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)

Ammonium perfluorooctanoate (C8) produced a dose-dependent increase in Leydig cell adenomas in Crl:CD BR (CD) rats fed 0, 30, or 300 ppm for 2 years. Administration of C8 to adult male CD rats, by gavage for 14 days, produced decreased serum and testicular interstitial fluid testosterone levels and increased serum estradiol levels. The C8-mediated decrease in the serum testosterone levels appeared to be due to a lesion at the level of the testis. These endocrine changes may play a role in the C8 induction of Leydig cell tumors. In the present work, C8 was examined for its ability to (1) directly affect Leydig cells in vitro using isolated Leydig cells from untreated rats and ex vivo using Leydig cells isolated from C8-treated rats, (2) affect testicular interstitial fluid hormone levels, and (3) induce aromatase activity. These studies were conducted to investigate the hypothesis that C8 produces an increase in estradiol by inducing cytochrome P450 XIX (aromatase), which converts testosterone to estradiol, and that the elevated estradiol levels ultimately produce Leydig cell hyperplasia and adenoma formation by acting as a mitogen or enhancing growth factor secretion. In the in vivo and ex vivo studies, adult male CD rats were gavaged with either 0 or 25 mg/kg/day C8 for 14 days. In addition to the ad libitum control, a second control group was pair-fed to the 25 mg/kg/day C8 group. In the in vitro and ex vivo studies, Leydig cells were isolated from testes of adult males by collagenase digestion followed by enrichment over Percoll gradients. A dose-dependent decrease in testosterone levels was observed in hCG-stimulated Leydig cells treated in vitro with C8 for 5 hr (IC50 approximately 200 microM). In contrast, ex vivo studies demonstrated an increase in testosterone production in hCG-stimulated Leydig cells from C8-treated rats when compared to Leydig cells isolated from either the ad libitum or pair-fed control rats. The in vitro data demonstrate that C8 directly inhibits testosterone release from Leydig cells, while the ex vivo data demonstrate that this inhibition is reversible.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Effects of ammonium perfluorooctanoate on Leydig cell function: in vitro, in vivo, and ex vivo studies. 767 54

In this study the localization and regulation of steady-state follistatin messenger ribonucleic acid (mRNA) levels in testicular cell cultures were examined with a solution-hybridization assay using a specific 32P-labelled cytosolic RNA antisense probe for follistatin and a 35S-labelled cytosolic RNA antisense probe for cyclophilin as internal standard. Testes from immature rats were dispersed with collagenase and fractionated in Sertoli and Leydig cell-enriched cultures. Follistatin mRNA was mainly localized to the Sertoli cell-enriched fraction and the expression of follistatin mRNA could be stimulated in vitro with fetal calf serum, epidermal growth factor or phorbol-12-myristate-13-acetate (an activator of protein kinase C), whereas follicle-stimulating hormone and forskolin (an activator of protein kinase A) had no effect. Neither prostaglandin E2, the synthetic glucocorticoid RU 28362 or all-trans-retinoic acid, which all regulate follistatin mRNA levels in non-testicular cell types, nor extracellular adenosine triphosphate (a purinergic receptor agonist) or testosterone had any obvious influence on follistatin mRNA levels in Sertoli cell-enriched cultures. From this study it is concluded that Sertoli cells are likely to be the source of follistatin expression in the rat testis, that follistatin mRNA levels in Sertoli cell-enriched cultures are subjected to regulation by epidermal growth factor and the protein kinase C-dependent pathway but are not regulated by extracellular adenosine triphosphate, follicle-stimulating hormone, all-trans-retinoic acid, prostaglandin E2, forskolin, testosterone or the glucocorticoid RU 28362 and that the regulation of follistatin mRNA is sex- and tissue-specific.
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PMID:Expression of follistatin messenger ribonucleic acid in Sertoli cell-enriched cultures: regulation by epidermal growth factor and protein kinase C-dependent pathway but not by follicle-stimulating hormone and protein kinase A-dependent pathway. 810 86

An in-vitro method was developed to study Sertoli-Leydig cell interactions in man, using testes removed at the time kidneys were removed for transplantation from 6 young adult men (aged 17-45 years) after cerebral death. After collagenase digestion of testicular tissue, Leydig cells were purified on discontinuous Percoll gradients. Two fractions of Leydig cells, 'L2' and 'L3' which differed in their buoyant density (1.05 g < L2 < 1.06 g and 1.06 g < L3 < 1.08 g), were obtained. The Sertoli cell-enriched preparation was obtained from seminiferous tubular fragments after sequential treatment with glycine buffer to remove peritubular-myoid cells, a second collagenase digestion, mechanical fragmentation and washes to remove germ cells. Purified Leydig cells were then cultured either alone or together with Sertoli cells in culture dishes coated with collagen, fibronectin and laminin in a chemically defined medium without serum. The influence of co-culture on basal testosterone secretion was examined in 3 successive 48 h periods.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Enhancement of testosterone secretion by normal adult human Leydig cells by co-culture with enriched preparations of normal adult human Sertoli cells. 846 93

The present study investigated the effects of aging in the testis interstitium in Sprague Dawley rats. Rats of 3, 6 and 24 months of age were used. Testes of rats (n = 5) were fixed by whole body perfusion using a fixative containing 2.5% glutaraldehyde in cacodylate buffer, processed and embedded in eponaraldite. Using 1 microns sections stained with methylene blue, qualitative and quantitative morphological studies were performed. Purified Leydig cell preparations, obtained by collagenase digestion followed by elutriation and density gradient centrifugation, were used to determine luteinizing hormone (LH; 100 ng/ml) stimulated testosterone secretory capacity per Leydig cell in vitro. Testosterone levels in the incubation medium, and testosterone and luteinizing hormone levels in serum of these three groups of rats were determined via radioimmunoassay. Morphological studies revealed that Leydig cells were more abundant in the testis interstitium at 6 and 24 months when compared to 3 months. Moreover, collagen fiber bundles were more frequently observed in the testis interstitium at older ages. Blood vessels of the testis interstitium in 24-month-old rats frequently showed partial and complete occlusion of their lumen and thickening of vessel walls. This feature was also present at 6 months, but less frequently. The results of the stereological studies revealed that the volumes of seminiferous tubules, interstitium and Leydig cells per testis was significantly higher (P < 0.05), at 6 and 24 months of age than those at 3 months. Moreover, volume of macrophages per testis was observed to be significantly higher (P < 0.05) at 6 months when compared to 3 and 24 months, and volume of connective tissue cells per testis was observed to be significantly lower (P < 0.05) at 6 and 24 months when compared to 3 months of age. No significant difference (P > 0.05) was observed for the volume of lymphatic space per testis in the three age groups studied. Volume of interstitial blood vessels per testis was not significantly different at 3 and 6 months of age, but a significantly greater (P < 0.05) volume was observed at 24 months. However, at 6 and 24 months, only 71% and 31% of the total blood vessel volumes respectively had completely open lumen in them; the rest of the blood vessels were either partially (12.5% at 6 months and 17% at 24 months) or completely (16.5% at 6 months and 52% at 24 months) occluded. The number of Leydig cells per testis was doubled at 6 and 24 months of age compared to 3 months. The average volume of a Leydig cell was not significantly different between 3 and 6 months of age, however, at 24 months a significantly lower (P < 0.05) value was observed. LH stimulated testosterone secretory capacity per Leydig cell in vitro was reduced by 50% at 6 months of age compared to 3 months; a further significant (P < 0.05) reduction was observed at 24 months. Serum testosterone and LH levels were not significantly different between 3 and 6 months of age but at 24 months a significantly lower (P < 0.05) value was observed for both of these hormones. In summary, the present study demonstrated many changes in the components of the testis interstitium in the aged Sprague Dawley rat. Modifications in the blood vessels and the occurrence of abundant collagen fibers in the interstitial space could possibly contribute to the reduced testosterone secretory capacity per Leydig cell with advancing in age. The observed Leydig cell hyperplasia could be suggested as a compensatory effort to maintain the normal androgen status of the aged rat, which is rather successful at 6 months but unsuccessful at 24 months. This investigation further revealed that these characteristic changes in the aged testis interstitium at 24 months are also present to some extent at 6 months of age in Sprague Dawley rats, suggesting that aging of the testis in this strain of rats commences early in life.
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PMID:Signs of aging are apparent in the testis interstitium of Sprague Dawley rats at 6 months of age. 857 59

In addition to the regulation of FSH secretion, it has been clearly shown that inhibin and activin have paracrine/autocrine effects in the gonads. We have studied the effect of human recombinant inhibin A and human recombinant activin A on immature porcine Leydig cells in vitro. Leydig cells were prepared by collagenase digestion of testes from 3-week-old piglets, purified on Percoll gradient, then cultured in a chemically defined medium. The cells were treated with increasing amounts of inhibin A or activin A (0.5-200 ng/ml). Direct application of either inhibin A or activin A on Leydig cells for 4 or 48 h did not stimulate basal testosterone secretion. Conversely, treatment of the cells for 48 h with either factor resulted in a dose-dependent increase in hCG-stimulated testosterone secretion (10[-9] M hCG, 2 h) with a maximal effect of 2.40 +/- 0.37- and 2.43 +/- 0.37-fold increases for inhibin A and activin A, respectively, and these changes were associated with a slight increase in LH/hCG-binding sites (1.37 +/- 0.19- and 1.24 +/- 0.11-fold increases). In addition, both inhibin A and activin A enhanced messenger RNA (mRNA) levels of LH/hCG receptor (2.75 +/- 0.40- and 2.53 +/- 0.60-fold increases) and cytochrome P450 17alpha-hydroxylase (6 +/- 1- and 3.5 +/- 0.6-fold increases), but had no effect on side-chain cleavage cytochrome P450 or cytochrome P450 aromatase mRNAs. 3beta-Hydroxysteroid dehydrogenase mRNA levels were increased (3.1 +/- 1.3-fold increase) by activin A, but not by inhibin A. However, inhibin A blocked the stimulatory action of activin A. In keeping with these changes in the steroidogenic enzyme mRNAs, both peptides enhanced the conversion of exogenous 22R-hydroxycholesterol and progesterone, but only activin A increased the conversion of dehydroepiandrosterone into testosterone. In conclusion, our findings demonstrate that both inhibin A and activin A have a stimulatory effect on immature porcine Leydig cell differentiated function in vitro. As inhibin has a stimulatory and activin has an inhibitory effect on rat Leydig cell function in vitro, the effects of these factors on Leydig cells seem to be species dependent.
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PMID:Stimulating effect of both human recombinant inhibin A and activin A on immature porcine Leydig cell functions in vitro. 934 6

Rat testicular cells dispersed by 0.03% collagenase digestion were seperated by discontinuous/continuous of Percoll, yielding highly pure Leydig cell fraction. 0.52 nmol/L hCG 0.1 mumol/L cholera toxin and 10 mumol/L forskolin signifcantly stimulated production of cAMP (Cholera toxin > Forskolin > hCG) and testosterone (no signifcient differences among cholera toxin, Forskolin and hCG). Ovalbumin glycopeptides prepared by extensive pronase digestion significantly inhibited production of cAMP and testosterone of rat testis Leydig cells stimulated by hCG, cholera toxin and forskolin. These results suggest that the carbohydrate moiety of hCG participate in the signal transduction among receptor, G-protein and adenylatecyclase which is inhibited by Asn-linked oligosaccharide chains from ovalbumin glycopeptides.
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PMID:[Inhibition of ovalbumin glycopeptides on the hCG signal transduction system]. 938 90

Estrogen sulfotransferase (EST) catalyzes the specific sulfonation and inactivation of estrogens. A common site for EST expression in mammalian species is the testicular Leydig cells. In previous in vivo studies, we have shown that testicular expression of EST is under the regulation of LH. Thus, EST expression in mouse Leydig cells was abolished by hypophysectomy, but could be restored by hCG injection. In this study, we have evaluated the downstream mechanisms by which LH exerts its regulatory effect on EST. Primary mouse Leydig cells were isolated and purified by collagenase digestion and Percoll density gradient centrifugation. They were cultured in serum-free medium at 32 C and treated with various agents for 24 or 48 h, and levels of EST messenger RNA and enzyme activity were determined. Consistent with the in vivo data suggesting an essential role of LH in regulating EST expression, treatment of primary mouse Leydig cells in vitro with 100 microM 8-bromo-dibutyryl cAMP [(Bu)2cAMP] increased EST expression 3- to 5-fold. The effect of (Bu)2cAMP was attenuated by the steroidogenesis inhibitor aminoglutethimide and was mimicked by the potent androgen 5alpha-dihydrotestosterone (5-DHT). The activity of 5-DHT in stimulating EST expression was blocked by the androgen receptor antagonist, hydroxyflutamide. These data suggested the involvement of androgen in (Bu)2cAMP-induced EST expression. Further evidence came from the study with interleukin-1beta, another agent known to suppress Leydig cell steroidogenesis by down-regulating P450c17 gene expression. Treatment of Leydig cells with 0.2 ng/ml interleukin-1beta inhibited (Bu)2cAMP-induced EST expression, which was overcome by the addition of 5-DHT. Finally, in the testis-feminized mouse (Tfm) in which the androgen receptor is nonfunctional due to a frameshift mutation, testicular EST expression is completely absent, whereas messenger RNAs of steroidogenic enzymes such as P450c17 and 3beta-hydroxysteroid dehydrogenase are relatively abundant. We conclude that, by acting as an autocrine or paracrine factor, androgen plays an essential role in the regulation of estrogen sulfotransferase expression in Leydig cell by LH and cAMP.
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PMID:Regulation of estrogen sulfotransferase expression in Leydig cells by cyclic adenosine 3',5'-monophosphate and androgen. 1006 24

Leydig cells were isolated from the perch testes belonging to the pre-spawning stage by collagenase treatment and mechanical separation followed by percoll gradient. They were incubated in vitro either for 5 h or at different times in the absence (control) or presence of piscine gonadotropin (GTH, 2 microg (1 x 10(6) cells)(-1)) or 3,5,3'-triiodothyronine (T3, 50 ng (1 x 10(6) cells(-1)) or T3-induced protein (TIP, 2 microg (1 x 10(6) cells)(-1)). 3Beta-hydroxysteroid dehydrogenase/delta5-delta4-isomerase (3beta-HSD) activity was determined by the conversion of [3H]delta5-dehydroepiandrosterone (DHEA) to [3H]delta4-androstenedione or [3H]delta5-pregnenolone to [3H]delta4-progesterone (P4) or by spectrophotometric estimation of NADH formation from NAD. T3 significantly increased (P < 0.01) both delta5-DHEA to delta4-androstenedione and delta5-pregnenolone to delta4-P4 conversion in Leydig cells indicating stimulation of 3beta-HSD activity. T3 stimulation of 3beta-HSD activity could be inhibited by cycloheximide (50 microg ml(-1)) suggesting the involvement of T3-induced protein (TIP) which was isolated and purified earlier in this laboratory from goat Leydig cells [15]. Addition of TIP or GTH significantly stimulated Leydig cell 3beta-HSD activity (P < 0.01). However, there was a difference between TIP and GTH stimulation in time kinetic study where TIP enhanced 3beta-HSD activity at 1 h (P < 0.05), reached its peak at 3 h (P < 0.01) and then plateaued till 8 h. GTH, on the other hand, did not show any stimulation of 3beta-HSD activity for 2 h, stimulation was marked only at 3 h (P < 0.05), reached a peak at 6 h (P < 0.01) and then leveled off. Determination of Km and Vmax of the enzyme showed an increase in the velocity of reaction by GTH with unaltered Km. TIP increased both velocity and affinity of the enzyme. GTH significantly increased the synthesis of 3beta-HSD protein at 3 h (P < 0.01) reaching maximal stimulation at 6 h which clearly coincided with the enzyme activity. In contrast, TIP had no effect on 3beta-HSD protein synthesis, but its direct addition to 3beta-HSD enzyme preparation in vitro caused significant augmentation of the enzyme activity (P < 0.01) suggesting thereby its modulatory effect on the enzyme. Results, therefore, show that although both T3 and GTH stimulated perch testicular Leydig cell 3beta-HSD activity, T3 effect was not direct but mediated via TIP and there is a clear distinction between GTH and TIP stimulation. GTH increased the enzyme activity by stimulating 3beta-HSD protein synthesis while TIP acts directly on the enzyme modulating it from less active to more active state.
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PMID:Differential regulation of Leydig cell 3beta-hydroxysteroid dehydrogenase/delta5-delta4-isomerase activity by gonadotropin and thyroid hormone in a freshwater perch, Anabas testudineus (Bloch). 1062 32

Gap junctions are intercellular protein channels which provide a pathway for the exchange of ions and small molecules. This exchange of materials allows metabolic coupling of cells. Gap junction channels are made up of connexins, integral membrane proteins encoded by a multigene family. Rat testes contain mRNAs for at least five different connexins: Cx26, Cx32, Cx33, Cx37 and Cx43. Immunocytochemical studies have shown that Cx43 assembles gap junctions between Leydig cells. The present study investigated the expression and regulation of the Cx43 gene in rat Leydig cells. Purified Leydig cells were obtained from 40- to 80-day-old Sprague-Dawley rats using a combination of arterial perfusion, collagenase digestion, centrifugal elutriation and Percoll gradient centrifugation. Leydig cells from 20- and 30-day-old rats were isolated without arterial perfusion or centrifugal elutriation. Cx43 mRNA was present in 20-day-old rat Leydig cells, reached a plateau at day 40, and remained at high levels in 65- and 80-day-old rat Leydig cells. To evaluate the regulation of Cx43 gene expression, Leydig cells were cultured overnight and then treated with human chorionic gonadotropin (hCG) for variable periods of time. Addition of hCG (10 ng/ml) increased cytochrome P450 side-chain cleavage and steroidogenic acute regulatory protein mRNA levels and testosterone formation. However, Cx43 mRNA levels were inhibited by hCG in a time- and dose-dependent manner. Cx43 mRNA levels decreased 27% as early as 2 h after the addition of hCG and decreased 60% by 24 h. Treatment of Leydig cells with 8-bromo-cAMP (0.1 mM) for 6 and 24 h also reduced Cx43 mRNA levels by 36 and 56% respectively. Primary cultured Leydig cells stained strongly positive with anti-Cx43 monoclonal antibody. Treatment with hCG for 24 h reduced Cx43 signals and caused Cx43 to redistribute to the periphery of the cells. To evaluate the regulation of Cx43 in vivo, rats were treated with hCG (300 ng i.p.) and testes were removed 24 h later. Frozen section of testes revealed that these interstitial cells stained positive for 3beta-hydroxysteroid dehydrogenase (3beta-HSD) by histochemical staining and were positive for Cx43 by immunofluorescence staining. The adjacent seminiferous tubules stained only weakly positive for Cx43. Twenty-four hours after hCG treatment, 3beta-HSD activity increased while Cx43 immunostaining of Leydig cells was reduced. In conclusion, gap junction channels of Leydig cells are regulated by hCG both in vivo and in vitro. hCG increased Leydig cell steroidogenesis and steroidogenic enzyme mRNA levels but caused a redistribution of Cx43.
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PMID:Expression and regulation of connexin43 in rat Leydig cells. 1092 34

Procedures for purification of Leydig cells have facilitated studies of their regulatory biology. A multistep procedure that includes a filtration with nylon mesh (100-micron pore size) to separate interstitial cells from the seminiferous tubules, combining centrifugal elutriation and Percoll density gradient sedimentation, has been used to obtain a 95% enrichment of rat Leydig cells. However, the number of recovered Leydig cells by this procedure represents only a small fraction of the 25 million, on average, that exist in the adult rat testis. The objective of this study was to test whether the yield of purified Leydig cells might be enhanced by substitution of unit-gravity sedimentation (S method) for the filter step (F method). We also asked whether a greater number of Leydig cell clusters, macrophages, or both would be recovered by this new method, and if the presence of Leydig cell clusters is associated with increased capacity for testosterone production in vitro. The number of purified Leydig cells was 1.9-fold higher for the S method than for the F method, with no differences in purity assessed by 3beta-hydroxysteroid dehydrogenase histochemical staining. Leydig cell clusters were also found in greater numbers with the S method both after collagenase dispersion and at the end of the purification. No differences were seen in testosterone production or in the number of macrophages present in the Leydig cells that were prepared by the 2 methods. These results indicate that the new method recovers greater numbers of Leydig cells by collecting clustered Leydig cells that are systematically eliminated when a filtration step is used.
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PMID:Purification of rat leydig cells: increased yields after unit-gravity sedimentation of collagenase-dispersed interstitial cells. 1145 64


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