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.