1. Retrotransposon expression and incorporation of cloned human and mouse retroelements in human spermatozoa.
- Author
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Lazaros, Leandros, Kitsou, Chrysoula, Kostoulas, Charilaos, Bellou, Sofia, Hatzi, Elissavet, Ladias, Paris, Stefos, Theodoros, Markoula, Sofia, Galani, Vasiliki, Vartholomatos, Georgios, Tzavaras, Theodore, and Georgiou, Ioannis
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RETROTRANSPOSONS , *SPERMATOZOA analysis , *HUMAN cloning , *REVERSE transcriptase polymerase chain reaction , *HUMAN genome , *ANIMAL experimentation , *CELL separation , *DNA , *FLOW cytometry , *GENES , *GENETIC techniques , *GENOMES , *INFERTILITY , *MICE , *MICROSCOPY , *POLYMERASE chain reaction , *PROTEINS , *PROTEOLYTIC enzymes , *RNA , *SPERMATOZOA - Abstract
Objective: To investigate the expression of long interspersed element (LINE) 1, human endogenous retrovirus (HERV) K10, and short interspersed element-VNTR-Alu element (SVA) retrotransposons in ejaculated human spermatozoa by means of reverse-transcription (RT) polymerase chain reaction (PCR) analysis as well as the potential incorporation of cloned human and mouse active retroelements in human sperm cell genome.Design: Laboratory study.Setting: University research laboratories and academic hospital.Patient(s): Normozoospermic and oligozoospermic white men.Intervention(s): RT-PCR analysis was performed to confirm the retrotransposon expression in human spermatozoa. Exogenous retroelements were tagged with a plasmid containing a green fluorescence (EGFP) retrotransposition cassette, and the de novo retrotransposition events were tested with the use of PCR, fluorescence-activated cell sorting analysis, and confocal microscopy.Main Outcome Measure(s): Retroelement expression in human spermatozoa, incorporation of cloned human and mouse active retroelements in human sperm genome, and de novo retrotransposition events in human spermatozoa.Result(s): RT-PCR products of expressed human LINE-1, HERV-K10, and SVA retrotransposons were observed in ejaculated human sperm samples. The incubation of human spermatozoa with either retrotransposition-active human LINE-1 and HERV-K10 or mouse reverse transcriptase-deficient VL30 retrotransposons tagged with an EGFP-based retrotransposition cassette led to EGFP-positive spermatozo; 16.67% of the samples were positive for retrotransposition. The respective retrotransposition frequencies for the LINE-1, HERV-K10, and VL30 retrotransposons in the positive samples were 0.34 ± 0.13%, 0.37 ± 0.17%, and 0.30 ± 0.14% per sample of 10,000 spermatozoa.Conclusion(s): Our results show that: 1) LINE-1, HERV-K10, and SVA retrotransposons are transcriptionally expressed in human spermatozoa; 2) cloned active retroelements of human and mammalian origin can be incorporated in human sperm genome; 3) active reverse transcriptases exist in human spermatozoa; and 4) de novo retrotransposition events occur in human spermatozoa. [ABSTRACT FROM AUTHOR]- Published
- 2017
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