Molecular Cloning And Analysis Of A Novel Mouse Gene Related To Spermatogensis

Mammalian Spermatogenesis is a unique cell differentiation process and can bedivided into three stages: mitosis, meiosis, and spermiogenesis. Primitive type Aspermatogonia differentiate to form primary spermatocytes. The primary spermatoeytesenter first meiosis after DNA replication for last time and give rise to two secondaryspermatocytes, which then undergo second meiosis without DNA replication and formhaploid round spermatids. Subsequently, these cells enter spermiogenesis and undergolfistone-protamine replacement reaction, nuclear condensation, and major morphologicalchanges to form completed spermatozoa. The spermatogenesis is strictly controled bymany different molecules including hormones and growth factors. The molecularmechanisms of spermatogenesis remain unclear and the underlying key genes controllingspermatogenesis have remained elusive. This study has focused on the identification ofnew genes related to spermatogenesis,and the findings may provide insight into molecularmechanisms of spermatogenesis. The thesis contains three parts:1. Molecular cloning of a novel mouse spermatogenesis-related gene (SRG-L). Wehappened to amplify a fragment that up-regulated during spermatogenesis when we studiedthe gene expression profiles in the spermatogenic cells with cDNA microarrays andRT-PCR. The BLAST program showed that it was high homologous to a rat and a monkeytestis gene cDNA in GenBank. But no report about their functions could be searched. Sowe were interested in this gene and designed several primer pairs according to thesequences of the rat and monkey homologous gene cDNAs and successfully amplified thefragments of 448 bp, 1122 bp, 280 bp, 523 bp and 561 bp by RT-PCR. By removing theoverlapping sequences, a 2584 bp cDNA was obtained. Then 5'-rapid amplification ofcDNA ends (5'-RACE) was used to complete the 5' end of the cDNA, and 3'-RACE for 3'end of cDNA. Finally, the full-length cDNA of this gene was acquired. It was a new mousegene in GenBank and named as SRG-L(spermatogenesis related gene expressed in the latestages of spermatogenic cells). The sequence of this cDNA has been deposited in GenBankwith the Accession No. AY352586. 2. Analyses of expression characteristics of SRG-L during mouse spermatogenesis.The results of RT-PCR and Northern Blotting indicated that no expression signal of thisgene could be observed in primitive type A spermatogonia and type B spermatogonia. Asignal first appeared in diplotene/pachytene spermatocytes, and was significantlyintensified in round spermatids and elongating spermatids, findly went down to beundetectable in mature spermatozoa. The tissue-specific expression analysis showed thatSRG-L expressed highly in testis. By Western Blotting and Immnunohistochemicalstatining, we further analysed expression of SRG-L in protein levels. SRG-L protein wasmainly observed in diplotene/pachytene spermatocytes, round and elongating spermatids.We can conclude this gene was activated during the early stage of meiosis, andtranscription of it kept high level during spermiogenesis. The results suggest that SRG-Lmay play specific roles in regulating differentiation of germ cells during spermatogenesis,particularly during meiosis and spermiogenesis.3. Structure analyses and functional predictions by Bioinformatics. BLAST inGenBank/Blast (http://www.ncbi.nlm.nih.gov/BLAST/) indicated that SRG-L was a novelmouse gene The sequence of this cDNA has been deposited in GenBank with theAccession No. AY352586. The full-length cDNA of SRG-L contains a 2625 nt openreading frame (ORF) with the start codon ATG at position 50 and stop codon TGA atposition 2674, corresponding to a deduced protein of 874 amino acids with a calculatedmolecular weight of approximately 104 kDa. There is a putative Kozak sequence, and apoly (A) tail from 2831 nt in the 3'-UTR. A canonical polyadenylation signal (AATAAA) ispresented at 29 nt upstream from the poly (A) tract. No classical promoters such as theCCAAT box (TTGCGCAAT) or TATA box were found. Blast programs(http://www.ncbi.nlm.nih.gov/BLAST/) were used for sequence homology searches atnucleic acid and deduced amino acid levels in public databases. SRG-L shares high levelsof homology with Rat, monkey, human and dog, displays a high conservation in evolution.BLAST program analyses showed that the SRG-L gene was mapped to chromosome8q33.1, spanning at least 29kb. It was split into eighteen exons and seventeen introns. Exon1 contains the 5' untranslated sequence (5' UTR), and exon 2 contains the initiating methionine (Met). The splicing sites of intron-exon boundaries are conformed to thestandard ag/gt rule. The analyses of Bioinformatics showed existence of 1 transmembranehelices at 184-207 aa of N-terminus and is a non-secretory protein, rich phosphorylationsites on serine, threonine and tyrosine residues and methylation sites. With PSORTprogram (http://www.psort.org/), we did not find of N-terminal signal peptide,mitochondrial targeting sequence, endoplasmic reticulum membrane retention signal,peroxisomal targeting signal, and RNA binding motif in the molecule. GenBank and NCBIConserved Domain Search analysis revealed that the SRG-L had two conserved regions,transglutaminase-like homologues domain and D-serine dehydratase domain at theC-terminus (723-766 aa). Take all results together, it is reasonable to conclude that SRG-Lcould play specific role in the late stage of spermatogenesis. It should be worthwhile tofurther study the function of SRG-L.