Spatio-temporal pattern for expression of galectin-3 in the murine utero-placental complex: evidence for differential regulation.

In mice, immunoreactive galectin-3 protein has previously been localized in uterine epithelial cells adjacent to implanting blastocysts as well as in the decidualized endometrium of implantation sites, uterine natural killer cells, and several types of placental trophoblast cells. Because galectin-3 is a soluble extracellular molecule, protein localization by immunohistochemical methods does not demonstrate its cellular origin. Therefore, the present study was undertaken to determine precisely which cell types in the utero-placental complex express galectin-3 mRNA. In situ hybridization results demonstrated that galectin-3 mRNA was expressed throughout the utero-placental complex in all cell types previously shown to contain immunoreactive protein, including uterine epithelium, decidualized endometrium, uterine natural killer cells, and placental trophoblasts. These results indicate that galectin-3 protein is not synthesized in a restricted cell type and translocated through the extracellular spaces to other tissue compartments. Furthermore, Northern blot analysis of total RNA prepared from separated fetal and maternal components of utero-placental complexes demonstrated different patterns of expression for galectin-3 mRNA in the uterus and placenta. Relative levels of galectin-3 mRNA peak at midgestation in the implantation site and during the second half of gestation remain elevated in the placenta but decline in the uterus. Separate mechanisms for regulating expression of galectin-3 on opposite sides of the feto-maternal interface are indicated.

Comparison of the ontogeny of specific cell surface determinants on normal and delayed implanting mouse embryos.

The ontogeny of immunospecific cell surface determinants on preimplantation mouse embryos was determined by means of an antibody-dependent complement-mediated cell lysis assay. The determinants conferring sensitivity to lysis in that assay were first observed at the late blastocyst stage on embryos recovered from normal pregnancy on day 5 or grown to an equivalent stage in vitro. Although blastocysts recovered during the dormant phase associated with delayed implantation were not lysed, those recovered following reactivation with an injection of oestrogen to the mother were sensitive. Furthermore, it was found that treating the dormant embryos with neuraminidase rendered them sensitive to lysis. These results demonstrate that the appearance of specific cell surface determinants on mouse embryos is temporally associated with the process of attachment to the uterus in both normal pregnancy and at termination of the dormant phase associated with delayed implantation. They also indicate that those determinants may be 'masked' with sialic acid during embryonic diapause. It is suggested that such cell surface determinants could be important for embryo attachment and that the mechanism responsible for their expression may explain some aspects of the synchronization between the preimplantation conceptus and its mother at the time of implantation.

Compartmentalized reactivity of M3/38 (anti-Mac-2) and M3/84 (anti-Mac-3) in the uterus of pregnant mice.

Changes in the expression of antigens on mouse uterine or embryonic tissues were examined by enzyme immunocytochemistry. Cryostat sections of uteri from days 1, 8 and 15 of pregnancy were probed with the monoclonal antibodies M3/38 and M3/84, originally defined by their reactivity with macrophage surface antigens (Mac-2 and Mac-3, respectively). In the uterus of pregnant mice, reaction of these antibodies was not limited to leucocytes. M3/38 did not react at detectable levels with cells in the uterus on day 1 but did react with decidual cells immediately surrounding the embryo on day 8. By day 15, the placenta, decidua basalis and metrial gland were intensely positive but the embryo was negative. M3/84 reacted with the luminal side of the endometrium on day 1, the entire decidual mass on day 8, and with all maternal and fetal tissues on day 15. M3/38 was detected in saline-soluble preparations of uterine tissue and had a molecular mass of approximately 32-35 kDa as determined by SDS-PAGE and western blot analysis. The pattern of expression of these molecules suggests a functional relationship to developmental changes that occur at the maternal-fetal interface.

Changes in surface antigens on preimplantation mouse embryos.

Changes in the expression of specific cell surface antigens on preimplantation mouse embryos were examined by immunocytochemistry. Embryos were recovered at various times during the preimplantation phase of normal pregnancy, and from pregnancies with experimentally induced delayed implantation, and were probed with a panel of monoclonal antibodies against murine leukocyte antigens. Antibodies directed against certain macrophage surface glycoproteins (i.e., Mac-2 and Mac-3) and those against lysosome-associated membrane glycoproteins (i.e., LAMP-1 and LAMP-2) reacted specifically with cell surface determinants on the embryos. Differences in the spatiotemporal patterns of antibody binding during normal and delayed implantation indicate that expression of the antigenic determinants recognized by these antibodies is regulated individually in response to intrinsic as well as extrinsic signals at the time of implantation, and thus they may be important for the process of embryo implantation.

Embryonic signaling at implantation in the mouse.

Signaling between the preimplantation embryo and its mother is generally presumed to be important for successful establishment of pregnancy in mammals. However, with the exception of the large domestic species, little information is available about either the mechanisms for embryonic signaling or the nature and significance of maternal responses. In mice it is the localized decidual transformation of endometrium, characterized in the early stages by development of the so called Pontamine Blue reaction, that is the most completely described maternal response to the embryo at the time of implantation. Although much interest has been focused on the induction of decidualization as an important example of embryonic signaling, the reaction can be elicited by a variety of nonspecific stimuli as well as by an implanting blastocyst and little progress has been made toward identifying the authentic signal. The present work was prompted by the recent finding that there are embryo dependent differences between normal implantation sites and experimentally induced deciduomata with respect to the patterns of production of uterine secreted proteins (i.e., stimulation of a subset of proteins with Mr 14,000-20,000). (Weitlauf and Suda-Hartman, 1988). It was reasoned that it should be possible to use this specific pattern of uterine protein production to dissect out and characterize the authentic signal factor from embryos. Blastocysts were recovered from delayed implanting mice after reactivation with estrogen and incubated in microdrops of tissue culture medium. The resulting embryo conditioned medium (CME) was instilled into the uteri of virgin mice that had been ovariectomized and given appropriate hormone replacement. Medium similarly conditioned by incubation without blastocysts was used as a control. The uteri were removed and incubated with 3H methionine (CME) or 35S methionine (control). The resulting samples of differentially labeled uterine proteins mixed together and subjected to two dimensional polyacrylamide gel electrophoresis (2D-PAGE). The radioactivity in individual spots was determined and patterns of relative differences in their production was determined by a dual label ratio method. It was found that the pattern of uterine protein production typically associated with development of a normal embryo dependent implantation site was induced by instillation of CME and that this response was absent with control medium. It is concluded that the mouse blastocyst, like that of the sheep, synthesizes and releases one or more soluble factors in the peri-implantation period that influence metabolic activity in the uterus.(ABSTRACT TRUNCATED AT 400 WORDS)

Changes in secreted uterine proteins associated with embryo implantation in the mouse.

A dual-label ratio method was used in conjunction with two-dimensional polyacrylamide gel electrophoresis to measure the relative changes in rates of production of individual secreted proteins by mouse uteri at the start of the process of decidualization. A characteristic pattern of differential changes in the rate of synthesis and secretion of the proteins was found to be associated with development of a positive Pontamine Blue reaction at the site of embryo implantation. These changes were compared with those associated with development of experimentally induced deciduomata and although the patterns were similar, presumably reflecting common processes in transformation of the endometrium, there was preferential enhancement of a subset of small (Mr 14,000-20,000) acidic proteins in the authentic implantation sites. It is suggested that this embryo-dependent modification of constitutive changes associated with decidualization reflects a form of embryo-maternal signal-response mechanism that may be important for the process of implantation in mice.

Synthesis and secretion of stage-specific proteins by peri-implantation mouse embryos.

Blastocysts were recovered from intact mice at various times on the fourth and fifth days of pregnancy and incubated in vitro with 35S-methionine. Labeled proteins synthesized by the embryos and secreted into the medium were separated in two-dimensions on polyacrylamide gels by electrophoresis and localized by fluorography. The array of proteins synthesized and secreted by late stage blastocysts was found to be qualitatively and quantitatively different from those released by embryos at earlier stages of development. Similar changes were also observed in secreted proteins when delayed-implanting embryos were reactivated after an injection of estrogen. Furthermore, there was a temporal correlation between the appearance of certain proteins secreted by the embryos and changes in specific proteins synthesized and released by the uterus. It is suggested that these various secreted proteins constitute a signal-response mechanism that is important for the process of embryo implantation in mice.

Implantation associated changes in uterine secreted proteins.

Cross sectional segments were removed from the uteri of pregnant and pseudopregnant mice and incubated in vitro with labeled amino acids. Proteins that were synthesized and secreted by these explants were separated by two-dimensional polyacrylamide gel electrophoresis and localized by fluorography. As expected, the overall amount of labeled protein secreted into the medium was greater with tissue removed from implantation sites, or an artificially induced deciduoma, than it was with tissue from non-decidualized areas. It was not possible, by means of visual examination of the gels, to determine whether qualitative changes occurred in the patterns proteins that were released. However, by means of dual label ratios, it was demonstrated that the synthesis and secretion of individual proteins was regulated differentially. Furthermore, it was found that when the stimulus for decidualization was a normal implanting embryo, the pattern of protein enhancement was different than it was with an intrauterine thread. The finding that the uterus can distinguish between these two deciduogenic stimuli implies that unique signal factors, which may be important for the establishment of pregnancy, are released by the mouse embryo before implantation.

Changes in the rate of translation with reactivation of delayed implanting mouse embryos.

The transient embryonic diapause associated with delayed implantation in mice is characterized by decreases in the rates of synthesis of RNA and protein as well as a cessation of development. The present experiments were undertaken to examine the possibility that controls on protein synthesis at the level of translation of mRNA provide a regulatory mechanism in this situation. Rates of peptide chain elongation were determined in dormant embryos as well as in embryos that were reactivated either in vivo by estradiol-17 beta or by incubation in vitro. In dormant embryos the rate of peptide elongation was found to be approximately half that in active embryos. Although this change in translational efficiency appears to be sufficient to account for previously observed differences in overall rates of protein synthesis in dormant and reactivated embryos, the possibility that some changes also occur at the level of transcription during reactivation is not ruled out.

Actin mRNA content in normal and delayed implanting mouse embryos.

Actin mRNA levels were measured in mouse eggs, early embryos, and delayed implanting blastocysts by a homologous, cloned recombinant DNA probe and "dot" blot methodology. A maternal store of 431 fg of actin mRNA was observed in the unfertilized eggs. This mRNA pool decreased 12-fold by the mid-two-cell stage. Actin mRNA levels were then observed to increase progressively from the eight-cell to the blastocyst stage on a basis proportional to cell number. Late blastocysts contained 2400 fg actin mRNA per embryo (22 fg per cell). The cellular level decreased by about 20% in embryos induced into delay of implantation by ovariectomy of donor females. Reactivation of the delayed implanting blastocysts through hormonal manipulation in vivo or culture in vitro was accompanied by reestablishment of the level of cellular actin mRNA observed in normal blastocysts.

Effects of metabolic substrates and ionic environment on in-vitro activation of delayed implanting mouse blastocysts.

The possibility that the embryonic diapause associated with delayed implantation in mice is maintained by limitation of an essential amino acid, energy substrate or concentration of ions was examined by comparing the rates of DNA synthesis in delayed implanting embryos that were 'reactivated' by incubation in 'complete' medium or in one of several specially formulated 'deficient' media. It was found, in agreement with earlier observations, that an increase in the rate of DNA synthesis could be detected within 12 h and continued through 72 h in complete medium. An identical pattern was found when embryos were incubated in medium deficient in amino acids and vitamins. Similar patterns of activation were observed in the absence of all metabolizable substrates, a drastically reduced concentration of Na+, and even in a medium consisting only of 25 mM-bicarbonate buffer, NaCl and KCl. The embryos incubated in the more drastically deficient media appeared to be damaged after 18-24 h. Nevertheless, the observation that the rate of DNA synthesis did not remain depressed suggests that such deficiencies are not the means by which embryonic dormancy is maintained in utero.

Regulation of glycolysis in the mouse blastocyst during delayed implantation.

The rate of oxidation of glucose is reduced in mouse embryos in the prolonged free living phase associated with delayed implantation and increases when the embryos are reactivated by estrogen. To determine how these changes in metabolism are regulated, several aspects of glucose metabolism were evaluated in dormant and reactivated blastocysts: 1) Embryos were exposed to 14C-pyruvate in vitro and evolved 14CO2 was measured. It was found that the rate of production of CO2 was equal in the two types of blastocysts, suggesting that aerobic pathways are fully functional during delayed implantation. 2) Production of lactate in the presence of O2 was measured and a decrease of 30% was found in delayed implanting embryos, suggesting that the overall regulatory mechanism for glucose metabolism resides in the glycolytic portion of the pathway. 3) Capacity for uptake and phosphorylation of glucose was evaluated using 3H-2-deoxyglucose and was found to be equal in the two types of embryos. 4) Total amounts of the rate-controlling enzymes for glycolysis (i.e., hexokinase and phosphofructokinase) in lysates of delayed and reactivated embryos were found to be equal, indicating that amounts of these enzymes are not limiting in delayed implantation. 5) Lactate production, measured under anaerobic conditions, was found to be equal, demonstrating that it is not the capacity for glycolysis but a difference in the degree of allosteric inhibition that is responsible for reduced glucose oxidation in delayed implantation. 6) Levels of ATP, ADP, and hexose-6-phosphates were found to be consistent with allosteric inhibition of the glycolytic pathway at phosphofructokinase during delay and a release of this inhibition with reactivation.(ABSTRACT TRUNCATED AT 250 WORDS)

Resumption of DNA synthesis in delayed implanting mouse blastocysts during activation in vitro.

The resumption of DNA synthesis in delayed implanting mouse embryos undergoing metabolic activation in vitro was examined. Blastocysts were recovered from ovariectomized mice, incubated for various intervals in basal Eagle's medium, exposed to 3H-thymidine, and prepared for light microscopic autoradiography. Following incubation the proportion of labeled cells increased from 4% at 1 hr to 30% by 24 hr. This increase in labeling was not uniform in all regions of the blastocyst, i.e., labeling was initially highest over the inner cell mass (ICM) but remained low over the polar and proximal mural trophoblast for 6 and 12 hr, respectively, and then began to increase. This pattern in the resumption of DNA synthesis during activation in vitro is similar to that reported in vivo (Given and Weitlauf, '81) and suggests that the mechanism responsible in intrinsic to the blastocyst rather than being a differential response to the intrauterine milieu. Furthermore, it appears that the ICM may play an essential role in the resumption of synthesis in the surrounding trophoblast.

In-vitro zona-lytic activity in uterine fluid from ovariectomized mice treated with oestradiol-17 beta and progesterone.

Mouse embryos collected before implantation were incubated in vitro for 24 h with fluid rinsed from the uteri of ovariectomized female mice injected with progesterone, oestradiol-17 beta + progesterone, oestradiol-17 beta + progesterone, or oestradiol-17 beta alone. Although none of the zonae was completely dissolved, those incubated in fluid from animals treated with oestradiol + progesterone were subsequently more soluble in sodium thiocyanate (NaSCN) than those incubated similarly in control buffer, indicating a sublytic change during the incubation with uterine washings. Zonae incubated in fluid from animals injected with either hormone alone did not undergo such a change.

Regional changes in the binding of [3H] concanavalin A to mouse blastocysts at implantation: an autoradiographic study.

Implanting and delayed-implanting mouse blastocysts were incubated in vitro with [3H] concanavalin A (Con A), and the distribution of binding on their surfaces was determined by light microscopic autoradiography. The density of binding was uniform on the trophectoderm of delayed-implanting embryos and was not changed on the polar surface of implanting embryos. However, binding was reduced on the proximal mural and distal mural trophectoderm of implanting blastocysts by 36% and 60%, respectively. These results suggest that there is a regional reduction in the density of mannose-like sugars on the surface of mouse blastocysts at the time of attachment and implantation.

Resumption of DNA synthesis during activation of delayed implanting mouse blastocysts.

Cell division ceases in mouse blastocysts during the extended dormant period associated with delayed implantation but resumes following activation of the embryos by administration of 17 beta-estradiol to the mother. To determine the temporal and spatial aspects of the resumption of DNA synthesis during activation, blastocysts were recovered from delayed implanting females at various intervals after an injection of 17 beta-estradiol, incubated with 3H-thymidine in vitro, and prepared for light microscopic autoradiography. Although less than 4% of the cells were labeled in delayed implanting embryos, the proportion of labeled cells increased soon after the administration of 17 beta-estradiol and reached a maximum of over 50% by 24 hours. This increase in labeling was not uniform in all regions of the embryo, i.e., the labeling index of the inner cell mass began a steady increase immediately after the injection of 17 beta-estradiol while labeling of the polar and proximal mural trophoblast remained depressed for 6 and 12 hours, respectively, and only then began to increase. No labeling was present over the distal mural trophoblast in delayed implanting or activated blastocysts although cytological changes characteristic of primary giant cell transformation were present in activated embryos. These results indicate that the resumption of DNA synthesis is part of the overall increase in metabolic activity associated with activation. Furthermore, the sequential pattern of resumption of synthesis suggests that the ICM may influence the initiation of DNA synthesis in the surrounding trophoblast.

The volume of uterine fluid in 'implanting' and 'delayed implanting' mice.

A variation of the dye dilution technique was used to determine the volume of uterine fluid in 'implanting' and 'delayed implanting' mice. The method involves rinsing Krebs-Ringer-bicarbonate buffer containing [methyl-14C]methylated-BSA through the uterine lumen and using the resulting decrease in concentration of the BSA to calculate the volume of uterine fluid. The results indicated that the volume of uterine fluid was essentially the same in 'implanting' and 'delayed implanting' mice (i.e. 300-400 nl/pair of uterine horns). The total amounts of a substance recovered by rinsing the uteri would therefore provide an estimate of relative concentrations in situ.

Lysis of the zona pellucida and attachment of embryos to the uterine epithelium in ovariectomized mice treated with oestradiol-17 beta and progesterone.

Preimplantation mouse embryos with intact zonae pellucidae were transferred into the uteri of ovariectomized females treated with progesterone, oestradiol-17 beta plus progesterone, or oestradiol-17 beta alone; the disappearance of zonae from the uterine lumen was used to determine the presence of 'zona-lytic' activity in situ. Lysis of zonae did not occur in animals treated with progesterone or oestradiol-17 beta alone. However, lysis did not occur when oestradiol-17 beta was combined with progesterone; 'zona-lytic' activity reached peak levels within 12 to 24 h, then decreased. Attachment of embryos to the uterine epithelium occurred only in animals treated with oestradiol-17 beta plus progesterone and was initiated at about the time of peak 'zona-lytic' activity. It is suggested that the chymotrypsin-like enzyme activity present in uterine fluid is involved with the initiation of implantation.