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TCTE1 Is a Conserved Component of the Dynein Regulatory Complex and Is Required for Motility and Metabolism in Mouse Spermatozoa

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TCTE1 Is a Conserved Component of the Dynein Regulatory Complex and Is Required for Motility and Metabolism in Mouse Spermatozoa TCTE1 is a conserved component of the dynein PNAS PLUS regulatory complex and is required for motility and metabolism in mouse spermatozoa Julio M. Castanedaa,b,1, Rong Huac,d,1, Haruhiko Miyatab, Asami Ojib,e, Yueshuai Guoc,d, Yiwei Chengc,d, Tao Zhouc,d, Xuejiang Guoc,d, Yiqiang Cuic,d, Bin Shenc, Zibin Wangc, Zhibin Huc,f, Zuomin Zhouc,d, Jiahao Shac,d, Renata Prunskaite-Hyyrylainena,g,h, Zhifeng Yua,i, Ramiro Ramirez-Solisj, Masahito Ikawab,e,k,2, Martin M. Matzuka,g,i,l,m,n,2, and Mingxi Liuc,d,2 aDepartment of Pathology and Immunology, Baylor College of Medicine, Houston, TX 77030; bResearch Institute for Microbial Diseases, Osaka University, Suita, Osaka 5650871, Japan; cState Key Laboratory of Reproductive Medicine, Nanjing Medical University, Nanjing 210029, People’s Republic of China; dDepartment of Histology and Embryology, Nanjing Medical University, Nanjing 210029, People’s Republic of China; eGraduate School of Pharmaceutical Sciences, Osaka University, Suita, Osaka 5650871, Japan; fAnimal Core Facility of Nanjing Medical University, Nanjing 210029, People’s Republic of China; gCenter for Reproductive Medicine, Baylor College of Medicine, Houston, TX 77030; hFaculty of Biochemistry and Molecular Medicine, University of Oulu, Oulu FI-90014, Finland; iCenter for Drug Discovery, Baylor College of Medicine, Houston, TX 77030; jWellcome Trust Sanger Institute, Hinxton CB10 1SA, United Kingdom; kThe Institute of Medical Science, The University of Tokyo, Minato-ku, Tokyo 1088639, Japan; lDepartment of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX 77030; mDepartment of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030; and nDepartment of Pharmacology, Baylor College of Medicine, Houston, TX 77030 Contributed by Martin M. Matzuk, May 15, 2017 (sent for review December 27, 2016; reviewed by George L. Gerton and Mary Ann Handel) Flagella and cilia are critical cellular organelles that provide a dynein is anchored to the outer microtubules and responsible for means for cells to sense and progress through their environment. generating the force required to produce the beating pattern of The central component of flagella and cilia is the axoneme, which flagella and cilia (6). The force generated by dynein causes comprises the “9+2” microtubule arrangement, dynein arms, ra- sliding of the microtubules among each other; however, the dial spokes, and the nexin-dynein regulatory complex (N-DRC). nexin complex anchors the microtubules in place. The nexin BIOLOGY Failure to properly assemble components of the axoneme leads complex [or nexin-dynein regulatory complex (N-DRC)] has DEVELOPMENTAL to defective flagella and in humans leads to a collection of diseases been observed in transmission electron micrographs for several referred to as ciliopathies. Ciliopathies can manifest as severe syn- decades as a thin filament between outer microtubule doublets, dromic diseases that affect lung and kidney function, central nervous but only recently has the N-DRC’s role in regulating flagellar system development, bone formation, visceral organ organization, function been elucidated (7). By preventing the sliding of mi- and reproduction. T-Complex-Associated–Testis-Expressed 1 (TCTE1) Chla- crotubules, the nexin complex turns the force generated by is an evolutionarily conserved axonemal protein present from dynein into a bending motion of both flagella and cilia. The mydomonas (DRC5) to mammals that localizes to the N-DRC. Here, we show that mouse TCTE1 is testis-enriched in its expression, with its mRNA appearing in early round spermatids and protein localized Significance to the flagellum. TCTE1 is 498 aa in length with a leucine rich repeat domain at the C terminus and is present in eukaryotes containing a Infertility is a global problem that afflicts 15% of couples, and in flagellum. Knockout of Tcte1 results in male sterility because Tcte1- 50% of cases, the attributing factor is linked to men. Among these null spermatozoa show aberrant motility. Although the axoneme is infertile men, 18% specifically exhibit decreased motility of sperm structurally normal in Tcte1 mutant spermatozoa, Tcte1-null sperm (asthenozoospermia). Sperm motility is dependent on the forma- demonstrate a significant decrease of ATP, which is used by dynein tion and functioning of the flagellum, a modified cilium used for motors to generate the bending force of the flagellum. These data locomotion. Cilia are present in almost every cell of vertebrates and provide a link to defining the molecular intricacies required for axo- are essential for proper organ functioning. Defects in cilia forma- neme function, sperm motility, and male fertility. tion lead to severe syndromic diseases, termed ciliopathies, af- fecting numerous tissues (e.g., polycystic kidney disease), wherein male infertility | asthenozoospermia | glycolysis | mutant mouse | male infertility is often comorbid. Advances in mouse genetics testis-specific gene implicate several genes responsible for ciliopathies observed in humans. Here, we identify a nonsyndromic flagellum protein, TCTE1, that is required for sperm motility in mice. lagella are ancient, analogous cellular structures used for Flocomotion and as sensory organelles present in all three Author contributions: J.M.C., R.H., M.I., M.M.M., and M.L. designed research; J.M.C., R.H., domains of life (bacteria, archaea, and eukaryotes). The advan- H.M., A.O., Y.G., Y. Cheng, T.Z., X.G., Y. Cui, B.S., Z.W., Z.H., Z.Z., J.S., R.P.-H., Z.Y., R.R.-S., tages conferred by this organelle are highlighted by the flagella’s M.I., M.M.M., and M.L. performed research; R.R.-S. contributed new reagents/analytic apparent independent evolution in all three domains (1–3). Of tools; J.M.C., R.H., H.M., A.O., Y.G., Y. Cheng, T.Z., X.G., Y. Cui, B.S., Z.W., Z.H., Z.Z., J.S., R.P.-H., Z.Y., R.R.-S., M.I., M.M.M., and M.L. analyzed data; and J.M.C. and R.H. wrote all of the different flagella present among eukaryotes, flagella the paper. attached to gametes play a critical function in uniting gametes Reviewers: G.L.G., Perelman School of Medicine, University of Pennsylvania; and M.A.H., for fertilization and the perpetuation of a species. Mammalian The Jackson Laboratory. spermatozoa have a specialized flagellum that contains a mid- The authors declare no conflict of interest. piece, principal piece, and end piece with the axoneme running Freely available online through the PNAS open access option. along the entire length (4). The flagellum equips sperm with the ’ Data deposition: The data reported in this article have been deposited in the ProteomeX- capability to deliver half of the male s genetic material to the change Consortium with identifier PXD005343. female gamete, the oocyte. In addition to flagella, eukaryotes 1J.M.C. and R.H. contributed equally to this work. contain another related structure called cilia. The defining fea- 2To whom correspondence may be addressed. Email: [email protected], ikawa@ ture of flagella and cilia is the axoneme, the “9+2” microtubule biken.osaka-u.ac.jp, or [email protected]. arraignment of two central pairs of microtubules surrounded by This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. nine pairs of microtubule doublets (5). The microtubule motor 1073/pnas.1621279114/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1621279114 PNAS Early Edition | 1of9 Downloaded by guest on September 25, 2021 N-DRC is a complex of up to 11 proteins that, in addition to TCTE1 Is Required for Progressive Movement in Spermatozoa. Male linking adjacent outer doublets, is also thought to regulate the infertility could result from defects in spermatogenesis up to di- motor activity in dynein (7). minished fertilization ability once the spermatozoa reaches the egg Failure to assemble or regulate flagellar function in sperma- (10). To determine if a block in spermatogenesis was the reason of tozoa leads to defective motility (asthenozoospermia) and fer- male infertility, we examined spermatogenesis in Tcte1 null males. tility defects, including male sterility (8). Mutations in multiple Gross examination of testis revealed no difference in appearance, genes in mouse models have been identified that disrupt the testis weight, and sperm counts between homozygous and control formation of the flagellum or the function of the flagellum (9, littermates (Fig. S3 A and B). Using the hematoxylin–eosin (H&E) 10). For example, mutations in Akap4, Tekt3, Tekt4, and Cabyr in staining method, no difference was observed in spermatogenesis mice lead to structural defects in the flagellum, whereas muta- between wild-type control and null testes (Fig. 1 F and G). Fur- tions in CatSper1, Pgk2, Gapdhs, and Ldhc lead to functional thermore, meiotic progression and acrosome formation did not re- defects in the flagellum, some of which cause defective metab- veal a difference between the two genotypes (Fig. S3 C and D). olism or glycolysis (11–18). In men, mutations in CATSPER, When examining epididymis sections, both genotypes contained DNAH1, DNAH11, and TEKT2 have been shown to lead to tubules full of spermatozoa in the cauda and caput regions of the asthenozoospermia and sterility (19–22). Here, we report that a epididymis (Fig. 1 H and I). In addition, spermatozoa morphology mutation in mouse T-Complex-Associated–Testis-Expressed 1 appears normal in Tcte1 KOs (Fig. 1 J and K). Taken together, these [Tcte1; the ortholog of the Chlamydomonas N-DRC component observations suggest that a block in spermatogenesis is not the Drc5 (23)] leads to male sterility characterized by asthenozoo- reason for male infertility observed in Tcte1 KOs. − − spermia and decreased metabolism within sperm. To determine the sperm quality in Tcte1 / males, we exam- ined sperm function using a Computer Assisted Sperm Analyzer Results (CASA). CASA analysis revealed decreased forwardly motile Tcte1 Is a Testis-Enriched Gene Required for Male Fertility.
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