- Background: Follicle selection in chickens refers to the process of selecting one follicle from a group of small yellow follicles (SY, 6 – 8 mm in diameter) for development into 12 – 15 mm hierarchical follicles (usually F6 follicles), which is an important process affecting laying performance in the poultry industry.
- In this study, we compared the proteomes and transcriptomes of SY and F6 follicles in laying hens and identified several genes involved in chicken follicle selection..
- Among the identified DEGs and DEPs, changes in the expression of seven genes, including VLDLR1, WIF1, NGFR, AMH, BMP15, GDF6 and MMP13, and nine proteins, including VLDLR, VTG1, VTG3, PSCA, APOB, APOV1, F10, ZP2 and ZP3L2, were validated.
- Conclusions: By comparing the proteomes and transcriptomes of SY and F6 follicles in laying hens, we identified several differentially expressed proteins/genes that might play certain roles in chicken follicle selection.
- These data may contribute to the identification of functional genes and proteins involved in chicken follicle selection..
- The ovary is a dynamic organ and a pivotal component of the reproductive system in hens.
- In the abdomen of laying hens, ovarian follicles of various sizes exist, in- cluding small white follicles that are less than 3 mm in.
- In the process of chicken.
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- Changes in the transcripts involved in steroidogenesis, paracrine signaling and transcription during the early stage of follicular growth and development were identi- fied by transcriptome analysis [7].
- Comparison among the transcriptomes of small white, F1 and postovulatory chicken follicles identified differentially expressed genes that are involved in the adhesion, apoptosis and steroid biosynthesis pathways [8].
- However, the dynamics of the transcrip- tome during chicken follicle development from the SY follicle to the F6 follicle are unclear..
- In addition, the mRNA abundance may not accurately predict the quantities of the corresponding functional proteins, while a proteomic approach can provide a sys- temic overview of protein levels [15].
- Proteomic analyses of ovarian function [17] and maturation of oocytes [18], such as polycystic ovarian syndrome (PCOS) and cancer [19], in human ovarian diseases and early embryonic develop- ment [20–22] in mammals were reported, while in chicken, 2889 proteins were identified in the white yolk and ovarian stroma of small white follicles in Bovan’s white laying hen [23].
- However, the temporal changes in the proteome during chicken follicle selection are un- known.
- In this study, we compared the proteomes and transcriptomes of 6–8 mm SY follicles and the smallest hierarchical follicles (F6) in laying hens and found sev- eral differentially expressed genes/proteins (DEGs/DEPs) that might play certain roles in chicken follicle selection..
- RNA-seq was used to compare the transcriptomes of three SY follicles and three of the smallest hierarchical follicles (F6), which are referred to here as S1, S2, and S3 and F1, F2 and F3, respectively.
- RNA-seq generated 61.66 Gb of clean data from the six samples of chicken follicles, and of the reads could be mapped to the chicken genome.
- For all six samples, at least 93.55% of the reads were equal to or exceeded Q30 (Table 1)..
- Detailed ana- lysis of the top 10 up−/downregulated DEGs is shown in Table 2.
- The GO functional analysis revealed that most of the DEGs were involved in circulatory system processes, cell differentiation and transition metal ion binding (Fig.
- KEGG pathway analysis of the DEGs showed that the most enriched pathways were those involved in TGF-β signaling, tyrosine me- tabolism and cytokine-cytokine receptor interactions (Fig.
- The proteins from three SY follicles and three of the smallest hierarchical follicles (F6) that were used for the above transcriptome analysis, i.e., the S1, S2, and S3 and F1, F2 and F3 follicles, were used for TMT labeling and HPLC fractionation followed by LC-MS/MS analysis..
- The distribu- tion of the mass error was close to zero, and most of the absolute values were less than 5 ppm, which meant that the mass accuracy of the MS data was compliant with.
- Table 1 Summary of the RNA-seq metrics for chicken follicles.
- In this study, a total of 5883 proteins were identified in the samples, and 5236 proteins were quantified.
- In the F6 follicles, the levels of 175 and 84 pro- teins were significantly increased and decreased, respect- ively, compared with those in the SY follicles.
- A detailed analysis of the top 10 up−/downregulated differentially expressed proteins (DEPs) is shown in Table 3.
- d KEGG signaling pathway enrichment analysis of DEGs.
- The pathways of the DEPs were constructed using KEGG software.
- Several important pathways were enriched in the F6 follicles compared with the SY folli- cles (Fig.
- 3d), including pathways involved in the ribo- some, neuroactive ligand-receptor interactions and cytokine-cytokine receptor interactions..
- 4) showed that the relative abundances of the peptides from the nine selected individual proteins were consistent with the proteome data..
- The association analysis of the proteomic and transcrip- tomic data of the F6 and SY follicles revealed a weak re- lationship between protein and mRNA expression with a Table 2 The top 10 up- and downregulated genes of chicken F6 vs SY follicles.
- The number of items for which “Transcript up and Protein up” and “Transcript down and Protein down” was 14 (1.3%) and 26 (2.4.
- In addition, the expression of two genes were inconsistent in terms of changes in the mRNA levels and protein levels.
- therefore, we further analyzed the expression of VLDLR mRNA in chicken tissues and found that it was predominantly expressed in the ovary (Fig.
- 6a), and VLDLR expression in the prehierarchical follicles was significantly higher than that in the hierarchical follicles (P <.
- In both the hierarchical and prehierarchical follicles, the VLDLR mRNA expression was significantly higher in the granulosa cells (GCs) than in the thecal cells (TCs) (P <.
- Follicle-stimulating hormone (FSH) treat- ment stimulated the expression of VLDLR in the GCs, in prehierarchical follicles, the effect was not significant at concentrations of ≤10 ng/ml (Fig.
- However, FSH treatment stimulated the expression of VLDLR in the.
- 3 TMT analysis of the DEP data for the chicken F6 and SY follicles.
- b The length distribution of the majority of the peptides.
- c Volcano plots of -log 10 (P value) versus log 2 (expression level) in the F6 vs SY follicles.
- d KEGG signal pathway enrichment analysis of the DEPs.
- Follicle selection is an important stage of follicle devel- opment that affects many egg production traits in the poultry industry.
- selection and the changes in the RNA N6-methyladenosine methylation profile [27] during chicken follicle selection;.
- however, high-throughput screening of functional genes at the protein level involved in chicken follicle selection is lacking.
- Therefore, in this study, by combined analysis of changes in the transcriptomic and proteomic profiles of fol- licles prior to and post selection in chickens, we revealed several DEGs and DEPs, including VLDLR, that may play important roles in chicken follicle selection..
- Table 4 Nine proteins selected for the parallel reaction monitoring analysis of the chicken follicle proteome data.
- During chicken follicle selection, VLDLR plays a pivotal role in the absorption of vitelin by oocytes, and without VLDLR, oocytes are unable to enter the rapid growth stage of follicle development [28].
- During the de- velopment of the small white follicle, VLDLR migrates to the follicular wall, enabling the endocytosis of vitello- genin into the yolk, followed by follicular differentiation [7].
- Studies have revealed that the expressed variant of VLDLR in chicken granulosa cells differs from the vari- ant expressed in the oocyte, which contains an O-linked sugar domain (VLDLR and the reduced level of VLDLR in granulosa cells is suggested to allow more VLDLR to reach the oocytes by passing through intercel- lular gaps rather than via receptor-mediated endocytosis into granulosa cells [31].
- found that VLDLR is mainly expressed in chicken ovar- ies, SW and SY follicles and the GCs of prehierarchical follicles, and its expression was stimulated by FSH in the GCs, especially in those of hierarchical follicles.
- Similarly, in geese, the expression of VLDLR mRNA is decreased concomitant with an increase in the follicular diameter [32].
- 4 The histogram of the nine significantly abundant proteins in F6 follicles (F) vs SY follicles (S) according to PRM (P <.
- The function of NGFR and WIF1 in chicken follicle selection requires further investigation..
- Hundreds of DEGs were identified by transcriptome comparison between SY and F6 follicles during chicken follicle selection.
- AMH is mainly expressed in granulosa cells of 1–5 mm follicles in the early stage of follicular development [1].
- Among the 259 DEPs identified by global proteome analysis of chicken SY and F6 follicles, the following pro- teins may play certain roles in chicken follicle selection..
- The increased expres- sion of APOV1 and APOB in F6 follicles is consistent with higher yolk incorporation after follicle selection..
- Vitellogenins are yolk precursor proteins produced by the liver and are essential for the growth of chicken ovarian follicles, and they circulate in the bloodstream until a follicle enters the stage of vitellogenesis, which triggers endocytosis of vitellogenins and transport into the yolk [6].
- In this study, we found that the expression levels of ZP2 and ZP3 were decreased to ap- proximately half of the level found in F6 follicles.
- These data may contrib- ute to the identification of the functional genes and pro- teins involved in chicken follicular development and selection..
- 6 Dynamics of the expression of chicken VLDLR mRNA and the effect of follicle-stimulating hormone (FSH) treatment on the VLDLR mRNA levels in the granulosa cells (GCs) of chicken ovarian follicles.
- c Expression of chicken VLDLR in the granulosa cells (pre-GCs) and theca cells (pre-TCs) of prehierarchical follicles and the GCs and TCs of hierarchical follicles.
- d Effect of FSH on VLDLR in the GCs of prehierarchical follicles.
- e Effect of FSH on VLDLR in the GCs of hierarchical follicles.
- Vaccination was performed according to the recommen- dations from Hy-line International.
- From each hen, small yellow follicles (6–8 mm in diameter, SY) and smallest hierarchical follicle (12–15 mm in diameter, F6) were separately collected, and the egg yolk was carefully squeezed out with twee- zers, washed with phosphate-buffered saline (Thermo Fi- scher Scientific, MA, USA), immediately frozen in liquid nitrogen and used for transcriptomic and proteomic analyses.
- This study was performed according to the Guidelines for Experimental Animals of the Ministry of Science and Technology of China.
- The index of the refer- ence genome was built and the paired-end clean reads were aligned to the Gallus gallus genome (ftp://ftp.ncbi..
- Differentially expressed genes (DEGs) between SY and F6 follicles were identified according to the cretiera of ad- justed P-value <.
- Gene ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes database (KEGG) pathway enrichment analysis of the DEGs were implemented by the cluster Profiler R package, and genes with P-values less than 0.05 were considered significantly enriched as differentially expressed [43].
- The proteins in the tissues were extracted with lysis buf- fer containing 8 M urea (Sigma Aldrich, MO, USA) and 1% protease inhibitor cocktail (Merck Millipore, MA, USA), their concentration was determined with a BCA kit (Beyotime, Shanghai, China) according to the manu- facturer’s instructions.
- Plus spectrometer (Thermo Fischer Scientific, MA, USA) coupled online to the UPLC system..
- The MS proteomics data have been de- posited in the ProteomeXchange Consortium (http://.
- Synthesis of the cDNA was performed using a PrimeScript RT reagent kit with 1 μg of the RNA pretreated with gDNA Eraser (TaKaRa, Dalian, China) according to the manufacturer’s protocol..
- Real-time quantitative PCR of the mRNA expression level of VLDLR, VLDLR1, WIF1, NGFR, AMH, BMP15, GDF6 and MMP13 was performed using a SYBR Premix Ex Taq™ II kit (TaKaRa, Dalian, China) with primers listed in Table S5 on a Light Cycler 480 real-time PCR system (Roche, Basel, Switzerland) as follows: 95 °C for 30 s, followed by 40 cycles of denaturation at 95 °C for 10 s and annealing and extension at 58 °C for 20 s.
- The melting curves were obtained, and quantitative analysis of the data was performed using the 2 −ΔΔ CT relative quantification method [46].
- The primers used in the experiments.
- The funding body had no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript..
- org/proteomes/ UP000000539, chicken proteome ID: UP000000539) database of the Gallus gallus were used in this study.
- The MS proteomics data have been deposited in the ProteomeXchange Consortium (http://www.proteomex change.org/) via the PRIDE partner repository with the dataset identifier PXD011470 (Username:
[email protected], Password: AfwLJZRb)..
- The animal experiments were carried out in accordance with the protocols of the ‘ Guidelines for Experimental Animals ’ of the Ministry of Science and Technology (Beijing, China) and all efforts were made to minimize suffering..
- Xiaoyan Yang who was the owner of the Hy-line brown hens..
- Ovarian follicle selection and granulosa cell differentiation..
- Transcriptome analysis on single small yellow follicles reveals that WNT4 is involved in chicken follicle selection.
- Bone morphogenetic protein 15 may promote follicle selection in the hen.
- Protein expression profile of the mouse metaphase-ii oocyte..
- Proteomic analysis of mouse oocytes reveals 28 candidate factors of the "reprogrammome".
- Follicle selection in the avian ovary.
- Profiling of RNA N6-methyladenosine methylation during follicle selection in chicken ovary.
- Signaling by the extracellular matrix protein reelin promotes granulosa cell proliferation in the chicken follicle.
- Basic fibroblast growth factor promotes prehierarchical follicle growth and yolk deposition in the chicken..
- Proteomic analysis of the chicken egg vitelline membrane..
- Changes in the sperm-zona pellucida binding properties during porcine oocyte maturation..
- Comparative analysis of the transcriptome and proteome during mouse placental development