PLZF的克隆及其对犏牛未分化精原细胞的增殖作用

doi:10.3864/j.issn.0578-1752.2024.02.013

摘要/Abstract

摘要：

【目的】犏牛作为牦牛与黄牛的种间杂交产物，具有优良的生产性能，但其杂种优势的进一步应用却受限于犏牛雄性不育。通过克隆犏牛PLZF，明确其在犏牛和牦牛睾丸组织和未分化精原细胞中的差异表达，并进一步揭示过表达该基因对犏牛未分化精原细胞活性的影响。为阐明犏牛生精停滞的作用机制提供理论基础。【方法】以24月龄公麦洼牦牛和F₁代公犏牛为实验动物，通过RT-PCR法克隆得到了犏牛PLZF的CDS序列，并进行了生物信息学分析；通过RT-qPCR法分析PLZF在犏牛和牦牛睾丸组织中的差异表达；采用同源重组的方法构建了PLZF的表达载体，并利用RT-qPCR检测了PLZF过表达效率及其下游靶基因的表达；通过PDT、CCK-8、EdU和免疫荧光检测了过表达PLZF对犏牛未分化精原细胞增殖活性的影响。【结果】克隆获得了犏牛PLZF的CDS区，并通过生物信息学分析发现该基因编码的蛋白序列不包含跨膜结构域和信号肽序列，其三级结构以α螺旋和无规卷曲为主。系统进化树分析表明犏牛PLZF与黄牛PLZF的亲缘关系更近。三级结构预测发现，虽然犏牛、牦牛和黄牛的PLZF蛋白三级结构高度相似，但牦牛PLZF蛋白在531—540位氨基酸处与犏牛和黄牛有较大差异。RT-qPCR发现，PLZF在犏牛睾丸组织和未分化精原细胞中的表达均显著低于牦牛（P<0.05），而在犏牛未分化精原细胞中过表达PLZF后，该基因的表达上调了13.8倍（P<0.01），且能显著增加犏牛未分化精原细胞的增殖活性（P<0.05），表明PLZF表达下调影响了犏牛未分化精原细胞增殖活性。此外，过表达PLZF后，犏牛未分化精原细胞中与增殖相关的基因（Etv5、Bcl6b、Pcna和c-fos）全部显著上调（P<0.05），与分化相关的基因（Stra8、Kit、Dmrt1和Sohlh2）全部显著下调（P<0.05），表明PLZF通过上调增殖相关基因、下调分化相关基因的表达促进犏牛未分化精原细胞增殖。【结论】 PLZF在犏牛未分化精原细胞中表达异常降低了犏牛未分化精原细胞增殖活性，导致其数量减少，影响了犏牛的精子发生。本试验为进一步阐明犏牛生精停滞的作用机制提供了理论基础，并为解决犏牛雄性不育问题提供了新的思路。

关键词: 犏牛, PLZF, 未分化精原细胞, 增殖

Abstract:

【Objective】 As the product of interspecific hybridization between yak and cattle, cattleyak has excellent production performance, but the further application of its heterosis is limited by the male sterility of cattleyak. The aim of the study was to clone the PLZF of cattleyak, and to identify its differential expression in the testicular tissue and undifferentiated spermatogonia of cattleyak and yak, and to further reveal the effect of expressing this gene on the activity of undifferentiated spermatogonia of cattleyak. This study could provide a theoretical basis for clarifying the mechanism of spermatogenesis stagnation in cattleyaks. 【Method】 In this study, 24-month-old male Maiwa yak and F₁ generation male cattleyak were used as experimental animals, and the CDS sequences of PLZF in cattleyak were cloned by RT-PCR and analyzed by bioinformatics. The differential expression of PZLF in the testis tissues of cattleyak and yak was analyzed by RT-qPCR. The expression vector of PLZF was constructed by homologous recombination, and the overexpression efficiency and the expression of downstream target genes were detected by RT-qPCR. The effect of overexpression of PLZF on the undifferentiated spermatogonia of cattleyak was detected by PDT, CCK-8, EdU, and immunofluorescence. 【Result】 The CDS region of the PLZF was cloned, and it was found by bioinformatics analysis that the protein sequence encoded by the gene did not contain transmembrane domain and signal peptide sequence, and its tertiary structure was mainly α helix and random curl. Phylogenetic tree analysis showed that the PLZF of cattleyak was more closely related to the PLZF of cattle. The prediction of tertiary structure showed that although the tertiary structure of PLZF protein of cattleyak, yak and cattle was highly similar, the PLZF protein of yak was quite different from that of cattleyak and cattle at amino acids 531-540. RT-qPCR found that the expression levels of PLZF gene in the testis tissue and undifferentiated spermatogonia of cattleyak were significantly lower than that in yak (P<0.05). After overexpression of PLZF, the expression of PLZF in the undifferentiated spermatogonia of the cattleyak was up-regulated by 13.8 times (P<0.01), and the proliferation activity of the undifferentiated spermatogonia of the cattleyak was significantly increased (P<0.05), which showed that the down-regulation of PLZF expression affected the proliferation activity of undifferentiated spermatogonia of cattleyak. In addition, after overexpression of PLZF, the proliferation activity of undifferentiated spermatogonia was significantly increased All proliferation-related genes (Etv5, Bcl6b, Pcna and c-fos) were significantly up-regulated (P<0.05), and all differentiation-related genes (Stra8, Kit, Dmrt1 and Sohlh2) were significantly down-regulated (P<0.05), indicating that PLZF could promote the proliferation of undifferentiated spermatogonia by up-regulating the expression of proliferation-related genes and down-regulating the expression of differentiation-related genes.【Conclusion】 The abnormal expression of PLZF in cattleyak undifferentiated spermatogonia reduced the proliferative activity of cattleyak undifferentiated spermatogonia, resulting in its decrease in number and affecting the spermatogenesis of cattleyak. This study provided a theoretical basis for further elucidation of the mechanism of spermatogenesis and stagnation in cattleyak, and a new idea for solving the problem of male sterility in cattleyak.

Key words: cattleyak, PLZF, undifferentiated spermatogonia, proliferation

张鹏, 王明秀, 敬科民, 李雨谦, 田园, 钟金城, 蔡欣. PLZF的克隆及其对犏牛未分化精原细胞的增殖作用[J]. 中国农业科学, 2024, 57(2): 390-402.

ZHANG Peng, WANG MingXiu, JING KeMin, LI YuQian, TIAN Yuan, ZHONG JinCheng, CAI Xin. Cloning of PLZF Gene and Its Effects on the Proliferation of Undifferentiated Spermatogonia in Cattleyak[J]. Scientia Agricultura Sinica, 2024, 57(2): 390-402.

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https://www.chinaagrisci.com/CN/Y2024/V57/I2/390

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参考文献 31

[1]	吴周林, 左玲, 徐弘扬, 王健蓉, 张仕民, 赵莉, 罗海艳. 犏牛杂种优势研究进展. 当代畜牧, 2018(6): 23-25.
	WU Z L, ZUO L, XU H Y, WANG J R, ZHANG S M, ZHAO L, LUO H Y. The research progress on heterosis in cattle-yak. Contemporary Animal Husbandry, 2018(6): 23-25. (in Chinese)
[2]	NIAYALE R, CUI Y, ADZITEY F. Male hybrid sterility in the cattle-yak and other bovines: a review. Biology of Reproduction, 2021, 104(3): 495-507. doi: 10.1093/biolre/ioaa207 pmid: 33185248
[3]	陈会友, 张建敏, 李柏森, 邓永琳, 张龚炜. 犏牛雄性不育的减数分裂基因表达与表观遗传调控研究进展. 遗传, 2020, 42(11): 1081-1092.
	CHEN H Y, ZHANG J M, LI B S, DENG Y L, ZHANG G W. Progress on meiotic gene expression and epigenetic regulation of male sterility in Dzo cattle. Hereditas(Beijing), 2020, 42(11): 1081-1092. (in Chinese)
[4]	张鹏, 王明秀, 敬科民, 彭巍, 田园, 李雨谦, 付长其, 舒适, 钟金城, 蔡欣. FGFs/FGFRs及其介导信号通路基因的异常表达影响犏牛未分化精原细胞增殖活性. 畜牧兽医学报, 2023: 1-15. (2023-03-14). https://kns.cnki.net/kcms/detail/11.1985.s.20230311.1755.004.html.
	ZHANG P, WANG M X, JING K M, PENG W, TIAN Y, LI Y Q, FU C Q, SHU S, ZHONG J C, CAI X. Abnormal expression of FGFs/FGFRs and their mediated signaling pathway genes affect the proliferative activity of undifferentiated spermatogonia in Cattleyak. Acta Veterinaria et Zootechnica Sinica, 2023: 1-15. (2023-03-14). https://kns.cnki.net/kcms/detail/11.1985.s.20230311.1755.004.html. (in Chinese)
[5]	DAVID G, ALLAND L, HONG S H, WONG C W, DEPINHO R A, DEJEAN A. Histone deacetylase associated with mSin3A mediates repression by the acute promyelocytic leukemia-associated PLZF protein. Oncogene, 1998, 16(19): 2549-2556. doi: 10.1038/sj.onc.1202043 pmid: 9627120
[6]	FISCHER S, KOHLHASE J, BÖHM D, SCHWEIGER B, HOFFMANN D, HEITMANN M, HORSTHEMKE B, WIECZOREK D. Biallelic loss of function of the promyelocytic leukaemia zinc finger (PLZF) gene causes severe skeletal defects and genital hypoplasia. Journal of Medical Genetics, 2008, 45(11): 731-737. doi: 10.1136/jmg.2008.059451 pmid: 18611983
[7]	BUAAS F W, KIRSH A L, SHARMA M, MCLEAN D J, MORRIS J L, GRISWOLD M D, DE ROOIJ D G, BRAUN R E. Plzf is required in adult male germ cells for stem cell self-renewal. Nature Genetics, 2004, 36(6): 647-652. doi: 10.1038/ng1366 pmid: 15156142
[8]	PHILLIPS B T, GASSEI K, ORWIG K E. Spermatogonial stem cell regulation and spermatogenesis. Philosophical Transactions of the Royal Society of London Series B, Biological Sciences, 2010, 365(1546): 1663-1678.
[9]	BABATABAR DARZI M, NEMATI F, AZIZI H, DEHPOUR JOUYBARI A. Immunohistochemistry and immunocytochemistry analysis of PLZF and VASA in mice testis during spermatogenesis. Zygote, 2023, 31(3): 273-280. doi: 10.1017/S0967199423000047
[10]	MIPAM T, CHEN X M, ZHAO W S, ZHANG P, CHAI Z X, YUE B L, LUO H, WANG J K, WANG H B, WU Z J, WANG J B, WANG M X, WANG H, ZHANG M, WANG H Y, JING K M, ZHONG J C, CAI X. Single-cell transcriptome analysis and in vitro differentiation of testicular cells reveal novel insights into male sterility of the interspecific hybrid cattle-yak. BMC Genomics, 2023, 24(1): 149. doi: 10.1186/s12864-023-09251-2
[11]	CHAN A L, LA H M, LEGRAND J M D, MÄKELÄ J A, EICHENLAUB M, DE SERAM M, RAMIALISON M, HOBBS R M. Germline stem cell activity is sustained by SALL4-dependent silencing of distinct tumor suppressor genes. Stem Cell Reports, 2017, 9(3): 956-971. doi: 10.1016/j.stemcr.2017.08.001
[12]	OATLEY J M, AVARBOCK M R, TELARANTA A I, FEARON D T, BRINSTER R L. Identifying genes important for spermatogonial stem cell self-renewal and survival. Proceedings of the National Academy of Sciences of the United States of America, 2006, 103(25): 9524-9529.
[13]	HOBBS R M, SEANDEL M, FALCIATORI I, RAFII S, PANDOLFI P P. Plzf regulates germline progenitor self-renewal by opposing mTORC1. Cell, 2010, 142(3): 468-479. doi: 10.1016/j.cell.2010.06.041 pmid: 20691905
[14]	MU H L, LI N, WU J, ZHENG L M, ZHAI Y X, LI B, SONG W C, WANG J L, ZHU H J, LI G P, HUA J L. PLZF-induced upregulation of CXCR4 promotes dairy goat male germline stem cell proliferation by targeting Mir146a. Journal of Cellular Biochemistry, 2016, 117(4): 844-852. doi: 10.1002/jcb.25371 pmid: 26365432
[15]	DI GIACOMO M, COMAZZETTO S, SAINI H, DE FAZIO S, CARRIERI C, MORGAN M, VASILIAUSKAITE L, BENES V, ENRIGHT A J, O’CARROLL D. Multiple epigenetic mechanisms and the PiRNA pathway enforce LINE1 silencing during adult spermatogenesis. Molecular Cell, 2013, 50(4): 601-608. doi: 10.1016/j.molcel.2013.04.026 pmid: 23706823
[16]	ZHANG P, WANG M X, CHEN X M, JING K M, LI Y Q, LIU X R, RAN H B, QIN J, ZHONG J C, CAI X. Dysregulated genes in undifferentiated spermatogonia and Sertoli cells are associated with the spermatogenic arrest in cattleyak. Molecular Reproduction and Development, 2022, 89(12): 632-645. doi: 10.1002/mrd.23653 pmid: 36409004
[17]	孙军伟. GDNF对奶山羊雄性生殖干细胞自我更新的调控[D]. 杨凌: 西北农林科技大学, 2011: 19-20.
	SUN J W. The mechanism of GDNF mediates self-renewal of dairy goat male germ-line stem cells[D]. Yangling: Northwest A & F University, 2011: 19-20. (in Chinese)
[18]	SHAMHARI A, JEFFERI N E S, ABD HAMID Z, BUDIN S B, IDRIS M H M, TAIB I S. The role of promyelocytic leukemia zinc finger (PLZF) and glial-derived neurotrophic factor family receptor alpha 1 (GFRα1) in the cryopreservation of spermatogonia stem cells. International Journal of Molecular Sciences, 2023, 24(3): 1945. doi: 10.3390/ijms24031945
[19]	AZIZI H, KORUJI M, SKUTELLA T. Comparison of PLZF gene expression between pluripotent stem cells and testicular germ cells. Cell Journal, 2020, 22(1): 60-65.
[20]	ISHII K, KANATSU-SHINOHARA M, TOYOKUNI S, SHINOHARA T. FGF₂ mediates mouse spermatogonial stem cell self-renewal via upregulation of Etv5 and Bcl6b through MAP2K1 activatio. Development, 2012, 139(10): 1734-1743. doi: 10.1242/dev.076539
[21]	OATLEY J M, AVARBOCK M R, BRINSTER R L. Glial cell line-derived neurotrophic factor regulation of genes essential for self-renewal of mouse spermatogonial stem cells is dependent on Src family kinase signaling. The Journal of Biological Chemistry, 2007, 282(35): 25842-25851. doi: 10.1074/jbc.M703474200
[22]	WU S X, MIPAM T, XU C F, ZHAO W S, ALI SHAH M, YI C P, LUO H, CAI X, ZHONG J C. Testis transcriptome profiling identified genes involved in spermatogenic arrest of cattleyak. PLoS ONE, 2020, 15(2): e0229503. doi: 10.1371/journal.pone.0229503
[23]	SONG W X, SHI X L, XIA Q, YUAN M, LIU J X, HAO K Y, QIAN Y J, ZHAO X D, ZOU K. PLZF suppresses differentiation of mouse spermatogonial progenitor cells via binding of differentiation associated genes. Journal of Cellular Physiology, 2020, 235(3): 3033-3042. doi: 10.1002/jcp.29208 pmid: 31541472
[24]	KOTAJA N, SASSONE-CORSI P. Plzf pushes stem cells. Nature Genetics, 2004, 36(6): 551-553. pmid: 15167928
[25]	SUZUKI H, AHN H W, CHU T J, BOWDEN W, GASSEI K, ORWIG K, RAJKOVIC A. SOHLH1 and SOHLH2 coordinate spermatogonial differentiation. Developmental Biology, 2012, 361(2): 301-312. doi: 10.1016/j.ydbio.2011.10.027 pmid: 22056784
[26]	穆海龙. PLZF通过靶向抑制miR-146a促进CXCR4表达调控奶山羊精原干细胞增殖[D]. 杨凌: 西北农林科技大学, 2015: 29.
	MU H L. PLZF up-regulatiion CXCR4 to promote dairy goat male germline stem cells proliferation via targeting miR-146a[D]. Yangling: Northwest A & F University, 2015: 29. (in Chinese)
[27]	ORNITZ D M, ITOH N. The fibroblast growth factor signaling pathway. Wiley Interdisciplinary Reviews Developmental Biology, 2015, 4(3): 215-266. doi: 10.1002/wdev.2015.4.issue-3
[28]	MENG X, LINDAHL M, HYVÖNEN M E, PARVINEN M, DE ROOIJ D G, HESS M W, RAATIKAINEN-AHOKAS A, SAINIO K, RAUVALA H, LAKSO M, PICHEL J G, WESTPHAL H, SAARMA M, SARIOLA H. Regulation of cell fate decision of undifferentiated spermatogonia by GDNF. Science, 2000, 287(5457): 1489-1493. doi: 10.1126/science.287.5457.1489 pmid: 10688798
[29]	GOERTZ M J, WU Z R, GALLARDO T D, HAMRA F K, CASTRILLON D H. Foxo1 is required in mouse spermatogonial stem cells for their maintenance and the initiation of spermatogenesis. The Journal of Clinical Investigation, 2011, 121(9): 3456-3466. doi: 10.1172/JCI57984
[30]	LOVASCO L A, GUSTAFSON E A, SEYMOUR K A, DE ROOIJ D G, FREIMAN R N. TAF4b is required for mouse spermatogonial stem cell development. Stem Cells, 2015, 33(4): 1267-1276. doi: 10.1002/stem.1914 pmid: 25727968
[31]	SNIPPERT H J, VAN DER FLIER L G, SATO T, VAN ES J H, VAN DEN BORN M, KROON-VEENBOER C, BARKER N, KLEIN A M, VAN RHEENEN J, SIMONS B D, CLEVERS H. Intestinal crypt homeostasis results from neutral competition between symmetrically dividing Lgr5 stem cells. Cell, 2010, 143(1): 134-144. doi: 10.1016/j.cell.2010.09.016 pmid: 20887898

基因 Gene	登录号 Accession No.	引物序列 Primer sequences (5′→3′)	引物长度 Primer Length (bp)	退火温度 Tm (℃)	产物长度 Product size (bp)
Bcl6b	XM_005906036	F：GAAAACGCATAGCCGCATCC R：GACTCTTGTGCCGGAAATGC	20 20	60.32 59.32	248
Etv5	XM_005910750	F：ACCGGCAAATGTCAGAACCT R：CGTCAAAGTACAACCGGGGA	20 20	59.89 59.97	276
c-fos	XM_005900989	F：GCCCGGACCTACAATGGCTA R：CTCTGCCTCCTGTCATGGTTT	20 21	61.69 60.00	150
Pcna	XM_005906528	F：AGCTGAAGATAACGCAGACAC R：ATCTCGGCATATACGTGCAA	21 20	58.39 57.19	178
Dmrt1	XM_005900007	F：CCCGTGCCTGATGATTGAGA R：GTACGGAAACAGAGACGGCT	20 20	59.82 59.76	211
Sohlh2	XM_002691802	F：CTATCCGTCCCAGGCATGAT R：ACCGATAGCCTTCTCTGACG	20 20	59.02 58.97	189
Kit	NM_001166484	F：AGAAAACGACACGCTGGTTC R：CTCGCGCTTCACATTTCTGA	20 20	59.06 58.93	151
Stra8	XM_005906930	F：TGTGGCAAGTTTCCTGGACA R：TCTCCGTGCACCTCAACATT	20 20	59.74 59.60	215
Plzf	XM_005891907	F：GTTCCATGCCCACCGGACT R：TGGCCTCCGTGTCATTGTCA	19 20	63.16 55.00	275
β-actin	DQ838049	F：CCCTGGAGAAGAGCTACGAG R：TAGTTTCGTGAATGCCGCAG	20 20	58.97 58.92	129

物种 Specics	登录号 Accession No.	CDS区长度 Length of CDS region (bp)	相似性 Percent identity (%)
牦牛 Bos Mutus	XM_005891907	2 019	98.96
黄牛 Bos Taurus	XM_005215851	2 022	99.80
瘤牛 Bos indicus	XM_019974560	2 022	99.75
猪 Sus scrofa	XM_021062870	2 022	94.07
山羊 Capra hircus	XM_018058857	2 022	97.23