Effects of transforming growth factors-α and -β on proliferation and apoptosis of rat theca-interstitial cells

Abstract

Ovarian development, follicular growth and atresia require mechanisms regulating proliferation and death of ovarian cells including theca-interstitial (T-I) cells. Transforming growth factors-α and -β (TGF-α and TGF-β) are well recognized local modulators of T-I function. This study was performed to evaluate the effects of TGF-α and TGF-β on ovarian T-I cell proliferation, differentiation and apoptosis. T-I cells from immature Sprague-Dawley rats were purified and incubated in chemically defined media. Proliferation was assessed by [3H]thymidine incorporation assay and by cell counting. Steroidogenically active cells were identified histochemically by detection of 3β-hydroxysteroid dehydrogenase (3β-HSD) activity. DNA was extracted and apoptosis was identified by detection of internucleosomal DNA cleavage producing the characteristic 'ladder pattern' of low-molecular weight (LMW) DNA following agarose gel electrophoresis. Quantification of apoptosis was carried out with the aid of 3′-end labeling of DNA fragments with [32P]-dideoxy-ATP. TGF-α and TGF-β stimulated [3H]thymidine incorporation by 2.2- to 3.1-fold and 1.7- to 3.4-fold respectively (P<0.005). A combination of TGF-α and TGF-β produced a synergistic increase in DNA synthesis by 6.7-fold (at 1 ng/ml of each TGF-α and TGF-β; P<0.001) and tenfold (at 10 ng/ml of each TGF-α and TGF-β; P<0.001). Cell counting revealed that TGF-α increased the total number of cells 2.8-fold and TGF-β 2.8-fold. The combination of TGF-α and TGF-β increased the total cell count 3.2-fold, compared with control (P<0.05). The percentage of the steroidogenically active cells was 37±9% (mean±S.E.M.) in the control cultures, 50±5% in the presence of TGF-α, 42±8% in the presence of TGF-β, and 47±13% in the presence of both TGF-α and TGF-β. TGF-α decreased apoptosis by 63±14% (P=0.02) while TGF-β had no statistically significant effect. TGF-α in combination with TGF-β produced the greatest inhibition of apoptosis by 73±8% (P=0.01). These findings demonstrate that TGF-α and -β stimulate proliferation of both steroidogenically active and inactive T-I cells. Furthermore, TGF-α alone and in combination with TGF-β protects T-I cells from apoptotic death. These effects of TGFs may be important in physiologic maintenance of ovarian mesenchymal growth and homeostasis as well as in pathophysiologic conditions associated with excessive growth of the T-I compartment, such as polycystic ovary syndrome.