Single-nucleus transcriptional and chromatin accessible profiles reveal critical cell types and molecular architecture underlying chicken sex determination.

INTRODUCTION: Understanding the sex determination mechanisms in birds has great significance for the biological sciences and production in the poultry industry. Sex determination in chickens is a complex process that involves fate decisions of supporting cells such as granulosa or Sertoli cells. However, a systematic understanding of the genetic regulation and cell commitment process underlying sex determination in chickens is still lacking. OBJECTIVES: We aimed to dissect the molecular characteristics associated with sex determination in the gonads of chicken embryos. METHODS: Single-nucleus RNA-seq (snRNA-seq) and ATAC-seq (snATAC-seq) analysis were conducted on the gonads of female and male chickens at embryonic day 3.5 (E3.5), E4.5, and E5.5. RESULTS: Here, we provided a time-course transcriptional and chromatin accessible profiling of gonads during chicken sex determination at single-cell resolution. We uncovered differences in cell composition and developmental trajectories between female and male gonads and found that the divergence of transcription and accessibility in gonadal cells first emerged at E5.5. Furthermore, we revealed key cell-type-specific transcription factors (TFs) and regulatory networks that drive lineage commitment. Sex determination signaling pathways, dominated by BMP signaling, are preferentially activated in males during gonadal development. Further pseudotime analysis of the supporting cells indicated that granulosa cells were regulated mainly by the TEAD gene family and that Sertoli cells were driven by the DMRT1 regulons. Cross-species analysis suggested high conservation of both cell types and cell-lineage-specific TFs across the six vertebrates. CONCLUSIONS: Overall, our study will contribute to accelerating the development of sex manipulation technology in the poultry industry and the application of chickens as a unique model for studying cell fate decisions.

Temperature-induced embryonic diapause in chickens is mediated by PKC-NF-κB-IRF1 signaling.

BACKGROUND: Embryonic diapause (dormancy) is a state of temporary arrest of embryonic development that is triggered by unfavorable conditions and serves as an evolutionary strategy to ensure reproductive survival. Unlike maternally-controlled embryonic diapause in mammals, chicken embryonic diapause is critically dependent on the environmental temperature. However, the molecular control of diapause in avian species remains largely uncharacterized. In this study, we evaluated the dynamic transcriptomic and phosphoproteomic profiles of chicken embryos in pre-diapause, diapause, and reactivated states. RESULTS: Our data demonstrated a characteristic gene expression pattern in effects on cell survival-associated and stress response signaling pathways. Unlike mammalian diapause, mTOR signaling is not responsible for chicken diapause. However, cold stress responsive genes, such as IRF1, were identified as key regulators of diapause. Further in vitro investigation showed that cold stress-induced transcription of IRF1 was dependent on the PKC-NF-κB signaling pathway, providing a mechanism for proliferation arrest during diapause. Consistently, in vivo overexpression of IRF1 in diapause embryos blocked reactivation after restoration of developmental temperatures. CONCLUSIONS: We concluded that embryonic diapause in chicken is characterized by proliferation arrest, which is the same with other spices. However, chicken embryonic diapause is strictly correlated with the cold stress signal and mediated by PKC-NF-κB-IRF1 signaling, which distinguish chicken diapause from the mTOR based diapause in mammals.

ATAC-seq and RNA-seq analysis unravel the mechanism of sex differentiation and infertility in sex reversal chicken.

BACKGROUND: Sex determination and differentiation are complex and delicate processes. In female chickens, the process of sex differentiation is sensitive and prone to be affected by the administration of aromatase inhibitors, which result in chicken sex reversal and infertility. However, the molecular mechanisms underlying sex differentiation and infertility in chicken sex reversal remain unclear. Therefore, we established a sex-reversed chicken flock by injecting an aromatase inhibitor, fadrozole, and constructed relatively high-resolution profiles of the gene expression and chromatin accessibility of embryonic gonads. RESULTS: We revealed that fadrozole affected the transcriptional activities of several genes, such as DMRT1, SOX9, FOXL2, and CYP19A1, related to sex determination and differentiation, and the expression of a set of gonadal development-related genes, such as FGFR3 and TOX3, by regulating nearby open chromatin regions in sex-reversed chicken embryos. After sexual maturity, the sex-reversed chickens were confirmed to be infertile, and the possible causes of this infertility were further investigated. We found that the structure of the gonads and sperm were greatly deformed, and we identified several promising genes related to spermatogenesis and infertility, such as SPEF2, DNAI1, and TACR3, through RNA-seq. CONCLUSIONS: This study provides clear insights into the exploration of potential molecular basis underlying sex differentiation and infertility in sex-reversed chickens and lays a foundation for further research into the sex development of birds.

Identification of crucial genes and metabolites regulating the eggshell brownness in chicken.

BACKGROUND: Protoporphyrin IX (Pp IX) is the primary pigment for brown eggshells. However, the regulatory mechanisms directing Pp IX synthesis, transport, and genetic regulation during eggshell calcification in chickens remain obscure. In this study, we investigated the mechanism of brown eggshell formation at different times following oviposition, using White Leghorn hens (WS group), Rhode Island Red light brown eggshell line hens (LBS group) and Rhode Island Red dark brown eggshell line hens (DBS group). RESULTS: At 4, 16 and 22 h following oviposition, Pp IX concentrations in LBS and DBS groups were significantly higher in shell glands than in liver (P < 0.05). Pp IX concentrations in shell glands of LBS and DBS groups at 16 and 22 h following oviposition were significantly higher than WS group (P < 0.05). In comparative transcriptome analysis, δ-aminolevulinate synthase 1 (ALAS1), solute carrier family 25 member 38 (SLC25A38), ATP binding cassette subfamily G member 2 (ABCG2) and feline leukemia virus subgroup C cellular receptor 1 (FLVCR1), which were associated with Pp IX synthesis, were identified as differentially expressed genes (DEGs). RT-qPCR results showed that the expression level of ALAS1 in shell glands was significantly higher in DBS group than in WS group at 16 and 22 h following oviposition (P < 0.05). In addition, four single nucleotide polymorphisms (SNPs) in ALAS1 gene that were significantly associated with eggshell brownness were identified. By identifying the differential metabolites in LBS and DBS groups, we found 11-hydroxy-E4-neuroprostane in shell glands and 15-dehydro-prostaglandin E1(1-) and prostaglandin G2 2-glyceryl ester in uterine fluid were related to eggshell pigment secretion. CONCLUSIONS: In this study, the regulatory mechanisms of eggshell brownness were studied comprehensively by different eggshell color and time following oviposition. Results show that Pp IX is synthesized de novo and stored in shell gland, and ALAS1 is a key gene regulating Pp IX synthesis in the shell gland. We found three transporters in Pp IX pathway and three metabolites in shell glands and uterine fluid that may influence eggshell browning.

The m6A methylation regulates gonadal sex differentiation in chicken embryo.

BACKGROUND: As a ubiquitous reversible epigenetic RNA modification, N6-methyladenosine (m6A) plays crucial regulatory roles in multiple biological pathways. However, its functional mechanisms in sex determination and differentiation during gonadal development of chicken embryos are not clear. Therefore, we established a transcriptome-wide m6A map in the female and male chicken left gonads of embryonic day 7 (E7) by methylated RNA immunoprecipitation sequencing (MeRIP-seq) to offer insight into the landscape of m6A methylation and investigate the post-transcriptional modification underlying gonadal differentiation. RESULTS: The chicken embryonic gonadal transcriptome was extensively methylated. We found 15,191 and 16,111 m6A peaks in the female and male left gonads, respectively, which were mainly enriched in the coding sequence (CDS) and stop codon. Among these m6A peaks, we identified that 1013 and 751 were hypermethylated in females and males, respectively. These differential peaks covered 281 and 327 genes, such as BMP2, SMAD2, SOX9 and CYP19A1, which were primarily associated with development, morphogenesis and sex differentiation by functional enrichment. Further analysis revealed that the m6A methylation level was positively correlated with gene expression abundance. Furthermore, we found that YTHDC2 could regulate the expression of sex-related genes, especially HEMGN and SOX9, in male mesonephros/gonad mingle cells, which was verified by in vitro experiments, suggesting a regulatory role of m6A methylation in chicken gonad differentiation. CONCLUSIONS: This work provided a comprehensive m6A methylation profile of chicken embryonic gonads and revealed YTHDC2 as a key regulator responsible for sex differentiation. Our results contribute to a better understanding of epigenetic factors involved in chicken sex determination and differentiation and to promoting the future development of sex manipulation in poultry industry.

Uterine Metabolomic Analysis for the Regulation of Eggshell Calcification in Chickens.

Eggshell quality is economically important for table eggs and functionally indispensable for hatching eggs. During the formation of eggshell in the uterus, organic matrixes in uterine fluid can control and modify the formation of calcified eggshell. At present, there are limited studies focusing on the effect of uterine organic metabolites on eggshell quality. In this study, an LC-MS-based metabolomic technology was performed to identify the crucial uterine metabolites that differently presented in hens producing eggs with divergent eggshell quality (eggshell strength, thickness, and weight). More than 1000 metabolites were identified in uterine fluid, and six putative metabolites, including phosphatidylcholine, diacylglycerol, verapamil, risedronate, coproporphyrinogen III, and biliverdin, were screened to play crucial roles in eggshell calcification. Then, two trials for oral administration and in vitro calcite crystal growth were conducted to verify the effect of potential different metabolites on the eggshell quality. Verapamil has a temporary effect on decreasing eggshell strength and eggshell thickness. Coproporphyrinogen III could induce smaller calcite crystals to improve eggshell strength while biliverdin could modify crystal morphology by forming rougher faces and rounder edges to strengthen the eggshell. The present study gives us new insight to understand the role of uterine fluid matrixes in eggshell calcification.

Screening and validation of candidate genes involved in the regulation of egg yolk deposition in chicken.

Eggs with the same total weight may have considerable differences in yolk weight. Eggs with a high percentage of yolk have a higher nutritional value, more flavor, and are more desirable to consumers. However, a large yolk proportion means more dry matter in the eggs, which reduces the feed efficiency. The elucidation of the genetic factors of yolk quantity in eggs is of scientific and practical significance. Through RNA sequencing, we explored the transcriptome of ovarian tissue from 12 Wenchang chickens, including 6 chickens that laid eggs with a high yolk percentage (32%) and 6 that laid low yolk percentage eggs (25%). We identified a total of 362 differentially expressed genes (P-value < 0.01, log2 fold change < -1, log2 fold change > 1), of which 220 were upregulated and 142 were downregulated in high yolk percentage hens. According to the Gene Ontology terms annotation and Kyoto Encyclopedia of Genes and Genomes enrichment analysis, the differentially expressed genes were associated with the regulation of various cell functions, cell differentiation and development, neuroactive ligand-receptor interactions, and calcium and ubiquitin-mediated proteolysis signaling pathways. To further filter for genes that were directly involved in yolk accumulation, the chicken quantitative trait loci database, genes within 100 kb upstream and downstream of the yolk weight trait SNP, and intersection genes in protein-protein interaction network diagrams were used to detect genes that overlapped with the differentially expressed genes. We found 7 candidate genes in total, MNR2, AOX1, ANTXRL, GRAMD1C, EEF2, COMP, and JUND, which affect female reproductive performance and the growth and development of follicles, supporting cell transport, cell proliferation, and differentiation. All candidate genes and several randomly selected genes were verified by quantitative real time PCR, and the results were consistent with the RNA sequencing. In conclusion, investigating the molecular mechanisms of high yolk percentage traits will allow breeding strategies to be optimized to alter the percentage of yolk in chicken eggs.

Differential proteomic analysis revealed crucial egg white proteins for hatchability of chickens.

For healthy development, an avian embryo needs the nutritional and functional molecules maternally deposited in avian eggs. Egg white not only provides nutritional components but also exhibits functional properties, such as defenses against microbial invasion. However, the roles of the more detailed messages in embryo development remain unclear. In this study, a tandem mass tag labeling quantitation approach was used to innovatively identify the differential proteins in the egg whites of fresh eggs produced by hens with divergent high/low hatchability and in the egg whites of embryonated eggs with healthy and dead embryos. A total of 378 proteins were quantified in egg white, which is the most complete proteome identified for egg white to date, and up to 102 differential proteins were identified. GO enrichment, pathway, and hierarchical clustering analysis revealed some of the differential proteins that are the main participants in several biological processes, including blood coagulation, intermediate filament, antibacterial activity, and neurodevelopment. A list of 11 putative protein biomarkers, such as keratin (KRT19, KRT12, KRT15, and KRT6A), which is involved in cell architecture, and fibrinogen (fibrinogen alpha chain, fibrinogen beta chain, and fibrinogen gamma chain), which is related to blood coagulation, were ultimately screened. The current study screened egg white proteins that can predict low hatchability and embryonic death and deciphered the role of these proteins in embryonic development, which is meaningful for the comprehensive understanding of embryonic growth.