TY - JOUR
T1 - Circadian clock gene BMAL1 controls testosterone production by regulating steroidogenesis-related gene transcription in goat Leydig cells
AU - Xiao, Yaoyao
AU - Zhao, Lijia
AU - Li, Weidong
AU - Wang, Xiaoyu
AU - Ma, Tiantian
AU - Yang, Luda
AU - Gao, Lei
AU - Li, Cuimei
AU - Zhang, Manhui
AU - Yang, Dan
AU - Zhang, Jing
AU - Jiang, Haizhen
AU - Zhao, Hongcong
AU - Wang, Yiqun
AU - Chao, Hsu Wen
AU - Wang, Aihua
AU - Jin, Yaping
AU - Chen, Huatao
N1 - Funding Information:
We are grateful to Dr. Steven A. Brown (University of Zurich, Zurich, Switzerland) for providing the pLV6‐‐ plasmid and Dr. Ximing Qin (Anhui University, Hefei, China) for providing the mCLOCK, mPER2, pRL‐CMV, and ‐ plasmids. We would like to thank Editage ( www.editage.com ) for their writing support of this manuscript. This study was supported by the National Natural Science Foundation of China Grants 31602125 and 31771301 (to Huatao Chen) and 31772817 (to Yaping Jin) and 31900838 (to Lijia Zhao), the China Postdoctoral Science Foundation Grants 2017M610658 and 2018T111112 (to Huatao Chen) and 2019M650279 (to Lijia Zhao), the Natural Science Basic Research Plan in Shaanxi Province of China Grant 2019JM‐038 (to Huatao Chen), the Shaanxi Postdoctoral Science Foundation Grant 2017BSHEDZZ105 (to Huatao Chen), The Scientific Research Foundation for Talents of Shaanxi Grant A279021712 (to Huatao Chen), The Scientific Research Foundation for Talents of Northwest A&F University Grant Z11021601 (to Huatao Chen). Bmal1 Luc PK2.8 Luc
Publisher Copyright:
© 2021 Wiley Periodicals LLC
PY - 2021
Y1 - 2021
N2 - Testosterone is produced by Leydig cells (LCs) and undergoes diurnal changes in serum levels in rats, mice, and humans, but little is known in goats. The present study revealed that goat serum testosterone levels displayed diurnal rhythmic changes (peak time at ZT11.2). Immunohistochemical staining showed that BMAL1, a circadian clock protein, is highly expressed in goat LCs. ELISA revealed that both hCG (0–5 IU/ml) and 22R-OH-cholesterol (0–30 μM) addition stimulated testosterone synthesis in primary goat LCs in a dose-dependent manner. Treating goat LCs with hCG (5 IU/ml) significantly increased intracellular cAMP levels. Additionally, real-time quantitative polymerase chain reaction (PCR) analysis revealed that the circadian clock (BMAL1, PER1, PER2, DBP, and NR1D1) and steroidogenesis-related genes (SF1, NUR77, StAR, HSD3B2, CYP17A1, CYP11A1, and HSD17B3) showed rhythmic expression patterns in goat LCs following dexamethasone synchronization. Several Bmal1-Luc circadian oscillations were clearly observed in dexamethasone-treated goat LCs transfected with the pLV6-Bmal1-Luc plasmid. BMAL1 knockdown significantly downregulated mRNA levels of PER2, NR1D1, DBP, StAR, HSD3B2, SF1, NUR77, and GATA4, and dramatically decreased StAR and HSD3B2 protein levels and testosterone production. In contrast, BMAL1 overexpression significantly increased the mRNA and protein expression levels of StAR and HSD17B3 and enhanced testosterone production. Reporter assays revealed that goat BMAL1, or in combination with mouse CLOCK, activated goat HSD17B3 transcription in vitro. These data indicate that BMAL1 contributes to testosterone production by regulating transcription of steroidogenesis-related genes in goat LCs, providing a basis for further exploring the underlying mechanism by which the circadian clock regulates ruminant reproductive capability.
AB - Testosterone is produced by Leydig cells (LCs) and undergoes diurnal changes in serum levels in rats, mice, and humans, but little is known in goats. The present study revealed that goat serum testosterone levels displayed diurnal rhythmic changes (peak time at ZT11.2). Immunohistochemical staining showed that BMAL1, a circadian clock protein, is highly expressed in goat LCs. ELISA revealed that both hCG (0–5 IU/ml) and 22R-OH-cholesterol (0–30 μM) addition stimulated testosterone synthesis in primary goat LCs in a dose-dependent manner. Treating goat LCs with hCG (5 IU/ml) significantly increased intracellular cAMP levels. Additionally, real-time quantitative polymerase chain reaction (PCR) analysis revealed that the circadian clock (BMAL1, PER1, PER2, DBP, and NR1D1) and steroidogenesis-related genes (SF1, NUR77, StAR, HSD3B2, CYP17A1, CYP11A1, and HSD17B3) showed rhythmic expression patterns in goat LCs following dexamethasone synchronization. Several Bmal1-Luc circadian oscillations were clearly observed in dexamethasone-treated goat LCs transfected with the pLV6-Bmal1-Luc plasmid. BMAL1 knockdown significantly downregulated mRNA levels of PER2, NR1D1, DBP, StAR, HSD3B2, SF1, NUR77, and GATA4, and dramatically decreased StAR and HSD3B2 protein levels and testosterone production. In contrast, BMAL1 overexpression significantly increased the mRNA and protein expression levels of StAR and HSD17B3 and enhanced testosterone production. Reporter assays revealed that goat BMAL1, or in combination with mouse CLOCK, activated goat HSD17B3 transcription in vitro. These data indicate that BMAL1 contributes to testosterone production by regulating transcription of steroidogenesis-related genes in goat LCs, providing a basis for further exploring the underlying mechanism by which the circadian clock regulates ruminant reproductive capability.
KW - BMAL1
KW - goat
KW - HSD17B3
KW - Leydig cells
KW - StAR
KW - testosterone
UR - http://www.scopus.com/inward/record.url?scp=85100874721&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85100874721&partnerID=8YFLogxK
U2 - 10.1002/jcp.30334
DO - 10.1002/jcp.30334
M3 - Article
AN - SCOPUS:85100874721
SN - 0021-9541
VL - 236
SP - 6706
EP - 6725
JO - Journal of Cellular Physiology
JF - Journal of Cellular Physiology
IS - 9
ER -