The feasibility of a less invasive method to assess endometrial maturation--comparison of simultaneously obtained uterine secretion and tissue biopsy.

OBJECTIVE: To compare the assessment of endometrial maturation parameters in endometrial secretion samples obtained by a novel minimally invasive technique with those assessed in tissue biopsies. DESIGN: Prospective study. SETTING: University Hospital. POPULATION: Healthy female volunteers attending a gynaecological outpatient clinic. METHODS: Endometrial secretion fluid and tissue sampling 5 days after a spontaneous ovulation assessed with ultrasound. MAIN OUTCOME MEASURES: Progesterone (P) receptor, Ki-67 expression and the Noyes criteria were used to date endometrial biopsies. In the endometrial fluid samples, glycodelin A (GdA), leukaemia inhibitory factor (LIF) and P levels were analysed, and protein content and electrophoresis patterns were determined. RESULTS: All data were correlated to estradiol (E2) and P serum concentrations. The dating according to histology and immunohistochemical staining patterns correlated significantly with GdA levels (r=0.376, P=0.048) in endometrial fluid samples as well with serum levels of E2 (r=0.568, P=0.001) and P (r=0.408, P=0.023). No correlation was observed between tissue dating and LIF levels and protein content in endometrial fluid samples. CONCLUSIONS: The measurement of GdA in endometrial secretion samples may provide a less invasive method for assessing endometrial maturation in potential conception cycles without disrupting implantation.

Impact of ovarian stimulation on mid-luteal endometrial tissue and secretion markers of receptivity.

The objective of this study was to investigate the effect of ovarian stimulation for IVF on endometrial secretion and tissue markers of receptivity in the mid-luteal phase. In 10 oocyte donors, endometrial secretions and biopsies were sampled 5 days after spontaneous ovulation and oocyte retrieval in consecutive cycles. Four subjects received progesterone in the luteal phase of the stimulated cycles. Mid-luteal endometrial maturation in the stimulated cycle was compared with the spontaneous cycle, by histological dating, Ki-67, oestrogen receptor (ER) and progesterone receptor (PR) expression, secretion levels of leukaemia inhibitory factor (LIF), glycodelin A (GdA) and progesterone, and protein profile. No significant differences in histological markers, expression of Ki-67, PR, ER, secretion protein profiles or concentrations of LIF, GdA, or progesterone were observed when comparing natural with stimulated cycles. Progesterone supplementation of stimulated cycles was associated with significantly lower Ki-67 (P = 0.03) and ER (P = 0.04) expression compared with the non-supplemented stimulated cycle. In this pilot study, ovarian stimulation was not demonstrated to alter the studied markers of endometrial maturation in the mid-luteal phase.

Indoleamine-dioxygenase is expressed in human decidua at the time maternal tolerance is established.

The semi-allogeneic fetus has to be tolerated by the maternal immune system. In mice, it has been shown that inhibiting indoleamine-dioxygenase (IDO) leads to fetal rejection, suggesting a central significance for IDO in establishing maternal tolerance. Consequently, we have analyzed IDO expression in human endometrium and decidua to determine whether it may be of significance in human reproduction. Endometrial (n=60) and decidual (n=68; first and second trimester) tissue samples and isolated cells were analyzed for IDO mRNA and protein expression by real-time PCR, Western blot and immunohistochemistry. IDO expression in the decidua of proven fertile women (n=34) was compared to women presenting with their first pregnancy (n=22) and women with a history of miscarriages (n=12). Expression of IDO was localized in glandular epithelial cells and scattered stromal leukocytes. Expression started at the mid-luteal phase in the menstrual cycle and was high until the second trimester of pregnancy. However, glandular expression of IDO decreased during the second trimester, whereas expression in villous trophoblast started at this time. There were no significant differences in decidual IDO expression between proven fertile women and women presenting with their first pregnancy or women with a history of miscarriages. From the expression pattern we conclude that IDO may play a central role in human pregnancies for the establishment of maternal tolerance of fetal antigens. Thereby, IDO expression may be needed in each pregnancy independently from prior pregnancies, and a history of miscarriage may not reflect a general deficiency in IDO expression.

Estradiol and medroxyprogesterone acetate regulated genes in T47D breast cancer cells.

Many mammary tumors express estrogen receptors (ER) and progesterone receptors (PR), and there is increasing evidence that progestins influence gene expression of breast tumor cells. To analyse the impact of progestins on breast cancer cells, we compared (a) the expression of two cytokines, involved in tumor progression, and searched (b) for differentially regulated genes by a microarray, containing 2400 genes, on T47D breast cancer cells cultured for 6 days with 17beta-estradiol (E2) or E2+medroxyprogesterone acetate (E2+MPA). Lower amounts of PDGF and TNFalpha were found in culture supernatants of E2+MPA treated T47D cells. MPA addition induced a 2.8-3.5-fold increase of the mRNA expression of (a) tristetraprolin, which is involved in the posttranscriptional regulation of cytokine biosynthesis, and (b) zinc-alpha2-glycoprotein and Na, K-ATPase alpha1-subunit, which both resemble differentiation markers of breast epithelium. In contrast, the mRNA expression of lipocalin 2, which promotes matrixmetalloproteinase-9 activity, was decreased five-fold in E2+MPA treated cells. Our data show that the expression of genes from various functional gene families is regulated differentially by E2 and E2+MPA treatment in T47D cells. This suggests that exogenous progestins applied for therapy and endogenous changes of the progesterone levels during the menstrual cycle both influence breast cancer pathophysiology.

Luteal control of endometrial receptivity and its modification by progesterone antagonists.

The objective of this study was to determine preimplantational effects of progesterone antagonists (PA) on the cell biology of the endometrium, on corpus luteum (CL) function and on the complex interactions between these two organs. The PA onapristone (ZK 98.299) or lilopristone (ZK 98.734) was given to pseudopregnant rabbits at days 5, 6, and 7 p.hCG. Three treatment protocols were investigated: Exp I, onapristone or lilopristone treatment only; Exp II, onapristone treatment after hysterectomy at day 1 p.hCG; Exp III, onapristone treatment together with 17 beta-estradiol, which represents the ultimate luteotropic hormone in the rabbit. In Exp I, onapristone and lilopristone gave rise to endometrial regression (inhibition of epithelial proliferation and differentiation, increase of apoptosis). Simultaneous addition of 17 beta-estradiol in Exp III did not alter these findings. A rapid luteolysis was found in Exp I. In Exp II and III, however, onapristone was unable to impair luteal development and function. Due to the unaffected CL in Exp III and due to the 17 beta-estradiol substitution, the endometrium was capable of starting a new transformation, which met all requirements for receptivity at day 12 p.hCG. Transfers of day 4 p.c. blastocysts from untreated donors into such delayed secretion recipient rabbits at days 12 p.hCG resulted in normal implantations and normal embryonic development. Contrary to Exp III, the missing of any luteotropic substitutions in Exp I resulted in a complete inhibition of further uterine transformation. The present findings suggest that PA can exert a direct inhibitory effect on the endometrium, which is followed by an indirect luteolytic effect via endometrial mediators. The simultaneous addition of a proper luteotropic signal to the PA protocol results in survival of CL. Furthermore, this prolongation of the CL life span can be interpreted as a functional dissociation of the endometrium from the CL.

Inhibition of the estradiol-mediated endometrial gland formation by the antigestagen onapristone in rabbits: relationship to uterine estrogen receptors.

There is evidence from previous studies that progesterone antagonists (antigestagens) modify estrogen responses at endometrial and myometrial levels without having affinity to the estrogen receptor (ER). The purpose of the present study was to investigate the influence of the antigestagen onapristone (ZK 98 299) on the uterus in ovariectomized (OVX) estradiol (E2)-substituted rabbits (3.0 micrograms/animal.day). The animals were treated for 8 days with different doses of onapristone (3.0, 10.0, and 30.0 mg/animal.day, sc). Uterine growth was not influenced by onapristone compared to that in OVX E2-substituted controls. However, morphological (light microscopy, transmission electron microscopy) and morphometric criteria indicated that there was a significant dose-dependent inhibition of the estrogen-induced gland formation within the endometrium and degenerative changes in glandular epithelial cells. By contrast, there were morphological signs of activation of the endometrial stroma (proliferation, increased capillarization, and vascularization, edema) above the level of E2-treated animals. A dose-dependent increase in the concentration of uterine cytosolic ER, nuclear ER, and ER mRNA (ER mRNA) was measured in uterine homogenates after onapristone treatment compared to values in OVX E2-substituted controls. Immunocytochemical analysis of ER in uterine sections suggests that the increase in ER after onapristone treatment took place predominantly in the myometrium and surface epithelium. To examine whether the observed interference was mediated via the progesterone receptor (PR), E2-substituted rabbits were treated, in a separate experiment, with onapristone (10.0 mg/animal.day, sc) and various doses of progesterone (1.0, 3.0, and 10.0 mg/animal.day, sc). Progesterone reversed all onapristone-induced changes, indicating that the observed effects were mediated via the PR. The data indicate that the antigestagen onapristone interacts with estrogen action in the absence of the natural PR ligand. The increase in ER and ER mRNA concentrations after onapristone treatment in OVX E2-treated animals suggests that this antigestagen abolished an inhibitory action of the unoccupied PR on ER biosynthesis.

Apoptosis of extravillous trophoblast cells limits the trophoblast invasion in uterine but not in tubal pregnancy during first trimester.

During the first trimester of pregnancy extravillous trophoblast cells (EVT) invade the maternal decidua. Invasion normally is reduced from the second trimester onwards and stops in the inner third of the myometrium. By contrast, in extrauterine tubal pregnancy, trophoblast invasion may even penetrate the tubal wall, which ultimately leads to the rupture of the fallopian tube. Induction of apoptosis of EVT cells, by maternal immune competent cells, may be an important mechanism to limit EVT invasion in uterine pregnancy. Tissue specimens from first and second trimester uterine pregnancy and first trimester tubal pregnancy were analyzed for apoptosis by TUNEL- and M30-staining. By immunohistochemical double labelling, maternal leukocyte subtypes were co-localized to apoptotic cells and in this context, the number of CD56(+)NK cells was analyzed. Our data show that apoptosis is confined to the decidua basalis. Most apoptotic cells are single cytokeratin-positive epithelial cells residing in the stromal compartment. Consequently these cells can only be EVT cells. Maternal leukocytes are not apoptotic. They are located in close contact to apoptotic cells. The number of apoptotic cells in the second trimester (1.8+/-0.7 per cent) is reduced compared to first trimester (5.6+/-0.7 per cent) of uterine pregnancy. In parallel, the number of NK cells declines from first (24.4+/-2.9) to second (12.4+/-1.8) trimester. Furthermore, apoptosis is significantly reduced in ectopic (0.9+/-0.3 per cent) compared to eutopic first trimester pregnancies. Consequently, we suggest that in first trimester uterine pregnancy, induction of EVT cell apoptosis by the maternal immune system is one mechanism to limit EVT invasion. During the second trimester, in parallel to declining numbers of NK cells, the mechanism changes. However, in tubal pregnancy due to differing immunological microenvironments at the ectopic implantation site, apoptosis induction fails, which deleteriously may result in uncontrolled invasion and penetration of the tubal wall.

Cytosol and nuclear progesterone-receptor concentrations in the rabbit endometrium during early pseudopregnancy under different treatments with estradiol and progesterone.

In an attempt to understand the mechanism of the antiprogestational action of estrogens during early pseudopregnancy we determined the cytosolic and nuclear concentrations of progesterone receptors in the endometrium of rabbits treated with hCG followed by various combinations of estradiol and progesterone. The progestational response of the endometrium was followed by quantitation of the uteroglobin content in the uterine lumen. In rabbits treated with hCG alone there was a clear progestational response (40% relative uteroglobin content), but only 16% of the progesterone receptors were located in the nucleus. After additional treatment with progesterone the progestational response remained high (45% relative uteroglobin content), the total cellular content of progesterone receptor increased, and 5% of the complexes were found in the nucleus. These findings suggest that a consumption of nuclear progesterone receptor is required for progestational action. Treatment of pseudopregnant rabbits with estradiol resulted in a marked increase not only of the total cellular progesterone receptor but also of the percentage of it located in the nucleus (35%). Concomitantly, the progestational response was markedly inhibited (5% relative uteroglobin content). These results confirm the relevance of nuclear consumption of progesterone receptor for progestational action, and suggest that some antiprogestational effects of estrogens may be due to their interference with the mechanism of progesterone receptor processing.

Expression and regulation of 17beta-hydroxysteroid dehydrogenase 7 in the rabbit.

We cloned partial cDNA sequences of rabbit 17beta-hydroxysteroid dehydrogenase 1 (17beta-HSD1) and 17beta-HSD7. We analyzed the tissue distribution of 17beta-HSD7 as well as the expression in corpus luteum and endometrium during pseudopregnancy. The obtained cDNA sequence of 17beta-HSD7 coded for all functional regions of the protein and showed 86 and 81% similarity to human and rodent sequences, respectively. The partial sequence of rabbit 17beta-HSD1 was 76 and 82% similar to rodent and human sequences. By Northern analysis 17beta-HSD7 expression was predominantly found in reproductive organs like ovary, oviduct, endometrium, placenta and mammary gland. Substantial expression was also apparent in the heart, stomach and cerebellum. The 17beta-HSD1 could be detected in placenta by reverse transcriptase-polymerase chain reaction (RT-PCR), so far. During rabbit pseudopregnancy 17beta-HSD7 expression was found to be regulated in corpus luteum as well as in endometrium. In the corpus luteum, strongest expression occurred from d10 to d14 of pseudopregnancy (p. hCG) and was downregulated on d16 p. hCG. In endometrium strongest expression of 17beta-HSD7 was found on d6 p. hCG, when the endometrium was differentiated to its implantation permissive status.

The discovery of uteroglobin and its significance for reproductive biology and endocrinology.

The discovery of uteroglobin resulted from investigations on the biochemical composition of oviductal and uterine secretions of the rabbit and other mammals. These determinations about physiological composition were urgently requested to prepare culture media for research on early mammalian development in vitro. Discovery of significant proteins during the sixties reflected the laboratory skills of that time. Protein characterization was achieved by isolation via Sephadex gels, electrophoresis on polyacrylamide gels, and finally immunoprecipitation using classical polyclonal antibodies. The molecular biology was not yet established. Uteroglobin could be found as the major protein component of rabbit uterine secretion. From the beginning, it was already identified as an unusually small, spheric uterine secretory molecule without any carbohydrates--hence its name. Uteroglobin was the first mammalian protein that turned out to be progesterone-regulated and, at the same time, released in mg amounts actually in one organ compartment. Moreover, uteroglobin and its gene proved to be a reliable model for the description of progesterone/progesterone receptor complex action at the DNA level. After its original observation in the uterus, however, uteroglobin was detected also in several other organs, for example, the epididymis, the seminal vesicle, and the lung. Initially, it could not be found in the blood, which challenged the hypothesis that uteroglobin specifically should operate by local activation rather than by a humoral or endocrine effect. Later, though, the human uteroglobin molecule, isolated from blood filtrate, was used for detailed structural analyses. The rabbit uteroglobin model certainly was beneficial for reproductive biological research. Experimental interference with steroid hormone regulation during preimplantation presented surprising effects, which led to the discovery of the transposition of the implantation window. The uterine secretion protein patterns, in particular the uteroglobin fraction and the beta-glycoprotein fraction, served as decisive marker profiles to identify the biological stage of the intrauterine microenvironment during preimplantation. This diagnostic procedure, using only protein parameters, enabled us to precisely predict the receptive stage of the endometrium for donated blastocysts to achieve implantation successfully.

Effect of clomiphene on the functional morphology of oviductal and uterine mucosa.

The effect of clomiphene on the functional morphology of the uterine and oviductal mucosa was studied in rabbits by means of light and electron microscopy (SEM and TEM). Tissues were obtained from mature nulliparous animals receiving subcutaneous doses ranging from 0.01-10 mg/kg per day. In all cases the effects were evaluated 2 days after termination of treatment. With 2 and 10 mg, effects were studied up to 12 and 7 days, respectively. Normally appearing oviductal and endometrial tissues, corresponding to various stages of the cycle, were observed with doses up to 5 mg. However, a burst of cellular secretory activity becomes evident with the administration of higher doses. Apical protrusions or cytoplasmic portions seem to be extruded, and draw attention. These cytologic events are concentrated near gland openings in the endometrium and can be seen abundantly among cilia of oviductal cells. Other ultrastructural changes are evident as well. These histologic changes may reflect nonsynchronous cellular activities which in turn interfere with oviductal and endometrial functions before implantation.

Hormone regulation and hormone antagonist effects on protein patterns of human endometrial secretion during receptivity.

Endometrial receptivity is a particular stage of maturation during the luteal phase to permit implantation. We have studied endometrial protein secretion and its patterns evaluated by SDS-PAGE, laser densitometry and Western blots. Uterine secretion electrophoresis (USE) permits highly sophisticated analyses of the intrauterine milieu and allows clinical determination of the receptive stage of the endometrium. This technique reveals direct parameters by patterns of numerous individual protein bands, mainly resolved between 68.0 and 6.5 kD. Characteristic bands appear during the typical functional states of the menstrual cycle presenting evidence on the diagnostic capacity of this method to identify stages of adequate (= normal) or inadequate (= defective) luteal phase maturation. Several individual protein bands appear as characteristic markers for the receptive stage of the luteal phase. We have isolated and molecularly identified several of these proteins: histones H2A, H2B, H3 and H4. In order to identify the endocrine dependency of the protein bands, which significantly contribute to the "receptive stage pattern," patients were treated with the progesterone antagonist RU 486 at day LH +2. The assessment 4 days later revealed deficient USE patterns, particularly diminished and missing bands of the H2A-, H2B-, and H3-histones. These results demonstrate progesterone-dependent components of the endometrium at the receptive stage, which can be used as useful markers for an improved precision in luteal phase diagnostics. On the other hand, essential parts of the protein pattern may serve as new targets for successful contraceptive interventions ("endometrial contraception").

Potential risk of light and room temperature exposure to preimplantation embryos.

Adverse effects of simultaneous exposure to visible light and room temperature were investigated in rabbit early cleavage stages and morulae. Routine laboratory conditions were mimicked as close as possible. Embryonic development was assessed by cell proliferation via incorporation of tritiated thymidine, by gross morphology, and by electron microscopy. Damage was detectable in both stages at 1-hour exposure by statistically significant impaired cell proliferation. Compared with single exposure to each individual stressor, combined exposure to light and room temperature amplified detrimental effects. Ultrastructural analysis of light-exposed cleavage stages revealed no indication of cell injury at 4-hour exposure. Gross morphology proved to be too inaccurate to evaluate damage imposed by exposure toward both physical factors investigated.

Effects of trophoblast invasion on the distribution of leukocytes in uterine and tubal implantation sites.

OBJECTIVE: To distinguish endocrine and paracrine influences on leukocyte subpopulations at uterine and tubal implantation sites. DESIGN: Retrospective immunohistochemical study. SETTING: Departments of Anatomy, and Obstetrics and Gynecology, School of Medicine, RWTH University of Aachen, Aachen, Germany. PATIENT(S): Ten women with a viable ectopic pregnancy (EP), 25 women who had undergone elective first-trimester termination of pregnancy, and 4 women who had undergone hysterectomy with adnexectomy. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Quantitative analysis of leukocyte subpopulations at the implantation sites and their corresponding noninvaded tissues, decidual tissue from patients with EP, and tubal mucosa from normal menstrual cycle. RESULT(S): Similar numbers and characteristic distribution patterns of macrophages, T cells, and B cells were found at both normal intrauterine and tubal implantation sites. Natural killer (NK) cells were always absent from tubal mucosa. The number and distribution of leukocytes within decidual tissue from women with EP corresponded to those in the noninvaded decidual compartment in intrauterine pregnancy (IUP). CONCLUSION(S): Leukocyte populations present in the tubal and uterine mucosa are an intrinsic characteristic of these tissues. The distinct leukocyte distribution pattern at the implantation sites suggests that the invading trophoblast exerts a paracrine influence on endometrial and endosalpingeal leukocytes. The absence of natural killer cells from the tubal wall may be one reason for the higher degree of invasiveness of the trophoblast at the tubal implantation site.

Advanced secretory changes in the proliferative human endometrial epithelium following clomiphene citrate treatment.

In an effort to characterize the effect of clomiphene citrate (CC) on the human endometrium, we took biopsy specimens of the endometrium 24 to 48 hours after CC treatment (100 to 250 mg/day for 5 consecutive days). Nineteen biopsy specimens were taken from 19 patients. Fifteen of the patients suffered from anovulatory infertility associated with oligomenorrhea or normal cycle length. The other four patients were amenorrheic, two in association with hypogonadotropic hypogonadism and two with hypergonadotropic hypogonadism. The histopathology of all samples was evaluated with the use of light microscopy, including periodic acid-Schiff (PAS) and PAS-diastase staining for glycogen demonstration. All samples were also examined with the use of scanning electron microscopy (SEM). Serum levels of estradiol (E2), progesterone (P), luteinizing hormone, and follicle-stimulating hormone were determined on the day of biopsy. In 10 of the 19 biopsy specimens, local or diffuse signs of early secretory events were demonstrated by the presence of subnuclear vacuolization and glycogen in the glandular epithelial cells. SEM corroborated these findings of advanced secretory changes by demonstrating apical protrusions at luminal epithelial cells and secretory products within the glands' openings. The E2 levels ranged between 110 and 1500 pg/ml (mean, 371 pg/ml) and P levels were either undetectable or less than 1.1 ng/ml. The two patients with hypogonadotropic hypogonadism both exhibited the same phenomena; those with primary ovarian failure had atrophic endometrium even after high-dose CC treatment. This observation, together with the low P levels detected, indicating the lack of luteinization, suggests a possible direct effect of CC on the endometrium.(ABSTRACT TRUNCATED AT 250 WORDS)

Direct effects of nicotine on rabbit preimplantation embryos.

The effects of various concentrations of nicotine on the in vitro development of 1-cell rabbit preimplantation embryos and on the DNA-synthesis of 4-day-old rabbit blastocysts are investigated. Exposure of rabbit preimplantation embryos to concentrations of nicotine higher than 1 X 10(-3) M results in a marked decrease in the in vitro development and in DNA-synthesis. Concentrations of nicotine below 1 X 10(-3) M have no effect on these early embryos. It can be concluded that the concentrations of nicotine which exert a direct embryotoxic effect are higher than the concentrations that may be expected in the blood circulation of humans considered to be "normal smokers".

The progesterone antagonist onapristone retards the advanced endometrial transformation after gonadotropin stimulation in rabbits.

Ovarian stimulation with gonadotropins (GN) during human in vitro fertilization and embryo transfer (IVF/ET) therapy alters the ovarian steroid output, especially that of progesterone. As a consequence, endometrial transformation is advanced, and embryo implantation is hampered. This study used the rabbit model to determine if the application of the progesterone antagonist (PA) onapristone (ONA) could retard endometrial development after GN-stimulation. Rabbits were GN-stimulated twice daily with 5 IU FSH and 5 IU LH on 3 consecutive days with a) hMG (n = 10) or b) with a mixture of recombinant FSH and LH (n = 10). The animals were then mated, and hCG was injected i.v. to ensure ovulation. This day is designated as day 0 post coitum (d 0 p.c.). On day 2 p.c., five animals of each group were treated with 20 mg ONA/kg body weight and five with vehicle for control. On d 5 p.c. endometrial transformation was analyzed by morphology, uteroglobin (Ugl)-mRNA expression, and proliferation. Embryos were flushed from the uteri. Their number and morphology was evaluated. The endometrium of GN-stimulated control animals demonstrated very long endometrial glands and narrow stromal septa. Ugl-mRNA expression was restricted to the cells at the bottom of the gland. 17.0 +/- 4.6% (mean +/- SD) of glandular cells and 6.0 +/- 5.3% of luminal epithelial cells proliferated. In ONA-treated animals, endometrial glands were significantly shorter, and the pattern of arborization was less pronounced. Endometrial gland cells and luminal epithelial cells expressed Ugl-mRNA. Furthermore, glandular and luminal cells proliferated with high intensity (38.6 +/- 6.8% and 36.4 +/- 9.3%, respectively). These results indicate that the status of endometrial differentiation was retarded after ONA-treatment. Nevertheless, the embryos of these ONA-treated animals were well developed. In conclusion, after GN-stimulation, ONA treatment retarded the advanced endometrial transformation in rabbits. Therefore, postovulatory administration of a PA might be a possible strategy to modulate the advanced endometrial development in IVF-cycles.

Progestins, progesterone receptor modulators, and progesterone antagonists change VEGF release of endometrial cells in culture.

The influences of the synthetic progestin, medroxyprogesterone acetate (MPA), the progesterone receptor modulator J867, and the antagonist ZK137316 were studied in vitro on isolated endometrial epithelial cells, as well as endometrial fibroblasts. We evaluated the expression of estrogen receptor alpha (ER) and the progesterone receptor (PR) by RT-PCR. ER and PR were strongly expressed in the fibroblasts and epithelial cells under treatment with 10(-8) M 17beta-estradiol (E(2)). Treatment with 10(-6) M J867 or ZK137316 upregulated the PR expression as did E(2), in contrast to treatment with 10(-6) M MPA, which caused a downregulation of PR in epithelial cells, but not in fibroblasts. In addition, the vascular endothelial growth factor (VEGF) release into the cell culture medium was analyzed by a VEGF-ELISA. VEGF which plays an important role in angiogenesis, is regulated by steroid hormones as well as hypoxia. E(2) stimulates VEGF release into the medium in both cell types. MPA reduces VEGF release significantly in the fibroblast cell culture, but increases it in the epithelial cell culture. ZK137316, in the presence or absence of E(2), reduces VEGF release in fibroblast cell culture. J867 increases the VEGF production in fibroblasts only in the presence of E(2). Both compounds show no significant effects, compared to E(2), in epithelial cell culture. The different results for the epithelial cells and fibroblasts indicate that the pharmacological effects of PR modulators (PRMs) and progesterone antagonists (PAs) may be cell specific and depend on the presence or absence of partial progestagenic agonistic activities. This observation opens up new perspectives for various clinical applications.

Immunocytochemical localization of uteroglobin in the rabbit endometrium.

Uteroglobin, the progesterone dependent pregnancy-characteristic endometrial protein in the rabbit, is found within the endometrial epithelium on the fourth and sixth day of pregnancy at the electron-microscopic level by use of the immunoperoxidase technique and a specific anti-uteroglobin serum from the sheep. As known from earlier studies, uteroglobin is the predominant protein synthesized of the endometrial secretion. In the present study, it is localized exclusively in the non-ciliated epithelial cells. A common route of secretory proteins within these cells is observed by uteroglobin labelling: rough endoplasmatic reticulum----Golgi complex----condensing vesicles----secretory products. Uteroglobin occurs in small vesicles on the trans-face of the Golgi complex, and in addition beneath the apical plasma membrane where it appears in membrane-bound vesicles, which apparently are extruded into the uterine lumen. Most of the uteroglobin is located in the luminal secretion. The distribution of intracellular uteroglobin is found only in cells of the basal endometrial gland, adjacent to the myometrium. The cytoplasm of uterine epithelial cells facing the cavum does not show uteroglobin reaction products.

Effect of in vitro culture on the dynamics of uteroglobin distribution in rabbit blastocysts.

The purpose of this study was to investigate the localization and transport of uteroglobin in normal rabbit blastocysts (day 4-day 6 p.c.) and in those cultured for 6-48 h in vitro, using a specific radioimmunoassay and immunocytochemistry. The results of the radioimmunoassay showed that in day 4 p.c. blastocyst tissue (based on homogenate measurements) a significant decrease of the uteroglobin content started after only 6 h of culture in vitro. A significant concomitant rise of uteroglobin was observed in the culture medium after 12 h of in vitro culture. Using immunocytochemistry it was not possible to detect uteroglobin in any compartment of the non-cultured or in vitro cultured day 4 p.c. blastocysts. The efflux of uteroglobin down a concentration gradient was confirmed by the immunocytochemistry in non-cultured and in vitro cultured day 5 p.c. and day 6 p.c. blastocysts. Uteroglobin immunoreactions were mainly detected in non-cultured blastocysts (day 5 and 6 p.c.) in large vesicles of the trophoblast cells. In addition endocytotic vesicles at the inside of the apical membrane of trophoblast cells, some cell debris within the perivitelline space and the neozona were labelled. During in vitro culture of day 5 and 6 p.c. blastocysts, uteroglobin labelling in the coverings did not change. In non-cultured and cultured day 5 and 6 p.c. blastocysts neither the compartments of the embryoblast, the endoderm cells nor the blastocyst cavity showed any uteroglobin immunoreactions. After only 6 h of in vitro culture, uteroglobin immunoreactions were no longer found within the trophoblast cells. The reaction did not reappear during the course of in vitro culture up to 48 h, suggesting a complete lack of de novo synthesis of uteroglobin by blastocysts.