Activity of the p90rsk in in vitro matured porcine oocytes was analysed with a kinase assay kit from the company Upstate biotechnology. This kit was used in another study analysing the p90rsk in porcine oocytes (SUGIURA et al., 2002). The kit was applied according to the manufacturer’s instructions and the results retrieved revealed an activity course during in vitro maturation very different from the expected.
The kit uses an S6 kinase specific substrate, not p90rsk specific. As there are two major S6 kinases in vivo, p90rsk and p70S6K (BANERJEE et al., 1990), one has to rule out the possibility of measuring the wrong kinase. SUGIURA et al. (1990) discuss that biochemical and molecular studies indicated that Rsk is the protein kinase responsible for S6 phosphorylation during meiotic maturation of Xenopus oocytes (ERIKSON and MALLER, 1985, 1986, 1989) and furthermore that p70S6K was inactive throughout the maturation period of Xenopus and mouse oocytes (GAVIN and SCHORDERET-SLATKINE; 1997; SCHWAB et al., 1999). Therefore the present and measured S6 kinase activity should be the activity of Rsk. On the basis
of the species specific differences that are already distinct comparing the phosphorylation pattern of a kinase, this argument is to be judged critically.
On top of this the company renamed the kinase assay kit during the course of the studies from ‘S6 kinase assay kit’ to ‘p70S6K assay kit’. An inquiry at the company confirmed this renaming but they also confirmed that it can still be used for analysis of the S6 kinases.
As our results, with the activity being high at the start of maturation and declining during the progress of maturation and also the same picture in in vitro matured oocytes with an addition of the inhibitor U0126, resemble the measured activity for p70S6K in Xenopus oocytes (SCHWAB et al., 1999) we conclude that the measured activity in our studies was not the p90rsk but the p70S6K. SCHWAB et al. (1999) observed that p70S6K activity is present in oocytes before induction of maturation by progesterone and a decrease in activity upon the induction.
As there is no specific substrate for the p90rsk available the method of the ‘In Gel’
renatured kinase assay was conducted to be able to differentiate between the activities of these two S6 kinases. The substrate is added directly into the acrylamide solution and becomes incorporated into the gel matrix. With the following electrophoresis a separation of the two kinases by their molecular weights would be possible and they could be analysed separately. Unfortunately the used protocol adapted from SAMPT et al. (2001) and RUBINFELD and SEGER (2004) did not give any results. These studies dealt with somatic cells which might be one reason for the difficulties in adaption. The protocol for the ‘In-Gel’ renatured kinase assay needs further changes for the analysis of oocytes. FISSORE et al. (1996) performed an ‘In-Gel’ renatured kinase assay for the analysis of MAPK in bovine oocytes. They used approximately 50 oocytes for each time point analysed. Therefore the problem might be the quantity of the protein as we only used 20 oocytes though this is unlikely as another study examining the MAPK activity in porcine oocytes only used 10 oocytes for each time point In an ‘In-Gel’ kinase assay (INOUE et al., 1995).
F Summary
Carolin Schuon
Analysis of the protein kinase p90rsk during in vitro and in vivo maturation of porcine oocytes and its dependence on the mitogen-activated protein kinase (MAPK)
The aim of this study was to elucidate the normal kinetics of the p90rsk during in vitro maturation of porcine oocytes, to analyse interdependence with the MAPK, and to compare the in vitro obtained results with the findings during in vivo oocytes maturation.
The overall success of IVP of embryos remains unsatisfactory low especially in the pig. Insufficient media compositions are thought to be one reason for these facts. To be able to improve the in vitro culture conditions it is necessary to throw light on the molecular basis of oocytes maturation. P90rsk and MAPK are two of the major protein kinases involved in the signal transduction pathway that leads to resumption of meiosis after its first arrest during oocyte maturation. Activation of these two protein kinases results from phosphorylation in which p90rsk is known to be activated by mitogen-activated protein kinase in vitro and probably in vivo via phosphorylation.
Cumulus oocyte complexes were collected from slaughtered pigs and matured in vitro (0, 22, 26, 30, 34, 46 h) without and also with an addition of the MEK-specific inhibitor U0126. For in vivo maturation gilts were stimulated with eCG (600-800 IU).
Maturation was induced 72 h later with hCG (500 IU). Oocytes were obtained surgically (0, 22, 30 h). The samples were submitted to electrophoresis and protein blotting analysis. Enhanced chemiluminescence was used for visualisation. The in vitro matured oocytes were further submitted to a radioactive kinase assay to determine the specific kinase activity.
For the in vitro maturation process it was shown that p90rsk exists in porcine cumulus cells as well as in oocytes and that this kinase becomes phosphorylated during in vitro maturation. With protein blotting analysis of p90rsk, two high-mobility bands were detected prior to GVBD showing that p90rsk is partially phosphorylated already immediately following recovery of the oocytes from the follicles (0 h). The lowest-mobility bands corresponding to full phosphorylation of the molecule were then detected around GVBD and remained present until the end of maturation culture. In addition it was demonstrated that the phosphorylation of p90rsk coincides with the phosphorylation of MAPK. These findings indicated a MAPK dependent phosphorylation of p90rsk. This indication was substantiated by the results of maturation with an addition of the inhibitor U0126 to the maturation medium. Under these conditions GVBD was completely inhibited as well as the phosphorylation of MAPK and p90rsk.
The kinase assay kit for activity analysis that was used in this study revealed an activity pattern resembling the activity pattern shown for the p70S6K in Xenopus and mouse oocytes. It was concluded that the kit is not appropriate to analyse specific p90rsk activity.
Results with in vivo matured oocytes also show a partial phosphorylation at 0 h with a further phosphorylation of p90rsk after 22 h. As expected the phosphorylation of the in vitro matured oocytes was seen at 90 kDa. Instead phosphorylation of the in vivo matured oocytes occurred little below 200 kDa. This is presumably a molecule complex with MAPK not being a component. Therefore the p90rsk molecule in vivo exists as a dimer.
It is concluded that fully grown porcine oocytes possess a partially phosphorylated p90rsk already at the GV stage with a further phosphorylation of the p90rsk molecule occurring between 22-24 h after initiation of cultivation respectively in vivo maturation.
G Zusammenfassung
Carolin Schuon
Analyse der Proteinkinase p90rsk während der In-vitro- und In-vivo-Reifung porziner Eizellen und ihre Abhängigkeit von der Mitogen-activierten Proteinkinase (MAPK)
Ziel dieser Untersuchung war es die Kinetik der p90rsk während der in vitro Reifung porziner Eizellen näher zu charakterisieren, eine gegenseitige Abhängigkeit mit der MAPK zu klären und die in vitro erhaltenen Ergebnisse mit den Abläufen während der in vivo Reifung der Eizellen zu vergleichen.
Der Gesamterfolg der IVP von Embryonen ist immer noch unbefriedigend, insbesondere beim Schwein. Als einer der Gründe hierfür gilt die unzureichende Zusammensetzung der Kulturmedien. Um die In-vitro-Kulturbedingungen verbessern zu können, ist es notwendig die molekularen Vorgänge während der Eizellreifung aufzuklären. Die p90rsk und die MAPK sind zwei der wichtigsten Proteinkinasen, die an dem Signaltransduktionsweg beteiligt sind, der zur Wiederaufnahme der Meiose nach ihrer ersten Arretierung führt. Die Aktivierung dieser beiden Kinase erfolgt durch ihre Phosphorylierung, wobei die p90rsk in vitro und vermutlich auch in vivo durch die MAPK phosphoryliert wird.
Kumulus-Oozyten-Komplexe wurden von Schlachttieren gewonnen und in vitro mit und ohne Zusatz des MEK-spezifischen Hemmstoffes U0126 gereift (0, 22, 26, 30, 34, 46 h). Für die In-vivo-Reifung wurden Jungsauen mit eCG (600-800 I.E.) stimuliert. 72 h später wurde die Reifung mit hCG (500 I.E.) induziert. Die Eizellen wurden chirurgisch gewonnen (0, 22, 30 h). Die Proben wurden mittels Elektrophorese und Immunoblot analysiert. Zur anschließenden Darstellung wurde ein verstärkte Chemilumineszenz eingesetzt. Die in vitro gereiften Eizellen wurden
weiterhin mittels eines radioaktiven Kinase-Assays untersucht, um die spezifische Kinaseaktivität festzustellen.
Für die In-vitro-Reifung konnte gezeigt werden, dass die p90rsk in porzinen Kumuluszellen, sowie in den Eizellen existiert, und dass sie während des Reifungsprozesses phosphoryliert wird. Durch die Untersuchung mittels Immunoblot konnten zwei Banden der p90rsk mit hoher Mobilität vor dem GVBD dargestellt werden. Das bedeutet, dass die p90rsk bereits in den Eizellen direkt nach der Isolierung aus dem Follikel (0 h) teilphosphoryliert vorliegt. Die Banden mit der geringsten Mobilität gleichbedeutend mit der vollständigen Phosphorylierung des Moleküls, waren erst um den Zeitpunkt des GVBD vorhanden und blieben bis zum Endpunkt der Reifung bestehen. Zusätzlich wurde gezeigt, dass die Phosphorylierung der p90rsk zeitlich mit der Phosphorylierung der MAPK zusammenfällt. Diese Ergebnisse wiesen auf eine MAPK-abhängige Phosphorylierung der p90rsk hin. Dieser Hinweis wurde durch die Ergebnisse einer Reifung mit Zusatz des Hemmstoffes U0126 zum Maturationsmedium bestätigt.
Unter diesen Bedingungen wurde der GVBD vollständig verhindert, ebenso wie eine Phosphorylierung der MAPK und der p90rsk.
Der Kinase Assay Kit der zur Untersuchung der spezifischen Kinaseaktivität verwendet wurde, ließ ein Aktivitätsmuster erkennen, das dem der p70S6K in Xenopus- und Mauseizellen ähnelt. Daraus wurde geschlossen, das sich dieser Kit nicht zur Untersuchung der spezifischen Aktivität der p90rsk eignet.
Die Ergebnisse der Untersuchung der in vivo gereiften Eizellen zeigten ebenfalls eine Teilphosphorylierung nach 0 h und eine zunehmende Phosphorylierung nach 22 h.
Wie erwartet war die Phosphorylierung bei den in vitro gereiften Eizellen bei etwa 90 kDa zu sehen, bei den in vivo gereiften Eizellen hingegen etwas unterhalb von 200 kDa. Es handelt sich vermutlich um einen Molekülkomplex, von dem gezeigt werden konnte, das die MAPK nicht beteiligt ist. Daher liegt das p90rsk Molekül in vivo als Dimer vor.
Es wurde geschlussfolgert, dass ausgewachsene porzine Eizellen eine bereits teilphosphorylierte p90rsk im GV Stadium enthalten. Eine weitere Phosphorylierung
des Moleküls erfolgt zwischen 22 und 24 h nach Beginn der Kultivierung bzw. Start der In-vivo-Reifung.
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