Retrotransposon expression and incorporation of cloned human and mouse retroelements in human spermatozoa
| Autor: | Chrysoula Kitsou, Georgios Vartholomatos, Charilaos Kostoulas, Leandros Lazaros, Elissavet Hatzi, Vasiliki Galani, Theodore Tzavaras, Paris Ladias, Sofia Bellou, Ioannis Georgiou, Sofia Markoula, Theodoros Stefos |
|---|---|
| Rok vydání: | 2017 |
| Předmět: |
Male
0301 basic medicine endocrine system Transcription Genetic Green Fluorescent Proteins Retrotransposon Cell Separation Minisatellite Repeats Biology Transfection Genome law.invention Green fluorescent protein Mice 03 medical and health sciences 0302 clinical medicine Plasmid Genes Reporter law Endopeptidases Animals Humans Cloning Molecular Polymerase chain reaction Microscopy Confocal Reverse Transcriptase Polymerase Chain Reaction Viral Proteases urogenital system Obstetrics and Gynecology Oligospermia Cell sorting Flow Cytometry Spermatozoa Sperm Molecular biology Long interspersed nuclear element Long Interspersed Nucleotide Elements 030104 developmental biology Gene Expression Regulation Reproductive Medicine 030220 oncology & carcinogenesis |
| Zdroj: | Fertility and Sterility. 107:821-830 |
| ISSN: | 0015-0282 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|