Assessment of sperm nuclear quality after in vitro maturation of fresh or frozen/thawed mouse pre-pubertal testes.

Abstract

Is nuclear quality of in vitro generated spermatozoa from fresh or frozen/thawed pre-pubertal mouse testes similar to that of their in vivo counterparts? The production of spermatozoa with aneuploidy, DNA fragmentation or chromatin condensation defects was not significantly increased in organotypic cultures compared to in vivo controls. Although murine spermatozoa have been produced in vitro from pre-pubertal testes, their nuclear DNA integrity has never been investigated. Fresh and frozen/thawed testicular fragments from 6 to 7 days postpartum (dpp) mice were cultured for 30 days. Testicular tissues were frozen by controlled slow freezing (CSF) or solid surface vitrification (SSV). In total, 30 fresh, 30 CSF, 30 SSV testes were used for in vitro maturation and 6 testes from 36 to 37 dpp mice were used as in vivo controls. Murine spermatozoa were extracted from pooled in vitro cultured testicular fragments and from in vivo controls. Sperm aneuploidy was analyzed by fluorescence in situ hybridization (FISH), DNA fragmentation by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling, chromatin condensation by aniline blue staining, telomere length and number by quantitative FISH, DNA oxidation by immunocytochemical detection of 8-hydroxy-2'-deoxyguanosine (8-OHdG). Because of the low spermatogenic yield in cultures, a hundred spermatozoa extracted from pooled tissues were examined and compared to their in vivo counterparts. Most of spermatozoa generated in vitro and in vivo were haploid, contained unfragmented DNA and normally condensed chromatin. A similar proportion of spermatozoa with aneuploidy, DNA fragmentation or chromatin condensation defects was found in cultures and in vivo. No significant difference in telomere length was found within the nuclei of in vitro and in vivo generated spermatozoa. However, the number of telomere spots was lower in gametes obtained from cultures of fresh, CSF and SSV testes than in their natural counterparts (P < 0.01). Moreover, the proportion of spermatozoa containing 8-OHdG was significantly increased in frozen/thawed tissues in comparison to fresh tissues and in vivo controls (P < 0.05). None. Further studies will be needed to enhance the production of spermatozoa in organotypic cultures while preserving their quality, to investigate epigenetic modifications and embryonic development. This is the first study comparing the nuclear quality of in vitro and in vivo generated murine spermatozoa. The organotypic culture system will have to be adapted for human tissue and extensive analyses of human gamete quality will have to be performed before potential clinical applications can be envisaged. This work was supported by Rouen University Hospital, Ligue contre le Cancer, Agence de la Biomédecine, Association Laurette Fugain, France Lymphome Espoir, and co-supported by European Union and Région Normandie. Europe gets involved in Normandie with European Régional Development Fund (ERDF). The authors declare that they have no conflict of interest.