Biol Reprod Keystone Symposia Conference on Frontiers in Reproductive Biology & Regulation of Fertility.
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Biology of Reproduction 65, 1260-1270 (2001)
© 2001 Society for the Study of Reproduction, Inc.

Regular Article

Expression of MSY2 in Mouse Oocytes and Preimplantation Embryos1

Junying Yua, Norman B. Hechtb, and Richard M. Schultz2,a,b

a Department of Biology and b Center for Research on Reproduction and Women's Health, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6018

ABSTRACT

Translational control plays a central role during oocyte maturation and early embryogenesis, as these processes occur in the absence of transcription. MSY2, a member of a multifunctional Y-box protein family, is implicated in repressing the translation of paternal mRNAs. Here, we characterize MSY2 expression in mouse oocytes and preimplantation embryos. Northern blot analysis indicates that MSY2 expression is highly restricted and essentially confined to the oocyte in the female mouse. MSY2 transcript and protein, as assessed by reverse transcription-polymerase chain reaction and immunoblotting, respectively, are expressed in growing oocytes, metaphase II-arrested eggs, and 1-cell embryos, but then are degraded by the late 2-cell stage; no expression is detectable in the blastocysts. During oocyte maturation, MSY2 is phosphorylated and following fertilization it is dephosphorylated. Quantification of the mass amount of MSY2 reveals that it represents 2% of the total protein in the fully grown oocyte, i.e., it is a very abundant protein. Both endogenous MSY2 and MSY2-enhanced green fluorescent protein (EGFP), which is synthesized following microinjection of an mRNA encoding MSY2-EGFP, are primarily localized in the cytoplasm, and about 75% of the MSY2 remains associated with oocyte cytoskeletal preparations. Results of these studies are consistent with the proposal that MSY2 functions by stabilizing and/or repressing the translation of maternal mRNAs.

developmental biology, early development, embryo, gamete biology, gene regulation

INTRODUCTION

Oocytes are unique cells because they are the only cells that can give rise to whole organisms. Oocytes acquire the ability to reprogram the pattern of gene expression directed by either the endogenous zygotic nuclei or transplanted somatic cell nuclei in cloning experiments only after they have undergone oocyte maturation. Although oocyte maturation occurs in the absence of transcription, dramatic changes in the pattern of protein synthesis occur that are due to both post-translational modifications, e.g., protein phosphorylation, and the recruitment of maternal mRNAs [1–4]. Little is known, however, regarding either the identity of maternal RNAs that are recruited or the molecular mechanisms that govern the timely recruitment of these mRNAs from messenger ribonucleoprotein (mRNP) particles that are masked from the translation machinery in the growing oocytes.

Subcellular localization, cytoplasmic polyadenylation, and Y-box proteins have emerged as leading candidates to regulate the translation of maternal mRNAs. In Drosophila, the asymmetric localization of specific mRNAs, e.g., oskar and nanos, in the developing egg or syncytial embryo regulates their translation. Sequences in the 3'-untranslated region (UTR) of these mRNAs are essential for targeting and translational activation and/or repression via RNA-protein interactions [5]. Likewise, the translation of Wnt-11 in Xenopus embryos is also subject to spatial regulation [6].

Cytoplasmic polyadenylation of maternal mRNAs is clearly implicated in recruiting maternal mRNAs during oocyte maturation, e.g., c-mos, tPA, cyclin B1, H1oo [7–10]. These maternal mRNAs have a U-rich cytoplasmic polyadenylation element (CPE) and the hexanucleotide AAUAAA in their 3'-UTRs. In growing oocytes, these mRNAs undergo deadenylation in the cytoplasm where they are packaged into mRNP particles. During oocyte maturation, cytoplasmic polyadenylation extends their poly(A) tail, which is mechanistically linked with their timely translation [7, 11, 12]. Factors common to this mechanism, such as the CPEB homolog, which is a CPE binding protein and required for cytoplasmic polyadenylation, are found in worms, clams, flies, Xenopus, and mice [13–17]. CPEB has a role in repressing the translation of some maternal mRNAs in the growing oocytes. Phosphorylation of this protein during oocyte maturation is required for CPEB to recruit the cytoplasmic polyadenyation complex and stimulate the translation of the same maternal mRNAs [18–22].

Y-Box proteins are highly conserved multifunctional proteins that are also implicated in translational regulation [23–25]. Generally these proteins consist of a variable N-terminus, a highly conserved cold-shock domain, and a C-terminal tail domain [26, 27]. The cold-shock domain has two RNA-binding motifs (RNP-1 and RNP-2) that are responsible for the protein's specificity for the Y-box (or reverse CCAAT box) present in the promoter of many genes [28–31]. The C-terminal tail domain contains four islands of basic/aromatic amino acids surrounded by acidic regions that contain potential sites for serine/threonine phosphorylation. This C-terminal domain is involved in nonspecific nucleic acid binding and protein-protein interactions [29, 31, 32]. Y-Box proteins regulate both transcription and translation [30, 33]. In addition, some members interact with the cytoskeleton, e.g., actin, suggesting that they may also function in the subcellular localization or transport of mRNAs [34].

The role of Y-box proteins in translational control is well studied for the germ-line member FRGY2 in Xenopus [25, 35]. This protein is a major component of mRNP particles, which packages about 80% of the maternal mRNAs synthesized in the Xenopus oocyte. At a high FRGY2:mRNA molar ratio (30:1), the FRGY2 recombinant protein can repress the translation of H1 mRNA both in rabbit reticulocyte lysates and in vivo [33]. FRGY2 may also stimulate the transcription from Y-box-containing promoters [26].

Due to the limited quantities of biological material, few maternal mRNAs packaged into mRNPs have been characterized in mouse oocytes. Consequently, little is known about the mechanisms that package maternal mRNAs into mRNP particles and how these particles are sequestered or masked from the translation machinery, which is highly active in the growing oocyte. Furthermore, maternal mRNAs have a very long half-life. For example, the half-life of total mRNAs during mouse oocyte growth is about 8–12 days [36]. Proteins in these particles likely play an important role in the assembly of maternal mRNAs into mRNP particles, as well as their translational repression and stability.

Several Y-box proteins, e.g., MSY1, MSY2, and MSY4, are expressed in female germ cells and early embryos soon after fertilization [27, 37, 38]. MSY2 is of particular interest because its expression is primarily restricted to the germ cells [27]. In the male, transcription ceases during the latter stages of spermatogenesis, when the haploid round spermatids differentiate into mature spermatozoa. The synthesis of proteins required for these morphogenetic changes depends on the timely recruitment of paternal mRNAs from mRNP particles that contain about 75% of the poly(A) mRNAs within the round spermatid [39–41]. MSY2 and its isoform MSY2a, which is an alternatively spliced shorter form, are first detected in pachytene spermatocytes, and the amount gradually increases and then peaks in the round spermatids where abundant paternal mRNAs are stored in mRNPs. The amount of MSY2 then declines as spermatids undergo nuclear condensation and elongation [27, 42, 43]. MSY2 is one of the major proteins binding to the nonpolysomal testicular mRNAs [44]. These data indicate that MSY2 likely plays a role in regulating the translation of paternal mRNAs. In a similar fashion, MSY2 may play a pivotal role in storing and stabilizing the maternal mRNAs and repressing their translation in growing oocytes.

We report here that MSY2 constitutes about 2% of the total protein in the fully grown oocyte. The protein is abundant in the growing oocyte and essentially degraded by the late 2-cell stage. These changes in protein are paralleled by changes in the relative abundance of MSY2 mRNA. The protein is phosphorylated during oocyte maturation and dephosphorylated following egg activation. Last, MSY2 is predominantly localized to the cytoplasm where about 75% is associated with Triton-insoluble structures.

MATERIALS AND METHODS

Collection of Oocytes and Embryos

Incompetent oocytes (55 µm in diameter) were collected from CF1 mice 12 days after birth in Ca2+, Mg2+-free CZB medium containing 1 mg/ml collagenase (Worthington Biochemical Corp., Lakewood, NJ) and 0.2 mg/ml DNase I (Sigma, St. Louis, MO). Fully grown oocytes (78 µm in diameter), ovulated metaphase II (MII) eggs, and 1-cell embryos were collected from adult CF1 mice (>6 wk old) as previously described [45]. Two-cell embryos were collected by culturing 1-cell embryos in vitro in CZB medium [46]. Blastocysts (3.5 days postfertilization) were collected by flushing uteri with bicarbonate-free minimal essential medium (Earle salt) containing pyruvate (100 µg/ml), gentamicin (10 µg/ml), polyvinylpyrrolidone (3 mg/ml), and 25 mM Hepes, pH 7.2 (MEM/PVP).

Tissue RNA Extraction and Northern Blotting

Total RNA from ovary, testis, and various other tissues was extracted using Trizol (Gibco, Rockville, MD) according to the manufacturer's protocol. Following RNA precipitation, the RNAs were digested with 2 µl of RQ1 DNase (1 U/µl; Promega, Madison, WI) at 37°C for 30 min. For Northern blotting, total RNA (20 µg each lane) from various tissues was subjected to electrophoresis in a 1% formaldehyde agarose gel. RNA was transferred to Hybond-N+ (Amersham, Piscataway, NJ) using a semidry capillary blotting procedure [47]. Church and Gilbert buffer (0.3 M sodium phosphate buffer, pH 7.2, 7% SDS, 1 mM EDTA) was used for both prehybridization and hybridization. The membrane was incubated in the prehybridization buffer at 65°C for 15 min. Hybridization was conducted at 65°C overnight with ~1–2 x 106 cpm/ml msy2 probe. After hybridization, the membrane was washed twice in 1x SSPE (0.18 M NaCl, 10 mM NaPO4, and 1 mM EDTA, pH 7.7):0.25% SDS for 15 min each at room temperature, followed by once in 1x SSPE:0.25%SDS for 15 min at 65°C. Stringency washing was done in 0.1x SSPE:0.1%SDS for 10 min at 65°C. After washing, the membrane was exposed to a phosphorimager cassette that was scanned with a Storm 860 system. As a control, a ß-actin probe was used to hybridize to the stripped membrane. All probes were made with the RadPrime DNA Labeling System (Gibco, Rockville, MD). The template for msy2 was the PCR product (638 base pairs [bp]) of msy2 cDNA with primer msy2F1: 5'-CCACCACCCTTCTTCTATCGA-3', and msy2R2: 5'-GGTGATGCCTCGGAACAATA-3'. The template for ß-actin was the PCR product (540 bp) with primer actin F: 5'-GTGGGCCGCTCTAGGCACCA-3', actin R: 5'-CTCTTTGATGTCACGCACGATTTC-3'.

Reverse Transcription-Polymerase Chain Reaction

RNA was extracted and reverse transcribed as previously described [45]. In each case, 0.03 pg of rabbit globin mRNA/oocyte was added prior to RNA extraction to normalize for RNA extraction and reverse transcription (RT) reaction efficiency. For each set of gene-specific primers, the linear region of semilog plots of the amount of polymerase chain reaction (PCR) product as a function of the cycle number was determined. The cycle numbers selected for msy2 [34] and {alpha}-globin mRNA [34] were in the linear region. The amount of PCR product under these conditions is proportional to the amount of specific mRNA present in the starting material, which permits quantification of relative changes in mRNA abundance.

RNA, isolated from 150 oocytes or embryos at different stages of development was reverse transcribed using random hexamers. For the PCR reactions, 10 oocyte/embryo-equivalents of cDNA were used as template for msy2, while 5 oocyte/embryo-equivalents of cDNA were used for {alpha}-globin. The msy2 primers used were msy2F2, 5'-CAGCCTATAGCCGCAGAGAC-3'; and msy2R2, 5'-GGTGATGCCTCGGAACAATA-3', which amplifies the 3'-end region of msy2. The {alpha}-globin primers used were F, 5'-ACCACCAAGACCTACTTTCCT-3'; and R, 5'-GTCAGCACGGTGCTCACAGA-3'. The PCR products were labeled with 2.5 µCi [{alpha}-32P]dCTP per 50-µl reaction (Amersham, Piscataway, NJ). The PCR amplification conditions were the same for both msy2 and {alpha}-globin: an initial denaturation at 94°C for 5 min, followed by 34 cycles of 94°C for 15 sec, 55°C for 30 sec, and 72°C for 1 min, followed by 72°C for 10 min. The PCR products were run on an 8% polyacrylamide gel. The gel was dried and exposed to a phosphorimager cassette that was scanned with a Storm 860 system and quantified using ImageQuant software (Molecular Dynamics, Sunnyvale, CA).

Immunoblotting

Oocytes and embryos were directly lysed in 2x SDS-PAGE sample buffer (100 mM Tris-HCl, pH 6.8, 200 mM dithiothreitol [DTT], 4% SDS, 0.2% bromophenol blue, 20% glycerol), and stored at -20°C until use. The samples were boiled for 3–5 min prior to being subjected to SDS-PAGE in a 9% gel. The proteins were then transferred to Immobilon P (Millipore, Bedford, MA), and the membranes were blocked with 10% fish-gelatin (in PBS) for 1 h at room temperature, or overnight at 4°C. After blocking, the membranes were washed twice in PBST (0.2% Tween-20) for 10 min each. The membranes were incubated with {alpha} -Xp54/56 (1:5000 dilution in PBS:3% BSA:0.1% Tween-20) for 1 h at room temperature; this antibody was made to Xenopus FRGY2 and was the generous gift of Mary Murray, Center for Molecular Medicine and Genetics, Wayne State University School of Medicine, Detroit, MI. Following incubation with the primary antibody, the membranes were washed six times in PBST for 15 min each. The membranes were then incubated with donkey anti-guinea pig alkaline phosphatase-conjugated secondary antibody (1:5000 dilution of a 0.6-mg/ml stock solution; Jackson Laboratory, West Grove, PA) for 1 h at room temperature. After washing in PBST for six times (15 min each), the membranes were developed using ECF substrate (Amersham), and scanned with a Storm 860 system.

Alkaline Phosphatase Treatment

Fully grown oocytes or MII eggs were frozen in an ethanol-dry ice bath, and stored at -80 °C until use. The samples were frozen and thawed three times in an ethanol-dry ice bath and then boiled for 5 min to inactivate proteases and phosphatases. The samples were then treated with calf intestine alkaline phosphase (CIAP) conjugated to agarose beads (Sigma) as follows. The 30-µl reaction contained 3 µl of 10x CIAP reaction buffer (1 mM MgCl2, 1 mM ZnCl2, 10 mM DTT, and 0.6 M Tris-HCl, pH 7.4), 0.3 µl 100 mM PMSF, 0.3 µl of 10 mg/ml leupeptin and pepstatin, with or without 8 units of CIAP. The reaction was incubated with rocking at room temperature for 45 or 60 min. After incubation, 5 µl of 6x SDS-PAGE buffer and 1 µl of ß-mercaptoethanol were added and the samples were boiled for 3–5 min before SDS-PAGE in a 9% gel.

Generation of MSY2 Recombinant Protein

Recombinant MSY2 protein bearing a T7 and His tag at the carboxyl terminus was prepared as follows. The primers (NdeI-msy2, 5'-GGAATTCCATATGAGCGAGGCGGAGGCGT-3'; XhoI-T7R-msy2R, 5'-CCGCTCGAGACCCATTTGCTGTCCACCAGTCATGCTAGCCATCTCCAGTATGGTGGTGGG-3' were used to generate the full-length msy2 cDNA while adding an NdeI restriction site at the 5' end, and a T7 tag and an XhoI restriction site at the 3' end. The PCR reaction contained 7.5% DMSO because of the high GC content of the msy2 cDNA, and Pfu polymerase was used to increase the fidelity of the PCR (Stratagene, La Jolla, CA). The PCR conditions were as follows: an initial denaturation at 94°C for 5 min, followed by 35 cycles of 94°C for 1 min, 55°C for 1 min and 72°C for 2 min, and followed by 72°C for 10 min. The msy2 PCR product was double-digested with NdeI and XhoI and cloned into pET-21a (+) (Novagen, Madison, WI). The plasmid was transformed into BL-21 (DE3) cells (Novagen). MSY2 expression was induced with 1 mM isopropyl-ß-D-thiogalactopyranoside at 30°C for about 3 h when the bacterial OD600 was between 0.8 and 0.9. The expressed MSY2 had a T7 tag (11 amino acids) and His tag (6 amino acids) at the C-terminus with two additional amino acids between the two tags.

The bacterially expressed protein was purified using the His-Bind Resin kit (with Bugbuster protein extract reagent) (Novagen) according to the manufacturer's protocol with the following modifications due to the protein's poor solubility. The bacterial pellet was resuspended in Bugbuster protein extract reagent (4 ml per 100 ml culture) containing benzonase (1 µl per 1 ml Bugbuster reagent). The suspension was incubated at room temperature for 15 min and then subjected to centrifugation at 16 800 x g at 4°C for 20 min. The majority of the MSY2 protein remained in the pellet. After removing the supernatant, the same volume of Bugbuster reagent was added to resuspend the pellet without benzonase. Freshly prepared lysozyme was added to the suspension (2 µl of 100 mg/ml lysozyme per 1 ml Bugbuster reagent). The suspension was again incubated at room temperature for about 15 min with occasional vortexing to dissolve completely the bacterial debris. The suspension was centrifuged at 16 800 x g at 4°C for 20 min and the supernatant was then loaded onto a nickel-binding column. The MSY2 was then purified according to the manufacturer's instructions with one additional washing step (6 volumes of 100 mM imidazole, 0.5 M NaCl, 20 mM Tris-HCl, pH 7.9). The purity of the recombinant protein, which was checked by Coomassie staining following SDS-PAGE, revealed it to be at least 95% pure (Fig. 1). The concentration of the eluted MSY2 protein was determined using the Bradford method (BioRad, Hercules, CA).



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  		FIG. 1. Coomassie staining of MSY2 recombinant protein. A T7 tag (11 amino acids) and a His tag (6 amino acids) were fused to the C-terminus of MSY2. The protein was purified using a nickel-binding column and run on a 9% SDS-PAGE gel. Lanes 1 and 2, 1.5 µg and 3 µg MSY2 recombinant protein, respectively

Quantification of Endogenous MSY2 Protein

To quantify the amount of endogenous MSY2 protein in the oocyte, two extracts were run on a 9% SDS-PAGE gel in parallel together with known amounts of recombinant MSY2. Western blotting was carried out as described above, and the amount of MSY2 protein was quantified with ImageQuant software as previously described [48].

Egfp-pXT7 and Msy2-egfp-pXT7 In Vitro Transcription Constructs

To make the egfp control construct for in vitro transcription, the HindIII/NotI fragment from plasmid pEGFP-N2 (Clontech, Palo Alto, CA) was inserted into plasmid pEGFP (Clontech). The egfp cDNA was excised with EcoRI, and inserted into plasmid pXT7, which has a Xenopus ß-globin 5'-UTR after the T7 promoter, and Xenopus ß-globin 3'-UTR and 33 A residues at the 3' end. The plasmid was the generous gift of Carmen Williams, School of Medicine, University of Pennsylvania, Philadelphia, PA. To make the msy2-egfp fusion cDNA construct, the full-length msy2 cDNA was inserted into the EcoRI and NotI site of egfp–pXT7 construct. The EagI fragment from egfp-pXT7 containing egfp cDNA was then reinserted into NotI site of msy2-pXT7 construct to generate the msy2-egfp fusion cDNA construct.

In Vitro Transcription

The egfp-pXT7 and msy2-egfp-pXT7 constructs were linearized at the SacI site that is present after the 33 A residues. Capped mRNAs were made by in vitro transcription with T7 RNA polymerase. The 100-µl reactions contained 20 µl of 5x transcription buffer, 10 µl of 100 mM DTT, 3 µl of 40 U/µl RNasin, 20 µl of 10 mM rNTP mix that contains 10 mM ATP, 10 mM CTP, 10 mM UTP, 2 mM GTP and 8 mM Cap analogue, 1 µg of linearized constructs, and 8 µl of 15 U/µl T7 RNA polymerase. The reactions were incubated at 37°C for 2 h. Following incubation, 1 µl RNase-free DNase (2 U/µl, Ambion, Austin, TX) was added and the samples were incubated at 37°C for an additional 15 min to digest the DNA. RNA was recovered by phenol:chloroform extraction and precipitated with 1 volume of isopropanol. A single band of the correct size was observed for each RNA on a 1% formaldehyde denaturing gel, and these RNAs encoded proteins of the correct size as assessed by in vitro translation (data not shown).

Microinjection

Oocytes were microinjected with 5–10 pl of either egfp mRNA (1 µg/µl) or msy2-egfp mRNA (1 µg/µl) as previously described [49]. The injected oocytes were cultured overnight in CZB containing 0.2 mM 2 mM 3-isobutyl-1-methylxanthine (IBMX) (to inhibit maturation) in an atmosphere of 5% CO2 at 37°C. Eggs arrested at MII were obtained by culturing the injected oocytes in IBMX-free CZB for an additional 16 h.

Laser-Scanning Confocal Microscopy

Live oocytes or eggs expressing enhanced green fluorescent protein (EGFP) or MYS2-EGFP were examined by laser-scanning confocal microscopy using a Leica microscope (Heidelberg, Germany). In addition, the cells were also examined following fixation with 3.7% paraformaldehyde for 20 min at room temperature. To determine the localization of the endogenous MSY2, oocytes or eggs were fixed with 3.7% paraformaldehyde for 20 min, followed by permeabilization in 0.1% Triton (in PBS) for 15 min. Cells were then washed twice in blocking buffer (PBS-0.1% BSA-0.01% Tween-20) and then incubated in the blocking buffer for 15 min. The incubation with primary MSY2 antibody (1:6000 dilution in the blocking buffer) was carried out for 1 h. Cells were washed in four drops of the blocking buffer for 15 min each before an incubation with Alexa Fluor 488 goat anti-guinea pig IgG antibody for 1 h (1:500 dilution of a 2 mg/ml stock solution; Molecular Probes, Eugene, OR). Cells were again washed in four drops of the blocking buffer for 15 min each before observation. All of these steps were carried out in a sealed humidified chamber at room temperature. Specificity of staining was established by incubating the diluted primary antibody with the MSY2 recombinant protein (5 µg/ml final concentration) overnight at 4°C prior to use.

Cytoskeletal Preparations

Cytoskeletal preparations used for confocal microscopy were prepared by treating the oocytes or eggs with 0.1% Triton X-100 containing 100 mM KCl, 5 mM MgCl2, 3 mM EGTA, 20 mM Hepes, pH 6.8, and 1% BSA (intracellular buffer, ICB) for 10 min at room temperature, followed by fixation in 3.7% paraformaldehyde for 20 min at room temperature [50].

RESULTS

Analysis of Msy2 Expression in Various Tissues

Msy2 is expressed in both male and female germ cells, but not in brain, spleen, liver, heart, and kidney [27]. A Northern blot analysis was performed to confirm and extend this expression pattern to include lung, intestine, muscle, skin, and uterus. As previously reported, the level of msy2 expression was highest in the testis, and low or undetectable levels of expression were observed in the other tissues (Fig. 2). RT-PCR of tissue mRNAs using msy2 gene-specific primers confirmed this expression pattern (data not shown). The weaker 4.2-kilobase (kb) band detected in the testis, which was not previously observed [27], may represent an alternatively spliced variant or unspliced species, or other Y-box mRNAs. The low level of msy2 expression in the ovary was anticipated, as oocytes represent a small population of the ovarian cells. The detection of msy2 in the ovary implied a high level of expression in the oocytes that was verified by direct measurement of the amount of MSY2 in the oocyte (see below).



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  		FIG. 2. Northern blot analysis of msy2 mRNA in 10 somatic tissues, ovary, and testis of adult mouse. Twenty micrograms of each tissue RNA was run on a 1% formaldehyde denaturing agarose gel, transferred to Hybond-N+ membrane, and hybridized with a 32P-labeled msy2 cDNA probe (top panel) or ß-actin cDNA that serves as a control (bottom panel)

Temporal Pattern of MSY2 Expression

The temporal pattern of msy2 expression during oocyte growth and preimplantation development was assessed by RT-PCR analysis using equal numbers of oocytes or embryos (Fig. 3). Compared to meiotically incompetent oocytes, the relative abundance of msy2 mRNA was about 2-fold greater in meiotically competent oocytes, MII-arrested eggs and 1-cell embryos (22.5 h post-hCG). The twofold increase in the amount of mRNA was consistent with the observation that oocytes reaching ~70% of their final volume have accumulated all of their mRNA and oocytes reaching ~35% of their final volume (oocytes 55 µm in diameter) have accumulated about 50% of their mRNA [51]. The amount of the msy2 transcript in the 2-cell embryo (47 h post-hCG) and blastocyst was about 15% and 5% of that present in the fully grown oocyte. Thus, the maternal msy2 transcript was degraded by the 2-cell stage and was apparently not reexpressed during preimplantation development. This expression profile contrasts with many other maternal mRNAs that are degraded following oocyte maturation and the 2-cell stage but are then reexpressed in the preimplantation embryo, e.g., MSY1 [37].



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  		FIG. 3. Reverse transcription-PCR analysis of msy2 RNA in incompetent oocytes (from 12-day-old mice), fully grown oocyte, MII eggs, 1-cell embryos (22.5 h post-hCG), 2-cell embryos (47 h post-hCG), and blastocysts (3.5 days old). Rabbit globin mRNA (0.03 pg/oocyte) was added prior to RNA extraction for normalization. Reverse transcriptions were randomly primed. {alpha}-32P-dCTP-labeled PCR reactions were run on an 8% polyacrylamide gel. The size of msy2 and {alpha}-globin amplicons is 302 bp and 257 bp, respectively

The changes in the msy2 transcript level during oocyte growth and early development were reflected in the MSY2 protein level. Immunoblotting experiments showed that the amount of MSY2 increased about 3-fold between meiotically incompetent oocytes (55 µm in diameter) and fully grown meiotically competent oocytes and was then undetectable in the blastocyst (Fig. 4A). The 3-fold increase during oocyte growth was in accord with the linear increase in protein as a function of oocyte volume [52], and the 2.9-fold increase in volume between the meiotically incompetent and competent oocytes. It should also be noted that when compared to the testis extract (Fig. 4A, lane 1), which contained 20 µg of protein, a very strong signal was obtained with about one fifth the amount of oocyte protein (Fig. 4A, lane 4) (150 fully grown oocytes contain about 3.75 µg of protein, using a value of 25 ng of protein/oocyte [53]). This result suggested that MSY2 was an abundant oocyte protein, which was confirmed below.



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  		FIG. 4. Western blot analysis of MSY2 protein in growing oocytes and early embryos. Samples were run on 9% SDS-PAGE gels. A) MSY2 protein expression in incompetent oocytes (55 µm in diameter), fully grown oocytes (78 µm in diameter), and blastocysts. Lane 1, 20 µg testis extract; lane 2, 150 incompetent oocytes from 12-day-old mice; lane 3, 150 fully grown oocytes; lane 4, 75 fully grown oocytes; lanes 5 and 6, 75 and 130 blastocysts (3.5 days old), respectively. B) MSY2 protein expression during oocyte maturation and immediately following fertilization. Seventy-five oocyte equivalents were loaded in each lane. Lane 1, fully grown oocytes; lane 2, oocytes that have undergone germinal vesicle breakdown (6 h in vitro maturation); lane 3, MII eggs from in vitro maturation; lane 4, MII eggs (in vivo); lane 5, 1-cell embryos (18 h post-hCG); lane 6, 1-cell embryos (30 h post-hCG); lane 7, 2-cell embryos (43 h post-hCG); and lane 8, 2-cell embryos (54 h post-hCG). The arrow points to the position that corresponds to an electrophoretic mobility of 50 000 Da. C). CIAP treatment of MSY2 in MII eggs. Oocyte and MII egg extracts were incubated with or without 8 U agarose-beaded CIAP for 45 or 60 min before being loaded being subjected to SDS-PAGE

Closer examination of the temporal changes in the amount of MSY2 revealed that during oocyte maturation protein species of slower electrophoretic mobility appeared following germinal vesicle breakdown and remained in the MII-arrested egg (Fig. 4B). These changes occurred whether the oocytes were matured in vitro or in vivo. Following fertilization, only the faster electrophoretic protein species was detected in the 1-cell embryo, and by the late 2-cell embryo MSY2 could not be detected (Fig. 4B). The changes in electrophoretic mobility that occurred during oocyte maturation and following fertilization were likely due, at least in part, to changes in phosphorylation, as treatment of extracts derived from MII-arrested eggs with alkaline phosphatase resulted in the formation of protein species of greater electrophoretic mobility (Fig. 4C). (Careful inspection of the gels reveals that phosphorylated MSY2 present in the MII-arrested egg was not converted to a form with an identical electrophoretic mobility as MSY2 present in the oocyte, for reasons that are not fully understood at this time. Nevertheless, the results do suggest that MSY2 is phosphorylated, although other modifications, e.g., ubiquitination or ribosylation, could contribute to the changes in electrophoretic mobility.) Thus, the amount of MSY2 paralleled changes in its transcript abundance that occurred during oocyte growth and early development. Moreover, the absence of MSY2 protein beyond the late 2-cell stage, at which time the maternal-to-zygotic transition has occurred, is consistent with a role for MSY2 in regulating translation and/or stability of maternal mRNAs during this window of time in which transcription is either absent or initiating.

Quantification of Endogenous MSY2 Protein

We determined the absolute amount of MSY2 protein in the oocyte to gain insights regarding its potential function as a regulator of translation. For example, a high molar ratio of MSY2 to the total amount of mRNA in an oocyte may suggest a global role for MSY2 in regulating translation of all mRNAs, whereas a low ratio may suggest that MSY2 regulates the translation of a subset of mRNAs. This latter possibility was of particular interest in light of the recent finding that binding of MSY2 to the 3'-UTR of protamine mRNA exhibits sequence specificity [38]. The amount of endogenous MSY2 protein was determined by immunoblotting using a standard curve constructed with recombinant MSY2 protein (Fig. 5). The amount of oocyte extract used gave a signal that fell within the linear range of the standard curve. Assuming that the antibody recognized the MSY2 recombinant protein and endogenous MSY2 protein equally well, results of these experiments indicated that the fully grown oocyte contains about 0.49 ng of MSY2 protein, which represents about 2.0% of total protein because the fully grown oocyte contains about 25 ng of protein [53]. The molar ratio of MSY2 to mRNA in the oocyte is about 73:1, as oocytes contain about 80 pg of total mRNA [54] (assuming an average mRNA length is 1500 nucleotides).



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  		FIG. 5. Quantification of endogenous MSY2 protein in the fully grown mouse oocyte. A) Immunoblot of two oocyte extracts (300 oocytes each) were run on a 9% SDS-PAGE gel together with 25 ng, 50 ng, 75 ng, 100 ng, 125 ng, 150 ng, 175 ng, and 200 ng MSY2 recombinant protein as standards. B) The quantification of the data shown in A. The solid circles correspond to the amount of MSY2 protein in two batches of 300 oocytes

Cellular Localization of MSY2

Many Y-box proteins function in both transcription and translation [26]. Accordingly, we localized MSY2 in the mouse oocyte to assess if it could function in either or both of these capacities. We first examined the localization of exogenously expressed MSY2 by monitoring the localization of an MSY2-EGFP fusion protein following injection of oocytes with MSY2-EGFP mRNA; control oocytes were injected with mRNA encoding EGFP. This expressed MSY2-EGFP fusion protein was also localized in MII-arrested eggs, by first expressing the protein in oocytes inhibited from resuming meiosis by culturing them overnight in the presence of IBMX and then allowing them to mature in IBMX-free medium. In both live and fixed oocytes, MSY2 was localized primarily in the cytoplasm (Fig. 6). The staining, which appeared punctate, seemed to be lower in the cortex than in the center of the oocyte. In contrast, EGFP was present in both the nucleus and cytoplasm, and did not reveal this gradient of staining. In the MII-arrested egg, MSY2-EGFP was more uniformly distributed in the cytoplasm and apparently excluded in the region of the spindle.



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  		FIG. 6. Subcellular localization of MSY2-EGFP fusion protein in the fully grown mouse oocytes and MII eggs injected with msy2-egfp mRNA. A) Localization of MSY2-EGFP in mouse fully grown oocytes. Injected oocytes were cultured in the presence of IBMX overnight to inhibit maturation. Oocytes were either subjected to confocal microscopy alive, or after fixation in 3.7% paraformaldehyde, or after treatment with 0.1% Triton followed by fixation in 3.7% paraformldehyde. B) Localization of MSY2-EGFP in MII eggs. Injected oocytes were cultured in the presence of IMBX overnight to allow the translation of injected mRNAs and their proper cellular localization. Then oocytes were washed to remove IBMX to allow in vitro maturation to MII-arrested eggs that were subjected to confocal microscopy similar to oocytes. The experiment was conducted three times, and in each experiment at least seven oocytes or eggs were analyzed for each group. Similar results were obtained for each experiment and shown are representative examples

MSY2 may associate with the cytoskeleton [34]. If this occurs, MSY2 would be expected to be retained in cytoskeletal preparations. Such appeared to be the case. MSY2 was readily detected in oocytes and eggs expressing the MSY2-EGFP fusion protein that were permeabilized with Triton prior to fixation. Although it is not possible to quantify accurately the signal obtained from each image due to the variability in the amount of mRNA injected, examination of a large number of injected oocytes revealed that a large fraction of the MSY2-EGFP fusion protein remained associated with these preparations (Fig. 6). The permeabilization was clearly effective, as no signal was observed in permeabilized cells expressing EGFP (Fig. 6).

The localization of endogenous MSY2 was then examined in oocytes, eggs, 1-cell, and 2-cell embryos. In the absence of Triton X-100 treatment, the amount of MSY2 decreased in the paraformaldehyde-fixed embryos, such that by the 2-cell stage, little signal above background was detected (Fig. 7A). These results confirmed the results of the immunoblotting experiments (see above). Moreover, the staining, which appeared punctate, was enhanced in the cortex. This was unlikely a fixation artifact, because the localization of the exogenously expressed MSY2 was not affected by the same fixation procedure (Fig. 6). Little or no endogenous MSY2 was detected in the nucleus. The localization of endogenous MSY2 was then determined following Triton X-100 permeabilization. The staining was again punctate and enhanced in the cortex. In addition, the signal intensity was similar to that obtained in the unpermeabilized cells. This was confirmed by conducting immunoblotting on treated and untreated oocytes, which revealed that about 75% of MSY2 was retained in the treated oocytes (Fig. 7B). It should be noted that MSY2 was present in the perivitelline space of the 1-cell and 2-cell embryo but not in the perivitelline space of the egg. Thus, the conditions for permeabilization did release a fraction of MSY2. The retention of MSY2 in the perivitelline space following fertilization may reflect changes in the permeability of the zona pellucida that occur as a consequence of the fertilization-induced changes in ZP2 and ZP3 [55–57].



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  		FIG. 7. Subcellular localization of endogenous MSY2 protein in the fully grown mouse oocytes, MII eggs, 1-cell embryos, and 2-cell embryos as detected by immunofluorescence. A) Fully grown oocytes (MII eggs) were collected directly from hormone-primed CF1 adult mice. One-cell embryos were collected about 21 h post-hCG, then cultured in vitro to about 30 h post-hCG, or 54 h post-hCG to obtain 2-cell embryos. Oocytes, MII eggs, and embryos were treated with or without 0.1% Triton before analysis by immunofluorescence. For preabsorption controls, the primary antibody was incubated with MSY2 recombinant protein (5 µg/ml final concentration) overnight at 4°C prior to use. The experiment was conducted two times and in each experiment at least 10 oocytes, eggs, or embryos were analyzed for each group. Similar results were obtained for each experiment and shown are representative examples. B) Western blot analysis of oocyte extracts treated with or without 0.1% Triton

DISCUSSION

Y-Box proteins are expressed in many organisms, and based on their tissue expression pattern, they are subdivided into somatic and germ cell-specific subfamilies; members within the same subfamily are usually more conserved than between subfamilies [27]. We report here that in the female mouse, MSY2 is preferentially expressed in the oocyte and is one of the most abundant oocyte proteins identified to date; MSY2 is either not expressed at detectable levels or expressed at levels below detection in a variety of somatic tissues. Following oocyte maturation and fertilization, MSY2 protein and mRNA are essentially absent by the late-2-cell stage and not reexpressed in the preimplantation embryo. This loss of MSY2 protein occurs during the maternal-to-embryonic transition, and hence its restricted window of expression is consistent with a role in the storage and translational regulation of maternal mRNAs. Transcripts for two other Y-box proteins, MSY1 and MSY4, are also present in the oocyte [37, 38]. The MSY1 transcript, like the MSY2 transcript, is degraded between the oocyte and 2-cell stages, but then is reexpressed in the 4-cell embryo; the expression pattern for MSY4 has not been determined. These differences in the pattern of expression for MSY1 and MSY2 suggest different functions for these two Y-box proteins. In the male mouse, MSY1, MSY2, and MSY 4 are highly expressed in germ cells in the testis, where their association with nonpolysomal mRNAs likely regulates the storage and timely translation of these mRNAs [38, 42, 44, 58]. MSY2 expression is essentially restricted to the testis, while MSY1 and MSY4 are expressed in the testis, as well as in a variety of somatic tissues [27, 38, 58]. Cross-linking studies suggest that MSY2 and MSY4 are present in a complex [38]. Whether such an association exists in the oocyte is not known.

MSY2, along with its shorter alternatively spliced isoform MSY2a, was initially cloned from mouse testis [27]. Results of immunoblotting experiments using an antibody specific for Xenopus FRGY2 demonstrate that both isoforms are found in testis extracts. MSY2 is the predominant isoform in the extracts from total testis and from earlier germ cell stages, while MSY2a is the predominant protein detected in the later postmeiotic stage germ cells [42]. We detect by immunoblotting only MSY2 and not the shorter MSY2a isoform, and efforts to amplify transcripts for this shorter isoform by RT-PCR were unsuccessful (unpublished observations). Thus, MSY2 is likely the major, if not only, isoform expressed in the mouse oocyte.

Phosphorylation is a commonly employed post-translational modification that regulates protein function, and we find that MSY2 is phosphorylated during oocyte maturation and dephosphorylated following fertilization. The effect of this phosphorylation on MSY2 function, however, is not fully resolved. For example, in Xenopus FRGY2 is phosphorylated both in vivo and in vitro by a casein kinase II (CK2) activity, and treating oocytes with specific inhibitors of CK2 or injecting inhibitory CK2 antibodies released about 20% of the masked mRNAs [59, 60]. Moreover, endogenous phosphorylation of FRGY2 correlates with increased binding to mRNA [25]. Nevertheless, CK2-mediated phosphorylation of recombinant FRGY2 that phosphorylates the same sites as in vivo does not alter its affinity for mRNA [60]. Thus, this phosphorylation may not directly regulate the ability of FRGY2 to bind mRNA but rather may influence its interactions with other proteins and thereby modulate translational repression-activation. Precedent for this exists. For example, phosphorylation of CPEB, the cytoplasmic polyadenylation element-binding protein, decreases its interaction with maskin, and thus allows translational activation of some maternal mRNAs [22]. In mouse testis, a kinase associated with nonpolysomal paternal mRNAs that is not CK2 can phosphorylate MSY2 and regulate its RNA-binding affinity [44]. Interestingly, treatment with CIAP did not result in any apparent change in electrophoretic mobility. As described above, we observe that MSY2 protein is phosphorylated during oocyte maturation and dephosphorylated following fertilization, based on the ability of CIAP to convert the slower-migrating electrophoretic forms to faster-migrating ones. The differential ability of CIAP to dephosphorylate testis and oocyte MSY2 suggests that the kinase that phosphorylates MSY2 in the mouse oocytes might not be the same as in the testis. Current experiments seek to identify potential kinase(s) that phosphorylate MSY2 in mouse oocytes and assess the effect of MSY2 phosphorylation on its RNA-binding properties.

MSY2 is one of the most abundant proteins in the mouse oocyte and represents about 2% of total oocyte protein. Only lactate dehydrogenase (5% of total oocyte protein) and creatine kinase (2% of total oocyte protein) rival this level of expression [61, 62]. Because the molar ratio of MSY2 protein to mRNA is 73:1, MSY2 is likely to have a role in the global packaging of maternal mRNAs that in turn affects their stability and/or translation. Consistent with such a role in mRNA stability is that mRNAs synthesized during oocyte growth are very stable, with a half-life of about 8–12 days [36]. In addition, in oocytes, the high MSY2:mRNA molar ratio may facilitate repression of translation of maternal mRNAs, as mRNA translation is inhibited when the ratio of FRGY2 to mRNA is 30 [33]. In somatic cells, the molar ratio of the somatic Y-box protein p50 to mRNA is 5–10:1, and overexpressing this protein inhibits protein synthesis [63]. Such a lower ratio is consistent with the apparent absence of large quantities of untranslated mRNAs in somatic cells, and the inhibition of protein synthesis at higher levels is consistent with a repressive role in translation. Interestingly, at a lower protein:RNA molar ratio, FRGY2 and p50 stimulate translation of mRNAs [33, 64]. The degradation of MSY2 that occurs following fertilization could relieve the repressive effects of MSY2 on translation and might even stimulate translation due to a lower MSY2:mRNA molar ratio. The 35% increase in the absolute rate of protein synthesis that occurs following fertilization is consistent with this proposal [2].

A global role in mRNA packaging implies that MSY2 interacts with mRNAs in a relatively sequence-independent manner. Consistent with this proposal is that MSY2 can package either luciferase RNA or brome mosaic virus RNA [44], and 75% of testis mRNA is associated with MSY2, as well as MSY4 [38, 44]. Nevertheless, RNA gel-shift assays using specific sequences in the 3'-UTR of the protamine 1 mRNA suggest that MSY2, as well as MSY4, can bind RNA with sequence specificity [38]. Thus, MSY2 may bind to mRNAs with different affinities. The short RNA sequences of the probe used in these in vitro assays may permit detection of such differential binding, which may not be readily apparent with full-length mRNAs. In particular, if binding of MSY2 to full-length mRNAs is cooperative, this may result in binding of relatively high affinity.

Many Y-box proteins function in both transcriptional and translational regulation [65]. Although MSY2 contains a potential nuclear localization signal in its C-terminal tail domain, no readily detectable signal is observed in the germinal vesicle of fully grown mouse oocytes or the nucleus of 2-cell embryo, i.e., MSY2 is essentially cytoplasmic. The endogenous MSY2 appears more concentrated in the oocyte cortex, whereas MSY2 expressed as an EGFP-fusion protein appears more centripetally concentrated. The cortical enrichment of endogenous MSY2, which is also observed with DNA methyltransferase 1 [66], suggests that MSY2 does not freely diffuse in the oocyte's cytoplasm but rather preferentially interacts with components enriched in the cortex. The enhanced concentration of the exogenously expressed MSY2 to the inner part of the oocyte's cytoplasm may then reflect the binding of the exogenously expressed MSY2 to the remaining sites present in the oocyte's interior. Consistent with the proposal that MSY2 does not diffuse freely is the observation that about 75% of the endogenous MSY2 is retained following Triton permeabilization, which generates cytoskeletal preparations and releases about 75% of the total oocyte protein (unpublished observations), as well as all of the exogenously expressed soluble EGFP. Although this result suggests that MSY2 is associated with the cytoskeleton, and treatment of mouse oocytes with nocodazole or cytochalasin D, or both, prior to Triton permeabilization does not result in any apparent decrease in the amount of MSY2 retained. (unpublished observations). Thus, the identity of the component(s) responsible for retaining MSY2 in the cytoplasm remains to be resolved.

The retention of MSY2 following Triton permeabilization raises an interesting question about the localization of mRNAs in the mouse oocyte. If, as in the testis, MSY2 packages repressed maternal mRNAs in the mouse oocyte, these maternal mRNAs would be associated with these cytoskeletal preparations, whereas mRNAs that are being translated would be in the soluble cytoplasmic fraction. Recruitment of untranslated maternal mRNAs during oocyte maturation or immediately following fertilization might then be accompanied by their release into the soluble fraction. We are currently testing this hypothesis.

ACKNOWLEDGMENTS

J.Y. thanks Dr. Peizhang Tang for the cloning of full-length msy2 cDNA.

FOOTNOTES

First decision: 23 May 2001.

1 This research was supported by grants from the National Institutes of Health to R.M.S. (HD 22681) and N.B.H. (HD 29125). Portions of this work are being submitted by J.Y. in partial fulfillment of the Ph.D. requirements at the University of Pennsylvania. Back

2 Correspondence: Richard Schultz, Department of Biology, University of Pennsylvania, 415 South University Avenue, Philadelphia, PA 19104-6018. FAX: 215 898 8780; rschultz{at}mail.sas.upenn.edu Back

Accepted: June 4, 2001.

Received: April 24, 2001.

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Absence of the DNA-/RNA-binding protein MSY2 results in male and female infertility
PNAS, April 19, 2005; 102(16): 5755 - 5760.
[Abstract] [Full Text] [PDF]

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J. Yang, S. Medvedev, P. P. Reddi, R. M. Schultz, and N. B. Hecht
The DNA/RNA-binding protein MSY2 marks specific transcripts for cytoplasmic storage in mouse male germ cells
PNAS, February 1, 2005; 102(5): 1513 - 1518.
[Abstract] [Full Text] [PDF]