Changes of connexin43 expression in non-pregnant porcine myometrium correlate with progesterone concentration during oestrous cycle.

Connexin43 (Cx43) is a major protein of myometrial gap junctions. The number of Cx43 gap junctions increase dramatically with the onset of labour in association with development of synchronized uterine contractions. The formation of myometrial gap junctions follows an increase in the oestrogen to progesterone ratio indicating an important role of steroid hormones in regulating Cx43 expression at term. However, no relationship has been established between the expression of Cx43 in the non-pregnant myometrium and concentration of steroid hormones during the oestrous cycle. Here, we used immunofluorescence and Western blotting to analyse the expression of Cx43 gap junctions in the myometrium of pre-pubertal pigs (n = 7) and mature pigs at pre-ovulatory (n = 7), luteal (n = 5) and late luteal (n = 3) stages of the oestrous cycle. The number of Cx43 gap junctions calculated per 1 mm(2) of the myometrial section was low in pre-pubertal pigs and significantly higher (p < 0.022) in pre-ovulatory animals. In relation to pre-ovulatory animals the number of myometrial gap junctions was significantly lower (p < 0.019) at the luteal phase and correlated with significantly higher (p < 0.005) concentration of endogenous progesterone. Phosphorylated isoforms of Cx43 protein were expressed in the myometrium of pre-pubertal pigs and mature animals at pre-ovulatory and late luteal phases, while they were down regulated at the luteal stage. These results indicate that changes of Cx43 expression in the porcine myometrium during the oestrous cycle may be regulated by progesterone concentration and may contribute to the modulation of uterine motility.

Capillary density and capillary-to-fibre ratio in vastus lateralis muscle of untrained and trained men.

Muscle fibre profile area (Af), volume density (Vv), capillary-to-fibre ratio (CF) and number of capillaries per fibre square millimetre (CD) were determined from needle biopsies of vastus lateralis of twenty-four male volunteers (mean +/- SD: age 25.4+/-5.8 years, height 178.6+/-5.5 cm, body mass 72.1+/-7.7 kg) of different training background. Seven subjects were untrained students (group A), nine were national and sub-national level endurance athletes (group B) with the background of 7.8+/-2.9 years of specialised training, and eight subjects were sprint-power athletes (group C) with 12.8+/-8.7 years of specialised training. Muscle biopsies of vastus lateralis were analysed histochemically for mATPase. Capillaries were visualized and counted using CD31 antibodies against endothelial cells. There were significant differences in the Vv of type I and type II muscle fibres in both trained groups, B (51.8%; 25.6%) and C (50.5%; 26.4%). However, in untrained group A that was treated as a reference group, the difference between Vv of type I and type II fibres was less prominent, nevertheless statistically significant (42.1%; 35.1%). There was also a significant difference in CF: 1.9 in group A and 2.1 in groups B and C. The number of capillaries per mm2 (CD) was 245 (group A), 308 (group B) and 325 (group C). Significant differences (P<0.05) in CF and CD, were found only between group A (1.9; 245) and both groups of trained men, B and C (2.1; 308 and 325). However, endurance athletes (group B), such as long-distance runners, cyclists and cross country skiers, did not differ from the athletes representing short term, high power output sports (group C) such as ice hockey, karate, ski-jumping, volleyball, soccer and modern dance.

Characteristics of myosin profile in human vastus lateralis muscle in relation to training background.

Twenty-four male volunteers (mean +/- SD: age 25.4+/-5.8 years, height 178.6+/-5.5 cm, body mass 72.1+/-7.7 kg) of different training background were investigated and classified into three groups according to their physical activity and sport discipline: untrained students (group A), national and sub-national level endurance athletes (group B, 7.8+/-2.9 years of specialised training) and sprint-power athletes (group C, 12.8+/-8.7 years of specialised training). Muscle biopsies of vastus lateralis were analysed histochemically for mATPase and SDH activities, immunohistochemically for fast and slow myosin, and electrophoretically followed by Western immunoblotting for myosin heavy chain (MyHC) composition. Significant differences (P<0.05) regarding composition of muscle fibre types and myosin heavy chains were found only between groups A (41.7+/-1.6% of MyHCI, 40.8+/-4.0% of MyHCIIA and 17.5+/-4.0% of MyHCIIX) and B (64.3+/-0.8% of MyHCI, 34.0+/-1.4% of MyHCIIA and 1.7+/-1.4% of MyHCIIX) and groups A and C (59.6+/-1.6% of MyHCI, 37.2+/-1.3% of MyHCIIA and 3.2+/-1.3% of MyHCIIX). Unexpectedly, endurance athletes (group B) such as long-distance runners, cyclists and cross country skiers, did not differ from the athletes representing short term, high power output sports (group C) such as ice hockey, karate, ski-jumping, volleyball, soccer and modern dance. Furthermore, the relative amount of the fastest MyHCIIX isoform in vastus lateralis muscle was significantly lower in the athletes from group C than in students (group A). We conclude that the myosin profile in the athletes belonging to group C was unfavourable for their sport disciplines. This could be the reason why those athletes did not reach international level despite of several years of training.

Glycoconjugates of the intestinal goblet cells of four cyprinids.

The aim of this work was to show differences in the terminal and subterminal sugar composition of carbohydrate chains of glycoconjugates produced by the goblet cells of the intestines of four cyprinids. We analysed intestines of two herbivorous species--sneep and grass carp--and two omnivorous ones--chub and common carp. We compared four intestinal regions of every studied species. In every region, the presence of neutral and acidic glycoconjugates was confirmed. The smallest amount of acidic glycoconjugates was present in the second region of sneep intestine. Sulphated glycoconjugates were absent in the third and fourth region of chub intestine. Lectin histochemistry provided evidence for the presence of beta-D-galactose, a-N-acetylgalactosamine, beta-N-acetylglucosamine and sialic acids. Additionally, the occurrence of alpha-L-fucose in the goblet cells of chub, grass carp and sneep was confirmed. We tried to correlate the pattern of glycoconjugate glycosylation with feeding habits of the studied fishes.

Multiple connexins localized to individual gap-junctional plaques in human myometrial smooth muscle.

The synchronous contractions of the uterus in labour depend on electrical coupling of myometrial smooth muscle cells by gap junctions. In the human myometrium, gap junctions are scarce in the non-pregnant uterus, but become abundant at term in preparation for labour. We have previously demonstrated that in the human myometrium at term, three different gap-junctional proteins are expressed, connexins 43, 45, and 40. These connexins are known to have distinctive functional capacities in in vitro expression systems but whether, in the human myometrium in vivo, they are co-assembled into the same gap junction or form different types of gap junction has previously been unclear. By applying triple immunogold labelling to sections of Lowicryl-embedded tissue for electron microscopy, together with complementary immunoconfocal microscopy, we demonstrate here that connexins 43, 45, and 40 are commonly present as mixtures within the same gap-junctional plaque. While all gap junctions contain connexin43, the relative signal for each connexin type varies between individual junctions. The presence within single gap-junctional plaques of three different connexins, each with the potential for conferring distinctive channel properties, suggests an inherent versatility for modulation of smooth muscle cell intercellular communication properties during human parturition.

Electron probe X-ray microanalysis of cultured myogenic C2C12 cells with scanning and scanning transmission electron microscopy.

Heterogeneity of the elemental content of myogenic C2C12 cultured cells was studied by electron probe X-ray microanalysis (EPXMA) with scanning (SEM EPXMA) and scanning transmission electron microscopy (STEM EPXMA). The best plastic substrate for growing cells was Thermanox. For STEM EPXMA, a Formvar film coated with carbon was found to be suitable substrate. The cells examined by scanning transmission electron microscopy showed great heterogeneity in their elemental content in comparison with the cells examined in the scanning electron microscope despite of an almost identical preparation procedure for EPXMA. Nevertheless the K/Na ratios obtained from both methods of EPXMA were very close (4.1 and 4.3). We conclude that the observed discrepancy in the elemental content obtained by the two methods may be due to differences in instrumentation and this must be taken into account when planning a comparative study.

Effect of progesterone and oestradiol on expression of connexin43 in cultured human myometrium cells.

In human myometrium, the formation of gap junctions at various stages of labour and in correlation with the concentration of progesterone and oestradiol in maternal blood was described previously by electron microscopy and laser confocal microscopy of immunohistochemically stained myometrial sections. The present investigation focused on the effect of continuous exposure of isolated myometrial tissue to progesterone and oestradiol on the number of gap junction plaques in human myometrium cells in vitro. The presence of gap junctions was evaluated by immunocytochemistry with antibodies against gap junction protein, connexin43 (Cx43). Human myometrial cells were isolated from biopsies obtained from term pregnant women who had an elective caesarean operation in the 37th or 40th week of pregnancy. The dispersed myometrial cells that were obtained by limited enzymatic digestion of the myometrial samples were maintained in monolayer culture for 1, 3 and 6 days in the presence of medium that contained foetal bovine serum and the steroids at different concentration. In primary culture, as well as after several passages, the characteristics of these cells were morphologically and biochemically similar to those of smooth muscle cells and myometrial tissue. The obtained results showed that the cells in culture responded synchronously to the increased concentrations of oestradiol/progesterone mixtures. The number of gap junctions increased significantly on days 1, 3 and 6 in culture and showed positive correlation (p < 0.05) with the cell number when the concentration of oestradiol was raised to 1 microgram/mL in the progesterone ratio (1.0 microgram/0.5 microgram/mL). No significant correlation, however, in connexin43 gap junction number versus cell number was observed between the six experimental groups treated with progesterone only.

Immunohistochemical analysis of connexin26 and 43 expression in the mouse alimentary tract.

The spatial pattern of connexins26 (Cx26) and 43 (Cx43) expressions were investigated in the mouse digestive tract by immunocytochemistry. High levels of connexin43 in the epithelium of the oesophagus, non-glandular part of the stomach, and the circular layer of duodenal and ilea muscularis externa were detected. Cx26 was expressed in stratum granulosum of oesophagal folds and in the non-glandular part of the stomach. A low level of immunoreactivity of Cx43 was observed in the circular, and very low in the longitudinal layer of the muscularis externa in the stomach and colon. No immunoreactivity for Cx26 and Cx43 was found in the entire muscularis externa of the oesophagus or in the longitudinal muscle layers of the duodenum and ileum.

Antiserum against connexin32 fragment reacts with a 32-kD protein localised in gap junctions of mouse and rat liver, endometrium and in the fish heart.

The expression of various connexins so far identified is metabolically and developmentally regulated. Examples include uterine endometrium where the expression of gap junction protein, connexin32 (Cx32) is regulated by steroid hormones. In this study we attempted to synthesise a short peptide which matches the portion of the amino acid sequence of the Cx32. Cx32 has primary structure predicted from the nucleotide sequence of cDNA clone. A fragment of Cx32 molecule was synthesised to produce anti-peptide antibody for detecting gap junction protein in mouse and rat liver and endometrium. The 12-peptide, plus Abu residue that corresponds to residues 108-119 (LEGHGDPLHLEE-Abu) of the rat Cx32 (283-mer) was synthesised by the solid phase method. Antibodies against this peptide were raised in rabbits, screened for reactivity and specificity using dot blot assays [15] and used for immunocytochemical staining at the light and at electron microscope levels. The antibodies also reacted with fish heart myocardium.

Identification of two further gap-junctional proteins, connexin40 and connexin45, in human myometrial smooth muscle cells at term.

The powerful synchronous contractions of the uterus in labor depend on electrical coupling of myometrial smooth muscle cells by gap junctions. In humans and other mammals, gap junctions are scarce in the myometrium of the non-pregnant uterus, but become abundant at term and/or with the onset of labor. Previous work has shown that the gap-junctional protein (connexin) expressed by human myometrial smooth muscle cells is connexin43, the same connexin type that predominates in cardiac muscle. Here we show that two further gap junctional proteins, connexin40 and connexin45, are expressed by the myometrial smooth muscle cells of the human uterus at term. Transcripts encoding the human isoforms of these connexins were demonstrated by Northern blot analysis, and immunoconfocal microscopy enabled precise localization of the corresponding proteins to punctate contact points (i.e., gap junctions) between interacting smooth muscle cells. Double labeling demonstrated that, while some fluorescent spots comprise only connexin43, both connexin40 and connexin45 predominantly colocalize to connexin43-positive fluorescent spots. Triple labeling revealed that where all three connexin types were expressed, they frequently localized to the same gap junction spot. As gap-junctional channels composed of different connexin types have been demonstrated in vitro to have different functional properties, multiple connexin expression may contribute to modulation of gap junction function in human myometrial smooth muscle cells in vivo.

Quantitative immunoconfocal analysis of human myometrial gap junction connexin43 in relation to steroid hormone concentrations at term labour.

The aim of this study was to quantify gap junction expression in the human myometrium in relation to progesterone and oestradiol concentrations, and to establish whether oxytocin-resistant dystocia is due to an abnormality in gap junction expression. Three groups of patients were investigated: (i) before labour (at term), (ii) normal labour and (iii) oxytocin-resistant dystocia (eight patients per group). For each patient, the concentrations of oestradiol and progesterone in maternal blood and in myometrial tissue were measured, and the number and area of immunostained connexin43 gap junctions per unit volume of tissue determined by quantitative analysis of digital images obtained by confocal microscopy. No significant difference in connexin43 gap junction content was observed between the three patient groups. When all groups were pooled, there was a significant positive correlation (P < 0.05) between the quantity of immunolabelled gap junctions and the oestradiol:progesterone ratio, but there was no significant difference in this correlation between the groups. Gap junction immunolabelling was not correlated with the progesterone or oestradiol concentration in the maternal blood or the myometrium. These data suggest that in human myometrium: (i) dystocia is not due to a reduced level of immunodetectable connexin43 gap junctions, (ii) onset of labour is not associated with a sudden increase in immunodetectable gap junction protein and (iii) gap junctions can be expressed in the presence of high progesterone concentrations.

Progesterone, oestradiol and oxytocin and their in vitro effect on maintaining the number of gap junction plaques in human myometrium at term.

Previous studies by electron microscopy and laser confocal microscopy of immunohistochemically stained sections have shown that during pregnancy the extent of gap junction formation in human myometrium is low, but that an increase occurs in the active stages of labour despite a high concentration of progesterone in maternal blood. The present investigation focused on the effect of in vitro exposure of isolated myometrial tissue to progesterone, oestradiol and oxytocin, on the number of gap junction plaques in human myometrium at term. Myometrial biopsies were obtained at term from 13 pregnant women who had an elective caesarean section in the 37th or 40th week of pregnancy. The biopsies were immersed immediately in Hepes buffer and buffer containing 0.5, 5.0 micrograms mL-1 of progesterone, and 0.1 micrograms mL-1 of oestradiol. The muscle biopsies were trimmed under a stereo microscope into strips along the bundles of smooth muscle cells and mounted in tissue baths, superfused with Hepes buffer supplemented with glucose (0.01 mM); subsequently the strips were exposed to buffer containing different concentrations of progesterone, oestradiol and oxytocin. Incubation of the muscle strips for 180 min resulted in a significant decrease of the number of gap junctions (P < 0.01). Neither oestradiol or oxytocin, alone or in combination, had a significant effect on the maintenance of the number of gap junctions. The progesterone concentration of 5.0 micrograms mL-1, combined with oxytocin, and with or without oestradiol has a significantly positive effect on the number of gap junction plaques in strips of human myometrium at term (P < 0.05 vs. buffer alone). The high concentration of progesterone in the superfusion medium during in vitro experiments may be responsible for the maintenance of high numbers of gap junction complexes in term human myometrium. This finding is of interest in the light of findings of persisting high progesterone levels in maternal blood during labour.

In vitro effect of Hepes buffer on maintaining the number of gap junction plaques in human myometrium at term.

In human myometrium, at various stages of labour and in correlation with the concentration of progesterone and estradiol in maternal blood the formation of gap junctions has been described previously by electron microscopy and laser confocal microscopy of immunohistochemically stained myometrial sections. The present investigation focused on the effect of continuous exposure of isolated human myometrium at term to Hepes buffer, on the number of gap junction plaques. Biopsies of myometrium were obtained from 5 pregnant women at term: they had an elective caesarean operation in the 37th or 40th week of pregnancy. The biopsies were immersed immediately in Hepes buffer, trimmed under a stereo microscope into small strips and kept in warm (37 degrees C) oxygenated Hepes buffer supplemented with glucose (0.01 mM). Then some strips of myometrium were incubated at 37 degrees C for 10, 20, 40, 55, and 180 min. The number of gap junction plaques were counted: it decreased in strips of myometrium after 10, 20, 40, 55, and 180 min of in vitro experiment (P < 0.01 vs control). In some of our experiments, the decrease in quantity of gap junction plaques was very dramatic and far below 50% of their numbers found in control specimens but never reached the zero value.

Morphometric analysis of gap junction density in human myometrium at term.

In the human myometrium, the formation of gap junctions in the various stages of labor and, in correlation with the concentration of progesterone in the maternal blood, has not been described adequately. The accepted hypothesis that raised concentrations of progesterone in maternal blood in animals prevent the formation of gap junctions was scrutinized in human myometrium. Myometrial tissue was examined by electron microscopy for the presence of gap junctions and analyzed morphometrically in 17 women who had an elective or emergency cesarean operation at the 38th or 39th week of pregnancy. The concentrations of progesterone and estradiol in the maternal and cord blood was measured by means of radioimmunoassay. The frequency and area of gap junctions varied in relation to the presence or absence of labor. In the myometrium of women in labor, gap junctions were most frequent (5.2/1000 microns of cell membrane), their area largest (0.32 microns 2/1000 microns of cell membrane) and their fractional area greatest (0.20%). When the women were not in genuine labor or when contractions were weak, the percentage fractional areas of gap junctions were low (0.020-0.090) and the gap junctions very small. In the pre- and active phases of labor, the concentration of progesterone in maternal blood was significantly higher (p < 0.025 and 0.03 respectively) than in women who were not in labor. Hence, despite high concentrations of progesterone in the maternal and cord blood at term, the myometrium presented numerous gap junctions of relatively large area.

Antisera against three connexin43 fragments react with a 43-kD protein localised to gap junctions in myocardium and human myometrium.

Gap junctions are thought to play an essential role in the control of parturition. In this study we attempted to synthesise three short peptides which match the portion of the amino acid sequence of the connexin43, protein predicted from the nucleotide sequence of cDNA clone and to produce an anti-peptide antibody which would detect gap junction protein in human myometrium. Three peptides were synthesized: (1) RKEEKLNKKEEE, (2) EJKKFKYGJEEH and (3) KNRVKGRSDPYH. Antibodies against these peptides were raised in rabbits, screened for reactivity and specificity using dot blot assays and used for immunocytochemical staining of heart and myometrial tissues.

Structural and functional studies of gap junctions in human myometrium.

Gap junctions are of importance in the coordinated contraction of uterine smooth muscle during labor. There are indications that in the human myometrium, gap junctions are much more numerous in the period immediately surrounding parturition. The presence and the functional state of gap junctions in human myometrium was studied by electron microscopical morphometry and by studying the distribution of an injected fluorescent dye. Electron microscopy showed that the number and size of gap junctions was significantly higher during the active phase of labor than during the pre-labor phase. Pressure injection of Lucifer Yellow into myometrial cells in vitro showed only weak coupling in the pre-labor stage. These data support the notion that coupling between myometrial cells develops in immediate association with parturition.

Gap junction density in human myometrium at term revealed by an anti-peptide antibody and laser scanning confocal microscopy.

The concept that the progesterone-estrogen ratio determines gap junction abundance in myometrium has developed from studies in laboratory animals. In the humans, however, gap junctions form in abundance at term despite a high progesterone-estrogen ratio. We have therefore examined the relationship between contractile activity, progesterone and estrogen levels and gap junction abundance in human myometrium at term. Myometrial tissue obtained from women who had an elective or emergency Cesarean operation at the 38th or 39th week of pregnancy was immunohistochemically examined using a polyclonal antiserum raised against a synthetic peptide matching part of the sequence of connexin43, the principal component of myometrial gap junctions. The concentration of progesterone and estradiol in the maternal blood was measured by means of radio-immunoassay. The number of gap junctions was estimated by counting of punctate staining patterns obtained in myometrial tissue as viewed by laser confocal microscopy and calculated per 1 microns 3 of tissue. The number of gap junctions measured was found to be related to the presence or absence of labor. In the myometrium of women in labor (active phase), the number of gap junctions was highest (45.25 +/- 35). When the myometrium was not contracting (non-labor) and when contractions were weak (pre-labor) the number of gap junctions was low (15.16 +/- 10-5.75 +/- 5). In the pre-and active phase of labor, the concentration of progesterone in maternal blood was significantly higher (P < 0.025 and 0.03, respectively) than in women who were not in labor. The present study shows the potential of using optical section series obtained by laser confocal microscopy for the quantification and mapping of the three-dimensional distribution of gap junctions in human myometrium at term.