Expression and localization of the chaperone protein MRJ in pig gametes and gonads

A group of specialized molecules, known as DnaJ proteins, plays a pivotal role in several cellular functions, promoting the folding and assembly of nascent proteins, their transport into cell organelles, and the formation of mature protein complexes. MRJ (Mammalian Relative of DnaJ) is a member of this family of chaperone proteins and has been shown to favor the remodeling of round spermatids to flagellate spermatozoa. Further evidence indicates the involvement of MRJ in chorioallantoic fusion and placenta development. However, only scattered information is available on the role of this protein in the female reproductive tract and female gametes and the data available are limited to murine and human species. Here we investigate the expression and localization of MRJ protein in pig spermatozoa, testis, cumulus oocyte complexes and ovaries. To this purpose, we extracted RNA from testis, granulosa cells and from pools of 5 oocytes. cDNA was amplified by Reverse Transcription-PCR (RT-PCR) using primers specifically designed for MRJ, based on sequence data bank available. The amplified products were separated on a 2% TAE agarose gel, purified, sequenced and aligned using ClustalW. Isolated spermatozoa protein extracts were resolved by SDS PAGE, immunoblotted and stained with an antibody for murine MRJ. In order to localize this protein in porcine spermatozoa, immunocytochemical analysis was carried out on pig semen using the previous antibodies and a suitable fluorescent secondary antibody. RT-PCR showed MRJ protein expression in pig testis, immature oocytes and granulosa cells. Comparison of the obtained pig cDNA sequence with databases revealed a degree of homology of 96%.with the human, 90% with the bovine and 86% with the mouse MRJ protein genes. Furthermore western blotting data demonstrated the presence of a porcine MRJ-like protein with a MW of 30KDa in mature spermatozoa protein extracts. Immunocytochemical results showed a specific localization of this protein on the acrosome surface, centrosomal region and tail principal piece of pig male gametes. Altogether our findings indicate expression of MRJ in pig gametes as well as in granulosa cells, with a highly conserved nucleotide sequence within mammalian species. The protein shows a specific localization in spermatozoa, consistent with what previously described in the mouse and human. Further investigation of the temporal and spatial regulation of MRJ expression in pig will be important to reveal the putative role of chaperones in porcine reproductive functions.