HSP60在双峰驼不同发育阶段睾丸及排卵前后生殖器官中的表达与定位

doi:10.7668/hbnxb.20195179

摘要/Abstract

摘要：

为探究热休克蛋白60(HSP60)对双峰驼不同发育阶段睾丸及排卵前后卵巢、子宫及输卵管的影响及调控作用,以双峰驼睾丸为材料,克隆HSP60的CDS区序列,利用ProParam及MEGA 7.0等软件对其进行生物信息学分析,利用聚合酶链式反应(PCR)、苏木精和伊红(H&E)染色、免疫组织化学法(IHC)、实时荧光定量PCR(qPCR)及蛋白免疫印迹(Western Blot)等探究其表达规律。克隆结果显示:HSP60的编码区(CDS)长度为1 722 bp,编码537个氨基酸,双峰驼HSP60基因与单峰驼及羊驼的核苷酸和氨基酸序列同源性较近。qPCR结果显示:HSP60在双峰驼不同发育阶段睾丸中均有表达,且性成熟后(3,5,7岁)睾丸中的表达水平显著高于3月龄;排卵前后卵巢、子宫及输卵管中均有HSP60的mRNA表达,且排卵后卵巢和子宫中的mRNA水平显著高于排卵前,而在输卵管中排卵前后无显著差异。Western Blot结果表明:HSP60在不同发育阶段睾丸中的表达趋势与mRNA水平相似,随着年龄的递增蛋白表达水平升高;而排卵后卵巢、子宫及输卵管中HSP60的蛋白水平显著高于排卵前。免疫组织化学法显示:HSP60蛋白主要定位于双峰驼的睾丸支持细胞、间质细胞及部分生精细胞,卵巢的颗粒细胞,子宫内膜的腺细胞以及肌细胞等。结果表明,HSP60参与双峰驼睾丸发育及精子生成过程,同时参与诱导排卵及减数分裂的调控过程。

关键词: 双峰驼, HSP60, 睾丸, 诱导排卵, 表达定位

Abstract:

The aim of this study was to explore the effects and regulatory roles of HSP60 on the testes and ovaries,uterus and fallopian tubes before and after ovulation in Bactrian camels at different developmental stages.The CDS region sequence of the HSP60 was cloned from the Bactrian camel testis and its bioinformatics analysis was analyzed using ProParam and MEGA 7.0 software,and its expression was explored by polymerase chain reaction(PCR),hematoxylin and eosin(H&E)staining,immunohistochemistry(IHC),Real-time Quantitative PCR(qPCR)and Western Blot.The cloning results showed that the coding sequence(CDS)region of the HSP60 was 1 722 bp in length and encoded 537 amino acids,and the HSP60 sequence of the Bactrian camel was similar to that of dromedary camel and alpaca.The qPCR results showed that HSP60 was expressed in the testes of Bactrian camels at different developmental stages,and the expression levels in the testes after sexual maturity(3,5 and 7 years old)were significantly higher than that at 3 months of age.The mRNA expression of HSP60 was found in the ovaries,uterus and fallopian tubes before and after ovulation,and the mRNA levels in the ovaries and uterus were significantly higher after ovulation than before ovulation,but there was no significant difference in the fallopian tube before and after ovulation.Western Blot results showed that the expression trends of HSP60 in the testes at different developmental stages were similar to that of mRNA,and the protein expression level increased with age.The protein levels of HSP60 in the ovaries,uterus and fallopian tubes were significantly higher after ovulation than before ovulation.Immunohistochemical staining revealed that the HSP60 protein was primarily located in Sertoli cells,Leydig cells,part of spermatogenic cells,granulosa cells of the ovaries,glandular cells of the endometrium and muscle cells.The results showed that HSP60 was involved in testicular development and spermatogenesis in Bactrian camels,as well as in the regulatory processes of ovulation induction and meiosis.

Key words: Bactrian camel, HSP60, Testis, Ovulation induction, Expression localization

成力童, 南京宏, 李天安, 颜秋, 王琪, 赵兴绪, 张勇. HSP60在双峰驼不同发育阶段睾丸及排卵前后生殖器官中的表达与定位[J]. 华北农学报, 2025, 40(2): 230-238. doi: 10.7668/hbnxb.20195179.

CHENG Litong, NAN Jinghong, LI Tianan, YAN Qiu, WANG Qi, ZHAO Xingxu, ZHANG Yong. Expression and Localization of HSP60 in Testis and Reproductive Organs of Bactrian Camels at Different Developmental Stages before and after Ovulation[J]. Acta Agriculturae Boreali-Sinica, 2025, 40(2): 230-238. doi: 10.7668/hbnxb.20195179.

http://www.hbnxb.net/CN/Y2025/V40/I2/230

图/表 8

表1 引物序列信息

Tab.1 Primer sequence information

基因 Gene	引物序列(5'—3') Primer sequence (5'—3')	退火温度/℃ Tm	基因登录号 GenBank number
HSP60-1	GGATCTTCCAGAGATATGCTTCGATTACCCGCAGTC	72	NW_011517963.1
	CTGCCGTTCGACGATTTAGAACATGCCGCCTCCC
HSP60-2	ATGCTTCGATTACCCGCAGTC	60	NW_011517963.1
	TTAGAACATGCCGCCTCCC
GAPDH	AACATCATTCCCTGCTTCTACC	56	NM_001357943.2
	ATGCCTGCTTCACTACCTTCT

图1 HSP60基因RT-PCR产物电泳分析 T1—T5.睾丸1—睾丸5;Marker.DL5000 DNA Marker。

Fig.1 Electrophoresis analysis of HSP60 gene RT-PCR products T1—T5.Testis 1—Testis 5;Marker.DL5000 DNA Marker.

图2 双峰驼HSP60生物信息学分析 A.HSP60蛋白的疏水性预测;B.HSP60蛋白的跨膜区域预测;C.双峰驼HSP60 CDS 区域的氨基酸组成;D.双峰驼HSP60蛋白的二级结构;E.双峰驼HSP60蛋白的三级结构:a.α-螺旋,b.延伸链,c.无规卷曲。

Fig.2 Bioinformatics analysis of HSP60 in Bactrian camel A.Hydrophobicity prediction of HSP60 protein;B.Prediction of transmembrane region of HSP60 protein;C.Amino acid composition in HSP60 CDS region of Bactrian camel;D.Secondary structure of HSP60 protein in Bactrian camel;E.Tertiary structure of HSP60 protein in Bactrian camel:a.α-helix,b.Extended chain,c.Random coil.

图3 不同物种HSP60核苷酸序列构建的进化树

Fig.3 Phylogenetic tree of HSP60 nucleotide sequences in different species

图4 HSP60的mRNA及蛋白水平检测 A.以GAPDH为持家基因,采用qPCR检测HSP60的mRNA水平,数据以平均值±标准差表示,n= 4;B.Western Blot检测HSP60的蛋白表达水平;C.Image J分析积分光密度值,数据以平均值±标准差表示。不同字母表示不同年龄差异显著(P<0.05)。

Fig.4 Detection of mRNA and protein levels of HSP60 A.The mRNA level of HSP60 was detected by qPCR using GAPDH as the housekeeping gene,and the data were presented as mean±s,n=4; B.The protein expression level of HSP60 was detected by Western Blot; C.Image J was used to analyze the integrated optical density value,and the data were presented as mean±s.Different letters indicated significant differences in different ages(P<0.05).

图5 双峰驼不同发育阶段睾丸的组织学鉴定及HSP60表达分布检测 A1—D1.不同年龄段(3月龄、3,5,7岁)睾丸的H&E染色鉴定;A2—D2.免疫组织化学法检测HSP60在不同年龄段(3月龄、3,5,7岁)睾丸的分布。S.精原细胞;SZ.精子;PS.初级精母细胞;LC.间质细胞;SC.支持细胞;ST.曲精小管;PMC.管周肌样细胞。

Fig.5 Histological identification of testes and detection of HSP60 expression distribution in Bactrian camels at different developmental stages A1—D1.H&E staining of testes of different age groups(3 months of age,3,5,7 years of age); A2—D2.Immunohistochemistry was used to detect the distribution of HSP60 in testes of different age groups(3 months of age,3,5,7 years of age).S.Spermatogonia; SZ.Spermatozoa; PS.Primary spermatocytes; LC.Mesenchymal cells; SC.Sertoli cells; ST.Carcospermous tubule; PMC.Peritubular myocytes.

图6 HSP60在双峰驼排卵前和排卵后不同组织中mRNA和蛋白水平检测 A.以GAPDH为持家基因,qPCR检测HSP60在双峰驼排卵前后不同组织中mRNA的表达水平,数据以平均值±标准差表示,n = 4;B.Western Blot检测HSP60在双峰驼不同组织中的蛋白表达水平;C.利用Image J 对HSP60在双峰驼排卵前后不同组织中蛋白表达的积分光密度值进行分析,数据以平均值±标准差表示,n=4,不同字母表示不同组织差异显著(P<0.05)。

Fig.6 Detection of mRNA and protein levels of HSP60 in different tissues of Bactrian camel before and after ovulation A.The mRNA expression level of HSP60 in different tissues of Bactrian camel before and after ovulation was detected by qPCR with GAPDH as the housekeeping gene,and data were presented as mean±s,n=4;B.Western Blot was used to detect the protein expression levels of HSP60 in different tissues of Bactrian camels;C.Image J was used to analyze the integrated optical density values of HSP60 protein expression in different tissues of Bactrian camels before and after ovulation,and the data were presented as mean±s,n=4,different letters indicating significant differences in different tissues(P<0.05).

图7 双峰驼各组织的形态学及HSP60蛋白的分布检测 SE.浆膜层;GEC.腺上皮细胞;SMC.平滑肌细胞;BV.血管壁;TM.子宫中膜;GE.生殖上皮;GC.卵泡颗粒细胞;IM.卵泡内膜。

Fig.7 Morphology of Bactrian camel tissues and the distribution of HSP60 protein SE.Serous layer; GEC.Glandular epithelial cell; SMC.Smooth muscle cells; BV.Vascular wall; TM.Memetrium;GE.Reproductive epithelium; GC.Follicular granule cells; IM.Follicular lining,original.

参考文献 32

[1]	Wang Q, Zhang Q W, Li Y N, Zhao X X, Zhang Y. RBP4 regulates androgen receptor expression and steroid synthesis in Sertoli cells from Bactrian camels[J]. Reproduction in Domestic Animals, 2022, 57(4):429-437.doi:10.1111/rda.14081.
[2]	李宗帅. 双峰驼诱导多能干细胞的建立、鉴定及特有诱导基因的筛选[D]. 兰州: 甘肃农业大学, 2022. doi:10.27025/d.cnki.ggsnu.2022.000049.
	Li Z S. Establishment and identification of induced pluripotent stem cells of Bactrian camel and screening of unique inducible genes[D]. Lanzhou: Gansu Agricultural University, 2022.
[3]	Bergfelt D R, Blum J L, Ratner J R, Ratto M H, O'Brien J K, Robeck T R. Preliminary evaluation of seminal plasma proteins and immunoreactivity of nerve growth factor as indicative of an ovulation inducing factor in odontocetes[J]. Journal of Zoo Biology, 2019, 2(1):21-29.doi:10.33687/zoobiol.002.01.2608.
[4]	Wang Q, Zhang Q W, Gan Z, Li H J, Yang Y, Zhang Y, Zhao X X. Screening for reproductive biomarkers in Bactrian camel via iTRAQ analysis of proteomes[J]. Reproduction in Domestic Animals, 2020, 55(2):189-199.doi:10.1111/rda.13607. pmid: 31840896
[5]	Ibrahim Z H, Al-Kheraije K A, Singh S K. Morphological and histochemical changes in the dromedary camel epididymis in relation to reproductive activity[J]. Histology and Histopathology, 2021, 36(5):485-504.doi:10.14670/HH-18-303. pmid: 33426635
[6]	Dioli M. Dromedary(Camelus dromedarius)and Bactrian camel(Camelus bactrianus)crossbreeding husbandry practices in Turkey and Kazakhstan:an in-depth review[J]. Pastoralism, 2020, 10:6.doi:10.1186/s13570-020-0159-3.
[7]	Cinone M, Albrizio M, Guaricci A C, Lacitignola L, Desantis S. Testicular expression of heat SHOCK proteins 60,70,and 90 in cryptorchid horses[J]. Theriogenology, 2024, 217:83-91.doi:10.1016/j.theriogenology.2024.01.019. pmid: 38262223
[8]	马鸣遥, 刘玉芬. 热休克蛋白与动物应激性反应的相关性研究进展[J]. 畜牧兽医科技信息, 2024(3):18-20.doi:10.3969/J.ISSN.1671-6027.2024.03.006.
	Ma M Y, Liu Y F. Research progress on the correlation between heat shock protein and stress response in animals[J]. Chinese Journal of Animal Husbandry and Veterinary Medicine, 2024(3):18-20.
[9]	张亚光, 王权胜, 黄子彦, 陈露, 宋吉祥, 林自立. 基于HSP60、HSP90表达探讨续断种子方对少弱精子症模型大鼠精液质量的影响[J]. 海南医学院学报, 2022, 28(20):1540-1545.doi:10.13210/j.cnki.jhmu.20220608.001.
	Zhang Y G, Wang Q S, Huang Z Y, Chen L, Song J X, Lin Z L. Based on the expression of HSP60 and HSP90,to explore the effect of Xuduan Zhongzi prescription on semen quality of oligoasthenospermia model rats[J]. Journal of Hainan Medical University, 2022, 28(20):1540-1545.
[10]	周乐乐, 黄建豪, 陈云, 张守全. 热休克蛋白90在雄性生殖中的研究进展[J]. 中华男科学杂志, 2021, 27(4):351-355.doi:10.13263/j.cnki.nja.2021.04.012.
	Zhou L L, Huang J H, Chen Y, Zhang S Q. Heat shock protein 90 in male reproduction[J]. National Journal of Andrology, 2021, 27(4):351-355.
[11]	Chen M, Yuan J X, Shi Y Q, Zhang X S, Hu Z Y, Gao F, Liu Y X. Effect of 43 ℃ treatment on expression of heat shock proteins 105,70 and 60 in cultured monkey Sertoli cells[J]. Asian Journal of Andrology, 2008, 10(3): 474-485. doi:10.1111/j.1745-7262.2008.00391.x. pmid: 18385910
[12]	Khosravanian N, Razi M, Farokhi F, Khosravanian H. Testosterone and vitamin E administration up-regulated varicocele-reduced Hsp70-2 protein expression and ameliorated biochemical alterations[J]. Journal of Assisted Reproduction and Genetics, 2014, 31(3):341-354.doi:10.1007/s10815-013-0165-0. pmid: 24395641
[13]	Srikanth K, Park J E, Ji S Y, Kim K H, Lee Y K, Kumar H, Kim M, Baek Y C, Kim H, Jang G W, Choi B H, Lee S D. Genome-wide transcriptome and metabolome analyses provide novel insights and suggest a sex-specific response to heat stress in pigs[J]. Genes, 2020, 11(5):540.doi:10.3390/genes11050540.
[14]	李庆伟, 徐静, 李瑶. 热休克蛋白进化及生物学功能研究进展[J]. 辽宁师范大学学报(自然科学版), 2024, 47(1):78-85.doi:10.11679/lsxblk2024010078.
	Li Q W, Xu J, Li Y. Research progress on function and evolution of heat shock protein[J]. Journal of Liaoning Normal University(Natural Science Edition), 2024, 47(1):78-85.
[15]	周方园, 郭嘉成, 解澎, 庞智文. 血清肿瘤标志物CEA、CA724及HSP60联合检测对胃癌的诊断价值[J]. 中国卫生工程学, 2021, 20(4): 646-647,649. doi:10.19937/j.issn.1671-4199.2021.04.043.
	Zhou F Y, Guo J C, Xie P, Pang Z W. Diagnostic value of combined detection of serum tumor markers CEA,CA724 and HSP60 in gastric cancer[J]. Chinese Journal of Public Health Engineering, 2021, 20(4):646-647,649.
[16]	金实, 孙仁斌, 吴胜春, 王忠裕, 关凤林, 王洪江. 胰腺癌组织中HSP60、HSP70表达及其意义[J]. 医学研究杂志, 2006, 35(10):50-52.doi:10.3969/j.issn.16733-548x.2006.10.025.
	Jin S, Sun R B, Wu S C, Wang Z Y, Guan F L, Wang H J. Expression and significance of HSP60 and HSP70 in pancreatic cancer[J]. Journal of Medical Research, 2006, 35(10):50-52.
[17]	唐耀香. HSP60与非小细胞肺癌的恶性表型及预后的相关性研究[D]. 长沙: 中南大学, 2022. doi:10.27661/d.cnki.gzhnu.2022.001752.
	Tang Y X. Correlation between HSP60 and malignant phenotype and prognosis of non-small cell lung cancer[D]. Changsha: Central South University, 2022.
[18]	王尧, 胡放荣, 张隆辉, 江明珠. 一种用于检测结直肠癌症标记物的太赫兹超材料传感器[J]. 桂林电子科技大学学报, 2023, 43(4):331-339.doi:10.16725/j.cnki.cn45-1351/tn.2023.04.012.
	Wang Y, Hu F R, Zhang L H, Jiang M Z. A terahertz metamaterial biosensor for detecting colorectal cancer marker[J]. Journal of Guilin University of Electronic Technology, 2023, 43(4):331-339.
[19]	印璞, 乔璐, 杨照微, 何成彦, 孙景春, 郑岚. HSPD1在肾癌中的表达及意义[J]. 中国实验诊断学, 2017, 21(10):1690-1692.doi:10.3969/j.issn.1007-4287.2017.10.005.
	Yin P, Qiao L, Yang Z W, He C Y, Sun J C, Zheng L. Expression and significance of HSPD1 in renal cell carcinoma[J]. Chinese Journal of Laboratory Diagnosis, 2017, 21(10):1690-1692.
[20]	詹昶. HSP60在糖尿病大鼠晶状体上皮细胞上的表达及意义[D]. 南昌: 南昌大学, 2014. doi:10.7666/d.D556198.
	Zhan C. Expression and significance of HSP60 in lens epithelial cells of diabetic rats[D]. Nanchang: Nanchang University, 2014.
[21]	Werner A, Meinhardt A, Seitz J, Bergmann M. Distribution of heat-shock protein 60 immunoreactivity in testes of infertile men[J]. Cell and Tissue Research, 1997, 288(3):539-544.doi:10.1007/s004410050839. pmid: 9134866
[22]	Kandil B, Kurtdede N, Bayraktaroglu A G. Immunohistochemical localization and expression of heat shock proteins(HSP27,HSP60,HSP70,and HSP90)during the oestrous cycle,pregnancy,and lactation in rat ovaries[J]. Acta Histochemica, 2024, 126(3):152157.doi:10.1016/j.acthis.2024.152157.
[23]	Feng H L, Sandlow J I, Sparks A E T. Decreased expression of the heat shock protein hsp70-2 is associated with the pathogenesis of male infertility[J]. Fertility and Sterility, 2001, 76(6):1136-1139.doi:10.1016/S0015-0282(01)02892-8. pmid: 11730740
[24]	Meinhardt A, Parvinen M, Bacher M, Aumüller G, Hakovirta H, Yagi A, Seitz J. Expression of mitochondrial heat shock protein 60 in distinct cell types and defined stages of rat seminiferous epithelium[J]. Biology of Reproduction, 1995, 52(4):798-807.doi:10.1095/biolreprod52.4.798. pmid: 7780001
[25]	张惟纲, 李锐, 张庆华, 刘威. 热休克蛋白及热休克转录因子在精子发育中的作用研究进展[J]. 中国男科学杂志, 2022, 36(5):94-98.doi:10.3969/j.issn.1008-0848.2022.05.017.
	Zhang W G, Li R, Zhang Q H, Liu W. Research progress on the role of heat shock protein and heat shock transcription factor in sperm development[J]. Chinese Journal of Andrology, 2022, 36(5):94-98.
[26]	阮崇美, 王相生, 张勇, 马友记, 胡俊杰, 赵兴绪. 天祝白牦牛HSP60基因的克隆及其在下丘脑-垂体-睾丸中的表达定位研究[J]. 核农学报, 2017, 31(7):1290-1298.doi:10.11869/j.issn.100-8551.2017.07.1290.
	Ruan C M, Wang X S, Zhang Y, Ma Y J, Hu J J, Zhao X X. Cloning and localization of heat shock protein 60 in hypothalamus-pituitary-testis axis of Tianzhu white yak[J]. Journal of Nuclear Agricultural Sciences, 2017, 31(7):1290-1298. doi: 10.11869/j.issn.100-8551.2017.07.1290
[27]	Paranko J, Seitz J, Meinhardt A. Developmental expression of heat shock protein 60(HSP60)in the rat testis and ovary[J]. Differentiation; Research in Biological Diversity, 1996, 60(3): 159-167. doi:10.1046/j.1432-0436.1996.6030159.x.
[28]	刘会玲, 高云荷, 温漓潮. 热休克蛋白在子宫颈鳞癌的表达及其意义[J]. 临床与实验病理学杂志, 2002, 18(5):511-514.doi:10.13315/j.cnki.cjcep.2002.05.016.
	Liu H L, Gao Y H, Wen L C. Expression of HSPs in cervical squamous carcinomas[J]. Chinese Journal of Clinical and Experimental Patholog, 2002, 18(5):511-514.
[29]	冯骏, 王利娟, 韩晓兵, 王星烨, 苟文丽. 热休克蛋白60在子宫颈癌组织中的表达及其临床意义[J]. 第三军医大学学报, 2010, 32(6): 554-557. doi:10.16016/j.1000-5404.2010.06.023.
	Feng J, Wang L J, Han X B, Wang X Y, Gou W L. Expression and clinical significance of heat shock protein 60 in cervical carcinoma[J]. Acta Academiae Medicinae Militaris Tertiae, 2010, 32(6):554-557.
[30]	刘耿华. 中药联合宫腹腔镜治疗H-HSP60抗体阳性输卵管炎性不孕患者的临床研究[D]. 广州: 广州中医药大学, 2011.
	Liu G H. Clinical study on the treatment of tubal inflammatory infertility with H-HSP60 antibody positive by traditional Chinese medicine combined with hysteroscopy and laparoscopy[D]. Guangzhou: Guangzhou University of Chinese Medicine, 2011.
[31]	Shen H, Fan X R, Zhang Z, Xi H M, Ji R, Liu Y H, Yue M S, Li Q H, He J P. Effects of elevated ambient temperature and local testicular heating on the expressions of heat shock protein 70 and androgen receptor in boar testes[J]. Acta Histochemica, 2019, 121(3):297-302.doi:10.1016/j.acthis.2019.01.009. pmid: 30723046
[32]	孙博. 靶向HSP60的金纳米颗粒及其对乳腺癌肿瘤细胞的光热杀伤作用研究[D]. 北京: 北京协和医学院, 2021. doi:10.27648/d.cnki.gzxhu.2021.000791.
	Sun B. Study on gold nanoparticles targeting HSP60 and its photothermal killing effect on breast cancer cells[D]. Beijing: Peking Union Medical College, 2021.

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