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Reproductive Biology and Endocrinology
Published in: Reproductive Biology and Endocrinology 1/2017

Open Access 01-12-2017 | Research

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

Authors: J. Isoler-Alcaraz, D. Fernández-Pérez, E. Larriba, J. del Mazo

Published in: Reproductive Biology and Endocrinology | Issue 1/2017

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Abstract

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.
Appendix
Available only for authorised users
Footnotes
1
The piRNA database - piRBase. http://regulatoryrna.org/database/piRNA/. Accessed June 2016.
 
2
Non-coding RNAs. http://www.ensembl.org/info/genome/genebuild/ncrna.html. Accessed June 2016.
 
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Metadata
Title
Cellular and molecular characterization of gametogenic progression in ex vivo cultured prepuberal mouse testes
Authors
J. Isoler-Alcaraz
D. Fernández-Pérez
E. Larriba
J. del Mazo
Publication date
01-12-2017
Publisher
BioMed Central
Published in
Reproductive Biology and Endocrinology / Issue 1/2017
Electronic ISSN: 1477-7827
DOI
https://doi.org/10.1186/s12958-017-0305-y

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