Histochemistry of some proteases in the normal rabbit, pig and ox corneas

The distribution of activities of membrane aminopeptidases (aminopeptidase M (APM), aminopeptidase A (APA), dipeptidyl peptidase IV (DPP IV), gamma-glutamyltransferase (GGT) and lysosomal exopeptidases (dipeptidyl peptidase I (DPP I), dipeptidyl peptidase II (DPP II)) was investigated in rabbit, ox and pig corneas. Cryostat sections of snap-frozen corneas treated with chloroform-acetone (4 degrees C) were used for the demonstration of membrane-bound enzymes and sections of corneas fixed in 4% paraformaldehyde (4 degrees C) for the demonstration of lysosomal enzymes. In activities of proteases species differences were found. The rabbit cornea was most active, followed by ox and pig corneas. Individual corneal layers reacted differently. Of membrane proteases a high APM activity was found in keratocytes, whereas epithelium and endothelium were negative. On the other hand, APA and GGT were active in the epithelium and endothelium. Their activities in keratocytes were less pronounced. DPP IV activity was demonstrated in some keratocytes beneath the epithelium only. Lysosomal enzymes DPP I and DPP II were active in all corneal layers. The epithelium displayed the highest activity. Differences in activities in the centro-peripheral and epithelio-endothelial directions were found. DPP I, DPP II, and APM were most active in the limbal region in all corneal layers.     

Histochemistry of some proteases in the normal rabbit,pig and ox corneas

Summary The distribution of activities of membrane aminopeptides (aminopeptidases M (APM), aminopeptidase A (APA), dipeptidyl peptidase IV (DPP IV), -gluamyltransferase (GGT) and lysosomal exopeptidases (dipeptidyl peptidase I (DPP I), dipeptidyl peptidase II (DPP II) was investigated in rabbit, ox and pig corneas. Cryostat sections of snap-frozen corneas treated with chloroform-acetone (4°C) were used for the demonstration of membrane-bound enzymes and sections of corneas fixed in 4% paraformaldehyde (4°C) for the demonstration of lysosomal enzymes.In activities of proteases species differences were found. The rabbit cornea was most active, followed by ox and pig corneas. Individual corneal layers reacted differently. Of membrane proteases a high APM activity was found in keratocytes, whereas epithelium and endothelium were negative. On the other hand APA and GGT were active in the epithelium and endothelium. Their activities in keratocytes were less pronounced. DPP IV activity was demonstrated in some keratocytes beneath the epithelium only. Lysosomal enzymes DPP I and DPP II were active in all corneal layers. The epithelium displayed the highest activity.Differences in activities in the centro-peripheral and epithelio-endothelial directions were found. DPP I, DPP II, and APM were most active in the limbal region in all corneal layers.     

Ultrastructural aspects of the antimesometral implantation in the rabbit

A scanning and transmission electron microscope study of blastocysts immediately prior to ovo implantation and of antimesometrial implantation sites was conducted. External membranes of the eggs showed at 6 days post coitum the imprint of the endometrial surface, pointing to the early establishment of contact between egg and uterus. At 7 days the antimesometrial region showed flattening with continued evidence of gland openings that appear to be the elective sites of attraction of the trophoblastic knobs. Attachment of the trophoblastic knobs followed on days 8 and 9.     

Glycoproteins in rabbit uterus during implantation

Summary The synthesis of glycoproteins in rabbit uterine epithelium during the late preimplantation period was studied using tritiated N-acetylglucosamine. In vivo labelling was achieved by the intra-uterine implantation of agar gel columns containing the precursor. Autoradiography showed the radioactivity to be predominantly localized in the apical cell surfaces, with single cells exhibiting an accumulation of silver grains in their supranuclear cytoplasm. After gel electrophoresis of uterine flushings, activity was mainly found in the -glycoprotein fraction. Fluorescein isothiocyanate (FITC)-conjugated wheat-germ agglutinin reacted with the apical cytoplasm and surfaces of the endometrial cells. However, FITC-conjugated concanavalin A exhibited a different binding pattern, reacting first with the basal cytoplasm, and later with the apical cytoplasm. After concanavalin-A staining, single cells exhibited positive vesicles in their lateral and apical parts. These cells may be released into the uterine lumen until 210 h post coitum. Neither of the lectins reacted with ciliated cells. Concanavalin A showed an affinity for the -glycoprotein fraction of the uterine secretion. The results indicate that, although all endometrial cells contain glycoproteins, only a few of these seem to be involved in the synthesis of secretory products.Supported by grants Ki 154/9-3 and 154/10-1 from the Deutsche Forschungsgemeinschaft     

The role of histamine in implantation in the rabbit.

When disodium cromoglycate, an inhibitor of histamine release, was instilled into the uterine lumen on Days 5 or 6 of pregnancy, the number of blastocysts implanting was significantly (P less than 0.002) reduced. Simultaneous instillation of histamine and disodium cromoglycate prevented the effect.     

Role of histamine and cyclic nucleotides in implantation in the rabbit

Summary The effect of histamine on cAMP and cGMP levels in day 6 (144 h post coitum) rabbit blastocysts was determined. Histamine at 200 M and 1000 M concentrations stimulated the increased formation of cAMP in vitro, whereas stimulation of cGMP occurred only in the presence of 1000 M histamine. Furthermore, intrauterine injection of RMI-12330A (50 g or 500 g/uterine horn), an inhibitor of adenylyl cyclase, on day 5 of pregnancy interrupted embryro development and implantation of the embryo. The drug was also effective in reducing the cAMP level in the endometrial cells in vitro. A relationship between histamine and cyclic nucleotide changes in embryo development and implantation is suggested.     

Blastocyst is the source of prostaglandins in the implantation site in the rabbit

The levels of prostaglandins (PGs) were measured by radioimmunoassay in the interimplantation and the implantation sites as well as in the implantation site without the blastocyst in the rabbit on day 7 of pregnancy (168h $\text{post coitum}$). The concentrations of PGs were also determined in the blastocyst (PGF:101.59_+?4.33 and PGE-A:29.74_+?3.11 ng/blastocyst, n=6) and the blastocel fluid (PGF:253.55_+?39.56 and PGE-A:83.29_+?6.60 ng/100 ul, n-4) on day 7. The levels of both PGF and PGE-A were significantly higher in the implantation site as compared to interimplantation site (PGF:73.63±6.68 vs. 0.59±0.21 and PGE-A:25.52±3.30 vs. 1.22±0.18 ng/100 mg wet weight n=8). The removal of the blastocyst from the implantation site drastically reduced the concentrations of PGs in this site (PGF:8.71±2.80 and PGE-A:1.64±0.12 ng/100 mg wet weight, n=8). The results provide evidence that the blastocyst is the major source of PGs which contribute to the hige concentration in the implantation site in the rabbit.     

Non-specific effects of passive immunization on implantation in the rabbit.

Passive immunization with goat anti-rabbit uteroglobin antiserum prevents implantation in the rabbit. The dose of antiserum was too low to neutralize all of the uteroglobin present on Day 5 of pregnancy, however, and the effect could not be shown to be specific, because 'control' treatments with goat antiserum to chick avidin or normal goat serum also prevented implantation. Non-specific antisera raised in rabbits had little or no effect on implantation. Partial purification of antibodies from the non-specific goat antisera reversed their effect, while anti-uteroglobin gamma globulin still reduced implantation. Fluorescein-labelled gamma globulin fractions of anti-avidin and anti-uteroglobin both bound to blastocysts, but pure FITC-IgG showed binding only of anti-uteroglobin. Both anti-avidin and anti-uteroglobin IgG prevented implantation. It is concluded that the effect on implantation is not necessarily achieved via a specific antigen.     

Histochemistry of the epimerases

Summary On the assumption that electron transfer is involved in the process of epimerization and by using tetrazolium salts as an indicator, a histochemical reaction for the demonstration of UDPGal-4-epimerase has been developed.By using UDPG or UDPGal as substrates it has been possible to ascertain the direction of the reaction catalysed by this enzyme in various tissues in normal physiological conditions.Biochemical tests support the concept that the histochemical reactions recorded were the result of UDPGal-4-epimerase activity.     

Effect of histamine receptor antagonists and indomethacin on implantation in the rabbit

Anti-inflammatory drugs were given to pregnant rabbits during the 24-h period prior to the increase in the uterine vascular permeability which occurs on Day 7. Their effect on the vascular response was monitored by quantifying the concentration of extravascular Evans blue dye. The increase in vascular permeability normally seen on Day 7 was inhibited by either indomethacin or a combination of H1-(mepyramine) and H2-(cimetidine) receptor antagonists. When given alone, neither cimetidine nor mepyramine was as effective as the combination in reducing vascular permeability. Prostaglandins and histamine may be acting together since simultaneous administration of lower doses of indomethacin and the antihistamines reduced vascular permeability below that observed following administration of either class of anti-inflammatory drugs alone. In a second experiment, anti-inflammatory drugs were administered during the peri-implantation period (Days 6-8) and their effect on the weights of maternal and fetal tissues, and on fetal viability were evaluated on Day 14 of pregnancy. Indomethacin had a more deleterious effect on both parameters than did the combination of histamine receptor antagonists. Results from these experiments suggest that both prostaglandins and histamine may participate in the uterine vascular response, whereas the overall process of implantation appears to be more dependent upon the synthesis of prostaglandins than the action of histamine.