Quantitative Proteomic Analysis of buffalo ovaries reveals the regulation of kinase and phosphatase during the Formation and Regression of Corpus Luteum

Background: Reproductive characteristics are made up of many complicated physiology procedures that have significant effects on the growth of the buffalo industry and are affected by numerous factors. The ovary is the most important sexual organ of female mammals, and during the oestrous cycles, its corpus luteum (CL) plays a significant part in mammalian reproduction. During the development and regression of corpus luteum, however, the differentially expressed proteins are less defined. In this study, we used a 6-plex tandem mass tag (TMT) strategy for quantitative proteomic comparison of three distinct ovary phases (corpus hemorrhagicum, corpus luteum and corpus hemorrhagicum) in buffalo. Results: A total of 148 differentially expressed proteins were identified, 32 of these proteins were identified as differentially expressed in the group CH (corpus hemorrhagicum) and 116 were identified as differentially expressed in the group CF (corpus fibrosum), with the group CL (corpus luteum) serving as the control group. Notably, we discovered that quite some enzymes such as kinase and phosphatase, are upregulated in the ovary CL phase, and three upregulated enzymes and proteins in the CL phase (PLK1, PGP, and HGS) were verified using Western blotting, quantitative RT-PCR, and immunohistochemistry analysis. The results of these validations were consistent with the quantitative analysis of the TMT-label, which indicated that they could play a crucial role during the CL's reproductive cycle. These buffalo results during the formation and regression of the corpus luteum are also shown to be a substantial reference value for comparable human and mouse studies after analyzing homologous BLAST cross-species. The expression information is accessible with the PXD009957 identifier via ProteomeXchange. Conclusions: Our research gives a deeper understanding of CL formation and regression during the oestrous cycles, which shows kinase and phosphatase regulation, and indicates some potential enzymes and proteins that may influence buffalo fertility. hepatocyte growth factor-regulated tyrosine kinase substratum, serine

4 Conclusions: Our research gives a deeper understanding of CL formation and regression during the oestrous cycles, which shows kinase and phosphatase regulation, and indicates some potential enzymes and proteins that may influence buffalo fertility. Keywords: corpus luteum, quantitative proteomic, oestrous cycles, ovary, cross-species Background Buffalo (Bubalusbubalis) is a specie with a single ovule, in its heat phase. Generally, only one follicle matures and ovulates, leading to a sharp decrease in fertility rates, which limits the buffalo industry to flourish. Reproductive characteristics are quantitative traits that are influenced by different factors.
The ovary is female mammals' most significant sexual organ. Its corpus luteum (CL) plays a vital role in mammalian reproduction during its formation and regression. substantial boost in places marked as binding to globin, endorphin 1B, and other 1B glycoproteins, and explained the use of PROT. Archaeology assumed by biomarkers in early diagnosis [1].Research on the proteomic characterization of bovine follicular granulosa cells conducted by Hao et al. has presented a theory analyzing that of these,259 proteins have a distinct expression in DF(dominant follicle) and SF(subordinate follicle). In 259, an aggregate quantity of 26 proteins was upregulated in DF (fold change range≥2), and 233 proteins were down-regulated in DF (fold change ≤0.5), pointing out that the research findings will provide valuable data to explore the follicle's growth and function [2].
Similar research methods were also used for bovine follicular liquid(bFF) and porcine follicular fluid (pFF). Ferrazza RdA et al. evaluated the FFs in a proteomic unit gathered from nine cows. They found that 143 proteins were recognized and split into multiple biological procedures that included responses to stimuli and metabolic processes. In his research, they outlined the various expression of proteins that provide inward-looking protein modifications that rely on size variables that may influence follicular function in the ovarian follicular microenvironment [3]. Bijttleier J et al. carried out a comparative proteome analysis of porcine follicular fluid (pFF). Their trials showed that they were prevalent to both liquids, of which 13 were considerably distinct in abundance (p<0.05), and they discovered that serum (2)-macroglobulin depletion in the serum could substantially compensate for the fluid development that is affected by the mature oocyte in the serum [4].
In recent years, sensitive proteome technique (nano HPLC tandem mass spectrometry) has enabled us to access the proteome from scarce materials, such as oocytes and blastocysts, which are isolated by micromanipulation. The Proteome analysis of pig oocytes during in vitro maturation indicated that UCH-L1 played a significant part in completing the first meiotic cycle and transitioning it to the and October of the same year, all ovaries were collected. Detailed ovary sample data was shown in Additional File 5: Table S5 and Additional File 6: Figure S1. Figure   5 shows the workflow of these studies. Six ovaries from three phases (CH, CL, CF) were used for sample preparing, and the six ovaries were split into two distinct samples for two biological replicates. The ovaries were transported in precooled physiological saline to the laboratory. The lysis buffer (7 M urea, 3 per cent CHAPS, 1.5 M thiourea, 1 per cent DTT, and 1 per cent v / v protease inhibitor cocktail) was used to remove the ovarian protein after liquid nitrogen grinding. After incubation with vortexing every 15 min for 60 min on ice, the samples were centrifuged at 4 °C at 12,000 / g for 30 min, and the supernatants of lysates were collected for the next experiment. The Bradford assay kit quantified the levels of the extracted proteins.

Digestion of Protein and TMT-labeling
One hundred micrograms of protein from each sample was precipitated overnight in the precooled acetone at −20 ° C and then centrifuged at 13,000 g for 30 min at 4 °C . The supernatant was removed, and precipitation was dissolved in a combination of 100 mM TEAB (triethylammonium bicarbonate), 5 uL 2 percent SDS, and 55 uL of ultrapure water. The protein reduction alkylation was performed at 55 ° C with the 5 uL of 200 mM trichloroethyl phosphate for 60 min, and then 5uL of 375 mM iodoacetamide was added in the dark and incubated at room temperature for 30 min. The sample was then precipitated for 3 hours in cold acetone (1:6) at −20 ° C and then placed in 100 uL of 100 mM triethyl ammonium bicarbonate. Finally, for 16-18 hours, each sample was digested with 2.5 uL of trypsin (1 ug / uL) at 37 ° C.
The TMT reagents were used to label the peptides in accordance with the manufacturer's instructions. Briefly, 41 uL of anhydrous acetonitrile was added to each TMT label tube (0.8 mg), and each sample was marked by adding 20 uL of TMT label and incubated at room temperature for 60 min. After that , 8 uL of 5 per cent   hydroxylamine was added to each sample and incubated for 15 min to end the   reaction. Samples of CH, CL, and CF buffalo ovaries were marked with 126, 127, and 130, respectively, for the first biological replicate; samples of CH, CL, and CF buffalo ovaries were marked with 128, 129, and 131, respectively, for the second biological replicate. The six samples with six tags were blended by equal quantity before downstream separation after labelling.

High pH Reverse Phase Separation and Nano-LC-MS/MS Analysis
Each sample was evaporated in a vacuum and resuspended in 50 μL buffer A (98% ddH2O, 2% acetonitrile; pH 10.0) and separated by high-pH reversed-phase liquid chromatography (RP-HPLC) column (2.1 ± 100 mm, three μm, 150 Å, C18). We used buffer A and buffer B (98 per cent acetonitrile, 2 per cent ddH20; pH 10.0) for a 60min linear gradient (4 per cent -20 per cent buffer B for 30 min, 20 percent -95 percent buffer B for 25 min, and 95 percent buffer B for 5 min). The fractions eluted from the column were collected every 1.5 min, and according to the maximum intensity, 20 fractions were gathered. The gathered fractions were desalted using a column of ZipTips C18 (Millipore, Billerica, MA, USA). The fractions were evaporated with a vacuum after desalination, and 10 uL of solvent A (2 per cent acetonitrile and 0.1 per cent formic acid) was used to resuspend the samples. In a trap column (PepMap RSLC C18 column, 50 um15 cm, 2 um nanoViper, Thermo Fisher Scientific, Bremen, Germany), a volume of 2 μl from each sample was loaded, and a maximum pressure (600 bar) was applied and a column for analysis (Acclaim ® PepMap100C18 column, 0.075 about 150 mm, 3μm, 100Å, Thermo Fisher Scientific, Bremen, Germany) was used for elution at a rate of 300 nL / min. Next, we used buffer A (2 percent acetonitrile and 0.1 percent formic acid) and buffer B (98 percent acetonitrile and 0.1 percent formic acid), followed by a gradient of 60 min

Proteomic Data Analysis and Bioinformatics
The raw information files were processed and quantified using Proteome Discoverer The GO (gene ontology) was used to annotate the recognized proteins, which consisted of BP (biological processes), CC (cellular elements) and MF (molecular functions), and the analysis was based on the UniProt-GOA database (http:/www.ebi.ac.uk/GOA/). The pathway analysis of differently expressed proteins was based on the KEGG (Encyclopedia of Genes and Genomes) database. The online KEGG Automatic Annotation Server (KAAS) service instruments were used to annotate the KEGG database description for each protein and to display the annotation outcomes using other online KEGG service instruments and the KEGG mapper. The interaction between groups of differential expression protein (DEPs) was extracted from the database of Search Tools for the Retrieval of Interacting Genes / Proteins (STRING). A high trust (0.7) was selected to draw the proteinprotein interaction map using Cytoscape 3.2.1 for the necessary interaction score and the active interaction sources, including text mining, tests, and databases.

The IDs conversion of identified proteins and homologous BLAST crossspecies
All buffalo protein IDs were transformed to Uniprot database mouse (Mus musculus) IDs and human (Homo sapiens) IDs using blast software (Blastall v2.2.26). The program name (comparison technique) is blastp, and the screening criteria is the expectation value < 1e-5. A similarity comparison of cross-species (buffalo, mouse and human) was also performed with the amino acid sequences of DEPs found in buffalo. In addition, the analysis of the GO and KEGG pathway was used to annotate the converted IDs from DEPs, including mouse and human.

Verification of Differentially Expressed Proteins Using Quantitative qRT-PCR
Total RNA obtained from ovaries at separate phases (CH, CL, and CF, n=3) was  Table S4.

Immunohistochemistry (IHC)
IHC was conducted on ovaries fixed with a polyoxymethylene (n=3). The ovaries embedded in paraffin were dewaxed and dehydrated, and 30 minutes of endogenous peroxidase activity was quenched with methanol comprising 3% H2O2. The paraffin parts were subjected to antigen extraction using microwave heating in the 0.01 M sodium citrate buffer (pH 6.0). These sections were blocked at room temperature for 2 hours using the 5 per cent BSA and then incubated overnight at 4 ° C with primary antibodies against PLK1, PGP, and HGS. The parts were then cleaned for 15min by PBS-TWeen-20, and this step was repeated three times. The parts were then incubated with secondary HRP-conjugated antibodies (CWBIO). The immunoreactive sites were visualized in brown after staining with diaminobenzidine (CWBIO), and the parts were stained with hematoxyl and assembled for observation using a bright-field microscope (Olympus, Japan). Instead of a primary antibody, negative checks were incubated with ordinary IgG.

Statistical Analysis
IBM SPSS Statistics version 17.0 and GraphPad Prism version 5.0 were used for statistical analysis. The research information was displayed as mean ± standard deviation (SD). A student t-test was used to compare the qRT-PCR outcomes of two groups (p < 0.05 was considered statistically significant).

Nano-LC−MS / MS analysis
We used the Bradford method to determine protein concentration and performed SDS-PAGE electrophoresis to check the impact of protein extraction. The findings showed that the electrophoretic bands had better differentiation degrees and were noticeable, indicating that protein extraction could meet the criteria of the downstream test (Fig. 1A). We then used the TMT sixplexTM Isobaric Label Reagent Set (Thermo ScientificTM, 90061, Labels from 126 to 131) to conduct this experiment. Samples of CH, CL, and CF buffalo ovaries were marked with 126, 127, and 130, respectively, for the first biological replicate; samples of CH, CL, and CF buffalo ovaries were marked with 128, 129, and 131, respectively, for the second biological replicate. After labelling, the six samples with six tags were mixed into a new pipe by equal quantity before downstream separation, which can decrease machine mistake (such as mass spectrometer and high pH inverse phase liquid chromatography tool) and experimental operation error. A graphic table of contents for this study is shown in Fig.5.
The specimens were then divided by high-pH reverse-phase fluid chromatography In all groups, a total of 2,103 proteins have been quantified. Taking CL as a control group, in the CH and CF groups respectively, proteins with TMT ratios > 1.5 or < 0.67 and P-values < 0.05 were regarded to be expressed differently. Through comparative study, 32 proteins with fold changes of ± 1.5 (log2 0.58) were recognized as differentially expressed in the CH group (Fig. 1C-I) and upregulated 21 proteins and downregulated 11 proteins (Additional File 1: Table S1). Of 116 proteins with a fold change of ≥ ± 1.5 (log2 0.58) recognized as differentially expressed in the CF group (Fig. 1C-II), 64 proteins were upregulated, and 52 proteins were downregulated (Additional File 1: Table S1).

Bioinformatic Analysis of Differentially Expressed Proteins
CH versus CL group differentially expressed proteins and CF versus CL group were categorized by gene ontology annotation based on three classifications: biological process, cellular component, and molecular function. The proteome of the GO annotation was obtained from the UniProt-GOA database (http:/www.ebi.ac.uk/GOA/) (additional file 2: table S2). After analyzing GO differentially expressed proteins from the CH vs CL group, we discovered that the biological procedures included wound healing, epidermal cell spreading, collagen biosynthetic process, endodermal cell differentiation, primary germ layer formation, and steroid biosynthetic process ( Fig. 2A). The molecular function of these proteins is essential in hydrolyase activity, carbon-oxygen lyase activity, and transferase activity (Fig. 2A). The findings of the cellular component analysis showed that these proteins were primarily a component of banded collagen fibril, a complex of collagen trimers, and mitochondrial nucleoid ( Fig. 2A).
A GO analysis was also performed on the CF versus CL group (Fig. 2B), and we discovered that the amount of GO conditions in this group was much higher than the CH versus CL group, which may imply that differentially expressed CF versus CL group proteins are more crucial than the CH vs CL group during the creation and regression of CL in water buffalo. An analysis of biological mechanisms recognized many mechanisms such as regulation of endodermal cell differentiation, cellular amine metabolic process, cytokine manufacturing involving the immune response and lipopolysaccharide-mediated signalling pathway, and prostanoid biosynthetic process (Fig. 2B). The molecular function of these proteins plays a significant part in the operation of motor microfilament and cargo receptors and in the binding of the protein complex, actin, cytoskeletal protein and antigen (Fig. 2B). In addition, the differentially expressed proteins from this group are primarily an element of collagen type V trimmer, fibrillar collagen trimmer, photoreceptor exterior section, and neuronal cell body (Fig. 2B). Interestingly, we discovered that many distinct kinds of enzymes appeared extremely expressed in the CL stage (Table 1), including kinase and phosphatase, which indicates that phosphorylated protein modification may play a significant role during ovarian CL formation. We performed validations for three proteins to further test our hypothesis: serine / threonine-protein kinase PLK1 (PLK1), glycerol-3-phosphate phosphatase (PGP), and hepatocyte growth factor-regulated tyrosine kinase substrate (HGS), all of which were upregulated in the CL stage.
Indeed, we evaluated differentially expressed proteins based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) database (Additional File 3: Table S3).
We discovered that the CH vs CL group's differentially expressed proteins were primarily enriched in autoimmune thyroid disease; hematopoietic cell lineage; valine, leucine and isoleucine degradation; PPAR signalling pathway; and phagosome (Fig. 2C). These pathways may be connected with CL formation. In addition, the associated processes were primarily engaged in the absorption and digestion of vitamins, protein, fat, and minerals in the CF vs CL group. Meanwhile, phagocytosis, cell cycle, steroid biosynthesis, and DNA replication also emerged during this era (Fig. 2D), indicating that these pathways may be involved in regulating regression in CL.

Analysis and Visualization of Identified Proteins
We also used the Cytoscape 3. Experiments are used for the input file in the Cytoscape 3.2.1 mapping software for high confidence (0.7) for the necessary interaction score and active interaction sources, including text mining. In addition, we performed KEGG's study online through the STRING database and exported significant pathways including actin cytoskeleton regulation, carbon metabolism, PI3K-Akt signalling pathway, oxidative phosphorylation, glycolysis/gluconeogenesis, and citrate cycle (TCA cycle). These pathway interactions were visualized using Cytoscape 3.2.1 mapping software (Fig.   3A). The BinGO package was used for gene ontology (GO) analysis for these recognized proteins and showed that the proteins were primarily involved in establishing protein localization (Fig. 3B), oxidation-reduction (Fig. 3C), and vesiclemediated transport (Fig. 3D). Notably, we discovered that six proteins were expressed differently. (Detailed data on these proteins appears in Table 2).

The homology and bioinformatic analysis of identified proteins among buffalo, human and mouse
To evaluate the conservation of the molecular system during the formation and regression of the corpus luteum in the ovary, we used homology and bioinformatic analysis to extend our outcomes to mouse and human. The GO and KEGG analysis was performed on the converted human IDs (Additional file 8: Figure S3A-B) and mouse (Additional file 8: Figure S3C-D), and we discovered that many biological procedures are involved in both species, such as metabolic process, reproductive process, cell aggregation, hormone secretion and cellular component organization or biogenesis. In addition, many pathways are enhanced in the converted protein IDs from humans and mouses, such as actin cytoskeleton regulation, MAPK signalling pathway, PI3K-Akt signalling pathway, PPAR signalling pathway and valine, leucine and isoleucine degradation. The findings of GO and KEGG pathway study disclosed that these pathways and biological procedures might be connected with human and mouse CL formation and regression. The visualization of proteinprotein interaction analysis from mouse and human transformed IDs was obtained from the database of Search Tools for Retrieving Interacting Genes / Proteins (STRING). High confidence (0.7) was selected to draw the protein-protein interaction map using Cytoscape 3.2.1 for the necessary interaction score and the active interaction sources, including text mining, tests, and databases. Also, we discovered that the same protein-protein interaction style of actin cytoskeleton regulation, oxidative phosphorylation and buffalo carbon metabolism were discovered both in humans (Additional file 8: Figure S3E) and mouse (Additional file 8: Figure S3F). We also examined the homologous of upregulated enzymes and proteins in the CL phase of ovary cross-species (Table 3), and it is essential to remember that these proteins demonstrate an elevated sequence identity (from 77.78 per cent to 95.65 per cent, Table 3) after homologous BLAST cross-species, indicating that these buffalo results appear during the creation and regression of corpus luteum.

Validation of Differentially Expressed Proteins
The three proteins (PLK1, PGP, HGS) that were upregulated in the CL phase were chosen for western blotting, RT-qPCR, and IHC. The findings of the three proteins ' western blot analysis are provided in Fig. 4B. Using Image J software, we evaluated the gray value of each western blot lane, and the findings were consistent with TMT quantification and transcription level analyses (Fig. 4C). All analyses used the CL group as control. We used immunohistochemistry localization analysis to investigate the prospective functions of the three further differentially expressed proteins because there is limited data about their location in the ovary at the CH, CL, and CF phases. As shown in the figure. 4A, PLK1 was articulated primarily around the follicle and in the ovary medulla ministry. PGP was expressed primarily in the follicle, particularly in the CL phase. PGP and HGS were both primarily situated in the surroundings of ovarian follicles and medulla ministry. The fluorescence intensity of the three proteins in the CL phase was higher compared to the CH and CF phase, particularly in the CL versus CF category, which suggests that the three proteins may play a significant part in the creation and regression of CL in the ovary, particularly for phosphorylated alteration during this biological process.

Discussion
In this research, a quantitative proteomics method based on TMT was implemented to screen differentially expressed proteins connected with CL formation and regression in the ovary of water buffalo. Three upregulated proteins in the CL phase (PLK1, PGP, and HGS) were verified using western blotting, quantitative RT-PCR, and ICH, indicating that they could play a vital part in the regular CL reproductive cycle.
Progesterone generated by the corpus luteum (CL) controls the synthesis of multiple endometrial proteins needed for embryonic implantation and growth [8]. The CL plays a vital role in regulating the oestrous cycle and maintaining pregnancy [9].
One of the most significant modifications during CL formation and regression is the shift in cellular reaction to external signals that enable the luteal cells to react to a fresh set of hormones. The most researched hormones are follicle-stimulating hormones, LH, prolactin (PRL), estrogen, and progesterone.
Fundamental understanding of the contribution of antioxidant enzyme mechanisms in progesterone synthesis and in maintaining luteal cell integrity in relation to pregnancy is still relatively limited. The processes engaged in CL formation, growth and regression are complicated and diverse in different mammalian species [8].
Proteomics can provide physicians and biologists with a better knowledge of the fundamental biological mechanisms engaged in maintaining CL's structural integrity and functional activity during the pre-implantation era [8]. In this research, we discovered that many kinds of enzymes, including kinase and phosphatase, emerged in the ovary CL phase (Table 1), which may imply that the formation and regression of CL are controlled by distinct enzymes, which may play a part in the activation of many signalling pathways. Many studies focused on activating signalling pathways during the formation and regression of CL.
The luteinization of luteal cells and cumulus cells mainly relies on the activation of the LH receptor (LH-R). Activation of LH-R binds to boost guanine nucleotide-binding protein Gs and emit adenylate cyclase (AC) signal, and cAMP is enhanced, and then cAMP-dependent PKA is activated [10]. Mitogen-activated protein kinases (MAPKs) comprise a superfamily kinase [11] recognized in multiple species CL. MAPK was involved in CREB phosphorylation. ERK1 and ERK2 have been recorded in porcine, bovine, rat, and human CL, and p38/MAPK has been shown to be expressed in rat and cow CL [12]. Jun N-terminal kinase was mostly researched in bovine CL [13].
PGF2α is essential for inhibiting progesterone synthesis in CL [14]. Although the uterus generates PGF2α, several studies reveal that the CL also generates PGF2α [15]. The expression levels of luteal COX-2 [16] and PGF2α [17] increase quickly and transiently in bovine animals after injection of PGF2α. This finding is also validated by the rise of COX-2 in mRNA levels [16]. Considering that PGF2α stimulates its own synthesis in CL, it can be thought that releasing PGF2α from the pulse of the uterus improves the production of PGF2α in CL, leading to an intensification in the luteolysis system.
In this research, we recognized many upregulated enzymes ( cytoplasmic, isovaleryl-CoA dehydrogenase, carnitine O-palmitoyltransferase 2. And prior trials have also discovered substantial proof that reactive oxygen species (ROS) are essential factors in determining the lifespan of CL [18] and that antioxidants play essential roles in CL physiology during the oestrous/menstrual cycle [19][20][21]. The rare in-vivo studies studying rat CL [22], women [19] and sheep Finally, we produced a validation of three recognized enzymes (PLK1, PGP and HGS) ( Fig. 4A-C) and they were upregulated in the luteal phase, and these enzymes may be engaged in regulating hormone secretion during luteal formation. We discovered that their expression modifications were compatible with proteomics outcomes, suggesting that during the formation and degradation of corpus luteum there was not only the involvement of different hormones earlier reported, but also that many enzymes play a part in this phase.
One differentially expressed kinase was PLK1, which was upregulated in the ovary's CL phase. PLK1 is a serine / threonine-protein kinase that has several significant tasks throughout the M phase of the cell cycle, including regulation of centrosome and spindle processing, removal of chromosome arm adhesions, inactivation of anaphase-promoting complex / cyclosome (APC / C) inhibitors, and regulation of mitotic manufacturing and cytokinesis [32]. Analysis of biological procedures linked to this protein showed that this kinase was involved in the G2/M transition during the mitotic cell cycle and in the negative regulation of cyclin-dependent protein serine/threonine kinase activity. It has long been thought that regulatory oocytederived molecules behave in the follicle to inhibit premature luteinization and limit progesterone biosynthesis. TGFβ superfamily members have the antiproliferative impacts of oocytes reported in vitro research [33]. The TGFβ superfamily has been proposed as luteinization inhibitors including activins and inhibins, TGFβ, BMPs (bone morphogenetic proteins), GDFs (growth/differentiation factors), and antimüller hormone [34]. Of these, the oocyte produces MP-15 [33] and GDF-9 [35].
Thus, we speculate that PLK1 upregulation may help with TGFβ-related enzymes by inhibiting luteinization during CL regression. Another chosen phosphatase in this study was PGP (phosphoglycolate phosphatase), which is a phosphatase engaged in such processes as glyoxylate and dicarboxylate metabolism. PGP acts as a particular Gro3P phosphatase (G3PP) and plays the central role in controlling glucose and lipid metabolism and signalling as well as a metabolic stress response in mammalian cells [36]. In CL, PGF2α works with the Gq-coupled receptor, which has been commonly discovered in mammalian species [37]. After activation, the Gqcoupled receptor creates PLC-mediated inositol triphosphate and diacylglycerol generation [38], followed by enhanced free intracellular calcium and PKC activity [39]. In addition, PGF2α activates the ERK1/2 signalling pathway in luteinized rat cells, which is dependent on enhanced free intracellular calcium [40]. The activation of PGF2α-and calcium-dependent ERK1/2 also results in the transcription factor JunD phosphorylate [40], which is mainly expressed in luteal cells [41]. Some studies report that, in relation to diacylglycerol and the MAPK signalling cascade, PGF2α can activate phospholipase D in luteal cells, generating phosphatidic acid [42]. These results indicate that PGP may play a significant role in the regression phosphorylation and dephosphorylation that happens during CL growth. HGS (hepatocyte growth factor-regulated tyrosine kinase substratum) was also a differentially expressed protein that was upregulated in the CL group in the current research. HGS is an evolutionarily preserved vesicular sorting protein belonging to ESCRT (an endosomal-sorting protein needed for transportation) [43]. HGS allows the molecule to anchor the inner surface of the lipid bilayer by having a FYVE domain [44] and by having a UIM domain it binds monoubiquitinated receptors [43].
There are reports that HGS can regulate hepatocyte growth factor or epidermal growth factor signalling through the endosomal/lysosomal pathway by associating with ubiquitinated receptors such as MET or EGFR [45]. HGS is also active in the transduction of cytokine-mediated intracellular signals and growth factors and is involved in BMP signalling through the phosphorylation of SMADs and TAK1 in early mouse embryos [46]. Targeted mutations in HGS cause immediate lethality to the early embryo after gastrulation [47]. Indeed, the connection between ACTR1 and SMAD2 is improved by HGS for ACTIVIN signalling [48]. These results show that HGS can be involved in a wide variety of extremely complex and multifaceted biological procedures connected with CL, ensuring a normal ovary reproductive cycle. We also performed the analysis of sequence similarity comparison and homology Blast acquired from buffalo, human and mouse, and discovered that the findings of this research in buffalo might have a substantial reference value for further study of the mechanism during the creation and regression of corpus luteum in human and mice ovaries. We speculate that regulatory processes for the creation and regression of corpus luteum in mammals may be conservative. Therefore, further studies are still needed to determine the role of antioxidant enzymes and protein phosphorylation-24 related enzymes in the function and integrity of CL during the peri-implantation era.   The interaction between the recognized proteins was studied using Cytoscape 3.2.1 software

Supplementary Files
This is a list of supplementary files associated with the primary manuscript. Click to download. Table S2.xlsx Table S1.xlsx Table S3.xlsx Table S4.xlsx Table S5.xlsx Figure S1.tif Figure S2.tif Figure S3.tif