Sperm calcineurin inhibition prevents mouse fertility with implications for male contraceptive

Miyata, Haruhiko; Satouh, Yuhkoh; Mashiko, Daisuke; Muto, Masanaga; Nozawa, Kaori; Shiba, Kogiku; Fujihara, Yoshitaka; Isotani, Ayako; Inaba, Kazuo; Ikawa, Masahito

doi:10.1126/science.aad0836

Cited by 145 publications

(196 citation statements)

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“…Despite diminished progressive movement, the sperm tails from null mice can beat; however, the range of flagellar beating is limited and appears to be asymmetric compared with that in wild type (Movies S1 and S2). Closer inspection of the sperm tails shows an inflexible midpiece (Movie S3) (30). The inflexible midpiece and asymmetric beating flagellum may account for the circular motion observed in KO sperm ( Fig.…”

Section: Resultsmentioning

confidence: 99%

TCTE1 is a conserved component of the dynein regulatory complex and is required for motility and metabolism in mouse spermatozoa

Castaneda

Hua

Miyata

et al. 2017

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

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Flagella and cilia are critical cellular organelles that provide a means for cells to sense and progress through their environment. The central component of flagella and cilia is the axoneme, which comprises the "9+2" microtubule arrangement, dynein arms, radial spokes, and the nexin-dynein regulatory complex (N-DRC). Failure to properly assemble components of the axoneme leads to defective flagella and in humans leads to a collection of diseases referred to as ciliopathies. Ciliopathies can manifest as severe syndromic diseases that affect lung and kidney function, central nervous system development, bone formation, visceral organ organization, and reproduction. T-Complex-Associated-Testis-Expressed 1 (TCTE1) is an evolutionarily conserved axonemal protein present from Chlamydomonas (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 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 flagellum. Knockout of Tcte1 results in male sterility because Tcte1-null spermatozoa show aberrant motility. Although the axoneme is structurally normal in Tcte1 mutant spermatozoa, Tcte1-null sperm demonstrate a significant decrease of ATP, which is used by dynein motors to generate the bending force of the flagellum. These data provide a link to defining the molecular intricacies required for axoneme function, sperm motility, and male fertility. male infertility | asthenozoospermia | glycolysis | mutant mouse | testis-specific gene F lagella are ancient, analogous cellular structures used for locomotion and as sensory organelles present in all three domains of life (bacteria, archaea, and eukaryotes). The advantages conferred by this organelle are highlighted by the flagella's apparent independent evolution in all three domains (1-3). Of all of the different flagella present among eukaryotes, flagella attached to gametes play a critical function in uniting gametes for fertilization and the perpetuation of a species. Mammalian spermatozoa have a specialized flagellum that contains a midpiece, principal piece, and end piece with the axoneme running along the entire length (4). The flagellum equips sperm with the capability to deliver half of the male's genetic material to the female gamete, the oocyte. In addition to flagella, eukaryotes contain another related structure called cilia. The defining feature of flagella and cilia is the axoneme, the "9+2" microtubule arraignment of two central pairs of microtubules surrounded by nine pairs of microtubule doublets (5). The microtubule motor dynein is anchored to the outer microtubules and responsible for generating the force required to produce the beating pattern of flagella and cilia (6). The force generated by dynein causes sliding of the microtubules among each other; however, the nexin complex anchors the microtubules in place. The nexin complex [or nexin-dynei...

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Section: Resultsmentioning

confidence: 99%

TCTE1 is a conserved component of the dynein regulatory complex and is required for motility and metabolism in mouse spermatozoa

Castaneda

Hua

Miyata

et al. 2017

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

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“…Alternatively, without a preceding functional assay in vitro, many testis-enriched genes proved to be essential, by KO, for male reproduction. For example, TEX14 is required for intercellular bridge formation and progression through meiosis (7), GASZ functions in the piRNA pathway and is required for suppression of retrotransposons (8), CATSPER1 is a voltage-dependent calcium channel required for sperm hyperactivated motility (9), CLGN is an endoplasmic reticulum chaperone protein essential for sperm migration into the oviduct and fertilization (10), and PPP3R2 is a testis-specific regulatory subunit of calcineurin required for normal motility of the sperm midpiece (11). Taking these facts into account, it would be better to know first whether a gene of interest is essential in vivo before deciding to invest valuable research dollars and experimental time and personnel efforts into the project.…”

Section: Significancementioning

confidence: 99%

Genome engineering uncovers 54 evolutionarily conserved and testis-enriched genes that are not required for male fertility in mice

Miyata

Castaneda

Fujihara

et al. 2016

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

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Gene-expression analysis studies from Schultz et al. estimate that more than 2,300 genes in the mouse genome are expressed predominantly in the male germ line. As of their 2003 publication [Schultz N, Hamra FK, Garbers DL (2003) Proc Natl Acad Sci USA 100(21):12201–12206], the functions of the majority of these testis-enriched genes during spermatogenesis and fertilization were largely unknown. Since the study by Schultz et al., functional analysis of hundreds of reproductive-tract–enriched genes have been performed, but there remain many testis-enriched genes for which their relevance to reproduction remain unexplored or unreported. Historically, a gene knockout is the “gold standard” to determine whether a gene’s function is essential in vivo. Although knockout mice without apparent phenotypes are rarely published, these knockout mouse lines and their phenotypic information need to be shared to prevent redundant experiments. Herein, we used bioinformatic and experimental approaches to uncover mouse testis-enriched genes that are evolutionarily conserved in humans. We then used gene-disruption approaches, including Knockout Mouse Project resources (targeting vectors and mice) and CRISPR/Cas9, to mutate and quickly analyze the fertility of these mutant mice. We discovered that 54 mutant mouse lines were fertile. Thus, despite evolutionary conservation of these genes in vertebrates and in some cases in all eukaryotes, our results indicate that these genes are not individually essential for male mouse fertility. Our phenotypic data are highly relevant in this fiscally tight funding period and postgenomic age when large numbers of genomes are being analyzed for disease association, and will prevent unnecessary expenditures and duplications of effort by others.

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“…Recently, Miyata et al (2015) demonstrated that a sperm specific calcineurin is responsible for the transition that releases the ODFs during epididymal maturation of the sperm. Furthermore, they showed that specific inhibition of the process by calcineurin inhibitors renders the sperm of normal mice stiff in the midpiece.…”

Section: How the Sperm Tail Workmentioning

confidence: 99%

Functional anatomy of the mammalian sperm flagellum

2016

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The eukaryotic flagellum is the organelle responsible for the propulsion of the male gamete in most animals. Without exception, sperm of all mammalian species use a flagellum for swimming. The mammalian sperm has a centrally located 9 1 2 arrangement of microtubule doublets and hundreds of accessory proteins that together constitute an axoneme. However, they also possess several characteristic peri-axonemal structures that make the mammalian sperm tail function differently. These modifications include nine outer dense fibers (ODFs) that are paired with the nine outer microtubule doublets of the axoneme, and are anchored in a structure called the connecting piece located at the base. The presence of the ODFs and connecting piece, and the absence of a basal body, dictate that physical forces generated by the dynein motors are transmitted to the base of the flagellum through the ODFs. Mammalian sperm flagella also possess a mitochondrial and a fibrous sheath that encircle most of the axoneme. These sheaths and the ODFs add mechanical rigidity to the flagellum creating the functional effect of increasing bend wavelength, which requires the entrainment of more dynein motors in the production of a single wave. The sheaths also act as a retinaculum and maintain the integrity of the central axoneme when large bending torques are generated by dynein. Large torque production is crucial to the process of hyperactivation and the unique motility transitions associated with effective fertilizing capacity. Consequently, these specialized anatomical features are essential for the effective interaction of sperm with the female reproductive tract and ovum. V C 2016 Wiley Periodicals, Inc.Key Words: axoneme; outer dense fibers; fibrous sheath; outer doublets; dynein; connecting piece; hyperactivation; sperm motility Introduction E ukaryotic cilia and flagella are highly conserved and ubiquitous organelles present in most animals, as well as many lower plants and eukaryotic protozoa. In the vertebrate lineage, the eukaryotic flagellum is universally employed to propel the male gamete. In the mammalian branch of the vertebrates, the sperm tail is always a modified flagellum with some characteristic accessory features that are added onto the basic 9 1 2 axoneme of microtubules found in most cilia and flagella. Figure 1 shows transmission electron micrographs (TEMs) of a mouse sperm flagellum in cross section at several positions along the length of the flagellum. The central axoneme is visible with its nine outer doublets surrounding a pair of single central microtubules (central pair or CP). Each of the outer microtubule doublets bears two rows of projections called the inner row and outer row of dynein arms. These arms are composed of the dynein motor proteins that power motility. The dynein arms are the source of the motive force that bends the flagellum. The dynein heavy chains (DHC) of the arms form bridges between the outer doublets and undergo a Mg-ATP driven power stroke that generates a sliding (or shearing) action between t...

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Sperm calcineurin inhibition prevents mouse fertility with implications for male contraceptive

Cited by 145 publications

References 38 publications

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 regulatory complex and is required for motility and metabolism in mouse spermatozoa

Genome engineering uncovers 54 evolutionarily conserved and testis-enriched genes that are not required for male fertility in mice

Functional anatomy of the mammalian sperm flagellum

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