Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.24.3 (collagenase)
18,340 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

To determine and compare the direct effects of prostaglandin F2a (PGF2a) and human chorionic gonadotropin (hCG) on luteal cell progesterone production in vitro, 9 human corpora lutea obtained at tubal ligation were minced and treated with collagenase to disaggregate luteal cells. Dispersed luteal cells (80% viable) were incubated in air at 37 degrees C in a shaking water bath for 3 h and total progesterone in the media and cells was determined by radioimmunoassay. Optimum progesterone production was obtained using 25,000 or more cells per incubate and an incubation time of 2-4 h. hCG-stimulated progesterone production increased significantly with 0.01 IU to as high as 100 IU. In the early luteal phase (days 1-5 post ovulation or days 15-20 of the luteal phase), PGF2a (10-1000 ng) significantly inhibited progesterone production but significantly stimulated progesterone production in the mid-luteal phase (days 21-25). PGF2a had no effect on luteal cell progesterone production in the late luteal phase (days 26-30). This age-dependent direct effect of PGF2a on human luteal cell progesterone production in vitro indicates a role for PGF2a in the total intragonadal regulation of progesterone output, possibly through a paracrine or autocrine manner directed towards synchronizing luteal progesterone secretion and endometrial preparation for nidation.
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PMID:Effect of human chorionic gonadotropin and prostaglandin F2a on progesterone production by human luteal cells. 260 39

Testis cells of the toad Xenopus laevis were dissociated with collagenase and the cell suspension was enriched for steroidogenic cells by Percoll gradients. Results suggested that cells should be preincubated during a 6-h period before stimulation with gonadotropin. Our results indicate that a 2-h incubation period with gonadotropin was necessary to obtain a significant response. Furthermore, the cells can be maintained in a functional state longer than mammalian testis cells. Different gonadotropins were used to stimulate androgen production, and their effects were compared in both dissociated cells and testicular explants. Cells were more sensitive to luteinizing hormone (LH) and follicle stimulating hormone (FSH) than the explants (ED50LH = 0.041 +/- 0.003 micrograms for cells and 0.097 +/- 0.002 micrograms for explants: ED50FSH = 0.41 +/- 0.03 micrograms for cells and 0.63 +/- 0.03 micrograms for explants). Moreover, human chorionic gonadotropin (hCG) which only stimulates testicular explants at high doses, failed to stimulate the androgen production of dissociated cells; this indicates a low sensitivity of amphibian testis to hCG and a possible damaging effect of collagenase on the receptors of isolated cells.
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PMID:Gonadotropin action on androgen synthesis in short-term incubation of explants and dispersed cells of the testis of Xenopus laevis. 283 21

Factors other than adrenocorticotropic hormone (ACTH) are thought to influence fetal adrenal steroidogenesis during primate pregnancy. Therefore, we determined the effects of prolactin (Prl), growth hormone (GH), and human chorionic gonadotropin (hCG) as well as ACTH on steroid secretion by collagenase-dispersed baboon fetal adrenal cells. Adrenal glands were obtained from seven baboon (Papio anubis) fetuses following cesarean section at Day 100-107 of gestation (term = Day 184). Tissue was minced with a fine scissors and cells were dispersed with 0.2% collagenase, then washed with Medium 199 containing penicillin/streptomycin. Cells (0.5 X 10(4)) were placed in 4 ml Medium 199 with or without 10 nmol ovine Prl, ovine GH, or ACTH, or 50 nmol hCG. After 18 h incubation (37 degrees C), cells were separated by centrifugation and the quantities of cortisol (F), dehydroepiandrosterone (DHA), and DHA-sulfate (DHAS) secreted into the medium were determined. In controls, DHA secretion [224 +/- 96 ng/(24 h X 10(5) cells] was greater (P less than 0.05) than that of DHAS (20 +/- 12) and F (14 +/- 12). Adrenocorticotropic hormone, Prl, and GH stimulated (P less than 0.05) DHA secretion by 370% +/- 71%, 215% +/- 61%, and 292% +/- 73%, respectively; hCG was not effective. Due primarily to the relatively low secretion rates, DHAS and F secretion were not altered by hormonal treatment. Moreover, addition of 20 nmol progesterone to the medium in the presence or absence of ACTH did not influence F production. These findings indicate that the baboon fetal adrenal at midgestation does not utilize placental progesterone for F synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Regulation of baboon fetal adrenal androgen production by adrenocorticotropic hormone, prolactin, and growth hormone. 299 43

A method has been developed to assay collagenase in ovarian extracts in the presence of tissue inhibitors. Rat ovarian tissue is first extracted with Triton X-100 and then heated to 60 degrees C in 50 mM Tris buffer containing 100 mM CaCl2. This extract contains collagenase activity and putative inhibitor(s). The inhibitory activity is removed by reduction with dithiothreitol and alkylation with iodoacetamide. Collagenase is then activated with aminophenylmercuric acetate and assayed using 3H-acetylated collagen from which the telopeptides have been removed. Identification of this activity as collagenase was performed by using the metalloprotease inhibitors EDTA and o-phenanthroline and by demonstration of the typical collagen cleavage fragments on sodium dodecyl sulfate-gel electrophoresis. To investigate the changes in collagenase activity associated with ovulation, immature rats received 20 IU of pregnant mare's serum gonadotropin and 52 h later 10 IU of human chorionic gonadotropin (hCG). After hCG administration, ovaries were removed at intervals from 0 to 20 h. Collagenase activity rose from 4.9 +/- 1.4% digestion of the 3H-collagen at 0 time to a maximum of 24.7 +/- 1.5% digestion at 8 h after hCG and remained high at 12 h (time of ovulation) and up to 20 h (18.7 +/- 1.9% and 16.1 +/- 1.6% digestion, respectively). These findings support a role of collagenase in the rupture of the follicle and they suggest a further role for this enzyme in the events following ovulation.
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PMID:The extraction of a tissue collagenase associated with ovulation in the rat. 300 8

We determined whether dehydroepiandrosterone (DHA) and androstenedione (A) were converted to testosterone (T) by the midgestation primate fetal testis in the absence of gonadotropins. Testes from six baboon (Papio anubis) fetuses, obtained by cesarean section at Day 100-107 of gestation (term = Day 184) were dispersed with 0.2% collagenase. Cells (1.1 X 10(6)) were suspended in 4 ml Eagle's Minimum Essential Medium containing penicillin/streptomycin (MEM) and incubated for 20 h (37 degrees C) with or without DHA, A, pregnenolone (P5), 17 alpha-hydroxypregnenolone (17OH-P5), progesterone (P4) or 17 alpha-hydroxyprogesterone (17OH-P4). Concentrations of T, A, P4, and 17OH-P4 in the medium and cells were measured by radioimmunoassay. Mean secretions of T and A, in the absence of exogenous substrates, were 0.5 +/- 0.2 and 0.8 +/- 0.3 ng/mg testis, respectively, and were not elevated by human chorionic gonadotropin (hCG). Addition of DHA at 100, 500, or 1000 ng/4 ml increased (p less than 0.05) the production of T to 6 +/- 0.6, 33 +/- 10, and 64 +/- 26 ng/mg testis and the production of A to 13 +/- 5.5, 54 +/- 10, and 67 +/- 22 ng/mg testis, respectively. Similarly, addition of A at 100, 500, or 1000 ng/4 ml increased (p less than 0.05) production of T to 27 +/- 5.3, 155 +/- 29, and 254 +/- 79 ng/mg testis, respectively. In contrast, production of T and A remained near baseline concentrations when cells were incubated with 1000 ng/4 ml of P5, P4, 17OH-P5, or 17OH-P4.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Testosterone production by collagenase-dispersed cells from baboon fetal testis. 302 Dec 48

Prepuberal (P) gilts were induced to ovulate with pregnant mare serum gonadotropin followed 72 h later by human chorionic gonadotropin (hCG). Three P gilts and three mature (M) gilts each were ovariectomized on d 10, 14, 18, 22 and 26 (d 0 = day of hCG for P gilts and onset of estrus for M gilts). Gilts ovariectomized on d 14, 18, 22 and 26 were hysterectomized on d 6 to ensure maintenance of the corpora lutea (CL). Two to five grams of minced luteal tissue were dispersed using collagenase and hyaluronidase in HEPES buffered salt solution supplemented with glucose and bovine serum albumin. Dispersed cells were rinsed in Dulbecco's Modified Eagle Medium (DMEM), counted (ratio of large to total number of luteal cells determined) and then incubated for 1 h in DMEM. With aliquots standardized to 2.5 X 10(4) viable, large cells (greater than 25 micron diameter) were incubated in 1 ml DMEM for 2 h in the presence of either 10, 50, 100 or 1,000 ng luteinizing hormone (LH); .1, 1, 10 or 100 ng hCG; 10, 100 or 1,000 ng norepinephrine (NE) or either .75, or 1.5 mM dibutyrl cyclic adenosine monophosphate (dbcAMP). Progesterone (P4) in the medium was quantified by radioimmunoassay. Basal P4 production (no P4 stimulator added to the medium) on d 10, 14, 18, 22 and 26 for P gilts was 246 +/- 9, 66 +/- 4, 64 +/- 6, 41 +/- 3 and 69 +/- 6 ng/ml medium, respectively, and for M gilts was 281 +/- 12, 128 +/- 8, 53 +/- 4, 82 +/- 6, 101 +/- 5 ng/ml medium, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Comparison of induced corpora lutea from prepuberal gilts and spontaneous corpora lutea from mature gilts: in vitro progesterone production. 303 Sep 95

The ability of 125I-labeled human chorionic gonadotropin (125I-labeled hCG) to bind and stimulate steroidogenesis was studied in light cells (density, 1.053-1.065 g/cm3) and heavier cells (density, 1.090-1.110 g/cm3) purified from collagenase-dispersed rat testicular interstitial cells by unit gravity sedimentation (Bhalla, V.K., Rajan, V.P., Burgett, A.C., and Sohal, G.S. (1987) J. Biol. Chem. 262, 5313-5321). Preferential localization of gonadotropin binding sites was demonstrated on light cells, and the heavier cells produced testosterone in response to hCG without occupancy of high affinity (Kd = 2.02 X 10(-10) M) binding sites. In this study, established methods for interstitial cell purification involving gradient centrifugation were utilized to demonstrate the cell heterogeneity. Light cells bound hCG with high affinity (Kd = 3 X 10(-10) M) without manifestation of steroidogenic response. The heavier cells responded to hCG with elicitation of steroidogenesis, but the occupancy was negligible. Stimulation of steroidogenesis by hCG in heavier cells was dose and time dependent. Dibutyryl and bromo cyclic AMP (1 mM) also promoted steroidogenesis comparable to a level stimulated by the tropic hormone (700% stimulation). The concept of spare receptors was tested in purified cell fractions. Upon cell purification, no saturable high affinity binding sites were observed in the heavier cell fraction. Autoradiographic analyses at the electron microscopical level supported this conclusion. Our data suggest that target cell activation is not preceded by hormone occupancy of high affinity binding sites. A model for defining the functional domains of the physiological receptor for hCG is presented.
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PMID:Interstitial cell heterogeneity in rat testes. II. Purification of cells by Percoll and metrizamide gradient centrifugation with preferential localization of gonadotropin binding sites in light cell fraction and hormone-induced steroidogenesis in heavier cell fraction. 303 Oct 50

During ovulation, enzymatic degradation of the extracellular matrix occurs within and around the graafian follicles. In this study, the activities of several different proteolytic enzymes were measured in the culture media of follicles taken from pregnant mare serum gonadotropin (PMSG)-primed immature rats. At 52 h after PMSG, the follicles were cultured for 2 to 15 h in media with or without human chorionic gonadotropin (hCG). Type I collagenase activity in hCG-stimulated follicles gradually increased within 6 h to 3.3-fold above that of the controls. Relatively pure populations of granulosa cells produced type I collagenase to a similar extent. Likewise, type IV collagenase increased 3.8-fold by 6 h after exposure of the follicles to hCG. In contrast, plasminogen activator activity increased by 3.9-fold at 2 h after hCG, but was negligible at 4, 6, and 15 h after incubation. These results suggest that plasminogen activator may activate both type I and type IV collagenase in hCG-stimulated ovulatory follicles.
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PMID:Types I and IV collagenolytic and plasminogen activator activities in preovulatory ovarian follicles. 303 95

In mammalian ovaries, luteinizing hormone (LH) induces androgen biosynthesis by theca interna cells, whereas both follicle-stimulating hormone (FSH) and LH stimulate aromatase activity by granulosa cells. The joint action of these two types of cell and pituitary hormones forms the basis of a 2-cell, 2-gonadotropin hypothesis for biosynthesis of estrogen. We tested the synergism between these cell types using antisera against the estrogen precursors progesterone and testosterone. Ovaries were obtained from immature rats two days after a single injection of pregnant mare serum gonadotropin (PMSG). Granulosa cells were obtained by puncturing the follicles, and ovarian remnants were dissociated with collagenase to derive cells from the theca interstitium. Granulosa and theca interstitial cells were cultured alone or in combination for 20 h. Granulosa cells secreted negligible amounts of estrogen and testosterone, but contained high aromatase activity. Treatment with both FSH and human chorionic gonadotropin (hCG) increased production of progesterone in granulosa cells. In contrast, theca interstitial cells had negligible aromatase activity. Treatment with hCG, but not FSH, increased androgen production by theca interstitium; the hCG action was further augmented by the inclusion of exogenous progesterone. Furthermore, co-culture of gonadotropin-treated granulosa and theca interstitial cells resulted in substantial increases in estrogen production, indicating synergism between the two types of cell. The increases in estrogen production in the co-cultures were accompanied by decreases in progesterone content. To test the possibility that progesterone released by granulosa cells may serve as a substrate for production of androgen in theca cells, specific antiserum was used to adsorb progesterone present in the medium. Addition of the progesterone antibody inhibited gonadotropin-stimulated production of testosterone (39% decrease) and estrogen (64% decrease) by the combined cell cultures. The inhibitory effect on estrogen production could be reversed by the addition of progesterone (10(-6) M) or testosterone (10(-6) M) but not by the addition of a synthetic progestin, R5020. Since testosterone released from theca cells may serve as the substrate for aromatases in granulosa cells, specific testosterone antiserum was also used. Production of estrogens by the combined cell cultures was inhibited (78%) by the testosterone antiserum, but the inhibitory effect was completely reversed by exogenous testosterone or progesterone. Thus, synergistic interactions between granulosa and theca interstitial cells are important in effecting maximal estrogen biosynthesis.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Synergism between granulosa and theca-interstitial cells in estrogen biosynthesis by gonadotropin-treated rat ovaries: studies on the two-cell, two-gonadotropin hypothesis using steroid antisera. 309 Nov 3

The present study was intended to examine the structure of the rat Leydig cell gonadotropin receptor. Leydig cell suspensions were prepared by either collagenase digestion or mechanical disruption of the testes. The cells were incubated with 125I-human chorionic gonadotropin (hCG) following which the bound 125I-hCG was covalently cross-linked to the cell surface receptor using a cleavable (dithiobis(succinimidyl propionate] and a noncleavable (disuccinimidyl suberate) cross-linking reagent. The extracted cross-linked membrane proteins were resolved on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing and nonreducing conditions and subjected to autoradiographic analysis. Under nonreducing conditions, three radiolabeled bands, in addition to intact hCG and its alpha-subunit, were detected with apparent molecular weights of 184,000, 136,000, and 103,000. However, under reducing conditions, three radiolabeled bands migrated on the gel corresponding to molecular weights of 144,000, 106,000, and 75,000. The binding of 125I-hCG to the receptor was inhibited by hCG and luteinizing hormone, but not by a number of other peptides or proteins. The radiolabeled bands were not detectable in hCG down-regulated Leydig cells. Furthermore, a similar autoradiographic pattern of 125I-hCG-linked complexes was seen when the 125I-linked receptor complex was subjected to immunoprecipitation with anti-hCG antibodies followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In addition, evidence was obtained indicating that these three labeled bands were derived from the same molecular species. The data suggests that the hCG receptor in Leydig cell is probably an oligomeric complex with a molecular weight of about 250,000, which is composed of three polypeptide chains of molecular weights 121,000, 83,000, and 52,000 held together through noncovalent forces. Additionally, collagenase treatment of Leydig cells does not appear to alter the autoradiographic pattern of the 125I-hCG-linked receptor.
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PMID:Characterization of rat Leydig cell gonadotropin receptor structure by affinity cross-linking. 333 12


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