| In the conservative perception the spermatozoon is a highly differentiated and specialized cell, which until recently was thought only to transport the paternal genome to the oocyte. However, recently more and more evidences have been accumulating that human ejaculate spermatozoa convincingly retain a complex and yet specific population of RNAs. For example, by using a complementary DNA (cDNA) microarray, about3500-5500mRNA species were detected in human spermatozoa. Also, in our lab we have already clustered and analyzed function of the complex population of RNAs by using an alternative approach of serial analysis of gene expression (SAGE). Among389SAGE tags of high abundance genes, the most obvious group claimed one fourth (96/389) of the general genes analyzed in spermatozoa as nucleus proteins related to transcription and transcription regulation (DNA-dependent). The rest included:ribosomal subunit (84/389) involving protein biosynthesis; the genes related to spermiogenesis process; proteolysis and peptidolysis; protein kinase (24/389); antigen presentation and cell adhere and immune (22/389); protein transportation and localization (18/389) and posttranslational modification (16/389), etc. Furthermore,54SAGE tags had no matches on the SAGEmap and therefore representing potential novel gene.Thus, it raised a puzzle why these mRNAs were reserved in such a ’quiescent’ specialized cell and what possible functions of those mRNAs could perform. And now, many evidences indicate that the spermatozoa RNA penetrates the oocyte during fertility and remains therein during the early embryonic development, it therefore appears that spermatozoa RNA are not merely non-functional remnants during spermatogenesis, but that they may play an important role in fertilization and early embryo development. As an accepted dogma of maternal effect, the maternal factors (including mRNAs and proteins) derived from oocytes regulate zygotic development before activation of zygotic genome. After fertilized by a sperm, in zygote the maternal factors initiate early embryonic development. Its approach is a cascade reaction process, in which maternal factors firstly activate zygotic genome transcription, expression of new genes and translating new factors, thus more cellular factors are generated to keep early embryonic development. Resembling the manner of maternal factors, whether does spermatozoon RNA penetrating the oocyte during fertility possible regulate early embryonic development? And whether is paternal effect possible necessary complementary with maternal effect during the early embryonic development? To clarify the function of spermatozoa RNA and validate the hypothesis about paternal effect, we want to identify and character the possible new genes from the54unmatched SAGE tags based on our SAGE library of spermatozoa mRNA, and then analyze their functions during the spermatogenesis and the early embryonic development. Through elucidating the function of these potential new genes, we will get more insight into the transcript complexity of human ejaculated spermatozoa.Part Ⅰ-Establishment of innovative method of3’-end amplification from SAGE tags (Semi-nested PCR analysis of unknown tags on serial analysis of gene expression).The serial analysis of gene expression (SAGE) technique is a high-throughput method, which can allows the construction of a comprehensive expression profile, in which each mRNA is defined by a specific14-mer. However, we could not get more genetic information from the tag than from its full-length gene because of short SAGE tag (14bp). Also it was difficult to indentify the full-length cDNA of gene by3’and5’ RACE technique due to its short tag. Hence, the special clone methods were developed to overcome the shortage of SAGE tag, such as RAST-PCR、GLGI、rSAGE. There methods share different characteristics and advantages. Yet there are many flaws about these methods, intensive labor; low clone efficiency; nonspecific amplification; what more, large amount requirement of initial mRNA, which limit the widespread use of them. In our study, it is hard to identify gene using the methods above because each human spermatozoon is estimated to contain just0.015pg of total RNA, only1/600of the amount of somatic total RNA. Therefore, we have developed a technique called the two-step analysis of unknown SAGE tags (TSAT-PCR) to generate the3’-longer cDNA ends. The first step is enrichment of cDNA template using PCR method with a pair of primes added to the5’and3’ends of cDNAs. The second step is to clone the3’-longer cDNA ends corresponding to SAGE tags based on semi-nested PCR with two downstream nested primers. Compared to analogous methods mentioned above, the TSAT-PCR method is significantly superior to them in the follow aspect, low amount of initial mRNA, high specific amplification, especially better effect for clone of low abundance genes.11of54unmatched tags were tested for the the TSAT-PCR method, and3’-longer cDNA ends corresponding to11tags were successfully amplified and cloned for following experiment.Part II-accomplishment of clone and functional study of a new gene from54unmatched SAGE tags using our new method above.Through the combined application of TSAT-PCR and5’-RACE, we have successfully amplied and identified a new gene QSA from54unmatched SAGE tags. BLAST result in NCBI web showed the new gene encoding28aa peptide, which we called QSA gene, consisted of three exons spliced from human chromosome19. Homology analysis of QSA gene disclosed there was no homologous nucleotide sequence blasting among other species, yet the28aa peptide encoding by QSA gene displayed high conservation during five species (pan troglodytes, dog, human, rattus norve, mus musculus). Besides homology analysis, we also had predicted the cellular localization of the28aa peptide was plasma membrane by online prediction software (website, http://www.cbs.dtu.dk/services/SignalP/). To investigate tissue distribution of QSA gene mRNA,24human tissues were tested and compared each other through RT-PCR and in situ hybridization. Among24tissues, its mRNA exists in great quantity at several glands, such as, parathyroid gland, pituitary gland, and prostate. In addition, the mRNA was detected at testicle, spermatozoon and zygote, while no QSA gene mRNA was detected at oocyte and blastula. Then we constructed prokaryotic expression plasmid (pET-28a-QSA) of QSA gene and it was expressed in E. coli BL21cells, the recombinant QSA fusion protein was purified and immunized in rabbit. After immunizing, polyclonal antibodies against recombinant protein QSA were generated in rabbit serum. According to result of in situ hybridization, several representative human tissues (parathyroid gland, pituitary gland, prostate, testicle, spermatozoon and ovary) were selected to screen tissue distribution of the peptide QSA using western-blot, immunohistochemistry, and indirect immunofluorescent technique. The case of peptide QSA distribution was in accordance with the result of in situ hybridization. And there was a significant case that peptide QSA was detected in human testicular spermatogenic cells at different stages, yet it was undetectable in other testicular cell, e.g., leydig cell and sertoli cell. To initially explore molecular function of peptide QSA, we tried to screen interacting protein of peptide QSA among16,000human proteins using protein chip technique. The result revealed Homo sapiens processing of precursor4(POP4), ribonuclease P/MRP variant subunit, interacted strongly with peptide QSA.In sum, the conclusion can be drawn from above two parts of studies:We developed a innovative method of3’-end amplification from SAGE tags, which possesses the advantages of being simple, rapid, low in cost, and highly efficient, a low amount of mRNA requirement, and what more, amplifying target PCR products from low-abundance transcripts. Using our new method, a new gene QSA was cloned and sequenced successfully. Molecular evolution analysis of QSA gene followed the neutral theory of molecular evolution, which if a population carries several different versions of a gene; odds are that each of those versions is equally good at performing its job-in other words, that variation is neutral. High expression of gene QSA was detected at several kinds of glands cell, such as, thyroid gland, pituitary gland, prostate, testicle, and spermatozoon. Result of protein chip showed peptide QSA was possibly associated with POP4, a variant subunit of ribonuclease P/MRP. Ribonuclease P participates in transcription of non-coding RNA genes including tRNA,5S rRNA, SRP-RNA and U6snRNA.Summarily, some inferences were drawn According to the results above.The case of peptide QSA distribution at several glands and testicle cell revealed that QSA gene possibly was related to synthesis and/or secretion of a hormone and played an important role during the early embryonic development. It is a speculated pathway that peptide QSA located at nuclear membranes mediates assembly of ribonuclease P/MRP through POP4; then, it would regulate synthesis and/or secretion of a hormone; last, the hormone acts on the early embryonic development. In conclusion, spermatozoa mRNA, a kind of paternal factors, may play a significant role in the early embryonic development, it is a obvious case of paternal effect. |
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