Epididymal cysteine-rich secretory proteins are required for epididymal sperm maturation and optimal sperm function

• Published in: Molecular human reproduction 2018-03, Vol.24 (3), p.111-122
• Main Authors: Hu, Jinghua, Merriner, D Jo, O’Connor, Anne E, Houston, Brendan J, Furic, Luc, Hedger, Mark P, O’Bryan, Moira K
• Format: Article
• Language: English
• Subjects: Acrosome - metabolism; Acrosome - physiology; Animals; Cell Line; Epididymis - metabolism; Female; Humans; Male; Membrane Glycoproteins - genetics; Membrane Glycoproteins - metabolism; Mice; Mice, Knockout; Seminal Plasma Proteins - genetics; Seminal Plasma Proteins - metabolism; Sperm Maturation - genetics; Sperm Maturation - physiology
• ISSN: 1360-9947; 1460-2407
• DOI: 10.1093/molehr/gay001

Abstract STUDY QUESTION What is the role of epididymal cysteine-rich secretory proteins (CRISPs) in male fertility? SUMMARY ANSWER While epididymal CRISPs are not absolutely required for male fertility, they are required for optimal sperm function. WHAT IS KNOWN ALREADY CRISPs are members of the CRISP, Antigen 5 and Pathogenesis related protein 1 (CAP) superfamily and are characterized by the presence of an N-terminal CAP domain and a C-terminal CRISP domain. CRISPs are highly enriched in the male reproductive tract of mammals, including in the epididymis. Within humans there is one epididymal CRISP, CRISP1, whereas in mice there are two, CRISP1 and CRISP4. STUDY DESIGN, SIZE, DURATION In order to define the role of CRISPs within the epididymis, Crisp1 and Crisp4 knockout mouse lines were produced then interbred to produce Crisp1 and 4 double knockout (DKO) mice, wherein the expression of all epididymal CRISPs was ablated. Individual and DKO models were then assessed, relative to their own strain-specific wild type littermates for fertility, and sperm output and functional competence at young (10–12 weeks of age) and older ages (22–24 weeks). Crisp1 and 4 DKO and control mice were also compared for their ability to bind to the zona pellucida and achieve fertilization. PARTICIPANTS/MATERIALS, SETTING, METHODS Knockout mouse production was achieved using modified embryonic stem cells and standard methods. The knockout of individual genes was confirmed at a mRNA (quantitative PCR) and protein (immunochemistry) level. Fertility was assessed using breeding experiments and a histological assessment of testes and epididymal tissue. Sperm functional competence was assessed using a computer assisted sperm analyser, induction of the acrosome reaction using progesterone followed by staining for acrosome contents, using immunochemical and western blotting to assess the ability of sperm to manifest tyrosine phosphorylation under capacitating conditions and using sperm-zona pellucida binding assays and IVF methods. A minimum of three biological replicates were used per assay and per genotype. MAIN RESULTS AND THE ROLE OF CHANCE While epididymal CRISPs are not absolutely required for male fertility, their production results in enhanced sperm function and, depending on context, CRISP1 and CRISP4 act redundantly or autonomously. Specifically, CRISP1 is the most important CRISP in the establishment of normally motile sperm, whereas CRISP4 acts to enhance capacitation-associ