scholarly journals Ovulatory Induction of SCG2 in Human, Nonhuman Primate, and Rodent Granulosa Cells Stimulates Ovarian Angiogenesis

Endocrinology ◽  
2018 ◽  
Vol 159 (6) ◽  
pp. 2447-2458 ◽  
Author(s):  
Patrick R Hannon ◽  
Diane M Duffy ◽  
Katherine L Rosewell ◽  
Mats Brännström ◽  
James W Akin ◽  
...  
Keyword(s):  
Granulosa Cells ◽  
Messenger Rna ◽  
Growth Factor Receptor ◽  
Endothelial Cell Migration ◽  
Receptor Signaling ◽  
Signaling Mechanism ◽  
Lh Receptor ◽  
Vitro Fertilization

Abstract The luteinizing hormone (LH) surge is essential for ovulation, but the intrafollicular factors induced by LH that mediate ovulatory processes (e.g., angiogenesis) are poorly understood, especially in women. The role of secretogranin II (SCG2) and its cleaved bioactive peptide, secretoneurin (SN), were investigated as potential mediators of ovulation by testing the hypothesis that SCG2/SN is induced in granulosa cells by human chorionic gonadotropin (hCG), via a downstream LH receptor signaling mechanism, and stimulates ovarian angiogenesis. Humans, nonhuman primates, and rodents were treated with hCG in vivo resulting in a significant increase in the messenger RNA and protein levels of SCG2 in granulosa cells collected early during the periovulatory period and just prior to ovulation (humans: 12 to 34 hours; monkeys: 12 to 36 hours; rodents: 4 to 12 hours post-hCG). This induction by hCG was recapitulated in an in vitro culture system utilizing granulosa-lutein cells from in vitro fertilization patients. Using this system, inhibition of downstream LH receptor signaling pathways revealed that the initial induction of SCG2 is regulated, in part, by epidermal growth factor receptor signaling. Further, human ovarian microvascular endothelial cells were treated with SN (1 to 100 ng/mL) and subjected to angiogenesis assays. SN significantly increased endothelial cell migration and new sprout formation, suggesting induction of ovarian angiogenesis. These results establish that SCG2 is increased in granulosa cells across species during the periovulatory period and that SN may mediate ovulatory angiogenesis in the human ovary. These findings provide insight into the regulation of human ovulation and fertility.

Effect of gonadotrophins on progesterone secretion by cultured granulosa cells obtained from human preovulatory follicles

1985 ◽  
Vol 110 (3) ◽  
pp. 401-407 ◽  
Author(s):  
T. Hillensjö ◽  
A. Sjögren ◽  
B. Strander ◽  
L. Nilsson ◽  
M. Wikland ◽  
...  
Keyword(s):  
Granulosa Cells ◽  
Maximal Effect ◽  
Tubal Infertility ◽  
Vitro Fertilization ◽  
Follicular Maturation ◽  
Progesterone Secretion ◽  
Preovulatory Follicles ◽  
Ultrasound Guided Puncture

Abstract. Granulosa cells were obtained from human preovulatory follicles in 31 women undergoing in vitro fertilization and embryo transfer due to tubal infertility. Follicular maturation was stimulated and synchronized by treatment with Clomiphene or human menopausal gonadotrophin (hMG), or both, plus human chorionic gonadotrophin (hCG). Follicles were aspirated by ultrasound guided puncture approximately 34–36 h after the hCG injection. The granulosa cells were washed and suspended in modified medium 199 containing 10% foetal bovine serum and cultured as monolayers for 6–8 days in the absence and presence of hormones and reactants. Progesterone formation was analyzed by RIA. In general, the cells underwent morphological luteinization and secreted high amount of progesterone. Under basal conditions the secretion of progesterone was highest during the first 2 days in culture and then gradually declined. Progesterone secretion was stimulated by human LH, hCG and the adenylate cyclase stimulator forskolin, with a maximal effect between days 2–6. The β-adrenergic agonist isoproteronol in preliminary experiments potentiated the stimulatory effect of hCG but had no own stimulatory effect. No clear differences in progesterone secretion or responsiveness to in vitro stimulation relating to the various in vivo stimulation protocols were found.

scholarly journals 21-Hydroxylase-Derived Steroids in Follicles of Nonobese Women Undergoing Ovarian Stimulation for In Vitro Fertilization (IVF) Positively Correlate With Lipid Content of Luteinized Granulosa Cells (LGCs) as a Source of Cholesterol for Steroid Synthesis

2014 ◽  
Vol 99 (4) ◽  
pp. 1299-1306 ◽  
Author(s):  
Marli Amin ◽  
Ariel Simerman ◽  
Michele Cho ◽  
Prapti Singh ◽  
Christine Briton-Jones ◽  
...  
Keyword(s):  
In Vitro Fertilization ◽  
Mrna Expression ◽  
Granulosa Cells ◽  
Lipid Content ◽  
Mineralocorticoid Receptor ◽  
Steroid Synthesis ◽  
Vitro Fertilization ◽  
Receptor Mrna

Context: Mineralocorticoid synthesis by the nonhuman primate periovulatory follicle enhances luteinization. Whether a similar event occurs in women undergoing in vitro fertilization (IVF) is unknown. Objective: The objective of the study was to determine whether human luteinized granulosa cells (LGCs) produce mineralocorticoids derived from 21-hydroxylase activity and also express mRNA for 21-hydroxylase and the mineralocorticoid receptor. Design: This was a prospective cohort study. Setting: The study was conducted at an academic center. Patients: LGC lipid content and follicle fluid (FF) hormone analysis was performed on 27 nonobese IVF women. LGCs from six additional nonobese IVF women were used for gene expression studies. Intervention: At oocyte retrieval, FF was aspirated from the first follicle (≥16 mm in size) of each ovary and pooled LGCs were collected. Main Outcome Measures: FF steroid analysis was performed by liquid chromatography-tandem mass spectrometry. LGCs were stained with lipid fluorescent dye BODIPY FL C16 to estimate lipid content by confocal microscopy as a cholesterol source for steroidogenesis in vivo. Quantitative real-time PCR was performed using LGCs to detect 21-hydroxylase and mineralocorticoid receptor mRNA expression. Pearson correlation coefficients determined associations between FF steroid levels and LGC lipid content. Results: FF levels of the 21-hydroxylase-derived steroids, 11-deoxycorticosterone [DOC, 39.97, median (13.94–63.02) ng/mL] and 11-deoxycortisol [11DOC, 2.07 (0.69–5.01) ng/mL], along with the 21-hydroxylase precursor 17-hydroxyprogesterone [1268.21 (493.26–3558.39) ng/mL], positively correlated with LGC lipid content (84 ± 43 fluorescent units/sample) (P ≤ .05, all steroids). 21-Hydroxylase and mineralocorticoid receptor mRNA expression was detected in LGCs. Conclusions: Human LGCs likely synthesize 21-hydroxylase-derived mineralocorticoids from cholesterol-containing lipid in vivo to promote postovulatory luteinization via mineralocorticoid receptor-mediated events.

scholarly journals Bisphenol S Impaired Human Granulosa Cell Steroidogenesis in Vitro

2020 ◽  
Vol 21 (5) ◽  
pp. 1821 ◽  
Author(s):  
Sarah Amar ◽  
Aurélien Binet ◽  
Ophélie Téteau ◽  
Alice Desmarchais ◽  
Pascal Papillier ◽  
...  
Keyword(s):  
Granulosa Cells ◽  
Messenger Rna ◽  
Cellular Proliferation ◽  
Structural Analog ◽  
Bisphenol S ◽  
Vitro Fertilization ◽  
Progesterone Secretion ◽  
Nanomolar Range ◽  
Follicular Fluids

Bisphenol S (BPS) is a structural analog of the endocrine disruptor bisphenol A (BPA); it is the main BPA replacement in the plastics industry. Previous studies have shown that BPA and BPS exhibit similar effects on reproduction in fish and rodent species. BPS reportedly alters steroidogenesis in bovine granulosa cells. Luteinised granulosa cells collected from 59 women who were undergoing an in vitro fertilization procedure were cultured for 48 h in the presence or absence of BPS (10 nM, 100 nM, 1 µM, 10 µM or 50 µM). BPS exposure was investigated by assessing follicular fluids from these 59 women for their BPS content. Culture medium, cells, total messenger RNA (mRNA) and total protein extracted from the luteinised granulosa cells were examined for oestradiol and progesterone secretion, cellular proliferation, viability, gene expression, steroidogenic enzyme expression and cell signaling. BPS was measured in follicular fluids using mass spectrometry. Exposure of granulosa cells to 10 or 50 µM BPS for 48 h induced a 16% (p = 0.0059) and 64% (p < 0.0001) decrease, respectively, in progesterone secretion; 50 µM BPS decreased oestradiol secretion by 46% (p < 0.0001). Ten µM BPS also tended to reduce CYP11A1 protein expression by 37% (p = 0.0947) without affecting HSD3B1 and CYP19A1 expression. Fifty µM BPS increased ERRγ expression. Environmental levels of BPS (nanomolar range) did not induce changes in steroidogenesis in human granulosa cells. The effects of BPS were observed after only 48 h of BPS exposure. These acute effects might be similar to chronic effects of physiological BPS levels.

The role of intraovarian regulators in the aetiology of polycystic ovarian syndrome

1996 ◽  
Vol 5 (3) ◽  
pp. 151-168 ◽  
Author(s):  
Ghanim Almahbobi ◽  
Alan O Trounson
Keyword(s):  
Granulosa Cells ◽  
Follicular Development ◽  
Aromatase Activity ◽  
Lh Receptor ◽  
Follicular Maturation ◽  
Developmental Capacity ◽  
Follicle Selection

The present review demonstrates that the availability of bioactive FSH and LH in PCOS is normal and that granulosa cells of PCO are not apoptotic and instead hyperexpress functional FSH receptors and may possess intact aromatase activity. Consequently, these cells respond excessively to exogenous FSH stimulation and produce high amounts of oestradiol both in vivo and in vitro. The altered developmental capacity of follicles from PCO in vivo is most likely due to the abnormal follicular milieu of PCO and the culminating effects of intrafollicular inhibitors and stimulators. The failure of ovarian oestradiol production and follicular maturation to dominance in vivo may be due to a mechanism that interferes with the function of FSH, such as intraovarian steroids and growth factors. It has previously been shown that EGF and TGFα have inhibitory actions on follicular development, aromatization and LH receptor formation. In contrast, EGF enhances early follicular recruitment and growth. Therefore, it is hypothesized that EGF/TGFα may have a causal relationship in the mechanisms of anovulatory infertility in women with PCOS. Thus, an aberration in the regulation of follicular fluid EGF and/or TGFα may result in reduced numbers of granulosa cells, cessation of follicle selection and ultimately in the creation and maintenance of PCOS. The exact mechanism by which the hyperfunction of EGF/TGFα occurs and the trigger for this hyperactivity in the ovary remain to be determined. An experimental animal model may be required to assist such investigations in the future.

Prior exposure to gonadotrophins prevents the subsequent antigonadotrophic actions of cloprostenol by a cyclic AMP-dependent mechanism in cultured human granulosa cells

1991 ◽  
Vol 131 (2) ◽  
pp. 319-325 ◽  
Author(s):  
A. E. Michael ◽  
G. E. Webley
Keyword(s):  
Cyclic Amp ◽  
Corpus Luteum ◽  
Granulosa Cells ◽  
Early Pregnancy ◽  
Prior Exposure ◽  
Vitro Fertilization ◽  
Human Granulosa Cells ◽  
Dependent Mechanism

ABSTRACT The antigonadotrophic action of a prostaglandin F2α analogue, cloprostenol, has been investigated in human granulosa cells obtained from cycles stimulated for in-vitro fertilization and induced to secrete luteal quantities of progesterone by culture in serum-supplemented medium. Cells were exposed to conditions which may mimic those occurring in early pregnancy to establish the roles of human chorionic gonadotrophin (hCG) versus LH and that of cyclic AMP (cAMP) in the anti-gonadotrophic action of cloprostenol. When human granulosa cells were cultured in the absence of treatment for 3 days, exposure to cloprostenol had no effect on basal progesterone production but inhibited hCG-stimulated progesterone (60% decrease; P<0·01), hCG-stimulated cAMP (40% decrease; P < 0·05) and the progesterone response to dibutyryl cAMP (dbcAMP; 70% decrease; P < 0·01), suggesting pre- and post-cAMP sites of cloprostenol action. The inhibitory actions of cloprostenol were prevented when the granulosa cells were either continuously exposed to treatment from the start of culture or pre-exposed for 3 days to maximum concentrations of LH, hCG, dbcAMP or 8-bromo-cAMP. We conclude that prior exposure either in vivo or in vitro to LH or hCG prevents the subsequent antigonadotrophic action of cloprostenol via a cAMP-dependent mechanism. Prevention of the antigonadotrophic action of cloprostenol after exposure to hCG may be a mechanism through which CG prevents regression of the corpus luteum in early pregnancy, while the suppressive effect of LH pretreatment may account for the refractory response of the early corpus luteum to cloprostenol following the midcycle LH surge. Journal of Endocrinology (1991) 131, 319–325

In vitro Stimulation of Granulosa Cells by a Combination of Different Active Ingredients of Unkei-to

2004 ◽  
Vol 32 (04) ◽  
pp. 569-578 ◽  
Author(s):  
Wen-Shu Sun ◽  
Atsushi Imai ◽  
Keiko Tagami ◽  
Michiyo Sugiyama ◽  
Tatsuro Furui ◽  
...  
Keyword(s):  
Granulosa Cells ◽  
Response Curves ◽  
Vitro Fertilization ◽  
Human Granulosa Cells ◽  
Estradiol Secretion ◽  
Paeoniae Radix ◽  
In Vivo Effects ◽  
Stimulation Of

Unkei-to is widely used in traditional Japanese herbal medicine for its ovulation-inducing effect. In the present study, we investigated the in vivo effects of Unkei-to and its compounds on the steroidogenesis and cytokine secretion in human granulosa cells. Unkei-to stimulated the secretions of 17β-estradiol and progesterone from highly luteinized granulosa cells obtained from in vitro fertilization patients; the stimulated effect on estradiol secretion occurred with 0.3 μg/ml, while a significant effect on progesterone secretion was obtained at 10 μg/ml. The Unkei-to stimulation of estradiol secretion could be accounted for by the effects of its ingredients, Shakuyaku (paeoniae radix, Paeonia lactiflora Pallas) and Keihi (cinnamomi cortex, Cinnamomum cassia Blume); while dose response curves for Unkei-to and Keihi to induce progesterone production were superimposable. Exposure of the cells to Unkei-to caused dose-dependent increases in the concentrations of interleukin (IL)-1β, IL-6 and IL-8 in the culture medium. Similar results were obtained when cells were incubated with the ingredient Ninjin (ginseng radix, Panax ginseng C.A. Meyer), but not Shakuyaku and Keihi. These results indicate that Unkei-to has direct stimulatory effects on human granulosa cells to stimulate the steroidogenesis and secretion of cytokines (IL-1β, IL-6 and IL-8). The various beneficial actions of Unkei-to on the ovary may result from a combination of different ingredient herbs with different stimulatory effects on both steroidogenesis and the ovulatory process within the ovary, as well as stimulatory effect on the hypothalamus-pituitary axis.

scholarly journals A role for gelsolin in actuating epidermal growth factor receptor-mediated cell motility.

1996 ◽  
Vol 134 (3) ◽  
pp. 689-698 ◽  
Author(s):  
P Chen ◽  
J E Murphy-Ullrich ◽  
A Wells
Keyword(s):  
Cell Motility ◽  
Membrane Fraction ◽  
Growth Factor Receptor ◽  
Cell Movement ◽  
Bound State ◽  
Cell Surface Receptor ◽  
Pharmacologic Agent ◽  
Signaling Mechanism

Phospholipase C-gamma (PLC gamma) is required for EGF-induced motility (Chen, P., H. Xie, M.C. Sekar, K.B. Gupta, and A. Wells. J. Cell Biol. 1994. 127:847-857); however, the molecular basis of how PLC gamma modulates the actin filament network underlying cell motility remains undetermined. We propose that one connection to the actin cytoskeleton is direct hydrolysis of PIP2 with subsequent mobilization of membrane-associated actin modifying proteins. We used signaling-restricted EGFR mutants expressed in receptor-devoid NR6 fibroblast cells to investigate whether EGFR activation of PLC causes gelsolin mobilization from the cell membrane in vivo and whether this translocation facilitates cell movement. Gelsolin anti-sense oligonucleotide (20 microM) treatment of NR6 cells expressing the motogenic full-length (WT) and truncated c'1000 EGFR decreased endogenous gelsolin by 30-60%; this resulted in preferential reduction of EGF (25 nM)-induced cell movement by &gt; 50% with little effect on the basal motility. As 14 h of EGF stimulation of cells did not increase total cell gelsolin content, we determined whether EGF induced redistribution of gelsolin from the membrane fraction. EGF treatment decreased the gelsolin mass associated with the membrane fraction in motogenic WT and c'1000 EGFR NR6 cells but not in cells expressing the fully mitogenic, but nonmotogenic c'973 EGFR. Blocking PLC activity with the pharmacologic agent U73122 (1 microM) diminished both this mobilization of gelsolin and EGF-induced motility, suggesting that gelsolin mobilization is downstream of PLC. Concomitantly observed was reorganization of submembranous actin filaments correlating directly with PLC activation and gelsolin mobilization. In vivo expression of a peptide that is reported to compete in vitro with gelsolin in binding to PIP2 dramatically increased basal cell motility in NR6 cells expressing either motogenic (WT and c'1000) or nonmotogenic (c'973) EGFR; EGF did not further augment cell motility and gelsolin mobilization. Cells expressing this peptide demonstrated actin reorganization similar to that observed in EGF-treated control cells; the peptide-induced changes were unaffected by U73122. These data suggest that much of the EGF-induced motility and cytoskeletal alterations can be reproduced by displacement of select actin-modifying proteins from a PIP2-bound state. This provides a signaling mechanism for translating cell surface receptor-mediated biochemical reactions to the cell movement machinery.

Toxicity and efficacy evaluation of multiple targeted polymalic acid conjugates for triple-negative breast cancer treatment

2020 ◽  
Author(s):  
Lungwani Muungo
Keyword(s):  
Triple Negative ◽  
Growth Factor Receptor ◽  
Engineered Nanoparticles ◽  
Efficacy Evaluation ◽  
Dual Action ◽  
Epidermal Growth ◽  
Laminin 411 ◽  
Immunologic Analysis

Engineered nanoparticles are widely used for delivery of drugs but frequently lack proof of safetyfor cancer patient's treatment. All-in-one covalent nanodrugs of the third generation have beensynthesized based on a poly(β-L-malic acid) (PMLA) platform, targeting human triple-negativebreast cancer (TNBC). They significantly inhibited tumor growth in nude mice by blockingsynthesis of epidermal growth factor receptor, and α4 and β1 chains of laminin-411, the tumorvascular wall protein and angiogenesis marker. PMLA and nanodrug biocompatibility and toxicityat low and high dosages were evaluated in vitro and in vivo. The dual-action nanodrug and singleactionprecursor nanoconjugates were assessed under in vitro conditions and in vivo with multipletreatment regimens (6 and 12 treatments). The monitoring of TNBC treatment in vivo withdifferent drugs included blood hematologic and immunologic analysis after multiple intravenousadministrations. The present study demonstrates that the dual-action nanoconju-gate is highlyeffective in preclinical TNBC treatment without side effects, supported by hematologic andimmunologic assays data. PMLA-based nanodrugs of the Polycefin™ family passed multipletoxicity and efficacy tests in vitro and in vivo on preclinical level and may prove to be optimizedand efficacious for the treatment of cancer patients in the future.

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