Prostaglandins regulate many physiological functions, including reproduction, by binding to specific plasma membrane receptors. In this study we evaluated the regulation of PGF_2α (FP) and PGE (EP₃ subtype) receptors in ovine corpora lutea. In the first study, tissue distribution of FP and EP₃ receptors was evaluated in 13 ovine tissues. FP receptor mRNA was present in 100-fold higher concentration in corpora lutea than in other tissues. Similarly, [³H]PGF_2α binding was much greater in luteal plasma membranes than in membranes from other tissues. In contrast, EP₃ receptor mRNA was more uniformly distributed, with high concentrations in adrenal medulla, inner myometrium, kidney medulla and heart. The distribution of [³H]PGE₁ binding was generally similar to EP³ mRNA, with the exception that ovarian stroma, endometrium and outer myometrium had high [³H]PGE₁ binding but low concentrations of EP³ receptor mRNA. The second study evaluated the action of PGF_2α on luteal mRNA encoding FP and EP₃ receptors. Ewes had PGF_2α or saline infused into the ovarian artery and corpora lutea were removed at 0, 1, 4, 12 and 24 h. FP receptor mRNA decreased by 50% at 12 and 24 h after infusion with PGF_2α, whereas EP₃ mRNA was unchanged. Treatment of large luteal cells with PGF_2α, phorbol didecanoate (protein kinase C activator), or ionomycin (calcium ionophore) decreased FP receptor mRNA after 24 h (P < 0.05). Glyceraldehyde 3-phosphate dehydrogenase mRNA was not changed by any treatment. These results show that EP₃ receptors are expressed in many tissues and expression is not regulated by PGF_2α. In contrast, FP receptors are primarily expressed in corpora lutea and expression is inhibited by PGF_2α.

Although the decrease of progesterone in serum and in luteal tissue during luteal regression is well characterized, relatively little is known about changes in proteins produced by the corpus luteum during this time. The first objective was to examine changes in patterns of protein secretion that might be associated with functional and structural luteal regression. The second objective was to characterize the expression of two major secretory products of regressing corpora lutea. Thirty normally cyclic heifers were randomly assigned at day 15–16 of the oestrous cycle (oestrus = day 0) to be ovariectomized at 0 h (no PGF_2α; n = 5) or at 4, 8, 12, 24 or 48 h after PGF_2α-induced luteal regression (n = 5 per time point). Total cellular RNA was isolated from tissue frozen at the time of ovariectomy. Thin slices (< 1 mm) of tissue were placed in methionine-deficient minimum essential media with [³⁵S]methionine and placed in a humidified CO₂ incubator at 38°C. Media and tissues were collected 6 h later. Changes in profiles of secreted proteins were analysed by one-dimensional SDS-PAGE. A number of proteins (relative molecular mass ranging from 14 300 to 200 000) were produced by luteal tissue at each time point (0–48 h). The major secretory proteins had relative molecular masses of approximately 21 500, 28 200, 43 700 and 46 000. Secretion of the relative molecular mass 46 000 protein(s) increased (P < 0.05) between 4 and 24 h after PGF_2α injection compared with the 0 h group. Western blot analyses with either tissue inhibitor of metalloproteinase-1 or tissue inhibitor of metalloproteinase-2 antisera detected immunoreactive proteins of relative molecular mass 28 200 and 21 500, respectively. Concentrations of mRNA encoding tissue inhibitor of metalloproteinases-1 increased (P < 0.01) by 8 h after PGF_2α injection, remained stable (P > 0.20) through 24 h and decreased (P < 0.05) by 48 h after PGF_2α. Concentrations of tissue inhibitor of metalloproteinases-2 mRNA were highest (P < 0.05) at 8 h after PGF_2α injection and lowest (P < 0.05) 48 h following induction of luteolysis. In summary, the profile of luteal protein production changed during luteolysis and two secretory products (tissue inhibitor of metalloproteinases-1 and -2) were identified. Metalloproteinase inhibitors may have an important role in tissue remodelling during structural luteolysis.

The regulation of mRNAs encoding insulin-like growth factor I (IGF-I) and the receptor for growth hormone (GH-R) in ovine luteal tissue by luteotrophic and luteolytic hormones was examined. In Expt 1, ewes were hypophysectomized (HPX) on day 5 of the oestrous cycle and administered saline (S), LH, GH, or LH + GH until day 12 of the oestrous cycle (n = 4 ewes per group). Concentrations of luteal mRNA encoding IGF-I in HPX + S ewes and pituitary-intact ewes at day 5 (n = 4) were approximately 60% (P < 0.05) of those in pituitary-intact ewes at day 12 (n = 4). Treatment of HPX ewes with GH or GH + LH, but not LH alone, increased concentrations of mRNA encoding IGF-I to values similar to those in pituitary-intact ewes at day 12. Hypophysectomy also reduced the mean concentration of mRNA encoding GH-R to approximately 60% (P < 0.05) of the values in pituitary-intact ewes (days 5 or 12). Treatment with LH, but not GH, increased (P < 0.05) concentrations of mRNA encoding GH-R to values observed in pituitary-intact ewes. In Expt 2, prostaglandin F_2α (PGF_2α; 1 μmole) injected into the ovarian artery on day 11 or day 12 of the oestrous cycle had no effect on luteal concentrations of mRNA for either IGF-I or GH-R. In Expt 3, concentrations of mRNA encoding IGF-I increased (P < 0.05) between days 3 and 6 and remained high for the duration (days 9, 12 and 15) of the oestrous cycle while luteal concentrations of mRNA encoding GH-R did not change. In conclusion, responsiveness of the corpus luteum to GH and luteal synthesis of IGF-I are likely regulators of luteal development and function. However, PGF_2α-induced luteolysis was not associated with a decrease in concentrations of mRNAs encoding either IGF-I or GH-R.

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.

Prostaglandin F_2α (PGF_2α) decreases secretion of progesterone from the corpus luteum in domestic ruminants. However, it is less effective during the early part of the oestrous cycle (Louis et al., 1973) and at the time of maternal recognition of pregnancy (Silvia and Niswender, 1984; Lacroix and Kann, 1986). Decreased luteal responsiveness may be due to failure of PGF_2α to activate fully its normal second messenger system, protein kinase C (PKC). Alternatively, increased resistance of the corpus luteum to PGF_2α might be attributable to greater concentrations of recently identified biological inhibitors of PKC. These possibilities were addressed by measuring steady-state concentrations of mRNA encoding PGF_2α receptor and two inhibitors of PKC, protein kinase C inhibitor-1 (PKCI-1) and kinase C inhibitor protein-1 (KCIP-1, brain 14-3-3 protein), in corpora lutea collected from ewes on days 4, 10 and 15 of the oestrous cycle (n = 5 per day) and day 15 of pregnancy (n = 7). There were no differences in mean concentrations of mRNA encoding PGF_2α receptor among the groups. However, concentrations of mRNA encoding both inhibitors of PKC were higher (P < 0.01) on day 4 of the oestrous cycle compared with the other groups. Treatment of ewes with a luteolytic dose of PGF_2α, which activates PKC, did not change concentrations of mRNA encoding either PKCI-1 or KCIP-I up to 24 h later. Luteal expression of mRNA encoding the PKC inhibitors and PGF_2α receptor was also examined in ewes treated with oestradiol in vivo for 16 h in the midluteal phase. High concentrations of oestradiol in serum (20 and 70 pg ml⁻¹) did not influence quantities of any of the mRNAs examined. Therefore, an increase in PKC inhibitors may be involved in resistance of the corpus luteum to PGF_2α during the early part of the oestrous cycle but does not appear to mediate the increased resistance of the corpus luteum to PGF_2α during maternal recognition of pregnancy. Neither PGF_2α nor oestradiol affected steady-state concentrations of mRNAs encoding PKCI-1 or KCIP-I.

IGFs are known to be key regulators of ovarian follicular growth in eutherian mammals, but little is known regarding their role in marsupials. To better understand the potential role of IGFs in the regulation of follicular growth in marsupials, expression of mRNAs encoding IGF1, IGF2, IGF1R, IGF-binding protein 2 (IGFBP2), IGFBP4 and IGFBP5 was localized by in situ hybridization in developing ovarian follicles of the brushtail possum. In addition, the effects of IGF1 and IGF2 on granulosa cell function were tested in vitro. Both granulosa and theca cells synthesize IGF mRNAs, with the theca expressing IGF1 mRNA and granulosa cell expressing IGF2 mRNA. Oocytes and granulosa cells express IGF1R. Granulosa and theca cells expressed IGFBP mRNAs, although the pattern of expression differed between the BPs. IGFBP5 mRNA was differentially expressed as the follicles developed with granulosa cells of antral follicles no longer expressing IGFBP5 mRNA, suggesting an increased IGF bioavailability in the antral follicle. The IGFBP protease, PAPPA mRNA, was also expressed in granulosa cells of growing follicles. Both IGF1 and IGF2 stimulated thymidine incorporation but had no effect on progesterone production. Thus, IGF may be an important regulator of ovarian follicular development in marsupials as has been shown in eutherian mammals.

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.