CRISPR/Cas9-based genome editing in mice uncovers 13 testis- or epididymis-enriched genes individually dispensable for male reproduction†

Zhou

et al. 2021

IJMS

The calcium-binding protein spermatid-associated 1 (Cabs1) is a novel spermatid-specific protein. However, its function remains largely unknown. In this study, we found that a long noncoding RNA (lncRNA) transcripted from the Cabs1 gene antisense, AntiCabs1, was also exclusively expressed in spermatids. Cabs1 and AntiCabs1 knockout mice were generated separately (using Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-Cas9 methods) to investigate their functions in spermatogenesis. The genetic loss of Cabs1 did not affect testicular and epididymal development; however, male mice exhibited significantly impaired sperm tail structure and subfertility. Ultrastructural analysis revealed defects in sperm flagellar differentiation leading to an abnormal annulus and disorganization of the midpiece–principal piece junction, which may explain the high proportion of sperm with a bent tail. Interestingly, the proportion of sperm with a bent tail increased during transit in the epididymis. Furthermore, Western blot and immunofluorescence analyses showed that a genetic loss of Cabs1 decreased Septin 4 and Krt1 and increased cyclin Y-like 1 (Ccnyl1) levels compared with the wild type, suggesting that Cabs1 deficiency disturbed the expression of cytoskeleton-related proteins. By contrast, AntiCabs1−/− mice were indistinguishable from the wild type regarding testicular and epididymal development, sperm morphology, concentration and motility, and male fertility. This study demonstrates that Cabs1 is an important component of the sperm annulus essential for proper sperm tail assembly and motility.

Section: Discussionsupporting

confidence: 93%

“…The calcium-binding properties of Cabs1 are well-established. A very recent study just showed that Cabs1-deficient mice are fertile to a certain extent [ 13 ]. However, the further physiological roles of Cabs1 in the male reproductive system have not been elucidated.…”

Section: Introductionmentioning

confidence: 99%

Cabs1 Maintains Structural Integrity of Mouse Sperm Flagella during Epididymal Transit of Sperm

Zhou

et al. 2021

IJMS

Proc. Natl. Acad. Sci. U.S.A.

“…To unveil the factor(s) involved in mitochondrial sheath formation, creating and analyzing male mice carrying null mutations is a feasible approach because we can observe protein function directly, and as yet no culture systems exist that produce fully functional spermatozoa in vitro. Therefore, we generated knockout (KO) male mice using CRISPR/Cas9-based genome engineering and screened their phenotypes ( 10 – 12 ) to find factors involved in mitochondrial sheath formation. In addition, we analyzed the functions of identified proteins to unveil the molecular mechanisms of mitochondrial sheath formation.…”

mentioning

confidence: 99%

ARMC12 regulates spatiotemporal mitochondrial dynamics during spermiogenesis and is required for male fertility

Shimada

Park

Miyata

et al. 2021

Self Cite

The mammalian sperm midpiece has a unique double-helical structure called the mitochondrial sheath that wraps tightly around the axoneme. Despite the remarkable organization of the mitochondrial sheath, the molecular mechanisms involved in mitochondrial sheath formation are unclear. In the process of screening testis-enriched genes for functions in mice, we identified armadillo repeat-containing 12 (ARMC12) as an essential protein for mitochondrial sheath formation. Here, we engineered Armc12-null mice, FLAG-tagged Armc12 knock-in mice, and TBC1 domain family member 21 (Tbc1d21)-null mice to define the functions of ARMC12 in mitochondrial sheath formation in vivo. We discovered that absence of ARMC12 causes abnormal mitochondrial coiling along the flagellum, resulting in reduced sperm motility and male sterility. During spermiogenesis, sperm mitochondria in Armc12-null mice cannot elongate properly at the mitochondrial interlocking step which disrupts abnormal mitochondrial coiling. ARMC12 is a mitochondrial peripheral membrane protein and functions as an adherence factor between mitochondria in cultured cells. ARMC12 in testicular germ cells interacts with mitochondrial proteins MIC60, VDAC2, and VDAC3 as well as TBC1D21 and GK2, which are required for mitochondrial sheath formation. We also observed that TBC1D21 is essential for the interaction between ARMC12 and VDAC proteins in vivo. These results indicate that ARMC12 uses integral mitochondrial membrane proteins VDAC2 and VDAC3 as scaffolds to link mitochondria and works cooperatively with TBC1D21. Thus, our studies have revealed that ARMC12 regulates spatiotemporal mitochondrial dynamics to form the mitochondrial sheath through cooperative interactions with several proteins on the sperm mitochondrial surface.

“…Fertile and without any phenotypic change(s) 2020 [31] FSH-follicle-stimulating hormone; LH-luteinizing hormone; FG-full-grown; hCG-human chorionic gonadotropin; 11-KT-11-Ketotestosterone; E2-estradiol; LHβ-luteinizing hormone beta polypeptide; MMAF-multiple morphological anomalies of the sperm flagella.…”

Section: Methodsmentioning

confidence: 99%

“…Among all one thousand genes assumed to have a role in fertility and hypotheses issued, partially proved to be dispensable since over forty-five genes knockout did not affect the host’s homeostasis [ 29 , 30 , 31 ]. For example, spermatogenesis associated 16 (Spata16), actin-like 7A (ACTL7A), or Zfy1/2-DKO mice are among the many others that are crucial for fertility.…”

Section: Crispr/cas9 and Infertilitymentioning

confidence: 99%

Mini-Review Regarding the Applicability of Genome Editing Techniques Developed for Studying Infertility

et al. 2021

Infertility is a highly debated topic today. It has been long hypothesized that infertility has an idiopathic cause, but recent studies demonstrated the existence of a genetic substrate. Fortunately, the methods of editing the human genome proven to be revolutionary. Following research conducted, we identified a total of 21 relevant studies; 14 were performed on mice, 5 on zebrafish and 2 on rats. We concluded that over forty-four genes in total are dispensable for fertility in both sexes without affecting host homeostasis. However, there are genes whose loss-of-function induces moderate to severe phenotypic changes in both sexes. There were situations in which the authors reported infertility, exhibited by the experimental model, or other pathologies such as cryptorchidism, cataracts, or reduced motor activity. Overall, zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 are techniques that offer a wide range of possibilities for studying infertility, even to create mutant variants. It can be concluded that ZFNs, TALENs, and CRISPR/Cas9 are crucial tools in biomedical research.