scholarly journals Concanavalin-A Induces Granulosa Cell Death and Inhibits FSH-Mediated Follicular Growth and Ovarian Maturation in Female Rats

Endocrinology ◽  
2013 ◽  
Vol 154 (5) ◽  
pp. 1885-1896 ◽  
Author(s):  
Ethel V. Velasquez ◽  
Mariana Ríos ◽  
María Elena Ortiz ◽  
Carlos Lizama ◽  
Elizabeth Nuñez ◽  
...  
Keyword(s):  
Cell Death ◽  
Granulosa Cell ◽  
Granulosa Cells ◽  
Concanavalin A ◽  
Ex Vivo ◽  
Female Rats ◽  
Carbohydrate Binding ◽  
Con A ◽  
Follicular Maturation

Abstract Reproductive success stems from a finely regulated balance between follicular maturation and atresia, in which the role of carbohydrate structure is poorly understood. Here, we describe for the first time a fraction of purified recombinant human FSH that is capable of bringing about the cell death of granulosa cells and preventing follicular maturation in a rat model. Further analysis by mass spectrometry revealed the presence of the lectin Concanavalin-A (Con-A) within this fraction of recombinant FSH. Using both the fractionated FSH and Con-A, the observed cell death was predominantly located to the granulosa cells. Ex vivo culture of rat follicles demonstrated that follicle degeneration occurred and resulted in the release of a denuded and deteriorated oocyte. Moreover, in vivo experiments confirmed an increase in atresia and a corresponding reduction confined to follicle in early antral stage. As a mechanism of action, Con-A reduces ovarian proliferation, Von Willebrand staining, and angiogenesis. Based on the observation that Con-A may induce granulosa cell death followed by follicle death, our results further demonstrate that follicular carbohydrate moiety is changing under the influence of FSH, which may allow a carbohydrate-binding lectin to increase granulosa cell death. The physiological consequences of circulating lectin-like molecules remain to be determined. However, our results suggest a potential exploitation of carbohydrate binding in fertility and ovarian cancer treatment. This work may shed light on a key role of carbohydrates in the still obscure physiological process of follicular selection and atresia.

scholarly journals Localization, Regulation and Possible Consequences of Apoptotic Protease-Activating Factor-1 (Apaf-1) Expression in Granulosa Cells of the Mouse Ovary

Endocrinology ◽  
1999 ◽  
Vol 140 (6) ◽  
pp. 2641-2644 ◽  
Author(s):  
Rodolfo Robles ◽  
Xiao-Jing Tao ◽  
Alexander M. Trbovich ◽  
Daniel V. Maravei ◽  
Ravit Nahum ◽  
...  
Keyword(s):  
Cell Death ◽  
Western Blot ◽  
Granulosa Cell ◽  
Granulosa Cells ◽  
Blot Analysis ◽  
Cell Apoptosis ◽  
Western Blot Analysis ◽  
C Elegans

Abstract The recent characterization of apoptotic protease-activating factor-1 (Apaf-1) in vertebrates as a putative homolog of the Caenorhabditis elegans gene, ced-4, indicates that the third major arm of the C. elegans programmed cell death machinery has also been conserved through evolution. Although apoptosis is now known to be important for ovarian follicular atresia in vertebrates, nothing is known of the role of Apaf-1 in ovarian function. Herein we show by immunohistochemical analysis that Apaf-1 is abundant in granulosa cells of early antral follicles whereas in vivo gonadotropin priming completely suppresses Apaf-1 expression and granulosa cell apoptosis. Western blot analysis of fractionated protein extracts prepared from granulosa cells before and after in vitro culture without hormonal support to induce apoptosis indicated that mitochondrial cytochrome c release, a biochemical step required for the activation of Apaf-1, occurs in granulosa cells cultured in vitro. Moreover, Western blot analysis of procaspase-3 processing, a principal downstream event set in motion by activated Apaf-1, indicated that healthy granulosa cells possess almost exclusively the inactive (pro-) form of the enzyme whereas granulosa cells deprived of hormonal support rapidly process procaspase-3 to the active enzyme. Lastly, we show that serum-starved granulosa cells activate caspase-3-like enzymes both prior to and after nuclear pyknosis, as revealed by a single-cell fluorescent caspase activity assay. These data, combined with previous observations regarding the role of homologs of the two other C. elegans cell death regulatory genes, ced-9 (Bc1-2 family members) and ced-3 (caspases), in atresia fully support the hypothesis that granulosa cell apoptosis is precisely coordinated by all three major arms of a cell death program conserved through evolution.

scholarly journals Sirtuin 1 and Sirtuin 3 in Granulosa Cell Tumors

2021 ◽  
Vol 22 (4) ◽  
pp. 2047
Author(s):  
Nina Schmid ◽  
Kim-Gwendolyn Dietrich ◽  
Ignasi Forne ◽  
Alexander Burges ◽  
Magdalena Szymanska ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Granulosa Cell ◽  
Granulosa Cells ◽  
Drug Targets ◽  
Growth Regulation ◽  
Sirtuin 1 ◽  
Ki 67 ◽  
Granulosa Cell Tumors ◽  
Novel Drug

Sirtuins (SIRTs) are NAD+-dependent deacetylases that regulate proliferation and cell death. In the human ovary, granulosa cells express sirtuin 1 (SIRT1), which has also been detected in human tumors derived from granulosa cells, i.e., granulosa cell tumors (GCTs), and in KGN cells. KGN cells are an established cellular model for the majority of GCTs and were used to explore the role of SIRT1. The SIRT1 activator SRT2104 increased cell proliferation. By contrast, the inhibitor EX527 reduced cell numbers, without inducing apoptosis. These results were supported by the outcome of siRNA-mediated silencing studies. A tissue microarray containing 92 GCTs revealed nuclear and/or cytoplasmic SIRT1 staining in the majority of the samples, and also, SIRT2-7 were detected in most samples. The expression of SIRT1–7 was not correlated with the survival of the patients; however, SIRT3 and SIRT7 expression was significantly correlated with the proliferation marker Ki-67, implying roles in tumor cell proliferation. SIRT3 was identified by a proteomic analysis as the most abundant SIRT in KGN. The results of the siRNA-silencing experiments indicate involvement of SIRT3 in proliferation. Thus, several SIRTs are expressed by GCTs, and SIRT1 and SIRT3 are involved in the growth regulation of KGN. If transferable to GCTs, these SIRTs may represent novel drug targets.

scholarly journals The role of IGFs in the regulation of ovarian follicular growth in the brushtail possum (Trichosurus vulpecula)

Reproduction ◽  
2010 ◽  
Vol 140 (2) ◽  
pp. 295-303 ◽  
Author(s):  
Jennifer L Juengel ◽  
Lisa J Haydon ◽  
Brigitta Mester ◽  
Brian P Thomson ◽  
Michael Beaumont ◽  
...  
Keyword(s):  
Granulosa Cell ◽  
Granulosa Cells ◽  
Cell Function ◽  
Antral Follicle ◽  
Brushtail Possum ◽  
Follicular Growth ◽  
Theca Cells ◽  
Ovarian Follicular Growth

IGFs are known to be key regulators of ovarian follicular growth in eutherian mammals, but little is known regarding their role in marsupials. To better understand the potential role of IGFs in the regulation of follicular growth in marsupials, expression of mRNAs encoding IGF1, IGF2, IGF1R, IGF-binding protein 2 (IGFBP2), IGFBP4 and IGFBP5 was localized by in situ hybridization in developing ovarian follicles of the brushtail possum. In addition, the effects of IGF1 and IGF2 on granulosa cell function were tested in vitro. Both granulosa and theca cells synthesize IGF mRNAs, with the theca expressing IGF1 mRNA and granulosa cell expressing IGF2 mRNA. Oocytes and granulosa cells express IGF1R. Granulosa and theca cells expressed IGFBP mRNAs, although the pattern of expression differed between the BPs. IGFBP5 mRNA was differentially expressed as the follicles developed with granulosa cells of antral follicles no longer expressing IGFBP5 mRNA, suggesting an increased IGF bioavailability in the antral follicle. The IGFBP protease, PAPPA mRNA, was also expressed in granulosa cells of growing follicles. Both IGF1 and IGF2 stimulated thymidine incorporation but had no effect on progesterone production. Thus, IGF may be an important regulator of ovarian follicular development in marsupials as has been shown in eutherian mammals.

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.

Expression and regulation of high mobility group AT-hook 1 (HMGA1) during ovulation and luteinisation in rat ovary

2019 ◽  
Vol 31 (4) ◽  
pp. 698 ◽  
Author(s):  
Hao-ran Li ◽  
Yan Li ◽  
Yu Liu ◽  
Jiao-jiao Yu ◽  
Fei-xue Li
Keyword(s):  
Mrna Expression ◽  
Granulosa Cell ◽  
Cell Cultures ◽  
Granulosa Cells ◽  
In Situ Hybridisation ◽  
High Mobility ◽  
High Mobility Group ◽  
Female Rats ◽  
Corpora Lutea ◽  
Hmga1 Expression

High mobility group AT-hook 1 (HMGA1) is able to regulate gene expression and function as a tumour suppressor. The spatiotemporal expression pattern of HMGA1 was investigated in this study. Immature female rats (22–23 days old) were treated with 10IU, s.c., pregnant mare’s serum gonadotrophin to stimulate follicular development, followed 48h later by injection with 5IU, s.c., human chorionic gonadotrophin (hCG). Whole ovaries or granulosa cells were collected at various times after hCG administration (n=3 per time point). Real-time polymerase chain reaction and western blot analysis revealed that HMGA1 was highly stimulated in the ovary by 4–12h after hCG treatment. In situ hybridisation analysis demonstrated that Hmga1 mRNA expression was induced in granulosa cells between 8 and 12h after hCG treatment. There was negligible Hmga1 mRNA signal observed in newly forming corpora lutea. In addition, the data indicated that both the protein kinase (PK) A and PKC pathways regulated Hmga1 expression in rat granulosa cells. In rat granulosa cell cultures, upregulation of Hmga1 was dependent on new protein synthesis because Hmga1 was inhibited by cycloheximide. Furthermore, Hmga1 mRNA expression in rat granulosa cell cultures was inhibited by AG1478, whereas NS398 and RU486 had no effect, suggesting that Hmga1 expression was regulated, in part, by the epidermal growth factor pathway. In summary, the findings of this study suggest that induction of Hmga1 may be important for theca and granulosa cell differentiation into luteal cells.

scholarly journals SMAD7 antagonizes key TGFβ superfamily signaling in mouse granulosa cells in vitro

Reproduction ◽  
2013 ◽  
Vol 146 (1) ◽  
pp. 1-11 ◽  
Author(s):  
Yang Gao ◽  
Haixia Wen ◽  
Chao Wang ◽  
Qinglei Li
Keyword(s):  
Granulosa Cell ◽  
Granulosa Cells ◽  
Transforming Growth Factor ◽  
Negative Regulator ◽  
Fine Tuning ◽  
Female Reproduction ◽  
Tgfβ Signaling ◽  
Tgfβ Superfamily

Transforming growth factor β (TGFβ) superfamily signaling is essential for female reproduction. Dysregulation of the TGFβ signaling pathway can cause reproductive diseases. SMA and MAD (mothers against decapentaplegic) (SMAD) proteins are downstream signaling transducers of the TGFβ superfamily. SMAD7 is an inhibitory SMAD that regulates TGFβ signalingin vitro. However, the function of SMAD7 in the ovary remains poorly defined. To determine the signaling preference and potential role of SMAD7 in the ovary, we herein examined the expression, regulation, and function of SMAD7 in mouse granulosa cells. We showed that SMAD7 was expressed in granulosa cells and subject to regulation by intraovarian growth factors from the TGFβ superfamily. TGFB1 (TGFβ1), bone morphogenetic protein 4, and oocyte-derived growth differentiation factor 9 (GDF9) were capable of inducingSmad7expression, suggesting a modulatory role of SMAD7 in a negative feedback loop. Using a small interfering RNA approach, we further demonstrated that SMAD7 was a negative regulator of TGFB1. Moreover, we revealed a link between SMAD7 and GDF9-mediated oocyte paracrine signaling, an essential component of oocyte–granulosa cell communication and folliculogenesis. Collectively, our results suggest that SMAD7 may function during follicular development via preferentially antagonizing and/or fine-tuning essential TGFβ superfamily signaling, which is involved in the regulation of oocyte–somatic cell interaction and granulosa cell function.

scholarly journals The influence of di-(2-ethylhexyl) phthalate on steroidogenesis by the ovarian granulosa cells of immature female rats

2007 ◽  
Vol 194 (3) ◽  
pp. 603-609 ◽  
Author(s):  
I Svechnikova ◽  
K Svechnikov ◽  
O Söder
Keyword(s):  
Granulosa Cells ◽  
Ex Vivo ◽  
Phthalate Esters ◽  
Primary Cultures ◽  
Serum Levels ◽  
Female Rats ◽  
Pituitary Cells ◽  
Once Daily ◽  
Hormonal Function ◽  
Ethylhexyl Phthalate

Phthalate esters are known to exert harmful effects on mammalian reproduction and fertility, but their potential adverse effects on the hormonal functions of the ovary have not yet been elucidated in detail. Here, we investigated the effects of di-(2-ethylhexyl) phthalate (DEHP) on the hypothalamic–pituitary–gonadal axis of young developing female rats, as well as on ex vivo steroidogenesis by granulosa cells (GCs) and secretion of LH by gonadotropes. Exposure of 20-day-old female rats to 500 mg DEHP by oral gavage once daily for 10 days reduced their serum levels of progesterone and estradiol, while tending to enhance levels of LH. Furthermore, primary cultures of GCs isolated from these rats exhibited an attenuated capacity to produce progesterone in response to stimulation by LH and FSH, as well as a lower degree of transport of endogenous cholesterol into mitochondria. Moreover, the abilityof primary cultures of pituitary cells isolated from DEHP-treated rats to produce and secrete LH in response to GnRH was significantly enhanced. In addition, 2-ethylhexanoic acid, a metabolite of DEHP, significantly potentiated GnRH-stimulated production of LH by cultures of pituitary cells isolated from untreated 20-day-old female rats. Together, these data indicate that DEHP exerts dual effects on the pituitary–gonadal axis, stimulating the hormonal function of the pituitary and, at the same time, by inhibiting steroidogenesis by GCs.

scholarly journals GATA-4 Regulates Bcl-2 Expression in Ovarian Granulosa Cell Tumors

Endocrinology ◽  
2008 ◽  
Vol 149 (11) ◽  
pp. 5635-5642 ◽  
Author(s):  
Antti Kyrönlahti ◽  
Maarit Rämö ◽  
Maija Tamminen ◽  
Leila Unkila-Kallio ◽  
Ralf Butzow ◽  
...  
Keyword(s):  
Cell Fate ◽  
Granulosa Cell ◽  
Granulosa Cells ◽  
Dominant Negative ◽  
Rt Pcr ◽  
Cyclin D2 ◽  
Granulosa Cell Tumors ◽  
Ovarian Granulosa Cell ◽  
Cell Cycle Regulator Cyclin

Excessive cell proliferation and decreased apoptosis have been implicated in the pathogenesis of ovarian granulosa cell tumors (GCTs). We hypothesized that transcription factor GATA-4 controls expression of the antiapoptotic factor Bcl-2 and the cell cycle regulator cyclin D2 in normal and neoplastic granulosa cells. To test this hypothesis, a tissue microarray based on 80 GCTs was subjected to immunohistochemistry for GATA-4, Bcl-2, and cyclin D2, and the data were correlated to clinical and histopathological parameters. In addition, quantitative RT-PCR for GATA-4, Bcl-2, and cyclin D2 was performed on 21 human GCTs. A mouse GCT model was used to complement these studies. The role of GATA-4 in the regulation of Bcl2 and ccdn2 (coding for cyclin D2) was studied by transactivation assays, and by disrupting GATA-4 function with dominant negative approaches in mouse and human GCT cell lines. We found that GATA-4 expression correlated with Bcl-2 and cyclin D2 expression in human and murine GCTs. Moreover, GATA-4 enhanced Bcl-2 and cyclin D2 promoter activity in murine GCT cells. Whereas GATA-4 overexpression up-regulated and dominant negative GATA-4 suppressed Bcl-2 expression in human GCT cells, the effects on cyclin D2 were negligible. Our results reveal a previously unknown relationship between GATA-4 and Bcl-2 in mammalian granulosa cells and GCTs, and suggest that GATA-4 influences granulosa cell fate by transactivating Bcl-2.

scholarly journals Intestinally Secreted C-Type Lectin Reg3b Attenuates Salmonellosis but Not Listeriosis in Mice

2012 ◽  
Vol 80 (3) ◽  
pp. 1115-1120 ◽  
Author(s):  
Marleen T. J. van Ampting ◽  
Linda M. P. Loonen ◽  
Arjan J. Schonewille ◽  
Irene Konings ◽  
Carolien Vink ◽  
...  
Keyword(s):  
Bacterial Infections ◽  
Salmonella Enteritidis ◽  
Ex Vivo ◽  
Protective Role ◽  
Carbohydrate Binding ◽  
Intestinal Infection ◽  
Gram Positive ◽  
Gram Negative ◽  
Content Type

The Reg3 protein family, including the human member designated pancreatitis-associated protein (PAP), consists of secreted proteins that contain a C-type lectin domain involved in carbohydrate binding. They are expressed by intestinal epithelial cells. Colonization of germ-free mice and intestinal infection with pathogens increase the expression of Reg3g and Reg3b in the murine ileum. Reg3g is directly bactericidal for Gram-positive bacteria, but the exact role of Reg3b in bacterial infections is unknown. To investigate the possible protective role of Reg3b in intestinal infection, Reg3b knockout (Reg3b−/−) mice and wild-type (WT) mice were orally infected with Gram-negativeSalmonella enteritidisor Gram-positiveListeria monocytogenes. At day 2 after oralListeriainfection and at day 4 after oralSalmonellainfection, mice were sacrificed to collect intestinal and other tissues for pathogen quantification. Protein expression of Reg3b and Reg3g was determined in intestinal mucosal scrapings of infected and noninfected mice. In addition,ex vivobinding of ileal mucosal Reg3b toListeriaandSalmonellawas investigated. Whereas recovery ofSalmonellaorListeriafrom feces of Reg3b−/−mice did not differ from that from feces of WT mice, significantly higher numbers of viableSalmonella, but notListeria, bacteria were recovered from the colon, mesenteric lymph nodes, spleen, and liver of the Reg3b−/−mice than from those of WT mice. Mucosal Reg3b binds to both bacterial pathogens and may interfere with their mode of action. Reg3b plays a protective role against intestinal translocation of the Gram-negative bacteriumS. enteritidisin mice but not against the Gram-positive bacteriumL. monocytogenes.

scholarly journals Glycophorin A interferes in the agglutination of human erythrocytes by concanavalin A Explanation of the requirement for enzymic predigestion

1987 ◽  
Vol 241 (2) ◽  
pp. 505-511 ◽  
Author(s):  
S M Gokhale ◽  
N G Mehta
Keyword(s):  
Sialic Acid ◽  
Correlation Coefficient ◽  
Concanavalin A ◽  
Binding Sites ◽  
Band 3 ◽  
Human Erythrocytes ◽  
The Other ◽  
Glycophorin A ◽  
Con A

Human erythrocytes become agglutinable with concanavalin A (Con A) after treatment with various proteinases or neuraminidase. The extent of agglutinability achieved with different enzymes is, however, different: Pronase, papain, trypsin, neuraminidase and chymotrypsin enhance the agglutinability in decreasing order, the last being barely effective. The actions of the enzymes on band 3, the Con A receptor, do not correlate with their abilities to increase the agglutinability: Pronase, papain and chymotrypsin cleave the protein, but not trypsin or neuraminidase. No significant differences are found in the number of Con A-binding sites or the affinities for the lectin between the normal and trypsin- or Pronase-treated cells. Thus the receptor does not seem to play a role in determining the Con A-agglutinability of erythrocytes. On the other hand, the cleavage of glycophorins, especially glycophorin A, and the release of sialic acid (in the peptide-bound form) are well-correlated with the enhancement in agglutination after the action of proteinases. The release of sialic acid by graded neuraminidase digestion and the increase in Con A-agglutinability show a correlation coefficient of 0.88. The major inhibitory role of glycophorin A in the process is indicated by the agglutination of En(a) heterozygous erythrocytes; the cells, known to bear about 50% glycophorin A molecules in their membrane, are agglutinated approximately half as well without proteolysis as are the trypsin-treated cells. Possible mechanisms by which glycophorin A could affect Con A-mediated agglutination are discussed.

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