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BHUSHAN K. GANGRADE, JEFFREY V. MAY, The Production of Transforming Growth Factor-β Porcine Ovary and its Secretion in Vitro, Endocrinology, Volume 127, Issue 5, 1 November 1990, Pages 2372–2380, https://doi.org/10.1210/endo-127-5-2372
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Porcine granulosa cells isolated from 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-β (TGFβ) inhibits FBS/EGF-stimulated proliferation in a dose-dependent manner. We have used this proliferation inhibitory property of TGFβ 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 TGFβ in the theca cell conditioned medium was quantitatively estimated by generating a TGFβ-dose-response curve (inhibition of FBS/EGF-stimulated proliferation of granulosa cells in monolayer culture) using authentic human TGFβ-1. Ovarian thecal cells isolated from small and large size follicles in the pig ovary secrete TGFβ-like activity in vitro. Medium conditioned by thecal cells in primary monolayer culture contains a latent form of TGFβ 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 TGFβ-like activity in the medium. Incubation of heat-activated thecal cell conditioned medium with a TGFβ-neutralizing antibody (which recognizes TGFβ-1 and 2) but not nonimmune serum attenuated the TGFβ-like activity in thecal cell conditioned medium suggesting that this activity is due to authentic TGFβ. Since many cell types secrete latent TGFβ in the medium when cultured in vitro, we next investigated whether thecal cell secretion of latent TGFβ was a function of cell culture or whether the ovarian thecal compartment actually contained detectable levels of TGFβ-like activity. To this end, we used an acid-ethanol extraction procedure to isolate thecal proteins from fresh-frozen tissue. The acidethanol extracted protein fraction was mixed with trace amounts of 125I-TGFβ for detection and chromatographed on Bio-Gel P-60 column under acidic conditions. Elution of TGFβ bioactivity from the Bio-Gel P-60 column as measured by inhibition of granulosa cell proliferation correlated with the elution of radioiodinated authentic TGFβ. Preincubation of TGFβ-like activitycontaining fractions with TGFβ-neutralizing antibody attenuated the proliferation-inhibitory activity in these fractions. TGFβ activity was also observed in fractions extracted from porcine corpora lutea. However, porcine follicular fluid collected from large size (>7 mm in diameter) follicles did not exhibit TGFβ activity. The occurrence of TGFβ in ovarian theca and corpora lutea, TGFβ secretion in vitro by thecal cells, and regulation of granulosa cell proliferation by TGFβ 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 TGFβ in the follicular fluid despite theca cell secretion of this growth factor suggests that TGFβ actions are mediated locally. (Endocrinology127: 2372–2380, 1990)
