scholarly journals Gene analysis of major signaling pathways regulated by gonadotropins in human ovarian granulosa tumor cells (KGN)†

2020 ◽  
Vol 103 (3) ◽  
pp. 583-598
Author(s):  
Patricia G Tremblay ◽  
Marc-André Sirard
Keyword(s):  
Gene Expression ◽  
Cell Differentiation ◽  
Signaling Pathways ◽  
Tumor Cells ◽  
Granulosa Cell ◽  
Granulosa Cells ◽  
Follicular Development ◽  
Reproductive Function ◽  
New Information ◽  
Pkc Signaling

Abstract The female reproductive function largely depends on timing and coordination between follicle-stimulating hormone (FSH) and luteinizing hormone. Even though it was suggested that these hormones act on granulosa cells via shared signaling pathways, mainly protein kinases A, B, and C (PKA, PKB, and PKC), there is still very little information available on how these signaling pathways are regulated by each hormone to provide such differences in gene expression throughout folliculogenesis. To obtain a global picture of the principal upstream factors involved in PKA, PKB, and PKC signaling in granulosa cells, human granulosa-like tumor cells (KGN) were treated with FSH or specific activators (forskolin, SC79, and phorbol 12-myristate 13-acetate) for each pathway to analyze gene expression with RNA-seq technology. Normalization and cutoffs (FC 1.5, P ≤ 0.05) revealed 3864 differentially expressed genes between treatments. Analysis of major upstream regulators showed that PKA is a master kinase of early cell differentiation as its activation resulted in the gene expression profile that accompanies granulosa cell differentiation. Our data also revealed that the activation of PKC in granulosa cells is also a strong differentiation signal that could control “advanced” differentiation in granulosa cells and the inflammatory cascade that occurs in the dominant follicle. According to our results, PKB activation provides support for PKA-stimulated gene expression and is also involved in granulosa cell survival throughout follicular development. Taken together, our results provide new information on PKA, PKB, and PKC signaling pathways and their roles in stimulating a follicle at the crossroad between maturation/ovulation and atresia.

scholarly journals AKT is involved in granulosa cell autophagy regulation via mTOR signaling during rat follicular development and atresia

Reproduction ◽  
2014 ◽  
Vol 147 (1) ◽  
pp. 73-80 ◽  
Author(s):  
JongYeob Choi ◽  
MinWha Jo ◽  
EunYoung Lee ◽  
DooSeok Choi
Keyword(s):  
Cell Death ◽  
Granulosa Cell ◽  
Granulosa Cells ◽  
Apoptotic Cell ◽  
Follicular Development ◽  
Negative Regulator ◽  
Metabolic Clearance ◽  
Akt Inhibitor ◽  
Western Blots

In this study, we examined whether granulosa cell autophagy during follicular development and atresia was regulated by the class I phosphoinositide-3 kinase/protein kinase B (AKT) pathway, which is known to control the activity of mammalian target of rapamycin (mTOR), a major negative regulator of autophagy. Ovaries and granulosa cells were obtained using an established gonadotropin-primed immature rat model that induces follicular development and atresia. Autophagy was evaluated by measuring the expression level of microtubule-associated protein light chain 3-II (LC3-II) using western blots and immunohistochemistry. The activity of AKT and mTOR was also examined by observing the phosphorylation of AKT and ribosomal protein S6 kinase (S6K) respectively. After gonadotropin injection, LC3-II expression was suppressed and phosphorylation of AKT and S6K increased in rat granulosa cells. By contrast, gonadotropin withdrawal by metabolic clearance promoted LC3-II expression and decreased phosphorylation of AKT and S6K. In addition,in-vitroFSH treatment of rat granulosa cells also indicated inhibition of LC3-II expression accompanied by a marked increase in phosphorylation of AKT and S6K. Inhibition of AKT phosphorylation using AKT inhibitor VIII suppressed FSH-mediated phosphorylation of S6K, followed by an increase in LC3-II expression. Furthermore, co-treatment with FSH and AKT inhibitor increased the levels of apoptosis and cell death of granulosa cells compared with the single treatment with FSH. Taken together, our findings indicated that AKT-mediated activation of mTOR suppresses granulosa cell autophagy during follicular development and is involved in the regulation of apoptotic cell death.

285 RELATIONSHIP BETWEEN FOLLICLE FLUID STEROID CONCENTRATIONS, GRANULOSA CELL GENE EXPRESSION, AND BOVINE OOCYTE DEVELOPMENTAL COMPETENCE

2010 ◽  
Vol 22 (1) ◽  
pp. 299
Author(s):  
S. Matoba ◽  
S. Mamo ◽  
E. Gallagher ◽  
A. G. Fahey ◽  
T. Fair ◽  
...  
Keyword(s):  
Gene Expression ◽  
Granulosa Cell ◽  
Granulosa Cells ◽  
Target Genes ◽  
Transcript Abundance ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Oocyte Competence ◽  
Cell Gene Expression ◽  
The Relationship

The ability to culture oocytes and embryos in an individually identifiable manner facilitates the study of the relationship between follicle param- eters and oocyte development, in order to identify markers of competent oocytes. The aim of this study was to examine the predictive value of intrafollicular steroid concentrations and granulosa cell transcript abundance on the ability of immature bovine oocytes to develop to the blastocyst stage in vitro. Individual follicles (n = 214, 11 replicates, 49 animals) were dissected from the ovaries of slaughtered animals. Following measure- ment of diameter, follicles were carefully ruptured under a stereomicroscope and the oocyte was recovered and individually processed through maturation, fertilization, and culture on the cell adhesive Cell-Tak (20 oocytes/100 μL; Matoba and Lonergan 2009 Reprod. Fertil. Dev. 21, 160). Cleavage and blastocyst rates were assessed on Days 2 and 9, respectively. Follicular fluid was recovered and stored at -80°C until analysis for concentrations of the steroids estradiol, progesterone, and testosterone by RIA. Granulosa cells were collected from each follicle for analysis of gene expression by quantitative RT-PCR. Primers were designed for 7 target genes (AMH, CYP19A, ESR1, ESR2, FSHR, HSD3B1 and LHCGR) and 2 reference genes (PPIA and H2AZ). Transcript abundance of target genes in granulosa cells associated with embryos that cleaved and developed to the blastocyst stage (competent) and those that cleaved but failed to develop (incompetent) was examined. Mean steroid concentrations were compared by ANOVA and Spearman correlations, and logistical regression were used to test the relationship between follicle size and steroid con- centration and the ability of steroid concentration to predict developmental competence. Gene expression data were analyzed using the delta-delta CT (cycle threshold) method. Values were normalized to the average values of the reference genes and means were compared by the Student’s t-test In total, 79.1% of oocytes cleaved after IVF and 28.3% developed to the blastocyst stage. The mean (±SEM) follicular concentrations of testosterone (62.8 ± 4.8 ng mL-1), progesterone (616.8 ± 31.9 ng mL-1), or estradiol (14.4 ± 2.4 ng mL-1 were not different (P ≥ 0.05) between competent and incompetent oocytes. Follicular diameter was negatively correlated with testosterone, progesterone, testosterone:estradiol, and pro- gesterone:estradiol (P ≤ 0.01) and positively correlated with estradiol (P ≤ 0.01). Logistical regression analysis showed that steroid concentrations or the ratio of steroids were not satisfactory predictors of oocyte competence. Transcript abundance of AMH, ESR1, ESR2, FSHR, and HSD3B1 was significantly higher (P ≤ 0.05) in granulosa cells associated with competent compared with incompetent oocytes. In conclusion, follicular steroid concentrations were not associated with oocyte development. In contrast, granulosa cell gene expression may be a useful predictor of oocyte competence. Supported by Science Foundation Ireland (07/SRC/B1156).

176 GENE EXPRESSION OF LUTEINIZING HORMONE RECEPTOR (LHR) ISOFORMS IN GRANULOSA CELLS OF FOLLICLES FROM NELLORE HEIFERS BEFORE, DURING, AND AFTER FOLLICULAR DEVIATION

2009 ◽  
Vol 21 (1) ◽  
pp. 187 ◽  
Author(s):  
C. M. Barros ◽  
R. L. Ereno ◽  
M. F. Machado ◽  
J. Buratini ◽  
M. F. Pegorer ◽  
...  
Keyword(s):  
Gene Expression ◽  
Granulosa Cells ◽  
Rna Extraction ◽  
Follicular Development ◽  
Sao Paulo ◽  
Luteinizing Hormone Receptor ◽  
São Paulo ◽  
Dominant Follicle ◽  
The Future ◽  
Group 2

During bovine follicular development, there is a phase known as follicular deviation in which the future dominant follicle grows faster than the other follicles and acquires LH receptors (LHR). In Nellore breed, deviation occurs 2.5 days after ovulation, and at this time, the dominant follicle has in average a diameter of 6.0 mm. Some authors believe that LHRs are present in the future dominant follicle before deviation and are essential for this process. However, others are convinced that LHRs are present only during or after follicular deviation. The aim of the present experiment was to evaluate the expression of 4 LHR isoforms (M1 to M4) in granulosa cells of follicles from Nellore heifers before, during, and after follicular deviation. At a random stage of the estrous cycle (D0), Nellore heifers (n = 21) received a progesterone intravaginal device (1.0 g, Primer®, Tecnopec, Sao Paulo, Brazil) and 2.5 mg of estradiol benzoate (EB, i.m., Estrogin®, Farmavet, Sao Paulo, Brazil). Eight days later (D8) PGF2α was administered (150 μg d-cloprostenol, i.m., Prolise®, ARSA S.R.L., Buenos Aires, Argentina), and the device was removed. Twenty-four hours after device removal, cows were treated with EB (1.0 mg, i.m.), and from this point in time, the growth of the dominant follicle growth was observed by ultrasonography (US, Aloka 900, Tokyo, Japan) every 12 h. The animals were allocated in 3 groups: Group 2 (G2, 2 days after ovulation, n = 7), Group 2.5 (G2.5, 2.5 days after ovulation, n = 7), and Group 3 (G3, 3 days after ovulation, n = 7), and were slaughtered 2, 2.5, and 3 days after ovulation, respectively, in order to remove the ovaries. The granulosa cells, obtained from ovarian follicles, were separated for total RNA extraction, and the gene expression of LHR isoforms was measured by semiquantitative RT-PCR. Since LHR expression was not detected in Group 2 (follicles with 4.5 to 6.7 mm), comparisons were performed between groups G2.5 and G3 by ANOVA. The LHR expression was detected only in 2 samples of Group G2 (7.0-mm follicles) and was significantly higher in Group G3 (63.6%; follicles from 8 to 14 mm, P < 0.05). In all samples that expressed LHR, the 4 isoforms were present. It is concluded that LHR expression is present in granulosa cells of follicles from Nellore heifers after follicular deviation. Support and fellowship from FAPESP (Sao Paulo, Brazil).We are grateful to Tecnopec (Sao Paulo, Brazil) for providing intravaginal devices used in the experiment.

scholarly journals Inhibitory effect of retinoic acid on the development of immature porcine granulosa cells to mature cells

2000 ◽  
Vol 25 (1) ◽  
pp. 53-61 ◽  
Author(s):  
M Hattori ◽  
K Takesue ◽  
N Nishida ◽  
Y Kato ◽  
N Fujihara
Keyword(s):  
Retinoic Acid ◽  
Cell Differentiation ◽  
Granulosa Cell ◽  
Granulosa Cells ◽  
Inhibitory Effect ◽  
Immature Stage ◽  
Mrna Levels ◽  
Lh Receptor ◽  
Receptor Mrna ◽  
Immature Cells

The present study investigated the effect of retinoic acid (RA) on the differentiation of granulosa cells prepared from porcine ovaries. The granulosa cells were precultured for 15 h, then cultured for 48 h with FSH and further treated for 24 h with LH in order to induce their transformation into luteal cells. After the cells had been exposed to 1 microM retinoids (RA, retinal and retinol) for 87 h, analysis of the LH receptor mRNA expression, an indicator of granulosa cell differentiation, was carried out by using semiquantitative RT-PCR. The results showed that there was a decrease in LH receptor mRNA levels, and that RA had a more potent effect on these levels than the other two retinoids. When cells were exposed to RA in the immature stage (before the addition of FSH) or the early stage of development (0-24 h after the addition of FSH), expression of LH receptor mRNA was greatly diminished. When the immature cells were cultured for 15 h with RA, then washed and cultured for 48 h with FSH and for 24 h with LH, the expression of LH receptor mRNA was not reversed. In the differentiated cells (24 h after the addition of FSH), however, RA no longer had any inhibitory effect. When the immature cells were exposed to RA, FSH-induced expression of c-fos mRNA was markedly decreased. In contrast, expression of c-jun and activating transcription factor-4 mRNAs remained constant. However, the expression of c-fos mRNA was not decreased by forskolin. The results indicate that RA is a potent inhibitor in the immature stage of porcine granulosa cell differentiation, probably through decreased expression of FSH receptor, but that RA does not inhibit differentiation in the mature stage of the cells.

scholarly journals The Phytoestrogen Quercetin Impairs Steroidogenesis and Angiogenesis in Swine Granulosa Cells In Vitro

2009 ◽  
Vol 2009 ◽  
pp. 1-8 ◽  
Author(s):  
Sujen Eleonora Santini ◽  
Giuseppina Basini ◽  
Simona Bussolati ◽  
Francesca Grasselli
Keyword(s):  
Granulosa Cell ◽  
Granulosa Cells ◽  
Follicular Development ◽  
Redox Status ◽  
Negative Influence ◽  
Animal Nutrition ◽  
Follicle Development ◽  
Vascular Endothelial ◽  
Endothelial Growth Factor

Experimental evidence documents that nutritional phytoestrogens may interact with reproductive functions but the exact mechanism of action is still controversial. Since quercetin is one of the main flavonoids in livestock nutrition, we evaluated its possible effects on cultured swine granulosa cell proliferation, steroidogenesis, and redox status. Moreover, since angiogenesis is essential for follicle development, the effect of the flavonoid on Vascular Endothelial Growth Factor output by granulosa cells was also taken into account. Our data evidence that quercetin does not affect granulosa cell growth while it inhibits progesterone production and modifies estradiol production in a dose-related manner. Additionally, the flavonoid interferes with the angiogenic process by inhibiting VEGF production as well as by altering redox status. Since steroidogenesis and angiogenesis are strictly involved in follicular development, these findings appear particularly relevant, pointing out a possible negative influence of quercetin on ovarian physiology. Therefore, the possible reproductive impact of the flavonoid should be carefully considered in animal nutrition.

scholarly journals Rapid effects of LH on gene expression in the mural granulosa cells of mouse periovulatory follicles

Reproduction ◽  
2009 ◽  
Vol 137 (5) ◽  
pp. 843-855 ◽  
Author(s):  
Martha Z Carletti ◽  
Lane K Christenson
Keyword(s):  
Gene Expression ◽  
Transcription Factors ◽  
Cell Signaling ◽  
Granulosa Cell ◽  
Granulosa Cells ◽  
Egf Receptor ◽  
Cell Function ◽  
Early Response ◽  
Cell Gene Expression

LH acts on periovulatory granulosa cells by activating the PKA pathway as well as other cell signaling cascades to increase the transcription of specific genes necessary for ovulation and luteinization. Collectively, these cell signaling responses occur rapidly (within minutes); however, presently no high throughput studies have reported changes before 4 h after the LH surge. To identify early response genes that are likely critical for initiation of ovulation and luteinization, mouse granulosa cells were collected before and 1 h after hCG. Fifty-seven gene transcripts were significantly (P<0.05) upregulated and three downregulated following hCG. Twenty-four of these transcripts were known to be expressed after the LH/hCG surge at later time points, while 36 were unknown to be expressed by periovulatory granulosa cells. Temporal expression of several transcripts, including the transcription factorsNr4a1,Nr4a2,Egr1,Egr2,Btg1, andBtg2, and the epidermal growth factor (EGF)-like ligandsAregandEreg, were analyzed by quantitative RT-PCR, and their putative roles in granulosa cell function are discussed. Epigen (Epgn), another member of the family of EGF-like ligands was identified for the first time in granulosa cells as rapidly induced by LH/hCG. We demonstrate thatEpgninitiates cumulus expansion, similar to the other EGF-receptor ligandsAregandEreg. These studies illustrate that a number of changes in gene expression occurin vivoin response to LH, and that many of the differentially expressed genes are transcription factors that we would predict in turn modulate granulosa cell gene expression to ultimately impact the processes of ovulation and luteinization.

scholarly journals Interaction between arachidonic acid and cAMP signaling pathways enhances steroidogenesis and StAR gene expression in MA-10 Leydig tumor cells

2002 ◽  
Vol 188 (1-2) ◽  
pp. 55-63 ◽  
Author(s):  
Xing Jia Wang ◽  
Matthew T. Dyson ◽  
Carolina Mondillo ◽  
Zoraida Patrignani ◽  
Omar Pignataro ◽  
...  
Keyword(s):  
Gene Expression ◽  
Arachidonic Acid ◽  
Signaling Pathways ◽  
Tumor Cells ◽  
Camp Signaling ◽  
Leydig Tumor Cells ◽  
Star Gene
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