title: Relationship between LTR methylation and gag expression of HIV-1 in human spermatozoa and sperm-derived embryos.
abstract: OBJECTIVE
  Studying the methylation status of long terminal repeats (LTR) and its relationship to gag expression of HIV-1 in order to explore regulation mechanism of HIV-1 gene expression in vertical transmission from sperm to embryo.
  
  
  RESULTS
  Sperm samples were collected from a healthy donor and seven patients with HIV/AIDS. Zona-free hamster ova were fertilized by donor's spermatozoa transfected with pIRES2-EGFP-LTR-gag and patient's spermatozoa to obtain zygotes and 2-cell embryos, respectively. Interspecific in vitro fertilization, bisulfite sequencing PCR (BSP), RT-PCR, nested RT-PCR, nested real-time qRT-PCR and 2(-△△Ct) method, indirect immunofluoresence (IF) assay were performed. For donor's samples, the methylation rates of HIV-1 LTR were 0.56%, 1.67%, 0.56%, 0.56% in plasmid, spermatozoa, zygotes and 2-cell embryos, respectively while spermatozoa were transfected with unmethylated plasmid, and were 95.0%, 84.44%, 3.3%, 1.67% while transfected with methylated plasmid. The positive bands for HIV-1 gag cDNA were detected in spermatozoa and 2-cell embryos. The positive signals for HIV-1 p24 Gag protein were detected in 2-cell embryos but not in spermatozoa. For patient's samples, methylation rates of HIV-1 LTR were different in spermatozoa among patients. After fertilization, CpG sites in HIV-1 LTR were highly demethylated in zygotes and 2-cell embryos. The gag transcription levels increased with decreasing of methylation rates of HIV-1 LTR, which showed a strong negative correlations between gag transcription levels and methylation rates of HIV-LTR ether in the spermatozoa (r = -0.9877, P<0.0001) or in the sperm-derived 2-cell embryos (r = -0.9092, P = 0.0045).
  
  
  CONCLUSIONS
  LTR methylation regulates expression of HIV-1 gag in vertical transmission from sperm to embryo.
authors: Guangdong Provincial Key Laboratory of Infectious Diseases and Molecular Immunopathology, Research Center for Reproductive Medicine, Shantou University Medical College, Shantou, Guangdong, China.
authors: FangZheng
authors: F
authors: Li
authors: LianBing
authors: L
authors: Li
authors: Ying
authors: Y
authors: Zhong
authors: QingDong
authors: Q
authors: Xie
authors: JiHua
authors: J
authors: Huang
authors: XiangJin
authors: X
authors: Kang
authors: Dian
authors: D
authors: Wang
authors: Lan
authors: L
authors: Xu
authors: TianHua
authors: T
authors: Huang
mesh_terms: D000818:Animals; D006224:Cricetinae; D019175:DNA Methylation; D005260:Female; D015870:Gene Expression; D015967:Gene Expression Regulation, Viral; D015658:HIV Infections / Q000378:metabolism / Q000821:virology; D016325:HIV Long Terminal Repeat / Q000235:genetics*; D015497:HIV-1 / Q000235:genetics*; D006801:Humans; D008297:Male; D010957:Plasmids; D014176:Protein Biosynthesis; D013094:Spermatozoa / Q000378:metabolism*; D014158:Transcription, Genetic; D014162:Transfection; D015053:Zygote / Q000378:metabolism*; D054301:gag Gene Products, Human Immunodeficiency Virus / Q000235:genetics*
chemical_list: D054301:gag Gene Products, Human Immunodeficiency Virus
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