Molecular Reproduction & Development 78:33–47 (2011)
Fer Tyrosine Kinase Is Required For Germinal Vesicle
Breakdown and Meiosis-I In Mouse Oocytes
1221LYNDA K. McGINNIS,* XIAOMAN HONG,LANE K. CHRISTENSON,AND WILLIAM H. KINSEY
1 Department of Anatomy and Cell Biology, University of Kansas Medical School, Kansas City, Kansas 2 Department of Molecular and Integrative Physiology, University of Kansas Medical School, Kansas City, Kansas
The control of microtubule and actin-mediated events that direct the physical arrangement and separation of chromosomes during meiosis is critical since failure to maintain chromosome organization can lead to germ cell aneuploidy. Our previous studies demonstrated a role for FYN tyrosine kinase in chromosome and spindle organization and in cortical polarity of the mature mammalian oocyte. In addition to Fyn, mammalian oocytes express the protein tyrosine kinase Fer at high levels relative to other tissues. The objective of the present study was to determine the function of this kinase in the oocyte. Feline encephalitis virus (FES)-related kinase (FER) protein was uniformly distributed in the ooplasm of small oocytes, but became concentrated in the germinal vesicle (GV) during oocyte growth. After germinal vesicle breakdown (GVBD), FER associated with the metaphase-I (MI) and metaphase-II * Corresponding author: (MII) spindles. Suppression of Fer expression by siRNA knockdown in GV stage Department of Anatomy and Cell Biology oocytes did not prevent activation of cyclin dependent kinase 1 activity or chromo- University of Kansas some condensation during in vitro maturation, but did arrest oocytes prior to GVBD or Medical Center during MI. The resultant phenotype displayed condensed chromosomes trapped in 3901 Rainbow Blvd. mail stop 3038 the GV, or condensed chromosomes poorly arranged in a metaphase plate but with an Kansas City, KS 66160 USA. underdeveloped spindle microtubule structure or chromosomes compacted into a E-mail: email@example.com tight sphere. The results demonstrate that FER kinase plays a critical role in oocyte meiotic spindle microtubule dynamics and may have an additional function in GVBD.
Mol. Reprod. Dev. 78: 33–47, 2011. ß 2010 Wiley-Liss, Inc. Published online 10 December 2010 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/mrd.21264 Received 6 October 2010; Accepted 15 November 2010
INTRODUCTION understood. The precise control of the microtubule and
actin-mediated events that direct the physical arrangement Maturation of the mammalian oocyte requires synchro- and separation of chromosomes during meiosis is critical nous progression and integration of many signaling path- since failure to maintain chromosome organization can lead ways. Historically, focus has been directed toward maturation promoting factor (MPF), mitogen-activated pro- tein kinases (MAPK), and other primary drivers that control Abbreviations: CDK1, cyclin-dependent kinase; FCH, FER-CIP1 homology meiosis (Masui and Markert, 1971; Su et al., 2002b; Peng domain; FER, FES-related kinase; FHM, ，ushing and holding medium; et al., 2007). Meiosis in oocytes also involves additional FES, feline encephalitis virus; GV, germinal vesicle; GVBD, germinal vesicle breakdown; hCG, human chorionic gonadotropin; IVM, in vitro maturation; oocyte-speci？c mechanisms involved in sister chromatid KSOM-MAT, KSOM medium with supplements for oocyte maturation; MAPK, cohesion (Hodges et al., 2005), chromosome condensation mitogen-activated protein kinase; MI, metaphase-I; MII, metaphase-II; MPF, (Swain and Smith, 2007), and spindle formation (Lindeman maturation promoting factor; PMSG, pregnant mare serum gonadotropin; PTK, and Pelegri, 2009) which are just now beginning to be protein tyrosine kinase; SH2, SRC homology domain.
ß 2010 WILEY-LISS, INC.
Molecular Reproduction & Development CGINNIS ET AL. M
to germ cell aneuploidy, a major risk to fertility especially in et al., 2003; Senis et al., 2003; Shapovalova et al., 2007) ageing women (Hunt and Hassold, 2008). Recent studies and its contribution remains an important question. have demonstrated that oocytes are highly specialized for Efforts to study FER following targeted gene knockout protein tyrosine kinase (PTK) signaling, with localized PTK have not been successful to date as a FES knockout activity occurring in the vicinity of spindle poles of proved to be embryonic lethal (Hackenmiller et al., 2000; metaphase-II (MII) oocytes as well as in the cortex of Hackenmiller and Simon, 2002). Successful generation of fertilized eggs (McGinnis et al., 2007; McGinnis and Alber- kinase dead mutant FER and FES mice, however, suggests tini, 2010). Studies revealed that the SRC family kinase that FER and FES perform some critical functions unrelated FYN plays an important role in maintaining meiotic spindle to kinase activity. Kinase dead FER or FES mutant females organization (Kinsey et al., 2003; Meng et al., 2006; McGin- exhibit reduced fertility (Senis et al., 2003) and double nis et al., 2007, 2009; Luo et al., 2009). Suppression of FYN mutant crosses exhibited reduced fertility and early repro- in oocytes by chemical inhibition, siRNA knockdown, or ductive senescence. Expression array data indicate that gene knockout led to the disorganization of metaphase-I oocytes express Fer at unusually high levels compared to (MI) and MII spindles that frequently correlated with meiotic other tissues, while Fes is barely detectable, suggesting arrest (McGinnis et al., 2009). In addition, Fyn-suppressed that Fer may play an important role in oocyte development. oocytes exhibited defects in cortical actin organization and The objective of the present study was to determine the polarity that correlated with reduced developmental com- function of FER in oocytes, and the results demonstrate that petence (Meng et al., 2006; McGinnis et al., 2007; this kinase plays an important role in meiosis. Experiments Tomashov-Matar et al., 2008; Luo et al., 2009). The above designed to test the function of this kinase during oocyte ？ndings highlighted the importance of identifying the control maturation indicated that the kinase is required for germinal mechanisms that direct spindle function in oocytes because vesicle breakdown (GVBD) and separately, for assembly of their importance to oocyte quality. The objective of the and function of the meiotic spindle. Suppression of Fer present study was to determine if other PTKs are critical to caused meiotic arrest, suggesting that FER activation may meiosis in oocytes. be a critical element of oocyte maturation and quality. One candidate family of protein kinases that may play a
role in meiotic spindle function is the Fer/Fes family. Fer and
Fes are the only known members of a distinct family of non- RESULTS receptor tyrosine kinases (Smithgall et al., 1998). Feline Expression of Fer Kinase in Oocytes encephalitis virus (FES)-related kinase (FER)-like proteins
Fer kinase is expressed at a low level in most cell types have been identi？ed in a diverse range of species including
humans and other mammals, Drosophila, C. elegans, birds, (Greer, 2002), but analysis of expression array data and marine sponges (Feldman et al., 1986; Pawson et al., (Novartis BioGPS, http://biogps.gnf.org; Su et al., 2002a) 1989; Katzen et al., 1991; Paulson et al., 1997; Cetkovic and http://amazonia.montp.inserm.fr (Assou et al., 2006, et al., 1998; Putzke et al., 2005). The FER proteins consist 2007; Wood et al., 2007) indicated the Fer transcripts are primarily of four domains: FER-CIP1 Homology (FCH), highly expressed in mouse (black bar Fig. 1A) and in human three-coiled coils, SRC homology domain (SH2), and (not shown). Quantitative RT-PCR analysis con？rmed ele-
C-terminal kinase domains. The kinase region includes an vated Fer expression in mouse oocytes (Fig. 1B). Fer atypical nuclear localization signal that requires the pres- mRNA was easily detectable in MII oocytes (Fig. 1B), while ence of the coiled coil and SH2 domains for regulation of Fes kinase mRNA was barely detectable above back- nuclear exit (Ben-Dor et al., 1999). This PTK family reg- ground (not shown). This con？rmed the expression array
ulates numerous cellular processes including cytoskeletal data and suggests that the oocyte is specialized for use of organization, cell adhesion, vesicle transport, and intracel- Fer and not Fes. Western blot analysis of whole ovarian lular signaling cascades (Greer, 2002). The close sequence tissue and oocytes demonstrated that both ovary (not homology between Fer and Fes suggests that these ki- shown) and oocytes (GV and MII) express FER protein nases have similar and sometimes redundant biological (Fig. 1C). The FER antibody detected a single band at functions (Smithgall et al., 1998; Greer, 2002). Several 94 kDa, the predicted molecular weight of full length FER. studies have described Fer (also called FerT2 in mouse) FES protein was detected in whole ovary, but was not expression in male germ cell maturation (Hazan et al., 1993; detectable in oocytes (not shown). The relative amount of Chen et al., 2003; Kierszenbaum et al., 2008), where a FER protein in each oocyte was determined by the ratio of truncated form lacking the N-terminal FCH and coiled coil FER to GAPDH within each set of oocytes. Although there domains participates in formation of the maturing sperm appeared to be a slight decrease in FER during maturation heads (Letwin et al., 1988; Pawson et al., 1989; Hazan et al., from GV to MII, this change was not statistically signi？cant
1993; Kierszenbaum et al., 2008). FER has been shown to (Fig. 1D; P > 0.05; four replicates). Immunohistochemistry associate with spindle microtubules in somatic cells, and indicated that FER was consistently concentrated in oo- can phosphorylate and promote elongation of microtubules cytes and associated granulosa cells of ovarian follicles in vitro (Kogata et al., 2003; Lee, 2005; Shapovalova et al., (Fig. 1E arrows), and was less abundant in stromal com- 2007). In spite of the strong evidence obtained in vitro, a ponents of the ovary and in the ovarian epithelium. Control requirement for FER in spindle function has not been sections (secondary antibody only) showed no labeling con？rmed in intact cells or gene mutant models (Kogata (data not shown). Detailed immunofluorescence analysis
34 Mol Reprod Dev 78: 33–47 (2011)
FER KINASE IN OOCYTE MEIOTIC MATURATION