X-linked inhibitor of apoptosis protein and active caspase-3 expression patterns in antral follicles in the sheep ovary

Abstract

X-linked inhibitor of apoptosis protein (XIAP) interacts with caspases to inhibit their activity, thereby providing a potential mechanism for regulation of granulosa cell apoptosis occurring during follicular atresia. The aim of this study was to determine the presence and localization of XIAP mRNA and protein content in the sheep ovary and compare these expression patterns with active caspase-3 protein in the same antral follicles. Romney ewe estrous cycles (nZ25) were synchronized with 2-3 Estrumate injections and ovarian tissue collected during the luteal and follicular phases of the cycle. The presence of XIAP mRNA was confirmed by RT-PCR using laser capture microdissected ovarian cell samples. XIAP mRNA was subsequently localized by in situ hybridization histochemistry and XIAP and active caspase-3 protein visualized by immunohistochemistry. In antral follicles extensive XIAP localization was evident in both granulosa and thecal cells. In contrast, mRNA expression was widespread in granulosa cells and only detected in thecal tissue from a small proportion of antral follicles. Active caspase-3 and XIAP comparative expression analysis showed positive XIAP mRNA expression in all late luteal phase (day 14) follicles, despite varying levels of active caspase-3 protein. A proportion of follicular phase (days 15 and 16) follicles, however, showed an inverse expression relationship at the protein and mRNA levels in both granulosa and thecal tissue, as did XIAP protein in day 14 follicles. These results suggest high XIAP may prevent activation of caspase-3, thereby regulating follicular atresia in antral follicles and could potentially be utilized as a marker of follicular health.

XIAP: a potential determinant of ovarian follicular fate

“…Moreover, subsequent studies in pigs and sheep have confirmed granulosa and thecal cell expression in antral follicles (Cheng et al 2008, Phillipps et al 2011. In sheep, XIAP has been detected at the protein level early in development in primary staged follicles up to large antral follicles (Phillipps et al 2011). This widespread expression across cell types and follicle stages suggests that XIAP and potentially IAPs in general are pivotal factors involved in regulating the dynamic environment within the ovary.…”

“…In rats, XIAP is present in granulosa cells, thecal tissue and oocytes within follicles from the preantral stage onwards (Li et al 1998). Moreover, subsequent studies in pigs and sheep have confirmed granulosa and thecal cell expression in antral follicles (Cheng et al 2008, Phillipps et al 2011. In sheep, XIAP has been detected at the protein level early in development in primary staged follicles up to large antral follicles (Phillipps et al 2011).…”

“…Furthermore, a transition from predominant nuclear to cytoplasmic expression in rat granulosa cells coinciding with the onset of atresia has been observed (Li et al 1998). This localisation change, however, did not occur in sheep, highlighting potential variation in XIAP expression patterning in relation to atresia in ruminants and rodents (Phillipps et al 2011). A decrease in XIAP mRNA and protein expression levels has been observed with increasing atresia as determined by in situ TUNEL and changes to antral follicular fluid ratios of 17b-oestradiol (E 2 )/progesterone in porcine ovarian follicles (Cheng et al 2008).…”

“…1), thecal cells ( Fig. 1), oocytes and luteal cells (Li et al 1998, Lareu et al 2003, Vischioni et al 2006, Cheng et al 2008, Phillipps et al 2011. Several studies have localised XIAP expression at different stages during folliculogenesis.…”

“…A decrease in XIAP mRNA and protein expression levels has been observed with increasing atresia as determined by in situ TUNEL and changes to antral follicular fluid ratios of 17b-oestradiol (E 2 )/progesterone in porcine ovarian follicles (Cheng et al 2008). Furthermore, a proportion of sheep antral follicles present during the follicular phase show decreasing XIAP expression levels with increasing active caspase-3 immunoreactivity (Phillipps et al 2011). In our laboratory, confocal imaging has also demonstrated protein colocalisation of active caspase-3 and XIAP in the cytoplasm of antral follicle granulosa cells, suggesting that direct caspase inhibition is a likely mechanism (Fig.…”