4 DISCUSSION
Sex chromosomes play a key factor in sex determination and reproductive function (Skaletsky et al., 2003). Other than genes located in the autosomal chromosome, sex chromosome genes lack corresponding alleles. Therefore, harmful mutations in sex chromosome genes have a direct impact on male infertility occurrence. To date, limited genetic causes in sex chromosomes have been acknowledged. For example, azoospermia factor (AZF), located on the long arm of the Y chromosome, is composed of AZFa, AZFb, and AZFc (Muller et al., 2008). Previous studies have revealed that AZF microdeletion is related to NOA and severe oligospermia (Yoshida et al., 2014; Navarro-Costa et al., 2010; Colaco et al., 2018). Deleted in azoospermia 1 (DAZ1 ),ubiquitin-specific peptidase 9 Y-linked (USP9Y), and RNA binding motif protein Y-linked family 1 member A1 (RBMY) in the AZF region are identified as candidate causative genes in spermatogenesis. Similar to the Y chromosome, the X chromosome contains genes with exclusive or predominant expression in the testis. Partial deletion oftestis expressed 11 (TEX11) leads to azoospermia due to meiotic arrest; mutations in androgen receptor (AR) attenuate AR regulation of target gene expression and cause oligozoospermia and azoospermia; dysfunction of adhesion G protein-coupled receptor G2 (ADGRG2) results in a buildup of fluid within the testis and an accumulation of spermatozoa within the efferent ducts; Dynein axonemal assembly factor 6 (DAAF6 ) is involved in the assembly of the dynein arm of the ciliary axoneme, and defects in this gene lead to primary ciliary dyskinesia (PCD). Hence, more sex chromosome gene pathogenicity and biological functions in male reproduction need to be explored.
A previous study on an animal model reported a necessary role ofCfap47 in spermatogenesis in mice, and their patients were infertile, characterized by abnormal sperm motility and morphology. However, the phenotype and genotype of CFAP47 in humans have not been comprehensively studied, and the underlying mechanism by whichCFAP47 regulates reproductive biology is limited. In the present study, we detected a novel recurrent missense mutation of CFAP47,which is located in the X chromosome, in two sterile patients from two unrelated families. Further in vitro experiments confirmed the pathogenicity of this mutation. By a comprehensive morphology analysis, we first suggested that CFAP47 mutation is linked to abnormal annulus and aberrations in sperm morphology either in the head or flagellum. More importantly, we explored the interacting proteins of CFAP47 and investigate the molecular mechanism of CFAP47 in sperm morphology. Interestingly, CFAP69 and CFAP65, known MMAF pathogenic genes, are required for flagella assembly and stability in sperm cells. Wang et al. reported that CFAP65 is expressed in the acrosome area and flagellar midpiece in normal human spermatozoa, and CFAP69 is localized to the sperm flagellum. We thus speculated that CFAP47 may be involved in spermatogenesis by interacting with CFAP65 and CFAP69. Moreover, damaged expression of CFAP65 and CFAP69 was detected in the sperm of the patients. Additionally, the co-expression of CFAP47 with CFAP65 and CFAP69 in various spermatogenic spermatids in the testes of humans and mice during spermatogenesis has been presented. These findings indicate a potential role of CFAP47 in regulating spermatogenesis through interacting with CFAP65 and CFAP69. Our study expands the mutational and phenotypic spectrum of CFAP47 and strongly elucidates the important role of CFAP47 in male reproduction.
In summary, our study identified a novel missense mutation inCFAP47 in two infertile male patients with various sperm morphology abnormalities. Our work presented more detailed information on the pathogenesis of CFAP47 -mutated MMAF and provided direct evidence that suggests the involvement of CFAP47 in spermatogenesis.