The Molecular Mechanism In Male Reproductive Tract Of The Prawn, Macrobrachium Rosenbergii

Sex and reproduction have received much attention in view of their importance in genetics, development, and evolution. Up to now, many studies are mainly focus on dealing with the human and biomodel organism, and a number of critical genes involved in sex differentiation and reproduction have been identified. But a little available and valuable gene information is shown in crustacean sex and reproduction, even the molecular cascade of sex and reproduction remain poorly understood. So it is of the great significance that researches about the molecular mechanism of sex and reproduction should be carried out in crustacean.In our present study, we selected the prawn, Macrobrachium rosenbergii, which is the commercially important organism, as the experimental materials. In crustacean, we firstly used suppression subtraction hybridization (SSH) to generate a subtracted cDNA library enriched for the male-specific transcripts. The subtracted and unsubtracted cDNA mixtures were used as probes in hybridization to screen the library. Thereby, some of male reproductive tract specific genes were obtained. After sequencing and by blast searches, one of them included an obvious open reading frame (ORF) and named as Mar-Mrr. Another one showed significant similarity to Kazal-type proteinase inhibitor and named as MRPINK.Then, full-length cDNA sequences for Mar-Mrr and MRPINK were determined by 3' and 5' RACE. The Mar-Mrr cDNA was found to contain 683 nucleotides including an ORF of 333 bp that encoded 110 amino acids peptide, with a molecular weight of 11.7 kDa, pI 5.574. The cDNA included a 82 bp 5' untranslated region (UTR) and a 268 bp 3' UTR that contained two potyadenylation signals (AATAAA) at 64 bp and 14 bp upstream from poly (A) tail in the mRNA, respectively. The deduced translation products contained a putative signal peptide of 24 amino acids, and a mature peptide of 86 amino acids. The amino acids composition of Gly, Ser and Ala was found to be relatively higher (15.4%, 10.9% and 9.1%). The full-length cDNA of MRPINK was found to contain 736 bp. The cDNA were found to include a 405 bp ORF, a 72 bp 5' UTR and a 259 bp 3' UTR that contained a polyadenylation signal (AATAAA). The ORF of the MRPINK cDNA was conceptually translated into a 134 amino acid residues precursor containing a 21 amino acid signal peptide, a 113 amino acid mature peptide with two Kazal-type domains, and a translation stop signal.By blast searches, the sequences of Mar-Mrr and the deduced protein had no significant homology with any known gene or protien in DDBJ/EMBL/GenBank database. This is the first time that a novel functional gene was found to relate with male reproduction in the prawn. As for MRPINK, comparison with several Kazal-type double-headed proteinase inhibitors revealed 15% - 32% identities. The two domains of MRPINK showed 19% - 52% identities with Kazal-type domains from other species. This is the first occasion in invertebrate in which male reproductive tract specific Kazal-type proteinase inhibitor was identified.By Northern blotting analysis, significant accumulation of Mar-Mrr transcript was observed in male reproductive tracts. The transcription of the gene was observed in all three parts of the male reproductive tract and the highest expression of the gene was found to be in the vas deferens. The expression level of Mar-Mrr was examined throughout the developmental process from the post-larva to the adult every two weeks. Results revealed the Mar-Mrr mRNA transcript appeared from the fourth week after post-larva development and the expression level evidently increased following 6, 8,10 and 12 weeks after the post-larva stage. Based on these results, we examined the expression of Mar-Mrr during the androgenic gland maturation. The result indicated the Mar-Mrr mRNA expression was significantly increased during the androgenic gland maturation. Thus, transcription of Mar-Mrr was shown to be a development-dependent regulated expression pattern. MRPINK mRNA expression was not detected in any other tissues, only in male reproductive tracts. Expression of MRPINK was detected in all the tested stages once the male reproductive tract formed. The semi-quantitative RT-PCR indicated that the expression of the gene was to be continuous in the shaped male reproductive tract. The results of Northern blotting showed that the MRPINK gene expression was dominant in vas deferens and quite low in the testis.In situ hybridization (ISH) was used to determine which cells within the male reproductive tract express the gene mRNA. We demonstrated Mar-Mrr and MRPINK mRNAs were localized only in the secretory epithelial cells of vas deferens. Using PCR with genomic DNA of M. rosenbergii as template, the results suggested that the two genes contained no introns. Southern blotting analysis indicated the existence of multiple gene copies of Mar-Mrr and MRPINK.In general, this is the first time to clone the two male specific genes from prawns, and to analyze their expression and localization. Moreover, it was proposedthat Mar-Mrr would play key roles in the male reproductive tract and MRPINK would play roles in maintaining internal surroundings stability rather than in directly regulating the onset of a specific stage in the male reproductive tract. These results will provide theoretical basis to have a better understanding of the molecular mechanism in male reproductive tract of the prawn.