Alterations in gap junction connexin43/connexin45 ratio mediate a transition from quiescence to excitation in a mathematical model of the myometrium

The smooth muscle cells of the uterus contract in unison during delivery. These cells achieve coordinated activity via electrical connections called gap junctions which consist of aggregated connexin proteins such as connexin43 and connexin45. The density of gap junctions governs the excitability of the myometrium (among other factors). An increase in gap junction density occurs immediately prior to parturition. We extend a mathematical model of the myometrium by incorporating the voltage-dependence of gap junctions that has been demonstrated in the experimental literature. Two functional subtypes exist, corresponding to systems with predominantly connexin43 and predominantly connexin45, respectively. Our simulation results indicate that the gap junction protein connexin45 acts as a negative modulator of uterine excitability, and hence, activity. A network with a higher proportion of connexin45 relative to connexin43 is unable to excite every cell. Connexin45 has much more rapid gating kinetics than connexin43 which we show limits the maximum duration of a local burst of activity. We propose that this effect regulates the degree of synchronous excitation attained during a contraction. Our results support the hypothesis that as labour approaches, connexin45 is downregulated to allow action potentials to spread more readily through the myometrium.     

Maternal Neutrophil Depletion Fails to Avert Systemic Lipopolysaccharide-Induced Early Pregnancy Defects in Mice

Maternal infection-induced early pregnancy complications arise from perturbation of the immune environment at the uterine early blastocyst implantation site (EBIS), yet the underlying mechanisms remain unclear. Here, we demonstrated in a mouse model that the progression of normal pregnancy from days 4 to 6 induced steady migration of leukocytes away from the uterine decidual stromal zone (DSZ) that surrounds the implanted blastocyst. Uterine macrophages were found to be CD206⁺ M2-polarized. While monocytes were nearly absent in the DSZ, DSZ cells were found to express monocyte marker protein Ly6C. Systemic endotoxic lipopolysaccharide (LPS) exposure on day 5 of pregnancy led to: (1) rapid (at 2 h) induction of neutrophil chemoattractants that promoted huge neutrophil infiltrations at the EBISs by 24 h; (2) rapid (at 2 h) elevation of mRNA levels of MyD88, but not Trif, modulated cytokines at the EBISs; and (3) dose-dependent EBIS defects by day 7 of pregnancy. Yet, elimination of maternal neutrophils using anti-Ly6G antibody prior to LPS exposure failed to avert LPS-induced EBIS defects allowing us to suggest that activation of Tlr4-MyD88 dependent inflammatory pathway is involved in LPS-induced defects at EBISs. Thus, blocking the activation of the Tlr4-MyD88 signaling pathway may be an interesting approach to prevent infection-induced pathology at EBISs.     

Dynamics of lipid droplets in the endometrium and fatty acids and oxylipins in the uterine lumen,blood, and milk of lactating cows during diestrus

Our objective was to investigate the lipid content of uterus, blood plasma, and milk at early, mid, and late diestrus. Lactating cows (n = 30) had the estrous cycle and ovulation synchronized by administration of exogenous hormones. Cows were blocked by parity and assigned randomly to receive transcervical uterine flushing and biopsy on d 5 (early diestrus), 10 (mid diestrus) or 15 (late diestrus) of the estrous cycle. Flushing and endometrial biopsy were performed in the uterine horn ipsilateral to the corpus luteum. The recovered flushing was used for analyses of lipid composition by liquid chromatography-tandem mass spectrometry and the biopsy was used for investigation of lipid droplet abundance in endometrial cryosections using a neutral lipid fluorescent dye. In addition, blood and milk samples were collected from all cows on d 5, 10, and 15. All blood samples were used to measure the concentration of progesterone in plasma, and all milk samples were used to determine milk composition. Subsamples of blood plasma and milk were also used to evaluate the composition of fatty acids and oxylipins using the same methodology used for uterine flushing samples. The abundance of lipid droplets in the endometrium increased 1.9-fold from d 5 to 10, and 2-fold from d 10 to 15. Concentration of long-chain fatty acids and oxylipins in uterine flushing were, on average, 2.2 and 2.5 times greater in samples collected on d 15 compared with those collected on d 5 and 10. These differences were not observed in blood and milk, suggesting that accumulation of fatty acids and oxylipins in the uterus is regulated locally. In addition to concentration, the profile of individual fatty acids and oxylipins in uterine lumen changed substantially during diestrus. The main categories with increased abundance at late diestrus were mono- and polyunsaturated fatty acids, and oxylipins derived from arachidonic acid, dihomo-γ-linolenic acid, and docosahexaenoic acid. In conclusion, fatty acids and oxylipins accumulate in the uterine lumen during diestrus and might work as a mechanism to supply these lipids to the developing conceptus at late diestrus, when the onset of elongation occurs and substantial synthesis of biomass and cell signaling by lipid mediators are required.     

The microvascular cast as a three-dimensional tissue skeleton: visualization of rapid morphological changes in tissues of the rat uterus

A new interpretation of microvascular corrosion casting is described using examples from casts of the rat uterus taken during the oestrous cycle and early pregnancy. The casts are considered as representing a three-dimensional model of the tissue vascular space; this characteristic model or tissue skeleton is used to demonstrate transient morphological changes that may be difficult to visualize by other techniques, that may be obscured by other structures, or destroyed by conventional aldehyde fixation procedures. This new application, when supplemented by conventional histology to determine vessel distribution within the tissue and to confirm observations first made by casting, provides a technique for visualizing three-dimensional morphological changes in tissues that is accurate, relatively simple and far more rapid than the laborious method of histological serial sectioning and three-dimensional tissue reconstruction.     

Melatonin and Glycine Reduce Uterus Ischemia/Reperfusion Injury in a Rat Model of Warm Ischemia

Ischemia/reperfusion injury (IRI) remains a significant problem to be solved in uterus transplantation (UTx). Melatonin and glycine have been shown to possess direct cytoprotective activities, mainly due to their antioxidative and anti-inflammatory properties. The aim of this study was to investigate the protective effects of melatonin and glycine and their combination on IRI in a rat model of warm ischemia. In this study, Sprague-Dawley rats were assigned to eight groups, including sham and IRI (n = 80). Melatonin and glycine alone or their combination were administered prior to 1 h of uterus ischemia followed by 1 h of reperfusion. Melatonin (50 mg/kg) was administered via gavage 2 h before IRI and glycine in an enriched diet for 5 days prior to intervention. Uterus IRI was estimated by histology, including immunohistochemistry, and biochemical tissue analyses. Histology revealed that uterus IRI was significantly attenuated by pretreatment with melatonin (p = 0.019) and glycine (p = 0.044) alone as well as their combination (p = 0.003). Uterus IRI led to increased myeloperoxidase expression, which was significantly reduced by melatonin (p = 0.004), glycine (p < 0.001) or their combination (p < 0.001). The decline in superoxide dismutase activity was significantly reduced in the melatonin (p = 0.027), glycine (p = 0.038) and combined treatment groups (p = 0.015) when compared to the IRI control group. In conclusion, melatonin, glycine and their combination significantly reduced oxidative stress-induced cell damage after IRI in a small animal warm ischemia model, and, therefore, clinical studies are required to evaluate the protective effects of these well-characterized substances in uterus IRI.     

CSH RNA Interference Reduces Global Nutrient Uptake and Umbilical Blood Flow Resulting in Intrauterine Growth Restriction

Deficiency of the placental hormone chorionic somatomammotropin (CSH) can lead to the development of intrauterine growth restriction (IUGR). To gain insight into the physiological consequences of CSH RNA interference (RNAi), the trophectoderm of hatched blastocysts (nine days of gestational age; dGA) was infected with a lentivirus expressing either a scrambled control or CSH-specific shRNA, prior to transfer into synchronized recipient sheep. At 90 dGA, umbilical hemodynamics and fetal measurements were assessed by Doppler ultrasonography. At 120 dGA, pregnancies were fitted with vascular catheters to undergo steady-state metabolic studies with the ³H₂O transplacental diffusion technique at 130 dGA. Nutrient uptake rates were determined and tissues were subsequently harvested at necropsy. CSH RNAi reduced (p ≤ 0.05) both fetal and uterine weights as well as umbilical blood flow (mL/min). This ultimately resulted in reduced (p ≤ 0.01) umbilical IGF1 concentrations, as well as reduced umbilical nutrient uptakes (p ≤ 0.05) in CSH RNAi pregnancies. CSH RNAi also reduced (p ≤ 0.05) uterine nutrient uptakes as well as uteroplacental glucose utilization. These data suggest that CSH is necessary to facilitate adequate blood flow for the uptake of oxygen, oxidative substrates, and hormones essential to support fetal and uterine growth.     

The effects of feeding fish oil on uterine secretion of PGF2alpha, milk composition, and metabolic status of periparturient Holstein cows

The objectives were to determine the effect of dietary fish oil (FO) on uterine secretion of PGF2alpha, milk production, milk composition, and metabolic status during the periparturient period. Holstein cows were assigned randomly to diets containing FO (n = 13) or olive oil (OO, n = 13). Cows were fed prepartum and postpartum diets that provided approximately 200 g/d from 21 d before the expected parturition until 21 d after parturition. The FO used contained 36% eicosapentaenoic acid (EPA, C20:5, n-3) and 28% docosahexaenoic acid (DHA, C22:6, n-3). Blood samples were obtained from 14 d before the due date until d 21 postpartum. A total of 6 FO and 8 OO cows without periparturient disorders were used in the statistical analyses of PGF2alpha-metabolite (PGFM) and metabolite concentrations. Length of prepartum feeding with OO or FO did not differ. Proportions of individual and total n-3 fatty acids were increased in caruncular tissue and milk of cows fed FO. The combined concentrations of EPA and DHA in caruncular tissue were correlated positively with the number of days supplemented with FO. Cows fed FO had reduced concentrations of plasma PGFM during the 60 h immediately after parturition compared with cows fed OO. Concentrations of prostaglandin H synthase-2 mRNA and protein in caruncular tissue were unaffected by diet. Production of milk and FCM were similar between cows fed the two oil diets. However, cows fed FO produced less milk fat. Feeding FO reduced plasma concentrations of glucose. Dietary fatty acids given during the periparturient period can reduce the uterine secretion of PGF2alpha in lactating dairy cows and alter the fatty acid profile of milk fat.     

Differential expression and regulation of PNA and UEA‐1 bindings in rabbit uterus during preimplantation period

Peanut agglutinin (PNA) and Ulex europaeus agglutinin‐1 (UEA‐1) were used as probes to study the distribution of β‐gal (1→3) ga1Nac and α‐l ‐Fucose in rabbit uterus during early pregnancy. PNA binding was mainly localized on the surface of uterine glandular and luminal epithelium. There were no positive signals on day 1 of pregnancy. PNA binding gradually increased from day 2 and reached its highest level on days 3 and 4. The distribution of PNA binding gradually declined from day 5 and reached a low level on day 7. However, UEA‐1 binding was only localized on the luminal epithelial during early pregnancy. A high level of UEA‐1 binding had been found on the luminal epithelium on day 1 of pregnancy and low level of positive signals had been found in the uterus on days 2 and 3. UEA‐1 binding increased gradually and reached its highest level on day 4. Then the distribution of UEA‐1 binding sharply declined and no positive signals were found on days 5–7. The distribution of PNA and UEA‐1 bindings in pseudopregnant uterus was similar to that in normal pregnant uterus. During estrus cycle, there was no detectable PNA binding signal in uterus. But, a high level of UEA‐1 binding was found in the luminal epithelium of estrus uterus. In ovariectomized rabbit uterus, progesterone significantly induced the expression of PNA binding, while estrogen stimulated UEA‐1 binding expression. These results suggested that the distribution of PNA and UEA‐1 bindings in rabbit uterus may be related to rabbit implantation. Microsc. Res. Tech. 76:398–403, 2013. © 2013 Wiley Periodicals, Inc.