Go 6983

Wilms’ tumour 1 (WT1) negatively regulates the expression of connexin 43 via a non-canonical Wnt signalling pathway in cultured bovine Sertoli cells

Xue Wang A,*, Ziming Wang A,*, S. O. Adeniran A, Fushuo Huang A, Mingjun Ma A, Han Zhang A, Xiaoyu Li A, Peng Zheng A and Guixue Zhang A,B


The gap junction protein connexin (Cx) 43 between adjacent Sertoli cells (SCs) is the main testicular factor regulating the growth and development of SCs, and plays a vital role in controlling cell differentiation and maturation. However, the endogenous testicular factors that regulate Cx43 and the downstream signalling pathways that mediate Cx43-dependent SC differentiation are unclear. In this study, high-purity SCs were isolated from newborn calves’ testes by differential adherence. The SCs were then cultured in vitro and treated with short interference RNA to knockdown endogenous Wilms’ tumour 1 (WT1). In WT1-knockdown SCs, Cx43 expression was upregulated. To elucidate the intracellular signalling mechanism of Cx43 in the differentiation and maturation of immature SCs, SCs were treated simultaneously with non-canonical Wnt signalling inhibitors CCG-1423 and GO-6983; in these SCs, Cx43 expression was upregulated. Together, these data indicate that WT1 negatively regulates the expression of Cx43 produced from SCs via a non-canonical Wnt signalling pathway in cultured bovine SCs.

Additional keywords: freshly isolated SCs, differentiation, non-canonical Wnt signalling inhibitors, WT1-knockdown. Received 13 March 2019, accepted 11 September 2019, published online 6 February 2020


In the testicular seminiferous epithelium, Sertoli cells (SCs) are called ‘mother’ or ‘nurse’ cells, providing a structural and phys- iological framework for the development, proliferation and maturation of germ cells (GCs) during spermatogenesis (Gerber et al. 2016). As puberty approaches, SCs form a complex network of specific intercellular junctions with each other and with adja- cent GCs (Yan et al. 2009; Cheng and Mruk 2012). Among these junctional complexes, the connexin (Cx)-based gap junctions are unique because they form cell membrane channels. In testicular cells, Cx43 is the most abundant gap junction protein (Risley et al. 1992) and allows intercellular communication. Cx43 coordinates SC metabolism and indirectly synchronises the proliferation and differentiation of GCs by metabolic signalling (Risley et al. 2002; Decrouy et al. 2004), which, in turn, plays a critical role in the control of cell proliferation and differentiation (Loewenstein and Rose 1992; Risley et al. 1992; Decrouy et al. 2004). Loss of Cx43 in SCs is associated with continuous SC proliferation and delayed maturation in adulthood (Brehm et al. 2007; Sridharan et al. 2007b). In addition, seminiferous tubules of mice with SC-specific Cx43 knockout contained only SCs and actively proliferating early spermatogonia, indicating that loss of Cx43 prevents initiation of spermatogenesis and leads to a significant reduction in GCs and infertility (Gilleron et al. 2006; Sridharan et al. 2007a; Rode et al. 2018).

Wilms’ tumour 1 (WT1) is a nuclear transcription factor that is expressed primarily in the kidney, gonad, spleen and mesoderm during embryo development (Reddy and Licht 1996). In mature individuals, WT1 expression is restricted to renal podocytes, ovarian granulosa cells and testicular SCs (Pelletier et al. 1991). WT1 is a stable marker of SCs (Mackay 2000; Sharpe et al. 2003) and plays an essential role in testicular development and spermatogenesis (Gao et al. 2006; Zheng et al. 2014). The loss of WT1 during embryo development has been reported to lead to testicular rupture and testicular hypoplasia (Gao et al. 2006). In addition, in vivo deletion of SC-specific WT1 led to the loss of adherence junctions, dysregulation of adherence junction-associated genes and impaired fertility (Rao et al. 2006). Wang et al. (2013) used WT1-conditional knockout mice to show that WT1 affected the development of GCs by regulating the polarity of SCs. In a previous study, we showed that WT1 had a regulatory effect on the polarity and tight junction proteins of bovine SCs and may be involved in the regulation of cell differentiation (Wang et al. 2019). However, the endogenous testicular factors that regulate Cx43 and the downstream signalling pathways that mediate Cx43-dependent SC differentiation remain unclear. In addition, the effects of Cx43 on bovine SCs in vitro has not been studied. Therefore, this study investigated the effects of knocking down the endogenous WT1 gene using short interference (si) RNA and the mechanisms underlying Cx43 regulation of WT1.

Materials and methods

Separation and culture of bovine SCs All animal studies were approved by the Animal Ethics Com- mittee of the North-east Agricultural University, Harbin, China, and were performed with strict adherence to the Guide for the Care and Use of Laboratory Animals (NRC 1996).
The testes of 20 Holland Holstein newborn calves were obtained from Harbin Modern Biological Technical, and SCs were isolated using the differential adherent selection method, as described previously (Adegoke et al. 2018; Wang et al. 2019). Briefly, testes were obtained from newborn calves and washed with phosphate-buffered saline (PBS). Following the removal of the tunica albuginea, the seminiferous tubules were cut into 1 mm3 pieces, placed in a centrifuge tube with 1.0 mg mL—1 collagenase IV/DNase solution (Sigma) and incubated at 348C in a humid environment with agitation for 20 min (110 oscilla- tions min—1). After washing with Dulbecco’s modified Eagle’s medium (DMEM)/F12, the tubules were further digested with 2.5 mg mL—1 trypsin for 20 min at 348C. After digestion, the mixture was passed through a 100-mm stainless mesh and washed with DMEM/F12. The samples were then centrifuged at 300g for 10 min at 348C to remove the enzyme solution, with the cell pellet resuspended in DMEM/F12 containing 10% fetal bovine serum (FBS). After 1–1.5 h at 378C, the medium containing floating cells was collected and plated in new culture dishes to get rid of any fibroblast cells that had adhered to the bottom of the culture dishes. After 4 h culture at 378C, the SCs had become attached to the bottom of the dishes and any floating contaminating GCs were removed by changing the medium. Fresh DMEM/F12 was added for the culture of cells at 378C in a humid environment containing 5% CO2. Vimentin immunostaining was used to determine the purity of the freshly isolated bovine SCs.

Reagents and chemicals
WT1 siRNA sequences were designed and synthesised by Thermo Fisher Scientific and were as follows: WT1-1 siRNA, 50-GCUCCAGCUCAGUGAAAUGTT-30 (sense) and 50-CAU- UUCACUGAGCUGGAGCTT-30 (antisense); WT1-2 siRNA, 50-GAUACAGCACGGUGACCUUTT-30 (sense) and 50-AAG-
GUCACCGUGCUGUAUCTT-30 (antisense); and negative control (NC) siRNA, 50-UUCUCCGAACGUGUCACGUTT-30 (sense) and 50-ACGUGACACGUUCGGAGAATT-30 (anti- sense). The non-canonical Wnt signal downstream Rho inhibitor CCG-1423 and the protein kinase (PK) C inhibitor GO-6983 were purchased from MCE. Rabbit anti-WT1 and anti-phosphorylated (p-) c-Jun N-terminal kinase (JNK) polyclonal antibodies (pAbs) were purchased from Bioss. Rabbit anti-Cx43 and anti-vimentin pAbs were purchased from Protein Tech. Lipofectamine 2000 transfection reagent and TRIzol were purchased from Invitrogen. The cDNA synthesis kit was purchased from Applied Biological Materials (ABM), Canada.

Cell culture

Bovine SCs were plated into six-well culture plates at a density of 3.0 105 cells well—1 in culture medium DMEM/F12 containing 10% FBS (Hyclone) and 1% penicillin–streptomycin (Corning) for 24 h. Then, the medium was replaced with serum-free DMEM/F12 basal medium without antibiotics and cells were transfected with the siRNA molecules using Lipofectamine 2000 transfection according to the manufacturer’s instructions. The transfection ratio of siRNA (pmol) to Lipofectamine 2000 (mL) was 1 : 0.05, and the final concentration of siRNA used was
60 pmol mL—1. After 48 h transfection, SCs were collected for detection of mRNA and proteins. Bovine SCs were then plated into 60-mm culture plates at a density of 8 105 cells in DMEM/F12 containing 10% FBS (Hyclone) and 1% penicillin–streptomycin (Corning) for 24 h. Following this SCs were treated with 10 mM CCG-1423 (a Rho inhibitor) and 100 nM GO-6983 (a PKC inhibitor) for 24 h under
serum-free conditions before being finally stimulated with 10% FBS for 15 min (Hayashi et al. 2014) at 378C and 5% CO2. The negative control group was cultured in medium containing DMSO, at the same volume as the treatment group. Each treatment was replicated three times. Cells were collected from each treatment after 24 h for western blot analysis.

RNA isolation and cDNA synthesis

Total RNA was extracted from the NC and WT1 siRNA-treated bovine SCs using TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. The RNA preparations were treated with AccurRT Reaction Mix 4 (ABM) to remove contaminat- ing genomic DNA and incubated at 428C for 2 min. Approxi- mately 2 mg total RNA was reversed transcribed to cDNA using 5 All In One RT MasterMix (ABM) in a total volume of 20 mL and incubated at 258C for 10 min and then at 428C for 50 min. The reaction was inactivated at 858C for 5 min and cooled on ice. RNA-free water was added to make samples up to 100 mL. Real-time quantitative polymerase chain reaction analysis Real-time quantitative polymerase chain reaction (qPCR) was performed using the comparative method (2—DDCT) to quantify the expression of target genes (Livak and Schmittgen 2001). The cDNA product (1 mL) was used as the template for qPCR. The reaction was performed on a QuantStudio Real-Time PCR
System (Applied Biosystems) using the FastStart Universal SYBR Master (ROX; Roche) according to the manufacturers’ instructions. The reaction mixture (10 mL) contained 5 mL FastStart Universal SYBR Master (ROX), 0.5 mL sense primer and 0.5 mL antisense primer. The thermal conditions were 948C for 5 min, followed by 40 cycles at 958C for 40 s, 608C for 40 s and 728C for 40 s. Fluorescence was measured following each cycle and analysed using QuantStudio Real-Time PCR software version 1.3 (Applied Biosystems). b-Actin (ACTB) was used as a reference gene. All primers used (WT1, Cx43, CTNNB1, Wnt4, Wnt11 and ACTB) are listed in Table 1. expressed in the WT1-2 siRNA and NC siRNA groups, which meant that the Wnt signalling pathway was activated in bovine SCs in vitro. An earlier study indicated that Wnt/b-catenin signalling enhanced transcription of the Cx43 gene in murine SCs (Lo´pez et al. 2019). Activation of aPKC in rat hepatic
epithelial cells increases Cx43 phosphorylation (Lampe et al. 2000), whereas the Wnt/Ca2þ signalling pathway in bovine SCs may inhibit Cx43 expression by PKC.

These results suggest that Cx43 functions differently in different cell types and is different from its cofactor. From this, we hypothesised that Cx43 in bovine SCs was regulated by WT1 through the Wnt/Ca2þ pathway. The maturation of SCs is a complex process involving massive changes in morphology and function (Sharpe et al. 2003; Brehm and Steger 2005). This process is characterised by inhibition or upregulation of the expression of specific proteins associated with SC differentiation (Sharpe et al. 2003), and the effect of WT1 on the expression of these marker proteins is not negligible. Our data indicate that WT1 negatively regulates the Western blot analysis of connexin (Cx) 43 expression 24 h after treatment of bovine Sertoli cells (SCs) with (þ) or without (–) the protein kinase C (PKC) inhibitor GO-6983 and the Rho inhibitor CCG-1423, alone or in combination (see text). The optical density of western blot bands was
determined using ImageJ (National Institutes of Health). Representative blots are shown along with a summary of the data from three independent experiments. Data are the mean s.e.m. **P , 0.01. Cx43 expression was upregulated in the PKCþ group (P , 0.01), was not affected in Rhoþ group (P . 0.05) and was upregulated in the PKCþ plus Rhoþ groups (P , 0.01). Dimethylsulfoxide at the same volume as the treatment group was used in the control groups (i.e. PKC– and Rho– groups). b-Actin was used as an internal reference standard. expression of Cx43, further demonstrating the essential of WT1 in regulating the differentiation of SCs.

This study illustrates that WT1 negatively regulates the expression of Cx43 via a non-canonical Wnt signalling pathway in cultured bovine SCs.

Conflicts of interest
The authors declare that they have no conflicts of interest.

This study was supported by the Heilongjiang Natural Science Foundation of China (C2017033) and National Key R&D Program of China (2017YFD0501903).


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