Summary. The introduction of rams to a group of previously isolated anoestrous ewes has been shown to stimulate ovarian follicular development and ovulation. The present experiment was carried out to determine the ability of follicles arising from this ram stimulus to produce steroids and bind hCG. Seasonally anoestrous Southdown ewes were exposed to rams for 24 h, 40 h, 3 days, 10 days or 20 days before ovariectomy. Steroid production and the concentration of hCG binding sites in follicles dissected from the ovaries were measured in vitro. The presence of a ram caused ovulation and enhanced oestradiol production by follicles, but had little effect on total androgen production or the number of hCG binding sites present in the follicles when compared to follicles from anoestrous ewes. The oestradiol concentrations in large follicles were not as high as in preovulatory follicles from cyclic ewes reported in other studies. Follicles continued to develop through the ram contact period and when incubated after 40 h and 10 days of ram contact produced high levels of progesterone, indicating partial luteinization, although the corpora lutea (CL) resulting from the induced ovulations regressed prematurely. We suggest that the lack of hCG binding sites in ram-induced follicles may be the cause of poor luteinization and suboptimal development of luteal tissue after induced ovulation in ewes during seasonal anoestrus.
S. Atkinson, P. Williamson, C. L. Kang, and R. S. Carson
R. S. Carson, D. M. Robertson, and J. K. Findlay
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
F. P. Sumbung, P. Williamson, and R. S. Carson
Summary. Prepubertal ewe lambs were treated with FSH after progesterone priming for 12 days (Group P), monensin supplementation for 14 days (Group M) or a standard diet (Group C). Serial blood samples were taken for LH and progesterone assay, and ovariectomy was performed on half of each group 38–52 h after start of treatment to assess ovarian function, follicular steroid production in vitro and the concentration of gonadotrophin binding sites in follicles. The remaining ewe lambs were ovariectomized 8 days after FSH treatment to determine whether functional corpora lutea were present.
FSH treatment was followed by a preovulatory LH surge which occurred significantly later (P < 0·05) and was better synchronized in ewes in Groups P and M than in those in Group C. At 13–15 h after the LH surge significantly more large follicles were present on ovaries from Group P and M ewes than in Group C. Follicles > 5 mm diameter from ewes in Groups P and M produced significantly less oestrogen and testosterone and more dihydrotestosterone, and had significantly more hCG binding sites, than did similar-sized follicles from Group C animals. Ovariectomy on Day 8 after the completion of FSH treatment showed that ewes in Groups P and M had significantly greater numbers of functional corpora lutea.
These results indicate that, in prepubertal ewes, progesterone priming and monensin supplementation may delay the preovulatory LH surge, allowing follicles developing after FSH treatment more time to mature before ovulation. This may result in better luteinization of ruptured follicles in these ewes, with the formation of functional corpora lutea.
R. S. Carson, L. A. Salamonsen, and J. K. Findlay
Summary. A 'double isotope' technique has been used to describe the temporal relationship between plasma and follicular concentrations of LH after injection of 51Cr and 125I-rat LH into immature rats. Radiolabelled LH was detectable in all follicles 1 min after injection. Concentrations in small antral and large preovulatory follicles were not significantly different at any time and reached a maximum of 34·2±3·0% of plasma concentrations at 40 min. Concentrations of LH in preovulatory follicles exposed to an ovulatory dose of hCG 4 h previously were significantly greater (P<0·05) than those in small antral and preovulatory follicles at all times, and reached a maximum of 46·2±1·7% of plasma concentrations after 1 h. Polyacrylamide gel electrophoresis and immunoprecipitation with an antibody specific for rat LH indicated that radioactivity in plasma and follicular fluid represented radio-iodinated LH. Steroidogenic activities, light microscopy and measurements of follicular volume of each class of follicle confirmed that small antral, preovulatory follicles and preovulatory follicles exposed to an ovulatory dose of hCG in vivo could be isolated specifically.
Based on these findings it is possible to calculate that, during an endogenous pulse of LH secretion, follicular concentrations of LH never exceed 20% of peak plasma concentrations. Pronounced increases in functional activities during antral growth were not correlated with increased follicular permeability. Only after acute exposure to an ovulatory dose of hCG in vivo was permeability significantly increased. We conclude that entry of LH into antral follicles is restricted and that exposure to an ovulatory dose of hCG results in greater amounts of LH entering preovulatory follicles.
C. G. Tsonis, R. S. Carson, and J. K. Findlay
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.
C. G. Tsonis, L. P. Cahill, R. S. Carson, and J. K. Findlay
Summary. Follicles of various sizes at the surface of the ovary were ablated by electrocautery at the time of cloprostenol-induced luteolysis in ewes and the interval from cloprostenol treatment to the onset of the LH surge determined as an index of the time from luteolysis to ovulation. When follicles 2–4 mm or > 4 mm diameter remained in the ovaries, the interval from cloprostenol treatment to the onset of the LH surge was similar to that in sham-operated (control) ewes (55–60 h), whereas when the only follicles remaining were < 2 mm, the interval was extended by 24 h (P < 0·05). This study demonstrates that follicles capable of ovulating can be selected from those ≥ 2 mm diameter at luteolysis, emphasizing the flexibility of the sheep ovary in its final selection of the ovulatory follicle.
R. S. Carson, Z. Zhang, L. A. Hutchinson, A. C. Herington, and J. K. Findlay
Medical Research Centre, Prince Henry's Hospital, St Kilda Road, Melbourne, Victoria 3004, Australia
Keywords: ovary; granulosa; theca; growth factors
The studies of the effects of epidermal growth factor (EGF) and fibroblast growth factor (FGF) on bovine granulosa cells under chemically defined conditions in vitro first indicated that these growth factors have a role in stimulation of follicle cell proliferation (Gospodarowicz et al., 1977). Even now, however, relatively few data in respect of the mitogenic action of these growth factors on follicle cells are available and most studies have concentrated on the role of growth factors in control of functional differentiation of follicle cells. This probably reflects the technical and interpretive difficulties associated with stimulation of follicle cell mitosis under defined conditions.
It is now evident that a range of growth factors influence not only proliferation but also functional differentiation of ovarian follicle cells. The growth factors of specific interest are