Atlas Construction and Regulatory Analysis of Duck Testicular Cell Development

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.

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.

More than 30 million high-quality sequencing data were obtained from the each six samples. After quality control, 54702 valid cells were obtained (27756 in the IMT group and 26946 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 4495 differential genes (1737 up-regulated and 2758 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.

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.