Phorbol ester increases plasminogen activator inhibitor accumulation in cultures of human granulosa cells

Proteolytic enzymes such as plasminogen activators (PAs) and collagenases are implicated in the process of ovarian follicle rupture. Data obtained in rats support the concept that PAs are both hormonally regulated and temporally related to the ovulatory process; however, such data are lacking in the human ovary. The recent identification of a family of PA inhibitors (PAIs) adds a new dimension to the control of PA activity, and in contrast to animal studies, the human preovulatory follicle is characterized by PA inhibitory activity. To initially examine the PA and PAI system in the human ovary, granulosa cells obtained from women undergoing gonadal hyperstimulation for in vitro fertilization were cultured in the presence or absence of the protein kinase-C activator, phorbol 12-myristate 13-acetate. Reverse fibrin autography of conditioned medium from control cells revealed the presence of a putative PAI with an apparent mol wt of 50,000. Phorbol ester stimulated the accumulation of this PAI in a specific and dose-dependent manner. Cumulus cells and noncumulus granulosa cells were similar in terms of presence and regulation of PAI. Immunoprecipitation with specific antisera revealed that this human granulosa cell PAI was immunochemically related to PAI-1. This identification was supported by quantitative analysis revealing a 3.7-fold increase in PAI-1 antigen, as assessed by a specific enzyme-linked immunoabsorbent assay. These findings are the first demonstration of the in vitro regulation of PAI activity in the human ovary.