Oxytocin secretion by bovine granulosa cells: effects of stage of follicular development, gonadotropins, and coculture with theca interna.

Oxytocin (OT) has been detected in ruminant preovulatory follicles. Bovine granulosa cells express the oxytocin/neurophysin I (OT/NP-I) gene and secrete OT in vitro. The objective of this study was to determine the developmental pattern of OT secretion by bovine follicle cells as they differentiate during the follicular phase and the preovulatory follicle approaches ovulation. Holstein heifers were injected with prostaglandin F2 alpha in midluteal phase to induce luteal regression and initiate a follicular phase. The ovary bearing the preovulatory follicle was obtained by ovariectomy early in the follicular phase, in midfollicular phase, or late in the follicular phase, after the LH/FSH surge (n = 4 heifers per group). Theca interna and granulosa cells were isolated and cultured for 5 days, individually or in coculture, in defined or serum-containing medium and with or without LH (300 ng/ml) or FSH (300 ng/ml). Media were collected and replaced completely every 24 h, and OT secreted into the media was measured by RIA. Granulosa cells isolated at all three time points during the follicular phase secreted measurable amounts of OT. However, total OT secretion by granulosa cells isolated after the LH/FSH surge was 18.9-fold (defined medium) to 64.8-fold (serum-containing medium) higher than OT secretion by granulosa cells isolated early in the follicular phase, and 14.6-fold (defined medium) to 170-fold (serum-containing medium) higher than OT secretion by granulosa cells isolated in midfollicular phase. Granulosa cells isolated before the LH/FSH surge responded to the addition of LH or FSH to the culture medium with an increase in OT secretion. Cocultures of granulosa cells and theca interna isolated before the LH surge secreted more OT than cultures of granulosa cells alone. When cells were isolated early in the follicular phase the effect of coculture was more than additive, but the effect of coculture was only additive when follicles were obtained in midfollicular phase. OT secretion by granulosa cells isolated after the LH/FSH surge was not affected by gonadotropins or by coculture with theca interna. In contrast to results for granulosa cells, theca interna secreted only small and variable amounts of OT, and responses to LH were inconsistent. These findings suggest that OT detected in cultures of theca interna may be produced by small and variable numbers of granulosa cells contaminating the theca interna preparation.(ABSTRACT TRUNCATED AT 400 WORDS)