Developmental Expression and Subcellular Localization of Mouse ...

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Endocrinology 145(3):1427–1434 Copyright © 2004 by The Endocrine Society doi: 10.1210/en.2003-031160

Developmental Expression and Subcellular Localization of Mouse MATER, an Oocyte-Specific Protein Essential for Early Development ZHI-BIN TONG, LYN GOLD, ANTO DE POL, KONSTANTINA VANEVSKI, HEIDI DORWARD, PAOLA SENA, CARLA PALUMBO, CAROLYN A. BONDY, AND LAWRENCE M. NELSON Developmental Endocrinology Branch, National Institute of Child Health and Human Development (Z.-B.T., K.V., C.A.B., L.M.N.), and Laboratory of Cellular and Developmental Biology, National Institute of Diabetes and Digestive and Kidney Diseases (L.G., H.D.), National Institutes of Health, Bethesda, Maryland 20892; and Department of Anatomy and Histology, Facolta di Medicina e Chirurgia, Universita di Modena e Riggio Emilia (A.D.P., P.S., C.P.), 41100 Modena, Italy We reported previously that Mater is a maternal effect gene that is required for early embryonic development beyond the two-cell stage in mice. Here we show the expressional profile of Mater and its protein during oogenesis and embryogenesis as well as its subcellular localization in oocytes. Mater mRNA was detectable earliest in oocytes of type 2 follicles, whereas MATER protein appeared earliest in oocytes of type 3a primary follicles. Both mRNA and protein accumulated during oocyte growth. In situ hybridization showed that Mater mRNA appeared progressively less abundant in oocytes beyond type 5a primary follicles. By ribonuclease protection assay, Mater mRNA was abundant in germinal vesicle oocytes, but was undetectable in all stages of preimplantation embryos. In con-

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ATERNAL EFFECT genes encode transcripts and proteins that are expressed during oogenesis. Their products are stored in the oocyte to function later in the completion of meiosis, initiation of mitosis, activation of the embryonic genome, and development of totipotential embryonic cells. A number of maternal effect genes have been identified in Drosophila melanogaster, Xenopus laevis, and Caenorhabditis elegans (1–3), but there have been relatively few maternal effect genes identified in mammals (4). We reported that Mater, a single copy gene located at the proximal end of mouse chromosome 7 (5), is a maternal effect gene (6). Activation of its locus appears restricted to oocytes during oogenesis (7). The primary structure of mouse MATER protein, deduced from the full-length cDNA, is composed of 1111 amino acids (125,502 Da) (7). We have shown that the development of mouse embryos derived from Mater-null female mice is arrested at the two-cell stage (6), the crucial time at which the major embryonic genome activation takes place in mice (8). Indeed, embryos without MATER fail to exhibit normal embryonic genome activation (6). Thus, MATER protein is required for early development in mice. The developmental arrest at the two-cell stage of embryos Abbreviations: DAB, Diaminobenzidene; hCG, human chorionic gonadotropin; HRP, horseradish peroxidase; KLH, keyhole limpet hemocyanin; nt, nucleotide; RNase, ribonuclease. Endocrinology is published monthly by The Endocrine Society (http:// www.endo-society.org), the foremost professional society serving the endocrine community.

trast, the protein persisted throughout preimplantation development. Immunogold electron microscopic analysis revealed that MATER was located in oocyte mitochondria and nucleoli, and close to nuclear pores. Taken together, our data indicate that Mater gene transcription and protein translation are active during oogenesis, but appear inactive during early embryogenesis. Thus, Mater and its protein are expressed in a manner typical of maternal effect genes. The presence of MATER protein in mitochondria and nucleoli suggests that it may participate in both cytoplasmic and nuclear events during early development. (Endocrinology 145: 1427–1434, 2004)

lacking MATER suggests that it may play a role in activation of the embryonic genome. Genome activation is a major event in early embryonic development (9). It represents a dramatic transition from a maternal to an embryonic developmental program. Species vary as to when embryonic transcription first begins (10). In mice, embryonic transcription begins in the late one-cell zygote stage and is required for embryonic development beyond the two-cell stage (11, 12). The molecular mechanisms governing embryonic genome activation are largely unknown. Identification of maternal effect genes opens avenues to address the role of preexisting maternal factors in initiating and maintaining activation of the embryonic genome. In addition to Mater, several maternal effect genes have been identified recently in mammals, such as Hsf1, Dnmt1o, Formin-2, Zar1 and Npm2 (13–17). As seen in embryos lacking MATER, embryos lacking products of these maternal genes exhibit defective development. Investigating the functions of these maternal effect genes and their regulatory mechanisms should advance our understanding of the molecular basis underlying embryonic development. The molecular mechanisms that activate maternal effect genes and regulate their products are largely unknown. To determine the timing of Mater activation and expression during oogenesis and embryogenesis, we examined its mRNA and protein in progressive stages of oocyte and embryonic development. Also, we used immunogold electron microscopy to define the subcellular localization of MATER protein in oocytes. Because ovarian folliculogenesis, meiotic matu-

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Tong et al. • Expression and Localization of Mouse MATER

FIG. 1. In situ hybridization of Mater mRNA in mouse ovarian sections. Ovarian sections of mice at age 5 d (A), 7 d (B and C), and 5 wk (D) were hybridized with the antisense (A, B, and D) and sense (C) probes for mouse Mater mRNA. Oocytes of type 2 follicles near the ovarian surface are indicated by arrows. Other stages of ovarian follicles are labeled as indicated (21). Scale bar, 20 ␮m.

ration, and ovulation are shown to progress normally in Mater-null female mice, MATER protein appears to play a role specific to early embryonic development. Materials and Methods Animals, oocytes, and embryos NIH Swiss mice were used for isolation of oocytes and embryos to analyze developmental expression of Mater mRNA and protein. Maternull mice were generated using stem cell mutagenesis technology and did not express Mater mRNA and protein in their oocytes (6). Under microscopy, inactive (10 –15 ␮m in diameter), smaller (40 –50 ␮m), and larger (55– 65 ␮m) germinal vesicle oocytes were collected by dissecting ovaries of newborn and 2- and 3-wk-old mice, respectively, and using a mouth-operated procedure. Ovulation was induced by injection with 5 IU pregnant mare serum gonadotropin (Sigma-Aldrich Corp., St. Louis, MO) and then 48 h later with 5 IU human chorionic gonadotropin (hCG; Sigma-Aldrich Corp.). Cumulus masses were flushed 14 h after hCG injection and treated with hyaluronidase (0.3 mg/ml) to release ova. One-cell zygotes, two-cell embryos, and morulae were flushed from the oviducts at 1, 2, and 3 d after mating, and blastocysts were collected from uteri at 4 d after mating, respectively. The oocytes, ova, and developing embryos were manipulated in M2 medium and cultured in M16 medium at 37 C in 5% CO2. They were either fixed in 1% paraformaldehyde for confocal microscopic analysis or frozen immediately at ⫺80 C for subsequent RNA and protein analysis.

Ribonuclease (RNase) protection assay Antisense RNA probes were prepared by in vitro transcription using T7 RNA polymerase, [␣-32P]UTP, and DNA templates (mouse Mater and Zp3). As described previously (18), the antisense RNA probes (2 ⫻ 104 cpm of each probe) were simultaneously hybridized at 42 C for 24 h with oocyte and embryo lysates in 20 ␮l hybridization buffer following the instructions of an RNase protection assay kit (Ambion, Inc., Austin, TX). After digestion with RNase A/T1 (1:1000) at 37 C for 1 h, the hybridization samples were precipitated for separation on 5% denaturing polyacrylamide gel. The protected signals were detected by autoradiography.

FIG. 2. Developmental expression of Mater mRNA in mouse oocytes and embryos. RNase protection assay was used to detect Mater mRNA in developing oocytes and embryos. 32P-labeled antisense probes of mouse Zp3 (257 nt) and Mater (180 nt) were hybridized with total RNA from different stages of oocytes and embryos as indicated. The first two lanes contain RNA markers and antisense probes; other lanes demonstrate the protection of mRNA from 50 normal oocytes or 50 embryos. Arrows indicate the probe-protected mRNA fragments of mouse Zp3 (205 bp) and Mater (139 bp), respectively. Molecular sizes (nt) are indicated on the left of the panel.

In situ hybridization NIH Swiss mouse ovaries were fixed in 4% paraformaldehyde and sectioned (5 ␮m). Both [35S]UTP-labeled sense and antisense probes were synthesized by in vitro transcription using MATER cDNA as templates [nucleotides (nt) 2149 –2831]. Short probes (200 – 400 nt) were

Tong et al. • Expression and Localization of Mouse MATER

prepared by alkaline hydrolysis and hybridized (60 C, 24 h) to the ovarian sections. After dipping in Kodak NTB-2 emulsion (Eastman Kodak Co., Rochester, NY), the slides were exposed for 5 d and developed in Kodak developer D-19 and Kodak Fixer (Molecular Histology, Inc., Gaithersburg, MD). The sections were stained with hematoxylin and eosin. Hybridization signals were examined under microscopy.

Immunohistochemistry and immunoblotting Primary antibodies against mouse MATER were raised by immunizing rabbits with a keyhole limpet hemocyanin (KLH)-conjugated MATER peptide (C terminus, amino acids 1093–1111). The fixed ovary (10% formalin) was embedded in paraffin and sectioned at 5-␮m thickness. For immunohistochemistry, the ovarian sections were incubated with the primary anti-MATER peptide antisera (1:200) after removing endogenous peroxidase activity in 3% H2O2. Horseradish peroxidase (HRP)-conjugated goat antirabbit IgG antibody (1:200) and diaminobenzidene (DAB) substrate were applied according to the instructions of the manufacturer (Vector Laboratories, Inc., Burlingame, CA). The sections were counterstained with hematoxylin and eosin. For immunoblotting, proteins from the isolated oocytes, ova, and developing embryos were separated on 10% SDS-PAGE and transferred onto a nitrocellulose membrane. The blot was incubated with anti-MATER peptide antibody (1:1000) for 2 h at room temperature. After washing, the primary antibody-bound proteins were detected by HRP-conjugated goat antirabbit IgG antibody using an enhanced chemiluminescence kit according to the manufacturer’s instruction (Amersham Pharmacia Biotech, Arlington Heights, IL).

ELISA Synthetic peptides of mouse ZP3 (ZP3328 –343, NH2-CSNSSSSQFQIHGPRQ-COOH) (19), P450 –17␣-hydroxylase (P450 –17␣344 –372, NH2-

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NDRTHLLMLEATIREVLRIRPVAPLLIPH-COOH) (20), and MATER (MATER 127–146 , NH 2 -TSETLQSKEEDEVTEADKDN-COOH; MATER1093–1111, NH2-VIDGDWYASDEDDRNWWKN-COOH) (7) were coated on 96-well ELISA plates at 5 ␮g/ml according to the instructions supplied with the Protein Detector ELISA kit (Kirkegaard & Perry Laboratories, Inc., Gaithersburg, MD). Different dilutions (1:100 – 10,000) of the rabbit preimmune serum or rabbit anti-MATER peptide antiserum were added for the antigen-antibody reaction. After washing, HRP-conjugated goat antirabbit IgG antibody (1:1,000) was used to react with the antigen-antibody complex. After adding peroxidase substrate solution with H2O2, the colorimetric reaction was read at an absorbance of 405 nm using automatic microplate reader.

Laser-scanning confocal microscopic analysis Oocytes, ova, and embryos were fixed in 1% paraformaldehyde and then treated with quench solution [1⫻ PBS, 2% goat normal sera, 3% BSA, saponin (1 mg/ml), and glycine (1.5 mg/ml)]. They were incubated with rabbit anti-MATER peptide antibody (1:100,000) at 4 C overnight. Cys5-conjugated goat antirabbit IgG antibody (1:200; Molecular Probes, Inc., Eugene, OR) was used as the secondary antibody to detect the embryo-bound primary antibody.

Immunogold electron microscopic analysis Ovaries from 10-d-old mice were fixed with 4% paraformaldehyde in 0.13 m phosphate buffer, pH 7.4, for 48 h and then transferred into 1% osmium tetroxide in 0.13 m phosphate buffer, pH 7.4, for 1 h. After dehydrating in graded ethanol, the ovaries were embedded in epoxy resin (Durcupan ACM, FLUKA, Buchs, Switzerland) and sectioned (80 nm) with a diamond knife mounted in an Ultracut microtome (Reichert, Wien, Austria). The sections were mounted on nickel grids for immunogold reactions. After washing with droplets of PBS, pH 7.4, for 10 min

FIG. 3. Specificity of rabbit antisera against MATER. A, Ovarian sections of mice at age 3 wk were incubated with the rabbit anti-MATER peptide antisera (a and b, 1:100) or the preimmune rabbit sera (c, 1:100), followed by incubation with (a and c) or without (b) HRP-conjugated goat antirabbit IgG antibody (1:200) and DAB substrates. The tissue sections were counterstained with hematoxylin and eosin. Scale bar, 50 ␮m. B, Ovarian sections of wild-type (a) and Mater-null (b) mice at age 8 wk were subjected to immunohistochemical staining using anti-MATER peptide antisera (1:100) as described above. Scale bar, 50 ␮m. C, ELISA was used to detect the specificity of anti-MATER antiserum using different mouse peptides. Peptides of mouse zona pellucida protein (ZP3328 –343), P450 –17␣ hydroxylase (P450 –17␣344 –372) and MATER (MATER127–146 and MATER1093–1111) were incubated with BSA (a); rabbit anti-MATER antisera diluted at 1:10,000 (b), 1:1,000 (c), and 1:100 (d); and rabbit preimmune serum (e; 1:100). HRP-conjugated goat antirabbit antibody (1:1000) and peroxidase substrates with H2O2 were used to detect reaction of the peptide-primary antibody. Values of absorbance at OD 405 nm were the means of triplicate determinations (SD, ⱕ0.005).

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Tong et al. • Expression and Localization of Mouse MATER

FIG. 4. Developmental expression of MATER protein in oocytes and embryos. A, Immunohistochemistry. Mouse ovarian paraffin sections were incubated with rabbit antisera against MATER peptide (1:500), followed by HRP-conjugated goat antirabbit antibody and DAB substrate reaction. The tissue sections were counterstained with hematoxylin and eosin. Scale bar, 50 ␮m. B, Confocal microscopy. Specific antibody against MATER (1:100,000) and Cy5-conjugated goat antirabbit IgG antibody were used to detect MATER protein in developing oocytes or embryos as indicated. Scale bar, 50 ␮m. C, Immunoblotting. Anti-MATER antibody (1:1,000) and HRP-conjugated goat antirabbit antibody were used as primary and second antibodies to detect MATER protein in developing oocytes and embryos as indicated. Each lane contained protein from 25 normal oocytes or 25 embryos. Molecular sizes (kilodaltons) are indicated on the left.

at 25 C, the sections were incubated with droplets of blocking buffer (1% BSA, 2.5% NaCl, 0.01% sodium azide, and 1% Tween 20 in PBS, pH 7.4) for 30 min. The grids were then incubated overnight at 4 C in droplets of rabbit anti-MATER antibody (1:1200). After the sections were washed with 0.2% BSA in 0.05 m Tris buffer (pH 7.4) and were incubated with droplets of 1% BSA in 0.05 m Tris buffer (pH 8.4) for 15 min, the grids were incubated with droplets of the gold-labeled (10 nm) goat antirabbit IgG (1:25; EY Laboratories, Inc., San Mateo, CA) for 2 h at 25 C. After rinsing with PBS (pH 7.4) and microfiltered double-distilled water, the grids were dried in air. A negative control was set without primary antibodies. After staining with 1% uranyl acetate and lead citrate, the ovarian sections were examined under a Zeiss EM109 electron microscope (Carl Zeiss, Inc., Thornwood, NY).

Results Mater mRNA during oogenesis and embryogenesis

Oocyte-specific expression of Mater has been demonstrated in the mouse (7). To determine when the Mater transcripts first appear during oogenesis, we performed in situ hybridization with ovarian sections of 5- and 7-d-old mice. Based on Pederson’s classifications of mouse oocytes and follicles (21), most oocytes of type 2 follicles had hybridization signals for Mater mRNA, although a few oocytes of type 2 follicles were negative for hybridization signal (Fig. 1, A and B). As follicle growth proceeds, Mater mRNA appeared increasingly abundant in oocytes, but then declined visually as the follicles mature. As shown in Fig. 1D, the mRNA appeared most abundant in oocytes of type 3b, 4, and 5a follicles and appeared to much less in oocytes beyond type 5a follicles. Thus, Mater mRNA appears to exhibit a dynamic profile during oogenesis. To further define the developmental profile of Mater

mRNA expression, we developed a sensitive RNase protection assay (Fig. 2). As an internal control, we used an oocytespecific probe for Zp3 mRNA, the expressional profile of which has been well documented (22). Although in situ hybridization revealed the presence of Mater mRNA in the oocytes of the majority of type 2 follicles, we were not able to detect Mater mRNA in oocytes from newborn ovaries using an RNase protection assay. At this stage, the 50 oocytes may not contain sufficient mRNA to be detected by this assay. In contrast, Mater mRNA was detected in 50 growing oocytes with germinal vesicle, and its amount appeared to be reduced in ovulated ova (M2 oocytes) and was undetectable during preimplantation development. The Mater mRNA expression profile was similar to expression of Zp3 mRNA. We have noted that early mouse embryo cDNA libraries contain clones of Mater sequences, which are included in established sequence tag databases (AA792079, AU021622, and AU044455). It is likely that a few Mater transcripts escaped degradation or came from contamination with fragmented eggs during the construction of these libraries. MATER protein during oogenesis and embryogenesis

To detect MATER protein, we raised the antiserum in rabbits immunized with mouse MATER peptide (residues 1093–1111) conjugated to KLH. The rabbit antiserum against MATER peptide, but not the preimmune rabbit sera, recognized oocyte protein in immunohistochemical staining (Fig. 3A). As expected, the antiserum failed to react with oocytes of Mater-null mice (Fig. 3B). Furthermore, an ELISA showed

Tong et al. • Expression and Localization of Mouse MATER

that the antiserum only recognized the MATER peptide that was conjugated with KLH to raise antisera, but did not react with a second MATER peptide (residues 127–146) or with ZP3 and P450 –17␣-hydroxylase peptides (Fig. 3C). Also, the immunoblotting showed that the anti-MATER peptide antiserum could detect an oocyte protein with 125 kDa, the expected size of mouse MATER protein (see below), but did not detect proteins of Mater-null oocytes (data not shown). Thus, we conclude that the antisera are specific to MATER protein. To determine the location of MATER protein in oocytes and early embryos, we conducted immunohistochemistry and confocal microscopic analyses. Using this antibody, MATER immunoreactivity was not detected in the oocytes of type 2 follicles, but was found in oocytes of type 3a small primary follicles (Fig. 4A). MATER protein was predominately localized in the oocyte cytoplasm, but some structures within the nuclei also appeared to have positive staining, as confirmed by immunogold electron microscopic observation (see below). As controls, preimmune serum did not stain the oocytes. Confocal microscopic analysis confirmed a predominant cytoplasmic location of the MATER protein in the developing oocyte and early embryo. MATER protein was also confirmed to be present in the early blastocyst. The protein was more evident in the trophectodermal outer layer cells than in the inner cells. However, MATER protein was barely detectable in the later stages of blastocyst development (Fig. 4B). Anti-MATER antisera diluted at 1:100,000 did not detect MATER protein in the nuclei of oocytes and preimplantation embryos examined by confocal microscopic analysis. To further examine the developmental profile of MATER protein expression, we used an immunoblotting assay in which total proteins from equal numbers of developing oocytes or embryos (25 each) were loaded. The 125-kDa MATER protein was detected in early growing oocytes and all stages of preimplantation embryos (Fig. 4C). Thus, this profile of MATER protein expression during oogenesis and embryogenesis, as determined by immunoblotting assay, was similar to that determined by confocal microscopic analysis. Subcellular localization of MATER

To localize MATER distribution in the subcellular organelles of oocytes, we conducted immunogold reactions using specific antibody and ultrathin sections of mouse ovaries. Immunogold particles, indicating the presence of MATER protein, were examined under transmission electron microscopy. Immunogold particles were seen within the oocytes of primary follicles, but were not detected in the surrounding granulosa cells. The immunogold particles were specifically located in the mitochondria, but not in other oocyte cytoplasmic organelles (Fig. 5). In addition, immunogold particles were detected within the nucleolus and close to oocyte nuclear pores (Fig. 6). Oocytes of primary follicles at different stages consistently exhibited MATER protein in the mitochondria and nucleolus (data not shown). The oocyte mitochondria of type 2 follicles did not show the presence of immunogold particles (Fig. 5D).

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FIG. 5. Localization of MATER protein in oocyte mitochondria. Using antimouse MATER antisera and ultrathin ovarian sections of 10-dold mice, immunogold reactions were examined by transmission electron microscopy. Black spots with arrows are immunogold particles indicating the presence of MATER protein. A, Oocytes and surrounding granulosa cells (⫻2,800). The positions of nucleus (n), cytoplasm (c), and granulosa cells (g) are indicated. B, Oocyte mitochondria with immunogold particles (⫻46,500). C, Mitochondium with immunogold particles at higher magnification (⫻100,200). D, Oocyte mitochondria without immunogold particles in an early type 2 follicle (⫻46,500).

Ovarian folliculogenesis and oocyte growth, maturation, and ovulation in Mater-null mice

Mater-null mice appear phenotypically normal. As stated previously (6), Mater-null female mice appear to undergo normal oogenesis and folliculogenesis as well as unimpaired oocyte maturation and ovulation. As shown in Fig. 7, Maternull ovarian sections indicate the presence of all stages of oogenesis and folliculogenesis, indistinguishable from the histology of wild-type ovaries. Also, typical corpora lutea indicating past ovulation were observed in adult ovaries. Mater-null female mice responded normally to exogenous gonadotropin stimulation, as evidenced by normal oocyte maturation and ovulation. The morphology and numbers of ovulated ova were indistinguishable between Mater-null and wild-type mice. Thus, the Mater-null mutation does not appear to affect ovarian development, oocyte maturation, or ovulation. Discussion

Maternal effect genes encode proteins that are produced during oogenesis and play a role during early embryogenesis. Previously, we showed that Mater is required for early embryonic development in mice (6). In the present study we

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FIG. 6. Localization of MATER protein in nucleoli and close to nuclear pores of oocytes. Using antimouse MATER antisera and ultrathin ovarian sections of 10-d-old mice, immunogold reactions were examined by transmission electron microscopy. Black spots with arrows are the immunogold particles indicating the presence of MATER protein. A, An oocyte with surrounding granulosa cells (⫻5,200). The positions of nucleus (n), cytoplasm (c), and granulosa cells (g) are indicated. The arrow indicates the nucleolus enlarged in B. B, Nucleolus with immunogold particles (⫻27,000). C, Nuclear pore with immunogold particles nearby (⫻45,000; arrows).

FIG. 7. Ovarian histology and ovulation. Ovarian sections from Mater-null mice, aged 4 (A) and 8 (B) wk, were stained with periodic acid-Schiff reagent. Scale bar, 200 ␮m. Mater-null mouse ovarian histology showed normal-appearing oocytes, follicles, and corpora lutea (CL), indistinguishable from those of wild-type mice (not shown). C, Normal-appearing morphology of ovulated ova from Mater-null mice after stimulation with exogenous pregnant mare serum gonadotropin and hCG. Scale bar, 50 ␮m. D, Numbers (mean ⫾ SEM) of ovulated ova from Mater-null and wild-type mice.

demonstrated that the temporal and spatial patterns of Mater expression are developmentally regulated in a manner typical of maternal effect genes. Mater mRNA was detected earliest in oocytes of type 2 follicles and appeared to accumulate during the growth phase of oogenesis. Similar to many oocyte transcripts, Mater transcription appeared to halt, and most of its mRNA was degraded in the process of meiotic maturation and ovulation. Mater mRNA was unde-

tectable in two-cell embryos and developing embryos throughout preimplantation development. This suggests that Mater mRNA is destroyed and the Mater gene is silenced in the process of embryonic genome activation. Thus, Mater appears to be transcribed only during oogenesis, and its product is derived from the maternal genome exclusively. MATER protein was first detected in oocytes of the type 3a early primary follicles in mice using immunohistochem-

Tong et al. • Expression and Localization of Mouse MATER

ical and immunogold techniques. The protein was not detected in resting oocytes of type 2 follicles by these histological methods or by immunoblotting. The fact that Mater mRNA is present in most oocytes of type 2 follicles, but the protein is not yet present suggests the possibilities of posttranscriptional regulation and transient uncoupling of transcription and translation. However, it is clear that MATER transcription-translation coupling still takes place during the growth phase of oogenesis. Thus, Mater mRNA belongs to the pool of transcripts ready for translation during oocyte growth, not to the pool of those stored for later translation during oocyte maturation and ovulation (23, 24). MATER protein accumulated during oogenesis and persisted during early embryogenesis. Mater-null oocytes and their associated follicles appeared morphologically normal. Ovulation and fertilization also appeared to proceed normally in Mater-null female mice. As there appears to be no detectable Mater mRNA in the preimplantation embryos, its protein appears to be made exclusively during oogenesis and stored in oocytes in preparation for supporting subsequent embryogenesis. In a phenotype, the absence of MATER protein has a specific detrimental effect solely at the time of expected embryonic genome activation. However, there still is the possibility of some subtle defects in oocyte or follicle function in Mater-null mice. Immunogold electron microscopy revealed that MATER protein is present in mitochondria and nucleoli, and close to nuclear pore complexes of mouse oocytes. As detected by immunohistochemical and confocal analyses, MATER protein is distributed predominantly in the cytoplasm. It is presumed that MATER protein is transported from mitochondria to nucleoli through nuclear pore machinery. As mouse MATER protein does not have signals of either mitochondrial targeting or nuclear localization, it is likely that other molecules interacting with MATER mediate its transport into these organelles. Indeed, MATER protein contains a leucinerich repeat domain and a short leucine zipper, both of which are known to mediate protein-protein interactions (25–27). It remains to be determined whether redistribution of MATER protein takes place during embryonic development. Localization of MATER protein in multiple organelles implies its involvement in different intracellular activities. Its presence in mitochondria suggests a role in cytoplasmic metabolism and signaling. Given their role in oxidative phosphorylation (ATP production) and cellular metabolism as well as programmatic cell death (apoptosis) (28 –30), mitochondria play important roles during oogenesis and embryogenesis (31). For example, mitochondria undergo remarkable microtubule-mediated redistribution during late oogenesis and early embryogenesis (32). Disruption of this process adversely affects chromosomal organization and segregation (33). Mitochondrial dysfunction can profoundly reduce ATP production and induce aberrant embryonic development (34). It has been proposed that apoptosis may be the default pathway for early embryos that fail to reach essential developmental milestones appropriately. Fragmentation of early embryos is accompanied by the activation of regulatory proteins inducing apoptosis, although the mitochondrial role in embryonic apoptosis is not yet defined (35). In addition to its potential role associated with the mito-

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chondria, the presence of MATER protein in nucleoli raises the possibility of a role in nuclear control of ribosome biogenesis, smaller RNA transcription, and cell cycling (36, 37). In the nuclear events of embryonic development, it has been reported that NPM2, a mammalian ortholog of Xenopus nucleoplasmin 2 protein, plays a role in embryonic nuclear and nucleolar organization (17). The absence of NPM2 protein results in structural anomalies of the nucleolus in oocytes and zygotes. This paradigm raises the possibility that nucleoli lacking MATER protein might result in oocyte or embryonic anomalies in nucleolar organization and chromatin function. Examining the function and morphology of mitochondria and nucleoli in Mater-null oocytes and embryos may provide insights into the function of MATER protein. In this report we present evidence for the exclusive maternal origin and developmental expression of Mater mRNA and its protein. The finding that MATER protein is localized to mitochondria and nucleoli provides intriguing clues to its potential function in oogenesis and embryogenesis. Its presence in the different compartmental organelles suggests that MATER protein may contribute to both cytoplasmic and nuclear controls of embryonic genome activation. Acknowledgments We are very grateful to Dr. Jurrien Dean for his invaluable help. We thank our colleagues for constructive discussions. Received September 4, 2003. Accepted December 4, 2003. Address all correspondence and requests for reprints to: Dr. Zhi-Bin Tong, Developmental Endocrinology Branch, National Institute of Child Health and Human Development, National Institutes of Health, Building 10 Room 10N262, Bethesda, Maryland 20892-1862. E-mail: tongz@ exchange.nih.gov.

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