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BOR - Papers in Press, published online ahead of print October 4, 2002.
Biol Reprod 2002, 10.1095/biolreprod.101.002550
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Biology of Reproduction 67, 1824-1831 (2002)
DOI: 10.1095/biolreprod.101.002550 © 2002 Society for the Study of Reproduction, Inc.

Male Reproductive Tract

Male-Enhanced Antigen-1 Gene Flanked by Two Overlapping Genes Is Expressed in Late Spermatogenesis1

Yasuhide Ohinataa,b, Shizuyo Sutoua, Masaaki Kondoc, Tomoko Takahashia, and Youji Mitsui2,a,b

a Institute of Molecular & Cell Biology (IMCB), National Institute of Advanced Industrial Science & Technology (AIST), Tsukuba, Ibaraki 305-8566, Japan b Institute of Applied Biochemistry, University of Tsukuba, Tsukuba, Ibaraki 305-0006, Japan c Central Research Institute, Itoham Foods, Inc., Moriya, Ibaraki 302-0104, Japan


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The male-enhanced antigen-1 gene (Mea1) was originally isolated from a murine testicular cDNA library using anti-H-Y antigen antisera and was assigned to chromosome 17. On analysis of its structure and expression, we found that the Mea1 genomic sequence is flanked by two other genes: Ppp2r5d present in its 3'-terminus in a tail-to-tail orientation and a novel gene called Peas in its 5'-terminus in a head-to-head orientation. The coding sequences of the two genes embedded in the Mea1 sequence are located on the opposite DNA strands of Mea1. Cap-site analysis of Mea1 revealed that it is transcribed from at least seven sites. Most splice variants of Mea1 were abundantly expressed in the testis; the d-type was weakly expressed in the other tissues. AP-2-binding motifs were detected in the transcription-initiation sites. In situ hybridization and immunohistochemical studies revealed Mea1 expression in pachytene spermatocytes. This expression was most prominent in spermatids and residual bodies. The Mea1 protein was also localized in the cytoplasm of elongated spermatids and residual bodies. Localization of the Mea1 suggests that it may function in the very late stages of spermiogenesis. The possibility that Mea1 is one of the serologically detectable male antigens is discussed.

sperm, sperm maturation, spermatid, spermatogenesis, testis


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The H-Y antigen was first described for a male-specific antigen during skin graft experiments in which male, but not female, skin was rejected by females [1]. It was considered as a candidate for the sex-determination factor (TDF in humans and Tdy in mice). It was proposed to work as a testis-inducing factor to entice the testes in the X-Y sex-determination system or the ovaries in the Z-W system [2]. However, this proposal was negated later [3]. On the basis of controversial data so far accumulated, Silvers et al. [4] argued that the H-Y antigen detected by T-cell-mediated systems (HY) must be different from serologically detectable male antigens (SDMs). This seems to be true. The first HY has been identified as Smcy [5], and several HYs have been identified subsequently. These include both major histocompatibility complex (MHC) class I-restricted [6] and MHC class II-restricted epitopes [7].

An antibody against male cells was found in females who had rejected male skin grafts or had been immunized with male cells [8]. The antibody detected SDM on heterogametic cells, that is, male (XY) in mammals, females (ZW) in chicks, females (ZW) in Xenopus frogs, males (XY) in leopard frogs, and male (but not female) cells in lobsters [9–11]. The SDM was identified on the acrosomal region of mouse sperm, but its chemical nature remained to be clarified [12]. Retrospectively, Müllerian-inhibiting substance (MIS) or anti-Müllerian hormone was the first molecule defined as an SDM [13]. The MIS is secreted by Sertoli cells of the testis during male organogenesis so that the Müllerian ducts degenerate as the Wolffian ducts develop. Because MIS is a product of testicular activities, it is reasonable that MIS is recognized as nonself by the immune surveillance of females.

The male-enhanced antigen-1 gene (Mea1) was initially isolated from a mouse prokaryotic expression cDNA library using pooled antisera against murine male cells [14]. Because Mea1 is located on an autosome in human (chromosome 6), mouse (chromosome 17) [14], and bovine (possibly chromosome 23) [15], Mea1 was considered to be different from HY. Molecular analyses indicated that the Mea1 gene is phylogenetically conserved throughout mammalian genomes and is expressed at relatively high levels in the testis [14, 15]. Because SDM is expressed on the surface of embryos of more than 8 cells [16], it was also used for sexing embryos [17]. Using reverse transcription-polymerase chain reaction (RT-PCR), Mea1 transcripts were detected from bovine embryos beyond the 8-cell stage; however, anti-bovine Mea1 monoclonal antibody did not identify Mea1 on the cell surface of bovine embryos. Therefore, this SDM expressed in early embryos must be different from Mea1 [15]. However, being a testis-specific protein, Mea1 has a high chance of being an SDM [18].

The cDNA sequences of human, mouse [14], and bovine [19] MEA1/Mea1 have been reported. The genomic structure of mouse Mea1 has not been resolved. Southern zoo-blot analyses revealed that Mea1 is phylogenetically conserved throughout mammals [14] but is absent in chicks [20]. Lau et al. [14] indicated that two unidentified genes must be located close to Mea1. In the present study, we aimed to elucidate the genomic structure and gene expression of mouse Mea1 and found that Mea1 exists as the central gene of three overlapping genes.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cloning of Mouse Mea1 Genomic DNA and Sequencing

Approximately 1.5 x 106 recombinants from a mouse genomic library (129SvJ {lambda} FIX II library; Stratagene, La Jolla, CA) were screened using a 32P-labeled, 0.9-kilobase EcoRI-XhoI fragment of Mea1 cDNA as a probe. Positive plaques were purified through two rounds of rescreening. The DNAs from several phage clones were mapped using T7 and T3 probes (FLASH nonradioactive gene mapping kit; Stratagene). Restricted fragments were subcloned into a pBluescript SK(-) vector (Stratagene). The DNA sequencing was performed with an ABI PRISM 310 genetic analyzer (PE Biosystems, Foster City, CA).

Determination of Transcription Start Sites

Transcription start sites were determined by 5'-RACE analysis using a mouse (BALB/c) testis cap-site cDNA kit (Cap Site cDNA dT; Nippon Gene, Toyama, Japan). The first PCR was performed using 1RDT and 1GSP primers (Table 1). The nested PCR was performed using 2RDT and 2GSP primers (Table 1). The PCR was conducted using Ex Taq polymerase (Takara, Shiga, Japan) with 30 cycles of amplification for 1 min each at 94, 55, and 72°C. Amplified products were separated by 12% (w/v) PAGE, and DNA bands detected with ethidium bromide were subcloned into the pCR2.1-TOPO vector (Invitrogen, Groningen, The Netherlands).


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  		TABLE 1. Oligonucleotide primers used in the present study.a

Detection of Mouse Mea1 Transcripts in Tissues

A multiple-tissue Northern blot (2 µg of poly(A)+ RNA from a variety of mouse tissues) was purchased from Ambion (Austin, TX). The membrane was hybridized with a random-primed, 32P-labeled DNA fragment of the full-length coding region of mouse Mea1 for 16 h at 45°C in a hybridization solution containing 50% (v/v) formamide. This was washed twice with 2x SSPE (single strength: 150 mM NaCl, 10 mM NaH2PO4, and 1 mM Na/EDTA)/0.1% SDS for 15 min at room temperature and for 10 min at 50°C with 1x SSPE/0.1% SDS and for 10 min at 50°C with 0.1x SSPE/0.1% SDS. Quantitative RT-PCRs were performed by using mouse RAPID-SCAN gene expression panels (OriGene, Rockville, MD) as templates. First-strand cDNAs had been loaded to 96-well PCR plates to a series of four amounts (1000x, 100x, 10x, and 1x), with the lowest amount (1x) being approximately 1 pg/well. A PCR to yield specific amplification of Mea1 transcripts was performed using specific combinations of primers (Table 1). The PCR with Ex Taq was performed using 30 cycles of amplification for 1 min each at 94, 55, and 72°C. The control PCR was performed using a ß-actin primer set (Table 1).

In Situ Hybridization

Paraffin sections (thickness, 3 µm) of the BALB/c mouse (age, 8 wk) testis were hybridized with antisense and sense digoxigenin (DIG)-11-UTP-labeled RNA probes that were prepared by in vitro transcription of a full-length DNA fragment of the coding region of Mea1 (525 base pairs [bp]) in a pCRBluntII vector (Invitrogen) using a DIG RNA labeling kit (T7/SP6) (Roche, Mannheim, Germany). Immunohistochemical detection of DIG-labeled RNA was carried out with alkaline phosphatase-conjugated anti-DIG antibody (Roche) and an alkaline phosphatase substrate kit VI (BCIP/NBT; Vector, Burlingame, CA). Counterstaining was performed with 0.1% safranin O.

Anti-Mea1 Antibody

Because the primary structure of bovine and mouse Mea1 are very similar to each other (92.5%) [19], we used the His x 6 tagged recombinant bovine Mea1 as an antigen. Rabbit polyclonal antisera against the bovine Mea1 were raised. The antisera were affinity-purified by column chromatography using a HiTrap NHS-activated column (Amersham Pharmacia Biotech, Uppsala, Sweden) linked with the antigen protein.

Western Blot Analysis

The 20 ng of His x 6 tagged recombinant antigen and the 10 µg of total mouse testis protein (BALB/c; age, 8 wk) were loaded per lane, separated by 12% (w/v) SDS-PAGE, and transferred to Immobilon polyvinylidene fluoride membrane (Millipore, Bedford, MA). The membrane was blocked with the blocking solution (3.7% [w/v] skim milk; Difco, Detroit, MI) and 1% (v/v) normal goat serum (Vector) in TBS-T (20 mM Tris-HCl, pH 7.4; 150 mM NaCl; 0.2% [v/v] Tween-20) for 1 h and reacted to the anti-Mea1 antibody (1:2000 dilution in the blocking solution) for 16 h at 4°C. The detection was performed by using goat anti-rabbit immunoglobulin (Ig) G antibody peroxidase conjugate (1:8000 dilution in the blocking solution; Sigma, St. Louis, MO) and enhanced chemiluminescence detection kit (Amersham Pharmacia Biotech).

Immunocytochemistry

HindIII and NotI sites were introduced to the mouse Mea1 coding fragment with PCR. The HindIII-NotI fragment was cloned into pcDNA3.1(+) vector (Invitrogen). The plasmid was purified by using Tip-500 (Qiagen GmbH, Hilden, Germany) and transfected to NIH3T3 cells in chamber slides by using SuperFect reagent (Qiagen). The cells were fixed in paraformaldehyde-PBS 24 h after transfection and using immunocytochemical assay. These cells were then blocked with the solution (5% [w/v] skim milk and 1% [v/v] normal goat serum in PBS-T [137 mM NaCl, 8.10 mM Na2HPO4, 2.68 mM KCl, 1.47 mM KH2PO4, 0.2% [v/v] Tween-20, pH 7.4]), reacted by 16-h incubation with anti-Mea1 antibody (1:1000 dilution in the blocking solution) at 4°C, visualized by using Cy3-labeled goat anti-rabbit antibody (Amersham Pharmacia Biotech; 1:500 dilution in the blocking solution), and counterstained for nuclei with 4',6'-diamidino-2-phenylindole (DAPI; 100 ng/ml in PBS). The slides were covered with a glass slip using Vectashield mounting medium (Vector) and were examined under a fluorescence microscope (Leica Microsystems, Wetzlar GmbH, Germany).

Immunohistochemistry

For indirect immunohistochemistry, paraffin sections (thickness, 3 µm) of the BALB/c mouse (age, 8 wk) testis were incubated with a blocking solution (normal goat serum [1:60; Vector] diluted with TBS-T) and probed with an anti-Mea1 rabbit polyclonal antibody diluted (1:250) with TBS-T. The signal was revealed using a Cy3-conjugated goat anti-rabbit IgG (1:200; Amersham Pharmacia Biotech) in TBS-T. Sections were costained with DAPI (100 ng/ml in PBS). The slides were covered with a glass slip using Vectashield mounting medium and were examined under a fluorescence microscope (Leica).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Genomic Structure of Human and Mouse Mea1

The genomic sequences of human and mouse MEA1/Mea1 loci (mouse: DDBJ accession no. AB074009) were analyzed; two genes were found to overlap with the Mea1 gene (Fig. 1). One is the mouse homologue of the PPP2R5D gene (mouse: accession no. AB074009) encoding a protein serine/threonine phosphatase 2A regulatory subunit B56{delta} isoform [21, 22]. The other is a novel gene that was named PEAS/Peas (human: accession no. AB055925; mouse: accession no. AB053465). In addition to the overlapping genes, a part of the Mea1-Ppp2r5d overlapping, noncoding region showed marked sequence conservation among human, bovine, and mouse (data not shown).



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  		FIG. 1. Positions and directions of three overlapping genes in mice, Mea1, Peas, and Ppp2r5d. The assembled sequence of clones 1 and 4 was registered to DDBJ (DNA Databank of Japan). The upper end of the mouse genomic Ppp2r5d has not been identified in the draft sequence of clone 8. Thick parts of lines indicate exons, and arrowheads represent 5'-ends of cDNA. The overlapping segment is also found in an intron of the outer dense fiber 2 gene (Odf2)

Alternative Exon 1 Variants of Mea1 Transcripts

Northern blot analyses of Mea1 showed that signals corresponding to Mea1 were rather broad, and alternative splice variants were expected. When transcription start sites were determined by the 5'-RACE using mouse testis cap-site cDNA as a template, seven major transcripts were detected (Fig. 2B). The splicing profile that generates these variants is schematically illustrated in Figure 2A. A 5'-flanking sequence of the mouse Mea1 gene and that of corresponding transcription start sites and exon 1 variants are presented in Figure 2C. In the 5'-flanking region, a CRE-binding motif and several SP-1 motifs were found. The most abundant transcriptional element binding motifs was AP-2, which was located at 5'-upstream of all transcripts.



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  		FIG. 2. Determination of transcription initiation sites of Mea1. A) Schematic illustration of alternative splice variants; the translation initiation ATG is assigned as position 1. B) Analysis of Mea1 transcripts containing transcription initiation sites by 12% PAGE. Lane 1: 100-bp ladder size marker; lane 2: 10-bp ladder size marker; lane 3: result of the first PCR; lane 4: results of nested PCR. C) AP-2 sites are underlined. SP-1 sites are double-underlined. A CRE-binding motif is boxed. Arrows indicate transcription-initiation sites. Uppercase letters indicate exons and lowercase letters introns. Colors in A correspond to those in C

Tissue-Specific Mea1 Expression

Northern blot analysis revealed that Mea1 expressed exclusively in mouse testis (Fig. 3A). However, RT-PCR revealed a low level of expression in most tissues examined (Fig. 3B). The expression levels of Mea1 in these tissues were one-hundredth or less than that of the testis. Therefore, it was concluded that Mea1 expression is enriched in testis. To learn alternative splice variant specificity, RT-PCR was conducted using exon 1 variant-exon 2 junction-specific primers (Fig. 3C). The results indicated that the d-type splice variant was the major type expressed in tissues other than testis. A low level of expression of the e-type was detected in brain, heart, and liver. In testis, all six types were detected; to present relative quantitation of the different types, RT-PCR data from four dilution series are shown for testis (Fig. 3C). We found that the d- and e-types were most abundant and were detected with the most diluted condition (1x). Of the two, the signal of the d-type was stronger than that of the e-type.



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  		FIG. 3. Comparison of expression of Mea1 in various tissues. A) Northern blot analysis with the full-length coding region of Mea1 as a probe. B) RT-PCR with a pair of mMea1 2-S and 2-A for Mea1 and that of ß-actin cont-S and cont-A for ß-actin (Table 1). C) Splice variant-specific RT-PCR. Primers for splice variant-specific RT-PCR were as follows: a, mMea1 JSP-a and 2-A; b, JSP-b and 2-A; c, JSP-c and 2-A; d, JSP-d and 2-A; e, JSP-e and 2-A; and f, JSP-f and 2-A (Table 1)

In Situ Hybridization Analysis

In situ hybridization with the sense probe did not show any signal (Fig. 4A). When antisense probe was used, Mea1 transcripts were abundantly detected in the late spermatids and residual bodies and moderately detected in pachytene spermatocytes (Fig. 4, B and C). These data revealed that Mea1 was mainly transcribed in the late stages of spermatogenesis.



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  		FIG. 4. Cell type- and stage-specific expression of Mea1 transcripts. In situ hybridization of mouse testis with sense (A, transverse section) and antisense (B, transverse section; C, longitudinal section). Bar = 100 µm (A and B) and 50 µm (C)

Anti-Mea1 Antibody

An anti-Mea1 antibody was raised against recombinant bovine Mea1 protein using rabbits. Amino acid sequences of human, bovine, and mouse MEA1/Mea1 are very similar to each other [19]. The amino acid sequence of the recombinant bovine Mea1 used as antigen was partially determined by high-performance liquid chromatographic purification, and its authenticity was confirmed. The cross-reactivity of rabbit anti-bovine Mea1 antibody was examined for endogenous protein from mouse testis (Fig. 5, I) and exogenous protein in NIH3T3 cells transiently expressing mouse Mea1 (Fig. 5, II). Endogenous protein was detected as two bands (34 and 36 kDa). This may be caused by the presence of multiple initiation codons, as has been suggested by Lau et al. [14], or could also represent endogenous degradation products. Marked signals ware observed only in the cytoplasm transfected with the vector harboring the mouse Mea1 gene (Fig. 5, II, D–I). No signals, however, were detected in the cells transfected only with the vector (Fig. 5, II, A–C).



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  		FIG. 5. Western blot and immunocytochemical analyses. I) Western blot analysis with the rabbit anti-Mea1 polyclonal antibody. The antibody detected His x 6 tagged recombinant Mea1 protein as a 32-kDa band, and two bands (34 and 36 kDa) were detected from mouse testis. II) Immunocytochemical staining of mouse Mea1 in NIH3T3 cells. Detection of mouse Mea1 with an anti-Mea1 antibody and a Cy3-conjugated secondary antibody (B, E, and H), DAPI staining (A, D, and G), merge of A and B (C), merge of D and E (F), and merge of G and H (I) are also shown. Bar = 100 µm (C and F) and 10 µm; (I)

Immunohistochemistry

Immunohistochemical examination showed that Mea1 was translated in the late stages of spermatogenesis (Fig. 6, A–D). Differential staining of seminiferous tubules at low magnification (Fig. 6, A–C) indicated that the expression of Mea1 was stage-specific and that Mea1 was translated later than developmental stage 13 of spermatids. In the cytoplasm of elongated spermatids, Mea1 was not evenly distributed; a rather granular appearance was observed. In contrast, sperm and epididymis lacked Mea1 expression (Fig. 6, D–F).



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  		FIG. 6. Immunohistochemical staining of Mea1 in the mouse testis. Detection of Mea1 with an anti-Mea1 antibody and a Cy3-conjugated secondary antibody (A and D), DAPI staining (B and E), merge of A and B (C), and merge of D and E (F) are shown. Bars = 100 µm (A–C) and 10 µm (D–F)


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The Mea1 is of interest from three viewpoints. First, Mea1 is a candidate SDM as an antigen detected by an anti-HY antibody. Second, Mea1, flanked by two overlapping genes, occupied the central position and was located on chromosome 6p21.1 (human) and 17 24.30-cM (mouse) in the vicinity of the HLA/H-2 (human leukocyte antigen/histocompatibility-2) complex loci. Third, Mea1 is expressed during very late spermatogenesis.

Mea1 as an SDM

Zenzes et al. [23] studied the SDM antigen in different cell fractions of the rat testis during pubescence and suggested that immature germ cells are SDM-negative until the late diploid stage and that late primary spermatocytes or spermatids become positive. In our study, however, Mea1 was not found in spermatocytes (Fig. 6). The SDM was detected on sperm [11], whereas all our anti-Mea1 monoclonal and polyclonal antibodies examined did not detect SDM in bovine and mouse sperm (data not shown). These apparently controversial findings nevertheless do not negate the possibility that Mea1 may be an SDM, especially with the understanding that more than one or different molecules could serve as an SDM [18]. In addition to Mea1, the male-enhanced antigen-2 gene (Mea2) was shown to be reactive to the anti-HY antigen monoclonal antibody [24]. Murine Mea2 cDNA has been cloned [25]. It is a Golgi protein localized in the Golgi apparatus of spermatocytes and spermatids. Its disruption resulted in spermatogenic failure [26]. GOLGIN160, a Golgi protein isolated from an expression library using serum from a patient with systemic lupus erythematosus [27], an autoimmune disease, was found to be a human homologue of murine Mea2 [25]. GOLGIN160 may be expressed normally in the testis and may become an autoantigen when it is erroneously expressed in women. The MIS, which is an SDM, is a product of Sertoli cells [13]. Taken together, these studies and our present data support the idea that SDM consists of several or many protein antigens involved in testicular activities or spermatogenesis and that Mea1 may be one of them [18]. All SDMs molecularly clarified so far are autosomal, whereas all HYs are linked to the Y chromosome. Here, we have suggested that investigations into SDM, one of which is Mea1, are important from the clinical viewpoint of autoimmune diseases. Presently, however, we do not know if Mea1 works as an autoantigen when expressed in females.

Mea1 as an Overlapping Gene

During the cloning of Mea1, Lau et al. [14] suggested the presence of two other genes in the vicinity of the Mea1 gene. Indeed, extensive analyses of genomic DNA around Mea1 revealed two genes: a mouse homologue of PPP2R5D and a novel gene called Peas (Fig. 1). These three overlapping genes are of interest, evolutionarily, because the topology of the three seems to be conserved throughout mammals, although Mea1 is not found in chicken. In addition, Mea1 is located close to the distal end of the MHC region (H-2) of mouse chromosome 17 (Mouse Genome Informatics, The Jackson Laboratory, Pos. 24.30 cM). Human MEA1 is located at 6p21.1 in chromosome 6, which harbors MHC (HLA). Interestingly, Shintani et al. [28] reported that two mouse genes, Acat2 (encoding acetyl-coenzyme A acetyltransferase 2) and Tcp1 (encoding T-complex protein-1), are overlapped in a tail-to-tail orientation in chromosome 17. Therefore, it has been postulated that this overlap has existed for more than 200 million years. Many testis-/embryo-expressed genes are clustered in the H-2K region [29]. Also, mouse chromosome 17 harbors the t-complex. Within a mouse t-haplotype, several hundred genes have been locked together by a series of chromosomal inversions that prevent recombination with wild-type chromosomes [30]. Males that are heterozygous for the t-haplotype preferentially transmit the t-chromosome to their progeny. Several distorter/sterility loci carried on the t-haplotype together impair flagellar function in all spermatozoa, whereas the responder, Tcr, rescues t-sperm but not wild-type sperm. The gene for Tcr, Smok, encodes a member of a protein kinase gene family, and the kinase is expressed in late spermiogenesis [31]. Human PPP2R5D is strongly expressed in the testis [21]. Human and mouse PEAS/Peas are also intensely expressed in the testis (unpublished data). Although our overlapping genes are located just outside of H-2, the three seem to belong to a large member of testis-, sperm-, and embryo-associated gene clusters that include the H-2 and t-complex. Furthermore, this short stretch of the overlapping segment was found in the mouse Odf2 (outer dense fiber 2 gene) (Fig. 1) locus. Details of the overlapping genes Mea1, Ppp2r5d, and Peas, will soon be reported.

Transcription and Translation of Mea1

The Mea1 is transcribed from several initiation sites (Fig. 2). The upstream regions of the transcription-initiation sites commonly had the AP-2-binding motif and were GC-rich (Fig. 2C). The GC-rich 5'-upstream regions frequently occur in testis-specific genes, and multiple transcription initiations are seen [32–35]. Thus, Mea1 seems to be under the typical control mechanisms of transcription in the testis.

Expression of the transcription factor AP-2 was enhanced by retinoic acid in a human teratocarcinoma cell line, and the AP-2-binding site-dependent transcriptional activity of a reporter gene was also elevated [36]. Retinoic acid supports the full development of spermatogenic cells [37]. Three retinoic receptors exist, RAR-{alpha}, RAR-ß, and RAR-{gamma}, and all are expressed in the testis. Disruption of RAR-{alpha} resulted in testicular degeneration [38]. Disruption of RAR-ß resulted in male sterility in mice [39]. Several genes are under the control of retinoic acid and RARs [40, 41]. The Mea1 seems to be one of the genes controlled by the AP-2 and exerts a role in spermatogenesis in a concerted fashion with the other genes.

The Mea1 is mainly expressed in the testis (Fig. 3A), with a weak expression in some other tissues (Fig. 3B). Although all six of the exon-1-variant types were detected in the testis, the d-type was the major transcript in all the tissues examined (Fig. 3C). The upstream region of the d-type might have some sequences that are responsive to transcription machinery in common with somatic organs. The dense occurrence of SP-1 sequences upstream of the transcription-initiation sites of the d- and e-types is of interest. Although Mea1 transcripts were detected in tissues other than the testis, the protein product was not detectable immunohistochemically.

During postnatal testis development, only somatic cells and primary spermatogonia are present in the testis before Day 8 postpartum (p.p.) [42, 43]. Meiosis initiates during Days 8–10 p.p., and secondary spermatocytes and round spermatids appear around Day 18 p.p. In the Northern blot analyses using testes from developing mice and the testes of mutant mice, Lau et al. [14] detected Mea1 transcripts at the haploid stage of spermatogenesis. On the other hand, Su et al. [24] fractionated spermatogenic cells and showed that Mea1 transcripts existed in pachytene spermatocytes, round spermatids, and residual bodies in the Northern blot analysis. To resolve the discrepancy between these results, we performed in situ hybridization in mouse testis (Fig. 4). Although Mea1 transcripts were detected abundantly in late spermatids and residual bodies, these also occurred moderately in pachytene spermatocytes.

On the other hand, Mea1 protein exists in late spermatids and residual bodies. A possibility that Mea1 transcripts may undergo posttranscriptional regulation is suggested from these findings. Present data that Mea1 protein exists specifically in late spermatogenesis suggest the important role(s) of Mea1 in late spermatogenesis, sperm movement, or fertilization processes.


   FOOTNOTES
 
1 Supported by special research grants to Y.M. from the National Institute of Advanced Industrial Science & Technology and from Tsukuba University. Back

2 Correspondence: Youji Mitsui, National Institute of Advanced Industrial Science & Technology, Institute of Molecular & Cell Biology, Central 6-5, Higashi 1-1-1, Tsukuba, Ibaraki 305-8566, Japan. FAX: 81 298 61 9498; y-mitsui{at}aist.go.jp Back

Received: 12 December 2001.

First decision: 6 January 2002.

Accepted: 25 June 2002.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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S. Sutou, K. Miwa, T. Matsuura, Y. Kawasaki, Y. Ohinata, and Y. Mitsui
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