Pulsatile oxytocin administered to ewes at 120 to 140 days gestational age increases the rate of maturation of the fetal electrocorticogram and nuchal activity.

Spontaneous, long lasting epochs of myometrial contractility, contractures, occur throughout the majority of pregnancy in sheep. Contractures are temporally related to a switch in fetal electroencephalogram (ECoG) patterns from low to high voltage. In late gestation, fetal ECoG increases in voltage. We have previously suggested that contractures may influence fetal ECoG maturation. In the present study, we hypothesized that a sustained increase in the frequency of myometrial contractures in pregnant sheep at 120-140 days gestation would accelerate maturation of the fetal ECoG. Five pregnant ewes were pulsed with oxytocin 600 microU.kg-1.min-1 intravenously for five minutes in every 30 minutes from 127.8 +/- 1.5 days gestational age for a minimum of six days. Six control ewes received pulses of saline. Fetuses of all eleven ewes were instrumented with bilateral electrodes to record fetal ECoG and nuchal muscle activity. Fetal high voltage (HV) ECoG increased in amplitude in both groups but the rate of increase was faster in the fetuses of ewes receiving oxytocin. There were no differences between the two groups in the duration of HV ECoG. The percentage increase in the amount of time the fetal nuchal muscles were active compared with the baseline day before infusion was only significant in the oxytocin infused group on the first day of oxytocin infusion. These findings support the hypothesis that myometrial activity during pregnancy has the capacity to influence fetal neural development.     

Prenatal glucocorticoid administration accelerates the maturation of fetal rat hepatocytes

Molecular Biology Reports - Prenatal glucocorticoid (GC) is clinically administered to pregnant women who are at risk of preterm birth for the maturation of cardiopulmonary function. Preterm and...     

Long noncoding RNA H19 accelerates tenogenic differentiation by modulating miR-140-5p/VEGFA signaling

Rotator cuff tear (RCT) is a common tendon injury, but the mechanisms of tendon healing remain incompletely understood. Elucidating the molecular mechanisms of tenogenic differentiation is essential to develop novel therapeutic strategies in clinical treatment of RCT. The long non-coding RNA H19 plays a regulatory role in tenogenic differentiation and tendon healing, but its detailed mechanism of action remains unknown. To elucidate the role of H19 in tenogenic differentiation and tendon healing, tendon-derived stem cells were harvested from the Achilles tendons of Sprague Dawley rats and a rat model of cuff tear was established for the exploration of the function of H19 in promoting tenogenic differentiation. The results showed that H19 overexpression promoted, while H19 silencing suppressed, tenogenic differentiation of tendon-derived stem cells (TDSCs). Furthermore, bioinformatic analyses and a luciferase reporter gene assay showed that H19 directly targeted and inhibited miR-140-5p to promote tenogenic differentiation. Further, inhibiting miR-140-5p directly increased VEGFA expression, revealing a novel regulatory axis between H19, miR-140-5p, and VEGFA in modulating tenogenic differentiation. In rats with RTC, implantation of H19-overexpressing TDSCs at the lesion promoted tendon healing and functional recovery. In general, the data suggest that H19 promotes tenogenic differentiation and tendon-bone healing by targeting miR-140-5p and increasing VEGFA levels. Modulation of the H19/miR-140-5p/VEGFA axis in TDSCs is a new potential strategy for clinical treatment of tendon injury.Key words: lncRNA, miRNA, tendon stem cell, rotator cuff tear repair     

Cortisol inhibits ACTH but not the AVP response to hypoxaemia in fetal lambs at days 123-128 of gestation

During acute hypoxemia in fetal sheep the elevation in ACTH concentration in the fetal circulation at days 125-129 is greater than that at term, but similar rises in AVP occur at both times. To examine whether the diminished ACTH response is due to elevated endogenous cortisol, and if there is differential control of ACTH and AVP release in hypoxemia, we infused either vehicle or cortisol (5 micrograms/min) into fetal sheep at days 123-128 for 5 h before and then during a 2-h period of acute hypoxemia (mean PaO2 decrease 8.2 mmHg) without acidemia. During cortisol infusion, plasma cortisol rose to 40-50 ng/ml, similar to values in term fetuses. In vehicle-infused fetuses, cortisol rose from 2.1 to 7.0 ng/ml at +1 to +2 h of hypoxemia. ACTH rose significantly during hypoxemia in the vehicle-infused fetuses, and this response was attenuated by cortisol infusion. In contrast, fetal AVP rose significantly during hypoxemia both in the presence and absence of cortisol infusion. Fetal breathing movements, and electroocular activity decreased during hypoxemia, and these responses were not altered by cortisol. We conclude that cortisol exerts differential negative feedback on ACTH but not on AVP release during hypoxemia. The maintained AVP response may facilitate cardiovascular adjustments of the fetus to hypoxemia even when endogenous cortisol is elevated, such as near term.     

Rapid-cooling and storage of plant nematodes at -140 degrees C

Low temperatures can assure the long-term or even indefinite preservation of important biological specimens. Nematode cryopreservation allows for the availability of large numbers of living nematodes at any one time, especially for experimental purposes. New isolates of Bursaphelenchus have recently been collected, including Bursaphelenchus eremus (Rühm) Goodey. This species was identified in north-central Italy on dying oak trees and from the bark beetle Scolytus intricatus Ratzeburg as dauer larvae. We therefore, sought to develop a cryopreservation technique for the long-term storage of all available Bursaphelenchus spp. The technique consists of a rapid-cooling protocol involving immersion in a liquid nitrogen bath before storage of the frozen samples in a mechanical freezer at -140 degrees C. The survival of nematodes subjected to this rapid-cooling protocol was higher than previously reported using slow-cooling methods and is suitable for several species of Bursaphelenchus and other phytoparasitic nematodes.     

The effect of hypothalamic paraventricular nuclear lesions placed at 108-110 days gestational age on plasma ACTH concentrations in the fetal sheep

Lesions that completely destroyed the paraventricular nucleus of the hypothalamus were placed in fetal sheep (n = 4) at 108-110 days of gestational age. These fetuses were then subjected to hypotension (50% of initial mean fetal arterial blood pressure), hypoxaemia (a decrease in fetal PaO2 greater than or equal to 5 torr) and bolus injection of corticotropin releasing factor (CRF-1.0 micrograms iv) in random order on successive days. The lesioned fetuses produced significantly less ACTH after hypotension (+10 min: 35.7 +/- 26.9 vs. 358.0 +/- 99.7 and +30 min: 28.2 +/- 12.2 vs. 238.0 +/- 73.0 pg.ml-1) (P less than 0.05), hypoxaemia (+40 min: 23.5 +/- 9.3 vs. 198.3 +/- 75.8 and +60 min: 32.3 +/- 18.8 vs. 295.3 +/- 99.9 pg.ml-1) (P less than 0.05) and intravenous administration of 1 microgram CRF (+15 min: 32.0 +/- 16.8 vs. 145.7 +/- 25.0 and +60 min: 33.0 +/- 23.3 vs. 161.3 +/- 43.1 pg.ml-1) (P less than 0.05). Our experiments suggest an important role for the fetal paraventricular nucleus in control of ACTH secretion. They also indicate that impairment of paraventricular nucleus function at this stage of fetal life may have a detrimental effect on the ability of the anterior pituitary to secrete ACTH in response to exogenous CRF.     

Effect of 48-hour infusion of the synthetic oxytocin antagonist, [1-beta-mercapto(beta-(CH2)5)1(OMe)Tyr2,Orn8]-oxytocin, on myometrial activity of pregnant sheep at 139-140 days of gestation.

Currently there is considerable interest in the actions of oxytocin antagonists on the pregnant myometrium. Few studies have been conducted involving long-term infusions of oxytocin antagonists to late-pregnant experimental animals. We set out to determine whether continuous infusion of an oxytocin antagonist ([1-beta-mercapto(beta-(CH2)5)1(OMe)Tyr2,Orn8]-oxytocin) would influence basal levels of myometrial activity of the contracture type and maternal prostaglandins in pregnant sheep. The antagonist was infused into a uterine vein at 80 micrograms.h-1 for 48 h starting at 139 days of gestational age. The antagonist significantly reduced total myometrial electromyogram activity and the frequency of contractures but did not change contracture duration. Antagonist infusion did not produce any significant alterations in maternal carotid or uterine vein 13,14-dihydro-15 keto prostaglandin F2 alpha concentrations. Contractures probably represent an intrinsic instability of the resting membrane potential of uterine muscle and these results suggest that oxytocin may play a role in regulating their frequency in sheep during the last third of gestation.     

Autonomous biochemical and morphological differentiation in fetal rat intestine transplanted at 17 and 20 days of gestation

The morphological and biochemical development of fetal rat intestine was examined for up to 5 weeks following transplantation to syngeneic hosts at 17 and 20 days of gestation. In transplants of both ages, normal villi bearing mature enterocytes developed. In addition, the disaccharidases lactase, maltase, and sucrase, as well as alkaline phosphatase, underwent normal patterns of development. Lactase activity, initially high, fell significantly, while maltase and sucrase activities increased significantly in the interval between 2 and 5 weeks following transplantation. During this same period, alkaline phosphatase developed the proximally located, high-activity form. The transplanted intestine also developed normal topographical distributions of enzyme activities. Measurement of corticosterone levels demonstrated that, except for a transient upsurge at the time of operation, hormone levels did not change significantly during the period of transplant maturation. These data indicate that the brush-border enzymes of the small intestine develop according to an intrinsic program which is already established as early as 17 days of gestation.     

Chromosome aberrations in in vitro-produced bovine embryos at days 2-5 post-insemination

Availability of embryos of high quality is required to obtain satisfactory embryonic developmental rates and normal calves following transfer of in vitro-produced (IVP) bovine embryos. One relevant quality parameter is the frequency of chromosome aberrations, which can be evaluated using multicolor fluorescent in situ hybridization (FISH) with chromosome 6- and chromosome 7-specific probes in cattle. In this study, interphase nuclei (n = 3805) were analyzed from 426 bovine IVP embryos. We found that 73%, 72%, 81%, and 58% of the embryos from Days 2, 3, 4, and 5 post-insemination (pi), respectively, displayed a normal diploid chromosome number in all cells. When looking at the types of chromosome aberrations, the percentages of mixoploidy at Days 2, 3, 4, and 5 pi were 22%, 15%, 16%, and 42%, respectively, whereas the percentages of polyploidy (i.e., all nuclei in an embryo were analyzed and were polyploid) were 5%, 13%, 3%, and 0%, respectively. In conclusion, numerical chromosome aberrations were detected as early as Day 2 pi. The development of polyploid embryos is slow and is apparently arrested during the third cell cycle, whereas the mixoploid embryos seem to continue development.     

Ovine fetal adrenal maturation at term and during fetal ACTH administration: evidence that the modulating effect of cortisol may involve cAMP

Exogenous ACTH1-24 promotes adrenal maturation in fetal sheep, and this effect appears to be modulated in part by cortisol (Challis et al. 1985). We have examined whether similar changes in adrenal metabolism of progesterone occur with ACTH-induced labour as at spontaneous term and whether the site of cortisol modulation is on adrenal steroidogenesis or at the level of cAMP generation. Chronically catheterized fetal sheep were infused in utero for 100 h between days 127 and 131 of pregnancy with P-ACTH, P-ACTH + metopirone, P-ACTH + metopirone + cortisol, or saline. After 100 h the metabolism of [3H]progesterone was measured in adrenal homogenates. Similar incubations were performed with adrenal tissue from fetal sheep at day 130 of pregnancy and at spontaneous labour. In the treatment groups of sheep, cAMP output by dispersed adrenal cells in response to ACTH added in vitro was also determined. Similar qualitative patterns of [3H]progesterone metabolism were found in adrenal homogenates after in vivo ACTH or at term. At both times there was an increase in cortisol and in total 17 alpha-hydroxycorticosteroid accumulation and also evidence for increased activity of 11 beta-hydroxylase enzyme. The formation of total 17 alpha-hydroxycorticosteroids was not affected significantly by concurrent treatment in vivo with metopirone +/- cortisol. The accumulation of cAMP in vitro was increased after in vivo ACTH, attenuated after ACTH + metopirone, but statistical significance over controls was restored after ACTH + metopirone + cortisol treatment. We conclude that ACTH-induced labour and spontaneous parturition in sheep is associated with qualitatively similar changes in progesterone metabolism by the fetal adrenal gland.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lack of correlation of severity of lung disease with the phosphatidylcholine concentration in fetal lung fluid from premature lambs at 133-136 days gestational age

Fetal lung fluid was collected following tracheotomy at the time of delivery of 40 premature lambs at 133-136 days gestational age. The concentration of phosphatidylcholine and saturated photophatidylcholine in fetal lung fluid was compared with the severity of lung disease of the lambs as assessed after 3 to 10 h of controlled mechanical ventilation with only peak inspiratory pressures varied to control the PCO2 values. Phosphatidylcholine concentration in fetal lung fluid did not correlate with the peak inspiratory pressures needed to ventilate the lambs, total lung compliance values, or the surfactant phosphatidylcholine pool sizes measured by alveolar wash after sacrifice. The ratio of saturated to total phosphatidylcholine was constant (0.55 +/- 0.02) and independent of concentration of phosphatidylcholine in the fetal lung fluid. The fetal lung fluid contained only about 0.7% of the final surfactant phosphatidylcholine pool released by the lambs to the alveoli after birth. Within a narrow gestational age range characterized by lung disease of widely varying severity, the phosphatidylcholine concentrations in fetal lung fluid were not predictive of the severity of lung disease.     

Distribution of lectin receptors in postimplantation mouse embryos at 6-8 days gestation

We have examined the pattern of binding of eleven lectins--BSL-II, WGA, LPA, Con A, DBA, SBA, LTA, UEA-I, MPA, PNA, and RCA-I, with specificity for a range of saccharides, to postimplantation mouse embryos from 6 to 8 days of gestation. The lectins were used to stain sections of ethanol-fixed paraffin-embedded and formaldehyde-fixed gelatin-embedded embryonic material. Our observations reveal a complex pattern of lectin binding to both cell surfaces and cytoplasm. Many of the lectins bind particularly to the outer surface of visceral endoderm (e.g., DBA, WGA, SBA, and RCA-I) and to the surface of the proamniotic cavity (e.g., RCA-I, PNA, and WGA). In the newly formed mesenchyme of primitive-streak-stage embryos, galactose and N-Ac-neuraminic acid are present but lectins with specificity for other sugars either did not bind to the cells or bound only in small amounts.     

Increased cardiovascular and renal risk is associated with low nephron endowment in aged females: an ovine model of fetal unilateral nephrectomy

Previously we have shown that ovariectomised (OVX) female sheep have reduced renal function and elevated blood pressure from 6 months of age following fetal uninephrectomy (uni-x) at 100 days of gestation (term  = 150 days). In the current study we examined if in intact female sheep the onset of decline in renal function and elevation in blood pressure was prevented. Studies were performed at 1 year, 2 and 5 years of age. Following fetal uni-x at 100 days, intact female sheep had ∼30% reduction in glomerular filtration rate (GFR) at 1 year, which did not exacerbate with age (P_treatment = 0.0001, P_age = 0.7). In contrast renal blood flow was similar between the treatment groups at 1 year of age but had declined in the uni-x animals at 5 years of age (P_{treatment × age} = 0.046). Interestingly, intact uni-x sheep did not develop elevations in arterial pressure until 2 years of age. Furthermore, uni-x animals had a similar capacity to respond to a cardiac challenge at 1 year and 2 years of age, however, cardiac functional reserve was significantly reduced compared to sham group at 5 years of age. Uni-x animals exhibited an increase in left ventricular dimensions at 5 years of age compared to the sham animals and compared to 2 years of age (P_treatment<0.001, P_{treatment × age}<0.001). In conclusion, the onset of renal dysfunction preceded the onset of hypertension in intact female uni-x sheep. Furthermore, this study showed that the intact females are protected from the impact of a reduced nephron endowment on cardiovascular health early in life as opposed to our findings in young male sheep and OVX uni-x female sheep. However, with ageing this protection is lost as evidenced by presence of left ventricular hypertrophy and impaired cardiac function in 5 year old uni-x female sheep.     

MiR-128-3p accelerates cardiovascular calcification and insulin resistance through ISL1-dependent Wnt pathway in type 2 diabetes mellitus rats

Vascular calcification is highly prevalent in patients with type 2 diabetes mellitus (T2DM), one of the most common chronic diseases with high morbidity and mortality. In recent years, microRNAs have been widely reported as potential biomarkers for the diagnosis and treatment of T2DM. We hypothesized that miR-128-3p is associated with cardiovascular calcification and insulin resistance (IR) in rats with T2DM by targeting ISL1 via the Wnt pathway. Microarray analysis was adopted to identify differentially expressed genes related to T2DM. T2DM models were induced in rats. Blood samples from normal and T2DM rats were used to detect islet β-cell function, islet sensitivity, and calcium content. Next, islet tissues were obtained to identify the expression of miR-128-3p, ISL1, and the Wnt signaling pathway- and apoptosis-related genes. Finally, apoptosis of islet β-cells was determined by flow cytometry. Through microarray analysis of GSE27382 and GSE23343, ISL1 was found to be downregulated in T2DM. In blood samples from T2DM rats, basic biochemical indicators, IR, and calcium content were increased, and islet sensitivity and islet β-cell function were decreased. Furthermore, upregulation of miR-128-3p and ISL1 gene silencing promoted the expression of Wnt-1, β-catenin, GSK-3β, and Bax and the phosphorylation of β-catenin and GSK-3β, inhibited c-fos, PDX-1, and Bcl-2 expression, and enhanced cell apoptosis. The key findings of our study demonstrate that miR-128-3p aggravates cardiovascular calcification and IR in T2DM rats by downregulating ISL1 through the activation of the Wnt pathway. Thus, miR-128-3p may serve as a potential target for the treatment of T2DM.     

Prostaglandin production in the umbilical and uterine circulations in pregnant sheep at 129-136 days gestation.

Prostaglandins circulating in the maternal and foetal blood have been implicated in important physiological systems. These functions include foetal adrenal function, maintenance of patency of the ductus arteriosus, regulation of uterine and umbilical circulations, and labor and delivery type myometrial contractions. The placenta is a major site of prostaglandin production in pregnancy. Limited data are available which combine measurements of veno-arterial differences across the uterine and umbilical circulations with blood flow in these circulations to enable calculation of umbilical-placental and utero-placental production rates for the prostaglandins. In chronically instrumented pregnant ewes, between 129 and 136 days of gestation, prostaglandin F2 alpha(PGF2 alpha), 13, 14 dihydro-15-keto prostaglandin F2 alpha (PGFM), prostaglandin E2 (PGE2) were measured in the maternal carotid artery and uterine vein. Foetal PGE2, and 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha) (the major metabolite of prostacyclin) were measured in umbilical venous and foetal descending aorta arterial plasma. Umbilical and uterine blood flow were measured using the diffusion-equilibrium technique. Uterine blood flow was 1693 +/- 137 ml.min-1 (mean +/- SEM); uterine production rates were 480 +/- 88 ng.min-1 for PGF2 alpha, 517 +/- 144 ng.min-1 for PGFM, and 165 +/- 27 ng.min-1 for PGE2. Umbilical blood flow was 147 +/- 17 ml.min-1.kg-1 foetal body weight. Umbilical production rates into the foetal circulation were 11 +/- 2 ng.min-1.kg-1 for PGE2 and 6 +/- 2 ng. ng.min-1.kg-1 foetal body weight for PGI2.     

Increased frequency of dividing cells of a phototrophic species of Cryptophyceae at a frontal structure off the Antarctic Peninsula

The hypothesis that enhanced phytoplankton growth explainedthe increase in phytc-plankton biomass observed at a frontalstructure off the Antarctic Peninsula during the summer of 1993was examined by analysing the phytoplankton cells undergoingmitosis. The frequency of dividing cells (FDC) of an unidentifiedphototrophic species of Cryptophyceae, which dominated phytoplanktonbiomass at the front, varied between 1.2 and 31.6%, with higherpercentages associated with the frontal structure. FDC valuesincreased as phytoplankton biomass increased, suggesting thatactive growth, rather than passive accumulation, was responsiblefor the enhanced phytoplankton biomass observed. The low temperatures(mean ± SE = 1.11 ± 0.52°C) that characterizedthe Antarctic waters sampled imposed an upper limit to the maximumFDC reached by the Cryptophyceae, but the relationship betweenFDC and temperature suggests a clear response of the maximalgrowth rate of this species to small changes in temperature.The stabilization of the water column, resulting in higher lightavailability and heating of the surface water in this frontalarea, appeared to promote the growth of the phototrophic speciesof Cryptophyceae and emphasizes the importance of mesoscaleprocesses as determinants of phytoplankton growth dynamics.     

Increased virulence of Puccinia coronata f. sp.avenae populations through allele frequency changes at multiple putative Avr loci

Pathogen populations are expected to evolve virulence traits in response to resistance deployed in agricultural settings. However, few temporal datasets have been available to characterize this process at the population level. Here, we examined two temporally separated populations of Puccinia coronata f. sp. avenae (Pca), which causes crown rust disease in oat (Avena sativa) sampled from 1990 to 2015. We show that a substantial increase in virulence occurred from 1990 to 2015 and this was associated with a genetic differentiation between populations detected by genome-wide sequencing. We found strong evidence for genetic recombination in these populations, showing the importance of the alternate host in generating genotypic variation through sexual reproduction. However, asexual expansion of some clonal lineages was also observed within years. Genome-wide association analysis identified seven Avr loci associated with virulence towards fifteen Pc resistance genes in oat and suggests that some groups of Pc genes recognize the same pathogen effectors. The temporal shift in virulence patterns in the Pca populations between 1990 and 2015 is associated with changes in allele frequency in these genomic regions. Nucleotide diversity patterns at a single Avr locus corresponding to Pc38, Pc39, Pc55, Pc63, Pc70, and Pc71 showed evidence of a selective sweep associated with the shift to virulence towards these resistance genes in all 2015 collected isolates.