Expression and Localization of HSP60 in Testis and Reproductive Organs of Bactrian Camels at Different Developmental Stages before and after Ovulation

doi:10.7668/hbnxb.20195179

Abstract

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

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.

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Figures/Tables 8

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

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

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.

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

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).

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.

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).

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.

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