Regional specialization of the cell membrane-associated, polymorphic mucin (MUC1) in human uterine epithelia.

The cell membrane-associated, polymorphic mucin, MUC1, has been proposed to hinder implantation by virtue of its anti-adhesive properties. Consistent with this proposal is the observation of a dramatic decrease in MUC1 protein and mRNA expression in the uterine epithelium of several species at the time of implantation. In contrast, little change in glandular epithelial expression of MUC1 protein or its mRNA during the peri-implantation period has been detected in humans. However, expression in the luminal epithelium, i.e. the epithelium involved in embryo attachment, has not been reported. Using tissue samples with a clearly defined luminal epithelium and antibodies directed against the cytoplasmic domain found in all cell-associated MUC1 species (CT-1) and against two MUC1 ectodomain epitopes, HMFG-1 and HMFG-2, we demonstrate that MUC1 expression in the luminal epithelium is maintained throughout the menstrual cycle. The staining observed with CT-1 correlates with that seen with HMFG-2, but not HMFG-1. HMFG-1 reactivity was high in all regions except basal glands in the mid proliferative endometrium and fell to very low levels throughout the tissue in the mid secretory phase. In all cases, HMFG-1 reactivity could be restored by predigestion with keratanase or neuraminidase which removes keratan sulphates and sialic acids, respectively. These observations suggest that regionally restricted glycosylation generates an altered external structure of MUC1. These alterations appear to decrease accessibility to the MUC1 protein core region and are maximal in luminal epithelium at the receptive phase. Due to their large highly extended structures, MUC1 ectodomains are very likely to be among the first cell surface components encountered during human blastocyst attachment to the luminal epithelium. Thus, MUC1 either must be locally removed during the attachment process or functions actually to promote the initial steps in embryo adhesion to the apical surface of the human uterine epithelium.

The progressive rise in the expression of alpha crystallin B chain in human endometrium is initiated during the implantation window: modulation of gene expression by steroid hormones.

Human endometrium undergoes sequential changes during the menstrual cycle and becomes receptive to implantation during a defined period in the secretory phase. We attempted to identify the genes expressed during this period by representational difference analysis (RDA). When the cDNAs of a proliferative endometrium were used as the driver and the cDNAs of a post-ovulatory day 5 endometrium were used as the tester, a number of bands were identified by RDA. DNA of the cloned RDA products revealed that the majority of the clones contained a fragment of a cDNA identical to that of a crystallin B chain. Northern blot analysis showed that the expression of the alpha crystallin B chain mRNA was absent during the proliferative phase. The expression of the mRNA of alpha crystallin B chain first appeared in the secretory phase, progressively increased during this phase and peaked in the late secretory endometria. The pattern of expression of alpha crystallin B chain mRNA in the endometrium of mature cycling baboons (Papio anubis) was similar to that seen in human endometrium. As revealed by Western blot analysis, the expression of the alpha crystallin B chain protein in human endometrium followed a pattern of expression similar to its mRNA. At the cellular level, the immunoreactive protein first appeared in the surface epithelial cells of human endometrium within the implantation window without significant immunoreactivity in the underlying glandular cells. During the mid- and late secretory phases, the intensity of staining in the epithelial cells was enhanced and an intense immunoreactivity was developed in the glandular epithelium, alpha crystallin B chain was virtually an epithelial product and no immunoreactivity for this protein was detectable in the stromal cells, endothelial cells or lymphoid cells. The expression of alpha crystallin B chain could be regulated, by medroxy progesterone acetate as well as by oestrogen withdrawal, in human endometrial carcinoma cells (EnCa-101), transplanted to nude mice. Based on the data presented here, the known function of alpha crystallin B chain and its distinct pattern of expression in human endometrium, we suggest that this protein is an important factor within the molecular repertoire that makes endometrium receptive to implantation.

Cell surface expression of HIP, a novel heparin/heparan sulfate binding protein, of human uterine epithelial cells and cell lines.

Previous studies established that uterine epithelial cells and cell lines express cell surface heparin/heparan sulfate (HP/HS)-binding proteins (Wilson, O., Jacobs, A. L., Stewart, S., and Carson, D. D. (1990) J. Cell. Physiol. 143, 60-67; Raboudi, N., Julian, J., Rohde, L. H., and Carson, D. D. (1992) J. Biol. Chem. 267, 11930-11939). The accompanying paper (Liu, S., Smith, S. E., Julian, J., Rohde, L. H., Karin, N. J., and Carson, D. D. (1996) J. Biol. Chem. 271, 11817-11823) describes the cloning of a full-length cDNA corresponding to a candidate cell surface HP/HS interacting protein, HIP, expressed by a variety of human epithelia. A synthetic peptide was synthesized corresponding to an amino acid sequence predicted from the cDNA sequence and used to prepare a rabbit polyclonal antibody. This antibody reacted with a protein with an apparent Mr of 24,000 by SDS-polyacrylamide gel electrophoresis that was highly enriched in the 100,000 x g particulate fraction of RL95 cells. This molecular weight is similar to that of the protein expressed by 3T3 cells transfected with HIP cDNA. HIP was solubilized from this particulate fraction with NaCl concentrations > or = 0.8 M demonstrating a peripheral association consistent with the lack of a membrane spanning domain in the predicted cDNA sequence. HIP was not released by heparinase digestion suggesting that the association is not via membrane-bound HS proteoglycans. NaCl-solubilized HIP bound to heparin-agarose in physiological saline and eluted with NaCl concentrations of 0.75 M and above. Furthermore, incubation of 125I-HP with transblots of the NaCl-solubilized HIP preparations separated by two-dimensional gel electrophoresis demonstrated direct binding of HP to HIP. Indirect immunofluorescence studies demonstrated that HIP is expressed on the surfaces of intact RL95 cells. Binding of HIP antibodies to RL95 cell surfaces at 4 degrees C was saturable and blocked by preincubation with the peptide antigen. Single cell suspensions of RL95 cells formed large aggregates when incubated with antibodies directed against HIP but not irrelevant antibodies. Finally, indirect immunofluorescence studies demonstrate that HIP is expressed in both lumenal and glandular epithelium of normal human endometrium throughout the menstrual cycle. In addition, HIP expression increases in the predecidual cells of post-ovulatory day 13-15 stroma. Collectively, these data indicate that HIP is a membrane-associated HP-binding protein expressed on the surface of normal human uterine epithelia and uterine epithelial cell lines.

Heat shock proteins in human endometrium throughout the menstrual cycle.

Human endometrium is a steroid-sensitive tissue and there is evidence that supports the viewpoint that heat shock proteins (HSP) are implicated in the regulation of steroid function. Therefore, in this study we examined the expression of various members of the heat shock family of proteins in the steroid-responsive human endometrium. Western blot analysis revealed that the expression of HSP90 showed minimal changes throughout the menstrual cycle. When normalized to the amount of HSP90, the expression of HSP27, HSP60 and the constitutive form of heat shock protein 70 (HSC70) increased progressively during the late proliferative and early secretory phases, and diminished in the mid- to late secretory and menstrual phases. In contrast, the inducible form of heat shock protein 70 (HSP70) did not undergo these changes. The cellular and subcellular localizations of these proteins were examined in human endometria by immunohistochemical staining. With the exception of HSP70, which was found primarily in the epithelial cells, the immunoreactivity for other heat shock proteins was found in both the stroma and the epithelium. Immunoreactivity for HSP27 was found in the lymphoid aggregates within endometrial stroma, and both HSP27 and HSP90 were found in endothelial cells. The immunoreactive heat shock proteins were found in the nuclei and/or cytoplasm of cells. However, no consistent nuclear versus cytoplasmic staining emerged, and such localization was irrespective of the site, the cell type or the phase of the menstrual cycle. Our findings show that endometrium has a full complement of heat shock proteins. The menstrual cycle-dependent changes in the amounts of heat shock protein suggest regulation by steroid hormones.

Progressive rise in the expression of interleukin-6 in human endometrium during menstrual cycle is initiated during the implantation window.

In order to be prepared for implantation, human endometrium undergoes a predictable series of proliferative and secretory changes. Cytokines play an important role in regulation of these changes. Therefore, in this study, we immunolocalized the cytokine, interleukin-6 (IL-6), its receptor and the signal transducer gp130 in human endometrium throughout the menstrual cycle. During the entire menstrual cycle, the IL-6 receptor and gp130 were found primarily in the endometrial glands and to a lesser extent in the stroma. The immunoreactivity of these proteins did not change in endometrial cells during the entire menstrual cycle with an exception of reduced immunoreactivity of gp130 in endometrial glands during menstrual phase. Immunostaining showed that immunoreactive IL-6 was weakly expressed in human endometrium during the proliferative phase. Strong immunoreactivity for IL-6 appeared in endometrium during the putative 'implantation window'. Expression was by far most pronounced both in the glandular and surface epithelial cells. The amount of immunoreactive IL-6 in the epithelium progressively increased during the secretory/menstrual phases. During the late secretory phase, only stromal cells in the upper functionalis exhibited immunoreactivity for IL-6. Western blot analysis corroborated the immunohistochemical data. Human endometrial IL-6 consisted of a protein with an apparent mobility of 26 kDa. The immunoreactive band of IL-6 was weak in the proliferative phase. The expression of this protein increased progressively during the secretory/menstrual phases. The findings show a cell-specific pattern of distribution for immunoreactive IL-6 in human endometrium. The menstrual cycle-dependent expression of IL-6 suggests that this cytokine may play a role in changes in endometrium that prepare this tissue for implantation and menstrual shedding.

The signals and molecular pathways involved in implantation, a symbiotic interaction between blastocyst and endometrium involving adhesion and tissue invasion.

Implantation is a complex process requiring the interaction of the blastocyst, and subsequently the developing embryo with the endometrium. Initially, the detailed cellular interactions implicated in this process were defined. More recently, many signals and molecular pathways are recognized that induce, or regulate the complex series of interactions required for implantation. In this review, the cellular and molecular interactions that take place during implantation are discussed.

Menstruation is associated with disordered expression of desmoplakin I/II and cadherin/catenins and conversion of F- to G-actin in endometrial epithelium.

Endometrium is unique since it is the only tissue that undergoes regular cyclic bleedings. Menstrual shedding is associated with the breakdown of endometrium, including the fragmentation of endometrial glands. To gain insight into the underlying basis of fragmentation of the endometrial epithelium during the menstrual phase, we examined the expression of proteins implicated in epithelial cell-cell binding in human endometria throughout the entire menstrual cycle. Western blotting failed to reveal differences in the relative amount of E-cadherin, alpha- or beta-catenin or actin in the menstrual endometria compared with those in the proliferative or secretory phases. However, specific changes in the expression pattern of these proteins as well as desmoplakin I/II were detected by immunohistochemical staining in epithelial cells of menstrual endometria. Desmoplakin I/II, E-cadherin, alpha- and beta-catenins and beta-actin were localized to intercellular borders as well as the luminal and basal regions of glandular epithelium during the proliferative and secretory phases. Immunoreactivity of E-cadherin and alpha-catenin was confined to epithelial cells, whereas beta-catenin and beta-actin were present in epithelial cells, as well as in stroma and endothelial cells. Binding of F-actin to fluorescein isothiocyanate-labelled phalloidin localized this form of actin to the intercellular borders, and the basal and luminal cytoplasm of epithelial cells in proliferative and secretory endometria. Menstrual shedding was associated with disorganization of the site-specific distribution of desmoplakin I/II, E-cadherin and alpha- and beta-catenins.(ABSTRACT TRUNCATED AT 250 WORDS)

Site and menstrual cycle-dependent expression of proteins of the tumour necrosis factor (TNF) receptor family, and BCL-2 oncoprotein and phase-specific production of TNF alpha in human endometrium.

Apoptosis in human endometrial epithelium progressively increases from early to late secretory/menstrual phases and remains consistently more prominent in the basalis. It has been suggested that tumour necrosis factor (TNF) alpha secreted during the secretory/menstrual phases plays a role in induction of programmed cell death in these cells. In the present study, we characterized expression of receptors of TNF alpha, Fas antigen and BCL-2 in endometrial cells to gain insight as to whether this type of cell death in endometrium may be related to differential or preferential expression of these proteins at specific phases of the menstrual cycle. In addition, to relate production of TNF alpha to the development of apoptosis, the amount of TNF alpha released by human endometrium was measured. Immunostaining demonstrated that the TNF receptor (TNFr; p55/60)-I, TNFr-II (p75/80) as well as Fas protein were expressed in endometrial epithelium throughout the entire menstrual cycle. This expression was progressively diminished from the basalis towards the upper functionalis. In the proliferative phase, the expression of BCL-2 was prominent in the endometrial glands particularly in those residing in the basalis. This expression became weak as early as the third post-ovulatory day and remained low during the remaining phases of the menstrual cycle. The amount of TNF alpha released by endometrial fragments obtained from various phases of the menstrual cycle was determined. The amount of TNF alpha released into the culture medium by the endometrium was low in the proliferative phase. However, the amount of released TNF alpha progressively increased in the secretory phase and peaked in the menstrual phase. TNFr-I, TNFr-II, Fas, BCL-2 and TNF alpha could be identified by Western blot analysis of proteins extracted from endometrium. Therefore, endometrial epithelium by virtue of expression of receptors of TNF alpha as well as Fas protein is properly poised to respond to ligand signals that regulate apoptosis. Induction of apoptosis in endometrial epithelium and menstrual shedding may be related to loss of the protective effect of BCL-2 as well as to the amount of TNF alpha.

Expression of adhesion molecules in human endometrial vasculature throughout the menstrual cycle.

In the present study, we examined the pattern of expression of human leukocyte antigen (HLA)-DR as well as several adhesion molecules implicated in leukocyte trafficking, including ICAM-1, E-selectin, and VCAM-1 in human endometrium. All of the vessels in endometrium exhibited HLA-DR and ICAM-1 throughout the menstrual cycle. In the proliferative phase, endothelial cells in the functionalis were weak to nonreactive for VCAM-1 and were E-selectin negative (E-selectin-). Endothelial cells of the vessels in the basalis and within myometrium were VCAM positive (VCAM-1+)/E-selectin-. In sharp contrast, in the secretory phase, endothelial cells in the basalis were VCAM-1+/E-selectin+. Surprisingly, endometrial glands, primarily those in the basalis, expressed E-selectin and VCAM-1 during the entire menstrual cycle. Stromal cells were ICAM-1+ and were focally HLA-DR+ around HLA-DR+ lymphoid cells during the entire menstrual cycle and were E-selectin-/VCAM-1- during the proliferative phase. Immunoreactivity for VCAM-1 and E-selectin, however, appeared in the stromal cells in the upper functionalis in the secretory phase. Immunoreactivity for VCAM-1 was the distinguishing feature that separated the lymphoid cells in the aggregates from other nonaggregated lymphoid cells. Recruitment of leukocytes to tissues is in part due to cytokine-regulated expression of specific molecules on endothelial cells. Therefore, we tested the effects of cytokines on the expression of these molecules in endothelial cells derived from microvasculature. ICAM-1 and VCAM-1 were inducible in a dose-dependent fashion in the endothelial cells by interleukin-1 alpha (IL-1 alpha), interferon-gamma (IFN gamma), and tumor necrosis factor-alpha (TNF alpha). Expression of HLA-DR in endothelial cells was inducible by IL-1 alpha and IFN gamma and not by TNF alpha. Expression of E-selectin on endothelial cells was induced only by IL-1 alpha, not by IFN gamma or TNF alpha. Cytokine treatment of endothelial cells significantly enhanced the binding of leukocytes to endothelial cells. The data show a heterogeneity in the vasculature of endometrium with respect to the expression of various adhesion molecules. This heterogeneity is potentially related to the type or amount of cytokine with which endothelial cells are activated. In addition, unique cell- and site-specific expression of adhesion molecules in human endometrium throughout the menstrual cycle may account for the distinct distribution pattern of leukocytes in this tissue.

Fetal lung maturation: amniotic fluid catecholamines, phospholipids, and cortisol.

The identification of beta-adrenergic receptors in the fetal lung led us to investigate amniotic fluid catecholamine levels in relation to other indices of fetal pulmonary maturity in late pregnancies (n = 62). Significant correlations were found between the percentage of phosphatidylglycerol and concentrations of norepinephrine (r = 0.44, p less than 0.001), its intraneuronal deaminated metabolite 3,4-dihydroxyphenylglycerol (DOPEG; r = 0.74, p less than 0.0001), epinephrine (4 = 0.43, p less than 0.001), and cortisol (r = 0.78, p less than 0.001). Highly significant elevations of these substances were noted with accelerated pulmonary maturation. Lecithin/sphingomyelin (L/S) ratios showed a significant correlation with cortisol levels (r = 0.50, p less than 0.001); however, significant associations between L/S ratios and catecholamine levels were found only when complicated pregnancies were excluded. These findings support the contention that fetal contention that fetal adrenergic activity participates in the process of pulmonary maturation.

Amniotic fluid catecholamines and metabolites in intrauterine growth retardation.

Simultaneous determinations of amniotic fluid levels of the catecholamines dopamine (DA), norepinephrine (NE), and epinephrine (E), and the intraneuronal metabolites of DA, 3,4-dihydroxyphenylacetic acid (DOPAC) and NE, 3,4-dihydroxyphenylglycol (DOPEG), were made, by radioenzymatic assay, in pregnancies resulting in growth-retarded (n = 14) and normal (n = 63) infants. Significant elevations in the mean concentration of NE (p less than 0.000005), E (p less than 0.005), and DOPEG (p less than 0.000001) as well as a significant decrease in the mean concentration of DOPAC (p less than 0.000001) were found in pregnancies resulting in growth-retarded infants as compared to pregnancies resulting in normal infants. Amniotic fluid DOPEG levels were found to be the most discriminative. As amniotic fluid catecholamines are predominantly of fetal origin, these findings suggest that an increase in adrenergic activity and a decrease in dopaminergic activity occur in intrauterine growth retardation as a response to chronic stress.

Maternal smoking and elevation of catecholamines and metabolites in the amniotic fluid.

To assess the effect of maternal smoking on fetal adrenergic activity, simultaneous measurements in amniotic fluid of the parent catecholamines, dopamine (DA), norepinephrine (NE), and epinephrine (E), as well as the specific intraneuronal deaminated metabolites of DA, 3,4-dihydroxyphenylacetic acid (DOPAC), and of NE, 3,4-dihydroxyphenylglycol (DOPEG), were made by radioenzymatic assay. In the second trimester, a significant (p less than 0.002) and selective elevation of the mean DOPEG concentration was noted in the amniotic fluid of smokers (N = 8) as compared to nonsmokers (N = 36). In the third trimester, significant elevations were found in the mean amniotic fluid concentration of E (p less than 0.0002) and NE (p less than 0.0005), as well as DOPEG (p less than 0.0002), of smokers (n = 12) when compared to nonsmokers (N = 12). There were no significant differences in amniotic fluid concentrations of DA and its deaminated metabolite DOPAC. Since compartmentalization of catecholamines exist between the maternal and fetal circulations, the elevated levels of NE, E, and DOPEG in the amniotic fluid of smokers suggests fetal adrenergic activation as a result of fetal hypoxia and/or by a direct effect of nicotine on the fetal adrenergic system.

Circadian rhythm in circulating concentration of dihydroxyphenylacetic acid in normal women.

The 24-h patterns of plasma concentration change in dopamine (DA) and its immediate deaminated metabolite, dihydroxyphenylacetic acid (DOPAC), were determined in 6 normal women (16 studies) by a modified radioenzymatic assay. Changes in DOPAC levels exhibited a marked circadian rhythm, with peak during the day and a nadir at night. At 1200 h, the DOPAC concentration increased significantly (P less than 10(-4)) to a peak value 62.9 +/- 8.4 ng/ml) 117% higher than the 24-h mean. At 2200 h, plasma DOPAC decreased (P less than 10(-4)) to a nadir concentration (10.0 +/- 3.3 ng/ml) 66% lower than the 24-h mean. The circadian rhythm of DOPAC could be reproducibly demonstrated over at least 4 successive days in individual subjects. There were no well defined circadian variations in plasma concentrations of DA. Since the plasma DOPAC concentration appears to reflect central nervous system dopaminergic neuronal activity, the present demonstration of a circadian rhythm of plasma DOPAC suggests that the activity of central nervous system DA-containing cells is higher during the day than at night.

An increase in catecholamines and metabolites in the amniotic fluid compartment from middle to late gestation.

By means of a modified radioenzymatic assay, simultaneous determinations of the parent catecholamines, epinephrine (E), norepinephrine (NE), and dopamine (DA), and their deaminated metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and 3,4-dihydroxyphenylglycol (DOPEG), were made in amniotic fluid obtained during the second (N = 44) and third (N = 20) trimesters of normal pregnancies. Significant positive correlations were noted between gestational age and concentrations of E, NE, DA, DOPAC, and DOPEG in amniotic fluid. These findings provide evidence of progressive fetal adrenergic maturation through pregnancy. In addition, our data suggest a parallel maturational event in the central nervous and peripheral catecholamine systems of the fetus, since DOPAC and DOPEG are more representative of catecholamine neuronal activity in the brain.

Local alcohol injection of the vulva: discussion of 35 cases.

During the past 20 years the senior author has, in selected cases, used alcohol injection of the anogenital area for the treatment of intractable pruritus. The initial experience of this treatment in 30 cases that had failed to respond to other appropriate local therapy was reported 6 years ago. During the past 5 years an additional 35 procedures have been performed. The results are similar to those reported previously and indicate that if the cases are carefully selected and have had appropriate but unsuccessful conservative treatment, the results of alcohol injection may prove to be satisfactory.

Placental histopathology of midtrimester termination.

Placentas spontaneously passed after second-trimester terminations of pregnancy using either hypertonic sodium chloride or hyperosmolar urea plus prostaglandin F2alpha (PGF2alpha) were examined to determine histologic characteristics. The placentas of hypertonic sodium chloride terminations demonstrated a type of "coagulation necrosis" that has been described previously, while placentas of pregnancies terminated by hyperosmolar urea plus PGF2alpha showed a similar pattern in about one half the cases but a histologic pattern of less severe damage in the remaining cases. The 2 groups showed no significant differences when characteristics such as injection-abortion interval or estimated hypertonicity of the fluid were examined.

PIP: Second trimester placentas, passed spontaneously in 17 abortions induced by hypertonic sodium chloride and in 45 abortions induced by hyperosmolar urea plus prostaglandin F, were histologically compared; in 16 of the 17 sodium chloride cases, a pattern of severe tissue damage (Type A) was observed, and in the urea cases, approximately half of the cases exhibited Type A damage and the other half had a less severe form of damage (Type B). Type A damage is characterized by a zone of "coagulation necrosis" in which cell structure is lost, vessels are thrombosed, and there is severe inflammation. In Type B damage, cell structure is maintained, vessels are not thrombosed, and inflammation is diffuse. In previous studies of sodium chloride induced abortions, Type A damage had been noted and attributed to the hypertonicity of the abortifacient agent. Since half of the urea cases also had Type A damage, an attempt was made to discover clinical differences between the Type A and Type B urea cases. No significant differences were found; however, a scatter diagram suggests that patients with high concentrations of urea and with a longer interval between injection and subsequent abortion tended to exhibit Type A damage, while those with a shorter interval between injection and abortion manifested Type B damage. Tables include clinical characteristics of age, parity, duration of gestation, injection abortion interval, and amount of amniotic fluid removed for urea cases and a scatter diagram depicting interval time and estimated urea concentration. Photographs of tissue sections depicting Type and Type B damage are also included.

Pregnancy success following abdominal myomectomy for infertility.

There were 46 patients with primary infertility (34 patients) or secondary infertility (12 patients) with no other detectable cause except myomas. After myomectomy, 38% of the patients with primary infertility had full-term pregnancies and 50% of those with secondary infertility. Preoperative distortion of the endometrial cavity was not impressively correlated with the postoperative prognosis.

The effect of magnesium sulfate on fetal heart rate baseline variability.

Variability of the baseline fetal heart rate is correlated with good fetal outcome, and loss of baseline variability has been observed as a sign of fetal distress. Central nervous system depressing drugs may also decrease fetal heart rate variability, and thus recognition of the effect of medication on the baseline fetal heart rate is important for accurate interpretation of fetal monitor tracings. In the cases reported, marked decrease in fetal heart rate variability was observed within 4 minutes of intravenous administration of magnesium sulfate, and fetal outcome was good in all cases.