scholarly journals Expression and effect of NAMPT (visfatin) on progesterone secretion in hen granulosa cells

Reproduction ◽  
2015 ◽  
Vol 150 (1) ◽  
pp. 53-63 ◽  
Author(s):  
Mélodie Diot ◽  
Maxime Reverchon ◽  
Christelle Ramé ◽  
Yannick Baumard ◽  
Joëlle Dupont
Keyword(s):  
Granulosa Cells ◽  
Specific Inhibitor ◽  
Follicular Development ◽  
Rt Pcr ◽  
Nicotinamide Phosphoribosyltransferase ◽  
Theca Cells ◽  
Protein Levels ◽  
Progesterone Secretion ◽  
Ovulatory Follicles

In mammals, nicotinamide phosphoribosyltransferase (NAMPT) is an adipokine produced by adipose tissue that is found in intracellular and extracellular compartments. The intracellular form of NAMPT is a nicotinamide phosphoribosyltransferase, whereas the extracellular form is considered an adipokine. In humans, NAMPT regulates energy metabolism and reproductive functions, such as ovarian steroidogenesis. To date, no study has investigated the role of NAMPT in hen ovaries. We investigated whether NAMPT is present in hen ovarian follicles and its role in granulosa cells. Using RT-PCR, western blotting and immunocytochemistry, we detected mRNA transcripts and proteins related to NAMPT in theca and granulosa cells from pre-ovulatory follicles. Using RT-PCR, we demonstrated that mRNA NAMPT levels were higher in granulosa cells than they were in theca cells and that during follicle development, theca cell levels decreased, whereas levels remained unchanged in granulosa cells. NAMPT protein quantities were significantly higher in theca cells than they were in granulosa cells, but they were unchanged during follicular development. Plasma NAMPT levels, as determined by ELISA and immunoblotting, were significantly lower in adult hens than they were in juveniles. In vitro, treatment with human recombinant NAMPT (100 ng/ml, 48 h) halved basal and IGF1-induced progesterone secretion, and this was associated with a reduction in STAR and HSD3B protein levels and MAPK3/1 phosphorylation levels in granulosa cells. These effects were abolished by the addition of FK866, a specific inhibitor of NAMPT enzymatic activity. Moreover, NAMPT had no effect on granulosa cell proliferation. In conclusion, NAMPT is present in hen ovarian cells and inhibits progesterone production in granulosa cells.

scholarly journals Regulation and action of fibroblast growth factor 17 in bovine follicles

2009 ◽  
Vol 202 (3) ◽  
pp. 347-353 ◽  
Author(s):  
M F Machado ◽  
V M Portela ◽  
C A Price ◽  
I B Costa ◽  
P Ripamonte ◽  
...  
Keyword(s):  
Growth Factor ◽  
Fibroblast Growth Factor ◽  
Granulosa Cells ◽  
Cumulus Cells ◽  
Mrna Abundance ◽  
Fibroblast Growth ◽  
Theca Cells ◽  
Progesterone Secretion ◽  
Igf I

Fibroblast growth factor 17 (FGF17) is a member of the FGF8 subfamily that appears to be relevant to folliculogenesis and oogenesis, as the prototype member FGF8 is an oocyte-derived protein that signals to cumulus cells. FGF8 has structural and receptor-binding similarities to FGF17, whose expression in the ovary has not been reported. In this study, we demonstrate localization of FGF17 protein to the oocyte of preantral follicles, and to the oocyte and granulosa cells of antral follicles. Real-time PCR demonstrated the presence of mRNA in oocytes and, to a lesser extent, in granulosa and theca cells. FGF17 mRNA abundance was low in granulosa and theca cells from healthy follicles and increased significantly in atretic follicles. Addition of FSH or IGF-I to granulosa cells in vitro decreased FGF17 mRNA abundance, and treatment with FGF17 inhibited estradiol and progesterone secretion from granulosa cells in relation to control cultures without these additives. We conclude that FGF17 is a potential mediator of granulosa cell differentiation.

scholarly journals sRAGE Downregulates the VEGF Expression in PCOS Ovarian Granulosa Cells

2020 ◽  
Author(s):  
Jingyan Wang ◽  
Yichun Guan ◽  
Yi Liu ◽  
Liang Wang ◽  
Zhan Zhang ◽  
...  
Keyword(s):  
Growth Factors ◽  
Growth Factor ◽  
Granulosa Cells ◽  
Ovarian Tissue ◽  
Rt Pcr ◽  
Vegf Expression ◽  
Vitro Fertilization ◽  
Protein Levels ◽  
Ovarian Granulosa Cells

Abstract Objective High expression of VEGF in ovarian tissue, serum and follicular fluid of PCOS women is involved in the physiological and pathogenesis processes of PCOS. Our objective was to investigate the effect of sRAGE on VEGF expression and EGF-like growth factor in PCOS ovarian granulosa cells.Methods We collected ovarian granulosa cells of PCOS patients who underwent in vitro fertilization (IVF). Then treatment ovarian granulosa cells with different concentrations of sRAGE. Levels of VEGF, AREG, BTC and EREG mRNA were examined by quantitative RT-PCR. The protein levels of VEGF, AREG, BTC and EREG were measured by ELISA.Results Treatment with sRAGE decrease the production of VEGF, and the effects were dependent on the concentrations of sRAGE (P < 0.05). Simultaneously, the expression of the EGF-like growth factors AREG, BTC and EREG were decreased, and the expression were dependent on the concentrations of sRAGE (P < 0.05).Conclusions sRAGE may downregulate VEGF expression in PCOS ovarian granulosa cells,and EGF-like growth factor pathway may be involved in this process.

scholarly journals Localization of gonadotrophin-releasing hormone I, bradykinin and their receptors in the ovaries of non-mammalian vertebrates

Reproduction ◽  
2007 ◽  
Vol 133 (5) ◽  
pp. 969-981 ◽  
Author(s):  
Padmasana Singh ◽  
Amitabh Krishna ◽  
Rajagopala Sridaran
Keyword(s):  
Granulosa Cells ◽  
Follicular Development ◽  
Cell Types ◽  
Physiological Significance ◽  
Rt Pcr ◽  
Releasing Hormone ◽  
Theca Cells ◽  
Gonadotrophin Releasing Hormone

GnRH I and its receptors have been demonstrated in the ovaries of various vertebrates, but their physiological significance in reproductive cascade is fragmentary. Bradykinin is a potent GnRH stimulator in the hypothalamus. In the present study, the presence of GnRH I and its receptor, and bradykinin and its receptor in the ovaries of non-mammalian vertebrates were investigated to understand their physiological significance. GnRH I immunoreactivity in the ovaries of fish, frog, reptile and bird were mainly found in the oocyte of early growing follicles and granulosa cells and theca cells of previtellogenic follicles. Vitellogenic follicles showed mild GnRH immunoreactivity. GnRH I-receptor and bradykinin were localized in the same cell types of the ovaries of these vertebrates. The presence of GnRH I, GnRH I-receptor and bradykinin in the ovaries of these vertebrates was confirmed by immunoblotting. The presence of GnRH I mRNA was demonstrated in the ovary of vertebrates using RT-PCR. The ovaries of reptiles and birds showed significantly higher intensity of immunoreactivity for GnRH I-receptor as compared with the fish and amphibian. This may have a correlation with the higher yolk content in the ovary of reptile and bird. These results suggest the possibility of GnRH I and bradykinin as important regulators of follicular development and vitellogenesis in the vertebrate ovary.

scholarly journals Expression of FOXL2 and RSPO1 in Hen Ovarian Follicles and Implication of Exogenous Leptin in Modulating Their mRNA Expression in In Vitro Cultured Granulosa Cells

Animals ◽  
2019 ◽  
Vol 9 (12) ◽  
pp. 1083 ◽  
Author(s):  
Weihe Niu ◽  
Izhar Hyder Qazi ◽  
Sichen Li ◽  
Xiaoling Zhao ◽  
Huadong Yin ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Granulosa Cells ◽  
Ovarian Follicles ◽  
Control Group ◽  
Follicle Development ◽  
Theca Cells ◽  
Increasing Trend ◽  
Group A ◽  
Ovulatory Follicles

In this study, using a laying hen model, we determined the expression of FOXL2 and RSPO1 in different central and peripheral tissue and ovarian follicles at different stages of development. At the same time, mRNA expression of both genes in granulosa and theca cells harvested from follicles at different stages of folliculogenesis was also evaluated. Finally, we assessed the effect of leptin treatment on expression of FOXL2 and RSPO1 in in vitro cultured granulosa cells harvested from 1–5 mm to F3–F1 follicles. Our RT-qPCR results revealed that a comparatively higher expression of FOXL2 and RSPO1 was observed in ovary, hypothalamus, and pituitary. Abundant mRNA expression of FOXL2 was observed in small prehierarchical follicles (1–1.9 and 2–2.9 mm follicles; p < 0.05), whereas mRNA expression of RSPO1 showed an increasing trend in large hierarchical follicles (F5–F1), and its abundant expression was observed in post-ovulatory follicles. FOXL2 mRNA expression was stable in granulosa cells harvested from 3–5 mm to F4 follicles, and exhibited a significantly higher expression in large hierarchical follicles. Conversely, relatively low mRNA expression of FOXL2 was observed in theca cells. RSPO1 mRNA expression was relatively lower in granulosa cells; however, theca cells exhibited a significantly higher mRNA expression of RSPO1 in F4 to F1 follicles. In the next experiment, we treated the in vitro cultured granulosa cells with different concentrations (1, 10, 100, and 1000 ng/mL) of exogenous leptin. Compared to the control group, a significant increase in the expression of FOXL2 was observed in groups treated with 1, 10, and 100 ng/mL leptin, whereas expression of RSPO1 was increased in all leptin-treated groups. When treated with 100 ng/mL leptin, FOXL2 and RSPO1 expression was upregulated in cultured granulosa cells harvested from both large hierarchical (F3–F1) and small prehierarchical follicles (1–5 mm). Based on these findings and evidence from mainstream literature, we envisage that FOXL2 and RSPO1 genes (in connection with hypothalamic-hypophysis axis) and leptin (via modulation of FOXL2 and RSPO1 expression) might have significant physiological roles, at least in part, in modulating the ovarian mechanisms, such as follicle development, selection, and steroidogenesis in laying hens.

RELATIONSHIP BETWEEN THE ENDOCRINE ENVIRONMENT WITHIN THE GRAAFIAN FOLLICLE AND THE SUBSEQUENT RATE OF PROGESTERONE SECRETION BY HUMAN GRANULOSA CELLS IN VITRO

1975 ◽  
Vol 66 (3) ◽  
pp. 391-400 ◽  
Author(s):  
K. P. McNATTY ◽  
R. S. SAWERS
Keyword(s):  
Steroid Hormones ◽  
Mitotic Activity ◽  
Granulosa Cells ◽  
Follicular Development ◽  
Human Granulosa Cells ◽  
Long Period ◽  
Progesterone Secretion ◽  
Graafian Follicle ◽  
High Concentrations

SUMMARY The steroidogenic potential of granulosa cells harvested from human Graafian follicles containing varying concentrations of pituitary and steroid hormones was examined. The mitotic activity and production of progesterone by granulosa cells in vitro was found to be correlated with their hormonal environment at the time of harvesting. Only cells from follicles containing some FSH and high concentrations of oestradiol underwent spontaneous mitosis in vitro. However, mitosis could be induced by adding FSH and high concentrations of oestradiol to the culture, provided that the concentration of LH was low. Cells harvested from follicles containing LH, FSH and high concentrations of oestradiol secreted significantly more progesterone than cells from follicles which did not contain all three hormones. It is suggested that after the initiation of follicular development by FSH, a long period of exposure (8–10 days) to both FSH and oestradiol is necessary before the maximum biosynthetic capacity of granulosa cells is achieved; this synthetic potential is then only realized under the influence of LH and prolactin. Premature exposure to LH inhibits both the mitotic activity and the steroidogenic potential of these cells.

scholarly journals The hedgehog-patched signaling pathway and function in the mammalian ovary: a novel role for hedgehog proteins in stimulating proliferation and steroidogenesis of theca cells

Reproduction ◽  
2009 ◽  
Vol 138 (2) ◽  
pp. 329-339 ◽  
Author(s):  
Leon J Spicer ◽  
Satoko Sudo ◽  
Pauline Y Aad ◽  
Lora Shuo Wang ◽  
Sang-Young Chun ◽  
...  
Keyword(s):  
Granulosa Cells ◽  
Follicular Development ◽  
Mrna Abundance ◽  
Rt Pcr ◽  
Theca Cells ◽  
Qrt Pcr ◽  
Hedgehog Proteins ◽  
Patched 1 ◽  
And Function ◽  
First Time

The expression of hedgehog (Hh) genes, their receptor, and the co-receptor in mice, rat, and bovine ovaries were investigated. RT-PCR of ovarian transcripts in mice showed amplification of transcripts for Indian (Ihh) and desert (Dhh) Hh, patched 1 (Ptch1), and smoothened (Smo) genes. Semi-quantitative RT-PCR and northern blot analyses showed that whole ovarianIhhandDhhtranscripts decreased 4–24 h after hCG versus 0–48 h after pregnant mares serum gonadotrophin treatment in mice, whereas mousePtch1andSmotranscripts were expressed throughout the gonadotropin treatments. Quantitative real-time RT-PCR (qRT-PCR) revealed that the expression of the Hh-patched signaling system withIhhmRNA abundance in granulosa cells was greater, whereasSmoandPtch1mRNA abundance was less in theca cells of small versus large follicles of cattle. In cultured rat and bovine theca-interstitial cells, qRT-PCR analyses revealed that the abundance ofGli1andPtch1mRNAs were increased (P<0.05) with sonic hedgehog (SHH) treatment. Additional studies using cultured bovine theca cells indicated that SHH induces proliferation and androstenedione production. IGF1 decreasedIhhmRNA abundance in bovine granulosa cells. The expression and regulation ofIhhtranscripts in granulosa cells andPtch1mRNA in theca cells suggest a potential paracrine role of this system in bovine follicular development. This study illustrates for the first time Hh activation of Gli1 transcriptional factor in theca cells and its stimulation of theca cell proliferation and androgen biosynthesis.

scholarly journals Two pathways for prostaglandin F2α synthesis by the primate periovulatory follicle

Reproduction ◽  
2008 ◽  
Vol 136 (1) ◽  
pp. 53-63 ◽  
Author(s):  
Brandy L Dozier ◽  
Kikuko Watanabe ◽  
Diane M Duffy
Keyword(s):  
Granulosa Cell ◽  
Granulosa Cells ◽  
Prostaglandin F2α ◽  
Follicular Development ◽  
Protein Levels ◽  
Human Granulosa Cells ◽  
Cell Conversion ◽  
Gonadotropin Surge ◽  
Cell Expression

Prostaglandin E2 (PGE2) has been identified as a PG necessary for ovulation, but the ovulatory gonadotropin surge also increases PGF2α levels in primate periovulatory follicles. To better understand the role of PGF2α in ovulation, pathways utilized for PGF2α synthesis by the primate follicle were examined. Monkeys were treated with gonadotropins to stimulate multiple follicular development; follicular aspirates and whole ovaries were removed before and at specific times after administration of an ovulatory dose of hCG to span the 40 h periovulatory interval. Human granulosa cells were also obtained (typically 34–36 h after hCG) from in vitro fertilization patients. PGF2α can be synthesized from PGH2 via the aldo-keto reductase (AKR) 1C3. AKR1C3 mRNA and protein levels in monkey granulosa cells were low before hCG and peaked 24–36 h after hCG administration. Human granulosa cells converted PGD2 into 11β-PGF2α, confirming that these cells possess AKR1C3 activity. PGF2α can also be synthesized from PGE2 via the enzymes AKR1C1 and AKR1C2. Monkey granulosa cell levels of AKR1C1/AKR1C2 mRNA was low 0–12 h, peaked at 24 h, and returned to low levels by 36 h after hCG administration. Human granulosa cell conversion of [3H]PGE2 into [3H]PGF2α was reduced by an AKR1C2-selective inhibitor, supporting the concept that granulosa cells preferentially express AKR1C2 over AKR1C1. In summary, the ovulatory gonadotropin surge increases granulosa cell expression of AKR1C1/AKR1C2 and AKR1C3. Both of these enzyme activities are present in periovulatory granulosa cells. These data support the concept that follicular PGF2α can be synthesized via two pathways during the periovulatory interval.

scholarly journals sRAGE downregulates the VEGF expression through EGF-like growth factor signal pathway in PCOS ovarian granulosa cells

2020 ◽  
Author(s):  
Jingyan Wang ◽  
Yichun Guan ◽  
Yi Liu ◽  
Liang Wang ◽  
Mingze Du ◽  
...  
Keyword(s):  
Growth Factor ◽  
Granulosa Cells ◽  
Ovarian Tissue ◽  
Signal Pathway ◽  
Rt Pcr ◽  
Vegf Expression ◽  
Vitro Fertilization ◽  
Protein Levels ◽  
Ovarian Granulosa Cells

Abstract Objective High expression of VEGF in ovarian tissue, serum and follicular fluid of PCOS women is involved in the physiological and pathogenesis processes of PCOS. Our objective was to investigate the effect of sRAGE on VEGF expression and EGF-like growth factor in PCOS ovarian granulosa cells. Methods We collected ovarian granulosa cells of PCOS patients who underwent in vitro fertilization (IVF). Then treatment ovarian granulosa cells with different concentrations of sRAGE. Levels of VEGF, AREG, BTC and EREG mRNA were examined by quantitative RT-PCR. The protein levels of VEGF, AREG, BTC and EREG were measured by ELISA. Results Treatment with sRAGE decrease the production of VEGF, and the effects were dependent on the concentrations of sRAGE ( P <0.05). Simultaneously, the expression of the EGF-like growth factors AREG, BTC and EREG were decreased, and the expression were dependent on the concentrations of sRAGE ( P <0.05). Conclusions sRAGE may downregulate VEGF expression via EGF-like growth factor pathway in PCOS ovarian granulosa cells.

scholarly journals AMP-activated protein kinase activation modulates progesterone secretion in granulosa cells from hen preovulatory follicles

2006 ◽  
Vol 190 (1) ◽  
pp. 85-97 ◽  
Author(s):  
Lucie Tosca ◽  
Sabine Crochet ◽  
Pascal Ferré ◽  
Fabienne Foufelle ◽  
Sophie Tesseraud ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Granulosa Cells ◽  
Cholesterol Metabolism ◽  
Specific Inhibitor ◽  
Negative Regulator ◽  
Star Protein ◽  
Theca Cells ◽  
Progesterone Secretion ◽  
Preovulatory Follicles ◽  
Amp Activated Protein Kinase

AMP-activated protein kinase (AMPK) is a fuel sensor in glucose, lipid, and cholesterol metabolism. Using RT-PCR and Western blot, AMPK subunits mRNAs (α1/2, β1/2, and γ1/2) and proteins (α1/2 and β1/2) can be found in the hen preovulatory follicles and precisely in both granulosa and theca cells. These preovulatory follicles are organized in a hierarchy according to their size (F5/6 to F1). The smallest number (F1) corresponds to the largest size and the latest mature stage. Phosphorylation of AMPKα on Thr172 and of acetyl-CoA carboxylase on Ser79 are higher in F4 and F3 than in F1 granulosa cells. However, they are not affected in F4–F1 theca cells. Treatment with 1 mM 5-amino-imidazole-4-carboxyamide-1-β-d-ribofuranoside (AICAR), an activator of AMPK, dose dependently increased phosphorylation of AMPKα on Thr172 in primary F3/4 and F1 granulosa cells. In the absence of FSH, AICAR treatment increased progesterone, P450 side chain cleavage and steroidogenic acute regulatory (StAR) production in both F3/4 and F1 granulosa cells. However, in the presence of FSH, AICAR treatment for 36 h increased progesterone secretion, StAR protein levels and reduced extracellular signal-regulated kinase (ERK)1/2 phosphorylation in F3/4 granulosa cells. Opposite data were observed in F1 granulosa cells. Adenovirus-mediated expression of dominant-negative AMPK totally restored the effects of AICAR on FSH-induced progesterone secretion, StAR protein production, and ERK1/2 phosphorylation in F3/4 and F1 granulosa cells. Using a specific inhibitor of ERK1/2 (U0126), we also showed that this kinase is a negative regulator of the FSH-induced progesterone secretion in F3/4 and F1 granulosa cells, suggesting that AICAR-mediated AMPK activation modifies FSH-induced progesterone secretion differently through the ERK1/2 signaling pathway in hen F3/4 and F1 granulosa cells.

Functional effects of Tribbles homolog 2 in bovine ovarian granulosa cells†

2020 ◽  
Vol 102 (6) ◽  
pp. 1177-1190
Author(s):  
Aly Warma ◽  
Kalidou Ndiaye
Keyword(s):  
Western Blot ◽  
Granulosa Cells ◽  
Developmental Stages ◽  
Polyclonal Antibodies ◽  
Differentially Expressed Gene ◽  
Follicular Development ◽  
Mapk Signaling ◽  
Tissue Growth ◽  
Ovulatory Follicles

Abstract Tribbles homologs (TRIB) 1, 2, and 3 represent atypical members of the serine/threonine kinase superfamily. We previously identified TRIB2 as a differentially expressed gene in granulosa cells (GCs) of bovine preovulatory follicles. The current study aimed to further investigate TRIB2 regulation and study its function in the ovary. GCs were collected from follicles at different developmental stages: small antral follicles (SF), dominant follicles (DF) at day 5 of the estrous cycle, and hCG-induced ovulatory follicles (OFs). RT-qPCR analyses showed greater expression of TRIB2 in GC of DF as compared to OF and a significant downregulation of TRIB2 steady-state mRNA amounts by hCG/LH, starting at 6 h through 24 h post-hCG as compared to 0 h. Specific anti-TRIB2 polyclonal antibodies were generated and western blot analysis confirmed TRIB2 downregulation by hCG at the protein level. In vitro studies showed that FSH stimulates TRIB2 expression in GC. Inhibition of TRIB2 using CRISPR/Cas9 resulted in a significant increase in PCNA expression and an increase in steroidogenic enzyme CYP19A1 expression, while TRIB2 overexpression tended to decrease GC proliferation. TRIB2 inhibition also resulted in a decrease in transcription factors connective tissue growth factor (CTGF) and ankyrin repeat domain-containing protein 1 (ANKRD1) expression, while TRIB2 overexpression increased CTGF and ANKRD1. Additionally, western blot analyses showed reduction in ERK1/2 (MAPK3/1) and p38MAPK (MAPK14) phosphorylation levels following TRIB2 inhibition, while TRIB2 overexpression increased p-ERK1/2 and p-p38MAPK. These results provide evidence that TRIB2 modulates MAPK signaling in GC and that TRIB2 could act as a regulator of GC proliferation and function, which could affect steroidogenesis during follicular development.

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