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Several studies have suggested that the granulated metrial gland (GMG) cells of the metrial gland (MG) may be natural killer (NK)-like cells. The cytotoxicity of NK cells involves secretion of a pore-forming protein termed perforin, which can polymerize on the target cell membrane to form transmembrane pores that are thought to be involved in target cell death. In the present study, we used an antiserum against perforin to determine whether this protein can be detected immunohistochemically in GMG cells. Mouse uteri were fixed by vascular perfusion with several fixatives on Day 14 of pregnancy, and tissue sections were labeled by an indirect immunofluorescence method. Specific perforin labeling was detected in GMG cells throughout the MG, in the decidua basalis, and in the labyrinthine placenta. The presence of perforin in GMG cells supports the suggestion that they may be NK-like cells.

A previous study demonstrated that mouse uterine horns contained bacteria of several species on the morning after mating, and immunolabeling showed that many of these bacteria were coated with immunoglobulins. In the present study we used an ELISA technique to detect naturally-occurring antibodies against bacteria in mouse uterine luminal fluid, vaginal washings and serum. Each fluid contained specific IgA and/or IgG antibodies to five of the six bacterial species recovered from the uterus after mating. The uterine fluid antibodies that bound to the bacteria were mainly IgA molecules, while those in the serum were mainly IgG. Naturally-occurring bacterial antibodies in mouse uterine luminal fluid may play a role in protecting the endometrium against microbial infections.

The mucosal immune system in the male rodent urogenital tract was studied by localizing secretory component (sc) in the rat and immunoglobulin A (IgA) in both rat and mouse by immunofluorescence. In the rat, bright labelling of sc was observed at several sites, including the ejaculatory ducts, excretory ducts of several accessory glands, and urethral glands in the pelvic and bulbous portions of the urethra. Pale labelling of sc was detected in epithelial cells of the ventral prostate gland. Plasma cells containing IgA were only observed in the urethral gland in the bulbous portion of the urethra in rats and mice. These results suggest that IgA may be transported into the urogenital tract of the male rat primarily at sites distal to the production of seminal fluid and spermatozoa. While locally synthesized IgA may be available in the bulbous urethra, it appears that serum may be the main source of IgA for transport into the rat urogenital tract at the other sites where its receptor, sc, was demonstrated.

Bacteria of several species were present in the mouse uterus on the morning after mating, as demonstrated by bacterial cultures and microscopic examination of Gram-stained uterine luminal contents. The similarity between bacteria cultured from the vagina before mating and from the uterus after mating suggested that bacteria were introduced into the uterus from the vagina, possibly by coitus. The bacteria were cleared from the uterus about two days after mating. Immunohistochemical labeling of smears of the luminal contents on the morning after mating demonstrated IgA, IgG, and possibly IgM bound to many of the bacteria. The bacteria were often agglutinated, and there was a correlation between the intensity of immunoglobulin labeling on bacteria and the extent of agglutination. The amount of antibody bound to bacteria in multiparous mice was about the same as in mice that had not been mated previously. We observed both IgG and IgA on bacteria when organisms from vaginal cultures were incubated for 60 min in the uteri of estrogen-primed, virgin, female mice. This indicated that the uterus was the source of at least part of the immunoglobulins bound to bacteria. We did not demonstrate that the immunoglobulins bound to bacteria were specific anti-bacterial antibodies, but the binding persisted through three washing steps and there was no immunoglobulin binding to sperm in the same preparations. Neutrophils in the uterine lumen on the day after mating contained phagocytosed bacteria. These results suggest that the secretory immune system in the female mouse reproductive tract may play a role in returning the uterus to an aseptic state after mating by at least three mechanisms: direct blocking of attachment sites involved in bacterial binding to mucosal epithelium, agglutination of bacteria and thus reduction in the number of organisms available for binding to the epithelium, and opsonization of bacteria for phagocytosis by neutrophils.

Throughout the second half of pregnancy in mice there were many plasma cells containing immunoglobulins A (IgA) and G (IgG) in the uterine endometrium. There was intense staining of IgA in uterine glands at all stages, but little staining of IgG. The staining of both immunoglobulins (Igs) in the luminal epithelium was moderate to dark on day 11, slight on day 14, and increased from day 16 to term. From day 14 to term the endometrium exhibited folds or villi around each placenta. The cores of the villi contained many plasma cells of both isotypes, and the staining of extracellular Igs in the villous cores was darker than in nonvillous endometrium. Both Igs were detected in the uterine lumen, and in visceral and parietal yolk sac endoderm cells at all stages. Near term, the staining of Igs in the visceral yolk sac was darkest in the peripheral villous portion adjacent to the endometrial villi. From day 14 to term IgG was present in the visceral yolk sac mesenchyme and embryo, consistent with its transfer from the uterine lumen to the embryo via the vitelline circulation. In contrast, IgA was not detected in yolk sac mesenchyme until day 19, when only slight staining was observed, and IgA was never detected in the embryo. Most trophoblast giant cells contained both Igs on day 11. During the remainder of pregnancy, there was staining of both Igs in labyrinthine trophoblast and in a few giant cells adjacent to the parietal yolk sac on the placenta, but there was negligible staining in the spongiotrophoblast region. Our observations suggest that the local immune system in the mouse uterus may protect the embryo during the second half of pregnancy by secreting anti-microbial immunoglobulins A and G into the uterine lumen surrounding the visceral yolk sac, and may at the same time contribute to the transfer of maternal IgG to the embryo via the yolk sac and vitelline circulation.

We have localized horseradish peroxidase (HRP) in the mouse uterus after intravenous administration on days 1 and 5 of pregnancy in an effort to understand how serum proteins reach the uterine lumen. Direct movement of HRP into uterine and glandular lumina was blocked by the epithelial tight junctions on both days. In luminal and glandular epithelial cells at both times, HRP was localized in endocytic vesicles along the basolateral membranes, multivesicular bodies (mvb), elongated dense bodies below the nucleus (bdb), and many small vesicles near the apical surface of the cells. The uptake of HRP was most extensive in the luminal epithelium on day 1: the number of tracer-containing apical vesicles and bdb was largest, and there were also clusters of vesicles containing the tracer above the nucleus. Acid phosphatase was localized on day 1 in mvb and bdb in both cell types, indicating that these structures are lysosomes. It appeared that HRP followed two pathways after basolateral endocytosis by the epithelial cells: it was transported to the apical region of the cells, where it was present in small vesicles that may release their contents into the uterine or glandular lumina, or it was transported to lysosomes. To investigate whether macromolecules may be transported from the uterine lumen to the stroma, we also studied endocytosis at the apical pole of luminal epithelial cells after intraluminal injection of HRP. There was no detectable uptake of HRP from the lumen on day 1, and no tracer was detected in the intercellular spaces or basement membrane region. On day 5, a large amount of HRP was taken up from the lumen into apical endocytic vesicles, mvb, and dense bodies, but tracer was not present in the Golgi apparatus, lateral intercellular spaces, or the basement membrane region at the times studied. These observations indicate that there was no transport of luminal macromolecules to the uterine stroma on day 1, while the possibility of transport on day 5 requires further study.

Mice were immunized with a protein antigen, horse ferritin, by eight different routes and the immune responses in the reproductive tract were compared by measuring specific IgA and IgG in vaginal fluid and by localizing anti-ferritin plasma cells in uterine horns, cervix and vagina. The eight routes of immunization were: subcutaneous with Freund's adjuvant (s.c.), intragastric (i.g.), intravaginal (i.v.), s.c.-i.g., s.c.-i.v., i.g.-i.v., i.v.-i.v. and s.c.-i.g.-i.v. The largest overall response, considering both IgA and IgG antibodies, was obtained by s.c. priming with ferritin in adjuvant followed by i.v. boosting. Intravaginal immunization also boosted priming by the i.g., s.c.-i.g. and i.v. routes, but the response to i.v. immunization alone was weak. All i.v. immunizations stimulated mainly IgA antibody responses in vaginal fluid. Specific plasma cells, mostly of the IgG isotype, were present in the vaginal fornix of several mice in the s.c.-i.v. and s.c.-i.g.-i.v. groups, but none were detected there in any other group and they were only rarely observed in the uterine horns. The results provide data on the relative effectiveness of different routes of immunization in producing a humoral immune response in vaginal fluid against a non-replicating antigen.

The present investigation studied the effects of sperm immunization in the gastrointestinal tract on anti-sperm antibody production and fertility in female mice. For comparative purposes, mice were also immunized with sperm intraperitoneally. Intraperitoneal immunization with 5 X 10(6) washed epididymal and vas deferens sperm 3 times per week for 7 wk produced anti-sperm IgG in plasma at 1:20,000 and in vaginal washings at 1:100 as determined by ELISA. Such mice have been shown previously to have reduced fertility. In comparison, mice immunized intragastrically with 5 X 10(6) sperm once per week for 11-14 wk had anti-sperm IgA in vaginal washings at only about 1:8 as determined by ELISA. After mating at the 14th wk these mice delivered 6.5 +/- 1.4 pups, which was not significantly different from the 7.1 +/- 1.1 pups delivered by an untreated control group. Mice immunized twice intragastrically and once intravaginally during a 25-day period had no detectable anti-sperm IgA in vaginal washings by ELISA. These mice delivered 9.7 +/- 1.2 pups after mating beginning on day 32, as compared to 9.7 +/- 0.8 pups in a PBS-sham immunized group. Mice immunized once intraduodenally and then once intraperitoneally 14 days later delivered 10.4 +/- 0.9 pups after mating 10-14 days after the second immunization, while a similar group of mice whose primary sperm immunization was directly into Peyer's patches delivered 9.0 +/- 1.4 pups. We could not detect anti-sperm IgG or IgA bound to sperm in the uterine or oviduct lumen using immunohistochemical labeling after any of the groups of immunized mice were mated.(ABSTRACT TRUNCATED AT 250 WORDS)

The ingestion of ferritin injected into the uterine lumen for 20 min was followed by light and electron microscopy. Uptake occurred on Days 4 to 6, with a distinct peak of activity on Day 5 when blastocysts are present in the uterine lumen. At this time, the antimesometrial cells took up more tracer than those on the mesometrial side. The ferritin was located in various types of organelles which contained acid phosphatase, indicating that the tracer entered the lysosomal system. Ferritin-containing lysosomes were essentially all located in the apical half of the cells at 1 h after the injection, but thereafter were distributed throughout the cell. There appeared to be some digestion of ferritin by 48 h, but no evidence for exocytosis of the tracer at the base of the cells was found with light or electron microscopy.

The transport of immunoglobulins into the lumen of the female reproductive tract is not well understood, especially in the case of IgG. In mice, there are conflicting reports concerning the presence of immunoglobulins in uterine luminal and glandular epithelial cells, and immunoglobulins have not been detected in the luminal epithelial cells of the oviduct. In the present study we detected both IgA and IgG in uterine luminal and glandular epithelial cells on day 1 of pregnancy by immunolabeling. Also, we observed that fluorescein-conjugated mouse and bovine IgG and other proteins were taken up into vesicles in uterine luminal and glandular epithelial cells after intravenous administration. These observations indicate that both kinds of epithelial cells take up immunoglobulins from the interstitial fluid on day 1 of pregnancy, and that the cells may therefore be involved in the transport of immunoglobulins and other proteins to the uterine lumen at that time. In the oviduct, we detected IgA and IgG in vesicles in the luminal epithelial cells of the preampulla by immunolabeling, and we observed fluorescein-conjugated IgA and IgG in similar vesicles after intravenous administration. The presence of IgA and IgG in vesicles in the epithelial cells of the preampulla, together with the previous demonstration of plasma cells of both isotypes and large amounts of interstitial immunoglobulins in the lamina propria of this segment, suggests that the preampulla of the oviduct may be an important site for the local immune system in the mouse female genital tract.

There was intense labelling of secretory component (sc) in the glandular and luminal epithelia of the uterine horns at pro-oestrus, oestrus and Day 1 of pregnancy, but at other stages labelling was weak or undetectable. There was also intense labelling of sc in the superficial layer of cells in the stratified epithelia of the cervix and vagina at pro-oestrus and Days 4-7 of pregnancy, but not at other stages. Plasma cells containing immunoglobulin A (IgA) were not observed in any region of the genital tract at any of the times studied. The presence of sc coupled with an absence of IgA-containing plasma cells suggest that IgA in genital tract secretions of the female rat may be derived mainly from serum.

An ultrastructural study of mouse and rat embryo implantation sites was undertaken to determine whether the uterine luminal epithelial cells surrounding the blastocyst exhibited the morphologic characteristics of apoptotic or necrotic cell death. In both species the epithelial cells exhibited all of the characteristics of apoptosis, including surface blebbing, shrinkage and fragmentation of the cells, condensation of chromatin, and indentation and fragmentation of nuclei. Cytoplasmic organelles remained morphologically intact, and the cytoplasm maintained normal or increased staining density. Also, the epithelial cells and cell fragments were phagocytosed by the adjacent trophoblast cells. The epithelial cells did not exhibit the characteristics of necrotic cell death, such as swollen cells and mitochondria, damaged surface membranes, and disintegrated cytoplasmic organelles. We conclude that uterine epithelial cells surrounding mouse and rat embryos during implantation undergo apoptotic cell death leading to their phagocytosis by trophoblast cells.

Prostaglandins (PGs) appear to have a role in the appearance of the increased uterine vascular permeability and subsequent decidualization observed at implantation in many species. However, the sites of production of these PGs have not been clearly established. To clarify the PG synthetic capacity of the blastocyst and the various types of cells in the uterus at implantation, we have studied the immunohistochemical localization of PG synthase in the rat blastocyst on Days 5 to 7 and uterus on Days 1, 4, 5, 6, and 7 of pregnancy. Labeling of PG synthase was negligible in the uterus on Day 1 of pregnancy. On Day 4, there was increased labeling in the luminal and glandular epithelium, in stromal cells adjacent to the luminal epithelium, and in blood vessels and some leukocytes. PG synthase was detected in the blastocysts on Days 5 to 7, but there was a gradual loss of label in the luminal and glandular epithelial cells during this period. Early differentiating stromal cells adjacent to the luminal epithelium in the implantation site on Day 5 showed bright labeling, whereas peripheral stromal cells were only slightly labeled. By Day 7, the differentiated cells of the primary decidual zone showed little or no label, but cells in the secondary decidual zone were brightly labeled. These results indicate that PG synthase is present in the rat blastocyst and in several kinds of uterine cells, and that its localization in uterine cells changed markedly during the implantation process.

Ovariectomized rats treated with progesterone and estradiol-17 beta in sequences that prepared the uterus for implantation showed marked increases in the number of endocytotic invaginations in the basal membranes of luminal epithelial cells and in the amount of intravenously administered horseradish peroxidase taken into the cells. Tight junctions between the epithelial cells blocked the direct passage of horseradish peroxidase into the uterine lumen. The number of basal pinocytotic invaginations in glandular epithelial cells was relatively small and did not increase following hormonal stimulation. The probable occurrence of a pathway for the transepithelial transport of macromolecules from the blood or stroma into the uterine lumen, and its sensitivity to stimulation by ovarian hormones, suggests a likely mechanism for altering the molecular environment of the embryo during the implantation period.