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.

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.

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

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.

The objective of this study was to develop a defined culture system in which bovine follicular and granulosa cells are grown in close contact with each other and with the extracellular matrix (ECM) component laminin. Granulosa and theca cells from follicles 4-6 mm in diameter were cultured on either side of laminin-coated BioCoat cell culture inserts in a serum-free medium containing 10 ng insulin ml(-1) at plating densities of 10(5) and 3 x 10(5) cells per membrane side. The cells adopted a clumped arrangement, maintained steroidogenic activity for at least 7 days and demonstrated paracrine communication by increased steroidogenesis and enhanced cell survival compared with cells in mono-culture. Co-cultured theca cells secreted significantly more androstenedione compared with cells in mono-culture. Granulosa cell viability was doubled by co-culture with theca cells. Co-cultures at both cell plating densities were responsive to treatment with physiological combinations of either FSH, LH and LR3 insulin-like growth factor I (IGF-I) (treatment A) or FSH, LR3 IGF-I and androstenedione (treatment B). Significantly more androstenedione was secreted in the presence of treatment A compared with controls. In contrast, oestradiol secretion was increased only by treatment B. Progesterone secretion was unaffected by treatment and did not increase during culture. Co-cultures at the higher plating density demonstrated higher theca cell survival and better maintenance of the follicular cell phenotype. In conclusion, this novel co-culture system provides a unique model for the study of paracrine communication between ovarian somatic cells and cell-ECM interactions during follicle growth.

UThe insulin-like growth factor binding proteins (IGFBPs) bind IGFs with high affinity and so regulate their access to the type 1 and 2 IGF receptors. This is the principal mechanism involved in regulating IGF bioavailability during folliculogenesis. IGFBPs undergo a number of post-translational modifications, including proteolytic cleavage, phosphorylation and glycosylation, which can regulate the affinity of IGFBPs for IGFs. However, the post-translational changes to IGFBPs that occur during folliculogenesis have not been fully characterized. The charge and size variants of the IGFBPs in bovine follicular fluid were examined by two-dimensional non-reducing SDS-PAGE followed by non-isotopic western ligand blot analysis, and immunoblot analysis during follicular development. The results demonstrate the presence of at least 51 IGFBP isoforms corresponding to IGFBP-1 to -6 in bovine follicular fluid from subordinate follicles, many of which were phosphorylated. The total number of IGFBPs was reduced in dominant follicles, whereas no gross changes in isoforms were observed during follicular development. These results demonstrate the high degree of conservation of IGFBP post-translational modifications between species, and from the in vitro dephosphorylation of these proteins it is hypothesized that these modifications may result in changes to IGF binding or susceptibility to proteolytic cleavage.

Although it has become increasingly clear that fertility in modern dairy cattle is declining in association with increased milk yields, the underlying mechanism is poorly understood. The first ovulation post partum is delayed in dairy cows undergoing selection for genetic merit for milk yield in association with lower circulating insulin concentrations. The aim of this study was to investigate whether feeding a diet to increase circulating insulin concentrations can overcome this delay in the first ovulation post partum. The experiment was a 2 x 2 factorial design (n = 10 per group) involving diet and genetic merit for milk yield. The dietary treatment started on the day of calving and lasted for 50 days. Plasma samples were collected each day and ovarian ultra-sonography was performed three times a week during the experimental feeding period. Milk yield was recorded each day, and body weight and body condition score were determined each week. Milk samples were collected three times a week from day 50 to day 105 post partum, and reproductive performance data were recorded for all the cows as part of the routine farm practice. The dietary treatment induced significant differences in plasma insulin concentrations in both high and low genetic merit cows. Although high genetic merit cows produced more milk, lost more body weight and had lower body condition scores during the experiment, no significant effect of diet was observed on these measurements. The high insulin inducing diet increased the proportion of cows ovulating within 50 days of calving and reduced the intervals from calving to first ovulation, and tended to reduce the intervals from calving to first service and to conception. These fertility parameters were also more favourable in low than in high genetic merit cows, but no interaction between diet and genetic merit was observed for any of these parameters. Genetic merit, but not diet, also affected the number of services required per conception and the conception rate. In conclusion, these results have confirmed that genetic selection for high milk yield is associated with a decrease in reproductive performance in dairy cows. More importantly, this study has demonstrated that it is possible to alleviate this problem by nutritional manipulation.

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.

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.

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