of culture although follicles of 100-mm diameter achieved a diameter of 120 mm, after 4 days of culture.
The overall follicular viability and morphology were better with treatments than the controls in Ž
. all cases; however, there was no significant difference
p 0.05 among them. From this experiment, FSH and FSH plus EGF may be recommended for in vitro culture of
Ž .
smaller 40, 60 and 80 mm follicles. q 2000 Elsevier Science B.V. All rights reserved.
Keywords: Cattle ovary; Follicles; In vitro culture; In vitro growth
1. Introduction
Thousands of small oocytes are contained in bovine ovaries, as in the case in other Ž
. mammalian ovaries, but about 99.9 of oocytes undergo atresia Erickson, 1966 . It
would be of great practical benefit if these follicles, destined to become atretic, could be rescued before they degenerate. Periodically, among the pool of primordial follicles, a
small population of non-growing oocytes, approximately 30 mm in diameter, begins to
Ž .
grow reaching a final size of 120 mm Fair et al., 1995 . The oocyte grows and the Ž
. surrounding granulosa cells proliferate as the follicle grows Hulshof et al., 1992 . In
cattle, a follicle that has formed an antrum needs 40 days to reach ovulatory size Ž
. Lussier et al., 1987 , and it is estimated that the preantral follicle requires at least
60–80 days to reach maturity. Ž
Growth after culture of preantral follicles could be observed in mice Carroll et al., .
Ž .
Ž 1991; Eppig, 1977; Nayudu and Osborn, 1992 , rats Daniel et al., 1989 , pigs Hirao et
. Ž
. al., 1992 , and cats Jewgenow and Pitra, 1993 .
Several endocrine and growth factors can stimulate the proliferation of granulosa cells from bovine preantral follicles. The ability of gonadotropins to stimulate the
proliferation of granulosa cells in primary follicles has been demonstrated in vivo Ž
. Ž
Chiras and Greenwald, 1978 and in vitro Roy and Greenwald, 1989; Carroll et al., .
Ž .
1991 . Follicle stimulating hormone FSH
has been reported to promote follicle Ž
. development in cultured bovine preantral follicles Wandji et al., 1996 . FSH has also
been demonstrated to be involved in proliferation and differentiation of preantral granulosa cells in vitro and thus in growth and normal in vitro development of preantral
Ž .
follicle of many species, including cows Ralph et al., 1995 . FSH binding and
Ž .
expression of the FSH receptor gene in granulosa cells of human Zheng et al., 1996 Ž
. and bovine Wandji et al., 1992 follicles and in oocytes of primordial follicle from
Ž .
small laboratory animals Roy, 1993 support the view of a physiological action of FSH Ž
. on preantral follicles Hurk et al., 1997 . FSH is an anti-apoptotic factor and helps to
Ž .
maintain the viability of granulosa cells Chun et al., 1996 . On the other hand, growth factors are ubiquitous peptides, acting in a paracrine andror an endocrine manner, and
are involved in regulation of cell proliferation, differentiation and survival. Epidermal Ž
. growth factor EGF may influence preantral folliculogenesis. EGF has been shown to
Ž .
be involved in proliferation of granulosa cells in pigs Morbeck et al., 1993 , hamsters Ž
. Ž
. Roy, 1993 and cows Wandji et al., 1996 . FSH and EGF are important for the in vitro
survival and growth of cultured preantral follicles. They may serve as chemotactic compounds for neo-vascularization or neo-innervation of small follicles, which provide
hormonal and neuropeptidergic compounds as well as cytokines and nutritional compo- Ž
. nents that initiate and maintain follicular growth Hurk et al., 1997 . There is report of
Ž .
using insulin–transferrin–selenium ITS to maintain granulosa cells within the follicu- lar microenvironment. Insulin is essential for follicle culture; lack of insulin results in
follicle degeneration. However, insulin alone is insufficient to maintain healthy follicles. Moreover, selenium acts as an acceptor of free radicals generated during cell metabolism
Ž .
and it improves culture conditions Roy and Treacy, 1993 . The presence of additives such as FSH, ITS, glutamine and sodium pyruvate greatly improved development of the
follicles. This was manifested by a twofold increase in the percentage of growth and Ž
survival time of follicles cultured in the supplemented medium Katska and Rynska, .
1998 . The number of morphologically normal oocytes increases when bovine preantral Ž
. follicles are cultured in hypoxanthine-supplemented medium Figueiredo et al., 1994 .
Ž .
Eppig and Downs 1987 demonstrated that hypoxanthine promoted the association of oocytes and accompanying granulosa cells during the culture of mouse preantral
follicles. The maintenance of oocyte–granulosa cell communication via gap junctions Ž
. has been shown to promote oocyte growth in vitro Buccione et al., 1990 . It is probable
that hypoxanthine improves the utilization of additional energy substrate by maintaining Ž
interactions between the oocyte and the surrounding granulosa cells Figueiredo et al., .
1994 . Larger variations in the increase of follicular diameter were observed among Ž
. follicle cultured in the absence of hypoxanthine Jewgenow, 1998 .
Ž There are several reports using wide range of culture media Daniel et al., 1989;
. Ž
Eppig, 1977; Torrance et al., 1989 , pyruvate Daniel et al., 1989; Eppig, 1977; Eppig .
Ž .
and Schroeder, 1989; Torrance et al., 1989 and glutamine Nayudu and Osborn, 1992 as energy substrate. The addition of pyruvate or glutamine to the culture medium
increased the percentage of intact follicle. It was shown that pyruvate was the predomi- Ž
nant substrate used by immature and mature bovine oocytes and zygotes Khurana and .
Ž .
Niemann, 1992 . Eppig 1976 reported that isolated growing mouse oocytes utilized exogenously administered pyruvate more efficiently than glucose, lactate or succinate. It
has been shown that glutamine is an efficient energy substrate for oocytes and embryos Ž
. Figueiredo et al., 1994 .
Cellular maturation and follicular development from the primary to tertiary stages and their regulation by endocrine and paracrine factors are areas of limited knowledge. The
ability to culture isolated preantral follicles constitute a new tool for investigation of paracrine and autocrine factors involved in early folliculogenesis. In this experiment, we
have studied the effects of some endocrine and growth factors on follicular growth by culturing preantral follicles over a period of 10 days.
2. Materials and methods
