Institute of Reproductive Medicine University of Veterinary Medicine Hannover ______
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)
THESIS
submitted in partial fulfilment of the requirements for the degree
DOCTOR OF PHILOSOPHY
-P H.D.-
in the field of Reproductive Medicine
at the University of Veterinary Medicine Hannover
by Carolin Schuon, née Bösebeck, Meppen, Germany
Hannover, Germany 2005 Supervisor: Prof. Dr. B. Meinecke Institute of Reproductive Medicine University of Veterinary Medicine Hannover, Foundation, Hannover, Germany
Advisory committee: Prof. Dr. B. Meinecke
Prof. Dr. G. Breves Institute for Physiology University of Veterinary Medicine Hannover, Foundation, Hannover, Germany
Prof. Dr. K.-D. Hinsch BfA-Reha-Klinik Borkum Riff Borkum, Germany
External evaluation: Prof. Dr. S. Kölle Institute for Veterinary Anatomy, Histology and Embryology, Justus-Liebig-University, Gießen
Oral examination: November 10 th 2005
This thesis was founded by the H. Wilhelm Schaumann Stiftung Es gibt nichts Schöneres als das Mysteriöse. Aus ihm entspringt alle wahre Kunst und Wissenschaft. Albert Einstein This study has been published in part: Conferences (Oral presentation): Bösebeck, C., S. Ebeling u. B. Meinecke Phosphorylation and MAPkinase-accociated activation of p90rsk during in vitro maturation of porcine oocytes. Vet. Med. Austria/ Wien. Tierärztl. Mschr. 91, Suppl. 2, S. 12-13, 2004
Conferences (Poster): Bösebeck, C., S. Ebeling u. B. Meinecke P90rsk and MAPKinase interdependence: Phosphorylation and activation patterns during in vitro maturation of porcine oocytes 38. Jahrestagung der Physiologie und Pathologie der Fortpflanzung, 30. Veterinär- Humanmedizinische Gemeinschaftstagung, 10.-11.02.Zürich Schweizer Archiv für Tierheilkunde 147 , 51, 2005 INDEX
A INTRODUCTION ...... 9 B LITERATURE REVIEW...... 12 1 OOCYTE DEVELOPMENT ...... 12 1.1 Oogenesis...... 12 1.2 Intercommunication between the oocyte and its surrounding somatic cells ...... 14 2 OOCYTE MATURATION ...... 16 2.1 Meiotic competence...... 16 2.2 Nuclear maturation...... 17 2.2.1 Morphological changes ...... 17 2.3 Cytoplasmic maturation...... 19 2.3.1 Morphological changes ...... 20 2.4 Biochemical changes ...... 21 2.4.1 Transcription and translation...... 21 2.4.2 Protein synthesis and phosphorylation...... 22 3 CELL CYCLE ASPECTS OF OOCYTE MATURATION ...... 23 3.1 Maturation-Promoting-Factor (MPF) ...... 24 3.1.1 MPF activation and inactivation ...... 25 3.1.2 Substrates and function of the MPF...... 31 3.2 Mitogen-activated protein kinase (MAPK) ...... 32 3.2.1 MAP-Kinase activation and inactivation ...... 33 3.2.2 Substrates and function of the MAP-Kinase...... 37 3.3 P90rsk...... 41 3.3.1 P90rsk activation and inactivation...... 42 3.3.2 Function and substrates of the p90rsk ...... 45 3.4 Interactions between MPF, MAP-Kinase, and p90rsk...... 47 C MATERIALS AND METHODS ...... 50 1 CELL CULTURE TECHNIQUES ...... 50 1.1 General handling and precautions ...... 50 1.2 Media ...... 50 1.2.1 Supplements...... 51 1.3 Preparation and selection of cumulus-oocyte-complexes ...... 52 1.4 Cultivation of cumulus-oocyte-complexes ...... 53 1.4.1 Regular cultivation conditions ...... 53 1.4.2 Cultivation conditions with addition of an inhibitor...... 53 1.5 Cytogenetic assessment of oocytes...... 54 1.5.1 Fixation and staining of oocytes...... 54 1.5.2 Assessment of the nuclear status of oocytes ...... 54 2 PROTEIN CHEMICAL TECHNIQUES ...... 55 2.1 Preparation and storage of oocytes and cumulus cells...... 55 2.2 One-dimensional electrophoresis...... 56 2.3 Immunoblot (Western Blot) ...... 57 INDEX
2.4 Chemiluminescence...... 57 2.5 P90rsk kinase assay ...... 59 2.5.1 Kinase assay kit ...... 59 2.5.2 ‘In-Gel’ renatured kinase assay...... 61 2.6 Autoradiography...... 62 2.7 Quantitative measurement of radioactivity ...... 62 3 ANIMAL EXPERIMENT ...... 62 3.1 Animals ...... 62 3.2 Experimental design...... 63 3.3 Surgical techniques...... 64 3.4 Preparation of oocytes and cumulus cells...... 65 3.5 Analysis of in vivo matured oocytes and cumulus cells...... 65 D RESULTS ...... 66 1 CONTROL OF MATURATION ...... 66 2 P90RSK IN OOCYTES AND CUMULUS CELLS ...... 67 3 MAPK IN OOCYTES ...... 68 4 PHOSPHORYLATION PATTERN OF P90RSK AND MAPK DURING IN VITRO MATURATION ...... 69 5 INHIBITION EXPERIMENTS ...... 71 6 KINASE ASSAY ...... 74 6.1 Kinase assay kit...... 74 6.2 ‘In-Gel’ renatured kinase assay...... 76 7 ANIMAL EXPERIMENT ...... 76 E DISCUSSION ...... 80 1 KINETICS OF NUCLEAR MATURATION ...... 80 1.1 Kinetics of nuclear maturation during in vitro maturation...... 80 1.2 Kinetics of nuclear maturation during IVM with an addition of U0126 ..... 81 1.3 Kinetics of nuclear maturation during in vivo maturation...... 81 2 P90RSK AND MAPK IN PORCINE OOCYTES AND CUMULUS CELLS .... 82 2.1 P90rsk in porcine oocytes and cumulus cells...... 82 2.2 MAPK in porcine oocytes and cumulus cells...... 82 3 PHOSPHORYLATION PATTERN OF SK AND MAPK ...... 83 3.1 Phosphorylation pattern of p90rsk during in vitro maturation ...... 83 3.2 Phosphorylation pattern of MAPK during in vitro maturation...... 83 3.3 Phosphorylation pattern of p90rsk during IVM with an addition of U0126 ...... 84 3.4 Phosphorylation pattern of p90rsk during in vivo maturation...... 85 4. P90RSK ACTIVITY DURING IVM WITH AND WITHOUT AN ADDITION OF U0126 ...... 86 F SUMMARY ...... 88 INDEX
G ZUSAMMENFASSUNG ...... 90 H REFERENCES ...... 93 I APPENDIX ...... 118 1 LIST OF ABBREVIATIONS ...... 118 2 CHEMICALS, DRUGS, AND EQUIPMENT ...... 122 3 MEDIA, BUFFERS, AND SOLUTIONS (ALPHABETICAL ORDER) ...... 125 4 INDEX OF TABLES ...... 132 ACKNOWLEDGEMENTS...... 134
INTRODUCTION
A Introduction
The central aim of biotechnologies in reproduction procedures is to increase the number of live and healthy offspring. Although new biotechnologies such as artificial insemination, embryo transfer, embryo production and mammalian cloning have been developed and are widely applied in reproduction procedures today, their success remains unsatisfactory low. The developmental potential of an embryo is dependent on the developmental potential of the oocyte from which it originates. The process of oocyte maturation is critical for the efficient application of biotechnologies. However, the overall efficiency of in vitro maturation remains low because oocytes matured in vitro have a lower developmental competence than oocytes matured in vivo (LAURINCIK et al., 1994; LONERGAN et al., 2003; RODRIGUEZ et al., 2004). Therefore it has become increasingly urgent to elucidate the basic mechanisms and pathways underlying oocyte maturation and fertilization to improve and optimize the in vitro culture conditions/protocols.
The number of successful pregnancies in all livestock species after the combination of the in vitro techniques, in vitro maturation (IVM), in vitro fertilization (IVF), and in vitro culture is still not satisfying. In sheep one reaches 25 %, in cattle 15 % and in the pig equal or even less than 5 % successful pregnancies (KIKUCHI et al., 1999). The in vitro production (IVP) of cattle embryos for example nowadays is a well established in vitro technique in breeding programs. Comparing the IVP of embryos with the in vivo situation the following data clearly demonstrate the loss. With a combination of the in vitro techniques one receives 1.9 calves per donor on average. After induction of ovulation and subsequent embryo transfer one receives 3.0 calves per donor on the average producing one more calf per donor with an improved embryo quality (GALLI and LAZZARI, 1996). One can conclude that the problem already lies in the suboptimal in vitro conditions. During oocyte maturation one has to distinguish between a first follicle-dependent and a following follicle-independent maturation period. Studies of BLONDIN et al. (2002) showed that with a gonadotropin injection before ovum pick up 80 % of the recovered oocytes formed
9 INTRODUCTION
blastocysts. These results highlight the importance of oocytes quality at the start of the in vitro embryo production process in determining the final outcome meaning that the oocytes need a certain developmental level for in vitro procedures to be successful. Specifying the problem one can conclude that IVM-conditions nowadays are not able to support the maturation of small-follicle-oocytes. But especially the maturation of these small oocytes could provide a new source of mature eggs for livestock production and assisted reproduction in humans and in endangered species (MIYANO, 2003). To improve these in vitro techniques it is necessary to throw light on the molecular mechanisms during gamete maturation which are, despite all the efforts in research, still mostly unknown.
Reasons to improve the in vitro culture conditions apart from increasing the quality of in vitro matured oocytes to provide in vitro matured embryos for embryo transfer, are the possibility to use the whole potential of gametes in ovaries of precious individual animals, for example transgenic animals, to provide matured oocytes for cloning of livestock animals and for preserving endangered species as well as for “producing” potential organ donors for human medicine regarding transgenic pigs.
To elucidate the underlying molecular mechanisms the oocyte is a very suitable object of studies. Because of its size in comparison with somatic cells it provides the possibility to easily study the structural and biochemical changes during the cell cycle. The research over the last decades revealed a lot of species specific differences so that the gained information in the classical laboratory animals as Xenopus , mice and rats needed to be completed with aspects from other species. Livestock species became more interesting even in basic research. As there are twenty million pigs slaughtered each year in Germany today a grand number of peripubertal ovaries can be obtained at relatively low costs what makes it possible to study oocyte maturation in a highly differentiated species without having to sacrifice the animals only for that purpose. Apart from this reason the pig oocyte is the ideal system in which to study the molecular mechanisms since pig oocytes require almost
10 INTRODUCTION
twice the time of other large domestic animals, such as cattle, to progress to and through the time span of interest.
The aim of the present study was to get a more detailed description of one of the kinases involved in the transduction pathway that restarts meiosis in oocytes during the maturation process. For the reasons described above and as previous research in the laboratory concentrated on pigs we decided to continue with this species. The choice of a gonadotropin induced in vitro maturation system has two reasons. First, oocytes that have been exposed to gonadotropins either in vivo or in vitro are more developmentally competent than oocytes matured in the absence of gonadotropins (IZADYAR et al., 1998; DELA PENA et al., 2002). Second, spontaneous maturation in the absence of gonadotropins can be considered as an artefact of in vitro culture (DOWNS 1995) and is not an appropriate system for studying the regulation of oocyte meiotic cell cycle (SU et al., 2001) as only the hormone-induced mechanism leads to meiotic resumption of mammalian oocytes under physiological conditions (FAN and SUN, 2004). Earlier studies focused on the mitogen-activated protein kinase , MAPK, and its involvement in oocyte maturation. The phosphorylating and activating mechanisms of this kinase and whether its activation is essential for the whole maturation process or only for parts of it were analyzed. The kinase of our interest is the p90rsk (protein 90 ribosomal S6 kinase) which was first discovered in Xenopus laevis oocytes phosphorylating the S6 protein of the 40S subunit of the ribosome. Rsk participates in meiosis I (first meiotic division) entry and may also down regulate the S-phase between meiosis I and II (second meiotic division). In addition it contributes to metaphase II (MII) arrest (SCHMITT and NEBREDA, 2002). In detail we wanted to analyse the phosphorylation and activation of the p90rsk during in vitro and in vivo maturation of porcine oocytes, and to find out whether this is a MAP Kinase dependent or independent phosphorylation.
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B Literature review
1 Oocyte development
1.1 Oogenesis Oogenesis is the lifespan of an oocyte starting with its first differentiation, followed by a period of mitotic multiplication, a growth phase and the meiotic divisions to reduce the diploid set of chromosomes to a haploid one. It ends with the fertilization of the oocyte. The germ line stem cells or primordial germ cells migrate from their extragonadal origin, the yolk sac, actively and selectively to the germinal ridge or the primitive gonad. In pigs they were noted here as early as 18 days post coitum (p.c.). Mitotic replication occurs during this time so that the number of germ cells increases from 5,000 at day 20 p.c. to a peak of 1,100,000 at 50 days (BLACK and ERICKSON, 1968). The primordial germ cells transform into oogonia within the ovary. This transformation is marked by cell growth and redistribution of cytoplasmic organelles (LEIBFRIED-RUTLEDGE et al., 1989). Followed by a period of overlapping mitotic replication and degeneration, or atresia the result is a population of approximately 500,000 germ cells at birth in pigs (BLACK and ERICKSON, 1968). During their growth phase the oogonia enter the prophase of the first meiotic division and from then on are called primary oocytes which are mitotically incompetent. The oocytes then progress through the first meiotic prophase consisting of the leptotene (chromosomal condensation), zygotene (pairing/synapsis of homologues), pachytene (genetic crossing over and recombination) and the diplotene (desynapsis). Upon reaching the late diplotene the so-called dictyate phase maturation of the oocytes arrests. The arrested oocytes, characterized by a large nucleus, the germinal vesicle (GV), are surrounded by a single layer of follicle cells and the basal membrane and constitute primordial follicles. This arrest occurs around the time of birth, prenatal in the horse, ruminants (SCHILLING, 1999) and the mouse (BORUM, 1966), shortly after birth in the pig (FULKA et al., 1972) and is maintained until puberty when the preovulatory gonadotropin surge takes place. During this meiotic
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block, also called the growth phase, the oocyte increases its volume 200times (MOOR et al., 1990) and is marked by a high ribonucleic acid (RNA)- and protein synthesis (SCHULTZ and WASSARMAN, 1977a; MOTLIK and FULKA, 1986) and forms the zona pellucida (ZP). The follicle cells enlarge and proliferate to form a multilayered envelop around the oocyte. When the size of the oocyte already remains constant, the follicle continues growth and develops a fluid-filled cavity, the antrum. The wall of an antral follicle is build of a multilayer of granulosa cells. At the end of this growth phase the oocyte is able to resume meiosis (THIBAULT et al., 1987). After activation through the gonadotropin stimulus meiosis is arrested again in the second reduction division (WASSARMAN and ALBERTINI, 1994). This second meiotic block is terminated after ovulation when the oocyte is fertilized or parthenogenetically stimulated. The majority of primary oocytes present in mammalian ovaries at birth fail to complete growth and to achieve meiotic competence. Only a small part of the fully grown oocytes from antral follicles restarts meiosis. Most of them become atretic and degenerate. Figure 1 gives a schematic overview of the stages during mammalian oogenesis.
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DEVELOPMENTAL EVENTS GERM CELL STATE MEIOTIC STAGE
Multiplication by mitosis PRIMORDIAL GERM Migration to genital ridge CELL
OOGONIA
DNA synthesis ACTIVATION I. MEIOTIC DIVISION PROPHASE
PRIMARY OOCYTE • LEPTOTENE •ZYGOTENE •PACHYTENE •DIPLOTENE BIRTH: MOST MAMMALS ARREST DICTYATE STAGE
GROWTH PHASE
PUBERTY ACTIVATION DIAKINESE METAPHASE I ANAPHASE I MATURATION TELOPHASE I SECONDARY OOCYTE
II. MEIOTIC DIVISION ARREST METAPHASE II
OVULATION: MOST MAMMALS FERTILIZATION ACTIVATION ANAPHASE II TELOPHASE II
PRONUCLEATE EGG
Figure 1: Schematic overview of mammalian oogenesis (modified from HAFEZ, 1993)
1.2 Intercommunication between the oocyte and its surrounding somatic cells The first evidence that follicular somatic cells support oocyte development came from the observation by PINCUS and ENZMANN (1935) that fully grown oocytes removed from antral follicles resume meiosis spontaneously in a gonadotropin-independent manner. Therefore they concluded that the follicular somatic cells maintained the oocytes in meiotic arrest. By now it has become evident that the communication in the mammalian ovarian follicle between oocyte and somatic cells is bidirectional and crucial for its development and function. In the recent literature this mutual
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dependence is described as an oocyte-granulosa cell regulatory loop (SU et al., 2004). The follicular somatic cells not only keep up the meiotic arrest but they also promote nuclear and cytoplasmic maturation (BUCCIONE et al., 1990) and participate in suppression of transcription (DE LA FUENTE and EPPIG, 2001). These cellular interactions are mediated via membrane gap junctions and paracrine factors. Several authors called this network of gap junctions uniting the mural and cumulus granulosa cells with the oocyte a structural and functional syncytium (LEIBFRIED-RUTLEDGE et al., 1989; EPPIG, 1991; DOWNS, 1993). FAIR et al. (1997) studied the oocyte ultrastructure in bovine primordial to early tertiary follicles. They found that gap junctions between the oocyte and its surrounding cumulus cells do not appear until the secondary follicle stage and this appearance coincides with the acquisition of competence to resume meiosis. It can be concluded that the integrity of this syncytium is essential for the resumption of meiosis. Furthermore, antisense silencing of Connexin 43 , the major protein gap junctions are composed of, resulted in a decrease in messenger(m)RNA expression of approximately 50 % and was associated with an inhibition of oocyte maturation (VOZZI et al. 2001). These findings substantiate the results of DE LA FUENTE and EPPIG (2001), that companion granulosa cells modulate the transcriptional activity of the oocyte. Until the secondary follicle stage the development is gonadotropin independent, but as soon as follicular antrum formation occurs it becomes gonadotropin dependent. With the antrum formation the oocyte becomes able to progress beyond diplotene of prophase and is called meiotically competent. At this time the granulosa cells divide into two main groups: the cumulus cells, directly surrounding the oocyte and the mural granulosa cells. Mural granulosa cells express luteinizing hormone (LH)- receptors, the more the closer to the basal lamina they are, cumulus cells express only few whereas the oocyte does not possess any LH-receptors. Therefore the response to the preovulatory gonadotropin surge is mediated by the somatic components.
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2 Oocyte maturation
The processes between resumption of meiosis and second meiotic arrest in metaphase II are called oocyte maturation. LEIBFRIED-RUTLEDGE et al., 1989, defined maturation as the process endowing the oocyte with the ability to complete meiosis, undergo fertilization, and prepare male and female haploid chromatin for syngamy and embryo development. The structural and regulatory proteins needed to complete these processes result largely from transcription at the earlier oocyte growth stage with translational or posttranslational modification. Apart from that they include characteristic changes of the nucleus (nuclear maturation) and the cytoplasm (cytoplasmic maturation) (SATHANANTHAN et al., 1991). Although several decades have passed since PINCUS and ENZMANN (1935) first observed spontaneous meiotic resumption in mammalian oocytes, the inhibitory factors involved in this maintenance of meiotic arrest, the regulatory proteins and mechanisms that cause their inactivation after the preovulatory gonadotropin surge are still mostly unknown.
2.1 Meiotic competence Meiotic competence is the ability of the oocyte to reinitiate meiosis. The oocytes acquire meiotic competence near the completion of their growth. At this stage the oocyte has drastically reduced its previously high level of transcriptional activities (reviewed by BACHVAROVA, 1985, 1988). In a species comparison two patterns for the acquisition of meiotic competence emerge. In rodents meiotic competence appears coincidentally at the time of antrum formation when the oocytes have reached their maximum growth (IWAMATSU and YANAGIMACHI, 1975: hamster; SORENSEN and WASSARMAN, 1976: mouse; BAR-AMI and TSAFRIRI, 1981: rat). In pig and cattle oocytes it is not strictly correlated with the appearance of antral cavity (MOTLIK and FULKA, 1986). In the porcine ovary, follicles less than 0.7 mm in diameter contain oocytes incapable of completing meiotic resumption. Developmental capacity increases as the diameter of the follicle increases and is acquired in some oocytes from follicles between 0.8 and 1.6 mm. But even not all
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oocytes from follicles > 1.7 mm progress to MII stage after 48 h of culture (reviewed by HUNTER, 2000). Similarly, in bovine oocytes, acquisition of meiotic competence does not occur until the oocyte diameter is greater than 100 µm. The majority of bovine oocytes exhibit full meiotic competence at a diameter of approximately 110 µm (FAIR et al., 1995). In the process of acquiring meiotic competence one has to distinguish two aspects of maturation, cytoplasmic and nuclear which are only partly independent of each other.
2.2 Nuclear maturation Nuclear maturation consists of the structural and biochemical changes concerning the nucleus and the chromosomes during maturation. EPPIG (1996) refers to nuclear maturation as the processes that reverse meiotic arrest at prophase I (PI) and drive the progression of meiosis to metaphase II. Further nuclear maturation is described as a two step process in which the ability to undergo germinal vesicle breakdown (GVBD) and continue to metaphase I (MI) is acquired earlier than the ability to reach metaphase II (THIBAULT, 1972; TSAFRIRI and CHANNING, 1975; SORENSEN and WASSARMAN, 1976; BAR-AMI and TSAFRIRI, 1981; MOTLIK et al., 1984; DE SMEDT et al., 1994). Similarly FAIR et al. (1995) describe this process in bovine oocytes in three stages: First the oocyte gains the capacity to undergo GVBD, second, it becomes capable of progressing to the MI and, finally, to the MII stage.
2.2.1 Morphological changes Meiosis starts in the foetal ovary but stops before or around the time of birth at the dictyate state of PI as already described in chapter 1.1. This state is a state of nuclear rest that may last until the oocyte is ovulated at puberty. During this growth phase not only the oocyte increases its volume also the nucleus increases its size. Thus resting oocytes have a large, prominent nucleus known as the germinal vesicle. Therefore this stage is also called germinal vesicle stage, GV stage. At this time the
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chromatin is distributed diffusely in the nuclear plasma (WASSARMAN & ALBERTINI, 1994). DAGUET (1980) describes a morphological change in the GV of the sow oocyte during the follicular phase preceding the ovulatory LH surge. The oocyte GVs of preovulatory follicles having a diameter of less than 2 mm showed uniformly dispersed chromatin with no condensation, while the GVs of oocytes from preovulatory follicles of more than 2 mm diameter presented condensed chromatin in the shape of a crown or horseshoe surrounding the nucleolus, and irregular chromatin clusters in the nucleoplasm; these clusters were usually found lying against the nuclear membrane. The GV is spherical and possesses either a single or more rarely double nucleoli (THIBAULT et al., 1987). The nucleoli express an intensive RNA synthesis and their compaction coincides with achievement of full size of oocytes in all species studied so far (MOTLIK and FULKA, 1986). After a species specific latent period following the overcome of the meiotic block GVBD occurs. Shortly before GVBD the GV moves peripherally (SUN et al., 2001). According to MOTLIK and FULKA, 1976, GVBD is characterized by the disassembly of the nuclear membrane, disappearance of the nucleolus and chromatin condensation. In their studies of pig oocytes GVBD in vivo was completed in most oocytes between 20 to 24 hours after human chorionic gonadotropin (hCG) injection whereas in a culture it was reached between 16 to 20 hours. For better characterization they divided the whole process into six light microscopically well-defined stages:
GV I: Nuclear membrane and nucleolus are clearly visible and chromatin forms a ring or horseshoe around the nucleolus. GV II: Nucleus and nucleolus are intact. A few orceinpositive structures (chromocenters, chromatin clumps) on the nuclear membrane can be detected. GV III: Nucleus and nucleolus are intact with slightly stained chromatin clumps or strands, localized especially around the nucleolus.
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GV IV: The nuclear membrane is less distinct and the nucleolus disappears completely. Chromatin is seen as an irregular network or as individual bivalents. Early diakinesis: The nuclear membrane disappeared; the bivalents lie in the region of the former nucleus. Late diakinesis: The chromosomes are condensed. SUN et al. (2004) added the GV0 stage to this classification. The GV0 configuration is distinct by the diffuse, filamentous pattern of chromatin in the whole nuclear area and is seen only in small follicles of diameters up to 2 mm.
Table 1: Kinetics of in vitro maturation in different species. Species MI AI TI MII Reference Mouse after 4 h after 9 h after 11 h after 11 h Donahue (1968) Cattle 10-15 h 15-16 h 16-18 h 18-24 h Sirard et al. (1989) Goat 8-15 h 14-18 14-18 16-24 h Pawshe et al. (1994) Horse 8-16 h n.d. n.d. 24-40 h Hinrichs et al. (1993) Pig after 24 h ~36 h ~36 h 36-48 h Christmann et al. (1994) MI=metaphase I, AI=anaphase I, TI=telophase I, MII=metaphase II, n.d.=not determined
During the further maturation process the chromosomes align on the metaphase I spindle, the homologous pairs separate and the first polar body is emitted before meiosis is arrested again at metaphase II.
2.3 Cytoplasmic maturation Cytoplasmic as well as nuclear maturation is essential for the capacity for fertilization and development to live offspring. Still the developmental programmes of both processes do not seem to be tightly linked as some aspects of cytoplasmic maturation occur without coordination with nuclear maturation. EPPIG (1996) refers to cytoplasmic maturation as the processes that prepare the egg for activation and preimplantation development. The production of maternal mRNAs and proteins in
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oocytes are critical for preimplantation development. Only some of these proteins are produced during the time of nuclear maturation and are, therefore, considered to be products of the process that is classically defined as cytoplasmic maturation, beginning at the time when nuclear maturation is initiated. But the term cytoplasmic maturation should include earlier stages of oocyte development, wherein maternal mRNAs and proteins critical for preimplantation development are synthesized and stored. These are the processes not linked to nuclear maturation in contrast to the aspects initiated as a result of the mixing of the GV contents with the ooplasm.
2.3.1 Morphological changes CRAN (1985) observed meiotic resumption after 20 to 30 h after hCG injection in porcine oocytes which coincided with a decline in the number of mitochondria which was due to fusion. Before the induction of maturation the mitochondria clustered at the periphery of the cell but dispersed with maturation. The number of lipid droplets increased maintaining a close spatial relationship with the endoplasmatic reticulum whereas the Golgi complexes decreased in size. For the first 18 h of in vitro maturation cortical granules (CGs) are distributed throughout the cortical cytoplasm. Thereafter, the CGs underwent centrifugal migration to form a monolayer next to the plasma membrane (YOSHIDA et al., 1993). This observation was confirmed by the results of SUN et al. (2001), who found that the CGs filled all the cytoplasm in a GV- stage oocyte but migrate to the cortical region and line up next to the plasma membrane as soon as the GV moves peripherally and GVBD occurs. Also the microfilaments were distributed in the entire cytoplasm in the GV-stage oocyte, concentrated where the chromosomes are located at the time of GVBD and finally distributed in the submembrane region. The enzymes released by the CGs cause the block to polyspermy at fertilization (DUCIBELLA, 1996). Similar results for the distribution and changes were found in cattle oocytes (KRUIP et al., 1983; HYTTEL et al., 1986).
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2.4 Biochemical changes At the biochemical level, changes in rates of RNA synthesis and protein synthesis (RODMAN and BACHVAROVA, 1976; WASSARMAN and LETOURNEAU 1976; SCHULTZ et al., 1978) as well as changes in protein synthesis patterns have been described during mammalian oocyte maturation (GOLBUS and STEIN, 1976; SCHULTZ and WASSARMAN, 1977b; MCGAUGHEY and VAN BLERKOM, 1977; WARNES et al., 1977; VAN BLERKOM and MCGAUGHEY, 1978). Also an extensive phosphorylation of several proteins before GVBD has been shown for different species (mouse: WASSARMAN et al., 1979; sheep: CROSBY et al., 1984; Xenopus: MALLER and SMITH, 1985; cattle: KASTROP et al., 1990). A progressive change in certain qualitative and quantitative aspects of protein synthesis is found in all mammalian oocytes, but ultrastructural as well as transcriptional and translational requirements to resume meiosis differ among the species.
2.4.1 Transcription and translation After oogonia complete their mitotic divisions they enter preleptotene, the interphase prior to meiosis. Deoxyribonucleic acid (DNA) synthesis and a number of cytological changes occur at this time, marking the transition from oogonium to primary oocyte. Transcription occurs in oogonia and in oocytes throughout PI (LEIBFRIED- RUTLEDGE et al., 1989) whereas the oocyte during the dictyate stage shows no DNA synthesis. In pig oocytes virtually no transcription is found during maturation (MOOR and DAI, 2001). DNA synthesis does not start until the first mitotic cell cycle of the zygote (LAURINCIK et al., 1995). The intensive RNA synthesis in the primary oocyte decreases at the end of the growth period and is almost not detectable during maturation (WASSARMAN and LETOURNEAU, 1976; STERNLICHT and SCHULTZ, 1981). At this point of time gene expression is regulated in the first place via translation and posttranslational mechanisms (BACHVAROVA and PAYNTON, 1988; DE VANTÉRY et al., 1997). These include differential recruitment of previously synthesized and stored RNAs and posttranslational modification of existing proteins involving phosphorylation (LEIBFRIED-RUTLEDGE et al., 1989). As the oocyte
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grows not all mRNA is translated but stored in large quantities as de-adenylated transcripts complexed with protein in the cytoplasm. This masked mRNA drives the process of maturation and all developmental events that occur before activation of the embryonic genome (MOOR and DAI, 2001). This study also describes that the relevant mRNAs for meiosis are recruited at different times, for different durations and at different levels.
2.4.2 Protein synthesis and phosphorylation The resultant protein synthesis is essential for meiotic progression in pig oocytes. De novo protein synthesis for GVBD is also necessary in other species (Xenopus: WASSERMAN and MASUI, 1975; cattle: HUNTER and MOOR, 1986; sheep: MOOR and CROSBY, 1986; KASTROP et al., 1991a). In mouse, rat, rabbit and starfish oocytes resumption of meiosis does not require active protein synthesis by the oocyte (WASSARMAN and SCHULTZ, 1977; EKHOLM and MAGNUSSON, 1979; PICARD et al., 1985). But mouse oocytes matured with an inhibited protein synthesis arrest before the MII stage; therefore for proceeding to MII de novo protein synthesis is necessary. Studying oocytes of different species it was demonstrated that changes of protein synthesis patterns are observed following GVBD (SCHULTZ and WASSARMAN, 1977; KASTROP et al., 1990), whereas extensive phosphorylation of some proteins mainly occurs preceding GVBD (KASTROP et al., 1990). The LH- surge is the trigger for meiotic resumption. The signal is transduced within the cell via activation and deactivation of specific proteins especially kinases. The activation and deactivation is caused by phosphorylation and/or dephosphorylation of these kinases. Comparison of the protein synthesis patterns of in vivo and in vitro matured oocytes demonstrated similar but not identical changes, when the oocytes were cultured as cumulus-oocyte-complexes (COC) and with the addition of gonadotropins to the culture medium (KASTROP et al., 1991b; MEINECKE and SCHRÖTER, 1996). The obvious differences in the protein synthesis patterns especially at the end of the maturation period might be the cause for the reduced developmental capacity of in
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vitro matured oocytes. The protein phosphorylation patterns of in vivo and in vitro matured oocytes were also similar but not identical (KASTROP et al., 1990).
FULKA et al. (1996) carried out cell fusion experiments during which they found a DNA synthesis inhibiting activity in maturing and matured but not yet activated mouse oocytes, which was missing in incompetent oocytes. They postulated the hypothesis that this activity takes part in suppression of the S-phase. SCHMITT and NEBREDA (2002) reported that the kinase Rsk might be this suppressing activity down regulating the S-phase between meiosis I and II.
3 Cell cycle aspects of oocyte maturation
The purpose of the cell cycle is to guarantee that the genetic material of a cell is distributed evenly when the cell divides. It consists of two different phases: M-phase (M=mitosis) and interphase. During M-phase cell division takes place; cell growth and protein production stop at this stage in the cell cycle. During interphase the cell is constantly synthesizing RNA, producing protein and growing in size. The interphase can be subdivided into three steps: S-phase (S=synthesis): DNA-synthesis G1-phase (G=gap): between end of M-phase and beginning of DNA-synthesis; Cells increase in size in Gap 1, produce RNA and synthesize protein. An important cell cycle control mechanism activated during this period (G1 Checkpoint) ensures that everything is ready for DNA synthesis. G2-phase: between end of S-phase and following M-phase; During the gap between DNA synthesis and mitosis, the cell will continue to grow and produce new proteins. At the end of this gap is another control checkpoint (G2 Checkpoint) to determine if the cell can now proceed to enter M-phase and divide. Figure 2 gives an image of the cell cycle.
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G2 M
G0
G1
S
Figure 2: The cell cycle G0=gap0 phase, G1=gap1 phase, G2=gap2 phase, S=DNA synthesis, M=mitosis
The meiotic block of the oocyte in the dictyate stage corresponds to an arrest between G2- and M-phase of the cell cycle (MOOR et al., 1990). Before the oocyte is able to resume meiosis two premises have to be fulfilled. It must have gained a certain cytoplasmic status to overcome the G2-arrest and the inducing stimulus, progesterone in amphibians and mammals, 17 α, 20 β-dihydroxy-4-pregnen-3-one in fishes, 1-methyladenine in starfish, and serotonin in some molluscs, has to influence the controlling systems of the cell cycle via a signal transduction cascade (NAGAHAMA and ADACHI, 1985; GUERRIER et al., 1990; WHITAKER, 1996). In this regulation of oocyte maturation phosphorylation and dephosphorylation of proteins plays a pivotal role. Responsible for phosphorylation and dephosphorylation are kinases, enzymes that transfer phosphorus from ATP to the target proteins. Three major kinases involved in the signal transduction cascade that restarts meiosis, are Maturation Promoting Factor (MPF), Mitogen-Activated Proteinkinase (MAPK) and Protein 90 ribosomal S6 Kinase (p90rsk).
3.1 Maturation-Promoting-Factor (MPF) MPF (maturation-promoting factor) was first described as a cytoplasmic activity present in amphibian metaphase II-arrested (mature) oocytes that could induce
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GVBD when microinjected into prophase-blocked (immature) oocytes without the need for protein synthesis (MASUI and MARKERT, 1971). The same activity was described in mouse and porcine oocytes where fusion of immature and mature oocytes resulted in chromosome condensation and GVBD in the immature oocytes (BALAKIER 1978; FULKA et al., 1985). It is present in dividing cells from almost all eukaryotes and is equally effective at promoting entry into M phase irrespective of the species of recipient or donor cells (RAO and JOHNSON, 1970; NURSE, 1990). MPF was therefore renamed “M-phase promoting factor”. LOHKA et al. (1988) purified Xenopus MPF and showed that it is a dimer containing a 32 kDa serine(Ser)/threonine(Thr) protein kinase subunit and a 45 kDa phosphoprotein. As well in 1988 ARION et al. found a 34 kDa protein with identical characteristics in starfish oocytes. The 32 kDa subunit was subsequently identified as cdc2 (cell division cycle), the Xenopus homolog of the cdc2+ gene product from the fission yeast Schizosaccharomyces (S.) pombe (DUNPHY et al., 1988; GAUTIER et al., 1988). Cdc genes control the cell cycle and their mutation results in an arrest of the cell cycle in the G2-phase (NURSE and BISSETT, 1981). The 45 kDa subunit was identified as a B-type cyclin (DRAETTA et al., 1989; LABBÉ et al., 1989; MEIJER et al., 1989). The catalytic subunit, the 34 kDa protein also named p34 cdc2 , and the regulatory subunit, the cyclin B, together form the inactive pre-MPF complex. As a result cdc2 kinase was the first discovered cyclin-dependent kinase (cdk). In all eukaryotes these cdks regulate each phase of the cell cycle. A synonym for MPF is histone H1 kinase because of its ability to phosphorylate histone H1.
3.1.1 MPF activation and inactivation The activity of MPF shows a biphasic course during oocyte maturation with two maxima in metaphase I and II (starfish: DORÉE et al., 1983; Xenopus: GERHART et al., 1984; HUCHON et al., 1993; ABRIEU et al., 2001; MALLER et al., 2001; mouse: HASHIMOTO and KISHIMOTO, 1988; FULKA et al., 1992; pig: MATTIOLI et al., 1991; NAITO and TOYODA, 1991; cattle: WU et al., 1997; rabbit: JELÍNKOVÀ et al., 1994). A scheme of MPF activity course is depicted in figure 3. MPF activity declines
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after activation of the oocyte through fertilization or a parthenogenetic stimulus (COLLAS et al., 1993; COLLAS et al., 1995; MACHATY et al., 1996).
enzyme activity
MPF
h of cultivation
GV GVBD MI AI TI MII
Figure 3: MPF fluctuations and cytological stages of meiotic maturation in Xenopus laevis as a typical example of a vertebrate oocyte (modified after TAIEB et al., 1997)
Three distinct posttranslational mechanisms regulate the activity of p34 cdc2 kinase: First, the binding of cyclin B to the catalytic subunit (MURRAY et al., 1989); second, the phosphorylation and dephosphorylation of p34 cdc2 (SOLOMON et al., 1992); and third, the phosphorylation and dephosphorylation of cyclin B (ROY et al., 1990). The details of these mechanisms have been studied extensively in invertebrates and amphibians especially Xenopus laevis. The results of these studies serve as a model for the situation in mammals and are described exemplarily in the following section. Figure 4 shows this model of the activating/deactivating mechanisms of MPF.
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Prophase
store
Thr14 Tyr15 Thr161 p34cdc2 P P P P Thr14 Tyr15 Thr161 Myt1 Thr14 Tyr15 Thr161 CAK p34cdc2 p34cdc2
Cyclin B Cyclin B Cyclin B Pre-MPF Cyclin B synthesis
GVBD P P P Thr14 Tyr15 Thr161 p34cdc2
Myt1 Cyclin B
Progesterone Pre-MPF cdc25 P Thr14 Tyr15 Thr161 p34cdc2 autoamplification Cyclin B
MPF
Figure 4: Accumulation of pre-MPF in G2-arrested oocytes and activation of MPF at GVBD in Xenopus (modified after TAIEB et al., 1997) In G2-arrested oocytes, cyclin B is continuously synthesized and binds to free cdc2 molecules. Cdc2 is phosphorylated on Tyrosine (Tyr) 15 and on Thr14 by Myt1 kinase and on Thr161 by CAK and accumulates as inactive pre-MPF. Progesterone stimulates synthesis of Myt1 inhibiting kinases and allows activation of cdc25 leading to an auto amplification loop between cdc25 and MPF. Thr=threonine, Tyr=tyrosine, P=phosphorus, CAK=cdc2-activating kinase
G2 arrested Xenopus oocytes contain a pre-formed stock of inactive p34 cdc2 /cyclinB complexes (pre-MPF). In these complexes p34 cdc2 kinase is phosphorylated on Thr14, Tyr15 and Thr161 (CYERT and KIRSCHNER, 1988; GOULD and NURSE,
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1989; SOLOMON et al., 1990; GAUTIER and MALLER, 1991; LEE et al., 1991;; KREK and NIGG, 1991). The activation of pre-MPF during maturation requires the dephosphorylation of p34 cdc2 on Thr14 and Tyr15 which is catalysed by the phosphatase Cdc25 (GAUTIER et al., 1991; STRAUSFELD et al., 1991; MALLER et al., 2001). In addition to an increasing cdc25 activity, inhibition of the kinases that phosphorylate p34 cdc2 is necessary for its activation. Two kinases phosphorylating and therefore inhibiting p34 cdc2 have been identified. Wee1 is known as the classic inhibitory kinase of p34 cdc2 in S. pombe , but the human and Xenopus Wee1 homologues phosphorylate p34 cdc2 only on Tyr15 but not Thr14. This supports the existence of a separate Thr14 kinase (PARKER and PIWNICA-WORMS, 1992; MCGOWAN and RUSSELL, 1993; MUELLER et al., 1995a). ATHERTON-FESSLER et al. (1994) detected a kinase activity that can phosphorylate p34 cdc2 on Thr14 in Xenopus egg extracts. This enzyme was later identified by MUELLER et al., 1995b, as Myt1, a membrane-associated inhibitory kinase that phosphorylates Cdc2 on both Thr14 and Tyr15 in Xenopus and was also shown in humans (BOOHER et al., 1997; LIU et al., 1997). Cdc25, Wee1 and Myt1 all become heavily phosphorylated during mitosis but whereas Cdc25 phosphorylation is accompanied by its increasing activity, Wee1 and Myt1 are inhibited by their phosphorylation contributing to the decrease in the inhibitory phosphorylation of p34 cdc2 (IZUMI et al., 1992; IZUMI and MALLER, 1993; HOFFMANN et al., 1993; TANG et al., 1993; MCGOWAN and RUSSELL, 1995; MUELLER et al., 1995a,b; BOOHER et al., 1997). Wee1 is not present in G2- arrested oocytes and is only synthesized upon progesterone stimulation (MURAKAMI and VANDE WOUDE, 1998). Thus, Myt1 probably is the MPF inhibitory activity in Xenopus oocytes. The depicted auto amplification loop between cdc25 and active MPF seems to be depending on the species. Whereas it was shown in Xenopus laevis and starfish oocyte as well as in bovine oocytes (IZUMI and MALLER, 1993; TATEMOTO and HORIUCHI, 1995) it was missing in porcine and murine oocytes (FULKA et al., 1988). The self-amplification of MPF was observed in human mitotic cells (HOFFMANN et al., 1993). The contribution of cyclin B phosphorylation to MPF activation is not yet fully understood. Although cyclin B1 phosphorylation is not required for cdc2 kinase
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activity, for binding to cdc2 protein, for stability of cyclin B1 before GVBD, or for destruction of cyclin B1 after GVBD or after egg activation (IZUMI and MALLER, 1991), it is required for Xenopus oocyte maturation (LI et al., 1995). Cyclin B1 phosphorylation might be necessary to control the targeting of cyclin B1 complexes to appropriate subcellular localizations, such as the nucleus (LI et al., 1997), and/or to specific substrates. Cyclin B degradation by a specific ubiquitin-dependent proteolytic system, the so called anaphase-promoting complex (APC), results in the drop of MPF activity between the two meiotic divisions (TAIEB et al., 1997; MADGWICK et al., 2004). At almost the same time when cyclin B is destructed, cyclin B synthesis is stimulated three- to fourfold (KOBAYASHI et al., 1991; RIME et al., 1994). While the drop in MPF activity can be accounted to the partial destruction of cyclins, the subsequent reactivation of MPF is probably due to the accumulation of newly synthesized proteins (TAIEB et al., 1997). But nevertheless one has to take account of notable interspecies differences that argue against a generalization of mechanisms regulating MPF activation in oocytes as the amounts of the two subunits during the cell cycle, differ depending on the species. The immature oocytes of several fishes (goldfish, carp, catfish, and lamprey) as well as some amphibians (Rana japonica and Bufo japonicus) do not contain cyclin B (HIRAI et al., 1992; TANAKA and YAMASHITA, 1995; YAMASHITA et al., 1995). In their maturing oocytes cyclin B is synthesized and binds to cdc2, being activated directly by Thr161 phosphorylation without inhibitory phosphorylation of Thr14 and Tyr15. These differences between amphibian species could depend on the annual reproduction cycles and the period during which the experiments are performed. Mouse oocytes in contrast to Xenopus oocytes do not require de novo protein synthesis to undergo GVBD and to activate MPF in vitro (WASSARMAN et al., 1979). Their concentration of cyclin B in meiotically incompetent and competent oocytes is similar whereas p34 cdc2 concentration is markedly less in incompetent oocytes. Only at the end of the growth period p34 cdc2 accumulates (CHESNEL and EPPIG, 1995a; DE VANTÉRY et al., 1996). The quantity of p34 cdc2 remains more or less constant throughout maturation with only slight fluctuations depending on the species (WU et
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al., 1997). KUBIAK et al. (1993) therefore conclude that cyclin B synthesis and degradation regulates MPF activity. Cyclin B synthesis in murine oocytes increases drastically until metaphase I and therefore newly synthesized cyclin B is not necessary to activate MPF, but it is necessary for the second increase of MPF activity after GVBD (HAMPL and EPPIG, 1995). Oocyte from other mammalian species, such as sheep, goat, pig, and cow, do not activate cdc2 kinase in the absence of protein synthesis (MOOR and CROSBY, 1986; HUNTER and MOOR, 1987; SIRARD et al., 1989; PROCHÁZKA et al., 1989; LE GAL et al., 1992). In porcine oocytes cyclin B is not synthesized for the first 23 hours after induction of maturation (NAITO et al., 1995). The amount of MPF complex and free cyclin increases during metaphase I until 35 hours of maturation and reaches its maximum in metaphase II. CHRISTMAN et al. (1994) demonstrated that growing and fully-grown porcine oocytes contain both subunits of MPF in comparable amounts, but the growing oocytes are not able to form the active complex. In bovine oocytes cyclin B synthesis was shown already 4 hours after induction of maturation with a maximum after 24 hours (LEVESQUE and SIRARD, 1996). WU et al. (1997) reported an oscillating course of cyclin B2 synthesis with an increase before metaphase I and II. Meiotic incompetence in goat oocytes is due to an absolute lack of cyclin B as cyclin B1 mRNA-synthesis does not start until the end of the growth period accompanied by acquisition of meiotic competence (HUE et al., 1997). Incompetent rat oocytes similar to pig oocytes express the catalytic subunit of MPF at amounts that are not different from that found in competent oocytes (GOREN et al., 1994). Interestingly, a similar pattern of MPF activation was found in male germ cells (CHAPMAN and WOLGEMUTH, 1994; GODET et al., 2000). High levels of cyclin B1 and cdc2 leading to high MPF activity were associated with the meiotic G2/M transition and decreased protein levels with no kinase activity at the exit of meiosis in rat and mouse male germ cells.
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3.1.2 Substrates and function of the MPF The MPF plays a critical role during oocyte maturation especially in the disassembly of the nuclear envelope membrane, chromosome condensation and reorganisation of the cytoskeleton (EPPIG, 1993; KRISCHEK and MEINECKE, 2001). Some of these changes are caused by a direct phosphorylation of structural components of the cell. Other changes are mediated indirectly by MPF through its action on a phosphatase/kinase system (PFALLER et al., 1991). The activated MPF complex is a protein kinase phosphorylating serine and threonine residues of its target proteins. Several substrates for MPF have been identified in vitro. To prove that they serve as in vivo substrates, they need to phosphorylate the same residues in vitro and in vivo and cause a following functional change in the cell. As the synonym of MPF, histone H1 kinase, already says, the histone H1 is one of the possible substrates. It is assumed that MPF phosphorylates DNA-binding proteins which thereafter dissociate from the DNA. This puts an end to transcription and allows DNA/chromosome condensation (MORENO and NURSE, 1990). Restricting these findings are the observations of several authors who only saw a transient rise in histone H1 kinase activity accompanying chromosome condensation respectively a certain degree of chromosome condensation was still seen when MPF activity was inhibited (rabbit: JELINKOVA et al., 1994; cattle: SIMON et al., 1989; TATEMOTO and HORIUCHI, 1995; pig: KUBELKA et al., 1995). Cyclin B as a substrate for MPF regulating p34 cdc2 activity has already been discussed in the previous section. The nuclear lamins form an intermediate filament-type network underlying the inner nuclear membrane. The disassembly of the nuclear membrane starts with a phosphorylation of serine residues of lamins (PETER et al., 1990; LUSCHER et al., 1991). But phosphorylation by p34 cdc2 alone is not sufficient; therefore another kinase(s) in addition seems to be involved (LUSCHER et al., 1991). CHOU et al. (1990) observed that protein vimentin is a substrate of p34 cdc2 . Its phosphorylation contributes to M phase reorganization of the intermediate filament network, the cytoskeleton.
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The elongation factor EF-1 gamma is an in vivo substrate of p34 cdc2 in Xenopus laevis as well as in goldfish oocytes (BELLE et al., 1989; TOKUMOTO et al., 2002). MADGWICK et al. (2004) postulated that sister chromatid attachment is maintained through MPF activity. When they blocked cyclin B1 degradation the chromatin remained condensed. With an addition of the MPF inhibitor roscovitine the separation of the sister chromatids was induced. They present a model in which MII arrest is maintained primarily by MPF levels only. Similar observations were recently made in Xenopus egg extract and HeLa (Henrietta Lachs) cells (STEMMANN et al., 2001; CHANG et al., 2003). A major microtubule-associated protein, p220, is phosphorylated by p34 cdc2 and MAPK in M-phase (SHIINA and TSUKITA, 1999). The phosphorylated p220 loses its microtubule binding and microtubule-stabilizing activities which is important for chromosome movement during anaphase. Further possible substrates are Nucleolin with a possible involvement in the dissolution of the nucleolus and RNA polymerase II whose inhibition via phosphorylation would lead to an inhibition of transcription (MORENO and NURSE, 1990; NORBURY and NURSE, 1992).
3.2 Mitogen-activated protein kinase (MAPK) Mitogen-activated protein kinases are ubiquitous and one of the most important kinases involved in intracellular signal transduction pathways in eukaryotic organisms playing a crucial role especially in cell proliferation and differentiation as well as cell cycle regulation (KYRIAKIS and AVRUCH, 1996; SUN et al., 1999). Members of the MAPK family include extracellular signal-regulated kinases (ERK1 and 2, 44-kDa and 42-kDa isoforms), which are activated in response to a large array of extracellular signalling molecules, notably growth factors and tumour promoters, via the Ras protooncogen. C-Jun N-terminal kinases (JNK) and p38 MAPKs constitute two other families, collectively known as stress-activated protein kinases (SAPK), since they are induced by UV radiation, heat shock, oxidative and osmotic stress or tumor- necrosis factor- α (TNF- α). As meiosis induction is mediated via extracellular signal-
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regulated kinases the following sections concentrate on this family of kinases and the term MAPK is used synonymously with the term ERK. SANGHERA et al. (1990) were the first to report about an involvement of MAPK in meiotic maturation in sea star and GOTOH et al. (1991) in Xenopus oocytes. Similar as with the MPF many studies since have been done on the role of MAPK cascade in the regulation of the amphibian oocytes meiotic cell cycle. It has been shown that the oocytes respond to reproductive hormones by activating MAPK cascades that lead to the activation of MPF and thus the resumption of meiosis (GOTOH and NISHIDA, 1995a, b). Therefore MAPK cascade is another principal regulatory system that functions parallel to and interacts with MPF in driving the meiotic cell cycle progression of oocytes. But increasing data showed that different organisms may use the MAPK cascade for different purposes, even within the meiotic cell cycle (MURRAY, 1998).The first report of a functional significance of MAPK in the mammalian oocytes came from Sobajima et al. in 1993, who described MAPK activation during mouse oocytes maturation.
3.2.1 MAP-Kinase activation and inactivation This family of kinases is characterized by their activation by MAPK kinases through dual phosphorylation of threonine and tyrosine residues in the activation loop and by their substrate specificity which is proline-directed phosphorylation of serine or threonine, meaning that they phosphorylate Ser or Thr residues that are neighbours of prolines. MAPK is activated by dual phosphorylation of the threonine 183 and tyrosine 185 residues; the phosphotyrosyl site is most probably regulatory (ANDERSON et al., 1990; PAYNE et al., 1991). The MAPK is part of a signalling cascade consisting of three to six tiers of protein kinases that sequentially activate each other by phosphorylation. Figure 5 illustrates this signalling cascade.
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Mitogens, hormones, others
plasma membrane Ras ?
Raf MOS
MEK Ser P
P P Tyr Thr P P Cytoplasmic targets MAPK Ser Thr RSK
Nuclear targets
Figure 5: Schematic representation of MAPK signalling pathway (modified after RUBINFELD and SEGER, 2004) MEK=MAPK/ERK Kinase, MOS=Moloney murine sarcoma virus, P=phosphorus, Raf=?, Ras=Rat sarcoma virus, RSK=Ribosomal S6 kinase, Ser=serine, Thr=threonine, Tyr=tyrosine
The induction of the MAPK cascade typically starts with binding of an extracellular ligand to a tyrosine specific receptor kinase. The activated receptor binds GTP proteins which in turn activate a small guanosine 5’-triphosphate (GTP)-binding protein (Ras family protein; oncogen, Rat Sarcoma Virus) or an adaptor protein, which transmits the signal either directly or through a mediator kinase to the MAPK kinase kinase level of the cascade. Raf and MOS (oncogen, Moloney Sarcoma Virus) are such MAPK kinase kinases. MOS is a 39 kDa germ cell specific Ser/Thr protein kinase that was first identified in cells transformed by Moloney murine sarcoma virus (PAPKOFF et al., 1982). Its mRNA is stored as maternal information in the growing oocytes and it is translated into protein which initiates MAPK cascade phosphorylation during oocytes maturation (GEBAUER and RICHTER, 1996). The MAPK kinase kinases transmit the signal down through serine phosphorylation of the
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MAPK/ERK kinases (MEKs). MEKs then activate the ERKs. Both of the already mentioned isoforms, ERK1 and ERK2, exist in mouse, rat, and porcine oocytes (VERLHAC et al., 1993; GOREN et al., 1994; INOUE et al., 1995). In bovine oocytes ERK1 is the dominant isoform (FISSORE et al., 1996). The amount of MAPK molecules in murine oocytes increases during the growth period of the oocytes reaching a maximum when they acquire meiotic competence. The accumulation of molecules in the oocytes is dependent on their contact to somatic cells (CHESNEL and EPPIG, 1995b). Since the first reports in 1993 by SOBAJIMA et al. MAPK activation during oocytes maturation has been shown for several mammalian species (porcine: INOUE et al., 1995; bovine: FISSORE et al., 1996; horse: GOUDET et al., 1998; rat: LU et al., 2001). MAPK activity increases with the resumption of meiosis and remains stably activated until activation of the oocytes in arrested MII. With the formation of the male and female pronucleus its activity decreases rapidly. This inactivation is induced via the dephosphorylation of the MAPK molecule (MOOS et al., 1996a, b).
enzyme activity
MAPK
GV GVBD MI AI TI MII PN Figure 6: Change of MAPK activity during gonadotropin-induced mammalian oocytes maturation (modified after FAN and SUN, 2004)
The extensive studies of MAPK activation, localization, regulation and function in mammalian oocytes revealed controversial findings in almost every step of the meiotic cell cycle. One reason might be the use of different in vitro maturation models: the spontaneous meiosis model and the hormone-induced meiosis model. In these two events different mechanisms regulate the progression of the meiotic cell
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cycle. As already described in chapter 2.4.2 protein synthesis is not required for spontaneous meiotic resumption in mouse and rat oocytes (MOTLIK and RIMKEVICOVA, 1990). Farm animals as pig, cow, sheep, and goat have difficulties in entering meiosis spontaneously and do require protein synthesis for GVBD (INOUE et al., 1996). In mouse and rat oocytes MAPK and p90rsk activation is not a prerequisite for the initial activation of MPF after spontaneous GVBD (VERLHAC et al., 1993; LU et al., 2001; TAN et al., 2001) but in FSH-induced meiotic resumption of mouse oocytes MAPK activity was detected before GVBD (SU et al., 2001; SU et al., 2002). FISSORE et al. (1996) reported a simultaneous activation of MAPK and MPF before GVBD in bovine oocytes, whereas DEDIEU et al. (1996) found that MAPK activity was delayed compared with MPF activity and the event of GVBD in goat oocytes. The increase of MPF activity concomitant with GVBD in bovine oocytes was also observed in our group whereas MAPK activity did not rise only until after GVBD (JANAS, 1997). In porcine oocytes no dramatic increase in kinase activity was observed even when more than 30 % of oocytes had undergone GVBD and an abrupt increase was observed at MI (INOUE et al., 1995). Results of our group confirmed the slight increase of MAPK activity when parts of the oocytes had already undergone GVBD and a rapid rise at MI (WEHREND, 1997). Similar patterns of MAPK activation were shown for mare, human, and rabbit oocytes (GOUDET et al., 1998; SUN et al., 1999; YU et al., 2002). Recent findings support the idea that MAPK activity is not required for the spontaneous meiotic resumption of denuded oocytes in mammals. In mouse denuded oocytes GVBD occurred normally when MAPK activity was inhibited by the MEK inhibitor U0126 (TONG et al., 2003). Oocytes of MOS knockout mice resume meiosis although MAPK failed to be activated (HASHIMOTO et al., 1994; COLLEDGE et al., 1994). Microinjection of mRNA encoding a specific MAPK phosphatase into GV-stage bovine oocytes did not prevent meiotic resumption (GORDO et al., 2001) and microinjection of MOS antisense RNA into pig oocytes failed to arrest them at GV stage (OHASHI et al., 2003). Spontaneous meiotic resumption occurred in denuded pig oocytes although MAPK phosphorylation was inhibited by U0126 a specific MEK inhibitor (FAN et al., 2003a). But U0126 as well as
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the MEK inhibitor PD98059 inhibited FSH-induced meiotic resumption in cumulus- enclosed mouse and pig oocytes (SU et al., 2001; SU et al., 2002; FAN et al., 2003a; MEINECKE and KRISCHEK, 2003). These findings led to the conclusion that MAPK activity in cumulus cells is necessary for the gonadotropin-induced meiotic resumption of oocytes. Phosphorylation of MAPK in porcine cumulus cells starts already after 30 min. of maturation culture (EBELING et al., 2004). Its activation in mouse and pig cumulus cells was seen 30 min. and 2 h after FSH stimulation (FAN and SUN, 2004). An increase of MAPK activity in pig cumulus cells followed maturation culture and peaked at 20 h of culture (SHIMADA et al., 2001; FAN et al., 2003a). Despite these recent findings the regulation and function of MAPK in the cumulus remains unknown. But it is obvious that in the process of gonadotropin- induced meiotic resumption, which is MAPK-dependent, cumulus cells are the primary targets of meiosis-inducing signal. Inactive MAPK in immature arrested oocytes is localized exclusively in the cytoplasm. Just before GVBD part of the MAPK is moved into the GV. This nuclear MAPK is the phosphorylated, active form whereas both, the active and inactive form, were present in the ooplasm at this time (INOUE et al., 1998). These results were confirmed by FAN et al., 2003a who also found that the MAPK migrated from the cytoplasm to the GV before GVBD and associated with the meiotic spindle at metaphase and anaphase. An association of MAPK with the spindle, especially the microtubule- organizing centres (MTOCs), was also found in mouse oocytes (VERLHAC et al., 1993).
3.2.2 Substrates and function of the MAP-Kinase The first found and best known physiological substrate of MAPK in oocytes is p90rsk (RSK). This protein kinase belongs to a family of Ser/Thr kinases that originally was identified phosphorylating the S6 protein of the 40S ribosomal subunit in maturing Xenopus oocytes (ERIKSON and MALLER, 1985; see next chapter). Main substrates apart from RSK are the downstream kinases MNK (MAPK signal- interacting kinases) and MSK (MAPK/Stress-activated protein kinase (SAPK)- activated kinase (RUBINFELD and SEGER, 2004). Upstream kinases as Raf and
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MEK are also substrates of MAPK which supports the hypothesis of a feedback loop regulating this protein kinase cascade (DAVIS, 1993; RUBINFELD and SEGER, 2004). The transcription factors Elk-1, c-Myc, and c-Jun are also MAPK substrates. Being involved in the regulation of their activation, MAPK might contribute to regulation of transcription. Another substrate is the cytosolic PLA 2 (phospholipase A 2), the rate- limiting enzyme in pathways involving arachidonic acid release. At the cell surface the EGF receptor is a target for MAPK phosphorylation, but the functional significance of this phosphorylation is still unclear. The phosphorylation of nuclear lamins, especially lamin B, by MAPK as well as the phosphorylation of the microtubule-associated protein tau, links this kinase to the regulation of the cytoskeleton (PETER et al., 1992; DAVIS, 1993; RUBINFELD and SEGER, 2004). Cyclin B, the regulatory subunit of the MPF complex, is also a substrate of MAPK (see previous chapter). These different substrates of MAPK already indicate the many and diverse functions of this kinase during oocytes maturation. Apart from the different substrates MAPK fulfils deviating functions in the different species. Table 2 shows a summary of the different functions.
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Table 2: Summary of MAPK cascade functions in meiotic cell cycle progression of mammalian and amphibian (Xenopus laevis as the representative) oocytes (modified after FAN and SUN, 2004)
Cell cycle stage Mammals Xenopus Meiotic resumption Necessary for FSH- Facilitate progesterone- induced m. r. (functions in induced m. r. (functions in cumulus cells); not for oocytes); involved in MPF spontaneous GVBD/MPF activation activation GVBD-MI Regulate normal meiotic Same as mammals spindle assembly MI-MII Ensure asymmetric division Inhibit APC activity; of MI; not determined in S- prevent complete cyclin B phase inhibition degradation and S-phase entry; promote cyclin B synthesis MII arrest Contribute to CSF activity; Contribute to CSF activity; maintain MII arrest and p90rsk is its sole mediator; normal spindle involved in spindle configuration assembly checkpoint PB2 emission Required for PB2 emission; No reports inhibit sperm aster development PN formation Incompatible with pro- Same as mammals nuclear membrane m. r. = meiotic resumption, APC = anaphase-promoting-complex, CSF = cytostatic factor, PB = polar body, PN = pronucleus
The necessity for MAPK activation during gonadotropin-induced meiotic resumption versus the spontaneous meiotic resumption without its activation has been discussed in the previous section as well as its involvement in MPF activation in the different species. The association of MAPK with the spindle and the fact that cytoskeletal elements, as lamin B, are substrates indicate its role in the regulation of the cytoskeleton. Explicitly MAPK is involved in the regulation of microtubule organization. It is present in the spindle and particularly associates with MTOCs present at the spindle poles as well as in the cytoplasm (VERLHAC et al., 1994). In mouse and pig oocytes, the active MAPK distributes to the spindle poles at metaphase and migrates to the middle of the
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spindle at anaphase. During polar body emission, MAPK is associated with the cytokinetic ring (LEE et al., 2000; HATCH et al., 2001). This pattern of distribution substantiates MAPK determining role in spindle assembly and microtubular configurations. It can be concluded that MAPK activity is up regulated by the spindle assembly (kinetochore attachment) checkpoint which is responsible for the arrest of cells in metaphase to prevent exit from mitosis/meiosis until all the chromosomes are aligned properly at the metaphase plate. The checkpoint operates by preventing activation of APC. Inhibition experiments resulted in further evidence for this hypothesis. Mouse oocytes injected with antibody against MOS failed to assemble a meiotic spindle (ZHAO et al., 1991), and the spindle shape was altered in maturing MOS-deficient oocytes (ARAKI et al., 1996; CHOI et al., 1996; VERLHAC et al., 1996). Bovine oocytes injected with mRNA of a specific MAPK phosphatase exhibited disorganized and diffuse spindles (GORDO et al., 2001). In porcine oocytes MAPK was colocalized with γ-tubulin, suggesting an involvement in microtubule nucleation (SUN et al., 2001). Inhibition of MAPK by the MEK-specific inhibitor U0126 resulted in inhibition of chromosome separation, first polar body emission, and MII spindle formation (LEE et al., 2000). Meiotic divisions of oocytes are typically asymmetric, resulting in the formation of a large oocytes and small polar bodies. The size difference between daughter cells is a consequence of asymmetric positioning of the spindle before cytokinesis. Normally the spindle forms at the centre of the cell and migrates to the cortex just before polar body extrusion. In murine MOS-deficient oocytes or oocytes treated with U0126, the spindle forms centrally and elongates but does not migrate to the cortex, resulting in the formation of an abnormally large polar body (Verlhac et al., 2000; TONG et al., 2003). Recently two new MAPK substrates were identified from a mouse cDNA library. The first was named MISS (MAPK-interacting and spindle-stabilizing protein) and accumulates only in MII, localizing to the spindle. The second, DOC1R (murine homologue of a potential human tumor suppressor gene), localizes to the microtubules. Inhibition of these proteins leads to severe defects of the spindle and microtubules during MII (LEFEBVRE et al., 2002; TERRET et al., 2003).
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In most vertebrates, unfertilized oocytes are arrested at MII by a cytostatic factor (CSF). CSF is defined as the activity that inhibits the transition from MII to AII in mature oocytes (MASUI and MARKERT, 1971). It is not a specific molecule but consists of many molecules of which only some have been identified. In Xenopus oocytes CSF activity appears to be downstream of the MOS/MEK/MAPK/p90rsk transduction pathway. There is no direct evidence proving the existence of CSF activity in mammalian oocytes. Fertilization and the release of MII arrest induce MAPK inactivation in Xenopus and other nonmammalian species as well as in mammalian oocytes (LIU et al., 1998; LIU and YANG, 1999; SUGIURA et al., 2001; TATEMOTO and MUTO, 2001). MAPK inactivation occurs after MPF inactivation therefore its activity seems to be necessary for maintenance of MPF activity. Further it can be concluded that its activation is essential for the maintenance of MII arrest. A possible explanation for the CSF arrested state is that MAPK-stimulated processes prevent active APC (see p.22) from ubiquitinylating its substrates and /or MAPK- dependent protein synthesis overweighs ubiquitin-dependent degradation. Under physiological conditions the release from MII arrest is mediated by Ca 2+ /calmodulin- dependent kinase II (CaMKII) which triggers degradation of M-phase cyclins and sister chromatid segregation (WINSTON and MARO, 1995; JOHNSON et al., 1998; TATONE et al., 2002; FAN et al., 2003b).
3.3 P90rsk The family of 90 kDa ribosomal S6 kinases also known as RSK, p90rsk or MAPK- activated protein kinase-1, MAPKAP-K1 was among the first substrates of ERK identified by STURGILL et al. already in 1988. RSK was first discovered in 1985 by ERICKSON and MALLER in Xenopus laevis oocytes as an intracellular kinase activity that phosphorylated the S6 protein of the 40 S subunit of the ribosome. The phosphorylation of S6 protein is believed to promote the translation of selected mRNAs (JEFFRIES et al., 1997). Two ribosomal S6 protein kinases (S6KI and S6KII) with a molecular size of 90 kDa were identified by fractioning Xenopus cell extracts, leading to the cloning of two cDNAs that predicted highly related proteins (~90% identity). Homologues of S6KI and S6KII (renamed p90rsk or RSK) were
41 LITERATURE REVIEW
subsequently cloned from several species (mouse and chicken: ALCORTA et al., 1989; rat: GROVE et al., 1993; human: MOLLER et al., 1994). Another S6 kinase of 70 kDa (p70 S6K ) has been identified which shows substantial sequence homology to the N-terminal kinase of RSK (BANERJEE et al., 1990; KOZMA et al., 1990). The p70 S6K is thought to be the major physiological S6 kinase (BALLOU et al., 1991; CHUNG et al., 1992; VON MANTEUFFEL et al., 1997) but it has been shown that it is not phosphorylated and subsequently activated during oocytes maturation and therefore probably has no function in the process of meiotic reinitiation (mouse: GAVIN and SCHORDERET-SLATKINE, 1997; Xenopus: SCHWAB et al., 1999). The RSK family belongs to the serine-threonine kinases, but it is unique having a special structure. It consists of two functional kinase domains that are each similar to distinct protein kinases (JONES et al., 1988). These authors postulated the possibility that the molecule has two ATP-binding sites and two catalytic centres. By now it became clear that the N-terminal kinase (NTK) is responsible for substrate phosphorylation and the C-terminal kinase (CTK) is involved in the activation mechanism of RSK (FRÖDIN and GAMMELTOFT, 1999; NEBREDA and GAVIN, 1999). The RSK family of kinases includes three isoforms named RSK1, RSK2 and RSK3 that are encoded by distinct genes and show 75-80 % amino acid identity (JONES et al., 1988; ALCORTA et al., 1989; MOLLER et al., 1994; ZHAO et al., 1995). RSK is located downstream of the Raf-MEK-MAPK signalling cascade and evidence from Xenopus oocytes suggests that p90rsk mediates most of the MAPK function in regulating the meiotic cell cycle progression (BHATT and FERRELL, 1999; GROSS et al., 1999; GROSS et al., 2000).
3.3.1 P90rsk activation and inactivation The signalling cascade that leads to activation of p90rsk is depicted in figure 5. To understand the activation mechanism of this molecule it is essential to know its structure. The three RSK isoforms show the same overall structure with two kinase domains, a linker region and short N-terminal and C-terminal tails (see figure 7). The six known phosphorylation sites are conserved in all isoforms. The N-terminal kinase is responsible for phosphorylating the substrates of RSK whereas the C-terminal
42 LITERATURE REVIEW
kinase regulates the activity of the NTK. The linker region is involved in activation of NTK. The CTK contains a short docking motif which is responsible for the specific association of RSK and ERK (ZHAO et al., 1996; SMITH et al., 1999; FRÖDIN and GAMMELTOFT, 1999).
3
PDK1 ERK ERK NTK? 1
S221 2 T573 T359 S363 S380 P P P P P S732 Activ linker Activ P NTK Loop CTK Loop
P regulating kinase activity P unknown function
Figure 7: Phosphorylation sites and protein kinases implicated in activation of RSK (modified after FRÖDIN and GAMMELTOFT, 1999) Activation of RSK in vivo is associated with increased phosphorylation at six sites, four of which modulate kinase activity. Four protein kinases have been implicated in phosphorylating the six sites: PDK1, ERK, CTK and NTK of RSK. A sequential activation mechanism (1-3) has been proposed. PDK1=3-phosphoinositide-dependent protein kinase-1, NTK=N-terminal kinase, CTK=C-terminal kinase, S=serine, T=threonine, P=phosphorus
RSK is phosphorylated at four serine and two threonine residues. Four of these residues, Ser221, 363,380 and Thr573, have been assigned to regulation of kinase activity. The function of Ser732 and Thr359 phosphorylation remains unclear. It was also shown that RSK has autophosphorylation activity stimulated by ERK (STURGILL et al., 1988; GROVE et al., 1993). The observations of BJØRBÆK et al. (1995) and FISHER and BLENIS (1996) led to the following conclusions: 1) NTK is
43 LITERATURE REVIEW
responsible for substrate phosphorylation; 2) ERK phosphorylates and activates the CTK; 3) activated CTK autophosphorylates RSK on sites that are important for activation of the NTK; 4) activated NTK contributes to autophosphorylation of RSK. Detailed analysis of the RSK1 isoform resulted in a proposed sequential activation mechanism for RSK1: ERK activates the CTK by phosphorylating Thr573 and contributes to activation of the NTK by phosphorylating Ser363. The activated CTK phosphorylates Ser380, which, together with phosphorylation of Ser363, activates the NTK (DALBY et al., 1998). This step by step phosphorylation of the molecule is represented in immunoblot analysis as protein bands of different mobility because of their increasing molecular weight (mouse: KALAB et al., 1996; rat: TAN et al., 2001; rabbit: YU et al., 2002; pig: SUGIURA et al., 2002; FAN et al., 2003). The phosphorylation pattern is reflected by the increasing kinase activity during oocytes maturation (Fig. 8). Although phosphorylation by MAPK is essential, complete RSK activation requires phosphorylation of a serine in the NTK by PDK1 (JENSEN et al., 1999; RICHARDS et al., 1999). Thus, RSK phosphorylation and activation integrates regulatory signals from both the MAPK- and the PDK1-signaling pathways. Though full phosphorylation of p90rsk is dependent on MAPK activity, it is already partially phosphorylated in mammalian oocytes at the GV stage through a MAPK-independent mechanism. This partially phosphorylated RSK is not an active kinase, and may be the result of autophosphorylation (GROVE et al., 1993). It was shown that fully grown GV oocytes have acquired the ability to phosphorylate p90rsk, but a phosphatase- dependent inhibitory mechanism prevents the activation. The same is true for MAPK (INOUE et al., 1998; FAN et al., 2003). After its first appearance, concomitantly with or shortly after GVBD, the activity of p90rsk increases with progression of meiosis with a maximum at the MI stage and remains high in MII-arrested mammalian oocytes (mouse: KALAB et al., 1996; TONG et al., 2003; rat: TAN et al., 2001; LU et al., 2001; rabbit: YU et al., 2002; pig: SUGIURA et al., 2002; FAN et al., 2003). RSK becomes slowly dephosphorylated in activated eggs by a phosphatase that is activated shortly before pronuclear formation.
44 LITERATURE REVIEW
enzyme activity p90rsk
h of cultivation
GV GVBDMI AI TI MII
Figure 8: Change of p90rsk activity during maturation of mammalian oocytes.
In pig oocytes it was observed that p90rsk shows the same pattern of distribution as MAPK. During the GV stage the kinase was almost evenly distributed in the oocytes, concentrating in the GV just before GVBD. In oocytes undergoing GVBD RSK was detected in the area around the condensed chromatin. In pre-MI or MI stage p90rsk was seen around the dispersed or aligned chromosomes and localized to the mid zone of the elongated spindle during PB1 emission. In MII eggs, p90rsk was present at the entire spindle except the equatorial region, whereas in activated eggs at anaphase II, p90rsk translocated to the centre of the elongating spindle (FAN et al., 2003). YU et al. (2002) observed a similar pattern of p90rsk distribution during the maturation of rabbit oocytes.
3.3.2 Function and substrates of the p90rsk Functional roles for p90rsk in the regulation of meiotic reinitiation have been studied in the past decade. During maturation of Xenopus oocytes, MAPK and p90rsk activity is not essential for entry into meiosis I but is required during the onset of meiosis II to suppress entry into S phase, to regulate the APC so as to support cyclin B accumulation, and to support spindle formation. P90rsk, as a substrate of MAPK, is sufficient to mediate these effects during oocytes maturation (GROSS et al., 2000). Whereas this group found the MAPK pathway not necessary for the entry into meiosis I, PALMER et al. (1998) observed contrary effects. They described a substrate of p90rsk, the MPF-inhibitory kinase Myt1. Activated p90rsk inactivates Myt1 and thereby promotes activation of MPF and entry into meiosis I. But they also
45 LITERATURE REVIEW
stressed the possibility of other, alternative pathways being activated in parallel during these processes. In Xenopus oocytes, RSK was shown to be the sole mediator of CSF arrest. A constitutively active form caused CSF arrest in the absence of active MAPK (GROSS et al., 1999) and depletion of p90rsk from egg extracts removed CSF activity, which could be restored by reading p90rsk (BHATT and FERRELL, 1999). In vertebrates the CSF is postulated to maintain elevated levels of MPF activity and therefore may be responsible for meiotic arrest at MII (SAGATA et al., 1989; MASUI, 1991). From their inhibition experiments with the specific MEK inhibitor U0126 FAN et al. (2003a) concluded that p90rsk activity might be necessary for the maintenance of MII arrest in porcine oocytes. Furthermore MEK/MAPK activity is essential not only for the phosphorylation of p90rsk, but also to keep its active state. In contrast to these results DUMONT et al. (2005) found that p90rsk is not involved in CSF arrest in mouse oocytes. In their studies oocytes from RSK knockout mice presented a normal CSF arrest. Another substrate that was identified in Xenopus oocytes is the protein kinase Bub1 (budding uninhibited in benzimidazole). Bub1 is an upstream component of the kinetochore attachment or spindle assembly checkpoint. It is activated during meiosis in a MAPK-dependent manner. P90rsk is sufficient to activate Bub1 in vivo and in vitro. These results indicate that in vertebrate eggs, spindle assembly checkpoint proteins, including Bub1, are downstream of p90rsk and may be effectors of APC inhibition and CSF-dependent metaphase arrest by p90rsk (SCHWAB et al., 2001). Whereas the rapid decrease of MPF activity is involved in the initiation of egg activation, little is known about the role of delayed inactivation of MAPK/p90rsk. TATEMOTO and MUTO (2001) suggested that MAPK plays an essential role in inducing PB2 emission and the transition from meiosis to mitosis. This function might be mediated via active p90rsk, supported by the finding that p90rsk concentrates in the spindle mid zone at anaphase of meiosis II (FAN et al., 2003a). These authors also implicated p90rsk as the mediator of MAPK during meiosis I/meiosis II transition leading to chromosome separation, spindle elongation, and cleavage furrow formation in pig oocytes.
46 LITERATURE REVIEW
The activation and nuclear translocation of RSK is concomitant with immediate-early gene expression indicating that RSK may phosphorylate and activate transcription factors (CHEN et al., 1992, 1993). Several transcription factors have been identified as RSK substrates: CREB (cAMP-response element-binding protein), CREB-binding protein (CBP), c-Fos, estrogen receptor, and NF κB/IκBα. RSK may regulate protein synthesis by phosphorylation of polyribosomal proteins and glycogen synthase kinase-3 and it also phosphorylates the Ras GTP-GDP-exchange factor Sos leading to feedback inhibition of the Ras-ERK pathway (FRÖDIN and GAMMELTOFT, 1999). Via all these different substrates RSK is likely to regulate cell cycle progression in various cell types.
3.4 Interactions between MPF, MAP-Kinase, and p90rsk A lot of evidence about the connections and interactions of MAPK, MPF, and p90rsk, which are summarized in Fig. 9, comes from experimental treatments that specifically inhibit MAPK activation or inhibit its dephosphorylation. In Xenopus oocytes the progesterone stimulus results in entry of meiosis I accompanied by an accumulation of MOS. MOS accumulation above a certain level activates the MAPK cascade leading to MPF activation. MPF activation is promoted by the MAPK substrate p90rsk which inactivates the MPF-inhibiting kinase Myt1. The three kinases interact resulting in entry of meiosis I (NEBREDA and HUNT, 1986; POSADA et al., 1993; ROY et al., 1996; PALMER et al., 1998). When MAPK activation is blocked GVBD is inhibited significantly (KOSAKO et al., 1994; CROSS et al., 1998). In mouse oocytes the regulation of MPF and therefore entry into meiosis I is not mediated via MOS/MAPK/p90rsk pathway. Oocytes of MOS-knock-out mice displayed a normal pattern of MPF activation but showed severe defects in spindle and polar body formation (ARAKI et al., 1996; CHOI et al., 1996; VERLHAC et al., 1996). FAN and SUN, 2004 suggested that MAPK is not involved in the initiation of MPF activation during meiotic resumption of mammalian oocytes under natural conditions, but artificial activation of MAPK in premature oocytes may lead to MPF activation and
47 LITERATURE REVIEW
GVBD by an undefined mechanism. But although evidence from several experiments documented that an artificial increase of MAPK activity accelerates GVBD (mouse: CHESNEL and EPPIG, 1995b; bovine: FISSORE et al., 1996; pig: SUN et al., 2002; rat: TAN et al., 2001; JOSEFSBERG et al., 2003) MAPK is only dispensable for spontaneous GVBD. Studies from our own group using the MAPK inhibitor PD98059 showed that MPF and GVBD are significantly inhibited (SCHILLING, 1999). The control of B-type cyclin levels is a major and universal role of MAPK. Thereby it contributes to meiosis II arrest by inhibiting APC-mediated degradation of cyclins. Paradoxically, in all oocytes, MAPK inactivation following fertilization is essential to allow MPF reactivation and completion of the first mitotic cell cycle.
MAPK activation
MAPK Cyclin B Synthesis
MOS Cyclin B
Inactive Active MAPK cyclin B-Cdc2 cyclin B-Cdc2 Cyclin B Cyclin B Cdc2 Cdc2 Cdc2 P P Active APC APC
(Wee1) Myt1 p90rsk y B MAPK p90rsk C c (First mitosis) MAPK
MAPK Inhibitory Phosphorylations Cyclin B Degradation
activation inhibition
Figure 9: Interactions between MAPK, MPF and p90rsk in oocytes (modified after ABRIEU et al., 2001) HSIAO et al. (1994) analysing Xenopus oocytes and eggs found that phosphorylated and active MAPK is monomeric whereas only half of the nonphosphorylated and inactive MAPK is monomeric and half is a component of a 110 kDa complex. The
48 LITERATURE REVIEW
other part of the complex was identified as RSK. Much or all of the cell’s RSK was coimmunoprecipitated with MAPK. This heterodimer dissociated upon activation.
49 MATERIALS AND METHODS
C Materials and Methods
This subunit describes the different scopes of work namely cell culture, protein chemistry and animal experiment. All instruments, drugs, materials and chemicals that were used are listed in the appendix together with the composition of all buffers, solutions and media.
1 Cell culture techniques
1.1 General handling and precautions The preparation of the media was done sterilely under the clean bench. All used glassware was sterilized for four hours at 180°C in a hot-air sterilizer after a washing step in a laboratory dishwasher. Plastic materials were, as far as possible, autoclaved for 15 minutes at 121°C. All chemicals w ere stored according to the manufacturers’ instructions. The water was taken from an ultra-pure water system and therefore fulfilled the criteria of the American Society of Testing and Materials. Only water with an electrical resistance higher than 15 MegOhm x cm was used. After composition of the media they were filtrated through a Cellulose-Acetate-filter with a size of pores of 0.22 µm and were stored at 4°C until used.
1.2 Media As a transportation medium for the ovaries 0.9% sodium chloride (NaCl) solution was used. The sodium chloride solution was autoclaved at 121°C for 15 minutes. For the flushing medium this 0.9% sodium chloride solution was supplemented with 1% heat-inactivated fetal calf serum (FCS). The cumulus-oocyte-complexes (COCs) were collected into a phosphate-buffered solution containing 10 % FCS (PBS + 10 % FCS) further referred to as collection medium. As a basic medium for the cultivation Tissue Culture Medium (TCM) 199 with Earle’s salts was used. Depending on the experimental design the different supplements
50 MATERIALS AND METHODS
were added at day of cultivation followed by an equilibration for two hours in the incubator at 39°C, in an atmosphere of 5 % CO 2 and saturated humidity.
1.2.1 Supplements (1) Fetal calf serum The serum was warmed up for 30 minutes at 56°C in a water bath to inactivate complement factors and immunoglobulins. It was stored at -20°C until use.
(2) Hormones The gonadotropines employed were porcine Luteinizing Hormone (pLH) and porcine Follicle Stimulating Hormone (pFSH). Both hormones were purchased from National Hormone & Peptide Program, Harbor-UCLA Medical Center, Torrance, California. They were solubilized in 0.9% NaCl solution containing 0.5% bovine serum albumin (BSA) and stored at -20°C until use. The BSA was added as a protective protein in the solubilizing solution to retard absorption of pFSH to glass.
Characterization of hormones: Description Lot LH-contamination FSH-contamination pLH, BIO AFP12389A < 0.02% pFSH-I-1, IOD/BIO AFP10640B 1.52%
They were added to the medium in the following concentrations: 5 µg/ml pLH and 2.5 µg/ml pFSH.
51 MATERIALS AND METHODS
(3) Inhibitor U0126 was used as a specific inhibitor of MEK1 and MEK2 a MAP kinase kinase.
Molecular formula C18 H16 N6S2 Molecular weight 380.48 (anhyd.) g/mol CAS Registry No. 109511-58-2 Chemical name 1,4-Diamino-2,3-dicyano-1,4-bis-(o- aminophenylmercapto)butadiene Physical properties Off-white photosensitive powder Storage Store tightly sealed at 4°C Solubility Soluble in dimethylsulfoxide (DMSO) (>10mg/ml). Stock solutions in DMSO should be used within a month.
1.3 Preparation and selection of cumulus-oocyte-complexes The collection of oocytes covered a period of three years from May 2002 to May 2005. Collection and following culture of all different time points was done during all seasons and covered all different slaughter days therefore an influence of season or slaughter day can be ignored. The ovaries from slaughtered peripubertal gilts were collected within about twenty minutes after the killing of the animals and kept in warm (~25°C) transportation medium. In the laboratory th ey were flushed and stored in fresh, warm (37°C) transportation medium until furt her treatment. For isolation of the COCs only follicles were chosen which macroscopically showed no signs of atresia (KRUIP und DIELEMANN, 1982; MEINECKE et al., 1982). COCs were obtained by slitting individual antral follicles (3-5 mm) and flushing each follicle antrum with prewarmed (39°C) phosphate buffered salt solution ( PBS). They were assessed under a stereo microscope with 10 to 70 fold magnification. Only COCs with several (at least three) compact and homogenous layers of cumulus cells and evenly granulated ooplasm were chosen. These selected COCs were picked up with a Pasteur pipette and stored intermediately in the collection medium in cell culture dishes on a heated table at 37°C. Before cultivatio n was started the cells were washed twice in the collection medium and once in the equilibrated culture medium. Preparing COCs which were to be cultivated with an inhibitor the collection medium
52 MATERIALS AND METHODS
was supplemented with this inhibitor in the same concentration as in the culture medium.
1.4 Cultivation of cumulus-oocyte-complexes The COCs were cultured in groups of ~20 for different time periods from 18 h up to 48 h in TCM containing 20 % (v/v) FCS, 0.02 mg/ml insulin, 0.08 mg/ml L-glutamine, 0.05 mg/ml gentamycin, 5 µg/ml LH and 2.5 µg/ml FSH. Culture was carried out in 1 ml medium in 4-well multidishes at 39°C, in an atmosphere of 5% CO 2 and saturated humidity.
1.4.1 Regular cultivation conditions To determine the existence, the phosphorylation and activation pattern of the p90rsk and MAPK molecule in vitro matured porcine oocytes COCs were incubated in the cultivation medium in different time intervals from 0 up to 48 h.
1.4.2 Cultivation conditions with addition of an inhibitor To further describe the suspected interdependence of p90rsk and MAPK the MEK- specific inhibitor U0126 was used. The inhibitor was dissolved in dimethylsulfoxide (DMSO) and added to the cultivation medium in a concentration of 10 µmol. To exclude an effect of the solvent on the maturation process, the solvent was added to the maturation medium instead of the inhibitor. A previous dissertation from our group (SCHILLING, 1999) showed that a concentration of DMSO of up to 0.5 % in the maturation medium has no effect on the maturation. In the present study a concentration of 0.2 % of DMSO in the maturation medium was used. A stock solution containing 5 mM U0126 was prepared (1 mg dissolved in 520 µl DMSO). From this stock solution 2 µl were added to 998 µl maturation medium or collection medium to gain a final concentration of 10 µM of U0126. COCs were cultivated in the inhibitor supplemented medium from 18 up to 46 h.
53 MATERIALS AND METHODS
1.5 Cytogenetic assessment of oocytes
1.5.1 Fixation and staining of oocytes The examination and description of the nuclear status was done modified after HANCOCK (1958). The oocytes were denuded by treatment with 0.25 % hyaluronidase for 5 min. at room temperature (RT) to digest the hyaluronic acid of the extracellular matrix followed by repeated pipetting with a fine drawn Pasteur pipette to mechanically remove the remaining cumulus cells. Afterwards the oocytes were incubated in approximately 50 µl of hypotonic 0.7 % potassium chloride solution (KCl) for five minutes. The oocytes were then mounted on a fat free slide in a ~50 µl drop of 0.7 % KCl and covered with a cover glass. A mixture of paraffin and vaseline served as a spacer between the slide and the cover glass. The oocytes were fixated by storage for at least 24 hours in a freshly prepared solution containing 25 % acetic acid and 75 % ethanol. Before staining with a 2 % orcein solution the oocytes were washed 30 min. in pure ethanol. The excess stain was removed with 25 % acetic acid.
1.5.2 Assessment of the nuclear status of oocytes The assessment of the nuclear status was done under a phase contrast microscope with 200-400 fold magnification according to the description of MOTLIK and FULKA (1976). Classification: GV I: Nuclear membrane and nucleolus are clearly visible and chromatin forms a ring or horseshoe around the nucleolus. GV II: Nucleus and nucleolus are intact. A few orceinpositive structures (chromocenters, chromatin clumps) on the nuclear membrane can be detected. GV III: Nucleus and nucleolus are intact with slightly stained chromatin clumps or strands, localized especially around the nucleolus.
54 MATERIALS AND METHODS
GV IV: The nuclear membrane is less distinct and the nucleolus disappears completely. Chromatin is seen as an irregular network or as individual bivalents. Early diakinesis: The nuclear membrane disappears; the bivalents lie in the region of the former nucleus. Late diakinesis: The chromosomes are condensed. Metaphase I: The nucleus is no longer existent. The chromosomes are maximally condensed and form clusters. Anaphase I: The chromosomes align in the equatorial plate. A spindle can be visualized. Telophase: The two groups of chromosomes drift apart. The meiotic spindle is clearly visible. Metaphase II: The chromosomes are divided into two distinct clusters. The membrane of the first polar body can be partly visualized.
2 Protein chemical techniques
2.1 Preparation and storage of oocytes and cumulus cells At the end of culture groups of 40 COCs per time were freed of attached cumulus cells by treatment with 0.25 % hyaluronidase and repeated pipetting. The denuded oocytes were then washed three times in protein free PBS and were frozen in groups of twenty to forty COCs in ~4µl of this protein free PBS until further processing. They were collected in 0.5 ml Eppendorf tubes and frozen at -80°C. The cumulus cells of 10 and 20 oocytes respectively were separated after the cultivation period from the oocytes by denudation. Therefore the COCs were incubated with 0.25 % hyaluronidase followed by repeated pipetting with a fine Pasteur pipette. The separated cumulus cells were collected in protein free PBS in a 0.5 ml Eppendorf tube, centrifuged 3 times with washing steps in protein free PBS in between. They were stored in ~4 µl protein free PBS and frozen at -80°C until analysis.
55 MATERIALS AND METHODS
2.2 One-dimensional electrophoresis In the first electrophoresis three different buffers for processing the oocytes were tested. Extraction buffer plus 2.5 x Laemmli buffer (reducing) (LAEMMLI, 1970), 2.5 x Laemmli buffer (reducing), and 2.5 x Laemmli buffer without β-mercaptoethanol (non reducing) was tested. Preparing the samples for electrophoresis 10-20 µl of modified 2.5 x Laemmli buffer (reducing and non-reducing without mercaptoethanol) was added to 20 (40) denuded oocytes or cumulus cells from 10 (20) COCs to extract the proteins. The samples were boiled for 5 minutes at 95°C, put on ice immed iately afterwards, centrifuged for at least one minute at 13000 rpm, put back on ice until they were filled into the gel pockets. The proteins were separated by discontinuous SDS-PAGE (sodium dodecyl sulphate-polyacrylamide gel electrophoresis) (LAEMMLI, 1970). The principle of this method is that the solubilized proteins bind SDS molecules and therefore all become negatively charged. Afterwards the separation of the proteins in the polyacrylamide matrix is possible according to their relative mass in an electrical field independent of the molecule-specific electrical charge. The gels consist of a stacking gel to concentrate the individual proteins and a separating gel for the actual separation. For the separation of the proteins 0.75 mm thick and 8.3 x 7 cm large SDS- polyacrylamide gels, with a 5 % stacking and a 9 % separation gel (‘shifting’ gel), were used. For the first experiments (existence of p90rsk in oocytes) a simple gel composition, not the ‘shifting’ gel was used (see Appendix 3, Media, buffers, and solutions). Gel pockets were formed by placing a thin plastic comb into the stacking gel. Electrophoresis was run in an electrophoresis chamber filled with electrode buffer. For the start 100 V for approx. 10 min. were applied in the stacking gel and 200 V for approx. 1 h in the separation gel. A biotinylated HMW (High Molecular Weight protein standard) or prestained protein mass standard was always run with the samples. The HMW was boiled with the samples before electrophoresis. Electrophoresis was stopped when the dye-front reached the end of the separation gel.
56 MATERIALS AND METHODS
2.3 Immunoblot (Western Blot) The reason for transferring proteins to membranes from gels is so as to be able to get at them more efficiently with various probes, as polyacrylamide is not particularly amenable to the diffusion of large molecules. The method was adapted from the description of TOWBIN et al. (1979). We used a ‘semi-dry’ or ‘horizontal’ blotting system. With this technique, one uses two plate electrodes (stainless steel or graphite / carbon) for uniform electrical field over a short distance, and sandwiches between of gel / membrane / filter paper assemblies, all well soaked in blotting buffer. The assembly is clamped between the two plate electrodes, and electrophoretic transfer proceeded in this position, using as blotting buffer only the liquid contained in the gel and filter papers in the assembly. Freshly-electrophoresed SDS-polyacrylamide gels are dipped into blotting buffer, and then laid flat on blotting-buffer wetted nitrocellulose paper supported on three layers of blotting buffer-wetted filter paper resting on the anode (positive electrode). The gel is overlaid with three wetted filter papers, and then the cathode (negative electrode). Care should be taken to exclude bubbles between gel and nitrocellulose, and between nitrocellulose and paper. The proteins were then transferred electrophoretically from the gel onto the nitrocellulose membrane for 2 h with a constant current of 1 mA/cm 2 at room RT. To prove the successful transfer the nitrocellulose membranes were stained with Ponceau Red for 5 min. and a destaining with Aqua bidest. This is a reversible stain which does not interfere with any following probe.
2.4 Chemiluminescence The most popular type of probe of immobilised proteins is an antibody. The attachment of specific antibodies to specific immobilised antigens can be visualised by indirect enzyme immunoassay techniques, usually using a chromogenic substrate which produces an insoluble product. Lately chemiluminescent substrates have begun to be used because of their greater detection sensitivity. Probes for the detection of antibody binding can be conjugated anti-immunoglobulins (e.g.: goat-
57 MATERIALS AND METHODS
anti-rabbit / human); conjugated staphylococcal Protein A (which binds IgG of various species of animal); or probes to biotinylated / digoxigeninylated primary antibodies (e.g.: conjugated avidin / streptavidin / antibody). The specific antibodies we used were a polyclonal rabbit anti-human for the RSK (Rsk-1 (C-21): sc-231 ; Santa Cruz Biotechnology) and a polyclonal rabbit anti-rat for the MAPK (ERK 1 (K-23): sc-94 ; Santa Cruz Biotechnology). Both of these primary antibodies detect the non- phosphorylated as well as the phosphorylated form of the specific molecule. The second antibody for detection of the specific antibody binding was a horse radish- peroxidase-conjugated goat anti-rabbit antibody (Goat Anti-Rabbit HRP-Conjugated, PIERCE). For visualization of the biotinylated standard horse radish-peroxidase- conjugated streptavidin was used. The transfer membrane was blocked with blocking solution (TBS/Tween 0.1 %, 5 % teleostean gelatine) to prevent further non-specific binding of proteins for at least two hours at RT. This is followed by washing of the membrane 3 times for 10 min. in washing solution (TBS, 1 % Tween) and incubation of the membrane in washing solution containing diluted antibody (1:200) over night at RT. Afterwards the membrane is washed again 3 times for 10 min. in washing solution before incubation in diluted conjugated probe antibody (1:500 up to 1:2000) for 1 hour at RT simultaneously with the streptavidin for visualization of the standard (dilution 1:200000 up to 1:400000), followed by 5 washing steps for 10 min., and final incubation with an enhancing chemiluminescence reagent for 5 min. The in this way prepared membrane is put into an X-ray cassette and an X-ray film (Kodak X-OMAT AR 18 x 24 cm) is exposed for various length of time (10 sec. up to 1 hour). The X- ray film was incubated in the developer solution for 1 min. followed by a short washing step in water and fixation in fixer solution for at least 1 min. Finally the film was watered for 5 min. before drying protected from light. The advantage of the technique lies in the simultaneous detection of a specific protein by means of its antigenicity, and its molecular mass: proteins are first separated by mass in the SDS-PAGE, and then specifically detected in the immunoassay step.
58 MATERIALS AND METHODS
Another advantage is that the membranes can be reused after a stripping step. The stripping removes all bound antibodies and the membrane can be used again for a chemiluminescence with different antibodies. Therefore the membranes were washed twice in washing solution and subsequently incubated in stripping solution for two hours at RT. Afterwards they are washed again three times in washing solution and were directly submitted to another chemiluminescence or stored at -20°C until further analysis. The successful stripping was controlled via washing, blocking, and incubation of the membrane solely with the peroxidase-conjugated probe antibody and a subsequent chemiluminescence.
2.5 P90rsk kinase assay
2.5.1 Kinase assay kit The assay kit is designed to measure the phosphotransferase activity of S6 kinase in immunoprecipitates and column fractions. Crude cell lysates may also be used but detergents/biochemicals contained in the cell lysis buffer may inhibit S6 kinase activity. Furthermore, although three inhibitors are included with the kit, editors may suggest other unknown kinases found in crude lysates are responsible for phosphorylation of the substrate peptide. The assay kit is based on phosphorylation of a specific substrate (AKRRRLSSLRA; modelled after the major phosphorylation sites in S6 kinase) using the transfer of the γ-phosphate of adenosine-5’-[ 32 P] triphosphate ([ γ-32 P] ATP) by S6 kinase. The phosphorylated substrate is then separated from the residual [ γ-32 P] ATP using P81 phosphocellulose paper and quantified by using a scintillation counter. The assay is linear for incubation times of up to 30 minutes and incorporation of up to 20% of total ATP. Further incubation or incorporation may not be linear and may therefore not be a true indication of S6 kinase activity in the sample extract. The enzyme assay is rapid, convenient and fairly specific for S6 kinase.
59 MATERIALS AND METHODS
Preparation of samples: 30 µl of PBS/PVA 2 % were added to the thawed samples. The oocytes of each sample were pipetted into a 50 µl drop of protein free PBS/PVA 2 %, followed by an incubation for 10 sec. in a 50 µl drop of 0.5 % Pronase to partly digest the zona pellucida. Afterwards the oocytes were washed at least three times in PBS/PVA 2 %. 10 µl of lysis-buffer were added to the samples. They were vortexed and subsequently centrifuged. In the following light microscopic control no intact oocytes should be visible.
Analysis of samples with S6 KINASE ASSAY KIT (Upstate) The S6 Kinase Assay was performed with the kit according to the manufacturers’ instructions. All steps were performed on ice to receive linear results. Suitable blanks should always be performed to correct for non-specific binding of [ γ-32 P] ATP and its breakdown products to the phosphocellulose paper. Controls for endogenous phosphorylation of proteins in the sample extract were performed by substituting ADB for substrate cocktail. 10 µl of ADB were added to an eppendorf tube followed by 10 µl of substrate cocktail (final concentration 50 µM), 10 µl of inhibitor cocktail, 10 µl of S6 Kinase (samples) and 10 µl of the diluted [ γ-32 P] ATP mixture (90 µl of the magnesium/ATP cocktail were added to 100 µCi [ γ-32 P] ATP. Therefore the final concentration of [ γ-32 P] ATP added to each sample was 10 µCi.) This mixture was incubated for 30 minutes at 30°C with agitation. 25 µl of the aliquot were then transferred onto the centre of a numbered P81 paper square. The radiolabeled substrate was allowed to bind to the filter paper for 30 sec. before immersing the paper into a 50 ml conical tube containing ~40 ml 0.75 % phosphoric acid. The squares were shaken gently for 5 min. This washing step was repeated 5 to 6 times followed by a final washing step in 20 ml acetone for 5 min. The assay squares were afterwards transferred to a scintillation vial and read in a scintillation counter.
60 MATERIALS AND METHODS
2.5.2 ‘In-Gel’ renatured kinase assay The protocol was adapted from SAMPT et al. (2001) and RUBINFELD and SEGER (2004). For the ‘In gel’ renatured kinase assay the samples that were frozen at -80°C were thawed on ice. The oocytes of each sample were pipetted into a 50 µl drop of protein free PBS/PVA 2 %, followed by an incubation for 10 sec. in a 50 µl drop of 0.5 % Pronase to partly digest the zona pellucida. Afterwards the oocytes were washed at least three times in PBS/PVA 2 %. To each sample 5 µl of extraction buffer were added and 3 times frozen and thawed at -80°C. 4-5 t imes concentrated Laemmli buffer was added. The gel for electrophoresis was prepared as usual, with an addition of 25-75 µg/ml of the substrate (RSK peptide) to the separation gel solution. The stacking gel was prepared without substrate. The samples and a prestained standard were loaded. To avoid heating 100 V were not exceeded during the whole run of the electrophoresis. After electrophoresis gels were freed of SDS by washing them 3 times with 100 ml 20 % isopropanol in Tris-HCl, pH 8, at RT for 1 h followed by two washing steps in renaturation buffer 30 min. each at RT. Gels were then washed in renaturation buffer containing 6 M guanidine-HCl at RT for 1 h with two changes of the buffer. The guanidine-HCl concentration was reduced in 3 steps by adding 50 ml of renaturation buffer containing 0.05 % Tween 20 to 50 ml 6 M guanidine-HCl Gels were washed at 4°C for 15 min. each followed by was hing steps three times 15 min. each with 100 ml of renaturation buffer supplemented with 0.05 % Tween 20. Gels were shook overnight in the cold room. Washing buffer was removed and the gels were incubated in 30 ml of in-gel kinase buffer at 30°C for 30 min. In gel kinase buffer was removed and 20 ml of in-gel kinase buffer containing 2 mM DTT, and 100 µCi of [ γ32 P]-ATP was added and incubate at 30°C for 2 h. The gels were washed extensively 4-5 times 15 min. each at RT with 5 % trichloroacetic acid (TCA) + 1 % sodium pyrophosphate (NaPPi). They were
61 MATERIALS AND METHODS
subsequently dried between two cellophane sheets clamped between gel drying frames over night at RT.
2.6 Autoradiography The dried gels were exposed to an X-ray film (Kodak X-OMAT AR 18 x 24 cm) at -80°C for several length of time (16 to 48 h). Afte r exposure at -80°C to suppress a diffusion of the bands the x-ray film was incubated in the developer solution for 1 min. followed by a short washing step in water and fixation in fixer solution for at least 1 min. Finally the film was watered for 5 min. before drying protected from light.
2.7 Quantitative measurement of radioactivity The incorporated radioactivity was measured with a liquid scintillation counter. Each phosphocellulose square was put in a scintillation vial to measure the emitted Cerenkov-radiation (ELRICK and PARKER, 1966). Each sample was measured twice for 10 min. The results were given in count per minute (cpm).
3 Animal experiment
The aim of the animal experiment was the collection of in vivo matured porcine oocytes to check if the in vitro obtained results concerning the phosphorylation pattern of the p90rsk molecule also apply for the in vivo situation. Therefore gilts were castrated after a preparing stimulation of the animals with eCG and hCG.
3.1 Animals Healthy, peripubertal hybrid gilts (Hennies, Wätzum) aged 7 to 9 months were introduced to the swine stall of the department of reproductive medicine, School of Veterinary Medicine Hannover foundation. The animals weighed between 90 and 120 kg. They were kept in groups of two to four animals without direct contact to the boars.
62 MATERIALS AND METHODS
3.2 Experimental design The animals were injected with 600-800 I.U. eCG (Pregmagon ®, IDT) at the beginning of the transport from the farmer to the department of reproductive medicine. ECG was injected to stimulate follicle development and to avoid transport heat. After 72 hours the gilts were tested for signs of heat. If they showed no signs of heat they were injected with 500 I.U. hCG (Ovogest ®, Intervet) to mimic the LH-surge and therefore induce oocytes maturation (RATKY et al., 2003a, b). The animals were castrated at selected time points according to the in vitro maturation intervals. The time points chosen were 0 h (at the regular time of hCG injection), 22 h, and 30 h after hCG injection corresponding to 0, 22, and 30 h of in vitro maturation. 20 oocytes for one sample per time point were necessary for a characterization of the protein via electrophoresis and immunoblot. With three repetitions per time point to prove reproducibility of the results a total of 60 oocytes were necessary for each time point. The anticipated quantity of oocytes per gilt was approx. 10. Therefore at least 6 animals were planned for each time point. Table 3 shows the actual number of necessary animals and the average number of oocytes retrieved per gilt.
Table 3: Actual numbers of necessary animals and average of oocytes retrieved per animal at the three different time points. hours 0 22 30 n of animals 9 9 7 ∅ oocytes/animal 7.1 7.6 9
A total number of 28 animals were operated as the outcome could not be exactly estimated and a surplus of 20 and 15 oocytes was retrieved for 22 and 30 h respectively.
63 MATERIALS AND METHODS
3.3 Surgical techniques In preparation for surgery feed was withheld for at least 12 hours, access to water ad libitum. As a preanaesthetic medication atropine (atropine 1 % 0.02-0.08 mg/kg b.m. i.m.) was given 30 min. before start of narcosis. The injectable anaesthetics Ketamin (Ursotamin ® 10 % (10-)15 mg/kg b.m. i.m.) and Stresnil ® (4 % 0.2 mg/kg b.m. i.m.) were used in combination with an epidural anaesthesia with a 2 % local anaesthetic (Minocain ®; 0.7 ml per 10 cm if injected epidurally, subdurally 0.5 ml per 10 cm). If necessary repeated dosing of Ketamin ® was performed intravenously into the ear vein. The animals were placed on the operating table with a pulley and fixed in the Trendelenburg position i.e. laying on their back the head about 30 cm higher than the hindquarter. The operation field was washed with water and soap, shaved if necessary, before being wiped twice with 20 % alcohol and 30 % iodine solution (Braunol ®). After a skin incision of about 15 cm preparation towards the linea alba was performed bluntly. The linea alba was cut through and the peritoneum was opened. The ovaries were pulled out of the abdomen and the ligature was prepared. After cutting off the ovaries the ligature was pulled tight. This was done to keep up the blood supply as long as possible to avoid hypoxia-caused changes of the ovaries. The ovaries were transported in prewarmed (37°C) tr ansportation medium directly to the laboratory. After an instillation of approx. 300 ml 0.9 % NaCl to reduce adhesions the wound closure was performed with a continuous suture (Kürschner). For the peritoneum a 4 metric Dexon thread was used, for the muscle layers a 5 metric Dexon thread and for the skin again the 4 metric Dexon thread. The wound was sprayed with CTC Blauspray ® containing a local antibiotic to minimize the risk of a wound infection. Additionally the animals were injected with a systemic antibiotic (Hostamox ® LA, 0.1 ml/kg b.m. i.m.). After the waiting time for the individual drugs was over the animals were slaughtered.
64 MATERIALS AND METHODS
3.4 Preparation of oocytes and cumulus cells Immediately after the ovaries arrived in the laboratory the oocytes and cumulus cells were prepared. The ovaries were placed in a glass dish filled with flushing medium. The individual follicles were opened with fine forceps under microscopic control and the oocytes normally well up out of the follicle. The oocytes were then aspirated with a Pasteur pipette and transferred into a cell culture dish containing collection medium. The cumulus cells were removed in a ~100 µl drop of collection medium by an addition of 0.25 % hyaluronidase. Oocytes and cumulus cells were washed three times in protein free PBS and collected into an eppendorf tube. They were frozen at -80°C until further analysis via electrophoresis an d immunoblot. At least two of the oocytes retrieved from one animal were examined for their nuclear status. Therefore they were treated as described under 1.5 ‘Cytogenetic assessment’.
3.5 Analysis of in vivo matured oocytes and cumulus cells
The in vivo matured oocytes and cumulus cells were analysed via electrophoresis, immunoblot, and chemiluminescence analogous to the in vitro matured oocytes and cumulus cells (see C 2.2 – 2.4).
65 RESULTS
D Results
1 Control of maturation
To control the in vitro maturation process a total of 716 oocytes was analysed according to the procedure described in ‘Materials and Methods’, chapter 1.5. Six time points (0, 22, 26, 30, 34, 46 h) throughout the maturation process were chosen and at least 80 oocytes per time point were fixed, stained, and their nuclear status was assessed. This was done to continuously check the maturation conditions and as well to have a reference for the samples that were submitted to further probes (electrophoresis, Immunoblot, kinase assay).
n=117 n=93 n=228 n=81 n=94 n=93 100%
80% MII 60% TI AI 40% D/MI
Nuclear status Nuclear GV 20%
0% 0 22 26 30 34 46 IVM (h)
Graph 1: Percentages of nuclear status in relation to the different time points during in vitro maturation; GV=Germinal Vesicle, D=Diakinesis, MI=Metaphase I, AI=Anaphase I, TI=Telophase I, MII=Metaphase II
Graph 1 shows the percentages of nuclear statuses in relation to the different time points analysed during in vitro maturation. For reasons of simplification GV stages I
66 RESULTS
to IV are summarized as GV. The concrete numbers of oocytes and their distribution to the different meiotic stages are listed in a separate table in the appendix. 46 h of culture was chosen as the end point of the maturation period because the M II stage is reached in almost all oocytes (90.3 %; see table 4). The frequency distribution to the various time points is significantly different between all of them on the basis of comparison of pairs (p < 0.05). At the start of maturation culture (0 h) 96.7 % of the oocytes are in the GV stage, meiotic resumption has not yet taken place. After 22 h of maturation 35.1 % of the oocytes resumed meiosis, GVBD therefore has taken place and they have proceeded to the diakinesis or metaphase I stage. The percentages of oocytes in the metaphase I to the metaphase II stage increase during the maturation period (26 h MI 64.5 %, MII 1.3 %; 30 h MI 80.2 %, MII 2.5 %; 34 h MI 62.8 %, MII 25.5 %) and reaches 90.3 % of MII stages after 46 h of maturation.
2 P90rsk in oocytes and cumulus cells
In a first step we demonstrated the existence of the p90rsk in porcine oocytes. This procedure also evaluated the used antibody (Rsk-1) and confirmed that the antibody works in porcine oocytes (Fig. 10). Afterwards this antibody was used to detect the p90rsk in porcine cumulus cells to demonstrate the existence of the kinase also in somatic cells (Fig. 11). For processing the oocytes different buffers were tested as indicated below the figures. As the non-reducing Laemmli buffer (without mercaptoethanol) gave the clearest band at all different time points, this buffer was used for all following experiments, also for the cumulus cells. In these first experiments the simple gel composition in contrast to the ‘shifting’ gel composition was used for electrophoresis. Samples of 40 oocytes from 0, 24, and 48 h of in vitro maturation were employed. Figure 10 exemplarily shows the resulting picture of the oocytes from the start of maturation (0 h). 24 and 48 h of maturation gave similar images.
67 RESULTS
Figure 10: Oocytes cultured for 0 h processed in different buffers Lane 1: 40 oocytes + extraction buffer + Laemmli buffer Lane 2: 40 oocytes + Laemmli buffer Lane 3: 40 oocytes + Laemmli buffer without Mercaptoethanol
Samples of cumulus cells of five COCs cultured for 0, 24, and 48 h were processed with the selected non-reducing Laemmli buffer. Their analysis via electrophoresis and immunoblot demonstrated the existence of the p90rsk also in the porcine cumulus cells and confirmed the possible use of the antibody.
Figure 11: Cumulus cells of 5 COCs at a time cultured for 0, 24 and 48 h Lane 1: 0 h, Lane 2: 24 h, Lane 3: 48 h
3 MAPK in oocytes
As well as for the p90rsk the existence of the MAPK was demonstrated in porcine oocytes. This procedure also evaluated the used antibody (ERK1) and confirmed that the antibody works in porcine oocytes (Fig.12). Samples of 30 oocytes from 0, 26, and 46 h of in vitro maturation were used, processed with the non-reducing Laemmli buffer and were subjected to electrophoresis with a ‘shifting’ gel followed by immunoblot. Figure 12 demonstrates the existence of the MAPK in porcine oocytes and shows that the used antibody works in these cells. The phosphorylation steps of
68 RESULTS
the MAPK that were examined more detailed later on can already be seen in this picture. Another project in our group concentrated on further analysis of the MAPK in cumulus cells.
Figure 12: MAPK in oocytes, 30 oocytes per lane, Antibody: ERK-1
4 Phosphorylation pattern of p90rsk and MAPK during in vitro maturation
Electrophoresis, immunoblot and subsequent chemiluminescence analysis revealed a three step phosphorylation pattern in the oocytes. These three steps are visible as three bands of p90rsk, which showed different mobility in electrophoresis (Fig. 13). Non phosphorylated molecules are smaller, therefore show a high mobility and are visible as the lowest bands. With increasing phosphorylation the molecule becomes bigger/increases in size and shifts upwards, meaning it does not diffuse into the gel as far as and therefore remains above the smaller, non phosphorylated form. In oocytes immediately following recovery from the follicles (0 h) until ~18 h of culture, p90rsk is observed as double bands. The band with the highest mobility corresponds to the non phosphorylated form of the kinase. Proceeding with maturation these two high mobility bands disappear whereas a lower mobility band appears. The lowest mobility band is seen here from 30 h on until the end of the culture period. This lowest mobility band represents the most strongly phosphorylated form of the kinase
69 RESULTS
Figure 13: Detailed phosphorylation pattern of p90rsk during in vitro maturation of porcine oocytes
For further analysis and comparison of the p90rsk and the MAPK phosphorylation pattern only some selected time points of maturation were chosen. The phosphorylation pattern and the observed shift of the p90rsk can also be seen in this overview (Fig. 14). At 22 h the two bands with the highest mobility, non phosphorylated form and first phosphorylation step, are seen. Until 34 h of culture the phosphorylation increases and the lowest mobility band is visible until 46 h.
Figure 14: Overview of p90rsk phosphorylation pattern; Antibody Rsk-1
Corresponding to the increasing phosphorylation of the p90rsk the MAPK phosphorylation increases during in vitro maturation. The phosphorylation patterns of
70 RESULTS
both kinases show a time coincidence. At 22 h of in vitro maturation the two non phosphorylated isoforms, ERK 1 and 2, are visible. After 26 h a first faint band of the phosphorylated form of the kinase becomes visible. A mixture of the phosphorylated and non phosphorylated form is visible at 30 and 34 h of maturation with the phosphorylated form becoming more and more prominent. At 46 h of in vitro maturation the phosphorylated form of both isoforms is the most prominent one and the non phosphorylated form is only weakly visible (Fig. 15).
Figure 15: Overview of MAPK phosphorylation pattern; same membrane as figure 14; Antibody ERK
5 Inhibition experiments
The nuclear status of the oocytes during this maturation process with an addition of the inhibitor was controlled at the same time points (0, 22, 26, 30, 34, 46 h) as the in vitro maturation without the addition of an inhibitor (D 1 Control of maturation). Again at least 80 oocytes were fixed, stained, and their nuclear status was assessed. Graph 2 shows the percentages of nuclear statuses in relation to the different time points analysed during in vitro maturation with an addition of the inhibitor to the maturation medium. For reasons of simplification GV stages I to IV are summarized as GV. The concrete numbers of oocytes and their distribution to the different meiotic stages are listed in a separate table in the appendix. At all selected time points of maturation until 46 h of maturation with the inhibitor over 90 % of the oocytes remain in the Germinal Vesicle (GV) stage whereas over 90 % of the oocytes cultured without an addition of the inhibitor have reached the M II stage after 46 h of IVM. Statistical analysis of the data showed that there is a significant
71 RESULTS
(p<0.05) difference in frequency distribution between 0 and 30, 26 and 30, and 30 and 46 h of IVM with the inhibitor. At all these time points over 90 % of the oocytes remain in the GV stage, but at 30 h of IVM a higher percentage of oocytes in the MII stage was found (30 h=6.7 %) whereas all the other time point showed no or only very low percentages of MII stage oocytes. A comparison of the results of the oocytes matured without the addition of the inhibitor and those with an addition of the inhibitor shows that all time points of the two groups are significantly different (p<0.05) except for the time point 0 h which shows no significant difference to all time points during IVM with an addition of the inhibitor. Therefore inhibition of GVBD is highly significant in COCs cultured in medium containing 10 µM U0126.
n=103 n=84 n=92 n=89 n=84 n=80 100%
80% MII 60% TI AI 40% D/MI
Nuclear status Nuclear GV 20%
0% 0 22 26 30 34 46 IVM (h)
Graph 2: Percentages of nuclear status in relation to the different time points during in vitro maturation with an addition of the MEK-inhibitor U0126 to the maturation medium; GV=Germinal Vesicle, D=Diakinesis, MI=Metaphase I, AI=Anaphase I, TI=Telophase I, MII=Metaphase II
Figure 16 shows the observed phosphorylation pattern of p90rsk during in vitro maturation carried out with an addition of the MEK-inhibitor U0126 to the culture
72 RESULTS
medium. In the pictures of the immunoblots one can clearly see the two high mobility bands that were already seen during culture conditions without the inhibitor from 18 until 22 h. Proceeding with the culture no lower mobility bands become visible, only the highest mobility band, corresponding to the non phosphorylated form of p90rsk, becomes more and more prominent. One can conclude that corresponding to the inhibition of GVBD also p90rsk phosphorylation is inhibited in these COCs.
Figure 16: Inhibition of phosphorylation of the p90rsk during in vitro maturation with U0126. 20 oocytes per lane; Antibody: RSK-1
Analysing the MAPK in the oocytes during maturation with medium containing U0126 was part of the other project in our group. It was shown that phosphorylation of MAPK inhibited completely. Only the non phosphorylated bands of the two isoforms were visible throughout the maturation process.
To exclude an effect of DMSO, the solvent of the inhibitor, on the in vitro maturation process, maturation controls were carried out with an addition of DMSO without inhibitor. These maturation controls were not significantly different from the regular maturation controls (Graph 3). Therefore an influence of DMSO on the maturation can be ignored.
73 RESULTS
n=80 n=93 n=115 100%
80% MII 60% TI AI 40% D/MI
Nuclear status Nuclear GV 20%
0% 46 + U0126 46 - U0126 46 + DMSO IVM (h)
Graph 3: Comparison of IVM for 46 h with an addition of the inhibitor U0126, without any addition, and with an addition of DMSO
6 Kinase Assay
6.1 Kinase assay kit To determine the enzyme activity of the p90rsk samples of 20 oocytes from the six time points that were also analysed before via electrophoresis and immunoblot (0, 22, 26, 30, 34, and 46 h IVM) were submitted to the procedure of the kinase assay kit from the company Upstate Biotechnology. This probe was performed as described in ‘Materials and Methods’, chapter 2.5.1 and repeated three times. The highest activity was measured at 0 h, before the start of maturation. A decrease in the activity was found at all other time points (22, 26, 30, 34, 46 h) during the following maturation process (Graph 4). Statistical analysis of the data revealed a significant difference between the activities at the start of maturation (0 h) and all other time points during the maturation except the time point 26 h.
74 RESULTS
1200
1000
800
600 cpm/20 cpm/20 oocytes 400
200
0 0 22 26 30 34 46 IVM (h)
Graph 4: Activity of p90rsk in porcine oocytes during the in vitro maturation period
In a second step samples of 20 oocytes each matured with an addition of the inhibitor U0126 to the culture medium were analysed. The same time points (0, 22, 26, 30, 34, and 46 h IVM + U0126) that were already analysed via electrophoresis and immunoblot were submitted to the procedure of the kinase assay kit from the company Upstate Biotechnology. This probe was performed as described in ‘Materials and Methods’, chapter 2.5.1 and repeated three times. Again the highest activity was measured at 0 h, before the start of maturation. A decrease in the activity was found at all other time points (22, 26, 30, 34, 46 h) during the following maturation process (Graph 5). Statistical analysis of the data revealed a significant difference between the activities at the start of maturation (0 h) and all other time points during the maturation.
75 RESULTS
1400
1200
1000
800
600 cpm/20 cpm/20 oocytes
400
200
0 0 22 26 30 34 46 IVM (h)
Graph 5: Activity of p90rsk in porcine oocytes during the in vitro maturation period with an addition of the MEK inhibitor U0126 to the cultivation medium
6.2 ‘In-Gel’ renatured kinase assay
Autoradiographic analysis of the dried gels from the ‘In-Gel’ renatured kinase assay revealed no bands corresponding to the [ γ-32 P] labelled substrate of the p90rsk. All tested adaptions of the used protocol did not result in an improvement. Bands of the radiolabeled substrate were never seen and therefore could not be excised and submitted to quantative measurement.
7 Animal experiment
The in vivo matured and afterwards retrieved oocytes (see C Materials and Methods, chapter 3) were also controlled concerning their nuclear status. Therefore a total number of at least 20 oocytes from each of the three time points (0, 22, and 30 h) chosen, were fixed, stained, and their nuclear status was assessed.
76 RESULTS
Graph 5 shows the percentages of nuclear statuses in relation to the different time points analysed during in vivo maturation. For reasons of simplification GV stages I to IV are summarized as GV. The concrete numbers of oocytes and their distribution to the different meiotic stages are listed in a separate table in the appendix.
n=22 n=25 n=20 100%
80% MII 60% TI AI 40% D/MI
Nuclear status Nuclear GV 20%
0% 0 22 30 in vivo maturation (h)
Graph 5: Percentages of nuclear status in relation to the different time points during in vivo maturation; GV=Germinal Vesicle, D=Diakinesis, MI=Metaphase I, AI=Anaphase I, TI=Telophase I, MII=Metaphase II
Analysis of the in vivo matured oocytes just like the in vitro matured via electrophoresis and immunoblot detected the p90rsk not at molecular weight of 90 but of little less than 200 kDa (Fig. 21). The phosphorylation pattern of the in vitro matured oocytes was also observed in the in vivo matured oocytes. Due to the smaller number of time points analysed during in vivo maturation the phosphorylation pattern could not be shown as detailed as during in vitro maturation. Although the same number of oocytes per time point were analysed the received bands in the immunoblots show very different intensity. Therefore figure 17 depicts different exposure times of the same immunoblot to the X-ray film (A: exposure time 1 min.; B: exposure time 5 min.). In all replicates the mobility shift of the bands
77 RESULTS
corresponding to the phosphorylation of the molecule was visible and at 0 h of maturation p90rsk was seen as double bands.
Figure 17: Phosphorylation pattern of in vivo matured porcine oocytes A and B pictures of the same immunoblot with different exposure times to the X-ray film; A: exposure time 1 min., B: exposure time 5 min.
Reprobing the immunoblots with the MAPK specific antibody detected no bands in the molecular range from 70 to 200 kDa.
In contrast to the in vivo matured oocytes detection of the p90rsk in the cumulus cells from these oocytes revealed the molecule at the expected molecular weight of around 90 kDa (Fig. 18). At 0 h of in vivo maturation the kinase can only be detected as a single band. Also longer exposure times did not reveal any further bands at this stage. At 22 h and 30 h of in vivo maturation a double band of the kinase is visible. A shift as in the oocytes is not observed.
78 RESULTS
Figure 18: Analysis of cumulus cells from ten in vivo matured oocytes per time point
79 DISCUSSION
E Discussion
Meiosis during oocytes maturation is arrested two times: first after segmental exchange of homologous chromosomes in prophase of the first meiotic division and after expulsion of the polar body in metaphase II of the second meiotic division. The overcoming of these arrests after an appropriate hormonal stimulus is dependent upon the interaction of a network of different enzymes. Most important for this is the signal transduction via kinases. The MPF was found to be the universal kinase promoting maturation in eukaryotic cells. Two of the most important kinases within the cell being responsible for the activation of MPF and therefore in this case the progress of meiosis are the MAPK and the p90rsk.
The objective of the present 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.
1 Kinetics of nuclear maturation
1.1 Kinetics of nuclear maturation during in vitro maturation In our in vitro maturation culture of porcine oocytes GVBD took place from 22 to 30 h of culture in more than 85 % of the oocytes. Most of the oocytes were in the MI stage after 30 h of IVM and the percentage of MII stage oocytes increased significantly after 34 h of culture and reached this stage in over 90 % of the oocytes after 46 h of IVM. These found kinetics are in agreement with previous findings in our group (WEHREND, 1997) as well as other authors (SUGIURA et al., 2002; FAN et al., 2003a).
80 DISCUSSION
1.2 Kinetics of nuclear maturation during IVM with an addition of U0126 During IVM with medium containing 10 µM U0126, a specific MEK inhibitor, GVBD was significantly inhibited. In our culture system over 90 % of the oocytes remained in the GV stage at all time points controlled. The higher percentage of MII stage oocytes after 30 h of IVM is probably due to a selection of oocytes that had already resumed meiosis at the start of the in vitro maturation period. FAN et al. (2003a) also found a significant inhibition of GVBD with the same concentration of the inhibitor in the medium, although still 23.66 % of the oocytes underwent GVBD after 44 h of IVM. This difference might be due to the different composition of the maturation medium. KAGII et al. (2000) in their experiments observed that MAPK phosphorylation and activation was only inhibited incompletely. They found 42 % of the oocytes undergoing GVBD. As these authors used medium containing 100 µM U0126 this difference in GVBD rate is not due to inhibitor concentration. It might be due to the selection of oocytes or/and the use of an inhibitor free collection medium before start of the culture. On the basis of our results one can conclude that the used concentration of the inhibitor reliably inhibits the IVM process under the applied conditions.
1.3 Kinetics of nuclear maturation during in vivo maturation With 84 % of the in vivo matured oocytes still being in the GV stage at 22 h after the oocytes maturation inducing hCG injection in vivo maturation appears to be slower compared to the situation during in vitro maturation. This picture changes during the progressing maturation process as after 30 h of in vivo maturation 80 % of the oocytes have reached the MI stage, same as during in vitro maturation (80.2 %), and 15 % have progressed to the MII stage, compared to 2.5 % during in vivo maturation. Therefore in our in vivo matured oocytes the maturation process was neither significantly faster nor slower compared to the in vitro matured oocytes. Early studies comparing the in vivo and in vitro maturation kinetics in porcine oocytes observed a delay in in vivo matured oocytes of four hours compared to the in vitro matured oocytes. This difference appeared at the beginning of nuclear maturation and
81 DISCUSSION
remained unchanged throughout the whole period studied (until 24 h) (MOTLIK and FULKA, 1976).
2 P90rsk and MAPK in porcine oocytes and cumulus cells
2.1 P90rsk in porcine oocytes and cumulus cells Before starting this work no work had been done dealing with the p90rsk in porcine oocytes. Therefore we decided to proof the existence of the p90rsk in porcine oocytes as a first step and to extend this proof on the somatic cell compartment. This analysis also validated the used antibody that was used in all following probes. Proving the existence of the p90rsk in porcine oocytes we simultaneously tested different buffers and decided on the basis of the results to continue to use a modified Laemmli buffer (non reducing) for the processing of the oocytes. The reducing buffers resulted in a splitting of the molecule and therefore produced a smeared band in the immunoblot. This modified Laemmli buffer was used for the processing of the oocytes during all further probes and can be recommended for such analysis. Existence of p90rsk had been proved and analysed before in several mammalian species (mouse: KALAB et al., 1996; rat: TAN et al., 2001; rabbit: YU et al., 2002), and during the course of our studies also in pigs (SUGIURA et al., 2002; FAN et al., 2003a).
2.2 MAPK in porcine oocytes and cumulus cells The existence of MAPK in porcine oocytes had been shown before but again to validate the used antibody and the experimental conditions for a further analysis of the MAPK molecule, we also looked for the MAPK. It was shown that the antibody works in the oocytes and it was used for all further probes. As another project in our group started to concentrate on the MAPK in porcine cumulus cells and by now also includes the p90rsk the cumulus cells were not further analysed.
82 DISCUSSION
3 Phosphorylation pattern of p90rsk and MAPK
3.1 Phosphorylation pattern of p90rsk during in vitro maturation Immunoblot analysis revealed three bands of different mobility of the p90rsk molecule. In oocytes immediately following recovery from the follicles (0 h) the p90rsk is already observed as double bands, meaning that the first phosphorylation step has already taken place. These are the two bands with the highest mobility that remain until about 18 h of IVM. After 22 h the intensity of the band corresponding to the first phosphorylation step increases and after 26 h the third and lowest mobility band appears. This lowest mobility band increases in intensity until the end of maturation whereas the two other high mobility bands disappear. P90rsk phosphorylation therefore starts around the time of GVBD. This observation is in agreement with the findings of other authors (SUGIURA et al., 2002; FAN et al., 2003a), who examined porcine oocytes. GROVE et al. (1993) proposed that this first phosphorylation step might be caused by autophosphorylation and that the partially phosphorylated p90rsk is not an active kinase. In mouse and rat oocytes p90rsk is also observed as double bands during the GV stage (mouse: KALAB et al., 1996; rat: TAN et al., 2001). In these species a phosphorylation of p90rsk visible as a shift of the bands takes place after GVBD and at least the first phosphorylation step is MAPK independent as MAPK phosphorylation occurs after this first phosphorylation of p90rsk. These findings exclude the necessity for MAPK and p90rsk phosphorylation for GVBD in mouse and rat oocytes. In rabbit oocytes on the other hand MAPK phosphorylation started around the time of GVBD but p90rsk phosphorylation is only seen clearly after GVBD (YU et al., 2002). MAPK therefore might well be involved in GVBD in this species whereas p90rsk is clearly not involved, though its phosphorylation might be MAPK dependent.
3.2 Phosphorylation pattern of MAPK during in vitro maturation The increase of phosphorylation of the p90rsk molecule during the progress of IVM timely correlates with the increase of MAPK phosphorylation. This finding indicated
83 DISCUSSION
interdependence between these two kinases. Each isoform of the MAPK (ERK 1+2) has a phosphorylated and a non phosphorylated form. Phosphorylation of MAPK also starts around the time of GVBD visible as a band of lower mobility for each isoform. These two lower mobility bands become weakly visible between 20 and 24 h of IVM and increase in intensity during the progress of IVM until the end. The higher mobility bands clearly visible from the start of IVM, almost completely disappear until the end. Similar results were obtained by FAN et al. (2003a) analysing porcine oocytes. In mouse and rat oocytes MAPK is necessary for full phosphorylation of p90rsk although the first seen phosphorylation of p90rsk after GVBD occurred independently of MAPK phosphorylation. As MAPK phosphorylation only takes place after GVBD it is not necessary for GVBD in these species (KALAB et al., 1996; TAN et al., 2001). MAPK phosphorylation is seen in rabbit oocytes before GVBD and also precedes p90rsk phosphorylation. MAPK phosphorylation therefore might be involved in GVBD in this species but further analysis is needed to substantiate this hypothesis. P90rsk phosphorylation occurs only after GVBD and therefore is not necessary for GVBD to take place (YU et al., 2002). In equine oocytes only one isoform (ERK 2) of MAPK was detected. This isoform was detected in GV stage oocytes as the non phosphorylated form and phosphorylated at MII stage (GOUDET et al., 1998). As the time around GVBD was not examined in detail in this study, MAPK involvement in GVBD needs further analysis. In bovine oocytes both isoforms of MAPK exist but the ERK 1 is only faintly detectable by immunoblotting. At 0 h of maturation MAPK was only observed as the non phosphorylated form. Full phosphorylation was seen as maturation progressed (FISSORE et al. 1996). ERK 2 is the only detectable isoform in Xenopus oocytes similar to the situation in cattle (GOTOH et al., 1991).
3.3 Phosphorylation pattern of p90rsk during IVM with an addition of U0126 Phosphorylation of the p90rsk as well as the nuclear maturation under our IVM conditions with medium containing 10 µM U0126 was completely inhibited. Only the two high mobility bands that are visible from the start of IVM remain until the end and become more and more intense/prominent. This result that the highly specific MEK
84 DISCUSSION
inhibitor U0126 completely inhibits the phosphorylation of the p90rsk molecule confirms the proposed interdependence of MAPK and p90rsk. It can be concluded that MAPK phosphorylation is necessary for the full phosphorylation of p90rsk. FAN et al. (2003a) also reported an inhibition of p90rsk phosphorylation using U0126 in the same concentration. In their studies this inhibition of phosphorylation of p90rsk and significant inhibition of GVBD was only observed in COCs. In denuded oocytes they still observed the inhibition of phosphorylation but GVBD occurred in the same percentage as in COCs or denuded oocytes cultured in the inhibitor free medium. KAGIi et al. (2000) also examined porcine oocytes cultured with an addition of U0126. They came to same conclusion that the inhibitory effects of U0126 needed the presence of cumulus cells.
3.4 Phosphorylation pattern of p90rsk during in vivo maturation
Two studies exist about p90rsk in vivo matured oocytes in Xenopus (ERIKSON and MALLER, 1989; HSIAO et al., 1994). ERIKSON and MALLER (1989) detected the molecule by immunoblotting around 90 kDa. As they only describe that the kinase was purified from unfertilized eggs it is difficult to timely assign the results to the maturation process and therefore to compare them with the results from HSIAO et al. (1994). HSIAO et al. (1994) found the p90rsk being complexed with MAPK in a heterodimer. It was shown that ERK 2 when phosphorylated and active is monomeric, but when it is non phosphorylated and inactive about half of it is monomeric and half is a component of a 110 kDa complex. The other part of the complex was identified as Rsk, and much or all of the cell’s Rsk was coimmunoprecipitated with ERK 2. Phosphorylation and activation of MAPK correlated with the release of ERK 2 from the complex. This study also reports about MAPK being part of a similar sized complex in murine somatic cells. Our observations instead indicate that the p90rsk exists as a dimer in in vivo matured oocytes. This dimer as well as the monomeric form which is seen in in vitro matured oocytes undergoes a mobility shift during the process of maturation due to the phosphorylation of the molecule.
85 DISCUSSION
MAPK being part of the complex can be excluded as the probe with the MAPK specific antibody revealed no bands in the immunoblot picture. In the cumulus cells of the in vivo matured oocytes the p90rsk was observed at the expected molecular weight of around 90 kDa. Earlier studies of cumulus cells from in vitro matured oocytes showed that p90rsk phosphorylation occurs earlier in the cumulus cells compared to the oocytes already beginning after 12 h of IVM and culminating at 24 h (FAN et al., 2003a). Analysis of p90rsk phosphorylation of cumulus cells after IVM in our laboratory, consistent with our results in the in vivo matured cumulus cells, revealed no mobility shift of p90rsk during the IVM period (EBELING et al., 2005). These differences in the phosphorylation might be due to different maturation conditions, especially the different additives to the culture medium.
4. P90rsk activity during IVM with and without an addition of U0126
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
86 DISCUSSION
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).
87 SUMMARY
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.
88 SUMMARY
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.
89 ZUSAMMENFASSUNG
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
90 ZUSAMMENFASSUNG
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
91 ZUSAMMENFASSUNG
des Moleküls erfolgt zwischen 22 und 24 h nach Beginn der Kultivierung bzw. Start der In-vivo-Reifung.
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117 APPENDIX
I Appendix
1 List of abbreviations
µ micro (10 -6 ) µCi micro Curie µg micro gram (10 -6 gram) µl micro litre (10 -6 litre) µm micrometer (10 -6 meter) APC anaphase promoting complex APS ammonium persulfate Aqua bidest. Aqua bidestillata ATP adenosine triphosphate b.m. Body mass Bub1 budding uninhibited in benzimidazole BSA bovine serum albumin cAMP cyclic adenosine monophosphate cdc cell division cycle Cdk cyclin dependent kinase cDNA copy DNA cm centimetre
CO 2 carbon dioxide COC cumulus oocyte complex cpm counts per minute CSF cytostatic factor DDT dithiothreitol DMSO dimethylsulfoxide DNA deoxyribonucleic acid eCG equine chorionic gonadotrophin
118 APPENDIX
EDTA edetic acid ERK extra cellular signal regulated kinase FCS foetal calf serum FSH follicle-stimulating hormone g gram G1 gap 1 G2 gap 2 GV germinal vesicle GVBD germinal vesicle breakdown h hour hCG human chorionic gonadotropin HCl hydrogen chloride i.m. intramuscular I.U. international units IVF in vitro fertilization IVM in vitro maturation KCl potassium chloride kDa kilo Dalton kg kilo gram
KH 2PO 4 potassium dihydrogen phosphate l litre LH luteinizing hormone m milli (10 -3 ) mm millimetre (10 -3 metre) M molar MI metaphase I MII metaphase II mA milli ampere (10 -3 ampere) MAPK mitogen activated protein kinase MAPKK MAPK kinase MEK MAP/ERK kinase, MAP kinase kinase
119 APPENDIX
mg milligram
MgCl 2 magnesium chloride min. minute ml millilitre mM milli molar MOPS 3-Morpholinopropanesulfonic acid MPF maturation promoting factor mRNA messenger ribonucleic acid NaCl sodium chloride
Na 2HPO 4 disodium hydrogen phosphate NaPPi sodium pyrophosphate
Na 3VO 4 sodium orthovanadate P70S6K protein 70 S6 kinase P90rsk protein 90 ribosomal S6 kinase PAGE polyacrylamide gel electrophoresis PBS phosphate buffered saline p.c. post coitum pFSH porcine FSH PKA protein kinase A PMSF phenylmethylsulfonyl fluoride PVA polyvinyl alcohol RNA ribonucleic acid rpm rounds per minute RSK ribosomal S6 kinase RT room temperature SDS sodium dodecyl sulphate Ser serine TBS TRIS-buffered saline TCA trichloroacetic acid TCM tissue culture medium TEMED N,N,N’,N’-Tetramethylethylenediamine
120 APPENDIX
Thr threonine TRIS tris(hydroxymethyl)amino methane Tyr tyrosine U0126 1,4-Diamino-2,3-dicyano-1,4-bis-(o-aminophenylmercapto) butadiene
121 APPENDIX
2 Chemicals, drugs, and equipment
All chemicals were purchased from SIGMA Aldrich unless otherwise indicated.
Acrylamide solution, Applichem Ammoniumpersulphate (APS), Applichem
Merck, Darmstadt: 3-(N-Morpholino)propanesulfonic acid Acetic acid Ethanol Magnesium chloride
Na 2HPO 4
NaHCO 3 Orcein Phosphoric acid
Roth, Karlsruhe: Glycerol Isopropyl alcohol Methanol Ponceau Rot
Serva, Heidelberg: Bovine serum albumine (BSA) Glycin
KH 2PO 4 TEMED
Paraffin, Sherwood Vaseline, Wirtschaftsgenossenschaft deutscher Tierärzte (WDT)
122 APPENDIX
S6 Kinase Assay Kit, Upstate, Lake Placid, NY S6 Kinase/Rsk, Substrate Peptide 1, upstate, Lake Placid, NY ERK 1 (K-23): sc-94, Santa Cruz Biotechnology Rsk-1 (C-21): sc-231, Santa Cruz Biotechnology Streptavidin, DIANOVA, Hamburg Goat Anti-Rabbit HRP-Conjugated, PIERCE SuperSignal ® West Dura Extended Duration Substrate, PIERCE Biotinylated SDS-PAGE Standards High Range, BIORAD Prestained Protein Ladder, Fermentas Porcine Luteinizing Hormone (pLH), National Hormone & Peptide Program, Harbor- UCLA Medical Center, Torrance, California Porcine Follicle Stimulating Hormone (pFSH), National Hormone & Peptide Program, Harbor-UCLA Medical Center, Torrance, California
Drugs: Ursotamin ®, Serumwerk Bernburg Pregmagon ®, 5x1000 IE, Impfstoffwerk Dessau-Tornau (IDT) Ovogest ®, 5x5000 IE, Intervet Hostamox ®, LA, Intervet Atropinum Sulfuricum 0,5 mg Injektionslsg., Eifelfango Minocain ®, 2 %, WDT Stresnil ® Lösung, Janssen
Equipment: Clean bench (Reinraumwerkbank); Edge Card Hood Laboratory dishwasher; Laborspülautomat G7783 Mielabor, Miele Hot-air sterilizer; Memmert Autoclave; Tischautoklav EK, Tuttnauer Ultra-pure water system Milli-Q ® UF Plus, Millipore Cellulose-Acetate-Filter Millex ®-GS Millipore Medium 199 Earle, Serva
123 APPENDIX
Incubator Cytoperm 8080, Heraeus Water bath, Köttermann Stereomicroscope SZH 10 Research Stereo, Olympus Phase contrast microscope BX 60, Olympus Cell culture dishes Heated table, Bachhofer 4-well multidishes, Nunclon Electrophoresis chamber, BIORAD Standard Power Pack P25, Biometra Fastblot B43, Biometra Centrifuge 5414, Eppendorf Refrigerator, Liebherr profi line Freezer (-20°C), Liebherr Comfort -85°C Ultralow Freezer, NUAIRE Thermomixer comfort, Eppendorf Eppendorf tubes X-ray film: X-OMAT AR 18 x 24 cm, Kodak X-ray cassette, Dr. Goos-Suprema GmbH, Heidelberg Hybond-ECL Nitrocellulose membrane, Amersham Biosciences Extra Cellophane Sheets For Gel Drying Frames, Sigma Gel Drying Frames, Promega USP 2 (5metric), SURGICRYL, 90 cm USP 3 (4metric), SURGICRYL, 90 cm
124 APPENDIX
3 Media, buffers, and solutions (alphabetical order)
Blocking solution TBS 0.1 % Tween 5 % Teleostean gelatine
Blotting buffer 0.038 M glycine 2.93 g solve first, before adding the 0.048 M Tris 5.81 g methanol 0.0375 % SDS 0.375 g 20 % methanol 200 ml Aqua bidest. Ad up to 1000 ml
Collection medium Dulbecco’s phosphate buffered saline 9.65 g solved in Aqua bidest. 300 ml CaCl 0.10 g solved in Aqua bidest. 50 ml mix both solution Gentamycin (50 mg/ml) 1 ml Aqua bidest. Ad up to 1000 ml Take 180 ml of this solution and add 20 ml heat-inactivated FCS.
Culture medium stock medium 40 ml heat-inactivated FCS 10 ml Insulin 0.001 g Store up to 14 days at 4°C.
125 APPENDIX
Destaining solution for gels Destaining solution for gels with glycerol 50 % methanol 5 % glycerol 7 % acetic acid 50 % methanol 43 % Aqua bidest. 7 % acetic acid 38 % Aqua bidest.
Electrode buffer 10times concentrated, pH 8.8: 25 mM Tris 30.28 g 192 mM glycine 144.13 g 0.1 % SDS (w/v) 10.0 g Aqua bidest. ad up to 1000 ml Diluted 1:10 with Aqua bidest. before use
Extraction buffer Final concentration 20 mM MOPS, pH 7.2 10 mM pNPP 20 mM betaGPP
0.1 mM Na 3VO 4 20 mM NaF 1 mM DTT 5 mM EGTA 0.1 mM EDTA 20 µg/ml Aproptinin 20 µg/ml Leupeptin 1 mM Benzamidine
Flushing medium 0.9 % NaCl solution 1 % heat-inactivated FCS
126 APPENDIX
Hyaluronidase Hyaluronidase 0.25 g Aqua bidest. Ad up to 100 ml Filtrate sterilely and store at -20°C.
In-gel kinase buffer 20 mM Tris-HCl, pH 8
10 mM MgCl 2
Laemmli-buffer (Laemmli 1970) 50 mM Tris/HCl, pH 6.8 10 % glycerin 2 % SDS (5 % β-mercaptoethanol) only for reducing conditions a tip of a spatula of Bromphenol blue
Lysis buffer for assay pH 7.0 for 10 ml 50 mM MOPS 20.93 mg
10 mM MgCl 2 47.61 mg 0.2 % Triton X-100 20 µl
0.1 mM Na 3VO 4 (in H 2O) 0.18 mg
20 µg/ml Leupeptin (in H 2O) 200 µg
10 µg/ml Aprotinin (in H 2O) 100 µg 10 µg/ml Pepstatin (in DMSO) 100 µg 10 µg/ml Chymostatin (in DMSO) 100 µg 2 mM PMSF (in ethanol) 3.48 mg
127 APPENDIX
PBS (Phosphate buffered saline) 10times 1l
Na 2HPO 4 9.10 mM 16.2 g 1.62 g
KM 2PO 4 2.71 mM 3.6 g 0.36 g NaCl 154.00 mM 90.0 g 9.00 g pH 7.4
Polyacrylamide gel 9 % separation gel (10 ml): 30 % acrylamide solution 2.5 ml 1.5 M Tris/HCl, pH 8.8 2.5 ml 10 % SDS (w/v) 100 µl Aqua bidest. 4.85 ml TEMED 5.0 µl 10 % APS 50 µl
Stock acrylamide solution for shifting gels: 29.7 g Acrylamide 3 g Bisacrylamide Add Aqua bidest. up to 100 ml
9 % ‘shifting’ separation gel (10 ml) 29.7 % acrylamide solution 3.0 ml 1.5 M Tris/HCl, pH 8.8 2.5 ml 10 % SDS (w/v) 100 µl Aqua bidest. 4.3 ml TEMED 7 µl 10 % APS 70 µl
128 APPENDIX
5.0 % stacking gel (5 ml): 30 % acrylamide solution 0.83 ml 1.0 M Tris/HCl, pH 6.8 0.63 ml 10 % SDS (w/v) 50 µl Aqua bidest. 3.4 ml TEMED 5.0 µl 10 % APS 50 µl
Ponceau Red solution Ponceau S 0.5 % 0.5 g Acetic acid 1 % 1.0 ml ad 100 ml Aqua bidest.
Renaturation buffer 100 mM Tris-HCl, pH 8 5 mM β-mercaptoethanol
Staining solution for gels 1 g Coomassie Brillante Blue in a mixture of 50 % methanol 7 % acetic acid 43 % Aqua bidest. Solve and filtrate
Stock medium Medium 199 Earle 1.92 g
NaHCO 3 0.44 g Gentamycin (50 mg/ml) 200 µl Aqua bidest. Ad 200 ml Store up to 4 weeks at 4°C
129 APPENDIX
Stripping-Reagent 2-mercaptoethanol 100 mM SDS 2 % Tris-HCl 62.5 mM pH 6.7 for 100 ml: 985 mg Tris-HCl 700 µl Mercaptoethanol 2 g SDS
TBS (Tris buffered saline) 10times 1 litre Tris 500 mM 60.57 g 6.05 g NaCl 1.5 M 87.66 g 8.76 g Adjust pH 7.5 with HCl
TBS/Tween TBS, 1 % Tween
Transportation medium 0.9 % NaCl solution
Washing buffer for assay pH 7.0 for 10 ml 50 mM MOPS 20.93 mg
10 mM MgCl 2 47.61 mg 0.2 % Triton X-100 20 µl
130 APPENDIX
Kit Components:
Assay Dilution Buffer I (ADBI): 20 mM MOPS, pH 7.2 25 mM β-glycerol phosphate 5 mM EGTA 1 mM sodium orthovanadate 1 mM dithiothreitol
Substrate Cocktail: 250 µM substrate peptide [AKRRRLSSLRA] in ADBI
P81 Phosphocellulose Squares
Inhibitor Cocktail: 20 µM PKC inhibitor peptide 2 µM PKA inhibitor peptide 20 µM Compound R24571 in ADBI
Magnesium/ATP Cocktail: 75 mM magnesium chloride 500 µM ATP in ADBI
131 APPENDIX
4 Index of tables
Table 1: Control of in vitro maturation; GV=Germinal Vesicle, D=Diakinesis, MI=Metaphase I, AI=Anaphase I, TI=Telophase I, MII=Metaphase II; the percentages are rounded to one decimal place, therefore the columns not always add up to 100 %. 0 h 22 h 26 h 30 h 34 h 46 h Total number 117 93 228 81 94 93 GV (I-IV) 99.2 % 59.1 % 32.0 % 14.8 % 2.2 % D/MI 0.9 % 35.5 % 66.2 % 80.3 % 62.8 % 6.5 % AI 0.4 % 1.2 % 1.1 % 1.1 % TI 1.2 % 10.6 % MII 5.4 % 1.3 % 2.5 % 25.5 % 90.3 %
Table 2: Control of in vitro maturation with an addition of the inhibitor U0126 to the maturation medium; GV=Germinal Vesicle, D=Diakinesis, MI=Metaphase I, AI=Anaphase I, TI=Telophase I, MII=Metaphase II; the percentages are rounded to one decimal place, therefore the columns not always add up to 100 % 0 h 22 h 26 h 30 h 34 h 46 h Total number 103 84 92 89 84 80 GV (I-IV) 97.1 % 95.2 % 98.9 % 92.1 % 97.6 % 100 % D/MI 2.9 % 1.2 % 1.1 % 1.1 % AI TI MII 3.6 % 6.7 % 2.4 %
132 APPENDIX
Table 3: Control of in vivo maturation; GV=Germinal Vesicle, D=Diakinesis, MI=Metaphase I, AI=Anaphase I, TI=Telophase I, MII=Metaphase II; the percentages are rounded to one decimal place, therefore the columns not always add up to 100 %
0 h 22 h 30 h Total number 22 25 20 GV (I-IV) 100 % 84 % 5 % D/MI 0 16 % 80 % AI 0 0 TI 0 0 MII 0 0 15 %
133 ACKNOWLEDGEMENTS
Acknowledgements
I would like to thank my supervisor Prof. Dr. Burkhard Meinecke for initiating this exciting project, his unrestricted support, his patience which was necessary mainly in the last year of the project, his academic guidance throughout every phase of my PhD study and his all time amusing and interesting little stories.
A special and warm thank to my lab team, Dr. Silja Ebeling, Edita Podhajsky, and Monika Gümmer for their friendship and all time provided support.
Furthermore, I thank my advisory committee, Prof. Dr. K.-D. Hinsch and Prof. Dr. G. Breves for their helpful suggestions and discussions.
Further, I would like to thank Prof. Dr. H. Naim from the Department of Physiological Chemistry and his team for offering the possibility to conduct my radioactive studies in their laboratory.
I thank Prof. Dr. E. Töpfer-Petersen and the whole ‘obere Abteilung’ for good collaboration.
Many thanks to the animal care takers and Christian and Heiko for their help with the surgeries.
Schließlich danke ich meiner Mutter für ihre bedingungslose und jederzeit gewährte Unterstützung.
And my husband Robert - for everything
134