Elevated Testosterone Reduces Uterine Blood Flow, Spiral Artery Elongation, and Placental Oxygenation in Pregnant Rats.

Elevated maternal testosterone levels are shown to cause fetal growth restriction, eventually culminating in sex-specific adult-onset hypertension that is more pronounced in males than in females. In this study, we tested whether uteroplacental and fetoplacental disturbances underlie fetal growth restriction and if these changes vary in male and female placentas. Pregnant Sprague-Dawley rats were injected with vehicle (n=16) or testosterone propionate (0.5 mg/kg per day from gestation day 15-19; n=16). On gestation day 20, we quantified uterine artery blood flow using microultrasound, visualized placental arterial network using x-ray microcomputed tomography, determined fetoplacental hypoxia using pimonidazole and hypoxia-inducible factor-1α, and used Affymetrix array to determine changes in placental expression of genes involved in vascular development. Plasma testosterone levels increased 2-fold in testosterone-injected rats. Placental and fetal weights were lower in rats with elevated testosterone. Uterine artery blood flow was lower, and resistance index was higher in the testosterone group. Radial and spiral artery diameter and length, the number of fetoplacental arterial branches, and umbilical artery diameter were reduced in the testosterone group. In addition, markers of hypoxia in the placentas and fetuses were elevated in the testosterone group. The magnitude of changes in placental vasculature and hypoxia was greater in males than in females and was associated with sex-specific alteration of unique sets of genes involved in angiogenesis and blood vessel morphogenesis. The results demonstrate that elevated testosterone during gestation induces a decrease in uterine arterial blood flow and fetal sex-related uteroplacental vascular changes, which may set the stage for subsequent sex differences in adult-onset diseases.

Prenatal Testosterone Exposure Leads to Gonadal Hormone-Dependent Hyperinsulinemia and Gonadal Hormone-Independent Glucose Intolerance in Adult Male Rat Offspring.

Elevated testosterone levels during prenatal life lead to hyperandrogenism and insulin resistance in adult females. This study evaluated whether prenatal testosterone exposure leads to the development of insulin resistance in adult male rats in order to assess the influence of gonadal hormones on glucose homeostasis in these animals. Male offspring of pregnant rats treated with testosterone propionate or its vehicle (control) were examined. A subset of male offspring was orchiectomized at 7 wk of age and reared to adulthood. At 24 wk of age, fat weights, plasma testosterone, glucose homeostasis, pancreas morphology, and gastrocnemius insulin receptor (IR) beta levels were examined. The pups born to testosterone-treated mothers were smaller at birth and remained smaller through adult life, with levels of fat deposition relatively similar to those in controls. Testosterone exposure during prenatal life induced hyperinsulinemia paralleled by an increased HOMA-IR index in a fasting state and glucose intolerance and exaggerated insulin responses following a glucose tolerance test. Prenatal androgen-exposed males had more circulating testosterone during adult life. Gonadectomy prevented hyperandrogenism, reversed hyperinsulinemia, and attenuated glucose-induced insulin responses but did not alter glucose intolerance in these rats. Prenatal androgen-exposed males had decreased pancreatic islet numbers, size, and beta-cell area along with decreased expression of IR in gastrocnemius muscles. Gonadectomy restored pancreatic islet numbers, size, and beta-cell area but did not normalize IRbeta expression. This study shows that prenatal testosterone exposure leads to a defective pancreas and skeletal muscle function in male offspring. Hyperinsulinemia during adult life is gonad-dependent, but glucose intolerance appears to be independent of postnatal testosterone levels.

Enalapril Normalizes Endothelium-Derived Hyperpolarizing Factor-Mediated Relaxation in Mesenteric Artery of Adult Hypertensive Rats Prenatally Exposed to Testosterone.

Prenatal exposure to elevated testosterone levels induces adult life hypertension associated with selective impairments in endothelium-derived hyperpolarizing factor (EDHF)-mediated relaxation in mesenteric arteries. We tested whether the angiotensin-converting enzyme inhibitor enalapril restores EDHF function through regulating the activities of small (Kcnn3) and intermediate (Kcnn4) conductance calcium-activated potassium channels in mesenteric arteries. Pregnant Sprague-Dawley rats were injected subcutaneously with vehicle or testosterone propionate (0.5 mg/kg/day from Gestation Day 15 to 19), and their 6-mo-old adult male offspring were examined. A subset of rats in these two groups was given enalapril (40 mg/kg/day) for 2 wk through drinking water. Blood pressures were assessed through carotid arterial catheter and endothelium-dependent mesenteric arterial EDHF relaxation, using wire myography. Ace and Kcnn3 and Kcnn4 channel expression levels were also examined. Renal and vascular Ace expression and plasma angiotensin II levels were increased in testosterone offspring. Blood pressure levels were significantly higher in testosterone offspring than in controls, and treatment with enalapril significantly attenuated blood pressure in testosterone offspring. EDHF relaxation in testosterone offspring was reduced compared to that in controls, and it was significantly restored by enalapril treatment. Kcnn4 channel expression and function were similar between control and testosterone rats, but it was not affected by enalapril treatment. Relaxation mediated by Kcnn3 was impaired in testosterone offspring, and it was normalized by enalapril treatment. Furthermore, enalapril treatment restored expression levels of Kcnn3 channels. These findings suggest that enalapril has a positive influence on endothelial function with improvement in EDHF relaxation through normalization of Kcnn3 expression and activity.

Enhanced mesenteric arterial responsiveness to angiotensin II is androgen receptor-dependent in prenatally protein-restricted adult female rat offspring.

Gestational protein restriction results in intrauterine growth restriction and hypertension in adult female growth-restricted rats. Enhanced vascular responsiveness to angiotensin II is observed, and blockade of the renin-angiotensin system abolishes hypertension in adult growth-restricted rats, suggesting that the renin-angiotensin system contributes to intrauterine growth restriction-induced hypertension. Moreover, growth-restricted adult rats have higher plasma testosterone levels, and antiandrogen treatment abolishes hypertension, indicating an important role for testosterone. We hypothesized that androgens may play a pivotal role in the enhanced responsiveness to Ang II and hypertension. Female offspring of pregnant rats fed 20% protein (control) or 6% protein diet (protein restricted), at 6 mo of age, were studied. Plasma testosterone and mean arterial pressure in protein-restricted offspring were significantly higher compared to controls. Flutamide treatment (10 mg/kg/day subcutaneously for 10 days) reduced mean arterial pressure in protein-restricted offspring but was without significant effect in controls. Vascular Agtr1/Agtr2 ratio was significantly higher in protein-restricted offspring, an effect that was reversed by flutamide. Flutamide treatment did not have any effect on Agtr1/Agtr2 ratio in controls. Enhanced contractile response to angiotensin II in mesenteric arteries was observed in protein-restricted offspring compared with control. Flutamide treatment reversed the enhanced contractile response to angiotensin II in protein-restricted offspring without significant effect in controls. Vascular reactivity to phenylephrine was similar between the control and protein-restricted offspring with and without flutamide treatment, suggesting that enhanced contractile response and flutamide's reversal effect is specific to angiotensin II. These results suggest that prenatally protein-restricted rats exhibit an enhanced responsiveness to angiotensin II that is testosterone-dependent.

Prenatal testosterone exposure induces hypertension in adult females via androgen receptor-dependent protein kinase Cδ-mediated mechanism.

Prenatal exposure to excess testosterone induces hyperandrogenism in adult females and predisposes them to hypertension. We tested whether androgens induce hypertension through transcriptional regulation and signaling of protein kinase C (PKC) in the mesenteric arteries. Pregnant Sprague-Dawley rats were injected with vehicle or testosterone propionate (0.5 mg/kg per day from gestation days 15 to 19, SC) and their 6-month-old adult female offspring were examined. Plasma testosterone levels (0.84±0.04 versus 0.42±0.09 ng/mL) and blood pressures (111.6±1.3 versus 104.5±2.4 mm Hg) were significantly higher in prenatal testosterone-exposed rats compared with controls. This was accompanied with enhanced expression of PKCδ mRNA (1.5-fold) and protein (1.7-fold) in the mesenteric arteries of prenatal testosterone-exposed rats. In addition, mesenteric artery contractile responses to PKC activator, phorbol-12,13-dibutyrate, was significantly greater in prenatal testosterone-exposed rats. Treatment with androgen receptor antagonist flutamide (10 mg/kg, SC, BID for 10 days) significantly attenuated hypertension, PKCδ expression, and the exaggerated vasoconstriction in prenatal testosterone-exposed rats. In vitro exposure of testosterone to cultured mesenteric artery smooth muscle cells dose dependently upregulated PKCδ expression. Analysis of PKCδ gene revealed a putative androgen responsive element in the promoter upstream to the transcription start site and an enhancer element in intron-1. Chromatin immunoprecipitation assays showed that androgen receptors bind to these elements in response to testosterone stimulation. Furthermore, luciferase reporter assays showed that the enhancer element is highly responsive to androgens and treatment with flutamide reverses reporter activity. Our studies identified a novel androgen-mediated mechanism for the control of PKCδ expression via transcriptional regulation that controls vasoconstriction and blood pressure.

Calcitonin gene related family peptides: importance in normal placental and fetal development.

Synchronized molecular and cellular events occur between the uterus and the implanting embryo to facilitate successful pregnancy outcome. Nevertheless, the molecular signaling network that coordinates strategies for successful decidualization, placentation and fetal growth are not well understood. The discovery of calcitonin/calcitonin gene-related peptides (CT/CGRP) highlighted new signaling mediators in various physiological processes, including reproduction. It is known that CGRP family peptides including CGRP, adrenomedulin and intermedin play regulatory functions during implantation, trophoblast proliferation and invasion, and fetal organogenesis. In addition, all the CGRP family peptides and their receptor components are found to be expressed in decidual, placental and fetal tissues. Additionally, plasma levels of peptides of the CGRP family were found to fluctuate during normal gestation and to induce placental cellular differentiation, proliferation, and critical hormone signaling. Moreover, aberrant signaling of these CGRP family peptides during gestation has been associated with pregnancy disorders. It indicates the existence of a possible regulatory role for these molecules during decidualization and placentation processes, which are known to be particularly vulnerable. In this review, the influence of the CGRP family peptides in these critical processes is explored and discussed.

Chronic binge alcohol exposure during pregnancy impairs rat maternal uterine vascular function.

BACKGROUND: Alcohol exposure during pregnancy results in an array of structural and functional abnormalities called fetal alcohol spectrum disorders (FASD). Alcohol dysregulates the exquisite coordination and regulation of gestational adaptations at the level of the uterine vasculature. We herein hypothesized that chronic binge-like alcohol results in uterine vascular dysfunction and impairs maternal uterine artery reactivity to vasoconstrictors and dilators. METHODS: We utilized a once-daily binge alcohol (4.5 g/kg body weight) exposure paradigm (gestational day 7 to 17) in a pregnant rat model system and investigated primary uterine artery function in response to vasoconstrictors and vasodilators utilizing wire myography. RESULTS: Alcohol (peak blood alcohol concentration, 216 mg/dl) produced uterine vascular dysfunction. Alcohol did not produce altered uterine vascular reactivity to α1 adrenergic agonist phenylephrine or the prostanoid thromboxane. However, alcohol specifically impaired acetylcholine (ACh)-mediated uterine artery vasodilation but exogenous endothelium-independent vasodilators like sodium nitroprusside exhibited no alcohol effect; ACh significantly decreased vessel relaxation (p = 0.003; ↓pD2 [negative log molar ACh concentration producing the half maximum response], -7.004 ± 0.215 vs. -6.310 ± 0.208; EMax [maximal ACh response], 92% vs. 75%). CONCLUSIONS: We conclude that moderate alcohol exposure impairs uterine vascular function in pregnant mothers. Alcohol specifically impairs agonist-induced uterine artery vasodilation. In summary, the maternal uterine compartment may play a significant role in the pathogenesis of FASD. Thus, the mechanistic targets of alcohol at the level of both the mother and the fetus need to be considered in order to develop effective therapeutic treatment strategies for FASD.

Gestational exposure to elevated testosterone levels induces hypertension via heightened vascular angiotensin II type 1 receptor signaling in rats.

Pre-eclampsia is a life-threatening pregnancy disorder whose pathogenesis remains unclear. Plasma testosterone levels are elevated in pregnant women with pre-eclampsia and polycystic ovary syndrome, who often develop gestational hypertension. We tested the hypothesis that increased gestational testosterone levels induce hypertension via heightened angiotensin II signaling. Pregnant Sprague-Dawley rats were injected with vehicle or testosterone propionate from Gestational Day 15 to 19 to induce a 2-fold increase in plasma testosterone levels, similar to levels observed in clinical conditions like pre-eclampsia. A subset of rats in these two groups was given losartan, an angiotensin II type 1 receptor antagonist by gavage during the course of testosterone exposure. Blood pressure levels were assessed through a carotid arterial catheter and endothelium-independent vascular reactivity through wire myography. Angiotensin II levels in plasma and angiotensin II type 1 receptor expression in mesenteric arteries were also examined. Blood pressure levels were significantly higher on Gestational Day 20 in testosterone-treated dams than in controls. Treatment with losartan during the course of testosterone exposure significantly attenuated testosterone-induced hypertension. Plasma angiotensin II levels were not significantly different between control and testosterone-treated rats; however, elevated testosterone levels significantly increased angiotensin II type 1 receptor protein levels in the mesenteric arteries. In testosterone-treated rats, mesenteric artery contractile responses to angiotensin II were significantly greater, whereas contractile responses to K(+) depolarization and phenylephrine were unaffected. The results demonstrate that elevated testosterone during gestation induces hypertension in pregnant rats via heightened angiotensin II type 1 receptor-mediated signaling, providing a molecular mechanism linking elevated maternal testosterone levels with gestational hypertension.

Elevated testosterone levels during rat pregnancy cause hypersensitivity to angiotensin II and attenuation of endothelium-dependent vasodilation in uterine arteries.

Elevated testosterone levels increase maternal blood pressure and decrease uterine blood flow in pregnancy, resulting in abnormal perinatal outcomes. We tested whether elevated testosterone alters uterine artery adaptations during pregnancy, and whether these alterations depend on endothelium-derived factors such as nitric oxide, endothelium-derived hyperpolarizing factor, and prostacyclin, or endothelium-independent mechanisms such as angiotensin II (Ang-II). Pregnant Sprague-Dawley rats were injected with vehicle (n=20) or testosterone propionate (0.5 mg/kg per day from gestation day 15 to 19; n=20). Plasma testosterone levels increased 2-fold in testosterone-injected rats compared with controls. Elevated testosterone significantly decreased placental and pup weights compared with controls. In endothelium-intact uterine arteries, contractile responses to thromboxane, phenylephrine, and Ang-II were greater in testosterone-treated rats compared with controls. In endothelium-denuded arteries, contractile responses to Ang-II (pD2=9.1±0.04 versus 8.7±0.04 in controls; P<0.05), but not thromboxane and phenylephrine, were greater in testosterone-treated rats. Ang-II type 1b receptor expression was increased, whereas Ang-II type 2 receptor was decreased in testosterone-exposed arteries. In endothelium-denuded arteries, relaxations to sodium nitroprusside were unaffected. Endothelium-dependent relaxation to acetylcholine was significantly lower in arteries from testosterone-treated dams (Emax=51.80±6.9% versus 91.98±1.4% in controls; P<0.05). The assessment of endothelial factors showed that nitric oxide-, endothelium-derived hyperpolarizing factor-, and prostacyclin-mediated relaxations were blunted in testosterone-treated dams. Endothelial nitric oxide synthase, small conductance calcium-activated potassium channel-3, and prostacyclin receptor expressions were significantly decreased in arteries from testosterone-treated dams. Hypoxia-inducible factor-1α, Ankrd37, and Egln were significantly increased in testosterone-exposed placentas. These results suggest that elevated maternal testosterone impairs uterine vascular function, which may lead to an increased vascular resistance and a decrease in uterine blood flow.

Gestational protein restriction impairs insulin-regulated glucose transport mechanisms in gastrocnemius muscles of adult male offspring.

Type II diabetes originates from various genetic and environmental factors. Recent studies showed that an adverse uterine environment such as that caused by a gestational low-protein (LP) diet can cause insulin resistance in adult offspring. The mechanism of insulin resistance induced by gestational protein restriction is not clearly understood. Our aim was to investigate the role of insulin signaling molecules in gastrocnemius muscles of gestational LP diet-exposed male offspring to understand their role in LP-induced insulin resistance. Pregnant Wistar rats were fed a control (20% protein) or isocaloric LP (6%) diet from gestational day 4 until delivery and a normal diet after weaning. Only male offspring were used in this study. Glucose and insulin responses were assessed after a glucose tolerance test. mRNA and protein levels of molecules involved in insulin signaling were assessed at 4 months in gastrocnemius muscles. Muscles were incubated ex vivo with insulin to evaluate insulin-induced phosphorylation of insulin receptor (IR), Insulin receptor substrate-1, Akt, and AS160. LP diet-fed rats gained less weight than controls during pregnancy. Male pups from LP diet-fed mothers were smaller but exhibited catch-up growth. Plasma glucose and insulin levels were elevated in LP offspring when subjected to a glucose tolerance test; however, fasting levels were comparable. LP offspring showed increased expression of IR and AS160 in gastrocnemius muscles. Ex vivo treatment of muscles with insulin showed increased phosphorylation of IR (Tyr972) in controls, but LP rats showed higher basal phosphorylation. Phosphorylation of Insulin receptor substrate-1 (Tyr608, Tyr895, Ser307, and Ser318) and AS160 (Thr642) were defective in LP offspring. Further, glucose transporter type 4 translocation in LP offspring was also impaired. A gestational LP diet leads to insulin resistance in adult offspring by a mechanism involving inefficient insulin-induced IR, Insulin receptor substrate-1, and AS160 phosphorylation and impaired glucose transporter type 4 translocation.

Interactive effects of in vitro binge-like alcohol and ATP on umbilical endothelial nitric oxide synthase post-translational modifications and redox modulation.

Alcohol dysregulates the regulation of reproductive vascular adaptations. We herein investigated chronic in vitro binge-like alcohol effects on umbilical endothelial nitric oxide synthase (eNOS) multi-site phosphorylation and related redox switches under basal (unstimulated) and stimulated (with ATP) states. Alcohol decreased endothelial excitatory (Pser1177)eNOS (P<0.001), whereas excitatory (Pser635)eNOS exhibited a main effect of alcohol (↓P=0.016) and ATP (↑P<0.001). Alcohol decreased (Pthr495)eNOS (P=0.004) levels, whereas inhibitory (Pser116)eNOS exhibited an alcohol main effect in both basal and stimulated states (↑P=0.005). Total eNOS was reduced by alcohol (P=0.038). In presence of ATP, alcohol inhibited ERK activity (P=0.002), whereas AKT exhibited no alcohol effect. Alcohol main effect on S-nitroso-glutathione reductase (↓P=0.031) and glutathione-S-transferase (↓P=0.027) were noted. Increased protein glutathiolation was noted, whereas no alcohol effect on GSH, GSSG, NOX2 or SOD expression was noted. Thus, alcohol effects on multi-site post-translational modifications and redox switches related to vasodilatory eNOS underscore the necessity for investigating alcohol-induced gestational vascular dysfunction.

Prenatal testosterone induces sex-specific dysfunction in endothelium-dependent relaxation pathways in adult male and female rats.

Prenatal testosterone (T) exposure impacts postnatal cardiovascular function, leading to increases in blood pressure with associated decreased endothelium-dependent vascular relaxation in adult females. Endothelial function in males is not known. Furthermore, which of the endothelial pathways contributes to endothelial dysfunction and if there exists sex differences are not known. The objective of this study was to characterize the relative contribution of nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF) to the impaired endothelium-dependent vasodilation in prenatal T-exposed adult males and females. Offspring of pregnant rats treated with T propionate or its vehicle were examined. Telemetric blood pressure levels and endothelium-dependent vascular reactivity were assessed with wire myography. Levels of nitric oxide synthase (NOS3) and Kcnn3 and Kcnn4 channel expression were examined in mesenteric arteries. Mean arterial pressure was significantly higher in T males and females than in controls. Endothelium-dependent acetylcholine relaxation was significantly lower in both T males and females. EDHF-mediated relaxation was specifically blunted in T males (Emax = 48.64% ± 3.73%) compared to that in control males (Emax = 81.71% ± 3.18%); however, NO-mediated relaxation was specifically impaired in T females (Emax = 36.01% ± 4.29%) compared with that in control females (Emax = 54.56% ± 6.37%). Relaxation to sodium nitroprusside and levcromakalim were unaffected with T-treatment. NOS3 protein was decreased in T females but not in T males. Kcnn3 expression was decreased in both T males and females compared to controls. These findings suggest that prenatal T leads to an increase in blood pressure in the adult offspring, associated with blunting of endothelial cell-associated relaxation and that the effects are sex-specific: EDHF-related in males and NO-related in females.

Testosterone alters maternal vascular adaptations: role of the endothelial NO system.

Sex steroid hormones estradiol and progesterone play an important role in vascular adaptations during pregnancy. However, little is known about the role of androgens. Plasma testosterone (T) levels are elevated in preeclampsia, mothers with polycystic ovary, and pregnant African American women, who have endothelial dysfunction and develop gestational hypertension. We tested whether increased T alters vascular adaptations during pregnancy and whether these alterations depend on endothelium-derived factors, such as prostacyclin, endothelium-derived hyperpolarizing factor, and NO. Pregnant Sprague Dawley rats were injected with vehicle (n=12) or T propionate [0.5 mg/Kg per day from gestation day 15-19; n=12] to increase plasma T levels 2-fold, similar to that observed in preeclampsia. Telemetric blood pressures and endothelium-dependent vascular reactivity were assessed with wire-myograph system. Phospho-endothelial NO synthase and total endothelial NO synthase were examined in mesenteric arteries. Mean arterial pressures were significantly higher starting from gestation day19 until delivery in T-treated dams. Endothelium-dependent relaxation responses to acetylcholine were significantly lower in mesenteric arteries of T-treated dams (pD(2) [-log EC(50)]=7.05±0.06; E(max)=89.4±1.89) compared with controls (pD(2)=7.38±0.04; E(max)=99.9±0.97). Further assessment of endothelial factors showed NO-mediated relaxations were blunted in T-treated mesenteric arteries (E(max)=42.26±5.95) compared with controls (E(max)=76.49±5.06); however, prostacyclin- and endothelium-derived hyperpolarizing factor-mediated relaxations were unaffected. Relaxation to sodium nitroprusside was unaffected with T-treatment. Phosphorylations of endothelial NO synthase at Ser(1177) were decreased and at Thr(495) increased in T-treated mesenteric arteries without changes in total endothelial NO synthase levels. In conclusion, increased maternal T, at concentrations relevant to abnormal clinical conditions, cause hypertension associated with blunting of NO-mediated vasodilation. T may induce the increased vascular resistance associated with pregnancy-induced hypertension.

Temporal alterations in vascular angiotensin receptors and vasomotor responses in offspring of protein-restricted rat dams.

OBJECTIVE: Examine temporal alterations in vascular angiotensin II receptors (AT(1)R and AT(2)R) and determine vascular response to angiotensin II in growth-restricted offspring. STUDY DESIGN: Offspring of pregnant rats fed low-protein (6%) and control (20%) diet were compared. RESULTS: Prenatal protein restriction reprogrammed AT(1a)R messenger RNA expression in male rats' mesenteric arteries to cause 1.7- and 2.3-fold increases at 3 and 6 months of age associated with arterial pressure increases of 10 and 33 mm Hg, respectively; however, in female rats, increased AT(1a)R expression (2-fold) and arterial pressure (15 mm Hg) occurred only at 6 months. Prenatal protein restriction did not affect AT(2)R expression. Losartan abolished hypertension, suggesting that AT(1a)R plays a primary role in arterial pressure elevation. Vasoconstriction to angiotensin II was exaggerated in all protein-restricted offspring, with greater potency and efficacy in male rats. CONCLUSION: Prenatal protein restriction increased vascular AT(1)R expression and vasoconstriction to angiotensin II, possibly contributing to programmed hypertension.

Prenatal testosterone exposure leads to hypertension that is gonadal hormone-dependent in adult rat male and female offspring.

Prenatal testosterone exposure impacts postnatal reproductive and endocrine function, leading to alterations in sex steroid levels. Because gonadal steroids are key regulators of cardiovascular function, it is possible that alteration in sex steroid hormones may contribute to development of hypertension in prenatally testosterone-exposed adults. The objectives of this study were to evaluate whether prenatal testosterone exposure leads to development of hypertension in adult males and females and to assess the influence of gonadal hormones on arterial pressure in these animals. Offspring of pregnant rats treated with testosterone propionate or its vehicle (controls) were examined. Subsets of male and female offspring were gonadectomized at 7 wk of age, and some offspring from age 7 to 24 wk received hormone replacement, while others did not. Testosterone exposure during prenatal life significantly increased arterial pressure in both male and female adult offspring; however, the effect was greater in males. Prenatal androgen-exposed males and females had more circulating testosterone during adult life, with no change in estradiol levels. Gonadectomy prevented hyperandrogenism and also reversed hypertension in these rats. Testosterone replacement in orchiectomized males restored hypertension, while estradiol replacement in ovariectomized females was without effect. Steroidal changes were associated with defective expression of gonadal steroidogenic genes, with Star, Sf1, and Hsd17b1 upregulation in testes. In ovaries, Star and Cyp11a1 genes were upregulated, while Cyp19 was downregulated. This study showed that prenatal testosterone exposure led to development of gonad-dependent hypertension during adult life. Defective steroidogenesis may contribute in part to the observed steroidal changes.

Maternal protein restriction regulates IGF2 system in placental labyrinth.

This study was to test the hypothesis that altered IGF2 system in the placental labyrinth zone (LZ) impairs feto-placental growth in response to maternal protein restriction. Rats were fed a 20% protein diet and an isocaloric 6 % protein diet (LP) from day 1 to days 14, 18, or 21 of pregnancy. The effects of diet, gender of placenta and fetus, and day of pregnancy on placental weight, fetal weight, and expression of the IGF2 axis in the placental LZ and amino acids in maternal plasma were analyzed. Growth restriction occurred in both female and male fetuses by LP, coincident with impaired LZ growth and efficiency. The expression of Igf2, Igf2P0, Igf1r, Igf2r, Insr, Igfbp1, and Igfbp2 in placental LZ were affected by diet, gender and/or day of pregnancy. Concentrations of total essential amino acids and total nonessential amino acids were reduced and increased, respectively, in maternal plasma of LP-fed rats. These results indicate that adaptation of the IGF2 system in rat LZ occurs in a sex- and time-dependent manner in response to maternal protein restriction; however, these adaptations cannot prevent the growth restriction of both male and female fetuses during late pregnancy.

Prenatal testosterone-induced fetal growth restriction is associated with down-regulation of rat placental amino acid transport.

BACKGROUND: Exposure of pregnant mothers to elevated concentrations of circulating testosterone levels is associated with fetal growth restriction and delivery of small-for-gestational-age babies. We examined whether maternal testosterone crosses the placenta to directly suppress fetal growth or if it modifies placental function to reduce the capacity for transport of nutrients to the fetus. METHODS: Pregnant rats were exposed to testosterone propionate (TP; 0.5 mg/kg) by daily subcutaneous injection from gestational days (GD) 15-19. Maternal and fetal testosterone levels, placental nutrient transport activity and expression of transporters and birth weight of pups and their anogenital distances were determined. RESULTS: This dose of TP doubled maternal testosterone levels but had no effect on fetal testosterone levels. Maternal daily weight gain was significantly lower only on GD 19 in TP treated dams compared to controls. Placental weight and birth weight of pups were significantly reduced, but the anogenital distance of pups were unaffected by TP treatment. Maternal plasma amino acids concentrations were altered following testosterone exposure, with decreases in glutamine, glycine, tyrosine, serine, proline, and hydroxyproline and increases in asparagine, isoleucine, leucine, lysine, histidine and arginine. In the TP dams, placental system A amino acid transport activity was significantly reduced while placental glucose transport capacity was unaffected. Decreased expression of mRNA and protein levels of slc38a2/Snat2, an amino acid transporter, suggests that reduced transporter proteins may be responsible for the decrease in amino acid transport activity. CONCLUSIONS: Taken together, these data suggest that increased maternal testosterone concentrations do not cross the placenta to directly suppress fetal growth but affects amino acid nutrient delivery to the fetus by downregulating specific amino acid transporter activity.

Raf-1 kinase regulates smooth muscle contraction in the rat mesenteric arteries.

We investigated the potential role of Raf-1 kinase in mesenteric arterial contraction. Inhibitors of Raf-1 kinase, GW5074, L779450 and ZM 336372 reversed phenylephrine (PE)-induced mesenteric vascular contraction. Studies in vivo in rats showed that GW5074 inhibited PE-induced increase in mean arterial pressure in adult female Sprague-Dawley rats. Isometric tension studies in mesenteric arteries of rats showed that GW5074 did not change the KCl-evoked contraction but significantly inhibited the contractions to PE, 5-HT, U46619, endothelin 1, angiotensin II and phorbol 12, 13-dibutyrate (PDBu). In mesenteric vascular smooth muscle cells (VSMCs), PE stimulated increase in Raf-1 phosphorylation which was inhibited by GW5074. Measurement of [Ca(2+)](i) with Fura-2 showed that GW5074-mediated inhibition of PE-induced contraction was not associated with decreases in [Ca(2+)](i). VSMCs treated with PE exhibited higher levels of the contractile proteins, p-MYPT1 and p-MLC(20), which was inhibited by GW5074. Similarly, PDBu induced increases in phosphorylation of Raf-1, MLC(20) and MYPT1 and this was inhibited by GW5074. However, GW5074 did not have any significant effect on PE/PDBu-induced MEK/ERK activation. The results indicate that Raf-1 kinase plays an important role in the regulation of vascular contractility through regulation of calcium sensitization.

Protein restriction during pregnancy induces hypertension and impairs endothelium-dependent vascular function in adult female offspring.

Intrauterine undernutrition plays a role in the development of adult hypertension. Most studies are done in male offspring to delineate the mechanisms whereby blood pressure may be raised; however, the vascular mechanisms involved in female offspring are unclear. Female offspring of pregnant Sprague-Dawley rats fed either a control (C; 18%) or a low-protein (LP; 6%) diet during pregnancy were used. Birth weight and later growth were markedly lower in LP than in C offspring. LP offspring exhibited impaired estrous cyclicity with increased mean arterial pressure. Hypotensive response to acetylcholine (ACh) and the hypertensive response to phenylephrine (PE) were greater in LP than in C rats. N-nitro-L-arginine methyl ester (L-NAME) induced greater hypertensive responses in C than in LP rats. Endothelium-intact mesenteric arteries from LP offspring exhibited increased contractile responses to PE and reduced vasodilation in response to ACh. In endothelium-denuded arteries, relaxation responses to sodium nitroprusside were similar in both groups. Basal and ACh-induced increase in vascular nitrite/nitrate production was lower in LP than in C offspring. L-NAME or 1H-1,2,4-oxadiazolo-4,3-quinoxalin-1-one inhibited ACh relaxations and enhanced PE contractions in C offspring, but had minimal effect in LP rats. The decreased NO-mediated vascular response might explain the increased vascular contraction and arterial pressure in female offspring with low birth weight.