• Asian-Australasian Journal of Animal Sciences
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• 제32권5호
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• pp.629-636
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• 2019

Objective: The study was designed to determine the cytotoxic effect of gallic acid (GA), obtained by the hydrolysis of tannins, on mice TM4 Sertoli cells apoptosis. Methods: In the present study, non-tumorigenic mice TM4 Sertoli cells were treated with different concentrations of GA for 24 h. After treatment, cell viability was evaluated using WST-1, mitochondrial dysfunction, cells apoptosis and necrosis was detected using JC-1, Hoechst 33342 and propidium iodide staining. The expression levels of Cyclin B1, proliferating cell nuclear antigen (PCNA), Bcl-2-associated X protein (BAX), and Caspase-3 were also detected by quantitative real-time polymerase chain reaction and Western-blotting. Results: The results showed that 20 to $400{\mu}M$ GA inhibited viability of TM4 Sertoli cells in a dose-dependent manner. Treatment with $400{\mu}M$ GA significantly inhibited PCNA and Cyclin B1 expression, however up-regulated BAX and Caspase-3 expression, caused mitochondrial membrane depolarization, activated Caspase-3, and induced DNA damage, thus, markedly increased the numbers of dead cells. Conclusion: Our findings showed that GA could disrupt mitochondrial function and caused TM4 cells to undergo apoptosis and necrosis.

• 한국발생생물학회지:발생과생식
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• 제9권2호
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• pp.115-121
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• 2005

본 연구는 Leydig 세포주와 Sertoli 세포주상에 bisphenol A(BPA)와 diethylstilbestrol(DES)의 영향을 알아보고자 수행하였다. 세포 종류에 따른 BPA의 영향을 알아보기 위해, BPA의 농도별로 두 세포주에 처리하여 세포생존율을 비교하였다. Sertoli 세포주가 Leydig 세포주에 비해서 저농도의 BPA에서 생존율이 유의하게 감소되는 것을 확인할 수 있어, Sertoli 세포가 Leydig 세포주에 비해 BPA에 더욱 민감한 것을 알 수 있었다. 또한 BPA나 DES를 처리했을 때 세포 내 분화 및 사멸 신호 전달에 관여하는 phospholipase D(PLD)의 활성이 현저하게 저하되는 것을 확인하였다. 역전사효소 연쇄 반응을 이용하여 세포막상의 세포자연사 신호전달자인 fas 와 fas ligand의 mRNA 발현을 확인해 본 결과, BPA의 처리에 의해 fas ligand의 발현이 다른 실험군에 비해 유의하게 증가하는 것을 확인할 수 있었다. 이상의 결과들을 정리해 볼 때, 내분비계 교란물질인 BPA는 Sertoli 세포 내 fas/fasL 신호전달계를 자극하여 PLD 활성을 저하시킴으로서, Sertoli 세포 내 세포자연사를 유발시키는 것으로 사료된다.

• 한국발생생물학회:학술대회논문집
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• 한국발생생물학회 2001년도 후기 제12차 학술대회 논문집
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• pp.38-38
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• 2001

TM4 Sertoli cell의 체외 관강형성 유도에 미치는 세포외기질 (ECM) 및 hepatocyte growth factor (HGF)의 역할과 세포분화 과정에서 MMP의 발현의 변화를 조사하였다. Matrigel bed(60%, v/v) 상에서 배양한 TM4 cell은 무혈청 조건하에서 chain 분화단계를 거쳐 cord의 구조로 분화하였다. 그러나 이후의 분화는 일어나지 않았다. TM4 cell에서 c-MET (HGF receptor)의 발현을 확인하였으며 HGF를 첨가한 배양액에서 분화가 촉진되었으며, cord에서 tubule로의 분화가 유도되었다. 또한 TM4 cell의 분화는 MMP-2 및 MMP-9의 발현이 증가를 수반하였으며 HGF는 MMPs의 발현을 증가시켰다. GFR-Matrigel과 성장인자인 HGF는 무혈청 배지에서 TM4 cell의 체외에서 관강형성에 필요한 환경을 제공하며, MMP-2 및 -9은 TM4 cell의 체외분화 과정에서 조절역할을 수행하는 것으로 사료된다.

• 한국발생생물학회지:발생과생식
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• 제25권1호
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• pp.15-24
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• 2021

Benzo[a]pyrene (B[a]P) is a potent carcinogen and is classified as an endocrine-disrupting chemical. In mammalian testes, Sertoli cells support spermatogenesis. Therefore, if these cells are negatively affected by exposure to xenotoxic chemicals, spermatogenesis can be seriously disrupted. In this context, we evaluated whether mouse testicular TM4 Sertoli cells are susceptible to the induction of cytotoxicity-mediated cell death after exposure to B[a] P in vitro. In the present study, while B[a]P and B[a]P-7,8-diol were not able to induce cell death, exposure to BPDE resulted in cell death. BPDE-induced cell death is accompanied by the activation of caspase-3 and caspase-7. Depolarization of the mitochondrial membrane and cytochrome c release from mitochondria were observed in benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE)-treated cells. These results indicate that TM4 cells are susceptible to apoptosis in a caspase-dependent manner. Western blot and reverse transcription-polymerase chain reaction (RT-PCR) analyses showed that aryl hydrocarbon receptor (AhR) expression was almost undetectable in TM4 cells and that its expression was not altered after B[a]P treatment. This indicates that TM4 cells are nearly AhR-deficient. In TM4 cells, the CYP1A1 protein and its activity were not present. From these results, it is clear that AhR may be a prerequisite for CYP1A1 expression in TM4 cells. Therefore, TM4 cells can be referred to as CYP1A1-deficient cells. Thus, TM4 Sertoli cells are believed to have a rigid and protective cellular machinery against genotoxic agents. In conclusion, it is suggested that tolerance to B[a]P cytotoxicity is associated with insufficient AhR and CYP1A1 expression in testicular Sertoli cells.

• 대한한의학방제학회지
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• 제26권2호
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• pp.103-111
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• 2018

Objectives : The purpose of this study was to examine the antioxidant effects of the extract of Rubi Fructus on TM4 Sertoli cells. Methods : The extract was studied for diphenyl-picryl-hydrazyl (DPPH) radical scavenging activity and cell viability assays on Sertoli cells. In addition, hydrogen peroxide-induced oxidative stress on Sertoli cells were examined by MTT assay. The antioxidant enzyme of Cu/Zn SOD, Mn SOD, catalase protein expression on Sertoli cells were also measured. Results : The results showed that the extract scavenged DPPH radical dose-dependent manner. The extract showed no cytotoxicity at concentration of 1, 5, 10, 50, $100{\mu}g/ml$. The hydrogen peroxide-induced cytotoxicity of Sertoli cells was protected to 88.3% by the extract at concentration of $100{\mu}g/ml$. Cu/Zn SOD and Mn SOD protein expression were significantly increased on Sertoli cells, but catalase protein expression was not significantly changed. Conclusions : In conclusion, the extract of Rubi Fructus has antioxidant effects on Sertoli cells and protect male reproductive system against oxidative stress.

• 대한본초학회지
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• 제36권5호
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• pp.117-123
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• 2021

Objectives : The aim of this study is to investigate the effect of wogonin on the production of hydrogen peroxide in polyinosinic:polycytidylic acid (poly I:C)-stimulated TM4 mouse sertoli cells. Methods : TM4 were treated with poly I:C (50 ug/mL) and wogonin at concentrations of 5, 10, 25, and 50 µM for 30 min, 2 hr, 12 hr, 18 hr, and 24 hr. The production of intracellular hydrogen peroxide was measured by dihydrorhodamine 123 assay. Results : For 30 min, 2 hr, 12 hr, 18 hr, and 24 hr treatment, wogonin significantly inhibited intracellular hydrogen peroxide productions of TM4 at the concentration of 5, 10, 25, and 50 µM (p<0.05). In details, production of hydrogen peroxide in poly I:C-stimulated TM4 treated for 30 min with wogonin at concentrations of 5, 10, 25, and 50 µM was 95.67%, 92.69%, 92.05%, and 91.97% of the control group treated with poly I:C only, respectively; the production of hydrogen peroxide for 2 hr was 94.44%, 94.41%, 93%, and 92.98%, respectively; production of hydrogen peroxide for 12 hr was 96.78%, 95.32%, 94.33%, and 93.17%, respectively; production of hydrogen peroxide for 18 hr was 94.7%, 93.4%, 93.38%, and 93.35%, respectively; and production of hydrogen peroxide for 24 hr was 95.75%, 94.77%, 94.58%, and 92.8%, respectively. Conclusions : Wogonin might have anti-viral property related with its inhibition of intracellular hydrogen peroxide production in poly I:C-stimulated TM4 cells.

• 한국동물생명공학회지
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• 제38권4호
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• pp.291-299
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• 2023

Background: Leydig cells, crucial for testosterone production, express ion channels like ANO1 that influence hormone secretion. This study investigates the expression and role of the Tandem of P domains in a weak inward rectifying K⁺ channel-related Acid-Sensitive K⁺-1 (TASK-1) channel in these cells, exploring its impact on testicular function and steroidogenesis. Methods: TASK-1 expression in Leydig cells was confirmed using immunostaining, while RT-PCR and Western Blot (WB) validated its expression in the TM3 Leydig cell line. The effect of a TASK-1 channel blocker on cell viability was assessed through live/dead staining and MTT assays. Additionally, the blocker's effect on testosterone secretion was evaluated by measuring testosterone levels. Results: Immunohistochemical analysis revealed a predominant presence of TASK-1, along with c-Kit and ANO-1, in Leydig cells adjacent to seminiferous tubules and also in Sertoli and spermatogenic cells. Expression levels of TASK-1 mRNA and protein were significantly higher in TM3 Leydig cells compared to TM4 Sertoli cells. In addition, blocking TASK-1 in TM3 cells with ML365 induced cell death but did not affect LH-induced testosterone secretion. Conclusions: These findings suggest that TASK-1 in Leydig cells is crucial for their viability and proliferation, highlighting its potential importance in testicular physiology.

• 한국발생생물학회:학술대회논문집
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• 한국발생생물학회 1998년도 제4차 학술발표대회 및 정기총회
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• pp.43-44
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• 1998

Effect of somatic cell coculture on hatching of mouse blastocyst was examined. Mid-blastocysts were cocultured with granulosa cell primary culture or Sertoli cell line ($TM_{4}$) derived from mouse testis for 48 hr. Blastocysts cultured in medium (10% FBS) started to hatch more faster than cocultured embryos during 12 hr of coculture. After then blastocysts cocultured with somatic cell hatched faster than control. Degeneration of embryos was also greately reduced by coculture. This result suggested the potentiation of hatching as well as embryonic viability by coculture with somatic cell and Sertoli cell line can be used for embryo coculture.