|
|
|
| Effect of CYP17A1 Gene Knockdown on Expression of Genes Related to Testosterone Synthesis and Testicular Development in Goat (Capra hircus) Leydig Cells |
| ZHANG Yuan1,2, CHEN Xiang1,2,*, ZHANG Yan1,2, FU Kai-Bin1,2, TANG Wen1,2, CHEN Jia-Jing1,2, JI Tao-Tao1,2, YANG Pei-Fang1,2 |
1 College of Animal Science, Guizhou University/Key Laboratory of Animal Geneties, Breeding and Reproduction in the Plateau Mountain Region, Ministry of Education, Guiyang 550025, China;
2 Guizhou Key Laboratory of Animal Genetics and Breeding, Guiyang 550025, China |
|
|
|
|
Abstract Cytochrome P450c17 (CYP17A1 ) is a rate-limiting enzyme in the pathway of cholesterol synthesis and plays a key role in androgen biosynthesis. It is of great significance to clarify the effect of CYP17A1 on testosterone synthesis and testicular development in goats (Capra hircus) for improving the fertility of rams. In this study, RNA interference vector of CYP17A1 was constructed and transfected into Leydig cells of Qianbei Ma goat. The knockdown effect of CYP17A1 gene was detected by qRT-PCR and Western blot. The effect of CYP17A1 knockdown on the proliferation of Leydig cells was detected by CCK-8 (Cell Counting Kit-8). The expression levels of proliferation-related genes, testosterone synthesis-related genes and testicular development-related genes were detected by qRT-PCR. The results showed that CYP17A1 was expressed in the testis and epididymis of each month old, and its gene knockdown effect was obvious, in which the expression of CYP17A1 gene and protein was significantly down-regulated (P<0.01), and the proliferation of Leydig cells was significantly inhibited (P<0.01). CYP17A1 gene knockdown significantly down-regulated the expression of proliferating cell nuclear antigen (PCNA) and cyclin E, CYP11A1, 3β-hydroxysteroid dehydrogenase type 1 (HSD3B1), HSD17B3 and CYP19A1, deleted in azoospermia like (DAZL) and insulin like growth factor 2 (IGF2)(P<0.01). This study provides basic data for further exploring the effect of CYP17A1 gene on the reproductive traits of Qianbei Ma goat.
|
|
Received: 22 December 2022
|
|
|
|
Corresponding Authors:
*xchen2@gzu.edu.cn
|
|
|
|
[1] 敖叶. 2020. 黔北麻羊产羔性状候选基因筛选及其功能验证[D]. 硕士学位论文, 贵州大学, 导师: 陈祥, pp. 2-3.
(Ao Y.2020. Screening and functional verification of candidate genes for lambing traits in Qianbei Ma goat[D]. Thesis for M.S., Guizhou University, Supervisor: Chen X, pp. 2-3.)
[2] Afedo S Y.2021. 生殖相关基因CYP17A1, MTHFR, DAZL, IGF2和H19的特征分析及其在牛睾丸中的表达研究[D]. 博士学位论文, 甘肃农业大学, 导师:崔燕, pp. 121-138.
(Afedo S Y.2021. Characterization of reproductive related genes CYP17A1, MTHFR, DAZL, IGF2 and H19 and their expression in bovine testis[D]. Thesis for Ph.D., Gansu Agricultural University, Supervisor: Cui Y, pp, 121-138.)
[3] 黄雅琼, 杨素芳, 农微, 等. 2008. 猪睾丸间质细胞的分离及培养[J]. 畜牧与兽医, 40(7): 21-25.
(Huang Y Q, Yang S F, Nong W, et al.2008. Isolation and culture of porcine interstitial cells[J]. Animal Husbandry and Veterinary Medicine, 40(7): 21-25.)
[4] 李讨讨, 马友记, 赵兴绪, 等. 2018. Dazl基因在绵羊不同发育期睾丸组织中的表达与细胞定位[J]. 农业生物技术学报, 26(03): 421-428.
(Li T T, Ma Y J, Zhao X X, et al.2018. Expression and cellular localization of Dazl gene in sheep testis at different developmental stages[J]. Journal of Agricultural Biotechnology, 26(03): 421-428.)
[5] 李忠邦. 2019. LYZL4, LYZL6和PCNA在牦牛睾丸中的表达定位及组织学分析[D]. 硕士学位论文, 西北民族大学, 导师: 阎萍, pp. 22-25.
(Li Z B.2019. Expression localization and histological analysis of LYZL4, LYZL6 and PCNA in yak testis[D]. Thesis for M.S., Northwest Minzu University, Supervisor: Yan P, pp. 22-25.)
[6] 尚明玉, 陈晨, 张立昌, 等. 2018. 种公鸡睾丸间质细胞的分离培养与鉴定[J]. 北京农学院学报, 33(4): 50-53.
(Shang M Y, Chen C, Zhang L C, et al.2018. Isolation, culture and identification of Leydig cells from testis of breeder cocks[J]. Journal of Beijing Agricultural University, 33(4): 50-53.)
[7] 史东杰, 谭政, 张强, 等. 2022. 细胞色素CYP17a1基因在锦鲤性腺不同发育时期的表达分析[J]. 河南农业科学, 51(2): 139.
(Shi D J, Tan Z, Zhang Q, et al.2022. Expression analysis of cytochrome CYP17a1 gene at different developmental stages of carp gonad[J]. Henan Agricultural Sciences, 51(2): 139.)
[8] 田志龙, 刘秋月, 王翔宇, 等. 2019. CYP11A1, CYP17A1, CYP19A1基因在小尾寒羊下丘脑-垂体-卵巢轴的表达[J]. 中国农业大学学报, 24(4): 87-93.
(Tian Z L, Liu Q Y, Wang X Y, et al.2019. Expression of CYP11A1, CYP17A1 and CYP19A1 genes in hypothalamus-pituitary-ovary axis of Small Tail Han sheep[J]. Journal of China Agricultural University, 24(4): 87-93.)
[9] 谭晓华. 2018.人结直肠癌组织中HIF-1α, Cyclin E和Cyclin D1蛋白表达变化及意义[D]. 硕士学位论文, 广西医科大学, 导师: 黄杰安, pp. 15-19.
(Tan X H.2018. Expression and significance of HIF-1α, Cyclin E and Cyclin D1 protein expression in human colorectal cancer tissues[D]. Thesis for M.S., Guangxi Medical University, Supervisor: Huang J A, pp. 15-19.)
[10] 王亚营. 2018. 牦牛Cyclin D1, E1基因克隆及IGF-I, TGF-β1对Cyclin D1在睾丸支持细胞表达的影响[D]. 硕士学位论文, 甘肃农业大学, 导师: 余四九, pp. 18-22.
(Wang Y Y.2018. Cloning of yak Cyclin D1/E1 gene and the effect of IGF-I/TGF-β1 on expression of Cyclin D1 in sertoli cell[D]. Thesis for M.S., Gansu Agricultural University, Supervisor: Yu S J, pp. 18-22.)
[11] 姚雅馨. 2017. 绵羊FecB突变高通量分型技术的建立及巴美肉羊多羔候选基因的筛选[D]. 博士学位论文, 中国农业科学院, 导师: 储明星, pp. 36-39.
(Yao Y X.2017. Establishment of high throughput genotyping methods for FecB mutation in sheep and screening for candidate genes of prolificacy[D]. Thesis for Ph.D., Chinese Academy of Agricultural Sciences, Supervisor: Chu M X, pp. 36-39.)
[12] 杨喜科, 姜平, 付莉萍. 2021. lncRNA CYP17A1-AS1对喉鳞癌细胞增殖、侵袭、迁移和凋亡的影响[J]. 安徽医药, 25(11): 2200-2204.
(Yang X K, Jiang P, Fu L P.2021. Effects of lncRNA CYP17A1-AS1 on proliferation, invasion, migration and apoptosis of laryngeal squamous carcinoma cells and its mechanism[J]. Anhui Medical and Pharmaceutical Journal, 25(11): 2200-2204.)
[13] 周志楠, 陈祥, 张艳, 等. 2021. CTSD对黔北麻羊卵泡颗粒细胞的调控机制及功能分析[J]. 畜牧兽医学报, 52(5): 1278-1292.
(Zhou Z N, Chen X, Zhang Y, et al.2021. Regulatory mechanism and functional analysis of CTSD on follicular granulosa cells of Qianbei Ma goat[J]. Acta Veterinaria Acet Zootechnica Siuica, 52(5): 1278-1292.)
[14] 曾琼, 史忠辉. 2011. 贵州省黔北麻羊的形成及利用[J]. 中国草食动物, 31(04): 75-77.
(Zeng Q, Shi Z H.2011. The formation and utilization of Qianbei Ma goat in Guizhou province[J]. Herbivores of China, 31(04): 75-77.)
[15] Bhardwaj V K, Purohit R.2021. Taming the ringmaster of the genome (PCNA): Phytomolecules for anticancer therapy against a potential non-oncogenic target[J]. Journal of Molecular Liquids, 337: 116437.
[16] Bi H, Li S, Qu X, et al.2015. DEC1 regulates breast cancer cell proliferation by stabilizing cyclin E protein and delays the progression of cell cycle S phase[J]. Cell Death & Disease, 6(9): e1891.
[17] Cao M, Liu H W, Ning S, et al.2020. High-level androgen enhances the differentiation of hESCs into male primordial germ cells[J]. National Journal of Andrology, 26(6): 487-498.
[18] Chandolia R K, Evans A C O, Rawlings N C.1997. Effect of inhibition of increased gonadotrophin secretion before 20 weeks of age in bull calves on testicular development[J]. Reproduction, 109(1): 65-71.
[19] Griffeth R J, Bianda V, Nef S.2014. The emerging role of insulin-like growth factors in testis development and function[J]. Basic and Clinical Andrology, 24(1): 1-10.
[20] Heinrich A, DeFalco T.2020. Essential roles of interstitial cells in testicular development and function[J]. Andrology, 8(4): 903-914.
[21] Janer G, LeBlanc G A, Porte C.2005. A comparative study on androgen metabolism in three invertebrate species[J]. General and Comparative Endocrinology, 143(3): 211-221.
[22] Khosla S, Monroe D G.2018. Regulation of bone metabolism by sex steroids[J]. Cold Spring Harbor Perspectives in Medicine, 8(1): a031211.
[23] Li T, Wang X, Zhang H, et al.2020. Gene expression patterns and protein cellular localization suggest a novel role for DAZL in developing Tibetan sheep testes[J]. Gene, 731: 144335.
[24] Liu C Y, Chang T C, Lin S H, et al.2022. Is a ketogenic diet superior to a high-fat, high-cholesterol diet regarding testicular function and spermatogenesis?[J]. Frontiers in Nutrition, 9(3): 805794.
[25] Merlet J, Racine C, Moreau E, et al.2007. Male fetal germ cells are targets for androgens that physiologically inhibit their proliferation[J]. Proceedings of the National Academy of Sciences of the USA, 104(9): 3615-3620.
[26] Nebert D W, Russell D W.2002. Clinical importance of the cytochromes P450[J]. Lancet, 360(9340): 1155-1162.
[27] Omoboyowa D A, Balogun T A, Saibu O A, et al.2022. Structure-based discovery of selective CYP17A1 inhibitors for castration-resistant prostate cancer treatment[J]. Biology Methods & Protocols, 7(1): 44-57.
[28] Ohta Y, Kawate N, Inaba T, et al.2017. Feeding hydroalcoholic extract powder of Lepidium meyenii (maca) enhances testicular gene expression of 3β-hydroxysteroid dehydrogenase in rats[J]. Andrologia, 49(10): e12792.
[29] Pott J, Bae Y J, Horn K, et al.2019. Genetic association study of eight steroid hormones and implications for sexual dimorphism of coronary artery disease[J]. Journal of Clinical Endocrinology & Metabolism, 104(11): 5008-5023.
[30] Rendic S, Guengerich F P.2015. Survey of human oxidoreductases and cytochrome P450 enzymes involved in the metabolism of xenobiotic and natural chemicals[J]. Chemical Research in Toxicology, 28(1): 38-42.
[31] Sechman A, Batoryna M, Antos P A, et al.2016. Effects of PCB 126 and PCB 153 on secretion of steroid hormones and mRNA expression of steroidogenic genes (STAR, HSD3B, CYP19A1) and estrogen receptors (ERα, ERβ) in prehierarchical chicken ovarian follicles[J]. Toxicology Letters, 264(2): 29-37.
[32] Smith L B, Walker W H.2014. The regulation of spermatogenesis by androgens[J]. Seminars In Cell & Developmental Biology, 30: 2-13.
[33] Stupp R, Mason W P, Van Den Bent M J, et al.2005. Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma[J]. New England Journal of Medicine, 352(10): 987-996.
[34] Tsujimura A.2013. The relationship between testosterone deficiency and men's health[J]. World Journal of Men's Health, 31(2): 126-135.
[35] Walker W H.2011. Testosterone signaling and the regulation of spermatogenesis[J]. Spermatogenesis, 1(2): 116-120.
[36] Yu M, Zhang L, Qiao P, et al.2015. Testicular oxidative stress and downregulation of CYP17a1 induced by di (N-butyl) phthalate inhibit synthesis of testosterone[J]. Journal of Hygiene Research, 44(3): 364-370.
[37] Zhao L, Zhang J, Yang L, et al.2021. Glyphosate exposure attenuates testosterone synthesis via NR1D1 inhibition of StAR expression in mouse Leydig cells[J]. Science of The Total Environment, 785: 147323.
[38] Zhao W, Wang H, Liu J, et al.2018. Positive PCNA and Ki-67 expression in the testis correlates with spermatogenesis dysfunction in fluoride-treated rats[J]. Biological Trace Element Research, 186(2): 489-497.
[39] Zhang M, Wei H, Liu T, et al.2020. Potential GnRH and steroidogenesis pathways in the scallop Patinopecten yessoensis[J]. Journal of Steroid Biochemistry and Molecular Biology, 204: 105756. |
|
|
|
