Growth, Antrum Formation, and Estradiol Production of Bovine Preantral Follicles Cultured in a Serum-Free Medium1

Abstract

The objective of this study was to identify factors that would allow the establishment of a serum-free culture system that could support follicular and oocyte growth, antrum formation, and estradiol-17beta (E(2)) production in preantral follicles of bovine ovaries. Large preantral follicles (145-170 micro m in diameter) were microsurgically dissected from ovaries, embedded in 0.15% type I collagen gels, and maintained in a serum-free medium for up to 13 days at 38.5 degrees C in 5% CO(2) in air. This culture environment allowed most preantral follicles to maintain a three-dimensional structure with the presence of a thecal layer and basement membrane surrounding the granulosa cells throughout the entire culture period. The effects of insulin, insulin-like growth factor (IGF)-I, IGF-II, FSH, and LH on preantral follicle growth were investigated in serum-free medium. Follicular diameters were significantly larger in the presence of insulin, IGF-I, IGF-II, or FSH after 13 days in culture. Oocyte diameters were also significantly larger in the presence of all hormones tested. The single addition of insulin, IGF-I, or FSH induced antrum formation between Days 7 and 13 of culture. Insulin progressively induced E(2) secretion by follicles after antrum formation, but IGF-I and FSH had no apparent effect. FSH and LH caused an increase in oocyte diameter in the presence of insulin. The addition of three hormones (insulin, FSH, and LH) initiated antrum formation and E(2) production earlier than insulin-containing medium alone. Furthermore, maximal E(2) secretion was maintained steadily between 7 and 13 days in this culture condition. From these results, we have demonstrated that insulin, FSH, and LH play substantial roles in the growth and development of bovine large preantral follicles in a serum-free medium.

Effects of IGF-I bioavailability on bovine preantral follicular development in vitro

“…Due to the protracted nature of preantral follicle development in large mammals, culture systems should be developed to accelerate oocyte growth, without inducing inappropriate follicular differentiation. A recent study has reported that recombinant IGF-I increased bovine preantral follicle and oocyte diameter during serum-free culture (Itoh et al 2002). By contrast, in a long-term culture system, LR3 IGF-I promoted bovine preantral follicle growth and antrum formation, but oocyte diameter was not increased .…”

Goat ovarian follicles express different levels of mRNA for inhibin-ßA subunit and activin-A stimulates secondary follicle growth in vitro

“…FSH is known to stimulate follicular growth and maintain the integrity of granulosa cells in sheep (CECCONI et al, 1999) and cattle (SAHA, et al, 2000). FSH treatment was shown to stimulate oocyte growth in bovine pre-antral follicles in-vitro (ITOH et al, 2002). Culture of ovarian tissue in medium containing FSH resulted in in-vitro growth of caprine pre-antral follicles, while their ultrastructural integrity was maintained (ROSSETTO et al, 2009).…”

In vitro development of primordial follicles after long-term culture of goat ovarian tissue

“…As there are FSH receptors in the granulosa cells of early follicles, it is possible that FSH may promote follicular growth and development (Méduri et al, 2002). Previous observations have showed that FSH promotes an increase in both oocyte (Itoh et al, 2002) and follicular diameter (Wright et al, 1999) after long-term culture.…”