Alkaline Phosphatase: An Important Regulator of Utero-Placental Phosphate Availability and Conceptus Development

Claire Stenhouse¹, Katherine M. Halloran², Robyn M. Moses², Nirvay Sah², Makenzie G. Newton², Larry J. Suva³,  Fuller W. Bazer², and Dana Gaddy⁴,
¹Department of Animal Science, Pennsylvania State University, University Park, Pennsylvania, 16802

²Department of Animal Science Texas A&M University, College Station, Texas, 77843

³Department of Veterinary Physiology and Pharmacology, Texas A&M University, College Station, Texas, 77843

⁴Department of Veterinary Integrative Biosciences, Texas A&M University, College Station, Texas, 77843

Abstract Text: Phosphate is essential for conceptus (embryo/fetus and associated extraembryonic membranes) development, yet the mechanisms that regulate its availability within utero-placental tissues remain poorly understood. Tissue-nonspecific alkaline phosphatase (TNSALP; encoded by the ALPL gene) has an important role in the regulation of phosphate homeostasis postnatally. However, the functional role(s) of TNSALP in the regulation of utero-placental phosphate availability and conceptus development and function is unclear. The first study aimed to determine the expression of ALPL mRNA, as well as cell-specific immunolocalization of TNSALP protein and enzymatic activity in endometria from Days 1, 9, and 14 of the estrous cycle and endometria and placentomes from Days 9, 12, 17, 30, 50, 70, 90, 110 and 125 of gestation. The expression of ALPL mRNA, immunolocalization of TNSALP protein, and quantification and localization of TNSALP enzymatic activity was performed on ovine endometria and placentomes. Day of the estrous cycle did not alter ALPL mRNA expression or enzymatic activity of TNSALP. TNSALP protein localized to uterine epithelial and stromal cells, blood vessels, myometrium, caruncular and cotyledonary stroma. TNSALP activity was localized to uterine epithelia, blood vessels, caruncular stroma (from Day 70 of gestation), and the apical surface of chorionic epithelia (from Day 50 of gestation). TNSALP protein and activity localized to the apical surface of uterine epithelia during the estrous cycle and in early pregnancy. Endometrial TNSALP enzymatic activity was downregulated on Day 17 and 30 of gestation (P < 0.05). Expression of ALPL mRNA decreased in late gestation in endometria and placentomes (P < 0.05). TNSALP activity peaked in placentomes on Days 70 and 90 of gestation. To further address the gap in knowledge of the role of TNSALP in the regulation of phosphate availability and conceptus development, a loss-of-function mutation in ALPL (c.1077 C >G; generated by CRISPR/Cas9) was introduced into the sheep genome. Pregnancies were generated and conceptus development was assessed in late gestation. In addition to gross assessment of conceptus development (fetal and placental weights and lengths, cotyledonary number, weights of fetal organs etc), maternal and fetal serum, as well as allantoic and amniotic fluid composition, was assessed to determine impacts of altered conceptus TNSALP activity on nutrient transport and utilization. Biochemical analyses were performed by an automated analyzer at the Texas A&M University Veterinary Medical Diagnostic Laboratory, and untargeted metabolomics was performed by the Huck Institute Metabolomics Core Facility. Collectively, our findings suggest loss of function mutations in ALPL alters conceptus development and the nutritional composition of fetal plasma, and allantoic and amniotic fluids, positioning TNSALP as an important regulator of conceptus development and function, far beyond what is widely accepted as simply regulating phosphate homeostasis postnatally. Acknowledgements: This work was supported by NIH R21-DE028076, Soft Bones Foundation, and the USDA National Institute of Food and Agriculture and Hatch Appropriations under Project PEN04995 and Multistate W4112. The co-authors would like to acknowledge the Huck Institutes’ Metabolomics Core Facility (RRID:SCR_023864) and Dr. Ashley Shay for her technical expertise.