Characterization of heat shock protein 70 in the red claw crayfish (Cherax quadricarinatus): evidence for its role in regulating spermatogenesis.

Heat shock protein 70 (HSP70) is a member of molecular chaperones and is important for reproductive biological processes in eukaryotes. In this regard, a full length HSP70 comprised of 2366 nucleotides from the red claw crayfish Cherax quadricarinatus (CqHSP70) was characterized, which with an open-reading frame of 1959 bp encoded 652 amino acid residues. Its mRNA transcript expression in the testes was measured by real-time quantitative PCR. CqHSP70 mRNA transcripts were detected in all organs especially high in the testes. Furthermore, the greatest transcript levels were found during the spermatogonial preparation phase, while the lowest levels were found during the resting stage of the reproductive cycle. In addition, its distribution in the testes was determined by Western blot and immunohistochemistry. Western blot results revealed a single immunoreactive band with an estimated molecular mass of 70 kDa in different phases of testes development. The strongest immunolabeling positive signal was found in spermatogonia, with lower positive staining in secondary spermatocytes, and weak or absent level in the mature sperm. Interestingly, CqHSP70 was mainly located in the cytoplasm of sperm cells. Thus, our results indicate that CqHSP70 is an essential protein in spermatogenesis, with a crucial reproductive function during sperm maturation in invertebrates.