Kerryn T. Westcott

Growth restriction before or after birth reduces nephron number and increases blood pressure in male rats

Impaired growth in utero predicts a low nephron number and high blood pressure later in life as does slowed or accelerated growth after a normal birth weight. We measured the effects of early postnatal growth restriction, with or without prenatal growth restriction, on blood pressure and nephron number in male rat offspring. Bilateral uterine artery and vein ligation were performed to induce uteroplacental insufficiency (Restricted) on day 18 of pregnancy. Postnatal growth restriction was induced in a subset of sham operated control animals by reducing the number of pups at birth to that of the Restricted group (Reduced Litter). Compared to Controls, Restricted pups were born smaller while Reduced Litter pups weighed less by postnatal day 3 and both groups remained lighter throughout lactation. By 10 weeks of age all animals were of similar weight but the Reduced Litter rats had elevated blood pressure. At 22 weeks, Restricted but not Reduced Litter offspring were smaller and the blood pressure was increased in both groups. Restricted and Reduced Litter groups had fewer glomeruli and greater left ventricular mass than Controls. These results suggest that restriction of both perinatal and early postnatal growth increase blood pressure in male offspring. This study also demonstrates that the early postnatal period is a critical time for nephron endowment in the rat.

Improved lactational nutrition and postnatal growth ameliorates impairment of glucose tolerance by uteroplacental insufficiency in male rat offspring

Intrauterine growth restriction and accelerated postnatal growth predict increased risk of diabetes. Uteroplacental insufficiency in the rat restricts fetal growth but also impairs mammary development and postnatal growth. We used cross fostering to compare the influence of prenatal and postnatal nutritional restraint on adult glucose tolerance, insulin secretion, insulin sensitivity, and hypothalamic neuropeptide Y content in Wistar Kyoto rats at 6 months of age. Bilateral uterine vessel ligation (restricted) to induce uteroplacental insufficiency or sham surgery (control) was performed on d-18 gestation. Control, restricted, and reduced (reducing litter size of controls to match restricted) pups were cross fostered onto a control or restricted mother 1 d after birth. Restricted pups were born small compared with controls. Restricted males, but not females, remained lighter up to 6 months, regardless of postnatal environment. By 10 wk, restricted-on-restricted males ate more than controls. At 6 months restricted-on-restricted males had increased hypothalamic neuropeptide Y content compared with other groups, and together with reduced-on-restricted males had increased retroperitoneal fat weight (percent body weight) compared with control-on-controls. Restricted-on-restricted males had impaired glucose tolerance, reduced first-phase insulin secretion, but unaltered insulin sensitivity, compared with control-on-controls. In males, being born small and exposed to an impaired lactational environment adversely affects adult glucose tolerance and first-phase insulin secretion, but improving lactation partially ameliorates this condition. This study identifies early life as a target for intervention to prevent later diabetes after prenatal restraint.

Cardio‐renal and metabolic adaptations during pregnancy in female rats born small: implications for maternal health and second generation fetal growth

Non‐technical summary  Low weight at birth, or being born small for gestational age, is associated with increased risk of a number of adult diseases, including cardiovascular and kidney disease and diabetes. Generally, low birth weight males have a greater risk of developing such diseases but females do present with subtle changes in organ structure and function that might render them susceptible to lifestyle challenges. We show, for the first time, that low birth weight females have largely normal cardiovascular and kidney adaptations to pregnancy but they do develop altered glucose control. We have shown that their own fetuses are growth restricted suggesting that low birth weight and risk of disease development can be passed on to subsequent generations. These results warrant close monitoring of pregnant women who were born small and shape future studies to focus on therapeutic strategies to minimize the transmission of low birth weight and adult disease risk.

The effect of prenatal exposure to carbon monoxide on breathing and growth of the newborn guinea pig

In utero hypoxia may affect the development of the brain and result in altered respiratory responses postnatally. Using a barometric plethysmograph, we examined the effects of exposing pregnant guinea pigs to 200 ppm carbon monoxide (CO) for 10 h/d from d 23-25 of gestation until term(≈68 d) on the ventilatory responses of their 4-5-d-old neonates at rest, and during progressive asphyxia and steady state hypercapnia. Exposure to this concentration of CO produced significantly higher levels of carboxyhemoglobin(COHb) in maternal (8.53 ± 0.6% versus 0.25 ± 0.1%) and fetal blood (13.0 ± 0.4% versus 1.6 ± 0.1%) from CO-treated animals when compared with controls. Hematocrit was significantly higher in the CO-treated neonates (46.3 ± 1.0% versus 41.3± 0.9%) at 5-6 d of age, although no difference existed between the groups for COHb at this time. There was no difference between the groups for length of gestation, litter size, or birth weight, but CO-treated neonates were significantly smaller at 4 d of age (102.4 ± 3.7 g) compared with controls (132.0 ± 5.0 g). At 4-5 d of age there was no difference between the groups for either tidal volume (VT), respiratory frequency (f), or minute ventilation (VE) at rest, but during steady state hypercapnia (4 and 6% CO2) the CO-treated neonates had a significantly greater VT and VE (but not f) than did controls. During progressive asphyxia, CO-treated animals had a significantly greater VT than did controls from 1-8% CO2. There was a significant fall in f at 1 and 3% CO2 in CO-treated animals; however, this effect did not persist, resulting in a significantly increased VE from 3 to 8% CO2. The inspiratory flow rate(VT/expiratory time) was significantly increased in the CO-treated neonates during progressive asphyxia; this occurred in the absence of a difference in inspiratory time between the groups. These results indicate that prenatal exposure to CO increases CO2 sensitivity in 4-5-d-old guinea pigs. This may be due to developmental alterations in the areas of the brainstem responsible for respiratory control.

RELATIVE CONTRIBUTION OF THE PRENATAL VERSUS POSTNATAL PERIOD ON DEVELOPMENT OF HYPERTENSION AND GROWTH RATE OF THE SPONTANEOUSLY HYPERTENSIVE RAT

1 To determine the relative roles of the prenatal and postnatal (preweaning) environment on the development of blood pressure and growth rate in the spontaneously hypertensive rat (SHR) of the Okamoto strain, we used combined embryo transfer and cross‐fostering techniques between SHR and normotensive Wistar‐Kyoto (WKY) rats to produce offspring whose development was examined during the first 20 weeks of life. 2 We measured litter sizes, bodyweights and tail‐cuff blood pressures in offspring at 4, 8, 12 and 20 weeks of age. We also recorded heart, kidney and adrenal weights at 20 weeks of age, when the study concluded. 3 We found that both the in utero and postnatal environments provided by the SHR mother could significantly affect WKY rat offspring growth rates, but blood pressure was unaffected in this strain. In SHR offspring, the SHR maternal in utero and suckling period both contributed to the rate of blood pressure development in the SHR, but not the final blood pressure of offspring at 20 weeks of age. This effect was greater for male than female offspring. Organ weights were largely unaffected by the perinatal environment in either strain. 4 We conclude that although the SHR maternal in utero and immediate postnatal environment both contribute to the rate of blood pressure development in the SHR, they do not appear to contribute to the final blood pressure of offspring at maturity. The SHR maternal environment also alters growth rate that may, in turn, underlie these effects on SHR blood pressure development, particularly in males.

Growth restriction in the rat alters expression of metabolic genes during postnatal cardiac development in a sex-specific manner

This study investigated the impact of uteroplacental insufficiency and growth restriction on the expression of genes related to mitochondrial biogenesis, glucose transport, and antioxidant defenses in cardiac tissue at embryonic day 20 (E20) and postnatal days 1, 7, and 35 in male and female Wistar rats (8-10 per group). Bilateral uterine vessel ligation to induce growth restriction (Restricted) or sham surgery was performed at pregnancy day 18. In male and female Controls, expression of most cardiac genes decreased during postnatal life, including genes involved in mitochondrial biogenesis regulation such as PGC-1α, NRF-2, and mtTFA and the glucose transporter GLUT-1 (P < 0.05). However, the pattern of gene expression during cardiac development differed in male and female Restricted rats compared with their respective Controls. These effects of restriction were observed at postnatal day 1, with female Restricted rats having delayed reductions in PGC-1α and GLUT-1, whereas males had exacerbated reductions in PGC-1α and mtTFA (P < 0.05). By day 35, cardiac gene expression in Restricted hearts was similar to Controls, except for expression of the antioxidant enzyme MnSOD, which was significantly lower in both sexes. In summary, during postnatal life male and female Control rats have similar patterns of expression for genes involved in mitochondrial biogenesis and glucose transport. However, following uteroplacental insufficiency these gene expression patterns diverge in males and females during early postnatal life, with MnSOD gene expression reduced in later postnatal life.

Progesterone Withdrawal, and Not Increased Circulating Relaxin, Mediates the Decrease in Myometrial Relaxin Receptor (RXFP1) Expression in Late Gestation in Rats

In pregnant rats, a significant decrease in myometrial relaxin family peptide receptor 1 (RXFP1) expression, indicative of a functional relaxin withdrawal for activation of myometrial contractions, occurs in late gestation and during spontaneous labor. This coincides with the highest level of circulating relaxin and a decrease in progesterone. We investigated the potential regulatory role of these two systemic factors on myometrial RXFP1 expression by examining the effects of the antiprogestin RU486 and a monoclonal antibody against rat relaxin (MCA1) in pregnant rats. Rats were injected with RU486 on Gestational Day (GD) 7, 16, or 19 and were killed on GD 8, 17, or 20. RU486 caused a significant reduction in myometrial RXFP1. Plasma progesterone and 17beta-estradiol levels were increased in RU486-treated animals compared with controls. RU486 treatment also caused significant increases in myometrial Esr1 and Vegf and a decrease in Esr2. MCA1 was administered i.v. to rats from GD 17 to GD 19. On GD 20, no significant effect of MCA1 treatment on myometrial RXFP1 expression was observed compared with controls. Furthermore, there was no change in Esr1 or Esr2. A significant reduction in myometrial Vegf, however, was observed. We suggest that blocking progesterone action with RU486 increases plasma 17beta-estradiol and myometrial Esr1 and results in decreased RXFP1 expression. In summary, myometrial RXFP1 expression is mediated mainly by progesterone and not circulating relaxin in pregnant rats.

Brain allopregnanolone in the fetal and postnatal rat in response to uteroplacental insufficiency.

Background/Aims: Allopregnanolone suppresses central nervous system activity and has neuroprotective actions in hypoxia-induced brain injury. In pregnant sheep allopregnanolone concentrations are high during fetal life and decline rapidly after birth. We investigated brain allopregnanolone concentrations of fetal and postnatal rats derived from normal and growth restricted pregnancies. Methods: Bilateral uterine vessel ligation (or sham) was performed at gestation day 18 to induce uteroplacental insufficiency in WKY rats (n = 7–8 per group). Brain allopregnanolone was measured by radioimmunoassay at 2 study ages, gestation day 20 (n = 6 per group) and postnatal day 6 (n = 6–8 per group), from control and growth-restricted pups. Results: Fetal brain allopregnanolone concentrations were higher in growth-restricted fetuses compared to control (p < 0.05). Allopregnanolone concentrations decreased at birth with a greater decline in growth restriction (p < 0.05). Postnatal day 6 brain allopregnanolone concentrations were lower in growth restriction (p < 0.05). Conclusions: Growth restriction is a potent stimulus for neurosteroid synthesis in the fetal brain in late pregnancy. The low concentrations of allopregnanolone in the growth-restricted postnatal brain suggest a delay in the capacity of the adrenal gland or brain to synthesize pregnane steroids or their precursors and may render the postnatal brain vulnerable to hypoxia-induced injury.

Maternal Progesterone Treatment Rescues the Mammary Impairment Following Uteroplacental Insufficiency and Improves Postnatal Pup Growth in the Rat

Uteroplacental insufficiency in the rat reduces maternal progesterone and impairs mammary function and pup milk intake, compromising postnatal growth. We determined whether progesterone administration to rat dams progesterone-deficient following uteroplacental insufficiency improves lactation and pup growth. Uteroplacental insufficiency (Restriction) or sham surgery (Control) was performed on day 18 of pregnancy in WKY rats. Pregnant dams were injected with progesterone or vehicle, and Control mothers with vehicle for three days and killed on day 20 of pregnancy or day 6 of lactation. Progesterone treatment in the Restricted group restored maternal progesterone with no effect on mammary Pgr mRNA expression. Uteroplacental insufficiency triggered early lactogenesis, with increased mammary Csn2, Lalba and Wap mRNA. Progesterone treatment following uteroplacental insufficiency increased mammary alveolar number and area. Pups from progesterone treated mothers had increased body weight when compared to Controls. Overall, maternal progesterone treatment following uteroplacental insufficiency improved postnatal growth by rescuing the mammary impairment.

Embryo transfer cannot delineate between the maternal pregnancy environment and germ line effects in the transgenerational transmission of disease in rats

Adverse conditions in utero can have transgenerational effects, in the absence of a subsequent insult. We aimed to investigate the contribution of the maternal pregnancy environment vs. germ line effects in mediating alterations to cardiorenal and metabolic physiology in offspring from mothers born small. Uteroplacental insufficiency was induced by bilateral uterine artery and vein ligation (Restricted group) or sham surgery (Control group) in Wistar-Kyoto rats. Restricted and control female offspring (F1) were mated with either breeder males (embryo donor) or vasectomized males (embryo recipient). Embryo transfer was performed at embryonic day (E) 1, whereby second-generation (F2) embryos gestated (donor-in-recipient) in either a control (Cont-in-Cont, Rest-in-Cont) or restricted (Cont-in-Rest, Rest-in-Rest) mother. In male and female offspring, glomerular number and size were measured at postnatal day (PN) 35, and systolic blood pressure, glucose control, insulin sensitivity, and pancreatic β-cell mass were measured in separate sibling cohorts at 6 mo. Rest-in-Rest offspring were hypothesized to have similar characteristics (reduced growth, altered metabolic control, and hypertension) to non-embryo-transferred Rest, such that embryo transfer would not be a confounding experimental influence. However, embryo-transferred Rest-in-Rest offspring underwent accelerated growth during the peripubertal phase, followed by slowed growth between 2 and 3 mo of age compared with non-embryo-transferred Rest groups. Furthermore, renal function and insulin response to a glucose load were different to respective non-embryo-transferred groups. Our data demonstrate the long-term effects of in vitro embryo manipulation, which confounded the utility of this approach in delineating between the maternal pregnancy environment and germ line effects that drive transgenerational outcomes.