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EXOC1 plays an integral role in spermatogonia pseudopod elongation and spermatocyte stable syncytium formation in mice

  1. Yuki Osawa
  2. Kazuya Murata
  3. Miho Usui
  4. Yumeno Kuba
  5. Hoai Thu Le
  6. Natsuki Mikami
  7. Toshinori Nakagawa
  8. Yoko Daitoku
  9. Kanako Kato
  10. Hossam Hassan Shawki
  11. Yoshihisa Ikeda
  12. Akihiro Kuno
  13. Kento Morimoto
  14. Yoko Tanimoto
  15. Tra Thi Huong Dinh
  16. Ken-ichi Yagami
  17. Masatsugu Ema
  18. Shosei Yoshida
  19. Satoru Takahashi
  20. Seiya Mizuno  Is a corresponding author
  21. Fumihiro Sugiyama
  1. Master’s Program in Medical Sciences, Graduate School of Comprehensive Human Sciences, University of Tsukuba, Japan
  2. Laboratory Animal Resource Center, Trans-border Medical Research Center, University of Tsukuba, Japan
  3. School of Medical Sciences, University of Tsukuba, Japan
  4. Ph.D Program in Human Biology, School of Integrative and Global Majors, University of Tsukuba, Japan
  5. Division of Germ Cell Biology, National Institute for Basic Biology, National Institutes of Natural Sciences, Japan
  6. Department of Basic Biology, School of Life Science, Graduate University for Advanced Studies (Sokendai), Japan
  7. Department of Comparative and Experimental Medicine, Nagoya City University Graduate School of Medical Sciences, Japan
  8. Doctoral program in Biomedical Sciences, Graduate School of Comprehensive Human Sciences, University of Tsukuba, Japan
  9. Doctoral program in Medical Sciences, Graduate School of Comprehensive Human Sciences, University of Tsukuba, Japan
  10. Department of Stem Cells and Human Disease Models, Research Center for Animal Life Science, Shiga University of Medical Science, Japan
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Cite this article as: eLife 2021;10:e59759 doi: 10.7554/eLife.59759

Abstract

The male germ cells must adopt the correct morphology at each differentiation stage for proper spermatogenesis. The spermatogonia regulates its differentiation state by its own migration. The male germ cells differentiate and mature with the formation of syncytia, failure of forming the appropriate syncytia results in the arrest at the spermatocyte stage. However, the detailed molecular mechanisms of male germ cell morphological regulation are unknown. Here, we found that EXOC1, a member of the Exocyst complex, is important for the pseudopod formation of spermatogonia and spermatocyte syncytia in mice. EXOC1 contributes to the pseudopod formation of spermatogonia by inactivating the Rho family small GTPase Rac1 and also functions in the spermatocyte syncytia with the SNARE proteins STX2 and SNAP23. Since EXOC1 is known to bind to several cell morphogenesis factors, this study is expected to be the starting point for the discovery of many morphological regulators of male germ cells.

Introduction

In mammalian testis, sperms are continuously produced in the seminiferous tubules throughout a male’s life. There are two types of cells in the seminiferous tubule, somatic cells (Sertoli cells) which support spermatogenesis and male germ cells which undergo various processes to become spermatogonia, spermatocytes, spermatids, and eventually spermatozoa. Structurally, the Sertoli cell tight junction (SCTJ) divides the seminiferous tubule into two compartments, the basal compartment and the luminal compartment (Tsukita et al., 2008). Spermatogonia cells are located in the basal compartment and undergo proliferation and differentiation by mitosis. The cells that have differentiated into spermatocytes migrate to the luminal compartment and differentiate into haploid spermatocytes by meiosis (Smith and Braun, 2012). These spermatocytes then undergo dynamic morphological changes to become mature sperms that are released into the lumen of the seminiferous tubule. As this entire differentiation process is cyclical and continuous, long-term stable spermatogenesis is supported by spermatogonial stem cells.

Murine spermatogonia are heterogeneous in their differential state and can be classified as undifferentiated and differentiating (Fayomi and Orwig, 2018; Suzuki et al., 2009). During spermatogenesis, germ cells undergo an incomplete cytoplasmic division. In most somatic cell types, daughter cells completely separate from each other, but in germ cells, the final separation is inhibited and they differentiate while maintaining intercellular bridges (ICBs) (Iwamori et al., 2010). Such structures, called syncytia, are thought to contribute to syncytial differentiation and maturation by the sharing of mRNAs and proteins that are halved during meiosis via ICB (Morales et al., 2002; Fawcett et al., 1959). Undifferentiated spermatogonia are a morphologically heterogeneous population that includes Asingle as isolated cells, Apair as two connected syncytia, and Aaligned as three or more connected cells. Furthermore, a large number of stem cells that contribute to long-term spermatogenesis are included within the GFRα1+ undifferentiated spermatogonia (mainly Asingle and Apair, and fewer Aaligned), although whether the stem cell compartment comprises of all or only a subset of GFRα1+ population remains under debate (Lord and Oatley, 2017). The GFRα1+ cells produce NGN3+/RARγ+ subset of undifferentiated spermatogonia (composed of more Aaligned, and fewer Asingle and Apair) (Nakagawa et al., 2007) that differentiate into differentiating spermatogonia (Kit+) in response to retinoic acid. These undifferentiated spermatogonia move within the basal compartment of the seminiferous tubules and regulate the balance between self-renewal and differentiation by competing for fibroblast growth factors (FGFs) that are secreted from deferent niches (Kitadate et al., 2019). Although transplantation studies using in vitro cultured spermatogonial stem cells have reported that Rac1-mediated cell migration is important for spermatogonial stem cell homing (Takashima et al., 2011), the molecules that are involved in spermatogonia migration and their mechanisms have not been clarified (Kanamori et al., 2019).

TEX14 is an important molecule for stability of ICBs, which are essential for maintaining the structure of syncytia. This protein localizes to germline ICBs and inhibits the final separation of the cytoplasm; in mice lacking this gene, the cytoplasm of male germ cells is completely separated causing spermatogenesis to be halted (Greenbaum et al., 2006). This result indicates that the formation of syncytium is essential for spermatogenesis. Seminolipids, which are sulfated glycolipids, are important for the sustenance of ICB in spermatocytes. This is evident in mice lacking the Ugt8a and Gal3st1, required for seminolipids synthesis, as they show an aggregation of spermatocytes (Fujimoto et al., 2000; Honke et al., 2002). Although the detailed molecular mechanism is unknown, a similar phenotype was observed in mice lacking the Stx2 gene (Fujiwara et al., 2013), which functions in the transport of seminolipids to the cell membrane. In humans, similar to that of mice, the loss of function of STX2 results in the failure of spermatogenesis with spermatocyte aggregation (Nakamura et al., 2018).

In this study, we focus on the exocyst complex, a heterodimeric protein complex composed of eight subunits (EXOC1–EXOC8) that are widely conserved from yeast to humans (Koumandou et al., 2007). Exocyst-mediated vesicle trafficking is crucial for various fundamental cellular phenomena such as cell division, morphogenesis, and cell migration. In cytokinesis, the exocyst is localized to ICB (Neto et al., 2013) and is required for the transport of factors required for the final separation of the plasma membrane (Kumar et al., 2019). The exocyst has also been implicated in cell migration via the reconstitution of the actin cytoskeleton (Liu et al., 2012; Parrini et al., 2011).

Although studies using gene-deficient mouse models have shown that the exocyst is important for embryo development (Mizuno et al., 2015; Friedrich et al., 1997; Fogelgren et al., 2015; Dickinson et al., 2016), its tissue-specific functions in adults are largely unknown. In Drosophila models, the exocyst plays an important role in the formation of both female and male gametes (Giansanti et al., 2015; Wan et al., 2019; Murthy and Schwarz, 2004; Mao et al., 2019). In mice, all exocyst subunits are expressed in male germ cells at each stage of differentiation (Green et al., 2018), and although it is predicted that these subunits may be involved in mammalian spermatogenesis, the function of these subunits is completely unknown. Therefore, in this study, we generated and analyzed a mouse model deficient in Exoc1, an exocyst subunit expressed in male germ cells.

Results

EXOC1 expression in mouse testis

The expression of Exoc1 mRNA has been observed in Sertoli cells and the germ cells of mouse testes at each stage: spermatogonia, spermatocytes, round spermatids, and elongating spermatids (Green et al., 2018; Figure 1—figure supplement 1A and Figure 1—figure supplement 2). To analyze the expression of EXOC1 at the protein level, we generated two types of genome-edited mice in which the PA-Tag sequence was knocked in at the C-terminus of Exoc1 (Exoc1PA-C) or the N-terminus (Exoc1PA-N) using the CRISPR-Cas system (Figure 1A) as immunofluorescence using commercially available antibodies for EXOC1 failed to detect any signal. The LG3 sequence, which is a flexible linker, was used as there was no successful case for producing a knock-in (KI) homozygous mutant implying that the functionality of EXOC1 has not been lost (Ahmed et al., 2018). In both strains, mice with the intended KI sequences were obtained (Figure 1—figure supplement 1B), and both heterozygous mutants showed no abnormal phenotypes. We attempted to produce homozygous mutants by intercrossing the hetero KI mutants of each strain. In the Exoc1PA-C strain, no homozygous mutants were obtained (0/20). By contrast, in the Exoc1PA-N strain, homozygous mutants were obtained (9/27), and no abnormal phenotypes were found in them. Furthermore, progenies were obtained from crosses between Exoc1PA-N homozygous mutants. As the Exoc1 knock-out (KO) mice are early embryonic lethal (Mizuno et al., 2015), this result suggests that the addition of the PA-tag connected to the N-terminus, linked by LG3, does not impair the function of EXOC1. We performed western blot analyses for the PA-tag in the testes of both Exoc1PA-C and Exoc1PA-N adult (10–20-week-old) mice and confirmed a band of the molecular size as expected (Figure 1B and Figure 1—source data 1). The signal intensity of the band, on the western blot, was lower for Exoc1+/PA-N than that for Exoc1+/PA-C. This intensity difference may be due to differences in their kinetics, structure, subcellular localization, and so on. Since the Exoc1PA-N homozygous mutant, unlike the Exoc1PA-C homozygous mutant, did not show a pronounced abnormal phenotype, we considered that EXOC1-PA-N is probably more similar in behavior and function to the wild-type EXOC1 than EXOC1-PA-C. In immunofluorescence, EXOC1-PA was also detected in all male germ cells observed in the Exoc1+/PA-C adult mice (Figure 1C and Figure 1—figure supplement 1C), while no signal could be detected in the Exoc1PA-N/PA-N mice using any method. In Exoc1+/PA-C adult mice, PA signals were also detected in Sertoli cells, which are located at the basal compartment of the seminiferous tubules and whose nucleus are euchromatic with a large nucleolus (França et al., 2016). These data indicate that EXOC1 protein is expressed in male mouse germ and Sertoli cells.

Figure 1 with 3 supplements see all
Confirmation of EXOC1 expression in testes using the PA-Tag knock-in mouse.

(A) Generation of PA-Tag knock-in mice. In the Exoc1PA-C allele, the LG3-linker connected PA-tag gene fragment was knocked-in just before the stop codon of Exoc1 using CRISPR-Cas9. In the Exoc1PA-N allele, the LG3-connected PA-tag gene fragment was knocked in just after the start codon of Exoc1 using CRISPR-Cas12a. Bold letters represent the CRISPR-Cas9 and Cas12a target sequence. (B) Western blotting of PA-Tag antibody demonstrated that each C- and N-terminal PA-tagged EXOC1 protein was expressed in the adult testes (n = 2 in each genotype). (C) Immunofluorescence with PA-Tag antibody. EXOC1 is observed in every cell in the adult testes. The arrowheads indicate Sertoli cells in which the nucleus is eurochromatin with a large nucleolus. Scale bars: 50 μm.

Impaired spermatogenesis in Exoc1 conditional KO mice

Exoc1 deficiency results in peri-implantation embryonic lethality in mice (Mizuno et al., 2015). In this study, we analyzed the function of Exoc1 in spermatogenesis using Exoc1 conditional knockout (cKO) mice obtained by crossing Exoc1-floxed mice (Figure 1—figure supplement 3) with Nanos3-Cre driver that expresses Cre in spermatogonia (Suzuki et al., 2008). To investigate the spermatogenic potential of Exoc1 cKO mice, PNA-lectin staining to detect acrosome was performed; however, no signal was detected in the Exoc1 cKO adult mice (Figure 2A). When cell morphology was observed by H and E staining, although almost no spermatids and spermatozoa were observed in the testes of Exoc1 cKO mice, large multinucleated cells were observed in the lumen of seminiferous tubules (Figure 2B and C). During spermatogenesis, male germ cells form syncytia and are interconnected via stable ICB (Iwamori et al., 2010). Detailed observations by scanning electron microscopy revealed ICB in control mice, while multinucleated cells that lacked ICB structures were observed in Exoc1 cKO mice (Figure 2D). As the observed multinucleated cells appeared to be aggregates of syncytia, they were named ‘AGS’ for the purpose of this study. To confirm the frequency of AGS in the Exoc1 cKO adult mice, H and E-stained sections of the seminiferous tubules were classified into three categories: sections containing neither AGS nor sperm, sections containing AGS, and sections containing sperm but no AGS; and counted (Figure 2—figure supplement 1A). More than 70% of the sections contained AGS, and approximately 20% contained neither AGS nor sperm (Figure 2—figure supplement 1B and C). Although less than 10% of the cross-sections contained sperms, these sperms were considered to be derived from spermatogonia that did not undergo Cre-LoxP recombination. We confirmed the genotype of the generated blastocysts by in vitro fertilization of wild-type oocytes with sperms from the Exoc1 cKO or control (Exoc1flox/-) mice and found embryos with the cKO allele in the control group but not in the Exoc1 cKO group (0/16) (Figure 2—figure supplement 1D). To determine at which stage of differentiation the AGS emerge from, immunofluorescence using male germ cell markers for each differentiation stage were used. γH2AX, a spermatocyte marker for the pachytene stage, was observed in the nuclei of AGS (Figure 2E). In contrast, no aggregation was observed in the differentiating spermatogonia, Kit+ syncytia (0/214) (Figure 2F and Supplementary file 1a). As the seminiferous tubules are divided into two compartments by the SCTJ, spermatogonia located within the basal compartment migrate to the luminal compartment when they become spermatocytes (Smith and Braun, 2012). In order to investigate the arrangement of AGS in the seminiferous tubules, H and E staining and immunofluorescence for CLDN11, a major component of SCTJ (Morita et al., 1999), were performed, and all of the 336 AGS in Exoc1 cKO adult mice were observed within the luminal compartment (Figure 2G). These data suggest that the AGS seen in Exoc1 cKO mice are spermatocytes.

Figure 2 with 2 supplements see all
Impaired spermatogenesis in Exoc1 cKO mice.

(A) PNA-lectin staining of the Exoc1 adult cKO testis. Signals of PNA-lectin, an acrosomal marker was not observed in the Exoc1 cKO testis. Scale bars: 50 μm. (B) The macroscopic images for H and E staining. Normal spermatogenesis was not observed in almost all of the seminiferous tubules in the Exoc1 cKO adult testis. Scale bars: 300 μm. (C) The mesoscopic images for H and E staining of the Exoc1 cKO testis. Large and circular cells, appearing to be aggregates of syncytia (AGS), containing multiple nuclei were observed in the lumen of seminiferous tubules (arrowheads). Scale bars: 100 μm. Control: Exoc1flox/wt:: Nanos3+/Cre adult mice. (D) SEM observation of the Exoc1 cKO adult testis. Intercellular bridges (ICBs) (arrows) were found in the syncytia in the control (Exoc1flox/wt:: Nanos3+/Cre) testis. There were no ICB observed in the AGS of Exoc1 cKO. Scale bars: 5 μm. (E) The serial section overlay image of Exoc1 cKO adult testis. There were γ-H2AX (marker of spermatocyte) signals in the AGS nucleus. Scale bars: 50 μm. (F) A representative immunofluorescence image of an Exoc1 cKO seminiferous tubule. Kit+ syncytia, which are observed in differentiating spermatogonia, have ICB. Scale bars: 50 μm. (G) H and E staining and immunofluorescence with CLDN11. CLDN11-positive Sertoli cell tight junction (SCTJ) divides the space between the basal and the luminal compartment, and AGS are present within the luminal compartment (arrowheads). Scale bars: 50 μm.

In the pachytene stage, chromosome synapsis occurs and the synaptonemal complex is formed (Heyting, 1996). To confirm this synapsis, chromosomes of spermatocytes from Exoc1 adult cKO mice were spread and immunofluorescence was performed using antibodies against SYCP1 and SYCP3, which are components of the synaptonemal complex. SYCP1 and SYCP3 signals were co-localized in autosomal pairs of the spermatocytes from Exoc1 cKO mice. These results suggest that meiosis proceeds normally to synapsis in Exoc1-deficient spermatocytes (Figure 2—figure supplement 2).

EXOC1 regulates ICB formation in cooperation with STX2 and SNAP23

The spermatocytes of Stx2repro34 mice, a null mutant of the Stx2 gene encoding the SNARE protein, exhibit multinucleated AGS (Fujiwara et al., 2013). Both Stx2repro34 and Exoc1 cKO mice show similar phenotypes of multinucleated AGS and normal chromosome synapsis (Fujiwara et al., 2013). In addition, in yeast, Sec3 (an ortholog of mouse Exoc1) promotes membrane fusion between transport vesicles and the cell membrane by coordinating with Sso2 (an ortholog of mouse Stx2) and another SNARE protein, Sec9 (an ortholog of mouse Snap23) (Yue et al., 2017). Therefore, we hypothesized that EXOC1 is required for normal syncytia formation in mouse spermatocytes to function in cooperation with STX2 and SNAP23. In yeast, Sec3 directly interacts with Sso2 and promotes the Sso2–Sec9 binary complex formation (Yue et al., 2017). As there were no previous reports regarding the interaction between EXOC1 and STX2 in mice, we analyzed the binding of EXOC1-STX2 and STX2-SNAP23 by the co-immunoprecipitation (Co-IP) of HEK293T cells overexpressing EXOC1, STX2, and SNAP23 from mice with FLAG, HA, and MYC epitope tags, respectively. The binding of the three proteins was confirmed in all in vitro experiments where each protein was immunoprecipitated (Figure 3A and Figure 3—source data 1). We then confirmed the binding of SNAP23 to EXOC1 in the adult Exoc1PA-N/PA-N testis with anti-Snap23 antibody available for immunoprecipitation. Consequently, it was confirmed that EXOC1 binds to SNAP23 in vivo (Figure 3B and Figure 3—source data 2). These results, consistent with those reported in yeast (Yue et al., 2017), suggested that mouse EXOC1 could bind to STX2 and STX2 to SNAP23.

Figure 3 with 2 supplements see all
EXOC1 regulates ICB formation in cooperation with STX2 and SNAP23.

(A) Co-immunoprecipitation of EXOC1-STX2-SNAP23 complex in vitro. FLAG-tagged mouse EXOC1, HA-tagged mouse STX2, and Myc-tagged mouse SNAP23 were co-overexpressed in HEK293T cells. The binding of the three factors was confirmed in all combinations of Co-IP experiments. FLAG-EGFP, V5-mCherry-HA, and E2 crimson-MYC-His were used as negative controls. (B) Interaction of EXOC1 with SNAP23 in vivo. PA-tagged EXOC1 was co-immunoprecipitated with endogenous SNAP23 in the adult Exoc1PA-N testis. The upper and middle panels show short and long period exposure images, respectively. Arrowhead indicates PA-EXOC1. (C) The macroscopic images for H and E staining. Sperms were found in frequent seminiferous tubules in adult Snap23 cKO mice. Scale bars: 300 μm. Control: Snap23flox/wt:: Nanos3+/Cre mice. (D) The mesoscopic images for H and E staining of Snap23 cKO adult testis. Large and circular cells containing multiple nuclei (arrowheads), appearing to be aggregates of syncytia (AGS) were observed. In contrast with Exoc1 cKO, every seminiferous tubule had sperms with elongated nuclei (arrows). Scale bars: 50 μm. Control: Snap23flox/wt:: Nanos3+/Cre mice. (E) The serial section overlay image of Snap23 cKO testis. Immunofluorescence signals of γ-H2AX were found in AGS. Scale bars: 50 μm. (F) PNA-lectin staining of Snap23 cKO testis. PNA-lectin that was used to detect the acrosome is observed in the lumen of the seminiferous tubule of Snap23 cKO testis. Scale bars: 50 μm.

Figure 3—source data 1

Raw data of the in vitro immunoprecipitation.

https://cdn.elifesciences.org/articles/59759/elife-59759-fig3-data1-v1.pptx
Figure 3—source data 2

Raw data of the in vivo immunoprecipitation.

https://cdn.elifesciences.org/articles/59759/elife-59759-fig3-data2-v1.pptx
Figure 3—source data 3

Occurrence rate of AGS per area in the extracted Section containing AGS.

https://cdn.elifesciences.org/articles/59759/elife-59759-fig3-data3-v1.xlsx

The Co-IP described above confirmed that EXOC1, STX2, and SNAP23 interact in mice, and given the hypothesis that EXOC1 functions in cooperation with STX2 and SNAP23 to form normal syncytium of spermatocytes. To test this hypothesis, we conducted a functional analysis of SNAP23. As Snap23 KO mice have been reported to be embryonically lethal (Suh et al., 2011) and the effect of Snap23 depletion on adult male germ cells was unknown, we generated Snap23 cKO mice specifically for male germ cells under the control of Nanos3 expression (Figure 3—figure supplement 1) and tested for the appearance of spermatocyte AGS in the adult testes. H and E staining and immunofluorescence showed that AGS with γH2AX signals were observed in Snap23 cKO mice (Figure 3C–E). However, as many spermatocytes did not aggregate in Snap23 cKO mice, spermatozoa were also observed by H and E and PNA-lectin staining (Figure 3D and F). We evaluated the appearance of AGS in Snap23 cKO adult mice by the same method as in the Exoc1 cKO mice and found sperms in more than 70% of the seminiferous tubule sections (Figure 3—figure supplement 2A and B). Although AGS were observed in about 10% of the sections, the number of AGS per area in those sections was approximately 10% of that in the Exoc1 cKO mice (Figure 3—figure supplement 2C and Figure 3—source data 3). This suggests that Snap23 is dispensable for spermatogenesis and that another protein in the SNAP family could be compensating for that function. Alternatively, although the analyzed ages, floxed distances (Figure 1—figure supplement 3 and Figure 3—figure supplement 1), and Cre driver strains were almost exactly the same, the possibility that this phenotypic difference was due to Cre-LoxP recombination efficiency could not be completely excluded. In any case, Snap23 is partially responsible for the formation or maintenance of the spermatocyte syncytium. These results suggest that EXOC1 regulates the formation of the correct syncytium structure in cooperation with STX2 and SNAP23.

EXOC1 regulates the pseudopod elongation via Rac1 inactivation

As Nanos3-Cre mice express CRE from spermatogonia (Suzuki et al., 2008), the Exoc1 cKO (Exoc1Flox/Flox:: Nanos3+/Cre) mice used in this study were models in which the Exoc1 gene was deficient from spermatogonia. Therefore, we investigated the effects of Exoc1 deficiency in spermatogonia. In a study of cultured cells, the exocyst complex was noted to contribute to cell migration by promoting actin assembly (Liu et al., 2012) and GFRα1+ undifferentiated spermatogonia were observed to be moving within the basal compartment of the seminiferous tubules (Hara et al., 2014). For these reasons, we carried out detailed morphological observations of GFRα1+ spermatogonia using confocal microscopy. The pseudopods of GFRα1+ spermatogonia in Exoc1 cKO adult mice were significantly shorter than that of control mice (Figure 4A). In the formation of syncytia in spermatocytes, Exoc1 is likely to function in concert with Stx2 (Figure 3). In contrast, as there are no reports on the function of Stx2 in the formation of pseudopods in spermatogonia, it is unclear whether Exoc1 cooperates with Stx2 in this phenomenon as well. Therefore, we generated Stx2 KO mice using the CRISPR-Cas9 system (Figure 4—figure supplement 1A and B) and performed detailed morphological observations of GFRα1+ spermatogonia. In the Stx2 KO mice generated in this study, as in the Stx2repro34 mice (Fujiwara et al., 2013), spermatocytes were observed to aggregate (Figure 4—figure supplement 1C). Pseudopod length was quantitatively assessed by Sholl analysis: GFRα1+ spermatogonia in Exoc1 cKO mice had significantly shorter pseudopods than those of wild-type and Stx2 KO (Figure 4B and Figure 4—source data 1). The Stx2 KO pseudopods tended to be shorter than wild-type pseudopods (p=0.052) (Figure 4A and B and Figure 4—source data 1). These results suggest that EXOC1 functions in the pseudopod elongation of GFRα1+ spermatogonia partially dependently, but not completely dependently of STX2. The GFRα1+ cell population, which should be a component of the spermatogonial stem cell pool, is mostly Asingle and Apair, with a minority of Aaligned (Hara et al., 2014). The elongation of the pseudopod in each of these morphological states was confirmed through whole-mount immunofluorescence staining with adult Exoc1 cKO mice (Figure 4C). In this experiment, pseudopod elongation was inhibited in GFRα1+ Asingle spermatogonia of the Exoc1 cKO group compared to the control group (Figure 4D and Figure 4—source data 2). The distance between the connected cells of Apair and Aaligned was significantly shorter in GFRα1+ Apair spermatogonia in the Exoc1 cKO group than in the control group (Figure 4E and F and Figure 4—source data 2).

Figure 4 with 1 supplement see all
EXOC1 regulates pseudopod elongation via Rac1 inactivation.

(A) A representative image of GFRα1+ undifferentiated spermatogonia in Exoc1 cKO and Stx2 KO. Pseudopod elongation was impaired in Exoc1 cKO, but not in Stx2 KO. Scale bars: 10 μm. (B) Pseudopod length quantification using sections. Average length of GFRα1+ spermatogonia pseudopods in Exoc1 cKO was shorter than that of Stx2 KO and wild type (n = 3 in each genotype, 25–36 cells in each mouse). *p=0.052, **p=1.8 × 10−6, ***p=9.5 × 10−9. one-way ANOVA. (C) Pseudopod length quantification through whole-mount immunofluorescence staining of adult testes. GFRα1+ spermatogonia with elongated pseudopod (white arrows) were frequently observed in control (Exoc1flox/cKO) mice, whereas they were rarely observed in Exoc1 cKO mice. Scale bars: 30 μm. (D) Measurement of the length of pseudopod of Asingle GFRα1+ cells based on whole-mount immunofluorescence images (n = 3 in each genotype, 20 cells in each mouse). *p=7.2 × 10−17, Student’s t-test. Control: Exoc1flox/cKO. (E, F) Measurement of intercellular length in connected Apair (n = 3 in each genotype, 20 intercellular distances in each mouse) or Aaligned (n = 3 in each genotype, 14–19 intercellular distances in each mouse) based on whole-mount immunofluorescence images. *p=3.6 × 10−12, **p=0.00014, Student’s t-test. Control: Exoc1flox/cKO. (G) A representative image of active-Rac1 in GFRα1+ spermatogonia of Exoc1 cKO adult testis. In control mice (Exoc1flox/wt:: Nanos3+/Cre), active-Rac1 signal was lower than the detection limit. Non-polar active-Rac1 signal was detected in Exoc1 cKO adult testis. Scale bars: 5 μm. (H) Quantification of signal intensity of active-Rac1 in GFRα1+ spermatogonia based on immunostaining images. The average intensity in each cell is higher in the Exoc1 cKO group than that in the control group (n = 3 in genotype, 8–10 cells in each mouse). *p=0.000036, Student’s t-test. Control: Exoc1flox/flox.

Figure 4—source data 1

Measurement of the length of the pseudopodia of GFRα1+ cells in section observation.

https://cdn.elifesciences.org/articles/59759/elife-59759-fig4-data1-v1.xlsx
Figure 4—source data 2

Measurement of the length of the pseudopodia of GFRα1+ cells in whole-mount observation.

https://cdn.elifesciences.org/articles/59759/elife-59759-fig4-data2-v1.xlsx
Figure 4—source data 3

Intensity of active Rac1 signal in each cell.

https://cdn.elifesciences.org/articles/59759/elife-59759-fig4-data3-v1.xlsx

We examined the molecular mechanism by which EXOC1 functions in GFRα1+ spermatogonia pseudopod elongation. Although the exocyst contributes to pseudopod formation by promoting Arp2/3-mediated actin assembly (Liu et al., 2012), Arp3 is not expressed in spermatogonia (Lie et al., 2010). Thus, we focused on the Rho family GTPase Rac1, which regulates pseudopod elongation and cell migration by regulating the reconstruction of the actin cytoskeleton (Raftopoulou and Hall, 2004). The exocyst is required for the transport of SH3BP1, which converts Rac1 from its active to inactive form, and when the exocyst is impaired in cultured human cells, Rac1 is over-activated and pseudopod elongation and cell migration are inhibited (Parrini et al., 2011). Therefore, immunofluorescence for active-Rac1 was performed to examine Rac1 activity in GFRα1+ spermatogonia of Exoc1 cKO adult mice, and as expected, the fluorescence intensity of active-Rac1 tended to be stronger for the plasma membrane of GFRα1+ spermatogonia of Exoc1 cKO mice compared to control mice (Figure 4G and H, and Figure 4—source data 3). Therefore, EXOC1 may function in GFRα1+ spermatogonia pseudopod elongation by negatively regulating the activity of Rac1.

Exoc1 functions to regulate the differentiation of spermatogonia

GFRα1+ cell migration dictates the state of spermatogonia differentiation (Kitadate et al., 2019). Therefore, we counted the numbers of GFRα1+ cells and RARγ+ cells, which are one differentiation step from GFRα1+ cells (Kitadate et al., 2019). Immunofluorescence with antibodies to GFRα1 and RARγ showed a significant increase in the number of GFRα1+ cells and a significant decrease in the number of RARγ+ cells in Exoc1 cKO adult mice compared to the control mice (Figure 5A and B and Figure 5—source data 1). In addition, no noticeable difference in the total number of both cells (Figure 5B). Additionally, the number of Kit+ differentiating spermatogonia significantly decreased in the Exoc1 cKO group (Figure 5—figure supplement 1 and Figure 5—source data 2). Taken together, these results suggest that Exoc1 strongly regulates spermatogonial differentiation but not proliferation as much.

Figure 5 with 1 supplement see all
The balance of spermatogonial differentiation is perturbed in Exoc1 cKO.

(A) Representative immunofluorescence image of adult Exoc1 cKO seminiferous tubule. GFRα1+ spermatogonia (arrow) density in the cKO was higher than that in control mice. RARγ+ spermatogonia (arrowhead) density decreased in the cKO. Control: Exoc1flox/flox mice. (B) The number of spermatogonia in a cross-section of seminiferous tubule (n = 3 in each genotype, 46–87 sections in each mouse). The number of GFRα1+ cell per section was significantly increased in the cKO. The number of RARγ+ cell in the cKO was significantly smaller than that in control (Exoc1flox/flox) mice. *p=1.9 × 10−7, **p=3.6 × 10−9, ***p=0.035, Student’s t-test.

Discussion

This study is the first to show that EXOC1 plays an essential role in spermatogenesis, in particular the formation of germ cell morphology, in the elongation of the pseudopod in spermatogonia, and in the formation of syncytium in spermatocytes.

Exoc1 cKO mice showed overactivity of Rac1 and disturbance of differentiation balance in spermatogonia. However, the presence of the next stage of differentiation, spermatocytes, suggests that abnormalities in the spermatogonia are not a direct factor in the failure of spermatogenesis exhibited by the Exoc1 cKO mice. In contrast, as spermatocytes failed to maintain ICB and aggregated, there were no germ cells observed after spermatid stage. Since chromosome synapsis is suggested to be normal in meiosis, the most significant factor in spermatogenesis failure may be the aggregation of spermatocytes. ICB complex proteins bind to small RNAs, suggesting that ICB may be involved in epigenetic regulation as well as the morphological formation of spermatocytes (Iwamori et al., 2020); in Exoc1 cKO spermatocytes, this epigenetic regulation may be responsible for the arrest of spermatogenesis.

There are three possible reasons for spermatocyte aggregation due to impaired vesicle transport via EXOC1, STX2, and SNAP23. First, the impaired transport of sulfated glycolipids: 90% of the glycolipids in the testes are seminolipids (Ishizuka, 1997), and there are spermatocyte aggregates even in mice lacking the genes required for seminolipid biosynthesis (Ugt8a and Gal3st1) (Fujimoto et al., 2000; Honke et al., 2002). As the localization of seminolipids to the plasma membrane is impaired in spermatocytes of Stx2repro34 mice (Fujiwara et al., 2013), similarly in Exoc1 cKO mice, impaired seminolipid transport may have caused for the aggregation of the spermatocytes. Second, insufficient membrane addition: cytokinesis requires the expansion of the surface area of the cell with the formation of cleavage furrow. The exocyst localizes to ICB for local membrane addition (Neto et al., 2013), and mutant Drosophila, which lacks Exoc8 function, shows little expansion of spermatocyte surface area causing the emergence of a defective cytokinetic ring in spermatocytes (Giansanti et al., 2015). This suggests that spermatocyte aggregation in Exoc1 cKO mice is due to an impaired supply of cell membranes to facilitate cytokinesis. Third, the impaired transport of endosomal sorting complex required for transport (ESCRT) proteins that functions in cell separation: in cytokinesis, cells are separated by ESCRT proteins after the cleavage furrow is squeezed by a contractile ring (Elia et al., 2011). In cultured human cells, depletion of EXOC3 and EXOC4 impairs the transport of CHMP2B and CHMP4B, subunits of the ESCRT III complex, to ICB and causes multinucleation (Kumar et al., 2019). This suggests that the exocyst complex is more important for recruitment of the ESCRT III complex to the ICB than for formation of ICB and that disruption of this recruitment in Exoc1 cKO spermatocytes may be responsible for the impaired secondary ingression event (Agromayor and Martin-Serrano, 2013) in cytokinesis. On the Mouse Genome Informatics database (http://www.informatics.jax.org/), there are 10 genes encoding the ESCRT III complex subunit, and two of them (Chmp2b and Chmp4c) KO mice are fertile (Dickinson et al., 2016). The function of the other eight genes in spermatogenesis has not been analyzed (Coulter et al., 2018; Lee et al., 2007). Further research will be needed to test these possibilities.

Analysis of the differentiation status of spermatogonia showed an increase in GFRα1+ spermatogonia and a decrease in RARγ+ spermatogonia in Exoc1 cKO mice, suggesting that spermatogonia are biased toward undifferentiated maintenance. In addition, the pseudopod of the GFRα1+ spermatogonia was shorter in the Exoc1 cKO mice and the fluorescence intensity of active-Rac1 was stronger. The exocyst is required for the transport of SH3BP1, which converts Rac1 from its active to inactive form, and over-activation of Rac1 inhibits cell migration (Parrini et al., 2011). Therefore, EXOC1 may regulate pseudopod elongation and migration in the undifferentiated spermatogonia by inactivating Rac1 via SH3BP1 transport; since there are no reports of Sh3bp1 KO mice, it will be necessary to investigate whether pseudopod elongation and migration are impaired when Sh3bp1 is deficient in undifferentiated spermatogonia. In addition, since undifferentiated spermatogonia move within the basal compartment of the seminiferous tubules and regulate the balance between self-renewal and differentiation by competing with each other for FGFs (Kitadate et al., 2019), undifferentiated spermatogonia migration may be important for maintaining the balance between self-renewal and differentiation. The disruption of this balance seen in Exoc1 cKO mice may have been caused secondary to impaired pseudopod elongation of the cells. In other words, undifferentiated spermatogonia, which normally move within the basal compartment of the seminiferous tubules, replicate themselves near FGFs-producing cells, and differentiate into differentiation-primed spermatogonia by leaving FGFs-producing cells via cell migration. In contrast, Exoc1 deficiency results in insufficient inactivation of Rac1 in undifferentiated spermatogonia and impaired its pseudopod elongation. As a result, undifferentiated spermatogonia that are restricted by migration may remain physically close to FGFs-producing cells and thus repeatedly self-renew, limiting their differentiation. It is still unclear whether all GFRα1+ cells are included in the self-renewing spermatogonial stem cell compartment (Lord and Oatley, 2017). Moreover, at present, it is unclear whether Exoc1 contributes to the fate determination of all spermatogonial stem cells, and thus, further analysis is required.

Materials and methods

Key resources table
Reagent type
(species) or
resource
DesignationSource or
reference
IdentifiersAdditional
information
Strain, strain background (M. musculus)C57BL/6JCharles River Laboratories JapanStock No: 000664
Strain, strain background (M. musculus)Crl:CD1(ICR)Charles River Laboratories Japan
Genetic reagent (M. musculus)Exoc1 floxThis paperThis is from the Exoc1tm1a(EUCOMM)Hmgu. IKMC Project #78575
Genetic reagent (M. musculus)Exoc1PA-CThis paper
Genetic reagent (M. musculus)Exoc1PA-NThis paper
Genetic reagent (M. musculus)Snap23 floxThis paperThis is from the Snap23tm1a(EUCOMM)Wts. Colony Name BLA3054
Genetic reagent (M. musculus)Stx2 KOThis paper
Genetic reagent (M. musculus)Nanos3tm2 (cre)YsaSuzuki et al., 2008Prof. Saga, RIKEN
BRC (RDB13130)
RBRC02568
Genetic reagent (M. musculus)B6;SJL-Tg(ACTFLPe)9205Dym/JThe Jackson LaboratoryStock No: 003800
Cell line
(Human)
HEK293T cellATCCATCC Sales Order: SO0623448FTA Barcode: STRB4056
AntibodyMonoclonal anti-PA-tag, Biotin conjugated (Rat)FUJIFILM Wako ChemicalsCat#017–27731WB (1:500)
AntibodyPolyclonal anti-b-actin (Rabbit)MEDICAL and BIOLOGICAL LABORATORIESCat#PM053WB (1:3000)
AntibodyMonoclonal anti-DYKDDDDK tag (Mouse)FUJIFILM Wako ChemicalsCat# 018–22381WB (1:2000)
AntibodyMonoclonal anti-DYKDDDDK tag (Rat)FUJIFILM Wako ChemicalsCat# 018–23621WB (1:2000)
AntibodyMonoclonal anti-HA-tag (Rabbit)Cell Signaling TechnologyCat#3724WB (1:2000)
AntibodyMonoclonal anti-HA-tag (Mouse)BioLegendCat#901513WB (1:1000)
AntibodyMonoclonal anti-Myc (Mouse)MEDICAL and BIOLOGICAL LABORATORIESCat#M192-3WB (1:1000)
AntibodyMonoclonal anti-SNAP23 (Mouse)Santa Cruz BiotechnologyCat#sc-166244WB (1:1000)
AntibodyAnti-Rat IgG, HRP-linked (Goat)GE HealthcareCat#NA935VWB (1:30000)
AntibodyAnti-Rabbit IgG, HRP-linked (Donkey)GE HealthcareCat#NA934VWB (1:30000)
AntibodyAnti-Mouse IgG, HRP-linked (Sheep)GE HealthcareCat#NA931VWB (1:30000)
AntibodyNormal IgG (Rabbit)FUJIFILM Wako ChemicalsCat#148–09551Co-IP
AntibodyPolyclonal anti-SNAP23 (Rabbit)AbcamCat#ab3340Co-IP
AntibodyMonoclonal anti-PA-tag (Rat)FUJIFILM Wako ChemicalsCat#016–25861IF (1:1000)
AntibodyMonoclonal anti-γH2AX (Mouse)Merck-MilliporeCat#05–636IF (1:100)
AntibodyPolyclonal anti-SYCP1 (Rabbit)Novus BiologicalCat#NB300-299IF (1:50)
AntibodyMonoclonal anti-SYCP3 (Mouse)Santa Cruz BiotechnologyCat#sc-74569IF (1:50)
AntibodyPolyclonal anti-GFRα1 (Goat)R and D systemsCat#AF560IF (1:400 for section)
IF (1:1000 for whole mount)
AntibodyMonoclonal anti-RARγ1 (Rabbit)Cell Signaling TechnologyCat#8965SIF (1:200)
AntibodyMonoclonal anti-active rac1 (Mouse)NewEast BiosciencesCat#26903IF (1:1000)
AntibodyPolyclonal anti-Exoc1 (Rabbit)ProteintechCat#11690–1-APIF (1:50)
AntibodyPolyclonal anti-Exoc1 (Rabbit)Atlas AntibodiesCat#HPA037706IF (1:50)
AntibodyAnti-Goat IgG, Alexa Fluor 488 (Chicken)Thermo Fisher ScientificCat#A21467IF (1:200 for section)
AntibodyAnti-Goat IgG, Alexa Fluor 594 (Chicken)Thermo Fisher ScientificCat#A21468IF (1:400 for whole mount)
AntibodyAnti-Rat IgG, Alexa Fluor 555 (Donkey)AbcamCat#ab150154IF (1:1000)
AntibodyAnti-Mouse IgG, Alexa Fluor 555 (Goat)Thermo Fisher ScientificCat#A28180IF (1:200)
AntibodyAnti-Mouse IgG, Alexa Fluor 555 (Donkey)Thermo Fisher ScientificCat#A31570IF (1:200)
AntibodyAnti-Rabbit IgG, Alexa Fluor 647 (Goat)Thermo Fisher ScientificCat#A27040IF (1:200)
Recombinant DNA reagentpcDNA3.1 (+) Mammalian Expression VectorInvitrogenV79020
Recombinant DNA reagentT7-NLS hCas9-pA plasmidYoshimi et al., 2016RIKEN BRC
(RDB13130)
Recombinant DNA reagentpCAG-FlpeMatsuda and Cepko, 2007Addgene (Plasmid #13787)
Recombinant DNA reagentpT7-Flpe-pAThis paperRIKEN BRC
(RDB16011)
Sequence-based reagentAll primers in Supplementary file 1bThermo Fisher Scientific
Sequence-based reagentAll primers in Supplementary file 1bThermo Fisher Scientific
Sequence-based reagentExoc1 PA-C ssODNIntegrated DNA TechnologiesGAATTCACTATTCAGGACATTCTGGATTATTGCTCCAGCATCGCACAGTCCCACGGCTCAACCAGCGGATCTGGTAAGCCAGGTAGTGGAGAAGGCAGCACCAAGCCTGGCGGCGTCGCCATGCCTGGAGCCGAGGATGATGTCGTGTAAGCCCTAGGAAAGAGGAGAAAGAAGTGAGCATGCATTCTCAGTCCAGCAAA
Sequence-based reagentExoc1 PA-N ssODNIntegrated DNA TechnologiesGGAGGGCAGTGGTTTTGAGAATTATTCTAAATGTTTTTCAGCTGAGAAAAGATGGGCGTCGCCATGCCTGGAGCCGAGGATGATGTCGTGGGCTCAACCAGCGGATCTGGTAAGCCAGGTAGTGGAGAAGGCAGCACCAAGCCTGGCACAGCAATCAAGCATGCGCTGCAGAGAGATATCTTCACACCAAATGATGAACG
Software, algorithmThe R Foundationhttps://www.r-project.org/foundation/
OtherStreptavidin-HRPNichirei BiosciencesCat#426061WB (1:1000 in 2% BSA/TBS-T)
OtherLectin from Arachis hypogaea, FITCSigma-AdrichCat#L7381Lectin staining (1:100)

Animals

The mice were maintained in plastic cages under specific pathogen-free conditions in 23.5 ± 2.5°C and 52.5 ± 12.5% relative humidity under a 14 hr light/10 hr dark cycle in the Laboratory Animal Resource Center at the University of Tsukuba. Mice had free access to commercial chow (MF diet; Oriental Yeast Co. Ltd) and filtered water. ICR and C57BL/6 mice were purchased from Charles River Laboratories Japan. Nanos3-Cre mice, Nanos3tm2 (cre)Ysa, were kindly gifted by Dr. Saga (Suzuki et al., 2008) through RIKEN BioResource Research Center (RBRC02568). Exoc1tm1a (EUCOMM)Hmgu and Snap23tm1a(EUCOMM)Hmgu mice were obtained from the International Knockout Mouse Consortium and the International Mouse Phenotyping Consortium (Skarnes et al., 2011). All male mice used in the experiment were 10–20 weeks old. The genetic background of all the genetically modified or wild-type mice used in the experiments was C57BL/6, except for the Nanos3-Cre mice. Since the genetic background of the Nanos3-Cre mice provided by RIKEN BRC was ICR, they were backcrossed to C57BL/6 for at least five generations before being used in the experiment.

Genome editing in mouse embryos

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The Exoc1 PA-C KI mice were generated by CRISPR-Cas9 based genome editing. The sequence (5′-GCA CAG TCC CAC TAA GCC CT-3′) was selected as the guide RNA (gRNA) target. The gRNA was synthesized and purified by the GeneArt Precision gRNA Synthesis Kit (Thermo Fisher Scientific, Waltham, Massachusetts) and dissolved in Opti-MEM (Thermo Fisher Scientific, Waltham, Massachusetts). The Exoc1 PA-N KI mice were generated by CRISPR-Cas12a-based genome editing. The following sequence was selected as the crRNA target and synthesized artificially (Integrated DNA Technologies): 5′-AGC TGA GAA AAG ATG ACA GCA-3′. In addition, we designed a 200-nt single-stranded DNA oligonucleotide (ssODN) donor; the LG3 linker (Kagoshima et al., 2007) and the PA tag sequence was placed between 55-nt 5′- and 53-nt 3′-homology arms. This ssODN was ordered as Ultramer DNA oligos (Integrated DNA Technologies) and dissolved in Opti-MEM. Two gRNA targets (5′-CAT AAA GTG GTT GCG CTC TT-3′ and 5′-GCA GAT GTG ATG CTC GGC TG-3′) located on intron 4 and 5 of Stx2, respectively, were selected for producing the Stx2 KO mouse. These two gRNAs were synthesized, purified, and dissolved in Opti-MEM as mentioned above.

The mixture of gRNA for Exoc1 PA-C (25 ng/μL), ssODN (100 ng/μL), and GeneArt Platinum Cas9 Nuclease (Thermo Fisher Scientific, Waltham, Massachusetts) (100 ng/μL) or the mixture of crRNA for Exoc1 PA-N (25 ng/μL), ssODN (100 ng/μL), and Alt-R A.s. Cas12a Nuclease V3 (Integrated DNA Technologies) (100 ng/μL) were electroplated to the zygotes of C57BL/6J mice (Charles River Laboratories Japan, Yokohama, Japan) by using the NEPA 21 electroplater (Nepa Gene Co. Ltd., Ichikawa, Japan) to produce the Exoc1 PA KI mouse (Sato et al., 2018). The mixture of two gRNAs for Stx2 (25 ng/μL, each) and GeneArt Platinum Cas9 Nuclease (100 ng/μL) were electroplated to zygotes for producing the Stx2 KO mouse. After electroporation, two cell embryos were transferred into the oviducts of pseudopregnant ICR female and newborns were obtained.

Electroporation of mice zygotes with Flpe mRNA

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A pT7-Flpe-pA plasmid (deposited in RIKEN BioResource Research Center, RDB16011) was constructed from a T7-NLS hCas9-pA plasmid, which was kindly gifted by Dr. Mashimo (Yoshimi et al., 2016), through RIKEN BioResource Research Center (RDB13130). Cas9 cDNA in the T7-NLS hCas9-pA was replaced with an Flpe cDNA from pCAG-Flpe. This pCAG-Flpe was kindly gifted by Dr. Cepko (Matsuda and Cepko, 2007) through Addgene (Plasmid #13787). Flpe mRNA was transcribed in vitro from NheI digested pT7-Flpe-pA by using mMESSAGE mMACHINE T7 ULTRA Transcription Kit (Thermo Fisher Scientific, Waltham, Massachusetts).

Female C57BL/6 mice (12 weeks old) were superovulated by intraperitoneal administration of 5 units of pregnant mare serum gonadotropin (ASKA Pharmaceutical Co. Ltd., Tokyo, Japan) and 5 units of human chorionic gonadotropin (ASKA Pharmaceutical Co. Ltd., Tokyo, Japan) with a 48 hr interval. In vitro fertilization was performed using the wild-type C57BL/6 oocytes and the Snap23tm1a(EUCOMM)Hmgu sperms according to standard protocols. Five hours later, Flpe mRNA (300 ng/μL) was electroplated to the zygotes by using the NEPA 21 electroplater. The poring pulse was set to voltage: 225 V, pulse width: 2 ms, pulse interval: 50 ms, and number of pulses:+4. The transfer pulse was set to voltage: 20 V, pulse width: 50 ms, pulse interval: 50 ms, and number of pulses: ±5 (attenuation rate was set to 40%). A day after electroporation, the developed two-cell embryos were transferred to pseudopregnant ICR mice.

Genotyping PCR

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For routine genotyping, genomic DNA was extracted from <0.5 mm tails of 3 weeks old mice. PCR was carried out using AmpliTaq Gold 360 Master Mix (Thermo Fisher Scientific, Waltham, Massachusetts) with the appropriate primers (Supplementary file 1b). For genotyping of blastocysts, they were treated with a proteinase K (F. Hoffmann-La Roche, Basel, Switzerland) solution prepared in DDW (0.02 mg/mL), at 55°C for 2 hr and then at 95°C for 7 min. This solution was used as a template, to which AmpliTaq Gold 360 Master Mix and appropriate primers (Supplementary file 1b) were added for PCR. For DNA sequencing, PCR products were purified with a FastGene Gel/PCR Extraction Kit (Nippon Genetics, Tokyo, Japan) and sequences were confirmed with a BigDye Terminator v3.1 Cycle Sequencing Kit (Thermo Fisher Scientific, Waltham, Massachusetts), FastGene Dye Terminator Removal Kit (Nippon Genetics, Tokyo, Japan), and 3500xL Genetic Analyzer (Thermo Fisher Scientific, Waltham, Massachusetts).

Western blot analysis and Co-IP of in vivo samples

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Mouse testes were homogenized in tissue protein extraction reagent (Thermo Fisher Scientific, Waltham, Massachusetts) and centrifuged at 14,000 rpm for 15 min at 4°C. Protein concentrations of the supernatants were determined using Quick Start Bradford assay kit (BioRad). The samples were mixed with sample buffer solution containing a reducing reagent (6×) for SDS-PAGE (Nacalai Tesque Inc, Kyoto, Japan), followed by incubation for 3 min at 100°C. The samples were subjected to SDS-PAGE and transferred to Immobilon-P PVDF membranes (Merck-Millipore, Burlington, Massachusetts). The membranes were blocked with 5% skim milk in Tris-buffered saline/Tween 20 (TBS-T) for 30 min at 20–25°C. The membranes were subsequently incubated with primary antibodies (Key Resources Table) for 1–3 hr at 20–25°C. The membrane was then incubated with streptavidin-HRP or HRP-linked secondary antibodies (Key Resources Table) for 1 hr at 20–25°C. All antibodies were diluted in TBS-T with 1% skim milk. The blots were developed by chemiluminescence using Luminata Forte Western HRP Substrate (Merck-Millipore, Burlington, Massachusetts) or ImmunoStar LD (FUJIFILM Wako Chemicals, Tokyo, Japan) and visualized by iBrightCL100 (Thermo Fisher Scientific, Waltham, Massachusetts).

For Co-IP assay, proteins were extracted from testes using lysis buffer (20 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1% triton X-100, and protease inhibitor cocktail). The lysates containing 1.5 mg of total protein were incubated with normal rabbit IgG or anti-SNAP23 antibody (Key Resources Table) for 1 hr at 4°C. The samples were further incubated with SureBeads Protein G Magnetic Beads for 1 hr at 4°C. After washing the beads, proteins were eluted by boiling in 1× SDS sample buffer and subjected to SDS-PAGE.

H and E staining

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Testes, with the tunica albuginea removed, were fixed using 10%-Formaldehyde Neutral Buffer Solution (Nacalai Tesque Inc, Kyoto, Japan) overnight. Fixed testes were then soaked in 70% ethanol and embedded into paraffin blocks. Tissues were sliced at 5 μm using a HM335E microtome (Thermo Fisher Scientific, Waltham, Massachusetts). After deparaffinization, xylene was removed from the sections with 100% ethanol and subsequently hydrated with 95% ethanol, 70% ethanol, and deionized distilled water. Hydrated sections were stained with Mayer's Hematoxylin Solution (FUJIFILM Wako Chemicals, Tokyo, Japan) and 1% Eosin Y Solution (FUJIFILM Wako Chemicals, Tokyo, Japan).

Electron microscope observation

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Testes were fixed in 1% OsO4 in 0.1 M phosphate buffer (pH 7.4), dehydrated in ethanol, and embedded in epoxy resin poly/Bed 812 (Polysciences Inc, Warrington, Pennsylvania). Subsequently, ultra-thin sections were made at 70–80 nm and stained with uranium acetate and lead citrate. A transmission electron microscope, JEM-1400 (JOEL Ltd., Tokyo, Japan), was used for the observation.

Immunofluorescence and lectin staining

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All immunofluorescence experiments except for active-Rac1, SYCP3, and SYCP1 were performed with paraffin sections. The paraffin sections were prepared similar to that of the H and E staining. After deparaffinization and rehydration, sections were permeabilized with 0.25% TritonX-100 in PBS and autoclaved (121°C, 10 min) with Target Retrieval Solution (Agilent Technologies, Santa Clara, California). Sections were incubated with Blocking One Histo (Nacalai Tesque Inc, Kyoto, Japan) for 15 min at 37°C. Primary antibodies (Key Resources Table) that were diluted with Can Get Signal Immunoreaction Enhancer Solution A (Toyobo Co. Ltd., Osaka, Japan) were applied and slides were incubated for 1 hr at 37°C. Alexa Fluor-conjugated secondary antibodies (Key Resources Table) were diluted with Can Get Signal Immunoreaction Enhancer Solution A, and applied for 1 hr at 37°C. Prolong gold antifade reagent with DAPI (Thermo Fisher Scientific, Waltham, Massachusetts) was used as a mounting media and for DAPI staining. Active-Rac1 immunofluorescence was performed with frozen sections. To prepare frozen sections, the tunica albuginea was removed from the testes, and the testes were fixed with 4% paraformaldehyde overnight at 4°C. Fixed testes were then soaked in 30% sucrose in PBS overnight at 4°C and embedded in Tissue-Tek O.C.T. Compound (Sakura Finetek, Tokyo, Japan). Tissues were sliced at 14 μm using HM525 NX Cryostat (Thermo Fisher Scientific, Waltham, Massachusetts). Permeabilization, blocking, and antibody reactions were performed similar to that used in immunofluorescence with paraffin sections. Whole-mount immunofluorescence of seminiferous tubules for GFRα1 (Key Resources Table) was performed as reported previously (Kitadate et al., 2019). Meiotic pachytene chromosome spread and immunofluorescence for SYCP1 and SYCP3 (Key Resources Table) was performed as reported previously (Peters et al., 1997). Samples were observed under a BZ-9000 fluorescence microscope (Keyence, Osaka, Japan), a SP8 Confocal Laser Scanning Microscopy (Leica microsystems, Wetzlar, Germany), and an LSM 800 with Airyscan (ZEISS, Oberkochen, Germany). PNA-lectin (Key Resources Table) staining was performed similar to that of immunofluorescence.

Transfection and Co-IP

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Testes RNA was extracted from C57BL/6 mouse using NucleoSpin RNA Plus (Nacalai Tesque Inc, Kyoto, Japan). cDNA was synthesized with PrimeScript RT Master Mix (Takara Bio, Kusatsu, Japan) and full-length cording sequences (CDS) of Exoc1, Snap23, and Stx2 were obtained with PrimeSTAR GXL DNA Polymerase (Takara Bio, Kusatsu, Japan) using the appropriate primers (Supplementary file 1b). Full-length CDSs were introduced into the pCDNA3.1 mammalian expression vector with epitope-tag sequences. Expression vectors for FLAG-EGFP, V5-mCherry-HA, and E2 crimson-MYC-His were used as negative controls. These vectors were transfected into HEK293T cells with PEI MAX (Polysciences Inc, Warrington, Pennsylvania) in 100 mm culture dish according to the manufacturer’s protocol. After 24 hr incubation, cells were lysed in lysis buffer (20 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1% triton X-100, protease inhibitor cocktail). The cell lysates were centrifuged at 14,000 rpm for 10 min at 4°C. The supernatants were used for immunoprecipitation. Co-IP was performed with a HA-tagged protein purification kit, DDDDK-tagged protein purification kit, and c-Myc-tagged protein mild purification kit ver.2 (Medical and Biological Laboratories Co., Nagoya, Japan) according to the manufacturer’s protocol. The antibodies for western blot are listed in Key Resources Table. HEK293T cells were obtained from The American Type Culture Collection (Manassas, Virginia). The HEK293T cell line has been authenticated by STR profilling and is negative of mycoplasma contamination testing.

Quantification of pseudopod elongation with thick sections

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Immunofluorescence for GFRα1 was performed using thinly sectioned (20 µm) paraffin sections. To include the entire cell, Z-stack photography was performed using a SP8 Confocal Laser Scanning Microscopy (Leica microsystems, Wetzlar, Germany). We applied Sholl analysis (Binley et al., 2014) to quantify pseudopod elongation, drawing concentric circles around the midpoint of cell body width and the intersection with the basement membrane and measuring the distance of the furthest elongated pseudopod.

Image analyses

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The elongation of the pseudopod and the distance between connected cells in whole-mount immunofluorescence assay, signal intensity of active-Rac1immunofluorescence, and the area of seminiferous tubule sections were calculated using ImageJ Fiji 1.53 c JAVA 1.8.0_172 (64-bit). All image data were selected randomly. For pseudopod extension analysis, the distance between the center (‘Centroid’) of the cell body and the tip of the pseudopod, at the furthest point from the cell body, was measured. For the analysis of the distance between connected cells, the distance between the centers (‘Centroid’) of each cell body was measured. For intensity analysis, we measured the intensity of active-Rac1 in GFRα1+ cells using immunofluorescence images. After selecting GFRα1+ cells, we calculated the signal intensity of active-Rac1, which was converted from RGB to 16-bit. The average intensity of the signal (‘Mean Gray Value’) was calculated so that the size of the cells would not affect the measurement.

Analysis of publicly available next-generation sequencing data

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The expression of each exocyst subunit in the respective germline differentiation stage was extracted from the open data source GSE112393 (Green et al., 2018). The expression of each exocyst subunit in Sertoli cells of adult mice was extracted from GSM3069461 (Green et al., 2018). The gene count was log-normalized and visualized in comparison with the expression levels of all genes. The R script used in the analysis of GSM3069461 can be downloaded from https://github.com/akikuno/Exoc_GSM3069461/blob/master/SupFig2.R.

Study approval

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All animal experiments were carried out in a humane manner with approval from the Institutional Animal Experiment Committee of the University of Tsukuba in accordance with the Regulations for Animal Experiments of the University of Tsukuba and Fundamental Guidelines for Proper Conduct of Animal Experiments and Related Activities in Academic Research Institutions under the jurisdiction of the Ministry of Education, Culture, Sports, Science, and Technology of Japan (Approval Number: 20–013).

Data availability

All data generated or analysed during this study are included in the manuscript and supporting files.

References

    1. Peters AH
    2. Plug AW
    3. van Vugt MJ
    4. de Boer P
    (1997) A drying-down technique for the spreading of mammalian meiocytes from the male and female germline
    Chromosome Research : An International Journal on the Molecular, Supramolecular and Evolutionary Aspects of Chromosome Biology 5:66–68.
    https://doi.org/10.1023/a:1018445520117

Decision letter

  1. Polina V Lishko
    Reviewing Editor; University of California, Berkeley, United States
  2. Anna Akhmanova
    Senior Editor; Utrecht University, Netherlands
  3. Robin Hobbs
    Reviewer; Monash University/ Hudson Institute, Australia
  4. Stefan Schlatt
    Reviewer; University of Munster, Germany, Germany

In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.

Acceptance summary:

Male germ cells undergo profound morphological changes during spermatogenesis, and highly orchestrated pieces of machinery are required for this process. These morphological changes require a formation of syncytia, and the failure of forming it results in the arrested spermatogenesis and causes male infertility. The authors have conducted a series of well-designed and labor-intensive experiments using genome-edited mouse lines and provided evidence that EXOC1 gene, which encodes a member of the Exocyst complex, is essential for the morphological changes of male germ cells during spermatogenesis. They showed that EXOC1 inactivates the Rho family small GTPase Rac1, and also plays a role in syncytia formation acting together with SNARE proteins.

Decision letter after peer review:

Thank you for submitting your article "EXOC1 regulates cell morphology of spermatogonia and spermatocytes in mice" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Anna Akhmanova as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Robin Hobbs (Reviewer #2); Stefan Schlatt (Reviewer #3).

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

As the editors have judged that your manuscript is of interest, but as described below that additional experiments are required before it is published, we would like to draw your attention to changes in our revision policy that we have made in response to COVID-19 (https://elifesciences.org/articles/57162). First, because many researchers have temporarily lost access to the labs, we will give authors as much time as they need to submit revised manuscripts. We are also offering, if you choose, to post the manuscript to bioRxiv (if it is not already there) along with this decision letter and a formal designation that the manuscript is "in revision at eLife". Please let us know if you would like to pursue this option. (If your work is more suitable for medRxiv, you will need to post the preprint yourself, as the mechanisms for us to do so are still in development.)

Summary:

The study by Osawa and colleagues highlights interesting and highly desired aspects of spermatogonial physiology and clonal expansion. Specifically, the role of EXOC1, a factor involved in syncytial organization of cells, is explored and many novel aspects are described. The authors have conducted a series of well-designed and labor-intensive experiments using genome-edited mouse lines, and successfully evidenced that germ cell EXOC1 in the testis is essential for their morphological changes during spermatogenesis therein. This manuscript is focused on an understudied and critical pathway in the male germline and would be of interest to the field and of relevance for fertility. However, in its current state, the insight provided by the manuscript is somewhat limited. The authors need to perform a much more detailed characterization of the mouse models generated and revisit the model proposed for exocyst function in the germline. The manuscript is well written and clear but short of mechanistic points. In contrast to the strong genetic studies, however, the referees see some weakness in their biochemical, histological, and physiological studies as indicated below.

Essential revisions:

1. The macroscopic images for HE stained testicular sections should be included otherwise it is difficult to judge the effects of genetic ablation. And the authors should present how efficiently genes are inactivated in these mice.

2. Line 134 (Figure 1C): Authors are recommended to distinguish germ- and somatic-cells in testis.

3. The authors only showed representative images. Quantitative data need to be included.

4. Figure 3A is the only data showing EXOC1, STX2, and SNAP23. The authors should include negative control such as other proteins tagged with HA. More importantly, the authors are strongly recommended to show the interaction in vivo (IP with HA then detect by IB or MS analysis).

5. Line 301: The authors suggest three possible reasons to explain the phenotype. As shown in the previous study, the author can easily address if lipids accumulate in mutant cells.

6. Expression pattern of Exoc1 in the germline requires additional analysis. Expression of the PA tagged EXOC1 from knock in mice should be compared to known markers of germ cell and somatic cell populations. Expression in these populations also requires quantification and comparison to seminiferous epithelium stages. Previously published testis single cell RNA-Seq datasets should be analyzed to assess expression of Exoc1 and other exocyst complex proteins during spermatogenesis and in supporting somatic cells. The western blot of Figure 1B requires additional replicates and molecular weights of bands indicated.

7. Multiple conditional knockout mouse models have been developed for the manuscript. However, the efficiency of gene knockout in germline cells needs to be confirmed for each model. Inefficient gene knockout can affect interpretation of phenotype. For example, the mild phenotype shown by Snap23 knockout model.

8. The PA tagged EXOC1 model is a novel line generated by CRISPR-based methods for the manuscript. Additional evidence needs to be provided that the genomic sequence is correctly modified and tag sequence in frame etc. Further, why is the LG3 linker region included?

9. Phenotype of the conditional knockout Exoc1 and Snap23 models are intriguing. However, no quantification is provided for the analyses. How frequently are the multinucleate meiotic spermatocytes observed? What ages of animals are studied? Both prepubertal and adult mice need to be analyzed to account for defects in postnatal germline development. Distinct germ cell populations need to be quantified by use of known markers (spermatogonia, spermatocytes, spermatids). How many KIT+ cells were analyzed to check for cell aggregation? How many chromosome spreads of spermatocytes were analyzed for the Exoc1 knockout? Representative images of knockout and control spreads should be provided in the figure.

10. Based on EM analysis of multinucleate spermatocytes from Exoc1 knockout testis (please provide quantification for this result), the authors suggest that intercellular bridge (ICB) formation in spermatocytes is specifically disrupted. This proposed model for exocyst complex function a little confusing. For formation of an ICB, cytokinesis is initiated at the end of mitosis but recruitment of TEX14 to the midbody blocks abscission and mid-body components remain as components of the ICB. The exocyst complex has a known role in cytokinesis, suggesting that cytokinesis is not initiated in the knockout and multinucleate cells are therefore formed. ICBs are then of course not present but this reflects the role of EXOC1 in the cytokinesis pathway rather than ICB formation per se. The authors should clarify this model.

11. For IP analysis in figure 3, the reciprocal pull-downs need to be performed. For instance, IP anti-FLAG and anti-Myc tag and test for interactions. Molecular weights for proteins need to be included.

12. Roles for EXOC1 in pseudopod formation in GFRa1-positive SSCs are suggested (Figure 4). While very interesting, it seems that correctly identifying pseudopods based on GFRa1 membrane staining of sections might be challenging. Particularly as GFRa1 cells can be present as interconnected pairs of cells (and some 4 cell chains), which can disrupt the ability to identify pseudopods of individual cells. The authors should repeat this analysis using wholemount staining of seminiferous tubules and compare the distinct GFRa1-positive cell populations (single, pairs and aligned). This will allow better quantification of cell extensions. Can specific markers of pseudopods be used (even just actin staining etc)? The active-Rac1 staining in panel D also needs to be quantified. Wholemount analysis can also reveal whether EXOC1 plays a role in cytokinesis of spermatogonial populations.

13. Defects in migration of Exoc1 knockout GFRa1-positive SSCs are suggested to result in SSC accumulation and defects in production of differentiation-primed undifferentiated spermatogonia (Figure 5). Images shown in panel A indicate that clumps of GFRa1-positive cells accumulate, with some cells pushed off the basal layer of the seminiferous tubules. This is highly unusual as all spermatogonia should be adherent to the basement membrane. It seems unlikely that this phenotype is due solely to defects in migration. Additional data should be provided to support this model and exclude other potential defects. For example, can functional data (e.g. in vitro analysis) be provided to confirm issues with SSC migration? Also, as fewer differentiation-primed cells are generated, the numbers of differentiating spermatogonia should be reduced in the knockout. Is this the case?

14. The manuscript text should be checked carefully for accuracy throughout. For instance, in the introduction, second paragraph, spermatogonia are stated to be divided into undifferentiated, differentiation-primed and differentiated fractions. However, differentiation -primed cells are found within the undifferentiated population as are SSCs. The significance of markers used for different spermatogonial populations (e.g. GFRa1) should be included in the introduction for clarity. The discussion should be modified to account for new datasets and models.

15. Many datasets are not quantified (see specific points above). Statistics cannot be evaluated as for most experimental settings the basic information (N number, means and coefficient of Variation… ) is not provided.

16. Additionally, the actual study design is not described. Therefore, the validity of the findings is partially questionable. No Information is provided on the number of mice analyzed. Nature and size of experimental groups and controls are not mentioned. Strategies for histological analysis are poorly described. Did the authors use random systematic sampling approaches on sufficient number of samples? The excellent micrographs must be considered individual observations as generalization of the descriptions would only be valid if repetitions are reported. Similar concerns apply to the blotting results. To check for validity and statistically Sound analysis, the authors need to provide a flow chart or table of all experimental animals and the endpoints analyzed in specific groups.

17. The mouse models are not as well defined as the authors Claim. Snap23 k/O mice Show germ cell development up to spermatids (Figure 3). Can the authors elaborate more on the presence of multinucleated cells and spermatids at the same time? Was that stable in all animals of this genotype?

[Editors' note: further revisions were suggested prior to acceptance, as described below.]

Thank you for resubmitting your work entitled "EXOC1 plays an integral role in spermatogonia pseudopod elongation and spermatocyte stable syncytium formation in mice" for further consideration by eLife. Your revised article has been reviewed by three peer reviewers and the evaluation has been overseen by a Reviewing Editor and Anna Akhmanova as the Senior Editor.

The manuscript has been significantly improved but there are some remaining issues (many could be amended with textual revisions) that need to be addressed, as outlined below:

1. Fluorescent images are still not clear.

2. Line 161: The authors mentioned "EXOC1 is observed in every cell in the adult testes" in the Figure 1 legend but described "EXOC1-PA was also detected in all male germ cells observed" in text. They may want to focus on germ cells, but it's better clarifying both Sertoli and germ cells are positive in the text if all cells are stained in testicular tubules.

The authors could not judge if Sertoli cells are positive with the current figure.

3. It was originally requested for the authors to assess the efficiency of floxed gene knockout in their models. While they have assessed whether remaining sperm in the Exoc1 conditional KO model are gene deleted, analysis of testis from this and other KO models described has not been performed. A simple qRT-PCR on total testis extracts or isolated testis cells could be used. It is important to define efficiency of gene knockout as the phenotype of some of the models is mild, e.g. Snap23 KO.

4. A new N-terminally PA tagged Exoc1 model is described in the revision as the C-tagged version clearly disrupts EXOC1 function, which is embryonic lethal. Confusingly, while the homozygous N-tagged mice are viable, indicating that EXOC1 function is retained, the authors could not detect expression of the N-tagged EXOC1 by immunostaining and suggest that it might be unstable. This seems counterintuitive and text should be modified to help resolve and discuss this issue. On a related point, lines 160-163 where rationale for use of the LG3 tag in the fusion construct is described – this should be reworded for clarity.

5. It is stated that while GFRa1+ spermatogonial pseudopods in the Exoc1 KO are shorter, those of the Stx2 KO are not, suggesting that EXOC1 operates independently of STX2 in this context. However, the pseudopods of Stx2 KO are strongly trending to be shorter and P value is quoted as 0.052. This suggests that a larger dataset may indeed show significant reduction and the conclusions from this data are overstated. Text should be changed appropriately to reflect this strong trend in data and/or more cells quantified to assess the robustness of the conclusions.

6. The abstract could be reworded for clarity. A sentence about role of EXOC1, SNAP23 etc in the exocyst complex should be included up-front to help non experts in the field. The term "high regulation" in the first sentence is ambiguous.

Reviewer #1:

The authors addressed most of the reviewer's concern, but there remains some remaining issues.

1. Fluorescent images are still not clear. The reviewer doesn't see the improvement.

2. Line 161: The authors mentioned "EXOC1 is observed in every cell in the adult testes" in the Figure 1 legend but described "EXOC1-PA was also detected in all male germ cells observed" in text. They may want to focus on germ cells, but it's better clarifying both Sertoli and germ cells are positive in the text if all cells are stained in testicular tubules.

The authors could not judge if Sertoli cells are positive with the current figure.

Reviewer #2:

The authors have performed substantial revisions to the manuscript, which is now significantly improved. In particular, they have included quantification of multiple key datasets as requested and included new analyses of exocyst complex gene expression patterns, spermatogonia cytoplasmic extensions, plus more thorough in vitro datasets. In general, they have responded very well to the comments.

The manuscript contains a wealth of data from genetic models although still lacks substantial mechanistic insight. However, it would be of interest to the field.

1. It was originally requested for the authors to assess the efficiency of floxed gene knockout in their models. While they have assessed whether remaining sperm in the Exoc1 conditional KO model are gene deleted, analysis of testis from this and other KO models described has not been performed. A simple qRT-PCR on total testis extracts or isolated testis cells could be used. It is important to define efficiency of gene knockout as the phenotype of some of the models is mild, e.g. Snap23 KO.

2. A new N-terminally PA tagged Exoc1 model is described in the revision as the C-tagged version clearly disrupts EXOC1 function, which is embryonic lethal. Confusingly, while the homozygous N-tagged mice are viable, indicating that EXOC1 function is retained, the authors could not detect expression of the N-tagged EXOC1 by immunostaining and suggest that it might be unstable. This seems counterintuitive and text should be modified to help resolve and discuss this issue. On a related point, lines 160-163 where rationale for use of the LG3 tag in the fusion construct is described – this should be reworded for clarity.

3. It is stated that while GFRa1+ spermatogonial pseudopods in the Exoc1 KO are shorter, those of the Stx2 KO are not, suggesting that EXOC1 operates independently of STX2 in this context. However, the pseudopods of Stx2 KO are strongly trending to be shorter and P value is quoted as 0.052. This suggests that a larger dataset may indeed show significant reduction and the conclusions from this data are overstated. Text should be changed appropriately to reflect this strong trend in data and/or more cells quantified to assess the robustness of the conclusions.

4. The numbers of cells scored in some of the datasets e.g. levels of activated Rac1, are very small – 8-10 cells per mouse. I would recommend scoring substantially more cells per mouse to improve robustness of the data.

5. The abstract could be reworded for clarity. A sentence about role of EXOC1, SNAP23 etc in the exocyst complex should be included up-front to help non experts in the field. The term "high regulation" in the first sentence is ambiguous.

Reviewer #3:

This is a valuable insight into spermatogonial biology. The mouse models provide fundamental evidence for the involvement if specific factors in germ cell eplitting and expansion. The findings are relevant for stem cell and infertility Research.

The authors provide relevant new mouse K/O models showing the involvement of EXOC 1 in spermatogonial pseudopod formation. This is an important observation. The authors have undertaken a solid revision of the paper. While some aspects of the histological analysis remain weak, the manuscript contains tremendous datasets of proven validity. The information will lead to a better understanding of spermatogonial physiology and initiation of spermatogenesis.

https://doi.org/10.7554/eLife.59759.sa1

Author response

Essential revisions:

1. The macroscopic images for HE stained testicular sections should be included otherwise it is difficult to judge the effects of genetic ablation. And the authors should present how efficiently genes are inactivated in these mice.

Thank you for your comments, and we agree with your suggestion. We have added the macroscopic image data to Figure 2C and Figure 3C. We also added Figure 2—figure supplement 1 and Figure 3—figure supplement 2 as data on the frequency of AGS and the gene ablation efficiency. We described the contents of these data in Lines 183-195 and Lines 256-262 in the main text.

2. Line 134 (Figure 1C): Authors are recommended to distinguish germ- and somatic-cells in testis.

We have added data to Figure 1—figure supplement 1C showing that it is expressed in germ cells. We have also added data on expression in Sertoli cells as Figure 1—figure supplement 2. We described the contents of these data in Lines 160-162 in the main text.

3. The authors only showed representative images. Quantitative data need to be included.

Thank you for your suggestion. Quantitative data for each experiment is added to Supplementary File1a, Figure 4I and Figure 2—figure supplement 1, Figure 3—figure supplement 2, and Figure 5—figure supplement 1.

Supplementary File1a: Number of c-Kit+ AGS in Exoc1 cKO

Figure 4I: Intensity of active Rac1

Figure 2—figure supplement 1: Frequency of AGS in Exoc1 cKO

Figure 3—figure supplement 2: Frequency of AGS in Snap23 cKO

Figure 5—figure supplement 1: The number c-Kit+ cells in Exoc1 cKO

4. Figure 3A is the only data showing EXOC1, STX2, and SNAP23. The authors should include negative control such as other proteins tagged with HA. More importantly, the authors are strongly recommended to show the interaction in vivo (IP with HA then detect by IB or MS analysis).

The in vitro Co-IP results for all combinations, including the negative control, are displayed as Figure 3A. We described the contents of these data in Lines 235-237 in the main text.

The in vivo co-IP data is presented in Figure 3B, and we also performed an experiment to detect SNAP23 by IP with PA, but we could not detect it, probably due to the low sensitivity. We described the contents of these data in Lines 237-241 in the main text.

We provided data showing that PA-tagged EXOC1 is functional in Lines 137-155 of the main text. We found that the C-terminal PA-tagged EXOC1 was not functional, so we generated a new N-terminal tagged mouse (Figure 1A).

5. Line 301: The authors suggest three possible reasons to explain the phenotype. As shown in the previous study, the author can easily address if lipids accumulate in mutant cells.

DI8 antibodies to detect target lipids are not commercially available, so we have not conducted this study due to difficulty in availability. The claim of this study is that EXOC1 is involved in the STX2 pathway, and studies on the downstream of this pathway may be a future consideration.

6. Expression pattern of Exoc1 in the germline requires additional analysis. Expression of the PA tagged EXOC1 from knock in mice should be compared to known markers of germ cell and somatic cell populations.

We found almost same comment in the Essential revisions comment 2. We answered there.

Expression in these populations also requires quantification and comparison to seminiferous epithelium stages. Previously published testis single cell RNA-Seq datasets should be analyzed to assess expression of Exoc1 and other exocyst complex proteins during spermatogenesis and in supporting somatic cells. The western blot of Figure 1B requires additional replicates and molecular weights of bands indicated.

We confirmed that all Exocyst subunits were expressed in both Germ and Sertoli cells. These data are shown in Figure 1—figure supplement 1A and 2.

7. Multiple conditional knockout mouse models have been developed for the manuscript. However, the efficiency of gene knockout in germline cells needs to be confirmed for each model. Inefficient gene knockout can affect interpretation of phenotype. For example, the mild phenotype shown by Snap23 knockout model.

Thank you for your suggestion. We have confirmed that spermatogonia lacking Exoc1 do not produce fertilizable sperm. The data was shown in Figure 2—figure supplement 1D. We described the contents of this data in Lines 189-195 in the main text. Since the distance of loxP in Snap23 is almost the same as that of Exoc1 (Exoc1 902 bp/Snap23 754 bp), it is considered that the Snap23 gene function is disrupted with the same efficiency as Exoc1.

8. The PA tagged EXOC1 model is a novel line generated by CRISPR-based methods for the manuscript. Additional evidence needs to be provided that the genomic sequence is correctly modified and tag sequence in frame etc.

We present the Sanger sequence data on Figure 1—figure supplement 1B.

Further, why is the LG3 linker region included?

This is to increase flexibility. The reason for this is described in Lines 142-145 in the main text.

9. Phenotype of the conditional knockout Exoc1 and Snap23 models are intriguing. However, no quantification is provided for the analyses. How frequently are the multinucleate meiotic spermatocytes observed?

Thank you for your suggestion.

We found almost same comment in the Essential revisions comment 2. We answered there.

What ages of animals are studied? Both prepubertal and adult mice need to be analyzed to account for defects in postnatal germline development.

We analyzed adult mice. We have stated this in the description of each experiment.

We have confirmed that AGS also appears in the first wave. Please see Author response image 1. Since the studies on the appearance of AGS due to the loss of STX2 and the involvement of migration in the differentiation state of sperm stem cells that were the basis of this study mainly used adults, we decided to present only adult data.

Author response image 1

Distinct germ cell populations need to be quantified by use of known markers (spermatogonia, spermatocytes, spermatids).

The frequencies of AGS-spermatocytes and Sperm are provided in Figure 2—figure supplement 1 and Figure 3—figure supplement 2. The frequencies of spermatogonia are shown in Figure 5 and Figure 5—figure supplement 1.

How many KIT+ cells were analyzed to check for cell aggregation?

This information is described in Supplementary File1a.

How many chromosome spreads of spermatocytes were analyzed for the Exoc1 knockout? Representative images of knockout and control spreads should be provided in the figure.

I have documented this information in Figure 2—figure supplement 2 and its figure legend.

10. Based on EM analysis of multinucleate spermatocytes from Exoc1 knockout testis (please provide quantification for this result).

Thank you for your suggestion.

We found almost same comment in the Essential revisions comment 2. We answered there.

The authors suggest that intercellular bridge (ICB) formation in spermatocytes is specifically disrupted. This proposed model for exocyst complex function a little confusing. For formation of an ICB, cytokinesis is initiated at the end of mitosis but recruitment of TEX14 to the midbody blocks abscission and mid-body components remain as components of the ICB. The exocyst complex has a known role in cytokinesis, suggesting that cytokinesis is not initiated in the knockout and multinucleate cells are therefore formed. ICBs are then of course not present but this reflects the role of EXOC1 in the cytokinesis pathway rather than ICB formation per se. The authors should clarify this model.

I appreciate your valuable suggestion. The following text is added to Lines 378-382 in the main text. “exocyst complex is more important for recruitment of the ESCRT III complex to the ICB than for formation of ICB and that disruption of this recruitment in Exoc1 cKO spermatocytes may be responsible for the impaired secondary ingression event (Agromayor and Martin-Serrano, 2013) in cytokinesis.”

11. For IP analysis in figure 3, the reciprocal pull-downs need to be performed. For instance, IP anti-FLAG and anti-Myc tag and test for interactions. Molecular weights for proteins need to be included.

Thank you for your suggestion. We found almost same comment in the Essential revisions comment 4. We answered there.

12. Roles for EXOC1 in pseudopod formation in GFRa1-positive SSCs are suggested (Figure 4). While very interesting, it seems that correctly identifying pseudopods based on GFRa1 membrane staining of sections might be challenging. Particularly as GFRa1 cells can be present as interconnected pairs of cells (and some 4 cell chains), which can disrupt the ability to identify pseudopods of individual cells. The authors should repeat this analysis using wholemount staining of seminiferous tubules and compare the distinct GFRa1-positive cell populations (single, pairs and aligned). This will allow better quantification of cell extensions. Can specific markers of pseudopods be used (even just actin staining etc)?

I have provided that information in Figure 4 D-G and described it in the main text, Lines 294-305. These are also GFRα1 immunostaining. We also performed WGA and actin immunostaining to get a clearer picture of the cell shape, but all the cells were stained and we could not measure the length of the pseudopodia in a single cell.

The active-Rac1 staining in panel D also needs to be quantified. Wholemount analysis can also reveal whether EXOC1 plays a role in cytokinesis of spermatogonial populations.

Thank you for your suggestion. The quantified signal intensity of active Rac1 is displayed as Figure 4I.

13. Defects in migration of Exoc1 knockout GFRa1-positive SSCs are suggested to result in SSC accumulation and defects in production of differentiation-primed undifferentiated spermatogonia (Figure 5). Images shown in panel A indicate that clumps of GFRa1-positive cells accumulate, with some cells pushed off the basal layer of the seminiferous tubules. This is highly unusual as all spermatogonia should be adherent to the basement membrane. It seems unlikely that this phenotype is due solely to defects in migration. Additional data should be provided to support this model and exclude other potential defects. For example, can functional data (e.g. in vitro analysis) be provided to confirm issues with SSC migration?

I would like to thank you for your valued remarks. We measured the occurrence of such a multilayered cell population, but it was very low. Please see Author response image 2A. Since such multiple layers are rare, we replaced the Exoc1 cKO figure used in Figure 5A. In Author response image 2B, we present all the cases (just only two) that we found. The top panel in Author response image 2B is the figures used in the Figure 5 of the first version. Considering the possibility that GFRα1+ spermatogonia in Exoc1 cKO mice are in a more undifferentiated state than those in control mice, we compared the expression level of GFRα1 between the Exoc1 cKO and control (Exoc1flox/flox) groups, but there was no noticeable difference (Author response image 2C). In addition, although it was very rare case, Kit+ cells were also multilayered (but not aggregated) in the Exoc1 cKO (Author response image 2D, white arrowhead). For these reasons, we consider that the reason for the appearance of such a multilayered cell population is not that the undifferentiated state is disorganized, but rather that the physical pressure is reduced by the emptying of the lumen of the seminiferous tubule. Since this is just a hypothesis and there is no evidence for it, we would like to analyze it in the future.

Author response image 2

Also, as fewer differentiation-primed cells are generated, the numbers of differentiating spermatogonia should be reduced in the knockout. Is this the case?

Yes, it is. I present this data in Figure 5—figure supplement 1 and describe in Lines 333-335 in the main text.

14. The manuscript text should be checked carefully for accuracy throughout. For instance, in the introduction, second paragraph, spermatogonia are stated to be divided into undifferentiated, differentiation-primed and differentiated fractions. However, differentiation -primed cells are found within the undifferentiated population as are SSCs. The significance of markers used for different spermatogonial populations (e.g. GFRa1) should be included in the introduction for clarity. The discussion should be modified to account for new datasets and models.

Following your advice, I have changed these notations to Lines 75-86, 295-298, 325-327, 386, 390, 417-421.

15. Many datasets are not quantified (see specific points above). Statistics cannot be evaluated as for most experimental settings the basic information (N number, means and coefficient of Variation… ) is not provided.

As I replied in Essential revisions comment 3, I have included the quantified data. The number of analyses and other details are described in each figure legends.

16. Additionally, the actual study design is not described. Therefore, the validity of the findings is partially questionable. No Information is provided on the number of mice analyzed. Nature and size of experimental groups and controls are not mentioned. Strategies for histological analysis are poorly described. Did the authors use random systematic sampling approaches on sufficient number of samples? The excellent micrographs must be considered individual observations as generalization of the descriptions would only be valid if repetitions are reported. Similar concerns apply to the blotting results. To check for validity and statistically Sound analysis, the authors need to provide a flow chart or table of all experimental animals and the endpoints analyzed in specific groups.

As I replied in Essential revisions comment 3, I have included the quantified data. The number of analyses and other details are described in each figure legends. The method of image analysis was described in Lines 626-640.

17. The mouse models are not as well defined as the authors Claim. Snap23 k/O mice Show germ cell development up to spermatids (Figure 3). Can the authors elaborate more on the presence of multinucleated cells and spermatids at the same time? Was that stable in all animals of this genotype?

As I replied in Essential revisions comment 3, the analysis frequency of AGS in Snap23 cKO was added to Figure 3—figure supplement 2.

[Editors' note: further revisions were suggested prior to acceptance, as described below.]

The manuscript has been significantly improved but there are some remaining issues (many could be amended with textual revisions) that need to be addressed, as outlined below:

1. Fluorescent images are still not clear.

We revised Figure 1C, Figure 2A, 2F, 2G and Figure 5A, which are fluorescence images.

2. Line 161: The authors mentioned "EXOC1 is observed in every cell in the adult testes" in the Figure 1 legend but described "EXOC1-PA was also detected in all male germ cells observed" in text. They may want to focus on germ cells, but it's better clarifying both Sertoli and germ cells are positive in the text if all cells are stained in testicular tubules.

The authors could not judge if Sertoli cells are positive with the current figure.

Thank you for your suggestion. We have corrected lines 167-172 and 893-895 as per your suggestion.

Lines 167-172

Before the revision:

“while no signal could be detected in the Exoc1PA-N/PA-N mice using any method. These data indicate that EXOC1 protein is expressed in male mouse germ cells.”

After the revision:

“while no signal could be detected in the Exoc1PA-N/PA-N mice using any method. In Exoc1+/PA-C adult mice, PA signals were also detected in Sertoli cells, which are located at the basal compartment of the seminiferous tubules and whose nucleus are euchromatic with a large nucleolus (Franca et al., 2016). These data indicate that EXOC1 protein is expressed in male mouse germ and Sertoli cells.”

Line 893-895

Before the revision:

“EXOC1 is observed in every cell in the adult testes.”

After the revision:

“EXOC1 is observed in every cell in the adult testes. The arrowheads indicate Sertoli cells in which the nucleus is eurochromatin with a large nucleolus.”

3. It was originally requested for the authors to assess the efficiency of floxed gene knockout in their models. While they have assessed whether remaining sperm in the Exoc1 conditional KO model are gene deleted, analysis of testis from this and other KO models described has not been performed. A simple qRT-PCR on total testis extracts or isolated testis cells could be used. It is important to define efficiency of gene knockout as the phenotype of some of the models is mild, e.g. Snap23 KO.

Thank you for your suggestion. This is due to our insufficient preparation, but we do not have live cKO mice, frozen samples, and mRNA stock, so we need more than 6 months to prepare for the experiments you kindly suggested. Although we thought your point was plausible, we believe that this RT-qPCR data is not necessarily needed in this study. The reasons for this are as follows.

EXOC1 has been shown to be expressed in spermatocytes and later stages of differentiation. These differentiated cells comprise the majority of all cells in the testis. Therefore, the fraction of each cell population that constitutes the testis drastically altered between the Exoc1 cKO and wild-type. In this situation, if RT-qPCR is performed with entire testis, it is not possible to determine whether the difference in Exoc1 expression between the cKO and wild-type is due to the deletion of the gene or to the alteration in the fraction of the germ cell population. A further complication is that the transcripts are shared among the connected cells that comprise the syncytium. If recombination occurs after Apair but not Asingle, there might be a syncytia with a dilute gene dosage of Exoc1 or Snap23. Moreover, although we cannot completely clarify whether it is complete or incomplete, we could well claim that the loss of Exoc1 leads to abnormal pseudopodia formation of spermatogonia and aggregation of spermatocytes in many cases based on the data that we have presented so far (such as the data on the counting of seminiferous tubules).

The results of Figure 3D and Figure 3—figure supplement 2B clearly showed that Snap23 was involved in the formation or maintenance of spermatocyte syncytia. Since the phenotyping of the Snap23 cKO was performed under almost the same conditions as that of the Exoc1 cKO (same Cre driver, same age at analysis, almost same floxed distance, and same genetic background), the requirement of Snap23 for the formation or maintenance of the syncytium may be lower than that of Exoc1. However, since we do not have experimental data to deny the possibility that the recombination efficiency of Exoc1 cKO and Snap23 cKO may differ, we have revised the text as follows to include both possibilities.

Line 270-279

Before the revision:

“This suggests that Snap23 is dispensable for spermatogenesis and that another protein in the SNAP family could be compensating for that function. […] These results suggest that EXOC1 regulates the formation of the correct syncytium structure in cooperation with STX2 and SNAP23.

After the revision:

“This suggests that Snap23 is dispensable for spermatogenesis and that another protein in the SNAP family could be compensating for that function. […] These results suggest that EXOC1 regulates the formation of the correct syncytium structure in cooperation with STX2 and SNAP23.”

We think that it is very important to know how often the deletion of Exoc1/Snap23 affects each phenotype, and to do so, we need to understand the efficiency of Cre-loxP recombination as you pointed out. However, to evaluate the magnitude of the impact of the loss of those genes, it is also essential to establish the challenging experimental techniques to analyze aggregated spermatocytes perfectly in 3D and the degree of Exoc1/Snap23 mRNA sharing in syncytium consisting of non-recombinant and recombinant cells. These issues are very fascinating and intriguing, but we think they are the next challenge.

4. A new N-terminally PA tagged Exoc1 model is described in the revision as the C-tagged version clearly disrupts EXOC1 function, which is embryonic lethal. Confusingly, while the homozygous N-tagged mice are viable, indicating that EXOC1 function is retained, the authors could not detect expression of the N-tagged EXOC1 by immunostaining and suggest that it might be unstable. This seems counterintuitive and text should be modified to help resolve and discuss this issue. On a related point, lines 160-163 where rationale for use of the LG3 tag in the fusion construct is described – this should be reworded for clarity.

Thank you very much for pointing this out. I have followed your suggestion and corrected lines 158-164.

Before the revision:

“The signal intensity of the band, on the western blot, was lower for Exoc1+/PA-N than that for Exoc1+/PA-C, which might be because EXOC1-PA-N is more easily decomposed.”

After revision:

“The signal intensity of the band, on the western blot, was lower for Exoc1+/PA-N than that for Exoc1+/PA-C. […] Since the Exoc1PA-N homozygous mutant, unlike the Exoc1PA-C homozygous mutant, did not show a pronounced abnormal phenotype, we considered that EXOC1-PA-N is probably more similar in

behavior and function to the wild-type EXOC1 than EXOC1-PA-C.”

5. It is stated that while GFRa1+ spermatogonial pseudopods in the Exoc1 KO are shorter, those of the Stx2 KO are not, suggesting that EXOC1 operates independently of STX2 in this context. However, the pseudopods of Stx2 KO are strongly trending to be shorter and P value is quoted as 0.052. This suggests that a larger dataset may indeed show significant reduction and the conclusions from this data are overstated. Text should be changed appropriately to reflect this strong trend in data and/or more cells quantified to assess the robustness of the conclusions.

Thank you very much for your valuable comments. We were preoccupied with our preconceived notions and were not able to make an accurate assessment. As you pointed out, we also think the Stx2 KO may well have a shorter pseudopod. Therefore, we have revised lines 299-307, lines 943-946.

Lines 299-307:

Before the revision:

“In the Stx2 KO mice generated in this study, as in the Stx2repro34 mice (Fujiwara et al., 2013), spermatocytes were observed to aggregate (Figure 4—figure supplement 1C), but no abnormalities in the length of the spermatogonia pseudopod of the Stx2 KO adult mice were observed (Figure 4A). […] These results suggest that EXOC1 functions in the pseudopod elongation of GFRα1+ spermatogonia independently of STX2.”

After the revision:

“In the Stx2 KO mice generated in this study, as in the Stx2repro34 mice (Fujiwara et al., 2013), spermatocytes were observed to aggregate (Figure 4—figure supplement 1C).[…] These results suggest that EXOC1 functions in the pseudopod elongation of GFRα1+ spermatogonia partially dependently, but not completely dependently of STX2.”

Lines 943-946

Before the revision:

“(B) Pseudopod length quantification using sections. Average length of GFRα1+ spermatogonia pseudopods in Exoc1 cKO was shorter than that of control (n = 3 in each genotype, 23–25 cells in each mouse). […] (n = 3 in each genotype, 40–44 cells in each mouse, *p = 0.052, student’s t-test).”

After the revision:

“(B) Pseudopod length quantification using sections. Average length of GFRα1+ spermatogonia pseudopods in Exoc1 cKO was shorter than that of Stx2 KO and wild type (n = 3 in each genotype, 25–36 cells in each mouse). *p = 0.052, **p = 1.8 × 10-6, ***p = 9.5 × 10-9. one-way ANOVA.”

6. The abstract could be reworded for clarity. A sentence about role of EXOC1, SNAP23 etc in the exocyst complex should be included up-front to help non experts in the field. The term "high regulation" in the first sentence is ambiguous.

We have revised the abstract according to your recommendations. Due to word count limitations, we were unable to describe the functions of these proteins, but we have tried to mention the families to which they belong.

https://doi.org/10.7554/eLife.59759.sa2

Article and author information

Author details

  1. Yuki Osawa

    Master’s Program in Medical Sciences, Graduate School of Comprehensive Human Sciences, University of Tsukuba, Tsukuba, Japan
    Contribution
    Investigation, Writing - original draft
    Contributed equally with
    Kazuya Murata
    Competing interests
    No competing interests declared
  2. Kazuya Murata

    Laboratory Animal Resource Center, Trans-border Medical Research Center, University of Tsukuba, Tsukuba, Japan
    Contribution
    Investigation, Methodology
    Contributed equally with
    Yuki Osawa
    Competing interests
    No competing interests declared
  3. Miho Usui

    School of Medical Sciences, University of Tsukuba, Tsukuba, Japan
    Contribution
    Investigation
    Competing interests
    No competing interests declared
  4. Yumeno Kuba

    Master’s Program in Medical Sciences, Graduate School of Comprehensive Human Sciences, University of Tsukuba, Tsukuba, Japan
    Contribution
    Investigation
    Competing interests
    No competing interests declared
  5. Hoai Thu Le

    Ph.D Program in Human Biology, School of Integrative and Global Majors, University of Tsukuba, Tsukuba, Japan
    Contribution
    Investigation
    Competing interests
    No competing interests declared
  6. Natsuki Mikami

    Ph.D Program in Human Biology, School of Integrative and Global Majors, University of Tsukuba, Tsukuba, Japan
    Contribution
    Investigation
    Competing interests
    No competing interests declared
  7. Toshinori Nakagawa

    1. Division of Germ Cell Biology, National Institute for Basic Biology, National Institutes of Natural Sciences, Okazaki, Japan
    2. Department of Basic Biology, School of Life Science, Graduate University for Advanced Studies (Sokendai), Okazaki, Japan
    Contribution
    Supervision, Methodology
    Competing interests
    No competing interests declared
  8. Yoko Daitoku

    Laboratory Animal Resource Center, Trans-border Medical Research Center, University of Tsukuba, Tsukuba, Japan
    Contribution
    Resources, Methodology
    Competing interests
    No competing interests declared
  9. Kanako Kato

    Laboratory Animal Resource Center, Trans-border Medical Research Center, University of Tsukuba, Tsukuba, Japan
    Contribution
    Resources, Methodology
    Competing interests
    No competing interests declared
  10. Hossam Hassan Shawki

    Department of Comparative and Experimental Medicine, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan
    Contribution
    Resources, Supervision
    Competing interests
    No competing interests declared
  11. Yoshihisa Ikeda

    Doctoral program in Biomedical Sciences, Graduate School of Comprehensive Human Sciences, University of Tsukuba, Tsukuba, Japan
    Contribution
    Investigation
    Competing interests
    No competing interests declared
  12. Akihiro Kuno

    1. Laboratory Animal Resource Center, Trans-border Medical Research Center, University of Tsukuba, Tsukuba, Japan
    2. Ph.D Program in Human Biology, School of Integrative and Global Majors, University of Tsukuba, Tsukuba, Japan
    Contribution
    Methodology
    Competing interests
    No competing interests declared
  13. Kento Morimoto

    Doctoral program in Medical Sciences, Graduate School of Comprehensive Human Sciences, University of Tsukuba, Tsukuba, Japan
    Contribution
    Investigation
    Competing interests
    No competing interests declared
  14. Yoko Tanimoto

    Laboratory Animal Resource Center, Trans-border Medical Research Center, University of Tsukuba, Tsukuba, Japan
    Contribution
    Resources, Methodology
    Competing interests
    No competing interests declared
  15. Tra Thi Huong Dinh

    Laboratory Animal Resource Center, Trans-border Medical Research Center, University of Tsukuba, Tsukuba, Japan
    Contribution
    Resources
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-1705-3865
  16. Ken-ichi Yagami

    Laboratory Animal Resource Center, Trans-border Medical Research Center, University of Tsukuba, Tsukuba, Japan
    Contribution
    Resources, Supervision, Funding acquisition
    Competing interests
    No competing interests declared
  17. Masatsugu Ema

    Department of Stem Cells and Human Disease Models, Research Center for Animal Life Science, Shiga University of Medical Science, Otsu, Japan
    Contribution
    Resources, Supervision
    Competing interests
    No competing interests declared
  18. Shosei Yoshida

    1. Division of Germ Cell Biology, National Institute for Basic Biology, National Institutes of Natural Sciences, Okazaki, Japan
    2. Department of Basic Biology, School of Life Science, Graduate University for Advanced Studies (Sokendai), Okazaki, Japan
    Contribution
    Supervision, Methodology
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-8861-1866
  19. Satoru Takahashi

    Laboratory Animal Resource Center, Trans-border Medical Research Center, University of Tsukuba, Tsukuba, Japan
    Contribution
    Resources, Supervision
    Competing interests
    No competing interests declared
  20. Seiya Mizuno

    Laboratory Animal Resource Center, Trans-border Medical Research Center, University of Tsukuba, Tsukuba, Japan
    Contribution
    Conceptualization, Funding acquisition, Writing - original draft, Project administration
    For correspondence
    konezumi@md.tsukuba.ac.jp
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-6740-5817
  21. Fumihiro Sugiyama

    Laboratory Animal Resource Center, Trans-border Medical Research Center, University of Tsukuba, Tsukuba, Japan
    Contribution
    Supervision, Funding acquisition
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-4744-3493

Funding

Ministry of Education, Culture, Sports, Science and Technology (17H03566)

  • Ken-ichi Yagami

Ministry of Education, Culture, Sports, Science and Technology (19H03142)

  • Seiya Mizuno

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

We would like to thank Tokuko Iwamori for advice on the experimental design for syncytia analyses. We are grateful to Narumi Ogonuki and Atsuo Ogura for their helpful discussions. We would like to thank Yoshihiro Miwa, Hiroyuki Sakuma, and Akio Sekikawa for advice on the experimental design for fluorescence imaging. We are grateful to Aya Ikkyu and Tomoyuki Fujiyama for advice on the experimental design for protein analyses. This work was supported by Scientific Research (B) (17H03566: KY and 19H03142: SM) from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT).

Ethics

Animal experimentation: All animal experiments were carried out in a humane manner with approval from the Institutional Animal Experiment Committee of the University of Tsukuba in accordance with the Regulations for Animal Experiments of the University of Tsukuba and Fundamental Guidelines for Proper Conduct of Animal Experiments and Related Activities in Academic Research Institutions under the jurisdiction of the Ministry of Education, Culture, Sports, Science, and Technology of Japan.

Senior Editor

  1. Anna Akhmanova, Utrecht University, Netherlands

Reviewing Editor

  1. Polina V Lishko, University of California, Berkeley, United States

Reviewers

  1. Robin Hobbs, Monash University/ Hudson Institute, Australia
  2. Stefan Schlatt, University of Munster, Germany, Germany

Publication history

  1. Received: June 7, 2020
  2. Accepted: April 21, 2021
  3. Version of Record published: May 11, 2021 (version 1)

Copyright

© 2021, Osawa et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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