Disruption of testis-enriched cytochrome c oxidase subunit COX6B2 but not COX8C leads to subfertility

Abstract

Mammalian sperm flagellum contains the midpiece characterized by a mitochondrial sheath that packs tightly around the axoneme and outer dense fibers. Mitochondria are known as the "powerhouse" of the cell, and produce ATP through the tricarboxylic acid (TCA) cycle and oxidative phosphorylation (OXPHOS). However, the contribution of the TCA cycle and OXPHOS to sperm motility and male fertility is less clear. Cytochrome c oxidase (COX) is an oligomeric complex localized within the mitochondrial inner membrane, and the terminal enzyme of the mitochondrial electron transport chain in eukaryotes. Both COX6B2 and COX8C are testis-enriched COX subunits whose functions in vivo are poorly studied. Here, we generated Cox6b2 and Cox8c knockout (KO) mice using the CRISPR/Cas9 system. We examined their fertility and sperm mitochondrial function to determine the significance of testis-enriched COX subunits in male fertility. The mating test revealed that disrupting COX6B2 induces male subfertility, while disrupting COX8C does not affect male fertility. Cox6b2 KO spermatozoa showed low sperm motility, but mitochondrial function was normal according to oxygen consumption rates. Therefore, low sperm motility seems to cause subfertility in Cox6b2 KO male mice. These results also indicate that testis-enriched COX, COX6B2 and COX8C, are not essential for OXPHOS in mouse spermatozoa.

Keywords: CRISPR/Cas9; cytochrome c oxidase subunit; mitochondria; sperm motility; subfertility.

Fig. 1.

Cox6b2-disrupted male mice are subfertile. (A) The expression of mouse Cox6b and Cox8 family genes was examined by RT-PCR using RNA isolated from various organs. Both Cox6b2 and Cox8c show testis-enriched expression. Actb was used as a loading control. (B, C) KO strategy for generating Cox6b2 (B) and Cox8c (C) KO mice. Panels show a diagram of each gene. Two gRNAs (green arrows) were designed to target the first and last exons. (D, E) The DNA sequence of the control and KO mouse lines in the Cox6b2 (D) and Cox8c (E) genes. A Cox6b2 KO mouse line that has 1,171-bp deletion and Cox8c KO mouse line that has 988-bp deletion were generated in the present study. (F, G) The graph (F) and table (G) indicate the number of litters born per plug detected. Males for each WT, Cox6b2 and Cox8c KO were mated with three WT females per male.

Fig. 2.

No abnormalities were detected in both Cox6b2 and Cox8c KO mouse testis. (A) Gross morphology of control and Cox6b2 KO testes. Scale bars: 3.0 mm. (B) Testis weight of control and Cox6b2 KO mice (N=4). Average weight of testis in control=77.5 ± 2.8 mg; Cox6b2 KO=76.7 ± 7.0 mg. P=0.7712, Student’s t-test; error bars represent SD. (C) PAS staining of testis samples from control and Cox6b2 KO male mice. Scale bars: 50 µm. (D) Gross morphology of control and Cox8c KO testes. Scale bars: 3.0 mm. (E) Testis weight of control and Cox8c KO mice (N=4). Average weight of testis was control=109.5 ± 13.0 mg; Cox8c KO=106.1 ± 4.8 mg. P=0.1718, Student’s t-test; error bars represent SD. (F) PAS staining of testis samples from control and Cox8c KO male mice. Scale bars: 50 µm.

Fig. 3.

Both Cox6b2 and Cox8c KO spermatozoa have no abnormalities in mitochondrial sheath formation and mitochondrial function. (A) Fluorescence images after transient expression of testis-enriched COX subunit genes in COS-7 cells. COS-7 cells were transiently expressed with mock vector, 8xHis- and 1D4-tagged COX6B2 expression vector or 3xHA-tagged COX8C expression vector, and stained with 1D4 or HA (green), TOM20 (red) and Hoechst 33342 (white) to visualize COX subunit, mitochondria and nuclei, respectively. Scale bars: 20 µm. (B) Sperm morphology of WT, Cox6b2, and Cox8c KO mice in the RBGS background, which express mitochondria-targeted DsRed2 (red). Nuclei were stained with Hoechst 33342 (blue). Scale bars: 20 µm. (C, D) Oxygen consumption rate (OCR) traces for spermatozoa collected from the cauda epididymis of WT, Cox6b2, and Cox8c KO mice (C), as well as quantification of basal respiration, ATP production, proton leak, maximal respiration, and spare capacity (D). Error bars represent SD, N=4.

Fig. 4.

Cox6b2 KO spermatozoa show low motility, but not Cox8c KO. (A, B) Sperm motility (A) and progressive sperm rate (B) from control and Cox6b2 KO mice. (C–E) The means of VAP (C), VCL (D) and VSL (E) from control and Cox6b2 KO mice. (F, G) Sperm motility (F) and progressive sperm rate (G) from control and Cox8c KO mice. (H–J) The means of VAP (H), VCL (I), and VSL (J) from control and Cox8c KO mice. *P<0.05, Student’s t-test; error bars represent SD (N=6). (K) Phosphorylation status of tyrosine residues of sperm proteins. β-actin was used as a loading control. (L) Flagellar waveforms were analyzed 10 min after incubation. Single frames throughout one beating cycle were superimposed.