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Specific polyclonal rabbit anti-human renin substrate-antibodies were used in order to study the distribution of renin substrate immunoreactivity in human fetal and placental tissue. Renin substrate was immunohistochemically detected in human decidua and placenta, as well as in 19 weeks old human fetal liver and kidney. The presence of renin substrate in fetoplacental tissue supports the concept of a locally functioning renin-angiotensin system.

An immunoaffinity purification system using C30N and C1Z Eupergit C beads was developed and optimized. Poly- and monoclonal antibodies were purified using immobilized antigens and antigens were purified using immobilized antibodies. Antigens were used that possess enzymic activities and the efficiency of antigen binding was determined from the enzymic activity of the matrix-bound immunocomplexes. High-performance immunoaffinity purification using Eupergit C beads proved to be highly specific, reproducible, free from protein leakage and possessed a low degree of non-specific adsorption of tissue proteins. These characteristics of the system were illustrated by the isolation of immunoglobulin G from serum and of human decidua proteins from the decidua tissue and from seminal plasma. These proteins were obtained at high purity in a single purification step, as shown by sodium dodecyl sulphate-polyacrylamide gel electrophoresis.

Decidual and placental concentrations of progesterone and oestrogen receptors in 15 women having medical terminations of pregnancy with a combination of the anti-progesterone steroid RU 38,486 and prostaglandin E1 analogue have been compared with 10 matched controls undergoing surgical aspiration of pregnancy. In the patients treated with RU 38,486, the mean (SD) decidual cytosolic and total progesterone receptor concentrations of 4.3 (5.6) and 7.6 (7.1) fmol/microgram DNA respectively were significantly lower than the levels of 18.5 (14.4) and 22.3 (17.4) fmol/microgram DNA in the control patients. Progesterone receptor concentrations in the placenta were very low in both groups. No significant change in oestrogen receptor concentrations occurred in the decidua or placenta after treatment with RU 38,486. These data indicate that there may be down-regulation of decidual progesterone receptor concentrations following RU 38,486 treatment.

The possible presence of gonadotropin receptors in nonpregnant human uterus and human fetoplacental unit was investigated by light microscope immunocytochemistry using a monoclonal antibody to rat luteal hCG/LH receptors. The receptor antibody cross-reacted with human and bovine hCG/LH receptors and appears to be directed against the receptor rather than other proteins, including HLA class I antigens. Uterus and fetoplacental unit contained receptor antibody-binding sites, which indicates the presence of hCG/LH receptors. In the endometrium these receptors were present in glandular and luminal epithelial cells as well as in stromal cells. In the myometrium the receptors were detected in circular and elongated myometrial smooth muscle and vascular smooth muscle. Comparison of immunostaining intensities, which indicates the presence of different amounts of receptors, revealed that luminal and glandular epithelial cells contained more receptors than stromal cells. These cells, in turn, contained more receptors than myometrial and vascular smooth muscle. All cells in secretory phase uterine specimens contained more receptors than corresponding cells from the proliferative phase of the cycle. Midpregnancy placenta, amniotic epithelium, chorionic cytotrophoblasts, and decidual cells contained hCG/LH receptors. At term pregnancy, while receptors in fetal membranes and decidua continue to be detected, placental tissues did not show any detectable receptors unless the tissues were pretreated with neuraminidase. This indicated that term pregnancy placenta contain hCG/LH receptors masked by sialic acid residues. Comparison of immunostaining intensities suggested that syncytiotrophoblasts contained more receptors than cytotrophoblasts at midpregnancy; mesenchymal cells or blood vessels contained no detectable receptors. There were more receptors in decidua than in fetal membranes at mid- and term pregnancy. While the amniotic epithelial receptors decreased, the receptors in chorionic cytotrophoblasts and decidual cells increased from mid- to term pregnancy. In summary, hCG/LH receptors were demonstrated in the nonpregnant human uterus, human placenta, fetal membranes, and decidua. This indicates that hCG/LH may directly regulate functions of these tissues by endocrine, autocrine, or paracrine mechanisms.

Immunocytochemistry and Northern analysis were used to show that relaxin is a product of intrauterine tissues of pregnancy. In addition, tissues from a patient without ovaries had similar results on both immunocytochemistry and Northern analysis as tissues from intact patients. The parietal decidua was clearly the major source of relaxin within the uterus and the relaxin mRNA (1.2 kilobases) from this tissue was detected with a 48-mer oligonucleotide probe designed to hybridize with both H1 and H2 relaxin gene transcripts. The mRNA isolated from the placental trophoblast was slightly smaller (1.1 kilobases), and the placental basal plate which has both maternal and fetal cells contained relaxin mRNAs of both sizes. Two monoclonal antibodies (Mabs) raised to synthetic human relaxin (H2) gave different patterns of localization in the fetal membranes, decidua and placenta. One Mab (RLX8) stained the chorionic cytotrophoblast in the fetal membranes and all of the cells in the placental basal plate. The other Mab (RLX6) stained the chorionic cytotrophoblast in some instances and selectively stained the decidua-like cells of the placental syncytiotrophoblast, whereas Mab RLX8 failed to detect this relaxin. Tissues obtained after spontaneous labor and delivery contained significantly less relaxin mRNA than tissues obtained at elective cesarean section without labor, but their hormone contents, as judged by immunocytochemistry, were not different. We conclude that the relaxin gene (H2) is expressed in intrauterine tissues, but that expression and hormone synthesis are not ubiquitous. Whether the relaxin gene H1 is expressed has not been determined.

A crude extract of pooled early-pregnancy decidual tissue was enriched for soluble decidual proteins by exhaustive affinity absorption with antibodies to human serum proteins immobilized on Eupergit C. The partly purified extract was used to prepare monoclonal antibodies. A monoclonal antibody was obtained recognizing an antigen present in extract of decidual tissue and not in extract of proliferative endometrium. The monoclonal antibody was used for immunoaffinity purification of the decidua-associated protein. By SDS-PAGE analysis, under reducing conditions it yielded 2 bands at apparent molecular weights of 55,000 and 25,000. Under non-reducing conditions a single protein band at apparent molecular weight of 200,000 was observed. The Mr 200,000 protein was named hDP200 and the Mr 55,000 protein was named hDP55. It is suggested that hDP55 is a subunit of the hDP200. The hDP200 did not react with polyclonal antibodies specific for PP12 and PP14. PP14 has been shown to be immunologically indistinguishable from PEP and alpha 2-PEG. Our data therefore suggest that hDP200 is a novel human decidua-associated protein.

Progesterone withdrawal as a mechanism for parturition in primates is controversial. The progesterone antagonist RU486, given in late pregnancy to rhesus monkeys at a dose of 47 mmol/kg.day (20 mg/kg.day), causes an increase in uterine activity, but not the expected increase in amniotic fluid prostaglandins or cervical dilatation. We, therefore, studied the effect of RU486 on estrogen receptor (ER) localization and concentration in reproductive tract tissues in rhesus monkeys during late gestation and after spontaneous labor at term. Distribution of ER in pregnant uterine tissues was studied by immunocytochemical techniques and quantified by a biochemical assay, both of which employed a monoclonal antibody specific for ER. ER was not present in amnion and chorion by immunocytochemical investigation; however, a significant increase in receptor staining was seen in decidua and myometrium after RU486 treatment compared to that in both pregnant control tissues and parturient tissues. Sucrose gradient assay of nuclear (n) and cytosolic (c) ER revealed a low level of ER (expressed as fmol of estradiol bound/mg of DNA) in pregnant and parturient decidua (pregnant: nER = 7.3 +/- 2.4, cER = 17.1 +/- 6.4; parturient, nER = 7.7 +/- 3.1, cER = 16.4 +/- 8.8) and myometrium (pregnant: nER = 21.7 +/- 4.1, cER = 20.8 +/- 5.3; parturient: nER = 30.0 +/- 2.8, cER = 10.7 +/- 6.7). In contrast, tissues collected from RU486-treated animals contained high levels of ER in decidua (nER = 52.3 +/- 16.8, cER = 240.5 +/- 145.3) and myometrium (nER = 77.0 +/- 19.2; cER = 66.5 +/- 31.6). We conclude that 1) the increase in ER in decidua and myometrium after RU486 treatment is the result of a decrease in the inhibitory action of progesterone on ER and documents the progesterone receptor antagonism by RU486 during induced myometrial contractility in late pregnant rhesus monkeys; 2) the absence of ER from amnion and chorion indicates that the normally observed increase in prostaglandin production by rhesus fetal membranes during labor is not mediated by ER; and 3) the absence of a change in the concentration of ER in decidua and myometrium from pregnant control monkeys and those in spontaneous labor indicates that an increase in ER (and, by inference, a withdrawal of receptor-mediated progesterone inhibition) is not part of the normal events in preparation for parturition in primates.

Conditioned media from cultures of human decidual explants and aqueous extracts of human decidual tissue contain a factor that causes a reversible dose-dependent inhibition of decidual PRL release in vitro. Decidual explants incubated for 30 min in medium containing 50, 100, and 250 micrograms/ml of a dialyzed and lyophilized preparation of decidual conditioned medium (DCM) released 32.4 +/- 2.7%, 70.9 +/- 4.5%, and 100.0%, respectively, less PRL than control explants. DCM, however, had no measurable effect on the synthesis of decidual PRL or the synthesis and release of trichloroacetic acid-precipitable 35S-labeled proteins. The effect was of short duration and completely reversible. The inhibition of decidual PRL release was not due to PRL, since 500 micrograms/ml human pituitary PRL (a PRL concentration 40 times that in the minimal effective dose of DCM) added to the incubation medium of decidual explants had no effect on the synthesis or release of decidual PRL or trichloroacetic acid-precipitable 35S-labeled decidual proteins. The inhibitory activity eluted from Sephadex G-200 with an apparent molecular weight of 38,000-45,000 daltons, was heat labile, was destroyed by treatment with trypsin, and was unaffected by extraction with acetone-ethanol. These results strongly suggest that the release of decidual PRL is under local control, regulated in part by a factor(s) other than PRL that is released by the decidua.

Maternal and fetal cellular relationships in the normal human term placental basal plate were investigated by single and double immunohistochemical labelling techniques. Extravillous fetal trophoblast in the basal plate was uniformly reactive with markers of low-molecular-weight cytokeratins. The predominant maternal leucocyte population in the basal plate consisted of leucocyte-common-antigen-positive, class II MHC-positive macrophages, which exhibited acid phosphatase activity. Double-labelling methods highlighted the close association of these macrophages with extravillous trophoblast: they often extended processes around the fetal cells and were also observed within islands of cytotrophoblast. Other leucocytes were uncommon, although aggregates of T cells were apparent in some tissues.

As shown by polyacrylamide gel disc electrophoresis, 21 fractions of soluble proteins were found in human endometrium at the proliferative step, whereas the decidual tissue contained only 13 fractions. Two specific protein components D1 and D2 with molecular weight 52,000 and 150,000 daltons, respectively, were observed in the cytosol of decidual tissue. The decrease in quantitative spectrum of soluble proteins from decidual tissue was apparently related to its narrow specialization during the differentiation process.

Relaxin, prolactin and prostaglandin synthase were localized by the avidin-biotin immunoglucose oxidase method in human amnion, chorion and decidua. Specimens from ten normal spontaneous deliveries and four elective Caesarean section deliveries with no labour were compared. Relaxin was found more consistently in the cells of the chorionic cytotrophoblast than in the cells of the parietal decidua adherent to the fetal membranes. Only half the tissues after spontaneous delivery contained positive relaxin-stained cells, whereas all the tissues from elective Caesarean sections contained cells positively stained with antiserum to relaxin. In both series of tissues prolactin was localized predominantly in the parietal decidual cells and was very infrequently found in the chorionic cytotrophoblast. Polyclonal antiserum to prostaglandin synthase was used to identify those cells producing prostaglandin in amnion, chorion and decidua. The cells of the amnion and chorion showed positive immunolocalization with no differences between tissues collected before or after labour. Double immunostaining using avidin-biotin immunoperoxidase for prolactin, followed by avidin-biotin immunoglucose oxidase for prostaglandin synthase, produced identical results in the same series of tissues examined with the single-staining method.

The concentration of human lactoferrin (LF) was measured by radioimmunoassay or non-competitive avidin--biotin assay in amniotic fluid, cord blood and in the decidua, trophoblast, fetal membranes and umbilical cord. Amniotic fluid was obtained by amniocentesis, and cord blood and tissue samples were taken after delivery or elective Caesarean section. No detectable concentration of LF was found in amniotic fluid before week 20 of pregnancy. A significant increase in the LF concentration was observed around week 30 and it remained high until term. In cord blood, an undetectable or low concentration of LF was measured. In tissue specimens the amount of LF was highest in the decidua (9-95 micrograms/g), a moderate concentration was assayed in the amniotic (2-37 micrograms/) and chorion (2-26 micrograms/g) membrane and in the trophoblast (5-35 micrograms/g). In the umbilical cord, the concentration was less than 1 microgram/g. These results suggest a decidual origin of LF. The role of LF during pregnancy is discussed.

The effects of extracellular osmolality and ions on the secretion of prolactin were examined in human decidual explants incubated for 4 h in modified Krebs-Ringer buffer. Explants incubated in media made hypersomotic (280-336 mosM/kg) with sodium, lithium or mannitol or in media made hypo-osmotic (280-224 mosM/kg) by decreasing the sodium concentration secreted the same amount of prolactin as explants incubated in control medium (280 mosM/kg). Explants incubated in calcium-deficient medium secreted 64 +/- 8% (P less than or equal to 0.001) less prolactin than controls (1.65 mmol Ca2+/1). Secretion was restored to control values by the addition of calcium (0.33 mmol/l) or barium (0.5, 1.0 or 2.0 mmol/l) to the medium. Prolactin secretion was unaffected by higher than control extracellular calcium concentrations, calcium ionophore A23187 (10-6, 10-9 mol/l) or lanthanum (0.5, 1.0 or 2.0 mmol/1). Changes in extracellular magnesium (0-20 mmol/l), potassium (0-55 mmol/l) or bicarbonate (0-32 mmol/l) had no effect on prolactin secretion. These results indicate that marked changes in extracellular osmolality and concentrations of sodium, magnesium and bicarbonate have no effects on decidual prolactin secretion. Calcium, however, is essential for the basal secretion of decidual prolactin.

Prostaglandin F2 alpha (PGF2 alpha) and its synthetic analog, 15-methyl-PGF2 alpha effects on the receptor content of estrogens and progesterone in the human decidual tissue were studied in vitro and in vivo experiments. The receptor content in vivo experiments was determined by Sketchard's technique and in vitro by the saturation method. Three hours after intravaginal administration of 15-methyl-PGF2 alpha suppositories to pregnant women the level of estrogen and progesterone receptors in decidual tissue cytosol significantly increased. Affinity of receptors with hormones remained unchanged. 10, 20, 50 and 100 mcg/ml concentrations of PGF2 alpha produced a significant and dose-dependent increase in the receptor content of estrogens following 15, 30, 60 and 180 minutes incubation of decidual tissue sections. The receptor level of progesterone remained unchanged. In experiments with inhibitors of protein and RNA biosynthesis, it was shown that an augmentation of the estrogen receptor content under PGF2 alpha action is not associated with biosynthesis of receptors de novo.

Human amniotic fluid samples obtained from normal pregnancies and supernatants of normal full-term placental and decidual tissues were assayed in a specific radioimmunoassay (RIA) and in a sensitive bioassay for tumor necrosis factor (TNF). TNF was detected in 91% of amniotic fluid samples and in all placental and decidual supernatants assayed by the RIA. Amniotic fluid samples collected during the second trimester of pregnancy contained significantly higher concentrations of TNF than samples collected during the third trimester, average concentrations being 2.5 ng/ml and 0.9 ng/ml, respectively. Parallel testing in the bioassay showed that most of the amniotic fluid TNF is biologically inactive, whereas placental and decidual supernatants had biologic activity correlating with TNF levels detected by the RIA. The quantities of TNF detected by the RIA ranged from 1.1 to 2.8 ng/ml in placental supernatants and from 3.9 to 8.5 ng/ml in decidual supernatants. The presence of TNF in normal amniotic fluids and in placental and decidual supernatants suggest that this cytokine has a physiologic role in human pregnancy.

A preparation of fertility alpha 2-microglobulin was isolated from an extract of decidual tissue with a purity about 95% and complete maintaining of native properties using chromatography on anion and cation exchange Sephadexes.

Using the PAP technique, the location of two new membrane-associated placental tissue proteins, MP1 and PP4 was studied in the placenta, its membranes, decidua and umbilical cord of human and cynomolgus monkeys. The results were the same throughout pregnancy. MP1 was located in the syncytiotrophoblast and trophoblastic cells of the reflected chorion. Other placental tissue components (cytotrophoblast, amnion, decidua, and umbilical cord) were negative. At present, MP1 appears to be specific for trophoblast. PP4 was located in the syncytiotrophoblast. Furthermore clear positive staining for PP4 was found in the villous cytotrophoblast, reflected and basal chorion, amnion, umbilical cord and decidua. In addition, PP4 was positive in some granulocyte-like blood cells in the intervillous space. Immunocytochemically, the most positive staining for both proteins was observed in the membrane of villous syncytiotrophoblastic cells. The findings in the placenta of cynomolgus monkeys were similar to those in women. The monkey could, thus, serve as a model for the investigation of these new membrane-associated placental tissue proteins.

The biotin-avidin immunoperoxidase staining method and antisera against highly purified porcine relaxin were used to localize relaxin in the genital tract of pregnant and nonpregnant women. Formalin-fixed tissue specimens from normal placenta, decidua, myometrium, vagina, corpus luteum, and Fallopian tubes were studied. In pregnant women, relaxin was found in the placental syncytiotrophoblast, decidua, and corpus luteum. In nonpregnant women, relaxin was identified in the corpus luteum and endometrium in the secretory, but not in the proliferative, phase. Myometrium, cervix, vagina, and Fallopian tubes were negative for relaxin. This is the first report describing relaxin in the nonpregnant corpus luteum, and we also confirm results of an early disputed study claiming that endometrium in the secretory phase contains relaxin. The origin and biological role of human endometrial relaxin remain to be studied.

Tissue polypeptide antigen (TPA) and cancer antigen 125 (CA125) were studied immunohistochemically by the avidin-biotin immunoperoxidase technique in human and cynomolgus monkey placentae, membranes, umbilical cords and decidua. In early human placentae, TPA was localized mainly in the cell membranes of villous syncytio- and cyto-trophoblast. The cytoplasm of those trophoblastic cells were weakly stained with TPA. The membrane of basal chorionic trophoblast cells was strongly stained with TPA and the cytoplasm stained weakly. In early cynomolgus placentae, similar immunostaining results were obtained. However, the positive stainings for TPA was more marked in the cytoplasm of villous syncytiotrophoblast and basal chorionic trophoblast, and less marked in the cell membrane of villous cytotrophoblast. In early human and cynomolgus placentae, CA125 was not demonstrated immunohistochemically in the villi and basal chorion. In human and cynomolgus term placentae, the villous syncytiotrophoblast and basal and reflected chorionic trophoblast showed similar immunostaining as the early placentae. In addition, TPA was found in the amniotic epithelium in both sorts of placentae. TPA was not detected immunohistochemically in the umbilical cord and decidual cells. While weakly positive stains for CA125 were observed in decidual cells, CA125 was localized mainly in the membrane and cytoplasm of amniotic epithelium in both human and cynomolgus term placentae. TPA and CA125 are thus oncoplacental antigens and the monkey could serve as a model for their investigation.

Immunostaining of baboon placental tissues with anti-human pregnancy-specific beta 1-glycoprotein (SPI) antibodies demonstrated that an SP1-like molecule was present in the syncytiotrophoblasts. Staining was observed on the membrane and in the cytoplasm, but the nucleus was devoid of any staining. Western blot analysis further demonstrated the presence of five protein species in baboon placental extract, whereas four protein bands were detected in human placental extract. Culture medium of baboon placental villi also contained five SP1-like molecules with sizes slightly different from those present in the placental extract. Amniotic fluid and culture medium of decidua basalis and chorioamniotic tissue contained lesser quantities and fewer species of SP1-like molecules. However, an 87 kDa band was present in all samples examined. Northern blot analysis of baboon placenta with a human placental SP1 cDNA probe demonstrated the presence of a 1.65 Kb band, whereas two hybridizing bands (1.65 Kb and 2.25 Kb) were present in human placenta. Southern blot analysis of baboon genomic DNA further demonstrated the presence of multiple bands hybridizing with a human placental SP1 cDNA probe. These results showed the presence in baboons of multiple genes encoding mRNAs and proteins highly similar to human placental SP1.

Radioimmunoassay, gel filtration and sodium dodecyl sulphate-polyacrylamide gel electrophoresis were used to study the content and properties of placental protein 12 (PP12) in the placenta, decidua and fetal membranes. The tissues were obtained from early pregnancy in 12 cases, and after normal term delivery in eight cases in seven of which chorion and amnion laeve were also studied. There was more PP12 in decidua and fetal membranes than in placenta. The decidua/placenta ratio of PP12 content ranged from 2 to 1154 (mean 193, SEM 66). These results suggest that PP12 is a decidual rather than placental protein.

Human alpha uterine protein (AUP) has been prepared from extracts of decudua by antibody affinity chromatography, DEAE Sepharose chromatography and by filtration through Sephadex G-150. This procedure yielded a protein fraction containing AUP, which was labelled with 125I by chloramine T. When analysed by SDS gel electrophoresis this radioiodinated protein fraction was found to contain predominantly a single species of protein which was precipitated by antibodies against AUP in antibody-antigen crossed electrophoresis. Rabbit anti-AUP precipitated 55-65% of the tracer in a double-antibody system. Sephadex G150 gel filtration of AUP obtained before and after affinity chromatography provided a molecular weight estimate of 50000. Since SDS gel electrophoresis revealed a polypeptide molecular weight of 23000-25000, it is suggested that AUP is a dimer.

Using an immunoperoxidase technique the localization of hCG, hPL, SP1, PAPP-A and PP5 within villous and extravillous trophoblast has been studied. The villous syncytiotrophoblast appears to be the sole significant source of hCG, SP1, PAPP-A and PP5 but hPL is also present in the infiltrating extravillous trophoblast. Within the interstitial extravillous trophoblast the proportion of cells staining positively for hPL increases progressively as the cells extend deeper into the uterine tissues but all the vascular extravillous trophoblast within the lumina of the spiral arteries stains positively for hPL at all levels within the decidua and inner myometrium. It is not clear why the synthetic capacity of the infiltrating trophoblast is limited to the production of hPL. We suggest that this might be indicative of selectivity of a specific subpopulation of cytotrophoblastic cells. It is unlikely that the physiological effects of infiltrating trophoblast are mediated by hPL, this substance being probably only a marker of a cell population with a particular invasive capacity.

Our recent finding that decidual luteotropin, a PRL-like hormone secreted by the rat decidua, is found primarily in the antimesometrial cells suggests strongly that the synthesis of this hormone may well be an important function of the antimesometrial tissue. The objective of this investigation was, therefore, 1) to determine whether antimesometrial tissue expresses mRNA for and actively secretes a protein(s) with PRL-like activity, and 2) to examine the pattern of protein production by the antimesometrial and mesometrial zones throughout decidual development. RNA obtained from decidual tissue of day 8 pseudopregnant rats was translated in a cell-free system. The translated products were subjected to PRL receptor affinity chromatography in the presence or absence of ovine PRL to assess binding specificity. The eluted 35S-labeled proteins were analyzed by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. A major 28K protein bound specifically to and was eluted from PRL receptor-enriched luteal membranes. This protein also cross-reacted with antibodies to human PRL. To determine where the mRNA for this 28K protein is expressed and whether this protein represents a prohormone, RNA isolated from both antimesometrial and mesometrial tissue was translated in the presence or absence of microsomal membranes. The 28K protein was synthesized specifically by RNA isolated from the antimesometrial zone. No apparent change in the relative mol wt of the 28K protein was observed when translation was performed in the presence of microsomal membranes. To determine whether this protein is a secreted product and to investigate the pattern of protein secretion by the mesometrial and antimesometrial decidua, tissue explants obtained from both zones between days 9-13 of pseudopregnancy were cultured in the presence of [35S]methionine. Antimesometrial tissue secreted one major 28K protein which was capable of binding to PRL receptors on luteal membranes and was immunoprecipitated by antibodies to human PRL, whereas the mesometrial tissue primarily secreted an approximately 180K protein. The overall pattern of protein synthesis and release not only differed between the mesometrial and antimesometrial tissues but also differed with each day of pseudopregnancy. The secretion of several proteins decreased with advancing gestational age, while the secretion of other proteins began abruptly after day 11, coincident with regression of the antimesometrial tissue. In summary, results of this investigation have established that rat decidual tissue synthesizes and selectively secretes proteins, and the specificity and rate of production of these distinct

Not only separation of native, two-stranded DNA from denatured molecules but more distinct fractionation of single-stranded DNA was demonstrated on unmodified powdery and microgranular cellulose. Denatured DNA, absorbed on cellulose, was eluted from a column as two asymmetric peaks by 0,12 M and 0.05 M NaCl (for I and II peaks, respectively). Absorption and fractionation of denatured DNA and poly-A containing RNA on unmodified cellulose was apparently achieved on the basis of the same principles.

By using an immunoglobulin enzyme bridge (PAP) technique, the location of two ubiquitous tissue proteins (PP8, PP9) and three placental specific tissue proteins (PP10, PP11, PP12) was investigated in ethanol/acetic acid-fixed paraffin-embedded human term placentae. PP8 was found mainly in both the cytoplasm and the nucleus of trophoblast cells (chorion) and syncytiotrophoblast (villi). PP9 was found in the cytoplasm of trophoblast cells (chorion), the fibrous part of interstitial connective tissues (villi), and the cytoplasm of histiocytes (villi, amnion, and decidua). PP10 was found in the cytoplasm of epithelium (amnion) and in both the cytoplasm and the nucleus of syncytiotrophoblast (villi). PP12 was found in the cytoplasm of histiocytes in decidua, amnion, and chorion and the cytoplasm of syncytiotrophoblast (villi). In addition, stains for PP8, PP10, and PP12 were taken up by the cytoplasm of histiocytes found in amnion, villi, the intervillous space, and decidua, in contrast to PP11, which was found to be specific to the cytoplasm of trophoblast cells (chorion and villi). Possible clinical applications of these findings are discussed.

The production of PRL by the human decidua is generally accepted, but the production of relaxin by this tissue is not. The two hormones were localized in decidual tissue using the avidin-biotin immunoperoxidase procedure with antisera to human PRL and to a synthetic 14-amino acid sequence of the connecting peptide of human relaxin (hCp14). The object of using the hCp14 antiserum was to verify relaxin production by the detection of C-peptide and/or prorelaxin. Cells of the parietal decidua adherent to the fetal membranes stained with both antisera, and immunostaining for both hormones in the same cell was seen. Also, the decidua-like cells of the placental basal plate stained with both antisera. The chorionic cytotrophoblast stained with the antiserum to hCp14, but not the antiserum to human PRL, whereas the placental syncytiotrophoblast stained for PRL and/or human placental lactogen (hPL), but not hCp14. The PRL staining in all tissues was lost when anti-PRL serum absorbed with human placental lactogen (hPL) was used. This finding suggests that the antiserum to PRL could not distinguish between PRL and hPL. It appears, therefore, that the parietal decidua cells and the decidua-like cells of the placental basal plate may be capable of producing both relaxin and PRL, while the syncytiotrophoblast produces hPL and possibly PRL.

Apparently Placenta-specific placental tissue proteins (PP14 and PP17) and solitary tissue proteins (PP16, 19, 20 and PP21) were investigated by avidin-biotin immunoperoxidase technique in the human and cynomolgus monkey placentae, membranes, decidua and umbilical cords. In human early placentae, PP14, 16, 17, 19 and PP21 were localized mainly in the cytoplasm of villous syncytiotrophoblast. PP20 was localized in the cytoplasm of basal chorionic trophoblasts. In human term placentae, positive stainings for PP16, 19 and PP21 were observed mainly in all kinds of trophoblastic cells, while positive stainings for PP14, 17 and PP20 were weakened in the trophoblastic cells. PP20 was clearly localized in the cytoplasm of Hofbauer-like cells in the villous stroma. The membrane of villous syncytiotrophoblast showed strongly positive stainings for PP21. PP21 was also localized in the membrane of amniotic and umbilical epithelium. The umbilical epithelium was cytoplasmically positive for PP14, 16 and PP20. Clear positive stainings for PP14 and PP21 were found in the cytoplasm of fetal polymorphonuclear neutrophils. All of the placental proteins were immunocytochemically positive in the decidual large cells. In the cynomolgus monkey placentae, similar immunostaining results were obtained. The monkey could, thus, serve as a model for the investigation of the placental proteins.

Protein synthesis in response to heat shock was induced in the proliferative and secretory human endometrium as well as in human decidua during a 2-h incubation period at 41 degrees C. A major 70 K stress protein and two minor stress proteins of 88 and 94 K were detected after [35S]methionine incorporation followed by SDS polyacrylamide gel electrophoretic separation and autoradiography of dried gels. Two dimensional isoelectric focusing followed by fluorography showed the major 70 K stress protein to consist of at least six polypeptides of pH 4.6 to 5.5, the 88 K to consist of at least four polypeptides, of pH 5.5 to 6.0, and the 94 K to consist of at least three polypeptides of pH 4.6 to 5.2. Stress proteins in the human endometrium and decidua may prove to be of physiological significance in reproductive events.

As a contribution to the aetiology of insufficiency of placenta carbohydrate-histo-chemical investigations of the kyema were per-formed from the 10 th to 14 th week of pregnancy. The studies were carried out on pregnancy products of women with affection of kidneys, circulatory failures, and under cytostatic therapy. For comparison purposes, cases were used where the pregnancy was without organic diseases of the pregnant women. The investigations performed did not prove evidence of an essential alteration of the regulation of carbohydrates in the kyema by maternal disorders or cytostatic treatment during early gravidity.

PGE2 and PGF2 alpha were isolated from the decidua of an early pregnancy, following medical termination. The biosynthetic capacity for 6-keto-F1 alpha was tested by incubation, using 3H-labelled arachidonic acid. Prostaglandin concentrations in the decidua differed strongly from steroid hormones in plasma. Steroid hormone concentrations increased with continuity, whereas prostaglandin values remained extremely low.

Immunohistochemical techniques have been used to investigate the expression of fetal trophoblast antigens and the maternal leucocytic response in molar pregnancy and choriocarcinoma. The antigenic phenotype of morphologically defined trophoblast populations in complete, partial and invasive moles was analogous to that in normal pregnancy. All trophoblast phenotypes described in normal pregnancy were also identified in choriocarcinoma, suggesting that extensive differentiation into heterogeneous subgroups occurs in malignant trophoblast. The maternal leucocytic infiltrate in molar pregnancy consisted of T lymphocytes and class II MHC-positive macrophages. CD2-positive, CD3-negative lymphocytes were identified in molar decidua but not in uterine tissue in choriocarcinoma. Similarly, endometrial granulocytes were present in molar decidua but not in choriocarcinoma; these cells were associated with decidualization rather than with fetal trophoblast.

Placental protein 12 (PP12) was originally isolated from term human placenta and adjacent membranes. Recently we found that the site of PP12 synthesis is decidua but not placenta. In this work, the purity of PP12 was first tested by sodium dodecyl sulfate polyacrylamide slab-gel electrophoresis and by reverse phase HPLC, and the N-terminal amino acid sequence of 15 residues was determined by a liquid-phase sequencer. A single amino acid sequence of Ala-Pro-Trp-Gln-Cys-Ala-Pro-Cys-Ser-Ala-Asp-Glu-Leu-Ala-Leu was obtained showing identity to the known N-terminal amino acid sequence of somatomedin-binding protein from human amniotic fluid. Like the latter, PP12 bound somatomedin (insulin-like growth factor I) as demonstrated in gel chromatography by a shift in the elution pattern of [125I]iodo-insulin-like growth factor I after incubation with PP12. These data show that PP12 is a somatomedin-binding protein and extend through previous literature on PP12 the existing knowledge on the physiology and pathophysiology of somatomedin-binding protein(s) in human reproduction and cancer.

We have studied the specific binding of human serum sex-hormone-binding protein and its complexes with estradiol and testosterone to an enriched membrane fraction isolated from human decidual endometrium. The specific binding of sex-hormone-binding protein to these membranes has been found to be dependent on it being bound with estradiol. When estradiol concentration in the medium is high enough to provide for the saturation of sex-hormone-binding protein with the steroid, the protein exhibits specific binding to the membranes with an apparent equilibrium constant, Kd = (3.5 +/- 2.0) X 10(-12) M. In the absence of estradiol, the binding protein devoid of steroid or complexed with testosterone does not interact with the membranes. At low concentrations of sex-hormone-binding protein and estradiol, when their complex is almost completely dissociated in solution, it can be reconstituted on the membranes with the optimum estradiol to binding protein ratio being 1:1 (mol/mol). It is proposed that, in plasma membranes of the estradiol target cells, there is a recognition system for the sex-hormone-binding protein-estradiol complex which may allow these cells to take up from blood not only free estradiol, but also estradiol complexed with the binding protein.

A human prolactin (PRL) cDNA clone was digested with restriction enzyme Pst I and the resultant fragments were cloned into bacteriophage M13mp9. Single stranded recombinant DNAs having a coding strand of the PRL cDNA were selected by hybridization with 125I-labeled PRL mRNA obtained from human prolactinoma tissue. One of the single stranded recombinant DNAs was purified by agarose gel electrophoresis and labeled with 125I to a specific activity of 1.4 X 10(8) cpm per microgram of DNA. The probe could be successfully used in RNA dot hybridization. Analysis of poly (A) RNAs from prolactinoma, liver and placental tissues revealed that this probe was specific to PRL mRNA sequence. Hybridization of poly (A) RNA from decidual tissue to this probe revealed the presence of PRL mRNA sequence. However, PRL mRNA in decidua was at least 20,000 times less than that in pituitary prolactinoma.

A study was made of the content of estrogen and progesterone receptors (ER and PR, respectively) in the cytosol and nuclei of the decidual tissue of women in induced and spontaneous abortion at 7-12 week pregnancy. Abortion was done surgically (control group) and in preliminary cervical dilatation with the help of intravaginal suppositories with a synthetic prostaglandin analog of E1-16.16-dimethyltrans-delta 2-PGE1 (ONO-802) group or laminaria. The level of ER in the cytosol and nuclei of the decidual tissue of the control group was twice as low as that of PR, and the ER/PR ratio in cell fractions as well as in general cell receptors was 0.58 +/- 0.09, 0.42 +/- 0.07 and 0.56 +/- 0.07, respectively. The suppositories with ONO-802 caused a statistically significant decrease in the PR content in each of the cell fractions and cytoplasmic ER resulting in an increase in the ER/PR ratio to 0.87 +/- 0.06 (P less than 0.05) in the cytosol, 0.09 +/- 0.24 (P less than 0.05) in the nuclei, and 0.86 +/- 0.8 (P less than 0.05) in the total cell. A decrease in the PR level in both cell fractions and the ER level in the fraction of the nuclei with an acute increase in the content of cytoplasmic ER was marked in spontaneous abortion. The ER/PR ratio also rose to ER and was 1.98 +/- +/- 0.2 (P less than 0.001) in the cytosol and 1.02 +/- 0.16 (P less than 0.05) in the total cell. It was concluded that an absolute and/or relative decrease in the PR level plays an important role in mechanism of abortion.

Placentas and membranes of seven healthy women after full-term gestation and spontaneous labor and delivery were studied in order to verify the presence and distribution of prolactin-containing cells. An unlabeled antibody-PAP-method, applied to formalin-fixed, paraffin-embedded tissue sections, showed varying degrees of cytoplasmic positivity in the elements of the amniotic epithelium, decidua and trophoblast. A comparison between these results and those previously reported in the literature is made.

1) The studies were performed to elucidate the mechanism of onset of labor. It was investigated in relation to the movement of prostaglandins in individual tissues of pregnant rabbits before and after labor with the onset of labor. 2) The rabbit uterus duplex used is convenient material to investigate the movement of PGs in the same rabbit at the different term. 3) The concentrations of PGs were measured by RIA. 4) The concentrations of PGs in blood and amniotic fluid were not essential to explain account for the mechanism of onset labor. 5) From the results of the PGs values before and after labor, we have been considered that the relative PGs at the onset of labor were PG E1 and PG E2 in the placenta. We infer that PG F2 alpha plays a leading role in uterine contraction, because the large amount of PG F2 alpha presented in decidual parietalis during delivery, and PG E1 and PG E2 play a role in uterine contraction, too.

Relaxin (RLX) has been purified from the ovary of the pregnant pig and rat but not from human tissues. Our study shows that tissue extracts and incubation media of in vitro culture of human decidua contain a substance with relaxin bioactivities: the inhibition of spontaneous uterine contractions and the elongation of the interpubic ligament. After chromatography on Sephadex G-50 the bioactivity was retained in a protein fraction of approximately 6000 daltons mol wt. The yield of RLX from decidua was 15--33.5 GPU/g fresh tissue. This opens the possibility of the isolation and purification of RLX in the human species.

The diameter of collagen fibrils was measured in different regions of the antimesometrial endometrium of mice on days 5, 6, and 7 of pregnancy as well as in the endometrium of virgin mice. The average diameter of fibrils of virgin mice was 39.18 nm (range: 20-80). In the region of fully decidualized cells, the averages and ranges were 45.32 nm (30-170), 89.39 nm (30-270), and 125.88 nm (20-370), respectively, on days 5, 6, and 7 of pregnancy. Thick fibrils larger than 70 nm had irregular profiles. Our results show that the increase in diameter is associated with the decidualization of the mouse endometrium.

Using cytophotometry of the Feulgen-stained nuclei, the quantity of DNA was measured in the nuclei of rat's large decidua cells (LDC) on tissue sections of the antimesometrial region within days 7-13 of gestation. The quantity of nuclear DNA was expressed in units of ploidy, the haploid DNA standard being the quantity of DNA in rat's spermatid nucleus. On different days of gestation, the nuclear DNA was seen to vary in cells located in different zones of decidua. The maximum DNA content was found in the LDC located on days 9-12 of gestation somewhat in the middle of the decidua thickness. On day 11, the quantity of nuclear DNA in these cells reached in average, 22c. The quantity of DNA in the nuclei of the least differentiated LDC located on the periphery of decidua never exceeded 4.9c, whereas that in the nuclei of the most differentiated LDC, located close to the embryo, varied from 2.9c to 9.3c. On days 10 and 11 of rat's false gestation, the maximum DNA contents in the nuclei were registered in the LDC located in the middle of the decidua thickness. 3H-thymidine incorporation into the nuclei of the most differentiated LDC located nearest to the embryo stopped starting from day 10 of gestation. Phenomena of lesser quantities of nuclear DNA in most differentiated LDC, compared to that in LDC in the previous steps of differentiation, are discussed.

The concentrations of prostaglandin E2 and F2 alpha have been measured by radioimmunoassay in portions of cord, placenta, amnion, chorion, decidua and myometrium. The samples were obtained at defined periods of pregnancy, and the results have been compared with those obtained from the analyses of endometrial and myometrial tissue removed from women during the secretory phase of a menstrual cycle. The results showed that during pregnancy the mean concentration of prostaglandin E2 was higher (27-518%) than the corresponding value for prostaglandin F2 alpha in all tissues. At term the concentration of prostaglandin E2 (ng/100 mg wet weight of tissue, mean +/- S.D.) was higher in the umbilical cord (5-54 +/- 0-88), decidua (4-02 +/- 1-78) and myometrium (4-19 +/- 1-06), than in the amnion (2-25 +/- 1-27), chorion (1-64 "/- 0-63) or placenta (1-04 +/- 0-25). During labour there was a significant rise (P less than 0.0005, Student's 't' test) in the concentration in decidua (10-76 +/- 4-45), and to a lesser extent (P less than 0-05) in the myometrium (5-84 +/- 2-65) and amnion (4-77 +/- 2-51). The overall concentration in decidua during the first trimester (3-09 +/- 1-02) was significantly lower (P less than 0-005) than in endometrial tissue(16-82 +/- 10-13). The concentration was lower in myometrial tissue from non-pregnant subjects (2-90 +/- 2-21), than in the corresponding tissue removed at term (4-19 +/- 1-06) or during labour 5-84 +/- 2-65). The results for prostaglandin F2 alpha showed a similar pattern, but the values were significantly lower in the umbilical cord, and the percentage changes in concentration in the decidua and myometrium were of a higher magnitude.

In an immunoperoxidase study, pregnancy-associated placental protein A (PAPP-A) was localized to the cytoplasm of decidual stromal cells, the villous syncytiotrophoblast and on the surface of placental trophoblast. A decidual and a trophoblastic origin is suggested for PAPP-A, and its presence on the surface of the syncytiotrophoblast is interpreted as a possible immunoprotective layer.

With a view to investigating the mechanism of the action of estrogen involved in nidation, the biologically active rat uterine RNA thereby derived was extracted and then administered in the uterine cavity of castrated rats to observe the ensuing morphological changes in the endometrium and compare them with those resulting from the administration of decidual RNA which is non-estrogenised. There was a tendency toward increasing proliferation noted in the endometrial epithelium of the castrated rats 48 hours after the administration of the biologically active uterine RNA, as in the endometrial epithelium of animals receiving estradiol-17beta. There was also an increase in the number of 3H-thymidine-labelled cells present in the endometrial epithelium of animals so treated. On the other hand, no such changes were observed following the administration of the RNA pretreated with RNase. In the case of castrated rats administered with the RNA in the uterine cavity under progesterone priming, the endometrial epithelium as the site of increase in the number of the radio-labelled cells at 24 hours was found to be superseded by the stromal cells at 48 hours. A similar phenomenon of transfer was seen with regard to the site of an increase in the mitosis index caused by pretreatment with colchicine. An observation of the endometrium of castrated rats 72 hours after the administration of decidual RNA disclosed that the stroma was mildly edematous with the stromal cells somewhat swollen, but without any such changes in the endometrial epithelium as seen following the administration of the biologically active uterine RNA. These findings led us to administer such biologically active substances as uterine RNA, decidual high molecular RNA, human chorionic RNA, rat placental RNA (L15) and early pregnant rat uterine RNA (L4) separately in the uterine cavity of delayed implantation rats to determine whether any one or more of these RNAs had a nidation promoting effect just like estrogen. As a result, nidation was found to be induced in animals administered with 1 approximately 100 microgram of the biologically active uterine RNA or 100 microgram of early pregnant rat uterine RNA. No nidation was provoked by other specimens of RNA. A study was then made of blastocysts recovered by the flush-out technique from the uterus of animals receiving 1 approximately 10microgram of human chorionic RNA or 10microgram of rat placental RNA, which failed to induce nidation. It was shown that these blastocysts were dormant morphologically. From these results it is apparent that both biologically active uterine RNA and early pregnant rat uterine RNA are effective in promoting nidation. It is also suggested that, in the process of implantation of fertilized eggs, nidatory estrogen may first promote RNA synthesis in the endometrium and then exhibit its action through the medium of RNA thus synthesized.