Epididymal proteins Binder of SPerm Homologs 1 and 2 (BSPH1/2) are dispensable for male fertility and sperm motility in mice

Marzieh Eskandari-Shahraki,Francis Bergeron,Puttaswamy Manjunath,Bruno Prud’Homme

doi:10.1038/s41598-020-66017-6

Marzieh Eskandari-Shahraki, Francis Bergeron + Show 2 more

Open Access

https://doi.org/10.1038/s41598-020-66017-6

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Abstract

The binder of sperm family of proteins has been reported to be indispensable for sperm maturation and capacitation. However, their physiological functions in fertility have only been studied in vitro. CRISPR/Cas9 genome editing was utilized to generate double knockout (DKO) mice by simultaneously targeting the two murine binder of sperm genes, Bsph1 and Bsph2. To confirm that the homologous genes and proteins were completely eliminated in the DKO mice, different methods such as reverse transcription polymerase chain reaction, digital droplet-polymerase chain reaction and liquid chromatography tandem mass spectrometry were applied. Bsph1/2 DKO male mice were bred by intercrossing. Compared to wild type counterparts, male Bsph1/2 null mice, lacking BSPH1/2 proteins, were fertile with no differences in sperm motility and sperm count. However, the weights of male pups were significantly increased in Bsph1/2 double knockout mice in a time dependent manner spanning days 6 and 21, as well as 6 weeks of age. No change was detected in the weights of female pups during the same period. Taken together, these data indicate that BSPH1/2 proteins are dispensable for male fertility in mice but may influence growth.

Highlights

Mammalian fertilization is a multi-step process that arises from complex and regulated cellular processes, and relies on highly specialized gametes; defects in which can lead to infertility[1]
The guide RNAs were designed, synthesized and ligated into the pX330 plasmid to target the early sequence of exons 2 and 1 of the Bsph[1] and Bsph[2] genes, respectively
Before injecting the constructs into mouse embryos, the designed guide RNAs (gRNAs) were validated in neuroblast N2 cells to ensure that they could target efficiently and cut the desired regions of Binder of SPerm (BSP) genes

Summary

Introduction

Mammalian fertilization is a multi-step process that arises from complex and regulated cellular processes, and relies on highly specialized gametes; defects in which can lead to infertility[1]. Taking into consideration that murine and human BSP proteins share many biochemical and functional characteristics such as the organ in which they are expressed, their binding properties, and a similar structure[12], studying BSP functions in mouse has the potential to provide invaluable information on the role of these proteins in human sperm function and fertility. Due to their co-localization on the same chromosome, generating a mouse model with the Bsph1/Bsph[2] double deletion via traditional knockout approaches presents important technical challenges. The present study is the first to investigate the endogenous functions of BSP proteins and their role in male fertility in vivo

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