Cloning and cellular expression of aquaporin 9 in ovine fetal membranes

Abstract

Amniotic fluid (AF) absorption across fetal membranes is essential for AF volume homeostasis, balancing fetal swallowing, urine flow, and lung liquid production. In sheep, AF is absorbed primarily across the amniotic membrane into fetal vasculature situated between the amnion and chorion. Aquaporins (AQPs) are cell membrane proteins that serve as water channels. Recent studies have demonstrated the expression of AQP 1, 3, 8, and 9 in human chorioamniotic membranes and placenta. As AF dynamics continued to be explored primarily in the ovine model, we sought to clone and characterize the expression of ovine AQP9 in fetal membranes. Ovine AQP9 gene was cloned with the use of homology reverse transcriptase-polymerase chain reaction (RT-PCR). RT-PCR and Northern analysis were used to determine AQP9 gene expression, and immunohistochemistry (IHC) used to localize AQP9 protein expression in ovine fetal membranes. A 2085-base pair (bp) full-length complementary DNA (cDNA) sequence of ovine AQP9 was cloned. The ovine AQP9 cDNA is 86%, 82%, and 82%, and the predicted amino acid sequence (295 amino acids) is 77%, 71%, and 69% identical to human, rat, and mouse AQP9, respectively. RT-PCR and Northern analysis detected AQP9 messenger RNA expression in ovine amnion and allantois, but not in placenta, chorion, or umbilical cord. Immunohistochemistry localized AQP9 protein in epithelia of amnion and allantois. The presence of significant AQP9 messenger RNA and protein expression in ovine fetal membranes suggests that AQP9 may be a major water channel for intramembranous AF resorption in sheep. The cloning of ovine AQP9 and the demonstration of AQP9 expression in amnion and allantois significantly enhances our understanding of ovine AF regulation and offers the potential for therapeutic approaches for the treatment of oligohydramnios and polyhydramnios.