Late Pregnant Animals Research Articles

Worsened Stroke Outcome in a Model of Preeclampsia is Associated With Poor Collateral Flow and Oxidative Stress.

Preeclampsia increases the incidence of maternal stroke, a devastating condition that is on the rise. We investigated stroke outcome in a model of experimental preeclampsia with and without treatment with clinically relevant doses of magnesium sulfate (experimental preeclampsia+MgSO4) compared to normal late-pregnant and nonpregnant rats. Transient middle cerebral artery occlusion was used to induce focal stroke for either 1.5 or 3 hours. Infarct volume and hemorrhagic transformation were determined as measures of stroke outcome. Changes in core middle cerebral artery and collateral flow were measured by dual laser Doppler. The relationship between middle cerebral artery perfusion deficit and infarction was used as a measure of ischemic tolerance. Oxidative stress and endothelial dysfunction were measured by 3-nitrotyrosine and 8-isoprostane, in brain and serum, respectively. Late-pregnant animals had robust collateral flow and greater ischemic tolerance of brain tissue, whereas experimental preeclampsia had greater infarction that was related to poor collateral flow, endothelial dysfunction, and oxidative stress. Importantly, pregnancy appeared preventative of hemorrhagic transformation as it occurred only in nonpregnant animals. MgSO4 did not provide benefit to experimental preeclampsia animals for infarction. Stroke outcome was worse in a model of preeclampsia. As preeclampsia increases the risk of future stroke and cardiovascular disease, it is worth understanding the influence of preeclampsia on the material brain and factors that might potentiate injury both during the index pregnancy and years postpartum.

THE RENIN-ANGIOTENSIN-ALDOSTERONE SYSTEM IN THE PREGNANT RAT

Objective: Preeclampsia is the leading cause of maternal and fetal morbidity and mortality in the United States, affecting ~2–5% of all pregnancies. Women with a history of preeclampsia have a 4-fold increased risk for developing hypertension, 2-fold increased risk for developing stroke, and 2-fold increased risk for developing ischemic heart disease later in life. Although it is known that intact renin-angiotensin-aldosterone system (RAAS) signaling is critical to a healthy pregnancy and that the RAAS is altered in preeclampsia the pathogenic mechanisms are largely unknown and a comprehensive examination of RAAS metabolism and receptor expression is lacking in the literature. The purpose of this study is to understand how the vasculature becomes refractory to the reported high circulating angiotensin II (ANGII) during pregnancy. This work provides basic information needed to understand blood pressure regulation and volume expansion in normal pregnancy, and why it fails to occur in women with preeclampsia, resulting in hypertension and fetal growth restriction. Design and method: We performed AT1 receptor autoradiography in kidneys of virgin (V) and late pregnant (LP) rats. We also performed equilibrium RAAS Fingerprinting in the plasma and kidneys of LP rats and V rats using a new LC/MS technique. Results: Using this new method, ANGII levels were decreased in the plasma and unchanged in the kidney of LP animals. This was surprising as it is inconsistent with historical data, however, other methods of measuring ANGII are problematic and often unreliable. There was a very high Aldosterone/ANGII-ratio (AA2) in both plasma and kidney of LP animals. The AA2-ratio provides information about the adrenal aldosterone-releasing response to ANGII. We also found that AT1R expression is decreased in the kidneys of LP compared to V rats. Conclusions: These results suggest a systemic down regulation of renal ANGII signaling with increased adrenal signaling. Continuing studies are assessing the RAAS signaling cascade to further validate these data, which may revolutionize the way we think about the RAAS in pregnancy, leading to improved treatment of women with preeclampsia.

Oxytocin, Prostaglandin F2α, and Prostaglandin E2 Induced Contractions in Isolated Uterine Smooth Muscle Strips from Non‐pregnant, Late Pregnant, and Postpartum Rats

Objective/IntroductionNumerous studies have investigated uterotonic agents (e.g., oxytocin) in uterine smooth muscle (USM) from non‐pregnant (NP) and late‐pregnant (LP) women and/or animal models of pregnancy. Surprisingly, few studies have investigated USM from the post‐partum period to determine whether USM contractility returns to its pre‐pregnant state. We designed a study in rodents to investigate whether USM contractility to oxytocin (OXY), prostaglandin F2α (PGF2α), and prostaglandin E2 (PGE2) returned to pre‐pregnant values within 30 days postpartum.MethodsStudies were performed in NP (n=12), LP (21d gestation, n=4), 3 days post‐partum (PP3, n=2), and 30 days post‐partum (PP30, n=4) Sprague Dawley rats. Uterine horns were cleaned of connective tissue and cut along the longitudinal axis allowing isolation of USM (width: 2mm, length: 4±1mm) oriented in the longitudinal muscle (LM) or circular muscle (CM) direction. CM or LM strips were mounted in tissue baths containing Krebs and equilibrated @ 37°C for 60 min prior to starting one of the two following protocols.Protocol 1 identified the optimal resting tension (in grams: 0.25, 0.50, 1.0, and 1.5) to measure spontaneous contraction (SCA), K+‐induced contraction (60mM K+), and cumulative OXY‐induced contraction (10‐11 to 10‐7) in CM and LM of NP rats (N=4‐6).Protocol 2 was designed assess SCA, K+‐induced contraction, cumulative concentration‐response relationships to OXY (10‐11 to 10‐7), PGF2α (10‐8 to 10‐4), or PGE2 (10‐8 to 10‐4) in CM and LM, of NP, LP, PP3, and PP30 rats (N=2‐6).Statistical comparisons were made using either a t‐test or two‐way ANOVA, and considered statistically different at P<0.05.ResultsProtocol 1‐ LM SCA was greater than CM SCA at all resting tensions. K+‐induced constrictions in LM and CM were similar at each resting tension. OXY‐induced contraction in LM was generally greater than CM (P<0.05 at 0.25g, 0.5g, and 1.5g tension, comparable at 0.5, 1.0, and 1.5g tension).Protocol 2 ‐ SCA in NP, LM was greater than NP, CM (P<0.05). There was no difference in SCA in CM vs LM in USM from LP, PP3, and PP30 animals. K+‐induced constrictions in CM and LM were similar in USM from NP, LP, PP3 and PP30 animals. OXY‐induced contractions were greater in LM than CM for all pregnancy states (P<0.05). PGE2 contractions were greater in LM vs CM in USM from NP rats (P<0.05), but were similar in USM from LP, PP3, and PP30 animals. In contrast, PGF2α‐contractions were similar in LM and CM for all pregnancy states.Summary/ConclusionOur preliminary data suggests that uterotonic agents be assessed at either 0.5g or 1.0g resting tension. In addition, uterotonic agents exhibit significantly greater contraction in longitudinal vs circular orientation. Finally, contractility to uterotonic agents returns to pre‐pregnant values by PP30.

DETECTION OF SUBCLINICAL PREGNANCY TOXEMIA AND EVALUATION OF GLYCAEMIC STATUS IN MALABARI DOES

Aim: The aim of the study was to screen Malabari does for subclinical pregnancy toxemia and to evaluate their glycaemic status. Materials and Methods: This study consist of 4 groups; Group I early pregnant does (n = 7), Group II late pregnant does with one fetus (n = 6), Group III late pregnant does with two fetuses (n = 8), and Group IV nonpregnant does (n = 8). The pregnancy was confirmed by 3.5 Megahertz B-mode ultrasonography. The concentration of beta-hydroxybutyrate (BHB) and glucose was analyzed in the blood samples. Results and Discussion: Significant increase in serum BHB concentration was found in Groups I and II when compared to control group. Group III showed highly significant increase when compared to control group. Among the four groups selected, late pregnant animals with two fetuses had a BHB level of 0.81 ± 0.10 mmol/l, which shows that those animals were in subclinical pregnancy toxemic stage. A highly significant decrease in plasma glucose concentration was found in Group I, Group II, and Group III; compared to controls. Hypoglycemia was seen in all the three groups studied. Conclusion: Hyperketonemia not only represents a disease sign but rather acts in a multifunctional way in promoting development of pregnancy toxemia.

Effect of Pregnancy on Autoregulation of Cerebral Blood Flow in Anterior Versus Posterior Cerebrum

Severe preeclampsia and eclampsia are associated with brain edema that forms preferentially in the posterior cerebral cortex possibly because of decreased sympathetic innervation of posterior cerebral arteries and less effective autoregulation during acute hypertension. In the present study, we examined the effect of pregnancy on the effectiveness of cerebral blood flow autoregulation using laser Doppler flowmetry and edema formation by wet:dry weight in acute hypertension induced by phenylephrine infusion in the anterior and posterior cerebrum from nonpregnant (n=8) and late-pregnant (n=6) Sprague-Dawley rats. In addition, we compared the effect of pregnancy on sympathetic innervation by tyrosine hydroxylase staining of posterior and middle cerebral arteries (n=5-6 per group) and endothelial and neuronal NO synthase expression using quantitative PCR (n=3 per group). In nonpregnant animals, there was no difference in autoregulation between the anterior and posterior cerebrum. However, in late-pregnant animals, the threshold of cerebral blood flow autoregulation was shifted to lower pressures in the posterior cerebrum, which was associated with increased neuronal NO synthase expression in the posterior cerebral cortex versus anterior. Compared with the nonpregnant state, pregnancy increased the threshold of autoregulation in both brain regions that was related to decreased expression of endothelial NO synthase. Lastly, acute hypertension during pregnancy caused greater edema formation in both brain cortices that was not attributed to changes in sympathetic innervation. These findings suggest that, although pregnancy shifted the cerebral blood flow autoregulatory curve to higher pressures in both the anterior and posterior cortices, it did not protect from edema during acute hypertension.

Cardiac vulnerability to ischemia/reperfusion injury drastically increases in late pregnancy

Although the murine late pregnant (LP) heart is speculated to be a better functioning heart during physiological conditions, the susceptibility of LP hearts to I/R injury is still unknown. The aims of this study were to investigate the cardiac vulnerability of LP rodents to ischemia/reperfusion (I/R) injury and to explore its underlying mechanisms. In vivo female rat hearts [non-pregnant (NP) or LP] or ex vivo Langendorff-perfused mouse hearts were subjected to I/R. The infarct size was approximately fourfold larger in LP animals compared with NP both in vivo and ex vivo. The heart functional recovery was extremely poor in LP mice compared with NP (~10% recovery in LP vs. 80% recovery in NP at the end of reperfusion, P < 0.01). Interestingly, the poor functional recovery and the larger infarct size in LP were partially restored one day post-partum and almost fully restored 1 week post-partum to their corresponding NP levels. Mitochondrial respiratory function and the threshold for opening of the mitochondrial permeability transition pore were significantly lower in LP compared with NP when they both were subjected to myocardial I/R injury [Respiratory control ratio = 1.9 ± 0.1 vs. 4.0 ± 0.5 in NP, P < 0.05; calcium retention capacity (CRC) = 167 ± 10 vs. 233 ± 18 nmol/mg protein in NP, P < 0.01]. Cardiac reactive oxygen species (ROS) generation, as well mitochondrial superoxide production, was approximately twofold higher in LP compared with NP following I/R. The phosphorylation levels of Akt, ERK1/2, and STAT3, but not GSK3β, were significantly reduced in the hearts from LP subjected to I/R. In conclusion, increased mitochondrial ROS generation, decreased CRC as well as impaired activation of Akt/ERK/STAT3 at reperfusion are the possible underlying mechanisms for higher vulnerability of LP hearts to I/R.

Cardiac Vulnerability to Ischemia/Reperfusion Injury Drastically Increases in Late Pregnancy

Although the murine late pregnant (LP) heart is speculated to be a better functioning heart during physiological conditions, the susceptibility of LP hearts to I/R injury is still unknown. The aims of this study were to investigate the cardiac vulnerability of LP rodents to ischemia/reperfusion (I/R) injury and to explore its underlying mechanisms. In vivo female rat hearts [non-pregnant (NP) or LP] or ex vivo Langendorff-perfused mouse hearts were subjected to I/R. The infarct size was approximately fourfold larger in LP animals compared with NP both in vivo and ex vivo. The heart functional recovery was extremely poor in LP mice compared with NP (~10 % recovery in LP vs. 80 % recovery in NP at the end of reperfusion, P < 0.01). Interestingly, the poor functional recovery and the larger infarct size in LP were partially restored one day post-partum and almost fully restored 1 week post-partum to their corresponding NP levels. Mitochondrial respiratory function and the threshold for opening of the mitochondrial permeability transition pore were significantly lower in LP compared with NP when they both were subjected to myocardial I/R injury [Respiratory control ratio = 1.9 ± 0.1 vs. 4.0 ± 0.5 in NP, P < 0.05; calcium retention capacity (CRC) = 167 ± 10 vs. 233 ± 18 nmol/mg protein in NP, P < 0.01]. Cardiac reactive oxygen species (ROS) generation, as well mitochondrial superoxide production, was approximately twofold higher in LP compared with NP following I/R. The phosphorylation levels of Akt, ERK1/2, and STAT3, but not GSK3β, were significantly reduced in the hearts from LP subjected to I/R. In conclusion, increased mitochondrial ROS generation, decreased CRC as well as impaired activation of Akt/ERK/STAT3 at reperfusion are the possible underlying mechanisms for higher vulnerability of LP hearts to I/R.

Effects of parturition on immediate early gene protein expression within the brains of female rats

The mother–infant interaction that occurs immediately postpartum period has important consequences including changes in protein expression in the astrocytes within cingulate cortex area 2 (Cg2). Because increased expression of basic fibroblast growth factor (bFGF) and glial fibrillary acidic protein (GFAP) has been associated with increased neuronal activity we used immediate early gene expression to evaluate the effects of parturition plus interaction with pups during and immediately after delivery to investigate patterns of neural activation in a number of brain areas including Cg2. In the first experiment, we compared levels of immunoreactivity of two immediate early genes: c-Fos and egr-1, among three groups of animals: Cycling, Late Pregnant, and rats perfused 1.5 h postpartum. The average number of Fos and egr-1-ir cells/section were calculated for the Parietal Cortex (ParCx), nucleus accumbens (NAc), medial amygdala (MEA), medial preoptic area (MPOA), supraoptic area (SON) and Cg2. In a second experiment, Fos-ir induction was compared between cycling rats and rats at hour 3 postpartum. Contrary to our hypothesis no changes in expression of either immediate early gene were observed in Cg2. In Experiment 1 however, an upregulation in both early genes was seen in the ParCx, and, of Fos-ir only in the MPOA of postpartum animals. A decrease in Fos-ir was seen in the NAc of late pregnant animals and no significant differences were seen in either immediate early gene expression within the MEA. Interestingly, areas that showed a significant increase in neuronal activation project to Cg2. The possibility of an interaction between these changes and glial cells in Cg2 is discussed.

Calorimetric investigation of rat myometrium as a function of gestational age

The aim of the present work was the characterization of the myometrial samples of healthy control and chronic inflammation-exposed rats as a function of gestational stage by means of differential scanning calorimetry (DSC). Non-pregnant, mid-pregnant (day 14) and late-pregnant (day 21) animals were included. The spontaneous, KCl-stimulated as well as maximal motor activity was additionally recorded in vitro. Calorimetric enthalpy (Δ H) normalised on wet mass significantly increased as pregnancy progressed in case of non-inflamed uteri. Inflammation resulted in a significant decrease in Δ H/ m of samples from non-pregnant till pregnant rats. Maximal contractility increased by inflammation in all tested stages of gestation. Our present results indicate that DSC can be considered as a useful tool for the investigation of the uterine function modified by gestation or a pathological condition.

Pregnancy Reverses Hypertensive Remodeling of Cerebral Arteries

Previous studies have shown that pregnancy prevents hypertensive remodeling of cerebral arteries. In the present study, we sought to determine whether pregnancy could reverse preexisting remodeling. Nonpregnant virgin Sprague-Dawley rats were treated with the NO synthase inhibitor nitro-l-arginine (0.5 g/L in drinking water) for 2 weeks before mating, after which treatment continued until late gestation for a total of 5 weeks. Pregnant animals with preexisting hypertension (n=6) were compared with nonpregnant animals that were treated with nitro-l-arginine for either 2 (n=8) or 5 (n=9) weeks and compared with nontreated controls (n=8). Blood pressure, passive and active diameters, wall thickness, media thickness, and passive distensibility of cerebral arteries were compared between groups. Treatment with nitro-l-arginine caused a significant increase in mean arterial pressure in all of the groups compared with controls that was sustained for the entire study: 103+/-3 versus 137+/-2, 141+/-4, and 140+/-7 mm Hg (P<0.01). Both 2 and 5 weeks of hypertension caused inward eutrophic remodeling in nonpregnant animals, characterized by decreased inner and outer lumen diameters and no change in media thickness. Pregnancy reversed this remodeling, because late-pregnant animals with preexisting hypertension had inner and outer diameters similar to controls. Passive distensibility was significantly less, and active myogenic tone increased in all of the hypertensive animals, independent of pregnancy. These results demonstrate that pregnancy reverses preexisting hypertensive remodeling of cerebral arteries without a decrease in blood pressure. This reversal of protective remodeling during hypertension in pregnancy may be detrimental by lowering the upper limit of autoregulation, whereas blood pressure remains elevated.

Effects of streptozotocin-induced diabetes on the uterine adrenergic nerve function in pregnant rats: A superfusion study

Pregnancy-induced diabetes mellitus poses one of the greatest challenges in obstetrical practice. The direct action of diabetes on the myometrial adrenergic functions has not been completely characterized. Accordingly, the present study relates to the impact of experimentally induced diabetes on the presynaptic functions of the rat uterus in relation to gestational age. Experiments were carried out on non-pregnant, early-pregnant (day 7), middle-pregnant (day 14) and late-pregnant (day 21) animals. Diabetes was induced with streptozotocin (60 mg/kg, i.v.) in virgin female or early-pregnant animals (on day 2 for the day 7 experiments and on day 5 for the experiments on the middle and late-pregnant animals). Myometrial samples were utilized for superfusion experiments. After saturation, [ 3H]noradrenaline perfusate fractions were collected and electric field stimulation was applied to determine the amount of transmitter liberated. Additionally, the total uptake capacity of each sample was assayed. Experimental diabetes decreases the transmitter uptake capacity both in virgin rats and at all stages of pregnancy. In early pregnancy (on day 7), this limitation in uptake is obvious as early as 5 days after the induction of diabetes. In non-pregnant animals, the electrically stimulated transmitter release is inhibited substantially, a similar decrease being observed only at mid-pregnancy (day 14). The present superfusion study proves that experimental diabetes depresses the presynaptic adrenergic functions (both the transmitter uptake and the stimulated release) in the myometrium of the rat. Since the effect of diabetes on the uptake capacity can be detected earlier than for generally accepted markers of peripheral neuropathies, superfusion can be suggested as a sensitive and reliable approach for investigations of hyperglycaemia-related functional deteriorations. We speculate that diabetes-induced functional deterioration of the adrenergic nerves could partially explain the anomalies of the reproductive functions found in diabetic patients if a similar mechanism is operative in humans.

Cerebral Blood Flow Autoregulation and Edema Formation During Pregnancy in Anesthetized Rats

Eclampsia is considered a form of hypertensive encephalopathy in which an acute elevation in blood pressure causes autoregulatory breakthrough, blood-brain barrier disruption, and edema formation. We hypothesized that pregnancy predisposes the brain to eclampsia by lowering the pressure of autoregulatory breakthrough and enhancing cerebral edema formation. Because NO production is increased in pregnancy, we also investigated the role of NO in modulating autoregulation. Cerebral blood flow autoregulation was determined by phenylephrine infusion and laser Doppler flowmetry. Four groups were studied: untreated nonpregnant (n=7) and late-pregnant (days 19 to 21; n=8) Sprague-Dawley rats and nonpregnant (n=8) and late-pregnant (n=8) animals treated with an NO synthase inhibitor (N(G)-nitro-l-arginine methyl ester; 0.5 to 0.7 g/L). Brain water content and blood-brain barrier permeability to sodium fluorescein were determined after breakthrough. Pregnancy caused no change in autoregulation or the pressure of breakthrough. However, treatment with the NO synthase inhibitor significantly increased the pressure of autoregulatory breakthrough (nonpregnant: 183.6+/-3.0 mm Hg versus 212.0+/-2.8 mm Hg, P<0.05; late-pregnant: 180.8+/-3.2 mm Hg versus 209.3+/-4.7 mm Hg, P<0.05). After autoregulatory breakthrough, only late-pregnant animals showed a significant increase in cerebral edema formation, which was attenuated by NO synthase inhibition. There was no difference in blood-brain barrier permeability between nonpregnant and late-pregnant animals in response to acute hypertension, suggesting that pregnancy may predispose the brain to eclampsia by increasing cerebral edema through increased hydraulic conductivity.

Pregnancy prevents hypertensive remodeling and decreases myogenic reactivity in posterior cerebral arteries from Dahl salt-sensitive rats: a role in eclampsia?

Previous studies have demonstrated that pregnancy prevents protective hypertension-induced remodeling of cerebral arteries using nitric oxide synthase (NOS) inhibition to raise mean arterial pressure (MAP). In the present study, we investigated whether this effect of pregnancy was specific to NOS inhibition by using the Dahl salt-sensitive (SS) rat as a model of hypertension. Nonpregnant (n = 16) and late-pregnant (n = 17) Dahl SS rats were fed either a high-salt diet (8% NaCl) to raise blood pressure or a low-salt diet (<0.7% NaCl). Third-order posterior cerebral arteries were isolated and pressurized in an arteriograph chamber to measure active responses to pressure and passive remodeling. Several vessels from each group were stained for protein gene product 9.5 to determine perivascular nerve density. Blood pressure was elevated in both groups on high salt. The elevated MAP was associated with significantly smaller active and passive diameters (P < 0.05) and inward remodeling in the nonpregnant hypertensive group only. Whereas no structural changes were observed in the late-pregnant hypertensive animals, both late-pregnant groups had diminished myogenic reactivity (P < 0.05). Nerve density in both the late-pregnant groups was significantly greater when compared with the nonpregnant groups, suggesting that pregnancy has a trophic influence on perivascular innervation of the posterior cerebral artery. However, hypertension lowered the nerve density in both nonpregnant and late-pregnant animals. It therefore appears that pregnancy has an overall effect to prevent hypertension-induced remodeling regardless of the mode of hypertension. This effect may predispose the brain to autoregulatory breakthrough, hyperperfusion, and eclampsia when MAP is elevated.

Effect of pregnancy and 5alpha-pregnan-3alpha-ol-20-one on atrial receptor afferent discharge in rats.

The atrial volume reflex is attenuated in pregnancy. This may be mimicked by chronic administration of 5alpha-pregnan-3alpha-ol-20-one (pregnan). We investigated whether afferent output from sensory receptors may be suppressed at this time. Vagal afferent nerve activity was measured during discrete localized stimulation of the atrial volume receptors by inflation of a balloon at the superior vena caval-right atrial junction. The receptors were classified as high- (HF) or low- (LF) frequency subtypes on the basis of their response to graded atrial distension. Although both HF (regression coefficient = 0.50 +/- 0.11 Hz/microl, r(2) = 0.47, P < 0.001) and LF (regression coefficient = 0.03 +/- 0.05 Hz/microl, r(2) = 0.009, P = 0.613) subtypes could be identified in virgin rats, only LF (regression coefficient = 0.09 +/- 0.05 Hz/microl, r(2) = 0.044, P = 0.099) receptors were found in late-pregnant animals. Similarly, in virgin rats treated chronically with pregnan (500 microg/24 h for 2 days), only LF receptors were identified (regression coefficient = -0.004 +/- 0.078 Hz/microl, r(2) = 0.000, P = 0.962), whereas both subtypes were present in the vehicle-treated animals (HF regression coefficient = 0.626 +/- 0.255 Hz/microl, r(2) = 0.317, P = 0.029; LF regression coefficient = -0.012 +/- 0.071 Hz/microl, r(2) = 0.002, P = 0.866). By contrast, acute intracardiac pregnan (2.6 microg/kg) did not alter vagal afferent nerve activity. In conclusion, stretch-induced discharge of high-frequency atrial receptors is suppressed during pregnancy, whereas that of low-frequency receptors is preserved. This effect may be mimicked by chronic, but not acute, pregnan. We propose that, during pregnancy, pregnan alters the transducer properties of the atrial volume receptors, thus allowing blood volume to increase.

Purinoceptor-mediated contractility of the perfused uterine vasculature of the guinea-pig: influence of oestradiol and pregnancy.

1. The effects of ATP, the stable ATP analogues alpha,beta-methylene ATP (alpha,beta-mATP), 2-methylthioATP (2meSATP) and adenosine tetraphosphate (ATP4), the pyrimidine nucleotide uridine 5'-triphosphate (UTP) and the alpha1-adrenoceptor agonist phenylephrine were examined on the isolated perfused uterine vasculature of dioestrous, oestradiol-treated, dexamethasone-treated and late-pregnant guinea-pigs. 2. The alpha1-adrenoceptor agonist phenylephrine elicited concentration-dependent vasoconstriction from preparations of perfused uterine vasculature from dioestrous, estradiol-treated and late-pregnant guinea-pigs. The mean maximal response to phenylephrine was unaffected by treatment of dioestrus guinea-pigs with oestradiol or dexamethasone, but was reduced in preparations from late-pregnant animals. 3. In perfused uterine arteries from dioestrous animals, the pyrimidine UTP, but not ATP4 and ATP, elicited vasoconstrictor responses. In preparations from oestradiol-treated animals, all three agonists elicited vasoconstriction, with a rank order of potency of ATP4 = UTP >> ATP, whereas in preparations from late-pregnant animals this order of potency was ATP4 >> UTP = ATP. In preparations from dexamethasone-treated animals, the vasoconstriction was similar to that seen in dioestrous animals. Vasoconstrictor responses to ATP4 were significantly greater in preparations of uterine vasculature from oestradiol-treated and pregnant animals than in preparations from dioestrous animals or dexamethasone-treated animals. 4. In preparations from dioestrous, oestradiol-treated, pregnant and dexamethasone-treated animals, alpha,beta-mATP was approximately two to three orders of magnitude more potent than 2meSATP. Compared with preparations from dioestrous animals, the maximal responses to alpha,beta-mATP were significantly greater in tissues from oestradiol-treated and pregnant animals. In preparations from dioestrous animals, the P2 purinoceptor antagonist suramin (100 micro mol/L) inhibited the responses to alpha,beta-mATP, but not those to ATP4. 5. The present study has demonstrated that pregnancy, but not the steroid treatment of dioestrous guinea-pigs with oestradiol or dexamethasone, reduces the sensitivity of the guinea-pig isolated perfused uterine vasculature to phenylephrine. In contrast, preparations from pregnant or oestradiol-treated guinea-pigs respond to ATP4 and to alpha,beta-mATP with significantly greater constrictions than those of dioestrous or dexamethasone-treated animals. These data indicate that the sensitivity of the uterine vasculature to adrenoceptor and purinoceptor agonists is differentially regulated by oestradiol and pregnancy, but not by the synthetic glucocorticoid dexamethasone.

Fat accumulation in the rat during early pregnancy is modulated by enhanced insulin responsiveness

Insulin sensitivity has been implicated in the variation of fat accumulation in early gestation by as-yet-unknown mechanisms. In the present study, we analyzed the insulin sensitivity of lipolysis and lipogenesis in lumbar adipocytes from rats at 0, 7, 14, and 20 days of gestation. In adipocytes of 7-day pregnant rats, we found a twofold decrease in both beta-agonist (isoproterenol and BRL-37344)-stimulated lipolysis and beta3-adrenoceptor protein but not in lipolysis initiated by forskolin or isobutylmethylxanthine, suggesting a modification of the lipolytic pathway at the receptor level. Whereas adipocytes from 7-day pregnant rats showed a twofold increase in fatty acid synthesis from glucose, those from 20-day pregnant animals displayed a decreased lipogenic activity. Insulin responsiveness of the lipolytic and lipogenic pathways was analyzed by dose-response experiments, giving evidence for the involvement of improved insulin responsiveness in the enhanced lipogenic and reduced lipolytic activities of adipocytes in early pregnancy. In contrast, insulin resistance is responsible for lower antilipolytic and lipogenic actions of insulin in late pregnant animals. In conclusion, the present study shows that enhanced adipose tissue insulin responsiveness during early pregnancy contributes to maternal fat accumulation, whereas decreased insulin responsiveness during late gestation modulates fat breakdown.

Effects of nitric oxide donors on non-pregnant and pregnant rat uterine and aortic smooth muscle.

To compare the effects of nitric oxide (NO) donors, diethylamine/nitric oxide (DEA/NO) and nitroglycerin (NTG), on isolated uterine and aortic tissues from non-pregnant, mid and late pregnant rats. The uterus and thoracic aorta were obtained from non-pregnant (estrous cycle) and pregnant Sprague-Dawley rats on day 14 and day 21. The uterine and aortic rings were incubated in organ chambers filled with Krebs-Henseleit solution bubbled with 5% CO2 in air for isometric tension recordings. Cumulative concentration-response relationships to DEA/NO and NTG were obtained in the aortic rings contracted with phenylephrine and in spontaneously contracting uterine rings. The sensitivity and the maximal inhibitory effects of DEA/NO did not differ in aortic tissues of any group. DEA/NO-induced Maximal inhibition of spontaneous contractions of uterine tissues from mid-pregnant rats was greater (although not significantly) than in the tissues from non-pregnant animals (with similar sensitivity), but it was significantly depressed in tissues from late pregnant rats. The sensitivity to and maximal inhibitory effects of NTG were less in aortic tissues from late pregnant versus mid-pregnant and non-pregnant rats. In uterine tissues from late pregnant rats the effect of NTG was negligible. The inhibitory action of both NO donors was much more pronounced in aortic versus uterine tissues. Uterine smooth muscle is less sensitive than vascular smooth muscle to NO. Uterine smooth muscle from late pregnant animals demonstrates refractoriness to both DEA/NO and NTG, while vascular smooth muscle from late pregnant animals demonstrates refractoriness to NTG, but not to DEA/NO.

Differential induction of transcription factors and expression of milk protein genes by prolactin and growth hormone in the mammary gland of rabbits.

Previously we demonstrated that administration of lactogenic hormones - prolactin (PRL) and growth hormone (GH) - to pregnant rabbits differentially induces expression of casein and whey proteins in the mammary gland. Now we extend these observations to transcription factors (TFs) that are responsive for differential induction of milk protein genes. Analysis of correlation between the number of putative TF binding sites in 5'-upstream sequences and the levels of induction of milk protein genes allowed preselection of the TFs involved. An electrophoretic mobility shift assay with nuclear proteins derived from rabbit mammary glands showed changes in the patterns of Stat5, MAF, NF1 and Oct1 DNA-protein binding during progression of pregnancy and transition to lactation. Administration of lactogenic hormones - PRL or GH - to early-pregnant rabbits induced DNA-protein complexes similar to those formed by nuclear proteins from the mammary glands of lactating (Stat5, MAF, NF1) or late-pregnant (Oct1) animals. Induction of milk protein genes by PRL was several-fold greater than that by GH. However, PRL and GH similarly induced MAF DNA-protein complexes, thus suggesting that the amount of MAF factor in the mammary gland can be limiting for expression of these genes. Our study for the first time provided the evidence that in the mammary gland both PRL and GH can induce DNA-binding activity of transcription factors other than Stats.

Epidemiology of Trypanosoma evansi infection in crossbred dairy cattle in Malaysia.

An investigation into the epidemiology of Trypansoma evansi infection in crossbred dairy cattle was conducted for a period of 12 months on a dairy cattle farm in Penninsular Malaysia. The prevalence of parasitaemia was highest in lactating animals (13.4%), followed by those in the dry herd (8.8%), late pregnant animals (8.1%), early pregnant animals (4.7%), calves (0.3%) and heifers (0.2%). The prevalence of antigenaemia was highest in the lactating animals (54.7%), followed by that in dry animals (53.7%), heifers (51.1%), late pregnant animals (47.7%), early pregnant animals (46.5%) and calves (24.2%).

Nitric oxide modulates placental prostanoid production from late pregnant non-insulin-dependent diabetic rat

Severe reproductive dysfunction has been described in non-insulin-dependent diabetes mellitus (NIDDM), correlated with high glucose levels in the plasma. We have characterized an abnormal prostanoid profile in tissues from NIDDM rats, and a tight correlation between nitric oxide (NO) levels and prostaglandin production. Likewise, we have determined that parturition is delayed in NIDDM rats compared to control animals. In order to characterize the events which precede delayed parturition in NIDDM rats, we evaluate (a) the arachidonic acid (AA) conversion in placental tissue obtained from control (day 21 and 22) and NIDDM (day 21, 22 and 23) late pregnant rats into prostaglandin E 2 (PGE 2) and F 2α (PGF 2α), thromboxane B 2 (TXB 2) and 6-keto-prostaglandin F 1α (6-keto-PGF 1α), and (b) NO synthase (NOS) activity in control and NIDDM late pregnant animals. Placental arachidonate conversion from control rats into different prostanoids, namely PGE 2, PGF 2α, and TXB 2, is higher in day 22 than in day 21, and radioconversion from diabetic rats into PGE 2, PGF 2α, TXB 2 and 6-keto-PGF 1α on day 23 is higher than in day 21 and 22. 6-keto-PGF 1α is lower and TXB 2 is higher in diabetic tissues than in control. Placental AA conversion of control diabetic tissues on the day of delivery is decreased by N G monomethyl-L-arginine (LNMMA) (600 mM), a well known NOS inhibitor, while prostanoid production remains unaltered on previous days. NOS activity is higher in control on day 22 when compared to day 21, and in diabetic on day 23 when compared to day 22 of pregnancy. We conclude that elevated NO placental levels are observed in control (day 22) and NIDDM (day 23) rats, and may increase placental prostaglandin production on the day of delivery.