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- Author: C A Bagnell x
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ABSTRACT
Northern analysis was used to identify relaxin gene expression in ovaries of prepubertal pigs primed with pregnant mare's serum gonadotrophin (PMSG) and human chorionic gonadotrophin (hCG). The cellular distribution of relaxin transcript in the developing follicle was localized by in-situ hybridization histochemistry. Three probes complementary to non-overlapping regions of the porcine prorelaxin molecule were used to identify relaxin gene expression in ovarian follicular tissue collected 0, 48, 60, 72 and 84 h after treatment with PMSG/hCG. A 1 kb transcript was detected in ovarian extracts of prepubertal gilts from 48 to 84 h after PMSG stimulation. This corresponds to the molecular size of the relaxin transcript reported in the pregnant sow ovary. Relaxin mRNA levels increased in ovaries from animals 48 through 84 h after PMSG. In-situ hybridization showed that the site of relaxin synthesis was the theca interna layer of the developing follicle. Relaxin mRNA was not observed in other follicular cell types, in small or atretic follicles or in follicles from unstimulated animals. The distribution and relative concentration of relaxin mRNA showed a good correlation with in-vitro production and immunohistochemical localization of relaxin previously reported in the developing pig follicle. The presence of both protein and mRNA for relaxin in the growing follicle supports a role for relaxin as a local regulator of ovarian function.
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ABSTRACT
Northern analysis and in-situ hybridization were used to follow the development of relaxin gene expression in the newly forming corpus luteum (CL) after ovulation and throughout luteal development. Alkaline phosphatase (AP) was used as a marker of theca-derived lutein cells and the relationship between AP-positive and relaxin mRNA-containing cells was assessed. Ovaries from prepubertal pigs treated with pregnant mares serum gonadotrophin (PMSG)/human chorionic gonadotrophin (hCG) were collected during the periovulatory period and at various times during 19 days after ovulation. In addition, CL from cyclic pigs on days 10 and 16 were used to monitor relaxin gene expression in small and large luteal cells. Northern analysis revealed that relaxin gene expression increased with CL development in the PMSG/hCG-treated pig, reaching maximal levels at around day 14 post-ovulation. Thereafter, as the CL regressed, the level of relaxin mRNA declined. In CL from cyclic pigs at day 10 of the cycle, only small luteal cells expressed relaxin mRNA. However, by day 16 of the cycle, large luteal cells were the source of relaxin gene expression. In-situ hybridization studies revealed that in the early CL (up to 30 h post-ovulation), the relaxin gene transcript was observed in cells along the margins of the CL and in the core of the infolding follicle wall corresponding to the AP-positive, luteinized theca cell layer. As luteinization progressed, the theca and granulosa cell layers could no longer be distinguished morphologically (from 54 h after ovulation until day 9). However, the pattern of relaxin hybridization persisted along the periphery in bands of cells penetrating the CL, and coincided with areas of AP staining, indicating that the theca lutein cells were the site of relaxin gene expression. At day 14, relaxin hybridization and AP staining were distributed throughout the luteal tissue. With CL regression both AP staining and relaxin hybridization declined. This pattern of relaxin hybridization in the CL of the gonadotrophin-primed pig was identical to that observed in cyclic pigs on days 10 and 16 of the cycle. These findings indicate that theca interna cells retain their ability to express the relaxin gene following ovulation and luteinization. In the early CL, the small theca-derived lutein cells are the source of relaxin transcript. However, as the CL becomes fully differentiated, the large granulosa-derived lutein cells acquire the capacity to express the relaxin message.
