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The in vitro effect of omentin-1 on the global proteome of granulosa cells from normal weight Large White and fat Meishan pigs

ABSTRACT: Granulosa cells (Gc) in the ovary play a vital role in nurturing oocyte development, producing estrogens, and maintaining the hormonal balance necessary for follicle maturation and successful ovulation. Omentin-1 (ITLN1) is an adipokine involved in energy metabolism and insulin resistance; its expression has been demonstrated in the ovary and varies depending on the degree of pig fatness. However, its effect on the global proteome of Gc has not been previously investigated. It was hypothesized that ITLN1 affects the abundance of proteins involved in key processes occurring in Gc during the mid-luteal phase of the estrous cycle in normal weight Large White and fat Meishan pigs, with potential variation based on the fat content of the pigs. Liquid chromatography‒tandem mass spectrometry (LC‒MS/MS) analysis of porcine Gc identified 208 statistically significant differentially abundant proteins (DAPs) in the Large White pigs, with 99 proteins upregulated, and 109 downregulated. In Meishan pigs, 42 statistically significant DAPs were identified, including 25 upregulated and 17 downregulated proteins. Western blotting and ELISA analyses of six selected DAPs (cadherin 5, farnesyl diphosphate synthase, fibroblast growth factor receptor 1, matrix metallopeptidase 2, phosphatidylinositol-5-phosphate 4-kinase type 2 gamma, and yes-associated protein 1) confirmed the accuracy and precision of the LC‒MS/MS methodology. The identified DAPs were associated with several key biological processes, including the estrogen signaling pathway, cell cycle and DNA replication, protein synthesis, transport and maturation, cytoskeleton dynamics, cell signaling, and hormonal regulation. The results indicate that ITLN1 has a modulatory influence on the porcine Gc proteome, which is dependent on fat content. This highlights the important role of ITLN1 in regulating ovarian functions.

INSTRUMENT(S):

ORGANISM(S): Sus Scrofa Domesticus (domestic Pig)

TISSUE(S): Ovary Cell Line

SUBMITTER: Agata Malinowska

LAB HEAD: Agnieszka Rak

PROVIDER: PXD061976 | Pride | 2025-07-23

REPOSITORIES: Pride

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			Action	DRS
	4032238resp_TMTpro_OVA_1.raw	Raw
	4032239resp_TMTpro_OVA_2.raw	Raw
	4032240resp_TMTpro_OVA_3.raw	Raw
	4032241resp_TMTpro_OVA_4.raw	Raw
	4032242resp_TMTpro_OVA_5.raw	Raw

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Project description:Background: The anterior pituitary plays a crucial role in endocrine regulation and homeostasis by orchestrating hormonal signaling, cellular growth, and functional adaptations. Adipokines regulate hypothalamic-pituitary-ovarian axis influencing hormone secretion and cellular processes. Omentin-1, an adipokine primarily expressed in visceral adipose tissue, is involved in metabolic and reproductive functions. However, its role in pituitary remains unknown. We hypothesize that omentin-1 modulates protein abundance in pituitary cells. This study aimed to investigate role of omentin-1 in regulating key pathways in anterior pituitary cells of pigs with different metabolic statuses: normal-weight Large White pigs and genetically obese Meishan pigs. Results: High-throughput proteomic analysis of porcine anterior pituitary cells exposed to omentin-1 revealed 230 differentially abundant proteins (30 downregulated and 3 upregulated in Large White pigs, as well as 107 downregulated and 90 upregulated in Meishan pigs), associated with 116 gene ontology terms. The most significantly altered proteins were associated with cell cycle and DNA replication, gene expression regulation and posttranscriptional/posttranslational modifications. Apoptotic and proliferative mechanisms varied between breeds, suggesting different metabolic adaptation strategies. In Large White pigs, CDK5RAP2 and SIX1 played a key role in transcriptional and proliferative regulation, whereas in Meishan pigs, stronger proliferative control was linked to AKT1S1 and RHOA. Moreover, Meishan pigs demonstrated a more dynamic regulation of apoptotic pathways (HTRA2, PARP2, DFFA), which may promote greater metabolic plasticity and better adaptation to physiological fluctuations. Additional in vitro experiments in Large White pigs showed that omentin-1 influenced key proliferative and apoptotic markers, downregulating cyclins D1, E1, A2, and B1, as well as caspase-3, while upregulating the anti-apoptotic protein BCL2, leading to an increased BCL2/BAX ratio. Further, omentin-1 modulated phosphorylation of selected kinases, including ERK1/2, AKT, AMPKα, and STAT3, in a time-dependent manner. It is also increased insulin receptor expression and promoted packaging and transport of secretory proteins. Conclusions: Proteomic and functional analyses revealed that omentin-1 regardless of breed exerts anti-proliferative and anti-apoptotic effects on porcine anterior pituitary cells, modulating key signaling pathways. These findings highlight that omentin-1 is a potential metabolic regulator influencing anterior pituitary function.

Project description:Porcine reproductive and respiratory syndrome caused by porcine reproductive and respiratory syndrome virus (PRRSV) is an infectious disease characterized by severe reproductive deficiency in pregnant sows, respiratory symptoms in piglets, and high mortality. In this study, we employed Affymetrix microarray chip technology to compare the gene expression profiles of lung tissue samples from Dapulian (DPL) pigs (a Chinese indigenous pig breed) and Duroc×Landrace×Yorkshire (DLY) pigs after infection with PRRSV. During infection with PRRSV, the DLY pigs exhibited the range of clinical features that typify the disease, while the DPL pigs exhibited only mild signs of the disease. The percentage of CD8+ T cells in the DPL pigs was significantly higher than that in the DLY pigs at 21 days post-infection (dpi) (p< 0.05). Interleukin (IL) 1 beta (IL-1β) and IL-2 levels showed significant differences between the DPL and DLY pigs at 0 and 7 dpi (p< 0.01). For IL-10, the DLY pigs had significantly higher values than the DPL pigs at 0 and 7 dpi (p< 0.01). Significant differences were apparent between the DPL and DLY pigs in terms of their tumor necrosis factor-alpha (TNF-α) and interferon (IFN)-gamma (IFN-γ) levels at 0 and 7 dpi (p< 0.01). Microarray data revealed 16 differentially expressed genes in the lung tissue samples from the DLY and DPL pigs (q≤5%), of which LOC100516029 and LOC100523005 were up-regulated in the PRRSV-infected DPL pigs, while the other 14 genes were down-regulated in the PRRSV-infected DPL pigs compared with the PRRSV-infected DLY pigs. The expression levels of 10 of the 16 genes, namely CCDC84, C6ORF52, THYMOSIN, PRVE, HSPCB, CYP2J2, AMPD3, TOR1AIP2, PTGES3, and ACOX3, were validated by real-time quantitative RT-PCR. This study provides a platform for further investigation of the molecular mechanisms underlying the differential immune responses to PRRSV infection in different breeds or lines of pig. We investigated the response of lung tissues from Dapulian (DPL) pigs (a Chinese indigenous pig breed) and Duroc×Landrace×Yorkshire (DLY) pigs infected with porcine reproductive and respiratory syndrome virus (strain JXA1) by using the Affymetrix Porcine Genome Array. Sixteen healthy 30-day-old weaned DPL pigs were selected from the Jiaxiang Dapulian farm, Jining City, China, and 15 healthy 30-day-old weaned DLY pigs were obtained from a commercial farm with high standards of animal health. These pigs were free from PRRSV, porcine circovirus type 2 (PCV2), pseudorabies virus (PRV), and classical swine fever virus (CSFV) as determined by ELISA tests for serum antibodies; the absence of PRRSV was also confirmed by real-time quantitative reverse transcription PCR (qRT-PCR). Pigs were randomly assigned into two groups and reared in separate places: the PRRSV-infected group consisted of 11 DPL and 10 DLY pigs, and the control group consisted of five DPL and five DLY pigs. Infections in the pigs proceeded via inoculation with 2 ml of a viral suspension of PRRSV (at a tissue culture infectious dose of 105) by dripping the solution into the nasal cavity of each pig. The control group was treated with an identical volume of PBS by the same method. Rectal temperatures and clinical examinations on the pigs were recorded daily during the experiment. Anticoagulant-treated blood and untreated blood samples were collected separately at 0, 7, 14, and 21 days post-infection (dpi) from the infected and control groups for assaying CD4+, CD8+, cytokine (interleukin (IL) 1 beta (IL-1β), IL-2, IL-10, interferon (IFN)-gamma (IFN-γ), tumor necrosis factor-alpha (TNF-α), and immunoglobulin G (IgG) protein levels. Lung samples for microarray analysis and real-time qRT-PCR analysis were collected from six infected DLY and DPL pigs (three pigs for each breed) immediately post-slaughter at 28 dpi. Total RNA was isolated from lung tissue samples and purified using an RNeasy Mini kit according to the manufacturer’s protocol. RNA was prepared using the GeneChip (AFF-900623) one cycle target for the labeling and control reagents, and the labeled RNA was hybridized in an Affymetrix Hybridization Oven 640 for sequencing.

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The in vitro effect of omentin-1 on the global proteome of granulosa cells from normal weight Large White and fat Meishan pigs

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