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Regular Article |
a Department of Immunology & Parasitology, Yamagata University School of Medicine, Yamagata 990-9585, Japan
b Section of Bioprocess Engineering, Faculty of Agriculture, Yamagata University, Tsuruoka 997-8555, Japan
c Departments of Anatomy and
d Obstetrics & Gynecology, Jichi Medical School, Tochigi 329-0498, Japan
e Department of Obstetrics & Gynecology, Center for Reproductive Biology Research, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-2633
f Department of Cell Biology, Institute for Virus Research, Kyoto University, Kyoto 606-8507, Japan
ABSTRACT
A monoclonal antibody, designated TES101, was raised by immunizing BALB/c mice with an allogenic mouse testicular homogenate followed by immunohistochemical selection as the initial screening method. By searching the expressed sequence tag (EST) database with the N-terminal amino acid sequence of TES101 reactive protein, we found that the predicted amino acid sequence encoded by a mouse testicular EST clone matched the TES101 protein sequence. Sequence analysis of the clone revealed no homologous molecule in the DNA/protein database. Based on data obtained from N-terminal amino acid analysis of the TES101 protein, the derived amino acid sequence contained a signal peptide region of 25 amino acids and a mature protein region of 225 amino acids, which translated into a protein with a molecular weight of 24 093. Northern blot analysis showed that mRNA of the TES101 protein was found in testis but not in any other mouse tissues examined. Western blot analysis revealed that TES101 reacted with a 38-kDa band on SDS-PAGE under nonreducing conditions, and this reactivity was abrogated under reducing conditions. Immunoelectron microscopic studies demonstrated that the molecule was predominantly located on the plasma membrane of spermatocytes and spermatids but not in Sertoli cells or interstitial cells, including Leydig cells. Thus, the TES101 protein is a novel molecule present primarily on the surface of developing male germ cells. TES101 protein may play a role in the processes underlying male germ cell formation.
spermatid, spermatocyte, spermatogenesis
INTRODUCTION
Spermatozoa form through a series of complex processes in the testis. The cell-to-cell interactions among the testicular cells and the microenvironment created by the endocrine and/or paracrine systems are thought to be important in accomplishing spermatogenesis [1]. Although mammalian spermatozoa acquire the ability to fertilize as they transverse through the epididymis in vivo [2–4], spermatogenesis itself occurs within the testicular seminiferous tubules [1]. Spermatogenesis, i.e., mitotic proliferation of spermatogonia, meiotic prophase, and division of spermatocytes as well as morphological alterations from haploid spermatids to mature sperm, results in the production of a cell that is highly specialized in structure and function. No other cell achieves such extreme morphological changes while undergoing both genetic recombination and a reduction in chromosome ploidy. Generally, it is believed that the testis has a unique mechanism to control spermatogenesis whereby certain molecules are activated and others are repressed during germ cell production.
To understand the molecular mechanisms regulating germ cell formation, it is important to investigate stage- and/or cell-specific molecular expression within the testis. Thus, several testicular molecules have been identified and characterized (for review, see Hecht [5]) utilizing different approaches. For example, molecular biological techniques, such as the generation of cDNA libraries using subtractive hybridization cDNAs or mRNA differential display procedures, have been used to identify the testis-specific molecules [6–11]. An alternative approach has been the production of testis-specific antibodies as an initial step in the characterization of testis-specific molecule(s). There are several reports to date concerning the generation of the antibodies specific for testicular germ cell antigens [12–18], although these procedures can be a lengthy in some cases. These specific antibodies could be used to characterize the antigen and may be helpful for designing experiments to uncover the role of these testicular antigens in the process of germ cell formation.
We are currently identifying mouse stage/cell-specific testicular antigen(s) to study the spermatogenetic process in more detail. Initially, we chose to produce monoclonal antibodies (mAbs) against testicular cells and to utilize immunohistochemical selection as a screening strategy. Data obtained by N-terminal amino acid analysis after immunoprecipitation using the mAbs established that one of the antibodies, designated TES101, reacted with a novel testicular protein as determined by searching the Expressed Sequence Tag (EST) database. Here, we report the molecular cloning and partial characterization of this testicular protein, which is primarily expressed in germ cells from spermatocytes to elongated spermatids in mice.
MATERIALS AND METHODS
Animals and Chemicals
Female/male ddy and BALB/c mice (6–8 wk old) were purchased from Japan SLC Inc. (Hamamatsu, Japan) and given free access to food and water. They were maintained and bred at the Animal Center, Yamagata University School of Medicine, under 12L:12D conditions.
Restriction endonucleases and modifying enzymes were purchased from either Roche Diagnostics Corp. (Indianapolis, IN) or Takara Shuzo Co. (Kyoto, Japan). Ultrapure grade chemicals were from Wako Pure Chemical Industries (Osaka, Japan), Nacalai Tesque (Kyoto, Japan), Sigma Chemical Co. (St. Louis, MO), Bio-Rad Laboratories (Hercules, CA), and Merck Co. (Darmstadt, Germany). The Taq dye primer cycle sequencing kit was from Applied Biosystems (Foster City, CA). Ten-nanometer colloidal gold particles were prepared by the tannic acid/citrate method and then conjugated with affinity purified goat anti-mouse IgG (Cappel, Durham, NC) according to the method described by Slot and Geuze [19]. All other chemicals were obtained commercially and were of the highest purity available.
Production of mAb
Preparation of immunizing antigen Two testes from a sexually mature (>8 wk old) ddy mouse were homogenized in 2 ml of PBS (pH 7.4) using a glass homogenizer, and the homogenate was allowed to stand for 1 min. The cell suspension, without tissue debris, was carefully transferred into a 1.5-ml tube and centrifuged for 5 min at 180 x g. The supernatant was then removed by aspiration. After washing with PBS, the cell suspension was divided into three equal parts, and each of the parts was used separately as immunizing antigen.
Immunization and cell fusion The testicular cells, mixed with an equal volume of complete Freund adjuvant (Difco Laboratories, Detroit, MI), were injected subcutaneously into female BALB/c mice (7–8 wk old). One week after the initial injection, three s.c. booster injections of the testicular cells with incomplete Freund adjuvant (Difco) were administered every other week. Four additional i.p. booster injections of the testicular cell suspension alone were carried out every 2 wk. The spleen was removed 4 days after the last immunization and was used for hybridoma production using a standard procedure as described previously [20–22]
Screening and cloning of antibody-producing cells Screening was carried out using an immunohistochemical method. Freshly isolated mouse testis or other organs were fixed with 0.2% (w/v) paraformaldehyde in PBS (pH 7.4) at 4°C for 2 h, then the tissue was treated overnight with acrylamide monomer solution (PBS, pH 7.0 containing 8.4% acrylamide, 0.014% N,N'-methylene-bis acrylamide and 0.7% N,N,N',N'-tetra methylethylenediamine) at 4°C according to the method described by Hausen and Dreyer [23]. After the treatment, the acrylamide solution was polymerized by the addition of ammonium persulfate solution for tissue embedment and then frozen with liquid nitrogen for preparation of cryosections. Cryosections were treated with 1% hydrogen peroxide in methanol for 5 min and then with 70% methanol for 1 min to abolish endogenous peroxidase activity. After washing in PBS with three changes, the sections were covered with 3% BSA in PBS for 1 h at 37°C. The sections were drained and incubated for 30 min in a humid chamber at room temperature with supernatants from the hybrydomas as the first antibody and biotin-labeled goat anti-mouse IgG (Vector Laboratories, Burlingame, CA) as the secondary antibody. Immunoreactivity was visualized using a Vectastain peroxidase-ABC kit (Vector Laboratories) with 3',3'-diaminobenzidine·4HCl as substrate. The antibody-producing hybridoma clone obtained was recloned twice by the dilution planting technique as previously described [20–22]. Isotyping of mAbs was determined using a mouse hybridoma isotyping kit (Roche) according to the manufacturer's protocol. The IgG class of mAb was purified as described previously [21].
Electrophoresis and Western Blot Analysis
Mouse testicular extracts were prepared as described previously [24] with slight modification. Mouse testis was homogenized with nine volumes of PBS (pH 7.2) containing 1 mM EDTA, 1 µg/ml leupeptin, and 1 µg/ml pepstatin. After sonication, the protein solution was centrifuged and the supernatant was used as the water-soluble fraction. The precipitated material was washed three times with phosphate buffer (pH 7.2) containing 0.5 M NaCl. After washing, the precipitate was resuspended in PBS containing 1% Triton X-100. After mixing several times by pipetting, the suspension was centrifuged and the supernatant was used as the Triton X-100-soluble membrane fraction. The insoluble material was washed and centrifuged as described above, dissolved in 0.1% SDS solution, and used as the Triton X-100-insoluble fraction. The protein concentration of each solution was determined by the Bio-Rad microprotein assay, using BSA as the standard. The protein solutions obtained from mouse testis were separated by the SDS-PAGE system of Laemmli [25] under reducing or nonreducing conditions. The molecular mass of the antigen bound by the mAb was determined by enzyme immunostaining of the protein after blotting to a polyvinylidene difluoride (PVDF) membrane (Immobilon-P, Millipore Corp., Bedford, MA) from the SDS-PAGE gel according to the standard method as described previously [22, 24, 26].
Amino Acid Sequence Analysis
The water-soluble or the Triton X-100-soluble testicular fraction was immunoprecipitated with TES101 antibody coupled to protein G-Sepharose (Amersham Pharmacia Biotech UK, Buckinghamshire, UK) beads. After washing several times with phosphate buffer (pH 7.2) containing 0.5 M NaCl, the beads were boiled in SDS-PAGE sample buffer (without ß-mercaptoethanol). The solubilized proteins were then separated by SDS-PAGE. The bands corresponding to the TES101 reactive antigen were excised and extracted in 100 mM Tris-HCl (pH 7.5) containing 0.1% SDS and then subjected to automated Edman degradation on a protein sequencer (model G1000A; Hewlett Packard, Boise, ID) equipped with an HP1090M on-line analyzer. Chromatographic data were collected and analyzed using a Vectra computer system (Hewlett Packard) as described elsewhere [27].
DNA Sequence Analysis
By screening the mouse EST database, an EST sequence (GenBank accession number AA066084) corresponding to the protein reactive with the TES101 mAb was identified. The EST cDNA clone 514549, subcloned into the EcoRI/XhoI site of the pBluescript II SK(-) vector, was obtained from Research Genetics (Huntsville, AL). The entire cDNA sequence of both strands was determined by the dideoxynucleotide termination method [28] using fluorescence-labeled primers (Applied Biosystems) according to the manufacturer's protocol. DNA sequence analysis, protein secondary structure prediction, amino acid sequence, and homology searches were performed as previously described [27, 29–31]. Searches for protein sequence motifs were carried out using the PROSITE database [32].
Northern Blot Analysis
Total RNA from various mouse tissues was prepared as described previously [29]. The RNAs (5 µg of each sample) were separated by 1% agarose/formaldehyde gel electrophoresis, transblotted to a positively charged nylon membrane (Hybond-N+; Amersham Pharmacia) by capillary blotting, and hybridized with the 32P-labeled TES101 cDNA probe. Following two washes in double strength standard saline citrate containing 0.1% SDS at 68°C, the blot was visualized using autoradiography.
Immunohistochemical Studies
Immunofluorescence microscopy The testes from adult (14 wk old) BALB/c mice were fixed in a mixture of 4% paraformaldehyde and 0.1% glutaraldehyde in 100 mM sodium cacodylate buffer (pH 7.4) containing 5% sucrose for 2 h at 22°C. After washing with the same buffer, the samples were embedded in Jung tissue freezing medium (Nuccloch, Germany). Immunofluorescent localization of TES101 was determined on 6- to 8-µm-thick cryostat sections. The sections were incubated with the TES101 mAb (8–16 µg/ml) for 1.5 h at 22°C. The secondary antibody employed was either Alexa Fluor 594-labeled (5–10 µg/ml) or fluorescein isothiocyanate-labeled goat anti-mouse IgG (33 µg/ml) (Molecular Probes, Eugene, OR). The sections were counterstained with Hoechst 33342 (Molecular Probes). Immunostained samples were examined with a fluorescence microscope equipped with a Provis AX80TR (Olympus, Tokyo, Japan). Control sections received the same treatment, with the exception that the primary antibody was either omitted or replaced with purified nonimmune mouse IgG.
Immunoelectron microscopy The testes from adult BALB/c mice were fixed using the same conditions as in the immunofluorescence microscopic study. After washing in the same buffer as described above, the samples were infiltrated with 2.3 M sucrose, cut as ultrathin cryosections, and then collected on formvar film-coated electron microscopy grids as previously described [33–35]. Cryosections mounted on these grids were incubated for 30 min at 22°C in a solution containing 1% BSA and 10% normal goat serum in 20 mM glycine-PBS to block nonspecific protein-binding sites. The grids were incubated with the TES101 mAb (8–16 µg/ml) for 1.5 h at 22°C. Sites of TES101 binding were detected with goat anti-mouse 10-nm colloidal gold particles (A520 was 0.479) or goat anti-mouse FluoroNanogold probes (8 µg Fab/ml) (Nanoprobes, Stony Brook, NY) for 1.5 h at 22°C and then rendered visible with the silver enhancement procedure developed by Burry and coworkers (for review, see Burry [36]) as reported previously [34, 35]. Control grids were incubated in the absence of the primary antibody or in nonimmune IgG. After silver enhancement, the ultrathin cryosections were refixed with 2% glutaraldehyde-PBS, washed in distilled water, negatively stained with aqueous 2% phosphotungstic acid (pH 7.0), simultaneously covered with a small square of thin formvar film by the method of Sakai et al. [37], and examined on an H-7000 electron microscope (Hitachi, Hitachinaka, Japan) operated at 100 kV.
RESULTS
TES101 mAb-Reactive Molecule Is a Novel Testicular Protein
A total of 12 hybridomas secreting mAbs (TES101–112) against mouse testicular molecules were established. Among the 12 antibodies, we found that one, designated TES101 (IgG1, 
light chain), recognized a novel testicular protein, based on data obtained from micro amino acid analysis after immunoprecipitation with the TES101 mAb. The N-terminal amino acid sequence of the TES101 reactive protein was determined to be XXXQVSQTLSLEDDPGRTF. A SWISSPLOT database search of the amino acid terminal sequence indicated that no significant match was found. By searching the EST database, we identified a mouse testis cDNA clone (GenBank accession number AA066084, clone ID514549) that had 100% sequence identity with that of the N-terminal sequence of the TES101 protein. We obtained EST clone 514549 and determined the entire nucleotide sequence of the insert. Sequence analysis of the insert revealed a cDNA of 1078 base pairs, containing an open reading flame encoding a putative protein of 250 amino acids, 5' and 3' untranslated regions and a stop codon (TGA), and a polyadenylation signal (AATAAA) with a poly(A)+ tail (Fig. 1). Nucleotide sequence analysis of the cDNA revealed no homologous molecules in the DNA database. Hydrophilicity/hydrophobicity plot analysis using the procedure reported by Kyte and Doolittle [38] revealed strong hydrophobicity at both the N- and C-terminal portions of TES101 protein (Fig. 2). According to micro amino acid sequence analysis, the N-terminus of the molecule appeared to be Thr-26. The calculated molecular weight of the predicted mature TES101 protein core (from Thr-26 to Pro-250) is 24093 and the isoelectric point is 5.25. It contains 4 potential N-glycosylation sites (Asn-Xaa-Ser/Thr) as well as several (>40) Ser/Thr residues, which could be possible O-glycosylation sites (Fig. 1).
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Immunoreactivity of the TES101 mAb Analyzed by Western Blotting
Mouse testicular proteins were separated by SDS-PAGE, transblotted to a PVDF membrane, and probed using the TES101 mAb (Fig. 3). The immunoreactivity migrated at an apparent molecular mass of 38 kDa as a distinct single band under nonreducing conditions, whereas the reactivity was abrogated under reducing conditions (Fig. 3A). Attempts were also made to identify the TES101 subcellular localization within the testis. Because the total amount of protein was nearly identical in both the water-soluble and the Triton X-100-soluble fractions, an equal amount (5 µg) of total protein from each fraction was analyzed by Western blotting. Although TES101 protein was detected in the testicular water-soluble fraction, the major reactivity was observed in the Triton X-100-soluble fraction (Fig. 3A).
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Postnatally, TES101 immunoreactivity was not detected in the testicular extract at Days 7, 11, and 14 but was first observed around Day 20. The testis from a 28-day-old mouse showed levels of protein expression almost identical to those seen in sexually mature mice (Fig. 3B).
Expression of the TES101 Protein
A 1.1-kilobase mRNA encoding the TES101 protein was detected in total RNA isolated from mature mouse testis by Northern blot analysis. This transcript was not detected in total RNA isolated from mouse heart, spleen, brain, kidney, intestine, liver, lung, skeletal muscle, lymph node, thymus, or stomach. In addition, detectable message for the TES101 protein was not expressed in other reproductive organs, such as the epididymis or the ovary (Fig. 4).
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Strong immunoreactivity for the TES101 protein was detected in the testis (Fig. 5A), with only weak staining of spermatozoa within the epididymis (data not shown). No antigen-positive cells were detected in other adult mouse tissues examined (epididymal cells, brain, heart, lung, liver, kidney, spleen, stomach, intestine, and ovary; data not shown) in agreement with the results of the Northern blot analysis.
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Immunofluorescent staining revealed that the TES101 mAb reacted primarily with cells in the seminiferous tubules and did not react with cells in the interstitial tissues, including the Leydig cells (Fig. 5A). To determine the stage of the seminiferous epithelium cycle during spermatogenesis, nuclear staining with Hoechst 33342 was carried out simultaneously with TES101 immunostaining. The intensity of TES101 immunofluorescence observed in the various stages of the seminiferous epithelium was almost identical (Fig. 5, A and B); however, it appeared to be dependent on the cell types in the seminiferous tubules. Spermatogonia attached to the basal lamina of the seminiferous tubules showed negative to faintly positive staining compared with the more advanced cells (stage V, Fig. 5C), whereas the TES101-positive cells attached to the basal lamina were leptotene or zygotene spermatocytes (stage X, Fig. 5E). Sertoli cells seemed to be TES101 negative at all stages (Fig. 5, C and E). These observations suggest that although all stages of seminiferous tubules are TES101 positive, positive staining appears to be associated with spermatocytes and more advanced cells but not with spermatogonia or Sertoli cells. Control preparations in which the primary antibody was omitted or replaced by nonimmune IgG did not show appreciable immunofluorescence (data not shown).
Immunoelectron Microscopic Localization of TES101 Protein in the Mouse Testis
To elucidate the subcellular localization of TES101 protein more precisely, immunoelectron microscopic studies were carried out. In agreement with the immunohistochemical data using light microscopy, immunogold particles were primarily found in spermatocytes and spermatids. Almost no labeling was seen on the cell surface or in the cytoplasm of the spermatogonia (Fig. 6). In the spermatocytes, strong immunoreactivity was observed on the surface of the cells (Fig. 7, A and B). In comparison, relatively weak reaction was observed in the Golgi area (Fig. 7B), although the Golgi area in some spermatocytes showed no immunoreactivity (data not shown). The cytoplasm of round spermatids was not labeled; however, the labeling intensity at the cell membrane was similar to that observed in spermatocytes (Fig. 8). In the elongated spermatids, strong immunoreactivity was present on the surface of spermatids and residual bodies and on the surface of the middle, principal, and end pieces of the flagellum (Fig. 9). Few gold particles were seen on the head portion of elongated spermatids that were attached to Sertoli cells (Fig. 10). In addition, the surface of the Sertoli cells was almost devoid of gold particles (Fig. 10). Control specimens, where TES101 was replaced by PBS or nonimmune IgG, were negative for gold labeling (data not shown).
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DISCUSSION
In this study, we identified a novel testicular germ cell-specific protein (TES101 protein), partially characterized its biochemical properties, and determined its testicular localization. Western blot analysis demonstrated that the TES101 mAb can only react with the antigen under nonreducing conditions, suggesting that the antigenic determinant is toward a domain that contains a disulfide bond(s). The TES101 protein may be hydrophilic; it is present in the testicular water-soluble fraction (Fig. 3). However, the major reactivity is found in the Triton X-100-soluble fraction (Fig. 3A), implying that the protein strongly associates with the membrane portion of the testicular cells after biosynthesis. The immunoelectron microscopic study demonstrated that the TES101 protein is located mainly on the plasma membrane of spermatocytes and spermatids and is weakly present in the Golgi area of the spermatocytes (Figs. 6–10). Alternatively, the TES101 protein may have two forms, i.e., a cytosolic form and a glycosylphosphatidyl inositol (GPI)-anchored form. The hydropathy plot analysis (Fig. 2) revealed two putative signal sequences at both the N- and C-terminals of the molecule, typically found in the GPI-anchored proteins [39–41].
Based on the sliding window/matrix scoring method and the -1, -3 rule for predicting a signal sequence cleavage site [42, 43], the amino acid position between Val-23 and Gln-24 was suggested to predict a site sensitive to cleavage by signal peptidase; however, the actual N-terminus of the molecule appeared to be Thr-26. At present, we cannot completely rule out the possibility that the difference comes from minor degradation during the isolation of the TES101 protein by immunoprecipitation. After removal of the putative 25-amino acid signal peptide at the N-terminus, the molecular mass of the mature form of the TES101 protein (225 amino acids) was 24 kDa, as calculated from the predicted amino acid sequence. However, Western blot analysis showed that TES101 protein had a molecular mass of 38 kDa under nonreducing conditions (Fig. 3). Presumably, this relatively large difference in the molecular mass might be due to glycosylation of the peptide. Four potential N-glycosylation sites and several possible O-glycosylation sites (Ser/Thr residues) were found in the deduced amino acid sequence of the TES101 protein (Fig. 1). Preliminary experiments were carried out to deglycosylate the protein using either N-glycanase, an enzyme known to cleave all types of N-linked oligosaccharide chains [44], or mild alkaline hydrolysis (ß-elimination), a chemical reaction known to be rather specific for O-linked oligosaccharide chains. However, no significant reduction in the size of the TES101 protein was detected by Western blot analysis under nonreducing conditions when either deglycosylation method was utilized (unpublished data). Our preliminary data suggested that another type of posttranslational processing might have occurred on the TES101 protein. In the case of glycoprotein deglycosylation, however, it is generally recommended to denature the molecule before the treatment using SDS and sulfydryl reagents such as ß-mercaptoethanol for complete deglycosylation. In addition, glycoproteins differ widely in their susceptibility to deglycosylation, so that specific conditions including denaturing conditions must be established in each case [44]. The results at this time are inconclusive because the TES101 mAb lacked immunoreactivity under reducing conditions (Fig. 3A); therefore, we could not use sulfydryl reagents for denaturing of the protein during deglycosylation procedure. Further biochemical studies will be necessary to clarify this issue.
Testis-specific mRNA expression (Fig. 4) and the results of the immunofluorescence and immunoelectron microscopic studies (Figs. 5–10) strongly suggest that the TES101 protein was expressed predominantly in testicular germ cells. Although immunofluorescence microscopic studies (Fig. 5) showed that the molecule is detected on the surface of spermatocytes to spermatozoa in all stages of seminiferous tubules, the immunoreactivity of TES101 in the Golgi area of spermatocytes was not always detectable (Fig. 7), suggesting that the TES101 protein might be produced at a specific stage of spermatogenesis. To identify the TES101 protein-producing cells and the stages of spermatogenesis at which it is produced will require further studies, including in situ hybridization and immunoelectron microscopic studies on the expression of the protein during various stages of spermatogenesis. Such studies are underway in our laboratories.
In mice, the first 3 wk of life are classified as the neonatal period [45, 46]. Testicular growth due to an increase in germ cell numbers during the expansion of spermatogenesis occurs from Day 10 to Day 60 after birth, and puberty occurs between 40 and 55 days of age [45]. Although some individual differences were noticed in the expression level of TES101 protein, detectable expression of the molecule in the testis seemed to start approximately 20 days after birth (Fig. 3B). An early study by Bellve et al. [46] indicated that pachytene spermatocytes were identified at 14 days of age and secondary spermatocytes and round spermatids were first observed at 18 days of age within the seminiferous tubules in mice. Because the expression of the TES101 protein is almost parallel to testicular growth, the molecule may have a significant physiological role in sperm formation. The immunohistochemical studies clearly demonstrate that the TES101 protein is primarily restricted to the spermatocytes and spermatids within the seminiferous tubules, suggesting that the protein may be involved with the process of spermatogenesis.
We have identified, cloned, and partially characterized a novel germ cell-specific protein that is primarily detected on the cell surface of spermatocytes and spermatids in the testis. Although the physiological function of the TES101 protein is unknown, these preliminary findings suggest that this novel protein is expressed during spermatogenesis and may be involved in sperm formation.
ACKNOWLEDGMENTS
We are deeply indebted to Drs. Keiji Mori (Yamagata University) and Kazuhiko Akatsuka (Nanyo City Hospital) for their technical support. We are grateful to Drs. Benjamin J. Danzo and Susan Kasper (Vanderbilt University) for critically reading the manuscript. The excellent secretarial assistance of Mrs. Loreita Little is gratefully acknowledged.
FOOTNOTES
1 This work was supported in part by Grant-in-Aid for General Scientific Research 11671593 and by project grants from the Center for Molecular Medicine of Jichi Medical School and the Ministry of Education, Science, Sports and Culture, Japan. 
2 Correspondence: Yoshihiko Araki, Department of Obstetrics & Gynecology, Center for Reproductive Biology Research, Vanderbilt University School of Medicine, 1161 21st Avenue South, Room U3305, Medical Center North, Nashville, TN 37232-2633. FAX: 615 343 7797; yoshihiko.araki{at}mcmail.vanderbilt.edu
3 The authors contributed equally to this work.
Accepted: October 23, 2000.
Received: May 17, 2000.
REFERENCES
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