Regulation of gap junction alpha 1 binding proteins in the adult rat epididymis and their influence on androgen-dependent gap junction alpha 1 localization

Edwards, Sara I. (2009). Regulation of gap junction alpha 1 binding proteins in the adult rat epididymis and their influence on androgen-dependent gap junction alpha 1 localization Mémoire. Québec, Université du Québec, Institut National de la Recherche Scientifique, Maîtrise en sciences expérimentales de la santé, 135 p.

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Résumé

Spennatozoa acquire motility and fertility by passing through the epididymis. The epididymal epithelium is maintained by androgens and testicular factors secreted by the testes. It is thought that epithelial regulation by these testicular factors influences gap junctional intercellular communication and involves signal transduction pathways. Little information exists regarding signal transduction pathways or on the regulation of gap junctions (GJ) in the epididymis. However, previous studies from this Jaboratory have shown that the targeting of the gap junction protein alpha 1 (GJA 1) is reliant on testicular androgens in the initial segment of the rat epididymis (Cyr et al. 1996). The first objective of this study was to characterize the expression and localization of signalling effectors cAMP-dependent protein kinase A catalytic subunit (PKAcat) and cellular Rous sarcoma oncogene (c-Src) m the adult, rat epididymis. Immunocytochemical and Western blot analyses demonstrated that c-Src and PKAcat were present in ali four segments of the epididymis. C-Src Jocalization was segment­ specifie. These data suggest that PKAcat and c-Src can mediate signalling cascades in the rat epididymis. The second objective of this study was to detern1ine the effect of androgens on GJA 1 protein synthesis and phosphorylation in the initial segment and caput epididymidis using an orchidectomy mode! and Western blot analyses. This was done in order to understand the androgen-dependent targeting of GJA 1. The proteins c-Src, PKAcat and tight junction protein 1 (TJP1) were a Iso investigated, since studies have shown that they regulate GJA 1 (Toyofuku et al. 1999; Giepmans et al. 2001 a; Giepmans et al. 2001 b; Duffy et al. 2004; Yogo et al. 2006). GJA 1 expression was testicular factor-dependent in the initial segment and caput, and its phosphorylation state was testicular factor­ dependent in the caput, but not the initial segment. These results indicate that GJA 1 targeting is independent of its protein synthesis and phosphorylation status. Western blot analyses demonstrated that c-Src was testicular factor-dependent in the initial segment and androgen-dependent in the caput. The ratio of active to inactive c-Src was androgen­ dependent m the caput, but remained constant m the initial segment. Immunocytochemical analysis indicated that c-Src cellular targeting was altered with orchidectomy and was dependent on testicular factors. These results indicate that despite a decrease in total c-Src, c-Src is still active in orchidectomized animais and therefore capable of regulating GJA1. PK.Acat expression decreased in orchidectomized animais maintained with testosterone in the caput. Orchidectomy did not influence the cellular localization of PK.Acat. No differences were observed in any treatment group for TJPI. These results suggest that neither PK.Acat nor TJP l influence GJA 1 intracellular targeting. The third objective of this study was to determine if a direct association between GJA l and c-Src, PKAcat or TJPI, played a role in GJA l 's intracellular targeting. Coimmunoprecipitation analyses were done on tissue lysates from the initial segment and caput epididymidis using an orchidectomy mode!. In the initial segment, an association was observed between GJAI and c-Src and GJAI and TJPI, which was treatment­ independent. In the ca put, a weak association was observed between GJA l and TJP l in ali treatment groups. There was no association between GJA l and PK.Acat in either segment. These results suggest that GJA 1 is regulated in a segment-specifie manner and that c-Src and TJPI may play a role in GJ inhibition. This study contributes severa! ideas to epididymal cell biology. It demonstrates that signalling pathways may contribute to segment-specifie physiological differences in the epididymis. The study suggests that tight and gap junctions could form a junctional nexus in the epididymis. The data provide evidence that gap junctions are regulated in a segment-specifie manner to contribute to epithelial diversity.

Type de document:	Thèse Mémoire
Directeur de mémoire/thèse:	Cyr, Daniel G.
Informations complémentaires:	Chapitre 4 : Synthèse de la mémoire redigé en français (sic)
Mots-clés libres:	epididyme ; androgene
Centre:	Centre INRS-Institut Armand Frappier
Date de dépôt:	27 sept. 2013 15:04
Dernière modification:	30 nov. 2015 19:59
URI:	https://espace.inrs.ca/id/eprint/199

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