Fig 1: Co-localization of omentin-1 (OMNT1) in porcine tropic cells on days 10–12 of the estrous cycle in Large White pigs. The immunoreactivity of all hormones was determined by fluorescent immunohistochemistry. Left column: OMNT1 expression, visualized by Alexa Fluor 488 as green fluorescence; middle column: tropic hormone expression, visualized by Alexa Fluor 594 as red fluorescence; right column: immunofluorescent double labeling of OMNT1 and GH (A–A″), PRL (B-B”), ACTH (C–C″), TSH (D–D″), LH (E-E″), and FSH (F–F″). Arrows indicate examples of dual-labeled cells or lack of co-expression between OMNT1 and ACTH (C–C′). Scale bar: 20 μm. Section thickness 5 μm.
Fig 2: Effect of GnRH, LH, and FSH at 100 ng/mL on omentin-1 (OMNT1) protein expression (A–C) and levels in the culture medium (A′–C′) of AP cells collected on days 2–3, 10–12, 14–16, and 17–19 of the estrous cycle of Large White pigs. The protein expression was analyzed using western blot, and the results are presented as a densitometric normalized ratio relative to the β-actin abundance. The OMNT1 concentration in the culture medium was evaluated using ELISA. The results of at least four independent replicates are presented as means ± SEM for each group. Data were compared by an unpaired two-tailed Student’s t test (***p < 0.001, ****p < 0.0001). Representative blots are attached as Supplementary Fig. 1.
Fig 3: The in vitro effect of visfatin on the secretion of gonadotropins: LH (A) and FSH (B) by porcine anterior pituitary cells after treatment with inhibitors of the INSR, AKT/PI3K, MAPK/ERK1/2, and AMPK signaling pathways. This study was conducted on anterior pituitary glands harvested from pigs (n = 5) during the mid-luteal phase (the phase of the highest corpus luteum activity throughout the cycle; days 10–12). After isolation, anterior pituitary cells were preincubated for 72 h and then incubated for 24 h with visfatin (VIS) at the physiological dose (10 ng/mL, VIS 10), or/and S961—an inhibitor of the insulin receptor pathway (INSR, 1 μM), or/and LY294002—an inhibitor of the protein kinase B/phosphatidylinositol 3-kinase pathway (AKT/PI3K, 20 μM), or/and U0126—an inhibitor of the mitogen-activated protein kinase/extracellular signal-regulated kinase pathway (MAPK/ERK1/2, 10 μM), or/and Dorsomorphin/Compound C (DMPH)—inhibitor of the adenosine monophosphate-activated protein kinase pathway (AMPK, 10 μM) or serum-free medium alone—CONTROL. The concentrations of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) in the culture media were determined using commercially available ELISA kits. Data were analyzed using a multifactorial analysis of variance (ANOVA) followed by Dunnett’s post hoc test. The results are presented as graphs (mean ± S.E.M.). Bars with different superscripts are significantly different at p < 0.05.
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