# Reverse transcriptase inhibitors enable the generation of fertile spermatids from fetal mouse testes in vitro

## TL;DR

Scientists generated fertile sperm cells from mouse fetal testes in the lab using reverse transcriptase inhibitors and hypoxia, enabling complete spermatogenesis.

## Contribution

A novel in vitro method for complete spermatogenesis from fetal mouse testes using RTIs and hypoxia is introduced.

## Key findings

- RTIs improve spermatogenesis efficiency by suppressing retrotransposon activity and preserving genomic integrity.
- E12.5 fetal testes cultured under hypoxia with RTIs produce fertile spermatids capable of generating offspring via microinsemination.
- The method successfully recapitulates full testicular development and spermatogenesis from an early fetal stage.

## Abstract

Organ culture systems enabling in vitro spermatogenesis from neonatal mouse testes exist, but differentiation from fetal testes shortly after sex determination remains unsuccessful. Here, we report the in vitro generation of fertile haploid cells from E12.5 fetal testes. While optimizing in vitro spermatogenesis protocols for neonatal testes, we find that supplementing the culture medium with reverse transcriptase inhibitors (RTIs) significantly improves the efficiency of spermatogenesis, by suppressing retrotransposon activity and protecting genomic integrity. Applying this approach, we successfully induce spermatogenesis through to the elongating spermatid by culturing E12.5 fetal testes under hypoxic conditions in RTI-supplemented medium. Notably, microinsemination using in vitro-derived spermatids produces healthy and fertile offspring, confirming their functional competence. These findings demonstrate the faithful in vitro recapitulation of testicular development and complete spermatogenesis from an early fetal stage, providing a valuable platform for investigating early germ cell development and reproductive biology.

Reverse transcriptase inhibitors combined with hypoxia enable complete in vitro spermatogenesis from E12.5 mouse fetal testes, producing functional spermatids and fertile offspring by suppressing retrotransposon activity during culture.