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R. Webb

Summary. Treatment of ewes with a 3β-hydroxysteroid dehydrogenase (3β-HSD) inhibitor (Epostane) resulted in a significant increase in both ovulation rate and in the mean number of lambs per ewe lambing. The progestagen sponge plus 3β-HSD inhibitor treatment also caused a significant increase in oestrous cycle duration of approximately 1·5 days. Treatment of ewes with the 3β-HSD inhibitor caused a significant decrease in peripheral progesterone concentrations, which were reduced even further when 3β-HSD inhibitor treatment was given to ewes after insertion of a progestagen sponge. However, mean oestradiol concentrations were significantly higher in the two treatment groups, both at the end of the luteal phase and during the follicular phase of the oestrous cycle. These results demonstrate that ovulation rate and the production of lambs per ewe lambing can be significantly increased by 3β-HSD inhibitor treatment.

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DG Armstrong and R Webb

Folliculogenesis is associated with the development of a group of follicles at various stages of maturation from which a species-specific number of follicles are selected for continued growth. These selected follicles, after being exposed to the requisite hormonal environment, ovulate in response to the preovulatory gonadotrophin surge. Follicular dominance is the mechanism by which the selected follicle(s) undergoes rapid development in an environment where growth and development of other follicles, recruited at a similar time, are suppressed. These processes are controlled by the interaction of endocrine signals and locally produced ovarian growth factors. The response of the two major follicular cell types, granulosa and theca cells, to gonadotrophins is regulated by the local production of growth factors. Mechanisms controlling growth factor action occupy a central role in the regulation of folliculogenesis. In this review, we highlight the influence of the extracellular matrix in this process by describing its involvement in regulating the activity of components of the insulin-like growth factor system, transforming growth factor beta superfamily, fibroblast growth factors and the epidermal growth factor/transforming growth factor alpha family. In addition, some recent studies on the role of protein factors produced by the dominant follicle in maintaining dominance and inhibiting the growth of subordinate follicles are described.

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C. A. Price and R. Webb

Summary. The aims of this study were to investigate whether treatment with a single ovulatory dose of hCG, between the day of oestrus and the end of the luteal phase, could induce extra ovulations in heifers and whether the presence of an existing corpus luteum (CL) affected the response. Heifers (N = 32) were injected with 1500 i.u. hCG or saline on a given day of the oestrous cycle. Treatments were repeated during subsequent cycles to provide a total of 71 observations, 57 of which followed an injection of hCG, given between Day 0 (oestrus) and Day 16, and 14 of which followed saline injections as controls. Ovulatory responses were noted by laparoscopy 2 days after hCG treatment. No heifers injected with saline produced additional CL. Of the hCG-treated cycles, 23 resulted in the formation of an additional CL, and this was significantly affected by the stage of the oestrous cycle when hCG was given; a greater response was observed during the early (Days 4–7) and late (Days 14–16) stages of the luteal phase than at the mid-luteal phase of the oestrous cycle. Two heifers were also treated with hCG on Days 17 or 18 of the oestrous cycle, but before oestrus; both had induced CL. There were no significant differences between the left–right orientation of the existing CL or the hCG-induced CL.

These results demonstrate that the large, luteal-phase follicle of the cow is capable of ovulating in response to hCG and that the induced CL is not affected by the presence of an existing CL.

Keywords: cattle; ovulation; hCG; follicle; luteal phase

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R. Webb and B. G. England

Summary. Ewes were ovariectomized before (Group 1, N = 5) or after (Group 2, N = 6) the peak of the preovulatory gonadotrophin surge. Ovarian secretion rates of oestradiol and testosterone were significantly higher in Group 1 than in Group 2. The presence of high levels of LH receptors in both thecal and granulosa cells was used to identify ovulatory from non-ovulatory follicles. There was a significant fall in the LH receptor concentration in the thecal and granulosa cells of ovulatory follicles after the peak of the preovulatory gonadotrophin surge. Ovulatory follicles in Group 1 produced significantly more oestradiol and testosterone in vitro than did those in Group 2. In both groups ovulatory follicles secreted significantly more oestradiol in vitro than did non-ovulatory follicles. Follicular fluid oestradiol concentrations were similar in pattern to the in-vitro oestradiol secretion activity in ovulatory and non-ovulatory follicles. However, follicular fluid testosterone concentrations were significantly higher in non-ovulatory follicles than in ovulatory follicles. Incubation of follicles with 250 ng testosterone/ml did not significantly alter the in-vitro oestradiol secretion rate in any of the groups of follicles except for Group 2 non-ovulatory follicles in which oestradiol accumulation increased. The number of thecal and granulosa cell LH receptors was significantly correlated with follicular fluid oestradiol concentrations in ovulatory follicles and with in-vitro oestradiol production by Group 1 ovulatory follicles. It is suggested that the fall in oestradiol secretion rates, which occurs after the peak of the preovulatory gonadotrophin surge, may be due to a decrease of aromatase activity associated with a fall in the concentration of LH receptors and is not due to a lack of the oestrogen precursor testosterone. The elevated concentration of testosterone and low oestradiol concentrations in non-ovulatory follicles compared with ovulatory follicles are probably due to an inactive aromatase system, perhaps associated with the lack of granulosa cell LH receptors.

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B. K. Campbell, R. J. Scaramuzzi and R. Webb

A serum-free ovine granulosa cell culture system is described that allows the induction of FSH-responsive oestradiol production by undifferentiated cells from small (< 3.5 mm) follicles (P < 0.001) and the maintenance of oestradiol production by differentiated cells from large (≥3.5 mm) follicles. Physiological doses of FSH stimulated (P < 0.01) proliferation of cultured granulosa cells from both small and large follicles. The synthesis of immunoreactive inhibin and progesterone by granulosa cells from small and large follicles increased (P < 0.01) with time of culture, and was not dependent on FSH. Inhibin secretion expressed on a per cell basis was not FSH responsive. Insulin and insulin-like growth factor I (IGF-I), in the presence of FSH, stimulated (P < 0.001) cell proliferation and oestradiol and inhibin production by granulosa cells from small and large follicles. There was a significant (P < 0.001) interaction between insulin and IGF-I in the stimulation of granulosa cell proliferation and differentiation. Both epidermal growth factor (EGF) and transforming growth factor α (TGF-α) in the presence of FSH stimulated cellular proliferation (P < 0.001) in a dose-responsive manner and concomitantly inhibited (P < 0.001) oestradiol and inhibin secretion. The development of this granulosa cell culture system will make it possible to study, in vitro, the cascade of events that controls granulosa cell differentiation and ultimately follicle selection in sheep.

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R. G. Gosden, M. I. Boulton, K. Grant and R. Webb

Cortical slices of either cat or sheep ovaries were grafted under the renal capsules of ovariectomized SCID mice. The grafts became vascularized and were still surviving with large follicles present at autopsy up to nine months later. As developing follicles undergo atresia during the period of ischaemia after ovarian grafting, those found in long-term grafts at autopsy had presumably started to grow from the primordial stage after transplantation. Some follicles had reached a diameter of 3 mm with a normal antrum and appeared to be cytologically normal, but the latent period for the emergence of antral follicles was shorter in cat compared with sheep grafts. Oestradiol production from grafts, as indicated by vaginal cornification and plasma measurements collected at autopsy, was not constant and circulating concentrations varied among animals, and were sometimes far in excess of the normal physiological range of the host. The vaginal smears never presented cytological patterns like those of the normal mouse oestrous cycle, and ovulation had not occurred in any of the grafts. These results demonstrate that ovarian xenografts in SCID mice can serve as experimental models for investigating follicle development in species in which follicle growth in vitro and studies of the parent animal are impracticable.

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M. A. Driancourt, R. Webb and R. C. Fry

Summary. The process by which a single follicle is selected to ovulate while others regress is unknown in ewes. If the dominant follicle secretes substances that directly inhibit the growth of other follicles, the superovulatory response to the administration of exogenous gonadotrophins may be blunted. Administration of 1250 iu pregnant mares' serum gonadotrophin (PMSG) before or after the emergence of the dominant follicle in the follicular phase, or 1000 iu PMSG in the presence or absence of a large healthy or atretic follicle during the luteal phase did not affect the induced ovulatory response. Comparisons between the ovary with or without the dominant follicle did not reveal any differences in ovulatory response to PMSG. The in-vitro features (i.e. mitotic index, oestradiol and testosterone production) of follicles ipsilateral or contralateral to the dominant follicle during the early and late follicular phases were also similar.

If the dominant follicle secretes substances detrimental to the other follicles, this could be mimicked in vitro. Co-culture of small follicles with the largest follicles in a closed system did not reduce their incorporation of 3H thymidine in granulosa cells, compared with small follicles cultured alone.

These data suggest that dominance is probably not operative in sheep. The administration of 500 iu of PMSG during the midfollicular phase increased ovulation rate in Merino ewes, indicating that dominance is essentially passive in ewes and can easily be overcome by raising gonadotrophin concentration.

Keywords: follicle; ovulation; gonadotrophin; paracrine regulation; sheep

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C. A. Price, B. A. Morris and R. Webb

Summary. Yearling heifers were actively immunized against 8 mg testosterone-3-carboxymethyloxime-ovalbumen in Non-Ulcerative Freund's Adjuvant, with or without the addition of Corynebacterium parvum (Groups A and B, respectively; N = 4 for each group). After the priming injection, Groups A and B were boosted twice at 4-monthly intervals. Control heifers (N = 9) were not injected. All treated animals except one gave a measurable antibody response, and all responding animals became anoestrous and displayed ovarian cysts after the first booster injection. There were no apparent differences between treatments, and so results for Groups A and B were pooled.

At 25 weeks after the second booster 3 of the 7 responding, anoestrous heifers resumed cyclicity; one with two consecutive double ovulations, and one with one double ovulation. The 3rd heifer showed 4 corpora lutea, then became anoestrous again. The 4 remaining acyclic heifers, and the control heifers, were intensively blood sampled; the anoestrous heifers showed significantly higher mean LH and significantly lower mean FSH concentrations and higher LH pulse frequency than did the control animals. These heifers remained anoestrous for 11 months after the second booster, at which point they were injected with GnRH and PGF-2α; only 1 heifer resumed ovarian cyclicity. These results indicate that it is possible to increase ovarian activity in cattle by active immunization against testosterone, but that there is a high incidence of anoestrus.

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R. Webb, I. K. Gauld and M. A. Driancourt

Summary. Morphological and functional features of large ovarian follicles from three breeds of sheep, with different ovulation rates (Finnish Landrace N = 12, Finnish Landrace × Scottish Blackface N = 16, Merino × Scottish Blackface N = 16) were compared by integrating three techniques; ink labelling, in-vitro oestradiol production and morphological classification. The follicles were removed at two stages of the follicular phase, 1 (PG + 1) or 2 (PG +2) days after PGF-2α treatment and compared after monitoring their rates of growth with the use of ink labelling. After ovariectomy all follicles ≥ 1 mm in diameter were dissected, and the 8 largest were incubated individually for 2 h to assess their ability to secrete oestradiol and testosterone. After incubation the follicles were processed for histological examination and checked for atresia. An analysis of the follicle population was based on in-vitro oestradiol secretion rates in all three breeds; an oestrogen-active population producing 500–8100 pg oestradiol/ml/h and an oestrogen-inactive population producing 0–499 pg oestradiol/ml/h. A comparison of the 3 approaches demonstrated agreement on 94·3 ± 1 ·2% of occasions. Ink-labelling demonstrated that all follicles identified as oestrogen-active were increasing in size. Within oestrogen-active follicles significant correlations were detected between oestradiol production and testosterone production (r = 0·42), oestradiol production and granulosa cell number (r = 0·45) and between oestradiol production and mitotic index (r = −0·38). A regression model fitting breed, stage of atresia, granulosa cell number, in-vitro testosterone production and mitotic index demonstrated that granulosa cell number is a characteristic which contributes significantly to the variation of in-vitro oestradiol production in oestrogen-active and oestrogen-inactive follicles. There was no significant difference between breeds in the mean number of ink-labelled follicles growing from Day PG − 1 to Day PG +1. There was a significant difference between the breeds in the number of ink-labelled follicles growing between Days PG + 1 and PG +2 (Days 1 and 2 of the follicular phase), the number being similar to the ovulation rate for the breed. The majority of the oestrogen-active follicles had been recruited by Day PG − 1, although in the Finnish Landrace genotypes more than 30% were recruited on or after Day PG + 1 compared to less than 10% in Merino × Scottish Blackface ewes. Although mean oestradiol production by oestrogen-active follicles was similar in all three breeds, oestrogen-active follicles in Finnish Landrace ewes had significantly reduced granulosa cell number and mitotic index compared to the other breeds.

These results demonstrate: (1) a good agreement between the three criteria used to characterize follicles, (2) that the mechanisms controlling follicular recruitment into the population of gonadotrophin-dependent follicles are different from the mechanisms controlling the selection of the follicles destined to ovulate and (3) that despite no

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BK Campbell, DT Baird, CJ Souza and R Webb

The aim of this study was to differentiate between pituitary and ovarian actions of the FecB gene by measuring the ovarian response to a standardized treatment with gonadotrophins designed to mimic the changes in FSH and LH that occur in the follicular phase of the ovarian cycle in ewes, with (Fec(B/-), n=6) and without (Fec(+/+), n=9) the gene, that were rendered hypogonadotrophic by pretreatment with a potent antagonist of GnRH. Ewes with ovarian autotransplants were used to facilitate the assessment of follicular function by the collection of ovarian venous blood and ultrasonography. The gonadotrophin regimen resulted in concentrations of FSH and LH that were similar to concentrations found in a normal cycle and did not differ between genotypes. Follicular development and ovulation occurred in all animals, and patterns of secretion of oestradiol, androstenedione and inhibin A were normal. Despite these endocrine similarities, the antral follicle population stimulated by FSH infusion retained the characteristic genotypic difference with the ovaries of Fec(+/+) animals containing a range of follicle sizes with decreasing proportions of small (<3.5 mm in diameter) and medium (3.5-4.5 mm in diameter) follicles as well as large follicles (> or =4.5 mm in diameter), whereas the ovaries of Fec(B/-) ewes contained no follicles of >4.5 mm in diameter. This genotypic difference was retained after ovulation with gene carriers having more preovulatory follicles/corpora lutea (3.8+/-0.3) of a smaller diameter (5.3+/-0.3 mm) than did non-gene carriers (1.7+/-0.3; 11.4+/-0.9 mm; P<0.05). As ewes carrying the FecB gene mutation were able to ovulate more follicles than non-gene carriers, despite identical concentrations and patterns of FSH and LH stimulation, the results of this study support the hypothesis that the FecB gene acts at the ovary to enhance ovarian sensitivity to gonadotrophic stimulation.