The Production of Transforming Growth Factor-β Porcine Ovary and its Secretion in Vitro*

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Abstract
Porcine granulosa cells isolated form small (1-3 mm in diameter) follicles proliferate rapidly in culture in response to 10% fetal bovine serum (FBS) and epidermal growth factor (EGF) (10 ng/ml). Transforming growth factor-.beta. (TGF.beta.) inhibits FBS/EGF-stimulated proliferation in a dose-dependent manner. We have used this proliferation inhibitory property of TGF.beta. to assay qualitatively, the presence of this growth factor in conditioned medium from cultured follicle cells as well as in partially purified preparations from porcine ovarian compartments. In addition, the concentration of TGF.beta. in the theca cell conditioned medium was quantitatively estimated by generating a TGF.beta.-dose-response curve (inhibition of FBS/EGF-stimulated proliferation of granulosa cells in monolayer culture) using authentic human TGF.beta.-1. Ovarian thecal cells isolated from small and large size follicles in the pig ovary secrete TGF.beta.-like activity in vitro. Medium conditioned by thecal cells in primary monolayer culture contains a latent form of TGF.beta. which can be activated by heat or acid treatment. In contrast, and unlike rat granulosa cells, porcine granulosa cells in primary monolayer culture do not secrete detectable levels of TGF.beta.-like activity in the medium. Incubation of heat-activated thecal cell conditioned medium with a TGF.beta.-neutralizing antibody (which recognizes TGF.beta.-1 and 2) but not nonimmune serum attenuated the TGF.beta.-like activity in thecal cell conditioned medium suggesting that this activity is due to authentic TGF.beta.. Since many cells types secrete latent TGF.beta. in the medium when cultured in vitro, we next investigated whether thecal cell secretion of latent TGF.beta. was a function of cell culture or whether the ovarian thecal compartment actually contained detctable levels of TGF.beta.-like activity. To this end, we used an acid-ethanol extraction procedure to isolate thecal proteins from fresh-frozen tissue. The acid-ethanol extracted protein fraction was mixed with trace amounts of 125I-TGF.beta. for detection and chromatographed on Bio-Gel P-60 column under acidic conditions. Elution of TGF.beta. bioactivity from the Bio-Gel P-60 column as measured by inhibition of granulosa cell proliferation correlated with the elution of radioiodinated authentic TGF.beta.. Preincubation of TGF.beta.-like activity-containing fractions with TGF.beta.-neutralizing antibody attenuated the proliferation-inhibitory activityin these fractions. TGF.beta. activity was also observed in fractions extracted from orcine corpora lutea. However, porcine follicular fluid collected from large size (> 7 mm in diameter) follicles did not exhibit TGF.beta. activity. The occurrence of TGF.beta. in ovarian theca and corpora lutea, TGF.beta. secretion in vitro by thecal cells, and regulation of granulosa cell proliferation by TGF.beta. in vitro suggest the possibility of an autocrine/paracrine role of this growth factor in folliculogenesis and overall ovarian function. The lack of detectable levels of TGF.beta. in the follicular fluid despite theca cell secretion of this growth factor suggests that TGF.beta. actions are mediated locally.

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