Factors Influencing the Rate of Preantral and Antral Growth of Mouse Ovarian Follicles in Vitro

The development of a culture system for individual mouse ovarian follicles using a low concentration of homologous serum, human follicle-stimulating hormone (hFSH) and a simple combination of growth factors is reported. Preantral follicles, 150 microns in diameter, with thecal cells attached were isolated mechanically. After 6-7 days on a Millicell membrane, a high proportion of the preantral follicles cultured individually with hFSH grew to morphologically normal large antral follicles (400-500 microns in diameter) with high oestradiol secretion. Without hFSH, the follicles grew to approximately 275 microns diameter in 6 days, but did not form antra or secrete oestradiol. The growth trajectory (overall pattern of growth formed by daily measurements of diameter) of each follicle was recorded and used as a measurement of response to experimental variation of culture conditions. The rapidly growing follicles were morphologically normal, but those that grew more slowly showed some abnormality or atresia and secreted less oestradiol. Follicles cultured in groups without being in direct contact with each other showed much poorer growth than those grown individually, but the inhibition was not uniform and some follicles grew larger than others in the group. Follicles that contacted each other directly in culture tended to fuse into one mass and their growth was substantially inhibited. Even under these conditions, one follicle often continued to grow slowly while the others degenerated. Such alteration of growth patterns suggests interfollicular paracrine control and may be a means of three-dimensional spacing of follicle growth within the ovary, as well as part of the mechanism of follicle selection. The dose-response curve based on the mean growth trajectory of follicles cultured individually, produced increasing rates of growth with 12.5-100 miu hFSH ml-1. Higher concentrations of hFSH did not increase growth rate further, but oestradiol secretion continued to increase with increasing hFSH up to the maximum used (2000 miu ml-1).