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. Author manuscript; available in PMC: 2021 Jul 22.
Published in final edited form as: FASEB J. 2020 Sep 22;34(11):15252–15268. doi: 10.1096/fj.202001104R

Maternal and fetal alkaline ceramidase 2 is required for placental vascular integrity in mice

Fang Li 1,2,3,#, Ruijuan Xu 1,2,#, Chih-Li Lin 1,2, Benjamin E Low 4, Louise Cai 2, Sally Li 2, Ping Ji 1, Liqun Huang 1, Michael V Wiles 4, Yusuf A Hannun 1,2, Lina M Obeid 1,2,5, Ye Chen 3, Cungui Mao 1,2
PMCID: PMC8296754  NIHMSID: NIHMS1720883  PMID: 32959379

Abstract

Sphingolipids have been implicated in mammalian placental development and function, but their regulation in the placenta remains unclear. Herein we report that alkaline ceramidase 2 (ACER2) plays a key role in sustaining the integrity of the placental vasculature by regulating the homeostasis of sphingolipids in mice. The mouse alkaline ceramidase 2 gene (Acer2) is highly expressed in the placenta between embryonic day (E) 9.5 and E12.5. Acer2 deficiency in both the mother and fetus decreases the placental levels of sphingolipids, including sphingoid bases (sphingosine and dihydrosphingosine) and sphingoid base-1-phosphates ( sphingosine-1-phophate and dihydrosphingosine-1-phosphate) and results in the in utero death of ≈ 50% of embryos at E12.5 whereas Acer2 deficiency in either the mother or fetus has no such effects. Acer2 deficiency causes hemorrhages from the maternal vasculature in the junctional and/or labyrinthine zones in E12.5 placentas. Moreover, hemorrhagic but not non-hemorrhagic Acer2-deficient placentas exhibit an expansion of parietal trophoblast giant cells with a concomitant decrease in the area of the fetal blood vessel network in the labyrinthine zone, suggesting that Acer2 deficiency results in embryonic lethality due to the atrophy of the fetal blood vessel network in the placenta. Taken together, these results suggest that ACER2 sustains the integrity of the placental vasculature by controlling the homeostasis of sphingolipids in mice.

Keywords: ACER2, ceramidase, placenta, sphingosine, sphingosine-1-phosphate, vasculature

INTRODUCTION

The rodent or human placenta contains cells of both fetal and maternal origin and can be divided into three distinct zones: the decidual zone (DZ), junctional zone (JZ), and labyrinthine zone (LZ) (1, 2). Trophoblast giant cells (TGCs), spongiotrophoblasts (spT), and syncytiotrophoblasts (SynTs) are the major cell types of fetal origin (1, 3). TGCs, which are the earliest differentiated cell type to form during embryogenesis in rodents, are essential for embryo implantation and maternal adaptations to pregnancy (1, 3). The SynT form two specialized cell layers (SynT-I and SynT-II) that encompass the entire surface of villous trees and the SynT-I cell layer is in direct contact with maternal blood in the maternal blood space (1, 3). The SynT layers prevent the exchange of large molecules and circulating cells between the fetus and the mother while enabling fetal nutrient uptake, waste elimination, and gas exchange through the maternal circulation (1, 3). Abnormalities in this maternal/fetal blood interface may result in various pregnancy-associated complications, such as recurrent miscarriage, preeclampsia (PE), and intrauterine growth restriction (IUGR) (1, 3). However, much remains unclear about the molecular mechanism by which the maternal and fetal blood interface is maintained during pregnancy.

Emerging evidence suggests that sphingolipids, a class of lipids containing a long-chain base (a sphingoid base) as a common moiety, have important roles in placental development and function. The biosynthesis of sphingolipids starts with the condensation of serine and palmitoyl-CoA through the action of serine palmitoyl-CoA transferase (SPT), which is followed by multiple sequential enzymatic steps . A recent study by Ding et al. (4) demonstrated that SPTLC1, SPTLC2, and SPTSSA, the genes encoding three SPT subunits, are markedly upregulated in mouse uterine stromal cells during decidualization and that inhibiting SPT with its inhibitor, I-cycloserine, results in defective decidualization and early pregnancy loss in mice. Sphingosine-1-phosphate (S1P), a sphingolipid metabolite, acts as a bioactive lipid that mediates various cellular responses (5), including cell proliferation (6–8), survival (8–10), differentiation (10), adhesion (11–13), and migration (14–17), mainly by activating one or more of its G protein-coupled receptors (GPCRs), S1P1-5 (aka S1PR1-5) (18–22). S1P also mediates various biological processes including the maturation of the fetal vasculature (23), the development of heart (24), and angiogenesis (25) in mice. As such, deficiency of either S1P (26) or its receptor S1PR1 (23) leads to embryonic lethality. Recent studies find that S1P may mediate placental differentiation, development, and immunity. Mizugishi et al. (26) found that blocking the formation of endogenous S1P in mice impairs endometrial decidualization and severely compromises uterine blood vessels, leading to early pregnancy failure in mice. Mizugishi et al. also showed that inhibiting the formation of S1P induces massive infiltration of inflammatory cells into the fetoplacental unit (27). Johnstone et al. (28) demonstrated that treatment with S1P inhibits the differentiation of primary human cytotrophoblasts into syncytiotrophoblasts in vitro. Yang et al. showed that S1P, when supplied exogenously, induces extravillous trophoblast cell invasion by activating S1PR1 (29). Westwood et al. demonstrated that S1P inhibits migration of human extravillous trophoblast cell lines (30). A recent study by Del Gaudio et al. (31) showed that S1P complexed with human cord blood-derived high-density-lipoprotein (HDL) preserves the integrity of the fetoplacental vasculature in an in vitro system. Similar to S1P, dihydrosphingosine-1-phosphate (DHS1P), a saturated analog of S1P, is abundant in blood (32) and can also activate S1PR1 in renal endothelial cells (33) although neither its role in placental development and function nor its regulation in the placenta are unclear. Although sphingolipids play important physiological roles in placental development and function, recent studies demonstrated that an aberrant elevation of placental sphingolipids may be implicated in pregnancy complications in humans, such as preeclampsia (PE) and intrauterine growth restriction (IUGR). Del Gaudio et al. (34) demonstrated that DHS, the immediate precursor of DHS1P, is increased in placental chorionic arteries and isolated endothelial cells from PE patients compared to healthy controls. Megan et al. (35) demonstrated that DHS is also increased in placentas from PE patients due to an increase in SPT activity. Chauvin et al. (36) found that the levels of sphingosine (SPH), the immediate precursor of S1P, are increased in IUGR placentas. Although the pathological role of SPH and DHS in placenta remains to be elucidated, both SPH and DHS, when accumulated in cells, have been shown to induce cell death (37, 38). Taken together, these results suggest that to ensure uneventful pregnancy, the metabolism of these bioactive sphingolipids in the placenta must be tightly regulated.

S1P and DHS1P are synthesized only from the phosphorylation of SPH and DHS, respectively, by the action of the SPH kinases, SPHK1 and SPHK2, in mammalian cells (39). SPH is in turn generated from the hydrolysis of ceramides by the action of ceramidases, which were classified into the acid, neutral, and alkaline ceramidase subtypes, respectively (40, 41). DHS is synthesized de novo from serine and palmitoyl-CoA by the sequential actions of serine palmitoyl transferase and ketodihydrosphingosine reductase or is generated from the hydrolysis of dihydroceramides by the action of ceramidase (40). We previously demonstrated that human alkaline ceramidase 2 (ACER2), a member of the alkaline ceramidase family that we identified initially from the yeast Saccharomyces cerevisiae and then from mammals, plays a key role in regulating S1P levels in cultured cells by catalyzing the generation of SPH, the rate-limiting step for S1P formation (42). Using genetically modified mouse models, we recently demonstrated that among the 5 known ceramidase genes, ACER2 plays the predominant role in regulating the homeostasis of SPH, DHS, S1P, and DHS1P in plasma and several peripheral tissues (43). We previously demonstrated that the human ACER2 is expressed at much higher levels in the placenta than in other major organs (44). However, it is unclear whether ACER2 and its mouse counterpart Acer2 play roles in regulating S1P and DHS1P levels in the placenta. Nor are clear its roles in placental development and function.

In this study, we demonstrate that the mouse Acer2 is highly expressed in the placenta, similar to the human ACER2, and that Acer2 plays a key role in regulating the placental levels of S1P and DHS1P by controlling the generation of SPH and DHS, respectively, in both maternal and fetal cells in the placenta. Moreover, we reveal a novel role for the ACER2 pathway in maintaining the integrity of the maternal vasculature in the placenta and thereby fetal survival.

MATERIALS AND METHODS

Animals

The mouse strain (C57BL/6J-Acer2em1Mvw/MvwJ JAX, stock 026793, abbreviated here to Acer2+/−) heterozygous for an Acer2 mutant allele in which exon 2 was deleted has been established in our previous study (43). The mouse strain (Acer2−/−) homozygous for the Acer2 mutant allele was generated by an intercross of Acer2+/− mice. Wild type C57BL/6 mice were purchased from The Jackson Laboratory. All mice used in this study were housed under conventional laboratory conditions of a constant room temperature (22°C), humidity level (55%), and 12-h light/dark photoperiod with mouse chow (W.F. Fisher & Son, Somerville, NJ) and water available ad libitum. For timed mating, male mice at ages of 2-5 months were housed individually for 1-2 weeks prior to mating with 2-5 month-old nulliparous female mice in proestrus or estrus. Female and male mice were paired at 6-7 pm and vaginal plugs were checked in the female mice next morning. If a copulatory plug was found, putative dams were separated from males. The morning that a plug was found was set as 0.5 days post coitum (0.5 dpc) or embryonic day 0.5 (E0.5). The plugged mice were weighed daily to confirm successful pregnancy. Female mice lacking copulatory plugs were re-paired with male mice until a copulatory plug was spotted. Mice were weaned at 21 days of age and genotyped by PCR as described (43). All animal studies were performed following procedures approved by the Institutional Animal Care and Use Committee (IACUC) at Stony Brook University (Stony Brook, NY) and comply with National Institutes of Health (NIH) guidelines.

Quantitative PCR (qPCR)

RNAs were extracted from tissues using a Trizol™ plus RNA purification kit with the catalog number (Cat. #) 12183555 from Thermo Fisher Scientific Inc. (Thermo-Fisher), Waltham, MA, USA, and reversely transcribed into cDNAs using Superscript™ III First-Strand Synthesis SuperMix (Cat. # 18080400, Thermo-Fisher) as described in our previous study (43). cDNAs were subjected to qPCR that was performed on an ABI Prism 7000 system (Thermo-Fisher). mRNA levels were analyzed using Q-Gene software which expresses data as mean normalized expression (MNE) (45). MNE is directly proportional to the mRNA levels of a target gene relative to those of the reference gene (β-actin). The primers for qPCR were synthesized by Integrated DNA Technologies, Inc. (Coralville, IA, USA) and listed in Table 1.

Table 1.

Primer pairs for qPCR

Gene Forward primer Reverse primer
Acer2 5’-GAGGACAACTACACTATCGTGCC-3’ 5’-TAGATGCCGCTGTTGAAGCACG-3’
Acer3 5’-GATTCACTGAGGAACTTTCG-3’ 5’-AGAGAAACTTCACTTTTGGC-3’
Ctsq 5’-GAGGCAGTAGTGGTCATCCC-3’ 5’-CAGTACTTCTTCCTCCGGACT-3’
Pecam1 5’-CCAAAGCCAGTAGCATCATGGTC-3’ 5’-GGATGGTGAAGTTGGCTACAGG-3’
Prl3b1 5’-CCAACGTGTGATTGTGGTGT-3’ 5’-TGCCACCATGTGTTTCAGAG-3’
Prl3d1 5’-CCCCTGTGTCATACTGCTTCCA-3’ 5’-TGAAAGACAACTCGGCACCTCA-3’
Prl7b1 5’-GGACACCAGTTTAGCAGCCTTT-3’ 5’-CATTTCGCTAACACCTGATCCA-3’
Prl8a2 5’-GGGAGAAAGCTGCATCAATTCCT-3’ 5’-GCTCTGAGAACCTCCTCATCACG-3’
Syna 5’-CTTTCCAAGGCTCTCTCGGACA-3’ 5’-CTCAGCCACAATGAGGTCCAGA-3’
Synb 5’-CAAACACTGCCATACCTCTCCG-3’ 5’-CACTGACATGGTAACAGGGTGG-3’
Tpbpa 5’-CCAGCACAGCTTTGGACATCA-3’ 5’-AGCATCCAACTGCGCTTCA-3’
Actb 5’-TGTTACCAACTGGGACGACA-3 5’-GGGGTGTTGAAGGTCTCAAA-3’
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Histology

Fetoplacental units were harvested at different dpc, fixed in 10% formalin (Thermo-Fisher, Waltham, MA), dehydrated in a graded series of ethanol (Thermo-Fisher) and xylene (Thermo-Fisher), followed by infiltration of melted paraffin (Thermo-Fisher) at 56°C in an automated processor. The tissues embedded in paraffin were sectioned vertically (the chorionic plate providing the theoretical horizontal plane) at a thickness of 7 μm as described (46), and tissue sections were mounted on SuperFrost® Plus slides (Thermo-Fisher Scientific). The tissue sections were baked, deparaffined, and rehydrated before being stained with a hematoxylin and eosin (H&E) solution (Sigma-Aldrich, St. Louis, MO) or with a Periodic acid–Schiff stain (PAS) solution (Cat. # 150680, Abeam, Cambridge, MA) as per the manusfecturer’s instructions. The stained tissue sections that were close to the placental midline and exhibited the largest area among the serial sections were imaged under an Olympus BX53 fluorescent microscope (Olympus Corporation of the Americas, Center Valley, PA).

In situ hybridization

Fetoplacental unit sections were prepared as described above and subjected to in situ hybridization with an RNAscope probe specific for the mouse gene Acer2 (Cat. # 493281) or Tpbpa (Cat. # 405511), which both were designed and manufactured by Advanced Cell Diagnostics (ACD (Newark, CA, USA). Briefly, tissue sections were baked and deparaffinized on the instrument, followed by epitope retrieval (using Leica Epitope Retrieval Buffer 2 at 95°C or at 88°C for 15 min) and protease treatment (15 min at 40°C). Probe hybridization, signal amplification, colorimetric detection, and counterstaining were subsequently performed using RNAscope® 2.5 HD reagent kits (ACD) as per the manufacturer’s instructions. The stained tissue sections were imaged under the Olympus BX53 fluorescent microscope. Brown or red dots in cells represent mRNA molecules of the genes Acer2 and Tpbpa, respectively.

Immunohistochemistry (IHC)

IHC was performed with paraffin-embedded sections as described in our previous study (47). Briefly, tissue sections were deparaffined, rehydrated, retrieved antigens, and quenched endogenous peroxidase activity. Sections were stained with 2 μg/ml of biotinylated Griffonia simplicifolia lectin I isolectin B4 (IB4) (Cat.# B-1205, Vector Laboratories, Burlingame, CA) in PBS, followed by HRP-conjugated streptavidin (1:4000) (Thermo-Fisher). Finally, the tissue sections were incubated with 1.1 mM 3,3’-Diaminobenzidine tetrahydrochloride hydrate (DAB) (Cat. # D5637, Sigma-Aldrich), and counterstained with hematoxylin (Cat. # HHS32, Sigma-Aldrich). The stained tissue sections were imaged under the Olympus BX53 fluorescent microscope.

Quantitative analyses of stained tissue sections

The software Fiji, an open source image processing package based on ImageJ (Media Cybernetics, Inc., Rockville, MD, USA), was used to measure the staining intensities or the area fractions of H&E, IHC, or ISH-stained tissue sections. Briefly, the microscopic image of each tissue section stained with H&E, IHC, or ISH was converted into an 8-bit grayscale image, the threshold of which was adjusted to match the stained areas in the original image. In the threshholded images, the total placental section area was marked using the ‘free-hand selections’ tool, and the area fraction, i.e., the percentage of the specifically stained area to the total tissue section area, was obtained using the ‘measure’ tool under the ‘Analyze’ panel in Fiji. To quantify ISH signals in cells in sections stained for Acer2 mRNA, raw integrated densities (IntDen) in individual cells were obtained in the thresholded images. The Acer2 mRNA levels in each cell were expressed as an average pixel intensity (pixels per cell).

Western blot analysis

Total cell membranes were isolated from mouse placental tissues as described in our previous study (47). Proteins were extracted from the cell membranes, measured using a Pierce™ BCA Protein Assay Kit (Cat.# 23225, Thermo-Fisher) and resolved on 12% polyacrylamide gels before being transferred to nitrocellulose (NC) membranes. The NC membranes were probed with the house-made anti-ACER2 antibody followed by the secondary antibody, goat anti-rabbit IgG antibody conjugated with horseradish peroxidase (HRP) (Cat.# 7074s, Cell Signaling Technology, Danvers, MA, USA). The ACER2 protein bands were detected with an Enhanced Chemiluminescence (ECL) kit (Cat.# PI32106, Thermo-Fisher). The ACER2 antibody was stripped off the NC membranes, which were re-probed with anti-GM130 antibody (Cat. # NBP2-53420, Novus Biologicals, Littleton, CO, USA) at a 1:1000 dilution, followed by the secondary antibody-HRP conjugate before the GM130 protein band was detected with the ECL kit. The ACER2 and GM130 protein bands were scanned and the density ratios of ACER2 protein bands to GM130 bands were measured using the software ImageJ. Briefly, the background was first subtracted from the scanned image using ImageJ’s built-in “subtract background” feature. Then the rectangle tool was used to select the ACER2 row as the first lane and then plot the lane. On the graph plot of the row, the lowest points of each peak were connected and the peak area was recorded using the wand tool. The same steps were repeated for the GM130 row to obtain the peak area of each GM130 band. Finally, the data from ImageJ were copied and pasted into an Excel spreadsheet, and the density ratio of the ACER2 protein band to the GM130 band from each sample was computed.

Sphingolipid analysis

Placental tissues collected from mice at different gestation days were subjected to LC-MS/MS analyses for various sphingolipids as described in our previous study (43). Levels of sphingolipids were normalized to total protein in each sample.

Statistical analysis

Data were presented as the mean ± SD and statistically analyzed by two-tailed and unpaired t-test or one-way ANOVA using the software GraphPad Prism 8 (GraphPad Software Inc., San Diego, CA, USA). A difference with a p value <0.05 is considered significant.

RESULTS

The mouse alkaline ceramidase 2 gene (Acer2) is highly expressed in multiple cell types in the placenta during placental development

With Northern blot analysis, we previously demonstrated that the human gene ACER2 is highly expressed in the placenta (44). To determine whether this is also the case with the mouse alkaline ceramidase 2 gene, Acer2, we compared Acer2 mRNA levels in the placenta to those in other major organs in mice. We isolated total RNA from placentas at embryonic day 12.5 (E12.5), a time when all the three cellular layers of the placenta have been well established, and other major organs from the mothers, including brain, heart, lung, liver, kidney, spleen, colon, stomach, and thymus from pregnant C57BL/6J females. The RNA samples were subjected to qPCR analysis with a primer pair specific for Acer2 or β-actin gene. The qPCR results showed that Acer2 mRNA levels are the highest in the placenta among the tissues that we examined (Fig. 1A), suggesting that similar to its human counterpart (ACER2), the mouse gene Acer2 is also highly expressed in the placenta.

Figure 1. Acer2 is highly expressed in the placenta at developmental stages.

Figure 1.

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A, total RNAs were extracted from placentas and major organs from pregnant mice (n=5) at 12.5 dpc and were subjected to qPCR analyses of Acer2 mRNA levels; B, total RNAs were extracted from placentas (n=5, 1 per dam) of pregnant mice (n=5) at 9.5, 10.5, 11.5, 12.5, 13.5, or 15.5 dpc and subjected to qPCR analysis for Acer2 mRNA levels as in A; C and D, total cell membranes were prepared from placentas (n=3 from 3 different pregnant mice at each time point) at different dpc and subjected to Western blot analyses using an antibody against ACER2 or GM130 (a Golgi complex marker as a sample-loading control). The image represents the results of 3 placentas (n=3) from 3 different pregnant mice. ACER2 and GM130 protein bands were revealed by the ECL kit (C) and the density ratio of the ACER2 vs GM130 band of each placenta was measured by the software Fiji (D). Data are presented as means ± SD and statistically analyzed by one-way ANOVA using the software Prims 8. *, p<0.05 vs other tissues (A) or time points (B and D).

For a better understanding of the role of Acer2 in placental development and function, we investigated spatiotemporal expression of Acer2 in the placenta. We first determined its temporal expression. Total RNA and protein were extracted from placentas at different embryonic days before Acer2 mRNA and protein levels were determined by qPCR and Western blot analysis, respectively. We found that placental Acer2 mRNA levels were high at E9.5 to E12.5 but nearly undetectable at E13.5 and E15.5 (Figure 1B). Consistently, ACER2 protein was readily detected in placentas at E9.5 to E12.5 but it was expressed only slightly in E13.5 placentas (Figure 1C and 1D). These results suggest that Acer2 is highly expressed in the placenta during its development. We then determined its spatial expression in the placentas by in-situ hybridization (ISH). Fetoplacental units at E11.5, a time when placental Acer2 mRNA levels peak, were dissected from Acer2+/+ pregnant dams crossed with Acer2+/+ males, fixed, paraffin-embedded, and sectioned. Sections from one of the fetoplacental units were processed for ISH, which was performed with the Acer2-specific ACD RNAscope probe. The results showed that Acer2 was highly expressed in decidual stromal cells (DC) (Figure 2A, 2B, and 2C), spongiotrophoblasts (JZT) (Figure 2A, 2B, and 2D), various trophoblast giant cells (TGCs) (Figure 2A, 2B, and 2E ), labyrinthine trophoblasts (LZT) (Figure 2A, 2B, and 2F), and but not fetal endothelial cells (EC) (Figure 2A, 2B, and 2G), suggesting that Acer2 is highly expressed in most major placental cell lineages in the placenta.

Figure 2. Acer2 is expressed in major cell types in the placenta.

Figure 2.

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Tissue sections with a 7 μm thickness were prepared from paraffin-embedded WT placentas at E11.5 as described in Materials and Methods. The tissue sections close to the placental midline were subjected to in situ hybridization (ISH) using the Acer2-specific RNAscope probe. Brown colored dots represent Acer2 mRNA molecules and nuclei were lightly counterstained blue with hematoxylin. A, an image of the placenta at a low microscopic magnification (50x); B, C, and D, the images were zoomed (200x) from the regions marked in A. B, the image showing decidual stromal cells (DC) above the green dotted line and P-TGCs below the dotted line and pointed by blue arrowheads; C, the image showing DC above the dotted line, spongiotrophoblasts (STB) below the dotted line, and P-TGCs pointed by blue arrowheads; and D, the image showing STB above the dotted line and labyrinthine trophoblasts (LTB) below the dotted line and fetal endothelial cells (FEC) pointed by red arrowheads. The images in A-D represent the results of 5 placentas from 5 different dams. E, 5 sections from each of 5 placentas from 5 dams were imaged at 200x magnification and the images of 5 microscopic fields of view per section were obtained. Ten cells of each cell type were randomly selected from each microscope field of view and quantified for ISH signals (the pixels of brown colored dots) that correlate with the Acer2 mRNA levels using Fiji as described in Materials and Methods. Each dot in the graph represents the average of ISH signals (pixels per cell) from 250 cells of each cell type. Data are presented as means ± SD of 5 placentas and statistically analyzed by one-way ANOVA. * p <0.05 vs. indicated groups.

Deficiency of Acer2 in both the mother and fetus reduces litter size

As the mouse placenta resembles the human placenta in both architecture and function (2), analyses of mutant mice deficient in the mouse Acer2 gene would enable a better understanding of the roles for this enzyme in placental development and function in mammals. We previously generated a mouse strain (Acer2+/−) heterozygous for the Acer2 mutant allele lacking exon 2 using the CRISPR/Cas9 genomic editing technology (43). We also demonstrated that intercrosses of Acer2+/− mice produced a similar number of live-born neonates per litter to intercrosses of wild-type (Acer2+/+) mice and that a similar number of offspring per litter (litter size) was weaned from intercrosses of Acer2+/− or Acer2+/+ (43). These results suggest that female heterozygotes are fertile and that total Acer2 deficiency in the placental cell lineages of fetal origin affects neither placental development and function nor fetal development and survival.

As the placenta consists of the cell lineages of both maternal and fetal origins (3), we next tested whether Acer2 deficiency in the mother impairs fetal development and survival by crossing Acer2−/− females with Acer2+/+ males. The crosses of Acer2−/− females and Acer2+/+ males delivered a similar number of live-born neonates per litter to the intercrosses of Acer2+/+ females and males (Figure 3), suggesting that ablating the Acer2 gene only from the maternal components of the placenta does not affect placental development and function either.

Figure 3. Acer2 deficiency in both the mother and embryo reduces litter size.

Figure 3.

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Acer2+/+, Acer2+/−, or Acer2−/− females (n=5) were crossed with Acer2+/+, Acer2+/−, or Acer2−/− males, respectively, and the number of live newborns from 2nd and 3nd litters from each mating pair were recorded and the average litter size (the number of live newborns per litter) was computed. The 1st litters were skipped because some inexperienced mothers ate newborns right after parturition. WT, Acer2+/+; KO, Acer2−/−. Data are presented as means ± SD and statistically analyzed by one-way ANOVA. * p <0.05 vs. indicated groups.

We then determined whether Acer2 deficiency in both mother and fetus impairs fetal development and survival by intercrosses of Acer2−/− mice. We observed that the intercrosses of Acer2−/− mice delivered 50% fewer live-born neonates per litter than the intercrosses of Acer2+/+ mice did (Figure 3), suggesting that Acer2 deficiency in both mother and fetus impairs fetal development and/or survival at 50% penetrance.

Acer2 deficiency in both mother and fetus results in placental hemorrhages and partial embryonic lethality

To investigate at what stage Acer2 deficiency impaired fetal development and/or survival, we performed necropsy of Acer2−/− and Acer2+/+ pregnant dams that were intercrossed with Acer2−/− and Acer2+/+ males, respectively, at different stages of pregnancy. By inspecting whole uteri, we found no differences in the number, size, gross morphology, and spacing of E10.5 or E11.5 fetoplacental units in the uteri of pregnant Acer2−/− and Acer2+/+ dams (Figure 4A). However, 50% of E12.5 fetoplacental units were dark-colored and being absorbed in the uteri of Acer2−/− pregnant dams compared to those in the uteri of Acer2+/+ pregnant dams (Figure 4A). By dissecting uteri and inspecting individual fetoplacental units, we found that all E11.5 Acer2−/− E11.5 Acer2+/+, and E12.5 Acer2+/+ fetuses had a heartbeat and thereby survived whereas ≈50% of E12.5 Acer2−/− fetuses died and/or absorbed (Figure 4B and 4C).

Figure 4. Acer2 deficiency causes death of embryos at E12.5.

Figure 4.

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Necropsy was performed on pregnant Acer2−/− females (n=5) crossed with Acer2+/+ males (n=5) or on pregnant Acer2+/+ females (n=5) crossed with Acer2+/+ males (n=5) at 10.5 dpc, 11.5 dpc, or 12.5 dpc. Uteri were dissected from pregnant mice (A), and embryos (B) were exposed from the uteri and the percentage of dead embryos in each dam was recorded (C). WT, Acer2+/+; KO, Acer2−/−. Data are presented as means ± SD and statistically analyzed by one-way ANOVA. * p <0.05 vs. indicated groups.

Macroscopic examination revealed numerous blood pools within 50% of E12.5 Acer2−/− placentas but not in any E12.5 Acer2+/+ placenta (Figure 5A), indicating that Acer2 deficiency causes hemorrhages in the placenta at E12.5. To define where hemorrhages occurred exactly in the placenta, we histologically analyzed hemorrhagic E12.5 placentas from Acer2−/− pregnant dams bred to Acer2−/− males and E12.5 placentas from Acer2+/+ pregnant dams bred to Acer2+/+. We observed the presence of massive hemorrhages of maternal blood in the Jz and Lz in the Acer2−/− placentas but not in Acer2+/+ placentas (Figure 5B, 5C, and 5D). Quantitative analysis with Image J showed that the bleeding area was markedly increased in the Acer2−/− placenta compared to the Acer2+/+ placenta (Figure 5E). These results suggest that Acer2 deficiency in both the mother and fetus results in partial embryonic lethality likely due to placental hemorrhages.

Figure 5. Acer2 deficiency causes hemorrhages of maternal blood in the junctional and labyrinthine zones.

Figure 5.

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Acer2+/+ (WT) and Acer2−/− E12.5 (KO) placentas at E12.5 (A) were processed into serial tissue sections as described in Materials and Methods. The tissue sections close to the placental midline were stained with a hematoxylin and eosin (H&E) solution and imaged under a microscope. The tissue sections (5 per placenta) were scanned under the microscope at 50x magnification (B). The areas in the rectangles in A were zoomed to reveal the bleeding areas (C). The images in A were converted to thresholded images (D) and total bleed areas were quantified with the software Fiji (E). The images represent the results of 5 placentas from 5 dams. The scale bars in B and D are 20 mm and those in C 20 μm. Data are presented as means ± SD and statistically analyzed by two-tailed Student’s t-test. *,p<0.05 for the difference in bleed areas per placenta between Acer2−/− and Acer2+/+ dams, which is considered significant.

Hemorrhages due to Acer2 deficiency result in expansion of P-TGC and atrophy of both junctional zone and fetal vascular network in the labyrinthine zone

The placenta contains various types of differentiated trophoblast cells, including trophoblast giant cells (TGC), spongiotrophoblasts, glycogen trophoblasts, and labyrinthine trophoblasts; each subclass trophoblasts has its unique gene expression signatures (3). To investigate whether Acer2 deficiency affects the differentiation of these trophoblast cell types, we investigated whether Acer2 deficiency affected the formation of the three distinct cellular layers of the placenta. Periodic acid-Schiff (PAS) staining showed that the population of P-TGCs was increased in the hemorrhagic E12.5 Acer2−/− placentas (Figure 6A, 6B, and 6E). The expansion of P-TGCs was confirmed with in situ hybridization (ISH) with an RNA probe specific for Prl2c2, a marker gene of P-TGCs (Figure 6C, 6D, and 6F). ISH with a Tpbpa-specific RNA probe showed that the area of the Jz was markedly reduced in the Acer2−/− placentas compared to the Acer2+/+ placentas and non-hemorrhagic Acer2−/− placentas (Figure 6G, 6H, and 6K). Labeling with biotinylated isolectin B4 (IB4), which specifically binds the endothelial cell surface of the blood vessels of fetal origin, revealed that the capillary network was strongly diminished in in the hemorrhagic Acer2−/− placenta compared to either Acer2+/+ or non-hemorrhagic Acer2−/− placenta (Figure 6I, 6J, and 6L). These results suggest that Acer2 deficiency causes the maternal blood hemorrhage, which in turn alters the placental architecture.

Figure 6. Acer2 deficiency alters the placental architecture due to hemorrhages from the maternal vasculature.

Figure 6.

Figure 6.

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A-D, Tissue sections were prepared from E12.5 fetoplacental units from Acer2−/− or Acer2+/+ dams (n=5) bred to Acer2−/− and Acer2+/+ males, respectively, as described in Materials and Methods. The tissue sections close to the placental midline were stained with a PAS solution (A and B) or were subjected to ISH analysis using a RNAscope probe specific for the gene Prl2c2 (C and D) or Tpbpa (G and H), or were labeled with the biotinylated lectin IB4 (I and J). The areas stained with the RNAscope probes were shown in red and those stained with the IB4 lection in brown. The P-TGC numbers (E) and the areas of the P-TGC layer (F) and Jz ( K) and capillary areas (L) were quantified using the software Fiji. The images in B, D, H, and J were zoomed from the regions framed by the yellow rectangles in A, C, G, and I, respectively. Data are presented as means ± SD and statistically analyzed by one-way ANOVA. *, p <0.05 vs. indicated group.

Acer2 deficiency does not affect the expression of genes that mark the major placental cell types during placental development

To investigate whether Acer2 deficiency impaired the differentiation of the placental cell lineages, we performed qPCR analyses of genes that mark major cell types in the placenta at E11.5, which is the time when all the three distinct cellular layers of the placenta have been established. We found no difference in the mRNA levels of placental cell marker genes between Acer2+/+ and Acer2−/− placentas, including the genes specific for decidual stromal cells (Prl8a2) (Figure 7A) and spiral artery TGCs (Prl7b1) (Figure 7B) in the Dz, parietal TGCs (Prl3d1) in the fetomaternal interface (Figure 7C), spongiotrophoblasts (Tpbpa) (Figure 7D), glycogen trophoblasts (Tpbpa) (Figure 7D), channel TGCs (Ctsq) (Figure 7E), and canal TGCs (Prl3b1) in the Jz (Figure 7F), sinusoidal TGCs (Ctsq) (Figure 7E), syncytiotrophoblast I (SynA) (Figure 7G), syncytiotrophoblast II (SynB) (Figure 7H), and fetal endothelial cells (Pecam1) in the Lz (Figure 7I), suggesting that Acer2 deficiency does not affect the differentiation of the placental cell lineages.

Figure 7. Acer2 deficiency does not affect the expression of the differentiation markers of placental trophoblasts.

Figure 7.

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E11.5 placentas (n=5, 1 per dam) were dissected from fetoplacental units from pregnant Acer2+/+ and Acer2−/− females (n=5 per genotype) mated with Acer2+/+ and Acer2−/− males, respectively. Total RNA was extracted from the placentas and subjected to qPCR analyses for mRNA levels of the indicated genes. Data are presented as means ± SD and statistically analyzed by two-tailed Student’s t-test. The p value for the difference in the mRNA levels of each marker between the Acer2+/+ and Acer2−/− placentas is greater than 0.05 and thereby considered insignificant.

Acer2 deficiency in both the mother and fetus reduces the placental levels of sphingolipids

To investigate whether Acer2 KO disrupts the integrity of the placental vasculature by breaching the homeostasis of sphingolipids in this extraembryonic organ, we measured the levels of sphingolipids, including ceramides, dihydroceramides, sphingoid bases, and sphingoid base phosphates in Acer2+/+ or Acer2−/− placentas. LC-MS/MS analyses demonstrated that knocking out Acer2 from both the mother and fetus significantly decreased the levels of SPH (Figure 8A), DHS (Figure 8B), S1P (Figure 8C), DHS1P (Figure 8D), and dihydroceramides (Figure 8E) without affecting the levels of ceramides (Figure 8F) in either hemorrhagic or non-hemorrhagic placentas at E12.5. Interestingly, the levels of both SPH and S1P were higher in the non-hemorrhagic placenta than in the hemorrhagic placenta. To investigate whether the differences in the levels of SPH and S1P resulted from a compensatory effect of another alkaline ceramidase 3 (ACER3) that is expressed in the placenta, we measured Acer3 mRNA levels in Acer2+/+ placentas, hemorrhagic and non-hemorrhagic Acer2−/− placentas. With qPCR analysis, we showed that the Acer3 mRNA levels were elevated in the non-hemorrhagic Acer2−/− placentas compared to either the Acer2+/+ placenta or the hemorrhagic Acer2−/− placenta (Figure 8G). These results suggest that Acer2 deficiency in both the mother and fetus alters the homeostasis of sphingoid bases (SPH and DHS) and their phosphates (S1P and DHS1P) in the placenta and that a compensatory upregulation of another alkaline ceramidase in the same protein family enables some but not all Acer2−/− placentas to develop and function normally.

Figure 8. Acer2 deficiency markedly reduces sphingoid bases, sphingoid base phosphates, and dihydroceramides in the placenta.

Figure 8.

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Placentas (n=5, 1 per mother) were harvested from pregnant Acer2−/− mice (n=5) bred to Acer2−/− or from Acer2+/+ mice (n=5) bred to Acer2+/+ mice at E12.5. The harvested placentas (n=5 per genotype) were subjected to LC-MS/MS analyses of sphingosine (SPH) (A), dihydrosphingosine (DHS) (B), S1P (C), DHS1P (D), ceramides (E), and dihydroceramides (F). Acer3 mRNA levels were measured by qPCR. WT, placentas from Acer2+/+ mice; KO-H, hemorrhagic plcentas from Acer2−/− mice; and KO-N, non-hemorrhagic placentas from Acer2−/− mice. Data are presented as means ± SD and statistically analyzed by one-way ANOVA. *, p<0.05.

DISCUSSION

The placenta in rodents and humans has both maternal and fetal vascular systems that come in close proximity to enable nutrient and gas exchange between the mother and fetus (3). Successful establishment and maintenance of both vasculature systems in the placenta are critical for fetal development and survival (48). S1P has been long known for its role in maintaining the integrity of the embryonic vasculature in mice (49) but neither its regulation in the placenta nor its role in regulating the placental vasculature were clear. In this study, we report two principle findings: 1) we identified the mouse ACER2 as a key regulator of the placental levels of S1P, DHS1P (the saturated analog of S1P), and their precursors (SPH and DHS); and 2) we demonstrated that ACER2 plays an important role in maintaining the integrity of the maternal vasculature in the mouse placenta .

Our previous and current data suggest that ACER2 is highly expressed in both human and mouse placentas. Via both Northern blot and qPCR analyses, we previously demonstrated that human ACER2 mRNA levels are the highest in the placenta among major organs that we examined (44). This is also true with mouse Acer2 mRNA levels, as demonstrated in this study. The placenta with the three cellular layers, the Dz, Jz, and Lz, is formed at E9.5 and fully mature by E14.5 (3). Both qPCR and Western blot analyses revealed that Acer2 is expressed in the placenta between E9.5 and E12.5 (Figure 1), suggesting that Acer2 is expressed in the placenta at its early developmental stage. Histology revealed that Acer2 deficiency does not affect the formation of the three distinct cellular layers in the placenta at E12.5 (Figure 5). Consistently, qPCR analyses revealed no difference in the mRNA levels of genes that mark the major cell lineages in the placenta at E11.5 (Figure 6), a time when all the three cellular layers have been well established but not fully mature. These results suggest that Acer2 expression in the placenta is not required for the early specification of the placental cell lineages. However, Acer2 deficiency in both the mother and fetus caused hemorrhages from the maternal but not fetal vasculature in an approximately 50% of E12.5 placentas (Figure 4), suggesting that Acer2 is required for the integrity of the maternal but not fetal vasculature in the placenta. Acer2 deficiency in either the mother or fetus does not impair fetal survival (Figure 3), indicating that Acer2 expression either in the mother or fetus is sufficient for maintaining the integrity of the maternal vasculature.

The placental dysfunction phenotype is not fully penetrant on this background. We analyzed the sex ratio of live pups from the intercrossing of Acer2−/− mice (6 pairs) and found that the ratio is 1:1 (data not shown). This suggests that the phenotype of hemorrhage is independent of fetal or placental sex. Our results indicate that ACER3, another member in the same protein family as ACER2, may compensate for loss of Acer2 in the placenta in mice that survive beyond E13.5. As a future direction, we will test whether loss of both Acer2 and Acer3 would increase penetrance in the placental defective phenotype in mice.

In primates and rodents, the fetal vasculature is lined with endothelial cells whereas the maternal vascular space in the placenta is unique in that, unlike other organs, placenta-derived trophoblast giant cells (TGCs) but not endothelial cells line the maternal side of the vasculature (1). There are several types of TGCs, including spiral artery TGCs (SpA-TGCs), canal TGCs (C-TGCs), channel TGCs (Ch-TGCs), and sinusoidal TGCs (S-TGCs) (1). ISH revealed high expression of Acer2 in fetal TGCs but not in fetal endothelial cells in the placenta (Figure 2). This cell type-specific expression is in line with its role in maintaining the integrity of the maternal vasculature but not the fetal vasculature in the placenta.

Interestingly, Acer2 deficiency leads to the expansion of P-TGCs with a concomitant atrophy of Jz and the fetal capillary network in Lz in hemorrhagic placentas but not in non-hemorrhagic placenta at E12.5 (Figure 5). These results suggest that the unregulated expansion of the P-TGC layer and the atrophy of both Jz and Lz may be the consequences rather than the causes of hemorrhages of the maternal blood in Acer2-deficient placentas. However, the atrophy of the Jz and Lz may limit the influx of nutrients from the mother to the fetus and the waste discharge from the fetus to the mother through circulation, thus leading to embryonic lethality.

As we previously demonstrated that ACER2 plays a critical role in the homeostasis of sphingoid bases (SPH and DHS) and sphingoid base phosphates (S1P and DHS1P) in several tissues in adult mice (43), its predominant expression in the placenta is expected to be pivotal for the homeostasis of these bioactive lipids in the this temporary organ. Indeed, in this current study, we demonstrated that generating nulls for both Acer2 alleles in both the mother and embryo markedly decreased the levels of SPH, DHS, S1P, and DHS1P in the placenta (Figure 7). In addition to the role of ACER2 in regulating S1P and DHS1P in peripheral tissues, we previously demonstrated that ACER2 plays a key role in regulating circulating SBPs (43). These results suggest that SBPs in the fetal components of the placenta are generated locally from major placental cell types and supplied systemically from the circulation. Plasma S1P has been shown to play a key role in the maturation of the fetal vasculature in mice by mediating the interaction between endothelial cells and mural cells (23) and the formation of adherens junctions between endothelial cells (50, 51). S1P mediates these biological processes mainly by activating S1PR1, one of the 5 S1P-specific G protein-coupled receptors (51). In contrast to S1P, the role of DHS1P in the vascular system is largely unclear. As DHS1P has been shown to activate S1PR1 in renal endothelial cells (33), we predict that DHS1P may have the same role as S1P in maintaining the integrity of the vasculature lined with endothelial cells. S1P and DHS1P may regulate the integrity of the maternal vasculature lined with TGCs in the placenta by activating S1PR1 in these cells. If this is the case, Acer2 deficiency may disrupt trophoblast and trophoblast adhesion by inhibiting the S1P/DHS1P-S1PR1 pathway, thus resulting in the leakage of the maternal blood space.

Most of pregnancy complications in humans, such as PE and IUGR, are associated with defects in the placental vasculature. As mentioned earlier, placental levels of SPH or DHS are elevated in patients with either IUGR (36) or PE (35). As both SPH and DHS are highly cytotoxic, their accumulation may lead to cell death in the placenta. In line with this notion, generating nulls for both Sphk1 alleles and a single Sphk2 allele impaired decidualization due to an aberrant accumulation of SPH and DHS, thus resulting in embryonic lethality (26). These results suggest that as the immediate precursors of sphingoid base phosphates, SPH and DHS are essential for the vasculature integrity whereas their aberrant increases may induce apoptosis of cells in the placenta, thus breaching placental integrity and/or placental atrophy.

These results suggest that the human ACER2, as a key regulator of both SPH and DHS, must be tightly regulated in the placenta during uneventful pregnancy. Therefore, it would be interesting to know whether an aberrant activation or upregulation of ACER2 contributes to the breaching of the homeostasis of these bioactive sphingolipids in the placentas in patients with PE, IUGR, or other pregnancy complications.

Although we have made great strides in understanding the role of ACER2 in regulating the hemostasis of bioactive sphingolipids in the placenta and the integrity of the placental vasculature, there are several limitations of this study. One limitation is that our current mouse model does not allow us to define the placental cell lineage-specific role of ACER2 in regulating the integrity of the placental vasculature as Acer2 is inactivated globally in Acer2−/− mice. Another limitation is that it is impossible to define the relative contribution of each of the bioactive lipids that are regulated by ACER2 to the integrity of the placental vasculature as several bioactive lipids, including S1P, DHS1P, and their precursors were perturbed in Acer2−/− knockout mice. Another limitation is that we do not know whether the adaptive upregulation of Acer3 is indeed accountable for the incomplete penetrance of the placental phenotype of Acer2−/− mice. As a future direction, these limitations will be addressed using more sophisticated animal models that we are planning to generate.

In conclusion, ACER2 is a key regulator of the homeostasis of placental bioactive sphingolipids, and its expression is important for the integrity of the maternal blood space in the placenta (Figure 9). This study may lead to novel approaches to prevention and/or treatment of many pregnancy complications, such as PE and IUGR.

Figure 9. ACER2 regulates the homeostasis of sphingolipids and the integrity of the maternal vasculature and the placenta.

Figure 9

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ACER2 is highly expressed in most placental cell lineages if not all and its expression plays a key role in regulating the placental levels of sphingosine-1-phosphate (S1P) and dihydrosphingosine-1-phosphate (DHS1P) by controlling the generation of sphingosine (SPH) and dihydrosphingosine (DHS). Loss of ACER2 impairs the integrity of the maternal vasculature in the placenta, resulting in massive hemorrhages in the junction zone (Jz) and labyrinthine zone (Lz), an aberrant expansion of the trophoblast giant cell (TGC) layer, thereby fetal death.

Supplementary Material

SM1

ACKNOWLEDGEMENTS

We thank Dr. Izolda Mileva and Dr. Ashley Snider for assistance with sphingolipid analyses and animal maintenance, respectively. This work was supported, in whole or in part, by National Institutes of Health Grants R01CA163825 (to C.M) and P01CA097132 (to Y.A.H and C.M), the National Natural Science Foundation of China grant 81770529 (to Y.C), and The Guangdong Gastrointestinal Disease Research Center grant 2017B020209003 (to Y.C). This work was also supported by the Sphingolipid Animal Cancer Pathobiology Shared Resource Core and the Lipidomics Shared Resource Core at Stony Brook University.

ABBREVIATIONS:

ACER2

alkaline ceramidase 2

ACER3

alkaline ceramidase 3

ASAH1

acid ceramidase

Cer

ceramide

dhCer

dihydroceramide

dhSPH

dihydrosphingosine

dhS1P

dihydrosphingosine-1-phophate

dpc

days post coitum

Dz

decidual zone

GPRC

G protein-coupled receptor

ISH

in-situ hybridization

IUGR

intrauterine growth restriction

Jz

junctional zone

Lz

labyrinthine zone

PE

preeclampsia

SBP

sphingoid base-1-phosphate

SPH

sphingosine

S1PR1

S1P receptor 1

S1P

sphingosine-1-phosphate

spT

spongiotrophoblasts

SynT

syncytiotrophoblasts

TGC

trophoblast giant cell

Footnotes

This work is dedicated to the memory of Professor Lina M. Obeid

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Supplementary Materials

SM1
NIHMS1720883-supplement-SM1.pdf (169.9KB, pdf)

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