enhance transcription only in the presence of the distal region.

Peroxisome proliferator-activated receptors in reproductive tissues: from gametogenesis to parturition.

Peroxisome proliferator-activated receptors (PPARalpha, PPARbeta/delta and PPARgamma) are a family of nuclear receptors that are activated by binding of natural ligands, such as polyunsaturated fatty acids or by synthetic ligands. Synthetic molecules of the glitazone family, which bind to PPARgamma, are currently used to treat type II diabetes and also to attenuate the secondary clinical symptoms frequently associated with insulin resistance, including polycystic ovary syndrome (PCOS). PPARs are expressed in different compartments of the reproductive system (hypothalamus, pituitary, ovary, uterus and testis). Conservative functions of PPARs in mammalian species could be suggested through several in vivo and in vitro studies, especially in the ovary and during placental development. Several groups have described a strong expression of PPARgamma in ovarian granulosa cells, and glitazones modulate granulosa cell proliferation and steroidogenesis in vitro. All these recent data raise new questions about the biologic actions of PPARs in reproduction and their use in therapeutic treatments of fertility troubles such as PCOS or endometriosis. In this review, we first describe the roles of PPARs in different compartments of the reproductive axis (from male and female gametogenesis to parturition), with a focus on PPARgamma. Secondly, we discuss the possible molecular mechanisms underlying the effect of glitazones on PCOS. Like other 'insulin sensitizer' molecules, such as metformin, glitazones may in fact act directly on ovarian cells. Finally, we discuss the eventual actions of PPARs as mediators of environmental toxic substances for reproductive function.

Apoptosis of Sertoli cells after conditional ablation of murine double minute 2 (Mdm2) gene is p53-dependent and results in male sterility.

Beside its well-documented role in carcinogenesis, the function of p53 family has been more recently revealed in development and female reproduction, but it is still poorly documented in male reproduction. We specifically tested this possibility by ablating Mdm2, an E3 ligase that regulates p53 protein stability and transactivation function, specifically in Sertoli cells (SCs) using the AMH-Cre line and created the new SC-Mdm2(-/-) line. Heterozygous SC-Mdm2(-/+) adult males were fertile, but SC-Mdm2(-/-) males were infertile and exhibited: a shorter ano-genital distance, an extra duct along the vas deferens that presents a uterus-like morphology, degenerated testes with no organized seminiferous tubules and a complete loss of differentiated germ cells. In adults, testosterone levels as well as StAR, P450c17 (Cyp17a1) and P450scc (Cyp11a1) mRNA levels decreased significantly, and both plasma LH and FSH levels increased. A detailed investigation of testicular development indicated that the phenotype arose during fetal life, with SC-Mdm2(-/-) testes being much smaller at birth. Interestingly, Leydig cells remained present until adulthood and fetal germ cells abnormally initiated meiosis. Inactivation of Mdm2 in SCs triggered p53 activation and apoptosis as early as 15.5 days post conception with significant increase in apoptotic SCs. Importantly, testis development occurred normally in SC-Mdm2(-/-) lacking p53 mice (SC-Mdm2(-/-)p53(-/-)) and accordingly, these mice were fertile indicating that the aforementioned phenotypes are entirely p53-dependent. These data not only highlight the importance of keeping p53 in check for proper testicular development and male fertility but also certify the critical role of SCs in the maintenance of meiotic repression.

Pit-1 binding sequences permit calcium regulation of human prolactin gene expression.

This study examines the regulation of the human PRL (hPRL) gene promoter by intracellular calcium. Deletants of the 5'-flanking region of the hPRL gene and constructs consisting of the thymidine kinase promoter linked to the first or second proximal Pit-1 binding site were fused to the bacterial chloramphenicol acetyl transferase (CAT) reporter gene. With the complete 5-kilobase pair (kbp) hPRL promoter sequence the calcium channel agonist Bay K8644 induced a significant 2-fold increase in CAT reporter gene expression and the antagonist verapamil a 4.5-fold reduction, using GH3 cells cultured in physiological levels of calcium. The transcriptional response to calcium influx was similar with a series of 5'-deleted hPRL-CAT constructs including those that comprised the proximal (up to 740 bp) or distal (-1300- to -1700-bp) sequences alone. When treating cells cultured in low calcium conditions the induction with the hPRL promoter increased to 5-fold on the addition of exogenous calcium and Bay K8644. The pituitary-specific expression of the hPRL gene is conferred by the interaction of the pituitary-specific factor Pit-1 with several binding sites located in the 5'-flanking DNA, of which three are located in the proximal region. This suggested that Pit-1 binding sites may be involved in the calcium response.(ABSTRACT TRUNCATED AT 250 WORDS)

Localization, characterization, and quantification of insulin-like growth factor-I-binding sites in the ewe ovary.

To assess a potential role of insulin-like growth factor-I (IGF-I) in the ewe ovary, the presence of IGF-I receptors and IGF-I-binding proteins was studied by binding assays performed on granulosa cell suspensions, in follicular fluid, and on ovarian sections. On the ovarian sections, labeling was quantified after autoradiography by microphotometry. Competition studies with IGF-I and insulin allowed us to estimate the relative proportions of binding proteins and type I receptors in the different compartments of the ewe ovary. Our results clearly show that saturable, specific, and high affinity IGF-I receptors are present on the ovine granulosa cells. At equilibrium for both granulosa cell suspensions and frozen sections, the Kd value was close to 2 nM. IGF-I binding proteins were also present in follicular fluid and stroma, thecal, and granulosa cells. At equilibrium for follicular fluid, the Kd value was 0.91 +/- 0.27 nM (mean +/- SE). Moreover, on frozen sections, it was shown that atresia of small follicles (less than 2 mm) was accompanied by a decrease in the number of IGF-I receptors and an increase in the number of IGF-I-binding proteins on granulosa cells. By contrast, this phenomenon was not observed in large follicles. These data indicate that granulosa cells of ewe ovary possess type I receptors, and IGF-I-binding proteins may modulate IGF-I action in the process of follicular growth and atresia.

Changes in insulin-like growth factor-I (IGF-I), IGF-II, and their binding proteins during growth and atresia of ovine ovarian follicles.

Levels of insulin-like growth factor-I (IGF-I), IGF-II, and IGF-binding proteins (IGFBPs) were studied in ovine follicular fluid and serum with a view to determining their respective endocrine and/or paracrine roles in ovarian folliculogenesis. Ovarian follicles of Ile-de-France ewes were dissected and measured individually, and their follicular fluid collected. Follicles were classified according to size and degree of atresia, assessed on the basis of microscopical examination of smears of granulosa cells. Follicular fluid and serum samples were assayed for IGF-I and IGF-II. Free IGF-binding activity was also determined, and the IGFBP profiles in serum and follicular fluid were examined by Western ligand blotting [44- to 42-kilodalton (kDa) doublet and 35-kDa, 28.5- to 32-kDa, and 24-kDa bands], followed by densitometric analysis of the autoradiographs. Finally, the effects of follicular fluid IGFBPs on granulosa cell responses to IGF-I were studied in vitro. The size and atretic stage of the follicles had little influence on the IGF-I concentrations in follicular fluid, but IGF-II concentrations were approximately 1.5 times higher in small than in large follicles (P < 0.01). IGF-I levels were lower in fluid from large normal (highly vascularized) follicles than in serum (P < 0.01). Follicular fluid and serum IGF-I levels were positively correlated (r = 0.55; P < 0.05). No significant difference was found between follicular fluid and serum IGF-II levels. Follicular growth was accompanied by a decrease in free IGF-binding activity (P < 0.0001), a slight increase in the intensity of the 44- to 42-kDa IGFBP doublet (P < 0.05), and a clear decrease in the intensities of the 35-kDa (P < 0.0001) and 24-kDa bands. By contrast, follicular atresia was characterized by a marked increase in free IGF-binding activity (P < 0.0001) and strongly increased intensities of the 35-kDa, 28.5- to 32-kDa, and 24-kDa bands (P < 0.0001). Low mol wt IGFBPs, particularly the 24-kDa species, were clearly more abundant in serum than in follicular fluid from large normal follicles. In vitro experiments showed IGF-I to be less active on granulosa cells in the presence of follicular fluid from atretic than from normal follicles. The action of the IGF-I analog [Gln3,Ala4,Tyr15,Leu16]IGF-I, which has a weak affinity for the IGFBPs, was, however, similar whether atretic or normal follicular fluid was tested.(ABSTRACT TRUNCATED AT 400 WORDS)

Insulin-like growth factor binding protein-4 proteolytic degradation in ovine preovulatory follicles: studies of underlying mechanisms.

The regulation of insulin-like growth factor binding protein (IGFBP)-4 proteolytic degradation by insulin-like growth factors (IGFs) has been largely studied in vitro, but not in vivo. The aim of this study was to investigate the involvement of IGFs, IGFBP-2, IGFBP-3, and IGFBP-3 proteolytic fragments in the regulation of IGFBP-4 proteolytic activity in ovine ovarian follicles. Follicular fluid from preovulatory follicles contains proteolytic activity degrading exogenous IGFBP-4. The addition of an excess of IGF-I enhanced IGFBP-4 proteolytic degradation, whereas addition of IGFBP-2 or -3 or monoclonal antibodies against IGF-I and -II dose dependently inhibited IGFBP-4 proteolytic degradation. IGF-I and IGF-II, but not LongR3-IGF-I, reversed this inhibition in a dose-dependent manner. C-terminal, but not N-terminal, proteolytic fragments derived from IGFBP-3 (aa 161-264), as well as heparin-binding domain-containing peptides derived from the C-terminal domain of IGFBP-3 and -5 also induced the inhibition of IGFBP-4 proteolytic degradation. Other heparin-binding domain-containing peptides derived from the connective tissue growth factor (CTGF) and from proteins not related to IGFBP, heparan/heparin interacting protein (HIP) and vitronectin, but not from p36 subunit of annexin II tetramer, inhibited IGFBP-4 degradation. Furthermore, IGFBP-3, mutated on its heparin-binding domain, was not able to inhibit IGFBP-4 proteolytic degradation. So, in ovine preovulatory follicles, IGFBP-4 proteolytic degradation both 1) depends on IGFs, and 2) is inhibited by IGFBP-3 via its C-terminal heparin-binding domain as well as by heparin-binding domain containing peptides. These data suggest that in early atretic follicles, the increase in IGFBP-2 participates in the decrease in IGFBP-4 degradation. In late atretic follicles, the increase in the levels of C-terminal IGFBP-3 proteolytic fragments, generated by IGFBP-3 degradation, as well as the increase in IGFBP-5 expression would strengthen the inhibition of IGFBP-4 degradation. This inhibition might be partly mediated by direct interaction of IGFBP-4 proteinase(s) and heparin-binding domain within the C-terminal region from IGFBP-3 and -5.

Proteolytic activity is involved in changes in intrafollicular insulin-like growth factor-binding protein levels during growth and atresia of ovine ovarian follicles.

In the sheep, follicular growth is characterized by both an increase and a decrease in the level of intrafollicular insulin-like growth factor-binding protein-3 (IGFBP-3) and IGFBPs less than 40 kDa (IGFBP-2, -4, and -5), respectively. In contrast, follicular atresia is associated with a decrease and a large increase in levels of IGFBP-3 and IGFBPs less than 40 kDa, respectively. To assess whether intrafollicular proteases are involved in such changes, follicular fluid from follicles of different sizes and degrees of atresia was incubated alone or with pure human IGFBP-3, -4, or -5 or serum (as a source of exogenous IGFBP-2) for 20 h at 37 C. Samples were then analyzed by Western ligand blotting and by immunoblotting using specific antisera. Ovine follicular fluid from different classes of follicles contained proteolytic activity degrading IGFBP-2, -3, -4, and -5. Degradation of IGFBPs was accompanied by the generation of small proteolytic fragments visualized by immunoblotting or after autoradiography using radiolabeled IGFBP-4. Moreover, follicular growth and atresia were characterized by changes in IGFBP proteolytic activity. Indeed, follicular growth (between 2 and 6 mm in diameter) was characterized by 1) a decrease in IGFBP-3 proteolytic activity and 2) a dramatic increase in proteolytic activity degrading IGFBP-4 and, to a lesser extent, IGFBP-2 and -5. Atresia, in contrast, was associated with a strong increase in IGFBP-3 proteolytic activity in small ( < 3-mm diameter) follicles and a decrease in IGFBP-4 and -5 proteolytic activity in large ( > 5-mm diameter) follicles. Regardless of the follicle class, IGFBP proteolytic activity was strongly inhibited by EDTA and 1,10-phenanthroline, but very slightly or not at all inhibited by tissue inhibitor of matrix metalloprotease-1 and-2 and BB-2116 (natural and synthetic inhibitors of matrix metalloproteases, respectively) as well as cysteine and serine proteases inhibitors, with the exception of phenylmethylsulfonylfluoride (1 mM) in atretic follicles. In addition, IGFBP proteolytic activity was dependent on the presence of zinc and calcium chloride. Zymography experiments showed the presence of 72- and 92- to 96-kDa gelatinases in follicular fluid; their levels were dramatically increased during follicular atresia. These results suggest that 1) changes in intrafollicular IGFBP proteolytic activity could be at least partly responsible for the changes in intrafollicular IGFBP levels that occur during follicular growth and atresia in the sheep; and 2) metalloprotease(s) in healthy and atretic follicles as well as serine protease(s) in atretic follicles are involved in IGFBP degradation.

Pregnancy-associated plasma protein-A (PAPP-A) in ovine, bovine, porcine, and equine ovarian follicles: involvement in IGF binding protein-4 proteolytic degradation and mRNA expression during follicular development.

IGF binding protein-4 (IGFBP-4) proteolytic degradation is a common feature of preovulatory follicles from human, ovine, bovine, porcine, and equine ovary. In all these species, the protease is a zinc-dependent metalloprotease and its ability to degrade IGFBP-4 is IGF dependent. The human intrafollicular IGFBP-4-degrading protease has recently been identified as pregnancy-associated plasma protein-A (PAPP-A). The aim of this study was to investigate whether PAPP-A is also involved in IGFBP-4 degradation in ovine, bovine, porcine, and equine preovulatory follicles and to study the expression of PAPP-A mRNA in bovine and porcine granulosa cells from different classes of follicles. Immunoneutralization and immunoprecipitation with polyclonal antibodies raised against human PAPP-A inhibited IGFBP-4 proteolytic degradation in preovulatory follicular fluid from the four species studied. As previously reported for the intrafollicular proteolytic activity degrading IGFBP-4, recombinant human PAPP-A generated in vitro 17- and 10-kDa IGFBP-4-proteolytic fragments. Recombinant PAPP-A activity was also shown to be IGF dependent and was inhibited by heparin-binding domain-containing peptides. In all mammalian species studied, the PAPP-A sequences showed high degree of identity. Moreover, the PAPP-A gene was localized on porcine chromosome 1 (1q29-1q213), in agreement with the localization of human PAPP-A gene on human chromosome 9q33.1. In bovine and porcine ovaries, real-time quantitative RT-PCR showed that PAPP-A mRNA expression in granulosa cells was maximal in fully differentiated follicles and was positively correlated with expression of P450 aromatase and LH receptor mRNAs. Overall, these data show that PAPP-A is responsible for IGFBP-4 degradation in ovine, bovine, porcine, and equine preovulatory follicles. The high expression of PAPP-A mRNA in granulosa cells from large, differentiated follicles suggest that it is a new functional marker of follicular development.

Growth kinetics of the granulosa cell population in ovarian follicles: an approach by mathematical modelling.

This paper describes, from a mathematical viewpoint, the cellular changes in the granulosa of ovarian follicles during their terminal development. A dynamic model takes into account the processes of (1) cell division, (2) exit from the cell cycle towards differentiation, and (3) apoptotic cell death. Proliferative cells leave the cycle in an irreversible way. The risk of entering apoptosis applies to non-cycling cells. Changes in the cell numbers and in the growth fraction are derived from differential equations. The transitions between the different cell states are ruled by time-dependent rates. Numerical applications of the model concern ovulating and degenerating ovarian follicles in the ewe. The main feature of the ovulating case is the progressive exhaustion of the proliferating compartment for the benefit of the non-cycling cells. From an initial mainly proliferative state the granulosa progressively switches to a highly differentiated state, so that the growth fraction continuously decreases. In the atretic cases, the pattern of changes in the total viable cell number is influenced by the follicular age at the onset of the apoptotic process and by the intensity of the cell death rate. As apoptosis affects the non-cycling cells, the growth fraction is no longer strictly decreasing. The sensitivity of the model to the parameters is studied in a more general framework than the granulosa cell population.

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.

Genotyping of Cre-lox mice and detection of tissue-specific recombination by multiplex PCR.

Conditional gene targeting, based on Cre-lox or other systems, requires frequent genotyping of transgenic mouse populations and monitoring of tissue-specific Cre recombinatory efficiency. This is currently achieved by Southern analysis from tail- and tissue-derived DNA. Multiplex PCR amplification of the floxed (flanked by loxP sites) genomic region, combined with the PCR detection of the Cre transgene, simplifies this task. Here, we show that complete genotyping of a floxed locus is possible with three appropriately placed primers and that this triplex PCR can be performed simultaneously with a universal PCR assay for the detection of Cre transgenes. Using this approach, we also determined the ratios of recombined versus non-recombined floxed genomic segments in genomic DNA samples. This allowed us to estimate the efficiency of in vivo conditional inactivation from biopsy material and tissue samples that were too small for Southern analysis. As many new conditional knockouts are spatiotemporally restricted, such assays will become increasingly useful. The proposed PCR strategy is flexible and may be adapted to the structural specificities of any target gene.

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.

Proteolytic degradation of IGF-binding protein (IGFBP)-2 in equine ovarian follicles: involvement of pregnancy-associated plasma protein-A (PAPP-A) and association with dominant but not subordinated follicles.

In the ovary of mammalian species, terminal follicular growth is accompanied by a decrease in intrafollicular levels of IGF-binding protein-2 (IGFBP-2) and IGFBP-4. The decrease in IGFBP-4 levels is essentially due to an increase in proteolytic cleavage by intrafollicular pregnancy-associated plasma protein-A (PAPP-A) in growing healthy follicles. The decrease in IGFBP-2 levels is partly due to a decrease in mRNA expression by follicular cells. In addition, we have recently shown that IGFBP-2 is also proteolytically cleaved by PAPP-A in bovine and porcine growing follicles. In the present work, we showed that follicular fluid from late dominant equine follicles (35 mm diameter) contains a proteolytic activity against IGFBP-2. First follicular fluid from dominant follicles contained lower levels of native IGFBP-2 than the corresponding serum, as assessed by Western ligand blotting. In contrast, immunoblotting experiments showed much higher levels of a 12 kDa proteolytic fragment in dominant follicular fluid than in the serum. Moreover, equine dominant follicular fluid was able to induce proteolysis of exogenous recombinant bovine (rb)IGFBP-2, this degradation being dose-dependently enhanced by IGFs. The proteolytic activity against IGFBP-2 in equine follicles was partially immunoneutralized by a polyclonal antibody raised against human PAPP-A. Moreover, cleavage of rbIGFBP-2 by equine follicular fluid was dose-dependently inhibited by a peptide derived from the heparin-binding domain of IGFBP-5, as well as by peptides derived from other heparin-binding domain-containing proteins such as connective tissue growth factor, vitronectin and heparin-interacting protein, previously shown to inhibit PAPP-A. Finally, the proteolytic activity was very low in subordinate follicles, was high in both early (25 mm diameter) and late (35 mm diameter) dominant follicles, and was slightly lower in preovulatory follicles recovered 35 h after human chorionic gonadotropin (hCG) treatment.Overall, these data show that in the equine ovary, the selection of dominant follicles is associated with an increase of the proteolytic degradation of IGFBP-2 by PAPP-A, as for IGFBP-4, and potentially other protease(s), probably contributing to the increase in IGF bioavailability. In atretic subordinate follicles, the decrease in the proteolytic degradation of IGFBP-2, probably due in part to a direct inhibition by peptides containing heparin-binding domains, contributes to the increase in IGFBP-2 levels and the decrease in IGF bioavailability. The expression of PAPP-A and IGFBP-2 mRNA during folliculogenesis remain to be investigated in the mare.

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.

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.

Alterations of somatotropic function in prion disease in sheep.

This study aimed at investigating the possible linkage between natural scrapie and alterations of the somatotropic axis. Scrapie-affected ewes exhibited 2-fold higher mean GH concentrations during both autumn and spring. GH pulse frequencies were higher in scrapie-affected ewes than in control animals (mean+/-S.E.M. number of pulses/24 h: 10.4+/-0.9 and 7.6+/-0.9 for scrapie-affected and control ewes respectively) suggesting the involvement of central mechanisms. GH secretion induced by administration of an alpha(2)-adrenergic agonist, which acts centrally to stimulate GH secretion, was similar between healthy and scrapie-affected ewes (ratios of the area under the curve (AUC) of GH concentration after to the GH AUC before the agonist administration were 3.6+/-1.6 and 4.9+/-1.0 for scrapie-affected and control ewes respectively). Finally, humoral markers and parameters of the metabolic status were determined to test the hypothesis that scrapie-associated alterations of GH secretion could be related to disruption of metabolic homeostasis. Glucose, insulin and urea plasma concentrations were higher in scrapie-affected than in healthy ewes. Neither leptin nor IGF-I levels were affected by scrapie. Total thyroxine (T4) was decreased in scrapie-affected ewes but free T4 and total and free triiodothyronine were not modified. In conclusion, our results showed the existence in scrapie-affected ewes of endocrine and metabolic alterations typical of acute illness proceeding, at least in part, from central mechanisms.