The Expression of Genes Related to Clathrin-mediated Endocytosis in Estrus Ovary of Congjiang Xiang Pig (Sus scrofa)
TANG Liang-Ting1,2, HUANG Shi-Hui1, WANG Jia-Fu1,*, RAN Xue-Qin1,*, LI Sheng1, NIU Xi1
1 Institute of Agri-bioengineering / College of Animal Science, Guizhou University, Guiyang 550025, China; 2 Guizhou Open University, Guiyang 550023, China
Abstract During the development of ovarian follicles, gap junctions between oocytes and surrounding granulosa cells mediate intercellular communication and material transport. Gap junction internalization refers to the clathrin-mediated endocytosis (CME) process. In order to study the effect of CME-related genes on ovary function of Congjiang Xiang pig (Sus scrofa), transcriptome sequencing technology was used to analyze the expression of CME-related genes and the alternative splicing of transcripts in Congjiang Xiang pigs ovaries at estrus stage by Limma, DESeq2, rMATs softwares and reverse transcription PCR (RT-PCR) method. Transcriptome changes at diestrus stage were used as controls. As a result, 8 CME genes were detected in the ovaries of Congjiang Xiang pigs, including DAB adaptor protein 2 (DAB2), adaptor related protein complex 2 subunit μ1 (AP2M1), clathrin light chain B (CLTB), clathrin light chain A (CLTA), dynamin-1 (DNM1), myosin Ⅵ (MYO6), dynamin-2 (DNM2) and clathrin heavy chain (CLTC). Among them, the differentially expressed genes were DAB2, AP2M1 and CLTB. Except for CLTB, the other 7 genes produced different alternative splicing events between estrous and diestrous ovaries, and the 7 transcripts produced by these alternative splicing events were the new transcripts not included in the Ensembl database up to now. It showed that alternative splicing was an important regulation mode of ovary in Congjiang Xiang pigs at estrus stage, and a specific network of protein interaction was formed by producing new transcripts and proteins during CME. The results of this study indicated that the internalization of gap junction was regulated by the expression of CME-related genes and splicing of transcripts at transcription and post-transcription levels, thus regulating the ovarian function of Congjiang Xiang pig. These researches would help to understand of the post-transcriptional regulation process and the molecular regulation mechanism of porcine ovarian function.
TANG Liang-Ting,HUANG Shi-Hui,WANG Jia-Fu, et al. The Expression of Genes Related to Clathrin-mediated Endocytosis in Estrus Ovary of Congjiang Xiang Pig (Sus scrofa)[J]. 农业生物技术学报, 2021, 29(9): 1741-1751.
[1] 侯建华, 岑尧, 马玉珍. 2012. 间隙连接蛋白在卵泡发育过程中的作用[J].现代生物医学进展,12(09):1791-1793. (Hou J H, Cen Y, Ma Y Z.2012. The role of gap junction proteins in the development of follicles[J]. Advances in Modern Biomedicine, 12(09): 1791-1793.) [2] 梁菁媛, 石德顺, 邓彦飞, 等. 2019. 哺乳动物原始卵泡体外培养与激活调控研究进展[J]. 中国畜牧杂志, 55(07): 5-9. (Liang J Y, Shi D S, Deng Y F, et al.2019. Research progress in vitro culture and activation and regulation of mammalian primordial follicles[J]. Chinese Journal of Animal Husbandry, 55(07): 5-9.) [3] 马骁, 王翀, 王金良, 等. 2019. AP2M1在狂犬病病毒侵入中的作用研究[J]. 中国预防兽医学报, 41(01): 6-11. (Ma X, Wang C, Wang J L, et al.2019. The role of AP2M1 in rabies virus invasion[J]. Chinese Journal of Preventive Veterinary Medicine, 41(01): 6-11.) [4] 裴悦, 周晓龙, 杨松柏, 等. 2017. 猪DNM2基因克隆,序列分析与组织表达谱研究[J]. 中国畜牧兽医, 09: 16-24. (Pei Y, Zhou X L, Yang S B, et al.2017. Porcine DNM2 gene cloning, sequence analysis and tissue expression profile study[J]. China Animal Husbandry and Veterinary Medicine, 09: 16-24.) [5] 周炎珍, 郑秀. 2006. Dab2/DOC-2基因的研究进展[J].医学综述, 12: 1473-1475. (Zhou Y Z, Zheng X.2006. Research progress of Dab2/DOC-2 gene[J]. Medical review, 12: 1473-1475) [6] Bruzzone R, White T W, Paul D L.1996. Connections with connexins: The molecular basis of direct intercellular signaling[J]. European Journal of Biochemistry, 238(1): 1-27. [7] Chu Q, Zhou B, Xu F, et al.2017. Genome-wide differential mRNA expression profiles in follicles of two breeds and at two stages of estrus cycle of gilts[J]. Scientific Reports, 7(1): 5052. [8] Cook T A, Urrutia R, Mcniven M A.1994. Identification of dynamin 2, an isoform ubiquitously expressed in rat tissues[J]. Proceedings of the National Academy of Sciences of the USA, 91(2): 644-648. [9] DE Jonge J J, Batters C, O'loughlin T, et al.2019. The MYO6 interactome: Selective motor-cargo complexes for diverse cellular processes[J]. FEBS Letters, 593(13): 1494-1507. [10] Delmar M, Coombs W, Sorgen P, et al.2004. Structural bases for the chemical regulation of Connexin43 channels[J]. Cardiovascular Research, 62(2): 268-275. [11] Ding W, Wang W, Zhou B, et al.2010. Formation of primordial follicles and immunolocalization of PTEN, PKB and FOXO3A proteins in the ovaries of fetal and neonatal pigs[J]. Journal of Reproduction and Development, 56(1): 162-168. [12] Downs S M, Daniel S A, Eppig J J.1988. Induction of maturation in cumulus cell-enclosed mouse oocytes by follicle-stimulating hormone and epidermal growth factor: Evidence for a positive stimulus of somatic cell origin[J]. Journal of Experimental Zoology, 245(1): 86-96. [13] EPPIG J J.1991. Maintenance of meiotic arrest and the induction of oocyte maturation in mouse oocyte-granulosa cell complexes developed in vitro from preantral follicles[J]. Biology of Reproduction, 45(6): 824-830. [14] Ferguson S M, Brasnjo G, Hayashi M, et al.2007. A selective activity-dependent requirement for dynamin 1 in synaptic vesicle endocytosis[J]. Science, 316(5824): 570-574. [15] Gumpert A M, Varco J S, Baker S M, et al.2008. Double-membrane gap junction internalization requires the clathrin-mediated endocytic machinery[J]. FEBS Letters, 582(19): 2887-2892. [16] Li Y, Liu H, Wu K, et al.2019. Melatonin promotes human oocyte maturation and early embryo development by enhancing clathrin-mediated endocytosis[J]. Journal of Pineal Research, 67(3): e12601. [17] Liu Y W, Surka M C, Schroeter T, et al.2008. Isoform and splice-variant specific functions of dynamin-2 revealed by analysis of conditional knock-out cells[J]. Molecular and Cellular Biology, 19(12): 5347-5359. [18] Lowther K M, Nikolaev V O, Mehlmann L M.2011. Endocytosis in the mouse oocyte and its contribution to cAMP signaling during meiotic arrest[J]. Reproduction, 141(6): 737-747. [19] Luo Z Y, Dai X L, Ran X Q, et al.2018. Identification and profile of microRNAs in Xiang pig testes in four different ages detected by Solexa sequencing[J]. Theriogenology, 117: 61-71. [20] Mcmahon H T, Boucrot E.2011. Molecular mechanism and physiological functions of clathrin-mediated endocytosis[J]. Nature Reviews Molecular Cell Biology, 12(8): 517-533. [21] Mihalas B P, Redgrove K A, Bernstein I R, et al.2020. Dynamin 2-dependent endocytosis is essential for mouse oocyte development and fertility[J]. FASEB Journal, 34(4): 5162-5177. [22] Piccinotti S, Whelan S P.2016. Rabies internalizes into primary peripheral neurons via clathrin coated pits and requires fusion at the cell body[J]. PLOS Pathogens, 12(7): e1005753. [23] Piehl M, Lehmann C, Gumpert A, et al.2007. Internalization of large double-membrane intercellular vesicles by a clathrin-dependent endocytic process[J]. Molecular Biology of the Cell, 18(2): 337-347. [24] Roychoudhury S, Massanyi P, Slivkova J, et al.2015. Effect of mercury on porcine ovarian granulosa cells in vitro[J]. Journal of Environmental Science and Health Part A-Toxic/Hazardous Substances & Environmental Engineering, 50(8): 839-845. [25] Sheng Z, Smith e R, He J, et al.2001. Chromosomal location of murine disabled-2 gene and structural comparison with its human ortholog[J]. Gene, 268(1-2): 31-39. [26] Tang L T, Ran X Q, Mao N, et al.2018. Analysis of alternative splicing events by RNA sequencing in the ovaries of Xiang pig at estrous and diestrous[J]. Theriogenology, 119: 60-68. [27] Tao W, Moore R, Smith E R, et al.2016. Endocytosis and physiology: Insights from disabled-2 Deficient Mice[J]. Frontiers in Cell and Developmental Biology, 4: 129. [28] Vaid K S, Guttman J A, Babyak N, et al.2007. The role of dynamin 3 in the testis[J]. Journal of Cellular Physiology, 210(3): 644-654. [29] White B R, Mclaren D G, Dziuk P J, et al.1993. Age at puberty, ovulation rate, uterine length, prenatal survival and litter size in Chinese Meishan and Yorkshire females[J]. Theriogenology, 40(1): 85-97. [30] Xu X X, Yang W, Jackowski S, et al.1996. Additions and corrections to cloning of a novel phosphoprotein regulated by colony-stimulating factor 1 shares a domain with the Drosophila disabled gene product[J]. Journal of Biological Chemistry, 271(22): 13292.
