Identification of Gasz, an Evolutionarily Conserved Gene Expressed Exclusively in Germ Cells and Encoding a Protein with Four Ankyrin Repeats, a Sterile-
Motif, and a Basic Leucine Zipper
Wei Yan,
Aleksandar Rajkovic,
Maria M. Viveiros,
Kathleen H. Burns,
John J. Eppig and
Martin M. Matzuk
Departments of Pathology (W.Y., M.M.M.), Department of Molecular and Cellular Biology (M.M.M.), Department of Molecular and Human Genetics (M.M.M., K.H.B.), Department of Obstetrics and Gynecology (A.R.), Baylor College of Medicine, Houston, Texas 77030; The Jackson Laboratory (M.M.V., J.J.E.), Bar Harbor, Maine 04609
Address all correspondence and requests for reprints to: Martin M. Matzuk, M.D., Ph.D., Stuart A. Wallace Chair and Professor, Department of Pathology, Baylor College of Medicine, One Baylor Plaza, Houston, Texas 77030. E-mail: mmatzuk{at}bcm.tmc.edu.
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ABSTRACT
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To discover causes of infertility and potential contraceptive targets, we used in silico subtraction and genomic database mining to identify conserved genes with germ cell-specific expression. In silico subtraction identified an expressed sequence tag (EST) present exclusively in a newborn mouse ovary library. The full-length cDNA sequence corresponding to this EST encodes a novel protein containing four ankyrin (ANK) repeats, a sterile-
motif (SAM), and a putative basic leucine zipper (bZIP) domain. Northern blot and semiquantitative RT-PCR analyses demonstrated that the mRNA is exclusively expressed in the mouse testis and ovary. The expression sites were localized by in situ hybridization to pachytene spermatocytes in the testis and oocytes in the ovary. Immunohistochemistry showed that the novel protein is localized to the cytoplasm in pachytene spermatocytes and early spermatids, oocytes at all stages of oogenesis, and in early preimplantation embryos. Based on its germ cell-specific expression and the presence of ANK, SAM, and basic leucine zipper domains, we have termed this novel protein GASZ. The mouse Gasz gene, which consists of 13 exons and spans 60 kb, is located on chromosome 6 between the Wnt2 and cystic fibrosis transmembrane conductance regulator (Cftr) genes. Using genomic database mining, orthologous genes encoding GASZ were identified in the rat, cow, baboon, chimpanzee, and human. Phylogenetic analyses reveal that the GASZ proteins are highly conserved among these species. Human and mouse GASZ proteins share 85.3% amino acid identity, and human and chimpanzee GASZ proteins differ by only 3 out of 475 amino acids. In humans, the GASZ gene resides on chromosome 7 and is similarly composed of 13 exons. Because both ANK repeats and the SAM domain function as protein-protein interaction modules that mediate signal transduction cascades in some systems, GASZ may represent an important cytoplasmic signal transducer that mediates protein-protein interactions during germ cell maturation in both males and females and during preimplantation embryogenesis.
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INTRODUCTION
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SPERMATOGENESIS AND OOGENESIS are complex processes under the control of both extragonadal and intragonadal signals. The exact molecular mechanisms and the factors that govern these events remain largely unknown. Studies over the past decades have demonstrated that both endocrine hormones (e.g. FSH and LH and locally generated paracrine factors affect these processes. The hypogonadal (hpg/hpg) mouse, which has reduced levels of FSH and LH due to a Gnrh mutation, develops an early block in folliculogenesis causing infertility (1, 2). Similarly, FSHß knockout (3) and FSH receptor knockout (4, 5) female mice are infertile due to an early block in folliculogenesis before antral follicle formation. On the other hand, oocytes secrete factors necessary for normal granulosa cell and theca cell function. Female mice lacking growth differentiation factor-9 (GDF-9), an oocyte-specific TGF-ß family member, are infertile owing to a block in folliculogenesis at the one-layer primary follicle stage (6, 7, 8). In contrast to kit ligand and other growth factors synthesized by the somatic cells that affect oocyte growth (7, 9), GDF-9 functions as an oocyte-derived growth factor required for somatic cell function.
This endocrine/paracrine model also applies to spermatogenesis. FSH acts on Sertoli cells, and Sertoli cells produce numerous factors that regulate germ cell maturation along the seminiferous epithelium. LH regulates Leydig cell production of testosterone, and testosterone acting through its receptor in Sertoli cells, and thus affects Sertoli cell function and thereby indirectly regulates germ cell proliferation and differentiation (10, 11, 12). Sertoli cell-produced kit ligand is regulated by FSH, and kit ligand acts through binding to its receptor on spermatogonia and spermatocytes to regulate male germ cell proliferation, differentiation, and survival (13, 14, 15). Male mice that fail to produce kit ligand or KIT receptor display disrupted spermatogenesis. Like oocytes, male germ cells can also secrete factors that can regulate the functional status of somatic cells in the testis (16). This reciprocal interaction between germ cells and somatic cells is a key determinant of synchronized germ cell growth and maturation in both sexes. Therefore, identification of genes that are involved in the paracrine interactions between germ cells and somatic cells may ultimately lead to the development of effective contraceptive measures and treatments for infertility.
We have discovered a number of novel genes that are specifically expressed in the ovary and the testis using in silico subtraction (17, 18, 19). Here, we report another novel germ cell-specific gene, Gasz, which we discovered through in silico analyses of expressed sequence tags (ESTs) from a murine newborn ovary cDNA library in the Unigene collection at the National Center for Biotechnology Information (NCBI). Furthermore, genomic database mining allowed us to electronically identify orthologous genes encoding GASZ. On the basis of its expression pattern, cellular localization, conserved domains, and high degree of phylogenetic conservation, we postulate that GASZ functions as an important cytoplasmic signaling protein in germ cells and early embryos.
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RESULTS
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Identification of a Novel Mouse EST by in Silico Subtraction
We identified 7577 ESTs from a murine newborn ovary cDNA library, which were deposited in the Unigene database at the NCBI (http://www.ncbi.nlm.nih.gov). In silico subtraction of the newborn ovary ESTs identified more than 100 ESTs that were present in newborn ovaries but absent in ESTs in libraries from other tissues. The expression patterns of the ESTs in multiple tissues were screened by semiquantitative RT-PCR, and only those detected in the ovary and/or the testis were chosen for further study. Among 26 ESTs that were selected, one EST matched a full-length cDNA that was deposited by RIKEN (AK016595). Using the full-length cDNA to search the entire NCBI database, we found that the majority of matched sequences were derived from the testis. Two independent EST clones (AK016595 and NM_023729.1) were purchased from ATCC (Manassas, VA) and sequenced to confirm the accuracy of the published cDNA sequence. One of the deposited EST sequences (AK016595) was inaccurate based on our sequence analysis of the purchased EST clones and RT-PCR generated cDNA fragments. Furthermore, we compared these cDNA sequences with two independently published mouse genomic fragments (AC068561 and AF162137) that contain the corresponding mouse gene. The full-length cDNA sequence is shown (Fig. 1
).

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Figure 1. The Mouse Gasz Full-Length cDNA and Its Encoded Protein
The mouse Gasz cDNA contains an ORF that encodes a protein of 475 amino acids. The mouse GASZ protein consists of four consecutively arranged ANK repeats (ANK14), a SAM, and a bZIP domain. The first ANK motif is underlined, the second ANK is double-underlined, the third ANK is dotted-underlined, and the fourth ANK is shaded. The SAM domain is framed, and the bZIP domain is both underlined and shaded.
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Mouse Gasz Encodes a Protein with 4 Ankyrin (ANK) Repeats, a Sterile-
Motif (SAM), and a Basic Leucine Zipper Domain (bZIP)
The identified cDNA contains a 1428-bp-long open reading frame (ORF), which encodes a predicted protein of 475 amino acids (Fig. 1
). Using the deduced protein sequence to search the Pfam protein family database (http://www.sanger.ac.uk/Software/Pfam/search.shtml), we found that this predicted protein is composed of 4 ANK repeats, a SAM, and a bZIP domain (Fig. 1
). Given that this gene is exclusively expressed in germ cells (see below) and the protein contains ANK, SAM, and bZIP domains, we have termed our gene Gasz.
The domain structure of the protein is schematically illustrated (Fig. 2A
). The alignment analysis shows that residues crucial for the arrangement of
-helices and ß-hairpins, which are characteristic of the ANK repeat architecture, are mostly conserved in all four ANK repeats at the N terminus of the GASZ protein (Fig. 2B
). A SAM domain is located in the middle of the GASZ protein (Figs. 1
and 2A
). Alignment of the GASZ sequence with several SAM-containing proteins shows that the hydrophobic residues necessary for formation of a small five-helix bundle are all highly conserved (Fig. 2C
). Lastly, the bZIP domain is located in the C terminus of the protein, and conserved residues are shown (Fig. 2D
).

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Figure 2. Genomic Structure of the Gasz Gene and Conserved Domains of the GASZ Protein
A, Schematic illustration of the mouse Gasz genomic structure, and the exon sequences that correspond to specific GASZ protein domains. The mouse Gasz gene consists of 13 exons (solid boxes) that are the following sizes: 124, 100, 123, 112, 112, 135, 125, 76, 56, 110, 106, 114, and 432 bp. Intron 4 is approximately 26 kb and intron 10 is about 16 kb. Four ANK repeats (ANK 14) are tandemly arranged in the N terminus of the protein. A SAM domain is located in the middle of the protein, and a bZIP motif resides at the C terminus of the protein. B, Protein alignment of the four ANK repeats (ANK 14). The critical ANK residues for arranging -helices and ß-hairpins are marked with asterisks. Regions of high conservation are framed. C, Sequence alignment of SAM domains. The positions of the residues used for alignment with the mouse (m) and human (h) GASZ amino acid are listed next to the names of each protein. The conserved hydrophobic residues are marked with asterisks, and the positions of the five helices (H15) are shown at the bottom of the alignment. All consensus residues were highlighted. The accession numbers of the sequences in this alignment are: p53squid (U43595), p63 (AF075430), p73(Y11416), BicaudalC (1085137), LAR-interacting protein 1(LAR1P; U22815), Neurabin (U72994), Diacylglycerol kinase (DGK, Q16760). D, Sequence alignment of the leucine zipper motif in mouse and human GASZ proteins. The conserved residues are highlighted.
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Identification of the Human GASZ Ortholog
In our in silico subtraction and database mining studies, we found several human ESTs (BG771896, BG720639, BI461419, BI560050, BG771697, BI561532, and BI561909) that share very high homology with the mouse Gasz cDNA. All of these ESTs were derived from human testis. On the basis of the 5'-end sequence (
780 bp) and the 3'-end sequence deduced from the human GASZ cDNA, we amplified the 5'- and 3'-end fragments of human GASZ by PCR using human testis cDNA. These PCR products were sequenced and compared with the partial cDNAs in the EST database and sequences deduced from human genomic fragments deposited into the NCBI database (see below). The complete human GASZ cDNA and deduced amino acid sequence is shown (Fig. 3
).

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Figure 3. Full-Length cDNA and Predicted Protein Sequences of Human GASZ
The human cDNA contains an ORF encoding a protein of 475 amino acids. Like mouse GASZ, human GASZ also contains four ANK repeats (ANK 14), a SAM, and a bZIP domain. The first ANK motif is underlined, the second ANK is double-underlined, the third ANK is dotted-underlined, and the fourth ANK is shaded. The SAM domain is framed and the bZIP domain is both underlined and shaded.
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Genomic Structure of the Mouse Gasz Gene
Using the mouse Gasz cDNA sequence to search the nonredundant database of NCBI, we found two mouse genomic fragments (AC068561 and AF162137) that contain the Gasz gene. The mouse Gasz gene consists of 13 exons that span approximately 60 kb on the long arm of chromosome 6 between the Wnt2 and cystic fibrosis transmembrane conductance regulator (Cftr) genes (20) (Figs. 2A
and 4
). Gasz and Wnt2 genes are transcribed in the same direction, opposite to the transcriptional orientation of Cftr (Fig. 4
). The distance between Gasz and Wnt2 is approximately 40 kb, and the distance between Gasz and Cftr is about 52 kb (Fig. 4
).

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Figure 4. Chromosomal Localization of the Mouse Gasz and Human GASZ Genes
The mouse Gasz gene is located between the Wnt-2 and Cftr genes on proximal mouse chromosome 6. A similar relationship between human WNT2, GASZ, and CFTR genes is also found on the long arm of human chromosome 7. Arrows represent transcriptional orientation of the genes.
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Identification of Orthologous Genes Encoding GASZ
Using the BLAST program (http://www.ncbi.nlm. nih.gov/BLAST) to search the entire NCBI nonredundant database, we identified genes encoding GASZ orthologs in other species including the rat (AC087112.2 and AC087213), cow (AC091728 and AC089992), baboon (AC087555.3 and AC089990.3), chimpanzee (AC087730 and AC087834.2), and human (AC002465 and AC021193.7). Two independent sequences from each species were downloaded and compared to confirm sequence accuracy. Exon/intron boundaries were identified by aligning mouse exons with these genomic fragments.
These GASZ ortholog genes are located between the WNT2 and CFTR genes (Fig. 4
). The human GASZ gene resides on human chromosome 7q31.31 (21). By analyzing the genomic structures of the GASZ orthologs, we found that the exon/intron boundaries of the GASZ genes were highly conserved among these species (Fig. 13, published as supporting information on The Endocrine Societys Journals Online web site at http://mend.endojournals.org/). All six genes are composed of 13 exons, and the sizes of each exon are identical in all the species with the exception of the last exon. Each GASZ protein is exactly the same length (475 amino acids), and alignment analysis of the deduced protein sequences revealed that the 6 proteins share high homology (Fig. 5
). Phylogenetic analysis suggests that the GASZ proteins of these different mammalian species have been highly conserved evolutionarily. GASZ protein identity ranges from 84.9% (mouse vs. chimpanzee) to 99.2% (chimpanzee vs. human). Mouse and rat GASZ proteins are 97.5% identical, and the mouse and human GASZ proteins share 85.3% identity (Fig. 14 of supporting information).

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Figure 5. Alignments of GASZ Proteins in Different Species
The mouse, rat, cow, baboon, chimpanzee, and human GASZ proteins are aligned and identical residues are shaded. As expected, the two rodent GASZ proteins are most similar, and the three primate GASZ proteins share highest identity. The orthologous GASZ genes appear to be single copy in all six mammalian species. Analysis of the 5' ends of cDNAs or genes in all cases fails to identify any additional upstream start codons.
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Expression Profile and Localization of Mouse Gasz mRNA and Human GASZ mRNA
The full-length mouse Gasz cDNA was used for Northern blot analysis of multiple mouse tissue, and it hybridized to a 1.9-kb transcript exclusively in mouse testis and ovary (Fig. 6A
). A minor 2.4-kb band could also be seen in the testis sample (Fig. 6A
). By sequence analysis, we found that the 2.4-kb transcript contains a longer 3' untranslated region sequence, whereas the entire ORFs of both cDNAs were identical (data not shown). Semiquantitative RT-PCR analysis confirmed that the Gasz mRNA is expressed exclusively in the testis and ovary (data not shown). Because Gasz mRNA was found to be gonad specific, we further analyzed the expression pattern of Gasz mRNA in developing mouse testes and ovaries by using Northern blot analysis. In the testis, Gasz mRNA is first detected at postnatal d 10, whereas its expression is much higher in the newborn and 2-wk-old ovaries than in the adult ovaries (Fig. 6
, A and B). Semiquantitative RT-PCR of human tissues showed that the human GASZ mRNA was also expressed exclusively in the gonads and at higher levels in the adult human testis (Fig. 6C
) compared with the ovary.

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Figure 6. Expression Profiles of the Mouse Gasz and Human GASZ Genes
A, Northern blot analysis of mouse Gasz mRNA expression in multiple mouse tissues. Total RNA isolated from adult mouse heart (He), liver (Li), spleen (Sp), kidney (Ki), brain (Br), stomach (St), small intestine (In), testis (Te), ovary (Ov) and uterus (Ut), as well as newborn (NB), 2-wk-old (2W), and adult (AD) ovaries was used for Northern blot analysis. Full-length Gasz cDNA labeled with 32P-dATP was used as a probe. The blots were rehybridized with an 18S rRNA cDNA probe, which served as a loading control. B, Gasz mRNA expression during testicular development. Total RNA from 5-d-old (5d), 10-d-old (10d), 15-d-old (15d), 20-d-old (20d), 35-d-old (35d), and 60-d-old (60d) testes were isolated for Northern blot analysis. An aliquot of 15 µg total RNA was loaded in each lane and the full-length mouse Gasz cDNA labeled with 32P-dATP was used as a probe. C, Human GASZ mRNA expression in multiple tissues as determined by semi-quantitative RT-PCR. Human cDNAs from multiple tissues were used as templates. As a loading control, human ß-ACTIN was amplified.
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In the postnatal d 5 mouse testis, the germ cell population within the seminiferous epithelium mainly consists of spermatogonia (Fig. 7F
), whereas spermatocytes are absent (22). By d 15, pachytene spermatocytes account for about 1/3 of the total cell population in the seminiferous epithelium (23, 24). Once the first cycle of spermatogenesis is accomplished by postnatal d 20, pachytene spermatocytes are present in every stage along the epithelial cycle. Absence of mRNA expression before d 5 (Fig. 6B
), a dramatic increase by postnatal d 10 (Fig. 6B
), and continued high levels of expression through the adult stage (Fig. 6B
) strongly suggested that the Gasz mRNA is expressed in pachytene spermatocytes in the mouse testis.

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Figure 7. Localization of Gasz mRNA in the Mouse Testis using in Situ Hybridization
Brightfield (A, C, E) and darkfield (B, D) images are shown. Specific hybridization signals were present in spermatocytes (Sp) of the 15-d-old testis (A, B, x200), which are located in the middle of the seminiferous tubules. In the adult testis, specific signals are confined to the adluminal compartment in all stages along the seminiferous epithelium (C, D, x100). High magnification (E, x640) view of a stage IX tubule shows that specific signals are confined to pachytene spermatocytes (Sp). No significant mRNA signal is seen over leptotene (L) spermatocytes or spermatids (Sd). F, Schematic illustration of Gasz mRNA expression sites during mouse testicular development. The frames represent cells present in the testes at each age. The solid bars inside the frames after 10 d represent the cell type (pachytene spermatocyte) that expresses Gasz mRNA. LC, Leydig cells; SC, Sertoli cells; Sg, spermatogonia; PL, preleptotene spermatocytes; P, pachytene spermatocytes; RSp, round spermatids; Esp, elongating spermatids; EdSp, elongated spermatids; Sz, spermatozoa.
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To determine the expression site of Gasz mRNA in both male and female gonads, we performed in situ hybridization analysis. Gasz mRNA was undetectable in the 5-d-old mouse testis (data not shown). In the 15-d-old testis, initial expression of Gasz is detected in pachytene spermatocytes (Fig. 7
, A and B). In the adult testis, the Gasz mRNA was detected exclusively in pachytene spermatocytes at all stages along the epithelial cycles (Fig. 7
, CE). Thus, Gasz mRNA is restricted to the pachytene spermatocytes in both the developing and adult testis (Fig. 7F
).
As mentioned above, the Gasz sequence was initially identified from a newborn ovary cDNA library. In situ hybridization showed that the Gasz mRNA is highly expressed in oocytes of primordial follicles in the 5-d-old ovary (Fig. 8
, A and B) and oocytes of primordial and primary follicles in the 2 wk ovary (Fig. 8
, C and D). In the adult ovary, the Gasz mRNA is expressed in growing oocytes of all follicles, but at a lower level (Fig. 8
, E and F). Thus, Gasz is expressed in a germ cell-specific pattern in the gonads of males and females.

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Figure 8. Localization of Gasz mRNA in the Mouse Ovary using in Situ Hybridization
Bright-field (A, C, E) and dark-field (B, D, E) images are shown. Specific signals were detected in oocytes of primordial follicles (PF) in the 5-d-old ovary (A, B; x400). In the 2-wk-old ovary, signals were confined to oocytes in primary follicles (1F) (C, D; x200). In the adult ovary, the levels of hybridization signals were much lower than in young ovaries. Hybridization signals were randomly detected in oocytes at different types of follicles (E, F; x50). AF, Antral follicle.
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Expression of GASZ Protein in Mouse Tissues
By using the pET protein production system, we produced a full-length mouse GASZ protein fused with 6-His tags at the C terminus of the protein. The protein was exclusively expressed in inclusion bodies in Escherichia coli (strain BL-21). Using a His affinity column purification method, we purified the recombinant GASZ protein. The purified GASZ protein was used to immunize rabbits to generate polyclonal antibodies. To test the specificity and confirm the tissue distribution of GASZ protein in the mouse, we performed Western blot analysis (Fig. 9
). The rabbit anti-GASZ antibody detects as little as 1 ng of recombinant GASZ protein, and preimmune serum did not react with the recombinant protein (data not shown). Western blot analysis revealed that the molecular size of GASZ protein is approximately 53 kDa, which is consistent with the size predicted on the basis of the Gasz cDNA. GASZ is exclusively expressed in gonads, and the protein level is higher in the testis than in the ovary. These results are consistent with our Northern blot (Fig. 6A
) and RT-PCR analysis.

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Figure 9. Western Blot Analysis of GASZ Protein in Multiple Mouse Tissues
A rabbit polyclonal antibody raised against full-length GASZ protein was used at a dilution of 1:1,000. The specific 53-kDa bands were only detected in the testis and ovary (upper panel). The membrane was stripped and subsequently blotted with an anti-ACTIN monoclonal antibody to monitor the loading (lower panel). Li, Liver; Sp, spleen; Lu, lung; St, stomach; In, intestine; Te, testis; Pr, prostate; Ov, ovary; Ut, uterus.
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Localization of GASZ Protein in the Testis and Ovary
We further analyzed GASZ protein expression and localization in the mouse gonads using immunohistochemistry. In the testis (Fig. 10
), the immunoreactivity was mainly detected in the cytoplasm of pachytene spermatocytes at all stages along the seminiferous epithelial cycle. Interestingly, GASZ protein expression displayed a stage-specific pattern (Fig. 10E
). GASZ protein expression was at a low level in spermatocytes at stages IVII. GASZ protein expression started to increase at stage VII and reached the highest levels at stages IXXII (Fig. 10
, A, B, and E). After meiotic division, GASZ protein expression persisted in step 1 round spermatids (Fig. 10C
) and gradually diminished in spermatids after stage II. Thus, the highest level of GASZ protein was observed mainly in late pachytene stages where meiotic cell division is taking place. GASZ protein was also detected in the cytoplasm of oocytes in primordial follicles of 5-d-old mice (Fig. 11A
). In the adult ovary (Fig. 11B
), GASZ was also detected in the cytoplasm of oocytes in follicles of all stages including primary, secondary, and antral follicles. In the Gdf9-/- ovary, folliculogenesis is blocked at the primary follicle stage (6). GASZ protein was expressed abundantly in the cytoplasm of oocytes in the blocked primary follicles of the knockout mice (Fig. 11C
).

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Figure 10. Immunohistochemical Detection of GASZ Protein in the Mouse Testis
GASZ protein is mainly expressed in pachytene spermatocytes and the expression displays a stage-specific pattern. GASZ protein is highly expressed in pachytene spermatocytes (Sp) at stage IX (A) and meiotically dividing spermatocytes (M) including secondary spermatocytes (Ssp) at stage XII (B). After the completion of meiotic division, GASZ protein expression is still present in step 1 spermatids (Sd) (C) but disappears after step 2 in spermatids. A control section stained with preimmune serum is shown (D). E, Schematic illustration of GASZ protein expression in the mouse seminiferous epithelium. The specific cell associations in the vertical columns represent specific stages (Roman numerals) of the epithelial cycle. Sc, Sertoli cells; A14, type A14 spermatogonia; In, intermediate spermatogonia; B, type B spermatogonia; Pl, preleptotene spermatocytes; L, leptotene spermatocytes; Z, zygotene spermatocytes; Di, diplotene spermatocytes; M, meiotically dividing spermatocytes; Sd, spermatids. GASZ-expressing cells are framed, and the width of the red frame represents the intensity of GASZ immunoreactivity.
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Figure 11. Immunohistochemistry of GASZ Protein in Mouse Ovaries
The cytoplasm of oocytes in primordial follicles (PF) of the 5 d mouse ovary are heavily stained (A). In the adult ovary (B), GASZ immunoreactivity was detected in the cytoplasm of oocytes in the primary (1F), secondary (2F), and antral follicles (AF). Folliculogenesis is blocked at primary follicle stage in Gdf9-/- mice (6 ). In the Gdf9-/- mouse ovary, GASZ immunoreactivity is detected in the cytoplasm of oocytes in all primary follicles (C). Primordial follicles in the adult wild-type (B) and Gdf9-/- (C) ovaries demonstrate reduced staining compared with the 5-d-old (A) mouse ovary. A control section stained with preimmune serum is shown in D.
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Expression of GASZ Protein in Oocytes and Early Cleavage Embryo
Indirect immunofluorescent labeling with the anti-GASZ antisera was used to assess protein expression in oocytes throughout meiotic maturation, as well as early preimplantation embryos. GASZ expression was evaluated using a confocal microscope. Fully grown oocytes were evaluated before the resumption of meiosis (GV stage), and after progression to metaphase II; at both of these stages, GASZ protein was localized throughout the cytoplasm (Fig. 12
, BD), a result consistent with our immunohistochemistry results above. No GASZ expression was discernable in the oocyte-associated granulosa cells (Fig. 12B
). GASZ protein assessment after fertilization demonstrated that the protein persists in early zygotes (Fig. 12E
), as well as early cleavage embryos at the 2-cell, 4-cell and 8-cell stages (Fig. 12
, FH). Thus, in females, GASZ could theoretically function at any stage of oogenesis and in early embryos.

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Figure 12. GASZ Protein Is Expressed in Oocytes throughout Meiotic Maturation and Early Preimplantation Embryos
Confocal microscopic analysis of GV-stage oocytes (B, C) and mature metaphase II eggs (D) demonstrates GASZ protein (shown in green) throughout the cytoplasm. Oocyte-associated granulosa cells exhibit bright (red) DNA staining, but no GASZ expression. GASZ protein persists in early zygotes collected 6 h post fertilization (E) and early cleavage embryos at the 2-, 4-, and 8-cell stage (FH). Negative control oocytes (A), tested with preimmune rabbit serum, showed no discernable protein expression. Double and single arrows indicate polar bodies and condensed sperm heads on the cell surface, respectively.
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DISCUSSION
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ESTs are short cDNA sequences derived from mRNAs of various sources. There are now more than 3 million human and 2 million murine ESTs available in the public database. These EST databases represent a tremendous resource for identifying tissue-specific transcripts. Using in silico subtraction analysis of the mouse ESTs in the databases (17), we previously identified an oocyte-specific gene, Nobox (18), a gonad-specific gene, Rfpl4 (19), and a family of germ cell-specific homeobox genes, the 6 member Obox family (25). In the present study, we report a novel germ cell and early embryo-specific gene, Gasz, that we identified using this strategy. This electronic sequence subtraction method provides a means for discovering novel gonad-specific genes. In addition, more and more genomic sequences from different species are being deposited to public databases. Most of these deposited sequences are bacterial artificial chromosome clones containing one or two known genes and possibly unknown genes as well. By genomic database mining, we identified GASZ orthologs in rat, cow, baboon, chimpanzee, and human.
The GASZ genes show high sequence conservation during species divergence, and their genomic structures, exon/intron boundaries, exon sizes, and predicted protein structures are nearly identical in the six mammalian species studied. This strongly suggests that GASZ fulfills important physiological functions in reproduction. Based on the expression of Gasz mRNA and GASZ protein, GASZ may mediate events of gametogenesis in both sexes as well as processes of early embryonic development. In the mouse testis, Gasz mRNA is expressed exclusively in developing pachytene spermatocytes, and GASZ protein is abundant in late pachytene spermatocyte and early round spermatid stages of spermatogenesis when meiotic division occurs. We interpret this stage- specific expression pattern as an indication of the potential of this gene product to regulate meiosis during this discrete window of spermatogenesis. The regulatory machinery of the meiotic cell cycle in the male testis is not clearly defined. A number of genes, including Tls, Atm, Abl, and Myb, have been suggested to play a role in prophase and metaphase of meiosis because disruption of these genes results in an arrest in either pachytene stage or metaphase I (26, 27). However, most of the meiotic genes are nuclear proteins, and they are probably involved in chiasma formation and stabilization, recombination, and meiotic cross-over. Because GASZ is a cytoplasmic protein, it may function through interacting with other cytoplasmic proteins or proteins that can shuttle between the cytoplasm and nucleus. Identification of its interacting partners will give us more clues on GASZ function during male meiosis.
In the mouse ovary, Gasz mRNA and GASZ protein are restricted to oocytes, but these exhibit a long-enduring expression from primordial follicles enriched in newborn ovaries to preovulatory follicles in mature mice. Thus, GASZ may act not only in germ cell and ovarian development but may additionally have multiple roles throughout folliculogenesis in females like GDF-9 (6, 7). Because GASZ protein persists in early cleavage stage embryos, it has the potential to play important roles in embryonic development as well as throughout female gametogenesis. The meiotic to mitotic transition and other complex events of early preimplantation embryonic development must rely on factors supplied by the oocyte (28). Disruptions of the maternal genome that cause phenotypes in developing offspring of homozygote females are termed maternal effect mutations, and two of these genes have been characterized in mice using knockout technology. In each case, the gene product is normally accumulated in growing oocytes and is present in early developing embryos. The first identified gene encodes MATER (maternal antigen that embryos require), which is necessary for development beyond the two-cell stage and has been implicated in establishing embryonic genome transcription (29). The second identified gene encodes DNMT1o, an oocyte-specific DNA methyltransferase critical for maintaining imprinting patterns established in the embryonic genome and the viability of the developing mouse during the last third of gestation (30). It is possible that GASZ is not required for gametogenesis, but rather is highly conserved phylogenetically because of critical functions in embryos. Thus, Gasz may represent a novel maternal effect gene in mammals.
The GASZ proteins from all six mammalian species analyzed contain three major domains, namely four ANK repeats, a SAM, and a bZIP domains. Literature and protein sequence searches of the public database failed to identify any protein with these three domains. Because these three domain structures are conserved in the GASZ proteins from all six species, GASZ is an extremely interesting novel protein. More than 400 proteins have been identified to contain ANK repeats, including p53-binding protein 2 (31), cyclin-dependent kinase inhibitor p19 (32, 33), nuclear factor-
B inhibitory protein I
B
(34), and the transcriptional regulator GABP-ß (35). ANK repeats are tandem repeats of about 33 amino acids. Crystal and solution structures of ANK motifs have been described, and they tend to be L-shaped, consisting a ß-hairpin and two
-helices that potentially facilitate protein-protein interaction (36, 37). Many ANK repeat regions are known to function as protein-protein interaction interfaces.
SAM is a protein module of approximately 70 amino acids originally found in a set of developmental proteins including yeast Ste4 and Byr2 and polyhomeotic proteins (38). Structural analyses have shown that the SAM domain is arranged in a small five-helix bundle with two large interfaces, which could allow SAM-containing proteins to form extended polymeric structures (39). SAM domains have been found in more than 60 proteins including Eph family receptors where the SAM domain is believed to play an important role in cell-cell contacts and signaling processes (39, 40). SAM domains can interact to form homo- and heterooligomers (38, 39). The tumor suppressor protein, p73, and its relative, p63, differ from their homolog p53 by the presence of C-terminal SAM domains (41, 42). Autosomal dominant mutations in the p63 gene have been found in patients with Hay-Wells syndrome, also known as ankyloblepharon-ectodermal dyslasia-clefting syndrome (43). Interestingly, all of the mutations that cause the ankyloblepharon-ectodermal dyslasia-clefting syndrome give rise to amino acid substitutions in the SAM domain and are believed to alter p63 protein-protein interactions. Thus, SAM domains play important roles in cellular integrity, and disruption of this domain can have dire consequences.
Although no known protein contains all three of the major domains present in GASZ, there are a few proteins that contain combinations of these domains. Somatostatin receptor interacting protein (called SHANK1 or SSTRIP) and SHANK3 contain six N- terminal ANK repeats and a C-terminal SAM (44, 45). As mentioned above, both ANK and SAM domains are proposed to function via mediating protein-protein interactions. In the case of SHANK1, the protein is believed to function as a cytoskeletal anchoring protein by interacting with the C terminus of the somatostatin receptor subtype 2. At post-synaptic sites of brain excitory synapse, SHANK1 and SHANK3 are believed to organize a cytoplasmic complex of signaling proteins that includes the
-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate-type glutamate receptor, the N-methyl-D-aspartate receptor, and the metabotropic glutamate receptor (46). The SAM domain of SHANK1 and SHANK3 are believed to multimerize as homomers or heteromers to permit cross-linking of several proteins at post synaptic sites. Likewise, several proteins also contain both a bZIP and a SAM. For example, ZAK (leucine-zipper and sterile-
motif kinase) is a mixed lineage kinase-like protein that appears to act as a MAPK kinase to specifically activate c-Jun N-terminal kinase and activate the transcription factor nuclear factor-kB (47). Although the C terminus of GASZ contains a bZIP domain, suggesting that GASZ may also function as a transcriptional regulator, the homology of this region to other bZIP domains is relatively low, and GASZ lacks any predicted nuclear localization signal sequences, suggesting that the conserved bZIP domain may function in an alternative manner. Given that both ANK and SAM domains presumably function through mediating protein-protein interactions, we hypothesize that GASZ functions as a germ cell and early embryo-specific cytoplasmic signaling protein, which may participate in the regulation of signal transduction pathways during germ cell maturation and early cleavage stage embryonic development.
In summary, we have identified a germ cell-specific protein in six mammalian species that contains four N-terminal ANK repeats, one SAM, and a C-terminal bZIP. Resolving the crystal structure of GASZ may help determine how the different protein domains affect its function. Additionally, identification of GASZ-interacting proteins would provide insight into its biological function during spermatogenesis and oogenesis. Given the high conservation of GASZ in evolution and its functional domains, GASZ may act as an important signaling protein and/or transcriptional regulator during germ cell maturation and early embryogenesis. Generation of Gasz null mice will allow us to define the physiological roles of this novel protein in reproduction.
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MATERIALS AND METHODS
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In Silico Subtraction
One newborn ovary library (Library 371) with 7577 ESTs was identified in the Unigene mouse sequence collection in the NCBI database. These sequences were downloaded, and each EST was individually searched using BLAST program (http://www.ncbi.nlm.nih.gov/blast) against all publicly available ESTs and nonredundant sequences. Newborn ovary ESTs that matched ESTs from other libraries were excluded if the expected frequency of chance occurrence was less than 10-15. Newborn ovary ESTs that were absent in cDNA libraries derived from other adult tissues were chosen for further screening of tissue specificity using a semiquantitative RT-PCR-based method. Primers were designed on the basis of the chosen EST sequences, and PCR was performed using cDNAs prepared from multiple tissues including heart, liver, spleen, lung, kidney, brain, stomach, small intestine, testis, ovary, and uterus.
Genomic Database Search for Mouse GASZ Orthologs
The full-length mouse Gasz cDNA sequence was used to search against the entire GenBank database, including the nonredundant and human genomic databases. The sequences of the bacterial artificial chromosome clones and genomic fragments that contained regions homologous to the mouse Gasz cDNA were downloaded and further analyzed through alignment with each exon of the mouse Gasz gene to locate exon/intron boundaries. The ORFs of the deduced cDNAs were determined using the ORF finder (http://www.ncbi.nlm.nih.gov/gorf/gorf.html) at the NCBI.
Protein Domain Structure and Alignment Analysis
We used the protein homology motif searching tool Pfam (http://www.sanger.ac.uk/Software/Pfam/search.shtml), located at the Sanger Center, to analyze the mouse and human GASZ protein sequences. The search results included the names of the conserved domains and displayed the alignments. Alignment of all GASZ proteins of different species was performed using the Megalign program of the DNASTAR software package (DNASTAR, Inc., Madison, WI). The sequence similarity and phylogentic tree were derived from the alignment analysis using the same program.
Northern Blot Hybridization
Total RNA (15 µg) isolated from multiple mouse tissues was fractionated on a 1.2% formaldehyde-agarose gel and transferred to Hybond-N nylon membrane (Amersham Pharmacia Biotech, Arlington Heights, IL). The full-length mouse Gasz cDNA was labeled with [
-32P] deoxy (d)-ATP using a Strip-EZ kit (Ambion, Inc., Austin, TX). The kit provides an easy stripping procedure allowing for rehybridization of used blots. Membrane hybridization, washing, and autoradiography, as well as stripping and reprobing were performed according to the manufacturers instructions. Blots were stripped and hybridized with a 18S rRNA cDNA labeled with [
-32P]dATP to control for equal RNA loading.
In Situ Hybridization
In situ hybridization was performed as described previously (48). Briefly, paraffin-embedded ovaries and testes were cut into 5-µm sections, dewaxed, fixed, hybridized, and washed. A 441-bp PCR fragment derived from the 3'-untranslated region (corresponding to nucleotides 870-1311 of the cDNA) was subcloned into a pGEM-T vector. Sense and antisense probes were generated by labeling with [
-35S] uridine phosphate using the Riboprobe Labeling System (Promega Corp., Madison, WI). Hybridization signals were detected by autoradiography using NTB-2 emulsion (Eastman Kodak Co., Rochester, NY). After development and fixation, the slides were counterstained with hematoxylin and mounted for photography. The Gasz sense probe did not generate a signal above background in either the testis or ovary sections.
Semiquantitative RT-PCR
The reverse transcription reaction was performed in a 50 µl volume containing 2 µg of total RNA, 1x RT Buffer (Promega Corp.), 100 pmol random primer (Promega Corp.), 1 mM deoxynucleoside triphosphate, 10 U RNase inhibitor RNasin (Promega Corp.), and 5 U avian myeloblastosis virus reverse transcriptase (Promega Corp.) at room temperature overnight. An aliquot (2 µl) of cDNAs synthesized above was used as templates for PCR. For amplifying the mouse Gasz cDNA, we used a pair of Gasz-specific primers (upstream primer: 5'-ATGGCCTTGGACTTGAACAT-3', downstream primer: 5'-GCAAAGTTAGCTTGCCGATG-3') spanning introns 10, 11, and 12 and yielding a 567-bp PCR product. For amplifying the human GASZ cDNA, a pair of primers (upstream primer: 5'-GCAGAAAATTCTGGCTGCTC-3', downstream primer: 5'-ACCGAATCCGCATATGGTAA) designed to cross introns 10, 11, and 12 were employed for RT-PCR. Human multiple tissue cDNAs (CLONTECH Laboratories, Inc., Palo Alto, CA) were used as templates. As a loading control, human ß-actin was amplified using a primer set purchased from CLONTECH Laboratories, Inc. To ensure that the PCR was in the exponential phase, different PCR cycles from 1530 were tested. Human ACTIN was amplified for 19 cycles. Thirty-five cycles were used for both mouse Gasz and human GASZ.
Production of GASZ Protein and Anti-GASZ Polyclonal Antibody
A pET protein production system (Novagen Inc., Madison, WI) was employed to produce mouse GASZ protein. The entire coding region (1428 bp) of Gasz cDNA was amplified by PCR using a pair of primers: 5'-GAATTCGCTGAGGGGCTTGGCAGT-3' (with EcoRI adaptor: GAATTC) and 5'-CTCGAGTTTTCTCTGCAAAGTTAG-3' (with XhoI adapter CTCGAG). The PCR products were subcloned into pGEM-T vector (Promega Corp.) and sequenced. The EcoRI/XhoI fragment was then subcloned into pET-23b vector (Novagen). Protein induction and purification were performed according to the manufacturers instructions. The 53-kDa fusion protein containing the full-length GASZ protein, N-terminal T7 flag and C-terminal histidine tag was used to immunize rabbits to produce the polyclonal antibody (Cocalico Biologicals, Inc., Reamstown, PA).
Immunoblotting and Immunohistochemistry
Proteins were isolated from multiple mouse tissues using T-PER Tissue Protein Extraction Reagent (Pierce Chemical Co., Rockford, IL) according to the manufacturers instruction. An aliquot of 100 µg of protein was fractionated on a 12.5% SDS-polyacrylamide gel and transferred onto a nitrocellulose membrane (Schleicher \|[amp ]\| Schuell, Inc., Keene, NH). Immunodetection was performed as described previously (48). The rabbit anti-GASZ polyclonal antibody was used at a dilution of 1:1000. The membrane was subsequently stripped and blotted with an anti-ACTIN monoclonal antibody (ICN Biomedicals, Inc., Aurora, OH) for monitoring the loading.
Sections (5 µm thick) were cut and mounted onto poly-lysine-coated slides. Microwave antigen retrieval was employed as described previously (48). After blocking, an aliquot of 100 µl primary antibody diluted at 1:1000 was applied to each section and incubated at 4 C overnight. Incubation with secondary antibody and visualization of positive cells were performed using Vectastain Elite-kit (Vector Laboratories, Inc., Burlingame, CA) according to the manufacturers instructions. Preimmune serum was used in control sections.
Immunofluorescence
The expression and subcellular distribution profile of GASZ protein was determined in fully grown oocytes as well as early preimplantation embryos from (C57BL/6J x SJL/J) F1 mice. All mice were injected with 5 IU equine CG (eCG) to stimulate preovulatory follicle development, and cumulus-enclosed oocyte complexes were isolated 4448 h later. GV stage oocytes were obtained by denuding the oocytes immediately after recovery from the ovary. An additional group of mice received 5 IU of human CG approximately 48 h after eCG, and metaphase II stage eggs were recovered from the oviduct 14 h post human CG treatment. One group of mature eggs was fixed for immunocytochemistry analysis, after the surrounding cumulus cells were removed by a brief exposure to 0.01% hyaluronidase (Sigma), whereas a second group was used for in vitro fertilization. Early zygotes were recovered and fixed at 6 h post fertilization. Two-cell, 4-cell, and 8-cell stage embryos were collected at 24, 48, and 72 h post fertilization, respectively.
Oocytes and embryos were fixed in 4% paraformaldehyde in PBS for 1 h at room temperature. The fixed cells were washed and transferred to PBS with 0.02% Triton-X for 10 min, rinsed several times in PBS with 1% serum, then placed in block solution (PBS with 10% serum) for 1 h. All subsequent incubations and washes were carried out in block solution. The oocytes and embryos were incubated with anti-GASZ antisera (diluted 1/1000) for 1 h, followed by three (15 min) washes, and then incubated with 3 µg/ml of FITC-conjugated goat antirabbit IgG (Jackson ImmunoResearch Laboratories, Inc., West Grove PA) for 45 min. DNA was counterstained with propidium iodide (1 µg/ml) for 10 min; the cells were rinsed three times and mounted onto poly-L-lysine-coated slides using Vectashield (Vector Laboratories, Inc.). Negative control samples were evaluated in which preimmune rabbit serum was substituted for the primary antibody. GASZ expression was detected using a TCS-NT laser scanning confocal microscope equipped with an air-cooled argon ion laser system (Leica Corp. Microsystems, Wetzlar, Germany).
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ACKNOWLEDGMENTS
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We thank Ms. Shirley Baker for help in manuscript formatting.
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FOOTNOTES
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This work was supported in part by NIH Grants HD-33438 (to M.M.M.) and HD-23839 (to J.J.E.).
Abbreviations: ANK, Ankyrin; bZIP, basic leucine zipper; CFTR, cystic fibrosis transmembrane conductance regulator; d, deoxy; eCG, equine CG; EST, expressed sequence tags; GASZ, a novel protein with germ cell-specific expression and the presence of ANK, SAM, and bZIP domains; GDF-9, growth differentiation factor-9; hpg, hypogonadal; NCBI, National Center for Biotechnology Information; ORF, open reading frame; SAM, sterile-
motif.
1 The GASZ cDNA sequences of mouse (AF459789), rat (AF461260), cow (AF461261), baboon (AF461262), chimpanzee (AF461263), and human (AF461259) have been deposited into GenBank of the NCBI. 
Received for publication December 21, 2001.
Accepted for publication March 25, 2002.
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