Inhibitory effects of epidermal growth factor on progesterone production of ovarian granulosa cells in Tsaiya duck (Anas platyrhynchos var. domestica)

Abstract

1. The purpose of this study was to investigate the effects of epidermal growth factor (EGF) on progesterone (P(4)) secretion from ovarian granulosa cells in Tsaiya ducks (Anas platyrhynchos var. domestica). 2. We obtained the largest (F1) follicle from Tsaiya duck, the granulosa layer was separated and the cells were isolated according to their proximity to the germinal disc. 3. The granulosa cells were cultured in vitro, the culture media and the cells were used to determine P(4) and steroidogenic enzyme concentrations, respectively. 4. P(4) concentrations were decreased in cultured granulosa cells taken proximal to the germinal disc (GD) compared to those distal to the germinal disc (NGD). 5. EGF inhibited both basal and ovine luteinising hormone (oLH)-induced P(4) concentrations. It also inhibited the P(4) secretion via protein kinase A (PKA) pathway when cultured with GD and NGD granulose cells (mixed together) in vitro. 6. Western blot results showed decreased concentrations of cytochrome P450 side-chain cleavage (P450scc) enzyme, 3β-hydroxysteroid dehydrogenase (3β-HSD) enzyme and steroid acute regulatory (StAR) protein expression, when the cells were co-treated with EGF and oLH. 7. The inhibitory effect of oLH-induced P(4) production was attenuated by EGF by the addition of MAP-erk kinase (MEK) inhibitor (PD98059) suggests EGF may inhibit P(4) production by affecting via the mitogen-activated protein kinase (MAPK) pathway.

Isolation and identification of ovarian theca‐interstitial cells and granulose cells of immature female mice

“…Multiple methods have been developed to isolate and culture TICs, one of which is Percoll density gradient centrifugation, as described by previous researchers (Magoffin and Erickson, ; Magoffin, ). Mechanical and enzymatic digestion methods have also been developed to collect GCs (Cannon et al, ; Chiu et al, ). Each method has its own advantages; however, it is important to develop methods for harvesting TICs and GCs from the same ovary.…”

Astaxanthin Protects Steroidogenesis from Hydrogen Peroxide-Induced Oxidative Stress in Mouse Leydig Cells

“…The progesterone assay was modified from a direct EIA using G7, an IgM monoclonal antibody with specific affinity of 1.1 × 10 10 M, as described previously [ 34 , 35 , 36 ]. G7 exhibits cross-reactivity of <0.01% with BSA and other steroids including pregnenolone, testosterone, estradiol, and estrone.…”

“…The separated proteins were transferred to a polyvinylidene fluoride membrane. The membrane was blocked by incubating in PBS containing 0.01% Tween-20 (PBST) and 2.5% BSA for 8 h at room temperature, followed by incubation with rabbit polyclonal primary antibodies specific for StAR (1:10,000 dilution) [ 36 , 39 ], CYP11A1 (1:5000 dilution), HSD3B1 (1:10,000 dilution) [ 36 , 40 ], and ACTB (1:5000 dilution) (MAB1501, Millipore, Temecula, CA, USA) in PBST overnight at 4 °C. After four washes with PBST, the membrane was incubated for 2 h with peroxidase conjugated goat anti-rabbit IgG (1:7000 dilution; Jackson ImmunoResearch, West Grove, PA, USA).…”

Curcumin downregulates 8-br-cAMP-induced steroidogenesis in mouse Leydig cells by suppressing the expression of Cyp11a1 and StAR independently of the PKA-CREB pathway

“…The separated proteins were transferred to a polyvinylidene fluoride membrane (Bio-Rad, Hercules, CA, USA) using a semi-dry blotter (Bio-Rad) at 25 V/0.4 A for 30 minutes. The membrane was blocked in TBST containing 5% skim milk for 1 hour at room temperature, and followed by an incubation with primary antibodies specific for StAR [12,13], CYP11A1 [12,14], phosphorylated-PKA substrate (Cell Signaling Technology, Boston, MA, USA), and ACTB (Millipore, Menlo Park, CA, USA), prepared in 1% BSA with TBST overnight at 4°C. After a threetime TBST wash, the membrane was incubated with horseradish peroxidase-conjugated anti-rabbit or antimouse IgG (Santa Cruz Biotechnology, Dallas, Texas, USA) in 5% skim milk with TBST for another 1 hour.…”

“…The antibodies for enzyme immunoassay of testosterone and progesterone were developed in our laboratory as described before [11,12]. Briefly, all samples and standards were co-incubated with horseradish peroxidaseconjugated progesterone or testosterone in antibodycoated 96-well plates.…”