Studies on tissue culture of equine ovarian cell types: effect of gonadotrophins and stage of cycle on steroidogenesis.

This research explores the activity of ovarian cells in horses (mares) over their oestrus cycle, including the way such cells respond to certain hormones and how they generate progesterone. The study also identifies how cell size and cycle stage influence this process.

Research Methodology

• The research involved harvesting granulosa cells from the follicles of mares at different stages of the oestrous cycle. These cells were then sustained in a tissue culture medium containing 15% horse serum, 30% medium ‘199’ and 55% Hanks’s solution.
• The granulosa cells were evaluated between days 4 and 10 of culture. Researchers investigated cells harvested from follicles of varying sizes and from mares at distinct stages of the oestrus cycle (midluteal phase, late and/or early oestrous phase).
• The team also studied the effect of human chorionic gonadotrophin (HCG) on progesterone secretion in these cultures. Pregnant mare serum gonadotrophin (PMSG) was also used to study the conversion of pregnenolone to progesterone and the aromatization of testosterone.

Key Findings

• Cells harvested from small follicles (1-2 cm diameter) of mares in the midluteal phase of the cycle had the least progesterone secretion (an average of 0.36 pg per cell/day).
• Cells from large follicles in mares at the late and early oestrous stages of the cycle showed a higher progesterone secretion (an average of 29.5 pg/cell/day). The largest progesterone secretion was observed in cells from large vascular follicles in mares during oestrus with an average of 173 pg/cell/day.
• 5 to 10 i.u. of HCG at each medium change (every 2-3 days) or for the first 4 days of culture enhanced progesterone secretion in cultures of mid-luteal phase cells. The stimulation of progesterone secretion was highest between 4 to 7 days.
• Pregnenolone was primarily converted into progesterone, 20α-hydroxypregn-4-en-3-one, and 17-hydroxyprogesterone. The metabolism wasn’t significantly changed by the addition of a mixture of 10 i.u. HCG and 10 i.u. PMSG.
• Cells harvested from mares in oestrus converted pregnenolone to progesterone in a higher yield compared to cells harvested from mares in the midluteal phase of the cycle. However, 10 i.u. of HCG/ml, or a combination of PMSG and HCG, only stimulated the aromatization of testosterone by ‘midluteal phase’ cultures and not by ‘oestrous phase’ cell cultures.

Conclusion

• This study shows that both the external environment (in vivo) and experimental conditions (in vitro) influence hormone production and steroidogenic activity of equine granulosa cell cultures. A better understanding of these variables can be beneficial in managing reproductive issues in mares.