Reduction of anionic sites on the rabbit uterine surface during the preimplantation period

Studies have been carried out to analyze distribution of anionic sites on the uterine epithelium of the rabbit, using cationized ferritin as a label. A negatively charged glycocalyx was demonstrated by transmission electron microscopy on the luminal cell surface during estrus and days 5–7 of pregnancy. There was a general reduction of labeling from estrus and day 5 to 7 of pregnancy. At estrus and on day 5 and 6 of pregnancy, the results were similar on the meso- and antimesometrial sides of uterine horns and at or between egg recovery sites. At day 7, anionic sites were no longer detected antimesometrially facing the eggs. These results suggested that the progressive loss of anionic sites during the preimplantation period was due to the combined actions of uterus and egg and that this loss might play a role in blastocyst antimesometrial implantation.     

Development of the implantation chamber in the pregnant bitch.

Uteri taken from 25 bitches at various times during the early stages of pregnancy were studies cytologically to determine how the implantation chamber developed and how fetal-maternal relations were established. On day 13 after the end of estrus, knobs of trophoblastic syncytium formed and became wedged between cells of the uterine luminal epithelium. The syncytium quickly spread along the uterine lumen and into the mouths of the glands, dislodging and surrounding maternal cells. As invasion continued trophoblastic villi, consisting of cores of cytotrophoblast covered by a continuous layer of syncytium, penetrated deeper into the endometrium. The syncytium spread to surround maternal vessels and decidual cells. By day 26 the trophoblast had extended down to the large lacunae. Here syncytial trophoblast covering tips of the villi degenerated, leaving cytotrophoblast exposed to the necrotic zone. These cells possessed characteristics of absorbing cells. Hematomas were formed by focal necrosis of fetal and endometrial tissue at the poles of the implantation sites. Large pools of extravasated blood accumulated and red blood cells were phagocytized by surrounding trophoblastic cells. Therefore, the endotheliochorial relationship in the canine placenta appeared to be established by syncytial trophoblast invading a cellular endometrium. In the necrotic zone and hematomas, cellular trophoblast may have lost its syncytial covering, but elsewhere maternal vessels and decidual cells in the placenta were in direct contact only with syncytial trophoblast.     

Light and electron microscopic examination of the mature decidual cells of the rat with emphasis on the antimesometrial decidua and its degeneration

Rat gestation sites were obtained on days 10 through 16 of normal pregnancy. Light and electron microscopic examination of day-10 sites revealed a consistent complex pattern of stromal cell morphologies. Six distinct regions were identified: an antimesometrial region of epithelioid decidual cells that form the gestation chamber containing the embryo and extraembryonic membranes; an abembryonic antimesometrial decidual region, the decidual crypt, where the cells are separated by large extracellular spaces; a mesometrial region with granule-containing cells and mesometrial decidual cells; a region of spiny cells that are lateral to the antimesometrial decidual cells and continuous with the mesometrial decidual cells; and a region of undifferentiated stromal cells adjacent to the myometrium. Between days 12 and 16, the antimesometrial decidua becomes thinner and is eventually sloughed into the newly formed uterine lumen. The role of the antimesometrial decidual cells is discussed with reference to trophoblast invasiveness, protein synthesis, and especially remodeling of the gestation chamber. Differences between decidua and deciduoma are considered.     

Chorioallantoic placenta formation in the rat: I. Luminal epithelial cell death and extracellular matrix modifications in the mesometrial region of implantation chambers.

On days 7 and 8 of pregnancy, mesometrial regions of rat gestation sites were examined by light microscopy and transmission electron microscopy to determine what changes occur before the chorioallantoic placenta forms in that region. By day 7, gestation sites contained a uterine lumen mesometrially and an antimesometrial extension of the uterine lumen, the implantation chamber. The implantation chamber consisted of a mesometrial chamber between the uterine lumen and the conceptus, an antimesometrial chamber that contained the conceptus, and a decidual crypt antimesometrial to the conceptus. Stromal cells that formed the walls of the implantation chamber were closely packed decidual cells, while those that surrounded the uterine lumen were loosely arranged. Late on day 7, a portion of the epithelium lining the mesometrial chamber was degenerating, but this area of initial degeneration was never adjacent to the antimesometrial chamber. By early day 8, most of the epithelial cells lining the mesometrial chamber were degenerating and were being sloughed into the chamber lumen. Although degeneration of these epithelial cells morphologically resembled necrosis, it was precisely controlled, since adjacent epithelial cells lining the uterine lumen remained healthy. The space that separated the denuded luminal surface of the mesometrial chamber from underlying decidual cells became wider and was occupied by an extracellular matrix rich in cross-banded collagen fibrils. Decidual cell processes, that earlier had penetrated the basal lamina beneath healthy epithelial cells, protruded into this matrix and penetrated the basal lamina at the luminal surface. By late day 8, large areas of denuded chamber wall were covered with decidual cell processes, little remained of the basal lamina, and cross-banded collagen fibrils were scarce in the area occupied by decidual cell processes. During the times studied, uterine tissues that formed the walls of the mesometrial chamber were not in direct contact with the conceptus. This study indicates that trophoblast does not play a direct role in epithelial degeneration, basal lamina penetration, or extracellular matrix modifications in the mesometrial region of implantation chambers where part of the chorioallantoic placenta forms, although trophoblast may be required to trigger or modulate some of the changes.     

Ultrastructure of the endometrial blood vessels during implantation of the rat blastocyst

The ultrastructure of the blood vessels of the endometrium was analysed during implantation of the blastocyst in rats, at the time of appearance of the Pontamine Blue Reaction. Vessels from implantation sites and from interimplantation sites were compared. In vessels from implantation sites the endothelial cells showed fenestrations covered by diaphragms. In addition, small interruptions (gaps) between the endothelial cells were observed. These features were present in vessels larger than 5 micrometers in diameter and more than 100 micrometers away from the uterine epithelium, both in the mesometrial and antimesometrial wall of the endometrium. Vessels from interimplantation sites displayed neither fenestrations nor interruptions. The endothelial cells of the implantation sites displayed morphological signs of metabolic activation. These consisted of increased numbers of polyribosomes, well developed Golgi complexes and prominent nucleoli. The fenestrations and gaps in the vessel wall were interpreted as constituting the morphological basis for the increase in vascular permeability and the consequent edema which characterize the Pontamine Blue Reaction.     

Conceptus-mediated integrity of endometrial epithelial cells and maintenance of relaxin synthesis in pregnant rabbits: effects of unilateral oviduct ligation

A previous study indicated rabbit endometrial relaxin synthesis is stimulated by blastocyst (Lee VH, Fields PA, Biol Reprod 1990; 40:737-745). To evaluate this hypothesis, unilateral oviduct ligations were placed (A) at the oviduct isthmus on Day 1 post-copulation and (B), in a separate group of rabbits, at the infundibulum before copulation. Blastocysts migrate into and implant in the uterine horn contralateral to the ligated oviduct only (conceptus-bearing uterus). The uterine horn ipsilateral to the ligated oviduct will be referred to as the non-conceptus-bearing uterus. Uteri and ovaries were removed on Days 4-28 of pregnancy and were evaluated for relaxin using guinea pig anti-porcine relaxin serum and avidin-biotin light microscopy immunohistochemistry. Results were identical for both models. Blastocysts first attach to the antimesometrial uterine surface by Day 7 post-copulation. Implantation on the mesometrial surface occurs on Days 8-11. Relaxin was observed in antimesometrial endometrial glands of both conceptus and non-conceptus-bearing uteri on Days 4-7 of pregnancy. Beyond Day 7, relaxin was observed in antimesometrial and mesometrial endometrial glandular and luminal epithelial cells at implantation sites of the conceptus-bearing uterus only. Relaxin was not found between implantation sites. Endometrial epithelial cells of the non-conceptus-bearing uterus were regressing by Day 9. These data indicate a conceptus-mediated maintenance of endometrial epithelial cells. Furthermore, the data suggest a paracrine maintenance of epithelial cell integrity and relaxin synthesis since these parameters are preserved only in the conceptus-bearing uterus. Cell-cell communication between conceptus and endometrium appears to be specific since endometrium between implantation sites does not contain relaxin. Uterine tissue from pseudopregnant rabbits (Days 1-16) was evaluated. Relaxin was observed in the antimesometrial glands on Day 7 only. Like the endometrium in the ligation model, endometrial epithelial cells of the pseudopregnant rabbit uterus were regressing by Day 9. These results indicate that pregnancy is not required for, but may enhance, relaxin synthesis. In addition, endometrial epithelial cells regress in the absence of pregnancy. Regression of endometrial epithelial cells on Day 9 suggests that maternal recognition of pregnancy occurs during the preimplantation period (Days 4-8).     

Apoptosis of rat decidual cells: site specific initiation and related biochemical changes

The artificially induced rat deciduoma serves as a model to study cellular changes associated with implantation in the endometrium. The stromal cells differentiate to form two types of decidual cells and are restricted to specific anatomical sites of the uterus. Programmed cell death starts in the antimesometrial area and expression of glutathione-S-transferase, an antioxidant enzyme, enhances in these cells as the deciduoma enters the regressive phase. The enzyme activity is significantly high compared with that of mesometrial decidual cells. Similarly, lipid peroxide content of antimesometrial decidual cells is high during this phase. DNA fragmentation, a feature of cells undergoing programmed cell death, is initiated in the antimesometrial area during regression of deciduoma.     

Granulated metrial gland cells in the pregnant uterus of mice expressing the collagen mutation tight-skin (Tsk/+)

Summary Influences of the extracellular matrix (ECM) on the differentiation and distribution of granulated metrial gland (GMG) cells, a uterine natural killer (NK)-like cell subset, were studied by histological examination of implantation sites in the mouse mutant Tsk/+. Tsk/+ mice overproduce collagens I and III. GMG cell differentiation appeared to progress normally in Tsk/+ mice between days 6.5 and 12.5 of gestation. The distribution of GMG cells, however, was abnormal. Significant numbers of GMG cells were found in the antimesometrial and lateral decidual regions at day 8.5 of gestation and in the regions between implantation sites until day 10.5 of gestation. Loss of GMG cells from these regions normally occurs by day 6.5 of gestation. These data suggest that alterations to the ECM change the migration properties or life span of GMG cells.     

Collagen concentrations in dissected tissue compartments of rat uterus on days 6, 7 and 8 of pregnancy.

Implantation and non-implantation sites were dissected into myometrial and stromal components; a decidual/embryonic region was obtained on Days 7 and 8 of pregnancy. The concentration of collagen (as a percentage of the dry weight of tissue), measured by hydroxyproline analysis, was significantly lower in the implantation regions than in the non-implant regions in all areas studied. The concentrations in the antimesometrial myometrium and stroma of the implantation region remained the same over the days studied. In contrast, the mesometrial collagen concentration in the implantation region declined from Day 6 to Day 8 of pregnancy. Collagen concentration was low within the decidual/embryonic tissue on Days 7 and 8 of pregnancy. Remodelling of collagen within the embryonic area appears to be an important feature of the uterine response to implantation in rats.     

Early postimplantation embryolethality in mice following in utero inhibition of adenosine deaminase with 2'-deoxycoformycin

Adenosine deaminase (ADA) catalyzes the hydrolytic deamination of adenosine (or 2'-deoxyadenosine) to inosine (or 2'-deoxyinosine). Previously, we have shown that ADA activity is subject to strong cell-specific developmental regulation in placental tissues of mice between days 6 and 11 of gestation (Knudsen et al.:Biology of Reproduction 39:937-951, 1988). In the present study, we examined the effects of intrauterine exposure to 2'-deoxycoformycin (dCF; pentostatin), a potent irreversible inhibitor of ADA, on early postimplantation development. Deoxycoformycin was administered to pregnant ICR mice as a single intraperitoneal injection at a dose of 5 mg/kg on one of days 6 through 11 of gestation (plug day 0). A marked increase in the incidence of implantation site resorptions was observed following treatment specifically on days 7 (61% resorbed) or 8 (78% resorbed). No effect was observed following treatment on days 6, 9, 10, or 11. ADA-immunoreactive protein was shown, by ABC-immunoperoxidase staining on days 7 or 8 of gestation, to be present at high levels in decidual cells of the antimesometrial region but at below-detectable levels in the embryo. Treatment of pregnant dams with dCF on day 7 produced a complete (greater than 99%) inhibition of ADA activity in the antimesometrial decidua by 30 min, induced excessive cell death in the prospective neural plate and primary mesenchyme of the trilaminar disc by 6 h, and arrested embryonic development at an early somite stage. These results suggest that the antimesometrial decidua plays a protective role in preventing an inappropriate accumulation of endogenous ADA substrates in the implantation site.     

Light and electron microscopic radioautography of DNA synthesis in the endometria of pregnant-ovariectomized mice during activation of implantation window.

Pregnant mice were ovariectomized at pre-implantation stage and exogenous nidatory estradiol was administered to evaluate the DNA synthesis of the endometrial cells during activation of uterine receptivity for blastocyst implantation. After 0, 3, 6, 12 and 18 hrs. of estradiol treatment, the animals received 3H-thymidine injection, sacrificed 1 hr. later, and the uteri were prepared for light and electron microscopic radioautography. At time 0, no labelled stromal or epithelial cells was found in the endometrium. According to the time-lapse after estradiol induction, a gradual increase of labelled stromal and endothelial cells was seen in the endometrium. The highest labeling index was observed at the antimesometrial side of the implantation sites and the lowest value was found at the interimplantation site. The cells found at mesometrial side of the implantation site showed an intermediate labeling index. Eighteen hrs. after estradiol treatment, the labelled stromal cells found near the implantation chamber resembled the morphology of decidual cells while those labelled cells localized at the interimplantation sites were similar to the fibroblast. The uterine luminal epithelial cells showed low DNA synthesis after estradiol treatment resulting in only a few labelled cells at the interimplantation sites and no labelled cells at the implantation sites. A similar labeling pattern was seen in the glandular epithelium. The distribution of labelled cells seen among the regions of pregnant endometrium under estradiol effect suggest that DNA synthesis related to uterine activation for blastocyst implantation is a focal reaction, where the luminal epithelium does nt proliferate while the stromal and endothelial cells around the conceptus increase the DNA synthesis to prepare the endometrial decidualization.     

Demonstration of ubiquitin thiolester formation of UBE2Q2 (UBCi), a novel ubiquitin-conjugating enzyme with implantation site-specific expression

We recently identified a differentially expressed gene in implantation stage rabbit endometrium encoding a new member of the ubiquitin-conjugating enzyme family designated UBE2Q2 (also known as UBCi). Its unusually high molecular mass, novel N-terminus extension, and highly selective pattern of mRNA expression suggest a specific function in implantation. This study analyzes its relationship to the E2 ubiquitin-conjugating enzyme superfamily, investigates its enzymatic activity, and examines its localization in implantation site endometrium. Construction of a dendrogram indicated that UBE2Q2 is homologous to the UBC2 family of enzymes, and isoforms are present in a broad range of species. In vitro enzymatic assays of ubiquitin thiolester formation demonstrated that UBE2Q2 is a functional ubiquitin-conjugating enzyme. The Km for transfer of ubiquitin thiolester from E1 to UBE2Q2 is 817 nM compared to 100 nM for other E2 paralogs; this suggests that the unique amino terminal domain of UBE2Q2 confers specific functional differences. Affinity-purified antibodies prepared with purified recombinant UBE2Q2 showed that the protein was undetectable by immunoblot analysis in endometrial lysates from estrous and Day 6(3/4) pregnant (blastocyst attachment stage) rabbits but was expressed in both mesometrial and antimesometrial implantation site endometrium of Day 8 pregnant animals. No expression was detected in adjacent interimplantion sites. Immunohistochemistry demonstrated UBE2Q2 expression exclusively in mesometrial and antimesometrial endometrial luminal epithelial cells of the Day 8 implantation chamber. Immunohistochemical localization of ubiquitin mirrored UBE2Q2 expression, with low-to-undetectable levels in implantation sites of Day 6(3/4) pregnant endometrium but high levels in luminal epithelial cells of Day 8 pregnant endometrium. This implantation site-specific expression of UBE2Q2 in luminal epithelial cells could play major roles in orchestrating differentiation events through the modification of specific protein substrates.     

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.     

Ovarian steroid receptors and activated MAPK in the regional decidualization in rats

Though the decidua serves a critical function in implantation, the hormonal regulated pathway in decidualization is still elusive. Here we describe in detail the regional distribution and the effects of progesterone receptors (PGR), estrogen receptors (ESR), and MAPK activation on decidualization. We showed an increase in PGR A, PGR B, ESR1, and phosphorylated MAPK3-1 proteins (p-MAPK3-1), but not in ESR2, in the decidual tissue up to Day 8 of pregnancy. PGR was predominantly found in the nuclei of mesometrial decidual cells and of undifferentiated stromal cells where it colocalizes with ESR2 and ESR1. In the antimesometrial decidua, all the receptors showed cytoplasmic localization. MAPK was activated exclusively in undifferentiated stromal cells of the junctional zone between the antimesometrial and mesometrial decidua and at the border of the antimesometrial decidua. Treatment with the progesterone antagonist onapristone and/or the estrogen antagonist faslodex reduced the extent of decidual tissue and downregulated the levels of PGR and ESR1. The expression level of ESR2 was affected only by the progesterone receptor antagonist, while neither the antiprogestin nor the antiestrogen significantly modified the p-MAPK3-1 level. The inhibition of MAPK3-1 phosphorylation by PD98059 impaired the extent of decidualization and the closure reaction of the implantation chamber, and significantly downregulated ESR1. These results confirm a role of both steroid receptors in the growth and differentiation of the different decidual regions and suggest a new function for p-MAPK3-1 in regulating expression levels of ESR1, thereby maintaining the proliferation capacity of stromal cells and limiting the differentiation process in specified regions of decidual tissues.     

Differential expression of genes in the endometrium at implantation: upregulation of a novel member of the E2 class of ubiquitin-conjugating enzymes

The process of embryo attachment and implantation is accompanied by dramatic cellular and functional changes in the endometrium, the control and mechanisms of which are not clearly understood. The cDNA cloning of differentially expressed genes, specifically at implantation sites in the rabbit endometrium, was used to identify genes controlling functional and remodeling changes. Tissue from the endometrium of Day 6(3/4) (preimplantation) and Day 8 (implantation initiation) pregnant rabbits was used to screen for differentially expressed genes by combined cDNA subtraction/suppressive hybridization. Twenty-nine differentially expressed genes were identified encoding protein modification enzymes, signaling proteins, structural proteins, and enzymes. One of these is a novel member of the E2 ubiquitin-conjugating enzyme family we have designated UBCi (i for implantation), which displayed dramatic nucleotide and deduced amino acid sequence conservation between rabbits, humans, and mice. In situ hybridization indicated UBCi expression exclusively in the luminal epithelium of the endometrium while glandular epithelium, trophoblast, and myometrium were negative. Expression was specific for epithelial cells at implantation sites and was not detected in non-implant-site endometrium. UBCi mRNA was detected in both the mesometrial and antimesometrial epithelial cells of the implantation sites, sites undergoing both differentiation and/or apoptosis. These results identify a group of differentially expressed genes in the endometrium including UBCi and provide new focal targets for studying processes controlling cellular remodeling during implantation. The important roles of ubiquitination in controlling the activities and turnover of key signaling proteins suggest potential roles in controlling critical aspects of implantation.     

Coexpression of cytokeratin and vimentin in mice trophoblastic giant cells

Trophoblastic giant cells reach their maximum size and exhibit a conspicuous synthetic and invasive activity during mouse placentation. The cytoskeleton, given the complex functions of the cells, shows a well-developed network of intermediate filament proteins. Immunohistochemistry combined with confocal and conventional immunofluorescence studies of intermediate filaments proteins cytokeratin and vimentin were performed in mice trophoblastic giant cells on days 9-11 of pregnancy. Specimens were fixed in phosphate-buffered formaldehyde and tissues were processed for routine paraffin embedding. Trophoblastic giant cells from antimesometrial, lateral or mesometrial uterine regions, through days 9-11 of pregnancy, expressed the same staining with both immunoperoxidase and immunofluorescent techniques. Cytokeratin filamentous structures were intensely immunoreactive and were detected throughout the cells cytoplasm; a few cells exhibited strongest fluorescence in the peripheral cytoplasm. Vimentin-positive staining was often distributed throughout the cells cytoplasm, most frequently and more intensely in the peripheral region; in some cells, it was present only in the peripheral regions. It is probable that expression of vimentin in midpregnancy trophoblastic giant cells may be associated with the rapid and conspicuous increase in size and synthetic activity of the cells and also with phagocytosis of degraded materials and invasion of decidual tissue.     

mt(1) Receptor-mediated antiproliferative effects of melatonin on the rat uterine antimesometrial stromal cells.

It has been shown that melatonin regulates uterine function. Our previous studies have demonstrated the presence of melatonin receptors in the rat uterine endometrium, indicating that melatonin may act directly on the uterus. In the present study, the histological localization of the rat uterine melatonin binding was revealed by autoradiography and the molecular subtyping was studied by in situ hybridization in the stromal cells. The signal transduction process and effects of melatonin on stromal cell proliferation was also investigated. Our autoradiograms showed that 2[(125)I]iodomelatonin binding sites were localized in the antimesometrial endometrial stroma. In situ hybridization with specific mt(1) receptor cDNA probe in the primary culture of antimesometrial stromal cells demonstrated the expression of mt(1) receptor mRNAs. Melatonin dose-dependently inhibited forskolin-stimulated cAMP accumulation, which was reversed by pertussis toxin. This indicates that the rat uterine melatonin receptors are negatively coupled to adenylate cyclase via pertussis toxin sensitive G(i) protein. Melatonin also inhibited the incorporation of [(3)H]thymidine in the rat uterine antimesometrial stromal cells, showing that melatonin has an anti-proliferative effect on the uterus. Our results suggest that melatonin may act directly on the mt(1) melatonin receptors in the rat uterine antimesometrial stromal cells to inhibit their proliferation. Its action may be mediated through a pertussis toxin-sensitive adenylate cyclase coupled G(i)-protein.     

Matrix metalloproteinase expression and activity in trophoblast-decidual tissues at organogenesis in CF-1 mouse

During early placentation, matrix metalloproteinases (MMPs) play important roles in decidualization, trophoblast migration, invasion, angiogenesis, vascularization and extracellular matrix (ECM) remodeling of the endometrium. The aim of our study was to analyze the localization, distribution and differential expression of MMP-2 and -9 in the organogenic implantation site and to evaluate in vivo and in vitro decidual MMP-2 and -9 activities on day 10 of gestation in CF-1 mouse. Whole extracts for Western blotting of organogenic E10-decidua expressed MMP-2 and -9 isoforms. MMP-2 immunoreactivity was found in a granular and discrete pattern in ECM of mesometrial decidua (MD) near maternal blood vessels and slightly in non-decidualized endometrium (NDE). Immunoexpression of MMP-9 was also detected in NDE, in cytoplasm of decidual cells and ECM of vascular MD, in trophoblastic area and in growing antimesometrial deciduum. Gelatin zymography showed that MMP-9 activity was significantly lower in CM compared to the active form of direct (not cultured) and cultured decidua. The decidual active MMP-9 was significantly higher than the active MMP-2. These results show differential localization, protein expression and enzymatic activation of MMPs, suggesting specific roles for MMP-2 and MMP-9 in decidual and trophoblast tissues related to organogenic ECM remodeling and vascularization during early establishment of mouse placentation.