# Spermatocyte injection into meiotic oocytes rescues diplotene, but not pachytene, arrest in azoospermic mutant mice

## TL;DR

Injecting spermatocytes into immature oocytes can rescue meiosis in some azoospermic mice, but only if the arrest occurs at the diplotene stage.

## Contribution

The study demonstrates that spermatocyte injection into meiotic oocytes can rescue diplotene-arrested spermatocytes but not pachytene-arrested ones in azoospermic mice.

## Key findings

- Spermatocytes arrested at the diplotene stage can resume meiosis and support full-term development when injected into immature oocytes.
- Pachytene-arrested spermatocytes failed to rescue embryo development, likely due to chromosomal abnormalities.
- Class 1 and some Class 2 spermatocyte arrest mutants produced viable offspring, while Class 3 mutants did not.

## Abstract

At which arrest stage can spermatocytes be rescued by injection into meiotic oocytes?

In mice, spermatocytes arrested at the diplotene stage, but not at the pachytene stage, can resume meiosis within immature oocytes and support full-term embryonic development.

In mice, at least some of the spermatocyte arrest mutations can be overcome by injecting spermatocytes into immature oocytes.

The study was carried out from October 2019 to April 2025. Adult azoospermic mice (at 4–26 weeks of age) from nine strains carrying spermatocyte arrest mutations were used as spermatocyte donors. Adult B6D2F1 females at 9–12 weeks of age were used as oocyte donors for spermatocyte injection. Adult ICR strain pseudopregnant females at 9–12 weeks of age were used as recipients for embryo transfer experiments.

The most advanced stage of spermatocytes from each mutant strain was assessed by chromosome spread analysis. These most advanced spermatocytes of each strain were injected into metaphase I (MI) oocytes. About half a volume of the ooplasm had been removed from the recipient oocytes to ensure more stable chromosome behaviours during meiosis. The spermatocyte-injected oocytes were allowed to mature in vitro to the metaphase II (MII) stage, and their ooplasm was refreshed with the ooplasm from intact MII oocytes. After activation with SrCl2, the reconstructed oocytes that reached the 2-cell stage were transferred into the oviducts of pseudopregnant females. On Day 19.5, recipient females were euthanized and their uteri were examined for live foetuses.

Based on spermatocyte spread analysis, sperm mutants were categorized into three classes: Class 1, arrest at mid-diplotene or later stage; Class 2, arrest at early diplotene stage; and Class 3, arrest at pachytene stage. All four Class 1 mutants could resume normal meiosis following injection into MI oocytes, as evidenced by births of normal offspring. Similarly, one of two Class 2 mutants could be rescued, but the other could not. By contrast, three Class 3 mutants did not support embryo development to term because of complete implantation failure, indicating that reconstructed embryos carried severe chromosomal aberrations.

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The number of mutant strains examined was limited. Nevertheless, the findings were consistent: the more advanced the arrest stage of spermatocytes, the higher the likelihood of a successful rescue.

In humans, a considerable proportion of spermatogenic arrest occurs at the primary spermatocyte stage. Spermatocyte injection might be an option to treat human male-factor infertility due to azoospermia in the future. However, numerous ethical and technical challenges remain to be addressed, and the reproductive physiological differences between mice and humans must be carefully taken into account.

This study was supported by Grants-in-Aid for Scientific Research (KAKENHI) from the Japan Society for the Promotion of Science to A.O. (grant number: JP19H05758), K.I. (grant number: 23H04956), M.I. (grant number: JP23K20043), and N.O. (grant number: 25H01372), and 2023 and 2025 grants of the University of Castilla-La Mancha for stays in foreign universities and research centres to E.C.-E. The authors declare that they have no conflicts of interest.