Of the ovarian follicles that develop during reproductive life, more than 99% do not ovulate and are eliminated from the ovary by follicular atresia. Atresia is achieved by the self destruction of thecal and granulosa cells that comprise the follicle, by the process of apoptosis. The objective of this study was to determine if activation of the Fas receptor could enact apoptosis of thecal cells, and to explore the signal transduction pathway involved. Primary cultures of thecal/interstitial cells isolated from immature rat ovaries were treated with anti-Fas monoclonal antibody (anti-Fas mAb) (2.5 microg/ml). Morphological changes indicative of apoptosis, such as, condensation of chromatin, nucleoplasmic segmentation and formation of apoptotic bodies, were observed by fluorescence microscopy following nucleic acid staining with Hoechst 33342 dye and propidium iodide. DNA analysis of cells after 10 h of treatment with anti-Fas mAb showed that DNA had been cleaved into fragments that were multiples of 180-300 bp in length; biochemical evidence of apoptosis. The sphingomyelin (N-acylsphingosine-1-phosphocholine, SM) pathway that is initiated by the hydrolysis of SM to ceramide (Cer) has been shown previously to be activated by the Fas ligand/receptor system in a number of different cell types. It was therefore possible that the intracellular transduction of Fas receptor activation of thecal/interstitial cells could also involve the SM-Cer pathway. Hence, we have measured the SM levels in control and treated thecal/interstitial cells. Extracts of untreated thecal/interstitial cells contained six major species of SM identified as d18:1/16:0 (sphingosine base/fatty acid), d18:1/18:0, d18:1/20:0, d18:1/22:0, d18:1/24:1, d18:1/24:0 by normal phase high performance liquid chromatography interfaced with electrospray mass spectrometry. Treatment with anti-Fas mAb (2.5 microg/ml) for 30 min caused significant hydrolysis of only two of the SM species, d18:1/16:0 and d18:1/24:1. The involvement of ceramide, the central lipid in this phospholipid second messenger system, was tested using the synthetic cell permeable Cer analog (N-acetyl-N-sphingosine, C2-Cer). C2-Cer (10 microM). This analog induced both morphological and biochemical changes in thecal/interstitial cells, that were characteristic of apoptosis, and the same as those induced by anti-Fas mAb. C2-dihydroceramide (10 microM), an inactive analog of C2-Cer, failed to induce apoptosis of thecal/interstitial cells. In conclusion, the sphingomyelin-ceramide cycle that can lead to cell suicide by apoptosis is functional and activated through the Fas ligand/receptor signal transduction pathway, not only in the immune system, but also in thecal/interstitial cells of the ovarian follicle.

Follicular atresia is characterized by the initial rapid loss of granulosa cells by apoptosis, followed by the loss of thecal cells at a slower rate. We have previously shown that treatment of subconfluent cultures of thecal/interstitial cells (T/I) with transforming growth factor (TGF) plus TGF caused chromatin condensation and internucleosomal fragmentation characteristic of apoptosis, whereas in the presence of either TGF or TGF alone the cells remained healthy. In this study we have examined the effect of TGF and TGF alone and in combination on the levels of mRNA encoding bcl-2 and interleukin-1 -converting enzyme (ICE) in T

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.

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