THE JOURNAL OF EXPERIMENTAL ZOOLOGY 242~351-354 (1987) zy Follicle Cell Regulation of Mammalian Oocyte zyx Growth ROBERTO BUCCIONE, SANDRA CECCONI, CARLA TATONE, FRANC0 MANGIA zyxwvut AND ROSELLA COLONNA zyxwv Dipartimento di Scienze e Tecnologie Biomediche e di Biometria, Uniuersitadell'Aquila, Cattedra di Biologia eZoologia Generule, Collemaggio, 67100-L'Aquila, Italy ABSTRACT To investigate mechanisms of follicle cell control on mamma- lian oocyte growth, preantral mouse oocytes free from surrounding follicle cells were individually cocultured with monolayers of different somatic cells com- petent to form gap junctions, and the rate of in vitro oocyte growth was directly correlated with the level of metabolic coupling on the same cells. The results indicate that 1) at a similar extent of metabolic coupling, mouse oocytes grew on follicle cells but not on 3T3 and Sertoli cell monolayers, and 2) the growth rate of oocytes cultured on follicle cells was dependent on the extent of meta- bolic coupling. It was concluded that gap-junction-mediated nutrition of ovar- ian mouse oocytes exerted by somatic cells is necessary but not sufficient to maintain oocyte growth. A specific regulatory role of follicle cells on mamma- lian oocyte growth is proposed. During oogenesis mammalian oocytes are invested by one or more layers of follicle cells which contact the oocyte by cytoplasmatic processes with formation of heterologous gap junctions (Anderson and Albertini, '76). These junctions provide channels for a direct passage of small molecules from one cell to another (Flagg-Newton et al., '79; Loewen- stein, '79) and mediate the so-calIed meta- bolic cooperativity or metabolic coupling, i.e., the transfer from follicle cells to oocytes of molecules such as energy substrates, nucleo- tides, amino acids, etc. (Heller and Schultz, '80; Moor et al., '80; Heller et al., '81; Brower and Schultz, '82; Osborn and Moor, '82; Co- lonna and Mangia, '83; Colonna et al., '83). Metabolic coupling is apparently essential for mammalian oocyte growth. In fact, ovar- ian mouse oocytes continue to grow in vitro only when intercellular communications with follicle cells are maintained (Eppig, '77, '79) or reestablished (Bachvarova et al., '80; Her- lands and Schultz, '84). In contrast, mouse oocytes do not grow on 3T3 cell monolayers even if functional gap junctions are estab- lished between the two cell types (Herlands and Schultz, '84). As a possible explanation for this finding, it was proposed that 3T3 cells do not form gap junctions with oocytes to the same extent as follicle cells do (Her- lands and Schultz, '84). The actual role of gap-junction-mediated nutritional function in mammalian oocyte growth is not clear. We approached this problem by directly correlat- ing oocyte growth and extent of metabolic cooperativity in the same individual oocyte coupled in vitro with different communica- tion-competent somatic cells such as follicle cells (Anderson and Albertini, '76), Sertoli cells (Eusebi et al., '85), and 3T3 cells (Heller and Schultz, zyxw '80). In the present report we provide evidence that the trophic function of folIicle cells cannot be per se responsibIe for oocyte growth and discuss these data in terms of a possible regulatory role specifically ex- erted on mammalian oocyte growth by folli- cle cells. MATERIALS AND METHODS CD1-COBS mice (Charles River Italia) were used in all experiments. Follicle cell in vitro culture Preantral follicles were isolated from ova- ries of 8-10-day-old mice in the presence of a HEPES-buffered medium (MH; Whitting- ham, '71). Follicles were then incubated in Dulbecco's phosphate-buffered saline (PBS) containing 0.05% collagenase (Cooper Biomedical) for 15 minutes at 37°C. Follicles were opened by repeated pipetting with a mouth-operated pippette, and escaped oo- cytes were discarded or eventually used for cocultures. Oocyte-free follicles were incu- bated further in Ca- and Mg-free PBS (CMFPBS) containing 0.25% trypsin (Difco) at 37"C, and after 20 minutes the digestion was blocked by addition of an excess of soy- z 0 1987 ALAN R. LISS, INC.