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

Abstract

Follicular atresia is believed to be largely regulated by apoptosis. To further understand how apoptosis can affect cumulus cells and oocytes we have evaluated the incidence and regulation of apoptosis affecting bovine cumulus oocyte complexes in vitro. Expression of components of the Fas signaling pathway was studied in both oocytes and cumulus cells by polymerase chain reaction after reverse transcription, immunoblotting, and indirect immunofluorescence. Furthermore, the Fas signaling pathway was activated in cumulus oocyte complexes with an agonistic anti-Fas antibody during in vitro maturation in the presence or absence of FSH. Viability and incidence of apoptosis in cumulus cells were evaluated by assessing membrane integrity and nuclear morphology. Oocyte nuclear maturation was also analyzed, as well as cleavage rates, blastocyst formation rates, and blastocyst quality, following in vitro fertilization. Fas mRNA and protein were expressed both in oocytes and cumulus cells. FasL protein was found in cumulus cells but could not be detected in oocytes, despite its mRNA expression. Both activation of the Fas pathway and presence of FSH during in vitro maturation increased the incidence of apoptosis in cumulus cells, affecting predominantly the middle and peripheral regions of the cumulus. The observed increase, however, had no effect on the developmental competence of the oocytes.

Pertinence of Apoptosis Markers for the Improvement of In Vitro Fertilization (IVF)

“…Fas ligand (Fas-L) is expressed in oocytes of primary, secondary and tertiary follicles, adding weight to the argument that the couple Fas/Fas-L is involved in the regulation of oocyte apoptosis [47]. Moreover, the activation of the Fas pathway during oocyte maturation in vitro increases the incidence of apoptosis of cells of the cumulus oophorus [48] and granulosum [49]. In addition, soluble Fas, a truncated form of Fas that acts as an inhibitor of Fas-induced apoptosis, is found at high concentration in follicular fluid and in the medium bathing the cumulus oophorus/mature oocyte complex [50].…”

Caspase‐2L, caspase‐9, and caspase‐3 during in vitro maturation and fragmentation of the mouse oocyte

“…In cattle, poor quality oocytes have been shown to have higher levels of the pro-apoptotic factor Bax, and lower levels of the anti-apoptotic factor Bcl-2. If the Fas/FasL system has been shown not to be involved in cattle oocyte death (Sakamaki et al, 1997;José de Los Santos et al, 2000;Rubio Pomar et al, 2004), an increase in caspase activity, cell fragmentation, and TUNEL labeling have been observed after a heat shock during IVM (Roth and Hansen, 2004a,b), and sphingosine 1-phosphate, which inhibits ceramide-induced apoptosis, protects oocytes against the effects of heat shock, as do caspase inhibitors (Roth and Hansen, 2004b). Moreover, these in vitro matured oocytes are more difficult to fertilize and often degenerate during embryo development (Yang and Rajamahendran, 2002).…”

Fas ligand expression and mediated activation of an apoptosis program in bovine follicular granulosa cells

“…Fas in testicular germ cells and ovary oocytes interacts with Fas ligand in sertoli cells and follicular granulosa cells, an interaction which could keep the spermatogenesis and oogenesis balanced (Geng et al, 2008;Rubio Pomar et al, 2004). During the development of the bovine oocytes, the Fas/FasL pathway induces apoptosis of ovarian granulosa cells by initiating an apoptosis signal, leading to follicular atresia.…”