Inhibin production by porcine granulosa and luteal cells: development and biological validation of a RIA

1989 ◽  
Vol 120 (4) ◽  
pp. 511-518 ◽  
Author(s):  
U. Michel ◽  
H. Jarry ◽  
M. Metten ◽  
W. Wuttke
Keyword(s):  
Detection Limit ◽  
Granulosa Cells ◽  
Follicular Fluid ◽  
Inhibin A ◽  
Serum Free ◽  
Luteal Cells ◽  
Biological Validation ◽  
Ovarian Cells ◽  
Progesterone Secretion ◽  
Porcine Granulosa Cells

Abstract. We describe the development and biological validation of a radioimmunoassay for immuno- and bioactive porcine inhibin. A synthetic 1-32 porcine inhibin peptide was used to raise an antiserum and Tyr-1-32 peptide as tracer. As standard we employed porcine follicular fluid calibrated with the 1-32 α-inhibin. Medium obtained from serum-free cultured porcine granulosa cells was chromatographed on Superose S-12 and Mono-Q. Resulting fractions were analysed for inhibin bio- and immunoreactivity. It is shown that granulosa cells produce at least two types of bioactive inhibins, one being also immunoactive in our RIA. We studied secretion of immunoreactive inhibin from porcine ovarian cells under various conditions: Inhibin secretion from mature and immature granulosa cells can be stimulated by FSH, whereas hCG enhances inhibin secretion only from mature granulosa cells. During extended time of culture, the capability of granulosa cells to secrete inhibin is reduced. In contrast, progesterone secretion from these cells increases; this is due to spontaneous functional luteinization. This assumption is supported by the low inhibin secretion of luteal cells in comparison to granulosa cells. Intracellular inhibin content in luteal cells is below detection limit of the RIA, whereas granulosa cells contain readily detectable amounts of this hormone.

DIRECT STIMULATION OF BOVINE OVARIAN PROGESTERONE SECRETION BY LOW CONCENTRATIONS OF a-INTERFERON

1992 ◽  
Vol 135 (2) ◽  
pp. R5-R8 ◽  
Author(s):  
M.R. Luck ◽  
J.A Shale ◽  
J.H. Payne
Keyword(s):  
Corpus Luteum ◽  
Granulosa Cells ◽  
Dna Content ◽  
Hormone Secretion ◽  
Direct Stimulation ◽  
Serum Free ◽  
Ovarian Cells ◽  
Progesterone Secretion ◽  
Low Concentrations ◽  
Stimulation Of

ABSTRACT The ruminant conceptus secretes proteins during early pregnancy which maintain the corpus luteum. These trophoblast proteins are related to the αII-interferons and prevent luteolysis indirectly by disrupting the secretion of endometrial prostaglandin. Although trophoblast interferons appear to be largely confined to the uterine lumen, it remains possible that they also act peripherally. This report describes in vitro studies which suggest that interferon may influence hormone secretion by the ovary directly. The study employed i) a well defined serum-free culture model in which bovine granulosa cells secrete the luteal hormones progesterone and oxytocin, and ii) serum-free and serum-supplemented cultures of cells from early CL. Dose-response experiments were performed using bovine recombinant α-interferon (brIFN). Progesterone and oxytocin secretions were measured over 4-5 days of culture and DNA content was also determined. Low concentrations of brIFN (10−15 mol/l to 10−11 mol/l) stimulated progesterone secretion by granulosa cells by up to three fold, without significantly affecting oxytocin concentrations or culture DNA content. Concentrations of 10−10 mol/l to 10−7 mol/l suppressed progesterone secretion in a log dose-related manner (r=0.97) with evidence of toxicity (lower oxytocin concentrations and significantly reduced DNA compared with controls). Progesterone secretion by luteal cells in serum-free culture was stimulated in the presence of 10−15 mol/l brIFN, whilst high concentrations again caused inhibition. The data show that ovarian cells can respond directly to low concentrations of interferon-like proteins. They also demonstrate an inhibition by high doses which may mask the stimulatory effect in this model. The data suggest that the early corpus luteum may be directly influenced by α-interferon. A stimulation of progesterone, but not of oxytocin, secretion from ovarian cells would be consistent with a role for conceptus proteins in maintaining the corpus luteum of pregnancy.

Effect of cortisol on neurophysin I/oxytocin and peptidyl glycine-α-amidating mono-oxygenase mRNA expression in bovine luteal and granulosa cells

2013 ◽  
Vol 16 (2) ◽  
pp. 231-239
Author(s):  
A. Ziolkowska ◽  
J. Mlynarczuk ◽  
J. Kotwica
Keyword(s):  
Mrna Expression ◽  
Granulosa Cells ◽  
Molecular Mechanisms ◽  
Hormone Secretion ◽  
Oestrous Cycle ◽  
Luteal Cells ◽  
Ru 486 ◽  
Ovarian Cells ◽  
Key Genes ◽  
Ovary Function

Abstract Cortisol stimulates the synthesis and secretion of oxytocin (OT) from bovine granulosa and luteal cells, but the molecular mechanisms of cortisol action remain unknown. In this study, granulosa cells or luteal cells from days 1-5 and 11-15 of the oestrous cycle were incubated for 4 or 8 h with cortisol (1x10-5, 1x10-7 M). After testing cell viability and hormone secretion (OT, progesterone, estradiol), we studied the effect of cortisol on mRNA expression for precursor of OT (NP-I/OT) and peptidyl glycine-α-amidating mono-oxygenase (PGA). The influence of RU 486 (1x10-5 M), a progesterone receptor blocker and inhibitor of the glucocorticosteroid receptor (GR), on the expression for both genes was tested. Cortisol increased the mRNA expression for NP-I/OT and PGA in granulosa cells and stimulated the expression for NP-I/OT mRNA in luteal cells obtained from days 1-5 and days 11-15 of the oestrous cycle. Expression for PGA mRNA was increased only in luteal cells from days 11-15 of the oestrous cycle. In addition, RU 486 blocked the cortisol-stimulated mRNA expression for NP-I/OT and PGA in both types of cells. These data suggest that cortisol affects OT synthesis and secretion in bovine ovarian cells, by acting on the expression of key genes, that may impair ovary function.

Increasing progesterone secretion in human granulosa-luteal cells induced by human follicular fluid*,**

1993 ◽  
Vol 8 (1) ◽  
pp. 46-52 ◽  
Author(s):  
Shalom Bar-Ami ◽  
Camellia Khoury ◽  
Efrat Zlotkin ◽  
Joseph M. Brandes
Keyword(s):  
Follicular Fluid ◽  
Human Follicular Fluid ◽  
Luteal Cells ◽  
Progesterone Secretion

scholarly journals Suppression of Notch Signaling Stimulates Progesterone Synthesis by Enhancing the Expression of NR5A2 and NR2F2 in Porcine Granulosa Cells

Genes ◽  
2020 ◽  
Vol 11 (2) ◽  
pp. 120
Author(s):  
Rihong Guo ◽  
Fang Chen ◽  
Zhendan Shi
Keyword(s):  
Notch Signaling ◽  
Granulosa Cells ◽  
Butyl Ester ◽  
Notch Pathway ◽  
Notch Signaling Pathway ◽  
Progesterone Secretion ◽  
Expression Of Genes ◽  
Porcine Granulosa Cells ◽  
Progesterone Synthesis ◽  
Progesterone Biosynthesis

The conserved Notch pathway is reported to be involved in progesterone synthesis and secretion; however, the exact effects remain controversial. To determine the role and potential mechanisms of the Notch signaling pathway in progesterone biosynthesis in porcine granulosa cells (pGCs), we first used a pharmacological γ-secretase inhibitor, N-(N-(3,5-difluorophenacetyl-l-alanyl))-S-phenylglycine t-butyl ester (DAPT), to block the Notch pathway in cultured pGCs and then evaluated the expression of genes in the progesterone biosynthesis pathway and key transcription factors (TFs) regulating steroidogenesis. We found that DAPT dose- and time-dependently increased progesterone secretion. The expression of steroidogenic proteins NPC1 and StAR and two TFs, NR5A2 and NR2F2, was significantly upregulated, while the expression of HSD3B was significantly downregulated. Furthermore, knockdown of both NR5A2 and NR2F2 with specific siRNAs blocked the upregulatory effects of DAPT on progesterone secretion and reversed the effects of DAPT on the expression of NPC1, StAR, and HSD3B. Moreover, knockdown of NR5A2 and NR2F2 stimulated the expression of Notch3. In conclusion, the inhibition of Notch signaling stimulated progesterone secretion by enhancing the expression of NPC1 and StAR, and the two TFs NR5A2 and NR2F2 acted as downstream TFs of Notch signaling in regulating progesterone synthesis.

scholarly journals Temporal and hormonal regulation of inhibin protein and subunit mRNA expression by post-natal and immature rat ovaries

2000 ◽  
Vol 166 (2) ◽  
pp. 339-354 ◽  
Author(s):  
AE Drummond ◽  
M Dyson ◽  
E Thean ◽  
NP Groome ◽  
DM Robertson ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Cell Cultures ◽  
Granulosa Cells ◽  
Inhibin B ◽  
Alpha Subunit ◽  
Inhibin A ◽  
Preantral Follicles ◽  
Subunit Mrna ◽  
Ovarian Cells ◽  
Old Rats

The contribution of specific follicle populations to dimeric inhibin production and inhibin subunit mRNA expression by the rat ovary has been investigated in two model systems, granulosa cells isolated from 25-day-old diethylstilboestrol (DES)-treated rats and post-natal rat ovaries, dispersed in culture or whole ovaries, using specific two-site immunoassays and 'real time' PCR. Media from FSH-stimulated granulosa cell cultures fractionated by gel filtration and RP-high performance liquid chromatography revealed two predominant peaks of alpha subunit activity which were attributed to alpha subunit and 31 k dimeric inhibin-A. The corresponding inhibin-B levels were low. FSH stimulation did not alter the ratio of inhibin-A:alpha subunit produced by granulosa cells. All three inhibin subunit mRNAs were expressed by granulosa cells, with eight-fold more alpha subunit mRNA relative to either of the beta subunits. Administration of DES to immature rats prior to the isolation of granulosa cells from the ovary led to beta(A) and beta(B) mRNA expression being down-regulated in the absence of any significant change in alpha subunit expression by the granulosa cells. Inhibin-A, -B and -alpha subunit were produced by basal and stimulated cultures of ovarian cells prepared from 4-, 8- and 12-day-old rats, indicating that primary, preantral and antral follicles contribute to total inhibin production. Consistent with these results, follicles within these ovaries expressed all three inhibin subunit mRNAs, with maximal expression observed in the ovaries of 8-day-old rats. The appearance of antral follicles in the ovary at day 12 led to a decline in the mRNA levels of each of the subunits but was most evident for the beta subunits. There was a profound influence of secondary preantral follicles on dimeric inhibin-A production, with FSH stimulation increasing inhibin-A relative to alpha subunit levels in cultures of ovarian cells prepared from 8-day-old rats. Thus, preantral follicles exposed to FSH contribute significantly to beta(A) subunit production by the ovary. In contrast, primary and preantral follicles did not produce inhibin-B in response to FSH stimulation. Transforming growth factor-beta (TGF-beta) enhanced, in a time-dependent manner, the production of the inhibin forms by ovarian cells in culture, although inhibin-B production was not responsive until day 8. The simultaneous treatment of ovarian cell cultures with FSH and TGF-beta elicited the greatest increases in production of all the inhibin forms. In summary, ovaries of 4-, 8- and 12-day-old rats expressed inhibin subunit mRNAs and produced dimeric inhibin-A and -B and free alpha subunit. Preantral follicles (day-8 ovarian cell cultures) were particularly sensitive to stimulation by FSH and TGF-beta and had a substantial capacity for inhibin production. The production of oestrogen by follicles may be instrumental in regulating inhibin production given that beta subunit mRNA expression was down-regulated by DES. The mechanisms by which inhibin-A and inhibin-B are individually regulated are likely to be similar during the post-natal period, when folliculogenesis is being established, and diverge thereafter, when inhibin-A becomes the predominant form in the fully differentiated ovary.

Porcine granulosa cells in suspension culture. II. Luteinization and hCG responsiveness

1981 ◽  
Vol 240 (6) ◽  
pp. E622-E629 ◽  
Author(s):  
A. R. LaBarbera ◽  
R. J. Ryan
Keyword(s):  
Adenylate Cyclase ◽  
Granulosa Cells ◽  
Culture Medium ◽  
Cultured Cells ◽  
Cyclase Activity ◽  
Adenylate Cyclase Activity ◽  
Short Term ◽  
Progesterone Secretion ◽  
Porcine Granulosa Cells ◽  
Ovine Prolactin

Granulosa cells from small follicles were cultured as suspensions in spinner flasks for 10 days in the absence or presence of follicle-stimulating hormone (FSH). With or without FSH, the cultured cells ultrastructurally resembled luteinized cells to different degrees. FSH increased progesterone accumulation in the culture medium. Ovine prolactin potentiated the effect of FSH in terms of the quantity of progesterone produced and the duration of accumulation. FSH increased acute human chorionic gonadotropin (hCG)-responsive progesterone secretion in short-term incubations of cultured granulosa cells. Responsiveness of FSH-cultured cells was maximal at day 4; that of control cultured cells was maximal at day 6. Adenylate cyclase activity of homogenates of cells cultured for 4, 6, or 8 days was measured. FSH induced in cultured cells an hCG sensitivity of the adenylate cyclase enzyme. These results indicate that FSH induced hCG-responsive progesterone secretion and hCG-responsive adenylate cyclase activity that correlate with ultrastructural signs of luteinization and with the previously reported FSH induction of hCG receptors.

Follicular fluid exosomes: Important modulator in proliferation and steroid synthesis of porcine granulosa cells

2021 ◽  
Vol 35 (5) ◽  
Author(s):  
Chenfeng Yuan ◽  
Zheng Li ◽  
Yun Zhao ◽  
Xin Wang ◽  
Lu Chen ◽  
...  
Keyword(s):  
Granulosa Cells ◽  
Follicular Fluid ◽  
Steroid Synthesis ◽  
Porcine Granulosa Cells
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