Types I and IV collagenolytic and plasminogen activator activities in preovulatory ovarian follicles

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.     

High degree of homology between primary structure of human lysosomal acid phosphatase and human prostatic acid phosphatase

Alignment of the amino-acid sequences of the human lysosomal acid phosphatase (LAP) and human prostatic acid phosphatase (PAP) yielded an extensive homology between the two mature polypeptide chains. In the overlapping part, which extends over the entire PAP sequence and the N-terminal 90% of the LAP sequence, the identity is 49.1%. The LAP has an additional C-terminal sequence, which is encoded by the last exon of the LAP gene. This sequence contains the transmembrane domain of LAP, which is lacking in the secretory PAP. All six cysteine residues as well as 20 out of 27 (LAP) and 26 (PAP) proline residues present in the overlapping part of the proteins are conserved, suggesting that they are involved in stabilization of the tertiary structure of both proteins. Only two out of 8 N-glycosylation sites in LAP and 3 in PAP are conserved, suggesting that the dense N-glycosylation of LAP is related to its function in lysosomes.     

Pre-lysosomal divergence of alpha 2-macroglobulin and transferrin: a kinetic study using a monoclonal antibody against a lysosomal membrane glycoprotein (LAMP-1)

We have used electron microscopic immunocytochemistry to compare the distribution of LAMP-1, a marker for lysosomal membranes, with the intracellular localization of alpha 2-macroglobulin (alpha 2-M) and transferrin at various time points after their endocytosis into cultured NIH 3T3 cells. The purposes of this study were (a) to determine how soon endocytic ligands reach lysosomal organelles, (b) to examine whether the intermediate endocytic vesicles gained lysosomal markers gradually or in a precipitous, discrete event, and (c) to examine the relationship, if any, between the pathway of recycling ligands and lysosomes. At early time points (0-5 min) after initiation of endocytosis, most structures containing alpha 2-M labeled with colloidal gold (receptosomes) were not labeled by anti-LAMP-1 detected using ferritin bridge or peroxidase immunocytochemistry. At late time points (greater than or equal to 15 min), the structures containing alpha 2-M (lysosomes) were strongly labeled by anti-LAMP-1. In contrast, transferrin that was directly labeled with ferritin was mostly located in LAMP-1-negative structures at all time points studied. The proportion of alpha 2-M-gold containing vesicles strongly labeled for LAMP-1 roughly paralleled the proportion of alpha 2-M-gold-containing structures positive for cytochemically detectable acid phosphatase. Our data indicate that ligands such as transferrin that are internalized through coated pits and receptosomes, but not delivered to lysosomes, do not traverse a lysosomal organelle compartment as marked by LAMP-1 content. Ligands such as alpha 2-M that are destined for lysosomal delivery reach a LAMP-1-positive organelle compartment only after they traverse LAMP-1-negative, non-lysosomal vesicles previously described as receptosomes.     

Relationships between unconjugated and sulphated steroids in porcine primary Leydig cell culture

Steroidogenesis in immature porcine Leydig cells was investigated in primary culture at 48-84 h under basal conditions and in the presence of hCG. The basal accumulation of unconjugated steroids was close to linear only during the first 4 h of study, whereas the sulphate-conjugated steroids accumulated essentially linearly over the 36 h experimental period. At the last time point, 95% of the steroids measured were sulphated. Stimulation with hCG (1 ng/ml) led to a still more pronounced sulphate conjugation, and approx 99% of the steroids measured were sulphated at 36 h. Under maximal stimulation with hCG (100 ng/ml) the sulphates accounted for 74% of the total steroids measured at 36 h. Testosterone, androstenedione, dehydroepiandrosterone, 5-androstene-3 beta, 17 beta-diol and estrone were usually quantitatively the most important unconjugated steroids, and sulphated dehydroepiandrosterone, estrone, testosterone and 5-androstene-3 beta, 17 beta-diol were the most important steroid sulphates, especially following maximal stimulation of the cultures. These data emphasize the importance of steroid sulphates in porcine testicular steroid metabolism. Under stimulation with hCG, there was a rapid response in testicular steroidogenesis, initially seen as a rapid increase in the secretion of unconjugated and sulphated steroids. At approx 4-12 h, the rate of sulphate conjugation appeared to reach or even to exceed that of steroid biosynthesis, which lead to stabilisation or a decrease in the concentrations of unconjugated steroids. Only high doses of hCG, 10-100 ng/ml, were then able to lead to a net accumulation of unconjugated steroids, at 24-36 h of incubation with hCG.     

Regulation of urokinase- and tissue-type plasminogen activator gene expression in the rat seminiferous epithelium

The secretion of plasminogen activator by seminiferous tubules at defined stages of the epithelial cycle is influenced both by neighboring spermatogenic cells and by hormones. We have used cRNA probes for urokinase-type (uPA) and tissue-type (tPA) plasminogen activators to analyze their mRNA levels in different stages of the epithelial cycle. Urokinase-type PA mRNA was most abundant in stages VII-VIII, while tPA mRNA levels showed smaller variations between the different stages. Both FSH and (Bu)2cAMP increased the steady-state level of tPA mRNA and tPA production without affecting those of uPA in stages VII-IX in vitro, whereas retinoic acid treatment selectively increased the concentration uPA mRNA and uPA production in stages II-VI. The results show that the expression of the uPA and tPA genes is differentially regulated in specific stages of the rat seminiferous epithelium.     

Human familial and sporadic breast cancer: analysis of the coding regions of the 17β-hydroxysteroid dehydrogenase 2 gene (EDH17B2) using a single-strand conformation polymorphism assay

17-Hydroxysteroid dehydrogenase (17HSD) is one of the key enzymes in estrogen metabolism, catalyzing the reversible reaction between estradiol and the less active estrogen, estrone. The gene encoding this enzyme, EDH17B2, has been mapped to chromosome 17, region q12–q21, in the vicinity of BRCA1, an as yet unidentified gene that appears to be involved in familial breast cancer and in familial ovarian cancer. The possibility that EDH17B2 gene is the same as BRCA1 was tested by screening for mutations in the coding regions of EDH17B2, using a polymerase chain reaction/single-strand conformation polymorphism method. An AG transition creating a new BstUI site at exon 6 was the only frequent sequence alteration found in the coding region of the gene. This mutation also led to an amino acid substitution of serine to glycine at position 312 (312^S312^G) in the 17HSD protein. Since the nucleotide change was detected both in specimens from patients with familial or sporadic cancer and in control samples, and at similar rates, this mutation appears to be of a polymorphic nature. In addition, a rare polymorphism located at intron 5 was detected. This CT substitution creates a BbvI site and is not thought to have any effect on 17HSD activity. The results indicate that there are no major alterations in the coding areas of EDH17B2 and thus studies testing the hypothesis that EDH17B2 may be the same as BRCA1 should be extended to the promoter and regulatory elements of EDH17B2.     

Exhaustive physical exercise and acid hydrolase activity in mouse skeletal muscle

Summary Adult, untrained NMRI mice were exhausted on a motor-driven treadmill by an intermittent-type running programme. Serial cryostate sections for the staining of NADH-tetrazolium reductase, -glucuronidase, -N-acetylglucosaminidase, and -glycerophosphatase activities and for making hematoxylin-eosin staining were cut from m. quadriceps femoris 1, 2, 3, 5, 7, and 15 days after physical exhaustion. A strong increase in the activities of -glucuronidase and -N-acetylglucosaminidase, was observed 7 days after exhaustion and the activity changes, which were similar for the both glycosidases, were more prominent in the highly oxidative red compared to less oxidative white fibres. Activity granules were more numerous in the perinuclear than the interfibrillar area of red fibres. Spots were arranged like longitudinal chains between myofibrils. Activity in connective tissue was usually observed only in animals exhausted 3–7 days earlier. Simultaneous activity in fibres exceeded that in connective tissue -Glycerophosphatase activity was not, by the method used, seen in histologically healthy or normal-looking fibres. in samples taken 2–5 days after exhaustion some degenerating and necrotic fibres were observed. Inflammatory reaction was also observed being at its strongest five days after loading when mononuclear cells were seen inside necrotic fibres. The number of regenerating muscle cells was most abundant 7 days after exhaustion. It is suggested that temporary hypoxia, which accompanies exhaustive physical exercise in skeletal muscle, upsets the energy metabolism and homeostasis of fibres and causes the observed histological and histochemical alterations, which posses features typical of both lethal and sublethal acute cell injury.     

A single bout of exercise with high mechanical loading induces the expression of Cyr61/CCN1 and CTGF/CCN2 in human skeletal muscle.

High mechanical loading was hypothesized to induce the expression of angiogenic and/or lymphangiogenic extracellular matrix (ECM) proteins in skeletal muscle. Eight men performed a strenuous exercise protocol, which consisted of 100 unilateral maximal drop jumps followed by submaximal jumping until exhaustion. Muscle biopsies were taken 30 min and 48 h postexercise from the vastus lateralis muscle and analyzed for the following parameters: mRNA and protein expression of ECM-associated CCN proteins [cysteine-rich angiogenic protein 61 (Cyr61)/CCN1, connective tissue growth factor (CTGF)/CCN2], and mRNA expression of vascular endothelial growth factors (VEGFs) and hypoxia-inducible factor-1alpha. The mRNA expression of Cyr61 and CTGF increased 30 min after the exercise (14- and 2.5-fold, respectively; P < 0.001). Cyr61 remained elevated 48 h postexercise (threefold; P < 0.05). The mRNA levels of VEGF-A, VEGF-B, VEGF-C, VEGF-D, or hypoxia-inducible factor-1alpha did not change significantly at either 30 min or 48 h postexercise; however, the variation between subjects increased markedly in VEGF-A and VEGF-B mRNA. Cyr61 protein levels were higher at both 30 min and 48 h after the exercise compared with the control (P < 0.05). Cyr61 and CTGF proteins were localized to muscle fibers and the surrounding ECM by immunohistochemistry. Fast fibers stained more intensively than slow fibers. In conclusion, mechanical loading induces rapid expression of CCN proteins in human skeletal muscle. This may be one of the early mechanisms involved in skeletal muscle remodeling after exercise, since Cyr61 and CTGF regulate the expression of genes involved in angiogenesis and ECM remodeling.