The bone morphogenetic protein (BMP) family is emerging as playing a crucial role in regulating normal follicle growth and determining ovulation rate. BMPs exert their effects via BMP receptors (BMPR-IA, -IB and -II). However, there is a paucity of information relating to the expression of the BMPRs within the ovary of large polyovular species such as the pig. Furthermore, there is a lack of information on the expression of BMPRs by fetal ovaries of any species. The purpose of this study was to investigate temporal and spatial expression of the BMPRs in the porcine ovary, at different developmental stages. Immunohistochemistry for BMPR-IA, BMPR-IB and BMPR-II was performed using sections from paraffin wax-embedded ovaries, obtained from fetal (n = 15), prepubertal (n = 3) and cycling postpubertal (n = 4) pigs. Results confirmed the presence of all three receptors in the fetal egg nests and in the granulosa cell layer of follicles ranging from primordial to late antral stages. Immunostaining was also observed in oocytes, theca layer, corpus luteum and ovarian surface epithelium. The expression of BMPRs by fetal ovaries may be related to follicle formation, whereas expression in pre- and post-pubertal animals indicates BMPs are involved in regulating porcine ovarian follicle growth.

There is considerable evidence for a mammalian ovarian renin-angiotensin system, which may influence ovulation, angiogenesis and steroidogenesis via the autocrine and/or paracrine actions of the biologically active product of the cascade, angiotensin II (AngII). There are two characterized AngII receptors -type 1, AT 1 and type 2, AT 2 .We report the localization of these receptor subtypes within porcine fetal, prepubertal and postpubertal ovaries. Positive staining for AT 1 and AT 2 receptors was observed in egg nests in all fetal ovaries studied, as well as in a defined two-cell layer at the ovarian periphery. In prepubertal tissue, positive AT 1 and AT 2 staining was localized to granulosa cells adjacent to the basement membrane of pre-antral and antral follicles, with no staining in the thecal layer. There was immunostaining for both receptors in prepubertal oocytes and zona pellucida. In postpubertal tissue, positive AT 1 and AT 2 immunostaining was localized to areas of putative neovascularization, the zona pellucida and the oocyte. Further AT 1 staining was located to the postpubertal antral follicle granulosa cells. The results indicate that there are higher densities of AT 1 receptors than AT 2 receptors in the porcine fetal, prepubertal and postpubertal ovary, and this has profound implications for the role of AngII in ovarian development.

A novel culture system is reported in which pig preantral follicles (< 300 microm in diameter) with an intact thecal cell layer were isolated and cultured in a serum-free medium for up to 30 days. The medium supported follicle culture after isolation, while maintaining both somatic cell and oocyte viability. Follicles were cultured in groups (n = 3 per group) on collagen-coated wells for 16 days, during which they retained a three-dimensional structure, maintained oocyte viability and increased in diameter and number of somatic cells. Follicle culture for 30 days resulted in a further increase in number of cells, oocyte viability was maintained, and a significant increase in follicle diameter was observed (P < 0.001), with 29% of follicles forming an antrum. Follicles synthesized measurable quantities of progesterone (168 pg per 100 microl per 48 h; no significant increase with time) and increasing quantities of oestradiol (136 pg per 100 microl per 48 h; P < 0.001 with time). Further supplementation of the medium with 100 micromol testosterone l(-1) at day 28 resulted in a significant increase in oestradiol secretion by both antral (P < 0.01) and preantral follicles (P < 0.05). Culture over 30 days in medium with 10(-10) mol angiotensin II l(-1) and further supplementation at day 28 with 100 micromol testosterone l-1 also increased oestradiol synthesis (P < 0.001). These results show that viable preantral follicles may be cultured for extended periods, and indicate that the possible role of angiotensin II in folliculogenesis and steroidogenesis in early development of pig follicles requires further investigation.

The aim of this study was to determine whether specific receptors for angiotensin II are present in prepubertal and postpubertal pig ovaries using an autoradiographic technique and computerized densitometry. Frozen sections were cut from prepared samples, and (125)I-labelled angiotensin II and the angiotensin II receptor subtype-specific nonpeptide antagonists for receptor subtype 1, AT(1) (GR117289) and subtype 2, AT(2) (PD123319) were used. In both pre- and postpubertal pig ovarian tissue, specific receptors for angiotensin II were demonstrated. These receptors had a density of 2487.6 (range: 267.5-5177.6, n = 4) and 3703.8 (range: 1819.9-5207.8, n = 4) fmol per mm(2), respectively, and dissociation constants of 130.0 and 26.3 nmol l(-1), respectively (prepubertal ovarian range: 106.0-165.4 nmol l(-1); postpubertal ovarian range: 26.1-100.3 nmol l(-1); P < 0.05, Mann-Whitney U test). AT(1) receptors with a K(i) for (125)I-labelled angiotensin II of 346.9 nmol l(-1) in the prepubertal and 268.1 nmol l(-1) in the postpubertal ovary were located predominantly in follicle wall tissue. Competitive inhibition studies using both angiotensin II antagonists resulted in a decrease in K(i) with prepubertal tissue (283.7 nmol l(-1)) and an increase in postpubertal tissue (293.9 nmol l(-1)). Immunocytochemistry using sections from paraffin wax-embedded prepubertal (n = 4) and postpubertal (n = 4) pig ovaries confirmed the presence of AT(1) receptors on the granulosa cell layer, but not the thecal cell layer, of antral follicles in both pre- and postpubertal pig ovarian tissue, and AT(2) receptors within the granulosa cell layer of prepubertal pig ovarian antral follicles. In summary, these results indicate that angiotensin II receptors are of higher affinity in postpubertal tissue than they are in prepubertal tissue, and indicate an active renin-angiotensin system within the pig ovary.