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.

Activin B and Activin C Have Opposing Effects on Prostate Cancer Progression and Cell Growth.

Current prognostic and diagnostic tests for prostate cancer are not able to accurately distinguish between aggressive and latent cancer. Members of the transforming growth factor-ß (TGFB) family are known to be important in regulating prostate cell growth and some have been shown to be dysregulated in prostate cancer. Therefore, the aims of this study were to examine expression of TGFB family members in primary prostate tumour tissue and the phenotypic effect of activins on prostate cell growth. Tissue cores of prostate adenocarcinoma and normal prostate were immuno-stained and protein expression was compared between samples with different Gleason grades. The effect of exogenous treatment with, or overexpression of, activins on prostate cell line growth and migration was examined. Activin B expression was increased in cores containing higher Gleason patterns and overexpression of activin B inhibited growth of PNT1A cells but increased growth and migration of the metastatic PC3 cells compared to empty vector controls. In contrast, activin C expression decreased in higher Gleason grades and overexpression increased growth of PNT1A cells and decreased growth of PC3 cells. In conclusion, increased activin B and decreased activin C expression is associated with increasing prostate tumor grade and therefore have potential as prognostic markers of aggressive prostate cancer.

A schizophrenia risk factor induces marked anatomical deficits at GABAergic-dopaminergic synapses in the rat ventral tegmental area: Essential evidence for new targeted therapies.

To develop new therapies for schizophrenia, evidence accumulated over decades highlights the essential need to investigate the GABAergic synapses that presynaptically influence midbrain dopaminergic neurons. Since current technology restricts these studies to animals, and evidence accumulated in recent decades indicates a developmental origin of schizophrenia, we investigated synaptic changes in male rat offspring exposed to maternal immune activation (MIA), a schizophrenia risk factor. Using a novel combination of lentiviruses, peroxidase-immunogold double labeling, three-dimensional serial section transmission electron microscopy and stereology, we observed clear anatomical alterations in synaptic inputs on dopaminergic neurons in the midbrain posterior ventral tegmental area (pVTA). These changes relate directly to a characteristic feature of schizophrenia: increased dopamine release. In 3-month-old and 14-month-old MIA rats, we found a marked decrease in the volume of presynaptic GABAergic terminals from the rostromedial tegmental nucleus (RMTg) and in the length of the synapses they made, when innervating pVTA dopaminergic neurons. In MIA rats in the long-term, we also discovered a decrease in the volume of the postsynaptic density (PSD) and in the maximum thickness of the PSD at the same synapses. These marked deficits were evident in conventional GABA-dopamine synapses and in synaptic triads that we discovered involving asymmetric synapses that innervated RMTg GABAergic presynaptic terminals, which in turn innervated pVTA dopaminergic neurons. In triads, the PSD thickness of asymmetric synapses was significantly decreased in MIA rats in the long-term cohort. The extensive anatomical deficits provide a potential basis for new therapies targeted at synaptic inputs on midbrain pVTA dopaminergic neurons, in contrast to current striatum-targeted antipsychotic drugs.

The Δ133p53ß isoform promotes an immunosuppressive environment leading to aggressive prostate cancer.

Prostate cancer is the second most common cancer in men, for which there are no reliable biomarkers or targeted therapies. Here we demonstrate that elevated levels of Δ133TP53ß isoform characterize prostate cancers with immune cell infiltration, particularly T cells and CD163+ macrophages. These cancers are associated with shorter progression-free survival, Gleason scores ≥ 7, and an immunosuppressive environment defined by a higher proportion of PD-1, PD-L1 and colony-stimulating factor 1 receptor (CSF1R) positive cells. Consistent with this, RNA-seq of tumours showed enrichment for pathways associated with immune signalling and cell migration. We further show a role for hypoxia and wild-type p53 in upregulating Δ133TP53 levels. Finally, AUC analysis showed that Δ133TP53ß expression level alone predicted aggressive disease with 88% accuracy. Our data identify Δ133TP53ß as a highly accurate prognostic factor for aggressive prostate cancer.

Role of activin C in normal ovaries and granulosa cell tumours of mice and humans.

Activins and inhibins play important roles in the development, growth and function of the ovary. Mice lacking inhibin develop granulosa cell tumours in their ovaries that secrete activin A, and these tumours are modulated by increased activin C expression. The aim of the present study was to identify where activin C is expressed in mouse and human ovaries and whether overexpression of activin C modulates normal follicular development in mice. Immunohistochemical staining for the activin ßC subunit was performed on sections from mouse and human ovaries and human adult granulosa cell tumours. Stereology techniques were used to quantify oocyte and follicular diameters, and the percentage of different follicular types in ovaries from wild-type mice and those underexpressing inhibin α and/or overexpressing activin C. Staining for activin ßC was observed in the oocytes, granulosa cells, thecal cells and surface epithelium of mouse and human ovaries, and in the granulosa-like cells of adult granulosa cell tumours. Overexpression of activin C in mice did not alter follicular development compared with wild-type mice, but it did modulate the development of abnormal early stage follicles in inhibin α-null mice. These results provide further evidence of a role for activin C in the ovary.

The Role of Oocyte Organelles in Determining Developmental Competence.

The ability of an oocyte to undergo successful cytoplasmic and nuclear maturation, fertilization and embryo development is referred to as the oocyte's quality or developmental competence. Quality is dependent on the accumulation of organelles, metabolites and maternal RNAs during the growth and maturation of the oocyte. Various models of good and poor oocyte quality have been used to understand the essential contributors to developmental success. This review covers the current knowledge of how oocyte organelle quantity, distribution and morphology differ between good and poor quality oocytes. The models of oocyte quality are also described and their usefulness for studying the intrinsic quality of an oocyte discussed. Understanding the key critical features of cytoplasmic organelles and metabolites driving oocyte quality will lead to methods for identifying high quality oocytes and improving oocyte competence, both in vitro and in vivo.

Over-Expression of Activin-ßC Is Associated with Murine and Human Prostate Disease.

Activins are members of the TGF-ß superfamily and have been linked to prostate cancer. There are four mammalian activin subunits (ßA, ßB, ßC, and ßE) that dimerize to form functional proteins. The role of activin-A (ßA-ßA) has been relatively well characterized and has been shown to generally inhibit growth in the prostate. In contrast, little is known about the biological function of the ßC and ßE subunits. Previous work indicated activin-C (ßC-ßC) to be an antagonist of activin-A. This is important because resistance to activin-A growth inhibition occurs during prostate cancer progression. This paradox is not currently well understood. Hence, we hypothesize that local expression of the activin-ßC subunit antagonizes activin-A-dependent growth inhibition and represents a key factor contributing to acquired insensitivity to activin-A observed in prostate cancer progression. To test our hypothesis, we characterized the ventral prostate lobes of 9-month-old transgenic mice over-expressing activin-ßC and examined the expression of activin-ßA, activin-ßC, and the activin intracellular signaling factor, Smad-2, in human prostate diseases. Prostate epithelial cell hyperplasia, low-grade prostatic intraepithelial neoplasia (PIN) lesions, alterations in cell proliferation, and reduced Smad-2 nuclear localization were evident in mice over-expressing activin-ßC. Increased activin-ßA and -ßC subunit immunoreactive scores and decreased Smad-2 nuclear localization were also evident in human prostate cancer. This study suggests that over-expression of activin-ßC is associated with murine and human prostate pathologies. We conclude that the activin-ßC subunit may have therapeutic and/or diagnostic implications in human prostate disease.

Early embryo loss, morphology, and effect of previous immunization against androstenedione in the ewe.

In a naturally mated cycle, ova and viable embryo number as well as embryo size were assessed on Day 4, 10, 14, 18, and 30 of gestation in Romney ewes (n = 38-44 per gestational group). For Days 4-18 of gestation, embryos were recovered by flushing the reproductive tract after slaughtering of the ewe. Ovulation rate was determined by counting the number of corpora lutea present on both ovaries. For the Day 30 group, number of ovulations was measured by laparoscopic examination of the ovaries at Day 9-12 of the cycle, and number of embryos present was determined by ultrasound examination at approximately Day 30 of pregnancy. Most of embryo loss occurred before Day 14 of gestation with 6% loss before Day 4, and 12% loss between Day 4 and 14 of gestation. A similar proportion of viable embryos per number of ova ovulated were recovered on Day 14 and 18 (82%) and Day 30 (81%) of gestation. Fertilization failure was estimated at 1%. Conceptus and embryo size was most variable on Day 14, representing a period of rapid growth (conceptus length ± standard deviation); Day 4 (169 ± 8 µm), Day 10 (379 ± 93 µm), Day 14 (23 ± 32 mm), Day 18 (embryo length ± standard deviation; 5.0 ± 0.7 mm). Vaccination with commercially available fertility vaccines targeting androstenedione (Androvax and Ovastim) in previous seasons resulted in reduced conceptus size compared with controls. However, no difference in the proportion of viable embryos was observed between treatments, signifying maternal tolerance for considerable variation at this stage of development. Furthermore, the finding that most of loss occurs within the first 14 days of gestation highlights the importance of both oocyte quality and the uterine environment for the embryo to successfully overcome the challenges leading up to and including pregnancy recognition in the ewe.

Activin A and follistatin during the oestrous cycle and early pregnancy in ewes.

The activin pathway has been postulated to be involved in regulation of multiple reproductive processes important for survival of the conceptus. These processes include luteinisation of the follicular cells and thus function of the corpus luteum, early embryo development and uterine function including implantation of the conceptus. Therefore, the aim of the current study was to determine whether the concentrations of activin A and follistatin (FST), an activin-binding protein, differed between ewes with a lifetime history of enhanced or reduced embryonic survival (ES). The mRNAs encoding FST and activin A (inhibin beta A subunit; INHBA) were present in the uterus and abundant in the uterine luminal or glandular epithelia by day 18 of gestation. A peak of activin A was observed in the systemic circulation around the time of oestrus, and activin A concentrations were elevated in animals with reduced ES during the oestrous cycle and early gestation. Concentrations of activin A in uterine fluid were approximately twofold greater on day 16 of gestation in ewes with reduced ES compared to those with enhanced ES. No consistent differences in FST were observed between these groups. Treatment of luteinising ovine granulosa cells with activin A in vitro suppressed progesterone secretion providing evidence of a potential pathway whereby increased concentrations of activin A may decrease ES.

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.

Activins and activin antagonists in the human ovary and ovarian cancer.

Activins are members of the transforming growth factor ß superfamily that play an important role in controlling cell proliferation and differentiation in many organs including the ovary. It is essential that activin signalling be tightly regulated as imbalances can lead to uncontrolled cell proliferation and cancer. This review describes the expression and function of the activins and their known antagonists in both normal and cancerous human ovaries.

Effects of acetyl-L-carnitine on lamb oocyte blastocyst rate, ultrastructure, and mitochondrial DNA copy number.

Viable lambs can be produced after transfer of in vitro-derived embryos from oocytes harvested from prepubertal lambs. However, this occurs at a much lower efficiency than from adult ewe oocyte donors. The reduced competence of prepubertal oocytes is believed to be due, at least in part, to deficiencies in cytoplasmic maturation. Differences in the cytoplasmic ultrastructure between prepubertal and adult oocytes have been described in the sheep, pig, and cow. Prepubertal lamb oocytes have been shown to have a different distribution of mitochondria and lipid droplets, and less mitochondria and storage vesicles than their adult counterparts. L-carnitine plays a role in supplying energy to the cell by transporting long-chain fatty acids into mitochondria for ß-oxidation to produce ATP. Both L-carnitine and its derivative acetyl-L-carnitine have been reported to increase the blastocyst rate of oocytes from mice, cows, and pigs, treated during IVM. L-carnitine has also been shown to increase mitochondrial biogenesis in adipose cells. Therefore, the aims of this study were to determine if treatment of oocytes from prepubertal lambs with acetyl-L-carnitine during IVM could increase the blastocyst rate and alter mitochondria, vesicle, or lipid droplet number, volume, or distribution. The blastocyst rate was doubled in prepubertal lamb oocytes treated with acetyl-L-carnitine when compared to untreated oocytes (10.0% and 4.6%, respectively; P = 0.028). Light microscopy, scanning electron microscopy, and stereology techniques were used to quantify organelles in untreated and acetyl-L-carnitine-treated lamb oocytes, and quantitative polymerase chain reaction methods were used to measure the mitochondrial DNA copy number. There were no differences in mitochondrial volume, number, or mitochondrial DNA copy number. Acetyl-L-carnitine treatment increased the cytoplasmic volume (P = 0.015) of the oocytes, and there were trends toward an increase in the vesicle volume (P = 0.089) and an altered distribution of lipid droplets (P = 0.076). In conclusion, acetyl-L-carnitine can be used to increase the in vitro blastocyst rate of juvenile oocytes and therefore to improve juvenile in vitro embryo transfer methods. These methods can be used for livestock improvement by increasing the rate of genetic gain. Further work is required to identify the contents of the vesicles and confirm the mode of action of acetyl-L-carnitine in improving oocyte competence.

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.

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.

Signalling pathways involved in the cooperative effects of ovine and murine GDF9+BMP15-stimulated thymidine uptake by rat granulosa cells.

Growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) are oocyte-secreted factors known to be involved in regulating the proliferation and differentiation of granulosa cells during follicular growth. The aims of this study were to determine the signalling pathways used by recombinant forms of murine and ovine GDF9 and BMP15 in combination (GDF9+BMP15) and the molecular complexes formed by combinations of these factors. Differences in the molecular forms of combinations of murine and ovine GDF9+BMP15 were observed by western blot analysis. Ovine GDF9+BMP15-stimulated (3)H-thymidine uptake was completely blocked by SMAD2/3 and nuclear factor-κB pathway inhibitors and partially blocked by a p38-mitogen-activated protein kinase (MAPK) inhibitor. Thymidine uptake by murine GDF9+BMP15 was reduced by the SMAD2/3 and extracellular signal-regulated kinase-MAPK pathway inhibitors and increased after addition of a c-Jun N-terminal kinase inhibitor. Stimulation of (3)H-thymidine uptake by GDF9+BMP15 from either species was not affected by the SMAD1/5/8 pathway inhibitor. In conclusion, both murine and ovine GDF9+BMP15-stimulated thymidine incorporation in rat granulosa cells was dependent on the SMAD2/3 signalling pathway but not the SMAD1/5/8 pathway. Divergence in the non-SMAD signalling pathways used by murine and ovine GDF9+BMP15 was also evident and may be due to the differences observed in the molecular complexes formed by these factors. These results are consistent with the hypothesis that the disparate cooperative functions of GDF9 and BMP15 in different species are mediated by divergent non-SMAD signalling pathways.

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.

The conflict between hierarchical ovarian follicular development and superovulation treatment.

In mammals with a low ovulation rate phenotype, ovarian follicular development is thought to be hierarchical with few, if any, antral follicles at similar stages of development. The hypothesis being tested herein was that if most follicles are in a functionally different state, then the application of exogenous hormones to increase ovulation rate will not overcome the hierarchical nature of follicular development. Using sheep as the experimental model, the functional states of all non-atretic antral follicles > or =2 mm diameter were assessed in individual ewes (N=10/group) during anoestrus with or without pregnant mare's serum gonadotrophin (PMSG) treatment, or after a standard superovulation regimen, or during the follicular phase of the oestrous cycle. The functional states of these follicles were assessed by measuring the FSH- or human chorionic gonadotrophin (hCG)-induced cAMP responses of granulosa cells in vitro. There were significant overall effects across the treatment groups on the responses of granulosa cells to either FSH or LH (both P<0.001). It was concluded that for anoestrous ewes with or without PMSG treatment, and ewes during the follicular phase, granulosa cell populations of many follicles (> or =2 mm diameter) did not share a similar cAMP response to FSH ( approximately 50% of follicles) or hCG (>90% of follicles) either on a per cell or total cell basis. After superovulation, < or =30 and 10% respectively of the granulosa cell populations shared similar responses to FSH and LH with regard to follicular diameter and cAMP output. Thus, exogenous hormone treatments used routinely for increasing oocyte yield do not effectively override the hierarchical pattern of ovarian follicular development during the follicular phase.

The cooperative effect of growth and differentiation factor-9 and bone morphogenetic protein (BMP)-15 on granulosa cell function is modulated primarily through BMP receptor II.

Growth and differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15, GDF9B) are oocyte-derived proteins essential for the growth and function of ovarian follicles. Moreover, ovine (o) GDF9 and oBMP15 cooperate to increase both (3)H-thymidine incorporation and alpha-inhibin production and to inhibit progesterone production by rat or ovine granulosa cells. Although the receptors through which these proteins act individually have been determined, the receptor(s) involved in mediating the cooperative effects of GDF9 and BMP15 is (are) unknown. In this study, the effects of the extracellular domains of the types I and II TGFbeta receptors on (3)H-thymidine incorporation by rat granulosa cells stimulated by oGDF9 and oBMP15 were investigated. Stimulation of (3)H-thymidine incorporation was completely blocked by the BMP receptor II (BMPRII) extracellular domain but unaffected by any other type II or any type I receptor. These results suggest that the initial interaction of oGDF9 and oBMP15 is with BMPRII and that a type I receptor is either recruited or already associated with BMPRII to mediate the cooperative effects of these growth factors.

The effects of immunizing sheep with different BMP15 or GDF9 peptide sequences on ovarian follicular activity and ovulation rate.

The aims of these studies were to determine the abilities of antisera against different regions of ovine bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9) to inhibit ovarian follicular activity, estrus (mating), and ovulation in sheep. The 9-15-mer peptides were conjugated to keyhole limpet hemocyanin (KLH) and used to generate antibodies against the flexible N-terminal regions of the mature protein as well as against regions in which dimerization of the protein or interaction with a type 1 BMP or a type 2 TGFB or BMP receptor was predicted to occur. Ewes (n = 10 per treatment group) were vaccinated with KLH or the KLH-BMP15 (n = 9 different peptides) or KLH-GDF9 (n = 10) peptides in Freund adjuvant at five consecutive monthly intervals. Overall, antisera generated against peptides that corresponded to amino acid residues 1-15 of the N-terminus of the BMP15 or GDF9 mature protein or GDF9 amino acid residues 21-34 were the most potent at inhibiting ovulation following primary and single booster vaccination. Several other BMP15 (8/9) or GDF9 (6/10) treatment groups, but not KLH alone, also produced significant reductions in the numbers of animals that ovulated, although 2, 3 or 4 booster vaccinations were required. Anovulation was commonly associated with the inhibition of normal ovarian follicular development and anestrus. The in vitro neutralization studies with IgG from the BMP15 or GDF9 immunized ewes showed that the mean inhibition of BMP15 plus GDF9 stimulation of (3)H-thymidine uptake by rat granulosa cells was approximately 70% for animals without corpora lutea (CL), whereas for animals with one to three CL or more than three CL, the inhibition was 24%-33% or 27%-42%, respectively. In summary, these data suggest that reagents that block the biological actions of BMP15 or GDF9 at their N-termini have potential as contraceptives or sterilizing agents.

The role of bone morphogenetic proteins 2, 4, 6 and 7 during ovarian follicular development in sheep: contrast to rat.

The intraovarian roles of BMP family members such as BMP2, 4, 6 and 7 are not well understood, particularly in species with low ovulation rates such as sheep. Therefore, the objectives of these experiments were to determine the expression patterns of mRNAs encoding BMP2, 4, 6 and 7 during ovarian follicular development in sheep, and to determine the effects of these growth factors on ovine granulosa cell functions in vitro. For comparative purposes, the effects of these BMPs were also determined in rat granulosa cells since these factors have been most widely studied in this poly-ovulatory species. As assessed by in situ hybridization, non-atretic ovine follicles expressed mRNA for BMP6 but not 2, 4 or 7. Furthermore, expression of BMP6 was limited to the oocyte of primordial as well as primary, pre-antral and antral follicles. Reverse transcription-PCR of granulosa cell mRNA detected low levels of all the BMPs in some pools of cells. BMP2, 4, 6 and 7 each inhibited progesterone production from ovine granulosa cells without affecting cellular proliferation/survival. Similarly, these BMPs inhibited progesterone production from rat granulosa cells. However, they also stimulated cellular proliferation/survival of the rat granulosa cells highlighting a species-specific difference for these growth factors. In conclusion, in sheep, BMP2, 4, 6 and 7 inhibit granulosa cell differentiation without affecting proliferation. However, as BMP2, 4 and 7 were not detectable by in situ hybridization in any cells of non-atretic ovarian follicles, it seems unlikely that these proteins would have an important intra-ovarian role in regulating follicular development in sheep. In contrast, localization of BMP6 mRNA in the oocyte suggests that this BMP family member may have a paracrine and/or autocrine role in regulating follicular growth in sheep, as has been shown for two other oocyte derived from members of the transforming growth factor superfamily, BMP15 and growth differentiation factor 9.