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Summary. Corpora lutea from cyclic ewes were dissociated by collagenase and trypsin/ EGTA treatments, and enriched fractions of small and large luteal cells were prepared on gradients of Ficoll. These fractions were incubated separately or remixed before incubation. Colchicine, cytochalasin B and the calcium channel-blocker verapamil significantly reduced progesterone production by both small and large luteal cell fractions, while isoprenaline stimulated an increase in progesterone production by large luteal cell fractions only. When fractions of small and large luteal cells were remixed, no more and no less progesterone was produced than would have been predicted from equivalent fractions incubated separately. There was therefore no evidence of synergism between small and large luteal cells in the production of progesterone. Prostaglandin F-2α, which can inhibit LH-stimulated progesterone production by ovine luteal tissue in vitro, had no effect on LH-stimulated progesterone production by small luteal cell fractions, but significantly inhibited that by enriched fractions of large luteal cells. Since large luteal cell fractions were contaminated with small luteal cells, which are probably responsible for the progesterone-secretory response of these fractions to LH, it was concluded that the inhibition of LH-stimulated progesterone production by small luteal cells is dependent on the presence of large luteal cells. Oxytocin added to large and small luteal cell fractions did not affect progesterone production by either fraction. It was therefore concluded that the inhibitory action of PGF-2α on LH-stimulated progesterone production may require the interaction of large and small luteal cells, but that oxytocin is not likely to be an intermediary in this interaction.
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Summary. Ovariectomized ewes were given a single injection (i.v.) of 100 μg oestradiol-17β. Nuclear oestrogen receptor values in the pituitary, as a function of total receptor concentrations, were 3·0 ± 1·0% in controls, 56·0 ± 5·4% at 1 h (P < 0·001) and 6·5 ± 2·1% at 6 h (P > 0·05) after oestradiol injection. There was a fall in plasma LH values from 5·7 ± 1·0 (preinjection) to 2·1 ± 0·3 ng/ml (P < 0·01) 4–6 h after oestradiol. At 13–21 h after injection plasma levels increased to 37 ± 8 ng/ml (P < 0·001). Plasma FSH levels declined from 840 ± 18 to 506 ± 48 ng/ml after 20–22 h (P < 0·001). Plasma prolactin concentrations fell from 90±16 ng/ml before injection to 29 ± 9 ng/ml at 1 h (P > 0·05), and then rose to a maximum of 1537 ± 24 ng/ml (P < 0·001) 11–14 h later. These results show that transient nuclear compartmentalization of oestrogen receptors after a bolus injection of oestradiol was associated with the feedback effects of oestradiol on LH, FSH and prolactin release.
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Summary. Corpora lutea from cyclic ewes were dissociated by collagenase digestion and trypsin/EGTA treatment. Enriched fractions of endothelial cells, small luteal cells and large luteal cells were prepared on a stepped gradient of Ficoll 400. Progesterone was measured by radioimmunoassay and the results corrected so that progesterone production by each cell type could be determined. Endothelial cells did not produce significant amounts of progesterone, with or without LH stimulation, and endothelial cell contamination of small and large luteal cell fractions did not influence progesterone production by these fractions. Mean ± s.e.m. basal progesterone production (n = 10) by large luteal cells was greater (P < 0·001) on a per cell basis than that by small luteal cells (1·16 ± 0·16 compared with 0·25 ± 0·06 pg/h/cell). However LH, which stimulated a maximal 3–4-fold increase in progesterone production by small luteal cells (LH ED50 = 0·14 ng/ml), had no significant effect on production by large luteal cells, when contamination by small luteal cells was taken into account. The response of small luteal cells was specific to LH, other hormones having had no significant effect.
Basal progesterone production by small luteal cells (0·12 ± 0·03 fg/h/μm3) calculated per unit volume of cell was not significantly different from that of large luteal cells (0·17 ± 0·02 fg/h/μm3). After LH stimulation, small luteal cells produced more progesterone than did large luteal cells (0·40 ± 0·09 compared with 0·18 ± 0·03 fg/h/μm3) (P < 0·05). When the amounts of progesterone produced per cell were multiplied by the absolute numbers of large luteal (1 × 107) and small luteal (5 x 107) cells in the intact corpus luteum, basal progesterone production by large luteal cells (11·6 ± 1·6 μg/h) was similar to that by small luteal cells (12·3 ± 3·0 μg/h). However, under LH stimulation, progesterone production by the small luteal cell type (39·9 ± 9·5 μg/h) was ∼ 3 times greater than that by the large luteal cell type (12·3 ± 1·6 μg/h) (P < 0·05).
We therefore conclude that small luteal cells may be the principal source of luteal progesterone production in the sheep.
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Summary. Ovariectomized Merino ewes were used to develop an in-vivo bioassay for purified bovine inhibin of M r 31 000. Various doses (0·25, 0·5, 1 or 2 ml) of bovine follicular fluid, given either by the intravenous (i.v.) or intracarotid route (i.c.) resulted in significant linear dose-related suppression of plasma FSH and interval to maximum suppression. Control ewes (1·0 ml steer plasma) showed no significant change in FSH over the same period. Doses of 470 and 2590 U of pure inhibin given i.v. caused a significant suppression of FSH in plasma in all ewes. The in-vivo potency estimate of the high dose (2760 U, 1420·4690 fiducial limits) agreed well with the in-vitro assay of potency. There were no significant changes observed in mean plasma LH after treatment with the higher dose of pure inhibin. There were no rebound effects of treatment with bovine follicular fluid or pure inhibin on FSH concentrations above that of controls. It is concluded that the form of bovine inhibin of M r 31 000, which is believed to be the predominant circulating form, is biologically active when administered in vivo.
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Summary. The concentrations of prostaglandin F (PGF) and its major metabolite, 13,14-dihydro-15-keto prostaglandin F-2α (PGFM), were measured in caruncular and intercaruncular endometrium of pregnant and non-pregnant ewes on Days 9,11,13 and 15 after mating (Day 0) and related to the content of PGF and PGFM in the uterine flushings. The tissue concentrations of PGF and PGFM increased with time after mating particularly on Days 13 and 15 and to a greater extent in pregnancy. However, the ratio of PGF to PGFM remained constant at 0·7, except on Day 15 in non-pregnant endometrium when it fell to 0·3 (P < 0·05), suggesting that synthesis rather than metabolism was limiting tissue concentrations of PGs.
The changes in tissue concentrations of PGF and PGFM were reflected in the contents of PGF and PGFM in the uterine flushings of non-pregnant, but not pregnant ewes. Pregnant ewes had relatively more PGF and less PGFM than did non-pregnant ewes on Day 15. Moreover, there was always 5–10-fold less PGFM than PGF in the uterine flushings. It is concluded that the increase in PG in the uterine lumen in pregnancy has its origin in the blastocyst, and that pregnancy may be associated with an increase in the synthesis and retention of PGs in the endometrium, rather than a redistribution towards the uterine lumen away from the uterine venous drainage.
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Summary. Groups of 5–6 ovariectomized ewes were treated with oestradiol-17β (100 mg implant) and/or bromocriptine (1 mg; 2 × daily) or with vehicle for 30 days during the anoestrous season. Oestradiol increased (P < 0·05) the priming effect of 2 × 1 μg LH-RH on LH release and decreased (P < 0·001) pituitary LH content, plasma LH levels, the frequency and amplitude of pulsatile LH release and the LH response to LH-RH. Bromocriptine decreased prolactin levels to <2·5 ng/ml (P < 0·001) and increased the LH response to LH-RH (P < 0·05). There were no significant treatment interactions. It is concluded that prolactin does not enhance the inhibitory effect of oestradiol on LH synthesis and release in the ewe.
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The effects of transforming growth factor α (TGF-α) on ovarian steroid secretion were investigated. Three crossbred ewes synchronized for oestrus with ovarian autotransplants were infused with TGF-α (30 μg in 12 h) via the ovarian artery for 12 h before withdrawal of progestagen pessary. Three ewes were used as controls. Jugular and ovarian venous blood samples were taken at intervals of 10 min at two stages during the follicular phase (21–27 h and 38–42 h after pessary withdrawal) and every 2 h from 44 to 86 h. Plasma LH and FSH concentrations, and ovarian secretion rates of inhibin, androstenedione, oestradiol and progesterone were determined using radioimmunoassays. LH pulse amplitude increased in ewes treated with TGF-α in the early follicular phase (0.92 ± 0.25 μg l−1 in controls versus 3.10 ± 0.35 μg l−1 in TGF-α treated ewes; P < 0.05) and remained high in the late follicular phase. Plasma FSH concentrations were high during the follicular phase in ewes treated with TGF-α (P < 0.05). The infusion of TGF-α had no significant effect on the ovarian rate of secretion of androstenedione and, although the secretion rates of oestradiol and inhibin were consistently lower in TGF-α-infused ewes, the differences were not significant. The ratio of secretion of androstenedione to oestradiol was greater during the follicular phase in TGF-α-treated ewes (P < 0.05), suggesting that the efficiency of aromatization had been impaired. Progesterone secretion was enhanced by treatment with TGF-α; progesterone secretion was detectable 52 h and 24 h after pessary withdrawal and had reached a secretion rate of 16.4 ± 17.4 ng min−1 and 119.6 ± 21.9 ng min−1 by 86 h after pessary withdrawal in control and TGF-α-treated ewes, respectively. An increase in progesterone secretion would suggest that TGF-α had induced either premature luteinization or atresia in the large antral follicles.
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Summary. Ovariectomized ewes were given 2 ml s.c. injections of ovine follicular fluid (oFF) (N = 3) or serum (N = 3) and blood samples were collected each day for 3 days. Follicular fluid caused a significant (P < 0·005) reduction in FSH within 1 day, but did not affect mean LH values. Two groups of 3 ewes were treated as above but sampled intensively (each 10 min for 6h) on Days 1 (before treatment) and 4; mean plasma FSH concentration and plasma LH pulse frequency and amplitude were ascertained. Significant (P < 0·005) reduction of FSH concentration was seen in the oFF-treated ewes. A non-specific reduction in LH pulse amplitude, but not pulse frequency, was noted in the control ewes. This experiment was repeated with 2 groups of 4 ewes that were conditioned to the experimental environment and effects on LH secretion were not observed in the controls given serum. Treatment with oFF caused a 70% reduction (P < 0·005) in plasma FSH and a small (30%) but significant (P < 0·005) reduction in mean LH concentrations. The latter was probably associated with a reduction in LH pulse amplitude in 3/4 animals (N.S.) with no change in LH pulse frequency. Treatment with oFF, as in Exp. 1, caused a 95% reduction in FSH values and significant (P < 0·01) reduction (32%) of LH pulse amplitude in ovariectomized ewes that had been subjected to hypothalamo-pituitary disconnection and in which gonadotrophin secretion was reinstated with pulses of 250 ng GnRH every 2 h.
These results suggest that proteins from the sheep follicular fluid, including inhibin, act at the pituitary level to inhibit FSH secretion and may have some effects on LH pulse amplitude.
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Summary. Aromatase activity was measured in granulosa cells using a 1-h in-vitro assay. This activity correlated with the concentration of oestradiol-17β and the ratio of oestradiol-17β to testosterone in follicular fluid of individual follicles ranging from 1·5 to 7·0 mm diameter. These data show an 8–10-fold difference in aromatase activity between small and large follicles and that aromatase activity per cell increased in small non-atretic follicles (<3·5 mm) whereas it remained relatively constant in large nonatretic follicles (≥3·5 mm). Aromatase activity was much lower in follicles at more advanced stages of atresia. Atresia was assessed using the morphological and the morphometric methods (% of maximum number of granulosa cells/follicle). Although the morphological method of assessment was preferable to the morphometric method, it did not differentiate a decrease in aromatase activity as a very early event in the atretic process. We believe this is due to the inability of these methods to detect follicles in the initial stages of atresia.
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Summary. The ability of antral follicular fluid obtained from sheep follicles to inhibit 3T3 fibroblasts maintained for 48 h in concentrations of fetal calf serum optimal for cell growth was examined. Addition of pooled follicular fluid to cultures resulted in a dose-dependent and reversible inhibition of [3H]thymidine incorporation. Serum from ovariectomized ewes, fetal calf serum, bovine inhibin, oestradiol-17β, testosterone, cortisol or progesterone were without effect over a range of doses. Treatment of pooled follicular fluid with charcoal–dextran did not reduce inhibitory activity which was only partly removed by heating at 85°C. Fluid obtained from large follicles (>5 mm) was more potent as an inhibitor than was fluid obtained from smaller (<5 mm) follicles. Gel chromatography of pooled fluid resolved two peaks of inhibitory activity associated with material of M r ∼ 180 000 and <10 000 respectively. No inhibitory activity was evident in fractions of serum from ovariectomized ewes chromatographed in an identical manner. These results indicate that ovine follicular fluid contains two components able to inhibit reversibly mitosis of 3T3 fibroblasts in vitro.
Keywords: fibroblast mitosis in vitro; inhibition; ovarian follicular fluid
