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 oocyteexpressed genes, BMP15 (GDF9B) and GDF9. Animals heterozygous for any of these mutations have higher ovulation rates (that is, 1 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. Shortterm (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.

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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 aim of this study was to test the hypothesis that the higher ovulation-rate in ewes heterozygous for a mutation in bone morphogenetic protein 15 (BMP15; FecX I ; otherwise known as Inverdale or IC ewes) is due to granulosa cells developing an earlier responsiveness to LH, but not FSH. To address this hypothesis, granulosa cells were recovered from every individual nonatretic antral follicle (O2.5 mm diameter) from IC and wild-type (CC) ewes during anoestrus and the luteal and follicular phases and tested for their responsiveness to FSH and human chorionic gonadotrophin (hCG; a surrogate for LH). For the FSH receptor (FSHR) binding study, granulosa cells were harvested in three separate batches from all antral follicles (R2.5 mm diameter) from IC and CC ewes. Using a highly-purified ovine FSH preparation, no evidence was found to suggest that IC ewes have a higher ovulation-rate due to enhanced sensitivity of granulosa cells to FSH with respect to cAMP responsiveness or to their FSHR binding characteristics (equilibrium K d or B max ). By contrast, a significantly higher proportion of follicles from IC ewes contained granulosa cells responsive to hCG. The higher proportion was due to cells from more small follicles (i.e. O2.5-4.5 mm diameter) developing a response to hCG. It is concluded that the mutation in the BMP15 gene in IC ewes leads to an earlier acquisition of LH responsiveness by granulosa cells in a greater proportion of follicles and this accounts for the small but significantly higher ovulation-rate in these animals.

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The putative negative feedback effects of IGF-I and IGF-II on GH secretion were tested by intracerebroventricular (icv) and intrapituitary administration to sheep. Over two consecutive days, serial jugular blood samples were taken at 10 min intervals for 6 h from ewes (n = 3/group) fitted with indwelling stainless steel cannulae into the lateral or third cerebral ventricles. The sheep were injected (icv) with either vehicle or purified ovine IGF-I (2, 4 or 8 micrograms). IGF-I injection had no effect on plasma GH secretion. Serial blood samples were taken from a second group of nine ewes in which ovine or recombinant human (rh) IGF-I was infused (2.5 micrograms/h for 2 h) into the third ventricle; once again, IGF-I failed to affect the episodic pattern of GH secretion. Three ewes fitted with indwelling stainless steel cannulae placed in the anterior pituitary gland were consecutively infused with either ovine or rhIGF-I (2.5 micrograms/h for 2 h) or vehicle. Plasma GH concentrations were suppressed in 3/3 sheep from 1-1.5 h after the commencement of infusion and GH levels remained low for the remainder of the sampling period. In another group of five ewes synergistic effects of IGF-I and IGF-II on GH secretion were tested by icv infusion of rhIGF-I, rhIGF-II, or rhIGF-I+rhIGF-II (5 micrograms/h for 2 h) or vehicle (sterile 10 mM HCl/saline). Each sheep received each treatment in a randomised design. Infusion (icv) of IGF-I and IGF-II alone or in combination failed to alter GH secretion.(ABSTRACT TRUNCATED AT 250 WORDS)

Competitive tracer binding studies using radioiodinated insulin-like growth factor-I and -II (125I-labelled IGF-I and 125I-labelled IGF-II) together with size exclusion chromatography and IGF-I affinity chromatography have been used to characterize IGF binding protein activity in ovine tissue fluids. Binding proteins of greater than 200, 150 and 40-50 kDa were revealed in these studies and shown to be widely distributed in body fluids. Thus, the greater than 200 kDa binding protein, which is IGF-II specific, is present in plasma from mature sheep, colostrum and follicular fluid as well as fetal sheep plasma. This may be the ovine equivalent of the soluble type-2 IGF receptor recently identified in rat serum. The presence of a 150 kDa binding protein, of mixed specificity for IGF-I and IGF-II, in fetal and mature sheep plasma was confirmed in these studies. This protein, previously believed to be restricted to vascular fluids, was also identified in mammary lymph, follicular fluid and as a minor component in vitreous humor. Binding proteins of 40-50 kDa were revealed in every fluid tested and multiple variants with distinct specificities were also suggested. This was investigated by IGF-I affinity chromatography using mature sheep plasma. Following passage through the affinity adsorbent, binding of 125I-labelled IGF-I to proteins in the 40-50 kDa region was abolished but when 125I-labelled IGF-II was used as tracer, a binding protein of 40-50 kDa was still observed. Thus sheep plasma contains at least two 40-50 kDa binding proteins. The competitive tracer binding studies indicated that one such protein demonstrates mixed specificity for IGF-I and -II while the other strongly favours IGF-II.

This study was undertaken to determine the effect of exogenous oxytocin on plasma concentrations of the prostaglandin (PG) F metabolite 13,14-dihydro-15-keto-PGF (PGFM) and the oxytocin-associated neurophysin (OT-N) during the estrous cycle and early pregnancy in the ewe. Ewes were given oxytocin (250 mU, i.v.) on Days 3 (n = 4), 8 (n = 5), 13 (n = 4) or 14 (n = 5) of the estrous cycle, and a further 6 ewes were injected on Days 13 (n = 2) and 14 (n = 4) of pregnancy. No significant rises in plasma concentrations of PGFM were observed on Days 3 and 8 of the estrous cycle and on Days 13 and 14 of pregnancy. A marked increase in plasma PGFM concentrations occurred on Day 14 of the estrous cycle with the PGFM levels rising from a mean basal value of 120 pg/ml to a mean maximum value of 415 pg/ml within 2-10 min of administering oxytocin (P less than 0.001). No increases in plasma OT-N concentrations were found in early pregnancy and only 1 of 4 ewes at Day 14 of the cycle showed any significant increase in OT-N concentrations. It is concluded that there is an increase in the responsiveness of the uterine-PGF secretory system to oxytocin during the latter stages of the estrous cycle. During early pregnancy this response was blocked by the presence of the embryo.(ABSTRACT TRUNCATED AT 250 WORDS)