Proteo-Transcriptomic Profiles Reveal Key Regulatory Pathways and Functions of LDHA in The Ovulation of Domestic Chickens

Nie et al.
Journal of Animal Science and

Journal of Animal Science and Biotechnology (2024) 15:68
https://doi.org/10.1186/s40104-024-01019-2 Biotechnology
RESEARCH Open Access
Proteo‑transcriptomic profiles reveal key

regulatory pathways and functions of LDHA
in the ovulation of domestic chickens (Gallus
gallus)
Ruixue Nie1, Wenhui Zhang1, Haoyu Tian1, Junying Li1, Yao Ling1, Bo Zhang1, Hao Zhang1* and Changxin Wu1
Abstract
Background In poultry, the smooth transition of follicles from the preovulatory-to-postovulatory phase impacts egg
production in hens and can benefit the poultry industry. However, the regulatory mechanism underlying follicular
ovulation in avians is a complex biological process that remains unclear.
Results Critical biochemical events involved in ovulation in domestic chickens (Gallus gallus) were evaluated
by transcriptomics, proteomics, and in vitro assays. Comparative transcriptome analyses of the largest preovulatory
follicle (F1) and postovulatory follicle (POF1) in continuous laying (CL) and intermittent laying (IL) chickens indicated
the greatest difference between CL_F1 and IL_F1, with 950 differentially expressed genes (DEGs), and the smallest
difference between CL_POF1 and IL_POF1, with 14 DEGs. Additionally, data-independent acquisition proteomics
revealed 252 differentially abundant proteins between CL_F1 and IL_F1. Perivitelline membrane synthesis, steroid
biosynthesis, lysosomes, and oxidative phosphorylation were identified as pivotal pathways contributing to ovulation
regulation. In particular, the regulation of zona pellucida sperm-binding protein 3, plasminogen activator, cathepsin
A, and lactate dehydrogenase A (LDHA) was shown to be essential for ovulation. Furthermore, the inhibition of LDHA
decreased cell viability and promoted apoptosis of ovarian follicles in vitro.
Conclusions This study reveals several important biochemical events involved in the process of ovulation, as well
as crucial role of LDHA. These findings improve our understanding of ovulation and its regulatory mechanisms in avian
species.
Keywords Chicken, Data-independent acquisition proteomics, LDHA, Ovulation, Regulatory mechanism,
Transcriptome
Background
Eggs are widely produced and globally consumed as a
cost-effective and high-quality source of protein. Accord-
ing to the National Bureau of Statistics of China, total
egg production in China reached 34.56 million tons in
*Correspondence:
Hao Zhang 2022, an increase of 31.0% from that in 2008, showing a
[email protected] continuous upward trend (http://www.stats.gov.cn). This
1
State Key Laboratory of Animal Biotech Breeding, Beijing Key rapid increase in egg production is mainly attributed to a
Laboratory for Animal Genetic Improvement, College of Animal Science
and Technology, China Agricultural University, Beijing 100193, China progressive increase in the scale of rearing [1] and signifi-
cant improvements in the egg production performance
© The Author(s) 2024. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which
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Nie et al. Journal of Animal Science and Biotechnology (2024) 15:68 Page 2 of 17
of laying hens. Egg production in laying hens is affected oligomenorrhea or amenorrhea resulting from chronic
by various factors, including genetic background [2], oligo-ovulation or anovulation [25]. The anovulatory
nutrition [3], environment [4], gut microbiota [5], and phenotype of patients with PCOS is similar to that of
follicular development in the ovary [6]. Poultry folli- intermittent laying hens; therefore, understanding the
cular development is a complex process achieved via ovulation regulatory mechanism in chickens could pro-
the regulation of many paracrine and autocrine factors vide a basis for prolonging the physiological egg-laying
and dynamic patterns of gene expression [7]. Over the ability of aged laying hens, and contribute to biomedical
past two decades, numerous studies have evaluated the modeling for human PCOS research.
molecular mechanisms underlying follicle selection [8, 9]; In this study, we performed transcriptome analysis to
however, little is known about the mechanisms regulating uncover the differences between F1s and POF1s in CL
follicle ovulation in laying hens. and IL chickens. Furthermore, we employed a proteomic
The transition from preovulatory to postovulatory fol- strategy combining data-independent acquisition (DIA)
licles plays a significant role in the ovulation rate and mass spectrometry to evaluate F1s and identify biologi-
egg production performance [10, 11]. In the domestic cal changes and candidate biomarkers in CL and IL hens.
chicken (Gallus gallus), ovulation occurs in the larg- To the best of our knowledge, this is the first study to
est preovulatory follicle, F1, which releases the oocyte elucidate the dynamic expression profile and potential
by rupturing along the stigma region [12]. Ovulation is regulatory network of chicken ovarian follicle ovulation
triggered by a luteinizing hormone (LH) surge, whereby through a comparison of CL and IL chickens. Our find-
LH induces a feedback control with gonadal steroids [13, ings expand the spectrum of relevant genes and provide a
14]. More specifically, LH can promote the production deeper understanding of the ovulation process in poultry.
of progesterone by granulosa cells (GCs), with proges-
terone in turn stimulating an increase of LH release by
the pituitary gland, forming a positive feedback loop in Methods
F1 [13, 15]. After F1 rupture, the remaining tissue, named Ethics statement
the postovulatory follicle (POF), rapidly regresses via All animal experimental protocols were approved by
apoptotic and autophagic processes and does not form the Animal Care and Use Committee of China Agricul-
a corpus luteum [16]. POFs remain in the granulosa and tural University and performed in accordance with the
theca layers and can secrete prostaglandins [17]. Dif- National Research Council’s Guide for the Care and Use
ferentially expressed genes (DEGs) screened from F1s of Laboratory Animals (AW80203202-1-1).
between different ovulatory stages are involved in cell
proliferation, lipid metabolism, and inflammatory pro-
cess [18]. Although hormonal secretion rhythms have Tissue collection
been monitored during ovulation, and transcriptomes A population of approximately 300 Yellow-bearded
have been analyzed during changes in LH levels [19–21], chickens, bred from crossing Huiyang Bearded chicken
the biochemical mechanisms underlying poultry ovula- with White Leghorn chickens, was raised at the Experi-
tion remain largely unknown. mental Chicken Farm of China Agricultural University
A decline in egg production in aged laying hens is very (Beijing, China) under standard conditions and with
common in layer raising farms and predominantly related ad libitum access to food and water. The daily egg pro-
to follicle dysplasia in the aging ovary [22, 23]. Previously, duction of the 300 individuals was recorded for 35 con-
we dissected and observed many aging hens and found secutive days from the age of 45 to 49 weeks (Additional
that continuous laying (CL) hens maintained daily release file 1). Subsequently, 5 hens exhibiting high egg produc-
of one oocyte from F1 into the oviduct, forming a POF1. tion and continuous laying of eggs were selected as the
Although intermittent laying (IL) hens exhibited a rela- CL group, whereas 5 hens with low egg production that
tively complete set of preovulatory follicles (F6/F5–F1) in also laid no eggs in the last several days at 50 weeks of
the ovaries, the F1 in these hens were unable to rupture age, indicating a temporary anovulation state, were
and form a POF1. Therefore, although we speculate that selected as the IL group (Fig. 1A). The 10 experimental
ovulation regulation may be linked to F1, its underlying hens were humanely euthanized and immediately dis-
mechanism remains unclear. Polycystic ovary syndrome sected to collect the follicles. After removing the yolk of
(PCOS) is common in the clinical and public health F1s, the remaining follicle walls of F1s and POF1s were
fields, affecting up to 20% of the women of reproductive washed with phosphate-buffered saline (Gibco, Gaith-
age [24]; however, its pathophysiology is complex and ersburg, MD, USA). All samples were snap-frozen in liq-
remains largely unclear [24]. Most patients with PCOS uid nitrogen and stored at –80 °C for RNA and protein
have ovarian dysfunction, which usually manifests as extraction.
Fig. 1 Global transcriptome patterns of largest preovulatory follicles (F1s) and postovulatory follicles (POF1s). A Record of egg production
in continuous laying (CL) and intermittent laying (IL) chicken. B Schematic representation of the research workflow. F1 of CL hens (CL_F1), POF1
of CL hens (CL_POF1), F1 of IL hens (IL_F1), and POF1 of IL hens (IL_POF1) were collected for RNA extraction and subjected to RNA-seq. C Principal
component analysis (PCA) of RNA-seq data for all samples. PCA plots of the expression patterns of highly expressed genes in CL_F1 and CL_POF1
(D), IL_F1 and IL_POF1 (E), and CL_POF1 and IL_POF1 (F). Blue-to-red gradient indicates low-to-high gene expression levels
Transcriptome sequencing and analysis 5 mmol/L dithiothreitol for 60 min at 37 °C and alkylated
Total RNA was extracted using TRIzol reagent (Invit- with 11 mmol/L iodoacetamide for 45 min at 25 ± 2 °C in
rogen, Carlsbad, CA, USA). RNA quality and quantity darkness. Finally, the peptides were desalted on a Strata
were assessed using gel electrophoresis, a NanoDrop X SPE column.
2000 spectrophotometer (Thermo Fisher Scientific, The tryptic peptides were dissolved and separated using
Wilmington, DE, USA), and an Agilent Bioanalyzer a NanoElute UHPLC system (Bruker Daltonics, Billerica,
2100 Bioanalyzer (Agilent Technologies, Santa Clara, MA, USA) at a constant flow rate of 1,000 nL/min. The
CA, USA). RNA-seq libraries were constructed using peptides were subjected to a capillary source, followed
RNAs extracted from CL_F1, CL_POF1, IL_F1, and by timsTOF Pro (Bruker Daltonics) mass spectrometry.
IL_POF1 (Fig. 1B); 5 replicates were designed for each Tandem mass spectrometry data were processed using
group. Sequencing was performed by Frasergen Infor- DIA-NN v1.8 and searched against the BLAST Gallus
mation Co., Ltd. (Wuhan, China). Raw RNA-seq data gallus database (27,535 entries). Trypsin/P was specified
were deposited in the National Center for Biotechnol- as a cleavage enzyme and up to one missing cleavage was
ogy Information Sequence Read Archive database under allowed. The false discovery rate (FDR) of the precur-
accession number PRJNA949555. sor was set to 1%. The protein results were exported for
Clean reads were obtained by removing adapters and further bioinformatic analyses. DIA mass spectrometry
low-quality reads using fastp v0.20.1 [26]. The clean reads measurements were performed using PTM Biolabs Co.,
were then mapped to the chicken reference genome, Ltd. (Hangzhou, China). Proteomic data were deposited
Gallus-gallus-6.0, using HISAT2 v2.2.1 [27]. Subse- in the ProteomeXchange Consortium (http://proteomece
quently, we assembled the mapped reads into transcripts ntral.proteomexchange.org) via the iProX partner reposi-
and quantified the gene expression before normalizing tory [31, 32] with the dataset identifier PXD041276.
the expression levels were normalized to fragments per
kilobase of transcript per million mapped fragments Protein–mRNA correlation analysis
using StringTie [28]. DEGs were identified using DESeq2 Following gene-wise protein–mRNA correlation analysis
v1.32.0 [29]. Gene Ontology (GO) and Kyoto Encyclope- for all genes detectable by both transcriptomic and pro-
dia of Genes and Genomics (KEGG) enrichment analyses teomic approaches, global Spearman’s correlation coeffi-
of DEGs were performed using the R package cluster- cients (rho) were calculated within the F1 of CL and IL.
Profiler [30] by importing a list of DEGs and converting FDR values were computed using the Benjamini–Hoch-
gene names from Ensembl ID to Entrez ID. Based on a berg procedure. Subsequently, a KEGG pathway enrich-
gene classification method of the GO corpus, the calcu- ment analysis was performed.
lated enrichment test for GO terms and KEGG pathways
was dependent on the hypergeometric distribution. Gene Quantitative real‑time PCR assay (qRT‑PCR)
Set Enrichment Analyses (GSEA) of all genes were per- Six DEGs, HSD3B1, LHCGR, NR5A1, CTSA, PTGS2, and
formed using the clusterProfiler package. RLN3, were selected to validate expression differences
using qRT-PCR. We hypothesized that these six genes
DIA mass spectrometry assay and data analysis may be involved in regulating follicular development or
Follicle samples were ground in liquid nitrogen and ovulation process [33–36]. The qRT-PCR assay was con-
lysed with a lysis solution (8 mol/L urea and 1% protease ducted as previously described [37]. Briefly, 2 μg of RNA
inhibitor cocktail). The homogenate was centrifuged at from each group (n = 5) was reverse-transcribed into
12,000 × g at 4 °C for 10 min, followed by sonication. The cDNA using FastKing gDNA Dispelling RT SuperMix
supernatant was collected, and the protein concentration (Tiangen, Beijing, China). qRT-PCR was performed on a
was detected using a BCA kit according to the manu- CFX96 Real-Time System (Bio-Rad, Hercules, CA, USA)
facturer’s instructions. The sample was slowly added to using 2 × SYBR Green Fast qPCR Mix (Abclonal, Wuhan,
obtain a final concentration of 20% (m/v) trichloroacetic China). The primers used to quantify gene expression
acid for protein precipitation, vortexed, and incubated were designed using Primer-BLAST (National Center
for 2 h at 4 °C. The precipitate was collected by centrif- for Biotechnology Information) [38] and synthesized by
ugation at 4,500 × g for 5 min at 4 °C. The precipitated Sangon Biotech Co., Ltd. (Shanghai, China). The 2−Ct
protein was then washed three times with pre-cooled method was used to calculate relative gene expression
acetone and dried for 1 min. Protein samples were then levels, and β-actin was used as a housekeeping gene [39].
redissolved in 200 mmol/L triethylammonium bicar-
bonate and ultrasonically dispersed. Trypsin was added Western blotting
at a trypsin-to-protein mass ratio of 1:50 for the first The relative expression levels of proteins in follicles
digestion overnight. The sample was then reduced with were detected by western blotting. Total tissue protein
was extracted using RIPA Lysis Buffer with a protease Statistical analysis and data visualization
inhibitor cocktail (Beyotime Biotechnology, Shanghai, All data are presented as mean ± standard error. The
China), and the concentration of the supernatant was two groups were compared via t-tests using SPSS v25
determined using the BCA Protein Assay Kit (Beyo- (SPSS Inc., Chicago, IL, USA). Significance was set to
time Biotechnology) [40] (n = 4). Proteins (50 μg) were P < 0.05, with extreme significance set to P < 0.01 or
separated by Bis–Tris SDS-PAGE and transferred onto P < 0.001. Visualization was performed using Graph-
polyvinylidene fluoride membranes (Bio-Rad). The Pad Prism v8 (GraphPad Software, San Diego, CA,
membranes were blocked in Blocking Buffer (Beyo- USA), ggplot2 [44], EVenn [45], TBtools v0.6673 [46],
time Biotechnology) for 20 min at 25 ± 2 °C and incu- and GSEA plot [47]. Schematics were generated using
bated overnight with primary antibody solutions at Figdraw (www.figdraw.com).
4 °C. The membranes were then further incubated with
a secondary antibody (Solarbio, Beijing, China) conju- Results
gated to horseradish peroxidase at room temperature Global gene expression characteristics of preovulatory and
(25 ± 2 °C) for 1 h. The primary antibodies of α-Tubulin postovulatory chicken follicles
(Absin, abs131993), ALDOB (Abclonal, A3728), LDHA RNA-seq generated 355.91 Gb of clean reads, 91.20%–
(Abclonal, A16394), FMOD (Abclonal, A6375), and 93.83% of which were mapped to the chicken reference
PTDSS1 (Abclonal, A13065) were diluted to a ratio of genome. For all samples, at least 91.4% of the reads had
1:1,000 according to the manufacturer’s instructions. quality scores equal to or exceeding Q30 (Additional
The secondary antibodies (Solarbio, Beijing, China) file 2). Principal component analysis (PCA) demonstrated
were diluted to a ratio of 1:5,000. α-Tubulin was used as comprehensive differences in gene expression among the
the reference protein, and protein bands were quanti- four groups. We observed clear separation between F1s
fied using ImageJ v2.0 [41]. and POF1s in both CL and IL groups, as well as distinct
physiologically specific clustering of F1s, whereas cluster-
ing of POF1s showed some overlap between CL and IL
Cell isolation and culture groups (Fig. 1C). Similar to the PCA results, the Pearson
GCs and theca cells (TCs) from F1 were isolated and correlation analysis showed the best intra-group cor-
cultured as described previously [42, 43] (n = 3). After relation coefficient was in CL_F1, while the inter-group
removing connective tissue from the follicle surface, correlation in POF groups was relatively high, and even
the GC and TC layers were subjected to enzymatic certain samples between CL_POF1 and IL_POF1 exhib-
digestion by collagenase type II (Sigma Aldrich, Inc., St. ited strong correlation (Additional file 3).
Louis, MO, USA) at 37 °C; GC and TC were digested Next, we observed gene expression abundance in each
for 5 min and 30 min, respectively. Cell suspensions group, which could be used as physiologically specific
containing GC or TC were filtered using cell strain- candidate markers to distinguish between CL and IL
ers (Biosharp, Hefei, Anhui, China) with a pore size of hens. The expression levels of the luteinizing hormone/
50 μm. The cells were maintained in a basal medium choriogonadotropin receptor (LHCGR) gene was notably
consisting of Dulbecco’s modified Eagle medium higher in CL_F1 and IL_F1 than in CL_POF1 and IL_
(Gibco, Gaithersburg, MD, USA) with 15% fetal bovine POF1 (Fig. 1D). The zona pellucida sperm-binding pro-
serum (Gibco) and 1% penicillin–streptomycin (Gibco) tein 3 (ZP3), inhibin alpha subunit, and NPC intracellular
in an incubator at 37 °C with a 5% CO2 humidified cholesterol transporter 2 were most abundant in CL_F1
atmosphere. The lactate dehydrogenase A (LDHA) and CL_POF1 groups (Fig. 1D). The extracellular fatty
inhibitor FX-11 (MedChemExpress, Monmouth Junc- acid-binding protein (EXFABP) and secreted phospho-
tion, NJ, USA) was diluted in dimethyl sulfoxide protein 1 (SPP1) were most abundant in IL_F1 and IL_
(Solarbio), which was used as a vehicle in the control POF1 (Fig. 1E). The Prostaglandin D2 synthase (PTGDS)
group. Various doses of FX-11 (5 μmol/L, 10 μmol/L, and prostaglandin-endoperoxide synthase 2 (PTGS2)
and 15 μmol/L) were used to pretreat GCs at 37 °C in exhibited high expression abundance in CL_POF1 and
an atmosphere of water-saturated 5% C O2. Lactate IL_POF1 (Fig. 1F).
dehydrogenase (LDH) activity assays were performed
using the LDH Activity Assay Kit (Solarbio), following
the manufacturer’s instructions. Cell Counting Kit-8 Transcriptional analysis of F1s and POF1s in continuous
(CCK8, Beyotime Biotechnology) and Annexin V-FITC and intermittent laying hens
Apoptosis Detection Kit (Beyotime Biotechnology) According to pairwise comparisons using log2 (fold change)
were used to analyze cell viability and apoptosis, > 2 and Padj < 0.05 as criteria, 950, 843, 469, and 14 DEGs
respectively, according to the manufacturer’s protocols. were identified in CL_F1 vs. IL_F1, CL_F1 vs. CL_POF1,
IL_F1 vs. IL_POF1, and CL_POF1 vs. IL_POF1, respec- (PVM) of chicken oocytes, ZP3 and uromodulin (UMOD),
tively (Additional file 4 and Fig. 2A, B). Most DEGs were showed high expression levels in CL_F1, suggesting
obtained in CL_F1 vs. IL_F1, whereas the fewest DEGs that the F1 of continuous laying hens acquired stronger
were obtained in CL_POF1 vs. IL_POF1, indicating that mechanical support to adapt to the long journey through
the greatest difference between CL and IL hens lies in the oviduct after ovulation (Fig. 1D, 2H) [49, 50]. The lev-
the preovulatory follicle, F1, rather than POF1. The Venn els of cathepsin A (CTSA) and glucosamine (N-acetyl)-
diagram showed that 204 DEGs were shared in the CL_ 6-sulfatase (GNS), both belonging to the lysosome family,
F1 vs. CL_POF1 and IL_F1 vs. IL_POF1 groups, and 246 as well as those of very low-density lipoprotein receptor
DEGs were shared in the CL_F1 vs. IL_F1 and CL_F1 (VLDLR) and fibrinogen-like 2 (FGL2), were significantly
vs. CL_POF1 groups, excluding the IL_F1 vs. IL_ POF1 reduced in IL_F1 (Fig. 2H).
group (Fig. 2A). The expression levels of lipid metabolism-related genes,
To validate the RNA-Seq results, we performed qRT- such as EXFABP, fatty acid-binding protein 4 (FABP4),
PCR to confirm the expression patterns of the six candi- and PTGS2, were significantly higher in POF1s than in
date DEGs. Primer information is shown in Additional F1s. Moreover, the expression levels of EXFABP and
file 5. The resulting expression differences were similar FABP4 were slightly higher in IL_POF1 than in CL_POF1
to those obtained by RNA-seq (Fig. 2C). For example, (Fig. 2H). The DEGs showing high expression levels in
hydroxy-delta-5-steroid dehydrogenase,3 beta- and ster- POFs, including fibronectin 1 (FN1), metallothionein 3
oid delta-isomerase 1 (HSD3B1) and LHCGRwere highly (MT3), metallothionein 4 (MT4), LDHA, suppressor of
expressed in CL_F1, and PTGS2 was highly expressed in cytokine signaling 3 (SOCS3), and angiopoietin-like 4
POF1s. Linear regression between RNA-seq and qRT- (ANGPTL4), contributed to increased fibrosis and energy
PCR results showed a positive correlation, with a correla- metabolism after ovulation (Fig. 2H).
tion coefficient (r) of 0.815, supporting the reliability of
the RNA-seq results (Fig. 2D). Signaling pathways in poultry ovulation determined
To further investigate the biological functions involved by functional annotation analyses
in ovulation, DEGs were evaluated by GO functional The F1-to-POF transition is a key step in poultry ovula-
enrichment analysis. In the CL_F1 vs. IL_F1 comparison, tion. Therefore, we performed KEGG analyses of DEGs
DEGs were enriched in chemokine proteolysis, integral in CL_F1 vs. CL_POF1 and IL_F1 vs. IL_POF1. The over-
components of membrane, proteolysis, and hydrolase lapping functional pathways during this transition in
activity (Fig. 2E). The DEGs of CL_F1 vs. CL_POF1 were both CL and IL included arachidonic acid metabolism,
clustered into hormone activity, steroid metabolic pro- steroid hormone biosynthesis, and TGF-beta signaling
cesses, lipid transport, and phospholipid biosynthetic pathway (Additional file 7, Additional file 8).
processes (Fig. 2F). The main GO categories in IL_F1 vs. To investigate new and key biological pathways
IL_POF1 were steroid biosynthetic processes, regulation involved in the F1-to-POF transition, we performed
of lipid metabolic processes, and extracellular regions GSEA of all genes expressed in CL_F1 and CL_POF1.
(Fig. 2G). DEGs in CL_POF1 vs. IL_POF1 were enriched According to strict detection criteria (P-value < 0.05 and
in the immune response, cytokine receptor binding, and FDR < 25%), we identified 82 gene sets (Additional file 9).
chemokine receptor binding (Additional file 6). Many of these pathways were also identified in the KEGG
Next, we investigated the expression patterns of ovula- analysis, thereby validating and supporting the GSEA
tion-related DEGs in these groups. As shown in Fig. 2H, results. We focused on four pathways involved in contin-
steroid hormone synthesis-related genes [48], such as uous ovulation: cytokine–cytokine receptor interaction,
HSD3B1, cytochrome P450 family 11 subfamily A member lysosomes, calcium signaling pathway, and apoptosis
1 (CYP11A1), and steroidogenic acute regulatory protein (Fig. 3). Ovulation regulation-related genes were involved
(STAR), were highly expressed only in CL_F1, indicating in the cytokine–cytokine receptor interaction pathway
that steroid hormone synthesis is most active in CL_F1. with a high normalized enrichment score, low P-value,
The major constituents of the perivitelline membrane and low FDR (Fig. 3A). Among the many genes identified
(See figure on next page.)

Fig. 2 Transcriptional characteristics and gene expression dynamics of F1s and POFs. A Venn diagram of differentially expressed gene (DEGs).
Criteria for DEGs filtering were |log2 (fold change)|> 2 and Padj< 0.05. B Heatmap of all DEGs in four comparisons. Blue to red colors indicates
the relative gene expression level from low to high, respectively. C–D Comparison and Pearson correlation analysis of fold change values in six DEGs
between qRT-PCR and RNA-seq analysis, respectively. Gene Ontology (GO) terms of DEGs in CL_F1 vs. IL_F1 (E), CL_F1 vs. CL_POF1 (F), and IL_F1 vs.
IL_POF1 (G). H Heatmap of DEGs involved in ovulation regulation
Fig. 2 (See legend on previous page.)

Fig. 3 Signaling pathways enriched in follicle ovulation by gene set enrichment analyses (GSEA). GSEA enrichment plots of significant pathways
in follicle ovulation transition, the cytokine–cytokine receptor interaction pathway (A), lysosomes (C), the calcium signaling pathway (E),
and apoptosis (G). The normalized enrichment score (NES), P-value, and FDR were determined using GSEA software and are indicated within each
enrichment plot. B, D, F, and H Box plots showing fragments per kilobase of transcript per million mapped fragments (FPKM) of the key component
genes in each pathway
in this pathway by GSEA, CXC motif chemokine recep- map was generated to depict differential protein expres-
tor 4 (CXCR4), a component of the CXC subfamily, was sion between groups (Fig. 4C).
highly expressed in CL_POF1 cells (Fig. 3B). Next, we Four DAPs, namely, phosphatidylserine synthase 1
considered the lysosomal pathway (normalized enrich- (PTDSS1), fibromodulin (FMOD), LDHA, and aldolase
ment score = 2.49, P < 0.01 and FDR < 0.01) (Fig. 3C). In and fructose-bisphosphate B (ALDOB) were selected to
this pathway, clathrin light chain A (CLTA) was down- verify the results of DIA proteomic analysis by western
regulated in CL_POF1 (Fig. 3D). In the calcium signal- blotting. Primary antibody information is listed in Addi-
ing and apoptosis pathways, adenylate cyclase 9 (ADCY9) tional file 11. The western blotting results exhibited a
and cathepsin O (CTSO) were expressed at lower levels remarkable degree of consistency with those of the quan-
in CL_POF1 cells than in CL_F1 cells (Fig. 3E–H). titative proteomic analyses (Fig. 4D).
Functional annotation of DAPs

Proteomic profiling of F1 membranes in continuous As determined by GO analysis, the upregulated DAPs
and intermittent laying hens in CL_F1 were mainly enriched in the processes of
The aforementioned results confirmed that factors proton-transporting V-type ATPase complex, proton
involved in regulating the frequency of poultry ovulation transmembrane transport, and active transmembrane
were expressed in F1 but not in POF1. To further ana- transporter activity. Downregulated DAPs were enriched
lyze the mechanism underlying the differences in ovula- in the extracellular matrix, carboxypeptidase activity,
tion frequency among laying chickens, we applied a DIA serine hydrolase activity, and metallopeptidase activity
quantitative proteomic approach to analyze samples from (Fig. 5A). DAPs were involved in various KEGG path-
CL_F1 and IL_F1 (n = 4). A total of 5,670 proteins were ways, including oxidative phosphorylation, synaptic vesi-
identified and 5,591 proteins were detected in CL_F1 vs. cle cycle, phagosomes, and chemokine signaling (Fig. 5B).
IL_F1 (Fig. 4A, Additional file 10). Proteins with a quan- GO and KEGG pathway enrichment analyses provide
titative fold change of > 1.5 or < 0.67 and P < 0.05 were valuable insights into the mechanism underlying con-
identified as differentially abundant proteins (DAPs). In tinuous ovulation in chickens. Carboxypeptidase X, M14
total, we identified 230 upregulated and 22 downregu- family member 1 (CPXM1), and carboxypeptidase Z
lated DAPs in the CL_F1 group compared with the IL_F1 (CPZ) levels were lower in IL_F1 than in CL_F1, indicat-
group (Fig. 4B, Additional file 10). Subsequently, a heat ing that carboxypeptidase activity was inhibited (Fig. 5C).
Fig. 4 Proteomic profiling of CL_F1 vs. IL_F1. A Comparison of peptides and proteins in data-independent acquisition strategies. B Volcano plot
of differentially abundant proteins (DAPs) in the CL_F1 vs. IL_F1 group. DAPs with P < 0.05 and quantitative fold change > 1.5 are marked in red; DAPs
with P < 0.05 and quantitative ratio < 0.67 marked in blue. C Heatmap of DAPs between CL_F1 vs. IL_F1. D, E Protein expression levels of PTDSS1,
FMOD, LDHA, and ALDOB in CL_F1 and IL_F1 groups. *P < 0.05, **P < 0.01
Fig. 5 Functional annotation analysis of DAPs. A Enriched Gene Ontology (GO) terms for DAPs in the proteomics analysis of CL_F1 and IL_F1
samples. B KEGG pathways enriched in DAPs indicated in the chord plot. C Heatmap of DAPs involved in ovulation regulation. D Protein–protein
interaction networks of DAPs
Phosphatidylserine synthase 1 (PTDSS1) and dipeptidyl members, including fatty acid binding protein 5 (FABP5)
peptidase 4 (FAP) levels were also lower in IL_F1 than in and fatty acid binding protein 3 (FABP3), were more
CL_F1, suggesting that peptidase activity was inhibited highly expressed in IL_F1 than in CL_F1. These results
(Fig. 5C). Lysosome-related proteins, including CTSH support the hypothesis that oxidative phosphorylation
and cathepsin C (CTSC), were overexpressed in IL_F1 was upregulated.
(Fig. 5C). ATP synthases, such as ATPase H + transporting A protein–protein interaction network analysis was
V1 subunit D (ATP6V1D) and ATPase H + transporting used to identify interactions between DAPs. Three
V1 subunit H (ATP6V1H), and fatty acid binding family proteins were identified as hubs in the network: ras-
related C3 botulinum toxin substrate 2 (RAC2),
Fig. 6 Integrated analysis of transcriptomics and proteomics data. A, B PCA of RNA and protein data in CL_F1 vs. IL_F1, respectively. C Histogram
showing gene-wise mRNA–protein Spearman’s correlations. D KEGG pathway enrichment for genes with a Spearman’s correlation between mRNA
and protein abundance (FDR < 0.05). E–H Line plots showing expression profile and correlation coefficient of mRNA and proteins: HK2 (E), CTSC (F),
LDHA (G), and FABP5 (H)
ribonuclease homolog (RSFR), and ubiquinone oxi- Integrated analysis of transcriptomics and proteomics data
doreductase subunit AB1 (NDUFAB1) (Fig. 5D). PCA showed clear separation between CL_F1 and IL_F1
In addition, heat shock protein family A member 9 tissues at both the RNA and protein levels, confirming
(HSPA9), ALDOB, ras homolog family member G the difference between IL_F1 and CL_F1 (Fig. 6A, B).
(RHOG), adenylate kinase 4 (AK4), and poly (ADP- A weak Spearman’s correlation was observed between
ribose) polymerase family member 14 (PARP14) were mRNA and protein abundance (Additional file 12).
identified as key proteins. Among 4,665 mRNA–protein pairs, 327 (7.0%) displayed
significant positive correlations with Spearman’s coef-
ficient > 0 and FDR < 0.05, whereas 112 (2.4%) displayed
significant negative correlations with Spearman’s coeffi- Zona pellucida family, as a major component of the PVM,
cient < 0 and FDR < 0.05 (Fig. 6C). provides strong mechanical regulation of F1 in CL hens
Genes involved in metabolism-related pathways were Birds are oviparous vertebrates, characterized by much
significantly enriched in propanoate metabolism, phe- larger egg sizes than in viviparous vertebrates [50]. In
nylalanine metabolism, glycolysis/gluconeogenesis, chickens, the largest preovulatory follicle (F1) ruptures
lysosome and pyruvate metabolism, and cofactor bio- from the left ovary and enters the oviduct; the oocyte
synthesis (Fig. 6D). Some components of the glycolysis/ is then wrapped in the albumin and shell during a long
gluconeogenesis pathway differed between groups. For oviduct journey [51]. A membrane structure with glyco-
example, hexokinase 2 (HK2) was consistently upregu- protein components surrounds the oocyte, known as the
lated in IL_F1 (Fig. 6E). In addition, LDHA, CTSC, and perivitelline layer or PVM [52]. The PVM not only plays a
FABP5 showed significant differences as key elements role in sperm binding for successful fertilization but also
in propanoate metabolism, lysosomes, and PPAR signal- physically protects the large oocyte, including the mass
ing pathways at the transcriptomic and proteomic levels of yolk, in the gravity field [50, 53]. The main constitu-
(Fig. 6F–H). ents of PVM are members of the ZP glycoprotein family.
Over the past few decades, various members of this gly-
Inhibition of LDHA induces cell death in chicken follicles coprotein family have been identified, such as zona pellu-
Both transcriptomic and proteomic analyses dem- cida glycoprotein 1 (ZP1), ZP2, ZP3, ZP4, and ZPD (also
onstrated that LDHA was significantly upregulated known as UMOD) [50, 54–56].
in the IL_F1 group compared to that in the CL_F1 In this study, ZP3 and UMOD were downregulated at
group (Fig. 6G). The expression levels of LDHA mRNA the mRNA level in the F1 samples of IL chickens com-
(Fig. 7A) and protein (Fig. 4D, E) were consistent with the pared to those of CL chickens. This suggests that the
results of omics data. LDHA is the main functional sub- PVM structure in CL chickens is sufficiently robust to
unit of LDH, which is a glycolytic rate-limiting enzyme. physically protect the oocyte from breaking during ovu-
After adding FX-11, a specific inhibitor of LDHA, to cul- lation. However, the thinner PVM of the IL chicken egg
tured chicken primary GCs, we found that the enzymatic was insufficiently robust, resulting in the suspension of
activity of LDH decreased in GCs in a dose-dependent ovulation. ZP family members have various synthetic
manner (Fig. 7B); 5 μmol/L of FX-11 was selected for pathways. Both ZP3 (Fig. 1D) and UMOD (Fig. 2H) were
subsequent experiments. LDHA reduction significantly exclusively expressed in the GC layer [50, 57, 58]. Thus,
inhibited the viability of GCs (Fig. 7C) and increased GC the GC layer plays an important role in the process of
death, which was characterized by increased labeling chicken ovulation.
of annexin V and propidium iodide (Fig. 7D). To verify
whether the inhibition of LDHA in GCs had an impact Sufficient progesterone may contribute to ovulation in CL
on TCs, we cultured TCs with GCs for 24 h using a Tran- hens
swell co-culture system (Fig. 7E). The viability of TCs Various steroid hormones, such as progesterone, estro-
decreased significantly, and the number of late apoptotic gen, and androgens, play a role in chicken follicle growth
cells increased significantly (Fig. 7F, G). and development [59]. In this study, the expression
of the progesterone synthesis-related genes HSD3B1,
Discussion CYP11A1, and STARdeclined significantly in the F1 of IL
Egg production performance is an important economic chickens, suggesting that progesterone deficiency occurs
trait in chickens. To improve the production perfor- during the suspension of ovulation (Fig. 2H). In 1987,
mance of laying hens, many studies have investigated cel- Tanaka et al. designed an in vitro perfusion device and
lular processes during ovarian follicle development [8, 9]. demonstrated the importance of progesterone in ovula-
Here, we integrated transcriptome and proteome data tion, which increased the ovulation rate to 80% in domes-
to reveal the molecular mechanisms underlying chicken tic fowl (compared with 0 in the control group) [60].
ovulation and comprehensively compared key genes In vivo, the steroid biosynthetic blocker aminogluteth-
and metabolic pathways between CL and IL hens. We imide phosphate can prevent an increase in progesterone
observed a clear difference in the frequency of egg-laying concentration and inhibit ovulation induced by LH [61].
within each population, which could reflect differences in These results confirm that progesterone may act directly
the ability of hens to ovulate and subsequently affect egg on ovulation in chickens.
production. These results elucidate the dynamic expres- Progesterone plays an important role in the regulation
sion profile and potential regulatory network chicken of follicular maturation, ovulation, and oviposition in
ovulation. domestic hens via the progesterone receptor (PGR) [62].
PGR is a nuclear receptor transcription factor present
Fig. 7 Inhibition of LDHA induces cell death in chicken ovarian follicles. A Expression levels of LDHA mRNA in CL_F1 and IL_F1. B Enzymatic activity
of LDH in granulosa cells (GCs) treated with FX-11. C CCK8 assay of chicken GCs treated with FX-11 and the control at 0, 6, 12, 24, and 48 h. D
Apoptosis rates of GCs after FX-11 treatment were assessed by flow cytometry. E Schematic diagram of the Transwell co-cultured system, with theca
cells (TCs) seeded in the inserts and GCs seeded in culture plates. F CCK8 assay of TCs 24 h after co-culturing with GCs treated with FX-11. G
Apoptosis rates of TCs 24 h after co-culturing with GCs treated with FX-11. *P < 0.05, **P < 0.01, ***P < 0.001
in TCs, GCs, and germinal epithelial cells, suggesting controlled by PGR, such as the synthesis of enzymes that
that these tissues are targets of progesterone in ovula- degrade the follicle wall, finally leading to the suspension
tion regulation [62, 63]. Moreover, in vitro and in vivo of ovulation in laying hens.
assays showed that PGR mediates progesterone-induced
ovulatory processes in macaques [64]. Similar results Proteases play a role in chicken ovulation
were obtained in experiments using mice [65]. In this During the complex process of chicken ovulation, in addi-
study, LHCGR (the receptor of LH) mRNA was highly tion to the establishment of PVM mechanical support
expressed in the F1 of CL chickens (Additional file 4), and the LH/progesterone surge, tissue degradation of the
and LH and progesterone secretion were induced by stigma region is equally important for rupturing the preo-
positive feedback [66]. Therefore, we propose that a pro- vulatory follicle [67, 68]. Proteases are involved in the deg-
gesterone deficiency inhibits the feedback loop between radation of collagen fibers and proteoglycans in chicken
LH and progesterone, which disrupts biochemical events follicle walls [69]. Plasminogen is a fibrinolytic protease of
the fibrinolytic system that can be activated by the urok- membrane tissue of CL_F1 will be degraded, so that the
inase-type plasminogen activator (PLAU) and tissue-type energy metabolism in follicle is weakened or suspended. In
plasminogen activator (PLAT) [70]. In our study, PLAU addition, we speculate that energy metabolism did not stop
was significantly downregulated specifically in the CL_F1 at the correct time before ovulation in IL_F1, which may
vs. IL_F1 comparison (Additional file 4), whereas PLAT have reduced follicle sensitivity to ovulation signals.
was not identified as a DEG, suggesting that PLAU plays Many human cancers show higher LDHA levels than
an important role in degradation of the follicle wall during those in normal tissues [79, 80]. LDHA is encoded by
the chicken ovulatory process. As a serine protease, PLAU a target gene of c-Myc, an oncogenic transcription fac-
is produced by the granulosa layer and is dependent on tor, and hypoxia-inducible factor (HIF-1) [81]. c-Myc can
stimulation by the theca layer in hens [71]. directly increase the LDHA expression level by transac-
Lysosomes are key degradative compartments of the tivating the LDHA promoter [82]. As a critical transcrip-
cell. They not only degrade proteins but are also involved tion factor in hypoxic adaptation, HIF-1 can bind to the
in membrane repair and other cellular processes [72]. LDHA sequence of the promoter [83]. Although hypoxia
Among lysosomal hydrolases, cathepsins play a major is commonly present in tumors, the relationship between
role as proteases [73]. They are a superfamily containing hypoxia and suspended ovulation in hens is poorly under-
cathepsin A (serine), B, C, H, and O (cysteine), or D and stood. Here, we report that the inhibition of LDHA activity
E (aspartate) [74], each of which has various functions. led to decreased cell viability and increased apoptosis on
CTSA expression is higher in carcinoma tissues and cultured GCs, as well as significantly decreased viability in
may participate in extracellular matrix degradation [75]. co-cultured TCs, which may be attributed to intercellular
In our study, CTSA mRNA was significantly reduced in communication. These results suggest that excessive glyco-
IL_F1 cells (Fig. 2H), which may explain abnormalities lysis in F1 of IL hens with suspended ovulation is caused
in ovulation. Jin et al. found that CTSD levels decreased by the abnormal upregulation of rate-limiting enzymes in
significantly in the ovaries of patients with PCOS [76], the glycolytic pathway. Therefore, restoring lower levels of
which is consistent with the regulatory mode of CTSA glycolysis may be key to promoting ovulation in chickens.
in IL chickens. Other members of the cathepsin fam-
ily, CTSH and CTSC, were both highly expressed in IL_ Conclusions
F1 cells (Fig. 5C), contrary to the expression pattern of In this study, we analyzed differences in the F1 and POF
CTSA. Research on CTSH and CTSC in the process of of continuous and intermittent laying chickens through
follicular development is limited; therefore, more experi- combined transcriptome and proteome analyses, thereby
ments are required to determine the role of these genes revealing the important biochemical events involved in
in the suspension of ovulation. We speculate that these ovulation. The synthesis of PVM with sufficient mechani-
two genes may reduce the sensitivity of follicles to PGR, cal strength, progesterone secretion, protease degradation
eventually leading to intermittent ovulation in chickens. of the follicle wall, and energy metabolism suspended at
the appropriate time point contribute to follicle ovu-
Excessive glycolysis metabolism in IL_F1 lation in CL hens. ZP3, CYP11A1, PLAU, CTSA, and
During follicle development and ovulation, a large sup- LDHA play vital roles during different phases of chicken
ply of adenosine triphosphate is produced via oxidative ovulation. The inhibition of LDHA promotes cell apop-
phosphorylation in the mitochondria to ensure sufficient tosis and decreases the viability of GCs and TCs, which
energy provision [77]. Similarly, glucose is an essential might promote ovulation. To the best of our knowledge,
energy source for animals [78]. To provide a substrate for our findings provide the first overview of the dynamic
energy metabolism in oocytes, glucose is first converted to expression profile and regulatory network in chicken fol-
pyruvate through glycolysis in GCs, and pyruvate is trans- licle ovulation. Therefore, this research not only extends
ported to the mitochondria of oocytes through the mono- the spectrum of relevant genes but also provides a deeper
carboxylic acid cycle [77]. Our proteomics analysis revealed understanding of the ovulation process in poultry.
that DAPs were enriched in the oxidative phosphorylation
Abbreviations
pathway (Fig. 5B), and the DAPs involved in this pathway ADCY9 Adenylate cyclase 9
were highly expressed in IL_F1 (Fig. 5C). More specifically, AK4 Adenylate kinase 4
ALDOB Aldolase and fructose-bisphosphate B
HK2 and LDHA, both glycolytic rate-limiting enzymes,
ANGPTL4 Angiopoietin-like 4
were upregulated in IL_F1, as shown in both transcrip- ATP6V1D ATPase H+ transporting V1 subunit D
tomic and proteomic analyses (Fig. 6E and 6G), indicating ATP6V1H ATPase H+ transporting V1 subunit H
CL Continuous laying
that glycolysis and pyruvate metabolism were enhanced in
CLTA Clathrin light chain A
IL_F1. When a follicle is about to undergo ovulation, the CPXM1 Carboxypeptidase X, M14 family member 1
CPZ Carboxypeptidase Z
CTSA Cathepsin A
CTSC Cathepsin C Additional file 6. Gene Ontology (GO) terms of DEGs in CL_POF1 vs.
CTSO Cathepsin O IL_POF1.
CXCR4 CXC motif chemokine receptor 4 Additional file 7. Venn diagram of KEGG results of DEGs in CL_F1 vs.
CYP11A1 Cytochrome P450 family 11 subfamily A member 1 CL_POF1 and IL_F1 vs. IL_POF1.
DAPs Differentially abundant proteins
Additional file 8. Kegg result of differentially expressed genes (DEGs).
DEGs Differentially expressed genes
DIA Data-independent acquisition Additional file 9. Gene set enrichment analyses results of all genes
EXFABP Extracellular fatty acid-binding protein expressed in CL_F1 and CL_POF1.
FABP3 Fatty acid binding protein 3
Additional file 10. Identification and quantitative results of data-inde-
FABP4 Fatty acid-binding protein 4
pendent acquisition mass spectrometry in CL_F1 and IL_F1.
FABP5 Fatty acid binding protein 5
FAP Dipeptidyl peptidase 4 Additional file 11. Primary antibodies for western blotting.
FGL2 Fibrinogen-like 2 Additional file 12. Results of Spearman’s correlation analysis between
FMOD Fibromodulin mRNA and protein abundance.
FN1 Fibronectin 1
GC Granulosa cells
GNS Glucosamine (N-acetyl)-6-sulfatase Acknowledgements
GO Gene Ontology We appreciate the assistance provided by the Institute of Biophysics, Chinese
GSEA Gene Set Enrichment Analyses Academy of Sciences for the flow cytometry experiments.
HK2 Hexokinase 2
HSD3B1 Hydroxy-delta-5-steroid dehydrogenase,3 beta- and steroid Authors’ contributions
delta-isomerase 1 RXN performed the experiments and prepared the manuscript. WHZ and HYT
HSPA9 Heat shock protein family A member 9 collected the samples and analyzed the data. JYL and YL visualized the results
IL Intermittent laying and provided comments on manuscript revision. BZ and HZ participated in
KEGG Kyoto Encyclopedia of Genes and Genomics the design of the study, provided critical discussion on the results, and revised
LDHA Lactate dehydrogenase A the manuscript. CXW conceived the study and provided overall supervision.
LH Luteinizing hormone All authors read and approved the final manuscript.
LHCGR Luteinizing hormone/choriogonadotropin receptor
MT3 Metallothionein 3 Funding
MT4 Metallothionein 4 This study was supported by the National Key Research and Development
NDUFAB1 Ubiquinone oxidoreductase subunit AB1 Program of China (2022YFD1600902), Key Research and Development
PARP14 Poly (ADP-ribose) polymerase family member 14 Program of Shandong (2022LZGC013), and China Agriculture Research System
PCOS Polycystic ovary syndrome (CARS-40).
PGR Progesterone receptor
PLAT Tissue-type plasminogen activator Availability of data and materials
PLAU Urokinase-type plasminogen activator The data for the current study are available from the corresponding author
POF Postovulatory follicle upon reasonable request.
PPI Protein–protein interaction
PTDSS1 Phosphatidylserine synthase 1
PTGDS Prostaglandin D2 synthase Declarations
PTGS2 Prostaglandin-endoperoxide synthase 2
PVM Perivitelline membrane Ethics approval and consent to participate
qRT-PCR Quantitative real-time PCR assay All animal experimental protocols were approved by the Animal Care and
RAC2 Ras-related C3 botulinum toxin substrate 2 Use Committee of China Agricultural University and performed in accordance
RHOG Ras homolog family member G with the National Research Council’s Guide for the Care and Use of Laboratory
RSFR Ribonuclease homolog Animals (AW80203202-1-1). All chickens were raised under equivalent environ-
SOCS3 Suppressor of cytokine signaling 3 mental conditions, and feed and water were provided ad libitum. In the view
SPP1 Secreted phosphoprotein 1 of animal welfare, all animals were slaughtered humanely, and all efforts were
STAR Steroidogenic acute regulatory protein made to minimize suffering.
TC Theca cells
UMOD Uromodulin Consent for publication
VLDLR Very low-density lipoprotein receptor Not applicable.
ZP1 Zona pellucida glycoprotein 1
ZP2 Zona pellucida glycoprotein 2 Competing interests
ZP3 Zona pellucida sperm-binding protein 3 The authors declare no conflict of interest.
ZP4 Zona pellucida glycoprotein 4
Received: 1 November 2023 Accepted: 3 March 2024

Supplementary Information
The online version contains supplementary material available at https://doi.
org/10.1186/s40104-024-01019-2.
Additional file 1. Egg laying records of all animals. References

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Proteo-Transcriptomic Profiles Reveal Key Regulatory Pathways and Functions of LDHA in The Ovulation of Domestic Chickens

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Proteo-Transcriptomic Profiles Reveal Key Regulatory Pathways and Functions of LDHA in The Ovulation of Domestic Chickens

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Nie et al.

Journal of Animal Science and

RESEARCH Open Access

Proteo‑transcriptomic profiles reveal key

(See figure on next page.)

Fig. 2 (See legend on previous page.)

Functional annotation of DAPs

Received: 1 November 2023 Accepted: 3 March 2024

Additional file 1. Egg laying records of all animals. References

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