Changes in Expression and Localization of X-linked Inhibitor of Apoptosis Protein (XIAP) in Follicular Granulosa Cells During Atresia in Porcine Ovaries

Abstract

Abstract. Follicular selection predominantly depends on granulosa cell apoptosis in porcine ovaries, but the molecular mechanisms regulating the induction of apoptosis in granulosa cells during follicular selection remain incompletely understood. To determine the role of X-linked inhibitor of apoptosis protein (XIAP), which suppresses caspase-3, -7 and -9 activities and acts as an endogenous inhibitor of apoptotic cell death, in the regulation of granulosa cell apoptosis during follicular atresia, we examined the changes in the expression level and localization of XIAP mRNA and protein in granulosa cells during follicular atresia using reverse transcription-polymerase chain reaction (RT-PCR), in situ hybridization, Western blotting and immunohistochemistry, respectively. High levels of XIAP mRNA and protein were noted in the granulosa cells of healthy follicles, and decreased levels were noted during follicular atresia. Ιn situ hybridization and immunohistochemistry demonstrated that XIAP mRNA and protein were strongly expressed in the granulosa cells of healthy follicles, but negative/trace stainings were noted in those of atretic follicles. The present findings strongly indicate that XIAP is a candidate molecule which acts as an anti-apoptotic/pro-survival factor by inhibiting intracellular apoptosis signaling and is involved in the regulation of apoptosis in porcine granulosa cells. Key words: Apoptosis, Caspase, Granulosa cell, Pig ovary, X-linked inhibitor of apoptotsis protein (XIAP) (J. Reprod. Dev. 54: [454][455][456][457][458][459] 2008) n mammalian ovaries, more than 99% of follicles disappear during follicular growth and development, and this appears to be primarily due to apoptosis of follicular granulosa cells, as the biochemical and morphological characteristics of apoptosis have been observed in the granulosa cells of atretic follicles [1][2][3]. Apoptotic stimuli and intracellular apoptosis-signal transduction pathways in granulosa cells have not yet been determined, and many researchers have attempted to confirm the primary trigger of apoptosis and how the intracellular apoptotic signal is transmitted in granulosa cells [4][5][6][7]. Many apoptosis-related factors are implicated in follicular atresia, including cell death ligands and receptors, intracellular pro-and anti-apoptotic molecules, cytokines and growth factors. In particular, cell death ligand-receptor signaling has been revealed to be the dominant regulatory system for apoptosis in granulosa cells [4][5][6][7]. To date, two major intracellular-signal pathways in cell-death ligand and receptor-dependent apoptosis (type I and type II: mitochondrion-independent anddependent pathways, respectively) have been found [8][9][10][11][12]. In type I and II apoptotic cells, apoptotic signaling is initially triggered by members of the cell death ligand and receptor superfamily [Fas ligand (FasL) and Fas (also called Apo-1, CD95 or TNFRsf6), tumor necrosis factor (TNF) α and its receptors (TNFRs), TNFrelated apoptosis-inducing ligand (TRAIL; ...

XIAP: a potential determinant of ovarian follicular fate

“…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). 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 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).…”

“…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.…”

Changes in the expression of FoxO1 and death ligand genes during follicular atresia in porcine ovary

“…The vast majority of follicles undergo atresia at various stages of development. Follicular atresia in pigs is regulated by many factors (Kimura et al, 1999;Wada et al, 2002;Matsuda-Minehata et al, 2005;Maeda et al, 2007;Cheng et al, 2005Cheng et al, , 2008Matsuda et al, 2008;Sugimoto et al, 2010). Manabe et al (2004) summarized five apoptosis signaling pathways and emphasized the importance of three cell death ligand-receptor systems, TNF-TNFR, Fas L-Fas, and TRAIL-TRAILR.…”

Effect of RNA Interference of BID and BAX mRNAs on Apoptosis in Granulosa Cell-derived KGN Cells

“…As previously reported [19,20], total RNA was extracted from cells using a QIAshredder and RNeasy Mini Kit (Qiagen, Chatsworth, CA, USA) according to the manufacturer's directions. First strand cDNA was synthesized from the total RNA using a Ready-To-Go T-Primed First-Strand Kit (GE Healthcare, Little Chalfont, UK).…”