Analysis of Reproduction Function Gene of Testicle in Different Size Black Cattle Based on Transcriptome Sequencing

doi:10.7668/hbnxb.20194430

Abstract

Abstract:

The aim of this experiment was to analyze the transcriptome data of testicle in different size of Shandong black cattle,and discover the key candidate genes affecting the reproductive function of testicles,in order to explore the molecular mechanism of maintaining the reproductive function in cattle testicle.Six bulls were divided into two groups according to different size testicle for transcriptome sequencing.The differential expression multiplier values |log₂(Fold Change)|≥1 and P-value≤0.01 were used as the conditions for selecting the differential expression genes (DEGs),and GO and KEGG analysis were performed to select the candidate gene of reproductive function of testicle.The CDS region of cattle candidate gene was cloned,and qRT-PCR,Western Blot,IHC were used for further confirmation.A total of 353 DEGs were obtained from the sequencing results,including 114 up-regulated genes and 239 down-regulated genes in small testicle.The results of GO functional annotation found that the items related to metabolism,development,reproduction and other processes.The results of KEGG pathway enrichment analysis showed that DEGs were mainly enriched in Wnt signal transduction,PI3K-Akt signaling pathway about reproductive function of testicle,further screening for CCN4 related to testicle reproductive function as candidate gene to validate.The qRT-PCR and Western Blot results showed that the relative expression of CCN4 gene and protein in big testicles was significantly lower than that in small testicle.The IHC results showed that CCN4 protein was mainly positive in the areas outside spermatids (especially sertoli cells) and the relative expression in big testicle was significantly lower than that in small testicle.It is speculated that the CCN4 gene may inhibit sertoli cells to affect cattle testicle function.This study provides a basic data for further study of reproductive function testicle in different size of cattle.

Key words: Cattle, Testicle size, Transcriptome, Differential gene, CCN4

MEI Yanfang, LIU Ruili, HAN Mingxuan, ZHANG Wenqi, CHEN Zhenpeng, WANG Xiuyuan, MIAO Xiuping, XIAO Chaozhu, ZHANG Renzheng, DONG Yajuan, BAI Xuejin. Analysis of Reproduction Function Gene of Testicle in Different Size Black Cattle Based on Transcriptome Sequencing[J]. Acta Agriculturae Boreali-Sinica, 2024, 39(1): 191-200. doi: 10.7668/hbnxb.20194430.

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Figures/Tables 11

Tab.1 Primes used for qRT-PCR

基因 Gene	引物序列(5'—3') Primer sequence (5'—3')	产物大小/bp Product length	退火温度/℃ Annealing temperature
CCN4	F: CACACATCAAGGAAGGGAAGAAG R: ACTTGTAGGGGATACAGCACCG	150	60
PFN4	F: ACAGTGCAGCCCTCATCAAAC R: TCGGACATCACTGGGCATTACA	83	61
ITGA4	F: AAGGGGAAACAGGCGATGTGA R: AGGCTGGACTTACAAACCCATGA	99	62
GAPDH	F: GATGCTGGTGCTGAGTATGT R: GCAGAAGGTGCAGAGATGAT	104	60
KCNU1	F: GTTCTGCGCCATCCCTTTCAG R: GCCTGGGCAACTAGAGGTGTT	144	62
RBL2	F: GTGGACCACAGCAGAAACAAGG R: GGCGGACACCTGCTAAGTGATA	114	62
STAR	F: CCCTCAAGGACCAAACTCACCT R: GCGAGAGGACCTGGTTGATGAT	85	62

Fig.1 Histological structure of HE staining of big and small testicular tissues IC.Interstitial cells;SS.Supporting cells;SP.Spermatogonia;P.Primary spermatocytes;SSP.Secondary spermatocytes; RS.Round spermatozoa;S.Mature spermatozoa.The same as Fig.10.

Fig.2 Differential genes volcano map

Fig.3 GO functional classification of differently expressed genes

Fig.4 KEGG enrichment of differential gene

Fig.5 qRT-PCR validation of differentially expressed genes

Fig.6 PCR electrophoretogram of CCN4 M.Marker 2 000;C.PCR product of CCN4 gene.

Fig.7 Relative expression of CCN4 gene in each tissue Testicle was used as control tissue,and different letters indicate significant differences between different tissues(P<0.05).The same as Fig.8—9.

Fig.8 Relative expression of CCN4 protein in each tissue A.Relative expression of CCN4 protein in each tissue;B.WB verification of protein expression of CCN4 and GAPDH in different tissues.

Fig.9 Relative expression of CCN4 in big and small testicle C.WB validation of protein expression of CCN4 and GAPDH in big and small testicle.

Fig.10 Immunohistochemical staining of CCN4 in big and small testicle(200×) A.The small testicle; B.The big testicle.

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