Role of gonadotrophins and ovarian steroids in the development of mouse follicles in vitro.

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.

Effects of epidermal growth factor on the growth and differentiation of cultured mouse ovarian follicles.

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.

Characterization of follicular energy metabolism.

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.

The effect of glucose metabolism on murine follicle development and steroidogenesis in vitro.

Previous studies have shown that mouse ovarian follicles produce large amounts of lactate during growth and maturation in vitro, suggesting a metabolic preference for glycolysis. Further investigations were therefore undertaken to determine firstly the effect of gonadotrophins on glucose uptake and secondly the role of glucose in follicle lactate production, development and steroidogenesis. Pre-antral mouse follicles were cultured individually in media containing different concentrations of glucose (1-5.5 mM) using a system which supported development to the pre-ovulatory stage within 5 days. Samples of media were removed every 24 h and analysed for oestradiol using an enzyme-linked immunosorbent assay technique and for glucose uptake and lactate production using a fluorimetric assay. Results showed that all the glucose consumed was converted to lactate, irrespective of the glucose concentration. Growth and steroidogenesis were also dependent on glucose concentration, and at concentrations below 2 mM, follicle development was significantly retarded. Results confirm that follicles adopt a predominantly glycolytic mode of energy production to sustain growth and steroidogenesis, and that the glycolytic rate is stimulated by gonadotrophins.

Mouse oocytes derived from in vitro grown primary ovarian follicles are fertile.

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.

Clonal analysis of chimaeric mouse ovaries using DNA in situ hybridization.

Analysis of chimaeric mouse ovaries using DNA in situ hybridization was undertaken to (i) investigate the morphogenesis of follicular cell clonal expansion, (ii) evaluate whether the different cell populations within the ovary are derived from the same or unrelated progenitor cells and (iii) estimate the number of progenitor cells giving rise to the different types of ovarian cell. Chimaeras were produced by aggregation of eight-cell morulae from normal mice and those transgenic for the beta-globin gene. Chimaeric blastocysts were transferred to pseudopregnant hosts and the ovaries of resultant adult offspring were prepared for in situ hybridization using a digoxigenin-labelled cDNA probe to the beta-globin gene. Results showed that follicles are constructed by the non-random, radial proliferation of granulosa cell clones, which form long, thin, unbranched columns across the follicle wall. Qualitative and quantitative studies revealed that both peripheral and central granulosa cells are derived from the same progenitor cells. Phenotypic differences may therefore be due to positional cues within the follicle rather than being cell lineage dependent. It is suggested that granulosa cells and germinal epithelium may be partly derived from the same progenitor cells and that theca externa is probably derived from interstitial tissue. However, results from this study did not support the contention that theca interna and theca externa-interstitial tissue have the same origin, and it is suggested that the former cell type may exist in an undifferentiated state from early stages of follicle development. Furthermore, granulosa and germinal epithelium appear to be derived from different progenitor cells from either theca interna or theca externa-interstitial cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Pattern of lactate production and steroidogenesis during growth and maturation of mouse ovarian follicles in vitro.

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.