Cloning Of A Novel Testis Gene And Its Expression In Cryptorchidism Model

Part 1 Cloning and sequence analysis of THEG-2, a novel human testis geneObjective:The cloning of genes related to spermatogenesis and development of testis, to lay a foundation for subsequent research of its regulatory mechanism of the meiosis and metamorphosis during the process of spermatogenesis Methods: The expressed sequence tags (EST) expressed in normal human testis was obtained from online EST database ZooDDD. Their highly homologous EST sequences were found through the dbEST database to construct contigs, and spliced by the biomedical software Biolign. The corresponding exons and introns within genome sequences were predicted by software GeneScan. Splice the exons into cDNA, BLAST it in human genome and find the homologous gene in human. According to the open reading frame, the primers were designed. RT-PCR was applied in the cloning of novel gene from mouse testis tissue and analyzing its expression pattern in various human normal tissues. The sequencing results of THEG-2 resort to bioinformatics analysis. Results: A novel gene THEG-2 was cloned from 49311047K-49368620K of human 22 chromosome, with full-length sequence of coding region of 267 bp. The open reading frame (ORF) is 267 bp in length and encodes a deduced amino acid sequence of 88 residues. The molecular weight of THEG-2 protein is 10601.09, the pI of THEG-2 is 4.32. RT-PCR demonstrated the correctness of ORF and high expression of THEG-2 in human testis. The GenBank accession number EU079024 was achieved. Subcellular localization of THEG-2 is predicted in nucleus, and it is assumed that it serve as a growth factor. Conclusions:The testis highly expressed gene THEG-2 is obtained, which lays a foundation for subsequent research of its biological function and expression regulation. Part 2 The cloning of mouse testis-specific expressed gene TSEG-2, and its expression in development stagesObjective: To study the function of THEG-2 in the development of testis based on prototype creature—mouse. Methods: The highly homologous EST sequences to THEG-2 were found through the dbEST database to construct contigs, and spliced by the biomedical software Biolign. The corresponding exons and introns within genome sequences were predicted by software GeneScan. According to the open reading frame, the primers were designed. RT-PCR was applied in the cloning of novel gene from mouse testis tissue and analyzing its expression pattern in various mouse organ tissues,different developmental stage of mouse testis,mouse cryptorchidism. The sequencing results of TSEG-2 resort to bioinformatics analysis. Results: The novel mouse testis gene TSEG-2 highly homologous to THEG-2 was successfully cloned, with full-length sequence of 451 bp. The open reading frame is 267 bp, coding a protein consisted of 88 amino acid residues, which was demonstrated by RT-PCR. TSEG-2 was distinctively expressed in mouse testis, but not in other tissues, and it expressed regularly in testes cDNA samples of different postnatal day. Meanwhile, the down regulation of TSEG-2 can be seen in mouse cryptorchidism model. No obvious homology with other mouse cDNA was found for TSEG-2. The GenBank accession number EU079025 was achieved. The function prediction shown that mouse TSEG-2 is probably a soluble non-secretary protein that locates at chromosome 15qE3. It is probably a kind of nucleoprotein with two phosphorylation sites of protein kinase C (PKC) and one phosphorylation sites of casein kinaseⅡ(CK2). Conclusion: The novel mouse testis specific gene TSEG-2 is cloned, which is excessively relevant to the stage of testis development . Part 3 Expression of testis-specific gene TSEG-2 in cryptorchidism modelObjective: To establish the murine cryptorchidism model validated by morphological observation, and to study the expression of gene TSEG-2 in cryptorchidism model .Methods: Thirty-five neonatal male BABL/C mice were randomly divided into three groups. In experimental group, fifteen mice were injected subcutaneously with 4μl (4μg/d) of 17-βestradiol from the postnatal day 3 to 30. In dissolvent control group, ten mice were injected with the same volume of propylene glycol. Ten untreated mice were chosen as normal controls. At the postnatal day 35, mice were harvested to observe the location and size of testes. HE staining and electron microscopy were applied to observe the morphological changes. The expression of gene TSEG-2 in cryptorchidism model was achieved by means of RT-PCR. Results: The incidence rates of cryptochidism in experimental group was 100%, while no cryptochidism occurred in normal and dissolvent control groups. Compared with normal controls, the size of testes were significantly reduced, with reduction of seminiferous tubules, disappearance of lumen, nuclear shrinkage of spermatocyte and spermatogenous cells, vacuolar degeneration of Sertoli cells and Leydig cells, and disappearance of sperms in experimental group. The results demonstrated that the expression of gene TSEG-2 weakened in group of cryptorchidism model, compared with the nomal and solvent control group highly expressed. Conclusions Estradiol can induce cryptorchidism in mice, it can be concluded that gene TSEG-2 can probably take a key role in spermatogenesis and the development of testis in mice based on the specific expression of it.