Follicle development, endocrine profiles and ovulation rate in adult Merino ewes: effects of early nutrition (pre- and post-natal) and supplementation with lupin grain.

In adult ewes, we tested whether ovarian function, including the response to short-term supplementation, was affected by the nutrition of their mothers during the pre-/post-natal period. A 2×2 factorial design was used with nutrition in early life (low or high) and a 6-day supplement (with or without) as factors. All ewes received three prostaglandin (PG) injections 7 days apart, and the supplement (lupin grain) was fed for 6 days from 2 days after the second until the third PG injection. We measured reproductive and metabolic hormones, studied follicle dynamics (ultrasonography), and evaluated granulosa cell numbers, aromatase activity and oestradiol (E2) concentrations in follicular fluid in healthy follicles at days 3 and 7 of supplementation. Ovulation rate was increased by 25% by exposure to high pre-/post-natal nutrition (1.5 vs 1.2; P<0.05), in association with a small decrease in FSH concentrations (P=0.06) and a small increase in insulin concentrations (P=0.07). The number of healthy antral follicles was not affected. Acute supplementation increased the number of granulosa cells (3.7±0.2 vs 3.0±0.2 million; P<0.05) in the largest follicle, and the circulating concentrations of E2 (4.6±0.3 vs 3.9±0.3 pmol/l; P<0.05) and glucose (3.4±0.03 vs 3.3±0.03 mmol/l; P<0.01). Both early life nutrition and acute supplementation appear to affect ovulation rate through changes in glucose-insulin homoeostasis that alter follicular responsiveness to FSH and therefore E2-FSH balance.

Effects of immunization against androstenedione or bone morphogenetic protein 15 (BMP15) on reproductive performance in sheep.

Partial neutralization of bone morphogenetic protein 15 (BMP15) bioactivity by immunization is known to increase ovulation rate in sheep. However, it remains uncertain whether BMP15 vaccination would be a suitable procedure for increasing lambing rate. The aim of this study was to compare the efficacy of a BMP15 vaccination treatment on lamb production to that of commercially-available androstenedione-based vaccines that are used for this purpose. Ewes were immunized for 3 yr against androstenedione, BMP15, or no antigen (control). Vaccination with androstenedione or BMP15 altered (P < 0.05) ovulation rate as well as litter size at midpregnancy, birth, and weaning compared with controls. No differences were detected in the proportions of ewes conceiving in the first cycle or partial failure of multiple ovulations. Both gender and litter size affected birth weight of the lamb (P < 0.05), but no effect of treatment was found. Growth rate was significantly affected (P < 0.05) by gender, birth weight, and the number of lambs raised, but not treatment. In conclusion, immunization against either androstenedione or BMP15 increased ovulation rate. Androstenedione vaccination also increased the number of lambs weaned (P < 0.05). Bone morphogenetic protein 15 vaccination altered the pattern of the number of lambs weaned, but no increase in lamb production was observed as more ewes produced zero or three lambs. Overall, androstenedione or BMP15 vaccination did not significantly affect embryo or fetal survival or lamb performance independently of the effects of these treatments on ovulation rate.

Long-term alteration of follicular steroid concentrations in relation to subclinical endometritis in postpartum dairy cows.

The focus of this study was to investigate the effect of subclinical endometritis (scEndo) on ovarian follicular steroid concentrations in early postpartum pasture-fed dairy cows. Mixed-age lactating dairy cows (n = 169) were examined to ascertain uterine health status on d 21 postpartum (±3 d). From this herd, a cohort of scEndo and uninfected cows (n = 47) were selected using uterine cytology to determine scEndo. To ensure cows with scEndo were selected for the study, a conservative threshold [>18% polymorphonuclear (PMN) cells among uterine nucleated cells] was chosen as a selection threshold. Ovarian follicular dynamics were assessed by ultrasonography on d 21, 42, and 63 postpartum. On the latter 2 d, all follicles >4 mm in diameter were ablated, and 4 d later, the largest (F1) and second largest (F2) follicles were measured and their follicular fluid aspirated. Hematological variables and plasma metabolites were measured also on these days to further characterize scEndo cows. On d 21, the prevalence of scEndo was approximately 9% in this herd; by d 42 infections had self-resolved in the majority (81%) of those cows classified as having scEndo on d 21. The scEndo cows had a delayed return to cyclicity; however, no effect was evident on ovarian follicle size or growth rate. Weeks after scEndo had self-resolved and cyclicity was restored, decreased (P = 0.07) testosterone and increased (P = 0.07) cortisol concentrations were evident in F1 follicles of scEndo compared with uninfected cows. Progesterone concentrations of F1 increased (P < 0.05) in 11- to 16-mm diameter follicles of scEndo cows, whereas estradiol, androstendione, and dehydroepiandrosterone concentrations were decreased (P < 0.05) in F1 8- to 10-mm diameter follicles of scEndo cows. These 3 steroids also differed (P < 0.05) between F1 follicle size categories of scEndo but not uninfected cows. On d 21, mean plasma albumin concentration was decreased (P = 0.02) in scEndo cows. In summary, early postpartum scEndo had surprisingly long-term influences on the steroid concentrations of ovarian follicles long after infections had self-resolved. This is likely to affect oocyte quality and may partially explain the reduced conception rates and longer interval between calving and conception that are often associated with scEndo, although more detailed investigations are required to substantiate this theory.

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.

Modelling female reproductive function in farmed animals.

Impaired reproduction in farmed animals is a major cost to agriculture, and this is exacerbated by the implementation of intensive production systems. Addressing this has been the focus of a significant body of research. While considerable advances have been made in biological experiments and understanding, a systems insight into the mechanisms that underlie reproductive function in mammals is needed. Mathematical modelling offers a means to develop a systems approach to reproduction by coalescing information and predicting outcomes of interventions. There has been steady progress in the development of mathematical models addressing various issues of reproduction over the last decade, from cell-signalling pathways through to herd management. We review these developments and their insights as well as their limitations. In addition, we identify other areas that need development, and how modelling might usefully contribute to these areas of reproduction science.

A mathematical model of pregnancy recognition in mammals.

In this paper we develop a mathematical model of the luteal phase of the reproductive cycle in mammals with the aim to generate a systems understanding of pregnancy recognition. Pregnancy recognition is initiated by the production of interferon tau (IFNtau) by the growing conceptus. This ensures that the maternal corpus luteum (CL) remains viable to secrete progesterone, which is critical for providing a uterine microenvironment suitable for embryonic growth. Our mathematical model describes the interactions among the CL, the reproductive hormones and the hormone receptors in the uterus. It also characterises the complex interactions amongst the uterine oestrogen, progesterone and oxytocin receptors that control the sensitivity of the uterus to oestrogen, progesterone and oxytocin, respectively. The model is represented by a dynamical system and exhibits qualitative features consistent with the known experimental results in sheep. A key factor identified was a time-dependent threshold for the IFNtau signal below which the presence of the embryo might not be recognised and thus pregnancy would likely fail. Furthermore, the model indicated that if the IFNtau signal is later than around day 13 of the cycle, then pregnancy will not be recognised irrespective of the IFNtau concentration. The thresholds in the concentration and time of the IFNtau signal is a screening mechanism whereby only embryos of sufficient quality are able to prevent luteolysis (i.e. regression of the CL). The effect of progesterone secretion rate from the CL on pregnancy recognition was investigated. The model suggests that if the secretion rate is low then the initiation of the IFNtau signal is delayed, which in turn compromises the likelihood of a pregnancy being recognised by the CL. Furthermore, pregnancy recognition does not occur below a critical threshold in the progesterone secretion rate. In summary, the model can be used to identify the most favourable conditions for pregnancy recognition.

Development of the ovary and ontongeny of mRNA and protein for P450 aromatase (arom) and estrogen receptors (ER) alpha and beta during early fetal life in cattle.

Estradiol-17beta is the predominant steroid produced during early stages of ovarian development in ruminants and steroid hormones have been hypothesized to regulate ovigerous cord formation, germ cell meiosis and ovarian vascular development. Therefore, the objective was to determine the presence and localization of mRNA and protein encoding cytochrome P450 aromatase (P450arom), and estrogen receptors alpha (ERalpha) and beta (ERbeta) during ovarian development in fetuses of cattle on days 35, 45, 60, 75, 90 and 105 after breeding (n=4/age) using in situ hybridization and immunohistochemistry. No ovarian tissue was found in the day 35 fetuses, but was found in all later ages studied. There appeared to be little organization of specific structures in ovaries on days 45 and 60, although germ cells could be identified. Evidence of the beginning of ovigerous cord formation was found on day 60. By day 75 of gestation, the ovigerous cords were more extensive and mesonephric-derived cell streams were detectable. By day 90 (and still present at day 105), both ovigerous cords and cell streams/rete tubules were definitive structures of the developing ovaries. Ovaries appeared to develop in "lobular" segments around the periphery of the ovary. Some lobes appeared to be at slightly different developmental stages, as assessed by the extent or definition of ovigerous cord formation. The localization of mRNAs for P450arom, ERalpha and ERbeta were closely associated with protein content. At days 45 and 60, mRNA and protein of P450arom and ERbeta were located throughout ovaries with signal in medulla being denser than in the cortex. P450arom mRNA or protein was punctate, but not evident in germ cells. From day 75, P450arom was increasingly becoming localized to cell streams or clusters of cells (rete tubules) in the medulla, and by days 90 and 105 of gestation, was more definitively localized to cell streams and/or rete tubules. Similar to P450arom, ERbeta mRNA and protein were observed in cells in the medulla, and also in germ cells, pre-granulosa cells and some surface epithelial cells. ERalpha mRNA and protein were predominately in the surface epithelium in ovaries of all ages with fainter signal for ERalpha protein also being observed in pre-granulosa and stromal cells including the cell streams/rete tubules. ERalpha protein was also detected in a few germ cells at days 90 and 105 of gestation. Thus, in cattle, estradiol-17beta has the potential to regulate, in an autocrine/paracrine manner, a number of different cell types during ovarian development.

Control of ovarian follicular development to the gonadotrophin-dependent phase: a 2006 perspective.

In sheep, as in other mammals, ovarian follicular growth is regulated mainly by intraovarian growth factors during early development with pituitary hormones increasingly important during the final phases to ovulation. Most follicles are present as primordial structures and these express many hundreds of genes that fulfil an array of housekeeping and signalling functions. Once growth has been initiated, at least two oocyte-derived growth factors, namely growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15), are critical for ongoing development to ovulation, most likely by regulating the proliferative and differentiative functions of adjacent follicular cells. In sheep, the granulosa cell populations double some 12-14 times and a well-defined thecal layer differentiates before antrum formation and the time taken to complete this process varies between 50 -150 days with very little follicular atresia. During preantral growth, FSH and LH receptors coupled to the cyclic AMP second messenger system develop in granulosa and thecal cells respectively. From the late preantral stage, GDF9, BMP15 and perhaps other factors are thought to regulate gene expression in cumulus cells to enhance metabolic cooperativity with the oocyte and in mural granulosa cells to regulate their responses to pituitary hormones. In sheep, antral follicular development is characterized by a much faster rate of growth, additional increases in the numbers of granulosa (4-5 more doublings) and thecal/cells, an increased level of steroid and inhibin secretion in response to FSH and LH, but also by most follicles undergoing atresia. The final number of follicles that go on to ovulate is dependent upon FSH as well as the intrafollicular concentrations of GDF9 and BMP15.

Oocyte-expressed genes affecting ovulation rate.

From examination of inherited patterns of ovulation rate in sheep, several breeds have been identified with point mutations in two growth factor genes (BMP15 and GDF9) and a related receptor (ALK6) that are expressed in oocytes. Five different point mutations have been identified in the BMP15 gene, one in GDF9 and one in ALK6. Animals heterozygous for these mutations or heterozygous for two of these mutations or homozygous for the ALK6 mutation have higher ovulation rates (i.e. +0.6-10) than their wild-type contemporaries. Animals homozygous for the BMP15 or GDF9 mutations are sterile due to arrested follicular development from the primary stage of growth. The BMP15 and GDF9 mutations are thought to result in reduced levels of mature protein or altered binding to cell-surface receptors. In sheep, GDF9 mRNA is present in germ cells before and after ovarian follicular formation as well as throughout follicular growth, whereas BMP15 mRNA is found in oocytes only from the primary stage of growth. Also ALK6 together with related cell-surface receptors such as ALK5 and BMPRII mRNA are present in oocytes at most, if not all, stages of follicular growth. Both GDF9 and BMP15 proteins are present in follicular fluid indicating that they are secreted products. Immunisation of sheep with GDF9 or BMP15 peptides shows that both growth factors are essential for follicular development, ovulation and/or corpus luteum formation. In animals with the ALK6 mutation, ovarian follicles undergo precocious maturation leading to three to seven follicles ovulating at smaller diameters without any increase above wild-types in the ovarian secretions of steroid or inhibin. One important consequence of the ALK6 mutation appears to be a decreased ability of some BMPs to inhibit differentiation of follicular cells. Current findings in sheep suggest that BMP15, GDF9 and ALK6 are targets for new methods of fertility regulation in some mammals.

The role of proteins of the transforming growth factor-beta superfamily in the intraovarian regulation of follicular development.

Ovarian follicular development occurs in a hierarchical manner with each follicle having a unique biochemical composition at any moment in time. It has long been understood that a precise coordination between the growth and maturation of the oocyte and adjacent follicular cells (i.e. somatic cells) is essential in order to produce an oocyte that is fully competent to undergo fertilization and embryo development. In addition to the critical endocrine signalling pathways between the hypothalamus, pituitary and ovary, it is now evident that the oocyte itself is important in influencing the microenvironment of the developing follicle by regulating, via paracrine and autocrine mechanisms, its own maturation as well as somatic cell proliferation, differentiation and ovulation rate. Several of the key oocyte-derived regulating factors are members of the transforming growth factor-beta (TGF-beta) superfamily and to date the best understood are growth differentiation factor 9 (GDF9), bone morphogenetic protein 15 (BMP15) and BMP6. Significant species differences appear to exist in the relative importance of these growth factors and much remains to be elucidated about their roles in the human ovary. More information on the roles of these factors during ovarian follicular development is likely to advance new therapeutic applications for management of fertility as well as our understanding of how better to assess oocyte quality.

The oocyte and its role in regulating ovulation rate: a new paradigm in reproductive biology.

Ovulation rate in mammals is determined by a complex exchange of hormonal signals between the pituitary gland and the ovary and by a localised exchange of hormones within ovarian follicles between the oocyte and its adjacent somatic cells. From examination of inherited patterns of ovulation rate in sheep, point mutations have been identified in two oocyte-expressed genes, BMP15 (GDF9B) and GDF9. Animals heterozygous for any of these mutations have higher ovulation rates (that is, + 0.8-3) than wild-type contemporaries, whereas those homozygous for each of these mutations are sterile with ovarian follicular development disrupted during the preantral growth stages. Both GDF9 and BMP15 proteins are present in follicular fluid, indicating that they are secreted products. In vitro studies show that granulosa and/or cumulus cells are an important target for both growth factors. Multiple immunisations of sheep with BMP15 or GDF9 peptide protein conjugates show that both growth factors are essential for normal follicular growth and the maturation of preovulatory follicles. Short-term (that is, primary and booster) immunisation with a GDF9 or BMP15 peptide-protein conjugate has been shown to enhance ovulation rate and lamb production. In summary, recent studies of genetic mutations in sheep highlight the importance of oocyte-secreted factors in regulating ovulation rate, and these discoveries may help to explain why some mammals have a predisposition to produce two or more offspring rather than one.

Physiology of GDF9 and BMP15 signalling molecules.

Two related oocyte-derived members of the transforming growth factor-beta (TGF-beta) superfamily, namely growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15, also known as GDF9B), have recently been shown to be essential for ovarian follicular growth. In addition, both proteins have been shown to regulate ovulation rate in sheep, and although it is evident that these growth factors interact both with one another and with other intra- and extra-ovarian factors, the precise mechanisms by which they influence follicular growth and ovulation rate have not been thoroughly elucidated.

Oocyte-derived growth factors and ovulation rate in sheep.

The physiological mechanisms controlling ovulation rate in mammals involve a complex exchange of endocrine signals between the pituitary gland and the ovary, and a localized exchange of intraovarian hormones between the oocyte and its adjacent somatic cells. The discoveries in sheep of mutations in bone morphogenetic protein 15 (BMP15) and bone morphogenetic protein receptor type IB (BMPR-IB) together with recent findings on the physiological effects of growth differentiation factor 9 (GDF9) and BMP15 on follicular development and ovulation rate highlight some important differences in the way in which the oocyte may function in mammals with different ovulation rate phenotypes. In sheep, BMP15 and GDF9 have each been shown to be essential for the early and later stages of follicular development. In addition, ovulation rate is sensitive to changes in the dose of either of these two oocyte-derived growth factors. These findings are in contrast to those reported for mice in which GDF9, but not BMP15, is essential for follicular development. The evidence to date is consistent with the hypothesis that the oocyte plays a central role in regulating key events in the process of follicular development and hence, is important in determining ovulation rate. Moreover, it appears that the mechanisms that the oocyte uses to control these processes differ between species with low and high ovulation rate phenotypes.

Production of biologically active tethered ovine FSHbetaalpha by the methylotrophic yeast Pichia pastoris.

The pituitary-derived glycoprotein hormone FSH plays a central role in controlling vertebrate gonadal function. In female mammals the maturation of ovarian follicles is critically dependent upon stimulation by FSH. Moreover, injection of exogenous FSH is used extensively to stimulate increased numbers of follicles to ovulate. Structurally FSH is a heterodimeric glycoprotein composed of two non-covalently associated polypeptide subunits. The tertiary structures of both the alpha- and beta-subunits are constrained by intramolecular disulphide bonds and are post-translationally modified with two N-linked carbohydrate moieties, the structure of which appears to modulate in vivo biological activity. Here we report the expression of ovine FSH (oFSH) as a biologically active single-chain polypeptide using the methylotrophic yeast Pichia pastoris. Sequences encoding the mature oFSH alpha- and beta-proteins were fused to form a gene encoding a fusion protein with the C-terminus of the beta-chain joined to the N-terminus of the alpha-chain, with the chains separated by a two amino acid linker sequence. This fusion gene was itself fused to two alternative Pichia leader sequences (mating factor alpha and acid phosphatase) and transformed into the Pichia strains GS115 and SMD1168. The recombinant fusion protein (oFSHbetaalpha) was expressed at approximately 0.1 microg/ml in 'shake-flask' cultures. The Pichia-expressed tethered protein was biologically active in an in vitro bioassay, had a molecular mass of 28 kDa, as determined by SDS-PAGE, and bound the bovine FSH receptor with a binding profile similar to that of native oFSH.

Expression of the FcRn receptor (alpha and beta) gene homologues in the intestine of suckling brushtail possum (Trichosurus vulpecula) pouch young.

The neonatal IgG transporter FcRn consists of two chains, FcRn alpha and beta (also known as beta(2) microglobulin), and is involved in transferring IgG molecules across both mammary and intestinal epithelial cells. Developmental changes in FcRn IgG alpha and beta chain mRNA levels were investigated in the gut of brushtail possum (Trichosurus vulpecula) pouch young (PY) using Northern hybridisation. FcRn alpha transcripts were detected in the PY proximal intestine at all times examined, between days 1 and 195 of post-natal life, with increased levels detected from around day 110. The beta(2) microglobulin transcript levels in the PY proximal intestine were low to undetectable until day 110 of post-natal life and then increased dramatically after day 159. Both the FcRn alpha and beta gene transcripts were detected in a wide range of tissues in the adult possum (>365 days). Genomic sequences located 5' to the start of transcription of the FcRn alpha and beta(2) microglobulin genes were cloned and analysed for predicted cis-acting transcription control elements. Both the FcRn alpha and beta(2) microglobulin genomic sequences contained STAT5 binding motifs consistent with the transcription of both genes being modulated by prolactin. Using in situ hybridisation, the FcRn alpha and beta(2) microglobulin transcripts were localised to the epithelial cells of the PY intestine. However, no prolactin receptor transcripts were detected in the same epithelial cells suggesting that the observed changes in FcRn alpha and beta(2) microglobulin gene expression in the proximal intestine are not modulated directly by prolactin. The results are consistent with the hypothesis that changes in FcRn alpha and beta(2) microglobulin gene expression take place in the possum PY intestine to accommodate changes in maternal milk composition to meet the changing immunological demands of the PY.

Bmp15 mutations and ovarian function.

BMP15, also known as growth and differentiation factor 9B (GDF9B), is a member of the transforming growth factor beta superfamily (TGFbeta) which in humans, rodents and sheep is expressed exclusively in the oocyte. BMP15 is closely related to GDF9, another oocyte-specific member of this superfamily which has been shown to be essential for early ovarian folliculogenesis. Inactivation of the BMP15 gene in mice has shown only minor effects on fertility. However, Inverdale and Hanna lines of sheep carry naturally occurring mutations in BMP15 which highlight differences in the action of this gene between mice and other mammals. Sheep which are heterozygous show an increase in ovulation rate whereas homozygotes are infertile. The granulosa cell receptor which mediates the BMP15 response has not yet been identified, but the discovery that a point mutation in the BMP1B receptor in Booroola sheep is responsible for increased ovulation rate highlights the importance of the TGFbeta signalling molecules in early folliculogenesis.

Variation in antral follicle development during the follicular phase of the oestrous cycle in red deer (Cervus elaphus) hinds.

The aim of this study was to quantify antral follicle populations in cyclic red deer hinds and to monitor follicle development leading to ovulation. Oestrus was synchronized with exogenous progesterone and ovaries were recovered approximately 0, 12, 24 or 36 h (follicular phase) or 10 days (luteal phase) after progesterone withdrawal (n = 5 per group). All follicles > or = 2 mm in diameter were dissected out, health status was assessed, follicular fluid oestradiol content was measured, granulosa cells were harvested and their capacity for oestradiol and cAMP production was determined. The time of oestrus and the preovulatory LH surge were monitored in five control hinds. Deer ovaries contained 26.6 +/- 3.45 (mean +/- SEM) follicles > or = 2 mm in diameter (range 4-81), with at least one large antral follicle (diameter: 8.3 +/- 0.38 mm) per hind. There was a strong correlation between follicle size and granulosa cell population (r(2) = 0.676). Approximately half (50.7%) of the follicles were classified as healthy, with the percentage classified as atretic decreasing with increasing follicle size. Neither the total number of antral follicles nor their size distribution differed significantly among groups. There were significantly more (P < 0.05) healthy follicles at 24 h after progesterone withdrawal than at 0 h, when large oestrogenic follicles had fewer granulosa cells, lower follicular fluid oestradiol concentrations and lower aromatase activity (P < 0.05) than did those from other groups. In summary, antral follicle development in red deer is similar to that in other monovulatory ruminants, and at least one large follicle is present at all stages of the oestrous cycle.

Ontogeny of steroidogenesis in the fetal sheep gonad.

The aim of this study was to determine 1) the time of onset and cellular localization of gene expression for steroidogenic factor-1 (SF-1), steroidogenic acute regulatory protein, 3beta-hydroxysteroid dehydrogenase/Delta(5),Delta(4) isomerase (3beta-HSD), and the cytochrome P450 enzymes for cholesterol side-chain cleavage (P450(scc)), 17alpha-hydroxylase (P450(17alphaOH)), and aromatase (P450(arom)) during gonadal development; and 2) the amount of progesterone, androstenedione, testosterone, and 17beta-estradiol present in the fetal sheep gonad. Fetuses were collected on Days 24, 26, 28, 30, 32, 35, 40, 55, and 75 of gestation, and gene expression was determined by in situ hybridization. The steroid content of gonads collected on Days 30, 35, 55, and 75 of gestation was determined by RIA. Developing gonads collected from both male and female fetuses were steroidogenically active around the time of morphological sexual differentiation. In the female, the steroidogenic cells were initially located at the boundary of the cortex and medulla but become increasingly restricted to the mesonephric-derived cell streams. In the male, once tubules were identifiable, steroidogenesis was restricted to the interstitial regions. Interestingly, expression of both SF-1 and 3beta-HSD was observed prior to morphological sexual differentiation. In addition, expression of both of these genes was more widespread than the other genes in both males and females.

Genes controlling ovulation rate in sheep.

Sheep provide a valuable model for studying the genetic control of ovulation rate. Recent progress includes the identification of mutations in BMP15 (bone morphogenetic protein 15) that increase ovulation rate in heterozygous carriers and block follicular development in homozygous carriers. The genes characterized to date appear to act principally within the ovary and result in earlier maturity of granulosa cells and reduced follicular size. There may also be other sites of action, and increased FSH concentrations appear to be important in the expression of the FecB phenotype. A new locus on the X chromosome in New Zealand Coopworth sheep increases ovulation rate by about 0.4 and is maternally imprinted. Results from studies in the Cambridge and Belclare breeds indicate that further genes remain to be characterized. Finding the first mutations leading directly to variation in ovulation rate is likely to speed up the identification and molecular analysis of these other genes. There is still much to learn about follicular development and the control of litter size from genetic models in sheep.

Highly prolific Booroola sheep have a mutation in the intracellular kinase domain of bone morphogenetic protein IB receptor (ALK-6) that is expressed in both oocytes and granulosa cells.

The Booroola fecundity gene (FecB) increases ovulation rate and litter size in sheep and is inherited as a single autosomal locus. The effect of FecB is additive for ovulation rate (increasing by about 1.6 corpora lutea per cycle for each copy) and has been mapped to sheep chromosome 6q23-31, which is syntenic to human chromosome 4q21-25. Bone morphogenetic protein IB (BMP-IB) receptor (also known as ALK-6), which binds members of the transforming growth factor-beta (TGF-beta) superfamily, is located in the region containing the FecB locus. Booroola sheep have a mutation (Q249R) in the highly conserved intracellular kinase signaling domain of the BMP-IB receptor. The mutation segregated with the FecB phenotype in the Booroola backcross and half-sib flocks of sheep with no recombinants. The mutation was not found in individuals from a number of sheep breeds not derived from the Booroola strain. BMPR-IB was expressed in the ovary and in situ hybridization revealed its specific location to the oocyte and the granulosa cell. Expression of mRNA encoding the BMP type II receptor was widespread throughout the ovary. The mutation in BMPR-IB found in Booroola sheep is the second reported defect in a gene from the TGF-beta pathway affecting fertility in sheep following the recent discovery of mutations in the growth factor, GDF9b/BMP15.