Biomedical subjects

B C Paria

Publications and source records attributed to B C Paria.

At least 55 records · Page 3Linked to original sources

PubMed1997

Activation of the chicken metallothionein promoter by metals and oxidative stress in cultured cells and transgenic mice.

Cis-acting elements in the chicken metallothionein promoter were tested for their ability to direct responses of reporter genes to metal ions and oxidative stress in transfected mouse cells and in transgenic mice. In addition, protein interactions with the promoter were analyzed by the electrophoretic mobility shift assay. In transient transfection assays and in transgenic mice, 107-bp of the chicken MT promoter was sufficient to direct responses to Zn. This promoter region also directed response to oxidative stress in transfected cells and transgenic mice, but in transgenic mice, maximal responsiveness to oxidative stress apparently involved other elements in the proximal promoter region (307-bp). The proximal 200-bp of the promoter contains sequences homologous to a metal response element (-47-bp), Sp1 binding sites (-70-bp and -161-bp), and an antioxidant response element (-189-bp). Electrophoretic mobility shift assay demonstrated that metal response element binding activity was low in control Hepa cell nuclear extracts, but was induced 6-fold after 45 min of H2O2 treatment. In contrast, Sp1 binding remained unchanged, and no evidence for specific binding to the core antioxidant response element consensus sequence was obtained. These studies demonstrate that cis-acting elements mediating induction of metallothionein gene expression by metals and oxidative stress are present in the chicken metallothionein promoter and suggest a role for increased binding of the transcription factor MTF-1 to the metal response element(s).

Animals↗

PubMed1997

Inappropriate expression of human transforming growth factor (TGF)-alpha in the uterus of transgenic mouse causes downregulation of TGF-beta receptors and delays the blastocyst-attachment reaction.

In the mouse, the initiation of the attachment reaction between the blastocyst trophectoderm and luminal epithelium of the receptive uterus occurs in the evening (2200-2300 h) of day 4 of pregnancy (day 1 = vaginal plug) and is followed by proliferation and differentiation of stromal cells into decidual cells at the sites of blastocyst attachment. This investigation demonstrates that an inappropriate expression of the human transforming growth factor alpha (hTGF-alpha) transgene in the uterus under the direction of a mouse metallothionein-I promoter downregulates uterine expression of TGF-beta receptor subtypes and delays the initiation of implantation (attachment reaction) resulting in delayed parturition. This delay in the attachment reaction is accompanied by deferred uterine expression of amphiregulin. The results suggest that a coordinated 'cross-talk' between the signaling pathways executed by epidermal growth factor-like growth factors and TGF-beta 5 is important for the normal implantation process.

Amphiregulin↗

PubMed1996

Immunolocalization of estrogen receptor protein in the mouse blastocyst during normal and delayed implantation.

We previously showed that estrogen receptor (ER) mRNA is present in preimplantation mouse embryos. The apparent synthesis of ER mRNA by the blastocyst at the time of implantation when estrogen is required was of special interest. A demonstration of the presence of ER protein would support the idea that estrogen can act directly on the embryo. The mouse embryo at the blastocyst stage is differentiated into two cell types, the trophectoderm and the inner cell mass. To determine whether ER mRNA is translated into ER protein and its cell-specific distribution, immunocytochemical analyses were performed in mouse blastocysts. ER protein was detected in all cell types of the normal, dormant, or activated blastocyst. To trace the fate of ER in these cell types, immunocytochemistry was performed in implanting blastocysts and early egg cylinder stage embryos developed in culture. Again, ER was detected in all cells of the implanting blastocyst. At the early egg cylinder stage, continued expression of ER was observed in cells derived from the inner cell mass or the trophoblast. In trophoblast giant cells, ER was concentrated in small regions of the nucleus, possibly the nucleoli, which was similar to that observed in dormant and activated blastocysts. The embryonic expression of ER at such early stages in a broad array of cells suggests that ER may have a general role during early development.

Animals↗

PubMed1996

Preimplantation mouse blastocysts fail to express CYP genes required for estrogen biosynthesis.

Implantation is initiated on day 4 in the mouse and on day 13 in the pig. The preimplantation pig blastocyst synthesizes steroid hormones, but whether preimplantation rodent embryos also have this ability has remained unresolved for the last two decades. In this study, the mRNAs encoding NADPH-cytochrome P450 reductase (P450-reductase), adrenodoxin, lanosterol 14-demethylase P450 (CYP51), 17 alpha-hydroxylase P450 (CYP17), cholesterol side-chain cleavage P450 (CYP11A1), sterol 27-hydroxylase P450 (CYP27), and aromatase P450 (CYP19) were examined in day 4 mouse blastocysts (day 1 = vaginal plug) and in day 13 and 16 pig blastocysts using reverse transcription-polymerase chain reaction (RT-PCR). In mouse blastocysts, mRNAs of P450-reductase, adrenodoxin, and CYP51, but not CYP17, CYP11A1, CYP27, and CYP19, were detected. In agreement with this finding, no aromatase protein could be detected by immunohistochemistry. By contrast, all these mRNAs were detected in the pig blastocyst. Furthermore, both the ovarian and placental types of aromatase (CYP19) mRNAs were detected in the pig blastocyst on days 13 and 16 of pregnancy, although the ovarian form was more abundant. Both forms of aromatase were much higher in day 13 than in day 16 pig blastocysts. The results provide definitive evidence that the preimplantation mouse blastocyst, as opposed to the pig blastocyst, has no ability to synthesize estrogen and no steroidogenic capacity. Maternal estrogen synthesis is essential for implantation of the mouse blastocyst.

Adrenodoxin↗

PubMed1996

The uterus is a potential site for anandamide synthesis and hydrolysis: differential profiles of anandamide synthase and hydrolase activities in the mouse uterus during the periimplantation period.

Arachidonoylethanolamide (anandamide) is an endogenous ligand for cannabinoid receptors. We demonstrated previously that ligand-receptor signaling with cannabinoids is operative in both the mouse embryo and uterus during the periimplantation period. In the present investigation, we provide evidence that mouse uterus has the enzymatic capacities to form (synthase) and hydrolyze (amidase) anandamide. These activities were primarily localized in uterine microsomes and were dependent upon pH, time, protein, and substrate concentrations. The rate of formation of anandamide was dependent on arachidonic acid (Km: 3.8 microM and Vmax: 2.5 nmol/h/mg protein) and ethanolamine (Km:1.2 mM and Vmax:4.1 nmol/h/mg protein) concentrations. The amidase activity showed an apparent Km of 67 microM and Vmax of 3.5 nmol/min/mg protein with anandamide as a substrate. While the synthase showed maximal activity at pH 9.0, the amidase activity was maximal at pH 8.5. As reported previously, phenylmethylsulfonyl fluoride (PMSF) or arachidonyl trifluoromethyl ketone (ATK) inhibited the amidase activity in a dose-dependent manner. In contrast, PMSF was not inhibitory to synthase activity, rather it stimulated synthase activity at lower concentrations. Further, inhibitory effects of ATK were only modest toward the synthase activity and the effects were not concentration-dependent. To determine whether uterine synthase and/or amidase activity have any physiological significance with respect to uterine receptivity and implantation during early pregnancy, profiles of synthase and amidase activities were analyzed in mouse uterine microsomes obtained during early pregnancy or pseudopregnancy. It should be noted that the synchronized development of the embryo to the blastocyst stage and differentiation of the uterus to the receptive state are critical to the embryo implantation process. In the mouse, the uterus becomes receptive for implantation only for a limited period during pregnancy or pseudopregnancy. The uterus becomes receptive on day 4 (the day of implantation) and by day 5, it becomes nonreceptive for blastocyst implantation (Paria et al., 1993: Proc Natl Acad Sci USA 90:10159-10162.). Both anandamide synthase and amidase activities remained virtually unaltered on days 1-4 of pregnancy. In contrast, while the synthase activity increased, the amidase activity decreased in the uterus on day 5 of pseudopregnancy (nonreceptive phase) as compared to those observed on day 4 of pregnancy or pseudopregnancy (receptive phase). The synthase and amidase activities in surgically separated implantation and interimplantation sites showed an interesting profile on days 5-7 of pregnancy; the synthase activity was lower in implantation sites as compared to that in interimplantation sites. In contrast, amidase activity was higher in implantation sites compared with that in interimplantation sites. Since we have shown previously that cannabinoids including anandamide interfere with preimplantation mouse embryo development, the local modulation of anandamide formation and hydrolysis by the implanting blastocysts could be critical for successful embryonic growth, implantation, and pregnancy establishment. The finding of increased synthase activity with concomitant decrease in amidase activity in the uterus on day 5 of pseudopregnancy, when the uterus in hostile to blastocyst survival and implantation, is consistent with this assumption. Further indomethacin, known to interfere with arachidonate metabolism and embryo implantation, stimulated the synthase activity, while inhibiting the amidase activity in the uterus in vivo and in vitro. Finally, considering the kinetics and profiles of these two enzymatic reactions during early pregnancy, the results suggest that synthase and amidase may be two separate enzymes in the mouse uterus. This investigation constitutes the first detailed studies on anandamide synthase and amidase activities in the female reproductive t

Amidohydrolases↗

PubMed1996

Activation of brain-type cannabinoid receptors interferes with preimplantation mouse embryo development.

The recent identification and cloning of guanine nucleotide regulatory protein-coupled brain-type and spleen-type cannabinoid receptors (CB1-R and CB2-R, respectively) provide evidence that many of the effects of cannabinoids are mediated via these receptors. Our recent observation of expression of both CB1-R and CB2-R genes in the preimplantation mouse embryo suggests that it could also be a target for cannabinoids. Indeed, cannabinoid agonists interfered with preimplantation embryo development in vitro. To examine whether cannabinoid effects on preimplantation embryos are mediated via CB1-R, we developed rabbit antipeptide antibodies against the N-terminal region of CB1-R and examined the receptor protein in the blastocyst by Western blotting and its spatiotemporal distribution in preimplantation mouse embryos by immunohistochemistry. Cannabinoid binding sites in the blastocyst were examined by Scatchard analysis, while the reversibility of cannabinoid-induced embryonic arrest in vitro was monitored using a specific antagonist to CB1-R, SR141716A. Western blot analysis detected a major band of approximately 59 kDa and a minor band of approximately 54 kDa in the blastocyst. Immunocytochemistry detected this receptor protein from the 2-cell through the blastocyst stages. Scatchard analysis using 3H-anandamide (an endogenous ligand) showed a single class of binding sites in Day 4 blastocysts with an apparent Kd of 1.0 nM and Bmax of 0.09 fmol/blastocyst. Considering the total number of cells (approximately 50) and total protein content (approximately 20 ng) of a blastocyst, it is apparent that the mouse blastocyst has many more high-affinity receptors than those in the mouse brain (Kd: 1.8 nM and Bmax: 18.8 pmol/mg membrane protein). Cannabinoid agonists and the CB1-R antagonist SR141716A effectively competed for anandamide binding in the blastocyst. To determine whether cannabinoid inhibition of embryonic development could be reversed by SR141716A, 2-cell embryos were cultured in the presence of cannabinoid agonists with or without SR141716A for 72 h. Most of the 2-cell embryos cultured in the absence of the agonists developed into blastocysts (approximately 90%). In contrast, the addition of cannabinoid agonists anandamide, Win 55212-2, or CP 55,940 in the culture medium severely compromised embryonic development: more than 60% of the 2-cell embryos failed to develop to blastocysts. A reduction in trophectoderm cell numbers was noted in those blastocysts that escaped the developmental arrest in the presence of cannabinoid agonists. However, this reduction was corrected when embryos were cultured simultaneously with an agonist and SR141716A. Furthermore, embryonic arrest was reversed when embryos were cultured simultaneously in the presence of an agonist and SR141716A. The addition of SR141716A alone in the culture medium apparently had no effects on embryonic development: more than 90% of the embryos developed into blastocysts. The results suggest that the CB1 receptors in preimplantation mouse embryos are biologically active and cannabinoid effects on them are primarily mediated by these receptors.

Analgesics↗

PubMed1996

Mouse preimplantation blastocysts adhere to cells expressing the transmembrane form of heparin-binding EGF-like growth factor.

Previous studies have shown that heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF) mRNA is synthesized in the mouse uterine luminal epithelium temporally, just prior to implantation, and spatially, only at the site of blastocyst apposition (Das, S. K., Wang, X. N., Paria, B. C., Damm, D., Abraham, J. A., Klagsbrun, M., Andrews, G. K. and Dey, S. K. (1994) Development 120, 1071-1083). HB-EGF is synthesized as a transmembrane protein (HB-EGF TM) that can be processed to release the soluble growth factor. An antibody that cross-reacts only with the transmembrane form detected HB-EGF TM in uterine luminal epithelium in a spatial manner similar to that of HB-EGF mRNA. HB-EGF TM is a juxtacrine growth factor that mediates cell-cell contact. To ascertain if HB-EGF TM could be an adhesion factor for blastocysts, a mouse cell line synthesizing human HB-EGF TM was co-cultured with mouse blastocysts. Cells synthesizing HB-EGF TM adhered to day-4 mouse blastocysts more extensively than parental cells or cells synthesizing a constitutively secreted form of HB-EGF. Adhesion of cells synthesizing HB-EGF TM to blastocysts was inhibited by excess recombinant HB-EGF but less so by TGF-alpha. Adhesion was also inhibited by the synthetic peptide P21 corresponding to the HB-EGF heparin binding domain, and by incubating the blastocysts with heparinase. In addition, adhesion to delayed implanting dormant blastocysts, which lack EGF receptor (EGFR), was diminished relative to normal blastocysts. These results suggested that adhesion between blastocysts and cells synthesizing HB-EGF TM was mediated via interaction with both blastocyst EGFR and heparan sulfate proteoglycan (HSPG). It was concluded that HB-EGF TM, which is synthesized exclusively in the luminal epithelium at the site of blastocyst apposition, and which is a juxtacrine adhesion factor for blastocysts, could be one of the mediators of blastocyst adhesion to the uterus in the process of implantation.

Animals↗

PubMed1996

Mechanisms of reduced fertility in Hoxa-10 mutant mice: uterine homeosis and loss of maternal Hoxa-10 expression.

The establishment of a receptive uterine environment is critical for embryonic survival and implantation. One gene that is expressed in the uterus during the peri-implantation period in mice and is required for female fertility is the homeobox gene Hoxa-10. Here we characterize the peri-implantation defects in Hoxa-10 mutant females and investigate functions of Hoxa-10 in the uterine anlage during morphogenesis and in the adult uterus during pregnancy. Examination of pregnancy in Hoxa-10 mutant females has revealed failure of implantation as well as resorption of embryos in the early postimplantation period. Morphologic analysis of the mutant uterus has demonstrated homeotic transformation of the proximal 25% into oviduct. Histology and molecular markers confirm this anterior transformation. Furthermore, in situ hybridization shows that this region coincides with the anterior limit of embryonic Hoxa-10 expression in the urogenital ducts and a parallel transformation is observed in Hoxa-10 mutant males at the junction of the epididymis and ductus deferens. Female fertility could be compromised by either the homeotic transformation or the absence of Hoxa-10 function in the adult during pregnancy. To distinguish between these two potential mechanisms of infertility, wildtype blastocysts were transferred into mutant uteri distal to the transformed region on day 2.5 of pseudopregnancy. This procedure did not rescue the phenotype, suggesting that adult uterine expression of Hoxa-10 is required during pregnancy. Moreover, when implantation was experimentally delayed, homozygous uteri were able to support survival of blastocysts comparable to wild-type controls, indicating that the requirement for Hoxa-10 is intrinsic to implantation. While expression of LIF and HB-EGF appears unaffected in the mutant uteri, a decrease is observed in the intensity and number of blue dye reactions, an indicator of increased vascular permeability in response to implantation. In addition, mutant uteri exhibited decreased decidualization in response to artificial stimuli. These results show that Hoxa-10 is required during morphogenesis for proper patterning of the reproductive tract and in the adult uterus for peri-implantation events.

Animals↗

PubMed1995

The preimplantation mouse embryo is a target for cannabinoid ligand-receptor signaling.

Using a reverse transcription-coupled PCR, we demonstrated that both brain and spleen type cannabinoid receptor (CB1-R and CB2-R, respectively) mRNAs are expressed in the preimplantation mouse embryo. The CB1-R mRNA expression was coincident with the activation of the embryonic genome late in the two-cell stage, whereas the CB2-R mRNA was present from the one-cell through the blastocyst stages. The major psychoactive component of marijuana (-)-delta-9-tetrahydrocannabinol [(-)-THC] inhibited forskolin-stimulated cAMP generation in the blastocyst, and this inhibition was prevented by pertussis toxin. However, the inactive cannabinoid cannabidiol (CBD) failed to influence this response. These results suggest that cannabinoid receptors in the embryo are coupled to inhibitory guanine nucleotide binding proteins. Further, the oviduct and uterus exhibited the enzymatic capacity to synthesize the putative endogenous cannabinoid ligand arachidonylethanolamide (anandamide). Synthetic and natural cannabinoid agonists [WIN 55,212-2, CP 55,940, (-)-THC, and anandamide], but not CBD or arachidonic acid, arrested the development of two-cell embryos primarily between the four-cell and eight-cell stages in vitro in a dose-dependent manner. Anandamide also interfered with the development of eight-cell embryos to blastocysts in culture. The autoradiographic studies readily detected binding of [3H]anandamide in embryos at all stages of development. Positive signals were present in one-cell embryos and all blastomeres of two-cell through four-cell embryos. However, most of the binding sites in eight-cell embryos and morulae were present in the outer cells. In the blastocyst, these signals were primarily localized in the mural trophectoderm with low levels of signals in the polar trophectoderm, while little or no signals were noted in inner cell mass cells. These results establish that the preimplantation mouse embryo is a target for cannabinoid ligands. Consequently, many of the adverse effects of cannabinoids observed during pregnancy could be mediated via these cannabinoid receptors. Although the physiological significance of the cannabinoid ligand-receptor signaling in normal preimplantation embryo development is not yet clear, the regulation of embryonic cAMP and/or Ca2+ levels via this signaling pathway may be important for normal embryonic development and/or implantation.

Animals↗

PubMed1995

Cannabinoid ligand-receptor signaling in the mouse uterus.

Using RNA (Northern) blot hybridization and reverse transcription-PCR, we demonstrate that the brain-type cannabinoid receptor (CB1-R) mRNA, but not the spleen-type cannabinoid receptor (CB2-R) mRNA, is expressed in the mouse uterus and that this organ has the capacity to synthesize the putative endogenous cannabinoid ligand, anandamide (arachidonylethanolamide). The psychoactive cannabinoid component of marijuana--delta 9-tetrahydrocannabinol (THC)--or anandamide, but not the inactive and nonpsychoactive cannabidiol (CBD), inhibited forskolin-stimulated cyclic AMP formation in the mouse uterus, which was prevented by pertussis toxin pretreatment. These results suggest that uterine CB1-R is coupled to inhibitory guanine nucleotide-binding protein and is biologically active. Autoradiographic studies identified ligand binding sites ([3H]anandamide) in the uterine epithelium and stromal cells, suggesting that these cells are perhaps the targets for cannabinoid action. Scatchard analysis of the binding of [3H]WIN 55212-2, another cannabinoid receptor ligand, showed a single class of high-affinity binding sites in the endometrium with an apparent Kd of 2.4 nM and Bmax of 5.4 x 10(9) molecules per mg of protein. The gene encoding lactoferrin is an estrogen-responsive gene in the mouse uterus that was rapidly and transiently up-regulated by THC, but not by CBD, in ovariectomized mice in the absence of ovarian steroids. This effect, unlike that of 17 beta-estradiol (E2), was not influenced by a pure antiestrogen, ICI 182780, suggesting that the THC-induced uterine lactoferrin gene expression does not involve estrogen receptors. We propose that the uterus is a new target for cannabinoid ligand-receptor signaling.

Animals↗

PubMed1995

Amphiregulin is an implantation-specific and progesterone-regulated gene in the mouse uterus.

A synchrony between the activated state of the blastocyst and differentiation of the uterus to the receptive state is essential to the process of implantation. This process is directed by progesterone (P4) and estrogen. The mechanism by which P4 differentiates the uterus, enabling estrogen to initiate implantation, is unknown but likely to involve localized induction of growth and differentiation factors. We have cloned the murine amphiregulin (AR) gene, a newly discovered member of the epidermal growth factor family, and demonstrate that its expression is implantation-specific and P4-regulated in the mouse uterus. A transient surge in AR mRNA levels occurred throughout the uterine epithelium on day 4 of pregnancy. With the onset of blastocyst attachment late on day 4, AR mRNA accumulated in the luminal epithelium exclusively at the sites of blastocysts. Thus, AR expression correlated first with rising P4 levels and then with the attachment reaction. The rapid induction of AR mRNA in the ovariectomized uterus only by P4 and abrogation of this induction by RU-486 (a P4 receptor antagonist) suggest that this uterine gene is regulated by P4. AR appeared to exhibit preferential phosphorylation of epidermal growth factor receptor in the uterus over that in the blastocyst. This is a first report of a P4-regulated uterine epithelial cell growth factor that is associated with epithelial cell differentiation during implantation. The association of AR in implantation is further documented by its down-regulation in the day 4 pregnant uterus in which uterine receptivity and implantation were disrupted by estrogen or RU-486 treatment on day 3. These results further indicate that the expression of the AR gene could serve as a molecular marker for the receptive state of the uterus for implantation.

Amino Acid Sequence↗

PubMed1994

Effects of chronic treatment with delta-9-tetrahydrocannabinol on uterine growth in the mouse.

The estrogenic responses of delta-9-tetrahydrocannabinol (THC), the major psychoactive component of marijuana, remain unsettled. We examined the effects of chronic treatment of THC on uterine wet weights and nuclear 3H-thymidine incorporation (DNA synthesis), and compared these responses with those of an acute treatment of estradiol-17 beta (E2) in ovariectomized (OVX) or hypophysectomized (HYPOX) mice. As shown previously, an injection of E2 increased uterine weights and nuclear 3H-thymidine incorporation in epithelial cells in OVX or HYPOX mice when examined 18 h after the injection. Injections of THC daily for 7 days also elicited uterine weight gains and nuclear 3H-thymidine incorporation in a dose dependent manner in OVX or HYPOX mice as compared to the vehicle (oil) treated controls. Like E2, THC induced nuclear 3H-thymidine incorporation primarily in luminal and glandular epithelial cells. However, effects of THC in these responses were much inferior to those elicited by an injection of E2. These data suggest that THC, given chronically in the mouse, behave as a weak uterotrophic agent, and THC effects are not mediated by the pituitary.

Animals↗

PubMed1994

Distribution of transforming growth factor alpha precursors in the mouse uterus during the periimplantation period and after steroid hormone treatments.

Temporal and cell-type specific distribution of transforming growth factor alpha (TGF alpha) precursor (proTGF alpha) was examined in the mouse uterus during the periimplantation period, and after steroid hormone treatments of ovariectomized adult mice by immunohistochemistry using antibodies that recognize the precursor forms of the growth factor. These studies were complemented by immunoblot analysis of proTGF alpha in separated uterine cell-type preparations. The specificity of the antibodies used in these studies was confirmed by use of pancreas or lactating mammary glands from transgenic mice in which mutated proTGF alpha, lacking recognition sites for proteolytic cleavages, was targeted for expression under a tissue-specific enhancer/promoter. Analysis of histochemical studies revealed accumulation of immunoreactive proTGF alpha primarily in luminal and glandular epithelial cells on Day 1 of pregnancy or pseudopregnancy followed by little or no accumulation on Days 2 and 3. However, immunoreactive proTGF alpha started to reappear in the luminal epithelium on the morning of Day 4 and became more prominent in the afternoon. In pregnant mice, immunostaining persisted in these cells at the implantation sites during the time of attachment reaction (2130 h on Day 4), but disappeared by morning of Day 5. Immunostaining appeared to be situated at the apical border of the luminal epithelium. No positive immunostaining could be detected in the nonreceptive uterus on Day 5 or 6 of pseudopregnancy. Consistent with the immunohistochemistry results, Western blot analysis detected two species of precursor proteins (14.5 and 17 kDa) in isolated luminal epithelial cell-enriched preparations on Day 4, but not on Day 5, of pseudopregnancy. The results suggest that proTGF alpha accumulates in the luminal epithelium of the receptive uterus prior to implantation. The effects of ovarian steroids on uterine accumulation of proTGF alpha were examined in ovariectomized adult mice by immunohistochemistry and immunoblotting. Whereas an injection of estradiol-17 beta (E2) or progesterone (P4) had little or a modest effect on epithelial accumulation of proTGF alpha, P4 priming for several days resulted in distinct accumulation of proTGF alpha in epithelial cells. The superimposition of an E2 treatment on P4 priming showed a biphasic response, with an initial gradual loss of immunostaining through 12 h followed by a return by 24 h of E2 treatment. The combined hormone treatment schedule employed here is similar to the situation of inducing implantation with E2 in P4-primed delayed implanting mice. The results suggest a paracrine/"juxtacrine" role for this growth factor in implantation.

Animals↗

PubMed1994

Expression of epidermal growth factor receptor in the preimplantation uterus and blastocyst of the western spotted skunk.

The western spotted skunk is unique in that its blastocysts undergo a 180-220-day period of arrested development before implantation. We investigated the potential role of epidermal growth factor (EGF)-related growth factors in regulating uterine and embryonic development in this species by studying the status of EGF receptor (EGF-R) in these tissues during delayed implantation and resumption of embryonic development. The cell-specific distribution of EGF binding sites and the expression of EGF-R mRNA were assayed by autoradiography and Northern blot analysis, respectively. The size of EGF-R was determined by affinity cross-linking studies, and its bioactivity was examined by determining EGF-dependent subcellular protein tyrosine kinase (PTK) activity. EGF binding sites were localized in the uterine luminal and glandular epithelium, endometrial stroma, myometrium, and blood vessels during both stages of pregnancy. As examined by Northern blot hybridization, a cRNA probe specific to mouse EGF-R hybridized to poly(A)+ RNA of skunk uteri. Transcripts similar to those of mouse uterine EGF-R were identified. [125I]-EGF was cross-linked to a 170-kDa protein both in the uterus and in blastocysts collected during the delayed implantation and periimplantation periods. However, EGF-induced PTK activity was significantly elevated above background levels during the period of renewed embryonic development, but not during arrested embryonic development. The results suggest that EGF-related growth factors may play an important role in regulating embryonic development in this species and that a change in the number and/or functional status of the EGF-R may be a prerequisite for blastocyst activation and implantation in the spotted skunk.

Animals↗

PubMed1994

Differential expression of epidermal growth factor receptor (EGF-R) gene and regulation of EGF-R bioactivity by progesterone and estrogen in the adult mouse uterus.

The present study examined several aspects of epidermal growth factor receptor (EGF-R) in the mouse uterus during the peri-implantation period and after ovarian hormone treatment of adult ovariectomized mice. The cell-specific distribution, regulation of expression, and binding kinetics were assessed by immunohistochemistry, Northern blot analysis, and ligand binding assays, respectively. Affinity cross-linking studies ascertained the size of the EGF-R, and its bioactivity was examined by determining EGF-dependent subcellular protein tyrosine kinase activity and receptor autophosphorylation. In the intact uterus and separated cell types, EGF-R was detected in the stroma, deciduum, and myometrium, but not in the luminal or glandular epithelium. Uterine EGF-R mRNA transcript profiles showed some differences between pregnant and ovariectomized mice regardless of steroid hormone treatments. Two major [6.5- and 2.7-kilobase (kb)] and two less abundant (9.6- and 5.0-kb) transcripts were detected in pregnant uterine poly(A)+ RNA. Three additional transcripts (< 2.0 kb) were detected in decidual poly(A)+ RNA, and a larger transcript (8.0 kb) was detected in uterine poly(A)+ RNA isolated from ovariectomized mice. Scatchard analysis of EGF binding also revealed apparent differences in binding kinetics between pregnant and ovariectomized mice, although EGF was cross-linked to a 170-kilodalton protein under these conditions. Two classes (Kd, approximately 0.2 and approximately 2.0 nM) of binding sites were noted in pregnant mice, whereas a single class (Kd, approximately 1.0 nM) was found in ovariectomized mice. 17 beta-Estradiol (E2) caused a rapid transient upregulation of uterine EGF-R mRNA levels and increased the number of EGF-binding sites in ovariectomized mice, as did an injection of progesterone (P4). However, the bioactivity of EGF-R could not be detected in uteri of ovariectomized mice treated with oil or P4. E2 treatment was found to be essential for EGF-R bioactivity. Taken together, the results suggest that in the adult mouse uterus, EGF-R status is influenced by factors other than P4 and E2, the epithelium is not the direct target for the actions of EGF-related growth factors as thought previously, the mitogenic effects of these growth factors on epithelial cells in vivo are perhaps mediated by other uterine cell-types expressing EGF-R, and, lastly, these growth factors are not likely to be functional in the uterus in the absence of estrogen. The present observations are supportive of the concept of paracrine or juxtacrine interactions between EGF-related growth factor ligands of luminal epithelial origin and blastocyst EGF-R in the process of implantation.

Animals↗

PubMed1994

Heparin-binding EGF-like growth factor gene is induced in the mouse uterus temporally by the blastocyst solely at the site of its apposition: a possible ligand for interaction with blastocyst EGF-receptor in implantation.

Heparin-binding EGF-like growth factor (HB-EGF) is a newly discovered member of the EGF family of growth factors. HB-EGF can bind to two loci on cell surfaces, heparan sulphate proteoglycans and EGF-receptor (EGF-R), and either one or both of these interactions could play a role in cell-cell interactions. In the rodent, increased endometrial vascular permeability at the site of blastocyst apposition is considered to be an earliest discernible prerequisite event in the process of implantation and this event coincides with the initial attachment reaction between the blastocyst trophectoderm and uterine luminal epithelium. This investigation demonstrates that the HB-EGF gene is expressed in the mouse uterine luminal epithelium surrounding the blastocyst 6-7 hours before the attachment reaction that occurs at 2200-2300 hours on day 4 of pregnancy. It was further demonstrated that this gene is not expressed in the luminal epithelium at the site of the blastocyst apposition during the progesterone-maintained delayed implantation, but is readily induced in the luminal epithelium surrounding an activated blastocyst after termination of the delay by an estrogen injection. In vitro studies showed that HB-EGF induced blastocyst EGF-R autophosphorylation, and promoted blastocyst growth, zona-hatching and trophoblast outgrowth. These results suggest possible interactions between the uterine HB-EGF and blastocyst EGF-R very early in the process of implantation, earlier than any other embryo-uterine interactions defined to date at the molecular level.

Animals↗

PubMed1993

Blastocyst's state of activity determines the "window" of implantation in the receptive mouse uterus.

The present investigation studied the influence of the blastocyst's state of activity on the "window" of implantation in the receptive uterus in the mouse. The receptive state of the uterus is defined as the limited time when the uterine milieu is favorable to blastocyst acceptance and implantation. In the mouse, implantation occurs on day 4 (day 1 = vaginal plug). Ovariectomy in the morning of day 4 prior to preimplantation estrogen secretion results in blastocyst dormancy and delayed implantation. These conditions are maintained by continued progesterone (P4) treatment but can be terminated with an injection of estrogen leading to blastocyst activation and subsequent implantation. Blastocyst transfers into intact pseudopregnant mice demonstrated that the window of implantation on day 4 remains open at least through 1800 h for normal day 4 blastocysts but only up to 1400 h for dormant blastocysts. These results suggested that the blastocyst's state of activity influenced the normally operative window of implantation in the receptive uterus. This finding was further confirmed by inducing conditions of delayed implantation in pregnant donors and pseudopregnant recipients. They were ovariectomized on the morning of day 4 and maintained with daily injections of P4 from days 5 to 7. On day 7, dormant blastocysts from P4-treated delayed donors were transferred into the uteri of P4-treated delayed pseudopregnant recipients at 1, 2, 4, or 8 h after an injection of 17 beta-estradiol (E2). Dormant blastocysts transferred into delayed recipients at 1 h after E2 treatment resulted in implantation in most of the animals as compared to complete failure of blastocysts to implant after transfer to P4-treated delayed recipients at 4 or 8 h after E2 treatment. However, implantation did occur in P4-treated delayed recipients at these later hours of E2 treatment when the P4-treated delayed donors also received E2 prior to blastocyst transfer. Furthermore, the majority of day 4 normal blastocysts implanted when transferred into P4-treated delayed recipients even at 16 h after E2 treatment. Interestingly, day 7 dormant blastocysts cultured for 8 or 24 h for in vitro activation failed to implant after transfer to P4-treated delayed pseudopregnant recipients at 4 ir 8 h after E2 treatment, although they did implant after transfer at 1 h after E2 treatment. As expected, normal day 4 blastocysts failed to implant after transfer to P4-treated delayed pseudopregnant recipients. Thus, these results establish that the blastocyst's state of activity alters the timing of implantation (window) in the receptive uterus. Thus, the window for successful implantation could be defined as a limited time span when the activated stage of the blastocyst is superimposed on the receptive state of the uterus. This window remains open for a shorter period for dormant blastocysts than for a normal or dormant blastocysts after E2 activation. Furthermore, dormant blastocysts, which apparently achieved metabolic activation in vitro, failed to attain the same status as blastocysts activated in utero by E2 for implantation into the receptive uterus. A key finding of this investigation is that E2 induces very rapidly, but transiently (1 h), a factor(s) in the P4-primed uterus that activates the dormant blastocysts for implantation in the receptive uterus.

Animals↗

PubMed1993

Stage-specific effects of cadmium on preimplantation embryo development and implantation in the mouse.

The effects of cadmium (Cd) exposure during the preimplantation period of pregnancy on the subsequent development and implantation of mouse embryos were examined. Injection of a high dose of Cd (38 mumol Cd/kg body wt.) on day 2 (D1 = vaginal plug), when the embryo is at the two-cell stage, had little effect on the initiation and maintenance of pregnancy when examined on D8. The initiation of implantation (localized sites of increased uterine vascular permeability) in a similarly treated group of mice was assessed in the morning of D5, and these sites were absent in 62% of the animals examined. Thus, Cd treatment on D2 delayed temporarily, but did not prevent implantation and was not embryolethal. In marked contrast, the same dose of Cd administered on D4 caused pregnancy failure in all mice examined on D8. No implantation sites were detected on D5 and the few blastocysts recovered were degenerating. To explore the mechanisms underlying these in vivo stage-specific effects of Cd, preimplantation embryos (two-cell, four-cell, eight-cell and morulae) were exposed in vitro to a high concentration of Cd (50 microM) for 8 h followed by reculture to monitor their potential to develop to the blastocyst stage. Two-cell embryos were remarkably resistant to Cd, but toxicity increased with development, and morulae readily degenerated after Cd exposure. Analysis of the accumulation of 109Cd (50 microM) by preimplantation embryos showed little or none in two-cell embryos, but rapid accumulation and efflux of this metal by blastocysts. Removal of the zona pellucida had no influence on Cd accumulation. Nifedipine (500 nM), a potent voltage-gated calcium channel blocker, and zinc (Zn; 100-fold molar excess) each significantly reduced (approximately 50% in 2 h) Cd accumulation by blastocysts, whereas N-ethylymaleimide (NEM; 20 microM) increased it. These results provide evidence that pregnancy failure after Cd exposure during the preimplantation period reflects a direct embryotoxic effect of Cd, although maternal injury by Cd may also contribute. Resistance to Cd at the two-cell stage (D2) reflects a lack of uptake of this metal, whereas sensitivity to Cd at the blastocyst stage (D4) reflects the ability to accumulate Cd.

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