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RESEARCH |
Key Laboratory of Animal Resistance, College of Life Science, Shandong Normal University, 88 East Wenhua Road, Jinan 250002, People’s Republic of China, 1 Cell Biology Laboratory, School of Basic Medicine, Shandong University of Traditional Chinese Medicine, Jinan 250014, People’s Republic of China and 2 State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100080, People’s Republic of China
Correspondence should be addressed to Y-L Li; Email: liyunlong1942{at}yahoo.com.cn
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Abstract |
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Introduction |
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Protein kinases have been shown to play an important role in this maturation process, serving to regulate key signal transduction pathways common to many other cell types. Protein tyrosine kinases (PTKs) are essential in extracellular signaling and actin cytoskeleton organization. A small group of PTKs have been identified in eggs of invertebrates, lower vertebrates and some mammals, and inhibitor studies have suggested that they are involved in egg activation (Sato et al. 2000, Runft et al. 2002, Talmor-Cohen et al. 2004). It has been suggested that protein tyrosine phosphorylation may be implicated in the regulation of mouse oocyte maturation. Genistein, an inhibitor of PTKs, inhibited GVBD in a dose-dependent manner (Kimura 1996). A recent study shows that Xyk, an egg Src-related protein tyrosine kinase, may be involved in accelerating meiotic maturation in Xenopus oocytes and its activity increases at the time of GVBD and remains elevated till the completion of maturation (Tokmakov et al. 2005). These data suggest that PTKs may have some functions during meiotic maturation process. However, little is known about the subcellular distribution and specific function of any of these PTKs during mammalian oocyte maturation. Proline-rich tyrosine kinase2 (Pyk2) (Lev et al. 1995), also known as cell adhesion kinaseß (CAKß) (Sasaki et al. 1995) or related adhesion focal tyrosine kinase (RAFTK) (Avraham et al. 1995), is a nonreceptor tyrosine kinase related to focal adhesion kinase (FAK). Pyk2, activated by multiple stimuli including extracellular matrix, hormones, growth factors, inflammatory cytokines, TNF-
, angiotensin, depolarization, u.v. irradiation, changes in osmolarity, stress-related signals, and increase in intracellular calcium concentration, is involved in the control of cell adhesion, migration and survival. Pyk2 has an autophosphorylation site at tyrosine 402 and this phosphorylated residue creates a binging site for the SH2 domain of Src, which in turn phosphorylates and activates Pyk2 (Dikic et al. 1996). Pyk2 also binds to proteins that interact with the cytoskeleton, such as paxillin (Salgia et al. 1996), p130cas, the Rho-GAP protein Graf (Ohba et al. 1998), and a LIM domain containing protein (Lipsky et al. 1998, Matsuya et al. 1998), suggesting its role in regulation of the cytoskeleton and cellular morphology in response to extracellular stimuli. Overexpression of Pyk2 in fibroblastic cells leads to reorganization of actin-associated cytoskeleton structures and cell rounding (Du et al. 2001). Co-localization of Pyk2 and actin filaments has been demonstrated in fibroblastic cell periphery and growing neurites (Wang et al. 2003, Haglund et al. 2004). Studies of cells derived from Pyk2-deficient mice indicate that Pyk2 is important for F-actin organization (Okigaki et al. 2003). Pyk2 exerts its effect on microfilaments by regulating other proteins, such as Cb1, ArgBP2, Src (Haglund et al. 2004), and gelsolin (Wang et al. 2003), which are implicated in cytoskeleton regulation. Pyk2 is also shown to regulate the activity of Rho family (Hall 1998) and the deficiency of Pyk2 leads to impaired Rho and PI-3 kinase activation in macrophages (Okigaki et al. 2003).
The aim of this study was to investigate the expression and localization of Pyk2 in rat oocyte and to analyze the functional significance of the interactions between Pyk2 and actin filaments, and in particular, the role of Pyk2 during rat oocyte maturation. By using Western-blot analysis, confocal microscopy, and antibody microinjection, we revealed the expression and localization of Pyk2 and its co-localization with actin filaments as well as its regulation on actin organization during rat oocyte maturation.
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Materials and Methods |
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Oocyte collection
Twenty-one-day-old female Wister rat was injected i.p. with 20 IU pregnant mare’s serum gonadotrophin (PMSG). Completely grown germinal vesicle-intact oocytes were collected approximately 48–50 h later from the ovaries and mechanically denuded of their cumulus cells with a thin-bore glass pipette and cultured in M2 medium supplemented with 60 µg/ml penicillin and 50 µg/ml streptomycin. All the cultures were carried out at 37 °C in a humidified atmosphere of 5% (v/v) CO2. Mature MII oocytes were obtained from female rat of the same strain. After i.p. injections of 20 IU of PMSG and hCG 54 h apart, the rats were killed and oviducts were removed at 15 h after hCG injection. Using a pair of fine forceps to tear the oviducts, cumulus masses were collected in M2 medium. To remove the cumulus cells, eggs were briefly exposed to 300 IU/ml hyaluronidase followed by three washes in M2 medium. The oocytes at different stages were collected for confocal microscopy.
Western-blot analysis
For the detection of Pyk2 expression in rat oocytes, proteins from a total of 300 GV or MII stage oocytes were extracted with SDS sample buffer and heated to 100 °C for 5 min. After cooling on ice and centrifuging at 10 000 g for 1 min, the total proteins were separated by SDS-PAGE with a 5% (w/v) stacking gel and a 6% (w/v) separating gel for 30 min at 70 V and for 1.5 h at 110 V respectively and then transferred onto PVDF transfer membrane for 2.5 h at 200 mA and 4 °C. The membrane was blocked for 2 h at room temperature in Tris-buffered saline (pH 7.4, 20 mM Tris, 137 mM NaCl) with 0.1% (v/v) Tween 20 (TBST) containing 5% (w/v) skimmed milk. The membrane was then incubated overnight at 4 °C with polyclonal Pyk2 antibody diluted 1:2000 in TBST containing 0.5% (w/v) skimmed milk. After three washes of 10 min each in TBST, the membrane was incubated for 1 h at 37 °C with goat anti-rabbit IgG (Santa Cruz Biotechnology, Santa Cruz, CA, USA) diluted 1:2000. The membrane was washed three times in TBST and then processed using the electrochemiluminescence (ECL) detection system
Confocal microscopy
Oocytes at the desired stages were fixed in 4% (w/v) paraformaldehyde in PBS for 30 min and permeabilized for 30 min in the incubation buffer (0.5% Triton X-100 in 20 mM Hepes, pH 7.4, 3 mM MgCl2, 50 mM NaCl, 300 mM sucrose and 0.02% NaN3) at room temperature. After blocking in PBS containing 1% (w/v) BSA for 1 h and incubated with polyclonal Pyk2 antibody or polyclonal Tyr 402 phospho-Pyk2 (PY402-Pyk2) antibody (Cell signaling technology, Inc., Hong Kong) diluted 1:200 for 1 h, the oocytes were washed in PBS with 0.1% (v/v) Tween 20 three times and incubated for 1 h with 1:200 fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG (Santa Cruz Bio-technology), followed by three washes and staining with 10 µg/ml propidium iodide. Finally, the oocytes were mounted on glass slides in 1,4-diazabicyclo (2.2.2) octane hydrochloride-containing (DABCO) medium, and examined using a laser scanning confocal microscope (Zeiss LSM510, Zeiss, Oberkochen, Germany). As a negative control, the first antibody was replaced with 1% BSA.
When oocytes were double labeled for microfilaments and Pyk2 or PY402-Pyk2, they were incubated with 1:200 TRITC-conjugated goat anti-rabbit IgG (Santa Cruz Biotechnology) for 1 h after incubation in rabbit anti-Pyk2 or PY402-Pyk2 antibody and three washes as described earlier. After another three washes, the eggs were incubated with 1:200 FITC-conjugated phalloidin for 1 h. Finally, after three washes, the eggs were mounted on glass slides and examined. The first antibody was replaced with 1% BSA in PBS as a negative control.
The actin filaments dynamics during the oocyte maturation as well as the F-actin and tubulin organization after antibody microinjection were examined by incubating the oocytes in 1:200 diluted FITC-conjugated phalloidin or FITC-conjugated -tubulin antibody for 1 h after blocking as described above. Then the chromosomes were marked using 10 µg/ml propidium iodide after washing thrice. Finally, the samples were added on glass slides and examined.
Antibody microinjection
In order to observe the role of Pyk2 during the maturation of rat oocyte, Pyk2 antibody (0.5 mg/ml in PBS without Ca2+, pH 7.4) was microinjected into the cytoplasm of GV oocytes as described by Dai et al.(2000). Each experiment was repeated three times, and 30–40 oocytes per group per experiment were used. To minimize the damage to oocyte, microinjection needle diameter was smaller than 1 µm. Hypoxanthine (HX, 4 mM) was added to the medium when GV stage oocytes were operated to prevent GVBD. A microinjection volume of about 7 pl per oocyte was used in all the experiments. The same amount of rabbit IgG (0.5 mg/ml) diluted in PBS was injected as control. After microinjection, the oocytes were incubated in 4 mM HX M2 medium for 30 min at 37 °C and then washed thoroughly with M2 medium. The oocytes were then cultured in M2 medium for the evaluation of GVBD and PB1 extrusion after 3 and 12 h respectively. The oocytes were collected for confocal microscopy at the same time.
Statistical analysis
All data on the rate of GVBD and the extrusion of the PB1 after microinjection were evaluated by 
2-test. If P < 0.05, the difference was considered as statistically significant.
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Results |
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, Pyk2 was detected by Western-blot analysis in rat oocytes. A polyclonal Pyk2 antibody recognized a single band at about 116 kDa both in GV stage and MII stage oocytes.
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and 3). In completely grown GV stage oocytes, Pyk2 and phospho-Pyk2 distributed around the germinal vesicles (Figs 2A and 3A). Pyk2 was also found in the germinal vesicle (Fig. 2A). After GVBD, when chromatin assembled to form chromosome clusters, both Pyk2 and phospho-Pyk2 were associated with chromosomes and in the centre of the cytoplasm (Figs 2B and 3B). At the beginning of the anaphase I, staining was localized right around the chromosomes and at the cortex, prominently in the area overlying the meiotic spindle (Figs 2C and 3C). In telophase I, both Pyk2 and its activated isoform concentrated in the PB1 cleavage furrow as well as the central cytoplasm (Figs 2D and 3D). At MII (Figs 2E and 3E), with the organization of chromosomes to the equatorial plate of second meiotic spindle, the immunoreactivity was observed to surround the spindle and in the ooplasm. A marked staining of phospho-Pyk2 was observed in the region overlying the metaphase spindle (Fig. 3E). The MII oocytes without primary antibody treatment were used as negative controls for Pyk2 and phospho-Pyk2 staining (Figs 2F and 3F).
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). After GVBD, a dense uniform F-actin layer was observed at the cell cortex in addition to its staining around the condensed chromosomes (Fig. 4B). In anaphase I and telophase I, in addition to the cytoplasm localization, brightening staining was observed around the cortex especially the area overlying the spindle and in the cleavage furrow respectively (Fig. 4C–E). In MII oocyte, F-actin was detected on the spindle, around the cortex, and in the centre of ooplasm (Fig. 4F).
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, the staining pattern of Pyk2 or phospho-Pyk2 was identical with that of phalloidin conjugated F-actin staining except some specific micro-domains. In GV-stage oocytes, Pyk2/p–Pyk2 co-localized with F-actin both in the oocyte cortex and the periphery of the germinal vesicle (Fig. 5A and a, B and b). Pyk2 was also found in the GV (Fig. 5A), but phospho-Pyk2 (Fig. 5B) and F-actin (Fig. 5a and b) did not exist in the GV. After GVBD, F-actin was detected in the centre and the cortex of oocytes and some microfilament arrays radiated from central cytoplasm to the cortex (Fig. 5c and d). The localization of Pyk2 and activated Pyk2 was consistent with F-actin as shown in Fig. 5C and D. At MII stage, Pyk2 and phospho-Pyk2 were distributed asymmetrically and evident staining was found to aggregate on one side of the cytoplasm and the cortex (Fig. 5E and F). F-actin was co-localized with Pyk2 in the cortex and cytoplasm (Fig. 5e and f). MII oocytes without primary antibody staining were used as a negative control for Pyk2 (Fig. 5G and g) and phospho-Pyk2 (Fig. 5H and h).
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). Actin filaments were detected around the chromosomes in the control group oocytes (Fig. 6B). At 12 h, the antibody- and sham-injected oocytes were scored for the emission of PB1. The rate of PB1 emission was decreased from 56.1% (32/57) in the control group to 24.7% (20/81) in the antibody microinjected group (P < 0.05). In the control group, more than half of the oocytes (56.1%) formed a typical telophase spindle and extruded the PB1 (Fig. 7A–C). F-actins were detected in the cytoplasm and in the cleavage furrow (Fig. 7C). While the antibody microinjected oocytes either did not form meiosis I spindle (Fig. 7D) or was blocked at MI stage (Fig. 7E). Some oocytes were at the anaphase stage, while the chromosomes separated in an abnormal direction, which was parallel instead of vertical to the plasma membrane (Fig. 7F). The cytoplasm micro-filaments in the experimental oocytes were destroyed, while the uniform cortex F-actins still could be detected (Fig. 7F).
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Discussion |
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Pyk2 is expressed at a high level in the brain, hematopoietic cells, osteoclasts and to a lesser extent in certain epithelial cells, smooth muscle cells, and fibroblasts (Avraham et al. 2000). Recent data show that Pyk2 is also expressed in prostate (Picascia et al. 2002) and testis (Chieffi et al. 2003). However, its expression and localization in oocyte is not known. Expression of Fyn, a member of Src kinase family (SFKs), is demonstrated and its activity is detected in rat eggs, which represents the first demonstration of a PTK in mammalian eggs (Talmor et al. 1998). Furthermore, other members of SFKs, including Src and Yes, were detected in rat eggs (Talmor-Cohen et al. 2004). In the present study, for the first time, we detected Pyk2 expression in mammalian eggs.
We have found that Pyk2 is co-localized with F-actin at various stages of oocyte maturation, suggesting that Pyk2 may be involved in regulating the polymerization and/or the organization of actin filaments during the oocyte maturation. The co-localization of Pyk2 and cortical F-actin is a common feature during rat oocyte maturation. The actin filaments that are associated with plasma membrane are important for generating cell-surface specialization areas and also provide the driving force for remodeling cell structure. This is achieved through the actin-binding proteins (Sun & Schatten 2006). Dmoesin, a member of ezrin-radixin-moesin (ERM) family, is required during oogenesis to anchor microfilaments to oocyte cortex in Drosophila (Polesello et al. 2002). Little is known about the proteins interacting with F-actin in the mammalian egg cortex. Immunostaining of Rho proteins shows that Rac1 and RhoB are present in the cortical ooplasm in mouse (Kumakiri et al. 2003). According to our results, the cortical localization of Pyk2 may imply that Pyk2 connects with F-actin and regulates its functions in the rat oocyte cortex. However, F-actin staining becomes weak but does not disappear after antibody microinjection and this may be due to the substitute pathways that regulate F-actin assembly which exist during oocyte maturation.
In GV stage oocyte, although expressed, Pyk2 is distributed in both cell periphery and cytoplasm including in the GV, activated Pyk2 is not located in the germinal vesicle. The staining pattern of activated Pyk2 is in close proximity with that of F-actin, suggesting that activated Pyk2 may contribute to the actin polymerization in GV oocyte. After GVBD, both Pyk2 and F-actin are aggregated around the condensed chromosomes. When GV oocytes are microinjected with Pyk2 antibody, the microfilaments are disrupted while GVBD happened. These results suggest that actin filaments have no effect on the GVBD and Pyk2 plays an important role in F-actin polymerization during the process of GVBD. Although F-actin is still detected co-localized with the chromosomes in the control group oocyte, its staining pattern is different from the normal culture oocyte. This may be due to the damage of the microinjection and the effect of the injected IgG. After GVBD, the condensed chromosomes organize to align at the metaphase plate and the meiotic spindle and then move peripherally. Coincident with the cortical localization of the chromosome is the formation of an area in the cortex rich in microfilaments and the polarization of the oocyte, which is required for asymmetric division. In our study, in both metaphase and the beginning of the anaphase, Pyk2 is asymmetrically associated with the chromosomes in ooplasm and the oocyte cortical area overlying the metaphase and the anaphase spindle. The distribution asymmetry and similarity with F-actin suggest that Pyk2 may contribute to polarity establishment in rat oocytes. Confocal microscopy results show that oocytes are arrested before MI stage or at anaphase after antibody injection. Some eggs separate the chromosomes with the spindle axis parallel instead of vertical to the plasma membrane. This may be due to the rotation of the spindle which is blocked. Although the cortical F-actin still exists after antibody injection, the polarity of the microfilament distribution disappears. They distribute uniformly in the cortex, which may contribute to the disability of the spindle rotation. However, how Pyk2 participates in polarity establishment is not known. A recent report indicates that PAR-3, a member of evolutionarily partitioning defective (PAR) proteins, is located within a central subdomain of the polarized actin cap, which overlies the spindle, prior to emission of the first and the second polar bodies. PAR-3 is phosphorylated during maturation. These results suggest a role for PAR-3 in establishing polarity in the egg and in defining the future site of polar body emission in mouse oocyte (Duncan et al. 2005). Our study also shows abundant distribution of phosphorylated Pyk2 in the cortical domain overlying the meiotic spindle, indicating that tyrosine phosphorylation may be involved in this process. We propose that Pyk2 may regulate the reorganization of F-actin in the spindle-associated cortical area by phosphorylating some microfilament regulatory proteins to form the polarity of the egg. In telophase, bundles of F-actin form a contractile ring and create a cleavage furrow to partition the oocyte into two daughter cells. Myosin II proteins also concentrate in the second polar body cleavage furrows of mouse egg (Simerly et al. 1998). Distinct Pyk2 staining is observed in meiosis I cleavage furrows in our study. Microinjecting Pyk2 antibody into the oocyte at GV stage results in an inhibition of the PB1 emission in our experiment. This may indicate that Pyk2 functions as a regulator of the proteins participating in contractile ring formation such as F-actin and/or myosin or a part of the contractile apparatus during the PB1 emission. However, the PB1 emission is lower in the IgG injection group (control group, 56.1%) compared with the normal cultured oocytes (about 75%) and the pattern of F-actin staining is quite different between the microinjected oocytes and the normal culture oocytes. It suggests that microinjection of IgG may injure the oocyte and have some effects on oocyte maturation. In our experiments, the PB1 emission is not totally inhibited by Pyk2 antibody microinjection, this is because Pyk2 is not a key regulator for F-actin or some other proteins or kinases participate in regulating F-actin together with Pyk2. Recently, p-MARCK (myristoylated alanine-rich C-kinase substrate), a major substrate for protein kinase C, is found to enrich in the periphery of the actin cap overlying the MI or MII spindle to form a ring-shaped subdomain (Michaut et al. 2005). In mitotic cells, contractile ring assembly is directed by the RhoA guanosine triphosphatase (GTPase), which activates myosin and actin filaments assembly (Glotzer 2005). RhoA and Cdc42 are found located in the cleavage furrow, and inhibition of RhoA and Cdc42 activation blocks polar body formation (Zhong et al. 2005, Ma et al. 2006). The mechanism of actin filament regulation during the polar body emission needs to be further elucidated in the mammalian eggs.
PY402 and its kinase activity are crucial for Pyk2 function on regulating polymerization of actin filaments (Wang et al. 2003). Our results reveal a consistent distribution of activated Pyk2 (Tyr 402) and F-actin during the process of oocyte maturation, which suggests an important role of tyrosine phosphorylation in regulating function of F-actin in oocyte. Phosphorylation of tyrosine 402 (tyr402), an auto-phosphorylation site in Pyk2 creates a binding site for the SH2 domains of Src family kinase. Src binding to tyr402 leads to activation of Src, which in turn phosphorylates and activates Pyk2 (Dikic et al. 1996). We have examined the immuno-activity of SFKs in rat oocyte, which are also associated with the chromosomes and localized in the cytoplasm and the cell cortex (data not shown), so it is intriguing to speculate that Pyk2 may regulate functions of RhoA or other proteins important for actin polymerization by the activation of Src.
Taken together, in this study, for the first time we show the expression and localization of Pyk2 during in vitro maturation of rat oocyte. We also detect the co-localization of Pyk2 and F-actin in the oocytes at various maturational stages. Furthermore, we find that the microinjection of Pyk2 antibody inhibits the organization of F-actin after GVBD and blocks the PB1 emission. These results lead us to propose that Pyk2 may regulate actin filaments polymerization and organization during rat oocyte maturation.
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Footnotes |
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Received 4 April 2006
First decision 11 May 2006
Revised manuscript received 10 July 2006
Accepted 1 September 2006
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References |
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