Transcriptomic Analysis of Big and Small Testis and Screening Genes Related to Reprodution in Blackette Black Cattle (Bos taurus)
WANG Xiu-Yuan1,2, LIU Rui-Li1,2, HAN Ming-Xuan1,2, ZHANG Wen-Qi1,2, CHEN Zhen-Peng1,2, MEI Yan-Fang1,2, MIAO Xiu-Ping1,2, XIAO Chao-Zhu2,3, Zhang Ren-Zheng1,2, DONG Ya-Juan1,2, BAI Xue-Jin1,2,*
1 College of Animal Science and Technology, Qingdao Agricultural University, Qingdao 266109, China; 2 Heiniu Seed Industry Technology Innovation Center, Qingdao 266109, China; 3 Shandong Zhaofu Animal Husbandry Technology Co., Ltd., Zibo 256306, China
Abstract Testis size is significantly related to semen quality and count in mammals, and male fertility can be predicted by testicular size in adulthood. In order to explore key candidate genes affecting bull fertility in differently testicular sizes, peripheral blood and testis of Blackette black cattle (Bos taurus) of 12 months old were collected and divided big testis and small testis groups according to testicular weight, long diameter and short diameter for hormone determination, histological study of testis and transcriptomic sequencing in this study. The differential expression multiplier value |log2 (Fold change)|≥1 and P-value≤0.01 were used as the conditions for selecting the differentially expressed genes (DEGs) and were subjected to Gene Ontology (GO) and KEGG analysis. Clone, qPCR, Western blot and immunohistochemistry (IHC) methods were used to detect and locate for candidate gene. The results showed that there were more testicular leydig cells and the development of spermatogenic tubules was better in big group. The concentrations of testosterone and estradiol of reproductive hormones in big testis were significantly higher than those in small testis group (P<0.05). A total of 1 006 DEGs were obtained from the transcriptome results, 297 genes were up-regulated and 709 genes were down-regulated in small testis. Functional analysis showed that the DEGs was enriched in cAMP and mitogen-activated protein kinase (MAPK) signaling pathways and played a role in reproduction and development; 9 reproduction-related candidate genes were screened, such as dual specificity phosphatase 4 (DUSP4), heat shock 70 kD protein 1-like (HSPA1L) and mitogen activated protein kinase kinase kinase kinase 4 (MAP4K4). Further study on MAP4K4 gene and protein showed that the expression of MAP4K4 in big testis was significantly lower than that in small testis (P<0.05). IHC found that MAP4K4 protein was highly expressed in sperm cells and spermatocytes and was mainly expressed in cytoplasm. Inhibition of MAP4K4 expression might promote spermatocyte proliferation and facilitate spermatoblast survival. This work provides reference for further understanding of the relationship between testis size and reproductive function in cattle.
WANG Xiu-Yuan,LIU Rui-Li,HAN Ming-Xuan, et al. Transcriptomic Analysis of Big and Small Testis and Screening Genes Related to Reprodution in Blackette Black Cattle (Bos taurus)[J]. 农业生物技术学报, 2024, 32(8): 1821-1835.
[1] 董雅娟. 2016. 牛[M]. 北京: 中国农业出版社, pp.20. (Dong Y J.2016. Cattle[M]. Beijing. China Agriculture Press, China, pp. 20.) [2] 李凌娟. 2019. 纳米氧化钛抑制大鼠睾丸间质细胞睾酮分泌的分子机制[D]. 硕士学位论文, 苏州大学, 导师:迮玉官, pp, 52. (Li L J.2019. Molecular mechanism of inhibition of testosterone secretion in rat leydig cells caused by nano titanium dioxide[D]. Thesis for M.S., Soochow University, Supervisor: Ze Y G, pp. 52 ) [3] 李龙龙, 朱燕玲, 曾斌, 等. 2019. 基于转录组学筛选miR-125b-2敲除小鼠睾丸发育相关基因及信号通路的研究[J].畜牧兽医学报, 50(10): 2022-2031. (Li L L, Zhu Y L, Zeng B, et al.2019. Screening of genes and signaling pathway related to testicular development in mir-125b-2 knockout mouse based on transcriptomics[J]. Chinese Journal of Animal and Veterinary Sciences[J], 50(10): 2022-2031.) [4] 刘佳美, 路婷婷, 姚婷, 等. 2022. 不同阴囊围度湖羊睾酮和雌二醇浓度及其合成相关基因的表达水平[J]. 草业科学, 39(07): 1452-1458. (Liu J M, Lu T T, Yao T, et al.2022. Serum testosterone, estradiol, and steroidogenesis-related gene expression levels in Hu sheep with different scrotal circumference[J]. Pratacultural Science, 39(07): 1452-1458.) [5] 罗静. 2020.湖羊睾丸发育和大小相关基因的鉴定及SNPs的筛选[D]. 硕士学位论文, 兰州大学, 导师: 乐祥鹏, pp, 81. (Luo J.2020. Identification of genes and SNPs related to testis development and size of Hu sheep[D].Thesis for M.S., Lanzhou University, Supervisor: Yue X P, pp. 81.) [6] 孙武. 2019. 整合RNA-seq和全基因组测序数据解析控制湖羊睾丸发育的基因和调控网络[D]. 博士学位论文, 兰州大学, 导师: 李发弟, pp, 17. (Sun W.2019. Identification of the candidate genes and regulatory network related to testicular development in Hu sheep based on RNA-seq and RE-sequencing data[D]. Thesis for Ph.D., Lanzhou University, Supervisor: Li F D, pp. 17.) [7] 孙武, 马世科, 孙永刚, 等. 2022. 睾丸发育过程概述及生殖生物学研究意义[J]. 青海畜牧兽医杂志, 52(03): 40-43. (Sun W, Ma S K, Sun Y G, et al.2022. The developmental process of testis and its significance in reproductive biology[J]. Chinese Qighai Journal of Animal and Veterinary Sciences[J]. 52(03): 40-43.) [8] 羊明智, 吴叶, 章亚东, 等. 2007. 钙调素及其依赖性激酶对即早基因c-fos表达的影响[J]. 中南医学科学杂志, 35(4): 529-532. (Yang M Z, Wu Y, Zhang Y D.2007. The effect of the CAM and CAM-dependent kinase on the expression of c-fos gene in the spinal cord matter[J]. Medical Science Journal of Central South China, 35(4): 529-532) [9] Abhari A, Zarghami N, Shahnazi V, et al.2014. Significance of microRNA targeted estrogen receptor in male fertility[J]. Iranian Journal of Basic Medical Sciences, 17(2): 81-86. [10] Abreu R, Penalva L O, Marcotte E M, et al.2009. Global signatures of protein and mRNA expression levels[J]. Molecular BioSystems, 5(12): 1512-1526. [11] Akbari S, Amiri F T, Naderi M, et al.2022. Sodium arsenite accelerates D-galactose-induced aging in the testis of the rat: Evidence for mitochondrial oxidative damage, NF-kB, JNK, and apoptosis pathways[J]. Toxicology, 470: 153148. [12] Arunachalam S, Nagoor Meeran M F, Azimullah S, et al.2022. α-Bisabolol attenuates NF-κB/MAPK signaling activation and ER-stress-mediated apoptosis by invoking Nrf2-mediated antioxidant defense systems against doxorubicin-induced testicular toxicity in rats[J]. Nutrients, 14(21): 4648. [13] Corte P F, Crociati M, Urli S, et al.2022. Environmental factors affecting the reproductive efficiency of italian simmental young bulls[J]. Animals (Basel), 12(18): 2476. [14] Dumasia K, Kumar A, Deshpande S, et al.2016. Differential roles of estrogen receptors, ESR1 and ESR2, in adult rat spermatogenesis[J]. Molecular and Cellular Endocrinology, 428: 89-100. [15] Er H, Tas G G, Soygur B, et al.2022. Acute and chronic exposure to 900 mhz radio frequency radiation activates p38/jnk-mediated mapk pathway in rat testis[J]. Reproductive Sciences, 29(5): 1471-1485. [16] Fadel F, Al-Kandari N, Khashab F, et al.2020. JNK inhibition alleviates oxidative DNA damage, germ cell apoptosis, and mitochondrial dysfunction in testicular ischemia reperfusion injury[J]. Acta Biochimica et Biophysica Sinica, 52(8): 891-900. [17] Fiedler L R, Chapman K, Xie M, et al.2019. MAP4K4 inhibition promotes survival of human stem cell-derived cardiomyocytes and reduces infarct size in vivo[J]. Cell Stem Cell, 24(4): 579-591. [18] França L R, Hess R A, Dufour J M, et al.2016. The Sertoli cell: One hundred fifty years of beauty and plasticity[J]. Andrology, 4(2): 189-212. [19] Golds G, Houdek D, Arnason T.2017. Male hypogonadism and osteoporosis: The Effects, clinical consequences, and treatment of testosterone deficiency in bone health[J]. International Journal of Endocrinology, 2017:4602129. [20] Grosebrinkhaus C, Storck L C, Frieden L, et al.2015. Genome-wide association analyses for boar taint components and testicular traits revealed regions having pleiotropic effects[J]. BMC Genetics, 16(1): 36-52. [21] Kastelic J P.2014. Understanding and evaluating bovine testes[J]. Theriogenology, 81(1): 18-23. [22] Lee W Y, Park H J.2023. T-2 mycotoxin Induces male germ cell apoptosis by ROS-mediated JNK/p38 MAPK pathway[J]. Ecotoxicology and Environmental Safety, 262: 115323. [23] Li X Y, Sun J, Ma L Y, et al.2023.The role of G3BP1 gene mediates p38 MAPK/JNK pathway in testicular spermatogenic dysfunction caused by cyfluthrin[J]. Toxics, 11(5): 451. [24] Lim P, Robson M, Spaliviero J, et al.2009. Sertoli cell androgen receptor DNA binding domain is essential for the completion of spermatogenesis[J]. Endocrinology, 150(10): 4755-65. [25] Llavanera M, Mateo-Otero Y, Delgado-Bermúdez A, et al.2021. Deactivation of the JNK pathway by GSTP1 is essential to maintain sperm functionality[J]. Frontiers in Cell and Developmental Biology, 25(9): 627140. [26] Mäkelä J A, Koskenniemi J J, Virtanen H E, et al.2019. Testis development[J]. Endocrine Reviews, 40(4): 857-905. [27] Palasz A T, Cates W F, Barth A D, et al.1994. The relationship between scrotal circumference and quantitative testicular traits in yearling beef bulls[J]. Theriogenology, 42(4): 715-726. [28] Patterson V, Ullah F, Bryant L, et al.2023. Abrogation of MAP4K4 protein function causes congenital anomalies in humans and zebrafish[J]. Science Advances, 9(17): eade0631. [29] Persson H, Ayer-Le Lievre C, Söder O, et al.1990. Expression of beta-nerve growth factor receptor mRNA in Sertoli cells downregulated by testosterone[J]. Science, 247(4943): 704-707. [30] Ricci G, Catizone A.2014. Pleiotropic Activities of HGF/c-Met system in testicular physiology: Paracrine and endocrine implications[J]. Frontiers in Endocrinology (Lausanne), 5: 38. [31] Robertson K M, O'Donnell L, Jones M E, et al.1999. Impairment of spermatogenesis in mice lacking a functional aromatase (cyp 19) gene[J]. Proceedings of the National Academy of Sciences of the USA, 96(14): 7986-7991. [32] Roth Flach R J, DiStefano M T, Danai L V, et al.2017. Map4k4 impairs energy metabolism in endothelial cells and promotes insulin resistance in obesity[J]. American Journal of Physiology-Endocrinology and Metabolism, 313(3): E303-E313. [33] Santhana Kumar K, Tripolitsioti D, Ma M, et al.2015. The Ser/Thr kinase MAP4K4 drives c-Met-induced motility and invasiveness in a cell-based model of SHH medulloblastoma[J]. Springerplus, 4: 19. [34] Schwaid A G, Su C, Loos P, et al.2015. MAP4K4 is a threonine kinase that phosphorylates FARP1[J]. ACS Chemical Biology, 10(12): 2667-2671. [35] Shepherd A, Brunckhorst O, Ahmed K, et al.2022. Botanicals in health and disease of the testis and male fertility: A scoping review[J]. Phytomedicine, 106: 154398. [36] Thumfart K M, Mansuy I M.2023. What are Sertoli cells? historical, methodological, and functional aspects[J]. Andrology, 11(5): 849-859. [37] Toe F, Rege J E, Mukasa-Mugerwa E, et al.2000. Reproductive characteristics of Ethiopian highland sheep. Ⅰ. Genetic parameters of testicular measurements in ram lambs and relationship with age at puberty in ewe lambs[J]. Small Ruminant Research, 36(3): 227-240. [38] Varela T, Conceição N, Laizé V, et al.2021. Transcriptional regulation of human DUSP4 gene by cancer-related transcription factors[J]. Journal of Cellular Biochemistry, 122(10): 1556-1566. [39] Von Kopylow K, Staege H, Schulze W, et al.2012. Fibroblast growth factor receptor 3 is highly expressed in rarely dividing human type A spermatogonia[J]. Histochemistry and Cell Biology, 138(5): 759-772. [40] Wang Y, Bi Y, Zuo Q, et al.2018. MAPK8 regulates chicken male germ cell differentiation through JNK signaling pathway[J]. Journal of Cellular Biochemistry, 119(2): 1548-1557. [41] Wright J H, Wang X, Manning G, et al.2003. The STE20 kinase HGK is broadly expressed in human tumor cells and can modulate cellular transformation, invasion, and adhesion[J]. Molecular and Cellular Biology, 23(6): 2068-82. [42] Yang N, Yang C, Tan T, et al.2022. Histology study and transcriptome analysis of the testis of loach (Misgurnus anguillicaudatus) in response to phenanthrene exposure[J]. Ecotoxicology and Environmental Safety, 242: 113950. [43] Yao Z, Zhou G, Wang X.S, et al.1999. A novel human STE20-related protein kinase, HGK, that specifically activates the c-Jun N-Terminal kinase signaling pathway[J]. Journal of Biological Chemistry, 274: 2118-2125. [44] Yin J, He W, Zhang M, et al.2023. Inhibition of spermatogenesis by hypoxia is mediated by V-ATPase via the JNK/c-Jun pathway in mice[J]. Reproductive Biology, 23(2): 100761. [45] Yuan J T, Gatti D M, Philip V M, et al.2018. Genome-wide association for testis weight in the diversity outbred mouse population[J]. Mammalian Genome, 29(2): 1-15. [46] Zhao T, Xiao T, Cao D, et al.2022. Sertoli cell PUMILIO proteins modulate mouse testis size through translational control of cell cycle regulators[J]. Biology of Reproduction, 107(1): 135-147. [47] Zou Y, Chen X, Tian X, et al.2023. Transcriptomic analysis of the developing testis and spermatogenesis in Qianbei Ma goats[J]. Genes (Basel), 14(7): 1334.
