Wnt5a Regulates Junctional Function of Sertoli cells Through PCP-mediated Effects on mTORC1 and mTORC2

Abstract

The blood–testis barrier (BTB) and apical ectoplasmic specialization (ES), which are synchronized through the crosstalk of Sertoli cells and Sertoli germ cells, are required for spermatogenesis and sperm release. Here, we show that Wnt5a, a noncanonical Wnt signaling pathway ligand, is predominately expressed in both the BTB and apical ES and has a specific expression pattern during the seminiferous epithelium cycle. We employed siRNA to knockdown Wnt5a expression in testis and Sertoli cells, and then identified elongated spermatids that lost their polarity and were embedded in the seminiferous epithelium. Moreover, phagosomes were found near the tubule lumen. These defects were due to BTB and apical ES disruption. We also verified that the expression level and/or location of BTB-associated proteins, actin binding proteins (ABPs), and F-actin was changed after Wnt5a knockdown in vivo and in vitro. Additionally, we demonstrated that Wnt5a regulated actin dynamics through Ror2-mediated mTORC1 and mTORC2. This study clarified the molecular mechanism of Wnt5a in Sertoli cell junctions through the planar cell polarity (PCP) signaling pathway. Our findings could provide an experimental basis for the clinical diagnosis and treatment of male infertility caused by Sertoli cell junction impairment.

Wnt5a, BTB, PCP, mTOR

Sertoli cells, as 1 of the most important types of somatic cells in the testis, are located at the basement membrane of seminiferous tubules and form the blood–testes barrier (BTB) through tight junctions (TJs), basal ectoplasmic specialization (ES), and gap junctions (GJs) and desmosomes (1). The BTB not only divides the seminiferous epithelium into the basal and apical compartments but also supplies nutrition for germ cells and prevents poisonous and harmful substances from entering the seminiferous tubules to damage germ cells (2). More importantly, BTB integrity is also a prerequisite for ensuring the process of meiosis (3).

At the apical compartment of the seminiferous epithelium, there is a special junction structure between Sertoli cells and elongated spermatids called apical ES. It is the only junction structure before sperm release and is involved in maintaining spermatid polarity, adhesion, and release (4).

TJs, GJs, basic ES, and apical ES are actin-based junctions, while desmosomes are intermediate filament–based junctions. Actin microfilaments, or F-actin, are a prerequisite for the maintenance of TJs, basal ES, GJs, and apical ES (2). The disorganization of actin microfilaments can directly disrupt the integrity of the BTB, preleptotene spermatocytes cannot pass through the BTB for meiosis, and spermatids lose polarity and are retained in the seminiferous epithelium, resulting in abnormal spermatogenesis (5). Therefore, to better understand the junctional function of Sertoli cells, we first clarified the regulatory mechanism of actin microfilaments in Sertoli cells.

Studies have shown that the BTB and apical ES display dynamic stability; in other words, they are “opening” and “closing” in the specific stage of the seminiferous epithelium cycle. In rat testes, there are XIV stages in a seminiferous epithelial cycle. The BTB only opens at stage VIII so that preleptotene spermatocytes can pass through, allowing meiosis to form sperm (6). Simultaneously, apical ES also disappears at this stage in readiness for sperm release. During BTB and apical ES “opening,” expression of actin binding proteins (ABPs), such as Arp2/3 and N-WASP (also are branched actin nucleation proteins), is increased; palladin, Eps8, and plastin3 (actin bundling proteins) levels are decreased; and the organization of actin filaments changes from “bundled” to “branched,” which alters the expression level and/or localization (transferred from the cell membrane to the cytoplasm) of junctional proteins between Sertoli cells and then leads to disruption of the BTB and apical ES. In contrast, during BTB and apical ES “closing,” Arp3 and N-WASP levels are decreased, palladin, Eps8, and plastin3 levels are increased, and the organization of actin filaments changes from “branched” to “bundled,” resulting in restructuring of the BTB and apical ES (7-11). Thus, the “bundled” and “branched” actin microfilaments determine the dynamic stability of the BTB and apical ES and ensure normal spermatogenesis, spermatid polarity maintenance, and sperm release (12). Notably, the organization of actin microfilaments is regulated by ABPs. However, the upstream regulatory molecules of these ABPs are still unclear.

The Wnt signaling pathway is highly conserved in invertebrates and vertebrates, and plays an important role in cell proliferation, survival, differentiation, polarization, and migration. The Wnt signaling pathway is divided into the canonical Wnt pathway and noncanonical Wnt pathway, which includes the planar cell polarity (PCP) pathway and Wnt/Ca²⁺ pathway (13). The PCP pathway contains core proteins (such as Vangl, Fzd, and Dvl), effectors (such as Fuzzy), ligands (such as Wnt5a), and signal proteins. The PCP pathway is crucial for tissue morphogenesis, organ development, and function (14). Vangl2 and Dvl3 have been proven to regulate ES dynamics through actin microfilaments; thus, our aim was to verify the role of Wnt5a in maintaining the dynamics of the BTB and apical ES and the mechanism underlying this process (15, 16).

Through in vivo and in vitro studies, we clarified that Wnt5a regulated the BTB and apical ES dynamics through the PCP-mediated mTOR signaling pathway, comprehensively determined the role of Wnt5a in spermatogenesis, and provided a scientific experimental basis for the prevention and treatment of male infertility caused by abnormal BTB function.

Materials and Methods

Animals

Adult male Sprague–Dawley (SD) rats (~300 g body weight) were purchased from the Experimental Animal Center of Chongqing Medical University (Chongqing, China). Animals were fed under specific pathogen–free (SPF) conditions and allowed free access to laboratory food and distilled water. Room temperature and a light/dark cycle were maintained at 25°C ± 2°C and 12 hours/12 hours, respectively. The Experimental Animal Committee of Chongqing Medical University approved for the handling of the animals (license number: 2017-0001).

Antibodies

The primary and secondary antibodies we used in the study are listed in Table 1, and their working dilutions used for immunofluorescence (IF), immunohistochemistry, and Western blot are also shown in Table 1.

Table 1.

Antibodies used for different experiments in this report

Antibody	Host species	Supplier	Catalog number	WB	IF/IHC	RRID
β-Actin	Mouse	ZSGB	TA-09	1:500		AB_2636897
ZO-1	Rabbit	Thermo Fisher Scientific	61-7300	1:1000	1:100	AB_2533938
Occluding	Rabbit	Proteintech	27260-1-AP	1:1000	1:100	AB_2880820
β-Catenin	Rabbit	Thermo Fisher Scientific	71-2700	1:1000	1:100	AB_2533982
N-cadherin	Rabbit	Abcam	ab18203	1:1000	1:100	AB_444317
Cx43	Rabbit	CST	3512S	1:1000	1:100	AB_2294590
Arp3	Rabbit	abcam	ab181164	1:1000	1:100	AB_2892539
Eps8	Mouse	BD	610143	1:1000	1:100	AB_397544
Wnt5a	Mouse	Santa Cruz	sc-365370	1:500	1:100	AB_10846090
rpS6	Mouse	Proteintech	66886-1-Ig	1:1000		AB_2882218
p-rpS6	Rabbit	CST	4858s	1:1000		AB_916156
Rictor	Rabbit	Proteintech	27248-1-AP	1:1000		AB_2880817
AKT	Rabbit	abcam	ab8805	1:1000		AB_306791
p-AKT	Rabbit	CST	4060T	1:1000		AB_2315049
MMP-9	Rabbit	Proteintech	10375-2-AP	1:500		AB_10897178
CDC42	Rabbit	abcam	ab187643	1:1000		AB_2818943
Goat antirabbit IgG		ZSGB	ZB-2301	1:5000		AB_2747412
Goat antimouse IgG		ZSGB	ZB-2305	1:5000		AB_2747415
Cy3-conjugated affinipure goat antimouse IgG		Proteintech	SA00009-1		1:200	AB_2814746
Cy3-conjugated affinipure goat antirabbit IgG		Proteintech	SA00009-2		1:200	AB_2890957

Abbreviations: WB, Western blot; IF, immunofluorescence; Ig, immunoglobulin; IHC, immunohistochemistry.

Adjudin and CdCl₂ Treatment

Nine adult male SD rats (~300 g body weight) were randomly divided into 3 groups: the control group, adjudin group (a single dose of adjudin was administered by gavage at 50 mg/kg body weight , and the testis was collected on day 4), and CdCl₂ group (a single dose of CdCl₂ was administered by intraperitoneal injection at 3 mg/kg body weight, and the testis was collected on day 3). Western blot was performed after extracting the protein from these testes.

Knockdown of Wnt5a In Vivo

On the basis of a published article, we used Polyplus in vivo-jetPEI (Polyplus-transfection S.A., PT-201-20G, France) as a transfection reagent to transfect Wnt5a siRNA duplexes that could knockdown Wnt5a in the testis (17). The target sequence of the Wnt5a siRNA (RiboBio, China) used in vivo was as follows: TCATGAACTTGCACAACAA. According to the manufacturer’s protocol, Wnt5a siRNA or nontargeting control siRNA (100 nM) and 0.34 μL of in vivo-jetPEI (the volume of an adult rat testis was approximately 1.6 mL) constituted 80 μL of the transfection solution, and then we injected the transfection solution using an insulin syringe with a 28-gauge needle. The right testis was transfected with the nontargeting control siRNA, and the left testis was transfected with the Wnt5a siRNA. Sixteen rats were transfected on days 1, 3, and 5, and the testes were collected on day 8 and fixed in 4% paraformaldehyde or Bouin’s solution for histologic analysis, or stored in liquid nitrogen for Western blot and IF analysis.

TM4 Cell Line Culture

The Sertoli cell line TM4 (CVCL_4327) was cultured in DMEM/F12 medium with 2.5% horse serum, 5% fetal bovine serum, and antibiotics. The cells were incubated in a humidified condition of 95% air/5% CO₂ at 37°C and passaged every 2 days. The cells were randomly transfected with nontargeting control siRNA or Wnt5a siRNA.

Knockdown of Wnt5a In Vitro

Wnt5a was knocked down in Sertoli cells by siRNA through using Lipofectamine™ RNAiMAX Transfection Reagent (13778150, Invitrogen). The target sequence of the Wnt5a siRNA (RiboBio, China) used in vitro was as follows: CCGAACGCTGTCATTGCAA. The transfection concentration of the nontargeting control siRNA and Wnt5a siRNA was 15 nM, with 2 transfections. The transfection procedure was performed according to the manufacturer’s instructions.

Hematoxylin–Eosin Staining

After dehydration and embedding in paraffin, testes were cut into 4-μm-thick sections and stained with hematoxylin–eosin to observe changes in the seminiferous tubules. The specific steps were performed according to our previous work (18). The slides were examined using a light microscope (Eclipse Ci-E, Nikon, Japan) with NIS-Elements D imaging software (Version 4.10.00, Nikon, Japan).

Western Blot

We extracted testis and cell proteins by using radioimmunoprecipitation assay lysis buffer (HY-K1001, MCE) and 10% protease inhibitor cocktail (HY-K0010, MCE), and the concentration was calculated using a bicinchoninic acid kit (P0012, Beyotime Biotech, China). The general steps of Western blot were performed according to our previously reported article (19). Image Lab (Version 6.0.0, USA) software was used to analyze images.

Immunofluorescence

Ten-micrometer-thick frozen sections of testes were used to perform IF. This process was performed according to our published article (20). For colocalization IF, we used primary antibodies from different species and combined them with the corresponding fluorescent secondary antibodies. The sections were observed with an A1R confocal microscopy system (Nikon, Japan) or a fluorescence microscope (Eclipse Ci-E, Nikon).

Immunohistochemistry Staining

Four-micrometer-thick sections were used to perform immunohistochemistry as previously described (18). The slides were counterstained with hematoxylin and were observed using an Olympus microscope (BX40).

BTB Integrity Assay

BTB integrity was assessed by injecting EZ-Link SulfoNHS-LC-Biotin (a membrane-impermeable biotinylation reagent with an Mr of 556.59) as previously described (21). Rats treated with CdCl₂ were used as a positive control. The sections were detected with a fluorescence microscope (Eclipse Ci-E, Nikon). When red fluorescence is only observed around the seminiferous tubules, BTB integrity is intact. In contrast, when red fluorescence is widely present across the seminiferous tubules, BTB integrity is disrupted.

Statistical Analysis

Data were analyzed using the SPSS 17.0 software (SPSS, Inc., Chicago) and are expressed as the mean ± standard deviation (SD). Statistical analysis was performed using Student’s t-test. All experiments were replicated at least 3 times with different samples. P < .05 was considered to be statistically significant.

Results

Wnt5a Expression Was Decreased After Treatment With Adjudin and CdCl₂

Adjudin and CdCl₂ are the most commonly used drugs to disrupt the apical ES and BTB integrity, respectively. We found that Wnt5a expression was strongly decreased in the testis after adjudin and CdCl₂ treatment (Fig. 1), suggesting that Wnt5a may have an important role in the junctional function of Sertoli cells.

Figure 1.

Wnt5a expression in testes after treatment with CdCl₂ or adjudin, as determined by Western blot. (A) Wnt5a expression was obviously decreased after treatment with CdCl₂. (B) Wnt5a expression was obviously decreased after treatment with adjudin. Data are presented as the mean ± SD, and all experiments were performed at least in triplicate. *P < .05, compared with the control siRNA group.

Wnt5a Expression Pattern During the Seminiferous Epithelial Cycle

We performed IF to observe the expression pattern of Wnt5a during the seminiferous epithelial cycle in normal testes. In the BTB, Wnt5a was highly expressed at stages VII and VIII (Fig. 2B). At the apical ES, Wnt5a was specifically expressed at stage VII and decreased or even disappeared at stage VIII (Fig. 2C), indicating that Wnt5a has a specific expression pattern during the seminiferous epithelial cycle.

Figure 2.

Wnt5a expression pattern during the seminiferous epithelium cycle. (A) The Wnt5a expression pattern in the BTB during the seminiferous epithelium cycle. Wnt5a was highly expressed at stages VII and VIII in the BTB. Scale bar, 100 μm. (B) The Wnt5a expression pattern in the apical ES during the seminiferous epithelium cycle. Scale bar, 25 μm. (C) Wnt5a was specifically expressed at stage VII and decreased or even disappeared at stage VIII in the apical ES. Scale bar, 25 μm. (D) Wnt5a colocalized with BTB-associated proteins, including ZO-1, N-cadherin, β-catenin, Cx43, and F-actin. Scale bar, 50 μm.

Wnt5a Colocalized With BTB-associated Proteins in the Seminiferous Epithelium

To confirm the location of Wnt5a, we used dual-label IF analysis to assess the colocalization of Wnt5a with F-actin, ZO-1 (TJ adaptor), N-cadherin (basal ES integral membrane proteins), β-catenin (basal ES adaptor), and Cx43 (GJ integral membrane protein), which are all localized at the BTB. Wnt5a was shown to colocalize with these junctional proteins, indicating that Wnt5a is localized at the BTB (Fig. 2D).

Wnt5a Knockdown In Vivo Induced Defects in Spermatogenesis

The expression of Wnt5a in the rat testes was specifically knockdown with siRNA by testicular injection, and we verified the efficiency of Wnt5a knockdown by Western blot and IF, and efficiency in testes was approximately 60% (Fig. 3A and 3B). Hematoxylin–eosin staining showed that in the testes of the control siRNA group, at stage VIII, the heads of the elongated spermatids were toward the seminiferous epithelium basement, and the tails were toward the seminiferous tubule lumen; at stage IX, there were no phagosomes in the apical compartment. However, in the testes of the Wnt5a siRNA group, at stage VIII, some spermatids were trapped in the seminiferous epithelium and failed to be transported to the luminal edge; at stage IX, phagosomes were also observed at the apical compartment (Fig. 3C). The results showed that Wnt5a knockdown caused spermatogenesis defects, and these defects may due to the BTB and apical ES disruption.

Figure 3.

Phenotype after Wnt5a knockdown in testes. (A) The knockdown efficiency of Wnt5a in the testis was determined by Western blot. Data are presented as the mean ± SD, and all experiments were performed at least in triplicate. *P < .05, compared to the control siRNA group. (B) The knockdown efficiency of Wnt5a in the testis by IF. (C) Histomorphologic changes in testes after Wnt5a knockdown. Some spermatids were trapped in the seminiferous epithelium and failed to be transported to the luminal edge at stage VIII (red arrowhead). Phagosomes were also observed at the apical compartment at stage IX (blue arrowhead).

Wnt5a Knockdown In Vivo Induced BTB-associated Protein Expression Changes

Because Wnt5a expression was localized at the BTB and was decreased after treatment with adjudin and CdCl₂, we confirmed that Wnt5a knockdown resulted in defects in spermatogenesis due to BTB disruption. Therefore, we examined BTB-associated protein expressions by Western blot and IF. Western blot results showed that the expressions levels of ZO-1, occludin, N-cadherin, β-catenin, and Cx43 were predominantly decreased after Wnt5a knockdown (Fig. 4A). IF showed the same expression trend as Western blot, and the localization of these proteins was not significantly different (Fig. 4B-4E and 4H). In addition, as the most important ABPs, Arp3 expression level was increased and Eps8 expression was decreased dramatically after Wnt5a knockdown in the testes (Fig. 4A). IF showed that at stage VII, Arp3 was expressed on the concave side of the spermatids at apical ES in the control siRNA group, but its expression disappeared after Wnt5a knockdown (Fig. 4F). Immunohistochemistry showed that in the control siRNA group Eps8 was expressed in the BTB at stage VII and became barely detectable at the Sertoli cell–elongated spermatid interface at stage VIII. However, in the Wnt5a siRNA group, Eps8 expression in the BTB was obviously decreased at stage VII and was still expressed at the Sertoli cell–elongated spermatid interface at stage VIII (Fig. 4H). Additionally, we examined the organization of actin microfilaments and found that the structure of F-actin was disordered (Fig. 4G). The results suggested that Wnt5a knockdown impaired the integrity of the BTB and apical ES.

Figure 4.

The expression level and localization of BTB-associated proteins and ABPs in the testis after Wnt5a knockdown. (A) BTB-associated proteins and Eps8 were decreased, and Arp3 was obviously increased after Wnt5a knockdown. Data are presented as the mean ± SD, and all experiments were performed at least in triplicate. *P < .05 compared with the control siRNA group. (B-H) The localizations of BTB-associated proteins and ABPs after Wnt5a knockdown. Scale bar, 50 μm.

Wnt5a Knockdown In Vivo Impaired the Integrity of the BTB

To further confirm that Wnt5a knockdown in the testes impaired the integrity of the BTB, we used biotin to verify BTB function. In the control siRNA group, red fluorescence was present at the basement of the seminiferous epithelium, and the lumens of the seminiferous tubules showed no red fluorescence. As a positive control, after treatment with CdCl₂, BTB integrity was seriously disrupted, and red fluorescence was predominantly observed in the whole tubule. Similarly, BTB function of the Wnt5a knockdown testes was impaired, and red fluorescence was also present in the tubules (Fig. 5).

Figure 5.

BTB integrity in testis after Wnt5a knockdown. BTB integrity was disrupted after Wnt5a knockdown, and red fluorescence was widely present in seminiferous tubules. CdCl₂ as a positive control. Scale bar, 100 μm.

Wnt5a Knockdown In Vitro Led to Alterations in BTB-associated Protein and ABP Expression

We used siRNA to knockdown Wnt5a expression in Sertoli cells, and examined the junctional function of Sertoli cells. Wnt5a knockdown efficiency was approximately 70% (Fig. 6A). Expression of BTB-associated proteins and ABPs in Wnt5a knockdown Sertoli cells showed a similar trend, except that Arp3, ZO-1, occludin, N-cadherin, β-catenin, Cx43, and Eps8 levels were predominantly decreased, but Arp3 expression was not different from that in the control siRNA group (Fig. 6B). Then we examined the localization of Arp3 in Sertoli cells, and we found that its localization was transferred from the cell membrane to the cytoplasm after Wnt5a knockdown (Fig. 6C). Moreover, we also detected organization of F-actin. Compared with the control siRNA group, F-actin organization was disordered in the Wnt5a siRNA group (Fig. 6D).

Figure 6.

The expression of BTB-associated proteins and ABPs in Sertoli cells after Wnt5a knockdown. (A) The knockdown efficiency of Wnt5a in Sertoli cells determined by Western blot. (B) The expression of BTB-associated proteins and ABPs was obviously decreased after Wnt5a knockdown. Data are presented as mean ± SD, and all experiments were performed at least in triplicate. *P < .05 compared with the control siRNA group. (C) The localization of Arp3 was changed in Sertoli cells after Wnt5a knockdown. Scale bar, 25 μm. (D) The F-actin organization was disordered in Sertoli cells after Wnt5a knockdown. Scale bar, 25 μm.

Wnt5a Regulated Sertoli Cell Junctions Through the PCP Signaling Pathway

The PCP pathway, as a noncanonical Wnt pathway, binds Wnt proteins to Frizzled and to coreceptors such as the receptor-tyrosine kinase-like orphan receptor 2 (Ror2) to regulate cell movement, cell cytoskeleton, and polarity (22-24). ROR2 was substantially decreased in vivo and in vitro after Wnt5a knockdown (Fig. 7A and 7B), suggesting that Wnt5a regulated BTB dynamics through the PCP pathway. According to a published article, Wnt5a can regulate the mTOR pathway, and the mTOR pathway has an important role in regulating BTB integrity (25, 26). mTOR binds with raptor or rictor to form 2 different complexes, mTORC1 and mTORC2. They have opposite roles in maintaining BTB dynamics: mTORC1 promotes BTB integrity, and mTORC2 disrupts BTB integrity (27-29). Thus, we examined the activity of mTORC1 and mTORC2. As shown in Fig. 7, we found that the expression level of p-rps6/rps6 (the downstream target of mTORC1) was significantly increased, and rictor (the component of mTORC2) levels were obviously decreased in vitro (Fig. 7A) and in vivo (Fig. 7B), indicating that Wnt5a knockdown perturbed the balance of mTORC1 and mTORC2. Cdc42, a polarity protein, was also decreased in Sertoli cells (Fig. 7A) and in the testis (Fig. 7B). The levels of pAkt/Akt and MMP9 were enhanced and diminished in Wnt5a knockdown Sertoli cells (Fig. 7A) and testes (Fig. 7B).

Figure 7.

The balance of mTORC1 and mTORC2 was perturbed in testis and Sertoli cells after Wnt5a knockdown. (A) After Wnt5a was knocked down in Sertoli cells, the expression levels of ROR2, rictor, MMP9, and CDC42 were obviously decreased, and the expression levels of p-rps6 and pAKT were significantly increased. (B) Consistent with the in vitro results, after Wnt5a was knocked down in the testis, the expression of ROR2, rictor, MMP9, and CDC42 was obviously reduced, and the expression levels of p-rps6 and pAKT were enhanced significantly. Data are presented as the mean ± SD, and all experiments were performed at least in triplicate. *P < .05 compared with the control siRNA group.

Discussion

The BTB and apical ES are critical for spermatogenesis, and the organization of actin microfilaments plays a crucial role in ensuring the integrity of the BTB and apical ES. Arp3 and Eps8, as the most important ABPs that regulate actin organization, showed changes in the testes and Sertoli cells after Wnt5a knockdown, indicating that Wnt5a is a regulator of ABPs and that it controls BTB and apical ES integrity by affecting ABPs. In the Wnt5a knockdown testis, Arp3, which should be expressed on the concave side of the spermatids at stage VII in the control testis, disappeared. However, the expression level of Arp3 was not increased in vitro. In the adjudin-treated SD rats model, mTORC1 led to abnormal distribution of Arp3, and the change in localization can also affect F-actin organization (5, 30). Thus, we examined the localization of Arp3 in Sertoli cells, and found that its localization was transferred from the cell membrane to the cytoplasm.

Additionally, we found that some spermatids were trapped in the seminiferous epithelium at stage VIII and failed to be transported to the luminal edge at stage IX, suggesting that apical ES was impaired in addition to BTB disruption. Apical ES is another special junctional structure between Sertoli cells and developing spermatids and is located at the apical apartment of the seminiferous epithelium (31). ES is the only anchoring structure before sperm release, is supported by actin filaments, and is involved in maintaining sperm polarity, adhesion, and release (4). There are 3 kinds of polarity protein complexes that regulate Sertoli cell and spermatid polarity: the CRB complex (CRB/PATJ/PALS1), Par complex (Par3/par6/αPKC/Cdc42), and Scribble complex (Scribble/Dlg/Lgl). The CRB and Par complexes localize at the apical compartment, and the Scribble complex localizes at the basal compartment (32). We demonstrated that the expression of Cdc42 was decreased after Wnt5a knockdown in vivo and in vitro. Moreover, localization of Arp3 and Eps8, ABPs regulating spermatid polarity, was changed after Wnt5a knockdown, suggesting that Wnt5a also has an important role in maintaining apical ES function.

Many genes are involved in the regulation of actin microfilament organization and BTB dynamics; however, the mechanism during this process remains unclear. The 2 most important signaling pathways in regulating the BTB are the mTOR (mTORC1 and mTORC2) and FAK (p-FAK-Tyr⁴⁰⁷ and p-FAK-Tyr³⁹⁷) pathways (6). Both mTORC1 and mTORC2, and p-FAK-Tyr⁴⁰⁷ and p-FAK-Tyr³⁹⁷ have opposite roles. mTORC1 and p-FAK-Tyr⁴⁰⁷ are known to support BTB integrity, and mTORC2 and p-FAK-Tyr³⁹⁷ have been demonstrated to restructure BTB integrity (26, 33). However, their upstream regulatory genes are unknown.

According to a recently published report, Wnt5a/ROR2 promotes myeloma cell interactions with the surrounding bone marrow through AKT and mTOR activation (25). Because the Wnt signaling pathway can regulate mTORC1 signaling function, in our results, ROR2 was decreased after Wnt5a knockdown in vivo and in vitro; therefore, we speculated that mTOR may be the downstream gene of Wnt5a (34). mTOR and raptor form mTORC1, which facilitates BTB restructuring through rps6/AKT-mediated MMP9 and Arp3. mTORC2, which is formed by mTOR and rictor, promotes BTB integrity through aPKC/Rac1 GTPase and GJs (such as Cx43). Both mTORC1 and mTORC2 have effects on actin filaments (6). In our study, we demonstrated that after knockdown of Wnt5a in testis and Sertoli cells, the ratio of p-rps6/rps6 and the expression levels of Arp3 (not in vitro, but the localization was altered) were obviously augmented; the expression levels of rictor and Cx43 were reduced significantly; and F-actin was disorganized after Wnt5a knockdown in testis. Nevertheless, when p-rps6 increased, the ratio of pAkt/Akt and the level of MMP9 should have declined and amplified, respectively. In fact, their expression levels were just the opposite. During the migration and invasiveness of rheumatoid arthritis fibroblast-like synoviocytes, Wnt5a positively regulated AKT and MMP9 (35). Wnt5a inhibition can also can upregulate MMP9 expression in malignant peripheral nerve sheath tumor cells (36). Combined with our results, we speculated that the regulatory relationship between Wnt5a and MMP9 was different in the different pathological processes. In the process of Sertoli cell junctional function disruption, Wnt5a may mainly regulate Arp3 through rps6 but not rps6/Akt, and the specific mechanism needs to be further explored. Although we know that mTORC1 and mTORC2 can regulate the “opening” and “closing” of the BTB, their upstream genes remain unclear. We found that knockdown of Wnt5a resulted in BTB and apical ES disruption, which was regulated by ROR2-mediated mTORC1 and mTORC2, indicating that Wnt5a is involved in actin filament organization through the imbalance of mTORC1 and mTORC2.

In conclusion, Wnt5a regulates the BTB and apical ES integrity through its receptor ROR2, which in turn mediates the balance of mTORC1 and mTORC2, ensuring correct “bundling” and “branching” of actin filaments and maintaining BTB and apical ES dynamics.

Abbreviations

Abbreviations
 
• ABP

  actin binding protein

 
• BTB

  blood-testis barrier

 
• ES

  ectoplasmic specialization

 
• GJ

  gap junction

 
• IF

  immunofluorescence

 
• PCP

  planar cell polarity

 
• SD

  Sprague–Dawley

 
• SD

  standard deviation

 
• SPF

  specific pathogen–free

 
• TJ

  tight junction

Acknowledgments

Financial Support: This study was supported by grants from the National Natural Science Foundation of China (Grant no. 81801521, 81771566), the Chongqing Science & Technology Commission (Grant no. cstc2018jcyjAX0270).

Additional Information

Disclosures: The authors have no potential conflicts of interest to declare.

Data Availability

Some or all datasets generated during and/or analyzed during the current study are not publicly available but are available from the corresponding author on reasonable request.