Immunofluorescence study of the extracellular matrix of the human placenta

Distribution of collagen types I, III, IV, V and fibronectin in human placental villi has been studied by indirect immunofluorescence. During 9-12 weeks of pregnancy the extracellular matrix of villi represents a network of filaments organized in bundles and aggregates that contain collagen types I and III and finer filaments of collagen types IV and V. Collagen type IV is regularly detected in basal membrane of capillaries and particularly in villous epithelium, collagen type V and fibronectin are occasionally detected there. Marked immunofluorescent reaction on collagen types IV and V and fibronectin, and weak reaction on collagen type III is observed in cellular islets around cytotrophoblasts. In the fetus born in term placental villi have uniform immunofluorescence in thick basal membranes of fetal capillaries and of chorionic epithelium. The immunofluorescent reaction specific for all collagen types is uniform in villous stroma. Distribution of different collagen types and fibronectin, including the unusual localization of membrane collagen type IV, in villous stroma and cellular islets of early and mature placenta is discussed.     

Genetical distances between nominative and paratypical forms of Colias crocea Fourc. and C. erate Esp. (lepidoptera, Pieridae) according to RAPD-PCR analysis

Genetic polymorphism of the two Palaearctic sulphur butterflies Colias crocea Fourc. and Colias erate Esp. was examined in serial samples using RAPD-PCR analysis of total cellular DNA. The primers OPA-01, OPA-03, OPA-04, OPA-08 were used. Taxon-specific molecular markers have been found for each species (markers OPA-08(250), OPA-08(380) were associated with rounded valve margin; marker OPA-04(400) was associated with angled valve margin). The genetic diversity of C. erate is considerably less than that of C. crocea. Genetic evidence for interspecific hybridization between C. crocea and C. erate is presented. Interspecific hybrids in question (paratypical forms such as f. chlorodona and f. pseudochrysodona) are situated at intermediate position with regard to the parental species on dendrograms (UPGMA) and maximal spanning trees (MST). Completely additional RAPD-spectra were not obtained though taxonomic status of these paratypical forms stay still vague. Only f. eratoides has DNA markers that are absent in spectra of other specimens tested. Probably it is a cryptic species unrevealed by common morphological and biochemical methods and its investigation by the methods of molecular genetics (sequence of cloned DNA) is very desirable.     

Immunohistochemistry of carbonic anhydrase in human placenta and fetal membranes

The localization of human carbonic anhydrase (CA) isoenzymes HCA I, HCA II, and rat CA II have been studied in human umbilical cord, chorion laeve including amnion and placenta from first and second trimester and also from term pregnancies. Detection techniques of immunofluorescence and immunoperoxidase were used in cryostat and paraffin sections. Both isoenzymes were found in the villous syncytiotrophoblast throughout pregnancy. HCA I staining patterns in the villous endothelium were highly variable whereas increasing immunoreactivity levels of endothelial HCA II were detected as pregnancy advances. The extravillous cytotrophoblast showed generally weaker levels of immunoreactivity. In amnionic epithelium of membranes, chorionic plate and umbilical cord, higher activities for HCA I, HCA II and rat CA II were found than in all other localizations. Our findings emphasize the importance of enzyme mediated bicarbonate/CO₂ removal from the feto-placental unit as opposed to simple bicarbonate diffusion or carrier mediated transport. As effective transfer routes should be considered not only umbilical cord — placental villi — intervillous space, but also fetal kidney — amnionic fluid — amnion — uterine vessels.     

Distribution of collagen types III and IV in human placental villi

Immunofluorescent examination showed more significant accumulation of interstitial collagen type III in the stroma of mature placenta compared with immature one. Localization of membrane collagen type IV was found neither in basal membranes of epithelium and villous vessels of mature term placenta, nor in their stroma. The described patterns of distribution of collagen types III and IV in human placenta villi were proved by immunoelectronmicroscopic method.     

Morphological changes in the rat uterine tissues exposed to pregna-D'-pentaran derivatives of progestins and anti-progestins

Changes in the uterus morphology of mature female rats were studied on the model of pseudopregnancy after treatment with the progestin 16 alpha,17 alpha-cyclohexanoprogesterone (PR) and the antirpogestins 5 alpha(H)-16 alpha,17 alpha-cyclohexano-4,5-dihydroprogesterone (APR1) and 5 beta(H)-16 alpha,17 alpha-cyclohexano-4,5-dihydroprogesterone (APR2). The rats were preliminarily estrogenized with 17 beta-estradiol at a dose of 1 microgram/(animal day) for 4 days and then treated with PR at a dose of 0.2 mg/(animal day) for 14 days. The first group was then left without any treatment, whereas APR1 and APR2 were injected at the dose of 0.2 mg/(animal day) for 4 days to the animals of the second and the third groups, respectively. Light and electron microscopy of the uterus preparations demonstrated that the PR action provoked a complete pseudopregnancy picture characterized by the endometrium functionalization and the myometrium hypertrophy. Subsequent treatment with APR1 and APR2 caused the hypertrophy to cease, which had a more pronounced effect in the case of APR1. At the same time, some indications of the endometrium functionalization remained observable after treatment with APR1 and APR2. The specific binding sites for 3H-labeled APR1 and APR2 were absent from the uterus cytosol for the rats gestagenized with PR.     

Manipulation of chromosome condensation by protein synthesis inhibitors and cyclic AMP during maturation of bovine oocytes

We investigated the effects of cycloheximide on bovine oocyte chromosomes during meiotic maturation in vitro. Bovine oocytes at Metaphase I (MI) of the meiotic maturation were treated with 10 mug/ml cycloheximide alone or in addition to 5 mM dibutyrylcAMP (dbcAMP) plus 1 mM isobutylmetylxantine (IBMX). A maturation period of 15 to 18 h followed by 12-h treatment with cycloheximide appeared to be most efficient to induce interphase (86% with 16 h maturation). About 60% of oocytes returned to a metaphase state 12 h after the oocytes were transferred to cycloheximide-free medium. In contrast, up to 73% of cycloheximide-treated oocytes at 17 h of maturation remained in interphase if dbcAMP plus IBMX was included in the cycloheximide-free medium. This shows that dbcAMP plus IBMX can inhibit the development of conditions in the oocytes that are required for the transition to metaphase. The chromosome decondensation induced by protein synthesis inhibition at Metaphase I is reversible. This study shows that transition to interphase in bovine oocyte depends on the stage of maturation of oocytes and is sensitive to cAMP levels.     

Morphological Changes in Rat Uterus Tissues under the Action of Progestins and Antiprogestins of the Pregna-D"-pentaran Series

Changes in the uterus morphology of mature female rats were studied on the model of pseudopregnancyafter treatment with the progestin 16,17-cyclohexanoprogesterone (PR) and the antirpogestins 5(H)-16,17-cyclohexano-4,5-dihydroprogesterone (APR1) and 5(H)-16,17-cyclohexano-4,5-dihydroprogesterone (APR2). The rats were preliminarily estrogenized with 17-estradiol at a dose of 1 g/(animal day) for 4 days and then treated with PR at a dose of 0.2 mg/(animal day) for 14 days. The first group was then left without any treatment, whereas APR1 and APR2 were injected at the dose of 0.2 mg/(animal day) for 4 days to the animals of the second and the third groups, respectively. Light and electron microscopy of the uterus preparations demonstrated that the PR action provoked a complete pseudopregnancy picture characterized by the endometrium functionalization and the myometrium hypertrophy. Subsequent treatment with APR1 and APR2 caused the hypertrophy to cease, which had a more pronounced effect in the case of APR2. At the same time, some indications of the endometrium functionalization remained observable after treatment with APR1 and APR2. The specific binding sites for ³H-labeled APR1 and APR2 were absent from the uterus cytosol for the rats gestagenized with PR.     

Isolated symplasts and stromal syncytial buds of the normal human placenta at 6 to 14 weeks of pregnancy

Formation of isolated symplasts (IS) ("deported trophoblast", according to English nomenclature) and stromal syncytial buds (SSB) has been studied in 10 normal patients at pregnancy of 6-14 weeks by means of the indirect immunoperoxidase method. Monoclonal antibodies against beta-subunit of the human chorionic gonadotropin (beta-HCG), serial paraffin slices, stained with hematoxylin-eosin, semithin slices, stained with toluidin blue-pyronine and scanning electron microscopy have been used. A great amount of syncytiotrophoblastic processes and IS, analogous by their structure to syncytiotrophoblast are revealed. Presence of beta-HCG in the syncytiotrophoblast and its absence in the cytotrophoblast are proved. Concentration of beta-HCG in the IS is higher than in the syncytiotrophoblast of the villi. Maximal concentration of beta-HCG in the SSB is described for the first time. The functional role of the IS and SSB is discussed in the system mother-placenta-fetus.