T-complex protein 1 subunit zeta-2 (CCT6B) deficiency induces murine teratospermia

Abstract

Background The CCT complex is an important mediator of microtubule assembly and intracellular protein folding. Owing to its high expression in spermatids, CCT knockdown can disrupt spermatogenesis. In the present report, we therefore evaluated the in vivo functionality of the testis-specific CCT complex component CCT6B using a murine knockout model system. Methods A CRISPR/Cas9 approach was used to generate Cct6b−/− mice, after which candidate gene expression in these animals was evaluated via qPCR and Western blotting. Testicular and epididymal phenotypes were assessed through histological and immunofluorescent staining assays, while a computer-assisted sperm analyzer was employed to assess semen quality. Results Cct6b−/− mice were successfully generated, and exhibited no differences in development, fertility, appearance, testis weight, or sperm counts relative to control littermates. In addition, no differences in spermatogenesis were detected when comparingCct6b+/+ and Cct6b−/− testes. However, when progressive motility was analyzed, the ratio of normal sperm was significantly decreased in Cct6b−/− male mice, with nuclear base bending being the primary detected abnormality. In addition, slight decreases in Cct4 and Cct7 expression were detected. Conclusion These data indicated that CCT6B is an important regulator of murine spermatogenesis, with the loss of this protein resulting in CCT complex dysfunction, providing a foundation for further studies.

Combined Use of Whole Exome Sequencing and CRISPR/Cas9 to Study the Etiology of Non-Obstructive Azoospermia: Demonstration of the Dispensable Role of the Testis-Specific Genes C1orf185 and CCT6B

“…In June 2021 and during the preparation of our manuscript, Yang et al published an interesting paper reporting the reproductive phenotype of Cct6b −/− mice generated by the CRISPR/Cas9 system by targeting the same exon (4/14). Concordant with what we describe here, they found that males exhibited no differences in development, fertility, testis weight, sperm counts, total motility, and spermatogenesis relative to control littermates, thus confirming our results [ 32 ].…”

DNALI1 deficiency causes male infertility with severe asthenozoospermia in humans and mice by disrupting the assembly of the flagellar inner dynein arms and fibrous sheath

“…WB was carried out as described previously [ 24 ], with slight modification. In brief, total protein extraction was done in a lysis buffer (7 M urea, 2 M thiourea, 2% (w/v) DTT) with 1% (v/w) protease inhibitor mixture (Pierce Biotechnology).…”

Forkhead-associated phosphopeptide binding domain 1 (FHAD1) deficiency impaired murine sperm motility

“…Previous studies in which testis-enriched genes were inactivated have successfully demonstrated the contributions of these genes to male infertility and the impairment of spermatogenesis in mice, underscoring their importance to reproductive outcomes. This approach has been used to evaluate many candidate testes-enriched genes to date, with many having been found to play detectable roles in the regulation of murine spermatogenesis ( Castaneda et al, 2017 ; Gao et al, 2020 ; Hua et al, 2019 ; Shah et al, 2021 ; Shibuya et al, 2015 ; Yamase et al, 2019 ; Yang et al, 2021 ; Zhang et al, 2019 ), whereas others are dispensable in this context, including FANK1 , Hspa1l , and Tex33 ( Wang et al, 2020 ; Zhang et al, 2019 ; Zhu et al, 2020 ). It has been suggested that the proteins encoded by these genes may have redundant functions in spermatogenesis.…”

“…These and related approaches have identified many key genes associated with male fertility, including Fbxo47, Tcte1, Majin, Dnali1 , and Meiob ( Castaneda et al, 2017 ; Hua et al, 2019 ; Rashid et al, 2006 ; Shibuya et al, 2015 ; Souquet et al, 2013 ). In contrast, other genes including Ttll3 , Ttll8 , and Cct6b , have also been found to play important regulatory roles in spermatogenesis, even though their knockout does not result in male infertility ( Gadadhar et al, 2021 ; Yang et al, 2021 ). As such, there is a clear need for further studies aimed at identifying other key proteins involved in the spermatogenic process to better clarify the mechanisms that govern male fertility.…”