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

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.

NC1‐peptide regulates spermatogenesis through changes in cytoskeletal organization mediated by EB1

“…In control testes, the MT‐specific minus (−) end directed motor protein dynein 1 (red fluorescence) and the PCP protein Dishevelled 3 (Dvl3, red fluorescence) co‐localized with MTs (visualized by α‐tubulin staining, green fluorescence) that stretched across the seminiferous epithelium as track‐like structures that laid perpendicular to the basement membrane (annotated by dashed white line; Figure ). Following NC1‐peptide overexpression, their spatial distribution was grossly disrupted.…”

“…For histological analysis, assessment of changes in MT organization (staining for α‐tubulin), and distribution of MARK4 and EB1, testes were fixed in modified Davidson fixative and embedded in paraffin to obtain sections (5 µm in thickness) using a microtome as described . For visualization of dynein 1, Dvl3 and their co‐localization with α‐tubulin, testes were fixed in Bouin's fixative and embedded in paraffin to obtain sections (5 µm in thickness) with a microtome . For visualization of distribution of F‐actin, Eps8 and Arp3 (and their co‐localization), frozen sections (5 µm thickness) were obtained the corresponding specific antibodies as noted in Table , and F‐actin was visualized using Alexa Fluor 488‐phalloidin conjugate as described .…”

“…[74][75][76][77] Based on these observations, we speculate that EB1 may be working in concert with the cell polarity proteins (such as Par6, Crbs3 33,36 ) and PCP proteins (such as Dvl3 30 ) to coordinate with NC1-peptide which exerts its effects on the cytoskeletal organization of MTs and F-actin since Par6, Crb3 and Dvl3 are earlier shown to exert their regulating effects in the testis through changes in MT and F-actin organization based on in vitro and/or in vivo studies. 30,33,36 Also, future investigations should also expand this observation to examine if EB2 and EB3, perhaps other +TIPs such as CLIP-170, 38 are also involved in NC1-peptide mediated effects on spermatogenesis in the testis, or EB1 is playing a more predominant role in NC1-peptide action in the testis.…”

“…16,28 For visualization of dynein 1, Dvl3 and their co-localization with α-tubulin, testes were fixed in Bouin's fixative and embedded in paraffin to obtain sections (5 µm in thickness) with a microtome. 29,30 For visualization of distribution of F-actin, Eps8 and Arp3 (and their co-localization), frozen sections (5 µm thickness) were obtained the corresponding specific antibodies as noted in Table 1, and F-actin…”

Combover interacts with the axonemal component Rsp3 and is required for sperm individualization

“…Orthologs of the PCP proteins Dsh and Vang are emerging as important regulators of mammalian spermatogenesis. For instance, knockdown of PCP proteins Vangl2 or Dvl1-3 in rat leads to defects in transport and maturation of sperm across the seminiferous epithelium due to disrupted actin and microtubule cytoskeletons in somatic Sertoli cells, which results at least in part from altered expression of cytoskeletal regulatory proteins Chen et al, 2018;Li et al, 2019). To our knowledge, no male fertility phenotype has been reported for PCP genes in Drosophila.…”