The extraction of a tissue collagenase associated with ovulation in the rat

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.     

Aroid scarabs in the genus Peltonotus Burmeister (Coleoptera,Scarabaeidae, Dynastinae): key to species and new distributional data

The southeast Asian scarab beetle genus Peltonotus Burmeister (Scarabaeidae, Dynastinae, Cyclocephalini) is reviewed. New country records for Peltonotus morio Burmeister (Myanmar and Vietnam), Peltonotus nasutus Arrow (southern China and Cambodia), and Peltonotus favonius Jameson and Wada (Myanmar) are reported, including a new record in the Palearctic/Sino-Japanese biogeographic region. The first female specimen of Peltonotus favonius is described. Biological associations with aroid inflorescences are reviewed, and human consumption of Peltonotus beetles is reported. A key to all species, paralectotype designations for Peltonotus nasutus, diagnoses, and distributions using dynamic mapping tools are included.     

Inhibition of follicle-stimulating hormone-induced ovulation by indomethacin in the perfused rat ovary

In isolated, perfused ovaries of rats treated with pregnant mare's serum gonadotropin (PMSG), purified preparations of ovine follicle-stimulating hormone (FSH) (oFSH-211B) and rat FSH (rFSH-I-6), 100 ng/ml, were found to induce ovulations (4.8 +/- 0.9, n = 4, and 6.4 +/- 2.0, n = 5, ovulations per ovary, respectively). Indomethacin (5 micrograms/ml) added to the perfusate inhibited this ovulatory effect and exogenous prostaglandin F2 alpha (PGF2 alpha) (1 microgram/ml), or prostaglandin E2 (PGE2) (0.5 microgram/ml), reversed the blockade. Ovine FSH and rFSH had only a weak stimulatory effect on estradiol release, and only rFSH caused a significant increase in progesterone accumulation. Indomethacin reduced the stimulatory effect of rFSH on progesterone release, and this effect was reversed by PGE2 but not by PGF2 alpha. In a 6-h incubation experiment with preovulatory rat follicles, we tested the biological activity of gonadotropins used to induce oocyte maturation. The concentration of FSH used in the perfusion experiments induced oocyte maturation in more than 88% of the oocytes studied. The data confirm earlier findings that FSH can induce ovulations and show that prostaglandins are involved in this process. The data also indicate that prostaglandins might be involved in the FSH-induced increase of progesterone levels.     

Excitation of rat hippocampal neurones by the stereoisomers of cis- and trans-1-amino-1,3-cyclopentane dicarboxylate

Intracellular recordings were obtained from rat hippocampal neurons during the microiontophoretic ejection of the stereoisomers of cis- and trans-1-amino-1,3-cyclopentane dicarboxylate into the dendritic region (stratum radiatum) of the impaled cells. L-(+)-cis-1-Amino-1,3-cyclopentane dicarboxylate, D(+)-trans-1-amino-1,3-cyclopentane dicarboxylate, and L-(-)-trans-1-amino-1,3-cyclopentane dicarboxylate all evoked patterns of excitation resembling that elicited by kainate. All of these responses were unaffected by D-(-)-2-amino-5-phosphonovalerate but were antagonized at comparable currents by kynurenate. The excitation produced by D-(-)-cis-1-amino-1,3-cyclopentane dicarboxylate was similar to that evoked by N-methyl-D-aspartate. At low ejection currents a slow depolarization triggered rhythmic burst firing, each burst consisting of a depolarizing shift in membrane potential upon which were superimposed four to five action potentials. These responses were antagonized both by D-(-)-2-amino-5-phosphonovalerate and by kynurenate. The results are discussed with respect to the conformational requirements considered to be necessary for interaction at the kainate and N-methyl-D-aspartate receptors on CA1 pyramidal neurones. It is important to note that the isopropylene side chain of kainate is absent from the 1-amino-1-3-cyclopentane dicarboxylate molecule.     

Immunohistochemical localization of prostaglandin endoperoxide synthase in human fetal membranes and decidua

Prostaglandins play an important role during the maintenance of pregnancy and the initiation of parturition. Prostaglandin endoperoxide synthase activity has been demonstrated in human fetal membranes and decidua. Using immunohistochemical techniques, we identified in these tissues the cell types that contain prostaglandin endoperoxide synthase. A total of 33 specimens, ranging from 8 wk to 42 wk gestation, were studied. Decidualized stromal cells stained the most intensely and consistently of all cell types. Cytotrophoblast of the chorion and early placental villi and syncytotrophoblast of all gestational ages demonstrated a lighter, more variable staining pattern. Regardless of gestational age, amnion stained in a heterogeneous fashion, with some cells demonstrating an intense staining and other cells having no staining. There were no observable differences in laboring compared to nonlaboring term specimens. In summary, the specific cell types that contain immunoreactive prostaglandin endoperoxide synthase have been identified in fetal membranes and decidua.     

Cellular localization of ovarian proopiomelanocortin messenger RNA during follicular and luteal development in the rat

Opioid peptides are expressed in the reproductive system and have been reported to regulate reproductive function. The present study used in situ hybridization to selectively localize ovarian cells containing high levels of proopiomelanocortin (POMC) mRNA, an opioid precursor, during different stages of ovarian development. Prepubertal rats were primed with PMSG to stimulate follicular development, followed by hCG to induce ovulation. Treatment groups consisted of control (no treatment), PMSG (2 days post-PMSG), 1 day corpus luteum (CL; 1 day post-hCG), and 8 day CL (8 days post-hCG). POMC mRNA-containing cells were present in antral follicles, CL, and the interstitial compartment. With gonadotropin treatment, the percentage of follicles containing heavily labeled cells increased in the PMSG and 1 day CL groups. The number of POMC mRNA-containing cells per follicle also increased in the 1 day CL group. In the CL, no difference was observed in the percentage of CL exhibiting labeled cells between the 1 day CL and 8 day CL groups; however, more labeled luteal cells per CL were present in the 1 day CL group. A marked increase in POMC mRNA-containing cells was observed in the interstitial compartment of the 1 day CL group. These results indicate that the number of POMC mRNA-containing cells increases with follicular development and CL formation; however, the ovarian distribution suggests that the labeled cells could be nonendocrine cells, possibly white blood cells. The in situ hybridization findings are indicative of low total concentrations of ovarian POMC mRNA, suggesting mainly an autocrine or paracrine role for POMC or POMC-derived peptides.(ABSTRACT TRUNCATED AT 250 WORDS)     

Expression and Secretion of a Cellulomonas fimi Exoglucanase in Saccharomyces cerevisiae

We used the yeast MEL1 gene for secreted alpha-galactosidase to construct cartridges for the regulated expression of foreign proteins from Saccharomyces cerevisiae. The gene for a Cellulomonas fimi beta-1,4-exoglucanase was inserted into one cartridge to create a fusion of the alpha-galactosidase signal peptide to the exoglucanase. Yeast transformed with plasmids containing this construction produced active extracellular exoglucanase when grown under conditions appropriate to MEL1 promoter function. The cells also produced active intracellular enzyme. The secreted exoglucanase was N-glycosylated and was produced continuously during culture growth. It hydrolyzed xylan, carboxymethyl cellulose, 4-methylumbelliferyl-beta-d-cellobiose, and p-nitrophenyl-beta-d-cellobiose. A comparison of the recombinant S. cerevisiae enzyme with the native C. fimi enzyme showed the yeast version to have an identical K(m) and pH optimum but to be more thermostable.     

Regulation of prodynorphin gene expression in the ovary: distal DNA regulatory elements confer gonadotropin regulation of promoter activity.

Examination of the regulation of prodynorphin (pro-DYN) promoter activity is limited by the absence of a good cell model. The discovery of pro-DYN mRNA and derived peptides in the reproductive tract led us to examine the cellular localization and hormonal regulation of ovarian pro-DYN expression and to evaluate normal granulosa cells as a model for studying pro-DYN gene regulation. Ovarian pro-DYN mRNA levels were significantly elevated in PMSG-primed immature rats 12-24 h after receiving an ovulatory dose of hCG. Levels peaked 2 days after hCG, remained elevated throughout the ensuing pseudopregnancy, and rose again at the end of pseudopregnancy. In situ hybridization localized pro-DYN expression predominantly to granulosa and luteal cells. Transfection of primary cultures of granulosa cells revealed that the activity of the rat pro-DYN promoter [-1858 to 133 base pairs (bp)] was increased 18- to 19-fold by hCG and human FSH treatments and 7-fold by cAMP analog treatment. Deletion analysis identified a 358 bp fragment as the primary hormone-responsive sequence (-1858 to -1500 bp; containing three potential cAMP-responsive elements); its deletion resulted in severely reduced FSH responsiveness, and its ligation to hormone-unresponsive basal promoter sequences completely restored FSH responsiveness. This is an unusually distal position for cAMP-responsive elements compared to other cAMP-regulated genes. These data demonstrate specific expression of pro-DYN in granulosa and luteal cells, which is under sensitive gonadotropin regulation, and identify a distal hormone-responsive sequence within the promoter.