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4 identification and functional characterization of heat shock protein 40 in pig ovary. Pennarossa G et al. Decreased fertility during the hot season is a common problem in pigs. Maternal hyperthermia reduces oocyte fertilizability and increases embryonic mortality. Cell biochemical thermoprotection mechanisms involve members of the heat shock protein (Hsp) family. Hsp40, also known as Mammalian Relative of DnaJ (MRJ) protein, plays a pivotal role as co-chaperone after heat shocks. This protein binds Hsp70 and, through ATP hydrolysis, induces the conformational changes needed by Hsp70 to bind and release heat unfolded proteins. However, no information is available on the role of Hsp40 in mammalian ovary. Here we investigate a) the expression and localization of Hsp40 in pig ovaries; b) its response to heat stress in oocytes and cumulus cells. To identify Hsp40 in pig, we extracted RNA from ovaries, granulosa cells and from pools of 5 oocytes. cDNA was amplified using primers specifically designed for Hsp40, based on sequence data available in other species. The amplified products were sequenced and aligned using ClustalW. The nucleotide sequence showed an homology of 95% with the human, 92% with the bovine and 84% with the mouse orthologs. A polyclonal antibody was raised against the mouse GST/MSJ1((145-242)) homologue protein in New Zealand rabbits and serum was affinity-purified using a GST-coupled Affigel-10 column. Whole ovary proteins were separated by SDS PAGE, immunoblotted and stained with the Hsp40 antibody. The protein displayed a MW of 38KDa, in agreement with results obtained in other species. Immunofluorescence studies showed that Hsp40 is found in oocytes, granulosa and theca cells of follicle at all developmental stages. Pig ovaries, collected at the slaughterhouse, were exposed to 42C for 1h, to verify whether Hsp40has a functional response to heat stress in this specie. Transcript level was compared with the control group, maintained at 38.5C.mRNA was extracted from cumulus cells and pools of 5 oocytes, isolated from follicles of 3 to 5mm. Semi-quantitative analysis was performed in the exponential phase of PCR amplification, using 28S as endogenous control. Data were analyzed with Quantity One (Bio-Rad) and Student-t statistical analysis was performed. Exposure of porcine ovaries to 42C for 1h resulted in a significant increase (P=0.05) of Hsp40mRNA levels (2.40.35 fold) in oocytes, while no significant raise was detected in cumulus cells. To our knowledge, this is the first demonstration that Hsp40 is expressed and responds to a thermal stress in pig ovary. Since this co-chaperone acts upstream to other heat shock protein-such as Hsp70- and it is specifically up regulated in the oocytes, our findings suggest that it may play an important protective role against heat stress infertility.
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