Glycoproteins in rabbit uterus during implantation

Summary The synthesis of glycoproteins in rabbit uterine epithelium during the late preimplantation period was studied using tritiated N-acetylglucosamine. In vivo labelling was achieved by the intra-uterine implantation of agar gel columns containing the precursor. Autoradiography showed the radioactivity to be predominantly localized in the apical cell surfaces, with single cells exhibiting an accumulation of silver grains in their supranuclear cytoplasm. After gel electrophoresis of uterine flushings, activity was mainly found in the -glycoprotein fraction. Fluorescein isothiocyanate (FITC)-conjugated wheat-germ agglutinin reacted with the apical cytoplasm and surfaces of the endometrial cells. However, FITC-conjugated concanavalin A exhibited a different binding pattern, reacting first with the basal cytoplasm, and later with the apical cytoplasm. After concanavalin-A staining, single cells exhibited positive vesicles in their lateral and apical parts. These cells may be released into the uterine lumen until 210 h post coitum. Neither of the lectins reacted with ciliated cells. Concanavalin A showed an affinity for the -glycoprotein fraction of the uterine secretion. The results indicate that, although all endometrial cells contain glycoproteins, only a few of these seem to be involved in the synthesis of secretory products.Supported by grants Ki 154/9-3 and 154/10-1 from the Deutsche Forschungsgemeinschaft     

Gap junction formation in rabbit uterine epithelium in response to embryo recognition

Gap junction formation was studied in the uterine epithelium of nonpregnant, pregnant, and pseudopregnant rabbits in the periimplantation phase (6, 7, 8 days post coitum/post human gonadotropin injection) using freeze-fracture and immunocytochemistry as well as intracellular Lucifer yellow injection. At implantation (7 days post coitum) the uterine epithelial cells of the implantation chamber become junctionally coupled as evidenced by all three methods used. Gap junction protein (26K) becomes detectable immunocytochemically with a monoclonal antibody at 6 days post coitum in the epithelium surrounding the blastocyst, i.e., in the forming implantation chamber. The same sequence of events, starting with the presence of the gap junction protein before cell-to-cell coupling becomes evident, was observed in the blastocyst-free segments 1 day later. In contrast, uterine epithelium of nonpregnant and pseudopregnant animals in comparable phases shows an extremely low degree of coupling. The presence of the blastocyst is a necessary condition for the induction of gap junctions as demonstrated by unilateral pregnancy produced by tubal ligation. Thus, gap junction formation is one of the first maternal responses to a locally acting signal of the blastocyst.     

Purification and immunohistology of a glycoprotein secreted from the rabbit uterus before implantation

Summary Two antigens of the -glycoprotein fraction from rabbit uterine secretion of the seventh day post coitum were purified firstly by gel filtration on Sephadex G 150, then either by ion exchange chromatography on DEAE Sephacel or chromatofocusing on PBE 94. By the use of a specific antiserum, raised in female sheep, two antigens with ₂- and ₂-mobility in agar gel electrophoresis could be demonstrated. Immunohistochemical staining of the uterine epithelium at the seventh and eighth day post coitum showed the antigens to be localized in a ciliated cell type of conspicuous shape, which is supposed to be the site of synthesis. Stain accumulated mainly in the apical part of the cell, but there were also small deposits around the nucleus.Supported by grant Ki 154/9-3 from the Deutsche Forschungsgemeinschaft.     

Circulating progesterone, progesterone-binding proteins and oestradiol-17 beta concentrations in the pregnant Cape porcupine, Hystrix africaeaustralis

Circulating concentrations of progesterone, progesterone-binding plasma proteins (PBPP) and oestradiol-17 beta in pregnant porcupines remained relatively low until Days 25-30 post coitum. Progesterone values peaked (102-180 ng/ml; N = 3) 42-60 days post coitum and the rapid increase in oestradiol-17 beta concentrations approximated that of progesterone with peak values (170-210 pg/ml) being attained 60-85 days post coitum. The pattern of PBPP synthesis, as suggested by circulating concentrations, was closely related to that of plasma progesterone, with values remaining low (less than 20 pmol/ml) until Day 31 post coitum, reaching peak levels at Days 50-56 and Days 73-77 post coitum. The production of PBPP during pregnancy is, as in related New World hystricomorph species, considered to be a mechanism which facilitates a reduction in the rate of progesterone metabolism during pregnancy.     

Immunofluorescent localization, by use of anti-idiotypic antibody, of monoclonal anti-progesterone antibody in the mouse uterus before implantation

Mouse monoclonal anti-progesterone IgG1 antibody designated DB3 has an anti-fertility effect when injected into female mice shortly after mating. In BALB/c mice, pregnancy is blocked, probably as a result of progesterone withdrawal with inhibition of implantation. Rabbit polyclonal anti-idiotype raised against DB3 has been used in an indirect immunofluorescence method on frozen tissue sections to demonstrate the presence of DB3 on the surface of uterine luminal and glandular epithelia before implantation. DB3 was only detectable 30-60 h after a single parenteral injection (9 nmol antibody per mouse i.p. or i.v. at 32 h post coitum). Immunolocalization was both pregnancy-dependent and anti-progesterone antibody-specific, as it was not seen in pseudopregnant mice or mice treated with P3 (mouse myeloma IgG1 protein, using polyclonal anti-P3 anti-idiotype as a probe) or saline. The immunofluorescent reaction was completely blocked by addition of DB3 idiotype in vitro. The results indicate that anti-progesterone antibody binds to an antigen associated with luminal and glandular epithelia which may locally inhibit the uterine uptake of progesterone and disrupt the process of implantation.     

Apical plasma membrane-bound enzymes of rabbit uterine epithelium

Summary In order to monitor changes in the apical cell membrane of rabbit uterine epithelium which are postulated to be a precondition for trophoblast attachment, the marker enzymes: alkaline phosphatase, aminopeptidase M, -glutamyl transferase and dipeptidyl peptidase IV were investigated during the periimplantation phase. Endometrium of early pregnancy (implantation chamber, interblastocyst endometrium; 5–8 days post coitum, d p.c.) was compared with specimens obtained at hCG-induced pseudopregnancy (p. hCG) to distinguish between membrane changes regulated by maternal plasma steroid hormones and such which might be induced locally by blastocyst-derived signals.All enzymes tested showed their main activity at 5 d p.c./p. hCG. The weakest reaction in this series of stages was generally found at 8 d p.c. (interblastocyst segments) or at 8 d p. hCG. In contrast to the rest of the epithelium, the implantation chamber retained high activity of dipeptidyl peptidase IV, and the activity of alkaline phosphatase even raised here again at 7 and 8 d p.c. indicating a direct local influence of the blastocyst on the luminal epithelium. The results suggest that 1) considerable changes occur in the composition of the apical plasma membrane of the uterine epithelium when the endometrium enters the receptive state, 2) the overall trend is towards a loss of apical-type characteristics of this membrane domain and 3) the changes are modulated both systemically (by plasma steroid hormone levels) and locally by signals from the implanting blastocyst.Abbreviations d p.c. days post coitum - d. p. hCG days post hCG injection - hCG human chorionic gonadotropin - aP alkaline phosphatase - ATPase adenosine triphosphatase - Ca²⁺-ATPase Ca²⁺-activated adenosine triphosphatase - APM aminopeptidase M - GGT -glutamyl transferase - DPP IV dipeptidyl peptidase IV - PCMB p-chloromercuric benzoate - DFP di-isopropylfluorophosphate - DMF dimethylformamide     

Adhesion molecule expression and infiltrating maternal leucocyte phenotypes during blastocyst implantation in the pig

During the implantation period (days 14 to 30 post coitum) in Large White pigs substantial numbers of endometrial sub-epithelial leucocytes were observed from about day 18 p.c. These were predominantly MHC Class II⁺, CD45⁺ and reactive for an accessory cell determinant carried by polymorphs and macrophages. Maternal lymphocytes of the CD2⁺, CD4^?, CD8^?, and a γδTcR⁺ subtype identified by the mAb 86D, were also present. Few CD4⁺, CD8⁺, or double-positive lymphocytes were seen. CD2^?CD4^?CD8^? γδTcR⁺ "Null" cells were seen in stroma adjacent to endometrial glands, as were CD2⁺ lymphocytes. There was no apparent upregulation of CD18, VLA-4 or CD25. E-selectin could not be detected on endothelium within uterine tissues or on trophectoderm, nor was a ligand for L-selectin detectable. The luminal aspect of the uterine epithelium was strongly reactive with anti-CD15 mAb, which was specifically blocked by lacto-N-fucopentaose III, indicating the presence of the Lewis^X determinant.     

Detection of estrogen alpha and progesterone receptors and cell proliferation in the uterus during early pregnancy in the goat

The objectives of this study were to investigate in the goat uterus the expression of estrogen-alpha (ER alpha) and progesterone receptors (PR) and their relationship to proliferation indices (Ki-67) during peri-implantation on Days 22 to 30 post coitum (pc). Immunohistochemical methods were used to quantify ER alpha and PR for luminal and deep regions of the endometrium and of the myometrium. On Day 22 pc cell proliferation was only observed in the luminal epithelium. On Day 24 pc, high cell proliferation indices were seen in luminal epithelium and proliferation began in the luminal stroma and glands. There was a positive correlation between Ki-67 and total ER alpha score in the luminal epithelium (r = 0.53, P < 0.01). Levels of PR scores were highly correlated with Ki-67 indices in luminal epithelium (r = 0.74, P < 0.01) and stroma (r = 0.70, P < 0.01). No Ki-67 expression was observed in deep glands, stroma or myometrium on any of the days studied. Results indicate that patterns of ER alpha and PR expression differ markedly, and that there was a high correlation between PR expression and cell proliferation in the caprine uterus during the peri-implantation period.     

Changes in endometrial vascular permeability during the periimplantation period in the ferret (Mustela putorius)

A highly localized increase in permeability of uterine blood vessels in the immediate vicinity of implanting blastocysts was first detected on the morning of the 12th day of pregnancy (290 h post coitum). The amount of extravasated dye which accumulated at implantation sites continued to increase through the evening of Day 13 (321 h p.c.). Blastocyst expansion, as indicated by small uterine swellings, preceded a detectable change in vascular permeability by about 10 h, suggesting that the timing of increased permeability is closely associated with initial blastocyst attachment to the uterine epithelium. The results do not support the hypothesis that prostaglandins are required for increased uterine vascular permeability as two doses of indomethacin (4 and 8 mg/kg body wt) administered 5 times/day failed to decrease endometrial vascular permeability. However, the 8 mg dose did cause a significant reduction in size and number of uterine swellings and delayed or inhibited attachment of the trophoblast to the uterine epithelium in 2 of 5 ferrets. These findings suggest that prostaglandins play an important role in the process of implantation that is unrelated to decidual formation as the ferret is an adeciduate species.     

Apoptosis in uterine epithelium and decidua in response to implantation: evidence for two different pathways

During the initial steps of implantation, the mouse uterine epithelium of the implantation chamber undergoes apoptosis in response to the interacting blastocyst. With progressing implantation, regression of the decidual cells allows a restricted and coordinated invasion of trophoblast cells into the maternal compartment. In order to investigate pathways of apoptosis in mouse uterine epithelium and decidua during early pregnancy (day 4.5–7.0 post coitum), we have investigated different proteins such as TNFalpha, TNF receptor1, Fas ligand, Fas receptor1, Bax and Bcl2 as well as caspase-9 and caspase-3 using immunohistochemistry. To detect cells undergoing apoptosis the Tunel assay was performed. Immunoreactivity for TNFalpha as well as for TNF receptor1 was observed exclusively in the epithelium of the implantation chamber and the adjacent luminal epithelium from day 4.5 post coitum onwards. In the developing decidua the Fas ligand, but not the Fas receptor, was expressed. Bax and Bcl2 revealed a complementary expression pattern with Bax in the primary and Bcl2 in the adjacent decidual zone. Strong immunolabelling for the initiator caspase-9 was restricted to the decidual compartment, whereas caspase-3 expression characterized the apoptotic uterine epithelium. Only some caspase-3 positive decidual cells were found around the embryo which correlated to the pattern of Tunel staining. Taken together, the apoptotic degeneration of the uterine epithelium seems to be mediated by TNF receptor1 followed by caspase-3, whereas the very moderate regression of the decidua did not show the investigated death receptor, but Bax and Blc2 instead and in addition caspase-9, which indicates a different regulation for epithelial versus decidual apoptosis.     

Effect of estradiol and progesterone on phosphatidylinositol metabolism in the uterine epithelium of the mouse

The effect of estradiol and progesterone on uterine phosphatidylinositol (PtdIns) metabolism was examined in whole uteri and separated uterine luminal epithelium of ovariectomized mice. Incorporation of [3H]myo-inositol in vitro, into inositol-containing phospholipids extracted from whole uteri, increased in mice injected with estradiol, with maximal incorporation at 9-12 h. The breakdown of PtdIns to inositol polyphosphates was also stimulated in whole uteri by estrogen, with an abrupt increase between 6 and 9 h. Comparable increases in both processes occurred in the uterine epithelium after estrogen stimulation and were inhibited by progesterone pretreatment which by itself had little or no effect. These results suggest that PtdIns metabolism is involved in the stimulation of uterine epithelial cell proliferation by estrogens, and its inhibition by progesterone.     

Progesterone receptor in the rat anterior pituitary: Effect of estrogen priming and adrenalectomy

The present study was done to determine if a progesterone receptor is present in rat pituitary. Cytosol was labeled with ³H-progesterone (3HP) or ³H-RS020 (³HR) and subjected to sucrose-glycerol density-gradient centrifugation. Serum progesterone was measured for correlation with progesterone receptor levels. Two ³HP-binding peaks (4S + 6S) were evident in uterine and pituitary cytosols. The 4S peak was eliminated by competition with unlabeled cortisol leaving a single 6S peak (progesterone receptor). Estradiol (E) priming of the male or female rat increased progesterone receptor levels in pituitary cytosol as demonstrated using ³HP and ³HR, and pituitary progesterone receptor bound ³HR with a higher affinity than ³HP. Following adrenalectomy of gonadectomized rats, progesterone receptor levels were increased in pituitary and uterine cytosol of both E-primed and unprimed groups. An inverse relationship was established between serum progesterone and progesterone receptor levels in the uterus and pituitary suggesting that stressinduced adrenal progesterone secretion significantly influences progesterone receptor levels in the rat. These results demonstrate an estrogen-inducible progesterone receptor in the rat pituitary with properties similar to those of the uterine progesterone receptor.     

Changes in Ovaries and Uterus after Aglepristone Administration in the Third Week of Luteal Phase of Non-Pregnant Bitches

Objective

The mechanism of aglepristone action in the placentation time in the bitch remains unclear. The aim of this study was to describe the mechanism by which aglepristone influences ovaries and uterus and to measure the levels of steroid sex hormones in non-pregnant bitches.

Materials and Methods

Fourteen bitches assigned to a study (n=9) and control (n=5) group were given aglepristone and saline solution, respectively, on the 19th and 20th day after LH peak. On the 26th day after LH peak an ovariohysterectomy was performed. Blood samples were screened for estradiol and progesterone concentrations. Ovaries and uterine horns and bodies were isolated for histological and morphometrical diagnosis and immunohistochemistry analysis of α-estrogen and progesterone receptor expression.

Results

A decrease of progesterone (p<0.01) and no differences in total estrogen level in the study group were observed. There were no significant differences either in the histomorphometry or α-estrogen and progesterone receptors expression in ovaries. Increase in expression of progesterone receptors in endometrium without surface epithelium of horns (p<0.05), endometrial surface epithelium (p<0.05), myometrium of uterine body (p<0.01) and estrogen receptors in endometrium without surface epithelium of horns (p<0.05) was observed. Elevated estrogen receptors probably increased sensitivity of tissues to estrogens in the bloodstream and led to notable inflammation, haemorrhages, and hyperplasia in endometrium with mononuclear immune cell infiltration. The myometrium of horns and endometrium of uterine body of study bitches were significantly thicker than in the control group (p<0.05 and p<0.01). Furthermore myometrium of uterine body was thicker than myometrium of horns (p<0.001) and expression of progesterone receptors was higher in uterine body (p<0.01). No differences were observed between endometrium of horns and body within groups.

Conclusion

To the knowledge of the authors this is the first study, which describes the inflammatory effect developing in uterus in response to aglepristone administration, and attempts to elucidate its mechanisms.     

Analysis of progesterone receptor binding in the ovine uterus

The appropriate conditions for the measurement of ovine uterine cytoplasmic progesterone receptors (PR) have been determined to be 20 nM ³H-progesterone (³H-P₄) with and without a 100-fold excess of non-radioactive progesterone (P₄) 0–4°C and 4 h of incubation. Under these conditions PR readily exchanged bound progesterone for progesterone added during the assay. This exchange occurred even when saturating concentrations of P₄ were present. The progestins, R5020 and P₄, effectively competed for the ovine uterine PR binding while non-progestin steroids and diethylstilbestrol failed to compete for the PR binding. The dissociation constant (K_d) measured for the ³H-P₄ binding was 1.60 × 10^?9 M indicating that the ³H-P₄ binding was of high affinity. The levels of PR and the dissociation constant measured using ³H-R5020 in place of ³H-P₄ were similar indicating a lack of corticosteroid binding globulin (CBG)-like binding in the ovine uterus.     

Expression of galectin-1 mRNA in the mouse uterus is under the control of ovarian steroids during blastocyst implantation

Galectin-1 is a member of β-galactoside-binding lectins expressed in a variety of mammalian tissues. We report here that galectin-1 mRNA is abundantly expressed in the mouse reproductive organs such as the uterus and ovary. Uterine expression of galectin-1 mRNA is specifically regulated in the embryonic implantation process. Its expression increased at a high level on the fifth day post coitum (dpc 5) when embryos hatched into the endometrial epithelial cells. In the absence of embryos, however, galectin-1 expression in the mouse uterus decreased on dpc 5. In the delayed implantation mice, galectin-1 mRNA level was augmented by the termination of the delay of implantation. Ovarian steroids progesterone and estrogen differentially regulated galectin-1 mRNA level in uterine tissues. Treatment with RU486, a progesterone receptor antagonist, blocked progesterone-induced galectin-1 mRNA level in uterine tissues of ovariectomized mouse. ICI182780, a pure estrogen receptor antagonist, clearly blocked the estrogen effect. Taken together, galectin-1 gene expression in the uterine tissues was regulated by ovarian steroids and this regulation correlated with the implantation process. Mol. Reprod. Dev. 48:261–266, 1997. © 1997 Wiley-Liss, Inc.     

Steroidal control mechanism of cell proliferation in mouse uterine epithelium

The percentage of labeled cells in the uterine luminal epithelium of cycling mice showed the different zonal distributions at each stage of estrous cycle after cumulative labeling with 3H-thymidine for 36 hr. It was estimated that the proliferating fraction in the epithelium at proestrus, estrus, metestrus, and diestrus was 100%, 100%, 40% and 5%, respectively. The percentage of labeled cells in the uterine luminal epithelium of cycling mice treated with progesterone remained below 10% level for at least 20 hr after injections of progesterone. Total labeling was attained in the uterine epithelium of castrated mice by the administration of estradiol-17beta. On the other hand, the cell proliferation in the uterine epithelium of castrated mice treated with estradiol and progesterone was markedly suppressed and the percentage of labeled cells remained approximately at 35%. The remaining cell population, however, still showed the mitotic potency when mice received estradiol. It is suggested from this study that the effect of progesterone is to suppress the epithelial cell proliferation and transfer cells into resting cell fraction which is still evoked to proliferate as the effect of estradiol and that a key factor controlling epithelial proliferation in mouse uterus during the estrous cycle is proliferating fraction rather than cell cycle time.     

Expression of different connexin genes in rat uterus during decidualization and at term.

The expression of different connexin genes (cx26, cx32, cx37, cx43) that code for the protein subunits of gap junctions, was investigated in various uterine tissues during the estrous cycle of nonpregnant rats, in pregnant rats at decidualization and at term. Connexin gene expression was studied at the mRNA level by Northern blot hybridization and at the protein level by immunocytochemistry. In gap junctions from uterine epithelium, stroma, or myometrium, connexin 26 and/or connexin 43 are much more abundant than connexins 32 and 37. The expression of connexin 26 and 43 appears to be modulated by maternal steroid hormones. High expression of these connexins is found in developing decidual cells by day 7 to 8 post coitum; furthermore, coexpression of connexins 26 and 43 in myometrium is observed just before delivery on day 21 post coitum. In both the decidua and the myometrium, the connexin 26 protein appears to be distributed in lower abundance than connexin 43. In uterine epithelium only connexin 26 is expressed throughout all of the reproductive phases investigated. The enhanced expression of this gene correlates with higher levels of maternal estrogen both in the proestrus/estrus phase and at term. The distinct spatial and temporal pattern of expression of connexins 26 and 43 in different uterine tissues suggests a physiological role for these proteins during embryo implantation and subsequent contraction of the uterus at birth.     

Changes in the surface morphology of the rabbit endometrium related to the estrous and progestational stages of the reproductive cycle

Summary Changes occurring on the surface of the uterine luminal epithelium of the rabbit during the estrous and progestational stages of the reproductive cycle were examined by scanning and transmission electron microscopy. The findings demonstrate that the uterine epithelium, or endometrium, contains two cell types: ciliated cells and nonciliated, microvillous cells. In estrous animals, ciliated cells, although not very numerous, were usually observed in small groups. However, at increasing intervals of time following mating, ciliated cells progressively disappeared from the endometrium until approximately eight to ten days post coitum, when they became scare. From several hours to four to five days following mating, extensive changes occurred on the surfaces of microvillous cells. When observed by TEM, these elements contained organelles typical of cells involved in the synthesis and secretion of glycoproteins. Furthermore, microvillous cells during this period displayed numerous apical protrusions of different sizes and shapes and containing material of varying electron density. Parallel SEM examinations of the same material confirmed the presence of these protrusions. Some of the protrusions appeared as spheroidal masses attached to the cytoplasm by means of a cytoplasmic strand. Other surface masses were clearly unattached to the cell surface and were distributed (1) on the surface of microvillous cells, (2) on the cilia of adjacent ciliated cells, and (3) on the surface of spermatozoa.Changes occurring on the luminal surface during the early postcoital period are interpreted as an expression of morphodynamic processes likely involving coupled secretion (exocytosis) and resorption (endocytosis) of luminal material. The observations presented here also demonstrate that between six and ten days post coitum, the rabbit endometrium contained increasing numbers of enlarged, nonciliated cells that probably arose by the fusion of smaller, microvillous elements.The work reported here was supported by C.N.R. contracts No. CT 760128809 and CT 77014239 (to P.M.) and NIH. Grant HD-04274 (to J.V.B.)