CEP164 is essential for efferent duct multiciliogenesis and male fertility

in Reproduction
View More View Less
  • 1 Molecular and Cellular Biology Graduate Program, Stony Brook University, Stony Brook, New York, USA
  • 2 Department of Pharmacological Sciences, Stony Brook University, Stony Brook, New York, USA
  • 3 Department of Comparative Biosciences, College of Veterinary Medicine, University of Illinois, Urbana, Illinois, USA

Correspondence should be addressed to K-I Takemaru; Email: ken-ichi.takemaru@stonybrook.edu
Restricted access

Cilia are evolutionarily conserved microtubule-based structures that perform diverse biological functions. Cilia are assembled on basal bodies and anchored to the plasma membrane via distal appendages. In the male reproductive tract, multicilia in efferent ducts (EDs) move in a whip-like motion to prevent sperm agglutination. Previously, we demonstrated that the distal appendage protein CEP164 recruits Chibby1 (Cby1) to basal bodies to facilitate basal body docking and ciliogenesis. Mice lacking CEP164 in multiciliated cells (MCCs) (FoxJ1-Cre;CEP164fl/fl) show a significant loss of multicilia in the trachea, oviduct, and ependyma. In addition, we observed male sterility; however, the precise role of CEP164 in male fertility remained unknown. Here, we report that the seminiferous tubules and rete testis of FoxJ1-Cre;CEP164fl/fl mice exhibit substantial dilation, indicative of dysfunctional multicilia in the EDs. We found that multicilia were hardly detectable in the EDs of FoxJ1-Cre;CEP164fl/fl mice although FoxJ1-positive immature cells were present. Sperm aggregation and agglutination were commonly noticeable in the lumen of the seminiferous tubules and EDs of FoxJ1-Cre;CEP164fl/fl mice. In FoxJ1-Cre;CEP164fl/fl mice, the apical localization of Cby1 and the transition zone marker NPHP1 was severely diminished, suggesting basal body docking defects. TEM analysis of EDs further confirmed basal body accumulation in the cytoplasm of MCCs. Collectively, we conclude that male infertility in FoxJ1-Cre;CEP164fl/fl mice is caused by sperm agglutination and obstruction of EDs due to loss of multicilia. Our study, therefore, unravels an essential role of the distal appendage protein CEP164 in male fertility.

Supplementary Materials

    • Fig S1. Sperm agglutination in the efferent ducts of FoxJ1-Cre; CEP164fl/fl mice. IF staining of ED sections with the lectin PNA (green). Nuclei were detected by DAPI. The boxed areas are magnified on the bottom. Scale bars, 500 µm and 50 µm (magnified images). Note that profound sperm agglutinations are commonly found in the EDs of FoxJ1-Cre;CEP164 mice (arrowheads). Arrows point towards the testis (T) or the epididymis (E).
    • Fig S2. RNA levels of FoxJ1 and CEP164 during spermatogenesis. The datasets for spermatogenic gene expression were retrieved from Ernst et al. (Ernst et al. 2019) through their website (https://marionilab.cruk.cam.ac.uk/SpermatoShiny/). The data are represented by a standard box-whisker plot with black dots representing outliers. SG, spermatogonia; eP, early-pachytene spermatocyte; mP, mid-pachytene spermatocyte; lP, late-pachytene spermatocyte; D, diplotene spermatocytes; M, meiosis; and S1-11, step 1-11 spermatids.
    • Fig S3. Validation of NPHP1 antibody. (A) Western blot analysis for ectopic NPHP1 in HEK293T cells either transfected with HA-tagged NPHP1 or empty vector. -Actin was used as a loading control. (B) Western blot analysis of endogenous NPHP1 in HEK293T cells and mouse testis. (C) IF staining of ED sections for either NPHP1 antibody, pre-adsorbed NPHP1 antibody, or buffer only. Nuclei were detected by DAPI. Scale bar, 10 µm.
    • Table S1. List of antibodies used for this study

 

     An official journal of

    Society for Reproduction and Fertility

 

Sept 2018 onwards Past Year Past 30 Days
Abstract Views 240 240 240
Full Text Views 43 43 43
PDF Downloads 57 57 57
  • Al Jord A, Spassky N & Meunier A 2019 Motile ciliogenesis and the mitotic prism. Biology of the Cell 111 199212. (https://doi.org/10.1111/boc.201800072)

    • Search Google Scholar
    • Export Citation
  • Anvarian Z, Mykytyn K, Mukhopadhyay S, Pedersen LB & Christensen ST 2019 Cellular signalling by primary cilia in development, organ function and disease. Nature Reviews: Nephrology 15 199219. (https://doi.org/10.1038/s41581-019-0116-9)

    • Search Google Scholar
    • Export Citation
  • Arbi M, Pefani DE, Kyrousi C, Lalioti ME, Kalogeropoulou A, Papanastasiou AD, Taraviras S & Lygerou Z 2016 GemC1 controls multiciliogenesis in the airway epithelium. EMBO Reports 17 400413. (https://doi.org/10.15252/embr.201540882)

    • Search Google Scholar
    • Export Citation
  • Badran HH & Hermo LS 2002 Expression and regulation of aquaporins 1, 8, and 9 in the testis, efferent ducts, and epididymis of adult rats and during postnatal development. Journal of Andrology 23 358373. (https://doi.org/10.1002/j.1939-4640.2002.tb02243.x)

    • Search Google Scholar
    • Export Citation
  • Blatt EN, Yan XH, Wuerffel MK, Hamilos DL & Brody SL 1999 Forkhead transcription factor HFH-4 expression is temporally related to ciliogenesis. American Journal of Respiratory Cell and Molecular Biology 21 168176. (https://doi.org/10.1165/ajrcmb.21.2.3691)

    • Search Google Scholar
    • Export Citation
  • Bork K, Chevrier C, Paquignon M, Jouannet P & Dacheux J 1988 Flagellar motility and movement of boar spermatozoa during epididymal transit. Reproduction, Nutrition, Development 28 13071315.

    • Search Google Scholar
    • Export Citation
  • Brooks ER & Wallingford JB 2014 Multiciliated cells. Current Biology 24 R973R982. (https://doi.org/10.1016/j.cub.2014.08.047)

  • Burke MC, Li FQ, Cyge B, Arashiro T, Brechbuhl HM, Chen X, Siller SS, Weiss MA, O'Connell CB & Love D et al. 2014 Chibby promotes ciliary vesicle formation and basal body docking during airway cell differentiation. Journal of Cell Biology 207 123137. (https://doi.org/10.1083/jcb.201406140)

    • Search Google Scholar
    • Export Citation
  • Clulow J, Jones RC, Hansen LA & Man SY 1998 Fluid and electrolyte reabsorption in the ductuli efferentes testis. Journal of Reproduction and Fertility: Supplement 53 114.

    • Search Google Scholar
    • Export Citation
  • Czarnecki PG & Shah JV 2012 The ciliary transition zone: from morphology and molecules to medicine. Trends in Cell Biology 22 201210. (https://doi.org/10.1016/j.tcb.2012.02.001)

    • Search Google Scholar
    • Export Citation
  • Dacheux JL & Dacheux F 2014 New insights into epididymal function in relation to sperm maturation. Reproduction 147 R27R42. (https://doi.org/10.1530/REP-13-0420)

    • Search Google Scholar
    • Export Citation
  • Dacheux JL, Belleannee C, Guyonnet B, Labas V, Teixeira-Gomes AP, Ecroyd H, Druart X, Gatti JL & Dacheux F 2012 The contribution of proteomics to understanding epididymal maturation of mammalian spermatozoa. Systems Biology in Reproductive Medicine 58 197210. (https://doi.org/10.3109/19396368.2012.663233)

    • Search Google Scholar
    • Export Citation
  • Danielian PS, Hess RA & Lees JA 2016 E2F4 and E2f5 are essential for the development of the male reproductive system. Cell Cycle 15 250260. (https://doi.org/10.1080/15384101.2015.1121350)

    • Search Google Scholar
    • Export Citation
  • de Kretser DM, Loveland KL, Meinhardt A, Simorangkir D & Wreford N 1998 Spermatogenesis. Human Reproduction 13 (Supplement 1) 18. (https://doi.org/10.1093/humrep/13.suppl_1.1)

    • Search Google Scholar
    • Export Citation
  • Ernst C, Eling N, Martinez-Jimenez CP, Marioni JC & Odom DT 2019 Staged developmental mapping and X chromosome transcriptional dynamics during mouse spermatogenesis. Nature Communications 10 1251. (https://doi.org/10.1038/s41467-019-09182-1)

    • Search Google Scholar
    • Export Citation
  • Fishman EL, Jo K, Nguyen QPH, Kong D, Royfman R, Cekic AR, Khanal S, Miller AL, Simerly C & Schatten G et al. 2018 A novel atypical sperm centriole is functional during human fertilization. Nature Communications 9 2210. (https://doi.org/10.1038/s41467-018-04678-8)

    • Search Google Scholar
    • Export Citation
  • Garcia TX, Farmaha JK, Kow S & Hofmann MC 2014 RBPJ in mouse Sertoli cells is required for proper regulation of the testis stem cell niche. Development 141 44684478. (https://doi.org/10.1242/dev.113969)

    • Search Google Scholar
    • Export Citation
  • Gervasi MG & Visconti PE 2017 Molecular changes and signaling events occurring in spermatozoa during epididymal maturation. Andrology 5 204218. (https://doi.org/10.1111/andr.12320)

    • Search Google Scholar
    • Export Citation
  • Goetz SC & Anderson KV 2010 The primary cilium: a signalling centre during vertebrate development. Nature Reviews: Genetics 11 331344. (https://doi.org/10.1038/nrg2774)

    • Search Google Scholar
    • Export Citation
  • Graser S, Stierhof YD, Lavoie SB, Gassner OS, Lamla S, Le Clech M & Nigg EA 2007 Cep164, a novel centriole appendage protein required for primary cilium formation. Journal of Cell Biology 179 321330. (https://doi.org/10.1083/jcb.200707181)

    • Search Google Scholar
    • Export Citation
  • Griswold MD 1998 The central role of Sertoli cells in spermatogenesis. Seminars in Cell and Developmental Biology 9 411416. (https://doi.org/10.1006/scdb.1998.0203)

    • Search Google Scholar
    • Export Citation
  • Griswold MD 2016 Spermatogenesis: the commitment to meiosis. Physiological Reviews 96 117. (https://doi.org/10.1152/physrev.00013.2015)

    • Search Google Scholar
    • Export Citation
  • Hess RA 2015 Small tubules, surprising discoveries: from efferent ductules in the turkey to the discovery that estrogen receptor alpha is essential for fertility in the male. Animal Reproduction 12 723.

    • Search Google Scholar
    • Export Citation
  • Hess RA 2018 Efferent ductules: structure and function. In Encyclopedia of Reproduction, 2nd ed. Ed Skinner MKOxford: Academic Press, pp. 270278. (https://doi.org/10.1016/B978-0-12-801238-3.64593-2)

    • Search Google Scholar
    • Export Citation
  • Horani A, Ferkol TW, Dutcher SK & Brody SL 2016 Genetics and biology of primary ciliary dyskinesia. Paediatric Respiratory Reviews 18 1824. (https://doi.org/10.1016/j.prrv.2015.09.001)

    • Search Google Scholar
    • Export Citation
  • James ER, Carrell DT, Aston KI, Jenkins TG, Yeste M & Salas-Huetos A 2020 The role of the epididymis and the contribution of epididymosomes to mammalian reproduction. International Journal of Molecular Sciences 21 5377. (https://doi.org/10.3390/ijms21155377)

    • Search Google Scholar
    • Export Citation
  • Knowles MR, Daniels LA, Davis SD, Zariwala MA & Leigh MW 2013 Primary ciliary dyskinesia. Recent advances in diagnostics, genetics, and characterization of clinical disease. American Journal of Respiratory and Critical Care Medicine 188 913922. (https://doi.org/10.1164/rccm.201301-0059CI)

    • Search Google Scholar
    • Export Citation
  • Lalioti ME, Arbi M, Loukas I, Kaplani K, Kalogeropoulou A, Lokka G, Kyrousi C, Mizi A, Georgomanolis T & Josipovic N et al. 2019 GemC1 governs multiciliogenesis through direct interaction with and transcriptional regulation of p73. Journal of Cell Science 132 jcs228684. (https://doi.org/10.1242/jcs.228684)

    • Search Google Scholar
    • Export Citation
  • Leigh MW, Pittman JE, Carson JL, Ferkol TW, Dell SD, Davis SD, Knowles MR & Zariwala MA 2009 Clinical and genetic aspects of primary ciliary dyskinesia/Kartagener syndrome. Genetics in Medicine 11 473487. (https://doi.org/10.1097/GIM.0b013e3181a53562)

    • Search Google Scholar
    • Export Citation
  • Lewis M & Stracker TH 2020 Transcriptional regulation of multiciliated cell differentiation. Seminars in Cell and Developmental Biology 110 5160. (https://doi.org/10.1016/j.semcdb.2020.04.007)

    • Search Google Scholar
    • Export Citation
  • Li FQ, Mofunanya A, Harris K & Takemaru K 2008 Chibby cooperates with 14-3-3 to regulate beta-catenin subcellular distribution and signaling activity. Journal of Cell Biology 181 11411154. (https://doi.org/10.1083/jcb.200709091)

    • Search Google Scholar
    • Export Citation
  • Li FQ, Mofunanya A, Fischer V, Hall J & Takemaru K-I 2010 Nuclear-cytoplasmic shuttling of chibby controls β-catenin signaling. Molecular Biology of the Cell 21 311322. (https://doi.org/10.1091/mbc.e09-05-0437)

    • Search Google Scholar
    • Export Citation
  • Li FQ, Siller SS & Takemaru KI 2015 Basal body docking in airway ciliated cells. Oncotarget 6 1994419945. (https://doi.org/10.18632/oncotarget.4609)

    • Search Google Scholar
    • Export Citation
  • Li FQ, Chen X, Fisher C, Siller SS, Zelikman K, Kuriyama R & Takemaru KI 2016 BAR domain-containing FAM92 proteins interact with Chibby1 to facilitate ciliogenesis. Molecular and Cellular Biology 36 26682680. (https://doi.org/10.1128/MCB.00160-16)

    • Search Google Scholar
    • Export Citation
  • Liu Q, Zhang Q & Pierce EA 2010 Photoreceptor sensory cilia and inherited retinal degeneration. In Retinal Degenerative Diseases, pp. 223232. Springer.

    • Search Google Scholar
    • Export Citation
  • Ma L, Quigley I, Omran H & Kintner C 2014 Multicilin drives centriole biogenesis via E2F proteins. Genes and Development 28 14611471. (https://doi.org/10.1101/gad.243832.114)

    • Search Google Scholar
    • Export Citation
  • Nakata H, Wakayama T, Takai Y & Iseki S 2015 Quantitative analysis of the cellular composition in seminiferous tubules in normal and genetically modified infertile mice. Journal of Histochemistry and Cytochemistry 63 99113. (https://doi.org/10.1369/0022155414562045)

    • Search Google Scholar
    • Export Citation
  • Nigg EA & Raff JW 2009 Centrioles, centrosomes, and cilia in health and disease. Cell 139 663678. (https://doi.org/10.1016/j.cell.2009.10.036)

    • Search Google Scholar
    • Export Citation
  • Quarmby LM & Parker JD 2005 Cilia and the cell cycle? Journal of Cell Biology 169 707710. (https://doi.org/10.1083/jcb.200503053)

  • Reiter JF & Leroux MR 2017 Genes and molecular pathways underpinning ciliopathies. Nature Reviews: Molecular Cell Biology 18 533547. (https://doi.org/10.1038/nrm.2017.60)

    • Search Google Scholar
    • Export Citation
  • Reiter JF, Blacque OE & Leroux MR 2012 The base of the cilium: roles for transition fibres and the transition zone in ciliary formation, maintenance and compartmentalization. EMBO Reports 13 608618. (https://doi.org/10.1038/embor.2012.73)

    • Search Google Scholar
    • Export Citation
  • Russell LD, Ettlin RA, Hikim APS & Clegg ED 1993 Histological and histopathological evaluation of the testis. International Journal of Andrology 16 8383. (https://doi.org/10.1111/j.1365-2605.1993.tb01156.x)

    • Search Google Scholar
    • Export Citation
  • Satir P & Christensen ST 2007 Overview of structure and function of mammalian cilia. Annual Review of Physiology 69 377400. (https://doi.org/10.1146/annurev.physiol.69.040705.141236)

    • Search Google Scholar
    • Export Citation
  • Schmidt KN, Kuhns S, Neuner A, Hub B, Zentgraf H & Pereira G 2012 Cep164 mediates vesicular docking to the mother centriole during early steps of ciliogenesis. Journal of Cell Biology 199 10831101. (https://doi.org/10.1083/jcb.201202126)

    • Search Google Scholar
    • Export Citation
  • Siller SS, Sharma H, Li S, Yang J, Zhang Y, Holtzman MJ, Winuthayanon W, Colognato H, Holdener BC & Li FQ et al. 2017 Conditional knockout mice for the distal appendage protein CEP164 reveal its essential roles in airway multiciliated cell differentiation. PLoS Genetics 13 e1007128. (https://doi.org/10.1371/journal.pgen.1007128)

    • Search Google Scholar
    • Export Citation
  • Spassky N & Meunier A 2017 The development and functions of multiciliated epithelia. Nature Reviews: Molecular Cell Biology 18 423436. (https://doi.org/10.1038/nrm.2017.21)

    • Search Google Scholar
    • Export Citation
  • Stubbs JL, Vladar EK, Axelrod JD & Kintner C 2012 Multicilin promotes centriole assembly and ciliogenesis during multiciliate cell differentiation. Nature Cell Biology 14 140147. (https://doi.org/10.1038/ncb2406)

    • Search Google Scholar
    • Export Citation
  • Terre B, Piergiovanni G, Segura-Bayona S, Gil-Gomez G, Youssef SA, Attolini CS, Wilsch-Brauninger M, Jung C, Rojas AM & Marjanovic M et al. 2016 GEMC1 is a critical regulator of multiciliated cell differentiation. EMBO Journal 35 942960. (https://doi.org/10.15252/embj.201592821)

    • Search Google Scholar
    • Export Citation
  • Terré B, Lewis M, Gil-Gómez G, Han Z, Lu H, Aguilera M, Prats N, Roy S, Zhao H & Stracker TH 2019 Defects in efferent duct multiciliogenesis underlie male infertility in GEMC1-, MCIDAS- or CCNO-deficient mice. Development 146 dev162628. (https://doi.org/10.1242/dev.162628)

    • Search Google Scholar
    • Export Citation
  • Yu X, Ng CP, Habacher H & Roy S 2008 Foxj1 transcription factors are master regulators of the motile ciliogenic program. Nature Genetics 40 14451453. (https://doi.org/10.1038/ng.263)

    • Search Google Scholar
    • Export Citation
  • Yuan S, Liu Y, Peng H, Tang C, Hennig GW, Wang Z, Wang L, Yu T, Klukovich R & Zhang Y et al. 2019 Motile cilia of the male reproductive system require miR-34/miR-449 for development and function to generate luminal turbulence. PNAS 116 35843593. (https://doi.org/10.1073/pnas.1817018116)

    • Search Google Scholar
    • Export Citation
  • Zhang Y, Huang G, Shornick LP, Roswit WT, Shipley JM, Brody SL & Holtzman MJ 2007 A transgenic FOXJ1-Cre system for gene inactivation in ciliated epithelial cells. American Journal of Respiratory Cell and Molecular Biology 36 515519. (https://doi.org/10.1165/rcmb.2006-0475RC)

    • Search Google Scholar
    • Export Citation
  • Zhou F, Narasimhan V, Shboul M, Chong YL, Reversade B & Roy S 2015 Gmnc is a master regulator of the multiciliated cell differentiation program. Current Biology 25 32673273. (https://doi.org/10.1016/j.cub.2015.10.062)

    • Search Google Scholar
    • Export Citation
  • Zhou W, De Iuliis GN, Dun MD & Nixon B 2018 Characteristics of the epididymal luminal environment responsible for sperm maturation and storage. Frontiers in Endocrinology 9 59. (https://doi.org/10.3389/fendo.2018.00059)

    • Search Google Scholar
    • Export Citation