A model culture system has been developed whereby individual, primary ovarian mouse follicles can be grown in vitro to the Graafian stage in the normal physiological time course, and then ovulated in response to luteinizing hormone. We report here on the successful fertilization and subsequent embryo development of the oocytes from such follicles. This is the first time that oocytes from in-vitro matured whole follicles have been fertilized and shown to produce viable offspring in host animals. The study demonstrates that the culture system mimics physiological conditions for normal follicle development.

A model culture system for the study of follicular metabolism has been developed from existing methods of whole-follicle culture. The modified system mimics the in vivo growth and maturation of mouse ovarian follicles from primary to preovulatory stages and the modulating influences of the LH and FSH. This is the first study to demonstrate ovulation in vitro from individual ovarian follicles. The pattern of follicular lactate production relative to steroidogenesis was studied throughout in vitro follicular development over a period of 6 days. Twenty-four-hour samples of medium from individual follicles were analyzed for lactate and pyruvate by an automated analytical technique, and for estradiol and progesterone by an immunoenzymatic method. Follicles produced remarkably large quantities of lactate and estradiol during FSH-stimulated development in vitro. LH further stimulated lactate production but resulted in a significant decrease in estradiol secretion and an ovulation rate of 30%. Progesterone production was not detectable throughout the culture period, and follicles showed no evidence of pyruvate uptake. These findings demonstrate the validity of using this model culture system for the study of follicular metabolism and provide new information on the pattern of carbohydrate metabolism relative to steroidogenesis during follicular growth and maturation.

A whole follicle culture system has been used to investigate the actions of gonadotrophic hormones, oestrogen and progesterone in the regulation of follicular development and steroidogenesis. Recombinant human FSH was required for the growth of preantral follicles and for Graafian morphogenesis, whereas recombinant LH was ineffective. While pure FSH was sufficient for growth and morphogenesis, production of oestrogen was greater when androstenedione or LH was present in combination with FSH, confirming that there is a two-cell mechanism for oestradiol production in the mouse follicle. When an antiserum to oestrogen or to progesterone or an oestrogen receptor antagonist were added to the culture medium, there was no significant effect on either follicular growth or oestradiol production. Thus, physiological concentrations of oestradiol are not needed for follicle development, although a role cannot be completely ruled out. In conclusion, the obligatory role of FSH was demonstrated. It appears to be sufficient for follicle development even in the absence of LH, and the paracrine or autocrine effects of oestradiol and progesterone, if any, appear to be minor in the mouse ovary.

The aim of this investigation was to determine the influence of epidermal growth factor (EGF) on follicular growth and steroidogenesis in mice. Follicles were cultured in medium containing human recombinant EGF at concentrations of 1-20 ng ml-1. Oestradiol production was assayed immunoenzymatically, and growth was measured by recording follicle diameter daily and by analysing the total DNA content of follicles. The effect of EGF on cumulus-oocyte complexes isolated from cultured follicles was also assessed. Results showed that EGF inhibited oestradiol production in a dose-dependent manner, but had no mitogenic effect. Despite almost complete inhibition of oestradiol production at concentrations of EGF > or = 10 ng ml-1, follicles were still able to achieve preovulatory size and morphology, although the incidence of atresia was increased over controls. Conversely, at a concentration of only 1 ng EGF ml-1, a significantly greater number of follicles reached the Graafian stage compared with control follicles. Cumulus expansion and meiotic maturation by isolated cumulus-oocyte complexes from cultured follicles was dramatically stimulated in the presence of EGF and FSH, but not by FSH alone. These findings suggest that EGF may have a modulatory effect on oestradiol production in vivo, and that follicular growth and differentiation may be uncoupled from steroidogenesis. Finally, ovulatory changes in the cumulus-oocyte complex may require the presence of this factor.

It has been shown that mouse ovarian follicles have a large glycolytic capacity, and this study was undertaken to determine whether follicles can develop normally using glycolysis alone. Pre-antral mouse follicles were grown using an in-vitro system which supports development to the preovulatory stage within 5 days. Cultures were maintained in either aerobic conditions in the presence of the inhibitor of oxidative phosphorylation, sodium malonate, or under anaerobic conditions. Samples of media were removed every 24 h and analysed for oestradiol using an enzyme-linked immunosorbent assay (ELISA) technique and for lactate and glucose using a fluorometric assay. Follicle size, oestradiol production and glycolytic rate were not significantly different between control and sodium malonate-treated follicles. Follicles cultured under anaerobic conditions showed significantly slower rates of growth and oestradiol production compared with controls. However, the rate of glycolysis was significantly higher during anoxia. Results indicated that anaerobic glycolysis may sustain limited periods of growth during the pre-antral phase, but that the presence of oxygen is vital to ensure normal development. It is concluded that pre-antral follicles can undergo development to the preovulatory stage using glycolysis alone, a feature which may allow them to conserve their limited supply of oxygen for other vital biosynthetic processes.