Fas-Induced Apoptosis in Rat Thecal/Interstitial Cells Signals Through Sphingomyelin-Ceramide Pathway*

Abstract

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.

Essential actions of melatonin in protecting the ovary from oxidative damage

“…Large amounts of ROS accompanied by reduced levels of antioxidant enzymes (e.g., SOD, glutathione peroxidase, and catalase) may likely contribute to oxidative stress-mediated apoptosis in atretic follicles [55]. Apoptosis is a physiological process regulated by endocrine factors such as FSH, LH, and paracrine factors [56]; they initiate the lethal stimulus (physiological or pathologic) leading to controlled cellular self-destruction [57], characterized by internucleosomal DNA fragmentation, cell shrinkage, plasma membrane blebbing, and formation of apoptotic bodies [58]. The role of mitochondria in regulating apoptosis as a consequence of the release of cytochrome c and other proapoptotic proteins has been documented [59].…”

Induction of apoptosis in thecal/interstitial cells: action of transforming growth factor (TGF) alpha plus TGF beta on bcl-2 and interleukin-1 beta-converting enzyme

“…Recently, we have shown that the ability of TGF plus TGF to cause death of T/I cells by an apoptotic mechanism was blocked by the reducing agent N-acetyl--cysteine, which is known to bind free radicals (Foghi et al 1996b). This suggested that the action of free radicals was necessary for the induction of apoptosis in T/I cells.…”

Role of Fas-Mediated Apoptosis and Follicle-Stimulating Hormone on the Developmental Capacity of Bovine Cumulus Oocyte Complexes In Vitro1

“…Throughout each follicular wave, promotion of the dominant follicle and atresia of the subordinate follicles occurs [1,2]. During early atresia in antral follicles, the cumulus cells and the oocyte remain apparently unaffected by the atretic changes, which are primarily manifested as apoptosis in the mural granulosa cells and, at a later stage, in the theca cells [3][4][5]. It has been suggested that apoptosis of granulosa cells is regulated by the Fas signaling pathway [6][7][8].…”

“…Two classes can be distinguished in the caspase family: initiator caspases and effector caspases. The activation of an effector caspase (e.g., caspase-3 in granulosa cells) [3,11] by initiator caspases ultimately results in nuclear fragmentation and cell death [12]. In the bovine ovary, Fas and FasL expression has been reported in theca and granulosa cells [3,6,7], with mRNA and protein levels being higher in atretic follicles than in healthy dominant follicles [7].…”

“…The activation of an effector caspase (e.g., caspase-3 in granulosa cells) [3,11] by initiator caspases ultimately results in nuclear fragmentation and cell death [12]. In the bovine ovary, Fas and FasL expression has been reported in theca and granulosa cells [3,6,7], with mRNA and protein levels being higher in atretic follicles than in healthy dominant follicles [7]. The presence of Fas has also been demonstrated in cumulus cells of various other species such as the pig [13], mouse [14], and human [15], although little is known about the role of the Fas system in the activation of apoptosis in these cells.…”