226. Expression of matrix metalloproteinases in bovine thecal cells

2005 ◽  
Vol 17 (9) ◽  
pp. 88
Author(s):  
L. Harland ◽  
H. F. Irving-Rodgers ◽  
S. E. Morris ◽  
R. J. Rodgers
Keyword(s):  
Extracellular Matrix ◽  
Matrix Metalloproteinases ◽  
Phorbol Ester ◽  
Granulosa Cells ◽  
Gelatin Zymography ◽  
Mrna Levels ◽  
Cell Culture Systems ◽  
Thecal Cells ◽  
Theca Interna ◽  
Free Media

As follicles grow the thecal layers expand. It is likely that extracellular matrix is remodelled in this process and possibly by matrix metalloproteinases (MMPs). A promising candidate to regulate MMPs is insulin-like factor 3 (INSL3). It is produced by thecal cells, its receptor, LGR8, is expressed in the theca interna (unpublished) and a related molecule, relaxin, regulates turnover of matrix in a number of tissues. For this reason we sort to examine the role of INSL3 in matrix turnover. However, in all thecal cell culture systems examined LGR8 receptors appear to be down regulated within 24 h. We therefore examined the effects of second messenger pathway activators. Thecal and granulosa cells were isolated and cultured and the levels of RNA for MMP2 and 9 quantitated by RT-PCR. MMP2 mRNA levels in thecal tissues were >10 fold higher than in granulosa cells (n = 19 follicles >10 mm). MMP2 levels were substantially greater than MMP9. At 12 h phorbol ester (100 nM phorbol 12,13-didecanoate) increased thecal expression of MMP 9 mRNA levels 11.5 fold (P < 0.001) and at 48 h MMP2 mRNA was increased 5 fold (P < 0.01). Pieces of whole follicle wall [follicles <5 mm in diameter, classified as healthy (n = 12) or atretic (n = 6)] were cultured in serum free media. Expression of the steroidogenic enzymes 17β HSD and P450scc but not 3β HSD were detected by immunohistochemistry even after 10 days. MMP activity on day 2 was analysed by gelatin zymography. Treatment with phorbol ester increased active MMP9 19 fold (P < 0.001). Treatment of thecal cells or follicle walls with 1 mM dibutyryl cAMP induced additional MMP activities at sizes of 110 and 122kDa. No effects on MMP2 activity were observed. In conclusion whilst we do not know the ligand inducers of the synthesis and activator of MMPs in thecal cells they can be regulated. Hence MMPs are candidates for remodelling the extracellular matrix of thecal layers.

Global gene expression profiles reveal an increase in mRNA levels of collagens, MMPs, and TIMPs in late radiation enteritis

2004 ◽  
Vol 287 (4) ◽  
pp. G875-G885 ◽  
Author(s):  
Carine Strup-Perrot ◽  
Denis Mathé ◽  
Christine Linard ◽  
Dominique Violot ◽  
Fabien Milliat ◽  
...  
Keyword(s):  
Gene Expression ◽  
Extracellular Matrix ◽  
Expression Profiles ◽  
Cdna Array ◽  
Muscularis Propria ◽  
Gelatin Zymography ◽  
Tissue Inhibitors Of Metalloproteinases ◽  
Radiation Enteritis ◽  
Mrna Levels ◽  
Fixed Tissue

Radiation enteritis, a common complication of radiation therapy for abdominal and pelvic cancers, is characterized by severe transmural fibrosis associated with mesenchymal cell activation, tissue disorganization, and deposition of fibrillar collagen. To investigate the mechanisms involved in this pathological accumulation of extracellular matrix, we studied gene expression of matrix components along with that of genes involved in matrix remodeling, matrix metalloproteinases (MMPs), and tissue inhibitors of metalloproteinases (TIMPs). Hybrid selection on high-density cDNA array, real-time RT-PCR, gelatin zymography and imunohistochemistry were used to characterize the mRNA expression profile, activity, and tissue location of extracellular matrix-related genes in radiation enteritis compared with healthy ileum. cDNA array analysis revealed a strong induction of genes coding for collagens I, III, IV, VI, and VIII, SPARC, and tenascin-C, extracellular-matrix degrading enzymes (MMP-1, -2, -3, -14, -18+19), and metalloproteinase inhibitors (TIMP-1, -2, plasminogen activator inhibitor-1) in radiation enteritis. This increase was correlated with the degree of infiltration of the mucosa by inflammatory cells, and the presence of differentiated mesenchymal cells in the submucosa and muscularis propria. Despite the fact that expression of collagens, MMPs, and TIMPs simultaneously increase, quantification of net collagen deposition shows an overall accumulation of collagen. Our results indicate that late radiation enteritis tissues are subjected to active process of fibrogenesis as well as fibrolysis, with a balance toward fibrogenesis. This demonstrates that established fibrotic tissue is not scarred fixed tissue but is subjected to a dynamic remodeling process.

scholarly journals Corticotropin-Releasing Factor Inhibits Luteinizing Hormone-Stimulated P450c17 Gene Expression and Androgen Production by Isolated Thecal Cells of Human Ovarian Follicles1

1998 ◽  
Vol 83 (2) ◽  
pp. 448-452
Author(s):  
H. F. Erden ◽  
I. H. Zwain ◽  
H. Asakura ◽  
S. S. C. Yen
Keyword(s):  
Gene Expression ◽  
Granulosa Cells ◽  
Inhibitory Effect ◽  
Corticotropin Releasing Factor ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Messenger Ribonucleic Acid ◽  
Estrogen Production ◽  
Thecal Cells ◽  
Crf System

Recently, we reported that the thecal compartment of the human ovary contains a CRF system replete with gene expression and protein for corticotropin-releasing factor (CRF), CRF-Receptor 1 (CRF-R1), and the blood-derived high affinity CRF-binding protein (CRF-BP). Granulosa cells are devoid of the CRF system. The parallel increases in intensity of CRF, CRF-R1, and 17α-hydroxylase messenger ribonucleic acid (mRNA) and proteins in thecal cells with follicular maturation suggest that the intraovarian CRF system may play an autocrine role regulating androgen biosynthesis, with a downstream effect on estrogen production by granulosa cells. The functionality of the ovarian CRF system may be conditioned by the relative presence of plasma-derived CRF-BP by virtue of its localization of protein, but not transcript in thecal cells and its ability to compete with CRF for the CRF receptor. To further these findings, in the present study we have examined the effect of CRF on LH-stimulated 17α-hydroxylase (P450c17) gene expression and androgen production by isolated thecal cells from human ovarian follicles (11–13 mm). During the 48-h culture, addition of LH (10 ng/mL) to the medium increased by 5- and 6-fold dehydroepiandrosterone and androstenedione production by thecal cells. Remarkably, the LH-stimulated, but not basal, androgen production was inhibited by CRF in a time- and dose-dependent manner. The half-maximal (ID50) effect dose of CRF occurred at 5 × 10−8 mol/L, and at a maximal concentration of 10−6 mol/L, CRF completely inhibited LH-stimulated androgen production. This inhibitory effect of CRF became evident at 12 h (45%), and by 24 h the effect was more pronounced, with a 70% reduction from baseline. As determined by Northern analyses, CRF dose dependently decreased LH-stimulated P450c17 mRNA levels, with a maximal inhibition of 85% P450c17 gene expression at a CRF concentration of 10−6 mol/L. With the addition of 10−6 mol/L of the antagonist α-helical CRF-(9–41), the inhibitory effect of CRF was partially reversed for both P450c17 mRNA (75%) and androgen production (50%), indicating the CRF-R1-mediated event. In conclusion, the present study demonstrated a potent inhibitory effect of CRF on LH-stimulated dehydroepiandrosterone and androstenedione production that appears to be mediated through the reduction of P450c17 gene expression. Thus, the ovarian CRF system may function as autocrine regulators for androgen biosynthesis in the thecal cell compartment to maintain optimal substrate for estrogen biosynthesis by granulosa cells. Further studies to define the role of CRF-BP in the endocrine modulation of the intraovarian CRF system are needed.

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 Localization of binding sites for IGF-I, insulin and GH in the sow ovary

1999 ◽  
Vol 163 (2) ◽  
pp. 363-372 ◽  
Author(s):  
H Quesnel
Keyword(s):  
Granulosa Cells ◽  
Stromal Cells ◽  
Binding Sites ◽  
Binding Proteins ◽  
Cell Function ◽  
Type I ◽  
Thecal Cells ◽  
Igf I ◽  
Theca Externa ◽  
Theca Interna

Binding sites for IGF-I, insulin, and GH were localized by in situ binding of (125)I-labelled hormones to the different compartments of the sow ovary. Binding sites for IGF-I were detected in oocytes, granulosa and thecal cells of healthy and atretic follicles as well as in the antrum and the stroma. Competition of (125)I-labelled IGF-I with IGF-I, insulin and an analogue of IGF-I (Long R(3)IGF-I), which allowed discrimination between binding to binding proteins from binding to type-I receptors, suggested that type-I receptors were present in granulosa cells of healthy follicles, whilst binding in other compartments was mainly due to binding proteins. Binding of insulin was revealed in oocytes, granulosa and theca interna cells of healthy preantral and antral follicles, and, to a lesser extent, in theca externa and stromal cells, and was still observed in granulosa cells of atretic follicles. Labelling with (125)I-labelled bovine GH was demonstrated in oocytes, granulosa cells, theca interna cells, and, although less intense, in theca externa and stromal cells. It disappeared in granulosa cells during atresia. Binding sites for GH were detected at all follicular stages, from preantral to preovulatory stages, but the intensity of labelling in granulosa cells was more intense in preantral than in large follicles. These data support the participation of insulin, GH and IGF-I in oocyte maturation, follicular growth and stromal cell function in swine.

scholarly journals Gene expression for LH receptor, 17α-hydroxylase and StAR in the theca interna of preantral and early antral follicles in the bovine ovary

Reproduction ◽  
2005 ◽  
Vol 129 (4) ◽  
pp. 453-461 ◽  
Author(s):  
R Braw-Tal ◽  
Z Roth
Keyword(s):  
Gene Expression ◽  
Granulosa Cells ◽  
Regulatory Protein ◽  
Morphological Differentiation ◽  
Lh Receptor ◽  
Antral Follicles ◽  
Thecal Cells ◽  
Key Enzyme ◽  
Theca Interna ◽  
Steroidogenic Acute Regulatory

The onset of gene expression for three proteins that play pivotal roles in theca interna function, namely the LH receptor (LH-R), cytochrome P450 17α-hydroxylase (17αOH) and the steroidogenic acute regulatory protein (StAR), was determined. Ovaries were obtained on day 9 of the oestrus cycle from mature synchronized dairy cows (n= 5) and gene expression in preantral and antral follicles up to 4 mm in diameter was evaluated byin situhybridization. LH-R and 17αOH mRNAs were observed first, in the theca interna of large preantral follicles (type 4), concurrent with its morphological differentiation. StAR mRNA appeared later during follicular growth, in follicles >1 mm in diameter (type 6). LH-R and 17αOH mRNAs were found exclusively in the thecal cells, whereas StAR mRNA appeared in thecal cells, granulosa cells of late atretic follicles and oocytes. In early atresia, thecal cells expressed all three mRNAs, and their expression decreased gradually as atresia progressed. Atresia in granulosa cells was characterized by massive apoptosis of periantral, but not peribasal cells, that differentiated into luteal-like cells expressing StAR.In summary, our study suggests that in spite of the presence of 17αOH, a key enzyme in steroidogenesis, the ability to produce steroids by bovine follicles smaller than 1 mm in diameter must be very limited due to the absence of StAR protein. During the early stages of atresia, thecal cells remain morphologically and functionally healthy, and continue to express all three studied mRNAs.

Matrix metalloproteinases and tissue inhibitor of metalloproteinase-2 in fetal rabbit lung

2000 ◽  
Vol 279 (3) ◽  
pp. L555-L561 ◽  
Author(s):  
Yuh Fukuda ◽  
Masamichi Ishizaki ◽  
Yasunori Okada ◽  
Motoharu Seiki ◽  
Nobuaki Yamanaka
Keyword(s):  
Extracellular Matrix ◽  
Epithelial Cells ◽  
Matrix Metalloproteinases ◽  
Late Stage ◽  
Lung Development ◽  
Gelatin Zymography ◽  
Fetal Lung ◽  
Tissue Inhibitor Of Metalloproteinase ◽  
Fetal Lung Development ◽  
Tissue Homogenates

Cell-extracellular matrix interaction and extracellular matrix remodeling are known to be important in fetal lung development. We investigated the localization of matrix metalloproteinases (MMPs) in fetal rabbit lungs. Immunohistochemistry for type IV collagen, MMP-1, MMP-2, MMP-9, membrane type (MT) 1 MMP, and tissue inhibitor of metalloproteinase (TIMP)-2 and in situ hybridization for MMP-9 mRNA were performed. Gelatin zymography and Western blotting for MT1-MMP in lung tissue homogenates were also studied. MMP-1 and MT1-MMP were detected in epithelial cells, and MMP-2 and TIMP-2 were detected in epithelial cells and some mesenchymal cells in each stage. MMP-9 was found in epithelial cells mainly in the late stage. Gelatin zymography revealed that the ratio of active MMP-2 to latent MMP-2 increased dramatically during the course of development. MT1-MMP was detected in tissue homogenates, especially predominant in the late stage. These findings suggest that MMPs and their inhibitors may contribute to the formation of airways and alveoli in fetal lung development and that activated MMP-2 of alveolar epithelial cells may function to provide an extremely wide alveolar surface.

scholarly journals Studies of granulosa cell maturation in dominant and subordinate bovine follicles: novel extracellular matrix focimatrix is co-ordinately regulated with cholesterol side-chain cleavage CYP11A1

Reproduction ◽  
2009 ◽  
Vol 137 (5) ◽  
pp. 825-834 ◽  
Author(s):  
H F Irving-Rodgers ◽  
M L Harland ◽  
T R Sullivan ◽  
R J Rodgers
Keyword(s):  
Extracellular Matrix ◽  
Granulosa Cells ◽  
Volume Density ◽  
Side Chain ◽  
Collagen Type Iv ◽  
Mrna Levels ◽  
Side Chain Cleavage ◽  
Type Iv ◽  
Chain Cleavage ◽  
Highly Correlated

During growth of antral ovarian follicles granulosa cells first become associated with a novel type of extracellular matrix, focimatrix, and at larger sizes follicles become either subordinate or dominant. To examine this, bovine subordinate (9.0±s.e.m.0.4 mm;n=16), partially dominant (12.0±0.6 mm;n=18) and fully dominant (15.0±0.4 mm;n=14) follicles were examined by real time RT-PCR analyses of granulosa cells and by immunohistochemistry of focimatrix. Changes in the expression of FSH receptor, LH receptor, cholesterol side-chain cleavage (CYP11A1), 3β-hydroxysteroid dehydrogenase, aromatase (CYP19A1) and inhibin-α and β-B were observed as expected for follicle sizes examined. After adjusting for size differences, onlyCYP11A1was significantly different between the groups, and elevated in dominant follicles. Also after adjusting for differences in size there were no significant differences in expression of focimatrix components collagen type IV α-1 (COL4A1), laminin β-2, nidogen 1 (NID1), and perlecan (HSPG2) or the volume density of NID1 and -2 and HSPG2. The volume density of focimatrix components in laminin 111 was significantly elevated in dominant follicles. Adjusting for analysis of more than one follicle per animal and for multiple correlations,CYP11A1mRNA levels were highly correlated with the focimatrix genesCOL4A1,NID1and -2andHSPG2. Thus, focimatrix may potentially regulateCYP11A1expression, and the regulation of both could be important in follicular dominance.

scholarly journals Induction of Heparanase in Bovine Granulosa Cells by Luteinizing Hormone: Possible Role during the Ovulatory Process

Endocrinology ◽  
2008 ◽  
Vol 150 (1) ◽  
pp. 413-421 ◽  
Author(s):  
Eyal Klipper ◽  
Ehud Tatz ◽  
Tatiana Kisliouk ◽  
Israel Vlodavsky ◽  
Uzi Moallem ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Luteinizing Hormone ◽  
Granulosa Cells ◽  
Prostaglandin F2α ◽  
Follicular Development ◽  
Mrna Levels ◽  
Ru 486 ◽  
Protein Levels ◽  
Gonadotropin Surge ◽  
Ovulatory Process

Follicular development, follicular rupture, and corpus luteum (CL) formation are accompanied by extensive tissue remodeling. We examined whether heparanase (HPSE), which cleaves heparan sulfate glycosaminoglycans, is induced during these processes. Prostaglandin F2α injection, which initiated luteolysis and the development of a preovulatory follicle, moderately increased HPSE mRNA in bovine granulosa cells (GCs). GnRH, used to induce gonadotropin surge, markedly augmented HPSE mRNA levels 12 h after its injection. The temporal pattern of HPSE gene expression in follicular-luteal transition was further examined in follicles collected before, and 4, 10, 20, 25, and 60 h after GnRH injection. HPSE mRNA increased transiently 10–20 h after GnRH injection to levels 10-fold higher than in untreated heifers. HPSE protein levels were similarly elevated 20 h after GnRH injection in GCs, but not in the theca layer. Cyclooxygenase-2 (PTGS2) mRNA peaked before ovulation when HPSE levels returned to baseline levels. HPSE mRNA abundance also remained low in the CLs. The antiprogesterone, RU-486, elevated HPSE levels in GC culture, suggesting that progesterone secreted by CLs may inhibit HPSE. HPSE immunostaining was more abundant in GCs than thecae. In cultured GCs, LH induced a transient increase in HPSE mRNA 3–6 h after its addition, but not at 24 h. However, PTGS2 mRNA was clearly induced at this time. These findings suggest that: 1) HPSE may play a role in ovulation but much less so during CL development, and 2) GC-derived HSPE may be a novel member of the LH-induced extracellular matrix-degrading enzyme family and may contribute to follicular rupture. Granulosa-derived heparanase is a novel member of the luteinizing hormone-induced extracellular matrix-degrading enzymes contributing to follicular rupture and ovulation.

scholarly journals Androgen Receptor Gene Expression in the Primate Ovary: Cellular Localization, Regulation, and Functional Correlations

1998 ◽  
Vol 83 (7) ◽  
pp. 2479-2485 ◽  
Author(s):  
S. J. Weil ◽  
K. Vendola ◽  
J. Zhou ◽  
O. O. Adesanya ◽  
J. Wang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Proliferation ◽  
Androgen Receptor ◽  
Granulosa Cell ◽  
Granulosa Cells ◽  
Stromal Cells ◽  
Cellular Localization ◽  
Specific Antigen ◽  
Mrna Levels ◽  
Theca Interna

Excess androgens are associated with a characteristic polyfollicular ovarian morphology; however, it is not known to what extent this problem is due to direct androgen action on follicular development vs. interference with gonadotropin release at the level of the pituitary or hypothalamus. To elucidate potential androgen effects on the ovary, we investigated the cellular localization of androgen receptor (AR) messenger ribonucleic acid (mRNA) in rhesus monkey using in situ hybridization. To investigate the regulation of ovarian AR gene expression, we compared the relative abundance of AR transcripts in monkeys during follicular and luteal phases of the menstrual cycle and in monkeys treated with testosterone. To assess potential functional consequences of AR expression in the primate ovary, we compared AR mRNA levels with indexes of follicular cell proliferation and apoptosis in serial sections from individual follicles. AR mRNA expression was most abundant in granulosa cells of healthy preantral and antral follicles in the primate ovary. Theca interna and stromal cells also expressed AR mRNA, but to a lesser degree than granulosa cells. No significant cycle stage effects were noted in AR mRNA levels; however, larger numbers of animals would be necessary to definitively establish a cycle stage effect. AR mRNA level was significantly increased in granulosa cells and was decreased in theca interna and stromal cells of testosterone-treated monkeys. Importantly, granulosa cell AR mRNA abundance was positively correlated with expression of the proliferation-specific antigen Ki-67 (r = 0.91; P &lt; 0.001) and negatively correlated with granulosa cell apoptosis (r = −0.64; P &lt; 0.001). In summary, these data show that primate ovary AR gene expression is most abundant in granulosa cells of healthy growing follicles, where its expression is up-regulated by testosterone. The positive correlation between granulosa AR gene expression and cell proliferation and negative correlation with programmed cell death suggests that androgens stimulate early primate follicle development.

Physical exercise can influence local levels of matrix metalloproteinases and their inhibitors in tendon-related connective tissue

2004 ◽  
Vol 96 (3) ◽  
pp. 861-864 ◽  
Author(s):  
S. O. A. Koskinen ◽  
K. M. Heinemeier ◽  
J. L. Olesen ◽  
H. Langberg ◽  
M. Kjaer
Keyword(s):  
Extracellular Matrix ◽  
Matrix Metalloproteinases ◽  
Inhibitory Activity ◽  
Gelatin Zymography ◽  
Treadmill Running ◽  
Type I ◽  
Tendon Tissue ◽  
Time Pattern ◽  
Local Levels

Microdialysis studies indicate that mechanical loading of human tendon tissue during exercise or training can affect local synthesis and degradation of type I collagen. Degradation of collagen and other extracellular matrix proteins is controlled by an interplay between matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs). However, it is unknown whether local levels of MMPs and TIMPs are affected by tendon loading in humans in vivo. In the present experiment, six healthy young men performed 1 h of uphill (3%) treadmill running. Dialysate was collected from microdialysis probes (placed in the peritendinous tissue immediately anterior to the Achilles tendon) before, immediately after, 1 day after, and 3 days after an exercise bout. MMP-2 and MMP-9 were measured in dialysate by gelatin zymography, and amounts were quantified by densitometry in relation to total protein in the dialysate. TIMP-1 and TIMP-2 were analyzed by reverse gelatin zymography and semiquantitated visually. Pro-MMP-9 increased markedly after exercise and remained high for 3 days after exercise. Pro-MMP-2 dropped from the basal level immediately after exercise and remained low 1 day after exercise but was slightly elevated 3 days after exercise. The MMP-2 inhibitory activity of TIMP-1 was clearly elevated 1 and 3 days after exercise, and the MMP-2 inhibitory activity of TIMP-2 rose 1 day after loading. The present findings demonstrate enhanced interstitial amounts of MMPs and TIMPs after exercise in the human peritendinous tissue in vivo, and the magnitude and time pattern of these changes may well indicate that MMPs and TIMPs are playing a role in extracellular matrix adaptation to exercise in tendon tissue.

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