XIAP, a potential regulator of follicular atresia in the sheep ovary
|dc.contributor.author||Douglas, Hollian Raphaelle|
|dc.identifier.citation||Douglas, H. R. (2011). XIAP, a potential regulator of follicular atresia in the sheep ovary (Thesis, Doctor of Philosophy). University of Otago. Retrieved from http://hdl.handle.net/10523/1635||en|
|dc.description.abstract||X-linked inhibitor of apoptosis (XIAP) is a strong inhibitor of initiator (-9) and executer (-3 and -7) caspase activity (Eckelman and Salvesen, 2006). In female reproductive research, XIAP has received minimal attention considering the ability to arrest caspases provides a potential mechanism for regulation of follicular atresia. This study aimed to test the hypotheses that XIAP is indicative of follicular health and regulates follicular atresia and to investigate the novel idea that prolactin (PRL) stimulates XIAP expression in the sheep ovary. Estrous cycles of adult Romney ewes (N=31) were synchronized with Estrumate®. Tissue and blood samples were subsequently collected on either days 14, 15 and 16 or at twelve hour intervals during the follicular phase. Initially analysis involved developing a plasma PRL profile by radioimmunoassay. The presence and localization patterns of XIAP and prolactin receptor (PRLR) isoforms protein and/or mRNA were determined by RT-PCR, in situ hybridization histochemistry and immunohistochemistry. This was followed by a comparative study evaluating levels of active caspase-3 immunoreactivity and XIAP mRNA (N=22) or protein (N=23) expression in adjacent sections containing the same day 14, 15 and 16 antral follicles. Triple label immunofluorescence and confocal microscopy were subsequently used to further clarify XIAP and active caspase-3 protein localization and to establish the presence of an inverse expression relationship in granulosa and thecal cells. Differing doses of Ovine FSH and PRL, as potential stimuli of XIAP upregulation, were trialed in cultured granulosa cells. Finally, a provisional attempt to develop a granulosa cell death model for determination of PRL’s ability to upregulate XIAP was undertaken. This study showed widespread XIAP expression during both luteal and follicular phases of the estrous cycle. XIAP protein was detected from the primary follicle stage onwards, whereas the mRNA was only evident in antral follicles, likely due to limited sensitivity of the in situ hybridization histochemical technique. This also proved problematic for detection of PRLR isoform mRNA, which was localized in luteal cells and three antral follicles only. In the comparative analysis, all day 14 antral follicles showed positive XIAP mRNA expression irrespective of active caspase-3 levels. Over 50% of the day 15 and 16 antral follicles scored, however, showed an inverse relationship between XIAP mRNA/protein and active caspase-3 protein levels in, as did XIAP protein in day 14 antral follicles. Interestingly, in healthy follicles XIAP expression in thecal tissue was widespread, becoming increasingly localized to interna layers as active caspase-3 immunopositive granulosa cells became prevalent, despite a lack of caspase activity in the theca itself. Confocal microscopy showed active caspase- 3 and XIAP colocalization in granulosa cells. No significant negative correlation in expression patterns was observed, possibly due to brief active caspase-3 expression at apoptosis onset and/or individual follicle properties. Increasing plasma PRL levels including two peaks were apparent during the follicular phase, however, limited granulosa cell culture lifespan prevented conclusive results concerning PRL-induced changes in XIAP levels in vitro. Overall the results indicate XIAP directly interacts with active caspase-3 in granulosa cells, thereby promoting follicle heath and functioning as a regulator of follicular atresia in the sheep ovary.|
|dc.publisher||University of Otago|
|dc.rights||All items in OUR Archive are provided for private study and research purposes and are protected by copyright with all rights reserved unless otherwise indicated.|
|dc.title||XIAP, a potential regulator of follicular atresia in the sheep ovary|
|thesis.degree.discipline||Anatomy and Structural Biology|
|thesis.degree.discipline||Anatomy and Structural Biology||en_NZ|
|thesis.degree.name||Doctor of Philosophy|
|thesis.degree.grantor||University of Otago|
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