Pseudopregnant mice generated from Piwil1 deficiency sterile mice

Abstract

Vasectomized mice play a key role in the production of transgenic mice. However, vasectomy can cause great physical and psychological suffering to mice. Therefore, there is an urgent need to find a suitable replacement for vasectomized mice in the production of transgenic mice. In this study, we generated C57BL/6J mice (Piwil1 D633A-INS99, Piwil1mt/mt) with a 99-base insertion in the Miwi (Piwil1) gene using CRISPR/Cas9 technology and showed that Piwil1mt/+ heterozygous mice were normally fertile and that homozygous Piwil1mt/mt males were sterile and females were fertile. Transplantation of normal fertilized eggs into wild pseudopregnant females following mating with Piwil1mt/mt males produced no Piwil1mt/mt genotype offspring, and the number of offspring did not differ significantly from that of pseudopregnant mice following mating and breeding with ligated males. The CRISPR‒Cas9 system is available for generating Miwi-modified mice, and provides a powerful resource to replace ligated males in assisted reproduction research.

Fig 1. Piwil1 gene editing mouse construct.

A. In situ injection of fertilized mouse eggs and embryo transfer. B. Mutant mouse gene construct. C. The mutant PIWIL1 protein predicted in Piwil1^mt/mt mice compared to the wild-type PIWIL1 protein.

Fig 2. PIWL1 protein expression.

A. PIWIL1 protein expression in the testes of Piwil1^mt/mt mice and wild-type mice. B. PIWIL1 protein immunofluorescence in the testes of Piwil1^mt/mt mice and wild-type mice. Scale bar, 20 μm.

Fig 3. Fertility analysis of Piwil1^mt/mt mice vs. vasectomized mice.

A. PAS staining of spermatogonia at all stages of testis development in Piwil1^mt/mt and. wild-type mice. Scale bar, 20 μm. B. HE staining of the epididymis in Piwil1^mt/mt and wild-type mice. Scale bar, 100 μm. C. Fertility statistics of Piwil1^mt/mt and wild-type mice. D. Line graph of spermatogonia to supporting cell ratio at all stages of testis development in Piwil1^mt/mt and wild-type mice. E. PAS staining of spermatogonia at all stages of testis development in ICR ligated and ICR wild-type mice. Scale bar, 20 μm. F. H&E staining of the epididymis in ICR ligated and ICR wild-type mice. Scale bar, 100 μm. G. Fertility statistics of ICR ligated and ICR wild-type mice. H. Line graph of the spermatogonia to supporting cell ratio at all stages of testis development in ICR ligated and ICR wild-type mice. PS, pachytene spermatocyte; ES, elongating spermatids; RS, round spermatids.

Fig 4. Reproductive accessories and fertility statistics of Piwil1^mt/mt and vasectomized mice.

A. Testes and epididymis tissues from Piwil1^mt/mt and wild-type mice at the same age. B. Testes and epididymis tissues from ICR wild-type and ICR ligated mice. C. Statistical analysis of the testes-to-body weight ratio of ICR wild-type and ICR ligated mice. D. Seminal vesicle glands of ICR wild-type and ICR ligated mice. E. The number of pups born to ICR wild-type sham-pregnant mice mated with Piwil1^mt/mt and ICR ligated mice. F. Table of statistical analysis for the number of pups born to ICR wild-type sham-pregnant mice mated with Piwil1^mt/mt and ICR ligated mice.