Glycerol kinase 2 is essential for proper arrangement of crescent-like mitochondria to form the mitochondrial sheath during mouse spermatogenesis

Abstract

The mitochondrial sheath is composed of mitochondria that coil tightly around the midpiece of sperm flagellum. These mitochondria are recruited from the cytoplasm to the flagellum late in spermatogenesis. Initially, recruited mitochondria are spherical-shaped but then elongate laterally to become crescent-like in shape. Subsequently, crescent-like mitochondria elongate continuously to coil tightly around the flagellum. Recently, disorganization of the mitochondrial sheath was reported in Glycerol kinase 2 (Gk2) disrupted mice. To analyze the disorganization of the mitochondrial sheath further, we generated Gk2-deficient mice using the CRISPR/Cas9 system and observed sperm mitochondria in testis using a freeze-fracture method with scanning electron microscopy. Gk2-disrupted spermatids show abnormal localization of crescent-like mitochondria, in spite of the initial proper alignment of spherical mitochondria around the flagellum, which causes abnormal mitochondrial sheath formation leading to exposure of the outer dense fibers. These results indicate that GK2 is essential for proper arrangement of crescent-like mitochondria to form the mitochondrial sheath during mouse spermatogenesis.

Keywords: Glycerol kinase; Male infertility; Mitochondrial sheath formation; Sperm mitochondria; Spermatogenesis.

Fig. 1.

Gk2 disrupted mice are male infertile because their spermatozoa cannot pass through the uterotubal junction (UTJ). (A) Design of sgRNA for generating Gk2 KO mice. Guide sequence is highlighted in green. (B) Control and Gk2^-7/-7 alleles. Red letters indicate 7 bp deletion site. (C) Putative protein product of the Gk2^-7/-7 allele. Red letters indicates differing amino acids due to a frame shift. *, stop codon. (D) Average litter size of control and Gk2 KO male mice. Error bars represent S.D. (E) Sperm morphology of control and Gk2 KO mice in the RBGS background, which express mitochondria-targeted DsRed2 (red). Nuclei were stained with Hoechst 33342 (blue). Arrowheads indicate abnormal-bending. Arrows indicate fragmented mitochondrial sheath. Scale bars are 10 µm. (F) Imaging of spermatozoa inside the female reproductive tract 2−3 h after observing vaginal plugs. Left panels display reproductive organs under normal bright field conditions. Middle panels show red fluorescence of RBGS spermatozoa in the female reproductive tract. Right panels show enlarged images of the boxed areas. Scale bars are 300 µm.

Fig. 2.

Mitochondrial disorganization of Gk2 disrupted spermatozoa appears before sperm bending. (A) Graphs indicate frequencies of bending spermatozoa collected from the three sections of epididymis. Lower panels show representative images of spermatozoa collected from the three sections of epididymis. Spermatozoon shape is categorized as Hairpin, Angular, or Straight (displayed in right panels). ** P < 0.01, Student’s t test. (B) Graph indicates the frequencies of fragmented mitochondrial sheath collected from the three sections of epididymis. Right panels are representative images of spermatozoon with or without a fragmented mitochondrial sheath. ** P < 0.01, Student’s t test, Error bars represent S.D.

Fig. 3.

Gk2 deficient spermatids show aberrant mitochondrial sheath formation caused by misalignment of crescent-like mitochondria. (A) Ultrastructural images of step 16 spermatids (stage VII) analyzed by transmission electron microscopy (TEM). Arrows indicate the midpiece not surrounded by mitochondria. Scale bars of cross section and longitudinal section are 1 µm and 500 nm, respectively. (B) Mitochondrial sheath development during spermatogenesis observed by scanning electron microscopy (SEM). During spermatogenesis, spherical mitochondria align around flagellum (left), and change their shape to crescent-like mitochondria to surround the flagellum (middle), and then the mitochondria continue to elongate to form mitochondrial sheath (right). Arrows show breaks of aligned mitochondria. Arrowheads show exposed outer dense fiber. Scale bars are 1 µm.