Induction of apoptosis in rat thecal/interstitial cells by transforming growth factor alpha plus transforming growth factor beta in vitro.

In each estrous cycle dominant follicles are selected from a growing pool to develop to the preovulatory stage and to ovulate. Those follicles that do not ovulate must be eliminated in order to maintain the constant mass and homeostasis of the ovary. Granulosa cells are lost by apoptosis at the onset of follicular atresia, whereas apoptotic thecal cells are identified at later stages of atresia. Since transforming growth factor (TGF) alpha and TGF beta 1 have been implicated in the regulation of thecal cell physiology we have localized these growth factors by immunohistochemistry in sections of ovaries from 25-day-old rats, an age at which the ovary exhibits a wave of atresia of preantral follicles. Thecal cells contained TGF alpha and TGF beta 1 throughout the entire process of follicular atresia. To determine if these growth factors could influence thecal cell death, thecal/interstitial cells were isolated from 25-day-old rats, and maintained in culture with growth factors. Subconfluent cultures treated with TGF alpha or TGF beta 1 alone remained healthy whereas in the presence of both TGF alpha and TGF beta 1 there was light microscopical evidence of rounding up of cells and detachment from the monolayer. Chromatin condensation and internucleosomal fragmentation, characteristic of apoptosis, were observed by nucleic acid staining and fluorescence microscopy of thecal/interstitial cells treated with TGF alpha plus TGF beta 1. Further evidence that these cells were undergoing apoptosis came from DNA analysis and the demonstration of DNA laddering. This response of thecal/interstitial cells to TGF alpha plus TGF beta 1 was density dependent; confluent cultures were protected from the induction of apoptosis under these conditions. We conclude that thecal cells are eliminated from atretic follicles by the active and strictly regulated process of involving the combined actions of TGF alpha and TGF beta 1.