Production of long term steroid-producing granulosa cell cultures by cell hybridization

Primary cultures of rat ovarian granulosa cells have been used extensively to study hormonal regulation of cellular function. To date, no long term cultures of ovarian cells which retain their differentiated functions have been developed. Hypoxanthine guanine phosphoribosyl transferase-deficient simian virus 40-transformed rat ovarian granulosa cells were fused with freshly prepared rat granulosa cells using inactivated Sendai virus. Putative hybrid cell strains obtained after selection in medium containing hypoxanthine, aminopterin, and thymidine were analyzed for progesterone synthesis. Neither the original simian virus 40-transformed granulosa cell nor its hypoxanthine guanine phosphoribosyl transferase-deficient derivative produced progesterone, but three of the hybrid strains produced progesterone at basal levels and in response to dibutyryl cAMP. One of these strains produced progesterone in a dose-responsive fashion when exposed to prostaglandin E2, cholera toxin, dibutyryl cAMP, and 2-chloroadenosine. Cell strains obtained by hybridization were remarkably similar to primary cultures of granulosa cells with respect to both the magnitude and temporal aspects of progesterone production in response to dibutyryl cAMP.