Plasminogen activator production by the granulosa layer is stimulated by factor(s) produced by the theca layer and inhibited by the luteinizing hormone surge in the chicken

The stages of follicular maturation of a preovulatory follicle in the hen can be divided into an extended proliferative phase (prior to LH surge) and a brief ovulatory phase (after LH surge). Previous studies suggest involvement of plasminogen activator (PA) in both the proliferative and ovulatory phases. The goals of the present study were 1) to determine whether PA production by granulosa and theca is dependent upon interaction of the two cell layers; 2) to investigate whether the structural difference of the stigma (site of follicular rupture) and nonstigma regions of the theca layer affect PA production; 3) to determine whether there is a change in the ability of the granulosa layer and stigma or nonstigma regions of the theca layer to produce PA as the follicle makes the transition from the proliferative to the ovulatory phase; and 4) to characterize the type(s) of PA produced by the hen follicle. Equal proportions of the granulosa layer (10-mm diameter) and stigma or nonstigma regions of the theca layer (10 mg) obtained from the F1 preovulatory follicle 8 h before ovulation (before LH surge) or 2 h before ovulation after LH surge) were incubated alone or in combination for 24 h. PA was measured in tissue homogenates and medium by use of the chromogenic substrate S-2251. The granulosa layer or stigma or nonstigma regions of the theca layer incubated alone and obtained either 8 h or 2 h before ovulation had very low amounts of PA activity in the medium and tissue homogenates. However, co-incubation of the granulosa layer with either stigma or nonstigma regions of the theca layer dramatically increased the amount of PA activity in the medium when obtained 8 h, but not 2 h, before ovulation. PA activity in the medium was similar whether the granulosa layer was co-incubated with stigma or nonstigma regions of the theca layer. The primary site of PA production was the granulosa layer, which was dependent upon stimulation by factor(s) secreted by the theca layer. To determine the possible role of LH in the regulation of PA activity, the granulosa layer or stigma and nonstigma regions of the theca layer were obtained 8 h before ovulation (before LH surge) and incubated alone or co-incubated in the presence or absence of ovine LH (25 ng/ml). LH substantially decreased the PA activity in the medium from co-incubation of the granulosa layer with stigma or nonstigma regions of the theca layer. LH had no effect on the ability of the granulosa layer or stigma or nonstigma regions of the theca layer to produce PA when incubated alone. The type(s) of PA produced by the granulosa layer during the proliferative phase was determined by polyacrylamide substrate gel electrophoresis and Western blot. Medium from co-incubation of the granulosa layer with stigma or nonstigma regions of the theca layer consistently showed only one band of plasminogen-dependent proteolytic activity with a molecular weight slightly lower than the human urokinase PA (uPA) standard. The Western blot showed one band of immunoreactive uPA, which coincided with partially purified chicken uPA. The present study demonstrates that in the hen 1) factor(s) from the theca layer stimulate the granulosa layer to produce uPA during the proliferative phase, and 2) the ovulatory LH surge suppresses PA activity resulting from the interaction of granulosa and theca layers. Therefore, the primary role of uPA may be for tissue remodeling during follicular growth and maturation.