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S K Dey

Publications and source records attributed to S K Dey.

At least 73 records · Page 4Linked to original sources
PubMed1999

Heparin-binding EGF-like growth factor interacts with mouse blastocysts independently of ErbB1: a possible role for heparan sulfate proteoglycans and ErbB4 in blastocyst implantation.

Blastocyst implantation requires molecular and cellular interactions between the uterine luminal epithelium and blastocyst trophectoderm. We have previously shown that heparin-binding EGF-like growth factor (HB-EGF) is induced in the mouse luminal epithelium solely at the site of blastocyst apposition at 16:00 hours on day 4 of pregnancy prior to the attachment reaction (22:00-23:00 hours), and that HB-EGF promotes blastocyst growth, zona-hatching and trophoblast outgrowth. To delineate which EGF receptors participate in blastocyst activation, the toxicity of chimeric toxins composed of HB-EGF or TGF-(&agr;) coupled to Pseudomonas exotoxin (PE) were used as measures of receptor expression. TGF-(&agr;) or HB-EGF binds to EGF-receptor (ErbB1), while HB-EGF, in addition, binds to ErbB4. The results indicate that ErbB1 is inefficient in mediating TGF-(&agr;)-PE or HB-EGF-PE toxicity as follows: (i) TGF-(&agr;)-PE was relatively inferior in killing blastocysts, 100-fold less than HB-EGF-PE, (ii) analysis of blastocysts isolated from cross-bred egfr+/- mice demonstrated that HB-EGF-PE, but not TGF-(&agr;)-PE, killed egfr-/- blastocysts, and (iii) blastocysts that survived TGF-(&agr;)-PE were nevertheless killed by HB-EGF-PE. HB-EGF-PE toxicity was partially mediated by cell surface heparan sulfate proteoglycans (HSPG), since a peptide corresponding to the heparin-binding domain of HB-EGF as well as heparitinase treatment protected the blastocysts from the toxic effects of HB-EGF-PE by about 40%. ErbB4 is a candidate for being an HB-EGF-responsive receptor since RT-PCR analysis demonstrated that day 4 mouse blastocysts express two different erbB4 isoforms and immunostaining with anti-ErbB4 antibodies confirmed that ErbB4 protein is expressed at the apical surface of the trophectoderm cells. It is concluded that (i) HB-EGF interacts with the blastocyst cell surface via high-affinity receptors other than ErbB1, (ii) the HB-EGF interaction with high-affinity blastocysts receptors is regulated by heparan sulfate, and (iii) ErbB4 is a candidate for being a high-affinity receptor for HB-EGF on the surface of implantation-competent blastocysts.

ADP Ribose Transferases↗
PubMed1999

Cyclin D3 in the mouse uterus is associated with the decidualization process during early pregnancy.

In the mouse, the attachment reaction between the blastocyst trophectoderm and the receptive uterine luminal epithelium occurs at 2200-2300 h on day 4 of pregnancy and is rapidly followed by transformation of stromal cells into decidual cells (decidual cell reaction). This process can also be induced experimentally (deciduoma) by intraluminal oil infusion in the uterus on day 4 of pseudopregnancy. The decidual cell reaction is associated with up- and down-regulation of many genes in a cell-specific manner. Using mRNA differential display, we identified cyclin D3 as one of the genes that is upregulated in the uterus at the sites of blastocyst apposition during the attachment reaction. The levels of expression were low in the morning of days 1-4 as determined by Northern hybridization. In situ hybridization analysis showed that on days 1 and 2, signals were primarily localized in uterine epithelial cells, while signals were detected in both the stromal and epithelial cells on days 3 and 4. In contrast, with the initiation and progression of decidualization on days 5, 6 and 7, the levels of cyclin D3 mRNA were remarkably upregulated in stromal cells both at the mesometrial and the antimesometrial poles. However, on day 8, signals were primarily localized in stromal cells at the mesometrial decidual bed. Implanting blastocysts on these days also expressed cyclin D3 mRNA. In the progesterone-treated delayed implanting mice, the uterine levels of cyclin D3 mRNA were modest at the sites of blastocyst apposition, but were upregulated with the onset of implantation by estradiol-17beta. However, the decidual expression of cyclin D3 mRNA was not dependent on the presence of blastocysts, since increased expression also occurred in experimentally induced deciduoma in the absence of blastocysts. The importance of cyclin D3 in decidualization was further examined in Hoxa-10-deficient mice which show defective decidualization. The expression of cyclin D3 mRNA in Hoxa-10(-/-) uteri on day 5 was severely compromised after application of a deciduogenic stimulus on day 4 of pseudopregnancy. Collectively, the results suggest that cyclin D3 could be important for the process of decidualization.

Animals↗
PubMed1999

Detection of azide in forensic samples by capillary electrophoresis.

Azide salts are highly toxic compounds that have been difficult to detect in forensic samples. Here, anion analysis by capillary electrophoresis with indirect spectrophotometric detection was applied to detect azide in forensic specimens from two suicide victims. Gastric specimens from the victims were shown to have high azide concentrations; azide represented one of the major anionic components and no corresponding component occurred in normal gastric juice. Samples of blood and bile had low concentrations of azide near the limits of detection. The method described for azide analysis used simple steps for sample preparation and analysis time was less than 10 min per sample. It offers a simple and reliable method for detecting azide in biological fluids.

Azides↗
PubMed1998

erbB genes in the mouse uterus: cell-specific signaling by epidermal growth factor (EGF) family of growth factors during implantation.

We previously described spatiotemporal expression of various epidermal growth factor (EGF)-like ligands and receptor subtypes, ErbB1 and ErbB2, during the peri-implantation period. To better understand the roles of these ligands and their possible signaling schemes in implantation, it is important to define the status of all the ligands and receptor subtypes in the uterus/embryo. No information is available about uterine and embryonic status of ErbB3 or ErbB4 during implantation. We cloned mouse erbB3 and erbB4 cDNAs and examined their expression and bioactivity in the peri-implantation uterus (days 1-8). Two erbB3 (cytoplasmic and extracellular) and three erbB4 (two cytoplasmic and one extracellular) clones were generated. Both forms of the erbB3 clone showed similar transcript profiles, while different transcript profiles were obtained with erbB4 clones. The steady-state levels of erbB3 and erbB4 mRNAs in whole uterine poly(A)+ RNA samples showed little changes during the peri-implantation period, while their unique cell-specific accumulation was noted. erbB3 is predominantly expressed in the epithelial cells, although decidual and embryonic cells also accumulate this mRNA. In contrast, the erbB4 mRNA is primarily expressed in the submyometrial stroma and myometrial connective tissues during this period. Additionally, the extracellular form of the erbB4 clone detected signals in a subpopulation of stromal cells. Autophosphorylation and immunoprecipitation studies provided evidence that uterine ErbB3 and ErbB4 are biologically active. This study provides a comprehensive analysis of possible ligand-receptor signaling schemes for EGF-like ligands in implantation.

Animals↗
PubMed1998

Abdominal B (AbdB) Hoxa genes: regulation in adult uterus by estrogen and progesterone and repression in müllerian duct by the synthetic estrogen diethylstilbestrol (DES).

Mice deficient for the Abdominal B (AbdB) Hox gene Hoxa-10 exhibit reduced fertility due to defects in implantation. During the peri-implantation period Hoxa-10 is sequentially expressed in the uterine epithelium and stroma. These observations, combined with the stringent regulation of uterine implantation by ovarian steroids, prompted us to test whether estrogen and progesterone directly regulate the expression of Hoxa-10 and other AbdB Hoxa genes. Here we show that Hoxa-10 expression in the adult uterus is strongly activated by progesterone. This activation is blocked by the progesterone receptor antagonist RU486 and is independent of new protein synthesis. In addition, Hoxa-10 expression is repressed by estrogen in a protein synthesis-independent manner. Analysis of adjacent AbdB Hoxa genes reveals that Hoxa-9 and a-11 are also activated in a colinear fashion by progesterone but differentially regulated by estrogen. These results suggest that the regulation of AbdB Hox gene expression in the adult uterus by ovarian steroids is a property related to position within the cluster, mediated by the direct action of estrogen and progesterone receptors upon these genes. We next examined whether the embryonic expression of Hoxa10 is regulable by hormonal factors. Previous work has demonstrated that perinatal administration of the synthetic estrogen diethylstilbestrol (DES) to mice and humans produces uterine, cervical, and oviductal malformations. Certain of these phenotypes resemble those in Hoxa-10 knockout mice, suggesting that Hoxa-10 gene expression might be repressed by DES during reproductive tract morphogenesis. Exposure of the developing female reproductive tract to DES, either in vivo or in organ culture, represses the expression of Hoxa-10 in the Müllerian duct. Thus, these data not only establish a direct link between ovarian steroids and AbdB Hoxa gene expression in the adult uterus, but also provide a potential mechanism for the teratogenic effects of DES on the developing reproductive tract.

Animals↗
PubMed1998

Expression of neu differentiation factor during the periimplantation period in the mouse uterus.

Complex cellular interactions occur between the blastocyst and the uterus during implantation. The expression of various polypeptide growth factors and their receptors in the uterus and/or blastocyst during the periimplantation period suggest that growth factors participate in the implantation process. Neu differentiation factor (NDF) is a member of the epidermal growth factor (EGF) family of growth factors and is represented by multiple conserved isoforms. The expression of several EGF-like ligands in the periimplantation uterus has been characterized, including EGF, heparin binding-EGF, transforming growth factor alpha, amphiregulin, betacellulin, and epiregulin. We analyzed the expression pattern of NDF in the periimplantation mouse uterus because of its mitogenic and differentiation-promoting effects. By using Northern analysis and isoform-specific polymerase chain reaction, we found that multiple isoforms are expressed in the periimplantation uterus. NDF displays a highly restricted temporal and spatial expression, with autoradiographic signals localized to the uterine stroma immediately surrounding the implanting blastocyst. NDF expression was absent in mice with delayed implantation but briefly reappeared with the same restricted distribution after termination of the delay by an injection of estrogen. Taken together, these results suggest that an activated blastocyst is required for the expression of NDF and that multiple isoforms may be involved in the complex network of cell-signaling events between the implanting blastocyst and the receptive uterus.

Animals↗
PubMed1998

Effects of cannabinoids on preimplantation mouse embryo development and implantation are mediated by brain-type cannabinoid receptors.

We examined the relative importance of G (Gi) protein-coupled brain-type (CB1-R) and spleen-type (CB2-R) cannabinoid receptors in preimplantation embryo development using agonists and antagonists specific to CB1-R and CB2-R. The results establish that endogenous cannabinoid ligands, anandamide and sn-2 arachidonoylglycerol, arrest embryo development in vitro, and this effect is reversed by CB1-R antagonists SR141716A or AM 251, but not by SR144528, a CB2-R antagonist. A CB2-R selective agonist AM 663 failed to affect embryo development. These results suggest that cannabinoid effects on embryo development are mediated by CB1-R. We also observed that delta9-tetrahydrocannabinol ([-]THC) infused in the presence of cytochrome P450 inhibitors interfered with blastocyst implantation. This adverse effect was reversed by coinfusion of SR141716A. The less active stereoisomer (+)THC plus the inhibitors failed to affect implantation. Analysis of tissue levels demonstrated that uterine accumulation of (-)THC occurred when it was infused in the presence of the P450 inhibitors. These results demonstrate that the uterus and perhaps the embryo have the cytochrome P450 enzymes to metabolize (-)THC and neutralize its adverse effects on implantation. Collectively, the present study demonstrates that cannabinoid effects on embryo development and implantation are mediated by embryonic and/or uterine CB1-R, but not CB2-R.

Animals↗
PubMed1998

Coordination of differential effects of primary estrogen and catecholestrogen on two distinct targets mediates embryo implantation in the mouse.

In the mouse, estrogen is essential for blastocyst implantation in the progesterone (P4)-primed uterus. The mechanism(s) by which estrogen initiates this response still remains elusive. The present investigation, using delayed implantation in the mouse, examined the differential role of estradiol-17beta (E2) and its catechol metabolite 4-hydroxy-E2 (4-OH-E2) in uterine and blastocyst activation for implantation. The conditions of delayed implantation were induced by ovariectomizing mice on day 4 (day 1 = vaginal plug) of pregnancy or pseudopregnancy and maintaining them with P4 from days 5-7. The binding of EGF to blastocysts was used as a marker for blastocyst activation. Our results show that whereas E2 fails to activate dormant blastocysts (with respect to EGF binding in vitro), 4-OH-E2, cAMP, or prostaglandin E2, is effective in this response. Further, whereas 4-OH-E2 induced-activation is not blocked by an antiestrogen, an inhibitor of PG synthesis, adenylyl cyclase or protein kinase A effectively blocks this activation. These results suggest that 4-OH-E2 effects on blastocysts are mediated by PGs, which, in turn, stimulate cAMP production and thus activation of protein kinase A. Two-fluoro-E2 is a poor substrate and an inhibitor of catecholestrogen synthesis, but it is estrogenic, with respect to uterine growth and gene expression. Using blastocyst transfer experiments, we observed that dormant blastocysts incubated with 4-OH-E2 in vitro, but not with E2, are capable of implanting in P4-treated delayed implanting mice receiving two-fluoro-E2. The results suggest that whereas E2 is necessary for preparation of the uterus, uterine-derived catecholestrogen is important for blastocyst activation for implantation. Indeed, the receptive uterus has the capacity to synthesize 4-OH-E2. Collectively, we demonstrate that the primary ovarian estrogen E2, via its interaction with nuclear estrogen receptors, participates in the preparation of the P4-primed uterus to the receptive state in an endocrine manner, whereas its metabolite 4-OH-E2, produced from E2 in the uterus, mediates blastocyst activation for implantation in a paracrine manner. Our results also establish that these target-specific effects of primary estrogen and catecholestrogen are both essential for implantation and that successful implantation occurs only when the activated stage of the blastocyst coincides with the receptive state of the uterus.

Animals↗
PubMed1998

Differential spatiotemporal regulation of lactoferrin and progesterone receptor genes in the mouse uterus by primary estrogen, catechol estrogen, and xenoestrogen.

Many xenobiotics are considered reproductive toxins because of their ability to interact with the nuclear estrogen receptors (ERalpha and ERbeta). However, there is evidence that these xenobiotics can regulate gene expression in the reproductive targets by mechanisms that do not involve these ERs. To examine this further, we compared the effects of estrogenic (o,p'-DDT [1-(o-chlorophenyl)-1-(p-chlorophenyl)2,2,2-trichloroethane] and Kepone, chlordecone) and nonestrogenic (p,p'-DDD [1,1-dichloro-2,2-bis(p-chlorophenyl)ethane], a metabolite of p,p'-DDT) xenobiotics with those of 17beta-estradiol (E2) and 4-hydroxyestradiol-17beta (4-OH-E2), a catechol metabolite of E2, on uterine expression of lactoferrin (LF) and progesterone receptor (PR). These genes are estrogen responsive in the mouse uterus. Normally, LF is expressed in the uterine epithelium, whereas PR is expressed in both the epithelium and stroma in response to estrogenic stimulation. Ovariectomized mice were injected with xenobiotics (7.5 mg/kg), E2 (10 microg/kg), 4-OH-E2 (10 microg/kg), or the vehicle (oil, 0.1 ml/mouse), and uterine tissues were processed for Northern blot and in situ hybridization. The pure antiestrogen ICI-182780 (ICI; 1 or 20 mg/kg) was used to interfere with estrogenic responses that were associated with the ERs. The results of Northern and in situ hybridization demonstrated increased uterine levels of PR and LF messenger RNAs (mRNAs) by all of these xenobiotics, but quantitatively the responses were much lower than those induced by E2 or 4-OH-E2. The results further showed that the E2-inducible epithelial LF mRNA accumulation was markedly abrogated by pretreatment with ICI (20 mg/kg). In contrast, this treatment retained the epithelial expression of PR mRNA, but down-regulated the stromal expression. In contrast, ICI had negligible effects on LF and PR mRNA responses to 4-OH-E2, indicating that this catechol estrogen exerted its effects primarily via a mechanism(s) other than the ERs. The heightened accumulation of LF mRNA in the epithelium in response to Kepone and o,p'-DDT was also severely compromised by pretreatment with ICI, but this antiestrogen had little effect on responses to p,p'-DDD. Similar to E2, Kepone increased the expression of PR mRNA in both uterine epithelium and stroma. However, pretreatment with ICI decreased stromal cell expression, whereas epithelial cell expression remained unaltered or increased. These responses were not noted in mice treated with o,p'-DDT or p,p'-DDD. Collectively, the results demonstrate that catechol estrogens or xenobiotics can alter uterine expression of estrogen-responsive genes by mechanisms that are not totally mediated by the classical nuclear ERs, and these alterations are cell type specific. We conclude that an interaction of a compound with the nuclear ERalpha and/or ERbeta is not an absolute requirement for producing specific estrogen-like effects in the reproductive target tissues.

Animals↗
PubMed1998

Histidine decarboxylase gene in the mouse uterus is regulated by progesterone and correlates with uterine differentiation for blastocyst implantation.

Cell-cell interactions between the blastocyst trophectoderm and uterine luminal epithelium are essential to the process of implantation. The factors that participate in these interactions or their mechanism of actions are poorly understood. Histamine has long been suspected as one of the factors that is involved in implantation. Histamine is formed from L-histidine by histidine decarboxylase (HDC). We examined the expression and regulation of HDC gene in the mouse uterus during early pregnancy and under steroid hormonal stimulation. Northern blot hybridization detected a 2.6-kb transcript of HDC messenger RNA (mRNA) in uterine poly(A)+ RNA samples. Maximum uterine accumulation of HDC mRNA occurred on days 3 and 4 of pregnancy, followed by marked declines on later days (days 5-8). In ovariectomized mice, uterine mRNA levels were up-regulated by an injection of progesterone (P4) by 6 h, and the levels were maintained through 24 h. In contrast, an injection of estradiol-17beta neither stimulated nor antagonized P4-induced HDC mRNA accumulation. P4-induced up-regulation was considerably abrogated by pretreatment with RU-486, a P4 receptor antagonist, suggesting involvement of P4 receptor. In situ hybridization detected HDC mRNA specifically in uterine epithelial cells but not in other cell types. Again, high epithelial accumulation occurred on day 4 of pregnancy. With the progression of implantation (days 5-8), HDC mRNA levels declined in the luminal epithelium surrounding the implanting blastocysts, as compared with that away from the blastocysts. Immunoreactive histamine and HDC were colocalized with HDC mRNA. Western blotting detected a 54-kDa protein in epithelial cell extracts, which also exhibited HDC activity. Expression of HDC in epithelial cells, preceding implantation on day 4, at lower levels after initiation of implantation on day 5, and its regulation by P4 suggest that this gene plays an important role in implantation.

Animals↗
PubMed1997

Estrogenic responses in estrogen receptor-alpha deficient mice reveal a distinct estrogen signaling pathway.

Estrogens are thought to regulate female reproductive functions by altering gene transcription in target organs primarily via the nuclear estrogen receptor-alpha (ER-alpha). By using ER-alpha "knock-out" (ERKO) mice, we demonstrate herein that a catecholestrogen, 4-hydroxyestradiol-17beta (4-OH-E2), and an environmental estrogen, chlordecone (kepone), up-regulate the uterine expression of an estrogen-responsive gene, lactoferrin (LF), independent of ER-alpha. A primary estrogen, estradiol-17beta (E2), did not induce this LF response. An estrogen receptor antagonist, ICI-182,780, or E2 failed to inhibit uterine LF gene expression induced by 4-OH-E2 or kepone in ERKO mice, which suggests that this estrogen signaling pathway is independent of both ER-alpha and the recently cloned ER-beta. 4-OH-E2, but not E2, also stimulated increases in uterine water imbibition and macromolecule uptake in ovariectomized ERKO mice. The results strongly imply the presence of a distinct estrogen-signaling pathway in the mouse uterus that mediates the effects of both physiological and environmental estrogens. This estrogen response pathway will have profound implications for our understanding of the physiology and pathophysiology of female sex steroid hormone actions in target organs.

Animals↗
PubMed1997

Multiple female reproductive failures in cyclooxygenase 2-deficient mice.

Cyclooxygenase (COX) is the rate-limiting enzyme in the synthesis of prostaglandins (PGs) and exists in two isoforms, COX-1 and COX-2. In spite of long-standing speculation, definitive roles of PGs in various events of early pregnancy remain elusive. We demonstrate herein that the targeted disruption of COX-2, but not COX-1, in mice produces multiple failures in female reproductive processes that include ovulation, fertilization, implantation, and decidualization. Using multiple approaches, we conclude that these defects are the direct result of target organ-specific COX-2 deficiency but are not the result of deficiency of pituitary gonadotropins or ovarian steroid hormones, or reduced responsiveness of the target organs to their respective hormones.

Animals↗
PubMed1997

Expression of betacellulin and epiregulin genes in the mouse uterus temporally by the blastocyst solely at the site of its apposition is coincident with the "window" of implantation.

In the mouse, the process of implantation is initiated by the attachment reaction between the blastocyst trophectoderm and uterine luminal epithelium that occurs at 2200-2300 h on day 4 (day 1 = vaginal plug) of pregnancy. Several members of the EGF family are considered important in embryo-uterine interactions during implantation. This investigation demonstrates that the expression of two additions to the family, betacellulin and epiregulin, are exquisitely restricted to the mouse uterine luminal epithelium and underlying stroma adjacent to the implanting blastocyst. These genes are not expressed during progesterone-maintained delayed implantation, but are rapidly switched on in the uterus surrounding the implanting blastocyst following termination of the delay by estrogen. These results provide evidence that expression of betacellulin and epiregulin in the uterus requires the presence of an active blastocyst and suggest an involvement of these growth factors in the process of implantation.

Animals↗
PubMed1997

Production and physiological actions of anandamide in the vasculature of the rat kidney.

The endogenous cannabinoid receptor agonist anandamide is present in central and peripheral tissues. As the kidney contains both the amidase that degrades anandamide and transcripts for anandamide receptors, we characterized the molecular components of the anandamide signaling system and the vascular effects of exogenous anandamide in the kidney. We show that anandamide is present in kidney homogenates, cultured renal endothelial cells (EC), and mesangial cells; these cells also contain anandamide amidase. Reverse-transcriptase PCR shows that EC contain transcripts for cannabinoid type 1 (CB1) receptors, while mesangial cells have mRNA for both CB1 and CB2 receptors. EC exhibit specific, high-affinity binding of anandamide (Kd = 27.4 nM). Anandamide (1 microM) vasodilates juxtamedullary afferent arterioles perfused in vitro; the vasodilation can be blocked by nitric oxide (NO) synthase inhibition with L-NAME (0.1 mM) or CB1 receptor antagonism with SR 141716A (1 microM), but not by indomethacin (10 microM). Anandamide (10 nM) stimulates CB1-receptor-mediated NO release from perfused renal arterial segments; a similar effect was seen in EC. Finally, anandamide (1 microM) produces a NO-mediated inhibition of KCl-stimulated [3H]norepinephrine release from sympathetic nerves on isolated renal arterial segments. Hence, an anandamide signaling system is present in the kidney, where it exerts significant vasorelaxant and neuromodulatory effects.

Amidohydrolases↗
PubMed1997

Changes in anandamide levels in mouse uterus are associated with uterine receptivity for embryo implantation.

Anandamide (N-arachidonoylethanolamine) is an endogenous ligand for both the brain-type (CB1-R) and spleen-type (CB2-R) cannabinoid receptors. This investigation demonstrates that the periimplantation mouse uterus contains the highest levels of anandamide (142-1345 pmol/micromol lipid P; 1-7 microg/g wet weight) yet discovered in a mammalian tissue. The levels fluctuate with the state of pregnancy; down-regulation of anandamide levels is associated with uterine receptivity, while up-regulation is correlated with uterine refractoriness to embryo implantation. Anandamide levels are highest during the nonreceptive phase in the pseudopregnant uterus and in the interimplantation sites, and lowest at the site of embryo implantation. The lower levels of uterine anandamide at the implantation sites may be a mechanism by which implanting embryos protect themselves from the detrimental effects of this endogenous ligand. We also observed a reduced rate of zona-hatching of blastocysts in vitro in the presence of anandamide, and inhibition of implantation by systemic administration of a synthetic cannabinoid agonist CP 55,940. These adverse effects were reversed by SR141716A, a specific CB1-R antagonist. Taken together, the results suggest that an aberrant synthesis of anandamide and/or expression of the cannabinoid receptors in the uterus/embryo may account for early pregnancy failure or female infertility.

Animals↗
PubMed1997

Expression of heparan sulfate proteoglycan (perlecan) in the mouse blastocyst is regulated during normal and delayed implantation.

Previous studies have shown that expression of the heparan sulfate proteoglycan, perlecan, on the external trophectodermal cell surfaces of mouse blastocysts increases during acquisition of attachment competence. However, it is not clear if this change in perlecan protein expression also is reflected at the level of perlecan mRNA expression. In the present investigation, the spatial and temporal patterns of perlecan mRNA expression in the mouse embryo during the periimplantation period were examined by in situ hybridization and reverse transcriptase-polymerase chain reaction. In addition, a delayed implantation model was used to determine the expression of perlecan mRNA and protein in dormant and estrogen-activated hatched blastocysts. The results demonstrate that perlecan mRNA expression is low in morulae, but increases in Day 4 blastocysts, attaining maximal expression in Day 4.5 attachment-competent blastocysts. In contrast, perlecan mRNA is detected in both the dormant and estrogen-activated delayed blastocysts; however, within 12 hr of blastocyst activation by estrogen, both perlecan protein and heparan sulfate chain expression markedly increase. Taken together, these results suggest that during normal development perlecan mRNA expression increases with the acquisition of attachment competence. Moreover, perlecan protein expression also is attenuated during delayed implantation and appears to increase in response to nidatory estrogen, perhaps via the increased translation of preexisting perlecan mRNA.

Animals↗
PubMed1997

Uterine preparation for implantation in the mouse is associated with coordinate expression of estrogen-responsive finger protein and estrogen receptor.

Estrogen-responsive finger protein (Efp) is a member of the RING finger-containing proteins. It is a putative transcription regulator that is speculated to amplify estrogen actions in the target organs. The present study examined the temporal and cell-type specific expression of Efp mRNA in the periimplantation mouse uterus (days 1-8) by Northern and in situ hybridization. Consistent with previous observation, a 6.0-kb transcript was detected in uterine RNA samples. The steady-state levels of Efp mRNA in whole uterine RNAs exhibited modest fluctuations during the periimplantation period. However, results of in situ hybridization showed cell-specific distribution of Efp mRNA in the periimplantation uterus in a temporal manner. On days 1-2 of pregnancy, distinct autoradiographic signals for Efp mRNA were detected in uterine luminal and glandular epithelia. However, on days 3 and 4, the accumulation of Efp mRNA occurred in stromal cells, in addition to its presence in the epithelium. After initiation of implantation on day 5, signal intensity was higher in stromal cells immediately surrounding the implantation chamber. However, on days 6-8, Efp mRNA was localized throughout the deciduum. To determine whether ovarian steroids influence the uterine expression of this gene, cell-specific localization of Efp mRNA was examined in the adult ovariectomized mouse uterus at 12 hr and 24 hr after an injection of estradiol-17 beta (E2) or progesterone (P4). An injection of E2 caused a modest increase in Efp mRNA levels in the luminal and glandular epithelia, while stromal cell accumulation occurred after an injection of P4. These results suggest that Efp is involved in P4/E2-mediated uterine cellular proliferation and/or differentiation. Since previous studies showed that Efp is colocalized with estrogen receptor (ER) in target cells, we also examined the cell-specific nuclear localization of ER in the periimplantation mouse uterus by immunohistochemistry. On days 1-2 of pregnancy, nuclear staining was distinct in the luminal epithelium and glandular epithelium. In contrast, nuclear staining was noted in stromal cells on days 3-4. However, glandular epithelium showed distinct staining during this period. On day 5, stromal cells surrounding the lumen at the mesometrial site were ER-positive. On days 6-8, the intensity of nuclear staining was very low, and limited to the cells adjacent to the luminal epithelium. The coordinate expression of Efp and ER in specific uterine cells during the preimplantation period (days 1-4) was consistent with the absolute requirement for estrogen in the preparation of the uterus for implantation. Since the amount of estrogen required for the preparation of the uterus is minuscule as compared to that of P4, these results suggest that the coexpression of Efp with ER is involved in amplifying the estrogen effects required for uterine cell proliferation and/or differentiation during implantation. In contrast, discoordinate expression of Efp and ER during the postimplantation period (days 5-8) suggests primary dependence of the decidualization process on P4, but not estrogen.

Animals↗