Altered downstream target gene expression of the placental Vitamin D receptor in human idiopathic fetal growth restriction

Fetal growth restriction (FGR) affects up to 5% of pregnancies and is associated with significant perinatal complications. Maternal deficiency of vitamin D, a secosteroid hormone, is common in FGR-affected pregnancies. We recently demonstrated that decreased expression of the vitamin D receptor (VDR) in idiopathic FGR placentae could impair trophoblast growth. As strict regulation of cell-cycle genes in trophoblast cells is critical for optimal feto-placental growth, we hypothesised that pathologically decreased placental VDR contributes to aberrant regulation of cell-cycle genes. The study aims were to (i) identify the downstream cell-cycle regulatory genes of VDR in trophoblast cells, and (ii) determine if expression was changed in cases of FGR. Targeted cell-cycle gene cDNA arrays were used to screen for downstream targets of VDR in VDR siRNA-transfected BeWo and HTR-8/SVneo trophoblast-derived cell lines, and in third trimester placentae from FGR and gestation-matched control pregnancies (n = 25 each). The six candidate genes identified were CDKN2A, CDKN2D, HDAC4, HDAC6, TGFB2 and TGFB3. TGFB3 was prioritised for further validation, as its expression is largely unknown in FGR. Significantly reduced mRNA and protein expression of TGFB3 was verified in FGR placentae and the BeWo and HTR-8/SVneo trophoblast cell lines, using real-time PCR and immunoblotting respectively. In summary, decreased placental VDR expression alters the expression of regulatory cell-cycle genes in FGR placentae. Aberrant regulation of cell-cycle genes in the placental trophoblast cells may constitute a mechanistic pathway by which decreased placental VDR reduces feto-placental growth.     

Plac1 (placenta-specific 1) is essential for normal placental and embryonic development

Plac1 is a recently identified, X‐linked gene whose expression is restricted primarily to cells of the trophoblast lineage. It localizes to a chromosomal locus previously implicated in placental growth. We therefore sought to determine if Plac1 is necessary for placental and embryonic development by examining a mutant mouse model. Plac1 ablation resulted in placentomegaly and mild intrauterine growth retardation (IUGR). At E16.5, knockout (KO) and heterozygous (Het) placentae of the Plac1‐null allele inherited from the mother (X^m?X) weighed approximately 100% more than wildtype (WT) placentae, whereas the corresponding embryos weighed 7–12% less. Histologically, Plac1 mutants exhibited an expanded spongiotrophoblast layer that invaded the labyrinth. By contrast, Het placentae that inherited the null allele from the father (XX^p?) exhibited normal growth and were histologically indistinguishable from WT placentae, consistent with paternal imprinting of Plac1. When examined across gestation, WT and X^m?X placental weights peaked at E16.5 and decreased slightly thereafter. KO placentae (X^m?X^p? and X^m?Y), however, continued to increase in weight after E16.5, consistent with a functional role for the paternal Plac1 allele. Subsequent analysis confirmed that the paternal allele partially escapes complete X‐inactivation and thus contributes to placental growth regulation. Additionally, although male Plac1 KO mice can survive, they exhibit decreased viability as a consequence of events occurring late in gestation or shortly after birth. Thus, Plac1 is a paternally imprinted, X‐linked gene essential for normal placental and embryonic development.Mol. Reprod. Dev. 79: 564‐572, 2012. © 2012 Wiley Periodicals, Inc.     

Transplantation antigen expression on murine trophoblast detection by induction of specific alloimmunity

Cell-mediated immune responses to murine embryonic trophoblast cells were investigated using lymphocyte trophoblast cultures (LTC) and cell-mediated lympholysis (CML). Spleen cells from CBA (H-2^k) or C57BL/6 (H-2^b) mice hyperimmunized with 3.5-day-old Balb/c (H-2^d) blastocysts did not undergo DNA synthesis after in vitro exposure to Balb/c blastocyst outgrowths nor were cytotoxic lymphocytes (CTL) generated against H-2^d alloantigens. Splenocytes from Balb/c mice presensitized with semiallogeneic (Balb/c female × C57BL/6 male) trophoblast cells derived from 17- to 20-day placental tissue expressed a weak proliferative response in the presence of semiallogeneic placental trophoblast and produced a moderate number of CTL against H-2^b (paternal strain) alloantigens when compared to mixed lymphocyte cultures (MLC) between Balb/c responder and semiallogeneic (stimulator) spleen cells. CTL were also generated in vitro after splenocytes from Balb/c mice hyperimmunized with semiallogeneic spleen cells were restimulated in vitro with placental trophoblast cells. These studies showing that early-stage trophoblast cells fail to evoke transplantation immunity and placental trophoblast is capable of generating alloimmunity only after combined in vivo hyperimmunization with in vitro restimulation suggest that these trophoblast cells are poorly immunogenic due in part to the relatively weak functional expression of major transplantation antigens.     

An EG-VEGF-Dependent Decrease in Homeobox Gene NKX3.1 Contributes to Cytotrophoblast Dysfunction: A Possible Mechanism in Human Fetal Growth Restriction

Idiopathic fetal growth restriction (FGR) is frequently associated with placental insufficiency. Previous reports have provided evidence that endocrine gland–derived vascular endothelial growth factor (EG-VEGF), a placental secreted protein, is expressed during the first trimester of pregnancy, controls both trophoblast proliferation and invasion, and its increased expression is associated with human FGR. In this study, we hypothesize that EG-VEGF-dependent changes in placental homeobox gene expressions contribute to trophoblast dysfunction in idiopathic FGR. The changes in EG-VEGF-dependent homeobox gene expressions were determined using a homeobox gene cDNA array on placental explants of 8–12 wks gestation after stimulation with EG-VEGF in vitro for 24 h. The homeobox gene array identified a greater-than-five-fold increase in HOXA9, HOXC8, HOXC10, HOXD1, HOXD8, HOXD9 and HOXD11, while NKX 3.1 showed a greater-than-two-fold decrease in mRNA expression compared with untreated controls. Homeobox gene NKX3.1 was selected as a candidate because it is a downstream target of EG-VEGF and its expression and functional roles are largely unknown in control and idiopathic FGR-affected placentae. Real-time PCR and immunoblotting showed a significant decrease in NKX3.1 mRNA and protein levels, respectively, in placentae from FGR compared with control pregnancies. Gene inactivation in vitro using short-interference RNA specific for NKX3.1 demonstrated an increase in BeWo cell differentiation and a decrease in HTR-8/SVneo proliferation. We conclude that the decreased expression of homeobox gene NKX3.1 downstream of EG-VEGF may contribute to the trophoblast dysfunction associated with idiopathic FGR pregnancies.     

Gestational age‐dependent gene expression profiling of ATP‐binding cassette transporters in the healthy human placenta

The ATP‐binding cassette (ABC) transporters control placental transfer of several nutrients, steroids, immunological factors, chemicals, and drugs at the maternal‐fetal interface. We and others have demonstrated a gestational age‐dependent expression pattern of two ABC transporters, P‐glycoprotein and breast cancer resistance protein throughout pregnancy. However, no reports have comprehensively elucidated the expression pattern of all 50 ABC proteins, comparing first trimester and term human placentae. We hypothesized that placental ABC transporters are expressed in a gestational‐age dependent manner in normal human pregnancy. Using the TaqMan^® Human ABC Transporter Array, we assessed the mRNA expression of all 50 ABC transporters in first (first trimester, n = 8) and third trimester (term, n = 12) human placentae and validated the resulting expression of selected ABC transporters using qPCR, Western blot and immunohistochemistry. A distinct gene expression profile of 30 ABC transporters was observed comparing first trimester vs. term placentae. Using individual qPCR in selected genes, we validated the increased expression of ABCA1 (P < 0.01), ABCA6 (P < 0.001), ABCA9 (P < 0.001) and ABCC3 (P < 0.001), as well as the decreased expression of ABCB11 (P < 0.001) and ABCG4 (P < 0.01) with advancing gestation. One important lipid transporter, ABCA6, was selected to correlate protein abundance and characterize tissue localization. ABCA6 exhibited increased protein expression towards term and was predominantly localized to syncytiotrophoblast cells. In conclusion, expression patterns of placental ABC transporters change as a function of gestational age. These changes are likely fundamental to a healthy pregnancy given the critical role that these transporters play in the regulation of steroidogenesis, immunological responses, and placental barrier function and integrity.     

Increase in placental 15-hydroxy-prostaglandin dehydrogenase in the first half of human pregnancy

NAD-dependent 15-hydroxy-prostaglandin dehydrogenase (PGDH) activity was measured in homogenates of 25 human placentae obtained between 7 and 17 weeks of gestation. PGDH activity, expressed in nanomoles PGF_2α metabolized per min, ranged from 0.2 to 5.4 nmoles per mg placental protein and from 1.5 to 80 nmoles per g wet weight. PGDH activity per mg protein and per g weight increased significantly in function of gestational age (p<0.001). Between 7–8 weeks' gestation and 15–16 weeks mean values increased tenfold from 0.4 to 3.0 nmoles per mg protein and from 2.7 to 36.6 nmoles per g wet weight. Per unit of weight these early placentae contained less PGDH activity than term controls, but this related mainly to their high water content. Per mg placental protein PGDH activities already equalled values found at term before the end of the first trimester. The data indicate that the development of terminal villi and the migration of trophoblast into the maternal spiral arteries is associated with a substantial increase in the placental capacity for prostaglandin metabolism.     

Expression of PIK3IP1 in the murine uterus during early pregnancy

The ovarian steroid hormones, estrogen (E2) and progesterone (P4), are essential regulators of uterine functions necessary for development, embryo implantation, and normal pregnancy. ARID1A plays an important role in steroid hormone signaling in endometrial function and pregnancy. In previous studies, using high density DNA microarray analysis, we identified phosphatidylinositol-3-kinase interacting protein 1 (Pik3ip1) as one of the genes up-regulated by ARID1A. In the present study, we performed real-time qPCR and immunohistochemistry analysis to investigate the regulation of PIK3IP1 by ARID1A and determine expression patterns of PIK3IP1 in the uterus during early pregnancy. The expression of PIK3IP1 was strong at the uterine epithelial and stromal cells of the control mice. However, expression of PIK3IP1 was remarkably reduced in the Pgr^cre/+Arid1a^f/f mice and progesterone receptor knock-out (PRKO) mice. During early pregnancy, PIK3IP1 expression was strong at day 2.5 of gestation (GD 2.5) and then slightly decreased at GD 3.5?at the epithelium and stroma. After implantation, PIK3IP1 expression was detected at the secondary decidualization zone. To determine the ovarian steroid hormone regulation of PIK3IP1, we examined the expression of PIK3IP1 in ovariectomized control, Pgr^cre/+Arid1a^f/f, and PRKO mice treated with P4 or E2. P4 treatment increased the PIK3IP1 expression at the luminal and glandular epithelium of control mice. However, the PIK3IP1 induction was decreased in both the Pgr^cre/+Arid1a^f/f and PRKO mice, compared to controls. Our results identified PIK3IP1 as a novel target of ARID1A and PGR in the murine uterus.     

A serum-free system for culturing human placental trophoblasts

Summary We have compared hormone production by early gestation and term human placental trophoblasts cultured in Ham's F10 medium containing 10% fetal bovine serum with that by cells cultured in serum-free HB102 medium. Mean daily production of progesterone on Days 3 to 7 was approximately 25% less by both early gestation and term cells cultured in HB102 as compared to Ham's F10, but production was maintained at a stable level for at least 7 d longer than the cells in Ham's. Estradiol production from 10⁻⁶ M dehydroepiandrosterone by both early gestation and term cells was comparable in both media. Human placental lactogen production on Days 3 to 7 was 40% less by cells cultured in HB102. Human chorionic gonadotropin (hCG) output by early gestation cells was also 50% less in HB102 but term cells in HB102 produced twice as much hCG as those in Ham's F10. 3B-Hydroxysteroid dehydrogenase (3BHSD) activity in early gestation and term cells and 11B-hydroxysteroid dehydrogenase (11BHSD) activity of early gestation cultures was comparable in the two media. 11BHSD activity was decreased in the term cultures, and this decrease was more marked in Ham's than in HB102. Sulfatase and aromatase activities in the early gestation cultures were comparable in both media; sulfatase activity was comparable and aromatase activity only 20% less in the term cells cultured in HB102. These results indicate that serum-free HB102 supports differential function of human trophoblast cells and is useful for studies of placental activity for as long as 14 d in culture.     

Colocalization of leptin receptor (OB-R) mRNA and placental lactogen-II in rat trophoblast cells: gestational profile of OB-R mRNA expression in placentae

The present study was designed to clarify the cellular localization and expression of leptin receptor(s) [OB-R(s)] mRNA including its splice variants and their correlation with the cells which secrete placental hormone, placental lactogen-II (PL-II), in rat placentae. By in situ hybridization analysis, hybridization signals for OB-Rb and the common extracellular domain of OB-R were first detectable in some cells of the labyrinth zone of the placentae on day 14 of pregnancy and then a lot of cells dispersed in the entire area of the labyrinth zone expressed OB-Rb during the latter half of pregnancy. However, no expression was observed in the decidua and the junctional zone of the placentae during pregnancy. Double staining study revealed that signals for OB-R expressing trophoblast cells showed PL-II immunoreactivity in the labyrinth zone of the placentae. In Northern blot analysis, two bands (2.8 kb and 5.1 kb) of OB-R mRNA expression were observed in the placentae from day 17 to 21 of pregnancy and the expression of both increased markedly up to day 21 of pregnancy. RT-PCR analysis revealed that OB-Rb, OB-Ra, and OB-Re are expressed in the placentae on days 19 and 21 of pregnancy. These results suggest that the OB-R may have a physiological significance in the placental function during the latter half of pregnancy.     

Murine junctional adhesion molecules JAM-B and JAM-C mediate endothelial and stellate cell interactions during hepatic fibrosis

Classical junctional adhesion molecules JAM-A, JAM-B and JAM-C influence vascular permeability, cell polarity as well as leukocyte recruitment and immigration into inflamed tissue. As the vasculature becomes remodelled in chronically injured, fibrotic livers we aimed to determine distribution and role of junctional adhesion molecules during this pathological process. Therefore, livers of naïve or carbon tetrachloride-treated mice were analyzed by immunohistochemistry to localize all 3 classical junctional adhesion molecules. Hepatic stellate cells and endothelial cells were isolated and subjected to immunocytochemistry and flow cytometry to determine localization and functionality of JAM-B and JAM-C. Cells were further used to perform contractility and migration assays and to study endothelial tubulogenesis and pericytic coverage by hepatic stellate cells. We found that in healthy tissue, JAM-A was ubiquitously expressed whereas JAM-B and JAM-C were restricted to the vasculature. During fibrosis, JAM-B and JAM-C levels increased in endothelial cells and JAM-C was de novo generated in myofibroblastic hepatic stellate cells. Soluble JAM-C blocked contractility but increased motility in hepatic stellate cells. Furthermore, soluble JAM-C reduced endothelial tubulogenesis and endothelial cell/stellate cell interaction. Thus, during liver fibrogenesis, JAM-B and JAM-C expression increase on the vascular endothelium. More importantly, JAM-C appears on myofibroblastic hepatic stellate cells linking them as pericytes to JAM-B positive endothelial cells. This JAM-B/JAM-C mediated interaction between endothelial cells and stellate cells stabilizes vessel walls and may control the sinusoidal diameter. Increased hepatic stellate cell contraction mediated by JAM-C/JAM-C interaction may cause intrahepatic vasoconstriction, which is a major complication in liver cirrhosis.     

Increase in placental 15-hydroxy-prostaglandin dehydrogenase in the first half of human pregnancy

NAD-dependent 15-hydroxy-prostaglandin dehydrogenase (PGDH) activity was measured in homogenates of 25 human placentae obtained between 7 and 17 weeks of gestation. PGDH activity, expressed in nanomoles PGF2 alpha metabolized per min, ranged from 0.2 to 5.4 nmoles per mg placental protein and from 1.5 to 80 nmoles per g wet weight. PGDH activity per mg protein and per g weight increased significantly in function of gestational age (p less than 0.001). Between 7-8 weeks' gestation and 15-16 weeks mean values increased tenfold from 0.4 to 3.0 nmoles per mg protein and from 2.7 to 36.6 nmoles per g wet weight. Per unit of weight these early placentae contained less PGDH activity than term controls, but this related mainly to their high water content. Per mg placental protein PGDH activities already equalled values found at term before the end of the first trimester. The data indicate that the development of terminal villi and the migration of trophoblast into the maternal spiral arteries is associated with a substantial increase in the placental capacity for prostaglandin metabolism.     

Vitamin D Receptor Gene Ablation in the Conceptus Has Limited Effects on Placental Morphology,Function and Pregnancy Outcome

Vitamin D deficiency has been implicated in the pathogenesis of several pregnancy complications attributed to impaired or abnormal placental function, but there are few clues indicating the mechanistic role of vitamin D in their pathogenesis. To further understand the role of vitamin D receptor (VDR)-mediated activity in placental function, we used heterozygous Vdr ablated C57Bl6 mice to assess fetal growth, morphological parameters and global gene expression in Vdr null placentae. Twelve Vdr ^+/- dams were mated at 10–12 weeks of age with Vdr ^+/- males. At day 18.5 of the 19.5 day gestation in our colony, females were euthanised and placental and fetal samples were collected, weighed and subsequently genotyped as either Vdr ^+/+, Vdr ^+/- or Vdr ^-/-. Morphological assessment of placentae using immunohistochemistry was performed and RNA was extracted and subject to microarray analysis. This revealed 25 genes that were significantly differentially expressed between Vdr ^+/+ and Vdr ^-/- placentae. The greatest difference was a 6.47-fold change in expression of Cyp24a1 which was significantly lower in the Vdr ^-/- placentae (P<0.01). Other differentially expressed genes in Vdr ^-/- placentae included those involved in RNA modification (Snord123), autophagy (Atg4b), cytoskeletal modification (Shroom4), cell signalling (Plscr1, Pex5) and mammalian target of rapamycin (mTOR) signalling (Deptor and Prr5). Interrogation of the upstream sequence of differentially expressed genes identified that many contain putative vitamin D receptor elements (VDREs). Despite the gene expression differences, this did not contribute to any differences in overall placental morphology, nor was function affected as there was no difference in fetal growth as determined by fetal weight near term. Given our dams still expressed a functional VDR gene, our results suggest that cross-talk between the maternal decidua and the placenta, as well as maternal vitamin D status, may be more important in determining pregnancy outcome than conceptus expression of VDR.     

Dual Benefit of Reduced Cx43 on Atherosclerosis in LDL Receptor-Deficient Mice

Paracrine cell-to-cell interactions are crucial events during atherogenesis, however, little is known on the role of gap junctional communication during this process. We recently demonstrated increased expression of Cx43 in intimal smooth muscle cells and in a subset of endothelial cells covering the shoulder of atherosclerotic plaques. The purpose of this study was to examine the role of Cx43 in the development of atherosclerosis in vivo. Atherosclerosis-susceptible LDL receptor-deficient (LDLR^?/?) mice were intercrossed with mice heterozygous for Cx43 (Cx43^+/?mice). Male mice with normal (Cx43^+/+LDLR^?/?) or reduced (Cx43^+/?LDLR^?/?) Cx43 level of 10 weeks old were fed a cholesterol-rich diet (1.25%) for 14 weeks. Both groups of mice showed similar increases in serum lipids and body weight. Interestingly, the progression of atherosclerosis was reduced by 50% (P < 0.01) in the thoraco-abdominal aorta and in the aortic roots of Cx43^+/?LDLR^?/?mice compared with Cx43^+/+LDLR^?/?littermate controls. In addition, atheroma in Cx43^+/?LDLR^?/?mice contained fewer inflammatory cells and exhibited thicker fibrous caps with more collagen and smooth muscle cells, important features associated, in human, with stable atherosclerotic lesions. Thus, reducing Cx43 expression in mice provides beneficial effects on both the progression and composition of the atherosclerotic lesions.     

Progesterone modulation of α5 nAChR subunits influences anxiety‐related behavior during estrus cycle

Smokers often report an anxiolytic effect of cigarettes. In addition, stress‐related disorders such as anxiety, post‐traumatic stress syndrome and depression are often associated with chronic nicotine use. To study the role of the α5 nicotinic acetylcholine receptor subunit in anxiety‐related responses, control and α5 subunit null mice (α5^?/?) were subjected to the open field activity (OFA), light–dark box (LDB) and elevated plus maze (EPM) tests. In the OFA and LDB, α5^?/? behaved like wild‐type controls. In the EPM, female α5^?/? mice displayed an anxiolytic‐like phenotype, while male α5^?/? mice were undistinguishable from littermate controls. We studied the hypothalamus–pituitary–adrenal axis by measuring plasma corticosterone and hypothalamic corticotropin‐releasing factor. Consistent with an anxiolytic‐like phenotype, female α5^?/? mice displayed lower basal corticosterone levels. To test whether gonadal steroids regulate the expression of α5, we treated cultured NTera 2 cells with progesterone and found that α5 protein levels were upregulated. In addition, brain levels of α5 mRNA increased upon progesterone injection into ovariectomized wild‐type females. Finally, we tested anxiety levels in the EPM during the estrous cycle. The estrus phase (when progesterone levels are low) is anxiolytic‐like in wild‐type mice, but no cycle‐dependent fluctuations in anxiety levels were found in α5^?/? females. Thus, α5‐containing neuronal nicotinic acetylcholine receptors may be mediators of anxiogenic responses, and progesterone‐dependent modulation of α5 expression may contribute to fluctuations in anxiety levels during the ovarian cycle.     

Expression and localization of estrogen receptor-alpha protein in normal and abnormal term placentae and stimulation of trophoblast differentiation by estradiol

Estrogens play an important role in the regulation of placental function, and 17-beta-estradiol (E2) production rises eighty fold during human pregnancy. Although term placenta has been found to specifically bind estrogens, cellular localization of estrogen receptor alpha (ER-alpha) in trophoblast remains unclear. We used western blot analysis and immunohistochemistry with h-151 and ID5 monoclonal antibodies to determine the expression and cellular localization of ER-alpha protein in human placentae and cultured trophoblast cells. Western blot analysis revealed a ~65 kDa ER-alpha band in MCF-7 breast carcinoma cells (positive control). A similar band was detected in five normal term placentae exhibiting strong expression of Thy-1 differentiation protein in the villous core. However, five other term placentae, which exhibited low or no Thy-1 expression (abnormal placentae), exhibited virtually no ER-alpha expression. In normal placentae, nuclear ER-alpha expression was confined to villous cytotrophoblast cells (CT), but syncytiotrophoblast (ST) and extravillous trophoblast cells were unstained. In abnormal placentae no CT expressing ER-alpha were detected. Normal and abnormal placentae also showed ER-alpha expression in villous vascular pericytes and amniotic (but not villous) fibroblasts; no staining was detected in amniotic epithelial cells or decidual cells. All cultured trophoblast cells derived from the same normal and abnormal placentae showed distinct ER-alpha expression in western blots, and the ER-alpha expression was confined to the differentiating CT, but not to the mature ST. Trophoblast cells from six additional placentae were cultured in normal medium with phenol red (a weak estrogen) as above (PhR+), or plated in phenol red-free medium (PhR-) without or with mid-pregnancy levels of E2 (20 nM). Culture in PhR- medium without E2 caused retardation of syncytium formation and PhR-medium with E2 caused acceleration of syncytium formation compared to cultures in PhR+ medium. These data indicate that the considerable increase in estrogen production during pregnancy may play a role, via the ER-alpha, in the stimulation of CT differentiation and promote function in normal placentae. This mechanism, however, may not operate in abnormal placentae, which show a lack of ER-alpha expression.     

Spatiotemporal expression of three gap junction gene products involved in fetomaternal communication during rat pregnancy.

The expression of three different members of the gap junction multigene family, alpha 1 (Cx43), beta 1 (Cx32), and beta 2 (Cx26), was analysed in the rat implantation chamber (a structural unit containing fetal, extraembryonic and maternal components within the pregnant uterus) during mid- and late stages of gestation as well as in the delivering, post-partum and non-pregnant uterus. A differential, spatiotemporal and cell-type-specific regulation of gap junctional coexpression was observed for beta 1 and beta 2 in all epithelia examined (visceral, luminal and glandular), as well as for alpha 1 and beta 2 in decidual cells and keratinocytes of the fetal epidermis. alpha 1 antigen was detected in the mesometrial stroma, mesometrial myometrium, connective tissue, mesothelia of the amnion and visceral yolk sac and in the allantoic mesodermal layer throughout gestation. In addition, expression of alpha 1 in the placental basal zone and trophoblast giant cells coincided with the differentiation of these cells. beta 2 expression was observed prominently in the chorionic villi of the placental labyrinth. The presence of beta 1 and beta 2 in the visceral epithelium (visceral yolk sac = the primary route for embryonic nourishment prior to the formation of the chorioallantoic placenta) and beta 2 in the chorionic villi (placental barrier = the major fetomaternal exchange route) suggests that gap junctions have an important role in fetomaternal communication.