Calcium channel TRPV6 is involved in murine maternal-fetal calcium transport

Abstract

Maternal-fetal calcium (Ca(2+)) transport is crucial for fetal Ca(2+) homeostasis and bone mineralization. In this study, the physiological significance of the transient receptor potential, vanilloid 6 (TRPV6) Ca(2+) channel in maternal-fetal Ca(2+) transport was investigated using Trpv6 knockout mice. The Ca(2+) concentration in fetal blood and amniotic fluid was significantly lower in Trpv6 knockout fetuses than in wildtypes. The transport activity of radioactive Ca(2+) ((45)Ca) from mother to fetuses was 40% lower in Trpv6 knockout fetuses than in wildtypes. The ash weight was also lower in Trpv6 knockout fetuses compared with wildtype fetuses. TRPV6 mRNA and protein were mainly localized in intraplacental yolk sac and the visceral layer of extraplacental yolk sac, which are thought to be the places for maternal-fetal Ca(2+) transport in mice. These expression sites were co-localized with calbindin D(9K) in the yolk sac. In wildtype mice, placental TRPV6 mRNA increased 14-fold during the last 4 days of gestation, which coincides with fetal bone mineralization. These results provide the first in vivo evidence that TRPV6 is involved in maternal-fetal Ca(2+) transport. We propose that TRPV6 functions as a Ca(2+) entry pathway, which is critical for fetal Ca(2+) homeostasis.

FIG. 1

Total calcium, phosphorus, and magnesium concentration in sera or amniotic fluid of wildtype, Trpv6 heterozygous, and Trpv6 knockout fetuses. All measurements were obtained from fetuses at 17.5 or 18.5 days of gestation (mean ± SE). +/+, wild types; +/−, heterozygotes; −/−, Trpv6 knockout fetuses. *p < 0.05, **p < 0.01 vs. wildtype group (Student's t-test).

FIG. 2

Maternal–fetal transports and ash weight in wildtype, heterozygous, and Trpv6 knockout fetuses. (A) Maternal–fetal Ca²⁺ transport. The ratio of ⁴⁵Ca/⁵¹Cr activity accumulated in fetuses was determined at 5 min after maternal administration of the isotopes. The mean heterozygote ⁴⁵Ca/⁵¹Cr ratio of each litter was set at 100% to allow the results of multiple litters to be compared. The number of observations is indicated in parentheses. All measurements were obtained from fetuses at 15.5–17.5 days of gestation (mean ± SE). +/+, wildtypes; +/−, heterozygotes; −/−, Trpv6 knockout fetuses. *p < 0.0001 (Student's t-test). (B) Ash weight in wildtype, heterozygous, and Trpv6 knockout fetuses. **p < 0.04 (Student's t-test) for both vs. wildtype and heterozygous fetues.

FIG. 3

RT-PCR with dissected mouse placenta and yolk sac. (A) A cartoon of mouse placenta, fetus, and yolk sac. Mouse fetus is covered with the inverted yolk sac bilayer, which is thought to represent another maternal–fetal transport system. A part of the yolk sac expands into the chorioallantioc placental disc and forms intraplacental yolk sac. YS, yolk sac; Pm, maternal side of placenta; Pc, central part of placenta; Pf, fetal side of placenta; (B) A representative RT-PCR result of TRPV6, TRPV5, calbindin D_9K, and β-actin.

FIG. 4

Localization of TRPV6 in mouse placenta and yolk sac. Tissue sections were taken from mice at 17 days of gestation. (A and B) In situ hybridization of TRPV6 using TRPV6 antisense RNA probe. A shows TRPV6 mRNA was predominantly localized in intraplacental yolk sac in wildtype placenta (arrows). Bar indicates 50 μm. B shows a representative result using Trpv6 knockout placenta. Bar indicates 50 μm. (C and D) Immunohistochemistry of TRPV6 in mouse yolk sac. C indicates a representative result in a wildtype mouse. The visceral layer of yolk sac was stained (arrows). Bar indicates 50 μm. D shows a result in wildtype mouse with TRPV6 antibody preincubated with the peptide used to raise the antibody. Bar indicates 50 μm. (E and F) Immunofluorescence of TRPV6 (E) and calbindin D_9K (F) in the same section. These were co-localized in the visceral layer of yolk sac (arrows). Bars indicate 100 μm.

FIG. 5

Developmental changes in TRPV6 and calbindin D_9k expression in mouse placenta. (A) TRPV6 (filled) or calbindin D_9k (open) expression were detected using real time RT-PCR from the placenta from pregnant mice at gestation days 15–19. The TRPV5 (B) and calbindin D_9K (C) mRNA expressions are shown in Trpv6 +/+, +/−, and −/− placenta (*p < 0.05, Student's t-test).