Protein Expression Of Human Spermatozoa-Mediated HBs And HBc Genes In Early Embryonic Cells

[BACKGROUND AND AIM] Hepatitis B is a public health problem worldwide. The governments and scientists in the world pay high attention to study on its transmission routes due to its high prevalence and harmful to human health. 1985, Hadchouel et al assumed that the presence of integrated sequences in spermatozoa suggested the possibility of true vertical transmission of HBV via germ line. Nobody confirmed their assumption in more than ten years, because neither experimental animals nor cell culture systems had been available. Study on vertical transmission of HBV genes using embryos from fertilization between human oocytes and human spermatozoa carrying HBV genes would be an ideal model, but such a system presents moral, ethical and methodological problems. The interspecific in vitro fertilization between zona-free golden hamster ova and human spermatozoa carrying HBV DNA was employed by Huang JM et al to avoid the mentioned problems and obtained the exciting results. They firstly provided the direct evidence that HBV DNA integrated into human sperm chromosomes and showed that human sperm carrying HBV genes can pass through oolemma to enter into the oocyte and complete fertilization normally. Furthermore, it was demonstrated that after fertilization the human sperm-mediated HBx gene was able to replicate itself and HBx, HBs and HBc gene were able to express their functions at mRNA level in the early embryonic cells by Ali et al using FISH and RT-PCR, respectively. The purpose of the present study is to investigate the protein expression of human sperm-mediated HBs and HBc genes in early embryonic cells, which has not been documented previously.[MATERIALS AND METHODS] 1) Materials:①recombinant plasmid pIRES2-EGFP -HBV;②spermatozoa from the healthy donor;③the matured oocytes from female golden hamsters. 2) Methods:①human spermatozoa were transfected with pIRES2-EGFP-HBV and then inseminated with zona-free hamster ova. Zygotes were cultured for reach the two-cell stage embryos.②two-cell embryos with green fluorescence which contein sperm-mediated HBV genes were collected under fluorescent microscope.③the sperm nuclei, pronuclei in zygotes and nuclei from two-cell embryos with green fluorescence were prepared for FISH with HBs and HBc DNA probes, respectively.④two-cell embryos with green fluorescence were collected to observe the expression of HBsAg and HBcAg proteins in the embryonic cells by immunofluoresence assay.⑤two-cell embryos with green fluorescence were collected to detect the amount of HBsAg and HBcAg proteins in the embryos by ELISA method.[RESULTS]①FISH: HBs and HBc DNA positive signals were detected in the sperm nuclei and each nucleus of two-cell embryos with green fluorescence.②immunofluoresence assay presented the positive signals for the expression of the HBsAg and HBcAg protein in some two-cell embryos.③ELISA analysis showed that HBsAg amount of individual two-cell embryo was about 0.064 ng/ml and the positive result for HBcAg was detected.[CONCLUSION]①HBs and HBc genes can be integrated into human sperm genome.②h uman sperm-mediated HBs and HBc genes are able to replicate themselves in the embryonic cells.③h uman sperm-mediated HBs and HBc genes are able to express their function at protein level in early embryonic cells.④Our results provided the direct evidences for viral protein expression in HBV vertical transmission via male germ line.