Fibronectin as a marker of granulosa cell cytodifferentiation

The hormonal regulation of fibronectin secretion by rat granulosa cells in culture was investigated: fibronectin was measured by a competitive enzyme-linked immunoadsorbant assay. Granulosa cells isolated from 25-day-old diethylstilbestrol-primed rats and cultured under defined conditions in the absence of hormones secreted low levels of fibronectin during the first 24 h of culture, after which there was a rapid increase in secretion until 72 h. In contrast, cultures treated with a combination of NIH-FSH-15 (200 ng/ml) and insulin (5 micrograms/ml) secreted low levels of fibronectin throughout the culture period. Subsequently, it was found that both FSH and insulin could independently suppress the increase in fibronectin secretion found in control cultures. Combined treatment with FSH and insulin resulted in a level of fibronectin which was the same as either FSH or insulin alone. The actions of FSH and insulin were dose dependent; 10 ng FSH/ml and 2.5 micrograms insulin/ml were required to produce a maximum suppression. The ability of (Bu)2cAMP (1.0 mM) to suppress fibronectin secretion suggested that the action of FSH on this parameter was mediated via the production of cAMP. Testosterone and estrogen alone did not influence secretion and did not modulate the actions of FSH and insulin. At the time at which FSH induces the cytodifferentiation of granulosa cells in culture, assessed by the increase in aromatase activity, fibronectin secretion is suppressed. The inverse relationship between fibronectin secretion and the induction of those granulosa cell functions essential for the development of the preovulatory follicle indicates that fibronectin may provide a useful marker for the stage of cytodifferentiation and follicular maturation.