鸭睾丸单细胞发育图谱构建与调控分析

doi:10.3864/j.issn.0578-1752.2026.02.016

摘要/Abstract

摘要：

【目的】 通过单细胞测序技术解析鸭睾丸发育成熟前后细胞异质性，构建鸭睾丸单细胞发育图谱，并进行比较分析，进一步揭示睾丸发育成熟过程和调控机制。【方法】 分别收集10周龄（未成熟睾丸组，IMT）和23周龄（成熟睾丸组，MT）鸭睾丸3份，制备单细胞悬液，进行单细胞测序。对获得的测序数据进行质控、标准化、降维和聚类分析，构建鸭不同发育时期睾丸图谱。对细胞亚群进行注释，并分析组间差异，筛选关键调控基因和信号通路。【结果】 6个样本平均获得超过30 000 000高质量测序数据，经质控获得54 702个有效细胞（IMT组27 756个，MT组26 946个）。通过细胞聚类和标记基因鉴定，成功鉴定21个细胞亚群，包含10个生殖细胞亚群和11个体细胞亚群。随着鸭睾丸发育成熟，精原干细胞、精原细胞、初级精母细胞、支持细胞1型和3型显著降低，中间型精母细胞、次级精母细胞、圆形精子细胞、长形精子细胞、支持细胞2型和4型显著增加。差异基因分析发现4 495个差异基因（上调1 737个，下调2 758个），其中生殖细胞中Gm614、SPATC1、TSSK6、ZIC1、PHF7、Armc12和 FAM166C等显著上调；在支持细胞中PKIA、SNX22、Wnt6、LAMA1、SCN4B、CDH6、FAM110C等显著下调。功能富集分析表明，TOP20 GO均属于细胞组分类别；KEGG分析提示内质网中的蛋白质加工和细胞衰老等信号通路具有关键调控作用；进一步对这两个信号通路中的差异基因进行蛋白互作网络分析显示，SEC63通过与ERLEC1、AMFR和RAD23B等相互作用调节内质网中的蛋白质加工过程，而CDK6通过CHEK2、CCNA2、CCNA1和CDC25等相互作用调节细胞衰老信号通路。【结论】 首次构建了鸭睾丸发育成熟前后单细胞转录图谱，揭示睾丸发育成熟过程中细胞亚群动态变化规律，发现了大量差异表达基因，且提示SEC63和CDK6可能分别通过调控内质网中蛋白质加工和细胞周期进程在睾丸成熟机制中发挥重要作用。

关键词: 鸭, 睾丸, 单细胞测序, 图谱构建, 调控分析

Abstract:

【Objective】 The aim of this study was to analyze the cellular heterogeneity of duck testes before and after maturation using single-cell sequencing technology, to construct a developmental map of duck testicular cells, and to further reveal the process and regulatory mechanisms of testicular maturation. 【Method】 Each three testes samples from 10 week (immature testis group, IMT) and 23 week old (mature testis group, MT) ducks were collected, and single-cell suspensions were prepared for single-cell sequencing. The quality control, standardization, dimensionality reduction, and cluster analysis on the obtained sequencing data were performed to construct testicular maps of ducks at different developmental stages. Cell subpopulations were annotated, intergroup differences were analyzed, and key regulatory genes and signaling pathways of testicular development and maturation were screened. 【Result】 More than 30 million high-quality sequencing data were obtained from the each six samples. After quality control, 54 702 valid cells were obtained (27 756 in the IMT group and 26 946 in the MT group). Through cell clustering and identification of marker genes, 21 cell sub-populations were successfully identified, including 10 germ cell sub-populations and 11 somatic cell sub- populations. During the development and maturation of duck testes, spermatogonial stem cells, spermatogonia, primary spermatocytes, and Sertoli cells type 1 and type 3 decreased significantly, while intermediate spermatocytes, secondary spermatocytes, round spermatids, elongated spermatids, and Sertoli cells type 2 and type 4 increased significantly. Differential gene analysis identified 4 495 differential genes (1 737 up-regulated and 2 758 down-regulated). Among them, genes such as Gm614, SPATC1, TSSK6, ZIC1, PHF7, Armc12 and FAM166C were significantly up-regulated in germ cells, and genes such as PKIA, SNX22, Wnt6, LAMA1, SCN4B, CDH6 and FAM110C were significantly down-regulated in Sertoli cells. Functional enrichment analysis showed that the top 20 GO terms all belonged to the cell component category. KEGG analysis indicated that signaling pathways such as protein processing in the endoplasmic reticulum and cellular senescence played key regulatory roles. Furthermore, protein-protein interaction network analysis of differentially expressed genes of these two signaling pathways showed that SEC63 regulated the protein processing process in the endoplasmic reticulum through interactions with ERLEC1, AMFR and RAD23B, etc., while CDK6 might regulate the cellular senescence signaling pathway through interactions with CHEK2, CCNA2, CCNA1 and CDC25, etc. 【Conclusion】 This study constructed the single-cell transcription map of duck testes before and after maturity for the first time, revealed the dynamic change rules of cell sub-populations during testicular development, discovered a large number of differentially expressed genes, and suggested that SEC63 and CDK6 might play important roles in the testicular maturation mechanism by regulating the endoplasmic reticulum stress response and cell cycle progression, respectively.

Key words: duck, testis, single-cell sequencing, atlas construction, regulatory analysis

陶志云, 徐文娟, 卢立志, 宋卫涛, 章双杰, 刘宏祥, 王志成, 顾昊天, 朱春红, 李慧芳. 鸭睾丸单细胞发育图谱构建与调控分析[J]. 中国农业科学, 2026, 59(2): 441-458.

TAO ZhiYun, XU WenJuan, LU LiZhi, SONG WeiTao, ZHANG ShuangJie, LIU HongXiang, WANG ZhiCheng, GU HaoTian, ZHU ChunHong, LI HuiFang. Atlas Construction and Regulatory Analysis of Duck Testicular Cell Development[J]. Scientia Agricultura Sinica, 2026, 59(2): 441-458.

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不同发育时期鸭睾丸细胞TOP20差异基因列表"

基因ID Gene ID	基因名称 Gene name	差异倍数 log₂FC	描述 Description	功能 Function
ncbi_113844722	Gm614	11.98	非典型蛋白CXorf65同源异构体X2 Uncharacterized protein CXorf65 homolog isoform X2	-
ncbi_119716153	SPATC1	11.15	精子发生和中心体相关1 Spermatogenesis and centriole associated 1	.预测能够实现r-微管蛋白结合活性 It is predicted to be capable of r-tubulin binding activity
ncbi_106017448	ncbi_106017448	10.38	-	-
ncbi_113844195	TSSK6	10.34	睾丸特异性丝氨酸/苏氨酸蛋白激酶6 Ttestis-specific serine/threonine-protein kinase 6	与雄性不育和精子形态异常有关 It is associated with male sterility and abnormal sperm morphology
ncbi-101805470	ZIC1	10.14	锌指蛋白ZIC 1 Zinc finger protein ZIC 1	在中枢神经系统器官发生的早期阶段以及脊髓背侧发育和小脑成熟过程中起着重要作用 It plays an important role in the early stages of central nervous system organogenesis, as well as in dorsal spinal cord development and cerebellar maturation
ncbi_101798232	PHF7	9.98	PHD手指蛋白7 PHD finger protein 7	在支持细胞中表达，与精子发生的转录调控有关 It is expressed in Sertoli cells and is associated with the transcriptional regulation of spermatogenesis
ncbi_113839899	Armc12	9.98	含犰狳重复序列蛋白12异构体X2 Armadillo repeat-containing protein 12 isoform X2	参与细胞生长和精子线粒体鞘组装的正向调节，与生精失败有关 It is involved in the positive regulation of cell growth and sperm mitochondrial sheath assembly, and is associated with spermatogenic failure
ncbi_101794437	ncbi_101794437	9.9	-	-
ncbi_119716622	ncbi_119716622	9.77	-	-
ncbi_101789599	FAM166C	9.74	UPF0573蛋白C2orf70同源物 UPF0573 protein C2orf70 homolog	位于轴丝微管内，预测与有鞭毛的精子运动有关 It is located within the axonemal microtubules and predicted to be associated with flagellated sperm motility
ncbi_101800754	PKIA	-5.11	cAMP依赖性蛋白激酶抑制剂α cAMP-dependent protein kinase inhibitor alpha	与PKA的Cα和Cβ催化亚基相互作用并抑制其活性 It interacts with and inhibits the activity of the Cα and Cβ catalytic subunits of PKA
ncbi_101799487	SNX22	-5.16	排序连接蛋白22 Sorting nexin-22	可能在细胞内运输中发挥作用 It may play a role in intracellular transport
ncbi_101803438	Wnt6	-5.84	蛋白Wnt-6亚型X1 Protein Wnt-6 isoform X1	参与控制未分化精原干细胞的增殖 It is involved in controlling the proliferation of undifferentiated spermatogonial stem cells
ncbi_106016481	ncbi_106016481	-5.87	-	-
ncbi_101791570	LAMA1	-6.19	层粘连蛋白亚基α-1 Llaminin subunit alpha-1	通过高亲和力受体与细胞结合，被认为通过与其他细胞外基质成分相互作用，在胚胎发育过程中介导细胞附着、迁移和组织到组织中 It binds to cells via high-affinity receptors and is thought to mediate cell attachment, migration, and organization into tissues during embryonic development by interacting with other extracellular matrix components
ncbi_101795249	SCN4B	-6.67	X1钠通道亚基β-4亚型X1 Sodium channel subunit beta-4 isoform	调节通道门控动力学 It regulates channel gating dynamics
ncbi_113843439JIGNY	ncbi_113843439	-6.96	未鉴定蛋白LOC113843439 Uncharacterized protein LOC113843439	-
ncbi_119715879	CDH6	-6.99	钙粘蛋白-6 Cadherin-6	在细胞分化和形态发生中起关键作用 It plays a critical role in cell differentiation and morphogenesis
ncbi_113845450	ncbi_113845450	-7.37	-	-
ncbi_101796927	FAM110C	-7.96	FAM110C蛋白 Protein FAM110C	激活a-微管蛋白活性。参与细胞迁移的正向调控 It activates α-tubulin activity and participates in the positive regulation of cell migration

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样本Sample	Reads数 Number of Reads	有效 Barcodes Valid Barcodes (%)	测序饱和度 Sequencing Saturation (%)	用于比对的Barcode序列中质量分数≥30的碱基百分比 Q30 Bases in Barcodes (%)	用于比对的Reads中质量分数≥30的碱基百分比 Q30 Bases in RNA Read (%)	UMI序列中质量分数≥于30的碱基百分比 Q30 Bases in UMI (%)	过滤前细胞数量 Before filter number of cells	过滤后细胞数量 After filter number of cells	检测到的总基因数 Total genes detected
IMT-1	332505029	94.20	76.30	95.70	94.60	96.90	7125	6567	21063
IMT-2	340882637	93.80	57.80	96.60	94.70	97.50	14059	12446	21483
IMT-3	336445006	94.00	67.90	96.20	94.20	97.20	9518	8743	21282
MT-1	327578469	97.20	44.80	96.60	91.30	96.20	11258	10109	21399
MT-2	343434190	97.30	48.20	96.80	92.50	96.40	9864	9010	21475
MT-3	300961645	96.90	47.40	96.80	92.10	96.30	8471	7827	21308

类群 Cluster	细胞类型 Cell type	未成熟睾丸组 Immature testis, IMT		成熟睾丸组 Mature testis, MT
类群 Cluster	细胞类型 Cell type	细胞数 Cell count	细胞比率 Cell ratio (%)	细胞数 Cell count	细胞比率 Cell ratio (%)
9	精原干细胞 Spermatogonial stem cell	1880	6.77	1070	3.97
0	精原细胞 Spermatogonia	4878	17.57	1599	5.93
10	初级精母细胞 Early spermatocytes	1383	4.98	549	2.04
6	中间型精母细胞 Middle spermatocytes	444	1.60	3582	13.29
2	次级精母细胞 Later spermatocytes	44	0.16	5830	21.64
15	圆形精子细胞 Round spermatids	12	0.04	339	1.26
11	长形精子细胞 Elongated spermatids	7	0.03	1497	5.56
3	未鉴定的生殖细胞1 Unidentified cell 1	2266	8.16	3199	11.87
5	未鉴定的生殖细胞2 Unidentified cell2	3652	13.16	493	1.83
7	未鉴定的生殖细胞3 Unidentified cell 3	2765	9.96	1132	4.20
8	支持细胞1型 Sertoli cell 3	4492	16.18	1832	6.80
4	支持细胞2型 Sertoli cell 2	1305	4.70	3902	14.48
1	支持细胞3型 Sertoli cell 1	2561	9.23	1157	4.29
17	支持细胞4型 Sertoli cell 4	1	0	194	0.72
12	T细胞T cell	737	2.66	58	0.22
13	巨噬细胞Macrophages	603	2.17	78	0.29
14	管周肌样细胞 Peritubular myoid cell	333	1.20	160	0.59
16	内皮细胞Endothelial cell	123	0.44	121	0.45
18	间质细胞Leydig cell	98	0.35	80	0.30
19	红细胞Red cell	107	0.39	22	0.08
20	粒细胞Granulocyte	65	0.23	52	0.19

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