Combined analysis of embryonic gonadal development differences of mulard duck and muscovy duck using long non-coding RNAs and mRNAs

doi:10.3785/j.issn.1008-9209.2022.07.071

Journal of Zhejiang University (Agriculture and Life Sciences)

2023, Vol. 49

Issue (5): 729-743 DOI: 10.3785/j.issn.1008-9209.2022.07.071

Animal sciences & veterinary medicines

Combined analysis of embryonic gonadal development differences of mulard duck and muscovy duck using long non-coding RNAs and mRNAs

Li LI^1,²(

),Linli ZHANG^1,²,Qingwu XIN^1,²,Zhongwei MIAO^1,²,Zhiming ZHU^1,²,Junzhi QIU³,Xiaona HAO⁴,Qinlou HUANG²(

),Nenzhu ZHENG^1,²(

)

^1.Institute of Animal Husbandry and Veterinary Medicine, Fujian Academy of Agricultural Sciences, Fuzhou 350013, Fujian, China
^2.Fujian Key Laboratory of Animal Genetics and Breeding, Fuzhou 350013, Fujian, China
^3.College of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou 350002, Fujian, China
^4.College of Animal Sciences (College of Bee Science), Fujian Agriculture and Forestry University, Fuzhou 350002, Fujian, China

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Abstract

The purpose of this study was to screen key mRNAs and long non-coding RNAs (lncRNAs) that affect gonadal development in duck embryos and to explain scientifically gonadal development defects in the mulard duck. Three male embryonic gonadal tissues of the mulard duck (BF₁, BF₂, BF₃) and the muscovy duck (F₁, F₂, F₃) were collected to extract RNA and perform high-throughput sequencing, and differential genes and lncRNAs were screened to predict target genes and perform functional annotations. Finally, the sequencing data were verified by real-time fluorescence quantitative polymerase chain reaction (qRT-PCR). The results showed that a total of 1 109 differentially expressed genes were screened from the gonadal tissues of the mulard duck and the muscovy duck. Compared with the muscovy duck, 857 genes were up-regulated and 252 genes were down-regulated in the mulard duck. Among them, the aldo-keto reductase family 1 member D1 gene (AKR1D1), 17β-hydroxysteroid dehydrogenase 3 gene (17β-HSD3), and cholesterol side-chain cleavage enzyme gene (P450scc) may be related to gonadal differentiation and development in the mulard duck. Meanwhile, 733 significantly differentially expressed lncRNAs were obtained. Compared with the muscovy duck, a total of 660 lncRNAs were significantly up-regulated and 73 lncRNAs were significantly down-regulated in the mulard duck. Target gene prediction analysis showed that a total of 136 down-regulated lncRNAs and 893 up-regulated lncRNAs may be involved in differential gene expression and had potential regulatory relationships, among which, TCONS_00246198 targeted 17β-HSD3, and TCONS_00229529 targeted tetraspanin-2 gene (TSPAN2), suggesting that the above lncRNAs may participate in duck embryonic gonadal development by targeting key genes. The qRT-PCR results showed that the expression levels of differential genes and lncRNAs were consistent with the expression trends in transcriptome sequencing, indicating that the data obtained by high-throughput sequencing are relatively reliable. RNA binding protein immunoprecipitation (RIP) assay results revealed that compared with IgG, the enrichment level of TCONS_00246198 reached 71.51 times. The above results indicate that TCONS_00246198 interacts directly or indirectly with the 17β-HSD3 protein, which means that they may have a targeting relationship. In summary, this study obtains a batch of key mRNAs and lncRNAs that may affect duck embryonic gonadal development, and it is speculated that the differential lncRNAs can regulate the expression of differential genes. This study provides a scientific basis for understanding the differences in duck embryonic gonadal development and the mechanisms of avian gonadal development.

Key words： mulard duck muscovy duck gonad infertility long non-coding RNAs (lncRNAs) mRNAs

Received: 07 July 2022 Published: 03 November 2023

CLC:

S834.89

Corresponding Authors: Qinlou HUANG,Nenzhu ZHENG E-mail: 576801792@qq.com;hql202@126.com;zhengnz@163.com

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	Li LI
	Linli ZHANG
	Qingwu XIN
	Zhongwei MIAO
	Zhiming ZHU
	Junzhi QIU
	Xiaona HAO
	Qinlou HUANG
	Nenzhu ZHENG

Cite this article:

Li LI,Linli ZHANG,Qingwu XIN,Zhongwei MIAO,Zhiming ZHU,Junzhi QIU,Xiaona HAO,Qinlou HUANG,Nenzhu ZHENG. Combined analysis of embryonic gonadal development differences of mulard duck and muscovy duck using long non-coding RNAs and mRNAs. Journal of Zhejiang University (Agriculture and Life Sciences), 2023, 49(5): 729-743.

URL:

https://www.zjujournals.com/agr/10.3785/j.issn.1008-9209.2022.07.071 OR https://www.zjujournals.com/agr/Y2023/V49/I5/729

利用长链非编码RNAs与mRNAs联合分析半番鸭及番鸭胚胎期性腺发育差异

本研究旨在筛选影响鸭胚胎期性腺发育的关键mRNAs和长链非编码RNAs（long non-coding RNAs, lncRNAs），科学解释半番鸭性腺发育缺陷问题。采集半番鸭（BF₁、BF₂、BF₃）和番鸭（F₁、F₂、F₃）各3个雄性胚胎性腺组织并提取其RNA，通过高通量测序筛选差异基因和lncRNAs，预测靶基因并进行功能注释，最后应用实时荧光定量聚合酶链反应（real-time fluorescence quantitative polymerase chain reaction, qRT-PCR）对测序结果进行验证。结果显示：从半番鸭和番鸭性腺组织中共筛选出1 109个差异基因；相对于番鸭，半番鸭中上调表达基因共857个，下调表达基因共252个，其中，醛酮还原酶家族1成员D1基因（aldo-keto reductase family 1 member D1 gene, AKR1D1）、17β-羟基类固醇脱氢酶3基因（17β-hydroxysteroid dehydrogenase 3 gene, 17β-HSD3）和胆固醇侧链裂解酶基因（cholesterol side-chain cleavage enzyme gene, P450scc）等可能与半番鸭性腺分化发育相关。同时，获得了733个显著差异表达的lncRNAs；相对于番鸭，半番鸭中显著上调的lncRNAs共660个，显著下调的lncRNAs共73个。靶基因预测分析显示，136个下调的lncRNAs和893个上调的lncRNAs可能参与差异基因表达并存在潜在的调控关系，其中，TCONS_00246198靶向17β-HSD3，TCONS_00229529靶向四次穿膜蛋白2基因（tetraspanin-2 gene, TSPAN2），说明以上lncRNAs可能通过靶向关键基因来参与鸭胚胎期性腺发育。qRT-PCR结果显示，差异基因和lncRNAs表达水平与转录组测序中的表达趋势一致，表明通过高通量测序获得的数据较为可靠。RNA结合蛋白免疫沉淀（RNA binding protein immunoprecipitation, RIP）试验结果发现，与IgG相比，TCONS_00246198富集水平达71.51倍，表明TCONS_00246198与17β-HSD3蛋白存在直接或间接的相互作用，即两者可能存在靶向关系。综上所述，本研究获得了一批可能影响鸭胚胎期性腺发育的关键mRNAs和lncRNAs，推测差异lncRNAs可以调控差异基因的表达，为了解鸭胚胎期性腺发育差异及禽类性腺发育机制提供了科学依据。

关键词： 半番鸭, 番鸭, 性腺, 不育, 长链非编码RNAs, mRNAs

名称 Name	GenBank登录号 GenBank accession No.	引物序列（5 $′$ →3 $′$ ） Primer sequence (5 $′$ →3 $′$ )	扩增长度 Size/bp	退火温度 Annealing temperature/℃
GAPDH	XM_005016745.2	F: GGAGCTGCCCAGAACATTATC R: GCAGGTCAGGTCCACGACA	141	60
AKR1D1	101802488	F: CGTGTCCTCCCCTCCTTTA R: CCTCGCTGGATGCTGAAAC	105	60
17β-HSD3	101799136	F: GGGACTGATCTGTTTCTGTGCT R: CCATCTCCTGCTCCTGTGA	138	60
LOC101794158	101794158	F: CCTTTGGCATCGGGATTGGA R: GCCGGATGACTCTGTTGGA	138	60
PTGFRN	101794936	F: CACAATGGGAGCTGGCATAAAGT R: GGTGGGGTCATCTGAAAACTTG	132	60
TCONS_00189165	XLOC_112996	F: GCTATGAAGAGGAAAAGGGATGTC R: GCGCTTAAACGAGAGGAACCA	79	60
XR_003494977.1	106018179	F: GGAACCATAATGTGCCAGCTGAA R: CCTTGCAGTTGATAAGACGATGGTA	96	60
TCONS_00087264	101791443	F: GAGCCACAGACACGAAGAGAA R: GGGGAATCAGTAACCCATAGC	96	60
XR_003498542.1	113844190	F: GAAGAGGACACCGATCAAGAG R: GTAACAACCTTCAGCCACTCAAAC	87	60

Table 1 qRT-PCR primer sequence information

Fig. 1 Female and male identification of the mulard duck and the muscovy duckbf/BF: Mulard duck; b: Pekin duck; f/F: Muscovy duck; M: DNA marker.

Table 2 Quality and alignment analysis of sequencing data

Fig. 2 lncRNAs differentially expressed in the mulard duck compared with the muscovy duck

Table 3 Differentially expressed genes at top 10 of fold changes

Table 4 Differentially expressed lncRNAs at top 10 of fold changes

Fig. 3 Venn diagram of differential lncRNAs targeting differential mRNAs

Fig. 4 GO enrichment process of differential genes and differential lncRNAs targeting genesA. Differential genes; B. Differential lncRNAs (co-expression); C. Differential lncRNAs (co-location).

Fig.5 KEGG enrichment process of differential genes and differential lncRNAs targeting genes

Fig. 6 Differential genes and lncRNAs in embryonic gonadal tissues of the mulard duck and the muscovy duck verified by qRT-PCR

Fig. 7 Enrichment analysis of lncRNAs TCONS_00246198 by 17β-HSD3 protein


[1]	郑嫩珠,李盛霖,陈晖.福建省白羽半番鸭及其母本选育[J].中国家禽,2010,32(14):5-8. DOI:10.16372/j.issn.1004-6364.2010.14.002 ZHENG N Z, LI S L, CHEN H. White mulard duck and female parent breeding in Fujian Province[J]. China Poultry, 2010, 32(14): 5-8. (in Chinese) doi: 10.16372/j.issn.1004-6364.2010.14.002

[2]	YOUSEFI H, MAHERONNAGHSH M, MOLAEI F, et al. Long noncoding RNAs and exosomal lncRNAs: classification, and mechanisms in breast cancer metastasis and drug resistance[J]. Oncogene, 2020, 39(5): 953-974. DOI: 10.1038/s41388-019-1040-y doi: 10.1038/s41388-019-1040-y

[3]	许玉德,智绪华.半番鸭(番鸭 ×家鸭 )雄性不育的辨析[J].厦门大学学报(自然科学版),1998,37(5):763-768. XU Y D, ZHI X H. Studies on male sterility of hybrid duck[J]. Journal of Xiamen University (Natural Science), 1998, 37(5): 763-768. (in Chinese with English abstract)

[4]	檀俊秩,陈晖,宋建捷,等.半番鸭繁殖性状的研究[J].福建农业学报,1998,13(2):41-45. TAN J Z, CHEN H, SONG J J, et al. Studies on reproductive character of mule duck[J]. Fujian Journal of Agricultural Sciences, 1998, 13(2): 41-45. (in Chinese with English abstract)

[5]	PENG Y D, CHANG L, WANG Y Q, et al. Genome-wide differential expression of long noncoding RNAs and mRNAs in ovarian follicles of two different chicken breeds[J]. Genomics, 2019, 111(6): 1395-1403. DOI: 10.1016/j.ygeno.2018.09.012 doi: 10.1016/j.ygeno.2018.09.012

[6]	AYERS K L, LAMBETH L S, DAVIDSON N M, et al. Identification of candidate gonadal sex differentiation genes in the chicken embryo using RNA-seq[J]. BMC Genomics, 2015, 16: 704. DOI: 10.1186/s12864-015-1886-5 doi: 10.1186/s12864-015-1886-5

[7]	REN J D, DU X, ZENG T, et al. Divergently expressed gene identification and interaction prediction of long noncoding RNA and mRNA involved in duck reproduction[J]. Animal Reproduction Science, 2017, 185: 8-17. DOI: 10.1016/j.anireprosci.2017.07.012 doi: 10.1016/j.anireprosci.2017.07.012

[8]	李丽,缪中纬,辛清武,等.半番鸭与番鸭精巢组织差异表达转录组测序分析[J].中国农业科学,2017,50(18):3608-3619. DOI:10.3864/j.issn.0578-1752.2017.18.016 LI L, MIAO Z W, XIN Q W, et al. Transcriptome analysis of differential gene expression associated with testis tissue in mule duck and muscovy duck[J]. Scientia Agricultura Sinica, 2017, 50(18): 3608-3619. (in Chinese with English abstract) doi: 10.3864/j.issn.0578-1752.2017.18.016

[9]	LI L, ZHANG L L, ZHANG Z H, et al. Comparative transcriptome and histomorphology analysis of testis tissues from mulard and Pekin ducks[J]. Archives Animal Breeding, 2020, 63(2): 303-313. DOI: 10.5194/aab-63-303-2020 doi: 10.5194/aab-63-303-2020

[10]	JIANG J Y, CHEN C, CHENG S Z, et al. Long noncoding RNA LncPGCR mediated by TCF7L2 regulates primordial germ cell formation in chickens[J]. Animals, 2021, 11(2): 292. DOI: 10.3390/ani11020292 doi: 10.3390/ani11020292

[11]	WU Y, XIAO H W, PI J S, et al. LncRNA lnc_13814 promotes the cells apoptosis in granulosa cells of duck by acting as apla-miR-145-4 sponge[J]. Cell Cycle, 2021, 20(9): 927-942. DOI: 10.1080/15384101.2021.1911102 doi: 10.1080/15384101.2021.1911102

[12]	ZUO Q H, JIN J, JIN K, et al. P53 and H3K4me2 activate N6-methylated LncPGCAT-1 to regulate primordial germ cell formation via MAPK signaling[J]. Journal of Cellular Physiology, 2020, 235(12): 9895-9909. DOI: 10.1002/jcp.29805 doi: 10.1002/jcp.29805

[13]	GAO W, ZHANG C, JIN K, et al. Analysis of lncRNA expression profile during the formation of male germ cells in chickens[J]. Animals, 2020, 10(10): 1850. DOI: 10.3390/ani10101850 doi: 10.3390/ani10101850

[14]	LI D, JI Y, WANG F, et al. Regulation of crucial lncRNAs in differentiation of chicken embryonic stem cells to sperma-togonia stem cells[J]. Animal Genetics, 2017, 48(2): 191-204. DOI: 10.1111/age.12510 doi: 10.1111/age.12510

[15]	LI X H, WANG Z, JIANG Z M, et al. Regulation of seminiferous tubule-associated stem Leydig cells in adult rat testes[J]. PNAS, 2016, 113(10): 2666-2671. DOI: 10.1073/pnas.1519395113 doi: 10.1073/pnas.1519395113

[16]	LO K C, LEI Z M, RAO C V, et al. De novo testosterone production in luteinizing hormone receptor knockout mice after transplantation of Leydig stem cells[J]. Endocrinology, 2004, 145(9): 4011-4015. DOI: 10.1210/en.2003-1729 doi: 10.1210/en.2003-1729

[17]	ZIRKIN B R， PAPADOPOULOS V. Leydig cells: formation, function, and regulation[J]. Biology of Reproduction, 2018, 99(1): 101-111. DOI: 10.1093/biolre/ioy059 doi: 10.1093/biolre/ioy059

[18]	ROBIC A, FARAUT T, PRUNIER A. Pathways and genes involved in steroid hormone metabolism in male pigs: a review and update[J]. The Journal of Steroid Biochemistry and Molecular Biology, 2014, 140: 44-55. DOI: 10.1016/j.jsbmb.2013.11.001 doi: 10.1016/j.jsbmb.2013.11.001

[19]	CHEN M Y, YANG W J, LIU N, et al. Pig Hsdl7b3: alternative splice variants expression, insertion/deletion (indel) in promoter region and their associations with male repro-ductive traits[J]. The Journal of Steroid Biochemistry and Molecular Biology, 2019, 195: 105483. DOI: 10.1016/j.jsbmb.2019.105483 doi: 10.1016/j.jsbmb.2019.105483

[20]	MENDONCA B B, GOMES N L, COSTA E M F, et al. 46, XY disorder of sex development (DSD) due to 17β-hydroxy-steroid dehydrogenase type 3 deficiency[J]. The Journal of Steroid Biochemistry and Molecular Biology, 2017, 165(Pt A): 79-85. DOI: 10.1016/j.jsbmb.2016.05.002 doi: 10.1016/j.jsbmb.2016.05.002

[21]	RHOUMA B B, KALLABI F, MAHFOUDH N, et al. Novel cases of Tunisian patients with mutations in the gene encoding 17β-hydroxysteroid dehydrogenase type 3 and a founder effect[J]. The Journal of Steroid Biochemistry and Molecular Biology, 2017, 165(Pt A): 86-94. DOI: 10.1016/j.jsbmb.2016.03.007 doi: 10.1016/j.jsbmb.2016.03.007

[22]	ENGELI R T, RHOUMA B B, SAGER C P, et al. Biochemical analyses and molecular modeling explain the functional loss of 17β-hydroxysteroid dehydrogenase 3 mutant G133R in three Tunisian patients with 46, XY disorders of sex development[J]. The Journal of Steroid Biochemistry and Molecular Biology, 2016, 155(Pt A): 147-154. DOI: 10.1016/j.jsbmb.2015.10.023 doi: 10.1016/j.jsbmb.2015.10.023

[23]	NURLIANI A, SASAKI M, BUDIPITOJO T, et al. An immunohistochemical study on testicular steroidogenesis in the Sunda porcupine (Hystrix javanica)[J]. Journal of Veterinary Medical Science, 2019, 81(9): 1285-1290. DOI: 10.1292/jvms.19-0167 doi: 10.1292/jvms.19-0167

[24]	MENG L H, YU H Y, NI F F, et al. Roles of two cyp11 genes in sex hormone biosynthesis in Japanese flounder (Paralichthys olivaceus)[J]. Molecular Reproduction and Development, 2020, 87(1): 53-65. DOI: 10.1002/mrd.23301 doi: 10.1002/mrd.23301

[25]	LIANG D D, FAN Z F, ZOU Y X, et al. Characteristics of Cyp11a during gonad differentiation of the olive flounder Paralichthys olivaceus [J]. International Journal of Molecular Sciences, 2018, 19(9): 2641. DOI: 10.3390/ijms19092641 doi: 10.3390/ijms19092641

[26]	KASHEF J, DIANA T, OELGESCHLÄGER M, et al. Expression of the tetraspanin family members Tspan3, Tspan4, Tspan5 and Tspan7 during Xenopus laevis embryonic develop-ment[J]. Gene Expression Patterns, 2013, 13(1/2): 1-11. DOI: 10.1016/j.gep.2012.08.001 doi: 10.1016/j.gep.2012.08.001

[27]	RENAULT L, PATIÑO L C, MAGNIN F, et al. BMPR1A and BMPR1B missense mutations cause primary ovarian insufficiency[J]. The Journal of Clinical Endocrinology & Metabolism, 2020, 105(4): dgz226. DOI: 10.1210/clinem/dgz226 doi: 10.1210/clinem/dgz226

[28]	EDSON M A, NALAM R L, CLEMENTI C, et al. Granulosa cell-expressed BMPR1A and BMPR1B have unique functions in regulating fertility but act redundantly to suppress ovarian tumor development[J]. Molecular Endocrinology, 2010, 24(6): 1251-1266. DOI: 10.1210/me.2009-0461 doi: 10.1210/me.2009-0461

[29]	AHMED K, LAPIERRE M P, GASSER E, et al. Loss of microRNA-7a2 induces hypogonadotropic hypogonadism and infertility[J]. Journal of Clinical Investigation, 2017, 127(3): 1061-1074. DOI: 10.1172/JCI90031 doi: 10.1172/JCI90031

[30]	ZHANG H, YU J Q, YANG L L, et al. Identification of genome-wide SNP-SNP interactions associated with important traits in chicken[J]. BMC Genomics, 2017, 18: 892. DOI: 10.1186/s12864-017-4252-y doi: 10.1186/s12864-017-4252-y

[31]	SCHINDLER K, SCHULTZ R M. CDC14B acts through FZR1 (CDH1) to prevent meiotic maturation of mouse oocytes[J]. Biology of Reproduction, 2009, 80(4): 795-803. DOI: 10.1095/biolreprod.108.074906 doi: 10.1095/biolreprod.108.074906

[32]	CHÁVEZ B, RAMOS L, GÓMEZ R, et al. 46, XY disorder of sexual development resulting from a novel monoallelic mutation (p.Ser31Phe) in the steroid 5α‐reductase type‐2 (SRD5A2) gene[J]. Molecular Genetics & Genomic Medicine, 2014, 2(4): 292-296. DOI: 10.1002/mgg3.76 doi: 10.1002/mgg3.76

[33]	ITTIWUT C, PRATUANGDEJKUL J, SUPORNSILCHAI V, et al. Novel mutations of the SRD5A2 and AR genes in Thai patients with 46, XY disorders of sex development[J]. Journal of Pediatric Endocrinology & Metabolism, 2017, 30(1): 19-26. DOI: 10.1515/jpem-2016-0048 doi: 10.1515/jpem-2016-0048

[34]	KUMAR A, SHARMA R, FARUQ M, et al. Spectrum of pathogenic variants in SRD5A2 in Indian children with 46, XY disorders of sex development and clinically suspected steroid 5α-reductase 2 deficiency[J]. Sexual Development, 2019, 13(5/6): 228-239. DOI: 10.1159/000509812 doi: 10.1159/000509812

[35]	ATA A, ÖZEN S, ONAY H, et al. A large cohort of disorders of sex development and their genetic characteristics: 6 novel mutations in known genes[J]. European Journal of Medical Genetics, 2021, 64(3): 104154. DOI: 10.1016/j.ejmg.2021.104154 doi: 10.1016/j.ejmg.2021.104154

[36]	ZHANG Y H, CUI Y, ZHANG X L, et al. Pig StAR: mRNA expression and alternative splicing in testis and Leydig cells, and association analyses with testicular morphology traits[J]. Theriogenology, 2018, 118: 46-56. DOI: 10.1016/j.theriogenology.2018.05.031 doi: 10.1016/j.theriogenology.2018.05.031

[37]	ESTIENNE A, REVERCHON M, PARTYKA A, et al. Chemerin impairs in vitro testosterone production, sperm motility, and fertility in chicken: possible involvement of its receptor CMKLR1[J]. Cells, 2020, 9(7): 1599. DOI: 10.3390/cells9071599 doi: 10.3390/cells9071599

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