Classen‐Linke I, Moss S, Gröting K, Beier H M, Alfer J & Krusche C A (2012) Histopathology 61, 955–965
Mammaglobin 1: not only a breast‐specific and tumour‐specific marker, but also a ...hormone‐responsive endometrial protein
Aims: The secretoglobin mammaglobin 1 (MGB1) is strongly expressed in breast tumours, and is therefore used to detect breast cancer metastases, although it has also been detected in other tissues. The aim of this study was to examine MGB1 expression and its hormonal regulation in human endometrium to further investigate the use of MGB1 as a marker molecule.
Methods and results: Mammaglobin 1 expression was assessed immunohistochemically in endometrial samples from 60 normal fertile patients throughout the menstrual cycle, in 49 endometriotic tissue samples, in 15 endometrial adenocarcinomas, and in 36 breast carcinomas. In addition, 25 endometrial samples were analysed by western blot and quantitative real‐time reverse transcription polymerase chain reaction. To prove hormonal regulation, primary endometrial epithelial cells were cultured with 17β‐oestradiol and promegestone. MGB1 was detected in human endometrial tissue, with peak expression during the luteal phase, in 31% of endometriotic samples, in 53% of endometrial adenocarcinomas, and in 64% of breast carcinomas. MGB1 mRNA expression was increased in vitro by hormonal treatment.
Conclusions: Our data show that MGB1 expression is not restricted to normal and malignant breast tissue. Besides its documented occurrence in endometriotic and malignant endometrial tissues, MGB1 is also expressed in normal human endometrium, and such expression is controlled by steroid hormones.
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 μM), 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 âsurvival factorsâ in preimplantation
development. Therefore, apoptosis was induced by slight UV irradiation (254 nm wave length; 11.8 W/m 2 ). 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 3 Hthymidine incorporation and blastocyst formation significantly. By immunohistochemistry we could show that addition of insulin
(0.68â68 nM) decreased apoptosis and increased cell proliferation in a dose-dependent manner, supporting blastocyst development
significantly.
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 α (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 17β-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.
After its original description as a steroid-dependent protein in the rabbit uterus, uteroglobin became one of the best characterized
proteins. However, detailed knowledge of its physiological role ...remains an enigma. In this study we investigate how its structure
is phylogenetically conserved in the horse compared to other mammalian species. Northern blot analysis showed that in horses,
the main expression of uteroglobin appears in lung, uterus, and prostate tissues. Western blot analysis demonstrated that
the dimeric form of uteroglobin is found predominantly in biological compartments. Using a RACE-PCR technique, we cloned and
sequenced the full-length cDNA (473 base pairs) that encodes equine uteroglobin. The nucleotide sequence was shown to characterize
the primary structure of this protein. This enabled us to add equine uteroglobin to a comparative amino acid alignment of
8 other uteroglobin molecules, and finally, to unravel 14 evolutionary completely conserved amino acids. We summarize these
results with a computer-based 3-D model of horse uteroglobin, and discuss new concepts on the physiological role of uteroglobin,
in particular as a specific binding protein.
Following attenuation of progesterone production corpora lutea are selectively cleared, a process associated with recruitment of macrophages. In the rabbit little is known about luteal immune cell ...phenotypes and expression of cytokines, which influence immune cells and resident luteal cells, during luteolysis. Consequently, we studied luteal immune cells by immunohistochemistry as well as luteal IL-10, TNFalpha, MCP-1, IFN-gamma, and IL-1beta mRNA expression by semiquantitative RT-PCR from day 8 to day 20 in pseudopregnant rabbits (d8-d20 p.hCG). Luteal function was assayed by serum progesterone levels. Functional luteolysis commenced by d14 p.hCG as indicated by attenuation of serum progesterone levels. X4(+) tissue macrophage levels increased transiently on d12 and d14 p.hCG, whereas CD5(+) T-cell levels transiently declined on these two days. CD68(+) macrophages increased progressively after d16 p.hCG. The luteal mRNA level of the anti-inflammatory cytokine IL-10 as well as the mRNA levels of the pro-inflammatory cytokines TNFalpha and MCP-1 increased after d16 p.hCG and remained elevated up to d20 p.hCG. IFN-gamma and IL-1beta mRNA expression did not vary systematically. In summary, luteolysis was associated with an initial transient increase of X4(+) macrophages and decrease of CD5(+) T-cells, and later recruitment of CD68(+) macrophages. During structural regression pro- and anti-inflammatory cytokines are upregulated possibly to control immune cell function.
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.
Besides typing and grading of breast cancer, pathologists are involved in the determination of biomarkers, such as steroid hormone receptors and HER2, which are of utmost importance in adjuvant ...therapy. There have been concerns with regard to security and reproducibility of the biomarker assays done on tissue sections applying either immunohistochemistry or in-situ hybridisation. In order to assure the quality of these biomarker assays, a number of measures are required, among them external proficiency testing. Therefore, external quality assurance trials have been implemented in Germany. In the period of 2002–2007, 5 consecutive trials were conducted with up to 180 participating laboratories. Tissue microarrays with 20–24 different breast cancer samples including cell lines enabled that a huge number of pathologists were challenged with identical samples which provides the prerequisite for comparability. Because there is no legal duress to undergo external proficiency testing in histopathology, all laboratories that took part volunteered to do so. These innovative quality assurance trials (Qualitätsinitiative Pathologie, QuIP) will be continued in the future on an annual or bi-annual basis. Participation is recommended for pathology departments involved in the service for breast units. The organisational frame work of the trials is described here.
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.
We cloned partial cDNA sequences of rabbit 17β-hydroxysteroid dehydrogenase 1 (17β-HSD1) and 17β-HSD7. We analyzed the tissue distribution of 17ß-HSD7 as well as the expression in corpus luteum and ...endometrium during pseudopregnancy. The obtained cDNA sequence of 17β-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 17β-HSD1 was 76 and 82% similar to rodent and human sequences. By Northern analysis 17β-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 17β-HSD1 could be detected in placenta by reverse transcriptase-polymerase chain reaction (RT-PCR), so far. During rabbit pseudopregnancy 17β-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 17β-HSD7 was found on d6 p. hCG, when the endometrium was differentiated to its implantation permissive status.
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