Expression of aldehyde dehydrogenase family 1, member A3 in glycogen trophoblast cells of the murine placenta.

INTRODUCTION: Retinoic acid (RA) signaling is a well known regulator of trophoblast differentiation and placental development, and maternal decidual cells are recognized as the source of much of this RA. We explored possible trophoblast-derived sources of RA by examining the expression of RA synthesis enzymes in the developing mouse placenta, as well as addressed potential sites of RA action by examining the ontogeny of gene expression for other RA metabolizing and receptor genes. Furthermore, we investigated the effects of endogenous RA production on trophoblast differentiation. METHODS: Placental tissues were examined by in situ hybridization and assayed for RARE-LacZ transgene activity to locate sites of RAR signaling. Trophoblast stem cell cultures were differentiated in the presence of ALDH1 inhibitors (DEAB and citral), and expression of labyrinth (Syna, Ctsq) and junctional zone (Tpbpa, Prl7b1, Prl7a2) marker genes were analyzed by qRT-PCR. RESULTS: We show Aldh1a3 is strongly expressed in a subset of ectoplacental cone cells and in glycogen trophoblast cells of the definitive murine placenta. Most trophoblast subtypes of the placenta express RA receptor combinations that would enable them to respond to RA signaling. Furthermore, expression of junctional zone markers decrease in differentiating trophoblast cultures when endogenous ALDH1 enzymes are inhibited. DISCUSSION: Aldh1a3 is a novel marker for glycogen trophoblast cells and their precursors and may play a role in the differentiation of junctional zone cell types via production of a local source of RA.

Ly6e expression is restricted to syncytiotrophoblast cells of the mouse placenta.

In the present study, we characterized the expression of lymphocyte antigen 6, locus E (Ly6e) in mouse placental trophoblast. We identified Ly6e mRNA expression in trophoblast stem (TS) cells by a gene expression screen. In vivo, Ly6e was first detectable by mRNA in situ hybridization in the chorion beginning at E8.5 with spatial expression similar to Syncytin a (Syna). At later stages of gestation, Ly6e was restricted to syncytiotrophoblast in the labyrinth. Northern blot confirmed that Ly6e was expressed in both undifferentiated and differentiated TS cell cultures but that its expression increased with differentiation. FACS analysis confirmed these results and allowed us to isolate LY6E⁺ cells, which we found to express Syna at a much higher level than did LY6E⁻ cells. Our findings suggest that LY6E is expressed in differentiated syncytiotrophoblast and may also be useful as an early marker, expressed in progenitors of this cell-type.

Nuclear localization of the type 1 PTH/PTHrP receptor in rat tissues.

The localization of PTH/PTH-related peptide (PTHrP) receptor (PTHR) has traditionally been performed by autoradiography. Specific polyclonal antibodies to peptides unique to the PTHR are now available, which allow a more precise localization of the receptor in cells and tissues. We optimized the IHC procedure for the rat PTHR using 5-microm sections of paraffin-embedded rat kidney, liver, small intestine, uterus, and ovary. Adjacent sections were analyzed for the presence of PTHR mRNA (by in situ hybridization) and PTHrP peptide. A typical pattern of staining for both receptor protein and mRNA was observed in kidney in cells lining the proximal tubules and collecting ducts. In uterus and gut, the receptor and its mRNA are present in smooth muscle layers (PTHrP target) and in glandular cuboidal cells and surface columnar epithelium. This suggests that PTH, or more likely PTHrP, plays a role in surface/secretory epithelia that is as yet undefined. In the ovary, PTHR was readily detectable in the thecal layer of large antral follicles and oocytes, and was present in the cytoplasm and/or nucleus of granulosa cells, regions that also contained receptor transcripts. PTHR protein and mRNA were found in the liver in large hepatocytes radiating outward from central veins. Immunoreactive cells were also present around the periphery of the liver but not within two or three cell layers of the surface. Clear nuclear localization of the receptor protein was present in liver cells in addition to the expected cytoplasmic/peripheral staining. PTHR immunoreactivity was present in the nucleus of some cells in every tissue examined. RT-PCR confirmed the presence of PTHR transcripts in these same tissues. Examination of the hindlimbs of PTHR gene-ablated mice showed no reaction to this antibody, whereas hindlimbs from their wild-type littermates stained positively. The results emphasize that the PTHR is highly expressed in diverse tissues and, in addition, show that the receptor protein itself can be localized to the cell nucleus. Nuclear localization of the receptor suggests that there is a role for PTH and/or PTHrP in the regulation of nuclear events, either on the physical environment (nucleoskeleton) or directly on gene expression.

Nuclear localization of the type 1 parathyroid hormone/parathyroid hormone-related peptide receptor in MC3T3-E1 cells: association with serum-induced cell proliferation.

We have recently demonstrated that the receptor for parathyroid hormone (PTH) and PTH-related peptide (PTHrP), PTHR, can be localized to the nucleus of cells within the liver, kidney, uterus, gut, and ovary of the rat. We set out to determine the localization of the PTHR in cultured osteoblast-like cells. MC3T3-E1, ROS 17/2.8, UMR106, and SaOS-2 cells were cultured in alpha-modified eagle medium containing 15% fetal calf serum under standard conditions. Untreated cells were grown on glass coverslips to 75-95% confluence and fixed in 1% paraformaldehyde. For experiments designed to examine cells synchronized by serum starvation, cells were grown on glass coverslips, starved of serum for 46 h, and then fixed at 2-h intervals for a total of 26 h after the addition of serum to the medium. Parallel sets of cells were pulsed with [3H]thymidine to track the DNA duplication interval. The PTHR was localized by immunocytochemistry using a primary antibody raised against a portion of the N-terminal extracellular domain of the PTHR. The results presented herein indicate that the PTHR attains a nuclear localization in each cell line examined. In UMR106 cells, PTHR immunoreactivity was restricted to the nucleolus. After cell synchronization, MC3T3-E1 cells double approximately 24 h after the addition of serum. Immunocytochemistry for the PTHR in these cells showed that the receptor staining is initially diffuse for the first 6 h, then becomes more perinuclear in distribution by 12-16 h. Nuclear localization of the receptor is achieved approximately 16-20 h after the addition of serum and remains there throughout the mitotic phase. Intense staining of mitotic and postmitotic cells was observed. No change in cell proliferation kinetics was observed in MC3T3-E1 cells cultured in the presence of 25 nM PTH(1-34). These data suggest an important role for the PTHR in the nucleus of MC3T3-E1 cells at the time of DNA synthesis and mitosis.