Crossing mice deficient in eNOS with placental-specific Igf2 knockout mice: a new model of fetal growth restriction.

We tested the hypothesis that crossing two mouse models of fetal growth restriction (FGR) of differing phenotype would induce more severe FGR than either model alone. Female endothelial nitric oxide synthase knockout mice (eNOS(-/-)) were mated with placental-specific Igf2 knockout males (P0). Resultant fetuses were no more growth restricted than those with P0 deletion alone. However, P0 deletion attenuated the reduced placental system A amino acid transporter activity previously observed in eNOS(-/-) mice. Manipulating maternal and fetal genotypes provides a means to compare maternal and fetal regulation of fetal growth.

Defining fetal growth restriction in mice: A standardized and clinically relevant approach.

The increasing number of mouse models of fetal growth restriction (FGR) make it crucial to standardize the way FGR is defined. By constructing growth curves in the placental-specific Igf2 knockout mouse (P0) it was demonstrated that 93% of P0 fetuses fell below the 5th centile of wild-type weights at E18.5, up from 44% at E16.5. This analysis, coupled with anthropomorphic measurements showing evidence of head sparing in P0 fetuses, allows clinical comparisons of FGR in mice through the use of clinically relevant growth curves. We suggest this as a standardized approach to defining FGR in mouse, and other animal models.

System A activity and vascular function in the placental-specific Igf2 knockout mouse.

OBJECTIVES: Deletion of the placental-specific P0 transcript of the insulin-like growth factor gene (Igf2) reduces placental growth from early pregnancy onwards. In Igf2 P0 knockout fetuses (P0), maternofetal flux of (14)C-methylaminoisobutyric acid ((14)C-MeAIB) mediated by system A amino acid transporter activity is increased at embryonic day 16 (E16), but this stimulation is not sustained, and by E19, fetal growth restriction (FGR) ensues. Here, we investigated whether upregulated (14)C-MeAIB transfer does occur concomitantly with a change in System A amino acid transporter activity and whether altered uteroplacental vascular function contributes to the FGR. We tested the hypothesis that FGR in P0 mice is attributable to altered nutrient transport rather than aberrant uteroplacental vascular function. METHODS: Plasma membrane vesicles were isolated from placentas of P0 and wild-type (WT) fetuses at E16 and E19. System A amino acid transporter activity was measured as sodium-dependent (14)C-MeAIB uptake over 60s. Wire myography was performed on uterine artery branches supplying P0 or WT implantation sites and agonist-induced constriction and dilation measured. RESULTS: Sodium-dependent uptake of (14)C-MeAIB (at 60s) was significantly (P < 0.05) higher in P0 compared to WT vesicles at E16; at E19 (14)C-MeAIB uptake was similar between P0 and WT. Uterine artery branch vascular reactivity was comparable between groups. CONCLUSIONS: System A activity in the maternal-facing plasma membrane of syncytiotrophoblast layer II underpins the adaptations observed in the transplacental MeAIB flux of P0 mice. Unaltered uterine artery vascular function suggests that the FGR phenotype of P0 fetuses is primarily due to deficient placental nutrient exchange capacity.

Placental-specific Igf2 knockout mice exhibit hypocalcemia and adaptive changes in placental calcium transport.

Evidence is emerging that the ability of the placenta to supply nutrients to the developing fetus adapts according to fetal demand. To examine this adaptation further, we tested the hypothesis that placental maternofetal transport of calcium adapts according to fetal calcium requirements. We used a mouse model of fetal growth restriction, the placental-specific Igf2 knockout (P0) mouse, shown previously to transiently adapt placental System-A amino acid transporter activity relative to fetal growth. Fetal and placental weights in P0 mice were reduced when compared with WT at both embryonic day 17 (E17) and E19. Ionized calcium concentration [Ca(2+)] was significantly lower in P0 fetal blood compared with both WT and maternal blood at E17 and E19, reflecting a reversal of the fetomaternal [Ca(2+)] gradient. Fetal calcium content was reduced in P0 mice at E17 but not at E19. Unidirectional maternofetal calcium clearance ((Ca) K (mf)) was not different between WT and P0 at E17 but increased in P0 at E19. Expression of the intracellular calcium-binding protein calbindin-D(9K), previously shown to be rate-limiting for calcium transport, was increased in P0 relative to WT placentas between E17 and E19. These data show an increased placental transport of calcium from E17 to E19 in P0 compared to WT. We suggest that this is an adaptation in response to the reduced fetal calcium accumulation earlier in gestation and speculate that the ability of the placenta to adapt its supply capacity according to fetal demand may stretch across other essential nutrients.

Isolation of plasma membrane vesicles from mouse placenta at term and measurement of system A and system beta amino acid transporter activity.

Placental amino acid transport is essential for optimal fetal growth and development, with a reduced fetal provision of amino acids being implicated as a potential cause of fetal growth restriction (FGR). Understanding placental insufficiency related FGR has been aided by the development of mouse models that have features of the human disease. However, to take maximal advantage of these, methods are required to study placental function in the mouse. Here, we report a method to isolate plasma membrane vesicles from mouse placenta near-term and have used these to investigate two amino acid transporters, systems A and beta, the activities of which are reduced in human placental microvillous plasma membrane (MVM) vesicles from FGR pregnancies. Plasma membrane vesicles were isolated at embryonic day 18 by a protocol involving homogenisation, MgCl(2) precipitation and centrifugation. Vesicles were enriched 11.3+/-0.5-fold in alkaline phosphatase activity as compared to initial homogenate, with minimal intracellular organelle contamination as judged by marker analyses. Cytochemistry revealed alkaline phosphatase was localised between trophoblast layers I and II, with intense reaction product deposited on the maternal-facing plasma membrane of layer II, suggesting that vesicles were derived from this trophoblast membrane. System A and system beta activity in mouse placental vesicles, measured as Na(+)-dependent uptake of (14)C-methylaminoisobutyric acid (MeAIB) and (3)H-taurine respectively confirmed localisation of these transporters to the maternal-facing plasma membrane of layer II. Comparison to human placental MVM showed that system A activity was comparable at initial rate between species whilst system beta activity was significantly lower in mouse. This mirrored the lower expression of TAUT observed in mouse placental vesicles. We conclude that syncytiotrophoblast layer II-derived plasma membrane vesicles can be isolated and used to examine transporter function.

In vitro assessment of mouse uterine and fetoplacental vascular function.

Adequate blood flow provision through alterations in maternal vascular function is essential during pregnancy for optimal fetal development. Abnormal uterine vasculature adaptation, resulting in aberrant blood flow to the placenta, has been implicated as a possible cause of fetal growth restriction (FGR). Our study aimed to develop strategies to evaluate murine vascular function in pregnancy using wire myography. Main uterine artery loop and branch vessels isolated from near-term pregnant mice showed significant contraction to phenylephrine (PE). Endothelial-dependent relaxation was noted with acetylcholine (ACH). U46619 elicited significant contraction of umbilical arteries and veins, but relaxation was only demonstrable with the nitric oxide (NO) donor sodium nitroprusside (SNP). In conclusion, our data suggest that murine uteroplacental and fetoplacental arteries show distinct responses to vasoactive agents. Furthermore, this study indicates that wire myography represents a robust technique for the assessment of murine uteroplacental and fetoplacental vascular function, which will aid evaluation of mouse genetic models of FGR.