Acquisition Of The Igy Antibody Against H-Y Antigen And Study Of The Y Chromosome Gene MRNA In Ejaculated Spermatozoa

In the conventional view, the only biological function of mature spermatozoa with minimal size (microgamete) is delivering the male haploid genome to the oocyte. Therefore, the ejaculated spermatozoa, which contain an extremely condensed nucleus of haploid genome, are considered as dormant cells. The nuclear DNA of these cells is protamine-bound, while numerous redundant organelles as well as almost all of the cytosol are shed in the latter stage of spermatogenesis. This indicates that there is almost no active transcription and translation in these cells. However, recent evidences suggest that the ejaculated spermatozoa contain a complex specific mRNA population. In fact, about 3500-5500 mRNA species were detected in human spermatozoa by using complementary DNA (cDNA) microarray. Similar result was derived based on the data obtained through serial analysis of gene expression (SAGE) from our laboratory. In addition, a functional analysis of 389 clustered genes in the ejaculate identified by SAGE reveals a highly selective categorization in the DNA-dependent transcription and in the regulation of the transcription. And a chromosomal distribution analysis of the overlapped 510 sperm-related genes reveals their uneven distribution among the two dozen chromosomes. The highest numbers of genes were located in chromosome 1, which harbor high frequency breakpoints in the infertile male. In contrast, only a single gene, DBY, is located in chromosome Y.Thus, why these mRNAs are reserved in such a highly differentiated terminal cells and what possible functions of those mRNAs could perform? Originally, they were simply believed as non- or immature sperm RNA contamination or delimited as a―by-product‖of the gradual shutdown in the nuclear transcriptional apparatus that mark the replacement of histones by protamines in spermatozoa chromatin. However, until recently the ejaculated spermatozoa have been proven capable to translate their mRNAs de novo by the mitochondrial ribosomes. Moreover, the spermatozoa RNA have been also indicated to penetrate the oocyte during fertility and remain therein up to the first cleavages of the early embryonic development. It therefore appears that spermatozoa RNA are not merely non-functional remnants during spermatogenesis, but may be translated to replace degraded proteins or for the specific needs of the capacitating spermatozoa. On the other hand, they also maybe play an important role in fertilization and early embryo development. Thus, to investigate whether the spermatozoa mRNAs are selectively retained in ejaculated spermatozoa and what the possible functions of these mRNA are, we selected Y chromosome genes which are least abundant in ejaculated spermatozoa as research objects. This study can be devided into two parts.Part 1-Acquisition of specific IgY antibodies against H-Y antigenIn order to give further investigation on SMCY gene, we have to obtain its specific antibody. It has been confirmed that H-Y antigen is encoded by SMCY gene. However, due to the low antigenicity and the phylogenetic conservation of H-Y antigen, in general, the conventional immunizing method can not produce good antibodies against H-Y antigen. Thus, to obtain a more specific antibody against H-Y antigen, we cloned three fragments of SMCY gene which contain four epitopes of H-Y antigen and ligated as a new recombinant. It will greatly improve the imunogenicity of H-Y antigen. Then this SMCY recombinant was sub-cloned into a bacterial expression vector of pET-28(a) resulting in a 6*His-SMCY fusion gene construction. This recombinant clone was over expressed in Escherichia coli BL-21 and the recombinant protein was purified by Ni-NTA column and dialyzed by urea gradient dialysis. The purified recombinant protein was then used in immunizing chicken to produce the specific IgY antibody.Then it was expressed in E. coli BL21 cells and the recombinant H-Y fusion protein was purified and immunized in chicken. Finally the IgY antibody against the H-Y recombinant was purified by affinity chromatography and then evaluated on the appetency to H-Y antigen by gel diffusion test and immunochemistry. The results showed that the H-Y antigen could be recognized by the IgY antibody against H-Y recombinant. Thus, the IgY antibody could be used in the following research.Part 2 - Study of the selective retention mechanism of Y chromosome gene mRNA in matured spermatozoa.To explore the possible mechanism of the selective retention of mRNA in matured spermatozoa, a panel of genes on the non-recombining region of Y chromosome (NRY) were selected and screened in testis and ejaculated spermatozoa of human and mouse by RT-PCR. The result shows that these NRY genes'mRNAs were all detected in testis, but that only DBY,SRY and RPS4Y can be detected in human ejaculated spermatozoa and only DBY can be detected in mouse ejaculated spermatozoa. Then these retained genes in human and mouse spermatozoa were analyzed and compared with another gene of SMCY that manifest contrasting functions with varying methods. Using in situ hybridization, transcripts of DBY, RPS4Y and SMCY were all detected in human testis sections. Moreover, DBY and RPS4Y mRNA was localized along the post-arcosome region which comprised nearly 50% of the spermatozoa. However, SMCY mRNA was undetectable in all spermatozoa. Similarly, tanscripts of DBY and SMCY were also detected in mouse testis sections, and only DBY could be detected in the post-arcosome region of nearly 50% of the spermatozoa. Then the expression pattern of the retained genes in ejaculated spermatozoa was test by western blot and immunohistochemistry. Interestingly, although DBY and RPS4Y transcripts were found in human ejaculated spermatozoa, no DBY and RPS4Y protein was detected in sperm by western blot analysis. On the contrary, immunohistochemistry analysis revealed that the H-Y antigen encoded by SMCY was detected in about half of the spermatozoa despite the fact that SMCY mRNA is not detectable in spermatozoa. The same procedures were also performed in mouse testis and spermatozoa and we also got the similar result. Functional analysis indicated that all these Y chromosome genes might be involved in the early zygotic and embryonic development. DBY which encodes a putative ATP-dependent RNA helicase may play an important role in DNA replication and translation initiation during embryogenesis; SRY is designated as the testis-determining factor (TDF) in mammals, it might play a role in controlling the gonadal ridge of XY embryos to trigger the male differentiation program during early zygotic and embryonic development; RPS4Y encodes ribosomal protein S4, a component of the 40S subunit. It may be responsible for accurate translation during early zygotic and embryonic development. Then in the subsequent experiment, DBY and SMCY antisense RNA were microinjected into male and female pronucleus of mouse zygotes, respectively, to evaluate their possible functions in embryonic development. The results demonstrate that antisense RNA against DBY can greatly affect the first cleavage of zygote and the sex ratio of the resulting developed embryos, while SMCY antisense RNA had no effect on zygote. This might imply that the spermatozoa mRNA is involved in the early zygotic and embryonic stage of reproduction.In summary, a few conclusions can be drawn from above two parts of studies:1. By cloning, expressing and purifying of the SMCY recombinant which contains the four H-Y epitopes, we obtained the purified specific IgY antibody against H-Y antigen.2. It is demonstrated that the transcripts of Y chromosome genes are selectively retained in ejaculated spermatozoa during spermatogenesis. In human, DBY, SRY and RPS4Y mRNA were selective retained in ejaculated sperm. In mouse, only DBY mRNA was found in ejaculated sperm. The transcripts of all these genes may be involved in the early zygotic and embryonic development.3. SMCY transcripts may be shed from spermatids with almost all of the cytosol during latter stage of spermatogenesis after they finished encoding H-Y antigen.4. What we have inherited from father is not only male haploid genome, but also the spermatozoa mRNA. And the spermatozoa mRNA also exerts important direct functions in early zygotic and embryonic development. Therefore, it is reasonable that the preparation of zygote formation and embryo development have begun at the initial stages of spermatogenesis.