Mouse Round Spermatid Injection

The final stage of spermatogenesis involves the transformation of a round spermatid into a fully developed spermatozoon, characterized by distinct head, neck, and elongated tail structures¹. This transformation encompasses significant changes in cell morphology, such as the condensation of chromatin in the nucleus, replacement of histones by protamine, acrosome formation, mitochondrial sheath development, centriole migration and loss, tail structure formation, and the removal of cellular residues².

In 1992, the first human fetus was successfully born through intracytoplasmic sperm injection (ICSI) technology³. Since then, researchers have been exploring the potential of utilizing round spermatids, which share the same haploid genetic composition as mature spermatozoa, to fertilize oocytes and sustain viable pregnancies^2,4. Subsequently, in 1996, the first human fetus conceived via round spermatid injection (ROSI) technology was delivered^5,6. It is worth noting that studies involving ICSI and ROSI in mice lagged behind those in humans due to the susceptibility of the mouse oocyte membrane to damage during the injection process. This issue was successfully resolved with the introduction of the Piezo membrane-breaking device. Consequently, in 1995, the first mouse conceived through ROSI technology was born. Additionally, research on ROSI in various other animals is also underway^7,8.

Currently, research on ROSI primarily centers around the following aspects: clinical application, mechanism elucidation, and strategies to enhance developmental efficiency, along with broader applications of ROSI technology. In the context of clinical applications, despite the birth of the first ROSI human fetus through ROSI in 1996, progress has been marked by a series of successes and failures^9,10,11,12. To date, ROSI technology has not achieved widespread clinical implementation, largely due to its low efficiency and the need for further validation regarding the safety of fetuses conceived through ROSI technology. Incomplete statistics indicate that globally, fewer than 200 human ROSI-conceived fetuses have been delivered. A turning point in understanding the potential of ROSI technology occurred in 2015 when Tanaka and colleagues reported on the successful birth of 14 fetuses through ROSI technology, instilling renewed confidence in its clinical application and feasibility^13,14. ROSI technology holds substantial promise for addressing reproductive biology challenges, particularly in non-obstructive azoospermia patients. In addition to its clinical applications, ROSI serves as a valuable tool for studying the intricate mechanisms of embryonic development^15,16,17.

Numerous animal studies have been conducted to investigate the underlying factors contributing to the low efficiency of ROSI in achieving full embryonic development. These factors encompass the choice of assisted oocyte activation (AOA) methods and their timings, abnormalities in genomic stability, and, particularly, abnormalities in epigenetic modifications. It is important to recognize that round spermatids are immature germ cells, differing significantly from mature spermatozoa in various physiological aspects. Mizuki Sakamoto and colleagues indicated that H3K27me3, derived from round spermatids, is associated with chromatin that is less accessible and leads to impaired gene expression in ROSI embryos¹⁸. In a related study by Jing Wang and colleagues, reprogramming defects in ROSI embryos at the pronuclear stages were predominantly associated with the misexpression of a cohort of the genes responsible for minor zygotic genome activation¹⁹. They also found that introducing a selective euchromatic histone lysine methyltransferase 2 inhibitor, A366, could potentially enhance the overall developmental rate by approximately twofold.

The mouse stands as one of the most valuable model animals for studying embryonic development. This article elaborates on how to perform ROSI on mice. This comprehensive protocol encompasses the selection of suitable mice, detailed ovulation induction procedures, AOA techniques, injection techniques, and the preparation of surrogate mice. Furthermore, we present a comparative analysis of the effects of two injection regimens on birth efficiency: AOA followed by ROSI (A-ROSI; first regimen) and ROSI followed by AOA (ROSI-A; second regimen). We aim to encourage researchers to conduct mouse ROSI experiments with greater precision, offering more robust support for their clinical application and the fundamental research of embryonic development mechanisms.