Ovariectomized Mouse Uterus Research Articles

Adam12 plays a role during uterine decidualization in mice

In mouse, decidualization is characterized by the proliferation of stromal cells and their differentiation into specialized type of cells (decidual cells) with polyploidy, surrounding the implanting blastocyst. However, the mechanisms involved in these processes remain poorly understood. Using multiple approaches, we have examined the role of Adam12 in decidualization during early pregnancy in mice. Adam12 is spatiotemporally expressed in decidualizing stromal cells in intact pregnant females and in pseudopregnant mice undergoing artificially induced decidualization. In the ovariectomized mouse uterus, the expression of Adam12 is upregulated after progesterone treatment, which is primarily mediated by nuclear progesterone receptor. In a stromal cell culture model, the expression of Adam12 gradually rises with the progression of stromal decidualization, whereas the attenuated expression of Adam12 after siRNA knockdown significantly blocks the progression of decidualization. Our study suggests that Adam12 is involved in promoting uterine decidualization during pregnancy.

Progesterone regulation of glutathione S-transferase Mu2 expression in mouse uterine luminal epithelium during preimplantation period

To investigate the differential expression and regulation of Gstm2 in mouse uterus during early pregnancy. Experimental animal study. University research laboratory. Sexually mature female Kunming white strain mice. Delayed and activated implantation, pseudopregnancy, hormonal treatment. The expression of Gstm2 mRNA was detected by in situ hybridization and reverse-transcription polymerase chain reaction (RT-PCR). By in situ hybridization, there were a low level of Gstm2 expression in luminal epithelium on day 3 and a strong level in the luminal epithelium on day 4 during early pregnancy. The expression pattern of Gstm2 in the pseudopregnant uterus was similar to that during early pregnancy. By RT-PCR, Gstm2 was strongly detected in the uteri on days 3 and 4 of pregnancy and pseudopregnancy. Gstm2 expression was strongly detected in the luminal epithelium under delayed implantation, but not seen after delayed implantation was activated by estrogen. In the ovariectomized mouse uterus, Gstm2 expression was strongly up-regulated by progesterone via progesterone receptor. The results showed that Gstm2 was highly expressed in the uterine luminal epithelium during preimplantation period and up-regulated by progesterone.

Morphologic effects of epithelial ion channels on the mouse uterus: differences between raloxifene analog (LY117018) and estradiol treatments

Estrogen regulates the expression of epithelial Na(+) channel (ENaC) and cystic fibrosis transmembrane conductance regulator (CFTR). Our purpose was to assess the effects of raloxifene analog LY117018 on the expression of ENaC and CFTR in ovariectomized mice. Three groups of 5 female ovariectomized mice were treated with 17beta-estradiol benzoate (E2), LY117018 (LY), or vehicle, respectively, for 4-12 weeks. Effects on the messenger ribonucleic acid expression levels of ENaC and CFTR channels in the uterus were studied using real-time reverse transcriptase-polymerase chain reaction. E2 treatment induced CFTR expression, repressed ENaC expression and resulted in fluid accumulation in the uterus. In contrast, LY induced CFTR expression, did not repress ENaC expression, and caused no fluid accumulation. Estradiol and LY117018 differentially regulate the expression of CFTR and ENaC in ovariectomized mouse uterus. This finding suggests that uterine fluid accumulation can be controlled mainly by targeting the ENaC.

275 EXPRESSION OF THE ACYL-COA SYNTHETASE 4 IN THE PERI-IMPLANTATION MOUSE UTERUS

Acyl-CoA synthetase 4 (ACS4) is an arachidonate-preferring enzyme abundant in steroidogenic tissues and postulated to modulate eicosanoid production. Arachidonate that is esterified predominantly in phospholipids is a precursor of eicosanoids. After its release by the action of cytosolic phospholipase A2 (cPLA2), arachidonate can be converted to prostaglandins, thromboxanes, and leukotrenes via the cyclo-oxygenase (COX1 and COX2) and lioxygenase pathways, respectively, depending on the cell type. It is reported that eicosanoids have influence on inflammation, vascularization, and parturition. Then again, free arachidonate released from the plasma membrane is reesterified into phospholipids to prevent constant synthesis of potent eicosanoids. In the rodent, vasoactive prostaglandins are implicated in the implantation process. To further clarify ACS4 gene expression during pregnancy, we examined developmental expression in the peri-implantation uterus of the normal mouse and regulation of ACS4 by steroid hormone in ovariectomized mice treated with estradiol-17β (E2) and/or progesterone (P4). Adult female mice (BDF1, 6 weeks old) were mated with fertile males of the same strain. The morning on which a vaginal plug was found was designated Day 0.5 of pregnancy. Mice were sacrificed between 09:00 and 10:00 h on Days 2.5 to 6.5 of pregnancy. To induce and maintain delayed implantation, mice were ovariectomized on the morning (09:00 to 10:00 h) of Day 3.5 of pregnancy and received a daily injection of P4 from Days 4.5 to 6.5. To terminate the delay and induce implantation, the P4-primed delayed mice were given an injection of E2 on Day 6.5. COX1, COX2, cPLA2, and ACS4 mRNA were analyzed by Northern blot analysis and real-time PCR. The expression of COX1 mRNA did not show much variation on the various days in the normal pregnancy and ovariectomized mice. The levels of COX2 mRNA were high on Day 4.5 of pregnancy and dramatically decreased on Day 5.5 of pregnancy. In the ovariectomized mouse uterus, COX2 mRNA levels declined rapidly at Day 4.5 of pregnancy. The expression of cPLA2 mRNA was not altered in the normal pregnancy. However, the expression was increased in the ovariectomized mouse as compared with that in the normal pregnancy mice. An ACS4 transcript was already present in the uterus at low level on Day 3.5 until the initiation of attachment reaction after which the expression was up-regulated. In the ovariectomized mouse uterus, ACS4 mRNA was increased at Day 4.5 of pregnancy as compared with the normal pregnancy mice. To determine whether ovarian steroids influenced the induction of ACS4 gene, ovariectomized mice were treated with (E2) and/or (P4). Treatment with P4 maintained the expression of ACS4 mRNA in the ovariectomized mouse uterus. In contrast, combined treatment with P4 and E2 modestly decreased the levels of ACS4 mRNA as compared with Day 7.5 of normal pregnancy. Overall, these results suggest that the ACS4 gene is regulated in the implantation process and influenced by ovarian steroids.

Expressions and regulation of endothelial and inducible nitric oxide synthases in mouse uterus during the estrous cycle and early pregnancy

Nitric oxide (NO) has been implicated in many cellular processes. We examined the temporal and spatial expressions of endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS) in mouse uteri during the estrous cycle and early pregnancy, as well as the regulation of eNOS and iNOS by estradiol (E2) and progesterone (P4) in ovariectomized mouse uteri using in situ hybridization and immunohistochemistry. Our results showed that positive eNOS and iNOS signals were localized in the uterine luminal epithelium and glandular epithelium during the estrous cycle. In ovariectomized mice, both E2 and P4 regulated the expression of eNOS and iNOS. During early pregnancy, eNOS and iNOS were detected not only in epithelium, but also in the primary decidual zone surrounding implanting embryos on day 6 of pregnancy, and in the whole decidualized stroma on day 7 of pregnancy. In conclusion, the results demonstrated that two NOS isoforms were localized in mouse uteri in specific temporal and spatial patterns during the estrous cycle and early pregnancy, and ovarian hormones can regulate their expression. Furthermore, the data suggest that the expression of NOS during the peri-implantation period might lead to enhance NO production, which could promote embryo implantation.

Analysis of estrogen-regulated genes in mouse uterus using cDNA microarray and laser capture microdissection.

The steroid hormone, estrogen, plays an important role in various physiological events which are mediated via its nuclear estrogen receptors, ERalpha and ERbeta. However, the molecular mechanisms that are regulated by estrogen in the uterus remain largely unknown. To identify genes that are regulated by estrogen, the ovariectomized mouse uterus was exposed to 17beta-estradiol (E2) for 6 h and 12 h, and the data were analyzed by cDNA microarray. The present study confirms previous findings and identifies several genes with expressions not previously known to be influenced by estrogen. These genes include small proline-rich protein 2A, receptor-activity-modifying protein 3, inhibitor of DNA binding-1, eukaryotic translation initiation factor 2, cystatin B, decorin, secreted frizzled-related protein 2, integral membrane protein 2B and chemokine ligand 12. The expression patterns of several selected genes identified by the microarray analysis were confirmed by RT-PCR. In addition, laser capture microdissection (LCM) was conducted to determine the expression of selected genes in specific uterine cell types. Analysis of early and late responsive genes using LCM and cDNA microarray not only suggests direct and indirect effects of E2 on uterine physiological events, but also demonstrates differential regulation of E2 in specific uterine cell types. These results provide a basic background on global gene alterations or genetic pathways in the uterus during the estrous cycle and the implantation period.

Expression of early response genes, Fra1 and Fra2, by 17β-Estradiol in the ovariectomized mouse uterus

Objective: The early response genes induced by 17β-estradiol(E2) include the important regulatory genes, such as transcription factors. The aim of this study was to investigate the early response gene induced by E2 in the ovariectomized(Ovx) mouse uterus. Design: Experimental laboratory study using a mouse model. Materials and Methods: The Ovx mice were treated with a single s.c injection of E2(300ng/0.1ml/mouse) dissolved in sesame oil, while the control mice received oil only. They were given the pure antiestrogen ICI 182,780(500ng/mouse) 30min before the injection of E2. The Ovx mice were killed at 2, 4, 6 and 12 hr after the injection, and their uteri were collected for RNA extraction. The expression of the 15 transcription factors was examined by reverse transcription-polymerase chain reaction(RT-PCR).The localization of these protein was investigated by immunohistochemistry. Results: The expression patterns of genes were divided into four groups: Group I, early response genes include Fra1, Fra2, CREB2 and GATA5; Group II, response maintained genes include CREB1, ATF1, GLI3 and E2F3; Group III, late response genes include E2F1; Group IV, down regulated genes include GLI1, GLI2, E2F5, GATA2, GATA3 and GATA6. Among these early resposne genes, the Fra1 and Fra2 were selected for protein localization. The levels of Fra1 mRNA peaked 2.3-fold at 4 hr, the levels of Fra2 mRNA peaked 3.2-fold at 2 hr compared with the control values, respectively and these mRNA levels returned to basal levels at 12 hr. In addition, The ICI 182,780 clearly blocked the effect of E2. The expression of Fra1 and Fra2 protein was stronger in the myometrium than in other regions. Conclusion: The results of this study suggest that expression of the Fra1 and Fra2 mRNA is up-regulated by E2 and activated via an estrogen nuclear receptor in the mouse uterus and these protein may play an important role in mediating the contraction and relaxation of the uterine smooth muscle, but the functional significance of these protein in the myometrium is not clear.

Estrogen regulation of aquaporins in the mouse uterus: potential roles in uterine water movement.

Estrogen stimulates water imbibition in the uterine endometrium. This water then crosses the epithelial cells into the lumen, leading to a decrease in viscosity of uterine luminal fluid. To gain insight into the mechanisms underlying this estrogen-stimulated water transport, we have explored the expression profile and functionality of water channels termed aquaporins (AQPs) in the ovariectomized mouse uterus treated with ovarian steroid hormones. Using immunocytochemical analysis and immunoprecipitation techniques, we have found that AQP-1, -3, and -8 were constitutively expressed. AQP-1 expression was restricted to the myometrium and may be slightly regulated by ovarian steroid hormones. AQP-3 was expressed at low levels in the epithelial cells and myometrium, whereas AQP-8 was found in both the stromal cells and myometrium. AQP-2 was absent in vehicle controls but strongly up-regulated by estrogen in the epithelial cells and myometrium of the uterus. This localization implicates all four isotypes in movement of water during uterine imbibition and, based on their localization to the luminal epithelial cells, AQP-2 and -3 in facilitating water movement into the lumen of the uterus. The analysis of the plasma membrane permeability of luminal epithelial cells by two separate cell swelling assays confirmed a highly increased water permeability of these cells in response to estrogen treatment. This finding suggests that estrogen decreases the luminal fluid viscosity, in part, by enhancing the water permeability of the epithelial layer, most likely by increasing the expression of AQP-2 and/or the availability of AQP-3. Together these results provide novel information concerning the mechanism by which estrogen controls water imbibition and luminal fluid viscosity in the mouse uterus.

Modulation of oestrogen receptor signalling by association with the activated dioxin receptor.

Environmental contaminants affect a wide variety of biological events in many species. Dioxins are typical environmental contaminants that exert adverse oestrogen-related effects. Although their anti-oestrogenic actions are well described, dioxins can also induce endometriosis and oestrogen-dependent tumours, implying possible oestrogenic effects. However, the molecular mechanism underlying oestrogen-related actions of dioxins remains largely unknown. A heterodimer of the dioxin receptor (AhR) and Arnt, which are basic helix-loop-helix/PAS-family transcription factors, mediates most of the toxic effects of dioxins. Here we show that the agonist-activated AhR/Arnt heterodimer directly associates with oestrogen receptors ER-alpha and ER-beta. This association results in the recruitment of unliganded ER and the co-activator p300 to oestrogen-responsive gene promoters, leading to activation of transcription and oestrogenic effects. The function of liganded ER is attenuated. Oestrogenic actions of AhR agonists were detected in wild-type ovariectomized mouse uteri, but were absent in AhR-/- or ER-alpha-/- ovariectomized mice. Our findings suggest a novel mechanism by which ER-mediated oestrogen signalling is modulated by a co-regulatory-like function of activated AhR/Arnt, giving rise to adverse oestrogen-related actions of dioxin-type environmental contaminants.

Estrogenic and progestational activity of 7 α-methyl-19-nortestosterone, a synthetic androgen

Synthetic androgens exhibit estrogenic/antiestrogenic and progestational activities in addition to their androgenic effects. To investigate the pharmacological action of the synthetic androgen, 7 α-methyl-19-nortestosterone (MENT), we examined its action in female rodents. The criteria employed for estrogenic/antiestrogenic effects were, uterine weight increase, vaginal cornification, induction of progesterone receptors (PR) synthesis and stimulation of peroxidase activity in the uteri of ovariectomized rats and mice. MENT increased uterine weight in a dose dependent manner, but did not cause vaginal cornification or stimulate PR synthesis in the uterus. The uterotropic activity of MENT was 200-fold lower than that of estradiol. Estrogen receptor (ER) bound [ 3H]-E 2 was displaced by E 2 and MENT with ED 50 values of 70 pg and 250 ng, respectively, a 3,500 fold difference in their binding affinity. The low binding of MENT to ER, in contrast to its relatively high uterotropic action, suggested that receptors other than ER may be involved in its action on the uterus. The progestational activity of MENT in immature rabbits using the McPhail index assay was comparable to that of progesterone. Binding affinities of MENT and progesterone to PR were also comparable. However, the action of MENT on the uterus does not seem to be a progestational effect since mifepristone, an antiprogestin, had no effect on MENT-induced uterine growth. Specific androgen receptors (AR) in uterine cytosol were demonstrated. The involvement of AR in MENT action was confirmed by using an antiandrogen (flutamide) and an antiestrogen (ICI-182) in ovariectomized mice. Although MENT did not block the uterotropic effect of E 2, it inhibited the E 2-induced cornification of vaginal epithelium, induction of uterine PR synthesis and increase in uterine peroxidase activity in ovariectomized rats. The antiestrogenic effect of MENT was also blocked by flutamide. These results suggest that the uterotropic and antiestrogenic effects of androgens are mediated via AR. It is concluded that the increase in uterine weight caused by MENT is attributable to its anabolic effects.

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.

Differential regulation of heparin-binding epidermal growth factor-like growth factor in the adult ovariectomized mouse uterus by progesterone and estrogen

Expression of heparin-binding epidermal growth factor-like growth factor (HB-EGF) was studied in the adult ovariectomized mouse uterus in response to progesterone (P4) and/or 17 beta-estradiol (E2) using Northern blotting, in situ hybridization, and immunohistochemistry. A 2.5-kilobase transcript of HB-EGF messenger RNA (mRNA) was detected in total uterine RNA samples. Although low levels of this mRNA were detected in uterine samples of oil-treated ovariectomized mice (control), an injection of E2 promptly up-regulated the levels. The mRNA levels peaked at 2 h and returned to basal levels after 12 h. Injection of P4 alone did not influence the basal levels; however, coinjection of E2 with P4 caused a rapid, but transient, up-regulation of the mRNA. The levels peaked between 2-4 h and declined 6 h after the hormone injections. Coinjection of E2 with P4 after 1 day of P4 priming also resulted in peak levels of HB-EGF mRNA at 2 h; however, the levels were not sustained thereafter. Because P4 and E2 differentially regulate heterogeneous uterine cell types, in situ hybridization was performed to determine cell-specific expression of HB-EGF mRNA in the ovariectomized uterus before and after steroid treatments. In the oil-treated uterine sections, very low levels of autoradiographic signals were observed in the luminal epithelium. In contrast, an injection of E2 resulted in a marked accumulation of HB-EGF mRNA primarily in uterine epithelial cells within 2 h. Although specific hybridization signals could not be detected in any uterine cell types after P4 treatment, combined treatment with P4 and E2 resulted in an accumulation of HB-EGF mRNA in stromal cells. To determine whether uterine HB-EGF mRNA was translated, cellular distribution of HB-EGF protein was investigated by immunohistochemistry. In oil-treated uterine sections, an overall weak immunostaining was noted, whereas no staining could be detected in uterine sections after P4 treatment. In contrast, positive immunostaining was noted in epithelial cells after E2 treatment. Coinjection of P4 with E2 caused immunostaining in the stroma. These results are consistent with those of in situ hybridization. The present investigation establishes that in the adult ovariectomized mouse uterus, E2 regulates HB-EGF expression in the epithelium, whereas expression of HB-EGF in the stroma is regulated by P4 and E2.

Differential regulation of heparin-binding epidermal growth factor-like growth factor in the adult ovariectomized mouse uterus by progesterone and estrogen.

Expression of heparin-binding epidermal growth factor-like growth factor (HB-EGF) was studied in the adult ovariectomized mouse uterus in response to progesterone (P4) and/or 17 beta-estradiol (E2) using Northern blotting, in situ hybridization, and immunohistochemistry. A 2.5-kilobase transcript of HB-EGF messenger RNA (mRNA) was detected in total uterine RNA samples. Although low levels of this mRNA were detected in uterine samples of oil-treated ovariectomized mice (control), an injection of E2 promptly up-regulated the levels. The mRNA levels peaked at 2 h and returned to basal levels after 12 h. Injection of P4 alone did not influence the basal levels; however, coinjection of E2 with P4 caused a rapid, but transient, up-regulation of the mRNA. The levels peaked between 2-4 h and declined 6 h after the hormone injections. Coinjection of E2 with P4 after 1 day of P4 priming also resulted in peak levels of HB-EGF mRNA at 2 h; however, the levels were not sustained thereafter. Because P4 and E2 differentially regulate heterogeneous uterine cell types, in situ hybridization was performed to determine cell-specific expression of HB-EGF mRNA in the ovariectomized uterus before and after steroid treatments. In the oil-treated uterine sections, very low levels of autoradiographic signals were observed in the luminal epithelium. In contrast, an injection of E2 resulted in a marked accumulation of HB-EGF mRNA primarily in uterine epithelial cells within 2 h. Although specific hybridization signals could not be detected in any uterine cell types after P4 treatment, combined treatment with P4 and E2 resulted in an accumulation of HB-EGF mRNA in stromal cells. To determine whether uterine HB-EGF mRNA was translated, cellular distribution of HB-EGF protein was investigated by immunohistochemistry. In oil-treated uterine sections, an overall weak immunostaining was noted, whereas no staining could be detected in uterine sections after P4 treatment. In contrast, positive immunostaining was noted in epithelial cells after E2 treatment. Coinjection of P4 with E2 caused immunostaining in the stroma. These results are consistent with those of in situ hybridization. The present investigation establishes that in the adult ovariectomized mouse uterus, E2 regulates HB-EGF expression in the epithelium, whereas expression of HB-EGF in the stroma is regulated by P4 and E2.

Effects of 9-ene-tetrahydrocannabinol on expression of β-type transforming growth factors, insulin-like growth factor-I and c- myc genes in the mouse uterus

Effects of cannabinoid on expression of β-type transforming growth factors (TGF-β1, -β2 and -β3), insulin-like growth factor-I (IGF-I) and c- myc genes in the uteri of adult ovariectomized mice were examined using Northern blot hybridization. Mice were exposed to 9-ene-tetrahydrocannabinol (THC) alone or in combination with an injection of estradiol-17β (E 2) and/or progesterone (P 4), and uteri were analyzed at various times thereafter. TGF-β isoform messenger RNAs (mRNAs) persisted in ovariectomized uteri and their levels were not altered after THC treatment, whereas an injection of E 2 caused a modest increase in TGF-β1 and -β3 mRNA levels at 24 h. Imposition of THC treatment advanced the stimulatory effects of E 2 by changing the timing for the peak of TGF-β3 mRNA levels to 12 h. In comparison, E 2 treatment substantially elevated the levels of TGF-β2 mRNA at 6 h, and THC potentiated this E 2 response without affecting the timing for the response. Imposition of P 4 treatment did not antagonize any of these responses. P 4 treatment alone or with THC had insignificant effects on mRNA levels for these TGF-β isoforms. Uterine levels of IGF-I and c- myc mRNAs were low in ovariectomized mice and THC did not alter these mRNA levels. In contrast, E 2 treatment induced a rapid, but transient, increase in IGF-I and c- myc mRNAs, and THC antagonized the rapid c- myc mRNA response and altered the timing of the IGF-I mRNA response. P 4 treatment alone also caused the transient induction of these mRNAs, but THC failed to antagonize these effects. An injection of P 4 plus E 2 resulted in further modest increases in IGF-I and c- myc mRNA levels as compared to E 2 or P 4 treatment alone. However, THC did not antagonize these transient stimulatory effects of the combined ovarian steroids. The data suggest that THC should not be classified as estrogenic or antiestrogenic. However, this compound can modulate (potentiate, antagonize and/or alter timing) the effects of ovarian steroids on uterine gene expression.

Expression of transforming growth factor-beta isoforms (beta 2 and beta 3) in the mouse uterus: analysis of the periimplantation period and effects of ovarian steroids.

Expression of beta-type transforming growth factor genes (TGF beta 2 and TGF beta 3) in the mouse uterus during the periimplantation period and in response to an acute exposure to 17 beta-estradiol (E2) and progesterone (P4) was studied using Northern blot hybridization and/or immunocytochemistry. Polyclonal antipeptide antibodies specific for TGF beta 2 or TGF beta 3 were employed for immunocytochemistry. In the preimplantation uterus [days (D) 1-4 of pregnancy; day 1 = vaginal plug], immunostaining for TGF beta 2 was observed in luminal and glandular epithelia as well as in myometrium and vascular smooth muscle. In the postimplantation period (D5-D8), TGF beta 2 immunostaining was also detected in decidual cells. In contrast, TGF beta 3 immunostaining was restricted to the myometrium and vascular smooth muscle throughout the periimplantation period (D1-D8). Antisense TGF beta 2 and TGF beta 3 RNA probes were employed for Northern blotting. Northern blot hybridization revealed four TGF beta 2 transcripts (approximately 6.0, 5.0, 4.0, and 3.5 kilobases) in total uterine poly(A)+ RNA on D1-D6 and in poly(A)+ RNA from the deciduum and myometrium collected on D7 and D8 of pregnancy. These TGF beta 2 transcripts were also detected in isolated samples of deciduomata or myometrium obtained from D8 pseudopregnant mice in which the decidual cell reaction was induced experimentally on D4. The levels of these transcripts remained relatively constant during the periimplantation period. Northern blot analysis detected a 3.8-kilobase TGF beta 3 transcript in total uterine poly(A)+ RNA on D1-D6. This transcript was detected in myometrial RNA samples on D7 and D8 of pregnancy or D8 of pseudopregnancy, but was not detected in RNA from the deciduum on D7 and D8 or in that from deciduomata on D8. The effects of ovarian steroids on TGF beta 2 and TGF beta 3 mRNAs were examined in uteri of adult ovariectomized mice. Uterine TGF beta 2 or TGF beta 3 mRNA persisted in ovariectomized mice. However, an injection of E2 induced a rapid (6 h), but transient, induction (approximately 3- to 4-fold) of TGF beta 2 mRNA. An injection of P4 had no effect on TGF beta 2 mRNA levels, and coinjection of P4 with E2 did not antagonize the E2-stimulated transient accumulation of TGF beta 2 mRNA. In comparison, neither an injection of E2 nor one of P4 exerted significant effects on TGF beta 3 mRNA levels.(ABSTRACT TRUNCATED AT 400 WORDS)

Expression of transforming growth factor-beta isoforms (beta 2 and beta 3) in the mouse uterus: analysis of the periimplantation period and effects of ovarian steroids

Expression of beta-type transforming growth factor genes (TGF beta 2 and TGF beta 3) in the mouse uterus during the periimplantation period and in response to an acute exposure to 17 beta-estradiol (E2) and progesterone (P4) was studied using Northern blot hybridization and/or immunocytochemistry. Polyclonal antipeptide antibodies specific for TGF beta 2 or TGF beta 3 were employed for immunocytochemistry. In the preimplantation uterus [days (D) 1-4 of pregnancy; day 1 = vaginal plug], immunostaining for TGF beta 2 was observed in luminal and glandular epithelia as well as in myometrium and vascular smooth muscle. In the postimplantation period (D5-D8), TGF beta 2 immunostaining was also detected in decidual cells. In contrast, TGF beta 3 immunostaining was restricted to the myometrium and vascular smooth muscle throughout the periimplantation period (D1-D8). Antisense TGF beta 2 and TGF beta 3 RNA probes were employed for Northern blotting. Northern blot hybridization revealed four TGF beta 2 transcripts (approximately 6.0, 5.0, 4.0, and 3.5 kilobases) in total uterine poly(A)+ RNA on D1-D6 and in poly(A)+ RNA from the deciduum and myometrium collected on D7 and D8 of pregnancy. These TGF beta 2 transcripts were also detected in isolated samples of deciduomata or myometrium obtained from D8 pseudopregnant mice in which the decidual cell reaction was induced experimentally on D4. The levels of these transcripts remained relatively constant during the periimplantation period. Northern blot analysis detected a 3.8-kilobase TGF beta 3 transcript in total uterine poly(A)+ RNA on D1-D6. This transcript was detected in myometrial RNA samples on D7 and D8 of pregnancy or D8 of pseudopregnancy, but was not detected in RNA from the deciduum on D7 and D8 or in that from deciduomata on D8. The effects of ovarian steroids on TGF beta 2 and TGF beta 3 mRNAs were examined in uteri of adult ovariectomized mice. Uterine TGF beta 2 or TGF beta 3 mRNA persisted in ovariectomized mice. However, an injection of E2 induced a rapid (6 h), but transient, induction (approximately 3- to 4-fold) of TGF beta 2 mRNA. An injection of P4 had no effect on TGF beta 2 mRNA levels, and coinjection of P4 with E2 did not antagonize the E2-stimulated transient accumulation of TGF beta 2 mRNA. In comparison, neither an injection of E2 nor one of P4 exerted significant effects on TGF beta 3 mRNA levels.(ABSTRACT TRUNCATED AT 400 WORDS)

Expression of peptidylarginine deiminase in the uterine epithelial cells of mouse is dependent on estrogen.

The effects of the steroid hormones estrogen and progesterone on peptidylarginine deiminase protein-L-arginine iminohydrolase, EC 3.5.3.15) levels in adult ovariectomized mouse uterus were studied. The amount of the enzyme in the uterus was considerably diminished by ovariectomy. When the mice were injected with a variety of estrogenic compounds, 17 beta-estradiol-3-benzoate, which was the most potent stimulator of uterine cell proliferation among the estrogens tested, dramatically elevated the enzyme formation of the uterus in a dose- and time-dependent fashion. Results of immunohistochemistry with the antiserum against mouse peptidylarginine deiminase demonstrated that the induction of the enzyme by the estradiol exclusively occurred at the luminal and glandular epithelia, corresponding with the previous findings in the normal estrous cycle. Furthermore, administration of the estradiol significantly increased the content of mRNA coding for peptidylarginine deiminase in uterus, indicating the evidence of regulation in pretranslation. On the other hand, progesterone alone did not restore the enzyme level of the ovariectomized mouse, but moderated the action of estrogen when given in concert with estrogen. Thus, the expression of peptidylarginine deiminase in luminal and glandular epithelia of mouse uterus is controlled by the amount of the steroid hormones estrogen and progesterone.

Cell type-specific localization of c-myc protein in the mouse uterus: modulation by steroid hormones and analysis of the periimplantation period.

Estrogen has been shown to induce a rapid transient increase in c-myc mRNA in the rat uterus. However, no studies of the cell specificity of c-myc expression in the uterus have been reported, and nothing is known about the expression of c-myc in response to other steroids or during normal uterine preparation for implantation. To this end, the cell type-specific localization of c-myc protein was determined in the ovariectomized mouse uterus after progesterone (P4) and/or 17 beta-estradiol (E2) injection as well as during the periimplantation period. After E2 injection, a rapid accumulation of c-myc protein was detected exclusively in the uterine luminal and glandular epithelial nuclei in the ovariectomized mouse. Essentially all of these cells contained immunoreactive c-myc by 12 h postinjection. In contrast, after P4 injection, rapid accumulation of c-myc was noted exclusively in some of the stromal cell nuclei. Pretreatment of the ovariectomized mouse for 4 days with P4 (P4 priming) followed by E2 injection resulted in an increase in the number of c-myc-positive stromal cells, but few, if any, c-myc-positive cells were detected in the luminal and glandular epithelia. These uterine cell type-specific localizations of c-myc protein, induced by E2 or P4 injection, were followed within 18-24 h by DNA synthesis ([ 3H]thymidine incorporation) restricted to the epithelia or stroma, respectively. c-Myc was detected in the nuclei of luminal and glandular epithelia during proestrus and on days 1 and 2 of pregnancy, a period when the uterus is under the influence of estrogen. c-Myc-positive cells were detected in the stroma on day 3, and by day 4 a large number of stromal cell nuclei were c-myc positive. These changes are coincident with increasing P4 levels during early pregnancy. At the implantation chamber on day 5, many cells in the primary decidual zone as well as some of the deeper stromal cells were c-myc protein positive, whereas on day 6, c-myc protein was detected only in the secondary decidual zone. During this period of uterine preparation for embryo implantation and subsequent decidualization, there was a positive correlation between c-myc protein localized in specific populations of uterine cells and subsequent DNA synthesis in those cell types. Thus, both E2 and P4 induce cell type-specific accumulation of c-myc protein in the uterus of the ovariectomized mouse, with E2 induction of c-myc being restricted to epithelia, and P4 induction restricted to stroma.(ABSTRACT TRUNCATED AT 400 WORDS)

Nuclear estrogen receptor molecular heterogeneity in the mouse uterus.

Holomeric estrogen receptor (ER) prepared from ovariectomized mouse uteri displays heterogeneous electrophoretic mobility when analyzed by NaDodSO4/PAGE. ER derived from nuclei (ERn) appears as a closely spaced doublet having apparent molecular masses of 66.4 and 65 kDa, while ER from the cytosolic compartment (ERc) has a single band of 65 kDa. Both partially purified ERc and the 8S form of unactivated ERc show only the 65-kDa band. The appearance of the ERn doublet is hormonally inducible, and the relative proportions of the two doublet bands are influenced by the type of hormone treatment, with weakly estrogenic compounds yielding the lower band as predominant while potent estrogens increase the proportion of the upper band. Steroid binding of the ERn doublet was determined by [3H]tamoxifen aziridine affinity labeling of both the 66.4- and the 65-kDa peptides; binding to the 65-kDa peptide was predominant. The ERn doublet displays a time dependency after estrogen administration with maximal amounts occurring in a bimodal fashion at 1 and 8 hr.

Experimental studies on enhanced effects of chemoendocrine therapy for breast cancer

Synthetic androgens exhibit estrogenic/antiestrogenic and progestational activities in addition to their androgenic effects. To investigate the pharmacological action of the synthetic androgen, 7α-methyl-19-nortestosterone (MENT), we examined its action in female rodents. The criteria employed for estrogenic/antiestrogenic effects were, uterine weight increase, vaginal cornification, induction of progesterone receptors (PR) synthesis and stimulation of peroxidase activity in the uteri of ovariectomized rats and mice. MENT increased uterine weight in a dose dependent manner, but did not cause vaginal cornification or stimulate PR synthesis in the uterus. The uterotropic activity of MENT was 200-fold lower than that of estradiol. Estrogen receptor (ER) bound [3H]-E2 was displaced by E2 and MENT with ED50 values of 70 pg and 250 ng, respectively, a 3,500 fold difference in their binding affinity. The low binding of MENT to ER, in contrast to its relatively high uterotropic action, suggested that receptors other than ER may be involved in its action on the uterus. The progestational activity of MENT in immature rabbits using the McPhail index assay was comparable to that of progesterone. Binding affinities of MENT and progesterone to PR were also comparable. However, the action of MENT on the uterus does not seem to be a progestational effect since mifepristone, an antiprogestin, had no effect on MENT-induced uterine growth. Specific androgen receptors (AR) in uterine cytosol were demonstrated. The involvement of AR in MENT action was confirmed by using an antiandrogen (flutamide) and an antiestrogen (ICI-182) in ovariectomized mice. Although MENT did not block the uterotropic effect of E2, it inhibited the E2-induced cornification of vaginal epithelium, induction of uterine PR synthesis and increase in uterine peroxidase activity in ovariectomized rats. The antiestrogenic effect of MENT was also blocked by flutamide. These results suggest that the uterotropic and antiestrogenic effects of androgens are mediated via AR. It is concluded that the increase in uterine weight caused by MENT is attributable to its anabolic effects.