Comparison of functional response of rat, macaque, and human ovarian cells in hormonally defined medium

A serum-free medium has been developed which supports in vitro function by ovarian cells derived from rat, monkey, and human tissue. This granulosa cell medium (GCM) consists of Dulbecco's Modified Eagle's Medium: Ham's F-12 medium (1:1, v:v) supplemented with insulin, transferrin, aprotinin, selenium, fibronectin, penicillin, and streptomycin. Ovarian cells from three species were compared: rat, macaque, and human. Four types of ovarian cultures were examined: 1) purified granulosa cell cultures and 2) co- cultures containing granulosa-theca-stroma cells, 3) luteal cells, and 4) granulosa-lutein (harvested from in vitro fertilization cultures) cells. Each cell type was characterized by its response to FSH or hCG when cultured in GCM. Morphologic responses to FSH were observed in GCM in rat granulosa and granulosa-theca-stroma cell cultures, macaque and human granulosa-lutein cells, and human granulosa-theca-stroma cell cultures. The FSH-stimulated cells retracted and became rounded, leaving long intercellular connections. Luteal cells did not retract in response to FSH, and the cells remained firmly attached to the fibronectin matrix. Steroidogenic regulation of the GCM-cultured ovarian cells was monitored following stimulation of the cultures with FSH. The ability of the cells to aromatize testosterone was first examined. Rat granulosa cell cultures and granulosa-theca-stroma cell cultures, macaque granulosa-lutein cell cultures, and human granulosa-theca- stroma cell cultures all accumulated estradiol when given FSH and testosterone for 48 h. Moreover, these cell types as well as human luteal cells were able to metabolize 25-hydroxy [1, 2-³H]cholesterol to various steroid metabolites. The data indicate that GCM supports normal granulosa cell morphologic response to FSH. Moreover, the aromatase and side-chain cleavage enzymes remain active and under gonadotropin regulation when the cells are maintained in GCM. Lastly, cells isolated from three species (rat, macaque, and human) respond similarly to FSH when maintained in GCM.