The ovarian follicle of ruminants: the path from conceptus to adult.

This review resulted from an international workshop and presents a consensus view of critical advances over the past decade in our understanding of follicle function in ruminants. The major concepts covered include: (1) the value of major genes; (2) the dynamics of fetal ovarian development and its sensitivity to nutritional and environmental influences; (3) the concept of an ovarian follicle reserve, aligned with the rise of anti-Müllerian hormone as a controller of ovarian processes; (4) renewed recognition of the diverse and important roles of theca cells; (5) the importance of follicular fluid as a microenvironment that determines oocyte quality; (6) the 'adipokinome' as a key concept linking metabolic inputs with follicle development; and (7) the contribution of follicle development to the success of conception. These concepts are important because, in sheep and cattle, ovulation rate is tightly regulated and, as the primary determinant of litter size, it is a major component of reproductive efficiency and therefore productivity. Nowadays, reproductive efficiency is also a target for improving the 'methane efficiency' of livestock enterprises, increasing the need to understand the processes of ovarian development and folliculogenesis, while avoiding detrimental trade-offs as greater performance is sought.

The follicular microenvironment in low (++) and high (I+B+) ovulation rate ewes.

Ewes with single copy mutations in GDF9, BMP15 or BMPR1B have smaller preovulatory follicles containing fewer granulosa cells (GC), while developmental competency of the oocyte appears to be maintained. We hypothesised that similarities and/or differences in follicular maturation events between WT (++) ewes and mutant ewes with single copy mutations in BMP15 and BMPR1B (I+B+) are key to the attainment of oocyte developmental competency and for increasing ovulation rate (OR) without compromising oocyte quality. Developmental competency of oocytes from I+B+ animals was confirmed following embryo transfer to recipient ewes. The microenvironment of both growing and presumptive preovulatory (PPOV) follicles from ++ and I+B+ ewes was investigated. When grouped according to gonadotropin-responsiveness, PPOV follicles from I+B+ ewes had smaller mean diameters with fewer GC than equivalent follicles in ++ ewes (OR = 4.4 ± 0.7 and 1.7 ± 0.2, respectively; P < 0.001). Functional differences between these genotypes included differential gonadotropin-responsiveness of GC, follicular fluid composition and expression levels of cumulus cell-derived VCAN, PGR, EREG and BMPR2 genes. A unique microenvironment was characterised in I+B+ follicles as they underwent maturation. Our evidence suggests that GC were less metabolically active, resulting in increased follicular fluid concentrations of amino acids and metabolic substrates, potentially protecting the oocyte from ROS. Normal expression levels of key genes linked to oocyte quality and embryo survival in I+B+ follicles support the successful lambing percentage of transferred I+B+ oocytes. In conclusion, these I+B+ oocytes develop normally, despite radical changes in follicular size and GC number induced by these combined heterozygous mutations.

Gestational nutrition 1: alterations to gestational nutrition can increase indicators of fertility in sheep.

The aim of this study was to examine the relationships between gestational nutrition, fetal ovarian development and offspring fertility in female sheep and to highlight the potential mechanisms underlying these relationships. Adult sheep (n = 79) were fed either a maintenance or 0.6 of maintenance plane of nutrition for the first 55 days of gestation and thereafter fed ad libitum. Fetuses were collected for analysis at days 55 and 75 of gestation. Female offspring were monitored from birth until 19 months of age. Effects of restricted nutrition were observed on maternal plasma concentrations of progesterone, creatinine, albumin and Ca2+ at day 55 and creatinine at day 75. Concentrations of metabolic factors and steroid hormones in day 75 fetal plasma were not affected by the restricted maternal plane of nutrition. At day 55 of gestation, fetal ovarian germ cell development was not affected by maternal plane of nutrition. At day 75 of gestation ovaries from fetuses whose dams were exposed to restricted nutrition contained more germ cells but had lower germ cell proliferation rates than controls. For female offspring at 8 months of age, the dams gestational plane of nutrition did not affect the onset of puberty, ovulation rate (OR) and antral follicle counts (AFC). At 19 months of age, ewes from dams exposed to the restricted plane of gestational nutrition had higher OR, AFC and progesterone concentrations while concentrations of FSH were lower. In conclusion, while effects on fertility per se are yet to be determined, a reduced maternal plane of gestational nutrition can improve indicators of fertility in female offspring.

Differential expression of CART in ewes with differing ovulation rates.

We hypothesised that cocaine- and amphetamine-regulated transcript (CARTPT) would be differentially expressed in ewes with differing ovulation rates. Expression of mRNA for CARTPT, as well as LHCGR, FSHR, CYP19A1 and CYP17A1 was determined in antral follicles ≥1 mm in diameter collected during the follicular phase in ewes heterozygous for the Booroola and Inverdale genes (I+B+; average ovulation rate 4) and ++ contemporaries (++; average ovulation rate 1.8). In ++ ewes (n = 6), CARTPT was expressed in small follicles (1 to <3 mm diameter), where 18.8 ± 2.5% follicles expressed CARTPT CART peptide was also detected in follicular fluid of some follicles of ++ ewes. In I+B+ ewes, 5/6 ewes did not have any follicles that expressed CARTPT, and no CART peptide was detected in any follicle examined. Expression pattern of CYP19A1 differed between I+B+ and ++ ewes with an increased percentage of small and medium follicles (3 to <4.5 mm diameter) but decreased percentage of large follicles (≥4.5 mm diameter) expressing CYP19A1 in the I+B+ ewes. Many of the large follicles from the I+B+ ewes appeared non-functional and expression of LHCGR, FSHR, CYP17A1 and CYP19A1 was less than that observed in ++ ewes. Expression of FSHR and CYP17A1 was not different between groups in small and medium follicles, but LHCGR expression was approximately double in I+B+ ewes compared to that in ++ ewes. Thus, ewes with high ovulation rates had a distinct pattern of expression of CARTPT mRNA and protein compared to ewes with normal ovulation rates, providing evidence for CART being important in the regulation of ovulation rate.

Ovarian characteristics in sheep with multiple fecundity genes.

Ewes heterozygous for combinations of the Inverdale (FecXI; I+), Booroola (FecB; B+) and Woodlands (FecX2W; W+) mutations have ovulation rates higher than each mutation separately. The aims of the experiments described herein were to examine the ovarian phenotypes in I+B+ and I+B+W+ ewes and to compare these with the appropriate ++ (controls), I+ and BB animals available for this study. The mean ± s.e.m. ovulation rates in the ++ (n = 23), I+ (10), I+B+ (7), I+B+W+ (10) and BB (3) animals were 1.8 ± 0.1, 2.5 ± 0.2, 6.6 ± 1.0, 9.6 ± 0.9 and 9.7 ± 0.9 respectively. The maximum number of granulosa cells per follicle in the ++ and I+ genotypes was accumulated after exceeding 5 mm diameter, whereas in I+B+, I+B+W+ and BB animals, this was achieved when follicles reached >2-3 mm. The number of putative preovulatory follicles, as assessed from those with LH-responsive granulosa cells, 24 h after the induction of luteolysis, was higher (P < 0.01) in the I+B+ and I+B+W+ compared to the ++ and I+ genotypes. The median follicular diameters of these follicles in the ++, I+, I+B+, I+B+W+ and BB genotypes were 6, 5, 3, 3 and 3 mm respectively. The total number of granulosa cells in the putative preovulatory follicles when added together, and total mass of luteal tissue, did not differ between the genotypes. Thus, despite large ovulation rate differences between animals with one or more fecundity genes, the total cell compositions over all preovulatory follicles and corpora lutea, when added together, are similar to that from the one or two such follicles in the wild types.

Signalling pathways involved in the synergistic effects of human growth differentiation factor 9 and bone morphogenetic protein 15.

Growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) act synergistically to regulate granulosa cell proliferation and steroid production in several species. Several non-Sma and mothers against decapentaplegic (SMAD) signalling pathways are involved in the action of murine and ovine GDF9 and BMP15 in combination, with the pathways utilised differing between the two species. The aims of this research were to determine if human GDF9 and BMP15 also act in a synergistic manner to stimulate granulosa cell proliferation and to identify which non-SMAD signalling pathways are activated. Human GDF9 with BMP15 (GDF9+BMP15) stimulated an increase in (3)H-thymidine incorporation (P<0.001), which was greater than the increase with BMP15 alone, while GDF9 alone had no effect. The stimulation of (3)H-thymidine incorporation by GDF9+BMP15 was reduced by the addition of inhibitors to the SMAD2/3, nuclear factor-KB (NF-KB) and c-Jun N-terminal kinase (JNK) signalling pathways. Inhibitors to the SMAD1/5/8, extracellular signal-regulated kinase mitogen-activated protein kinase (ERK-MAPK) or p38-MAPK pathways had no effect. The addition of the BMP receptor 2 (BMPR2) extracellular domain also inhibited stimulation of (3)H-thymidine incorporation by GDF9+BMP15. In conclusion, human GDF9 and BMP15 act synergistically to stimulate granulosa cell proliferation, a response that also involves species-specific non-SMAD signalling pathways.

Mitochondria and vesicles differ between adult and prepubertal sheep oocytes during IVM.

Oocytes from prepubertal animals have a reduced ability to undergo normal embryo development and produce viable offspring. The correct quantity, activity and cytoplasmic distribution of oocyte organelles are essential for oocyte maturation, fertilisation and subsequent embryo development. The aim of this study was to quantify the ultrastructural differences between oocytes from prepubertal lamb and adult ewes using electron microscopy and stereology. We also determined whether quantitative polymerase chain reaction (qPCR) methods give comparable estimates of mitochondrial number to stereology. Mean storage vesicle volume was greater in adult compared with lamb oocytes before IVM and decreased during maturation in both adult and lamb oocytes. Mitochondrial volume and number increased in adult oocytes during maturation; however, no increase was observed in lamb oocytes. Mitochondrial DNA copy number measured by qPCR showed no differences between adult and lamb oocytes. A different distribution of mitochondria was observed in lamb oocytes before maturation, while the percentage of hooded mitochondria increased during maturation in adult oocytes and decreased in the lamb. In conclusion, the present study has identified differences in the vesicles and mitochondria between adult and lamb oocytes from ewes that may contribute to reduced developmental competence in prepubertal oocytes.

Creation of DNA aptamers against recombinant bone morphogenetic protein 15.

The oocyte-derived growth factor bone morphogenetic protein (BMP) 15 plays important roles in fertility, but its mechanism of action differs between species. Generation of BMP15-binding molecules, as an essential investigation tool, would be helpful to provide valuable insight into the underlying biological features of BMP15. The BMP15-binding molecules could be antibodies or aptamers. Aptamers have many advantages over antibodies as macromolecular ligands for target proteins. DNA aptamers can be obtained by a method of Systematic Evolution of Ligands by EXponential enrichment (SELEX) beginning with a pool of random sequences. However, the success of this technique cannot be guaranteed if the initial pool lacks candidate sequences. Herein, we report on the creation of DNA aptamers by means of modified SELEX. The modification included enhanced mutation and progressive selection during an in vitro evolutionary process. As a proof-of-principle, we started from a single sequence instead of a multiple-sequence pool. Functional aptamers against the recombinant BMP15 were successfully created and identified.

Oocyte expression, secretion and somatic cell interaction of mouse bone morphogenetic protein 15 during the peri-ovulatory period.

Bone morphogenetic protein 15 (BMP15) is a key intraovarian growth factor regulating mammalian fertility, yet expression and localisation of different BMP15 protein forms within ovarian follicles around the time of the preovulatory LH surge remains unclear. Using immunoblotting and immunocytochemistry, the present study identified that post-translationally processed BMP15 proregion and mature proteins are increasingly expressed and localised with cumulus and granulosa cells from mice treated with pregnant mare's serum gonadotropin (PMSG) + human chorionic gonadotrophin (hCG). However, this increased expression was absent in cumulus-oocyte complexes matured in vitro. Pull-down assays further revealed that the recombinant BMP15 proregion is capable of specific interaction with isolated granulosa cells. To verify an oocyte, and not somatic cell, origin of Bmp15 mRNA and coregulated growth differentiation factor 9 (Gdf9), in situ hybridisation and quantitative polymerase chain reaction results confirmed the exclusive oocyte localisation of Bmp15 and Gdf9, regardless of treatment or assay method. Relative oocyte expression levels of Bmp15 and Gdf9 decreased significantly after PMSG + hCG treatment; nevertheless, throughout all treatments, the Bmp15:Gdf9 mRNA expression ratio remained unchanged. Together, these data provide evidence that the preovulatory LH surge leads to upregulation of several forms of BMP15 protein secreted by the oocyte for putative sequestration and/or interaction with ovarian follicular somatic cells.

Mutations in the leptin receptor gene associated with delayed onset of puberty are also associated with decreased ovulation and lambing rates in prolific Davisdale sheep.

The aim of this study was to determine if single nucleotide polymorphisms (SNPs) in the leptin receptor (LEPR) gene associated with delayed onset of puberty are associated with changes in other reproductive traits in adult ewes. The ovulation rate of ewes homozygous for the SNPs was ~15% lower (PPLEPR SNPs than their wild-type or heterozygous contemporaries. Partial failure of multiple ovulations was also increased (PLEPR had on average 0.2 fewer lambs at mid-pregnancy and at birth compared with the wild-type or heterozygous ewes (PLEPR were strongly associated with poorer reproductive performance in Davisdale ewes, which is likely to be linked to both a reduced number of ova available for fertilisation and an increased number of ewes failing to become pregnant. Increased partial failure of multiple ovulations in ewes with high ovulation rates (i.e. 3 or greater) may also contribute to the poor reproductive performance.

Single-nucleotide polymorphisms in the LEPR gene are associated with divergent phenotypes for age at onset of puberty in Davisdale ewes.

Attainment of puberty is a key developmental event influenced by genetic and environmental factors. In examining age at attainment of puberty, we observed closely related rams from the Davisdale line whose daughters differed in age at which they attained puberty. A candidate gene approach was used to identify mutations that may underlie these observed differences. Four rams with divergent phenotypes for their daughter's age at onset of puberty were selected for whole-genome sequencing. The coding regions of genes with known roles in regulating reproductive function were searched for single-nucleotide polymorphisms (SNPs) that altered the amino acid sequence of the protein. Of interest were three SNPs in the leptin receptor gene (LEPR). A Sequenom assay was developed to determine the genotype of these SNPs in daughters of 17 sons of a founding sire. A higher percentage of ewe lambs homozygous for the LEPR mutations failed to undergo puberty before 1 yr of age, and those that did undergo puberty during the first breeding season on average were approximately 17 days older than homozygous wild-type ewes. Heterozygous ewes were intermediate for both measurements. Given the predicted change in protein function produced by the mutation in LEPR and the strong associations between the genotype and onset of puberty phenotypes, we propose that this mutation in LEPR underlies the observed difference in age at onset of puberty in the Davisdale line. Furthermore, these animals will likely provide a useful model to better understand the role of leptin in the regulation of puberty.

The role of the oviduct environment in embryo survival.

CONTEXT: Declining fertility is an issue in multiple mammalian species. As the site of fertilisation and early embryo development, the oviduct plays a critical role in embryo survival, yet there is a paucity of information on how the oviduct regulates this process. AIMS: We hypothesised that differences in steroid hormone signalling and/or immune function would be observed in a model of poor embryo survival, the peripubertal ewe. METHODS: We examined expression of steroid hormones in systemic circulation, oviductal expression of oestrogen receptorαand genes important in steroid hormone signalling, and immune function in pregnant and cyclic peripubertal and adult ewes on day 3 after oestrus. KEY RESULTS: Concentrations of progesterone, but not oestradiol, were decreased in the peripubertal ewe compared to the adult ewe. Oestrogen receptorαprotein expression was increased in the peripubertal ewe, but pathway analysis of gene expression revealed downregulation of the oestrogen signalling pathway compared to the adult ewe. Differential expression of several genes involved in immune function between the peripubertal and adult ewe was consistent with an unfavourable oviductal environment in the peripubertal ewe lamb. Oestradiol concentration was positively correlated with the expression of multiple genes involved in the regulation of immune function. CONCLUSIONS: Differences in the immune environment of the oviduct, potentially linked to differential modulation by steroid hormones, may partially underly the poor fertilisation and early embryo survival observed in the peripubertal ewe. IMPLICATIONS: A unfavourable oviductal environment may play an important role in limiting reproductive success.

Evolution of cyclin B3 shows an abrupt three-fold size increase, due to the extension of a single exon in placental mammals, allowing for new protein-protein interactions.

Cyclin B3 evolution has the unique peculiarity of an abrupt 3-fold increase of the protein size in the mammalian lineage due to the extension of a single exon. We have analyzed the evolution of the gene to define the modalities of this event and the possible consequences on the function of the protein. Database searches can trace the appearance of the gene to the origin of metazoans. Most introns were already present in early metazoans, and the intron-exon structure as well as the protein size were fairly conserved in invertebrates and nonmammalian vertebrates. Although intron gains are considered as rare events, we identified two cases, one at the prochordate-chordate transition and one in murids, resulting from different mechanisms. At the emergence of mammals, the gene was relocated from chromosome 6 of platypus to the X chromosome in marsupials, but the exon extension occurred only in placental mammals. A repetitive structure of 18 amino acids, of uncertain origin, is detectable in the 3,000-nt mammalian exon-encoded sequence, suggesting an extension by multiple internal duplications, some of which are still detectable in the primate lineage. Structure prediction programs suggest that the repetitive structure has no associated three-dimensional structure but rather a tendency for disorder. Splice variant isoforms were detected in several mammalian species but without conserved pattern, notably excluding the constant coexistence of premammalian-like transcripts, without the extension. The yeast two-hybrid method revealed that, in human, the extension allowed new interactions with ten unrelated proteins, most of them with specific three-dimensional structures involved in protein-protein interactions, and some highly expressed in testis, as is cyclin B3. The interactions with activator of cAMP-responsive element modulator in testis (ACT), germ cell-less homolog 1, and chromosome 1 open reading frame 14 remain to be verified in vivo since they may not be expressed at the same stages of spermatogenesis as cyclin B3.

Using sheep lines with mutations in single genes to better understand ovarian function.

Livestock populations have been subjected to strong selection pressure to improve reproductive success, and this has led to the identification of lines of animals with increased fecundity. These animals provide a rich biological resource for discovery of genes and regulatory mechanisms that underpin improved reproductive success. To date, three genes, all related to the transforming growth factor ß pathway, have been identified as having mutations that lead to alterations in ovulation in sheep. In addition, several other sheep lines have been identified with putative mutations in single genes with major effects on ovulation rate. This review is focused on the identification of the mutations affecting ovulation rate and how these discoveries have provided new insights into control of ovarian function.

Active immunization against the proregions of GDF9 or BMP15 alters ovulation rate and litter size in mice.

The transforming growth factor ß (TGFB) superfamily proteins bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9), are essential for mammalian fertility. Recent in vitro evidence suggests that the proregions of mouse BMP15 and GDF9 interact with their mature proteins after secretion. In this study, we have actively immunized mice against these proregions to test the potential in vivo roles on fertility. Mice were immunized with either N- or C-terminus proregion peptides of BMP15 or GDF9, or a full-length GDF9 proregion protein, each conjugated to keyhole limpet hemocyanin (KLH). For each immunization group, ovaries were collected from ten mice for histology after immunization, while a further 20 mice were allowed to breed and litter sizes were counted. To link the ovulation and fertility data of these two experimental end points, mice were joined during the time period identified by histology as being the ovulatory period resulting in to the corpora lutea (CL) counted. Antibody titers in sera increased throughout the study period, with no cross-reactivity observed between BMP15 and GDF9 sera and antigens. Compared with KLH controls, mice immunized with the N-terminus BMP15 proregion peptide had ovaries with fewer CL (P<0.05) and produced smaller litters (P<0.05). In contrast, mice immunized with the full-length GDF9 proregion not only had more CL (P<0.01) but also had significantly smaller litter sizes (P<0.01). None of the treatments affected the number of antral follicles per ovary. These findings are consistent with the hypothesis that the proregions of BMP15 and GDF9, after secretion by the oocyte, have physiologically important roles in regulating ovulation rate and litter size in mice.

Effects of species differences on oocyte regulation of granulosa cell function.

The aims were to investigate whether oocyte-secreted growth factors from a high (i.e. rat) and low (i.e. sheep) ovulation rate species could stimulate (3)H-thymidine incorporation in granulosa cells (GC) from antral follicles from the same or across species. Denuded oocytes (DO) were co-incubated with GC with or without specific antibodies to growth differentiating factor 9 (GDF9) or bone morphogenetic protein 15 (BMP15). Co-incubations of DO-GC from the same or across species significantly increased thymidine incorporation in GC with increasing numbers of DO. GDF9 immuno-neutralisation reduced thymidine incorporation in rat GC co-incubated with either rat or ovine DO and in ovine GC co-incubated with ovine or rat DO. BMP15 immuno-neutralisation only reduced thymidine incorporation when ovine DO were co-incubated with either ovine or rat GC. Western blotting of oocytes co-incubated with GC identified GDF9 and BMP15 proteins for sheep and GDF9 protein for rats in oocyte lysates and incubation media. With respect to rat BMP15, a promature protein was identified in the oocyte lysate but not in media. Expression levels of GDF9 relative to BMP15 mRNA in DO co-incubated with GC were highly correlated (R (2)=0.99) within both species. However, the expression ratios were markedly different for the rat and sheep (4.3 vs 1.0 respectively). We conclude that during follicular development, rat oocytes secrete little, if any, BMP15 and that GDF9 without BMP15 can stimulate proliferation of rat and ovine GC. In contrast, ovine oocytes secrete both BMP15 and GDF9, and both were found to stimulate proliferation in ovine and rat GC.

Booroola BMPR1B mutation alters early follicular development and oocyte ultrastructure in sheep.

Booroola ewes homozygous (BB) for a mutation in the bone morphogenetic protein receptor-1b (BMPR1B) gene exhibit higher ovulation rates, have larger diameter oocytes at earlier stages of follicular development (i.e. Type 3) and smaller diameter follicles at ovulation than wild-type (++) sheep. However, it is not known when BMPR1B is first expressed in the developing ovary or the cell types involved. In addition, the effects of the BMPR1B mutation on primordial (Type 1) follicles or during growth to the Type 3 stage are unknown. In the present study, BB and++fetal ovaries at Days 30-135 of gestation were screened by in situ hybridisation for BMPR1B mRNA. Ovaries from BB and++lambs were examined by microscopy to measure follicular and oocyte ultrastructural characteristics in Type 1-3 follicles. BMPR1B mRNA was observed in ovaries from Day 35 of gestation and was evident in oocytes of newly forming and fully formed Type 1 follicles. In BB animals, the Type 1 follicles had larger mean follicular and oocyte diameters, a greater volume of mitochondria, smooth endoplasmic reticulum and ribosomes and a greater surface area of junctions with the granulosa cells compared with++animals. It is concluded that the BMPR1B mutation alters follicular development from the onset of follicular formation.

Identification of a line of sheep carrying a putative autosomal gene increasing ovulation rate in sheep that does not appear to interact with mutations in the transforming growth factor beta superfamily.

Sheep lines with mutations in single genes that have major effects on ovulation rate have been very useful in gaining a better understanding of pathways important in controlling follicular development and ovulation rate. To date however, all known mutations are in the transforming growth factor beta (TGFB) superfamily. Ovulation rates were measured in 720 progeny of 20 rams that were descendants of a single prolific ewe. Evaluation of ovulation rates of daughters of closely related sires suggests the presence of a segregating major gene Fecundity Davisdale (FECD) that increases ovulation rate between 0.4 and 0.8 in heterozygous daughters. Key features of mutations in genes of the TGFB superfamily pathway, such as synergistic interactions with other family members, infertility in homozygous carriers, and increased responsiveness to exogenous gonadotropins, were not observed in this line; thus, the mutation does not appear to be acting in the TGFB pathway. Hence, there is likely a novel mutation being carried in this line of sheep that alters ovulation rate. Future identification of the causative mutation may provide new insights into regulation of follicular development and ovulation rate.

Effects of immunizing ewes against bone morphogenetic protein 15 on their responses to exogenous gonadotrophins to induce multiple ovulations.

Sheep with a heterozygous inactivating mutation in the bone morphogenetic protein 15 (BMP15) gene experience an increased ovulation rate during either a natural oestrous cycle or a cycle in which exogenous FSH and eCG (gonadotrophins) are given to induce multiple ovulations. The primary aim of these studies was to determine whether ewes immunised against BMP15 would also show an improved superovulation rate following exogenous gonadotrophin treatment. A secondary aim was to determine the effects of BMP15 immunisation on ovarian follicular characteristics. In most ewes (i.e. > 75%) immunised with a BMP15-keyhole limpet haemocyanin peptide in an oil-based adjuvant in order to completely neutralise BMP15 bioactivity, there was no superovulation response to exogenous gonadotrophins. In ewes treated with exogenous gonadotrophins following a BMP15-BSA peptide immunisation in a water-based adjuvant to partially neutralise BMP15 bioactivity, the ovulation rate response was similar to the control superovulation treatment groups. Characterisation of follicular function revealed that the water-based BMP15-immunised animals had fewer non-atretic follicles 2.5-3.5 or > 4.5  mm in diameter compared with controls. Basal concentrations of cAMP were higher in granulosa cells from animals immunised against BMP15 than control animals. There were no significant differences in the concentrations of cAMP between granulosa cells from BMP15- and control-immunised animals when given FSH or hCG, although there were differences in the proportions of follicles in different size classes that responded to FSH or hCG. Thus, immunisation against BMP15 may have been causing premature luteinisation and thereby limiting the numbers of follicles recruited for ovulation following treatment with exogenous gonadotrophins.

Oocytes in sheep homozygous for a mutation in bone morphogenetic protein receptor 1B express lower mRNA levels of bone morphogenetic protein 15 but not growth differentiation factor 9.

The aim of this study was to test the hypothesis that the high ovulation rate in ewes (BB) homozygous for a mutation in the bone morphogenetic protein receptor type 1B (BMPR1B) gene is linked to lower BMP15 and/or GDF9 mRNA in oocytes compared with those in wild-type (++) ewes. Cumulus cell-oocyte complexes (COC) and granulosa cells (GC) were recovered from ≥1 mm diameter follicles of BB and ++ ewes during a prostaglandin-induced follicular phase. Expression levels of GDF9 and BMP15 were measured by multiplex qPCR from individual COC. The gonadotropin-induced cAMP responses of the GC from each non-atretic follicle were measured following treatment with FSH or human chorionic gonadotropin. In a separate validation experiment, GDF9 and BMP15 expression was present only in oocytes and not in cumulus cells. There was no effect of follicular diameter on oocyte-derived GDF9 or BMP15 mRNA levels. The mean expression levels of BMP15, but not GDF9, were significantly lower in all non-atretic follicles, including the subsets containing either FSH- or LH-responsive GC in BB, compared with ++, ewes. No genotype effects were noted for FSH-induced cAMP production by GC either with respect to dose of, or number of follicles responding to, FSH. However, ovaries from BB ewes contained significantly more follicles responsive to LH, with respect to cAMP production in GC. We propose that these findings are consistent with the hypothesis that the higher ovulation rate in BB sheep is due, at least in part, to lower oocyte-derived BMP15 mRNA levels together with the earlier onset of LH-responsiveness in GC.