title: In situ viability detection assays induce heat-shock protein 70 expression in spermatozoa without affecting the chromatin integrity.
abstract: To differentiate dead spermatozoa from viable but immotile spermatozoa, several techniques are being used during ICSI. As processed spermatozoa from poor-quality ejaculate are confronted with a higher risk of experiencing stress on exposure to altered osmotic conditions or chemicals, this study was undertaken to determine the expression of stress response gene Hsp70 and chromatin integrity in spermatozoa subjected to in situ viability assays such as hypo-osmotic swelling (HOS) test, modified hypo-osmotic swelling (M-HOS) test and pentoxifylline in 25 fresh and frozen-thawed asthenozoospermic ejaculates. RT-PCR and immunofluorescence detection of Hsp70 were performed to elucidate the expression and localisation of Hsp70 in spermatozoa, whereas DNA fragmentation analysis was performed by sperm chromatin dispersion assay. Exposure of fresh and frozen-thawed asthenozoospermic spermatozoa to M-HOS and pentoxifylline significantly increased Hsp70 expression as evidenced by increased RNA expression and immunolocalisation of Hsp70 protein in sperm head (P < 0.05-0.001). However, chromatin integrity was not significantly affected in any groups until 6 h of post-exposure time period. Our results suggest that conventional HOS may be preferred for the in situ detection of the viability as there was no immediate stress response and chromatin instability in the exposed spermatozoa.
authors.affiliation: Division of Clinical Embryology, Department of Obstetrics and Gynecology, Kasturba Medical College, Manipal University, Manipal, India.
authors.forename: Y
authors.initials: Y
authors.lastname: Asokan
authors.affiliation: Division of Clinical Embryology, Department of Obstetrics and Gynecology, Kasturba Medical College, Manipal University, Manipal, India.
authors.forename: S D
authors.initials: SD
authors.lastname: Honguntikar
authors.affiliation: Division of Clinical Embryology, Department of Obstetrics and Gynecology, Kasturba Medical College, Manipal University, Manipal, India.
authors.forename: S
authors.initials: S
authors.lastname: Uppangala
authors.affiliation: Division of Clinical Embryology, Department of Obstetrics and Gynecology, Kasturba Medical College, Manipal University, Manipal, India.
authors.forename: S R
authors.initials: SR
authors.lastname: Salian
authors.affiliation: Division of Clinical Embryology, Department of Obstetrics and Gynecology, Kasturba Medical College, Manipal University, Manipal, India.
authors.forename: D
authors.initials: D
authors.lastname: Kumar
authors.affiliation: Division of Clinical Embryology, Department of Obstetrics and Gynecology, Kasturba Medical College, Manipal University, Manipal, India.
authors.forename: G
authors.initials: G
authors.lastname: Kalthur
authors.affiliation: Division of Clinical Embryology, Department of Obstetrics and Gynecology, Kasturba Medical College, Manipal University, Manipal, India.
authors.forename: S K
authors.initials: SK
authors.lastname: Adiga
mesh_terms: D053627:Asthenozoospermia / Q000175:diagnosis / Q000503:physiopathology; D002843:Chromatin / Q000502:physiology*; D053938:DNA Fragmentation; D005455:Fluorescent Antibody Technique; D018840:HSP70 Heat-Shock Proteins / Q000032:analysis*; D006801:Humans; D007248:Infertility, Male / Q000175:diagnosis; D008297:Male; D010431:Pentoxifylline / Q000009:adverse effects / Q000494:pharmacology; D011446:Prospective Studies; D020133:Reverse Transcriptase Polymerase Chain Reaction; D055101:Semen Analysis / Q000009:adverse effects* / Q000379:methods; D013077:Sperm Head / Q000737:chemistry; D013081:Sperm Motility / Q000502:physiology; D013094:Spermatozoa / Q000737:chemistry* / Q000502:physiology
chemical_list: D002843:Chromatin; D018840:HSP70 Heat-Shock Proteins; D010431:Pentoxifylline
keywords: Hsp70; hypo-osmotic swelling test; pentoxifylline; sperm chromatin integrity; sperm viability