The Pre-Implantation Embryo Induces Uterine Inflammatory Reaction in Mice.

It has been well established that uterine function during the peri-implantation period is precisely regulated by ovarian estrogen and progesterone. The embryo enters the uterine cavity before implantation. However, the impact of pre-implantation embryo on uterine function is largely unknown. In the present study, we performed RNA-seq analysis of mouse uterus on day 4 morning of natural pregnancy (with embryos in the uterus) and pseudo-pregnancy (without embryos in the uterus). We found that 146 genes were upregulated, and 77 genes were downregulated by the pre-implantation embryo. Gene ontology and gene network analysis highlighted the activation of inflammatory reaction in the uterus. By examining the promoter region of differentially expressed genes, we found that NF-kappaB was a causal transcription factor. Finally, we validated 4 inflammation-related genes by quantitative RT-PCR. These 4 genes are likely the main mediators of the inflammatory reaction in the uterus triggered by the pre-implantation embryo. Our data indicated that the pre-implantation embryo causes uterine inflammatory reaction, which in turn might contribute to the establishment of uterine receptivity and embryo implantation.

The influence of mating on estrogen receptor alpha protein level in spleen and uterine macrophages in female mice.

Macrophages are antigen-presenting cells that have a key role in the regulation of immune phenomena and are responsible for the recognition of paternal antigens during pregnancy. The aim of this study was to investigate changes in the estrogen receptor alpha (ERalpha) protein level in splenic and uterine mature (F4/80(+)MHC II(+)) and immature (F4/80(+)MHC II(-) ) macrophages in female mice during time corresponding to the preimplantation period. C57BL/6J females in estrus were mated with Balb/c male mice or were mechanically stimulated through the vagina to achieve pseudopregnancy. Uterine and spleen cells were isolated on days 0.5 and 3.5 after mating or after uterine cervix stimulation. ERalpha content in macrophages was measured by flow cytometry and expressed as mean fluorescence intensity (MFI). The ERalpha level in splenic macrophages on 0.5 day after mating was higher than that in splenic macrophages of pseudopregnant mice on 0.5 day after stimulation. The ERalpha level was also higher in mature than in immature macrophages present in both the spleen and uterus, especially in mated mice. In the spleen, a correlation was found between the percentage of mature macrophages and ERalpha level in these cells. In conclusion, the elevated alpha level observed shortly after mating in splenic but not in uterine macrophages indicates an early systemic response to male antigens.

Cytokine production of in vitro stimulated peripheral lymphocytes during the course of pregnancy and pseudopregnancy in the rat.

PROBLEM: Does maternal lymphocyte cytokine production after in vitro stimulation vary with the stage of pregnancy in the rat? METHOD OF STUDY: Blood samples were taken during the estrus cycle in rats (n = 11). Thereafter, rats were rendered pregnant (n = 6) or pseudopregnant (n = 5) and blood samples were taken at days 4, 8, 11, 15, and 20 of pregnancy and pseudopregnancy. White blood cell (WBC) count was measured and whole blood was stimulated with phorbol 12-myristate 13-acetate and calcium ionophore; interferon-gamma (IFNgamma) and interleukin-4 (IL-4) production as well as (sub)populations of lymphocytes were measured using flow cytometry. RESULTS: We observed an increase of WBC in the second week of pregnancy and a slowly decreasing percentage of lymphocytes during the course of pregnancy. The percentage IFNgamma producing T-lymphocytes after in vitro stimulation was increased during pregnancy (for Th-lymphocytes only in the second week of pregnancy, for Tc-lymphocytes at all days). This increased IFNgamma production in pregnant T-lymphocytes was accompanied by an increase during pseudopregnancy, and therefore may result from increased sex hormone concentrations. The percentage IFNgamma producing natural killer (NK) cells after in vitro stimulation was decreased on day 20 of pregnancy. No effect of pregnancy or pseudopregnancy was seen on percentage IL-4 producing lymphocytes after in vitro stimulation. CONCLUSION: In the rat the IFNgamma production after in vitro stimulation varies during pregnancy and is increased, rather than decreased, during pregnancy.

Total white blood cell counts and LPS-induced TNF alpha production by monocytes of pregnant, pseudopregnant and cyclic rats.

Pregnancy in the rat may be associated with an activated innate immune system. Therefore, we investigated monocyte function as well as total white blood cell (WBC) counts during the follicular phase of the ovarian cycle, pregnancy and pseudopregnancy in the rat. Rats were equipped with a permanent jugular vein cannula, and 0.43 ml blood samples were taken from this cannula during the 4 days of the regular oestrus cycle of the rat (n=12). Thereafter, six rats were rendered pregnant, and the other six rats were rendered pseudopregnant according to standard methods. Blood samples were withdrawn from the cannula on days 4, 7 and 11 of pseudopregnancy and on days 4, 7, 11 and 20 of pregnancy. From each blood sample, 0.4 ml was stimulated with lipopolysaccharide (LPS) and monocyte intracellular cytokine production measured using flow cytometry. 30 microl of the blood was used to measure WBC counts and differential WBC counts. The results showed that the number of WBC was significantly increased only on day 11 of pregnancy compared with the follicular phase, and that this was due to the increased numbers of polymorphonuclear (PMN) cells. The percentage of TNF alpha-producing monocytes was increased on all days of pseudopregnancy and on day 11 of pregnancy. The fact that the percentage of monocytes producing TNF alpha upon an LPS stimulus was increased during the post-implantation phase of pregnancy and during pseudopregnancy as compared to the follicular phase may indicate that these conditions are proinflammatory conditions. For the post-implantation phase of pregnancy, this is once more stressed by the increased numbers of WBC and PMN.

Distribution patterns of leucocyte subpopulations expressing different cell markers in the cumulus-oocyte complexes of pregnant and pseudopregnant mice.

The nature of leucocyte subpopulations expressing different cell markers around the cumulus-oocyte complex (COC) of pregnant and pseudopregnant mice was investigated in the present study. Immunolabelling for CD4, CD8, CD14, CD45 and CD163 and transmission electron microscopy were used to determine whether leucocytes differ between pregnant and pseudopregnant mice. Sexually mature female BALB/c mice (n = 36; 18 pregnant, 18 pseudopregnant) were stimulated to superovulate with pregnant mare's serum gonadotropin and human chorionic gonadotrophin, then mated with either fertile or vasectomised males. Postovulatory oocytes were collected after mating. The cumulus cell masses of the pregnant group contained spermatozoa between cells and were more variable than COCs of the pseudopregnant group. Streptavidin-biotin-peroxidase immunohistochemical labelling of the cell markers CD4, CD8, CD14, CD45 and CD163 showed that there were fewer leucocytes in the COCs of the pseudopregnant group compared with the pregnant group. Transmission electron microscopy revealed that often there were macrophage-like cells containing spermiophagic bodies between the cumulus cells. These observations suggest that, together with other cumulus cells and oviducal cells, these macrophage-like cells may be involved in removing unsuitable or excess spermatozoa and, therefore, in maintaining a suitable microenvironment for normal fertilisation.

Functional and structural regression of the rabbit corpus luteum is associated with altered luteal immune cell phenotypes and cytokine expression patterns.

Following attenuation of progesterone production corpora lutea are selectively cleared, a process associated with recruitment of macrophages. In the rabbit little is known about luteal immune cell phenotypes and expression of cytokines, which influence immune cells and resident luteal cells, during luteolysis. Consequently, we studied luteal immune cells by immunohistochemistry as well as luteal IL-10, TNFalpha, MCP-1, IFN-gamma, and IL-1beta mRNA expression by semiquantitative RT-PCR from day 8 to day 20 in pseudopregnant rabbits (d8-d20 p.hCG). Luteal function was assayed by serum progesterone levels. Functional luteolysis commenced by d14 p.hCG as indicated by attenuation of serum progesterone levels. X4(+) tissue macrophage levels increased transiently on d12 and d14 p.hCG, whereas CD5(+) T-cell levels transiently declined on these two days. CD68(+) macrophages increased progressively after d16 p.hCG. The luteal mRNA level of the anti-inflammatory cytokine IL-10 as well as the mRNA levels of the pro-inflammatory cytokines TNFalpha and MCP-1 increased after d16 p.hCG and remained elevated up to d20 p.hCG. IFN-gamma and IL-1beta mRNA expression did not vary systematically. In summary, luteolysis was associated with an initial transient increase of X4(+) macrophages and decrease of CD5(+) T-cells, and later recruitment of CD68(+) macrophages. During structural regression pro- and anti-inflammatory cytokines are upregulated possibly to control immune cell function.

Natural cytotoxic and antibody-dependent cellular cytotoxic activity of cells in the decidua basales and metrial glands of pseudopregnant rats with deciduomata.

Cytotoxic cells are present in the uterine wall of pregnant rats. To determine if the cytotoxic activity arises in response to semen or the products of conception, the profile of cytotoxic activity in deciduomata of pseudopregnant rats was examined. To examine NK activity, Yac-1 cells were used as targets in chromium release cytotoxicity assays and an antibody to Yac-1 cells was included in some assays to determine antibody-dependent cellular cytotoxic (ADCC) activity. Cells from the metrial glands and deciduae of deciduomata of rats at days 10 and 13 of pseudopregnancy did not show NK activity but ADCC activity was present. To examine natural cytotoxic (NC) activity, Wehi 164 cells were used as targets in chromium release cytotoxicity assays. Cells isolated from the metrial glands and deciduae of rats at day 10 of pseudopregnancy were able to kill Wehi 164 cells after 21 h assays, thus demonstrating NC activity. The profile of cytotoxic activity in the uterine wall of pseudopregnant rats with deciduomata is similar to that found in pregnancy and is thus independent of semen or the products of conception.

Administration of thymocytes derived from non-pregnant mice induces an endometrial receptive stage and leukaemia inhibitory factor expression in the uterus.

We have previously reported that intravenous administration of splenocytes prepared from mice in the early stages of pregnancy promoted embryo implantation in pseudopregnant mice. Since a T-lymphocyte-rich, but not a monocyte-rich preparation from splenocytes enhanced embryo implantation, similar effects of thymocytes from non-pregnant mice on implantation were examined in this study. Thymocytes were prepared from immature 21 day old ICR female mice and the supernatant of a thymocyte suspension (Th-sup) was used as the control. Thymocytes or Th-sup were injected into the caudal vein of recipient mice on pseudopregnancy day 2, and blastocysts were transferred into the endometrial lumen. The implantation rates per recipient were significantly higher in the thymocyte-treated group. ICR mice were then oophorectomized on pseudopregnancy day 3. After 3-day progesterone supplementation, blastocysts were transferred with intravenous injection of thymocytes or Th-sup. Under progesterone supplementation, successful implantations were observed in the thymocyte-treated group, but not in the Th-sup-treated group. Reverse transcriptase-polymerase chain reaction analysis revealed that mRNA expression of leukaemia inhibitory factor in the uterus was induced by thymocyte administration, but not by Th-sup. Thymocytes were divided into two populations, CD4(+/-)CD8(-) group and CD4(-)CD8(+/-) group, by separation columns. On pseudopregnancy day 2, the separated thymocytes in each group or their supernatant were injected into the endometrial stroma of the recipient mice, and blastocysts were transferred into the endometrial lumen. The administration of CD4(+/-) CD8(-) lymphocytes significantly promoted implantation rates, but no effect was observed in the CD4(-) CD8(+/-) group. These findings showed that thymocytes, especially CD4-positive lymphocytes, facilitate embryo implantation, probably by regulating endometrial differentiation.

The human corpus luteum: reduction in macrophages during simulated maternal recognition of pregnancy.

It has been shown that immune cells, particularly macrophages, accumulate in the corpus luteum during luteolysis. This study aimed to investigate the effect of maternal recognition of pregnancy on the localization and numbers of macrophages in the human corpus luteum. Corpora lutea (n = 12) were obtained from normally cycling women at the time of hysterectomy and were dated on the basis of serial urinary luteinizing hormone (LH) estimation. In addition, corpora lutea (n = 4) were collected from women who had received daily doubling doses of human chorionic gonadotrophin (HCG) to mimic the hormonal changes of early pregnancy. Macrophages were localized by immunohistochemistry using an anti-CD68 antibody. Steroidogenic cells, steroidogenic cells of thecal origin and endothelial cells were identified on serial sections by immunohistochemistry for 3beta-hydroxysteroid dehydrogenase, 17alpha-hydroxylase and von Willebrand factor, respectively. The luteal cells capable of responding directly to HCG were identified by isotopic in-situ hybridization for messenger RNA encoding LH/HCG receptors. Macrophages were localized primarily to the vascular connective tissue and theca-lutein areas of the corpus luteum, although some were found in the granulosa-lutein cell layer. Macrophage numbers increased throughout the luteal phase to a maximum in the late-luteal phase (P < 0.05). Luteal 'rescue' with HCG was associated with a marked reduction in the numbers of tissue macrophages when compared with those of the late-luteal phase (P < 0.001). One of the effects of HCG during maternal recognition of pregnancy is to prevent the normal influx of macrophages into the corpus luteum. As LH/HCG receptors localized to the steroidogenic cells, this implies a fundamental role for steroidogenic cell products in the control of macrophage influx into the human corpus luteum.

Phagocytotic activity of macrophages separated from corpus lutea of pseudopregnant rabbits.

Macrophage activity as indicated by phagocytosis in the corpus luteum at the luteolysis stage was examined in rabbits during pseudopregnancy. The day of induction of pseudopregnancy was regarded as day 0, and the phagocytotic activity of macrophages obtained from luteal tissues was examined on days 7 and 16. When sheep red blood cells (SRBCs) were used to assess phagocytotic activity there was no difference in phagocytosis between days 7 and 16 but, when sensitized SRBCs were used to assess phagocytotic activity, the activity of macrophages on day 16 was more than double that on day 7 (P < 0.05). These results indicate that immunomediated phagocytosis of luteal macrophages is enhanced at the luteolysis stage (day 16). This phenomenon may contribute to the process of luteolysis.

Elevated endometrial natural killer cell activity during early porcine pregnancy is conceptus-mediated.

This study investigated an extended time course of endometrial NK cell activity during gestation and the mechanisms underlying changes in uterine NK cell activity in pigs. Endometrial tissues were collected from cyclic, pseudopregnant and pregnant nulliparous pigs on various days post-estrus, and from pigs 10 days after insemination with seminal plasma or killed spermatozoa. NK effector cells were isolated from each endometrial sample, size fractionated and tested for cytolytic activity against NK target cells (K562) using chromium release assays and immunocytochemically for the frequency of perforin-positive cells. Various cell fractions showed different levels of NK activity and had different proportions of cells expressing perforin. Morphologically, cells in the fraction with maximal NK activity almost all showed typical lymphocyte size and shape. Substantially elevated NK cell activity was recorded in pregnant pigs on days 10 and 20 of gestation. By day 30, the cytolytic activity declined dramatically to an almost undetectable level. Very little activity was found in uterine cells isolated from cyclic, pseudopregnant, and seminal plasma or killed spermatozoa inseminated animals, and no differences were detected either between follicular and luteal phases of the estrous cycle or between different days of pseudopregnancy. These results indicate that elevated NK cell activity during early porcine pregnancy cannot be attributed to contributions from either the maternal systemic endocrine status or from components of boar semen. The changes in NK cell activity observed in porcine endometrial tissues during early pregnancy must therefore be associated with the actual presence of conceptuses.

Maternal alloimmunization in rat pregnancy: in vivo and in vitro studies of T-cell-dependent immunity to mating and third party alloantigens.

Rat pregnancy, experimental pseudopregnancy, and experimental decidua induction promote skin allograft survival of paternal and unrelated skin donors by specific and nonspecific mechanisms. Experimental pseudopregnant females were given allogeneic cells by two routes: intraperitoneal (IP) and in utero (IU). The females injected by the IU route, with allosensitized spleen cells, tolerated paternal skin allografts (average 26 days) better than those from other experimental groups and even from allopregnant females bearing a paternal allograft. The orthotopic immunization by the IU route, as shown by an enhancing effect of in utero culture medium injection, seemed important. In vitro studies revealed that the ability of subscapular lymph node (SCLN) cells draining the allograft to destroy Wistar/Furth (W/Fu) target cells decreases markedly in parallel with prolongation of skin allograft survival. Suppression of lymphocyte-mediated cytotoxicity against W/Fu target cells exceeded 70% when SCLN cells were used as responder cells. We propose that maternal T-cell immunization (via in utero) against fetal antigens occurs in pregnancy and plays a role in maintaining fetal tolerance, while specific cell-mediated cytotoxicity is impaired. Both specific and nonspecific local factors may promote such an enhancement of allograft survival.

Effect of treatment with methylprednisolone on duration of pseudopregnancy and on macrophages and T lymphocytes in rabbit corpora lutea.

The potential role of macrophages and T lymphocytes in the destruction of the corpus luteum at the end of the luteal phase was investigated by treating pseudopregnant rabbits with the immunosuppressant glucocorticoid methylprednisolone. Eleven specific pathogen-free New Zealand White rabbits were injected with pregnant mares' serum gonadotrophin (40 iu, i.m.), followed 2 days later by human chorionic gonadotrophin (40 iu, i.v.) to stimulate ovulation. The following day (day 1 of pseudo-pregnancy) all animals had an oestradiol-filled Silastic capsule implanted s.c., to ensure that oestradiol, the luteotrophic hormone in this species, would not be limiting. From day 10 of pseudopregnancy, three animals were injected with a low dose of methylprednisolone (2 mg kg-1 per day) until day 20. Three other animals were injected with a higher dose of methylprednisolone (20 mg kg-1 per day) from day 13 of pseudopregnancy until day 19. Five animals served as control, vehicle-injected animals. Blood samples were taken at intervals and assayed for progesterone. Immunofluorescence was used to stain luteal tissue for macrophages, T lymphocytes and class II antigens, and positive cells were counted under high-power magnification. Methylprednisolone treatment reduced (by about 70%), but did not eliminate, the macrophages in the regressing corpora lutea. In contrast, the high dose of methylprednisolone essentially eliminated T lymphocytes, and reduced (by about 90%) the number of cells expressing class II antigen in the luteal tissue. Despite the effects of methylprednisolone on these cells, serum progesterone profiles were not altered by treatment with methylprednisolone, and pseudopregnancy was of normal duration.(ABSTRACT TRUNCATED AT 250 WORDS)

Activation and distribution of inflammatory cells in the mouse uterus during the preimplantation period.

Northern blot analysis of mouse uterine RNA showed that IL-1 (alpha and beta), and TNF-alpha mRNA were abundant on day (D) 1 of pregnancy, reduced on D2, and remained basal throughout the remainder of the preimplantation period (D3 and D4). Elevated IL-1 beta and TNF-alpha mRNA levels on D1 were accompanied by increased levels of immunoreactive protein in uterine cytosol preparations as determined by ELISA. In situ hybridization detected IL-1 beta mRNA in cells located in the endometrial stroma and concentrated in subepithelial regions on D1. Immunocytochemical localization of IL-1 beta and TNF-alpha identified cells scattered throughout the endometrial stroma, but more concentrated in the subepithelial region on D1. On D3 and D4, cytokine-immunopositive cells decreased in number and became located predominantly at the endometrial-myometrial junction. Histochemical localization of peroxidase as a marker predominantly for eosinophils showed an abundance of these cells in the D1 uterus. The distribution of peroxidase-positive cells in the uterus followed the same temporal and spatial changes as cytokine-immunopositive cells during the preimplantation period. These data document the occurrence of an inflammatory response in the uterus on D1 of pregnancy, and demonstrate that as the preimplantation period progresses the distribution of inflammatory cells changes from the subepithelial region of the endometrial stroma to the periphery of the uterus at the endometrial-myometrial junction. Mechanisms regulating the uterine inflammatory response on D1 were investigated. Cytokine mRNA levels were not significantly elevated during the estrous cycle or after treatment of adult ovariectomized mice with estradiol-17 beta. In contrast, mating with vasectomized males resulted in an inflammatory response on D1 of pseudopregnancy similar to that on D1 of normal pregnancy, whereas mechanical stimulation of the uterine cervix failed to elicit such a response. These results strongly suggest a role for some factor(s) in the ejaculate, other than spermatozoa, in the initiation of a uterine inflammatory response after mating, but an effect of the act of mating cannot be excluded.

Leukocyte distribution in the pseudopregnant mouse uterus.

Pseudopregnancy, induced by mating females with vasectomized males, is a frequently used model for studying pregnancy-like uterine changes in the absence of an embryo. Leukocytes make a significant contribution to uterine cellularity during pregnancy. The present study was designed to determine whether changes in numbers and distribution of leukocytes in the uterus during pseudopregnancy and following intraluminal injection of a deciduogenic stimulus parallel changes observed during the first eight days of pregnancy. Common leukocyte antigen positive (CLA+) cells, macrophages (F4/80+ cells), and granulocytes were assessed between days 1 and 8 of pseudopregnancy using qualitative and quantitative immunohistochemistry. High numbers of CLA+ leukocytes were present on days 1 and 2. Those were comprised primarily by macrophages, polymorphonuclear leukocytes, and eosinophils. High concentrations of leukocytes were detected in the endometrium, and some granulocytes were observed migrating through the luminal epithelium. Leukocytes, principally macrophages, were reduced in number and were distributed throughout the endometrium on days 3 and 4. Introduction of oil to the uterine lumen on day 4 stimulated primary decidualization. Decidual cells were CLA- and F4/80-, and, as decidualization proceeded, CLA+ and F4/80+ cells decreased in number in the anti-mesometrial uterus and were detected primarily in the deep endometrium. Later, a secondary decidualization zone developed in the mesometrial aspect of the uterus. Unlike the initial decidual reaction, which was relatively free of leukocytes, the secondary decidual zone contained very high numbers of CLA+ and F4/80+ cells. The uninjected uterine horn remained relatively unchanged from days 3 through 8.(ABSTRACT TRUNCATED AT 250 WORDS)

Secretory function of cells in the rat metrial gland.

The immunochemical determination of antigens, products of the secretory activity of metrial gland cells, in the serum of pregnant rats is described. In order to prepare a specific immune antiserum, rabbits were immunised with homogenates of the rat metrial gland removed at day 15 of pseudopregnancy. The resultant immune antisera were adsorbed by glutaraldehyde-polymerised sera from nonpregnant rats until the Ouchterlony test ceased to give a positive reaction with sera of nonpregnant rats. These adsorbed specific sera continued to react with sera of pregnant and pseudopregnant animals, indicating that the latter both contain one or more antigens identical to those produced by the cells of the metrial gland, and against which the specific antisera had been raised. When 10% and 15% fresh serum from nonpregnant females was added to molten agar or was initially dropped into wells in the agar, the positive reaction between the specific antisera and the sera of pregnant and pseudopregnant rats was not eliminated. An antigen identical to the serum antigen was also found in amniotic fluid, in sera, and in extracts of metrial glands from pregnant and pseudopregnant laboratory animals. The data obtained indicate that cells in the metrial gland have a secretory function. It is proposed that the serum antigen of pregnant and pseudopregnant rats is secreted by the granular cells in the metrial gland.

Hormonal control of a carbohydrate epitope involved in implantation in mice.

Ovariectomy and hormone replacement of mice were used to examine the hormonal control of expression on the uterine surface of a carbohydrate determinant (lacto-N-fucopentaose I, LNF I), involved in the initial interaction between the blastocyst and the endometrial epithelium at implantation. Pseudopregnant mice mated with sterile males were also used to elucidate the impact of embryonic signals on the expression of this antigen on the uterine surface. Two groups of fucosylated structures could be distinguished; one group was predominantly dependent on maternal oestrogen and progesterone, while the other group appeared to be less influenced by the hormonal milieu.

Alteration in platelet count during early pregnancy in the mouse.

Published reports of pregnancy associated thrombocytopenia in mice have utilized the Quackenbush strain. The inability of some laboratories to verify this observation in other mouse strains prompted us to report our findings by using Swiss Albino ICR mice. In Exp. 1, pregnant and pseudopregnant mice were bled prior to mating (time 0) and daily on day 1 (vaginal plug) through day 7. In Exp. 2, media from 24 hr cultures of 2-cell mouse embryos or media from unfertilized oocytes were injected into splenectomized mice. Animals were bled at time 0 (before injection) and at 30, 60, and 120 min after injection. In Exp. 3, splenectomized mice were treated with either media from 2-cell stage embryos or with media supplemented with synthetic platelet-activating factor (PAF: 0.05, 0.1 or 0.2 micrograms). Animals were bled as in Exp. 2. Platelet numbers were determined in duplicate from each blood sample by using a hemacytometer. In Exp. 4, antagonist (SRI 63-441) or vehicle was administered to mated mice on days 1 through 4 of pregnancy. Animals were examined on day 8 to determine number of developing conceptuses. In Exp. 1-3, data were analyzed by using ANOVA for repeated measures, and in Exp. 4 data were analyzed by chi-square analysis. In Exp. 1, there was a treatment x time interaction (P less than .06) due to transient thrombocytopenia in pregnant but not pseudopregnant mice.(ABSTRACT TRUNCATED AT 250 WORDS)

Modulation of the immunosuppressive activities by blastocoelic fluid during rabbit pregnancy.

Temporal variation in immunosuppressive activity was determined in biological samples such as embryo-foetal fluids (blastocoelic- or amino-allantoic fluid) and blood collected from pregnant and pseudopregnant rabbits. Each of the fluids to be analyzed was pre-incubated with mitogen stimulated human lymphocytes for 48 h and then inhibition of [3H]thymidine incorporation or IL-2 receptor expression was estimated. Both means of assessing immunosuppression indicated variations in the suppressive activity throughout pregnancy. This was observed in embryo-foetal fluids but not in autologous peripheral blood nor in homologous pseudopregnant blood. At days 9-13 of pregnancy, the immunosuppressive effects of blastocoelic fluids were higher than that of the autologous sera, reached a peak at days 12 and 13 and declined thereafter, to reach the lowest levels. In order to further characterize the biological activity of day-12 blastocoelic fluid and autologous serum, they were submitted to ultracentrifugation. No suppressive activity could be demonstrated in the lipoprotein fractions. But all the activity was found in the protein fraction. Precipitation with cold ethanol confirmed that the biologically active compound was a protein. Furthermore, results obtained after ultrafiltration suggest biologically active compounds of high mol. wt (greater than 300 kDa). From the above findings, we can suggest that in the rabbit, there is no pregnancy specific systemic immunosuppression. We can also infer that (1) the immuno-tolerance of the mother towards the embryo is more due to a localized effect; (2) this effect decreases with the progression of gestation and (3) a high mol. wt factor is responsible for the immunosuppression.

Defective anti-listerial responses in deciduoma of pseudopregnant mice.

Two different hormonal regimens to induce pseudopregnancy resulted in a pronounced increase in the susceptibility of the murine uterus to intraluminal injections of Listeria monocytogenes. Preimmunization, which profoundly augments systemic listeria resistance, had no effect on this increased uterine susceptibility. Anti-listerial responses in other organs were unaffected by pseudopregnancy. Animals manifesting increased susceptibility formed distinct uterine swellings in response to the combination of hormones and uterine listeria. These swellings correspond to previously described deciduoma and closely mimic the decidualized endometrium of pregnancy. The nature of the defective response to listeria was investigated by immunocytochemistry. Increased bacterial titers were correlated with an inability of macrophages and T lymphocytes to reach tissue listeria in discrete regions of deciduoma-bearing uteri. Control uteri showed a normal granulomatous pattern of inflammation. These findings closely parallel previous findings in the murine decidua basalis and suggest that properties of decidualized endometrial stromal cells regulate local immune responsiveness.