Summary. Systemic intravenous infusion of physiological concentrations of PGF-2α and its major metabolite, 13,14-dihydro-15-keto-PGF-2α (PGFM) into non-pregnant ewes possessing a corpus luteum induced the release of oxytocin—neurophysin. These results suggest that, during luteolysis, endogenous release of uterine PGF-2α would be able to stimulate the release of ovarian oxytocin and oxytocin—neurophysin from the ovary.
L. G. Moore, V. J. Choy, R. L. Elliot, and W. B. Watkins
Summary. Frequent blood samples were removed from a utero-ovarian vein, a jugular vein and a femoral artery of 5 ewes during luteolysis. Analysis of these samples for oxytocin-associated neurophysin revealed a significant venous—arterial difference across the ovary and uterus but not across the head. This occurred during the pulsatile surges as well as when levels were basal and confirms the corpus luteum as a major source of the pulsatile surges of oxytocin-associated neurophysin and oxytocin that occur during CL regression and also of the elevated luteal phase concentrations of both hormones. The pulsatile surges of oxytocin-associated neurophysin measured in the utero-ovarian vein were accompanied by the release of an approximately equimolar amount of oxytocin.
The concentration of PGF-2α in the utero-ovarian vein samples began to increase before the levels of oxytocin and oxytocin-associated neurophysin started to increase. This suggests that uterine PGF-2α initiates the release of ovarian oxytocin and oxytocin-associated neurophysin during luteolysis in the ewe.
D. J. Phillips, L. G. Moore, N. L. Hudson, S. Lun, and K. P. McNatty
This study investigated the effects of short-term (20 days) ovariectomy, the effects of FSH assay (radioimmunoassay, receptor assay or in vitro bioassay) and of FecBB genotype on the characteristics of pituitary FSH from Booroola ewes. Pituitary extracts were obtained from ovariectomized homozygous carriers (BB) and non-carriers (++, n = 8 per genotype) and ovary-intact controls (n = 4 per genotype). The extracts (n = 4 per genotype per treatment) were subjected to agarose suspension electrophoresis and the eluates were assayed by the three FSH methods. There were significant effects of ovariectomy (P < 0.01) and assay system (P < 0.05) but not of genotype on the median charge of FSH isoforms. The mean ± sem migration rates for FSH in intact and ovariectomized ewes were 0.469 ± 0.006 and 0.439 ± 0.006 albumin mobility units, respectively (P < 0.01), indicating a shift to more basic isoforms after short-term ovariectomy. When the pituitary extracts were subjected to anion-exchange HPLC, there was a significant (P < 0.01) shift to more basic isoforms in the ovariectomized ewes as shown using agarose electrophoresis, and no gene effects were noted. When the pituitary extracts (n = 4–8 per group) were injected into mature female mice, there were no significant effects of ovariectomy or genotype on the circulating half-lifes of the pituitary FSH isoforms. These results indicate that after short-term ovariectomy, the increase in plasma FSH concentrations is accompanied by a shift in the median charge of pituitary FSH isoforms without any observable change in their metabolic clearance rates. Moreover, the FecBB gene has little effect on the median charge or half-life of pituitary FSH.
Kenneth P McNatty, Derek A Heath, Norma L Hudson, Stan Lun, Jennifer L Juengel, and Lloyd G Moore
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; FecXI; otherwise known as Inverdale or I+ 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 (>2.5 mm diameter) from I+ and wild-type (++) 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 (≥2.5 mm diameter) from I+ and ++ ewes. Using a highly-purified ovine FSH preparation, no evidence was found to suggest that I+ 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 I+ ewes contained granulosa cells responsive to hCG. The higher proportion was due to cells from more small follicles (i.e. >2.5–4.5 mm diameter) developing a response to hCG. It is concluded that the mutation in the BMP15 gene in I+ 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.
P M Lokman, Y Kazeto, Y Ozaki, S Ijiri, R Tosaka, M Kohara, S L Divers, H Matsubara, L G Moore, and S Adachi
In order to study the regulation of the growth differentiation factor-9 (gdf9) gene in a primitive teleost with semelparous life history, we cloned a cDNA encoding shortfinned eel Gdf9, expressed a partial peptide in Escherichia coli, and raised an antiserum to evaluate changes in Gdf9 expression during its pituitary homogenate-induced reproductive cycle. The effects of in vivo and in vitro exposure to the androgen 11-ketotestosterone (11-KT), known to affect previtellogenic (PV) oocyte growth, were also determined. Furthermore, we investigated whether Gdf9 expression was metabolically gated by treating PV fish with recombinant GH in vivo. Immunoreactive proteins of ca. 52 and 55 kDa were identified by western blot analysis. Gdf9 message and protein were most abundant in PV oocytes, and peaked slightly earlier for mRNA than for protein. Captivity resulted in reduced gdf9 mRNA levels, which were restored following pituitary homogenate treatment. As oocytes progressed through induced oogenesis, Gdf9 expression decreased. Neither 11-KT nor GH treatment affected gdf9 mRNA levels in PV fish, although GH could partially restore handling- or captivity-induced decreases in gdf9 mRNA levels. Semelparous eels thus show an expression pattern of Gdf9 during oogenesis that is similar to that seen in other vertebrates, that appears responsive to handling or captivity stress, and whose control remains to be elucidated.
K P McNatty, L G Moore, N L Hudson, L D Quirke, S B Lawrence, K Reader, J P Hanrahan, P Smith, N P Groome, M Laitinen, O Ritvos, and J L Juengel
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.