Characterization of Temporal Changes in Tissue Non-specific Alkaline Phosphatase Activity and Phosphate Concentration in Utero-placental Tissues and Fetal Fluids in Cattle

Maria F. Tyree^1,2;Yvette E. Wolpo¹; Alaina M. Helbus¹; Morgan S. Clemens¹; Camilla H.K. Hughes¹; Claire Stenhouse¹  

< !1. Department of Animal Science, The Pennsylvania State University, University Park, United States  

2.  2. Huck Institute of Life Sciences, The Pennsylvania State University, University Park, United States  

Abstract Text:

Phosphate has important roles in many cellular signaling and metabolic pathways that are essential to conceptus development, including nucleic acid and ATP synthesis.  Postnatally, tissue non-specific alkaline phosphatase (TNSALP) is a membrane-bound glycoprotein that hydrolyzes pyrophosphate substrates to generate inorganic phosphate. Previous research suggests that TNSALP plays an important role in uterine receptivity in sheep.  Additionally, dysregulation of alkaline phosphatases has been associated with adverse pregnancy outcomes in humans.  While the mechanisms regulating phosphate availability postnatally are well appreciated, we have a limited understanding of prenatal phosphate availability in utero-placental tissues and the extent to which TNSALP contributes to phosphate availability throughout gestation.  This study aimed to characterize 1) TNSALP activity and 2) phosphate concentration in fetal fluids and utero-placental tissues of cattle during early to mid-gestation.  Due to the increased phosphate demand of the growing fetus, it was hypothesized that TNSALP activity and phosphate concentration would 1) be greater in the allantoic fluid (ALF) compared to the amniotic fluid (AMF) and 2) increase in utero-placental tissues with gestational age.  Fetal fluids (n=91) and uterine (n=95) and placental (n=88) tissues were collected from a local slaughterhouse.  Crown-rump length was used to estimate gestational age.  Samples were categorized into 7-day gestational age intervals, ranging from day 46 to day 136, to assess differences across specific gestational stages (n=3-10/group).  Cross sections of the uterus and placentomes were fixed and embedded in paraffin wax.  Placentomes and endometria were snap-frozen, and protein was extracted.  TNSALP activity and phosphate concentration in fetal fluids, endometrium, and placentomes were quantified using spectrophotometric assay.  Immunohistochemistry was used to determine the localization of TNSALP protein in uterine tissues.  TNSALP activity in AMF peaked on day 70 and then decreased as gestation progressed (P < 0.0001).  In ALF, TNSALP activity peaked on day 56 and then remained consistently low for the remainder of gestation (P < 0.01).  The concentration of phosphate in AMF decreased as gestation progressed (P < 0.0001).  In contrast, the concentration of phosphate in ALF increased as gestation progressed (P=0.0001).  TNSALP activity was greater in AMF than ALF on gestational days 60, 77, and 84 (P < 0.01).  Phosphate concentration was greater in ALF than AMF on gestational days 91, 98, 112, 119, and 133 (P < 0.05).  Gestational day affected TNSALP activity in the endometrium (P < 0.0001) but not in placentomes (P=0.11). TNSALP activity peaked around day 63 in the endometrium and then decreased with gestational age while TNSALP activity in the placentomes was consistently low throughout gestation.  TNSALP was greater in the endometrium than placentomes at all gestational stages (P < 0.0001).  TNSALP protein immunolocalized to the endometrial stratum spongiosum stroma, endothelium, luminal epithelium, and apical surface of the glandular epithelia.  Epithelia and utero-placental vasculature have critical roles in the regulation of nutrient availability.  The expression of TNSALP in these cell types suggests TNSALP is a regulator of phosphate availability in bovine utero-placental tissues.  These data suggest dynamic regulation of both TNSALP activity and phosphate concentration in utero-placental tissues in early and mid-gestation, suggesting a potential role in the regulation of fetal and placental development.  An improved understanding of phosphate regulation in utero-placental tissues could reveal key physiological processes in both human and livestock pregnancies that may lead to improved maternal-fetal health and agricultural production.  This project was funded by NIH Grant T32GM154124 and the Society for the Study of Reproduction.