The bovine placenta as a source and target of steroid hormones : aspects on the role of androgens and sulfonated steroids
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As a temporary endocrine organ, the placenta is capable of synthesizing and secreting a broad range of hormones and other bioactive molecules. Like in many other mammalian species, also in cattle the placenta exhibits a considerable steroidogenic activity with estrone sulfate (E1S) and progesterone being the main products from a quantitative point of view. However, the biological role of placental steroidogenesis in cattle is widely unclear as the placenta contributes only negligibly and temporarily to peripheral maternal progesterone blood levels, and the main estrogenic product E1S does not interact with classical nuclear estrogen receptors. Based on the observation that receptors for progesterone and estrogens are expressed in bovine placentomes the concept of placental steroids as local regulators of placental growth, differentiation and functions has been put forward. However, data from functional studies to corroborate this concept are not yet available, and apart from the functions of bovine placental steroids, there are still many open questions concerning synthesis and transport especially of sulfonated estrogens in cattle. Thus, this study focuses on three aspects of bovine placental steroidogenesis: 1) the expression of the estrogen-specific sulfotransferase (SULT1E1) to identify the sites of estrogen sulfonation in the pregnant cow 2) to further characterize the expression of the sodium-dependent organic anion transporter (SOAT; syn.: SLC10A6) which is considered as a putatively relevant transporter of estrogen sulfates 3) to test for the possibility that steroids other than progesterone and estrogens e.g. androgens - are functionally important products of bovine placental steroidogenesis.In order to localize SULT1E1 in bovine placentomes on a cellular level and to assess its expression quantitatively throughout gestation, immunocytochemical and real-time RT-PCR methods were established, respectively. In immunocytochemisty (ICC) two different primary antibodies were applied: a rabbit antiserum against bovine recombinant SULT1E1 (generously provided by Dr. R. Sullivan, Centre de Recherche en Biologie de la Reproduction and Departement d Obstetrique-Gynecologie, Faculte de Médecine, Université Laval, Quebec, Canada) and a commercial murine antiserum against the human enzyme. Specificity of these antibodies was confirmed in western blot. They yielded virtually identical results in ICC. Different from previous data published in the literature based on ICC and in situ hybridization, where SULT1E1 in bovine placentomes was localized in trophoblast giant cells (TGC), strong specific cytoplasmic staining was only found in uninucleated trophoblast cells (UTC) and SULT1E1 was rapidly down-regulated when trophoblast cells showed characteristics of TGC differentiation. Throughout gestation, distinct immunostaining for SULT1E1 was found in UTC of the chorionic plate. A gradient of staining intensity was observed along the chorionic villous tree with a decrease of mean staining intensities between the basal parts of stem villi and the tertiary villi. With real-time RT-PCR, in the course of pregnancy a significant increase of SULT1E1-mRNA expression was found in the last trimester and at parturition (p=0.0043), which confirms earlier studies by other investigators. Consistent with the increase of SULT1E1-mRNA in real-time RT-PCR in immunohistochemistry an increase of overall staining intensity was detected during late gestation and at parturition with the antiserum against bovine SULT1E1 but this was not obvious with the antiserum against the human enzyme. Moreover, with the antiserum against the bovine enzyme a weak cytoplasmic signal was obtained in the caruncular epithelium in a part of placental samples in addition to the distinct signals in UTC. When screening bovine organs for SULT1E1-mRNA expression by real-time RT-PCR, highest levels were found in the placenta (2851 relative units, RU; day 272), followed by fetal liver (day 185: 748 RU; day 210: 816 RU). In adult bovine organs, SULT1E1 expression was clearly lower with highest levels in adrenal (144 RU) and skin (91 RU) and was only minimal in the remaining organs investigated including liver (21 RU).The results give strong evidence that different from results of earlier studies the main sites of SULT1E1 expression in bovine placentomes are the UTC and not the TGC. They suggest that SULT1E1 may protect UTC from the high levels of free estrogens produced by TGC and it may be involved in the control of TGC differentiation.The aim of the second part of this study was to further characterize the expression of the five isoforms identified so far of the SOAT in the bovine placentome and other organs. The SOAT standard form (variant 1) cloned from bovine placentome has been recently shown in vitro to efficiently mediate the cellular import of E1S and thus may be a physiologically relevant transporter for the large amount of E1S produced in the trophoblast during bovine gestation. Virtually no information was available so far on the expression pattern and functions of the remaining four isoforms. Variant-specific conventional RT-PCR methods could be established for all of the five variants. With these methods, placental tissue samples from different stages of gestation were screened for SOAT isoform expression. For comparison, a broad spectrum of other tissues and organs was also included into the study. Specific amplicons were obtained in all (variants 1, 2, 5) or most (variant 3) of the tissue samples. For variant 4, bands were only weak or absent in a considerable proportion of placental or adult tissue samples. Real-time RT-PCR methods (SYBR green) could be established for variants 1, 2, 3 and 5. Expression of all these variants did not change significantly in bovine placentomes during early and midgestation but was more than 10 fold higher in the maternal part of placentomes from cows at normal term suggesting that their expression is up-regulated in the prepartal period. When screening various bovine organs quantitatively for expression of mRNA specific for SOAT isoforms, for variants 1, 2 and 5 expression in testis (1253 RU, 8195 RU, 4575 RU, resp.) exceeded by far expression measured in other organs or tissues. Other significant sites of SOAT-variant 1 expression are skin (734 RU), udder (282 RU) and colon (255). Besides in the testis, variant 2 was significantly expressed in the skin (1410). Highest expression for variant 3 was also found in the testes (158 RU), which however- was only slightly higher than in the ovarian stroma (113 RU). The spectrum of measurable SOAT isoforms varied considerable between tissues and organs with testis, placenta and corpus luteum being the only organs where all four isoforms assessed were expressed in measurable levels. Expression levels in placentome for variants 1 (5.3 RU), 2 (31.8 RU), 3 (2.5 RU) and 5 (9.6) were all above the limit of detection but only minimal compared to the testis. These observations question the hypothesis of SOAT variants as important transporters of the high amounts of sulfonated estrogens in bovine placentomes. Moreover, the results suggest that in cattle standard SOAT (variant 1) and its other isoforms play an important role especially in the testis, an organ, which however in the bovine species does not produce considerable amounts of sulfated estrogens.In addition to estrogens and progesterone, of which no biological role has been definitely identified yet in bovine placenta, in the bovine trophoblast androgens may also be produced and may have effects in bovine placentomes. In order to identify putative target cells of placental androgens, an immunohistochemical method was established to detect androgen receptor (AR) in bovine tissues using an antiserum raised against the N-terminus of human AR. Specificity of the primary antibody applied for bovine AR was confirmed by western blot and by control experiments using bovine epididymis as a positive control tissue. Throughout gestation, distinct nuclear signals were found in invasive TGC. As assessed by quantitative evaluation using an immunoreactive score, in TGC situated in the trophoblast, immature TGC, UTC, stromal cells of the chorionic villi, caruncular epithelial and stromal cells AR expression was low at early and midgestation but significantly increased during late gestation (p<0.01, resp.). Expression of AR was qualitatively confirmed on the mRNA-level by conventional RT-PCR. With real-time RT-PCR (taqman method) only a trend for an increased AR expression in the prepartal phase and at parturition was observed, which however was not statistically significant. Radioimmunological measurement of testosterone concentrations in bovine placental tissue yielded concentrations that must be considered sufficient to activate local ARs, and showed a significant increase (p<0.01) of mean testosterone concentrations from values slightly above background level (0.1 ng/g tissue) between days 50-100 to mean concentrations of 0.9 ng/g tissue during late gestation. The results suggest that androgens may be active products of bovine placental steroidogenesis and that they may be involved in the control of TGC differentiation. However, as steroid receptors are in part constitutionally active, it cannot be ruled out the ARs detected in bovine placentomes may have functions independent from the binding of steroidal ligands.In conclusion, results obtained in these studies provide new information on different aspects of bovine placental steroids and give starting points for new concepts on their functions.Verknüpfung zu Publikationen oder weiteren Datensätzen
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