Mammalian egg and embryo coats are primarily represented by the zona pellucida which, however, undergoes biochemical and structural changes during fertilization and embryo development. It serves several functions, from ovulation until shortly before implantation. Initially the zona pellucida induces sperm-oocyte interaction, acrosome reaction and prevents polyspermy. Later, it prevents disaggregation of the noncompacted blastomeres and the premature attachment to the oviductal and endometrial surface. Additionally, it protects the embryo against toxins and xenobiotics, as well as bacteria, viruses and phagocytes. As the embryo is covered by the zona pellucida until immediately before implantation, all signals of embryo-maternal signalling have to pass the zona and are detectable within it. Logically we may define the zona pellucida as a mailbox of the embryo-maternal signalling, especial for investigating these messages. Oviductal, uterine and embryonic proteins are incorporated into the zona pellucida as embryonic development goes on and change the zona's morphological and biochemical properties. Whether a protein is able to penetrate the zona, whether it accumulates within the zona or whether it is rejected by the zona depends on its biochemical properties. Three specific proteins have been detected within the embryonic coats (IGFBP3, HB-EGF, P19). New insights into the physiology of the zona pellucida might present new achievements in the in vitro culture of embryos, and present new aspects as to how to prevent zona hardening. Furthermore, knowledge of the zona proteins enables the development of immunocontraceptive vaccines. Consequently, the zona pellucida is not only significant with regard to fertilization, but also during early embryonic development. Investigations of the zona pellucida will yield new insights into early embryo-maternal signalling which in turn may lead to improvements in classic IVF and new approaches to in vitro culture.

Animals↗

PubMed1999

Expression of uteroglobin in the human endometrium.

Uteroglobin is a progesterone binding protein, a member of the antiflammin gene family and possibly a novel cytokine. Initially, uteroglobin was identified as the major protein of rabbit uterine secretion during the phase of preimplantation. Counterparts of the rabbit uteroglobin or its gene are described in rat, mouse, hamster, hare, pig, horse and human. While uteroglobin appears as one of the most extensively studied proteins, particularly its physico-chemical properties, including its crystal structure and its gene, the true physiological role of this protein still remains to be unravelled. Essential to understanding the significance of human uteroglobin in reproductive organs, particularly in the endometrium, is a knowledge of the spatial and chronological expression of this secretory protein. Our studies on 115 volunteers combined reverse transcription-polymerase chain reaction (RT-PCR), immunohistochemistry and quantitative assessment by an enzyme-linked immunosorbent assay for uteroglobin. The expression, localization and release of uteroglobulin in the human endometrium are presented. Secretory uteroglobin is found in endometrial tissue homogenates in highest levels of expression during the early luteal phase (days 15-19, 340 pg/mg total protein). In turn, uteroglobin is released into the uterine lumen in peak amounts during the receptive phase of the menstrual cycle (mid-luteal phase, days 20-23, secretion level 833.4 pg/mg total protein). Our immunohistochemical studies match with these results, as uteroglobin is located during the early and mid-luteal phase in the apical compartments of endometrial gland cells. These observations strongly suggest an involvement of uteroglobin in endometrial preparations for implantation.

Endometrium↗

PubMed1999

Tumour necrosis factor-alpha (TNF-alpha) in human endometrium and uterine secretion: an evaluation by immunohistochemistry, ELISA and semiquantitative RT-PCR.

Tumour necrosis factor-alpha (TNF-alpha is a pleiotropic cytokine synthesized throughout the female reproductive tract. Even though evidence has accumulated that supports its role in autocrine and paracrine processes, its expression and function in the human endometrium are still not completely understood. To gain a better understanding of the synthesis and release of TNF-alpha in the endometrium and how this relates to concentrations in uterine secretion, its expression throughout the menstrual cycle was investigated by three different techniques. Samples of endometrial tissue and uterine secretions were collected from patients undergoing abdominal and vaginal hysterectomy for benign reasons. The mRNA expression of TNF-alpha was investigated in homogenized endometrial tissue by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) (n = 18). An assessment of the cellular TNF-alpha protein localization in the endometrial glands was performed immunohistochemically (n = 39). The concentrations of the secreted TNF-alpha protein in endometrial secretion were determined by enzyme-linked immunosorbent analysis (n = 30). All three methods gave similar results on the temporal expression of TNF-alpha mRNA and TNF-alpha protein during the cycle. Concentrations of endometrial TNF-alpha mRNA in tissue samples and TNF-alpha protein in uterine secretion were quite low at the beginning of the cycle, rose sharply in the mid- to late proliferative phase and decreased towards the end of the cycle. The concentrations of TNF-alpha protein in the endometrial glands, as shown by immunohistochemical investigation, stayed high throughout the secretory phase at values slightly below those of the late proliferative phase.

Endometrium↗

PubMed1999

Nuclear and cytoplasmic maturation of mouse oocytes after treatment with synthetic meiosis-activating sterol in vitro.

Synthetically produced meiosis-activating sterol, a sterol originally derived from follicular fluid (FF-MAS), induces meiotic maturation of mouse oocytes in vitro. We therefore compared FF-MAS-induced maturation of naked mouse oocytes arrested in prophase I by either hypoxanthine (Hx) or forskolin (Fo) with spontaneous maturation of naked oocytes. FF-MAS-treated oocytes overcame the meiotic block by Hx or Fo, although germinal vesicle breakdown was delayed by 11 h and 7 h, respectively. We also investigated the influence of FF-MAS on chromosome, microtubule, and ultrastructural dynamics in Hx-cultured oocytes by immunocytochemistry and electron microscopy. Similarly to spontaneously matured oocytes, chromosomes became aligned, a barrel-shaped spindle formed, and overall organelle distribution was normal in FF-MAS-matured oocytes. The number of small cytoplasmic asters was elevated in FF-MAS-treated oocytes. Although the number of cortical granules (CGs) was similar to that in spontaneously matured oocytes, the overall distance between CGs and oolemma was increased in the FF-MAS group. These observations suggest that the initiation of meiotic maturation in FF-MAS-treated oocytes in the presence of high cAMP levels leads to a delayed but otherwise normal nuclear maturation. FF-MAS appears to improve oocyte quality by supporting microtubule assembly and by delaying CG release, which is known to contribute to reduced fertilization.

Animals↗

PubMed1998

The receptive endometrium is characterized by apoptosis in the glands.

Apoptosis in the human endometrium up to now has been detected during the mid to late luteal phase and therefore connected to the onset of the menstrual shedding. However, there is increasing evidence that regulated apoptosis may be important during decidualization and implantation. To investigate a possible role for apoptosis in the human endometrium and its regulation, we correlated the immunolocalization of the apoptosis regulatory protein bcl-2 and the proliferation marker Ki67 to the in-situ nuclear DNA fragmentation - a key feature of apoptosis - detected by using the terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labelling (TUNEL) method during the menstrual cycle. Whereas proliferation and bcl-2-expression were predominantly detected in the glandular compartment during the proliferative phase, only single apoptotic cells could be shown during this period. During the transformation of the endometrium (days 15-19) proliferation and bcl-2 expression decreased markedly and there was no sign of apoptosis. At the beginning of the implantation window (days 19-20) we could detect the first signs of apoptosis in the glandular epithelia in the basalis, which extended to the functionalis during the luteal phase. Proliferation and bcl-2 expression are limited to the stromal compartment comprising the large granular lymphocytes - during this time, and extend in parallel with apoptosis from the basal to the functional layers. Apoptosis therefore may be related to the loss of the protective effect of bcl-2 and may have significance for the establishment of an endometrium adequately prepared for successful implantation.

Adult↗

PubMed1998

Insulin and insulin-like growth factor-I promote rabbit blastocyst development and prevent apoptosis.

Insulin as well as insulin-like growth factor-I (IGF-I) promote early embryo development, and IGF-I binds to the coats of preimplantation rabbit embryos. As the IGF-I receptor is expressed from the morula stage onwards, the embryos are capable of responding to insulin and IGF-I, which is present in the oviductal and uterine secretions that surround them. The embryonic coats were removed to exclude any influence by IGF-I bound to the coats. The in vitro development of such embryos under classical conditions appears to be retarded. Addition of IGF-I (68 pM-6.8 nM) or insulin (68 nM-6.8 microM), however, promotes blastocyst formation. Embryo development under such conditions is not significantly different from that of embryos cultured with intact coats. In contrast, coat-free embryos cultured without IGF-I or insulin supplementation show apoptosis. Because IGF-I stimulates cell proliferation and prevents apoptosis, we investigated whether insulin or IGF-I may act as "survival factors" in preimplantation development. Therefore, apoptosis was induced by slight UV irradiation (254 nm wave length; 11.8 W/m2). Compared to the untreated controls, embryos displaying retarded development or degeneration were increased by 22% and 14%, respectively. Addition of IGF-I or insulin to the culture medium of UV-irradiated embryos improved [3H]thymidine incorporation and blastocyst formation significantly. By immunohistochemistry we could show that addition of insulin (0.68-68 nM) decreased apoptosis and increased cell proliferation in a dose-dependent manner, supporting blastocyst development significantly.

Animals↗

PubMed1997

Establishment of a human endometrial cell culture system and characterization of its polarized hormone responsive epithelial cells.

Uterine epithelial cells from normal human endometrium were cultured as a primary cell culture in a dual-chambered system. The epithelial cells were isolated from endometrial tissue of the proliferative phase obtained by hysterectomy. The epithelial cells were seeded on Millicell CM filters coated with the extracellular matrix Matrigel. Depending on the culture conditions, the epithelial cells formed a polarized cell monolayer on Matrigel or gland-like structures in Matrigel. The epithelial cell polarity was maintained during culture, which could be proved by electron microscopy. The progesterone and estrogen receptors as typical marker molecules for physiologically intact endometrial epithelial cells could be detected immunohistochemically as well as by RT-PCR in vitro and were down-regulated by medroxyprogesterone acetate (MPA) used as progesterone analogue. As this cell culture system exhibits morphological and immunohistochemical characteristics, typical for the in vivo situation, and since it can be modulated by hormone treatment under the in vitro conditions described, it represents a valuable tool for investigating processes that are essential for endometrial differentiation and reproductive functions.

Cell Division↗

PubMed1997

Binding of IGF-I to preimplantation rabbit embryos and their coats.

It is known that insulin-like growth-factor I (IGF-I) promotes early embryonic development from the morula to the blastocyst stage in rabbits (28). Therefore we used autoradiography to investigate whether IGF-I binds to preimplantation embryos and its coats. From Day 3 after mating onwards, a clear binding of IGF-I to the embryos was observed. There was no difference in binding to the embryoblast or trophoblast cells. Using ligand blot, several IGF-binding proteins (IGFBP; 31 kDa, 33 kDa, 36 kDa, three overlapping bands at 40 to 55 kDa) were obvious in the embryoblast and trophoblast. A 120 to 130 kDa protein was observed exclusively in the embryoblast. Significant binding of (125)I IGF-I to the coats of embryos older than 3 d was detected, and IGF-I was bound via a 38 kDa protein, as detected by ligand blot. To investigate the origin of this protein, the patterns of IGFBP were determined in the oviductal and uterine fluids of pregnant animals (Days 0 to 6). The following binding proteins were observed regularly in the oviductal and uterine flushings: 28 kDa, 32 kDa and 3 overlapping bands in the area of 40 to 55 kDa. In the oviduct the main IGF binding protein was the 32 kDa band (38.7% to 45.9%), while in the uterus it was the 3 overlapping bands at 40 to 55 kDa (42.5% to 24.1%). Because IGF-I is produced in the oviduct and uterus (27), IGFBPs are found in oviductal and uterine fluids, IGF-I is stored in the coats, IGF-I binds to preimplantation embryos and IGF-I promotes early embryonic development (28), the IGF system seams to have a function in the maternal-embryonic interaction.

Journal Article↗

PubMed1997

Cervical ripening in guinea-pigs after a local application of nitric oxide.

In humans cervical ripening is an inflammatory reaction accompanied by an infiltration of white blood cells and the remodelling of the extracellular matrix. Similar changes occur in guinea-pigs during cervical ripening. Nitric oxide (NO) is thought to be important in the maintenance of pregnancy because it is synthesized by the uterus and inhibits contractility. Previous studies in rats also demonstrated that an NO generating system is present in the cervix and is up-regulated during labour. We studied the local effect of the NO donor sodium nitroprusside (SNP) on cervical ripening in guinea-pigs during advanced pregnancy. SNP (5 mg/injection) was administered into the cervical canal in 0.2 ml phosphate-buffered saline containing 3% hydroxycellulose twice a day either for 1 [on day 42 post coitum (p.c.)] or 2 consecutive days (days 42-43 p.c.; term day 67 + 3 p.c.). The effects were assessed 24 h after treatment by both extensibility measurements (force resistance to incremental stretch) and morphological evaluation (light and electron microscopy after in-situ fixation). The control animals were treated with the vehicle. In another experiment, the guinea-pigs were subcutaneously (s.c.) treated on day 43 p.c. with either the progesterone antagonist onapristone (3 and 10 mg/animal s.c.) or with prostaglandin E2 (PGE2) (1 and 3 mg/animal s.c.) and the PGE2 analogue sulprostone (0.03 and 0.1 mg/animal s.c.). The cervical extensibility was measured 24 h later. One-day SNP treatment tended to reduce cervical resistance, but not significantly, whereas 2 day treatment with SNP led to a significant increase in cervical extensiblity (P < 0.05) with little effect on cervical dilatation (indirect evidence of the absence of uterine contractions). The effects on cervical resistance were comparable to those achieved with 10 mg onapristone and high-dose prostaglandins (PG)s (3 mg PGE2 and 0.1 mg sulprostone) treatment. An electron microscope study of the SNP-treated animals revealed a dissolution of collagen fibres, stromal oedema, arterial dilatation, and the infiltration of macrophages, lymphocytes and granulocytes. Numerous mast cells were also present. The morphological effects of SNP were similar to those observed during normal cervical ripening at term. We conclude that the local application of a NO donor effectively induces cervical ripening without inducing labour in pregnant guinea-pigs.

Animals↗

PubMed1996

Isolation of human uteroglobin from blood filtrate.

The purpose of this study was to assess the possibility of isolating biologically active peptides from human blood using large volumes of blood filtrate, which are available from patients undergoing extracorporeal ultrafiltration because of renal insufficiency. This filtrate was submitted to six chromatographic separation steps, yielding one purified peptide which was completely analysed in its primary structure. It was found to be strikingly similar to proteins, described initially as rabbit uteroglobin (or blastokinin) and, more recently, from human bronchial lavage as the '10 kDa Clare cell protein', as well as from human urine as 'protein-1'. The natural molecule contains two chains of identical amino acid sequences of 70 residues which are arranged as an antiparallel dimer due to the disulphide bonds between two cysteines at positions 3 and 69. Mass analysis of the molecular forms yielded molecular weights from 15827 Da (non-oxidized form) to 15859 Da (bi-oxidized form). We conclude that this peptide isolated from the filtrate represents the human uteroglobin, and we demonstrate for the first time that this peptide may be involved as a humoral factor in reproductive or other physiological functions.

Amino Acid Sequence↗

PubMed1995

Expression of gap junction connexins in the human endometrium throughout the menstrual cycle.

Expression of connexins, the proteins which comprise gap junction channels, is regulated by ovarian hormones in the female reproductive tract of rodents. In order to determine if these hormones also affect connexin expression in the human uterus, the distribution patterns of different connexins (cx26, cx32, cx43) were investigated by immunohistochemistry in human endometrial tissue collected throughout the menstrual cycle. During the early proliferative phase of the cycle extremely low staining for connexin 43 was observed and connexin 26 antigens could not be detected. An increase in the amount of connexin 43 in stromal cells and of connexin 26 in glandular and luminal epithelial cells was seen from days 11-15 of the cycle. Following ovulation, the expression of both connexins was suppressed and was completely abolished in the late secretory phase. Weak staining for connexin 32 was found mainly in the late proliferative and the early secretory phase and was restricted to the basal membrane region of the glandular cells. These results suggest that the different connexins could represent cell biological markers for the proliferation and differentiation of the human endometrium throughout the menstrual cycle.

Connexin 26↗

PubMed1995

Identification of cyclophilin A from human decidual and placental tissue in the first trimester of pregnancy.

The protein patterns of tissue homogenates from human decidua and placenta of first trimester pregnancies were investigated. Particular attention was paid to the low molecular weight components of these tissues, since substantial evidence has accumulated that some of these smaller proteins show a characteristic cyclic and pregnancy expression. Two specific bands were purified from homogenates of first trimester decidua and placenta using gel filtration and anion exchange chromatography. These bands were separated by gel electrophoresis and blotting onto polyvinylidendifluoride membrane. Partial amino acid sequencing of both proteins revealed sequences identical to human cyclophilin A. One protein was sequenced V-N-P-T-V-F-F-D-I-A-V-D-G-E-P-L-G-R-(X)-S-F-E-L-F-A-D-K-V-P and identified as the 17 kDa isoform of cyclophilin A. The other protein was sequenced V-N-P-T-V-F-F-D-I-A and identified as the 18 kDa isoform of cyclophilin A.

Amino Acid Isomerases↗

PubMed1994

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").

Animals↗

PubMed1994

Localization of uteroglobin mRNA by nonradioactive in situ hybridization in the pregnant rabbit endometrium.

The distribution of uteroglobin mRNA has been investigated in the endometrial compartments of the rabbit uterus during early pregnancy (day 0.5 p.c.--day 12 p.c.) using nonradioactive in situ hybridization. Digoxigenin-dUTP labeled oligodesoxy-nucleotide-probes (24mer) and an anti-digoxigenin-antibody conjugated with alkaline phosphatase were developed and used. It could be shown, that uteroglobin mRNA localization is restricted to the endometrial epithelium. There are differences in the extent of uteroglobin mRNA expression within the epithelial cells, which is in accord with our interpretation on the existence of different epithelial cell populations. From day 0.5 p.c. to day 9 p.c. the cells of the basal glands express uteroglobin mRNA continuously, whereas the proliferating surface epithelium shows a remarkable fluctuating pattern of uteroglobin mRNA expression. On day 2 p.c. the whole surface epithelium starts to express the uteroglobin message, and up to day 5 p.c. all these cells show a high level of uteroglobin mRNA expression, which drops significantly on day 6 p.c., when significant changes in the cyto-morphology of the surface epithelium for implantation occur. On day 7 p.c., there is no more uteroglobin mRNA expression in the surface epithelium, however remaining expression in the basal glands. The latter is evident up to day 9 p.c. From day 10 p.c. onwards, neither the luminal nor the deep glandular epithelium express any uteroglobin mRNA. Our observations on the cellular level have been continued in parallel studies on endometrial homogenates by Northern Blot analysis of uteroglobin mRNA (600 bases). Finally, it is discussed whether Uteroglobin mRNA may be an useful marker for the differentiation of various endometrial epithelial cell populations.

Animals↗