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T. D. Braden, H. R. Sawyer and G. D. Niswender

Summary. The ability of sheep luteal cells from the first corpus luteum formed after parturition (Group F) to secrete progesterone in the presence or absence of LH was compared with that of luteal cells obtained from normal cyclic ewes (Group C). Luteal concentrations of receptors for LH and prostaglandins (PG) F-2α (PGF-2α) and the cellular composition of corpora lutea from Groups F and C were also compared. Luteal cells from Group F secreted less progesterone in either the presence or absence of LH (P < 0·01). There was no difference in the number of receptors for LH or PGF-2α per luteal cell between Groups F and C (P > 0·1), nor was there a difference in the number of large or small steroidogenic luteal cells (P > 0·1). It was concluded that, if short-lived corpora lutea are insensitive to gonadotrophins, this response is not mediated by decreased numbers of receptors for LH. In addition, if the first corpus luteum formed post partum in ewes is more sensitive to the luteolytic effects of PGF-2α, this effect is not mediated by an increased number of receptors for PGF-2α or an increased proportion of PGF-2α-sensitive large luteal cells.

Keywords: corpora lutea; post partum; sheep; LH receptors; PGF-2α receptors

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C. E. Farin, T. M. Nett and G. D. Niswender

Summary. To examine the effect of purified LH on development and function of luteal cells, 27 ewes were assigned to: (1) hypophysectomy plus 2 μg ovine LH given i.v. at 4-h intervals from Days 5 to 12 of the oestrous cycle (oestrus = Day 0; Group H + LH; N = 7); (2) hypophysectomy with no LH replacement (Group N–LH; N = 6); (3) control (no hypophysectomy) plus LH replacement as in Group H + LH (Group S + LH; N = 7); (4) control with no LH treatment (Group S–LH; N = 7). Blood samples were collected at 4-h intervals throughout the experiment to monitor circulating concentrations of LH, cortisol and progesterone. On Day 12 of the oestrous cycle corpora lutea were collected and luteal progesterone concentrations, unoccupied receptors for LH and number and sizes of steroidogenic and non-steroidogenic luteal cell types were determined. Corpora lutea from ewes in Group H–LH were significantly smaller (P < 0·05), had lower concentrations of progesterone, fewer LH receptors, fewer small luteal cells and fewer non-steroidogenic cells than did corpora lutea from ewes in Group S–LH. The number of large luteal cells was unaffected by hypophysectomy, but the sizes of large luteal cells, small luteal cells and fibroblasts were reduced. LH replacement in hypophysectomized ewes maintained luteal weight and the numbers of small steroidogenic and non-steroidogenic luteal cells at levels intermediate between those observed in ewes in Groups L–LH and S–LH. In Group H + LH ewes, luteal and serum concentrations of progesterone, numbers of luteal receptors for LH, and the sizes of all types of luteal cells were maintained. Numbers of small steroidogenic and non-steroidogenic cells were also increased by LH in hypophysectomized ewes.

In Exp. II, 14 ewes were assigned to: (1) sham hypophysectomy with no LH replacement therapy (Group S–LH; N = 5); (2) sham hypophysectomy with 40 μg ovine LH given i.v. at 4-h intervals from Day 5 to Day 12 of the oestrous cycle (Group S + LH; N = 5); and (3) hypophysectomy plus LH replacement therapy (Group H + LH; N = 4). Experimental procedures were similar to those described for Exp. I. Treatment of hypophysectomized ewes with a larger dose of LH maintained luteal weight, serum and luteal progesterone concentrations and the numbers of steroidogenic and non-steroidogenic luteal cells at control levels. Based on the results of the two experiments it is concluded that: (1) LH has important trophic actions for steroidogenic and non-steroidogenic luteal cells; and (2) LH appears to be the only pituitary gonadotrophin required to maintain luteal function in ewes hypophysectomized during the oestrous cycle.

Keywords: sheep; LH; luteal cells

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R. H. Schwall, H. R. Sawyer and G. D. Niswender

Introduction

In a nonfertile cycle, the corpus luteum of the ewe secretes progesterone for about 2 weeks, and then regresses and is resorbed within the ovary. Luteolysis does not occur, however, if fertilization takes place. In fact, luteolysis must be prevented for pregnancy to be successful. The most important function of the corpus luteum appears to be the secretion of progesterone, since removal of the corpus luteum in early gestation causes abortion, but abortion is prevented by progesterone therapy (Amoroso & Perry, 1977).

Since progesterone plays a crucial role in the establishment and maintenance of early pregnancy, elucidation of the factors which regulate progesterone secretion is important for understanding the mechanisms involved in the maternal recognition of pregnancy. Perhaps the most important factors involved in the regulation of progesterone secretion are luteinizing hormone (LH) and the prostaglandins. LH stimulates progesterone secretion by luteal tissue and can maintain luteal function in

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L. M. Harrison, N. Kenny and G. D. Niswender

Summary. Corpora lutea were collected from sheep on Days 6,10, and 15 of the oestrous cycle and Day 25 of pregnancy and dissociated into single cell suspensions. Purified preparations of large and small luteal cells were prepared by elutriation on all days except Day 6. Basal progesterone production by large cells was 6–8-fold higher than by small cells (36–65 vs 6–9 fg/cell/min). Oxytocin secretion was maximal on Day 6(1·0 fg/cell/min) and declined thereafter. The number of receptors for LH increased between Day 6 and Day 10 and the two cell types had an equal number of receptors on Days 10 and 15 (19 000–23 000). Large cells on Day 25 of pregnancy had fewer receptors (12 000) than did small cells (26 000). Progesterone secretion by small luteal cells from all days examined was stimulated by LH (0·01–1000ng/ml) in a dose-dependent manner; maximum sensitivity to LH occurred on Day 10. Despite the presence of receptors for LH on large cells, LH failed to stimulate progesterone production. Basal production of progesterone by large and small cells, and the response of small cells to LH, was not influenced by day examined. Re-combinations of large and small cells from Day 10 synergized to increase progesterone secretion. Prostaglandin E-2 (0·1–1000 ng/ml) did not stimulate progesterone secretion by large or small cells.

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S. Edwards, J. F. Roche and G. D. Niswender

Summary. Treatment of 97 suckling beef cows at about 30 days post partum with doses of 0·25, 0·5, 1·0, 2·5, 3·0, or 5·0 μg Gn-RH/pulse for period of 2–4 days at 1-h or 2-h intervals in three different experiments did not increase the number of cows ovulating when compared to untreated controls. Treatment with 0·25 or 0·5 μg Gn-RH/pulse resulted in little or no LH release, whereas higher doses gave discernible LH releases after each Gn-RH pulse. Pretreatment with progesterone did not affect the ovulatory or LH responses.

These data indicate that pulsatile injections of Gn-RH, at the dose levels and frequencies used, cannot reactivate ovarian cyclicity in a majority of cows. We suggest that the degree of follicular development at the time of treatment is a major factor determining the ovulatory response to such pulsatile Gn-RH regimens.

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G. D. Niswender, K. P. McNatty, P. Smith, K. D. Niswender, C. E. Farin and H. R. Sawyer

Summary. In Exp. 1 ovulation rates, plasma concentrations of progesterone, mean individual and total CL weights were determined on Days 4, 10 and 12 after oestrus of Booroola Merino ++ ewes and FF ewes. Mean ovulation rates ranged from 1·5 to 1·8 in ++ ewes and from 5·3 to 6·2 in FF ewes (P < 0·01). There were no differences in plasma concentrations of progesterone or total luteal weight between the two groups on any of the days studied. Individual CL were smaller (P < 0·01) in FF ewes than in ++ ewes. In Exp. 2 the numbers of luteal cells in CL collected from 5 ++ and 5 FF ewes on Day 10 of the oestrous cycle were morphometrically determined. The CL from FF ewes were smaller (P < 0·01) and had fewer total steroidogenic cells (P < 0·01), fibroblasts (P < 0·01), and capillary endothelial cells and pericytes (P < 0·05). However, the luteal cell volume density, number of cells/g tissue, average cell diameter or average cell volume was not different between the two groups of ewes for any cell type studied. It is concluded that the 5–6 CL in FF ewes function in an identical fashion to the 1–2 CL in ++ ewes.

Keywords: Booroola ewes; luteal cells; steroidogenesis

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T. D. Bradent, M. E. King, K. G. Odde and G. D. Niswender

Summary. Oestrus, expected to be followed by a short luteal phase, was induced in post-partum cows by weaning their calves at 35 days after parturition. Ovaries containing the first preovulatory follicles (Type F) formed after parturition were collected 3 h after the onset of oestrus. For comparison, preovulatory follicles (Type C) were collected 3 h after the onset of oestrus in normally cycling cows. The number of granulosa cells was determined and the concentrations of receptors for follicle-stimulating hormone (FSH) and luteinizing hormone (LH) in granulosa cells and for LH in theca cells were measured. Concentrations of oestradiol-17β, testosterone, androstenedione and progesterone in follicular fluid were also measured. Type F follicles contained about twice the number of granulosa cells (based on DNA) as did Type C follicles (45·8 ± 11·3 and 24·5 ± 3·9 μg DNA/follicle, respectively; P < 0·05) but these cells had fewer receptors for LH (0·13 ± 0·02 vs 0·29 ± 0·03 fmol/μg DNA; P < 0·01) and FSH (0·61 ± 0·08 vs 1·3 ± 0·29 fmol/μg DNA; P < 0·08) than did those from Type C follicles. Additionally, there were fewer receptors for LH in theca tissue from Type F than from Type C follicles (28·3 ± 5·2 vs 51·3 ± 6·1 fmol/follicle; P < 0·01). Concentrations of oestradiol-17β (475·8 ± 85·6 vs 112·9 ± 40·0 ng/ml; P < 0·01) and androstenedione (214·1 ± 48·7 vs 24·7 ± 7·7 ng/ml; P < 0·01) in follicular fluid were higher in Type C than in Type F follicles. There were therefore more granulosa cells in Type F follicles which contained fewer receptors for LH and FSH and accumulated less oestradiol-17β in the follicular fluid. We suggest that the characteristics of preovulatory follicles may influence the life-span of the subsequently formed corpus luteum and short luteal phases may de due, in part, to altered follicular development.

Keywords: cow; post partum; anoestrus; follicle; gonadotrophin receptors

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S-J. Tsai, L. E. Anderson, J. Juengel, G. D. Niswender and M. C. Wiltbank

Prostaglandins regulate many physiological functions, including reproduction, by binding to specific plasma membrane receptors. In this study we evaluated the regulation of PGF (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 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 on luteal mRNA encoding FP and EP3 receptors. Ewes had PGF 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, whereas EP3 mRNA was unchanged. Treatment of large luteal cells with PGF, 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. In contrast, FP receptors are primarily expressed in corpora lutea and expression is inhibited by PGF.

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J. L. Juengel, T. M. Nett, R. V. Anthony and G. D. Niswender

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 (PGF; 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-induced luteolysis was not associated with a decrease in concentrations of mRNAs encoding either IGF-I or GH-R.

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J. L. Juengel, M. H. Melner, J. A. Clapper, A. M. Turzillo, G. E. Moss, T. M. Nett and G. D. Niswender

Prostaglandin F (PGF) 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 to activate fully its normal second messenger system, protein kinase C (PKC). Alternatively, increased resistance of the corpus luteum to PGF 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 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 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, 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 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 during the early part of the oestrous cycle but does not appear to mediate the increased resistance of the corpus luteum to PGF during maternal recognition of pregnancy. Neither PGF nor oestradiol affected steady-state concentrations of mRNAs encoding PKCI-1 or KCIP-I.