Cellular and molecular characterization of gametogenic progression in ex vivo cultured prepuberal mouse testes

Reproductive Biology and Endocrinology, Oct 2017

Recently, an effective testis culture method using a gas-liquid interphase, capable of differentiate male germ cells from neonatal spermatogonia to spermatozoa has been developed. Nevertheless, this methodology needs deep analyses that allow future experimental approaches in basic, pathologic and/or reprotoxicologic studies. Because of this, we characterized at cellular and molecular levels the entire in vitro spermatogenic progression, in order to understand and evaluate the characteristics that define the spermatogenic process in ex vivo cultured testes compared to the in vivo development. Testicular explants of CD1 mice aged 6 and 10 days post-partum were respectively cultured during 55 and 89 days. Cytological and molecular approaches were performed, analyzing germ cell proportion at different time culture points, meiotic markers immunodetecting synaptonemal complex protein SYCP3 by immunocytochemistry and the relative expression of different marker genes along the differentiation process by Reverse Transcription - quantitative Polymerase Chain Reaction. In addition, microRNA and piwi-interactingRNA profiles were also evaluated by Next Generation Sequencing and bioinformatic approaches. The method promoted and maintained the spermatogenic process during 89 days. At a cytological level we detected spermatogenic development delays of cultured explants compared to the natural in vivo process. The expression of different spermatogenic stages gene markers correlated with the proportion of different cell types detected in the cytological preparations. In vitro progression analysis of the different spermatogenic cell types, from both 6.5 dpp and 10.5 dpp testes explants, has revealed a relative delay in relation to in vivo process. The expression of the genes studied as biomarkers correlates with the cytologically and functional detected progression and differential expression identified in vivo. After a first analysis of deep sequencing data it has been observed that as long as cultures progress, the proportion of microRNAs declined respect to piwi-interactingRNAs levels that increased, showing a similar propensity than which happens in in vivo spermatogenesis. Our study allows to improve and potentially to control the ex vivo spermatogenesis development, opening new perspectives in the reproductive biology fields including male fertility.

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Cellular and molecular characterization of gametogenic progression in ex vivo cultured prepuberal mouse testes

Isoler-Alcaraz et al. Reproductive Biology and Endocrinology Cellular and molecular characterization of gametogenic progression in ex vivo cultured prepuberal mouse testes J. Isoler-Alcaraz D. Fernández-Pérez E. Larriba 0 J. del Mazo 0 0 Equal contributors Department of Cellular and Molecular Biology, Centro de Investigaciones Biológicas (CIB-CSIC) , 28040 Madrid , Spain Background: Recently, an effective testis culture method using a gas-liquid interphase, capable of differentiate male germ cells from neonatal spermatogonia to spermatozoa has been developed. Nevertheless, this methodology needs deep analyses that allow future experimental approaches in basic, pathologic and/or reprotoxicologic studies. Because of this, we characterized at cellular and molecular levels the entire in vitro spermatogenic progression, in order to understand and evaluate the characteristics that define the spermatogenic process in ex vivo cultured testes compared to the in vivo development. Methods: Testicular explants of CD1 mice aged 6 and 10 days post-partum were respectively cultured during 55 and 89 days. Cytological and molecular approaches were performed, analyzing germ cell proportion at different time culture points, meiotic markers immunodetecting synaptonemal complex protein SYCP3 by immunocytochemistry and the relative expression of different marker genes along the differentiation process by Reverse Transcription - quantitative Polymerase Chain Reaction. In addition, microRNA and piwi-interactingRNA profiles were also evaluated by Next Generation Sequencing and bioinformatic approaches. Results: The method promoted and maintained the spermatogenic process during 89 days. At a cytological level we detected spermatogenic development delays of cultured explants compared to the natural in vivo process. The expression of different spermatogenic stages gene markers correlated with the proportion of different cell types detected in the cytological preparations. Conclusions: In vitro progression analysis of the different spermatogenic cell types, from both 6.5 dpp and 10.5 dpp testes explants, has revealed a relative delay in relation to in vivo process. The expression of the genes studied as biomarkers correlates with the cytologically and functional detected progression and differential expression identified in vivo. After a first analysis of deep sequencing data it has been observed that as long as cultures progress, the proportion of microRNAs declined respect to piwi-interactingRNAs levels that increased, showing a similar propensity than which happens in in vivo spermatogenesis. Our study allows to improve and potentially to control the ex vivo spermatogenesis development, opening new perspectives in the reproductive biology fields including male fertility. Spermatogenesis; Ex vivo culture; Gametogenic progression; Testis; Meiosis; microRNA Background Spermatogenesis is a complex cell differentiation process ending in male gamete generation and which entails three main phases: spermatogonial proliferation and renewal, meiosis and spermiogenesis [ 1 ]. During the first step, spermatogonial stem cells (SSCs) proliferate generating a stem cell pool or differentiating into spermatogonial cells. In mice, this occurs around the third postnatal day [ 2–4 ]. Spermatogonial cells go through different stages being the most representative type A spermatogonia, intermediate spermatogonia and type B spermatogonia. These last differentiate into primary spermatocytes which initiates the meiotic process [ 5 ] ending in generation of haploid cells (round spermatids) [ 3, 6 ]. Finally, in spermiogenesis, the round and elongated spermatids suffer deep morphological and physiological changes that include the replacement of histones by nuclear protamines, the elimination of nearly all cytoplasm, a nuclear condensation and the acrosome and flagellum formation that lead into spermatozoa genesis [ 3, 7, 8 ]. The spermatogenic process requires a regulated finetuning, which includes genetic, metabolic and hormonal regulations and even specific physiological conditions such as an optimal testes temperature control, lower to the body temperature in placental mammals [ 9, 10 ]. This germ line regulation is also mediated by the participation of testicular somatic cells as the Leydig cells, located in the intertubular regions, the myoid cells organizing the tubular wall and the Sertoli cells inside of the seminiferous tubules orchestrating the cell-cell interactions with the germ cells [ 2, 11, 12 ]. Defined gene expression patterns regulate the spermatogenic process both at a coding [ 13–16 ] and non-coding gene level. Currently, it is known that small non-coding RNA (sncRNA) participation is crucial as a posttranscriptional regulator. Amongst the sncRNA types, microRNAs (miRNAs) and Piwi-interacting RNAs (piRNAs) are being studied as regulatory elements essential in gamete differentiation and in pathologies leading to inf (...truncated)


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J. Isoler-Alcaraz, D. Fernández-Pérez, E. Larriba, J. del Mazo. Cellular and molecular characterization of gametogenic progression in ex vivo cultured prepuberal mouse testes, Reproductive Biology and Endocrinology, 2017, pp. 85,