Growth and Antrum Formation of Bovine Preantral Follicles in Long-Term Culture In Vitro1

Abstract

Culture of preantral follicles has important biotechnological implications through its potential to produce large quantities of oocytes for embryo production and transfer. A long-term culture system for bovine preantral follicles is described. Bovine preantral follicles (166 +/- 2.15 micrometer), surrounded by theca cells, were isolated from ovarian cortical slices. Follicles were cultured under conditions known to maintain granulosa cell viability in vitro. The effects of epidermal growth factor (EGF), insulin-like growth factor (IGF)-I, FSH, and coculture with bovine granulosa cells on preantral follicle growth were analyzed. Follicle and oocyte diameter increased significantly (P < 0.05) with time in culture. FSH, IGF-I, and EGF stimulated (P < 0.05) follicle growth rate but had no effect on oocyte growth. Coculture with granulosa cells inhibited FSH/IGF-I-stimulated growth. Most follicles maintained their morphology throughout culture, with the presence of a thecal layer and basement membrane surrounding the granulosa cells. Antrum formation, confirmed by confocal microscopy, occurred between Days 10 and 28 of culture. The probability of follicles reaching antrum development was 0.19 for control follicles. The addition of growth factors or FSH increased (P < 0.05) the probability of antrum development to 0.55. Follicular growth appeared to be halted by slower growth of the basement membrane, as growing follicles occasionally burst the basement membrane, extruding their granulosa cells. In conclusion, a preantral follicle culture system in which follicle morphology can be maintained for up to 28 days has been developed. In this system, FSH, EGF, and IGF-I stimulated follicle growth and enhanced antrum formation. This culture system may provide a valuable approach for studying the regulation of early follicular development and for production of oocytes for nuclear/embryo transfer, but further work is required.

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

“…In contrast, the other treatments (FSH-7d and FSH + FGF-2) were effective in maintaining follicle ultrastructural integrity up to day 7. Our light histological results confirm earlier studies in which FSH maintained preantral follicle viability after short (Matos et al, 2007a andMatos et al, 2007c) or long-term culture (Cortvrindt et al, 1997, Wright et al, 1999and Gutierrez et al, 2000. It is suggested that the absence of FSH in the medium could compromise the diffusion of several essential chemical and physical factors through the basement membrane (Hsueh et al, 1994).…”

“…Therefore, an extended culture period may be required for a follicle to develop to the preovulatory stage. Few long-term in vitro culture studies have achieved follicular activation, i.e., the transition from primordial to primary follicles (ovine: Muruvi et al, 2005; human: Otala et al, 2002 andSadeu et al, 2006) or the development of antral follicles (bovine: Gutierrez et al, 2000;mouse: Mitchell et al, 2002). Recently, Gupta et al (2008) produced buffalo embryos after the long-term culture of isolated large secondary follicles.…”

Interaction between growth differentiation factor 9, insulin-like growth factor I and growth hormone on the in vitro development and survival of goat preantral follicles

“…There is a growing body of evidence showing that IGF-I plays a key role in the development of preantral follicles and in the process of follicular atresia. When added during in vitro culture of preantral follicles, IGF-I has been shown to stimulate follicular growth in humans (12), cattle (13), rats (14), and mice (15) in synergy with follicle-stimulating hormone. In the rat, IGF-I added during in vitro preantral follicular culture significantly increased follicular diameter and DNA content (14).…”

Interaction between ascorbic acid and follicle-stimulating hormone maintains follicular viability after long-term in vitro culture of caprine preantral follicles

“…To our knowledge, this is the first report that demonstrates the maintenance of caprine preantral follicle survival using histological and ultrastructural analysis, even after longterm culture (14 d) in domestic animals. Other studies related antrum formation in goats [28], cattle [29], and mice [30], and in the production of embryos [31] from in vitro culture of preantral follicles. It is important to mention that these studies used the large, advanced stage of secondary preantral follicles.…”