Assessment of an AI-based tool for population-wide collection of placental morphological data.

OBJECTIVES: Automated placental assessment could allow accurate and timely morphological/pathological measurements at scale. We undertook a pilot study using an artificial intelligence-based assessment system (AI-PLAX) to ascertain the potential of a state-wide rollout as part of Generation Victoria, assessing the impact of time post-delivery, user, and technology used for image capture, on a range of derived placental data. STUDY DESIGN: Ten placentas were imaged by three different users and imaging technologies (iPad, iPhone, Samsung) at (0 h), 24 h, and 48 h post-delivery. Using AI-PLAX, disc size (short and long length, perimeter, area), shape (normal, abnormal), cord insertion type (central, eccentric), cord coiling, abruption (retroplacental hematoma), and meconium staining were determined. RESULTS: When analysing the maternal surface of the placenta, time in cold storage post-delivery had modest effects on placental dimensions, with decreases in the short length (24-48 h: -3.7 %), disc area (0-24 h: 4.7 % and 0-48 h: -7.4 %), and perimeter (0-48 h: -3.8 %) observed. There was marginal impact on placental dimensions when the placenta was imaged by different users, including long length (+1.9 %), disc area (+2.9 %), and perimeter (+2.0 %). Measures of placental size were not impacted by the type of technology used to capture the images. When analysing the fetal surface of the placenta, more variance in placental size measures were observed between users. Abruption detection was not affected by any parameter. Time between delivery and imaging impacted apparent meconium staining - likely reflecting changes in fetal surface colour over time. Meconium staining was not affected by technology or user. CONCLUSIONS: This study supports the feasibility of the collection of placenta images for later morphological analysis by AI-PLAX, with measures obtained minimally influenced by time in cold storage, user imaging the placenta, or technology to capture the images.

Pregnant biglycan knockout mice have altered cardiorenal adaptations and a shorter gestational length, but do not develop a pre-eclamptic phenotype.

INTRODUCTION: Pre-eclampsia complicates 4.6% of pregnancies and is linked to impaired placentation; likely due to dysregulated vasculogenesis/angiogenesis. Proteoglycans, such as biglycan, are located on the endothelial surface of fetal capillaries. Biglycan is reduced in the placenta of pregnancies complicated by fetal growth restriction and pre-eclampsia. Importantly, biglycan stimulates angiogenesis in numerous tissues. Therefore, this study investigated whether biglycan knockdown in mice results in a pre-eclamptic phenotype. METHODS: Wild-type (WT) and Bgn-/- mice underwent cardiorenal measurements prior to and during pregnancy. One cohort of mice underwent post-mortem on gestational day 18 (E18) and another cohort underwent post-mortem on postnatal day 1 (PN1), with maternal and offspring tissues of relevance collected. RESULTS: Bgn-/- dams had increased heart rate (+9%, p < 0.037) and reduced systolic (-11%, p < 0.001), diastolic (-15%, p < 0.001), and mean arterial (-12%, p < 0.001) pressures at all ages investigated compared to WT. Additionally, Bgn-/- dams had reduced urine flow rate (-64%, p < 0.001) as well as reduced urinary excretions (-49%, p < 0.004) during late gestation compared to WT. Bgn-/- pups had higher body weight (+8%, p = 0.004; E18 only) and a higher liver-to-brain weight ratio (+43%, p < 0.001). Placental weight was unaltered with only minor changes in vasculogenic and angiogenic gene abundances detected, which did not correlate to changes in protein expression. DISCUSSION: This study demonstrated that total knockdown of biglycan is not associated with features of pre-eclampsia.

The transgenerational effect of maternal and paternal F1 low birth weight on bone health of second and third generation offspring.

Low birth weight programs diseases in adulthood, including adverse bone health. These diseases can have intergenerational and transgenerational origins, whereby transmission to subsequent generations occurs via both parental lines. Uteroplacental insufficiency surgery (Restricted) or sham surgery (Control) was performed on gestational day 18, in F0 Wistar-Kyoto rats. F1 Restricted males and females mated with breeders in order to generate F2 offspring of maternal and paternal lineages. F2 males and females were randomly selected for breeding to generate F3 offspring. F2 and F3 offspring did not have differences in birth weight irrespective of F1 low birth weight and parental line. Maternal line females had minor alterations to trabecular content and density at 6 months, these differences were not sustained at 12 months. Maternal line males had changes to trabecular content at 6 and 12 months; however, differences were no longer present at 16 months. Despite altered bone geometry at 12 and 16 months, bending strength remained unaffected at both ages. Bone health of paternal line females was not affected at 6 and 12 months. Paternal line males at 6 months had changes to trabecular and cortical content; cortical thickness, periosteal circumference and bending strength; however, these differences were no longer sustained at 12 and 16 months. Our data demonstrate that there is no transgenerational transmission of adverse bone health in F2 and F3 offspring, derived from low F1 birth weight females and males. Our results are novel, as bone health across generations and both parental lines has not been investigated in a model of low birth weight due to uteroplacental insufficiency.

Fetal growth restriction shortens cardiac telomere length, but this is attenuated by exercise in early life.

BACKGROUND AND AIMS: Fetal and postnatal growth restriction cause a predisposition to cardiovascular disease (CVD) in adulthood. Telomeres are repetitive DNA-protein structures that protect chromosome ends, and the loss of these repeats (a reduction in telomere length) is associated with CVD. As exercise preserves telomere length and cardiovascular health, the aim of this study was to determine the effects of growth restriction and exercise training on cardiac telomere length and telomeric genes. METHODS AND RESULTS: Pregnant Wistar Kyoto rats underwent bilateral uterine vessel ligation to induce uteroplacental insufficiency and fetal growth restriction ("Restricted"). Sham-operated rats had either intact litters ("Control") or their litters reduced to five pups with slowed postnatal growth ("Reduced"). Control, Restricted, and Reduced male rats were assigned to Sedentary, Early exercise (5-9 wk of age), or Late exercise (20-24 wk of age) groups. Hearts were excised at 24 wk of age for telomere length and gene expression measurements by quantitative PCR. Growth restriction shortened cardiac telomere length ( P < 0.001), but this was rescued by early exercise ( P < 0.001). Early and Late exercise increased cardiac weight index ( P < 0.001), but neither this nor telomere length was associated with expression of the telomeric genes Tert, Terc, Trf2, Pnuts, or Sirt1. DISCUSSION AND CONCLUSIONS: Growth restriction shortens cardiac telomere length, reflecting the cardiac pathologies associated with low birth weight. Exercise in early life may offer long-term protective effects on cardiac telomere length, which could help prevent CVD in later life.

Sustained cardiac programming by short-term juvenile exercise training in male rats.

KEY POINTS: Cardiac hypertrophy following endurance-training is thought to be due to hypertrophy of existing cardiomyocytes. The benefits of endurance exercise on cardiac hypertrophy are generally thought to be short-lived and regress to sedentary levels within a few weeks of stopping endurance training. We have now established that cardiomyocyte hyperplasia also plays a considerable role in cardiac growth in response to just 4 weeks of endurance exercise in juvenile (5-9 weeks of age) rats. The effect of endurance exercise on cardiomyocyte hyperplasia diminishes with age and is lost by adulthood. We have also established that the effect of juvenile exercise on heart mass is sustained into adulthood. ABSTRACT: The aim of this study was to investigate if endurance training during juvenile life 'reprogrammes' the heart and leads to sustained improvements in the structure, function, and morphology of the adult heart. Male Wistar Kyoto rats were exercise trained 5 days week-1 for 4 weeks in either juvenile (5-9 weeks of age), adolescent (11-15 weeks of age) or adult life (20-24 weeks of age). Juvenile exercise training, when compared to 24-week-old sedentary rats, led to sustained increases in left ventricle (LV) mass (+18%; P < 0.05), wall thickness (+11%; P < 0.05), the longitudinal area of binucleated cardiomyocytes (P < 0.05), cardiomyocyte number (+36%; P < 0.05), and doubled the proportion of mononucleated cardiomyocytes (P < 0.05), with a less pronounced effect of exercise during adolescent life. Adult exercise training also increased LV mass (+11%; P < 0.05), wall thickness (+6%; P < 0.05) and the longitudinal area of binucleated cardiomyocytes (P < 0.05), despite no change in cardiomyocyte number or the proportion of mono- and binucleated cardiomyocytes. Resting cardiac function, LV chamber dimensions and fibrosis levels were not altered by juvenile or adult exercise training. At 9 weeks of age, juvenile exercise significantly reduced the expression of microRNA-208b, which is a known regulator of cardiac growth, but this was not sustained to 24 weeks of age. In conclusion, juvenile exercise leads to physiological cardiac hypertrophy that is sustained into adulthood long after exercise training has ceased. Furthermore, this cardiac reprogramming is largely due to a 36% increase in cardiomyocyte number, which results in an additional 20 million cardiomyocytes in adulthood.

Uteroplacental insufficiency in rats induces renal apoptosis and delays nephrogenesis completion.

AIM: Uteroplacental insufficiency in rats reduces nephron endowment, leptin concentrations and programmes cardiorenal disease in offspring. Cross-fostering growth-restricted (Restricted) offspring onto a mother with normal lactation restores leptin concentrations and nephron endowment. This study aimed to determine whether the reduced nephron endowment in Restricted offspring is due to delayed glomerular formation and dysregulation of renal genes regulating branching morphogenesis, apoptosis or leptin signalling. Furthermore, we aimed to investigate whether cross-fostering Restricted offspring onto Control mothers could improve glomerular maturation and restore renal gene abundance. METHODS: Uteroplacental insufficiency was induced by bilateral uterine vessel ligation (Restricted) or sham (Control) surgery on gestation day 18 (E18). Kidneys were collected at E20, postnatal day 1 (PN1) and PN7. An additional cohort was cross-fostered onto separate mothers at birth and kidneys collected at PN7. RESULTS: Kidneys were lighter in the Restricted group, but weight was restored with cross-fostering. At E20, abundance of Bax, Flt1 and Vegfa was increased in Restricted offspring, while Ret and Bcl2 transcripts were increased only in Restricted females. At PN7, abundance of Gdnf and Ret was higher in Restricted offspring, as was Casp3. Restricted offspring had a wider nephrogenic zone with more immature glomeruli suggesting a delayed or extended nephrogenic period. Cross-fostering had subtle effects on gene abundance and glomerular maturity. CONCLUSION: Uteroplacental insufficiency induced apoptosis in the developing kidney and delayed and extended nephrogenesis. Cross-fostering Restricted offspring onto Control mothers had beneficial effects on kidney growth and renal maturity, which may contribute to the restoration of nephron endowment.

Respiratory modulation of sympathetic nerve activity is enhanced in male rat offspring following uteroplacental insufficiency.

Sympathetic nerve activity to the cardiovascular system displays prominent respiratory-related modulation which leads to the generation of rhythmic oscillations in blood pressure called Traube-Hering waves. An amplification of this respiratory modulation of sympathetic activity is observed in hypertension of both genetic, the spontaneously hypertensive rat, and induced, chronic intermittent hypoxia or maternal protein restriction during gestation, origin. Male offspring of mothers with uteroplacental insufficiency, induced by bilateral uterine vessel ligation at 18 days of gestation, are also hypertensive in adulthood. In this study we examined whether these male offspring display altered respiratory modulation of sympathetic activity at pre-hypertensive ages compared to controls. Respiratory, cardiovascular and sympathetic parameters were examined using the working heart-brainstem preparation in 35 day old male rats that had reduced birth weight due to uteroplacental insufficiency. Whilst all respiratory parameters were not different between groups, we observed an enhanced respiratory-related burst of thoracic sympathetic nerve activity and amplified Traube-Hering waves in the growth-restricted group. This group also showed an increased sympathetic and bradycardic response to activation of peripheral chemoreceptors. The observations add support to the view that altered respiratory modulation of sympathetic activity represents a common mechanism involved in the development of several forms of hypertension.

The role of maternal nutrition, metabolic function and the placenta in developmental programming of renal dysfunction.

The intrauterine environment is critical for the development of the foetus. Barker and colleagues were the first to identify that adverse perturbations during foetal development are associated with an increased risk of developing diseases in adulthood, including cardiorenal disease. Specifically for the kidney, perturbations in utero can lead to nephron deficits and renal dysfunction by a number of mechanisms. Altered programming of nephron number is associated with an increased risk of developing kidney disease via glomerular hypertrophy and reduced vasodilative capacity of the renal blood vessels; both of which would contribute to hypertension in adulthood, with males being more susceptible to disease outcomes. Additionally, alterations in the renin-angiotensin system (RAS) such as an upregulation or downregulation of specific receptors, depending on the nature of the insult, have also been implicated in the development of renal dysfunction. Sex-specific differences in the expression of the RAS during late gestation and in the early postnatal environment have also been identified. Extensive research has demonstrated that both uteroplacental insufficiency and maternal malnutrition alter renal development in utero. Equally, exposure to maternal diabetes and maternal obesity during development are also associated with an increased risk of developing renal disease, however, the mechanism behind this association is poorly understood. Therefore, identifying the link between an adverse intrauterine environment and the programmed kidney disease risk in adulthood may facilitate the development of strategies to alleviate the epidemics of cardiorenal disease worldwide, in addition to understanding why males are more susceptible to adult-onset cardiovascular diseases.

Growth restriction in the rat alters expression of cardiac JAK/STAT genes in a sex-specific manner.

Uteroplacental insufficiency resulting in intrauterine growth restriction has been associated with the development of cardiovascular disease, coronary heart disease and increased blood pressure, particularly in males. The molecular mechanisms that result in the programming of these phenotypes are not clear. This study investigated the expression of cardiac JAK/STAT signalling genes in growth restricted offspring born small due to uteroplacental insufficiency. Bilateral uterine vessel ligation was performed on day 18 of pregnancy to induce growth restriction (Restricted) or sham surgery (Control). Cardiac tissue at embryonic day (E) 20, postnatal day (PN) 1, PN7 and PN35 in male and female Wistar (WKY) rats (n=7-10 per group per age) was isolated and mRNA extracted. In the heart, there was an effect of age for males for all genes examined there was a decrease in expression after PN1. With females, JAK2 expression was significantly reduced after E20, while PI3K in females was increased at E30 and PN35. Further, mRNA expression was significantly altered in JAK/STAT signalling targets in Restricteds in a sex-specific manner. Compared with Controls, in males, JAK2 and STAT3 were significantly reduced in the Restricted, while in females SOCS3 was significantly increased and PI3K significantly decreased in the Restricted offspring. Finally, there were specific differences in the levels of gene expression within the JAK/STAT pathway when comparing males to females. Thus, growth restriction alters specific targets in the JAK/STAT signalling pathway, with altered JAK2 and STAT3 potentially contributing to the increased risk of cardiovascular disease in the growth restricted males.

Periconceptional alcohol consumption causes fetal growth restriction and increases glycogen accumulation in the late gestation rat placenta.

INTRODUCTION: Alcohol consumption is a common social practice among women of childbearing age. With 50% of pregnancies being unplanned, many embryos are exposed to alcohol prior to pregnancy recognition and formation of the placenta. The effects of periconceptional (PC) alcohol exposure on the placenta are unknown. METHODS: Sprague-Dawley rats were exposed to alcohol (12.5% v/v ad libitum) from 4 days prior to 4 days after conception and effects on placental growth, morphology and gene/protein expression examined at embryonic day (E) 20. RESULTS: PC ethanol (EtOH)-exposed fetuses were growth restricted and their placental/body weight ratio and placental cross-sectional area were increased. This was associated with an increase in cross-sectional area of the junctional zone and glycogen cells, especially in PC EtOH-exposed placentas from female fetuses. Junctional Glut1 and Igf2 mRNA levels were increased. Labyrinth Igf1 mRNA levels were decreased in placentas from both sexes, but protein IGF1R levels were decreased in placentas from male fetuses only. Labyrinth mRNA levels of Slc38a2 were decreased and Vegfa were increased in placentas following PC EtOH-exposure but only placentas from female fetuses exhibited increased Kdr expression. Augmented expression of the protective enzyme 11ßHsd2 was found in PC EtOH-exposed labyrinth. DISCUSSION: These observations are consistent with a stress response, apparent well beyond the period of EtOH-exposure and demonstrate that PC EtOH alters placental development in a sex specific manner. CONCLUSION: Public awareness should be increased to educate women about how excessive drinking even before falling pregnant may impact on placental development and fetal health.

No change in calreticulin with fetal growth restriction in human and rat pregnancies.

INTRODUCTION: Calreticulin is a ubiquitously expressed protein that was detected in the circulation and is significantly increased in maternal blood during human pregnancy compared to the non-pregnant state. Calreticulin is further increased in the plasma of women with the pregnancy-related disorder pre-eclampsia compared to normotensive pregnancy. The aims of this study were to compare calreticulin in human pregnancy with calreticulin in rat pregnancy, and to compare calreticulin during fetal growth restriction with normal control pregnancies. METHODS: Women were recruited who either had normal pregnancies or had pregnancies complicated with fetal growth restriction; maternal blood samples and placentas were collected. Blood was also taken from women who were not-pregnant. Growth restriction was induced in pregnant rats by uterine vessel ligation; blood and placental samples were collected. Blood was also taken from non-pregnant rats. Western blot was used to quantify the placental expression of calreticulin and the concentrations of calreticulin in plasma. RESULTS: Although calreticulin was significantly increased in maternal plasma during human pregnancy compared to the non-pregnant state; it did not increase in plasma during rat pregnancy. These results suggest that there may be differences in the role of extracellular calreticulin in human compared to rat pregnancy. Calreticulin was not significantly altered in either placental extracts or maternal plasma in both the human and rat pregnancies complicated by fetal growth restriction compared to gestational matched control pregnancies. CONCLUSION: This study found that there was no change in calreticulin during human pregnancy complicated with fetal growth restriction or when growth restriction is induced in rats.

Stage of perinatal development regulates skeletal muscle mitochondrial biogenesis and myogenic regulatory factor genes with little impact of growth restriction or cross-fostering.

Foetal growth restriction impairs skeletal muscle development and adult muscle mitochondrial biogenesis. We hypothesized that key genes involved in muscle development and mitochondrial biogenesis would be altered following uteroplacental insufficiency in rat pups, and improving postnatal nutrition by cross-fostering would ameliorate these deficits. Bilateral uterine vessel ligation (Restricted) or sham (Control) surgery was performed on day 18 of gestation. Males and females were investigated at day 20 of gestation (E20), 1 (PN1), 7 (PN7) and 35 (PN35) days postnatally. A separate cohort of Control and Restricted pups were cross-fostered onto a different Control or Restricted mother and examined at PN7. In both sexes, peroxisome proliferator-activated receptor (PPAR)-γ coactivator-1α (PGC-1α), cytochrome c oxidase subunits 3 and 4 (COX III and IV) and myogenic regulatory factor 4 expression increased from late gestation to postnatal life, whereas mitochondrial transcription factor A, myogenic differentiation 1 (MyoD), myogenin and insulin-like growth factor I (IGF-I) decreased. Foetal growth restriction increased MyoD mRNA in females at PN7, whereas in males IGF-I mRNA was higher at E20 and PN1. Cross-fostering Restricted pups onto a Control mother significantly increased COX III mRNA in males and COX IV mRNA in both sexes above controls with little effect on other genes. Developmental age appears to be a major factor regulating skeletal muscle mitochondrial and developmental genes, with growth restriction and cross-fostering having only subtle effects. It therefore appears that reductions in adult mitochondrial biogenesis markers likely develop after weaning.

Growth restriction alters adult spatial memory and sensorimotor gating in a sex-specific manner.

In Western society, impaired uteroplacental blood flow is the major cause of human intrauterine growth restriction. Infants born small and who experience late childhood accelerated growth have an increased risk of developing adult diseases. Recent studies also suggest a link between birth weight and altered adult behavior, particularly relating to motor function, learning and memory, depression and schizophrenia. The aim of this study was to determine the relative influence of prenatal and postnatal growth restriction on adult behavioral outcomes in male and female rats. Uteroplacental insufficiency was induced in Wistar Kyoto rats by bilateral uterine vessel ligation on day 18 of gestation producing growth-restricted offspring (Restricted group). The Control group had sham surgery. Another group underwent sham surgery, with a reduction in litter size to five at birth equivalent to the Restricted litter size (Reduced Litter group). At 6 months of age, a series of behavioral tests were conducted in male and female offspring. Growth restriction did not impair motor function. In fact, Restricted and Reduced Litter males showed enhanced motor performance compared with Controls (P < 0.05). Spatial memory was greater in Restricted females only (P < 0.05). The Porsolts test was unremarkable, however, males exhibited more depressive-like behavior than females (P < 0.05). A reduction in sensorimotor gating function was identified in Reduced Litter males and females (P < 0.05). We have demonstrated that growth restriction and/or a poor lactational environment can affect adult rat behavior, particularly balance and coordination, memory and learning, and sensorimotor gating function, in a sex-specific manner.

Review: Sex specific programming: a critical role for the renal renin-angiotensin system.

The "Developmental Origins of Health and Disease" hypothesis has caused resurgence of interest in understanding the factors regulating fetal development. A multitude of prenatal perturbations may contribute to the onset of diseases in adulthood including cardiovascular and renal diseases. Using animal models such as maternal glucocorticoid exposure, maternal calorie or protein restriction and uteroplacental insufficiency, studies have identified alterations in kidney development as being a common feature. The formation of a low nephron endowment may result in impaired renal function and in turn may contribute to disease. An interesting feature in many animal models of developmental programming is the disparity between males and females in the timing of onset and severity of disease outcomes. The same prenatal insult does not always affect males and females in the same way or to the same degree. Recently, our studies have focused on changes induced in the kidney of both the fetus and the offspring, following a perturbation during pregnancy. We have shown that changes in the renin-angiotensin system (RAS) occur in the kidney. The changes are often sex specific which may in part explain the observed sex differences in disease outcomes and severity. This review explores the evidence suggesting a critical role for the RAS in sex specific developmental programming of disease with particular reference to the immediate and long term changes in the local RAS within the kidney.

Cross-fostering and improved lactation ameliorates deficits in endocrine pancreatic morphology in growth-restricted adult male rat offspring.

Uteroplacental insufficiency and poor postnatal nutrition impair adult glucose tolerance and insulin secretion in male rat offspring, which can be partially ameliorated by improving postnatal nutrition. Uteroplacental insufficiency was induced in the WKY rat on day 18 of pregnancy (Restricted) compared to sham-operated Controls. Pups were then cross-fostered onto Control or Restricted mothers one day after birth resulting in: (Pup-on-Mother) Control-on-Control, Control-on-Restricted, Restricted-on-Control and Restricted-on-Restricted. Endocrine pancreatic morphology and markers of intrinsic ß-cell function and glucose homeostasis were assessed in male offspring at 6 months. Pancreatic and hepatic gene expression was quantified at postnatal day 7 and 6 months. Restricted pups were born 10-15% lighter than Controls and remained lighter at 6 months. Relative islet and ß-cell mass were 51-65% lower in Restricted-on-Restricted compared to Controls at 6 months. Non-fasting plasma C-reactive protein levels were also increased, suggestive of an inflammatory response. Overall, the average number of islets, small islets and proportion of ß-cells per islet correlated positively with birth weight. Intrinsic ß-cell function, estimated by insulin secretion relative to ß-cell mass, was unaffected by Restriction, suggesting that the in vivo functional deficit was attributable to reduced mass, not function. Importantly, these deficits were ameliorated when lactational nutrition was normalized in Restricted-on-Control offspring, who also showed increased pancreatic Igf1r, Pdx1 and Vegf mRNA expression at 7 days compared to Control-on-Control and Restricted-on-Restricted. This highlights lactation as a critical period for intervention following prenatal restraint, whereby deficits in endocrine pancreatic mass and associated impaired in vivo insulin secretion can be ameliorated.

Growth restriction before and after birth increases kinase signaling pathways in the adult rat heart.

To investigate the mechanisms for the previously reported development of adult cardiac hypertrophy in male rats following growth restriction, the levels of oxidative stress and activation of signaling kinases were measured in the left ventricle (LV) of adult rat offspring. In experiment one, bilateral uterine vessel ligation to induce uteroplacental insufficiency and growth restriction in the offspring (Restricted) or sham surgery was performed during pregnancy. Litters from sham mothers had litter size either reduced (Reduced Litter), which also restricted postnatal growth, or were left unaltered (Control). In males, Reduced Litter offspring had increased LV phosphorylation of AMPKα, p38 MAPK and Akt compared with Restricted and Controls (P < 0.05). In females, both Restricted and Reduced Litter adult offspring had increased LV phosphorylation of p38 MAPK and Akt, however, only Restricted offspring had increased phosphorylation of AMPKα (P < 0.05). In addition, only Restricted male offspring displayed LV oxidative stress (P < 0.05). Experiment two investigated in mothers exposed to uteroplacental insufficiency or sham surgery the effects of cross-fostering offspring at birth, and therefore the effects of the postnatal lactational environment. Surprisingly, the cross-fostering itself resulted in increased LV phosphorylation of AMPKα and Akt in females and increased phosphorylation of Akt in males compared with Control non-cross-fostered offspring (P < 0.05). In conclusion, kinase signaling in the adult LV can be programmed by uteroplacental insufficiency induced growth restriction in a gender-specific manner. In addition, the heart of adult rats is also sensitive to programming following the postnatal intervention of cross-fostering alone as well as by postnatal growth restriction.

Identification of the Alzheimer's disease amyloid precursor protein (APP) and its homologue APLP2 as essential modulators of glucose and insulin homeostasis and growth.

The amyloid precursor protein (APP), the source of the neurotoxic amyloid beta (A beta) peptide involved in Alzheimer's disease (AD), belongs to a conserved family of related proteins. In mammals, the APP family contains amyloid precursor-like protein 1 (APLP1) and amyloid precursor-like protein 2 (APLP2). Whilst a number of activities have been attributed to the APP family, an overall function has not been definitively established. While ablating either the APP or APLP2 gene in mice produces minimal phenotypic change, the combined knockout of these genes in mice causes postnatal mortality. Postnatal survival therefore requires a shared but unknown function of APP and APLP2. To investigate the biochemical basis for the postnatal lethality, plasma was analysed from double knockout mice (APP-/- APLP2-/-) 2 days before birth, at gestational day E17, and from mice at 12-16 h after birth. The postnatal double knockouts had 66% lower plasma glucose levels than their wild-type controls and 50% lower than their single knockout counterparts. Interestingly, the postnatal double knockouts displayed hyperinsulinaemia, as shown by inappropriate plasma insulin levels, given their degree of hypoglycaemia. The single knockout mice also showed hyperinsulinaemia and had 31% lower plasma glucose than the wild-types. While the double knockouts did not survive more than 24 h after birth, the single knockouts reached adulthood and their hypoglycaemia continued. Therefore, APP and APLP2 expression modulates plasma insulin and glucose concentrations. Plasma calcium, magnesium and phosphate were also significantly reduced in the double knockouts compared to the wild-types, and they showed distinctive growth restriction, suggesting the involvement of a metabolic impairment. These results link the expression of the APP and APLP2 genes with glucose homeostasis and growth and therefore identify a novel function for the APP family.

Effect of nuclear factor-kappa B inhibitors and peroxisome proliferator-activated receptor-gamma ligands on PTHrP release from human fetal membranes.

Parathyroid hormone-related protein (PTHrP) has been implicated in many processes during normal and pathological pregnancies. In the human fetal membranes, PTHrP exhibits cytokine-like actions. We have recently shown that inhibitors of the nuclear factor-kappa B (NF-kappaB) and activators of the peroxisome proliferator-activated receptor (PPAR)-gamma signalling pathways down-regulate cytokine release from human gestational tissues. Therefore, the aim of this study was to determine whether NF-kappaB and PPAR-gamma also regulate PTHrP release from human fetal membranes. Human amnion and choriodecidua explants were incubated in the absence (control) or presence of two known NF-kappaB inhibitors (1, 5 and 10 mM sulphasalazine (SASP) or 5, 10 and 15 mM N-acetyl-cysteine (NAC)), and two PPAR-gamma ligands (15 and 30 microM 15-deoxy-Delta(12,14)-PGJ(2) (15d-PGJ(2)) or 15 and 30 microM troglitazone), under basal conditions. After 18 h incubation, the tissues were collected and NF-kappaB p65 DNA binding activity in nuclear extracts was assessed by ELISA, and the incubation medium was collected and the release of PTHrP was quantified by RIA. Treatment of amnion and choriodecidual tissues with SASP concentrations greater than 5 mM, 15 mM NAC, 30 microM 15d-PGJ(2) and 30 microM troglitazone significantly reduced the release of PTHrP (p < 0.05). This study demonstrates that PTHrP release from human fetal membranes is regulated by inhibitors of NF-kappaB, and ligands of PPAR-gamma.

PTH/PTHrP receptor and mid-molecule PTHrP regulation of intrauterine PTHrP: PTH/PTHrP receptor antagonism increases SHR fetal weight.

Parathyroid hormone-related protein (PTHrP) has important roles in fetal growth and development through stimulation of placental calcium transport, vasodilatation of the uteroplacental vasculature and regulation of cellular growth and differentiation. The growth restricted spontaneously hypertensive rat (SHR) has reduced fetal plasma, placental and amniotic fluid PTHrP concentrations compared to its progenitor, the Wistar Kyoto (WKY) rat. The aim of this study was to determine whether intrauterine PTHrP infusions can restore PTHrP levels and promote SHR fetal growth. PTHrP(1-34), midmolecule PTHrP(67-94), the PTH/PTHrP receptor antagonist [Asn(10), Leu(11)]-PTHrP(7-34) or vehicle were infused via a mini-osmotic pump between 10 and 20 days of gestation into the uterine lumen of SHR and WKY rats. Uterine, placental, amniotic fluid and plasma (fetal and maternal) PTHrP were measured via N-terminal radioimmunoassay. PTH/PTHrP receptor antagonism and mid-molecule PTHrP(67-94) induced endogenous intrauterine PTHrP production with receptor antagonism eliciting a greater and more wide spread effect. The PTH/PTHrP receptor antagonist [Asn(10), Leu(11)]-PTHrP(7-34) acting through a receptor other than the PTH/PTHrP receptor increased SHR fetal and placental weights above vehicle (P<0.05) to that of the WKY and restored SHR amniotic fluid volume (P<0.05). This was associated with a highly significant up regulation of placental, uterine and plasma (fetal and maternal) PTHrP (P<0.05). Modest increases in placental and uterine PTHrP (P<0.05) following intrauterine infusions of PTHrP(1-34) and PTHrP(67-94) had no effect on WKY and SHR fetal weight. Effective growth promoting actions of increased endogenous PTHrP were observed following PTH/PTHrP receptor antagonism rather than exogenous PTHrP administration. A novel finding was that mid-molecule PTHrP also up regulates endogenous intrauterine N-terminal PTHrP production supporting the existence of a mid-molecule receptor. This study highlights that an increase in endogenous uterine, placental and fetal plasma PTHrP following PTH/PTHrP receptor antagonism was associated with increased SHR fetal growth presumably by improving placental growth and function.

Angiotensin II influences ovarian follicle development in the transgenic (mRen-2)27 and Sprague-Dawley rat.

There is accumulating evidence that local renin-angiotensin systems (RASs) influence cell growth and organ function in a variety of tissues including the ovary. The first aim of this study was to characterise the cellular location of RAS components in the rat ovary. This was facilitated by the use of the hypertensive transgenic (mRen-2)27 rat which overexpresses renin and angiotensin in extra-renal tissues. Comparisons were made with normal Sprague-Dawley (SD) rats. The second aim was to determine if the upregulated RAS of the transgenic (mRen-2)27 rat and infusion of angiotensin II (ANG II) in SD rats influences follicle number and litter size. Gene expression, immunohistochemical and autoradiographic techniques were used to identify a discrete RAS including ANG II receptors in the ovarian stroma, follicles (particularly atretic) and to a lesser extent corpora lutea. The RAS at these sites was most abundant in homozygous (HMZ) followed by heterozygous (HTZ) (mRen-2)27 rats and then SD rats. Large antral and preovulatory follicles and litter size were reduced in (mRen-2)27 rats. In HMZ (mRen-2)27 rats and SD rats infused with ANG II, angiotensin 1a (AT(1a)) receptor mRNA in the ovarian stroma was lower than control SD rats and was associated with a reduction in large antral and preovulatory follicles. These findings indicate that upregulation of the ovarian RAS in the rat influences follicular development and, potentially, reproductive capacity.