Mechanisms involved in calcitonin gene-related Peptide-induced relaxation in pregnant rat uterine artery

Human and rodent studies have demonstrated that calcitonin gene-related peptide (CGRP), a potent vasodilator, relaxes uterine tissue during pregnancy but not during labor. The vascular sensitivity to CGRP is enhanced during pregnancy, compared to nonpregnant human uterine arteries. In the present study, we hypothesized that uterine artery relaxation effects of CGRP are enhanced in pregnant rats compared to nonpregnant diestrus rats (NP-DE) and that several secondary messenger systems are involved in this process. We also hypothesized that the expression of CGRP-A receptor components, calcitonin receptor-like receptor (CRLR), receptor activity-modifying protein (RAMP1), and CGRP-B receptors are greater in pregnant rats. For vascular relaxation studies, uterine arteries from either NP-DE or Day 18 pregnant rats were isolated, and responsiveness of the vessels to CGRP was examined with a small vessel myograph. CGRP-A and CGRP-B receptor expressions were assessed by RT-PCR and Western immunoblotting, respectively. CGRP (10(-10)--10(-7) M) produced a concentration-dependent relaxation of norepinephrine-induced contractions in both NP-DE and Day 18 pregnant rat uterine arteries. Pregnancy increased the vasodilator sensitivity to CGRP significantly (P < 0.05) compared to NP-DE rats. CGRP receptor antagonist, CGRP8-37, inhibited CGRP-induced relaxation of pregnant uterine arteries. The CGRP-induced relaxation was not affected by NG-nitro-l-arginine methyl ester (L-NAME) (nitric oxide inhibitor, 10(-4) M) but was significantly (P < 0.05) attenuated by inhibitors of guanylate cyclase (ODQ, 10(-5) M) and adenylate cyclase (SQ 22536, 10(-5) M). CGRP-induced vasorelaxation was significantly (P < 0.05) attenuated by potassium channel blockers KATP (glybenclamide, 10(-5) M) and K(CA) (tetraethylammonium, 10(-3) M). The expression of CRLR and RAMP1 was significantly (P < 0.05) elevated during pregnancy compared to nonpregnant diestrus state (NP-DE). However, CGRP-B receptor proteins in uterine arteries were not altered with pregnancy compared to those of NP-DE. These studies suggest that CGRP-induced increases in uterine artery relaxation may play a role in regulating blood flow to the uterus during pregnancy and, therefore, in fetal growth and survival.     

Estrogen-induced disruption of neonatal porcine uterine development alters adult uterine function

In the pig, estradiol-17beta valerate (EV) exposure from birth (Postnatal Day [PND] 0) disrupts estrogen receptor-alpha (ER)-dependent uterine development and increases embryo mortality in adults. To determine effects of neonatal EV exposure on adult uterine morphology and function, 36 gilts received corn oil (CO) or EV from PND 0 to PND 13. Cyclic and pregnant (PX) adults from each treatment group were hysterectomized on Day 12 after estrus/mating. Treatment and pregnancy effects were determined for uterine weight and horn volume, uterine luminal fluid (ULF) protein and estradiol content, endometrial incorporation of 3H-leucine (3H-Leu) into nondialyzable product, and endometrial mRNA levels for ER, progesterone receptor (PR), uteroferrin (UF), retinol-binding protein (RBP), and keratinocyte growth factor (KGF). Adults cycled normally and had similar numbers of corpora lutea. Uteri of PX gilts contained tubular/filamentous conceptuses, and ULF estradiol content was unaffected by treatment. However, pregnancy increased uterine weight and size only in CO gilts (Treatment x Status, P < 0.01). Treatment reduced ULF protein content (P < 0.01), endometrial 3H-Leu incorporation (P < 0.05), and the pregnancy-associated increase in ULF protein (Treatment x Status, P < 0.01). Treatment did not affect endometrial ER or PR mRNA levels but attenuated the pregnancy-associated increase in UF mRNA (Treatment x Status; P < 0.01), increased RBP (P < 0.10), and decreased KGF mRNA levels (P < 0.05). These results establish that transient postnatal estrogen exposure affects porcine uterine responsiveness to potentially embryotrophic signals and that estrogen-sensitive postnatal uterine organizational events are determinants of uterine size and functionality.     

Identification of a uterine elastase in the pregnant rat uterus

During development of the pregnant rat uterus there is a several fold increase in elastin content. Using Verhoeff's elastic fiber stain, we have shown that a significant proportion of these elastin fibers are in the extracellular matrix of the myometrium. They do not appear as an organized structure but rather in a variety of partially extended, random configurations. An elastase was identified in both the pregnant and the postpartum uterus. Partial characterization of the enzyme indicated that it is a serine protease with a molecular weight around 24,500 and a pH optimum of 8.5. In addition to the enzyme, relatively high levels on an elastase inhibitor were found in the uterine extracts. The inhibitor did not inhibit trypsin, indicating that it was not alpha-1-antiprotease. The data suggest that the elastase and inhibitor are uterine tissue derived and perhaps important in the normal remodeling process of uterine connective tissue.     

Gonadotropin-releasing hormone alters the T helper cytokine balance in the pregnant rat

The interactions between immune-endocrine and reproductive systems are heightened during pregnancy as an adaptive mechanism, and are regulated by a complex array of hormones and cytokines that control the survival of a semiallogeneic conceptus. GnRH can exert direct effects on the immune system via its receptor (GnRH-R) on lymphoid cells. In the present study, we employed in vitro, ex vivo, and in vivo approaches to investigate the role of GnRH in the modulation of T helper cytokines in pregnant rats undergoing termination of pregnancy. Day 8 pregnant rats were infused with a GnRH agonist (GnRH-Ag) for 24 h using an osmotic minipump. Sham control rats were infused with the vehicle, saline. Lymphocytes were isolated from sham and treated rats and polyclonally stimulated with immobilized anti-CD3 antibody. The levels of the signature T helper 1 (Th-1) cytokines (interferon-gamma [IFN-gamma] and interleukin-2 [IL-2]) and Th-2 cytokines (IL-4 and IL-10) were measured in culture supernatants. Using immunoflourescence confocal microscopy, we demonstrated for the first time the spatial localization of GnRH-R protein on the surface of lymphocytes. We observed a marked increase in IFN-gamma and inhibition of IL-4 production from lymphocytes of pregnant rats treated in vitro with different doses of GnRH-Ag. Further, the responsiveness of lymphocytes to produce IFN-gamma was markedly increased in cells cultured ex vivo from GnRH-Ag infused rats, whereas the capacity of lymphocytes to produce IL-4 was significantly inhibited. In addition, GnRH-Ag infusion in pregnant rats induced a shift toward Th-1 cytokines in the serum. We did not observe any significant difference in IL-2 and IL-10 production in response to GnRH-Ag. Our results suggest an additional function for GnRH as a Th-1 inducer and Th-2 inhibitor. GnRH can thus skew the cytokine balance to predominantly Th-1 type in pregnancy, leading to the termination of pregnancy in rats.     

Fetectomy alters maternal pituitary-adrenal function in pregnant rhesus macaques.

The interplay between the fetus and mother may play a key role in the regulation of primate pregnancy and parturition. This study was designed to test the hypothesis that fetectomy alters maternal pituitary-adrenal function. Between 117 and 122 days of gestation (term = 167 days), six rhesus macaques underwent surgery for catheter implantation. At surgery the fetuses were removed while the membranes and placenta were left in situ. Six additional intact catheterized pregnant animals served as controls. Animals were maintained under a 12L:12D cycle with lights-on from 0700 to 1900 h. Beginning at least 1 wk after surgery, maternal arterial blood samples were collected at 3-h intervals for 24 h for hormone and catecholamine analysis. This sampling protocol was repeated at weekly intervals until cesarean delivery at 151-157 days of gestation. Following fetectomy, plasma ACTH, dehydroepiandrosterone sulfate (DHEAS), and cortisol levels were significantly lower (36%, 35%, and 44%, respectively) compared with control animals (P;lt 0.05). Despite a significant reduction in overall levels, the rhythm in maternal plasma cortisol was maintained following fetectomy. Plasma dopamine and norepinephrine were also depressed (P;lt 0.05), whereas epinephrine remained unaffected. Our data clearly demonstrate the role of the fetus in the regulation of the maternal pituitary-adrenal axis during gestation. This interaction plays a significant role in the regulation of maternal endocrine function that may influence the initiation of labor.     

Maternal n-3 PUFA deficiency alters uterine artery remodeling and placental epigenome in the mice

The maternal n-3 polyunsaturated fatty acid (PUFA) deficiency on decidual vascular structure and angiogenesis in mice placenta was investigated. Namely, we studied uterine artery remodeling, fatty acid metabolism, and placental epigenetic methylation in this animal model. Weanling female Swiss albino mice were fed either alpha-linolenic acid (18:3 n-3, ALA) deficient diets (0.13% energy from ALA) or a sufficient diet (2.26% energy from ALA) throughout the study. The dietary n-3 PUFA deficiency altered uteroplacental morphology and vasculature by reversing luminal to vessel area and increased luminal wall thickness at 8.5-12.5gD. Further, placentas (F0 and F1) showed a significant decrease in the expression of VCAM1, HLAG proteins and an increase in MMP9, KDR expression. The conversion of ALA to long-chain (LC) n-3 PUFAs was significantly decreased in plasma and placenta during the n-3 deficiency state. Reduced n-3 LCPUFAs increased the placental expression of intracellular proteins FABP3, FABP4, and ADRP to compensate decreased availability of these fatty acids in the n-3 deficient mice. The N-3 PUFA deficiency significantly increased the 5-methylcytosine levels in the placenta but not in the liver. The alteration in DNA methylation continued to the next generation in the placental epigenome with augmented expression of DNMT3A and DNMT3B. Our study showed that maternal n-3 PUFA deficiency alters placental vascular architecture and induces epigenetic changes suggesting the importance of n-3 PUFA intake during the development of the fetus. Moreover, the study shows that the placenta is the susceptible target for epigenetic alteration in maternal deficiency n-3 fatty acids.     

Predicted SAR in Sprague-Dawley rat as a function of permittivity values

Specific absorption rate (SAR) value is dependent on permittivity value. However, variability in the published permittivity values for human and animal tissue and the development of sophisticated 3-dimensional digital anatomical models to predict SAR values has resulted in the need to understand how model parameters (permittivity value) affect the predicted whole body and localized SAR values. In this paper, we establish the partial derivative of whole body SARs and localized SAR values (defined as SAR for individual organs with respect to a change in the permittivity values of all tissue types, as well as for those tissues with the most variable permittivity values. Variations in the published permittivity values may substantially influence whole body and localized SAR values, but only under special conditions. Orientation of the exposed object to the incident electromagnetic wave is one of the most crucial factors. Published 2001 Wiley-Liss, Inc.     

Effects of estradiol and progesterone on uterine prostaglandin levels in the pregnant rat

Prostaglandin D₂ was found to be a potent inhibitor of B-16 melanoma cell replication $\text{in vitro}$. The inhibition was dose-dependent between 3×10^?9M and 3×10^?6M (IC_50～ 0.3 μM after 6 days). On a molar basis, PGD₂ was a better inhibitor than PGA₂ or 16,16-dimethyl-PGE₂-methyl ester (di-M-PGE₂) and in higher concentrations (10^?6?10^?7M), comparable to retinoic acid. In higher concentrations, PGD₂ inhibited DNA, RNA and protein synthesis. The B-16 melanoma cell line which we used synthesized arachidonic acid metabolites which comigrated with PGA₂, PGD₂, PGE₂ and PGF_2α on a thin layer chromatography system.     

Possible mechanisms underlying pregnancy-induced changes in uterine artery endothelial function

The last 10 years has seen a dramatic increase in our understanding of the mechanisms underlying the pregnancy-specific adaptation in cardiovascular function in general and the dramatic changes that occur in uterine artery endothelium in particular to support the growing fetus. The importance of these changes is clear from a number of studies linking restriction of uterine blood flow (UBF) and/or endothelial dysfunction and clinical conditions such as intrauterine growth retardation (IUGR) and/or preeclampsia in both humans and animal models; these topics are covered only briefly here. The recent developments that prompts this review are twofold. The first is advances in an understanding of the cell signaling processes that regulate endothelial nitric oxide synthase (eNOS) in particular (Govers R and Rabelink TJ. Am J Physiol Renal Physiol 280: F193-F206, 2001). The second is the emerging picture that uterine artery (UA) endothelial cell production of nitric oxide (NO) as well as prostacyclin (PGI2) may be as much a consequence of cellular reprogramming at the level of cell signaling as due to tonic stimuli inducing changes in the level of expression of eNOS or the enzymes of the PGI2 biosynthetic pathway (cPLA2, COX-1, PGIS). In reviewing just how we came to this conclusion and outlining the implications of such a finding, we draw mostly on data from ovine or human studies, with reference to other species only where directly relevant.     

Effects of the fetal-placental unit on uterine prostaglandin levels at term in the pregnant rat

Uterine prostaglandins (PGs) increase markedly at term in the pregnant rat. To assess the contribution of the fetal-placental unit (FPU) on uterine tissue and uterine venous blood PG concentrations, each uterine horn of 14 unilaterally pregnant rats at day 21 of pregnancy were compared. In addition, 7 bilaterally pregnant rats were studied. Uterine tissue and uterine venous plasma PGF, PGE, 6-Keto-PGF1 (6KF) and thromboxane B2 (TxB2) and systemic plasma progesterone, estradiol and estrone were determined by radioimmunoassay. Uterine concentrations of PGs (ng/mg DNA) were always greater on the pregnant side of unilaterally pregnant rats (p less than .05) although the PGF levels were elevated to a lesser extent than were PGE, TxB2 or 6KF. However, no differences were detected between uterine tissue from the pregnant side of unilaterally pregnant compared to bilaterally pregnant rats. In addition, no differences were found in uterine venous plasma PGs adjacent or opposite the pregnant uterine horn and in systemic plasma progesterone, estradiol and estrone levels in unilaterally vs bilaterally pregnant rats. These data suggest that the presence of the FPU is associated with an increased capacity of uterine tissue to produce PGE, TxB2 and 6KF, and to a lesser degree PGF, and thus may contribute to the increase in uterine PGs periparturition.     

Effects of the fetal-placental unit on uterine prostaglandin levels at term in the pregnant rat

Uterine prostaglandins (PGs) increase markedly at term in the pregnant rat. To assess the contribution of the fetal-placental unit (FUP) on uterine tissue and uterine venous blood PG concentrations, each uterine horn of 14 unilaterally pregnant rats at day 21 of pregnancy were compared. In addition, 7 bilaterally pregnant rats were studied. Uterine tissue and uterine venous plasma PGF, PGE, 6-Keto-PGF₁ (6KF) and thromboxane B₂ (TxB₂) and systematic plasma progesterone, estradiol and estrone were determined by radioimmunoassay. Uterine concentrations of PGs (ng/mg DNA) were always greater on the pregnant side of unilaterally pregnant rats (p<.05) although the PGF levels were elevated to a lesser extent than were PGE, TxB₂ or 6KF. However, no differences were detected between uterine tissue from the pregnant side of unilaterally pregnant compared to bilaterally pregnant rats. In addition, no differences were found in uterine venous plasma PGs adjacent or opposite the pregnant uterine horn and in systematic plasma progesterone, estradiol and estrone levels in unilaterally vs bilaterally pregnant rats. These data suggest that the presence of the FPU is associated with an increased capacity of uterine tissue to produce PGE, TxB₂ and 6KF, and to a lesser degree PGF, and thus may contribute to the increase in uterine PGs periparturition.     

Cyclooxygenase and lipoxygenase inhibitors--induced changes in the distribution of glycosaminoglycans in the pregnant rat uterine cervix

In order to study the hormonal control mechanisms of cervical maturation, we investigated cyclooxygenase and 5-lipoxygenase inhibitors-induced changes in the distribution of glycosaminoglycans (GAG) in pregnant Wistar rat uterine cervices at term. The GAG were measured in a control (n = 11), in a Diclofenac (cyclooxygenase inhibitor) treated group (n = 8), in a BW 755C (dual inhibitor of cyclooxygenase and 5-lipoxygenase) treated group (n = 6), and a L 651392 (5-lipoxygenase inhibitor) treated group (n = 9). The results of these studies suggest, that cervical hyaluronic acid metabolism and cervical hydration are controlled in association by prostaglandins and leukotrienes (and perhaps by other phospholipids metabolites), whereas heparan sulphate metabolism is obviously controlled by prostaglandins. Nevertheless complete and normal cervical maturation is probably controlled in association by arachidonic acid metabolites and other factors (steroids and peptides).     

Obesity alters the uterine environment before pregnancy

Obesity is a metabolic disorder that predisposes to numerous diseases and has become a major global public health concern. Cafeteria diet (CAF) is the animal model used for the study of obesity that more closely reflects Western diet habits. Previously, we described that CAF administration for 60 days induces obesity in female rats and their fetuses develop macrosomia. Given that, in our model, rats are not genetically modified and that obese mothers were fed standard chow during pregnancy, the aim of the current study was to test the hypothesis that obesity alters the intrauterine environment prior to pregnancy, and this may explain the exacerbated fetal weight gain. We found that uteri from obese rats during the estrous phase developed insulin resistance through mechanisms that involve the induction of uterine hypoxia and the down-regulation of the insulin receptor gene. Moreover, uterine cell proliferation was induced by obesity concomitantly with the reduction in the uterine contractile response to a β2 AR agonist, salbutamol, and this may be consequence of the down-regulation in the uterine β2 AR expression. We conclude that CAF-induced obesity alters the uterine environment in rats during the estrous phase and may cause the fetal macrosomia previously described by us in obese animals. The lower sensitivity of the uterus to a relaxation stimulus (salbutamol) is not a minor fact given that for implantation to occur the uterus must be relaxed for embryo nidation. Thus, the alteration in the uterine quiescence may impair implantation and, consequently, the foregoing pregnancy.     

Ontogeny of uteroplacental progestagen production in pregnant mares during the second half of gestation

In pregnant mares during late gestation, little, if any, progesterone (P4) is found in the maternal circulation. Hence, quiescence of the equine uterus is believed to be maintained by metabolites of pregnenolone and P4 known as progestagens, which are produced by the uteroplacental tissues. However, little is known about the ontogeny, distribution, or actual rates of uteroplacental progestagen production in pregnant mares and their fetuses during the second half of pregnancy. Therefore, the present study measured the rates of uteroplacental uptake and output of eight specific progestagens in chronically catheterized, pregnant pony mares from 180 days to term. No significant uteroplacental uptake of any of the eight individual progestagens was observed from the uterine circulation. In contrast, significant uteroplacental uptake was observed for five of the eight individual progestagens from the umbilical circulation, and the uptakes increased toward term. The major uteroplacental progestagen outputs were 5 alpha-pregnane-3,20-dione (5 alphaDHP) and 20 alpha-hydroxy-5 alpha-pregnan-3-one (20 alpha 5P). These were released into both the umbilical and uterine circulations at rates that increased toward term. The majority of the total uteroplacental 20 alpha 5P output was distributed into the uterine circulation at all gestational ages studied. In contrast, distribution of the total uteroplacental 5 alphaDHP output switched from preferential delivery into the uterine circulation before 220 days of gestation to release predominantly into the umbilical circulation after 260 days. These findings demonstrate that uteroplacental progestagen production changes during the second half of gestation, which may have important implications for the maintenance of pregnancy and the onset of labor in the mare.     

Chronic hypoxia augments uterine artery distensibility and alters the circumferential wall stress-strain relationship during pregnancy.

Pregnancy-associated increases in uterine artery (UA) blood flow are due, in part, to vasoactive and growth-related changes that enlarge UA diameter. Although active and passive mechanical factors can contribute to this enlargement, their role is less well understood. We hypothesized that pregnancy increased UA distensibility and/or decreased myogenic tone. Given the fetal growth restriction and lower UA flow seen under chronic hypoxia, we further hypothesized that chronic hypoxia opposed these normal active and passive mechanical changes. UA were isolated from 12 nonpregnant and 12 pregnant (0.7 gestation) guinea pigs housed under normoxia or chronic hypoxia (3,960 m) and studied by pressure myography. Pregnancy increased UA diameter similarly under normoxia and hypoxia. Although chronic hypoxia raised resting tone in UA from nonpregnant guinea pigs to approximately 20% and tone was greater in preconstricted pregnant chronically hypoxic vs. normoxic UA (both P<0.01), there was an absence of myogenic response (i.e., an increase in tone with rising pressure) in all groups. Pregnancy increased UA distensibility 1.5-fold but did not change stiffness or the stress-strain relationship. Compared with vessels from normoxic pregnant animals, hypoxic pregnancy raised UA distensibility fourfold, decreased stiffness (rate constant b=3.80+/-1.06 vs. 8.92+/-1.25, respectively, P<0.01), lowered elastin by 50%, and shifted the stress-strain relationship upward such that four times as much strain was present at a given stress. We concluded that increased distensibility and low myogenic tone contribute to enlarging UA diameter and raising UA blood flow during pregnancy. Chronic hypoxia exaggerates the rise in distensibility and alters the stress-strain relationship in ways that may provoke vascular injury.