3.2 Presentation of defective sperm flagellum and head in two infertile patients
To confirm the two patients’ detailed phenotypes, we performed semen analysis according to WHO guidelines. The results indicated dramatic decreases in sperm motility and morphological abnormalities in both patients (Table 1). Papanicolaou staining and SEM were further used to analyze sperm morphology. Compared to normal controls, the spermatozoa from patients exhibited a typical MMAF phenotype, including absent, short, and coiled flagella (Figure 2a, b). Noticeably, a defect was observed in the conjunction between the midpiece and principal piece (Figure 2b). To further confirm whether this disruption is involved in the sperm annulus, we detected the expression and localization of SEPTIN4, as a marker of the sperm annulus, by immunofluorescence staining in the patients’ sperm (Kissel et al., 2005). As expected, the SEPTIN4 signal was located in the annulus in normal control but was almost absent in patients (Figure 3a), suggesting that CFAP47 might also be related to the sperm annulus formation.
TEM was performed to investigate the ultrastructure of spermatozoa. In contrast with the well-organized peri-axonemal and axonemal structures in the sperm flagella from normal control, the sperm flagella of patients showed severe disorganization of the peri-axonemal and ‘9 + 2’ axonemal arrangement, the absence of central or peripheral microtubules in the sperm flagella and the disorganized arrangement of mitochondrial sheaths and dense fibers around the axoneme (Figure 2c). Strikingly, typically swollen mitochondria were present in the middle piece (Figure 2c). Based on this phenomenon, COXIV, as a marker of mitochondrial sheath integrity, was used to analyze defects in the mitochondrial sheath (Böttinger et al., 2013). In control sperm, COXIV localized to the midpiece of sperm flagella, but it disappeared completely in the sperm of patients (Figure 3c). Intriguingly, most patients’ spermatozoa exhibited abnormally shaped heads, and the nuclei in most sperm of the patients were irregular as well as more unconsolidated than those of the control. Additionally, the majority of the acrosomes were small or even absent in the sperm heads of the patients (Figure 2C). Immunofluorescence staining of PNA also demonstrated acrosomeless spermatozoa or disrupted acrosomes in the sperm of patients (Figure 3b). All evidence indicates that CFAP47 mutation contributes not only to the MMAF phenotype but also to abnormalities in the sperm head and annulus.