STUDIES ON TISSUE CULTURE OF EQUINE OVARIAN CELL TYPES: EFFECT OF GONADOTROPHINS AND STAGE OF CYCLE ON STEROIDOGENESIS

Abstract

SUMMARY: Granulosa cells were harvested from follicles of mares at various stages of the oestrous cycle and maintained in a tissue culture medium containing 15% horse serum, 30% medium '199' and 55% Hanks's solution. Between days 4 and 10 of culture the granulosa cells harvested from small follicles (1–2 cm. diam.) of mares in the midluteal phase of the cycle secreted an average of 0·36 pg. progesterone/cell/day. Cells harvested from large follicles of mares in the late and/or early oestrous stage of the cycle secreted an average of 29·5 pg. progesterone cell/day; the cells harvested from the large vascular follicles found at oestrus secreted an average of 173 pg./cell/day. The small, poorly vascularized follicles found adjacent to the large vascular follicles of mares in oestrus yielded cells which secreted less progesterone than those from the larger follicles. Addition of 5 to 10 i.u. human chorionic gonadotrophin (HCG)/ml. at each medium change (every 2–3 days) or for the first 4 days of culture brought about a marked stimulation of progesterone secretion in cultures of ' mid-luteal phase' cells which was maximal after 4 to 7 days. Pregnenolone was converted primarily to progesterone, 20α-hydroxypregn-4-en-3-one and 17-hydroxyprogesterone; the metabolism was not significantly altered by the addition of a mixture of 10 i.u. HCG plus 10 i.u. pregnant mare serum gonadotrophin (PMSG). Cells harvested from mares in oestrus converted pregnenolone to progesterone in a higher yield compared with cells harvested from mares in the midluteal phase of the cycle. Addition of 10 i.u. HCG/ml. or PMSG plus HCG (10 i.u. each/ml.) stimulated aromatization of testosterone by 'midluteal phase' cultures but not by 'oestrous phase' cell cultures. These results demonstrate that the in vivo environment as well as the in vitro conditions influence the steroidogenic activity of equine granulosa cell cultures.