Mouse Round Spermatid Injection

Published: January 26, 2024

doi:

Haibo Zhu², Yuting Jiang², Haiwei Quan², Leyi Li², Jiarui Wei², Ruizhi Liu², Ruixue Wang²

¹Center of Reproductive Medicine,First Hospital of Jilin University, ²Center of Prenatal Diagnosis,First Hospital of Jilin University

Summary

Here, we introduce a method for performing round spermatid injection (ROSI) in mice, a technique with promising clinical applications and utility for investigating the mechanisms underlying embryonic development.

Abstract

Round spermatids, characterized by their haploid genetic content, represent the precursor cells to mature spermatozoa. Through the innovative technique of round spermatid injection (ROSI), oocytes can be successfully fertilized and developed into viable fetuses. In a groundbreaking milestone achieved in 1995, the first mouse fetus was born through ROSI technology. ROSI has since emerged as a pivotal tool for unraveling the intricate mechanisms governing embryonic development and holds significant potential in various applications, including the acceleration of mouse generation and the production of genetically modified mice. In 1996, a milestone was reached when the first human fetus was born through ROSI technology. However, the clinical applications of this method have shown a fluctuating pattern of success and failure. To date, ROSI technology has not found widespread application in clinical practice, primarily due to its low birth efficiency and insufficient validation of fetal safety. This article provides a comprehensive account of the precise methods of performing ROSI in mice, aiming to shed new light on basic research and its potential clinical applications.

Introduction

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 pregnancies²^,⁴. Subsequently, in 1996, the first human fetus conceived via round spermatid injection (ROSI) technology was delivered⁵^,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 underway⁷^,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 failures⁹^,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 feasibility¹³^,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 development¹⁵^,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.

Protocol

B6D2F1 (C57BL/6 x DBA/2), C57BL/6, and ICR mice used in this experiment were purchased from Beijing Vital River Laboratory Animal Technologies Co. Ltd. (Beijing, China). All animal treatments adhered to the experimental procedures and standards approved by the Experimental Animal Ethics Committee of the First Hospital of Jilin University (approval number: 20200435). 1. Preparation of relevant reagents Acquire some reagents commercially and self-prepare the remainin…

Representative Results

We initiated our investigation by examining AOA's effect on embryos' developmental capability. A schematic illustration of the experimental design is shown in Figure 1A. Before the spermatozoon injection, the oocytes underwent either AOA (A-ICSI) or remained untreated (ICSI). Detailed data on embryonic development is presented in Table 1. The results revealed no significant differences in cleavage, blastocyst, or birth rates between the A-ICSI and ICSI groups (P<…

Discussion

Assisted oocyte activation
A critical prerequisite for ROSI is AOA since round spermatids alone cannot initiate oocyte activation. Currently, the most established method in mice involves the use of strontium chloride²³^,²⁴, while the most advanced human application employs electrical activation¹³^,¹⁴. The timing of oocyte activation is also of great significance. As reported in the li…

Disclosures

The authors have nothing to disclose.

Acknowledgements

We extend our gratitude to Wenjie Zhao for her invaluable assistance in sorting round spermatids through flow cytometry and to Fang Wang for her expertise in mouse embryo transfer. This work received partial support from the Natural Science Foundation of Jilin Province (No. YDZJ202301ZYTS461). We thank Bullet Edits Limited for the linguistic editing and proofreading of the manuscript.

Materials

CaCl₂2H₂O	Sigma	C7902	Preparation of CZB
Glucose	Sigma	G6152	Preparation of CZB
HEPES-Na (basic)	Sigma	H3784	Preparation of CZB
Hoechst 33342	Beyotime	C1025	FACS
human chorionic gonadotropin (HCG)	Ningbo Second Hormone Company	HCG	Ovulation promoting drugs
Hyaluronidase	Sigma	H3506	Removing granulosa cells around the oocyte
KCl	Sigma	P5405	Preparation of CZB
KH₂PO₄	Sigma	P5655	Preparation of CZB
KSOMaa	Caisson Labs	IVL04-100ML	Potassium simplex optimized medium supplemented with amino acids
L-glutamine	Sigma	G8540	Preparation of CZB
M2	Sigma	M7167-50ML	Operating fluid
MgSO₄7H₂O	Sigma	M1880	Preparation of CZB
Na₂-EDTA2H₂O	Sigma	E5134	Preparation of CZB
NaCl	Sigma	S5886	Preparation of CZB
NaHCO₃	Sigma	S5761	Preparation of CZB
Na-lactate 60% syrup d = 1.32 g/L	Sigma	L7900	Preparation of CZB
Na-pyruvate	Sigma	P4562	Preparation of CZB
Piezo drill tips (ICSI)	Eppendorf	piezoXpert	Piezoelectric membrane rupture
pregnant mare serum gonadotropin (PMSG)	Ningbo Second Hormone Company	PMSG	Ovulation promoting drugs
PVA	Sigma	P8136	Preparation of CZB

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