Knockout mouse model generated by CRISPR technology to study the function of BSP proteins on male fertility in vivo

Infertility is a worldwide problem that affects 15% of couples, with male and female factors contributing equally to cases of infertility. The cause of male infertility in half of the cases is unknown. A series of coordinated cellular events are responsible for sperm-egg interaction and successful fertilization. Therefore, elucidating the mechanisms involved in sperm production and maturation is critical to facilitate the diagnosis and treatment of male infertility, as well as the regulation of fertility. Over the last three decades, the laboratory of Dr. P. Manjunath has been extensively investigating a particular family of proteins that is expressed specifically in the male reproductive tract, called the Binder of SPerm (BSP) proteins. This protein family was first identified in bovine seminal plasma and subsequently, BSP proteins were identified in other mammalian species such as stallion, boar, ram and goat. Depending on the species, these small ubiquitous proteins are expressed by the seminal vesicles and/or epididymis. Systematic work done by Manjunath’s group has demonstrated that these proteins bind to choline phospholipids on the outer leaflet of the sperm membrane upon ejaculation and/or during passage through the epididymis and promote sperm capacitation. In 2007, BSP-homologous genes were identified in human (BSPH1) and mouse (Bsph1 and Bsph2) epididymis. Further studies indicated that the interaction of BSP proteins with the sperm membrane results in lipid (phospholipid and cholesterol) alterations and facilitates epididymal sperm maturation. Additional research on BSP proteins suggested that these proteins may play an important role in male fertility. However, the exact role of BSP proteins in fertility has remained largely unknown. In the current study, I generated the first single and double BSP knockout mouse model to investigate whether the absence of BSP proteins could affect male fertility. To target and generate single and double knockout Bsph1 and Bsph2 mice models, the Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR/Cas9) approach was utilized. I used RT-PCR, Digital Droplet-PCR (dd-PCR) and Liquid Chromatography tandem Mass Spectrometry (LC-MS/MS) techniques to examine Bsp gene expression at the mRNA and protein levels and confirm whether BSP proteins were completely eliminated in knockout animals. The knockout BSP mouse lines were used to evaluate sperm parameters of fertility. No obvious abnormalities were observed in the fertilizing ability nor in the number of pups for knockout compared to wild type male mice. Sperm count and motility analyses indicated no significant differences between BSP-null mice compared to control. Interestingly, significant differences in terms of the pup’s body weight were observed in double knockout compared to wild type. Based on these results, we conclude that BSP proteins, individually or together, are not essential for proper sperm function and fertilization in mice, and the absence of these proteins does not affect male fertility. Further studies could provide a better understanding of the mechanisms involved and possible pathways by which BSP proteins could be involved in regulating pup weight.

Keyphrases: Male infertility, Binder of sperm proteins, Epididymal proteins, Knockout mice