睾酮在体外对人卵巢颗粒细胞SVOG凋亡的影响及其内质网应激机制

doi:10.13481/j.1671-587X.20230507

Abstract

Abstract:

Objective To discuss the inductive effect of testosterone on the apoptosis of the granulosa cells in vitro，and to clarify its mechanism. Methods The inhibitory rates of proliferation of human ovarian granulosa cells SVOG were detected by methyl thiazolyl tetrazolium（MTT） assay. After screening the optimal concentrations of the drugs， the SVOG cells were divided into control group， testosterone group （1.0×10^－5 mol·L^－1 testosterone），testosterone+fluorothiamine group（1.0×10^－5 mol·L^－1 testosterone+ 1.0×10^－5 mol·L^－1 fluorothiamine），and testosterone+tauroursodeoxycholic acid （TUDCA）group （1.0×10^－5 mol·L^－1 testosterone+1.5 g·L^－1 TUDCA）. Real-time fluorescence quantitative PCR （RT-qPCR） method was used to detect the expression levels of spliced X-box binding protein 1 （XBP1s）， activating transcription factor 4 （ATF4）， transcription factor C/EBP homologous protein （CHOP）， and death receptor 5 （DR5） mRNA in the SVOG cells in various groups； flow cytometry was used to detect the apoptotic rates of the SVOG cells in various groups. Results Compared with control group， the SVOG cells in testosterone group showed morphological changes， the number of atypical cells was increased， the granularity in the cytoplasm was increased， the cell fragments in the culture medium was increased， and the inhibitory rate of proliferation of the SVOG cells was increased （P<0.05）. Compared with testosterone group， the morphological changes of the cells in testosterone +fluorothiamine group and testosterone+TUDCA group were decreased and inhibitory rates of proliferation of the cells were decreased（P<0.05）. The flow cytometry results showed that compared with control group， the apoptotic rate of the SVOG cells in testosterone group was significantly increased （P<0.05）. Compared with testosterone group， the apoptotic rates of the SVOG cells in testosterone + TUDCA group and testosterone + fluorothiamine group were significantly decreased （P<0.05）. The RT-qPCR analysis results showed that after treated for 48 h， compared with control group， the expression levels of XBP1s， ATF4， CHOP， and DR5 mRNA in the SVOG cells in testosterone group were increased （P<0.05）. Compared with testosterone group， the expression levels of XBP1s，ATF4，CHOP， and DR5 mRNA in the SVOG cells in testosterone+TUDCA group and testosterone+fluorothiamine group were decreased （P<0.05）. Conclusion Testosterone can induce the apoptosis of human ovarian granulosa cells and cause follicular atresia，and its mechanism may be that androgen combined with adrogen receptor（AR） activates the CHOP-DR5 pathway， induces endoplasmic reticulum stress（ERS）， and promotes the apoptosis of the granulosa cells.

Key words: Testosterone, Granulosa cells, Endoplasmic reticulum stress, Androgen receptor, Transcription factor C/EBP homologous protein, Death receptor 5

CLC Number:

R711.75

Xiaoli TONG,Minghui FAN,Jia MENG,Jihong ZHU,Minjia SHENG. Effect of testosterone on apoptosis of human ovarian granulosa cells SVOG in vitro and its endoplasmic reticulum stress mechanism[J].Journal of Jilin University(Medicine Edition), 2023, 49(5): 1154-1160.

Figures/Tables 5

Tab.1

Tab.2

Tab.3

Fig. 1

Fig. 2

References 26

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Group	Inhibitory rate of proliferation
Group	0 mol·L^－1testosterone	0.1×10^－5 mol·L^－1testosterone	0.5×10^－5 mol·L^－1 testosterone	1.0×10^－5 mol·L^－1 testosterone	5.0×10^－5 mol·L^－1 testosterone	10.0×10^－5 mol·L^－1 testosterone
24 h	1.08±1.15	2.04±1.48	10.98±2.24^△	30.07±0.80^△#	43.64±5.55^△#○	50.10±6.68^△#○▲
48 h	10.75±1.87^*	19.15±0.37^*	32.19±1.63^*△	45.91±2.17^*△#	70.77±2.42^*△#○	75.85±2.38^*△#▲
^*P<0.05 vs 24 h;^△P<0.05 vs 0 mol·L^－1 testosterone;^#P<0.05 vs 0.5×10^－5 mol·L^－1 testosterone;^○P<0.05 vs 1.0×10^－5 mol·L^－1 testosterone;^▲P<0.05 vs 5.0×10^－5 mol·L^－1 testosterone.

Group	Inhibitory rate of proliferation
Group	0 mol·L^－1flutamide	0.1×10^－5 mol·L^－1 flutamide	0.5×10^－5 mol·L^－1 flutamide	1.0×10^－5 mol·L^－1 flutamide	5.0×10^－5 mol·L^－1 flutamide	10.0×10^－5 mol·L^－1 flutamide
24 h	35.31±0.77	33.36±8.39	18.67±1.63^△	13.32±0.87^△#	21.04±0.37^△#○	26.05±2.70^△#○▲
48 h	47.15±2.69^*	45.91±2.17^*	33.68±3.86^*△	21.99±1.51^*△#	36.15±4.11^*△#○	49.89±4.09^*△#○▲
^*P<0.05 vs 24 h;^△P<0.05 vs 0 mol·L^－1 flutamide;^#P<0.05 vs 0.5×10^－5 mol·L^－1 flutamide;^○P<0.05 vs 1.0×10^－5 mol·L^－1 flutamide;^▲P<0.05 vs 5.0×10^－5 mol·L^－1 flutamide.

Group	Inhibitory rate of proliferation
Group	0 g·L^－1 TUDCA	0.5 g·L^－1TUDCA	1.0 g·L^－1TUDCA	1.5 g·L^－1 TUDCA	2.5 g·L^－1 TUDCA	5.0 g·L^－1 TUDCA
24 h	38.50±5.43	27.07±5.47^△	21.03±4.09^△#	11.22±1.47^△#○	21.17±4.35^△▲	35.78±5.37^▲●
48 h	46.91±1.03^*	40.08±3.62^*△	32.49±5.15^*△#	19.95±1.19^*△#○	39.43±1.05^*#▲	43.98±5.31^*▲●
^*P<0.05 vs 24 h;^△P<0.05 vs 0 g·L^－1 TUDCA;^#P<0.05 vs 0.5 g·L^－1 TUDCA;^○P<0.05 vs 1.0 g·L^－1 TUDCA;^▲P<0.05 vs 1.5 g·L^－1 TUDCA;^●P<0.05 vs 2.5 g·L^－1 TUDCA.

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Effect of testosterone on apoptosis of human ovarian granulosa cells SVOG in vitro and its endoplasmic reticulum stress mechanism

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