PARENT SESSION
Follicular Development

DIFFERENTIATION OF BOVINE GRANULOSA CELLS IN VITRO.

Sahmi, Malha¹, Hamel, Mélanie ¹, Price, Christopher¹, ¹

ABSTRACT- Messenger RNA levels for steroidogenic proteins change during follicle development in cattle. The most marked changes are observed for granulosa cell levels of P450 aromatase (P450arom), 3-hydroxysteroid dehydrogenase (3-HSD) and luteinizing hormone receptor (LHr) mRNA. We have shown that in granulosa cells from small bovine follicles, P450arom mRNA levels increase with time of culture in a pattern similar to that observed in vivo. Here, we determined if 3-HSD mRNA and functional LHr are also induced in this culture system. Granulosa cells were recovered from small (2-5mm dia) follicles, and cultured in serum-free MEM medium supplemented with follicle-stimulating hormone (FSH), insulin and insulin-like growth factor-1. Cells were recovered after 2, 4 or 6 days of culture. Before culture, these cells have very low levels of P450arom and undetectable levels of 3-HSD mRNA. Northern analysis confirmed that a marked increase in P450arom mRNA (P<0.05) occurs with time in culture, whereas P450 cholesterol side-chain cleavage mRNA levels were stable. 3-HSD mRNA was detectable in cultured cells, and relative abundance increased significantly with time of culture (P<0.01), and was evident in fresh granulosa cells of large (>8mm) but not medium follicles (5-8mm). On day 6 of culture, binding assays detected specific binding of FSH but not LH to cells. LH or human chorionic gonadotropin (hCG) did not stimulate progesterone (P) secretion. As estradiol (E), FSH and cadherin-mediated cell-cell interaction are required for LHr induction in rats, we added excess androstenedione (A₄) precursor or calcium to cultured cells. The addition of calcium decreased overall steroidogenesis. Addition of A₄ at 10^-6 or 10^-5 M increased, whereas A₄ at 10^-4 M inhibited E secretion and P450arom mRNA compared to A₄ at 10^-7 M. The addition of LH at the end of culture still did not stimulate P secretion irrespective of A₄ dose used. We conclude that although this cell system enables induction of P450arom and 3-HSD expression, some critical factors(s) are missing for the expression of LHr. (Supported by NSERC).

KEY WORDS: granulosa cell, aromatase, LH receptor, 3-hydroxysteroid dehydrogenase