Planar cell polarity protein Dishevelled 3 (Dvl3) regulates ectoplasmic specialization (ES) dynamics in the testis through changes in cytoskeletal organization

Li et al. Cell Death and Disease (2019)10:194 https://doi.org/10.1038/s41419-019-1394-7 ARTICLE Cell Death & Disease Open Access Planar cell polarity protein Dishevelled 3 (Dvl3) regulates ectoplasmic specialization (ES) dynamics in the testis through changes in cytoskeletal organization 1234567890():,; 1234567890():,; 1234567890():,; 1234567890():,; Linxi Li1,2, Baiping Mao1,2, Ming Yan2,3, Siwen Wu1,2, Renshan Ge1, Qingquan Lian1 and C. Yan Cheng 1,2 Abstract In the mammalian testes, such as in rats, the directional alignment of polarized elongating/elongated spermatids, in particular step 17–19 spermatids, across the plane of seminiferous epithelium resembles planar cell polarity (PCP) found in hair cells of the cochlea. It is obvious that spermatid PCP is necessary to support the simultaneous development of maximal number of elongating/elongated spermatids to sustain the daily production of > 50 million sperm per adult rat. Studies have shown that the testis indeed expresses multiple PCP proteins necessary to support spermatid PCP. Herein, using physiological and biochemical assays, and morphological analysis, and with the technique of RNA interference (RNAi) to knockdown PCP protein Dishevelled (Dvl) 1 (Dvl1), Dvl2, Dvl3, or Dvl1/2/3, Dvl proteins, in particular Dvl3, it was shown that Dvl3 played a crucial role of support Sertoli cell tight junction (TJ)permeability barrier function through changes in the organization of actin- and microtubule (MT)-based cytoskeletons. More important, an in vivo knockdown of Dvl1/2/3 in the testis, defects of spermatid polarity were remarkably noted across the seminiferous epithelium, concomitant with defects of spermatid adhesion and spermatid transport, leading to considerably defects in spermatogenesis. More important, Dvl1/2/3 triple knockdown in the testis also impeded the organization of actin- and MT-based cytoskeletons owing to disruptive spatial expression of actin- and MT-regulatory proteins. In summary, PCP Dishevelled proteins, in particular, Dvl3 is a regulator of Sertoli cell blood–testis barrier (BTB) and also spermatid PCP function through its effects on the actin- and MT-based cytoskeletons in Sertoli cells. Introduction During spermatogenesis, developing step 17–19 spermatids in the rat testis displays conspicuous planar cell polarity (PCP)1. It is noted that the alignment of polarized developing spermatids in stage V–VIII tubules, with their heads and tails point towards the basement Correspondence: Qingquan Lian () or C. Yan Cheng () 1 The Second Affiliated Hospital and Yuying Children’s Hospital, Wenzhou Medical University, Wenzhou, Zhejiang 325027, China 2 The Mary M. Wohlford Laboratory for Male Contraceptive Research, Center for Biomedical Research, Population Council, 1230 York Ave, New York 10065, USA Full list of author information is available at the end of the article. These authors contributed equally: Linxi Li, Baiping Mao Edited by E. Candi membrane and seminiferous tubule lumen, respectively, across the plane of the seminiferous epithelium1,2, resembles the directional alignment of hair from hair cells of the inner ear in mammals known as PCP3–5. This unusual alignment of developing spermatids across the epithelium thus packs the maximum number of spermatids in a restricted surface area of the epithelium to support the production of millions of sperm on a daily basis from an adult male2,6. As such, the fixed population of Sertoli cells in adult testes7 can nurture the simultaneous development of millions of germ cells with a Sertoli:germ cell ratio of ~1:30–1:508. It is also necessary to provide orderly interactions between Sertoli cells and spermatids in the microenvironment of the epithelium © The Author(s) 2019 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Official journal of the Cell Death Differentiation Association Li et al. Cell Death and Disease (2019)10:194 behind the blood–testis barrier (BTB) to support the developing germ cells structurally, functionally, and nutritionally2,6,9. Studies have shown that the testis is equipped with multiple PCP proteins necessary to confer spermatid PCP, such as the PCP core proteins Van Goghlike (Vangl) proteins (e.g., Vangl2), Dishevelled (Dvl) (e.g., Dvl2, Dvl3), and Frizzled (Fzd) class receptors (e.g., Fzd3, Fzd5)10. It is now established that PCP protein Vangl2 is necessary to support spermatogenesis through its regulatory effects on actin- and microtubule (MT)based cytoskeletons10. More important, Vangl2 knockdown in the testis in vivo was found to perturb spermatogenesis considerably, including spermatid exfoliation, but also unwanted retentions of spermatid 19 spermatids in post-stage VIII tubules as spermatid 19 spermatids were found in the epithelium together with step 9, 10, and 11 spermatids in stage IX, X, and XI tubules10. Studies from other animal models (in particular insects, worms, and flies) and epithelia have shown that Vangl2/ Prickle and Fzd/Dvl are two primary PCP protein complexes wherein Vangl2 and Fzd are integral membrane proteins whereas Prickle and Dvl are the corresponding primary adaptor proteins; and these two PCP protein complexes are mutually exclusive regarding their distribution and functionally11–14. To better understand the role of PCP proteins in spermatogenesis, we reported herein results of a series of experiments that delineated the role of Dvl3 (i.e., the adaptor proteins of the integral membrane protein family Fzd) in the testis. The selection of Dvl3 instead of Dvl1 and Dvl2 for more detailed analysis was based on initial observations that its knockdown by RNAi led to considerably more disruptive effects on the Sertoli cell TJ-barrier function compared to Dvl1 and Dvl2. However, for our in vivo studies, Dvl1/2/3 were simultaneously silenced by RNAi to confirm changes in phenotypes, correlating the function of Dvl to support spermatogenesis. Materials and methods Animals Adult Sprague–Dawley rats at 250–275 gm b.w. and male pups at 16 days of age were obtained from Charles River Laboratories (Kingston, NY). Adult rats were housed in groups of two in the same cage, and 10 male pups will be housed with a fos (...truncated)