Novel mineral regulatory pathways in ovine pregnancy: I. phosphate, klotho signaling, and sodium-dependent phosphate transporters

Abstract

Appropriate mineralization of the fetal skeleton requires an excess of phosphate in the fetus compared to the mother. However, mechanisms for placental phosphate transport are poorly understood. This study aimed to identify phosphate regulatory pathways in ovine endometria and placentae throughout gestation. Suffolk ewes were bred with fertile rams upon visual detection of estrus (Day 0). On Days 9, 12, 17, 30, 70, 90, 110, and 125 of pregnancy (n = 3–14/Day), ewes were euthanized and hysterectomized. Phosphate abundance varied across gestational days in uterine flushings, allantoic fluid, and homogenized endometria and placentae (P < 0.05). The expression of mRNAs for sodium-dependent phosphate transporters (SLC20A1 and SLC20A2) and klotho signaling mediators (FGF7, FGF21, FGF23, FGFR1–4, KL, KLB, ADAM10, and ADAM17) were quantified by qPCR. Day 17 conceptus tissue expressed SLC20A1, SLC20A2, KLB, FGF7, FGF21, FGF23, FGFR1, and FGFR2 mRNAs. Both sodium-dependent phosphate transporters and klotho signaling mediators were expressed in endometria and placentae throughout gestation. Gestational day influenced the expression of SLC20A1, ADAM10, ADAM17, FGF21, FGFR1, and FGFR3 mRNAs in both endometria and placentae (P < 0.05). Gestational day influenced endometrial expression of FGF7 (P < 0.001), and placental expression of FGF23 (P < 0.05). Immunohistochemistry confirmed that both FGF23 and KL proteins were expressed in endometria and placentae throughout gestation. The observed spatiotemporal profile of KL-FGF signaling suggests a potential role in the establishment of pregnancy and regulation of fetal growth. This study provides a platform for further mechanistic investigation into the role for KL-FGF signaling in the regulation of phosphate transport at the ovine maternal-conceptus interface.

Phosphate, calcium, and vitamin D signaling, transport, and metabolism in the endometria of cyclic ewes

“…Studies in both pigs and sheep indicate that calcium and phosphate increase in uterine flushings during the peri-implantation period of pregnancy, and are more abundant in uterine flushings from pregnant than cyclic animals during the mid-luteal phase of the estrous cycle [ 18 , 19 , 27 , 28 ]. Further, temporal changes in the abundance of minerals in the uterine lumen during the peri-implantation period are accompanied with striking alterations in the expression of molecules with regulatory roles in phosphate, calcium, and vitamin D transport and signaling [ 18 , 19 ]. Administration of P4 (the hormone of pregnancy) and/or interferon tau (IFNT, the pregnancy recognition signal in ruminants) to cyclic ewes results in alterations in circulating phosphate and 25-hydroxyvitamin D (25(OH)D; vitamin D metabolite) abundance [ 29 ].…”

Uptake of Phosphate, Calcium, and Vitamin D by the Pregnant Uterus of Sheep in Late Gestation: Regulation by Chorionic Somatomammotropin Hormone

“…The concentrations of calcium in caruncular and cotyledonary homogenates were quantified using a colorimetric assay (Sigma Aldrich; MAK022), as described previously [ 17 ]. The concentrations of phosphate in uterine and umbilical plasma samples, and caruncular and cotyledonary homogenates were quantified using a colorimetric assay (Abcam, Cambridge, MA, USA; ab65622), as described previously [ 16 ]. Additionally, the concentrations of the vitamin D metabolite 25(OH)D in uterine and umbilical plasma samples were quantified by ELISA (Eagle Biosciences, Amherst, NH, USA; VID91-K01), as described previously [ 17 ].…”

“…The relative expression of candidate genes in cotyledonary and caruncular samples was quantified by quantitative polymerase chain reaction (qPCR) as described previously [ 16 , 17 ]. To investigate the effects of CSH on phosphate signaling in the sheep placentome, the mRNA expression of the sodium dependent phosphate transporters solute carrier family 20 member 1 ( SLC20A1 ) and SLC20A2 , and components of the fibroblast growth factor-klotho signalling cascade (fibroblast growth factor receptor 1 [ FGFR1 ], FGFR2 , klotho [ KL ], a disintegrin and metalloproteinase domain-containing protein 10 [ ADAM10 ], and ADAM17 ) was quantified by qPCR.…”

“…These hormones regulate mineral metabolism via the expression of regulatory molecules such as calcium-transporting ATPases, calcium-binding proteins, sodium dependent phosphate transporters, and the transient receptor potential vanilloid (TRPV) family members [ 6 , 10 , 11 , 12 , 13 , 14 , 15 ]. Emerging evidence suggests that many of these molecules with important roles in the regulation of mineral transport postnatally are expressed at the ovine maternal-conceptus interface throughout gestation [ 16 , 17 ]. As such, these molecules regulate the active transport of minerals from the maternal circulation to the fetal-placental circulation where they are essential for the appropriate regulation of fetal development [ 1 , 18 ].…”

“…The relative abundance of phosphate and calcium in ovine fetal fluids (allantoic and amniotic fluids) in late gestation have been reported [ 16 , 17 ]. However, the concentration of phosphate, calcium, and vitamin D in uterine and umbilical vessels and how these nutrients are utilized by the fetus, placental membranes, and uterus are not well documented.…”