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Analysis of LINE-1 Retrotransposition at the Single Nucleus Level

Analysis of LINE-1 Retrotransposition at the Single Nucleus Level

Published on: April 23, 2016

Retrotransposon expression and incorporation of cloned human and mouse retroelements in human spermatozoa

Leandros Lazaros¹, Chrysoula Kitsou², Charilaos Kostoulas²

¹Laboratory of Medical Genetics of Human Reproduction, Medical School, Ioannina University, Ioannina, Greece; Medical Genetics and Assisted Reproduction Unit, Department of Obstetrics and Gynecology, Ioannina University Hospital, Ioannina, Greece; Genesis-Genoma Lab, Chalandri-Athens, Greece.

Fertility and Sterility

|February 1, 2017

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.

Keywords:

HERV-K10 LINE-1 human spermatozoa retroelements retrotransposition

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