Abstract
Abstract
Tissue non-specific alkaline phosphatase (TNSALP) regulates postnatal phosphate homeostasis, but its role in utero-placental phosphate availability remains poorly understood. Gilts were bred and hysterectomized on Day 60 or Day 90 of gestation (n = 6/day). Phosphate was less abundant in allantoic and amniotic fluids on Day 90 compared to Day 60. TNSALP protein was immunolocalized, and enzymatic activity was quantified and localized in endometrial and chorioallantois tissues. Day had no effect on TNSALP activity in the chorioallantois. In contrast, endometrial TNSALP activity was lower on Day 90 compared to Day 60. Phosphate abundance in allantoic fluid correlated positively with endometrial TNSALP activity on Day 60 but not Day 90. TNSALP protein was abundantly expressed in the endometrium and chorioallantois on both days investigated, with localization to the endometrial, chorionic, and areolar epithelia, as well as stromal cells and endothelium. TNSALP activity was detected in the endothelium of the blood vessels in both the endometrium and chorioallantois, and on the basal surface of the endometrial glands on Day 60 but not Day 90. The endometrial stratum compactum stroma had strong TNSALP activity on Day 60. Weak TNSALP activity was present in the areolar epithelium, with a modest increase in activity on Day 90 compared to Day 60. TNSALP activity was present in the columnar chorionic epithelial cells, with an apparent decrease in activity in the chorioallantois on Day 90 compared to Day 60. These data reveal spatiotemporal changes in TNSALP localization and activity, suggesting its involvement in regulating phosphate availability at the utero-placental interface in swine.
Lay Summary
Phosphate is an essential nutrient for fetal growth, but how it is managed during pregnancy is not fully understood. This study explored the role of an enzyme called tissue non-specific alkaline phosphatase (TNSALP) in regulating phosphate availability in the uterus and placenta in pigs in mid- and late pregnancy. Phosphate levels decreased in the fluids surrounding the fetus in late pregnancy. TNSALP was present in the uterus and placenta, and the amount of the enzyme varied depending on the tissue and stage of pregnancy and correlated with changes in phosphate levels. These findings suggest that TNSALP plays a key role in managing phosphate transport from the mother to the fetus in pregnancy to support fetal development.
Introduction
The endometrium and placenta have critical roles in the exchange of minerals, gases, amino acids, sugars, and proteins, while producing regulatory molecules such as cytokines, growth factors, and hormones that are crucial for the establishment and maintenance of pregnancy and regulation of fetal growth (Sinowatz & Friess 1983, Roberts & Bazer 1988, Bazer et al. 2012, Maltepe & Fisher 2015, Johnson et al. 2021, Stenhouse et al. 2022a ). Pigs have a true epitheliochorial placenta, wherein the endometrial luminal epithelium (LE) remains intact throughout pregnancy, and the trophectoderm directly attaches to the LE (Johnson et al. 2021). In addition, the fetus is surrounded by amniotic and allantoic fluids, which contribute to fetal development by serving as reservoirs for nutrients transported across the utero-placental interface.
Phosphorus is one of the most abundant minerals in the body, with approximately 85% of phosphorus stored in bone in the form of hydroxyapatite (Ca10[PO4]6[OH]2) (Mitchell et al. 1945, Goretti et al. 2012). Inorganic phosphate has crucial roles in the regulation of cellular processes important for conceptus (embryo/fetus and associated extraembryonic membranes) development, including DNA and RNA synthesis, phospholipid formation, nucleotide and creatine phosphate generation, and cell signaling through phosphorylation and dephosphorylation (Wu 2018). Thus, significant amounts of phosphate must be transported from the mother for utilization by the conceptus (Taylor-Miller & Allgrove 2021). Yet, there is a limited understanding of the mechanisms regulating phosphate availability in utero-placental tissues during gestation.
Postnatally, the regulation of phosphate homeostasis is complex, with several factors and endocrine hormones modulating homeostasis, including vitamin D, parathyroid hormone, sex steroids, alkaline phosphatases, and fibroblast growth factor 23 (Suva & Friedman 2020). In addition, there is regulation of phosphate transport by sodium-dependent phosphate transporters (SLC20, SLC34, and SLC37 family members) (Hernando et al. 2021). Importantly, postnatal regulation of phosphate homeostasis relies upon interactions among the kidneys, intestine, and skeletal system. In contrast, during gestation, the uterus and placenta must regulate phosphate availability locally to ensure sufficient phosphate for fetal growth and development.
Alkaline phosphatases are membrane-bound glycoproteins that generate phosphate by hydrolyzing phosphocompound substrates, including inorganic pyrophosphate (PPi), pyridoxal-5′-phosphate (PLP), and phosphoethanolamine (PEA) (Whyte et al. 1995, Millán 2006). Tissue non-specific alkaline phosphatase (TNSALP, encoded by the ALPL gene) is an important regulator of phosphate homeostasis postnatally, with well-characterized regulatory roles in many critical processes, including skeletal mineralization (Millán 2006). In postnatal pigs, abundant TNSALP activity has been reported in several vital organs, including the kidney, liver, intestine, lung, and heart (Khailova et al. 2020), suggesting an important role in regulating animal health. However, the role of TNSALP in conceptus development and utero-placental phosphate regulation remains uninvestigated in pigs.
We recently i) characterized the expression of ALPL mRNA; ii) localized TNSALP protein; and iii) quantified and localized TNSALP enzymatic activity in endometria of cyclic ewes and ovine endometria and placentomes at multiple stages of gestation (Stenhouse et al. 2023). These findings highlighted a potential role of TNSALP in regulating phosphate transport and homeostasis at the maternal–conceptus interface in ruminants.
Alkaline phosphatases are expressed in human and sheep utero-placental tissues, and alterations in activity have been associated with adverse pregnancy outcomes, including gestational diabetes, preterm birth, stillbirths, fetal distress in utero, intrauterine growth retardation, and preeclampsia (Posen et al. 1969, Meyer et al. 1995, Whyte et al. 1995, Leitner et al. 2001, Moawad et al. 2002, Safarova et al. 2007, Kovacs 2014, 2015, McErlean & King 2019, Correia-Branco et al. 2020, Stenhouse et al. 2022b , 2023, Paquette et al. 2023, Titaux et al. 2023). Local regulation of phosphate homeostasis in porcine utero-placental tissues has been suggested (Choi et al. 2014, Lee et al. 2021, Stenhouse et al. 2022b ), but the potential role of TNSALP in regulating utero-placental phosphate availability remains poorly understood. We hypothesized that there would be: a) abundant TNSALP activity in porcine utero-placental tissues and b) spatiotemporal alterations in TNSALP expression and activity in utero-placental tissues that would correlate with alterations in phosphate in allantoic and amniotic fluid. This study characterized i) the abundance of phosphate in allantoic and amniotic fluid, ii) TNSALP protein localization, and iii) TNSALP enzymatic activity in endometrial and chorioallantois samples from gilts on Days 60 and 90 of gestation.
Materials and methods
All experimental procedures followed the Guide for the Care and Use of Agricultural Animals in Research and Teaching and were approved by the Institutional Animal Care and Use Committee of Texas A&M University.
Experimental animals and sample collection
Yorkshire x Landrace gilts (n = 12) were group housed and fed a diet to meet the National Research Council nutritional requirements (National Research Council 2012). Gilts were observed daily for signs of estrus using a boar and were bred with a boar when detected in estrus (Day 0) and 12 and 24 h later. Gilts were euthanized on either Day 60 or Day 90 of gestation (n = 6 gilts per day). Following confirmation of death, a mid-ventral incision was made to expose the reproductive tract, which was removed from the body cavity and dissected immediately. Samples of allantoic and amniotic fluids were collected using a syringe with an 18G needle. Total volumes of allantoic and amniotic fluid were calculated by measuring the volume of each fluid in a graduated cylinder. Allantoic and amniotic fluids were stored at −20°C following centrifugation (10,000 g for 10 min). Uterine and chorioallantois samples were obtained from the first two fetuses closest to the ovarian ends of each uterine horn. Fetal sex was determined by gross examination of the external genitalia and was recorded. Uterine cross sections and samples of dissected chorioallantois were fixed overnight in 4% paraformaldehyde (Electron Microscopy Services, USA) and stored in 70% ethanol before embedding in paraffin wax. Samples of chorioallantois and endometrium dissected from the underlying myometrium were frozen in liquid nitrogen and stored at −80°C.
Quantification of phosphate abundance in allantoic and amniotic fluids
The concentrations of phosphate in allantoic and amniotic fluid were quantified using a colorimetric assay (Abcam, USA; ab65622) as previously described (Stenhouse et al. 2021). In brief, samples and the phosphate standards (200 μL total volume) were pipetted in duplicate and incubated with the phosphate reagent for 30 min at room temperature (∼25°C), protected from light. The absorbance of the plate was read on a spectrophotometric plate reader (SynergyH1, BioTek) at 650 nm. Phosphate abundance was calculated relative to the known standards and expressed as total phosphate (volume of fluid x concentration of phosphate).
Immunohistochemical localization of TNSALP protein in utero-placental tissues
Cell-specific immunohistochemical localization for TNSALP protein in uterine and chorioallantois paraffin sections was performed as previously described (Stenhouse et al. 2023). In brief, tissues were fixed overnight in 4% paraformaldehyde (Electron Microscopy Services), dehydrated through a graded series of ethanol, cleared in xylene, and embedded in paraffin wax (Surgipath Paraplast, Leica Biosystems, USA). Paraffin-embedded tissues were sectioned at 5 μm using a microtome, mounted on charged glass slides (Fisher Scientific, USA), and dried overnight at 37°C. Sections were deparaffinized using CitriSolv (Decon Laboratories Inc, USA) and rehydrated through a graded series of ethanol to double-distilled water. Heat-induced epitope retrieval was performed in Tris-buffer (pH 9.0; Vector Laboratories, USA). Slides were incubated for 15 min in hydrogen peroxide (0.3%; Sigma Aldrich) in methanol to block endogenous peroxidase activity. Non-specific binding sites were blocked by incubation with normal horse serum (Vector Laboratories) for 1 h at room temperature. Sections were incubated with a primary antibody (11187, Proteintech, USA, 1.38 μg/mL) or with rabbit immunoglobulin G (rIgG, 1.38 μg/mL, Vector Laboratories) as an isotype control, diluted in 1% bovine serum albumin in phosphate-buffered saline (PBS), overnight in a humidified chamber at 4°C. Following washes in PBS, the slides were incubated for 1 h at 37°C in a humidified chamber with a biotinylated anti-rabbit IgG secondary antibody (Vectastain Elite ABC kit; Vector Laboratories) at 0.005 mg/mL in PBS containing 1.5% normal horse serum. Sections were incubated with Vectastain Elite ABC reagent (Vectastain Elite ABC kit; Vector Laboratories) for 30 min at 37°C in a humidified chamber. Slides were washed in PBS, followed by a wash in 0.05 mol/L Tris–HCl. Slides were then incubated with diaminobenzidine-tetrahydrochloride hydrate (Sigma Aldrich) in 0.05 mol/L Tris–HCl containing hydrogen peroxide (Sigma Aldrich) before being counterstained with hematoxylin (Ricca Chemical, USA). Slides were dehydrated through a graded series of ethanol and CitriSolv (Decon Laboratories Inc) before coverslips were affixed using Permount mounting medium (Fisher Scientific). Slides were visualized using a MoticEasyScan One scanner (Motic Instruments, Canada), and six non-overlapping representative images from the complete slide scan were exported at 10× or 20× magnification.
Quantification of TNSALP enzymatic activity in homogenates
TNSALP activity was quantified in endometrial and chorioallantois tissue homogenates as previously described (Stenhouse et al. 2023). In brief, about 100 mg of frozen endometrial and chorioallantois tissue were homogenized using a mechanical rotor-stator homogenizer in 1 mL of lysis buffer (60 mmol/L Tris–HCl (Sigma Aldrich, USA), 1 mmol/L Na3VO4 (Fisher Scientific), 10% glycerol (Fisher Scientific), 1% sodium dodecyl sulfate (BioRad, USA)) containing an EDTA-free protease inhibitor (Roche, USA). Following centrifugation of the homogenates (14,000 g for 15 min at 4°C), the supernatant was used for subsequent assays. The enzymatic activity of TNSALP was quantified spectrophotometrically (SynergyH1, BioTek, USA; Abs 405 nm) utilizing a TNSALP enzymatic activity assay (ab83369, Abcam, USA). Concentrations of protein in the homogenates were determined using a protein assay dye reagent (BioRad; 500-0006) according to the manufacturer’s instructions and quantified spectrophotometrically (SynergyH1, BioTek). Enzymatic activity was expressed relative to protein concentration in the tissue homogenates.
Localization of TNSALP enzymatic activity
TNSALP enzymatic activity was localized in paraffin-embedded tissue sections as described previously, with some modifications (Stenhouse et al. 2023). In brief, tissue sections were deparaffinized in CitriSolv (Decon Laboratories Inc) and rehydrated through a graded series of ethanol to double-distilled water. Sections were incubated with an alkaline phosphatase substrate solution (SK-5300; Vector Laboratories) in 100 mmol/L Tris–HCl (pH 8.3) for 3 h at room temperature. As a negative control, sections were incubated with only 100 mmol/L Tris–HCl. Slides were counterstained for 5 min in 0.5% methyl green in 0.1 mol/L sodium acetate, washed in 95% ethanol, rehydrated, and mounted with aqueous mountant (ab104131; Abcam). Digital images of representative fields from six non-overlapping areas per section were recorded under brightfield illumination using a Nikon Eclipse microscope and NIS-Elements AR 4.30.02 64-bit Software (Nikon Instruments Inc, USA).
Statistical analysis
All statistical analyses were performed using GraphPad Prism (version 10; GraphPad Software Inc, USA). Mean values were calculated for each individual sample for each parameter investigated, and normality of distribution of data was assessed using the Anderson-Darling test (P > 0.05). Outliers identified by a robust regression and outlier removal (ROUT; Q = 1%) test were excluded (Motulsky & Brown 2006). Supplementary Table 1 (see section on Supplementary materials given at the end of the article) summarizes the number of samples analyzed after outlier removal for each analysis performed. To assess the effect of gestational day or sex within gestational day, t-tests were performed. Two-way ANOVA was performed to assess the interaction of gestational day and fetal sex on the parameters investigated. Pearson’s correlations were performed to correlate tissue TNSALP activity with phosphate abundance in fetal fluids. Results were considered significant at P < 0.05.
Results
Quantification of phosphate in allantoic and amniotic fluid
The abundance of phosphate in both allantoic and amniotic fluid was affected by gestational day (Fig. 1), with less phosphate present in both allantoic (P < 0.05) and amniotic (P < 0.01) fluids on Day 90 than Day 60 of gestation. There was no effect of fetal sex (P > 0.05) phosphate abundance in allantoic and amniotic fluids (data not presented).
Effect of day of gestation on abundances of phosphate in allantoic and amniotic fluids. Mean values presented ± S.E.M. n = 10, Day 60, and n = 11, Day 90. *P < 0.05. **P < 0.01.
Citation: Reproduction and Fertility 6, 2; 10.1530/RAF-25-0005
Quantification of TNSALP enzymatic activity in endometrial and chorioallantoic homogenates
There was no effect of gestational day on TNSALP activity in homogenates of chorioallantois (Fig. 2). Endometria from Day 90 of gestation had less TNSALP activity than endometria from Day 60 of gestation (P < 0.001; Fig. 2). Fetal sex did not affect TNSALP activity in utero-placental tissues on Day 60 or Day 90 of gestation (Fig. 2).
Effect of day of gestation and fetal sex on TNSALP activity in homogenates of porcine chorioallantois (A and B) and endometria (C and D). Mean values presented ± S.E.M. n = 10–12 per day (A and C), n = 4–7 per sex per day (B and D). ***P < 0.001.
Citation: Reproduction and Fertility 6, 2; 10.1530/RAF-25-0005
Correlation of phosphate abundance in allantoic and amniotic fluids with TNSALP activity in utero-placental tissues
Phosphate abundance in allantoic fluid was positively correlated with endometrial TNSALP activity on Day 60 (P < 0.05) and when combining data from both Day 60 and Day 90 samples (P < 0.001. Table 1). Similarly, phosphate abundance in amniotic fluid was positively correlated with endometrial TNSALP activity (P < 0.05) when combining data from both Day 60 and Day 90 samples, but not when considering only one of the days of gestation. No significant (P < 0.05) correlations between chorioallantoic TNSALP activity and phosphate abundance in allantoic or amniotic fluids were detected (Table 1). However, phosphate abundance in allantoic fluid tended to be positively correlated with chorioallantoic TNSALP activity (P = 0.076) on Day 90.
Correlations between total phosphate in allantoic and amniotic fluids and TNSALP activity in homogenates of endometrial and chorioallantois tissues.
| Fluid | Tissue | Gestational day | RSq | P value |
|---|---|---|---|---|
| Allantoic | Endometria | 60 + 90 | 0.594 | <0.0001 |
| Allantoic | Endometria | 60 | 0.496 | <0.05 |
| Allantoic | Endometria | 90 | 0.101 | >0.10 |
| Amniotic | Endometria | 60 + 90 | 0.204 | <0.05 |
| Amniotic | Endometria | 60 | 0.284 | 0.113 |
| Amniotic | Endometria | 90 | 0.005 | >0.10 |
| Allantoic | Chorioallantois | 60 + 90 | 0.015 | >0.10 |
| Allantoic | Chorioallantois | 60 | 0.151 | 0.302 |
| Allantoic | Chorioallantois | 90 | 0.619 | 0.076 |
| Amniotic | Chorioallantois | 60 + 90 | 0.088 | >0.10 |
| Amniotic | Chorioallantois | 60 | 0.008 | >0.10 |
| Amniotic | Chorioallantois | 90 | 0.184 | >0.10 |
Bold P values indicate P < 0.05.
Localization of TNSALP protein in utero-placental tissues
TNSALP protein was abundantly expressed in the uterus and chorioallantois on both days investigated, with TNSALP localized to the endometrial LE, endometrial glandular epithelium, chorionic and areolar epithelium, as well as stromal cells and endothelium in the endometrium, myometrium, and chorioallantois (Fig. 3). There was an apparent decrease in immunoreactivity for TNSALP protein in the endometrial stratum compactum stroma and chorionic epithelium on Day 90 compared to Day 60 of gestation.
Representative images of immunolocalization of TNSALP protein in porcine endometria (A, B, C, D) and chorioallantois (F, G, H, I, K, L, M, N). Rabbit IgG (RIgG) controls were included at equivalent concentrations of protein to the TNSALP antibody as a negative control. BV, blood vessels; LE, luminal epithelium; GE, glandular epithelium; SC, stratum compactum stroma; AE, areole; CE, chorionic epithelium. Scale bars represent 100 μm in A, F, K, C, H, M, E, J, and O or 60 μm in B, G, L, D, I, and N. n = 12 utero-placental units on Day 60 and Day 90.
Citation: Reproduction and Fertility 6, 2; 10.1530/RAF-25-0005
Localization of TNSALP enzymatic activity in utero-placental tissues
TNSALP activity was present in the endothelium of blood vessels in both the endometrium and chorioallantois on both Days 60 and 90 of gestation (Fig. 4), with an apparent increase in activity in endometrial blood vessels closest to the luminal-chorionic epithelial bilayer when compared to those deeper in the endometria or chorioallantois. Enzymatic activity was present on the basal surface of the endometrial glands, perhaps in the basement membrane and fibroblasts that surround the glands, on Day 60 of gestation but not Day 90. The endometrial stratum compactum stroma had strong TNSALP activity on Day 60 of gestation, which was not detected on Day 90 of gestation. TNSALP activity was present in columnar cells at the bottom of the chorioallantoic troughs on both Day 60 and Day 90 of gestation, with an apparent decrease in activity in the chorioallantois on Day 90 as compared to Day 60. Weak TNSALP activity was present in the areolar epithelium, with a modest increase in activity on Day 90 when compared with Day 60.
Representative images of localization of TNSALP enzymatic activity in porcine endometria and chorioallantois. Sections incubated with 100 mmol/L Tris–HCl were utilized as a negative control. BV, blood vessels; LE, luminal epithelium; GE, glandular epithelium;, SC, stratum compactum stroma; AE, areole; CE, chorionic epithelium. Scale bars represent 100 μm. n = 12 utero-placental units on Day 60 and Day 90.
Citation: Reproduction and Fertility 6, 2; 10.1530/RAF-25-0005
Discussion
Despite the appreciation of important roles of phosphate in the regulation of conceptus growth and development, mechanisms regulating utero-placental phosphate availability are under-investigated. The results of this study provide evidence for i) expression and activity of TNSALP in porcine utero-placental tissues in mid- and late gestation, and ii) spatiotemporal changes in TNSALP localization with corresponding alterations in abundances of phosphate in allantoic and amniotic fluids.
In this study, we investigated two distinct stages of gestation: Days 60 and 90. The porcine placenta undergoes significant proliferation from approximately Day 20–60 to increase in size (Marrable 1971, Knight et al. 1977, Wu et al. 2005, Wright et al. 2016), after which its growth plateaus (Marrable 1971). In addition, during this period, there is extensive remodeling of the luminal-chorionic epithelial bilayer to maximize surface area available for nutrient exchange (Friess et al. 1980, Dantzer 1985, Rootwelt et al. 2012, 2013, Seo et al. 2020). In contrast, Day 90 is a period of exponential fetal growth and rapid skeletal mineralization, which occurs during the last trimester of gestation (Marrable 1971, Wrathall et al. 1974, Connolly et al. 2004). As both stages of gestation are associated with high nutritional demands, it was hypothesized that there would be an accumulation of phosphate in the allantoic and amniotic fluids at these stages of gestation in pigs. Although phosphate was abundant in both allantoic and amniotic fluids on Days 60 and 90 of gestation, there was an effect of day of gestation on abundances of phosphate in allantoic and amniotic fluids, with less phosphate in fetal fluids on Day 90 than on Day 60 of gestation. It could be speculated that this significant decrease in phosphate abundance occurs due to utilization by the fetus, which would be anticipated to have high phosphate requirements to sustain exponential fetal growth for the synthesis of ATP, generation of nucleotides and creatine phosphate, cell signaling pathways (phosphorylation and dephosphorylation), and skeletal mineralization, all of which are key for growth and development of the conceptus (Wu 2018).
The decrease in phosphate abundance in fetal fluids was associated with decreased TNSALP activity in the endometrium on Day 90. However, no effect of gestational day on TNSALP activity in the chorioallantois was detected. This suggests that TNSALP may regulate the availability of phosphate from the gilt for utilization by the conceptus.
TNSALP enzymatic activity was located on the basal surface of the endometrial glandular epithelium on Day 60 of gestation. This differs from what has been reported for sheep (Stenhouse et al. 2023, 2024), where there is strong TNSALP activity on the apical surface of the endometrial luminal and glandular epithelia, which may generate phosphate that is secreted into the uterine lumen to contribute to histotroph. This suggests that in contrast to sheep, the endometrial glandular epithelia in swine may utilize TNSALP to generate phosphate for the regulation of their own cellular functions, and not for secretion into the uterine lumen (Wu et al. 2022).
Strong TNSALP activity was present in the endothelium of blood vessels in the porcine endometrium, particularly in blood vessels in close proximity to the luminal-chorionic epithelial bilayer. This suggests that TNSALP acts in the endometrial blood vessels to generate phosphate, which may then be utilized by a) the endometrium itself, b) the chorioallantois for cellular signaling pathways, or c) the chorioallantois for transport, sequestration in fetal fluids, or utilization by the fetus. This finding emulates our recent findings from studies of sheep utero-placental tissues (Stenhouse et al. 2023, 2024), suggesting conservation of this function across livestock species with different types of placentation. Interestingly, the finding of abundant TNSALP activity in endometrial endothelial cells was not found in the vasculature of the chorioallantois, suggesting that TNSALP may not regulate phosphate availability in the placental vasculature in pigs.
The pig has non-invasive epitheliochorial placentation in which the trophectoderm attaches to the endometrial LE and, for the duration of gestation, the endometrial LE and placental chorionic epithelium remain intact, forming a unique epithelial bilayer interface (Seo et al. 2020, Johnson et al. 2021, Stenhouse et al. 2022a ). Within the folded epithelial bilayer, differences in expression of molecules with important roles in nutrient transport have been reported (Steinhauser et al. 2016, Kramer et al. 2020, Seo et al. 2022, Johnson et al. 2023, Lefevre et al. 2024). Although TNSALP protein localized throughout the endometrial LE and the chorionic epithelium, TNSALP activity localized specifically to the columnar chorionic epithelial cells on both Day 60 and Day 90 of gestation, with an apparent decrease in activity in the chorioallantois on Day 90 as compared to Day 60. While the physiological significance of differences in the expression of molecules with roles in nutrient transport in the folded epithelia remains poorly understood, it is generally thought that the tall columnar chorionic epithelial cells are important for nutrient transport across the uterine–placental interface (Vallet & Freking 2007, Vallet et al. 2009). Thus, the specific localization of activity in these specific areas of the chorionic epithelium suggests differential regulation of TNSALP activity and function at the folded epithelial interface to regulate phosphate availability across gestation.
In addition to nutrient transport across the luminal-chorionic epithelial bilayer, nutrients are also transported via invaginations of the chorioallantois called areole, that form over the openings of the endometrial glands to sequester and transport nutrients and macromolecules present in histotrophic secretions from the endometrial glands into the conceptus vasculature (Friess et al. 1981, Dantzer & Leiser 1994, Johnson et al. 2021, Stenhouse et al. 2022a ). TNSALP protein and enzymatic activity were present in the areolar epithelium on both Day 60 and Day 90 of pregnancy, with an apparent upregulation on Day 90 as compared to Day 60. This finding suggests that, in addition to the greater activity of TNSALP in other cell types in utero-placental tissues, TNSALP may act in the areole to convert phosphocompound substrates such as PPi, PLP, and PEA present in histotrophic secretions into inorganic phosphate for entry into the fetal-placental circulation. Areolar-specific gene expression in the chorioallantois of pigs has previously been reported, and aquaporin 5 (AQO5) is a genetic marker for areolar cells before their invagination into areole (Song et al. 2010, Steinhauser et al. 2016, McLendon et al. 2022, Johnson et al. 2023).
The present study highlights the importance of establishing cell-specific localization of TNSALP protein and enzymatic activity. Striking differences in the localization of TNSALP protein and enzymatic activity in porcine utero-placental tissues were detected (Figs 3 and 4). Human TNSALP has several post-translational modifications, including five putative N-glycosylation sites (Asn140, Asn230, Asn271, Asn303, and Asn430), and an undetermined O-glycosylation site (Weiss et al. 1986, Nosjean et al. 1997), which may contribute to differential enzymatic activities. Post-translational modifications of TNSALP have not been investigated in the pig, but it would be reasonable to anticipate that they would be present and have an impact on enzymatic activity.
Conclusion
This study provides evidence for a potential role of TNSALP in the regulation of phosphate availability at the porcine utero-placental interface, which warrants further investigation. Our findings suggest cell- and time-specific alterations in the regulation of utero-placental phosphate availability, which also warrant further investigation.
Supplementary materials
This is linked to the online version of the paper at https://doi.org/10.1530/RAF-25-0005.
Declaration of interest
C Stenhouse is an Associate Editor of Reproduction and Fertility. C Stenhouse was not involved in the review or editorial process for this paper, on which she is listed as an author. The authors declare that they have no competing interests.
Funding
This project was supported by Agriculture and Food Research Initiative Competitive Grant no. 2016-67015-24958 from the USDA National Institute of Food and Agriculture awarded to FWB, GAJ, and GW, USDA National Institute of Food and Agriculture and Multistate/Regional Research Appropriations under Project PEN04775 and Accession number 7001112, and the USDA National Institute of Food and Agriculture and Hatch Appropriations under Project PEN04995 and Accession number 7007748.
Author contribution statement
The animal experimentation and sample collections were planned and executed by FWB, GAJ, GW, JWC, CS, NS, KMH, RMM, MGN, and HS. Sample analyses were performed by CS and NS. The first draft of the manuscript was written by CS and FWB, and edited by NS, KMH, RMM, MGN, JWC, HS, GW, and GAJ.
Acknowledgments
The contributions of undergraduate students, graduate students, and faculty of Texas A&M University and the Pennsvylania State University to this study are gratefully acknowledged.
References
Bazer FW , Song G , Kim J , et al. 2012 Uterine biology in pigs and sheep. J Anim Sci Biotechnol 3 1–21. (https://doi.org/10.1186/2049-1891-3-23)
Choi Y , Seo H , Shim J , et al. 2014 Klotho: expression and regulation at the maternal-conceptus interface in pigs. J Anim Reprod Biotechnol 29 375–383. (https://doi.org/10.12750/jet.2014.29.4.375)
Connolly SA , Jaramillo D , Hong JK , et al. 2004 Skeletal development in fetal pig specimens: MR imaging of femur with histological comparison. Radiology 233 505–514. (https://doi.org/10.1148/radiol.2332030131)
Correia-Branco A , Rincon MP , Pereira LM , et al. 2020 Inorganic phosphate in the pathogenesis of pregnancy-related complications. Int J Mol Sci 21 1–11. (https://doi.org/10.3390/ijms21155283)
Dantzer V 1985 Electron microscopy of the initial stages of placentation in the pig. Anat Embryol 172 281–293. (https://doi.org/10.1007/bf00318976)
Dantzer V & Leiser R 1994 Initial vascularisation in the pig placenta: I. Demonstration of nonglandular areas by histology and corrosion cases. Anat Rec 238 177–190. (https://doi.org/10.1002/ar.1092380204)
Friess AE , Sionwatz F , Skolek-Winnisch R , et al. 1980 The placenta of the pig I. Finestructural changes of the placental barrier during pregnancy. Anat Embryol 158 179–191. (https://doi.org/10.1007/bf00315905)
Friess AE , Sinowatz F , Skolek-Winnisch R , et al. 1981 The placenta of the pig II the ultrasound of the areolae. Anat Embryol 163 43–53. (https://doi.org/10.1007/bf00315769)
Goretti M , Penido MG & Alon US 2012 Phosphate homeostasis and its role in bone health. Pediatr Nephrol 27 2039–2048. (https://doi.org/10.1007/s00467-012-2175-z)
Hernando N , Gagnon K & Lederer E 2021 Phosphate transport in epithelial and nonepithelial tissue. Physiol Rev 101 1–35. (https://doi.org/10.1152/physrev.00008.2019)
Johnson G , Bazer F & Seo H 2021 The early stages of implantation and placentation in the pig. Adv Anat Embryol Cell Biol 234 61–89. (https://doi.org/10.1007/978-3-030-77360-1_5)
Johnson GA , Seo H , Bazer FW , et al. 2023 Metabolic pathways utilized by the porcine conceptus, uterus, and placenta. Mol Reprod Dev 90 673–683. (https://doi.org/10.1002/mrd.23570)
Khailova L , Robison J , Jaggers J , et al. 2020 Tissue alkaline phosphatase activity and expression in an experimental infant swine model of cardiopulmonary bypass with deep hypothermic circulatory arrest. J Inflamm 17 1–13. (https://doi.org/10.1186/s12950-020-00256-2)
Knight JW , Bazer FW , Thatcher WW , et al. 1977 Conceptus development in intact and unilaterally hysterectomized-ovariectomized gilts: interrelations among hormonal status, placental development, fetal fluids and fetal growth. J Anim Sci 44 620–637. (https://doi.org/10.2527/jas1977.444620x)
Kovacs CS 2014 Bone development and mineral homeostasis in the fetus and neonate: roles of the calciotropic and phosphotropic hormones. Physiol Rev 94 1143–1218. (https://doi.org/10.1152/physrev.00014.2014)
Kovacs CS 2015 Calcium, phosphorus, and bone metabolism in the fetus and newborn. Early Hum Dev 91 623–628. (https://doi.org/10.1016/j.earlhumdev.2015.08.007)
Kramer AC , Steinhauser CB , Gao H , et al. 2020 Steroids regulate SLC2A1 and SLC2A3 to deliver glucose into trophectoderm for metabolism via glycolysis. Endocrinology 161 1–19. (https://doi.org/10.1210/endocr/bqaa098)
Lee S , Jung MH , Song K , et al. 2021 Failure to maintain full-term pregnancies in pig carrying klotho monoallelic knockout fetuses. BMC Biotechnol 21 1–11. (https://doi.org/10.1186/s12896-020-00660-9)
Lefevre CM , Cain JW , Kramer AC , et al. 2024 Evidence for metabolism of creatine by the conceptus, placenta, and uterus for production of adenosine triphosphate during conceptus development in pigs. Biol Reprod 111 694–707. (https://doi.org/10.1093/biolre/ioae088)
Leitner K , Szlauer R , Ellinger I , et al. 2001 Placental alkaline phosphatase expression at the apical and basal plasma membrane in term villous trophoblasts. J Histochem Cytochem 49 1155–1164. (https://doi.org/10.1177/002215540104900909)
Maltepe E & Fisher SJ 2015 Placenta: the forgotten organ. Annu Rev Cell Dev Biol 31 523–552. (https://doi.org/10.1146/annurev-cellbio-100814-125620)
Marrable AW 1971 The Embryonic Pig: A Chronological Account. London: Pitman Medical.
McErlean S & King C 2019 Does an abnormally elevated maternal alkaline phosphatase pose problems for the fetus? BMJ Case Rep 12 e229109. (https://doi.org/10.1136/bcr-2018-229109)
McLendon BA , Kramer AC , Seo H , et al. 2022 Temporal and spatial expression of aquaporins 1, 5, 8, and 9: potential transport of water across the endometrium and chorioallantois of pigs. Placenta 124 28–36. (https://doi.org/10.1016/j.placenta.2022.05.007)
Meyer RE , Thompson SJ , Addy CL , et al. 1995 Maternal serum placental alkaline phosphatase level and risk for preterm delivery. Am J Obstet Gynecol 173 181–186. (https://doi.org/10.1016/0002-9378(95)90187-6)
Millán JL 2006 Mammalian Alkaline Phosphatases. From Biology to Applications in Medicine and Biotechnology. Wiley VCH. (https://doi.org/10.1002/3527608060)
Mitchell H , Hamilton T , Steggerda F , et al. 1945 The chemical composition of the adult human body and its bearing on the biochemistry of growth. J Biol Chem 168 625–637. (https://doi.org/10.1016/s0021-9258(19)51339-4)
Moawad AH , Goldenberg RL , Mercer B , et al. 2002 The preterm prediction study: the value of serum alkaline phosphatase, alpha-fetoprotein, plasma corticotropin-releasing hormone, and other serum markers for the prediction of spontaneous preterm birth. Am J Obstet Gynecol 186 990–996. (https://doi.org/10.1067/mob.2002.121727)
Motulsky HJ & Brown RE 2006 Detecting outliers when fitting data with nonlinear regression – a new method based on robust nonlinear regression and the false discovery rate. BMC Bioinf 7 1–20. (https://doi.org/10.1186/1471-2105-7-123)
National Research Council 2012 Nutrient Requirements of Swine. Washington DC, USA: National Academies Press. (https://doi.org/10.17226/13298)
Nosjean O , Koyama I , Goseki M , et al. 1997 Human tissue non-specific alkaline phosphatases: sugar-moiety-induced enzymic and antigenic modulations and genetic aspects. Biochem J 321 297–303. (https://doi.org/10.1042/bj3210297)
Paquette AG , MacDonald J , Bammler T , et al. 2023 Placental transcriptomic signatures of spontaneous preterm birth. Am J Obstet Gynecol 73 1–18. (https://doi.org/10.1016/j.ajog.2022.07.015)
Posen S , Cornish CJ , Horne M , et al. 1969 Part V. Clinical enzymology. Placental alkaline phosphatase and pregnancy. Ann N Y Acad Sci 166 733–744. (https://doi.org/10.1111/j.1749-6632.1969.tb54313.x)
Roberts RM & Bazer FW 1988 The functions of uterine secretions. J Reprod Fertil 82 875–892. (https://doi.org/10.1530/jrf.0.0820875)
Rootwelt V , Reksen O , Farstad W , et al. 2012 Associations between intrapartum death and piglet, placental, and umbilical characteristics. J Anim Sci 90 4289–4296. (https://doi.org/10.2527/jas.2012-5238)
Rootwelt V , Reksen O , Farstad W , et al. 2013 Postpartum deaths: piglet, placental, and umbilical characteristics. J Anim Sci 91 2647–2656. (https://doi.org/10.2527/jas.2012-5531)
Seo H , Li X , Wu G , et al. 2020 Mechanotransduction drives morphogenesis to develop folding during placental development in pigs. Placenta 90 62–70. (https://doi.org/10.1016/j.placenta.2019.12.011)
Safarova A , Bige Ö , Doğan E , et al. 2007 Origin and significance of extremely elevated serum alkaline phosphatase activity during normal pregnancy. Turkiye Klinikleri J Gynecol Obst 17 405–408.
Seo H , Kramer AC , McLendon BA , et al. 2022 Elongating porcine conceptuses can utilize glutaminolysis as an anaplerotic pathway to maintain the TCA cycle. Biol Reprod 107 823–833. (https://doi.org/10.1093/biolre/ioac097)
Sinowatz F & Friess AE 1983 Uterine glands of the pig during pregnancy. Anat Embryol 166 121–134. (https://doi.org/10.1007/bf00317948)
Song G , Bailey DW , Dunlap KA , et al. 2010 Cathepsin B, cathepsin L, and cystatin C in the porcine uterus and placenta: potential roles in endometrial/placental remodeling and in fluid-phase transport of proteins secreted by uterine epithelia across placental areolae. Biol Reprod 82 854–864. (https://doi.org/10.1095/biolreprod.109.080929)
Steinhauser CB , Landers M , Myatt L , et al. 2016 Fructose synthesis and transport at the uterine-placental interface of pigs: cell- specific localization of SLC2A5, SLC2A8, and components of the polyol Pathway1. Biol Reprod 95 108. (https://doi.org/10.1095/biolreprod.116.142174)
Stenhouse C , Halloran KM , Newton MG , et al. 2021 Novel mineral regulatory pathways in ovine pregnancy: I. phosphate, klotho signaling, and sodium-dependent phosphate transporters. Biol Reprod 104 1084–1096. (https://doi.org/10.1093/biolre/ioab028)
Stenhouse C , Seo H , Wu G , et al. 2022a Insights into the regulation of implantation and placentation in humans, rodents, sheep, and pigs. Adv Exp Med Biol 1354 25–48. (https://doi.org/10.1007/978-3-030-85686-1_2)
Stenhouse C , Suva LJ , Gaddy D , et al. 2022b Phosphate, calcium, and vitamin D: key regulators of fetal and placental development in mammals. Adv Exp Med Biol 1354 77–107. (https://doi.org/10.1007/978-3-030-85686-1_5)
Stenhouse C , Halloran KM , Newton MG , et al. 2023 Characterization of TNSALP expression, localization, and activity in ovine utero-placental tissues. Biol Reprod 109 954–964. (https://doi.org/10.1093/biolre/ioad113)
Stenhouse C , Halloran KM , Hoskins EC , et al. 2024 Progesterone regulates tissue non-specific alkaline phosphatase (TNSALP) expression and activity in ovine utero-placental tissues. J Anim Sci Biotechnol 15 1–14. (https://doi.org/10.1186/s40104-024-01048-x)
Suva L & Friedman P 2020 PTH and PTHrP actions on bone. Handb Exp Pharmacol 262 27–46. (https://doi.org/10.1007/164_2020_362)
Taylor-Miller T & Allgrove J 2021 Endocrine diseases of newborn: epidemiology, pathogenesis, therapeutic options, and outcome “current insights into disorders of calcium and phosphate in the newborn”. Front Pediatr 9 600490. (https://doi.org/10.3389/fped.2021.600490)
Titaux C , Ternynck C , Pauchet M , et al. 2023 Total alkaline phosphatase levels by gestational age in a large sample of pregnant women. Placenta 132 32–37. (https://doi.org/10.1016/j.placenta.2022.12.005)
Vallet JL & Freking BA 2007 Differences in placental structure during gestation associated with large and small pig fetuses. J Anim Sci 85 3267–3275. (https://doi.org/10.2527/jas.2007-0368)
Vallet JL , Miles JR & Freking BA 2009 Development of the pig placenta. Soc Reprod Fertil Suppl 66 265–279.
Weiss MJ , Henthorn PS , Lafferty MA , et al. 1986 Isolation and characterization of a cDNA encoding a human liver/bone/kidney-type alkaline phosphatase. Proc Natl Acad Sci U S A 83 7182–7186. (https://doi.org/10.1073/pnas.83.19.7182)
Whyte MP , Landt M , Ryan LM , et al. 1995 Alkaline phosphatase: placental and tissue-nonspecific isoenzymes hydrolyze phosphoethanolamine, inorganic pyrophosphate, and pyridoxal 5′-phosphate. Substrate accumulation in carriers of hypophosphatasia corrects during pregnancy. J Clin Investig 95 1440–1445. (https://doi.org/10.1172/jci117814)
Wrathall AE , Bailey J & Hebert CN 1974 A radiographic study of development of the appendicular skeleton in the fetal pig. Res Vet Sci 17 154–168. (https://doi.org/10.1016/s0034-5288(18)33675-0)
Wright EC , Miles JR , Lents CA , et al. 2016 Uterine and placenta characteristics during early vascular development in the pig from day 22 to 42 of gestation. Anim Reprod Sci 164 14–22. (https://doi.org/10.1016/j.anireprosci.2015.11.002)
Wu G 2018 Principles of Animal Nutrition. Boca Raton, FL: CRC Press. (https://doi.org/10.1201/9781315120065)
Wu G , Bazer FW , Hu J , et al. 2005 Polyamine synthesis from proline in the developing porcine placenta. Biol Reprod 72 842–850. (https://doi.org/10.1095/biolreprod.104.036293)
Wu G , Bazer FW , Satterfield MC , et al. 2022 L-Arginine nutrition and metabolism in ruminants. Adv Exp Med Biol 1354 177–206. (https://doi.org/10.1007/978-3-030-85686-1_10)
