Einfluss von endogenen Sexualsteroidhormonen auf die
Wachstumshormon-Rezeptor-Signaltransduktion in der Leber im physiologischen Zyklus bei Färsen
Es ist bekannt, dass sich die endokrine metabolische und reproduktive Achse gegenseitig regulieren. Hierbei spielt die somatotrope Achse eine Schlüsselrolle, da sie sowohl den Metabolismus als auch die Fruchtbarkeit der Tiere beeinflusst. Zum vermuteten Einfluss von endogenen Östrogenen auf die Komponenten der somatotropen Achse rund um die Kalbung liegen bereits mehrere Studien vor. Es konnte gezeigt werden, dass nach der Kalbung die GH-Konzentration im Blut steigt und die von IGF-I sinkt, was vermutlich durch die Abnahme des GHR und zusätzlich durch die Zunahme von SOCS2 (Inhibitor des GHR-Signaltransduktionsweges) in der Leber bedingt sein könnte (Piechotta et al. 2013; Winkelman et al. 2008; Radcliff et al. 2003; Lucy et al. 2001). Andererseits wurde auch gezeigt, dass die Konzentration von IGF-I zur Ovulation hin steigt (Kawashima et al. 2007). Die Gabe von Östradiol führte bei ovarektomierten Kühen ferner zu einer Erhöhung von GH und IGF-I im Blut, während parallel dazu die hepatische GHR-Expression anstieg (Colak et al. 2011). Dies impliziert, dass endogene Sexualsteroidhormone post partum und im bovinen Zyklus in unterschiedlicher Weise auf die hepatische GH-Signaltransduktion wirken könnten. Momentan liegen jedoch noch keine Untersuchungen des Einflusses von endogenen Sexualsteroidhormonen auf den hepatischen GHR-Signaltransduktionsweg im physiologischen Zyklus des Rindes vor. Ziel der vorliegenden Dissertation war es daher den Einfluss von Sexualsteroidhormonen auf die GHR-Signaltransduktion in vivo zu untersuchen, sowie in vitro Modelle zur Analyse des Lang- und Kurzzeit-Einflusses von E und P4
auf die hepatische GHR-Signaltransduktion auf ihre Eignung zu untersuchen. Im in vivo Versuch wurde während des physiologischen Zyklus (n=56) von 30 Holstein Friesian Färsen täglich eine transrektale, ultrasonographische Untersuchung der Ovarien durchgeführt und so die Follikelphase (nach mindestens 48 Stunden
149 Brunstsymptomen, direkt ante ovulationem, n=7; post ovulationem, n=49) und die Lutealphase (Tag 12 ± 1 post ovulationem) terminiert. Zu diesen Zeitpunkten wurden Blutproben und Leberbiopsien entnommen. Im Blut wurden die Konzentrationen von P4, E, IGF-I, IGF-II und GH gemessen und in den Leberbiopsien die mRNA-Expression von ERα, GHR, JAK2, STAT5B, SOCS2, SOCS3, IGF-I und IGF-II mittels qPCR bestimmt. In den in vitro Versuchen wurden humane HepG2-Zellen über 48 Stunden oder 3 Wochen hinweg mit E1, E2, P4 oder der Kombination dieser Hormone kultiviert und abschließend mit GH stimuliert. Überdies wurden primäre Rattenhepatozyten in einer Monolayer-Kultur über maximal zwei Tage mit den zuvor genannten Sexualsteroidhormonen kultiviert und ebenfalls durch GH-Zugabe einem Stimulationsversuch unterzogen. Während der in vitro Versuche wurden Proben des Mediums und der kultivierten Zellen zu Analyse entnommen. Im Medium wurde die Konzentration von P4, E, bGH, IGF-I und IGF-II bestimmt und in den HepG2-Zellen die IGF-II mRNA-Expression gemessen, während bei den primären Rattenzellen die mRNA-Expression von IGF-I mittels qPCR bestimmt wurde. Im in vivo Versuch war die E-Konzentration im Blut wie erwartet vor der Ovulation am höchsten. Die IGF-I- und GH-Konzentrationen waren in der Lutealphase niedriger als in der Follikelphase.
Die IGF-II-Konzentration war hingegen in der Lutealphase höher als in der Follikelphase. Dagegen war kein Unterschied der GH-, IGF-I- und IGF-II-Konzentrationen zwischen den ante und post ovulationem entnommenen Proben erkennbar. Die relativen Menge der hepatischen GHR und SOCS2 mRNA war ante ovulationem höher als post ovulationem und in der Lutealphase. Die relativen Mengen der mRNA von ERα, JAK2, STAT5B, SOCS3, IGF-I und IGF-II waren zu allen Zeitpunkten vergleichbar. Während der in vitro Versuche zeigte sich, dass sich HepG2-Zellen und primäre Rattenzellen weder durch GH noch durch Sexualsteroidhormone zur IGF-Produktion stimulieren ließen. Die weiteren Untersuchen ergaben, dass sich HepG2-Zellen nicht als Modell zur Analyse des GH-Signaltransduktionsweges beim Rind eignen, da ihr IGF-System durch die Tumorgenese der Zellen stark verändert ist. Des Weiteren wurde festgestellt, dass sich auch eine Monolayer-Kultur von primären Rattenzellen nicht als Modell eignet, da diese Kulturform keine ausreichende Funktionalität der Zellen im Hinblick auf die
150
zu untersuchende endokrine Komponente gewährleistete. Es kann geschlussfolgert werden, dass die Etablierung einer Sandwich-, 3D- oder Mikrofluid-Biochip-Kultur primärer Rinderhepatozyten angestrebt werden sollte, um in vitro die GH-Signaltransduktion des Rindes untersuchen zu können. Die Ergebnisse des in vivo Versuches bestätigten, dass E2 im bovinen Sexualzyklus die hypophysäre GH-Ausschüttung, den GHR, den nachgeschalteten Signaltransduktionsweg und damit auch die hepatische IGF-I-Ausschüttung stimuliert. Direkt ante ovulationem war nicht nur die GHR mRNA, sondern auch die SOCS2 mRNA–Expression in der Leber erhöht. Trotz der stark erhöhten IGF-I-Konzentration im Blut während der Follikelphase, konnte keine Steigerung der IGF-I mRNA-Expression nachgewiesen werden. Es kann also spekuliert werden, dass die gesteigerte SOCS2-Expression zu diesem Zeitpunkt bereits die Expression von IGF-I supprimiert haben könnte, obwohl die IGF-I-Konzentration im Blut noch durch eine verlängerte Halbwertszeit durch die Bindung von IGF-I an Bindungsproteine erhöht war. Die Ergebnisse lassen die Schlussfolgerung zu, dass SOCS2 wesentlich sensibler und zeitnaher durch E2
reguliert wird als momentan in der Literatur angenommen. Dieser Nachweis stellt somit ein wichtiges Bindeglied für die Erforschung der Regulationsmechanismen des GH-Signaltransduktionsweges im Zyklus und in der späten Trächtigkeit beim Rind dar.
151 7 Summary
Kirsten Mense
Influence of endogenous sexual steroid hormones on the hepatic growth hormone receptor signalling pathway in heifers during estrus cycle
It is already known that the metabolic and reproductive endocrine axes regulate each other. The somatotropic axis, which influences metabolism and reproduction, could probably be a key factor for this mutual regulation. The influence of endogenous estrogens on the somatotropic axis during late pregnancy and post partum has been investigated in several studies. It has been shown that the blood concentration of GH increased, while the IGF-I blood concentration decreased post partum. This is probably caused by the reduction of GHR and further attenuated by the increase of SOCS2 (inhibitor of the GH signalling pathway) in the bovine liver (Piechotta et al.
2013; Winkelman et al. 2008, Radcliff et al. 2003; Lucy et al. 2001). However, it has been shown that the plasma IGF-I concentration increases during the follicular phase of Japanese Black and lactating Holstein cattle (Kawashima et al. 2007). The IGF-I and growth hormone (GH) blood concentrations as well as the hepatic GHR expressions were increased by E2 injection in ovariectomized cows (Colak et al., 2011). These findings indicate that the influence of endogenous sexual steroid hormones could affect the hepatic GH signalling pathway differently during estrus cycle and post partum. However, the effect of sexual steroid hormones, especially on the GH signalling pathway during the estrus cycle, has not been investigated in detail yet. Therefore, the aim of this study was to examine GH, IGF-I and IGF-II blood concentrations as well as hepatic mRNA expression of the GH signalling pathway components during the physiological cycles (n=56) of 30 Holstein Friesian heifers in vivo. Moreover, the aim was to investigate the short term and long term effect of sexual steroid hormones on the GH signalling pathway in vitro.
Daily ovarian ultrasound was performed to determine the follicular phase (after at least 48 hours of clinical estrus signs, ante ovulationem, n=7; post ovulationem, n=49) and the luteal phase (day 12 ± 1 after ovulation, n=56) for the in vivo experiments. At these defined cycle stages, blood samples and liver biopsies were
152
taken. The serum concentrations of P4, E, IGF-I, IGF-II and GH were measured and in the liver biopsies the mRNA expression of ERα, GHR, JAK2, STAT5B, SOCS2, SOCS3, IGF-I and IGF-II were determined by qPCR. For the in vitro experiments, human HepG2 cells were cultivated with E1, E2, P4 or the combination for either 48 hours or three weeks, before GH stimulation was performed. Furthermore, primary rat hepatocytes were cultivated with these hormones in a monolayer-culture for a maximum of 48 hours and were also stimulated by addition of GH to the medium.
Samples of the cell culture medium and cells were taken. Concentrations of P4, E, bGH, IGF-I and IGF-II were measured in the medium. While in HepG2 cells the relative abundance of IGF-II mRNA was determined, the relative abundance of the IGF-I mRNA was analysed in the primary rat hepatocytes by qPCR.
As expected, a high estrogen concentration was detected ante ovulationem in vivo. The IGF-I and GH concentrations were lower during the luteal phase compared to the follicular phase (P < 0.001) whereas the IGF-II concentrations were higher during the luteal phase compared to the follicular phase (P < 0.001). No differences in the GH, IGF-I or IGF-II blood concentrations were observed when comparing samples taken ante ovulationem versus post ovulationem. The hepatic GHR expression was higher ante ovulationem compared to post ovulationem and the luteal phase (P <
0.05). Interestingly, the expression of SOCS2 was also higher ante ovulationem compared to post ovulationem and the luteal phase (P < 0.05). During the in vitro experiments it was obvious that the IGF-I and -II secretion of HepG2 cells and primary rat cells could not be increased by GH or influenced by sexual steroid hormone addition to the medium. Furthermore, it was shown that neither HepG2 cells nor monolayer cultured primary rat hepatocytes were a suitable model to investigate the hepatic GH signalling pathway in cattle. The IGF system of HepG2 cells is modified by malignant transformation of the cells. In addition, a monolayer culture of primary rat hepatocytes did not provide the full hepatocyte functions of the cell in vivo. A sandwich, 3D or microfluid biochip culture of bovine primary hepatocytes should be established in future studies to investigate the bovine GH signalling pathway in vitro.
153 Based on the results of the in vivo experiment, it can be concluded that the pituitary GH release, the GH signalling pathway and the hepatic IGF release were influenced by increased E2 concentrations during the follicular phase of the bovine estrus cycle.
In addition to the GHR mRNA, also the SOCS2 mRNA expression was increased ante ovulationem. In contrast to high IGF-I plasma concentrations, the IGF-I mRNA expression was not increased during the follicular phase. It can be speculated that SOCS2 expression had already inhibited the IGF-I expression, but plasma concentrations were still high due to a prolonged half-life of IGF-I by binding to its binding proteins.
It can be concluded that SOCS2 is probably regulated more sensitively and promptly by estrogen than assumed in the literature before.
154
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