Expression of nidogens in rat uterus and embryo during decidualization and implantation

The purpose of this study was to demonstrate the expression of nidogen-1 and nidogen-2 and their possible role in decidualization and implantation events during early pregnancy in rats. The tissue samples were examined from pregnant animals between gestational days 1-8 using immunocytochemistry. The uterine luminal epithelium, the glandular epithelium, and the myometrial smooth muscle cells stained strongly from gestational days 1-8 with both nidogen antibodies. At day 4 the decidual reaction areas began to appear in the stromal matrix and immunostaining of both nidogens revealed that the basement membrane of the surface epithelium was discontinuous. The differentiation of stromal cells into decidual cells was seen at gestational day 5 and both nidogens were weakly expressed in the decidualizing cells. At day 6, nidogen-2 immunoreactivity was higher in the primary decidual cells close to the embryo than nidogen-1, and during development of the decidual tissue both nidogens appeared in the endometrial stromal cells. At day 7, while expression of both nidogens declined in the primary decidual cells, their expression was markedly observed in the secondary decidual cells close to the myometrium. At day 8, expression of both nidogens was also observed to increase in the primary decidual cells. While nidogen-2 expression was seen in the parietal endoderm and primary ectoderm of the rat embryos at this developmental stage, nidogen-1 expression was only detected in the parietal endoderm. These results indicate that nidogen-1 and nidogen-2 could play important roles during embryogenesis, decidualization, and implantation in the endometrium of rat uterus.     

Developmental expression of adenosine deaminase during decidualization in the rat uterus

Adenosine deaminase (ADA) is expressed in high concentrations at the fetal-maternal interface during postimplantation stages of gestation in the mouse. The experiments reported here were designed to identify the specific uterine cells that express ADA subsequent to implantation in the rat and to determine if embryonic cells contribute to ADA expression. The results of biochemical analysis demonstrate that ADA-specific activity increases to very high levels in implantation sites, beginning approximately 72 h after blastocyst attachment. Immunocytochemical analysis localized this ADA expression to the decidualized stromal cells in the antimesometrial region of the pregnant uterus. In experimentally induced deciduoma, these cells were capable of synthesizing high levels of both ADA and mRNA for ADA in the absence of embryos. The enzyme first appeared in decidual cell cytoplasm, approximately 72 h after induction of decidualization, and later was localized in the decidual cell nuclei. Since the expression of ADA and its mRNA in decidual cells follows the appearance of desmin, a protein marker for decidualization, by at least 48 h, ADA appears to be involved in the functioning of mature decidual cells rather than in stromal cell differentiation. The expression of ADA, but not desmin, was restricted to the antimesometrial decidual cells and decreased when these cells regressed. At mid-gestation ADA activity increased and was localized principally in the fetal placenta. The results presented here demonstrate that ADA is localized to the antimesometrial decidual cell and that its expression is consequent to differentiation of the uterine stromal cell and independent of any embryonic stimulus.     

Glucose transporter expression in rat embryo and uterus during decidualization, implantation, and early postimplantation.

Efficient transfer of glucose from the mother to the embryonic compartment is crucial to sustain the survival and normal development of the embryo in utero, because the embryo's production of this primary substrate for oxidative metabolism is minimal. In the present study, the temporal sequence of expression of the sodium-independent facilitative glucose transporter isoforms GLUTs 1, 3, 4, and 5 was investigated in the developing rat uteroembryonic unit between conception and Gestational Day 8 using immunohistochemistry. The GLUTs 1, 3, and 4 were expressed in the embryonic tissues after the start of implantation, being colocalized in the parietal endoderm, visceral endoderm, primary ectoderm, extraembryonic ectoderm, and the ectoplacental cone. In the uterus, a faint GLUT1 labeling emerged, but not until Gestational Day 3, in the luminal epithelium, endometrial stroma, and decidual cells. The intensity of GLUT1 staining increased in the latter population with progressing decidualization. Endometrial glands and myometrial smooth muscle cells stained neither for GLUT1 nor for GLUT3 until postimplantation. During all developmental stages examined, GLUT4 was visualized throughout the pregnant rat uterus, as was GLUT3 (with the above-mentioned exceptions). The density of GLUT5 was generally less than the sensitivity of the immunohistochemical detection method in all tissues investigated. In conclusion, the data point to a significant expression of the high-affinity glucose transporters GLUTs 1, 3, and 4 in the rat uteroembryonic unit, providing supportive evidence for an important role of facilitative glucose diffusion during peri-implantation development.     

Expression of matrix metalloproteinases 2 and 9 in the mouse uterus during implantation and oil-induced decidualization

During implantation, matrix metalloproteinases are believed to play roles in the tissue remodelling that accompanies decidualization in the endometrium and in embryo invasion. The objective of this study was to characterize further the expression of matrix metalloproteinases 2 and 9 in the mouse uterus during early pregnancy and oil-induced decidualization. mRNA encoding matrix metalloproteinase 2 was detected in pregnant uteri and uteri undergoing oil-induced decidualization by northern blot analyses. The steady-state concentrations of mRNA encoding matrix metalloproteinase 2 did not change significantly in implantation compared with inter-implantation areas on days 5-8 of pregnancy but were significantly lower in stimulated compared with non-stimulated uterine horns during artificially induced decidualization. mRNA encoding matrix metalloproteinase 9 was also detected in uteri undergoing oil-induced decidualization but not in pregnant uteri. Its concentration was significantly greater in uterine horns undergoing oil-induced decidualization compared with control horns. Immunoreactive matrix metalloproteinases 2 and 9 were detected in the uterus during early pregnancy and oil-induced decidualization by immunohistochemistry, localized to the endometrial stroma, but the staining progressively became weaker and was absent in areas that had undergone decidualization. By day 8 of pregnancy and 72 h after the induction of decidualization, matrix metalloproteinase 2 and 9 proteins remained mainly in the region of non-decidualized stromal cells adjacent to the myometrium. In implantation segments, they were also localized to the region of the trophoblast giant cells. The second objective of the present study was to determine whether endometrial stromal cells isolated from uteri sensitized for decidualization express matrix metalloproteinases 2 and 9. Northern blot analyses and gelatin zymography showed that these cultured cells expressed matrix metalloproteinase 2 and 9, and that transforming growth factor beta1 significantly increased matrix metalloproteinase 9 expression. The results of the present study further characterize matrix metalloproteinases 2 and 9 expression in the uterus during implantation and artificially induced decidualization.     

Expression of c-fos and c-myc genes in uterus and nonreproductive organs during the implantation period in the rat.

The c-fos and c-myc mRNA levels were investigated in the uterus and nonreproductive organs of rats during the implantation period (from day 5 to 7 of pregnancy). They were determined by densitometric analysis of slot blots and the mean values (n = 4) at a defined age of pregnancy were compared with those observed in nonpregnant control rats (NP group). No significant variations of c-fos level were observed in the liver and brain of pregnant rats. In the uterus, the c-fos level decreased, reached a minimum on day 6 (4-fold decrease compared with the NP group), and then increased. The c-fos level significantly increased in the kidney (2- and 2.5-fold on days 6 and 7), and in the lung (2.5-fold on day 7). The c-myc level did not significantly vary in the uterus, liver, and lung. It significantly increased in the kidney (3-fold on day 7) and in the brain (4.1-, 3.8-, and 6-fold, on days 5, 6, and 7). The changes in c-fos and (or) c-myc gene expression in the uterus and nonreproductive organs could be due to sexual steroids and (or) systemic factors from uterine cells or blastocysts.     

Expression and regulation of chemokine genes in the mouse uterus during pregnancy

Leukocytes accumulate in the pregnant mouse uterus following mating, during implantation and during placental development. Changes in leukocyte number are primarily due to recruitment from the blood, not local proliferation, but the underlying recruitment mechanisms are poorly understood. Mating-induced granulocyte and macrophage recruitment is due in part to pro-inflammatory and chemotactic factors present in seminal plasma. Accumulation of macrophages later in pregnancy appears to be caused in part by ovarian hormone-stimulated CSF-1 production and in part by other as yet unidentified uterine chemotactic factors. The current study was performed to assess chemokine production in the uterus during pregnancy. Northern blotting was used to demonstrate NSI/KC (KC), macrophage chemotactic protein-1 (MCP-1), macrophage inflammatory protein one alpha (MIP1alpha) and regulated inactivation, normal T expressed and secreted protein (RANTES) mRNA in the uterus. Oestrogen and progesterone induced intrauterine production of all four chemokines and may have done so through the autocrine/paracrine activities of IL-1. The data suggest that C-C chemokines play a role in accumulation of macrophages in the uterus during pregnancy.     

Expression of stathmin family genes in the murine uterus during early pregnancy

Stathmin, a cytosolic phosphoprotein that regulates microtubule dynamics during cell-cycle progression, is abundantly expressed at embryo implantation sites in rats. Here, we characterized the expression of stathmin and its family genes in the murine uterus during the peri-implantation period. Stathmin protein was expressed in the glandular and luminal epithelium, blood vessels, and stromal cells on day 3 of pregnancy. On the day of implantation (day 5), stathmin was mainly localized in blood vessels in the endometrium. On day 7, intense stathmin expression was limited to capillary vessels and secondary decidual cells. Stathmin expression was higher at implantation sites than at uterine segments between implantation sites and increased during oil-induced decidualization. Although the artificially-induced deciduoma weights and number of implantation sites were similar between stathmin-knockout (KO) and wild-type (WT) mice, the stathmin-KO mice had fewer newborn pups (reduced by 30%). The expression of alkaline phosphatase, desmin, and cyclin D3 was attenuated in decidual zones of stathmin-KO mice. Messenger RNA level of the stathmin family gene, SCG10, was high at the time of decidualization in WT and stathmin-KO mice. In contrast, the others of stathmin family members, SCLIP and RB3 were highly expressed in stathmin-KO mice compared to WT mice. These results suggest that stathmin and stathmin family genes are expressed in the murine endometrium with enhanced expression in the implantation or the decidualization process.     

Differential expression and anti-oxidant function of glutathione peroxidase 3 in mouse uterus during decidualization

Glutathione peroxidase 3 (GPX3) is an important member of antioxidant enzymes for reducing reactive oxygen species and maintaining the oxygen balance. Gpx3 mRNA is strongly expressed in decidual cells from days 5 to 8 of pregnancy. After pregnant mice are treated with GPX inhibitor for 3 days, pregnancy rate is significantly reduced. Progesterone stimulates Gpx3 expression through PR/HIF1α in mouse endometrial stromal cells. In the decidua, the high level of GPX3 expression is closely associated with the reduction of hydrogen peroxide (H₂O₂). Based on our data, GPX3 may play a major role in reducing H₂O₂ during decidualization.     

Regulation of cyclooxygenase-2 induction in the mouse uterus during decidualization. An event of early pregnancy

The infertility phenotype of cyclooxygenase-2 (Cox-2)-deficient female mice establishes the important role of Cox-2 in pregnancy. Cox-2 deficiency results in defective ovulation, fertilization, implantation, and decidualization; the latter of which can be restored in part by the prostacyclin analog carbaprostacyclin. Uterine Cox-2 expression during early pregnancy shows distinct localization and kinetics in the uterine luminal epithelium and underlying stromal cells, suggesting that expression is tightly regulated. Several intracellular signaling cascades including ERK, p38, and JNK are implicated in vitro as critical components of regulated Cox-2 expression in response to mitogens, growth factors, and cytokines. We investigated the involvement of these signaling pathways during Cox-2 induction in vivo by monitoring uterine kinase activity after intraluminal application of a deciduogenic stimulus. Our results show that the ERK and p38 pathways are activated in uterine preparations as early as 5-min post-stimulation. ERK activation was sustained for several hours with a return to baseline levels by 4 h. p38 activation was rapid with a peak at 5-min post-stimulation and returned to near baseline levels after 45 min. Systemic administration of a MEK inhibitor completely inhibited ERK activation, but did not affect early (2 h) luminal epithelial or late (24 h) stromal Cox-2 expression and only modestly affected decidualization. In contrast, administration of a p38 inhibitor modestly inhibited early Cox-2 expression in the luminal epithelium, while dramatically diminishing late stromal expression. In parallel, induced stromal peroxisomal proliferator activated receptor-delta (PPARdelta) expression is blunted by p38 inhibition. p38 inhibition also significantly inhibited decidualization. These results suggest that p38, but not ERK, activation is required for induced Cox-2 and PPARdelta expression during decidualization. In addition, inhibition of p38 led to decreased decidualization suggesting that an intracrine prostanoid pathway consisting of Cox-2, prostacyclin, and PPARdelta is required for maintenance of early pregnancy.     

Peroxisome proliferator-activated receptor delta expression and regulation in mouse uterus during embryo implantation and decidualization

The aim of this study was to examine the expression and regulation of peroxisome proliferator-activated receptor (PPAR) PPARdelta gene in mouse uterus during early pregnancy by in situ hybridization and immunohistochemistry. PPARdelta expression under pseudopregnancy, delayed implantation, hormonal treatment, and artificial decidualization was also investigated. There was a very low level of PPARdelta expression on days 1-4 of pregnancy. On day 5 when embryo implanted, PPARdelta expression was exclusively observed in the subluminal stroma surrounding the implanting blastocyst. No corresponding signals were seen in the uterus on day 5 of pregnancy. There was no detectable PPARdelta signal under delayed implantation. Once delayed implantation was terminated by estrogen treatment and embryo implanted, a strong level of PPARdelta expression was induced in the subluminal stroma surrounding the implanting blastocyst. Estrogen treatment induced a moderate level of PPARdelta expression in the glandular epithelium, while progesterone treatment had no effects in the ovariectomized mice. A strong level of PPARdelta expression was seen in the decidua on days 6-8 of pregnancy. PPARdelta expression was also induced under artificial decidualization. These data suggest that PPARdelta expression at implantation sites require the presence of an active blastocyst and may play an essential role for blastocyst implantation.     

Loss of collagen type VI from rat endometrial stroma during decidualization.

The expression of collagen type VI in the extracellular matrix of rat uterine endometrial stroma after a decidual stimulus was examined by immunolocalization and immunoblotting. The intermediate filament protein, desmin, was used as a marker to identify decidual cells. Tissue was examined from pregnant animals and from ovariectomized, hormone-treated rats in which decidualization had been induced artificially. In undifferentiated tissue from both groups of animals, collagen type VI was abundant, and desmin was present only in vascular smooth muscle cells. By 72 h after a decidual stimulus, however, collagen type VI had essentially disappeared from the matrix of the antimesometrial stromal compartment, and desmin was highly expressed in the decidualizing cells. During regression of the decidual tissue, collagen type VI began to reappear in the stromal matrix, whereas desmin expression declined as decidual cells degenerated. These results indicate that remodeling of the uterine extracellular matrix in response to embryo implantation is a function of the differentiating decidual cell.     

Subtilisin proprotein convertase-6 expression in the mouse uterus during implantation and artificially induced decidualization

During implantation, a balance of factors regulates the invasive properties of the embryo and the anti-invasive properties of uterine decidua. Although antiproteinases such as the metalloproteinase inhibitor TIMP-3 are thought to play critical roles in preventing the overaggressive invasion of trophoblasts, the mechanism of antiproteinase regulation is unknown. Recently, the prohormone convertase SPC-6 has been found to be co-expressed in embryo-proximal decidua in association with TIMP-3. As members of this serine proteinase family are known to activate latent TGFbeta family members which regulate decidual TIMP-3 levels, we sought to characterize the expression of SPC-6 during pregnancy and artificial decidualization. In this study, we demonstrate that the zone of SPC-6 gene expression exhibits a great degree of temporal and spatial overlap with TIMP-3 gene expression in uterine decidua from E5.5 through to E8.5. Like TIMP-3, we demonstrate that SPC-6 expression is induced during the decidual cell response using an in vivo model of artificial decidualization. Both the secreted and membrane bound forms of SPC-6 are expressed throughout the period of decidualization, suggesting that SPC-6 may play multiple roles during this developmental period. This is confirmed by our observation of the movement of SPC-6 expression to the presumptive placental region, as TIMP-3 expression regresses at the implantation site.     

Myometrial connexin 43 trafficking and gap junction assembly at term and in preterm labor.

Modulation of connexin 43 (cx43) in the myometrium of timed pregnant rats was studied using enzyme-linked immunosorbent assay (ELISA), immunocytochemical localization, and immunoblot. These techniques utilized site-specific antibodies directed against a portion of the carboxyl tail of cx43. We found that cx43 is synthesized several days prior to labor but accumulates within the cytoplasm until parturition, when it is rapidly transported to the plasma membrane and assembled into gap junction plaques at the cell surface. These cx43-positive gap junctions begin to disappear from the plasma membrane within hours of delivery of the last pup and are completely absent within 24 hr following delivery. These structures are apparently internalized and degraded within the cytoplasm. ELISA documents a reduction of total cellular cx43 to baseline levels within 5 days following parturition. While the timing of synthesis, cytoplasmic storage, concentration in apparent Golgi vesicles, and transport to and assembly in the plasma membrane are accelerated in three models of preterm labor, the sequence of these events and the correlation of parturition with the formation of gap junctions are identical to those documented in normal labor. These results support the hypothesis that effective labor requires the synthesis and assembly of cx43 into functional gap junctions at the myometrial cell surface.     

Expression of gap junction protein connexin43 in the adult rat cochlea: comparison with connexin26.

To elucidate whether the two different gap junction proteins connexin43 (Cx43) and connexin26 (Cx26) are expressed and localized in a similar manner in the adult rat cochlea, we performed three-dimensional confocal microscopy using cryosections and surface preparations. In the cochlear lateral wall, Cx43-positive spots were localized mainly in the stria vascularis and only a few spots were present in the spiral ligament, whereas Cx26-positive spots were detected in both the stria vascularis and the spiral ligament. In the spiral limbus, Cx43 was widely distributed, whereas Cx26 was more concentrated on the side facing the scala vestibuli and in the basal portion. In the organ of Corti, Cx43-positive spots were present between the supporting cells but they were fewer and much smaller than those of Cx26. These data demonstrated distinct differences between Cx43 and Cx26 in expression and localization in the cochlea. In addition, the area of overlap of zonula occludens-1 (ZO-1) immunolabeling with Cx43-positive spots was small, whereas it was fairly large with Cx26-positive spots in the cochlear lateral wall, suggesting that the differences are not associated with the structural difference between carboxyl terminals, i.e., those of Cx43 possess sequences for binding to ZO-1, whereas those of Cx26 lack these binding sequences.     

Expression of peroxisome proliferator-activated receptor isoforms in the rat uterus during early pregnancy

Peroxisome proliferator-activated receptors (PPARs) play an important role in different compartments of the female reproductive system in rodents and humans. However, expressional profiles and physiological functions of PPARs in the endometrium prior to the placentation are not well understood. In this study, we determined expressional profiles of the PPARs during early pregnancy. Immunocytochemistry revealed that both PPARα and PPARβ/δ were strongly detected in the endometrial stroma on days 4.5–6.5 of pregnancy, which is just a starting time of implantation. Delayed implantation animal model showed that the expressions of PPARα and PPARβ/δ occurred after the initiation of implantation in the endometrial stroma. Moreover, an in vitro decidualization model further revealed that the expression of PPARα increased in the cultured rat endometrial stromal cells at 24 h after the decidualization treatment, but the expression of PPARβ/δ was delayed and increased at 48 h after the treatment. PPARγ was expressed in the endometrial stroma and its expression decreased significantly at 2.5 days post-coitum and maintained a low level of expression during the period of implantation. These results indicate that PPARα is expressed and induced by the initiation of implantation, prior to the expression of PPARβ/δ in decidualized endometrium. Increasing expression of PPARγ during fertilization and its decline during the period of implantation further suggest that PPARs may play important roles during early pregnancy.     

Enzymic activity in rat uterus during early pregnancy.

Total protein, RNA and DNA content and the activity of acid and alkaline phosphatases, 5'-nucleotidase and isocitrate dehydrogenase were studied in rat uterus during the first 8 days of pregnancy. Isocitrate dehydrogenase activity showed marked fluctuations from day to day. Nucleotidase and acid phosphatase activities showed a significant increase on day 8. The most marked change in activity was that of alkaline phosphatase which showed a 10-fold increase between days 6 and 8, due largely to an increase in the activity of this enzyme in the decidual nodule. The rise in alkaline phosphatase activity did not occur in rats ovariectomized on days 1, 2 or 4 of pregnancy and was markedly decreased in those ovariectomized on day 6. [3H]-uridine incorporation into RNA showed a significant increase between days 2 and 6 whereas [3H]-thymidine incorporation into DNA showed a significant increase on day 6.     

Expression of desmin, laminin and fibronectin during in situ differentiation (decidualization) of rat uterine stromal cells

Immunocytochemical analysis of frozen rat uterine sections containing decidual tissue, formed in response to normal or artificial stimulation of uteri sensitized by endogenous or exogenous hormonal regimens, demonstrated an elevated expression of the intermediate filament protein desmin in decidual cells. Changes in the expression of extracellular matrix (ECM) components were coordinated with the elevated expression of desmin as stromal cells underwent decidualization. In parallel with the pattern of regional decidualization, as determined by elevated desmin expression, laminin accumulated in ECM of decidual cells while an apparent decrease in fibronectin was associated with altered organization at the decidual cell surface. The in situ observations confirm previous results, which indicated that the expression of desmin in decidual cells formed in vivo or in vitro is a valid marker of their differentiation, and resolve questions unanswered in the previous study: (a) desmin (and laminin) appear to be constitutively expressed in non-decidualized stroma at barely detectable levels, (b) desmin is a valid marker of stromal cell differentiation because it is expressed similarly in decidual cells, irrespective of varying experimental protocols for uterine sensitization and stimulation, and (c) desmin expression follows the same regional progression described for the process of decidualization in morphological and histochemical studies.