Pregnant Phenotype Analysis Of AQP3 Null Mice And AQP8 Null Mice

Aquaporins (AQPs) were found as a family of intrinsic membrane protein that function as special water-transporting channels at the membrane of eukaryocyte and prokaryocyte. Till now, thirteen members have been defined in this family from mammals and the study on their gene structure, expression regulation, chromosome location, tissue distribution, and physiological function has been on study. Large number of studies showed that the water channel proteins play an important role in the body liquid transit by the knock-out mice. The current studies demostrated that both AQP3 and AQP8 expressed in the placenta and fetal membrane of human and mice, suggesting that AQP3, AQP8 may play a key role in pregnant physiology. This study investigated the pregnant phenotype changes of AQP3-KO mice (or AQP8-KO mice) , to find the direct evidences for the AQP3,s (or AQP8,s) role at fetal growth and maternal-fetal fluid blance,PartⅠPregnant phenotype analysis of AQP3 null miceObjective: To detect expression level and distribution of aquaporin 3(AQP3)in placenta and fetal membrances and try to find out the significance of AQP3 in homeostasis of maternal-fetal fluid exchange, and to investigated the pregnant phenotype changes of AQP3 null mice, to find the direct evidence of AQP3,s role at fetal growth and maternal-fetal fluid blance.Materials and Methods:1. The homozygous AQP3 knockout mice were mated. The day a copulation plug was found was designated as 1 gestational day (GD). The number of pregnant mice was record daily. The pregnant mice were divided into 5 subgroups respectively (7GD, 13GD, 16GD, 18GD and full term) depended on gestational age, with KM pregnant mice as the control group.2. Samples of placenta and fetal membrane (3 cases) were collected from KM mice at 16GD. The expression of AQP3 mRNA reveal was detected by RT-PCR.3. Fixing placentae at 13GD in 4% paraformaldehyde for HE (hematoxylin and eosin stain) staining and Immunohistochemistry.4. Total embryo and atrophic embryo numbers were recorded in each subgroup. Fetal weight, placental weight and area, were recorded at 13GD, 16GD, 18GD. The placentae at 13GD, 16GD and 18GD were used to measure water content.The amniotic fluid in each sac at 13GD, 16GD, 18GD was measured, and amniotic fluid at 16GD was collected to detection the composition and osmolality of amniotic fluid.5. Data analysis:Continuous data with normal distribution are given as mean±SEM. One-way analysis of variance (ANOVA) on ranks was used to determine differences in amniotic fluid volume, placenta weight and the placenta water contents. The difference incidence rate of abnormal embryo and the conception rate were assessed by chi-square test. Statistical significance was accepted at P<0.05.Results:1. RT-PCR revealed the presence of AQP-3 mRNA in placenta and fetal membranes. Immunohistochemistry performed on placenta revealed that AQP3 was associated with trophoblast cell of placenta and epithelial cell of amnion.2. The pregnant phototype changes of the AQP3-KO mice are as follows:(1) The conception rate of AQP3-KO mice was lower than that of the KM mice.The number of AQP3-KO pregnant mice embryo was much lesser than that in KM pregnant mice (P=0.001) at the early trimester of pregnancy (7GD).(2)The fetal weight of AQP3 null mice was significantly lighter than that of KM mice at 16GD and 18GD and also at the newborns.(3)The placenta of AQP3-KO mice was signi ficantly heavier than that of KM pregnant mice at 13GD and 16GD, due to increased placenta water content.(4) Amniotic fluid volume is decreased in AQP3-KO mice compared to KM pregnant mice at 16GD and 18GD.The AF Urea, Ka+, Na+and osmolality of AQP3 null mice were also significantly higher than that of KM pregnant mice at 16GD.(5) Pathology of placenta (hematoxylin and eosin stain) showed the structure of labyrinthine trophoblasts with disordered, mixed with many trophoblast giant cells and spongiotrophoblasts cells in AQP3-KO mice.Conclusions:1. AQP3 expressed on both placenta and fetal membranes tisseus in RNA and protein level.2. The pregnant phenotype of AQP3 knock-out mice were quite differrent from KM mice pregnant mice.3. The pregnant phenotype of AQP3 knock-out mice exhibit descended conception rate, low fetal weight, decreased amniotic fluid volume and placenta change.4. This result provides the direct evidences that AQP3 play an important role in pregnancy, fetal growth and maternal-fetal fluid blance.PartⅡPregnant phenotype analysis of AQP8 null miceObjective: To detect expression level and distribution of aquaporin 8(AQP8)in placenta and fetal membrances, try to find out the significance of AQP8 in homeostasis of maternal-fetal fluid exchange,and to investigated the pregnant phenotype changes of AQP8 null mice, to find the direct evidence of AQP8,s role at fetal growth and maternal-fetal fluid blance.Materials and Methods:1. The homozygous AQP8 knockout pregnant mice were divided into 5 subgroups respectively (7GD, 13GD, 16GD, 18GD and full term) depended on gestational age, with C57 pregnant mice as the control group.2. The methods and data analysis were the same as the PartⅠ. Results:1. RT-PCR revealed the presence of AQP-8 mRNA in placenta and fetal membranes. Immunohistochemistry performed on placenta revealed that AQP8 was associated with trophoblast cell of placenta and epithelial cell of amnion.2. The pregnant phototype changes of AQP8-KO mice are as follows:(1)The fetal weight of AQP8 null mice was significantly greater than that of the C57 pregnant mice at 16GD and 18GD and also the newborns(P=0.001;0.000;0.000).(2)The placenta of AQP8-KO mice was heavier than that of C57 mice at 16GD and 18GD, due to increased water content.(3)The amniotic fluid volume is increased in AQP8-KO mice compared to C57 mice at 13GD, 16GD, 18GD. The concentration of K+, Na+, Cl-, urea and osmolality of AQP8 null mice were also significantly higher than that of C57 mice at 16GD.(4) Pathology of placenta (hematoxylin and eosin stain) showed no obvious abnormalities of the placenta structure in AQP8 null mice under microscope.Conclusions:1. AQP8 expressed on both placenta and fetal membrances tissues in RNA and protein level.2. The pregnant phenotype of AQP8 knock-out mice were quite differrent from C57 pregnant mice.3. The pregnant phenotype changes of AQP8 knock-out mice exhibit greater fetal weight, increased amniotic fluid.4. These results provide the evidences that AQP8 plays an important role in the homeostasis of maternal-fetal fluid, and also play a direct or indirect role in fetal growth.