EGF receptor activities in mammalian development

The receptor for epidermal growth factor (EGF) and its analog transforming growth factor alpha (TGF alpha) is ubiquitous, implying quite general roles for EGF/TGF alpha in cell viability and tissue maintenance in adult tissues. There is also evidence that the EGF receptor is active in promoting wound healing and tissue regeneration in adult organs, such as skin, liver, and intestinal epithelium. It is likely that EGF receptors have more specific roles during the gestation period. For example, we have detected EGF receptors on the 3.5-day blastocyst (trophectoderm) surface and since TGF alpha-like mRNA sequences and peptides have been detected at this time (Rappolee et al., Science 241:1823, 1988), there is a strong implication for autocrine stimulation in pre- and peri-implantation stage embryos. Paracrine stimulation between the embryo and maternal tissues is also likely since both receptors and TGF alpha are present in decidual cells. Therefore EGF receptors may take part in growth regulation of the early embryo and in the process of implantation. Other examples where EGF receptors may play specific roles during embryonic development are discussed.     

Temporal expression of tissue inhibitors of metalloproteinases in mouse reproductive tissues during gestation

Tissue inhibitors of metalloproteinases (TIMPs) appear to play an important regulatory role in tissue remodelling and invasion by malignant cells. Since pregnancy involves morphological changes in existing maternal tissues, as well as a strictly controlled invasion by fetal trophoblasts, we have examined the temporal expression of TIMP-1, TIMP-2, and specific metalloproteinases in the mouse uterus, decidua, placenta, amnion, and ovaries throughout gestation by examining mRNA levels on northern and slot blots. Maximal levels of TIMP-1 mRNA were observed from day 6 to day 10 in the uterus, decidua, and placenta. In clear contrast to the early burst of TIMP-1 mRNA accumulation, the level of TIMP-2 mRNA increased steadily throughout gestation in the uterus, decidua, and amnion, while in the placenta it showed a sevenfold increase after day 14. In amnion, TIMP-1 was induced specifically on day 18. Interestingly, the normally high level of TIMP-1 mRNA seen in the ovaries of virgin mice was low during gestation, until day 18 and postpartum, when a sixfold increase over the levels in virgin ovary was observed. In contrast, ovarian TIMP-2 mRNA showed a marginal increase during gestation. The temporal pattern of 72 kDa gelatinase type A followed that of TIMP-2 in the decidua and ovary. Stromelysin-2 mRNA was detected at term only in ovary and decidua. Our data show that the temporal accumulation of TIMP-1 and TIMP-2 mRNA is precisely coordinated in each of the tissue compartments and is independently regulated during the in vivo remodelling of reproductive tissues in gestation. The peak of TIMP-1 mRNA levels in the uterus, decidua, and placenta at midgestation is associated with the most invasive period of embryo development. © 1993 Wiley-Liss, Inc.     

Regulated temporal and spatial expression of the calcium-binding proteins calcyclin and OPN (osteopontin) in mouse tissues during pregnancy.

In situ hybridization and northern/slot blot analyses were used to quantify the expression of calcyclin (2A9, 5B10), osteopontin (opn, secreted phosphoprotein, 2ar) and calmodulin mRNAs in mouse tissues that support pregnancy. High-to-moderate levels of the mRNAs of all three genes were detected at discrete locations in the uterus, decidua and placenta as a function of gestation time. Calmodulin expression was constant in these tissues; calcyclin mRNA was high during early pregnancy and declined after day 8-9 of gestation; and opn mRNA was undetectable before day 7, with maximal levels on days 9-12 in each of these tissues. Calcyclin, but not opn, expression was also observed in the chorioamnion after day 12. Calcyclin was expressed throughout the decidua on day 8 but became restricted to the primary (antimesometrial) decidual zone and decidua lateralis on day 9, and the decidua capsularis after day 9. By contrast, opn mRNA was localized on day 9 to the mesometrial triangle, which contains a large population of granulated metrial gland cells, and to the decidua basalis. These two genes may serve as markers for the two types of decidual tissue. We suggest that one function of OPN, which may be an indicator of cells in the decidua that have a bone marrow genealogy, is to mediate the flux of calcium from the maternal circulation to the developing embryo.     

The development of rat alpha 2-macroglobulin. Studies in vivo and in cultured fetal rat hepatocytes

During inflammation and tissue injury, there is an increase in the plasma concentration of several proteins, the acute-phase proteins. The levels of some acute-phase proteins have been reported to increase in pregnant and tumour-bearing animals. Rat alpha 2-macroglobulin is classified as an acute-phase protein. In this study we report the expression of alpha 2-macroglobulin in various tissues during development of the rat embryo by analysis of mRNA. The tissues studied are liver, visceral yolk sac, placental labyrinth, decidua and trophoblast. In addition, the sites of alpha 2-macroglobulin expression are localized by in situ hybridization of cDNA for alpha 2-macroglobulin to mid-sagittal cryosections of rat embryos. The level of mRNA coding for alpha 2-macroglobulin is determined in the liver of rats aged between 12 days gestation and 2 days postnatal. alpha 2-Macroglobulin mRNA is first observed in fetal liver from 12 days of gestation and increases after day 17, reaching a maximum on day 20. At this time the level is greater than that found in the liver of an adult rat suffering from acute inflammation. alpha 2-Macroglobulin mRNA is detectable in the yolk sac, placental labyrinth, trophoblast tissue and decidua. In the decidua the alpha 2-macroglobulin message is first detected at 8 days of gestation, with high levels observed from 10 to 21 days of gestation. These observations are supported by in situ hybridization studies. Experiments using cultured hepatocytes show that cells derived from rats at 15 days and 19 days of gestation are capable of synthesizing and secreting alpha 2-macroglobulin. Both synthesis and secretion can be induced by the addition of dexamethasone to the culture medium.     

Developmental regulation of human cytomegalovirus receptors in cytotrophoblasts correlates with distinct replication sites in the placenta

Cytomegalovirus (CMV), the major viral cause of congenital disease, infects the uterus and developing placenta and spreads to the fetus throughout gestation. Virus replicates in invasive cytotrophoblasts in the decidua, and maternal immunoglobulin G (IgG)-CMV virion complexes, which are transcytosed by the neonatal Fc receptor across syncytiotrophoblasts, infect underlying cytotrophoblasts in chorionic villi. Immunity is central to protection of the placenta-fetal unit: infection can occur when IgG has a low neutralizing titer. Here we used immunohistochemical and function-blocking methods to correlate infection in the placenta with expression of potential CMV receptors in situ and in vitro. In placental villi, syncytiotrophoblasts express the virion receptor epidermal growth factor receptor (EGFR) but lack integrin coreceptors, and virion uptake occurs without replication. Focal infection can occur when transcytosed virions reach EGFR-expressing cytotrophoblasts that selectively initiate expression of alphaV integrin. In cell columns, proximal cytotrophoblasts lack receptors and distal cells express integrins alpha1beta1 and alphaVbeta3, enabling virion attachment. In the decidua, invasive cytotrophoblasts expressing coreceptors upregulate EGFR, thereby dramatically increasing susceptibility to infection. Our findings indicate that virion interactions with cytotrophoblasts expressing receptors in the placenta (i) change as the cells differentiate and (ii) correlate with spatially distinct sites of CMV replication in maternal and fetal compartments.     

Developmental expression of rat transforming growth factor-alpha mRNA.

Expression of the gene encoding transforming growth factor-alpha (TGF alpha) was examined in developing rat embryos by using a cloned TGF alpha cDNA as a hybridization probe. Northern blot analysis of RNA isolated from whole fetuses revealed that TGF alpha mRNA was present at relatively high levels in 8- through 10-day-old embryos and then declined to the low or undetectable level, which is characteristic of adult tissues before birth. The level of TGF alpha mRNA present during early gestation was similar to that present in retrovirus-transformed cells in culture, suggesting that TGF alpha expression is not highly localized in the embryo. These observations are consistent with the hypothesis that TGF alpha plays a role in development, possibly as a fetal growth factor.     

The expression of alpha(1)-acid glycoprotein mRNA during rat development. High levels of expression in the decidua

During the acute phase response to inflammation the plasma concentration of some proteins, such as alpha(1-acid glycoprotein (AGP), increases dramatically. Since breakdown and remodeling of tissue is common to both nidation and inflammation we studied the tissue distribution and regulation of AGP mRNA levels during the embryonic development of the rat. High levels of mRNA coding for AGP were detected in the placenta during early fetal development. Expression of this mRNA was confined to the decidua and was first observed approximately 1 day after implantation when proliferation of the decidua is already well advanced. Maximum levels were attained about 5 days after implantation, after which the levels decreased rapidly. In contrast to the high levels of AGP mRNA in the decidua only very low levels were detected in fetal liver and visceral yolk sac, and there was only a small increase in the levels in maternal liver. Corticosteroid hormone responsiveness of AGP mRNA synthesis by hepatocytes appeared 3 days before birth. It is likely that the synthesis of AGP by the cells of the decidua is important in establishing the precisely controlled interaction between mother and embryo during nidation.     

Cell-specific metallothionein gene expression in mouse decidua and placentae

Oligodeoxyribonucleotide excess solution hybridization, Northern blot and in situ hybridization were used to analyze metallothionein gene expression in mouse decidua and placentae during gestation. Metallothionein (MT) -I and -II mRNA levels were constitutively elevated, 11- and 13-fold, respectively, relative to the adult liver, in the deciduum (D8), and decreased coordinately about 6-fold during the period of development when the deciduum is replaced by the developing placenta (D10-16). Coincident with this decline, levels of MT mRNA increased dramatically in the visceral yolk sac endoderm. In situ hybridization established that MT-I mRNA was present at low levels in the uterine luminal epithelium (D4), but was elevated at the site of embryo implantation exclusively in the primary decidual zone by D5, and then in the secondary decidual zone (D6-8). Although low levels of MT mRNA were detected in total placental RNA, in situ hybridization revealed constitutively high levels in the outer placental spongiotrophoblasts. Analysis of pulse-labeled proteins from decidua and placentae established that these tissues are active in the synthesis of MT. The constitutively high levels of MT mRNA in decidua were only slightly elevated following injection of cadmium (Cd) and/or zinc (Zn), whereas in placentae they increased several-fold. MT mRNA levels were equally high in decidua and experimentally induced deciduomata (D8) which establishes that decidual MT gene expression is not dependent on the presence of the embryo or some embryo-derived factor. Although the functional role of MT during development is speculative, these results establish the concept that, from the time of implantation to late in gestation, the mouse embryo is surrounded by cells, interposed between the maternal and embryonic environments, which actively express the MT genes. This suggests that MT plays an important role in the establishment and maintenance of normal pregnancy.     

Epidermal growth factor-like ligands and erbB genes in the peri-implantation rabbit uterus and blastocyst

Molecular cloning of the partial cDNA coding sequences of the four erbB receptors and the epidermal growth factor (EGF)-like ligands EGF, transforming growth factor alpha (TGF), and heparin-binding EGF (HB-EGF) has provided the basis for a comprehensive analysis of the spatiotemporal expression pattern of the EGF receptor/ligand system during the peri-implantation period in the rabbit. Employing nonradioactive in situ hybridization and immunolocalization, we observed differential expression of erbB1-erbB3 within the trophectoderm of the blastocyst. ErbB1 was strongly expressed in the cytotrophoblast but was downregulated upon syncytium formation. ErbB3 was a product of both the cyto- and syncytiotrophoblast. Despite the expression of erbB2 mRNA, the trophectoderm was devoid of immunoreactive ErbB2. ErbB4 gene activity was exclusively detected in the trophoblast at midpregnancy. The luminal and glandular epithelium and stroma of the nonpregnant, pseudopregnant, and pregnant rabbit uterus at Day 6 of gestation also expressed ErbB1-ErbB3. In the peri-implantation period, gene activities of erbB1-erbB3 were upregulated upon decidualization. At the site of implantation, uterine luminal epithelial cells apposing the preimplantation blastocyst displayed a distinct membrane immunolocalization of ErbB2, identifying the uterine epithelium as target for EGF, TGFalpha, and HB-EGF derived from both the embryonic trophectoderm and the uterine epithelium. In the luminal epithelium at the antimesometrial uterine site, HB-EGF gene activity was upregulated at the time of blastocyst attachment, but this upregulation was not reflected in an increase in immunoreactive HB-EGF. The detection of tyrosine phosphorylated ErbB2 in the rabbit placenta indicated the presence of a functional ErbB/EGF-like system in the pregnant rabbit uterus. This study provides strong evidence for a role of the ErbB/EGF-like system in embryo/maternal interactions during the peri-implantation period in the rabbit.     

Uterine and placental expression of TRPV6 gene is regulated via progesterone receptor- or estrogen receptor-mediated pathways during pregnancy in rodents

Transient receptor potential cation channel, subfamily V, member 6 (TRPV6) is an epithelial Ca²⁺ channel protein expressed in calcium absorbing organs. In the present study, we investigated the expression and regulation of uterine and placental TRPV6 during gestation in rodents. Uterine TRPV6 peaked at pregnancy day (P) 0.5, P5.5 and, P13.5 and was detected in uterine epithelium and glands of rats, while placental TRPV6 mRNA levels increased in mid-gestation. Uterine and placental TRPV6 mRNA levels in rats appear to cyclically change during pregnancy, suggesting that TRPV6 may participate in the implantation process. In addition, uterine TRPV6 mRNA is only expressed in placenta-unattached areas of the uterus, and uterine TRPV6 immunoreactivity was observed in luminal and glandular epithelial cells. In the placenta, TRPV6 was detected in the labyrinth and spongy zone. These results may indicate that TRPV6 has at least two functions: implantation of the embryo and maintenance of pregnancy. To investigate the pathway(s) mediating TRPV6 expression in rodents, anti-steroid hormone antagonists were injected prior to maximal TRPV6 expression. In rats, TRPV6 expression was reduced by RU486 (an anti-progesterone) through progesterone receptors, and ICI 182,780 (an anti-estrogen) blocked TRPV6 expression via estrogen receptors in mice. The juxtaposition of uterine and placental TRPV6 expressed in these tissues supports the notion that TRPV6 participates in transferring calcium ions between the maternal and fetal compartments. Taken together, TRPV6 gene may function as a key element in controlling calcium transport in the uterus between the embryo and the placenta during pregnancy.     

Molecular cloning and expression study of Xenopus latent TGF-beta binding protein-1 (LTBP-1)

Latent transforming growth factor β binding protein-1 (LTBP-1) is important in regulating the localization and activation of transforming growth factor β. In this paper is reported the isolation of the full-length Xenopus LTBP-1 cDNA from screening a neurula embryo cDNA library. Sequence analysis of XLTBP-1 cDNA revealed an open reading frame of 4518 bp encoding a 1398 amino acid protein with a molecular mass of 154.1 kDa and an isoelectric point of 4.65. The Xenopus XLTBP-1 shares 61 and 65% amino acid identity with the mouse and human LTBP-1, respectively. It contains 17 epidermal growth factor-like motifs and four eight-cysteine repeats (8-Cys). RNase protection assay revealed that XLTBP-1 is a maternal and zygotic gene, while whole-mount in situ hybridization analysis performed on embryos at different stages showed that during early Xenopus development, XLTBP-1 mRNA is expressed in the Spemann organizer, prechordal and chordal mesoderm, and later on in the organizer derived tissues. These findings suggest an important role for XLTBP-1 in embryo axis formation.     

Ontogeny of adenosine deaminase in the mouse decidua and placenta: immunolocalization and embryo transfer studies

This study has determined the cellular site of adenosine deaminase (ADA) expression in the mouse during development from Days 5 through 13 (day vaginal plug was found = Day 0) of gestation. Developmental expression of ADA progressed in two overlapping phases defined genetically (maternal vs. embryonal) and according to region (decidual vs. placental). In the first phase, ADA enzyme activity increased almost 200-fold in the antimesometrial region (decidua capsularis + giant trophoblast cells) from Days 6 through 9 of gestation but remained low in the mesometrial region. Immunohistochemical staining revealed a major localization of ADA to the secondary decidua. In the second phase, ADA activity increased several-fold in the placenta (labyrinth + basal zones) from Days 9 through 13 of gestation but remained low in the embryo proper. Immunohistochemical staining revealed a major localization of ADA to secondary giant cells, spongiotrophoblast, and labyrinthine trophoblast. Regression of decidua capsularis and growth of the spongiotrophoblast population accounted for an antimesometrial to placental shift in both ADA enzyme activity and a 40-kDa immunoreactive protein band. To verify a shift from maternal to fetal expression, studies were performed with two strains of mice (ICR, Eday) homozygous for a different ADA isozyme (ADA-A, ADA-B). Blastocysts homozygous for Adab were transferred to the uterus of pseudopregnant female recipients homozygous for Adaa. The isozymic pattern in chimeric embryo-decidual units analyzed at Days 7, 9, 11, and 13 revealed a predominance of maternal-encoded enzyme at Days 7 through 11 of gestation and a shift to fetal-encoded enzyme by Day 13. Thus, maternal expression of ADA in the antimesometrial decidua may play a role during establishment of the embryo in the uterine environment, whereas fetal expression of ADA in the trophoblast might be important to placentation.