Mouse Round Spermatid Injection

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 (I....

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

1. Acquire some reagents commercially and self-prepare the remainin.......

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 chloride^23,24, while the most advanced human application employs electrical activation^13,14. The timing of oocyte activation is also of great significance. As reported in the li.......

Materials

Name	Company	Catalog Number	Comments
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