Round Spermatid-specific Gene Rsb66 Encoded Protein And Testicular Gene The Hsd14 Functional Studies

In order to identify novel genes in spermatogenesis,tens of thousands of rat spermatocytes and round spermatids had been isolated by Laser Capture Microdisseetion (LCM) in our laboratory,and total RNA of these two cells had been extracted.Then the Suppression Subtracted Hybridization(SSH) library of round spermatid-specific cDNAs against those of primary spermatocyte(RSB) had been constructed,rsb66 which is investigated in present study was one of those genes obtained from RSB subtracted library.Its full-length cDNA comprises 564bp and was assigned the GenBank accession number AY121839.The open reading frame encodes a polypeptide consisting of 168 amino acid residues.Northern blot showed that rsb66 was specifically expressed in rat testis.The coding sequences of rsb66 and its human homolog showed 88.2%homology on the nucleotide and 92.3%identity on the amino acid level.Such highly homolog between human and rodents suggests that this gene is conserved in the evolution.In previous studies,a Yeast Two-Hybrid system had been used to screen the interaction partners of full-length RSB66 in human testis eDNA library.As a result,two positive clones were identified and designated as hsd45(AY604176) and hsd46(AY604177).The full-length cDNA of hsd45 comprises 985bp and the open reading frame encodes a polypeptide consisting of 221 amino acid residues.The result of BLASTP analysis showed that HSD45 was identical to INCA1(Inhibitor of CDK interacting with Cyelin A1),a novel cyclin A1/CDK2 interaction partner in a Yeast-Triple Hybrid approach.In our study the interaction between RSB66 and HSD45 were firstly eomfirmed both in vitro and in vivo by GST-pull down and co-immunoprecitetation,respectively.According to the previous results in crystal structure of RSB66,Tyr-117 and His-119 residues may form an active site which is essential for the function of RSB66.To investigate the influence of these two sites on the function of RSB66,one dot mutant mRSB66(117Ala119Ala) and tow deletions(106RSB66 and 128RSB66) were constructed.Results of co-immunoprecitetation showed that mRSB66 had weaker interaction with HSD45 compareing with wild-type RSB66, while the interaction between two deletions of RSB66 and HSD45 could not be detectable.It indicated Tyr-117 and His-ll9 residues had effected on the interaction between RSB66 and HSD45,and theα-helix in C-terminal was also crucial for the interaction.By immunoflouresence we found HSD45 located in both nuclear and cytoplasm of HeLa,it co-localized with RSB66 in cytoplasm.All the mutants showed similar localization to wild-type RSB66 which indicated that the mutants had no apparent effect on the localization of the protein.It was reported that HSD45(INCA1) can interacted with eyclin A1 and therefore inhibit the kinase activity of Cdk.This suggested its participation in regulation of the cell cycle.We presumed that RSB66 could also regulate the cell cycle through its interaction with HSD45. Results of flow cytometry showed that obvious G2/M delay was induced by overexpression of RSB66 in HeLa,which was also found in overexpression of mRSB66.However,the degree of G2/M delay was alleviated in the latter one.In vitro kinase avtivity assay using histone H1 as substrates demonstrated that cyclin A1/Cdk2 kinase activity was inhibited by purified RSB66.And this down-regulation of kinase activity did not due to the variation of the expression levels of cyclin A1,Cdk2 and HSD45. Moreover,the kinase activity was influenced by putified mRSB66,106RSB66 and 128RSB66 in different levels.Our results indicated that this inhibition in kinase activity might be cause of G2/M delay in cell cycle of HeLa.hsd14 was a novel gene obtained from a human testis-specific EST by silicon cloning.Its full-length eDNA comprises 1107bp and was assigned the Genbank accession number AY251164.The open reading frame encodes a polypeptide consisting of 288 amino acid residues and contains three dsRNA binding domain.The result of BLASTP analysis showed that amino acid sequence of HSD14 was identical to PACT(PKR activating protein) except the absence of 25 amino acids in the N-terminal.It indicated that HSD14 and PACT might be products of the same gene due to selected splicing.It was reported that PACT can activate PKR,inhibit protein synthesis,and therefore induce apoptosis of cells.It may also be involved in RNA silencing pathway.In this study,we try to explore the function of HSD14 in spermatogenesis on the base of the function of PACT.We immuned BALB/c mice with purified His6-HSD14 and injected mice testis with anti-HSD14 antibodies.To our surprise,the testes of mice were in severe disorder. Degeneration and apoptosis were observed in many germ cells.Moreover,anti-growth phenotype was observed in yeast when we screen for interaction partner of HSD14 in a Yeast-Two Hybrid system using HSD14 as bait.We constructed stable RNAi cell line of endogenous prkra(hsd14 mouse homologue) successfully using GC-1spg.Our group has established a novel technique of microinjection into seminiferous tubules of mouse and spermatogonial cell line(GC-1spg) transplantation.Our work of serum microinjection and constructin of stable RNAi cell line will lay a foundation for our dedication to function of HSD14 in spermatogenesis through the novel technique.