Differential effects of ovarian steroids and triphenylethylene compounds on macromolecular uptake and thymidine incorporation in the mouse uterus

In the rodent uterus, estrogen elicits a biphasic response i.e. an early phase (Phase I) and a late phase (Phase II). Estradiol-17 beta (E2) and estriol (E3), as well as triphenylethylene (TPE) compounds, CI-628 and clomiphene citrate (CC), were used to characterize Phase I and Phase II responses in uterine preparation for implantation in the mouse. While uterine macromolecular uptake (vascular permeability), a Phase I response, was studied in progesterone (P4)-primed animals, uterine [3H]thymidine incorporation (DNA synthesis), a Phase II response, was investigated with and without P4-priming. In the P4-primed uterus, all compounds, except CC, significantly increased uterine macromolecular uptake as determined by interstitial tissue accumulation of [125I]bovine serum albumin [( 125I]BSA). DNA synthesis as determined by cellular incorporation of [3H]thymidine was modulated by P4, estrogens and TPE compounds in a cell-type specific and temporal manner. As a single injection and in the absence of P4, E2 induced [3H]thymidine incorporation in the luminal and glandular epithelium at 18 and 24 h. E3 was inferior to E2 in this response. On the other hand, treatment with P4 for 1 day or 4 days induced [3H]thymidine incorporation primarily in stromal cells. However, stromal cell incorporation was potentiated when P4 treatment was combined with estrogens or TPE compounds. These results reveal the relative importance of Phase I and cell-type specific Phase II responses in uterine preparation for implantation.     

Effects of 9-ene-tetrahydrocannabinol on uterine estrogenicity in the mouse.

Several early (Phase I) and late (Phase II) estrogenic effects of 9-ene-tetrahydrocannabinol (THC) were examined in the adult mouse uterus. An injection of THC (2.5 or 10 mg/kg body wt) in ovariectomized mice neither stimulated uterine water imbibition or accumulation of [125I]bovine serum albumin (Phase I responses) at 6 h, nor antagonized these Phase I responses elicited by estradiol-17 beta (E2). With respect to Phase II responses, although single injections of THC (2.5, 5.0 and 10 mg/kg body wt) alone were ineffective in influencing uterine weight at 24 h or incorporation of [3H]thymidine at 18 h, this drug interfered with these responses elicited by E2 in a dose-dependent manner. In contrast, an injection of THC in progesterone (P4)-primed ovariectomized mice modestly enhanced (61%) uterine incorporation of [3H]thymidine. However, E2-stimulated uterine thymidine incorporation in P4-primed ovariectomized mice was antagonized by THC treatment. Effects of THC on blastocyst implantation were examined. Single or multiple injections of various doses of THC neither induced implantation in P4-primed delayed implanting mice, nor interfered with E2-induced implantation. Furthermore, daily injections of THC (10 mg/kg body wt) during the peri-implantation period had no apparent adverse effects on implantation, or on experimentally induced decidualization (deciduomata). The data suggest that THC is neither pro- nor antiestrogenic with respect to Phase I responses. However as regards Phase II responses, THC is modestly pro-estrogenic in the P4-treated uterus, but is anti-estrogenic in the presence of E2. These estrogen agonistic/antagonistic effects of THC on uterine Phase II responses do not adversely affect the process of implantation and decidualization.     

CD43 is relocated from the basal to the apical plasma membrane of rat uterine epithelial cells by progesterone

Adhesion molecules play an important part in preparing uterine epithelial cells for receptivity to the implanting embryo, and their rearrangement is crucial in allowing successful implantation. CD43 is an adhesion molecule which has previously been suggested to take part in implantation in mice. Indirect immunofluorescence microscopy localising CD43 was performed on uterine tissue during early pregnancy, and tissue obtained from ovariectomised rats administered with ovarian hormones. Western blotting was performed during early pregnancy on isolated epithelial cells and ovariectomised rats for comparison of the amount of CD43. Immunofluorescence microscopy showed CD43 was situated basally in uterine luminal epithelial cells on day 1 of pregnancy and during oestrogen administration, corresponding to a 95-kDa band of CD43 seen in western blotting. At the time of implantation, and during progesterone or progesterone plus oestrogen combined treatment, CD43 is apical in uterine luminal epithelial cells, resulting in an 85-kDa form of CD43. We suggest that a de-glycosylated form of CD43 moves from basally to apically at the time of implantation, thus facilitating blastocyst attachment to uterine epithelial cells as well as their removal.     

Hormonal control of a carbohydrate epitope involved in implantation in mice.

Ovariectomy and hormone replacement of mice were used to examine the hormonal control of expression on the uterine surface of a carbohydrate determinant (lacto-N-fucopentaose I, LNF I), involved in the initial interaction between the blastocyst and the endometrial epithelium at implantation. Pseudopregnant mice mated with sterile males were also used to elucidate the impact of embryonic signals on the expression of this antigen on the uterine surface. Two groups of fucosylated structures could be distinguished; one group was predominantly dependent on maternal oestrogen and progesterone, while the other group appeared to be less influenced by the hormonal milieu.     

Inhibitory influence of uterine secretions on mouse blastocysts decreases at the time of blastocyst activation

Uterine flushings from artificially 'pseudopregnant', pseudopregnant and pregnant mice and those with 'diapausing' embryos were tested for their effect on [3H]uridine incorporation by mouse blastocysts. An inhibitor of [3H]uridine incorporation was detected in the uterine fluid of the mice with diapausing embryos and the activity of the inhibitor was significantly reduced 6.25 h after an injection of oestrogen. This reduction of the inhibitory activity was dependent on the presence of blastocysts in utero, since a similar reduction did not occur in uterine fluids of pseudopregnant mice. The results support the suggestion that 'delayed' implantation in mice is caused by the presence of inhibitors of blastocyst metabolism and that activation, after an increase in oestradiol, is due to an embryo-dependent loss of activity of the inhibitors.     

Ovarian hormones regulate expression of the focal adhesion proteins,talin and paxillin,in rat uterine luminal but not glandular epithelial cells

During early pregnancy in the rat, focal adhesions disassemble in uterine luminal epithelial cells at the time of implantation to facilitate their removal so that the implanting blastocyst can invade into the underlying endometrial decidual cells. This study investigated the effect of ovarian hormones on the distribution and protein expression of two focal adhesion proteins, talin and paxillin, in rat uterine luminal and glandular epithelial cells under various hormone regimes. Talin and paxillin showed a major distributional change between different hormone regimes. Talin and paxillin were highly concentrated along the basal cell surface of uterine luminal epithelial cells in response to oestrogen treatment. However, this prominent staining of talin and paxillin was absent and also a corresponding reduction of paxillin expression was demonstrated in response to progesterone alone or progesterone in combination with oestrogen, which is also observed at the time of implantation. In contrast, the distribution of talin and paxillin in uterine glandular epithelial cells was localised on the basal cell surface and remained unchanged in all hormone regimes. Thus, not all focal adhesions are hormonally dependent in the rat uterus; however, the dynamics of focal adhesion in uterine luminal epithelial cells is tightly regulated by ovarian hormones. In particular, focal adhesion disassembly in uterine luminal epithelial cells, a key component to establish successful implantation, is predominantly under the influence of progesterone.     

Increase in uterine peroxidase activity in the rat uterus during oestrogen hyperaemia.

The administration of oestrogen results in increased arterial blood flow in all mammalian species studied to date, but its mechanism of action has not been elucidated. Because an interval of 30-60 min is observed between oestrogen injection and uterine hyperaemia, it has been suggested that a vasoactive intermediate is involved and recent evidence suggests that catechol oestrogens are the vasoactive oestrogen intermediates. Uterine peroxidase catalyses the conversion of oestrogens to their catechol forms and thus may play an important role in oestrogen-induced uterine hyperaemia. The present studies evaluated the time course and dose-response effects of oestrogen on uterine peroxidase activity and related these to changes in uterine blood volume, an index of uterine hyperaemia in immature rats. These data demonstrated that the minimal effective hyperaemic dose of oestradiol also increased (P less than 0.05) uterine peroxidase activity. The oestradiol-induced increase in uterine peroxidase activity preceded significant increases in uterine blood volume (1 h versus 2 h, respectively). These data are consistent with a role for peroxidase-mediated conversion of oestradiol to catechol oestradiol in facilitating uterine hyperaemia in rats.     

Emerging principles for the development of resistance to antihormonal therapy: implications for the clinical utility of fulvestrant

We seek to evaluate the clinical consequences of resistance to antihormonal therapy by studying analogous animal xenograft models. Two approaches were taken: (1) MCF-7 tumors were serially transplanted into selective estrogen receptor modulator (SERM)-treated immunocompromised mice to mimic 5 years of SERM treatment. The studies in vivo were designed to replicate the development of acquired resistance to SERMs over years of clinical exposure. (2) MCF-7 cells were cultured long-term under SERM-treated or estrogen withdrawn conditions (to mimic aromatase inhibitors), and then injected into mice to generate endocrine-resistant xenografts. These tumor models have allowed us to define Phase I and Phase II antihormonal resistance according to their responses to E₂ and fulvestrant. Phase I SERM-resistant tumors were growth stimulated in response to estradiol (E₂), but paradoxically, Phase II SERM and estrogen withdrawn-resistant tumors were growth inhibited by E₂. Fulvestrant did not support growth of Phases I and II SERM-resistant tumors, but did allow growth of Phase II estrogen withdrawn-resistant tumors. Importantly, fulvestrant plus E₂ in Phase II antihormone-resistant tumors reversed the E₂-induced inhibition and instead resulted in growth stimulation. These data have important clinical implications. Based on these and prior laboratory findings, we propose a clinical strategy for optimal third-line therapy: patients who have responded to and then failed at least two antihormonal treatments may respond favorably to short-term low-dose estrogen due to E₂-induced apoptosis, followed by treatment with fulvestrant plus an aromatase inhibitor to maintain low tumor burden and avoid a negative interaction between physiologic E₂ and fulvestrant.     

Implantation in ovariectomized mice treated with dibutyryl adenosine 3',5'-monophosphate (dibutyryl cyclic AMP).

Experiments are described that demonstrate that uterine intraluminal injection of a 1-25 mM-solution of dibutyryl cyclic AMP (dcAMP) in phosphate buffered saline (PBS) induced implantation in ovariectomized pregnant mice. Pretreatment with progesterone was essential for this effect. When PBS was injected alone, it did not induce implantation in mice treated with progesterone. Bilateral adrenalectomy had no effect on the ability of dcAMP to substitute for oestradiol, showing that the effect was not due to dcAMP-induced oestrogen synthesis in the adrenal cortex. It is suggested that the dcAMP may act at the level of the uterus, the embryo, or both.     

Phase I and phase II of short-term mechanical restitution in perfused rat left ventricles

We examined the contributions of the Ca(2+) channels of the sarcolemma and of the sarcoplasmic reticulum to electromechanical restitution. Extrasystoles (F(1)) were interpolated 40-600 ms following a steady-state beat (F(0)) in perfused rat ventricles paced at 2 or 3 Hz. Plots of F(1)/F(0) versus the extrasystolic interval consisted of phase I, which occurred before relaxation of the steady-state beat, and phase II, which occurred later. Phase I exhibited a period of enhanced left ventricular pressure development that coincided with action potential prolongation. Phase I was eliminated by -BAY K 8644 (100 nM) and FPL 64176 (150 nM), augmented by 3 microM thapsigargin plus 200 nM ryanodine and unaffected by KN-93 and KB-R7943. Phase II was accelerated by the Ca(2+) channel agonists and by isoproterenol but was eliminated by thapsigargin plus ryanodine. The results suggest that phase I of electromechanical restitution is caused by a transient L-type Ca(2+) current facilitation, whereas phase II represents the recovery of the ability of the sarcoplasmic reticulum to release Ca(2+).     

Metabolism of [4-14C]oestradiol by oestrogen-induced uterine peroxidase

1. An enzyme that catalyses the metabolism and binding of [4-(14)C]oestradiol to protein and to other high-molecular-weight substances in the presence of H(2)O(2) was shown to be absent from the uteri of immature rats and to be induced by physiological doses of oestrogen or pregnant-mare-serum gonadotrophin. 2. The pH optimum, stability to heat and other characteristics of the uterine enzyme system as well as its subcellular distribution were determined. 3. The increase in the ability of uterine preparations to convert [4-(14)C]oestradiol into water-soluble products as a result of oestrogen treatment was accompanied by an increase in peroxidase and NADH oxidase activities and was inhibited by actinomycin D and cycloheximide. 4. The results support the proposal that the increase in peroxidase activity after oestrogen treatment might be part of an adaptive response of the uterus permitting it to bind and inactivate oestrogens and thus limit the duration of their effect upon this target tissue.     

Induction of dual-specificity phosphatase 1 (DUSP1) by pulsatile gonadotropin-releasing hormone stimulation: role for gonadotropin subunit expression in mouse pituitary LbetaT2 cells

The adherens junction (AJ) is important for maintaining uterine structural integrity, composition of the luminal environment, and initiation of implantation by virtue of its properties of cell-cell recognition, adhesion, and establishment of cell polarity and permeability barriers. In this study, we investigated the uterine changes of AJ components E-cadherin, beta-catenin, and alpha-catenin at their mRNA and protein levels, together with the cellular distribution of meprinbeta, phospho-beta-catenin, and active beta-catenin proteins, in hamsters that show only ovarian progesterone-dependent uterine receptivity and implantation. By in situ hybridization and immunofluorescence, we have demonstrated that uterine epithelial cells expressed three of these AJ proteins and their mRNAs prior to and during the initial phase of implantation. Immunofluorescence study showed no change in epithelial expression patterns of uterine AJ proteins from Days 1 to 5 of pregnancy. With advancement of the implantation process, AJ components were primarily expressed in cells of the secondary decidual zone (SDZ), but not in the primary decidual zone (PDZ). In contrast, we noted strong expression of beta-catenin and alpha-catenin proteins in the PDZ, but not in the SDZ, of mice. Taken together, these results suggest that AJ proteins contribute to uterine barrier functions by cell-cell adhesion to ensure protection of the embryo. In addition, cleavage of E-cadherin by meprinbeta might contribute to weakening uterine epithelial cell-cell contact for blastocyst implantation. We also report that the nuclear localization of active beta-catenin from Day 4 onward in hamsters implies that beta-catenin/Wnt-signal transduction is activated in the uterus during implantation and decidualization.     

The relationship of the oestrogen and progestin receptors in the abnormal uterus of the adult anovulatory rat. Effects of neonatal treatment with testosterone propionate or clomiphene citrate.

The neonatal administration of testosterone propionate to Wistar rats resulted in anovulatory adults in persistent vaginal oestrus. Clomiphene citrate had a similar effect. In both groups of adults, hyperplasia of the uterine epithelium and occasional metaplasia was observed. The uterine nuclear and cytosol oestrogen and progestin receptors of these anovulatory rats were found to have affinities for their respective ligands similar to those of normal females. The nuclear oestrogen receptor comprised occupied and unoccupied components, as in normal females. The content of the nuclear oestrogen receptor was comparable with that of females in the late dioestrous or pro-oestrous phase. This content was higher in the clomiphene-treated group. Despite the relatively high nuclear oestrogen receptor content the content of progestin receptors, a putative index of the oestrogenic response, was lower in the treated rats than in normal adult females throughout the cycle. Administration of oestradiol to both treatment groups resulted in depletion of cytosol oestrogen receptor content 1 h later, which, however, was not reflected by an increase in the content of nuclear oestrogen receptors. There was no measurable increase in progesterone receptor content in treated rats after daily administration of oestrogen (5 microgram/rat) for 3 days. These changes in sex-hormone-receptor interactions involving an impairment of the normal oestrogenic response may be associated with the abnormal differentiation of the uterus in these sterile, anovulatory animals.     

Cyclic AMP and the preparation of the mouse uterus for implantation.

Experiments are described that demonstrate the ability of dibutyryl cyclic AMP (dcAMP) to induced oestrogen-like changes in the uteri of ovariectomized mice treated with progesterone. A single intraluminal injection of 6-14 micrograms dcAMP in 0-02 ml phosphate-buffered saline promoted cell division in the uterine stroma and sensitized the endometrium so that a decidual reaction could be induced by a subsequent injection of arachis oil into the uterine lumen. Since dcAMP has already been shown to induce implantation of diapausing mouse blastocysts in the absence of oestrogen, it is suggested that cyclic AMP is involved in egg implantation in rodents.     

Progesterone regulation of the mammalian ortholog of methylcitrate dehydratase (immune response gene 1) in the uterine epithelium during implantation through the protein kinase C pathway

Implantation requires coordination between development of the blastocyst and the sex steroid hormone-regulated differentiation of the uterus. Under the influence of these hormones, the uterine luminal epithelium becomes receptive to attachment of the hatched blastocyst. In this study we sought to identify genes regulated by progesterone (P4) in the uterine epithelium. This resulted in the identification of one novel P4-regulated gene that had been previously found in lipopolysaccharide-stimulated macrophages and called immune response gene-1 (Irg1) and which is the mammalian ortholog of the bacterial gene encoding methylcitrate dehydratase. In adult mice Irg1 expression was limited to the uterine luminal epithelium where it is expressed only during pregnancy with a peak coinciding with implantation. Irg1 mRNA expression is regulated synergistically by P4 and estradiol (E2) but not by E2 alone. In macrophages Irg1 is induced by lipopolysaccharide through a protein kinase C (PKC)-regulated pathway. Now we demonstrate that the PKC pathway is induced in the uterine epithelium at implantation by the synergistic action of P4 and E2 and is responsible for the hormone induction of Irg1. These results suggest that the PKC pathway plays an important role in modulating steroid hormone responsiveness in the uterine luminal epithelium during the implantation window and that Irg1 will be an important marker of this window and may play an important role in implantation.     

Cyclooxygenase-2 differentially directs uterine angiogenesis during implantation in mice

Increased vascular permeability and angiogenesis at the site of blastocyst apposition in the uterus are two hallmarks of the implantation process. The present investigation shows that although the proangiogenic vascular endothelial growth factor (VEGF) and its receptor, Flk-1, are primarily important for uterine vascular permeability and angiogenesis prior to and during the attachment phase of the implantation process, VEGF in complementation with the angiopoietins and their receptor, Tie-2, directs angiogenesis during decidualization following implantation. Mice with null mutation for the gene encoding cyclooxygenase-2 (COX-2), a rate-limiting enzyme in prostaglandin (PG) biosynthesis, show implantation and decidualization failure. Using reporter and mutant mice, we show here that COX-2-derived prostaglandins (PGs) are important for uterine vascular permeability and angiogenesis during implantation and decidualization, suggesting that one cause of the failure of these latter processes in Cox-2-/- mice is the deregulated vascular events in the absence of COX-2. The attenuation of uterine angiogenesis in these mice is primarily due to defective VEGF signaling and not due to the defective angiopoietin system.     

Human T cell leukemia/lymphoma virus type I infection of a CD4+ proliferative/cytotoxic T cell clone progresses in at least two distinct phases based on changes in function and phenotype of the infected cells

The effect of human T cell leukemia/lymphoma virus type I (HTLV-I) infection on the function and the phenotype of a human proliferating/cytotoxic T cell clone, specific for tetanus toxin, was investigated. During the period after infection, two distinct phases were observed, based on growth properties, phenotype, and functional activity of the infected cells. Phase I HTLV-I infected cells (0 to about 150 days after infection) proliferated in an IL-2-dependent way, but without the requirement for repetitive antigenic stimulation. No differences in expression of the CD2, CD3, CD4, Tp103, and CD28 Ag between these cells and the parental cells could be demonstrated, with the exception of the expression of IL-R p55 and HLA-DR Ag, which were constitutively expressed on the phase I cells. The phase I HTLV-I-infected cells, as well as the parental 827 cells reacted with a mAb specific for an epitope on the variable part of the TCR beta-chain, indicating that the TCR was not altered after HTLV-I infection. Like the parental clone, the phase I cells proliferated in response to tetanus toxin, but the tetanus toxin-specific response of the phase I cells did not require the presence of APC. Results of experiments, in which the levels of intracellular Ca2+ were measured, indicated that HTLV-I cells can acquire the capability to process Ag and present that to themselves. Phase I HTLV-I-infected T cells had lost their cytotoxic activity which was likely to be due to an effect on the lytic machinery rather than on Ag recognition by the TCR, inasmuch as it was found that phase I HTLV-I-infected T cells did no longer contain N-alpha-benzyloxy-L-lysine thiobenzylester-serine esterase activity. Furthermore, it was found that phase I HTLV-I-infected T cells had a diminished capacity to form conjugates with target cells. From a period of about 200 days after HTLV-I infection, phase II cells emerged that proliferated strongly in the absence of IL-2 and that had lost all functional activity. These cells did not express the CD3/T cell receptor complex on their surface. Phase I as well as phase II HTLV-I-infected cells were targets for CTL raised in the autologous donor.     

Alpha-tocopherol modulates genes involved in hepatic xenobiotic pathways in mice

Hepatic proteins involved in xenobiotic pathways (Phases I, II and III) are responsible for the metabolism and disposition of endogenous and exogenous compounds including dietary phytochemicals. To test the hypothesis that elevated alpha-tocopherol intakes alter gene expression of hepatic xenobiotic pathways, mice were fed diets supplemented with either 1000 IU (+E) or 35 IU (E) all-rac-alpha-tocopheryl acetate for 4 months; liver RNA was isolated, and gene expression was determined using both whole genome microarray and real-time quantitative polymerase chain reaction analyses. Hepatic alpha-tocopherol (173+/-18 vs. 21+/-1 nmol/g, mean+/-S.E.) and its metabolite (2,5,7,8-tetramethyl-2-(2'-carboxyethyl)-6-hydroxychroman, 0.232+/-0.046 vs. 0.031+/-0.019 nmol/g) concentrations were approximately eightfold higher following the +E dietary treatment. In +E relative to E mice, gene expression of Phase I enzymes, P450 oxidoreductase and cytochrome P450 3a11 increased 1.6- and 4.0-fold, respectively; two Phase II genes, sulfotransferase 2a and glutathione S-transferase mu 3, increased 10.8- and 1.9-fold respectively, and a Phase III biliary transporter, Abcb1a, doubled. Thus, consumption of high-level dietary alpha-tocopherol simultaneously coordinated Phase I, II and III gene expression. These data demonstrate that increased hepatic alpha-tocopherol modulates its own concentrations through increasing xenobiotic metabolism, a process that may alter metabolism of other foreign compounds, such as therapeutic drugs and phytochemicals, in humans.     

Mechanical alternans and restitution in failing SHHF rat left ventricles

We examined mechanical alternans and electromechanical restitution in normal and failing rat hearts. Alternans occurred at 5 Hz in failing versus 9 Hz in control hearts and was reversed by 300 nM isoproterenol, 6 mM extracellular Ca(2+), 300 nM -BAY K 8644, or 50 nM ryanodine. Restitution curves comprised phase I, which was completed before relaxation of the steady-state beat, and phase II, which occurred later. Phase I action potential area and developed pressure ratios were significantly reduced in the failing versus control hearts. Phase II was a monoexponential increase in relative developed pressure as the extrasystolic interval was increased. The plateau of phase II was significantly elevated in failing hearts. Thapsigargin (3 microM) plus ryanodine (200 nM) potentiated phase I to a significantly greater extent in control versus failing hearts and abolished phase II in both groups. The results suggest that both regulation of Ca(2+) influx across the sarcolemma and Ca(2+) release by the sarcoplasmic reticulum may contribute to altered excitation-contraction coupling in the failing spontaneously hypertensive heart failure prone rat heart.