Bovine granulosa cells express extracellular matrix proteins and their regulators during luteinization in culture

1996 ◽  
Vol 8 (2) ◽  
pp. 259 ◽  
Author(s):  
Y Zhao ◽  
MR Luck
Keyword(s):  
Extracellular Matrix ◽  
Granulosa Cells ◽  
Culture Media ◽  
Gelatin Zymography ◽  
Fold Increase ◽  
Tissue Inhibitor Of Metalloproteinase ◽  
Subunit Gene ◽  
Gelatinase Activity ◽  
Cell Extracts

This study investigated the ability of bovine granulosa cells to express and secrete collagen, metalloproteinase (MMP) activity and a tissue inhibitor of metalloproteinase (TIMP-1) during luteinization in vitro. Cells from mature (1-2 mL fluid volume) bovine follicles were cultured over 4 days in serum-free medium. Their luteinization during culture was confirmed by a 10-fold increase in progesterone secretion. Samples of cell extracts, culture media and follicular fluid were subjected to Western blotting to identify secreted proteins and to gelatin zymography to detect enzyme activity. Poly A+ RNA, isolated from cells before and after culture, was probed to detect expression of collagen alpha 1(I), collagen alpha 3(IV) and TIMP-1. The results revealed that: (1) the collagen alpha 1(I) subunit gene was expressed in cells before culture but with greater intensity by Day 4 culture; collagen I protein, on the other hand, was not detectable in culture medium; (2) the collagen alpha 3(IV) subunit gene was expressed at a low level in uncultured cells and could be detected on Day 4 of culture; low amounts of the protein were detected in medium; (3) a 92-kDa band of gelatinase activity (presumed MMP-9) was present in all medium samples, together with bands of unidentified activity; and (5) the TIMP-1 gene was expressed in uncultured cells but its expression increased markedly up to Day 4 of culture. These results show that granulosa luteinization is associated with an increase in the expression of collagen, collagen-degrading enzymes and TIMP-1. Collagen protein, however, may be only poorly synthesized in this culture model. The results suggest that granulosa-derived cells are a likely source of components of the extracellular matrix during post-ovulatory remodelling of early luteal tissue.

scholarly journals Wdnm1-like, a new adipokine with a role in MMP-2 activation

2008 ◽  
Vol 295 (1) ◽  
pp. E205-E215 ◽  
Author(s):  
Yu Wu ◽  
Cynthia M. Smas
Keyword(s):  
Adipose Tissue ◽  
Kinase Inhibitor ◽  
Culture Media ◽  
Gelatin Zymography ◽  
Fold Increase ◽  
Transcript Level ◽  
P38 Map Kinase ◽  
Extracellular Milieu ◽  
Tumor Microenvironments

White adipose tissue functions in energy storage and as an endocrine organ. DNA microarray analysis led us to identify Wdnm1-like, a distant member of the whey acidic protein/four-disulfide core (WAP/4-DSC) family, as a differentiation-dependent gene in white and brown adipogenesis. Wdnm1-like is a novel 6.8-kDa protein, and Western blot analysis reveals secretion into culture media. Wdnm1-like transcript is selectively expressed in adipose tissue and liver and is enriched ∼500-fold in white adipose depots vs. brown. Cellular fractionation of WAT demonstrates Wdnm1-like transcript expression is restricted to the adipocyte population. Studies in 3T3-L1 preadipocytes, an in vitro model of white adipogenesis, indicate Wdnm1-like transcript increases within 6 h of adipogenic induction with an ∼17,000-fold increase by day 7. Dramatic upregulation of Wdnm1-like also accompanies white adipogenesis of ScAP-23 preadipocytes and primary preadipocytes. TNF-α treatment of 3T3-L1 adipocytes increased Wdnm1-like transcript level 2.4-fold and was attenuated by pretreatment with the p38 MAP kinase inhibitor SB203580. A number of WAP/4-DSC family proteins function as protease inhibitors. This, taken with the role of extracellular remodeling in adipogenesis, led us to address effects of Wdnm1-like on matrix metalloproteinase (MMP) activity. Gelatin zymography of HT1080 fibrosarcoma cells transfected with a Wdnm1-like expression construct revealed markedly increased levels of active MMP-2. Our findings identify a new member of the adipocyte “secretome” that functions to enhance MMP-2 activity. We postulate that Wdnm1-like may play roles in remodeling of the extracellular milieu in adipogenesis, as well as in tumor microenvironments where adipocytes are key stromal components.

scholarly journals Lysyl Oxidase Gene Expression and Enzyme Activity in the Rat Ovary: Regulation by Follicle-Stimulating Hormone, Androgen, and Transforming Growth Factor-β Superfamily Members in Vitro

Endocrinology ◽  
2003 ◽  
Vol 144 (1) ◽  
pp. 154-162 ◽  
Author(s):  
Christopher R. Harlow ◽  
Mick Rae ◽  
Lindsay Davidson ◽  
Philip C. Trackman ◽  
Stephen G. Hillier
Keyword(s):  
Extracellular Matrix ◽  
Enzyme Activity ◽  
Granulosa Cells ◽  
Transforming Growth Factor ◽  
Lysyl Oxidase ◽  
Enzymatic Reaction ◽  
Follicular Development ◽  
Fold Increase ◽  
Oxidase Gene

Abstract Lysyl oxidase (LOX) catalyzes the final enzymatic reaction required for cross-linking of collagen and elastin fibers and therefore has a crucial role in regulating the formation and maintenance of extracellular matrix in the ovary. LOX mRNA is abundantly expressed in rat granulosa cells. To examine how regulation of LOX in the ovary might influence follicular development, we studied LOX mRNA expression and enzyme activity in rat granulosa cells from late preantral/early antral follicles in vitro. FSH dose dependently inhibited LOX mRNA and enzyme activity (50% reduction at 10 ng/ml) in vitro, and FSH action was mimicked by 8-bromo-cAMP, suggesting FSH action via elevation of cAMP. Dihydrotestosterone alone enhanced LOX mRNA and enzyme activity, but potentiated the effect of FSH, causing a further reduction. TGFβ1 alone dose dependently enhanced LOX mRNA (5-fold increase at 10 ng/ml) and activity (1.5-fold increase). FSH dose dependently inhibited the increase in LOX mRNA and activity caused by TGFβ1 (by up to 84% and 80%, respectively). Growth differentiation factor-9 (GDF-9) and activin A, at the same concentration as TGFβ1 (10 ng/ml), stimulated LOX mRNA and activity within 6 h, although overall expression was higher at 48 h. All three factors when combined with FSH further reduced both mRNA and enzyme activity (by up to 60%) compared with FSH alone. These findings indicate control of LOX at endocrine, paracrine, and autocrine levels within the ovary and suggest coordinated regulation of ovarian extracellular matrix during follicular development, with FSH determining whether local factors act as stimulators or inhibitors of LOX.

scholarly journals Effect of dexamethasone as osteogenic supplementation in in vitro osteogenic differentiation of stem cells from human exfoliated deciduous teeth

2021 ◽  
Vol 32 (1) ◽  
Author(s):  
Mariane Beatriz Sordi ◽  
Raissa Borges Curtarelli ◽  
Izabella Thaís da Silva ◽  
Gislaine Fongaro ◽  
Cesar Augusto Magalhães Benfatti ◽  
...  
Keyword(s):  
Stem Cells ◽  
Extracellular Matrix ◽  
Osteogenic Differentiation ◽  
Culture Media ◽  
Deciduous Teeth ◽  
Free Calcium ◽  
Alizarin Red ◽  
Matrix Mineralization ◽  
Media Supplementation

AbstractIn in vitro culture systems, dexamethasone (DEX) has been applied with ascorbic acid (ASC) and β-glycerophosphate (βGLY) as culture media supplementation to induce osteogenic differentiation of mesenchymal stem cells. However, there are some inconsistencies regarding the role of DEX as osteogenic media supplementation. Therefore, this study verified the influence of DEX culture media supplementation on the osteogenic differentiation, especially the capacity to mineralize the extracellular matrix of stem cells from human exfoliated deciduous teeth (SHED). Five groups were established: G1—SHED + Dulbecco’s Modified Eagles’ Medium (DMEM) + fetal bovine serum (FBS); G2—SHED + DMEM + FBS + DEX; G3—SHED + DMEM + FBS + ASC + βGLY; G4—SHED + DMEM + FBS + ASC + βGLY + DEX; G5—MC3T3-E1 + α Minimal Essential Medium (MEM) + FBS + ASC + βGLY. DNA content, alkaline phosphatase (ALP) activity, free calcium quantification in the extracellular medium, and extracellular matrix mineralization quantification through staining with von Kossa, alizarin red, and tetracycline were performed on days 7 and 21. Osteogenic media supplemented with ASC and β-GLY demonstrated similar effects on SHED in the presence or absence of DEX for DNA content (day 21) and capacity to mineralize the extracellular matrix according to alizarin red and tetracycline quantifications (day 21). In addition, the presence of DEX in the osteogenic medium promoted less ALP activity (day 7) and extracellular matrix mineralization according to the von Kossa assay (day 21), and more free calcium quantification at extracellular medium (day 21). In summary, the presence of DEX in the osteogenic media supplementation did not interfere with SHED commitment into mineral matrix depositor cells. We suggest that DEX may be omitted from culture media supplementation for SHED osteogenic differentiation in vitro studies.

scholarly journals Suppressive Activity of Vitamin D3 on Matrix Metalloproteinase Production From Cholesteatoma Keratinocytes In Vitro

2005 ◽  
Vol 2005 (4) ◽  
pp. 210-215 ◽  
Author(s):  
Hitome Kobayashi ◽  
Kazuhito Asano ◽  
Ken-ichi Kanai ◽  
Harumi Suzaki
Keyword(s):  
Extracellular Matrix ◽  
Matrix Metalloproteinase ◽  
Vitamin D3 ◽  
Biological Activities ◽  
Tissue Inhibitor Of Metalloproteinase ◽  
Suppressive Activity ◽  
Tissue Specimens ◽  
Culture Supernatants ◽  
Dose Dependent

There is much evidence that degradation of the extracellular matrix is essential for the development of cholesteatomas and that this is induced by activation of matrix metalloproteinases (MMPs). Vitamin D3 (VD3) has several well-recognised biological activities, including suppression of MMP production. The present study, therefore, was undertaken to examine whether VD3 could suppress MMP production from cholesteatoma keratinocytes in vitro. Keratinocytes (2.5×105cells/mL) induced from cholesteatoma tissue specimens were cultured with various concentrations of VD3. After one hour, lipopolysaccharide was added to the cell cultures at 100μg/mL. The culture supernatants were then collected and assayed for MMP-1 and MMP-3 by ELISA. We also used ELISA to measure the levels of both TIMP (tissue inhibitor of metalloproteinase)-1 and TIMP-2 in culture supernatants. Addition of VD3 into keratinocyte cultures caused the suppression of MMP and TIMP production, which was increased by LPS stimulation. This was dose-dependent. The present results showing the suppressive activity of VD3 on the production of MMPs, which are responsible for tissue remodeling, strongly suggest that VD3 would be a good candidate for an agent in the medical treatment of, or prophylaxis for, cholesteatomas.

scholarly journals Absence of leptin expression and secretion by human luteinized granulosa cells

2003 ◽  
Vol 31 (1) ◽  
pp. 233-239 ◽  
Author(s):  
M Karamouti ◽  
P Kollia ◽  
E Karligiotou ◽  
A Kallitsaris ◽  
N Prapas ◽  
...  
Keyword(s):  
Gene Expression ◽  
Granulosa Cells ◽  
Messenger Rna ◽  
Limit Of Detection ◽  
Culture Media ◽  
Point Of View ◽  
In Vitro Fertilisation ◽  
Ob Gene ◽  
Human Granulosa Cells

Whether leptin is secreted by the human ovary is not known. The available data on leptin gene (ob gene) expression by human granulosa cells are conflicting. The aim of the present study was first to re-examine the expression of leptin messenger RNA (mRNA) by human granulosa cells and second to investigate if these cells have the ability to secrete leptin in cultures. Human luteinized granulosa cells were obtained from normal women undergoing in vitro fertilisation treatment after ovarian stimulation and follicle aspiration. The expression of ob gene was studied by Reverse-Transcriptase Polymerase Chain Reaction (RT-PCR) both in primary granulosa cells treated immediately after oocyte recovery and in cells cultured up to 24 h under baseline and hormonally stimulated conditions (FSH: 100 ng/ml, LH: 100 ng/ml). ob mRNA transcripts were not detected in luteinized granulosa cells, while they were present in adipose tIssue cDNA. Actin gene expression was detected in all studied samples. Using a sensitive radioimmunoassay (lower limit of detection 0.05 ng/ml), leptin was undetectable in the culture media at all points during the 72 h cultures, while at the same time significant amounts of oestradiol and progesterone were produced particularly after the addition of androstendione (1 microM) to the incubation media. These results demonstrate for the first time that leptin is not secreted by human luteinized granulosa cells in cultures. From a physiological point of view, this may contribute to the development of the optimal follicular environment for oocyte maturation during the preovulatory period.

scholarly journals Role of ascorbic acid in promoting follicle integrity and survival in intact mouse ovarian follicles in vitro

Reproduction ◽  
2001 ◽  
pp. 89-96 ◽  
Author(s):  
AA Murray ◽  
MD Molinek ◽  
SJ Baker ◽  
FN Kojima ◽  
MF Smith ◽  
...  
Keyword(s):  
Ascorbic Acid ◽  
Basement Membrane ◽  
Growth And Development ◽  
Culture Media ◽  
Membrane Integrity ◽  
Tissue Inhibitor Of Metalloproteinase ◽  
Follicular Growth ◽  
Intact Mouse

Ascorbic acid has three known functions: it is necessary for collagen synthesis, promotes steroidogenesis and acts as an antioxidant. Within the ovary, most studies have concentrated on the role of ascorbic acid in luteal formation and regression and little is known about the function of this vitamin in follicular growth and development. Follicular growth and development were investigated in this study using an individual follicle culture system that allows the growth of follicles from the late preantral stage to Graafian morphology. Follicles were isolated from prepubertal mice and cultured for 6 days. Control media contained serum and human recombinant FSH. Further groups of follicles were cultured in the same media but with the addition of ascorbic acid at concentrations of either 28 or 280 micromol l(-1). Addition of ascorbic acid at the higher concentration significantly increased the percentage of follicles that maintained basement membrane integrity throughout culture (P < 0.001). Ascorbic acid had no effect on the growth of the follicles or on oestradiol production. Metalloproteinase 2 activity tended to increase at the higher concentration of ascorbic acid and there was a significant concomitant increase in the activity of tissue inhibitor of metalloproteinase 1 (P < 0.01). Follicles cultured without the addition of serum but with FSH and selenium in the culture media underwent apoptosis. Addition of ascorbic acid to follicles cultured under serum-free conditions significantly reduced apoptosis (P < 0.05). From these data it is concluded that ascorbic acid is necessary for remodelling the basement membrane during follicular growth and that the ability of follicles to uptake ascorbic acid confers an advantage in terms of granulosa cell survival.

87 EVALUATION OF THE NUMERICAL CHROMOSOMAL ABNORMALITIES ON IN VITRO EARLY BOVINE EMBRYOS: EFFECT OF THE CELL CO-CULTURE WITH GRANULOSA CELLS

2014 ◽  
Vol 26 (1) ◽  
pp. 157
Author(s):  
S. Demyda-Peyrás ◽  
M. Hidalgo ◽  
J. Dorado ◽  
M. Moreno-Millan
Keyword(s):  
Embryo Development ◽  
Granulosa Cells ◽  
Chromosomal Abnormalities ◽  
Culture Media ◽  
Gradient Centrifugation ◽  
Early Embryo ◽  
Bovine Embryos ◽  
Humid Atmosphere ◽  
Early Embryos

Chromosomal numerical abnormalities (CNA) were described as a major cause of developmental failures in in vitro-produced (IVP) embryos. It has been described that CNA are influenced by the post-fertilization culture environment of the embryo. Furthermore, it was demonstrated that the use of different culture media affects the CNA rates. The addition of granulosa cells during early embryo development is a well-known procedure to simplify the culture of bovine IVP and cloned embryos. This technique avoids the use of culture environments saturated with N2 (tri-gas chambers). The aim of this study was to determine the effect of the addition of granulosa cells in the chromosomal abnormalities of IVP cattle embryos. Cumulus–oocyte complexes (COC) were matured in TCM-199 medium, supplemented with glutamine, sodium pyruvate, FSH, LH, oestradiol, and gentamicin during 20 h at 38.5°C in a 5% CO2 humid atmosphere. Subsequently, matured oocytes were fertilized in IVF-TALP medium using 1 × 106 spermatozoa mL–1, selected through a Percoll gradient centrifugation. After fertilization, zygotes were divided in 2 groups and cultured in TCM-199 medium for 48 h, with (TCM-GC) or without (TCM) the addition of 1 × 106 granulosa cells. These cells were obtained by centrifuging and washing the follicular fluid remaining from searching dishes and adjusted to the working concentration. After culture, a total of 106 early embryos (72 hpi) were cytogenetically evaluated following our standard laboratory techniques. Embryos showing normal development were individually fixed onto a slide, disaggregated into blastomeres with acetic acid, and stained with Giemsa solution. Chromosomal numerical abnormalities were evaluated by direct observation at 1250× magnification in a brightfield microscope. Percentage of normal diploid embryos (D) and abnormal haploid (H), polyploid (P), or aneuploid (A) embryos were determined. Results were statistically compared between treatments using a Z test for proportions. Results were: D = 81.4%, H = 7.2%, P = 7.2%. and A = 3.6% in TCM and D = 84.3%, H = 3.9%, P = 9.8%, and A = 1.9% in TCM-GC. No significant differences (P > 0.05) were found between culture media in the chromosomal abnormality rates. According to our results, the use of somatic cells in co-culture during embryo development did not influence the appearance of abnormal complements in the produced embryos. This would allow the use of GC as a potential complement to simplify the techniques used in the culture of bovine embryos until Day 3.

scholarly journals Orexin-A Exerts Equivocal Role in Atherosclerosis Process Depending on the Duration of Exposure: In Vitro Study

Nutrients ◽  
2019 ◽  
Vol 12 (1) ◽  
pp. 53
Author(s):  
Narjes Nasiri Ansari ◽  
Flora Spentza ◽  
Georgios Dimitriadis ◽  
Aphrodite Daskalopoulou ◽  
Angeliki Karapanagioti ◽  
...  
Keyword(s):  
Gelatin Zymography ◽  
In Vitro Study ◽  
Tissue Inhibitor Of Metalloproteinase ◽  
Intermittent Fasting ◽  
Mrna Levels ◽  
High Concentration ◽  
Orexin A ◽  
Monocyte Chemoattractant Protein 1 ◽  
Feeding Regulation

Orexin-A is a peptide hormone that plays a crucial role in feeding regulation and energy homeostasis. Diurnal intermittent fasting (DIF) has been found to increase orexin-A plasma levels during fasting hours, while Ramadan fasting which resembles DIF, has led to beneficial effects on endothelial function. Herein, we aimed to investigate the effects of orexin-A on the expression of molecules involved in the atherogenesis process: Monocyte chemoattractant protein-1 (MCP-1), matrix metalloproteinases 2 and 9 (MMP-2 and MMP-9) and tissue inhibitor of metalloproteinase-1 and 2 (TIMP-1 and TIMP-2), in human aortic endothelial cells (HAECs). HAECs were incubated with orexin-A at concentrations of 40 ng/mL, 200 ng/mL and 400 ng/mL for 6, 12 and 24 h. The mRNA levels of MCP-1, MMP-2, MMP-9, TIMP-1, and TIMP-2 and orexin-1 receptor were measured by real-time qPCR. We also evaluated the MMP-2, p38, phospho-p38, NF-κΒ/p65 as well as TIMP-1 protein levels by Western blot and ELISA, respectively. MMP-2 activity was measured by gelatin zymography. Short-term 6-h incubation of HAECs with orexin-A at a high concentration (400 ng/mL) decreased MCP-1, MMP-2 expression, MMP-2/TIMP-1 ratio (p < 0.05), and MMP-2 activity, while incubation for 24 h increased MCP-1, MMP-2 expression (p < 0.05), MMP-2/TIMP-1 and MMP-2/TIMP-2 ratio (p < 0.01 and p < 0.05, respectively) as well as MMP-2 activity. The dual effects of orexin-A are mediated, at least in part, via regulation of p38 and NF-κΒ pathway. Orexin-A may have an equivocal role in atherosclerosis process with its effects depending on the duration of exposure.

scholarly journals Insulin and Insulin-Like Growth Factor Stimulation of Vascular Endothelial Growth Factor Production by Luteinized Granulosa Cells: Comparison between Polycystic Ovarian Syndrome (PCOS) and Non-PCOS Women

2007 ◽  
Vol 92 (7) ◽  
pp. 2726-2733 ◽  
Author(s):  
Meghan B. Stanek ◽  
Sherri M. Borman ◽  
Theodore A. Molskness ◽  
Janine M. Larson ◽  
Richard L. Stouffer ◽  
...  
Keyword(s):  
Vascular Endothelial Growth Factor ◽  
Growth Factor ◽  
Granulosa Cells ◽  
Polycystic Ovarian Syndrome ◽  
Culture Media ◽  
Vascular Endothelial ◽  
Vitro Fertilization ◽  
Endothelial Growth Factor ◽  
Ovarian Syndrome

Abstract Context: Vascular endothelial growth factor A (VEGF-A) is a potent cytokine that promotes angiogenesis and vascular permeability. After controlled ovarian stimulation (COS) for in vitro fertilization (IVF), excessive VEGF-A production can occur, particularly in women with polycystic ovarian syndrome (PCOS); however, it is unclear whether the regulation of VEGF-A production is different between PCOS and non-PCOS women. Objective: The aim of this study was to determine whether there were differences in the dose- and time-dependent effects of insulin and IGFs on VEGF-A production by luteinized granulosa cells (LGCs) from women with and without PCOS. Design and Setting: A prospective comparative experimental study was conducted at an institutional practice. Patients: Patients included six PCOS and six non-PCOS women undergoing COS and IVF. Interventions: Interventions included COS for IVF. Main Outcome Measures: VEGF-A levels in culture media were collected daily for 3 d from LGCs after incubation with variable doses of insulin, IGF-I, and IGF-II in the presence and absence of LH. Results: In both study groups, exposure to LH alone did not alter VEGF-A levels. However, insulin or IGF increased VEGF-A levels within 1 d and appeared to synergize with LH at 3 d. VEGF-A production by non-PCOS LGCs was more sensitive to IGF exposure, whereas PCOS cells were more sensitive to insulin. Although an increase in DNA content (P &lt; 0.05) was noted in cultures of PCOS cells, progesterone levels were lower compared with non-PCOS LGCs. Conclusion: Insulin and IGFs promote VEGF-A production in LGCs, but the response patterns are different when cells from PCOS and non-PCOS women are compared.

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