Effects of transforming growth factors-alpha and -beta on proliferation and apoptosis of rat theca-interstitial cells

Ovarian development, follicular growth and atresia require mechanisms regul ating proliferation and death of ovarian cells including theca-interstitial (T-I) cells. Transforming growth factors-alpha and -beta (TGF-alpha and TG F-beta) are well recognized local modulators of T-I function. This study wa s performed to evaluate the effects of TGF-alpha and TGF-beta on ovarian T- I cell proliferation, differentiation and apoptosis. T-I cells from immatur e Sprague-Dawley rats were purified and incubated in chemically defined med ia. Proliferation was assessed by [H-3]thymidine incorporation assay and by cell counting. Steroidogenically active cells were identified histochemica lly by detection of 3 beta -hydroxysteroid dehydrogenase (3 beta -HSD) acti vity. DNA was extracted and apoptosis was identified by detection of intern ucleosomal DNA cleavage producing the characteristic 'ladder pattern' of lo w-molecular weight (LMW) DNA following agarose gel electrophoresis. Quantif ication of apoptosis was carried out with the aid of 3'-end labeling of DNA fragments with [P-32]-dideoxy-ATP. TGF-alpha and TGF-beta stimulated [H-3] thymidine incorporation by 2.2- to 3.1-fold and 1.7- to 3.4-fold respective ly (P <0.005). A combination of TGF-alpha and TGF-beta produced a synergist ic increase in DNA synthesis by 6.7-fold (at 1 ng/ml of each TGF-alpha and TGF-beta; P <0.001) and tenfold (at 10 ng/ml of each TGF-alpha and TGF-beta ; P <0.001). Cell counting revealed that TGF-alpha increased the total numb er of cells 2-8-fold and TGF-beta 2-8-fold. The combination of TGF-alpha an d TGF-beta 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-alp ha, 42.8% in the presence of TGF-beta, and 47 +/- 13% in the presence of bo th TGF-alpha and TGF-beta. TGF-alpha decreased apoptosis by 63 +/- 14% (P=0 .02) while TGF-beta had no statistically significant effect. TGF-alpha in c ombination with TGF-beta produced the greatest inhibition of apoptosis by 7 3.8% (P=0.01). These findings demonstrate that TGF-alpha and -beta stimulat e proliferation of both steroidogenically active and inactive T-I cells. Fu rthermore, TGF-alpha alone and in combination with TGF-beta protects T-I ce lls from apoptotic death. These effects of TGFs may be important in physiol ogic maintenance of ovarian mesenchymal growth and homeostasis as well as i n pathophysiologic conditions associated with excessive growth of the T-I c ompartment, such as polycystic ovary syndrome.