Biomedical subjects

S K Dey

Publications and source records attributed to S K Dey.

At least 91 records · Page 5Linked to original sources

PubMed1997

Expression of matrix metalloproteinases and tissue inhibitors of metalloproteinases in the mouse uterus during the peri-implantation period.

The attachment of the blastocyst to the uterine luminal epithelium and the subsequent invasion by trophoblast cells through the stroma and deciduum occur in a highly regulated manner by remodeling of the extracellular matrix. We investigated the temporal and spatial expression of mRNAs for four matrix metalloproteinases (MMPs; MMP-2 [gelatinase A], MMP-3 [stromelysin 1], MMPs; MMP-2 [gelatinase B], and MMP-13 [collagenase 3]) and tissue inhibitors of metalloproteinases (TIMPs; TIMP-1, TIMP-2, and TIMP-3) in the mouse uterus from days 1 to 8 of pregnancy. Northern blot analyses showed the transcripts for MMP-2, MMP-3, RNA on these days. However, MMP-13 mRNA was not detected in the uterus, and only weak signals for MMP-3 mRNA were detected in the myometrium. Striking expression was observed with MMP-2 mRNA in the subepithelial stroma on days 3-5. With the progression of decidualization on day 6, signals were primarily in the secondary decidual zone. On day 8, MMP-2 mRNA was localized at the site of placenta formation in the mesometrial pole. Signals for MMP-9 mRNA were first detected in a small population of stromal cells exclusively at the site of implantation on day 5 at the antimesometrial pole. However, the most pronounced expressed was noted in trophoblast giant cells on day 8. TIMP-1 mRNA was present in the myometrium on day 1. On days 2-5, modest signals were detected in the stroma, and on days 6 and 8, they were in the secondary decidual zone. Localization of TIMP-2 mRNA was similar to that of TIMP-1 except it was restricted to the stroma on day 1. The regulation of TIMP-3 was more pronounced. While a gradual increase in signals was observed in stromal cells from days 1 to 4, strong signals were detected in antimesometrial stromal cells at the sites of blastocyst attachment on day 5. On days 6 and 7, even stronger signals were present in the primary decidual zone surrounding the embryo, and on day 8 signals were localized primarily in the mesometrial decidual bed. These results suggest that MMP-2 may participate in the early phase of decidualization and neovascularization required for placentation. The restricted MMP-9 expression in stromal cells on day 5 and in trophoblast giant cells on day 8, coupled with the expression of TIMP-3 in the stroma surrounding the embryo, suggests that a fine balance between MMP-9 and TIMP-3 may regulate trophoblast invasion in the uterus.

Animals↗

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

Potential sites of prostaglandin actions in the periimplantation mouse uterus: differential expression and regulation of prostaglandin receptor genes.

Prostaglandins (PGs), especially PGE2 and PGF2alpha, are considered important for blastocyst spacing, implantation, and decidualization in the rodent uterus. However, information regarding uterine sites of PG actions in these processes is lacking. PGE2 or PGF2alpha interacts with specific G protein-coupled membrane receptors. PGE2 receptors are classified into four subtypes, EP1, EP2, EP3, and EP4. While EP1 is coupled to Ca2+ mobilization, activation of EP2 and EP4 triggers stimulation of adenylyl cyclase. In contrast, activation of EP3 inhibits adenylyl cyclase. PGF2alpha receptor (FP) is coupled to stimulation of phospholipase C-inositol trisphosphate (IP3) pathway and Ca2+ mobilization. This investigation demonstrates that PGE2 and PGF2alpha receptor genes are expressed in a temporal and cell-specific manner in the periimplantation mouse uterus. In the mouse, the attachment reaction occurs in the evening (2200-2300 h) of Day 4 of pregnancy and is preceded by embryo spacing, uterine edema, and luminal closure resulting in an intimate apposition of the blastocyst with the uterine luminal epithelium. Expression of EP3 and FP primarily in the circular muscle of the myometrium on Days 3-5 of pregnancy suggests that the circular muscle, not the longitudinal muscle, is the primary target for PG-mediated uterine contractions required for embryo transport, spacing, and/or accommodation in the uterus. In contrast, expression of EP3 in a subpopulation of cells in the stromal bed at the mesometrial side, and of EP4 in the epithelium and stroma on these days, suggests that PGE2 effects on uterine preparation for implantation (such as epithelial cell differentiation, stromal cell proliferation, uterine edema, luminal closure, and increased localized endometrial vascular permeability at the sites of blastocyst attachment) are mediated by these receptor subtypes. Similar expression patterns of EP3 and EP4 in the Day 4 pseudopregnant mouse uterus or in the ovariectomized uterus under combined treatment with estrogen and progesterone suggest that these genes are regulated by ovarian steroids rather than by the embryo during the preimplantation period (Days 1-4). In contrast, the expression of these genes during the postimplantation period (Days 5-8) is associated with the onset of decidualization.

Animals↗

PubMed1997

Expression of vascular endothelial growth factor (VEGF) and VEGF-receptor messenger ribonucleic acids in the peri-implantation rabbit uterus.

The endometrial vasculature undergoes expansion during preimplantation stages and, even more prominently, after implantation. In addition to angiogenesis, vascular hyperpermeability accompanies the attachment and invasion of blastocysts into the uterine lining. Vascular endothelial growth factor (VEGF) is an angiogenic factor expressed in mammalian uteri that also has potent activity in inducing vascular permeability. Rabbit uteri were examined using Northern and in situ hybridization to assess the temporal and spatial expression of VEGF and its receptor (Flk-1, Flt-1) mRNAs during the pre- and peri-implantation periods (Days 0-8). Steady-state levels of VEGF mRNA were highest in endometrium at estrous and peri-implantation stages (Days 6-8). In situ hybridization revealed a shift from uniform expression of VEGF transcripts throughout the uterus at estrus and Day 4, to an endometrial epithelial localization just before and during implantation. At implantation sites, a pronounced signal was present in the trophoblastic knobs, the syncytial aggregates that attach to and invade the endometrium. VEGF protein was detected by immunoblot analysis in peri-implantation-stage uteri but was below the limit of detection in estrous endometrium. VEGF receptor mRNAs were expressed in the uterus at all stages examined, with high levels of Flk-1 and Flt-1 at estrus and again just before implantation, 6-3/4 days pregnant. The high level just before implantation correlates with in situ hybridization results showing a prominent, but transient, signal for Flk-1 mRNA in the endometrial epithelium. During implantation, Flk-1 mRNA was associated with blood vessels of the endometrial stroma. We conclude that VEGF is a candidate factor for the induction of vascular hyperpermeability at implantation in the rabbit and in the angiogenic process that follows.

Animals↗

PubMed1997

Prostaglandin E2 receptor subtype EP2 gene expression in the mouse uterus coincides with differentiation of the luminal epithelium for implantation.

Among the PGs, PGE2 is considered especially important for implantation and decidualization. Four major PGE2 receptor subtypes, EP1, EP2, EP3, and EP4, mediate various PGE2 effects via their coupling to distinct signaling pathways. Previously, we have shown that the EP1, EP3, and EP4 genes are expressed in the periimplantation mouse uterus in a spatio-temporal manner, suggesting compartmentalized actions of PGE2 during this period. In this study, we examined the expression of the EP2 gene in the mouse uterus during the periimplantation period (days 1-8) and during experimentally induced progesterone (P4)-maintained delayed implantation and its resumption by 17beta-estradiol (E2). We also examined its regulation in the uterus by ovarian steroid hormones. Our results establish that EP2 messenger RNA (mRNA) is expressed exclusively in the luminal epithelium primarily on day 4 (the day of implantation) and day 5 (early implantation) of pregnancy. In (P4)-maintained delayed implanting mice, EP2 mRNA was present in the luminal epithelium, and the expression was further enhanced regardless of the location of the blastocysts after reinitiation of implantation. This observation suggests little or no embryonic influence in regulating EP2 expression and, instead, shows its regulation by P4 and E2. Indeed, treatment with E2 and/or P4 exhibited unique regulation of this gene. The treatment of adult ovariectomized mice with E2 down-regulated the basal levels of EP2 mRNA, whereas that with P4 up-regulated its levels in the luminal epithelium. The up-regulation of EP2 mRNA levels by P4 was further augmented by superimposition of the E2 treatment, suggesting a synergistic interaction between E2 and P4 in regulating this gene in the uterus. Collectively, the results suggest that EP2 could be a potential mediator of PGE2 actions in regulating luminal epithelial differentiation and serve as a marker for uterine receptivity for implantation.

Animals↗

PubMed1997

Differential expression of the erbB2 gene in the periimplantation mouse uterus: potential mediator of signaling by epidermal growth factor-like growth factors.

Ligand-receptor signaling with the epidermal growth factor (EGF) family of growth factors in the uterus and embryo is considered to be important for implantation. The EGF family includes EGF, transforming growth factor-alpha, heparin binding-EGF, amphiregulin, beta-cellulin, epiregulin, and heregulins, whereas the receptor family (the erbB genes) consists of erbB1 (EGF-receptor, EGF-R), erbB2, erbB3, and erbB4. Interactions of uterine EGF-R with EGF-like ligands have been examined, but limited information is available regarding the status of other receptor subtypes. Thus, we examined the expression of the erbB2 gene in the mouse uterus during the periimplantation period (days 1-8 of pregnancy) and after 17 beta-estradiol and/or progesterone stimulation. Northern blot hybridization detected two transcripts (approximately 4.0 and 5.0 kb) of erbB2 messenger RNA (mRNA) in day 1-8 uterine polyadenylated RNA samples. In situ hybridization experiments showed unique uterine cell-specific erbB2 mRNA distribution. On days 1-4, unlike the full-length erbB1 mRNA which is not expressed in the uterine epithelium, the erbB2 mRNA was detected primarily in epithelial cells; the day 1 uterus showed the highest accumulation. On day 5, the epithelium and the decidualizing stromal cells around the implanting blastocyst exhibited accumulation of this mRNA. On days 6-8, the accumulation persisted in the epithelium at both the implantation and interimplantation sites in addition to modest levels of signals in the secondary decidual zone. On days 7 and 8, accumulation of the erbB2 mRNA was also prominent in the trophoblastic giant cells. Western blotting detected a predicted protein of 185 kDa in day 4 uterine membrane preparations. Results of immunocytochemistry demonstrated colocalization of the erbB2 protein with its mRNA in the periimplantation uterus. The uterine ErbB2 underwent phosphorylation by several members of the EGF family. Treatment of adult ovariectomized mice with 17 beta-estradiol, but not progesterone, up-regulated the expression of the erbB2 mRNA by more than 3.5-fold, as determined by quantitative reverse transcription-PCR, and this increase was limited to the epithelium, as revealed by in situ hybridization. Collectively, the results place ErbB2 as a potential candidate receptor subtype for interaction with the EGF-related ligands in epithelial cell proliferation/differentiation during the preimplantation period and stromal cell proliferation/decidualization during the postimplantation period.

Amino Acid Sequence↗

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

Regulation of interleukin-6 and interleukin-1 beta gene expression in the mouse deciduum.

Expression and regulation of interleukin-6 (IL-6) and IL-1 beta were examined in the mouse deciduum and in experimentally induced deciduoma from 6 to 8 days postcoitum (1 dpc = vaginal plug), as well as in cultured mouse decidual cell preparations. Levels of these mRNAs in the deciduum and deciduoma were below the limits of detection by Northern blotting. However, enzymatic dispersion and culture of decidual cells and/or exposure to bacterial endotoxin-lipopolysaccharide (LPS) induced these mRNAs. IL-6 levels that accumulated in the culture medium (3990 pg/3 x 10(6) cells/day) were about 90-times higher than those of IL-1 beta (45 pg/3 x 10(6) cells/day). Progesterone (10(-7) M) modestly (40%) reduced the levels of IL-6 mRNA and protein during culture, whereas LPS dramatically (8-fold) and rapidly induced IL-6 and IL-1 beta mRNAs and proteins. In vivo, few IL-1 beta immunopositive cells were localized by immunohistochemistry in the 8 dpc deciduum. In contrast, IL-6 mRNA was localized by in situ hybridization in dispersed clusters of a few cells in the mesometrial deciduum near the center of the implantation site. LPS rapidly induced interleukin mRNAs in the deciduum and deciduoma. After LPS injection, IL-1 beta immunopositive cells were dispersed in the myometrium and mesometrial deciduum. In contrast, after LPS injection (2 h), IL-6 mRNA was abundant in 'cords' of cells that traverse the mesometrial deciduum longitudinally, as well as in cells dispersed throughout the myometrium. Thus, the IL-1 beta and IL-6 genes are expressed and regulated in distinct subsets of cells in the decidual bed. The pattern of F4/80 immunostaining is consistent with macrophages as the major, if not only, source of decidual IL-1 beta. IL-6 is also expressed in these cells. However, IL-6 gene expression is regulated in a distinct subset of cells located in the mesometrial decidual bed of the mouse.

Animals↗

PubMed1996

Evidence against a significant role for mast cells in blastocyst implantation in the rat and mouse.

Rats were treated with the highly potent stabilizer of mast cells, FPL 55618, before and during the first seven days of pregnancy to establish whether stabilization of mast cells resulted in impaired blastocyst implantation. There was no significant reduction in either the number of ovulations or the number of implantation sites in treated rats compared with controls; 11 of 15 treated rats were pregnant compared with 5 of 6 control rats. The distribution of mast cells was examined in uterine tissues, implantation sites and interimplantation sites in both rats and mice using highly sensitive immunohistochemical techniques. Virtually all of the mast cells in rat uterine tissue stained for rat mast cell protease-I (RMCP-I; connective tissue type), whereas few stained for RMCP-II (mucosal type). Most of the mast cells were present in the myometrium with very sparse distribution in the endometrium and there were no differences in numbers of mast cells between implantation and inter-implantation sites on Day 7 of pregnancy. In tissue sections of mouse uteri sampled from Day 1 to Day 8 of pregnancy there were virtually no mast cells in the endometrium or deciduum adjacent to implantation sites. Mouse uterine mast cells also stained predominantly for the connective tissue-type mast cell protease MMCP-4, the murine equivalent of RMCP-I. Thus, mast cells and their products appear to play little, if any, role in blastocyst implantation in murid rodents. Since mast cells are a prominent feature of human endometrium, this study emphasizes the important consideration of species differences when choosing animal models for implantation studies.

Animals↗

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

Differential expression of the full-length and truncated forms of the epidermal growth factor receptor in the preimplantation mouse uterus and blastocyst.

The present investigation examined the differential expression of the full-length (fl) and the truncated (tr) forms of the epidermal growth factor receptor (EGFr) in the preimplantation mouse uterus and blastocyst. Northern blot hybridization using a complementary RNA probe specific to the full-length form (EGFr-fl) detected a 6.5-kb transcript, whereas that of the truncated form (EGFr-tr) detected a 2.7-kb transcript in the preimplantation mouse uterus on days 1 and 4 of pregnancy (day 1 = vaginal plug). In situ hybridization using these probes detected the EGFr-fl transcripts only in the stroma and myometrium, but not in the epithelium, whereas EGFr-tr transcripts were detected in all major uterine cell-types. To confirm the results of in situ hybridization, RT-PCR was performed on RNA isolated from separated uterine cell-types on day 4 of pregnancy using sequence specific primers for the two forms of the receptor. The results concur that the EGFr-tr transcript is expressed in the epithelium, stroma and myometrium, whereas that of the EGFr-fl transcript is not expressed in the epithelium. In the preimplantation blastocyst, RT-PCR detected the EGFr-fl messenger RNA, but not the EGFr-tr messenger RNA. These results suggest that the blastocyst, not the uterine epithelium, is the target for EGF family of growth factors in embryo-uterine interaction during implantation.

Animals↗

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↗

PubMed1996

Developmental expression of the cyclo-oxygenase-1 and cyclo-oxygenase-2 genes in the peri-implantation mouse uterus and their differential regulation by the blastocyst and ovarian steroids.

Cyclo-oxygenase (COX) is a rate-limiting enzyme that converts arachidonic acid to prostaglandins (PGs) and exists in two isoforms, COX-1 and COX-2. In the rodent, increased uterine vascular permeability at sites of blastocyst apposition is one of the earliest prerequisite events in the implantation process. This event is preceded by generalized uterine edema and luminal closure, and coincides with the initial attachment reaction between the trophectoderm and luminal epithelium. Vasoactive PGs are implicated in these processes. Here we demonstrate that COX genes are differentially regulated in the peri-implantation mouse uterus. During the preimplantation period (days 1-4), the COX-1 gene was expressed in the uterine epithelium mainly on day 4 until the initiation of attachment reaction in the evening after which the expression was downregulated. This COX-1 expression coincides with the generalized uterine edema required for luminal closure. In contrast, the COX-2 gene was expressed in the luminal epithelium and subepithelial stromal cells at the anti-mesometrial pole exclusively surrounding the blastocyst at the time of attachment reaction on day 4 and persisted through the morning of day 5. This uterine gene was not expressed at the sites of blastocyst apposition during progesterone (P(4))-treated delayed implantation, but was readily induced in the uterus surrounding the activated blastocysts after termination of the delay by estradiol-17beta (E(2)). The results suggest that PG synthesis catalyzed by COX-2 is important for localized increased uterine vascular permeability and attachment reaction. The COX-1 gene that was downregulated from the time of attachment reaction on day 4 was again expressed in the mesometrial and anti-mesometrial secondary decidual beds on days 7 and 8. These results suggest that PGs generated by COX-1 are involved in decidualization and/or continued localized endometrial vascular permeability observed during this period. In contrast, the COX-2 gene, expressed at the anti-mesometrial pole on days 4 and 5, switched its expression to the mesometrial pole from day 6 onward. These results suggest that PGs produced at this site by COX-2 are involved in angiogenesis for the establishment of placenta. In the ovariectomized mice, the COX-1 gene was induced in the epithelium by a combined treatment with P(4) and E(2). However, P(4) and/or E(2) treatments failed to influence the uterine COX-2 gene. Overall, the results suggest that the uterine COX-1 gene is influenced by ovarian steroids, while the COX-2 gene is regulated by the implanting blastocyst during early pregnancy.

Amino Acid Sequence↗

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↗