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Department of Obstetrics and Gynecology,4 Jichi Medical School, Tochigi 329-0498, Japan
Department of Molecular Anatomy,5 Nippon Medical School, Tokyo 113-8602, Japan
Institute for Environmental and Gender-Specific Medicine,6 Juntendo University Graduate School of Medicine, Chiba 279-0021, Japan
Department of Obstetrics and Gynecology,7 Juntendo University School of Medicine, Tokyo 113-8421, Japan
Department of Otolaryngology–Head and Neck Surgery,8 Gunma University Graduate School of Medicine, Gunma 371-8511, Japan
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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developmental biology, gametogenesis, oocyte development, spermatogenesis, testis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Prospermatogonia and oogonia play central roles in the process of prespermatogenesis and oogenesis, respectively. Although the outlines of the process have been documented, the details of prospermatogonia and oogonia have not been fully elucidated. The identification and characterization of molecular events within the gonads constitute an approach to understanding the molecular mechanisms of germ cell development and differentiation during prespermatogenesis. Recently, we have focused on the production of monoclonal antibodies (mAbs) that react with mouse stage/cell-specific testicular antigens; this focus has facilitated studies of the molecular mechanism or mechanisms of gametogenesis. One of these mAbs, designated TES101, reacted with a novel testicular protein of 38 kDa (TES101 reactive protein, or TES101RP), which is located, predominantly, on the plasma membranes of spermatocytes and spermatids, but not in Sertoli cells or interstitial cells, including Leydig cells, in adult mice [10]. The protein TES101RP is also known as protein TEX101 on the Mouse Genome Informatics (MGI) Database.
In the present study, we investigated the expression of TEX101 on germ cells in developing mouse gonads using immunofluorescence microscopy, and we confirmed the results using reverse transcription-polymerase chain reaction (RT-PCR) and in situ hybridization (ISH). During gametogenesis, germ cell death occurs in parallel with cellular proliferation [11, 12]. Therefore, we also characterized the TEX101-positive cells in terms of proliferation and cell death using bromodeoxyuridine (BrdU) labeling and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL). The results of this study provide new information on the activities of germ cells during gonadal development.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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All the animal experiments were conducted according to the Guide for the Care and Use of Laboratory Animals published by Jichi Medical School and Nippon Medical School. BALB/c mice were purchased from Japan SLC (Hamamatsu, Japan). The mice were housed and bred at 25°C in a 12L:12D cycle and given food and water ad libitum. Observation of the vaginal plug was considered as 0 dpc. For neonatal mice, the day they were born was regarded as 1 dpp. Male and female embryos at Days 12, 14, 16, 18, and 19 of gestation, and male and female mice at 1, 2, 4, 6, 8, 10, 12, 14, 17, 21, and 28 days of age were used in the following experiments. For each age, three to eight embryos and postnatal mice were examined.
TES101 mAb Production
The TES101 mAb specific to TEX101 was prepared and purified as described previously [10, 13].
Immunohistochemistry
The testes and ovaries from BALB/c mice were cut into small pieces and fixed for 2 h at room temperature in a mixture of 4% paraformaldehyde and 0.1% glutaraldehyde in 100 mM sodium cacodylate buffer (pH 7.4) that contained 5% sucrose. After washing with the same buffer, the samples were embedded in Jung tissue-freezing medium (Leica, Nuccloch, Germany) or Tissue-Tek O.C.T. compound (Sakura Finetechnical, Tokyo, Japan) in aluminum foil molds, flash-frozen in liquid nitrogen, and then stored at –80°C until use. Tissue sections (5–6 µm in thickness) were made with the Jung Frigocut 2800E cryostat (Leica) or the Microm HM 550 cryostat (Microm, Walldorf, Germany), mounted on round glass coverslips (13 mm diameter, No. 1 thickness; Matsunami, Osaka, Japan), coated with 2% 3-aminopropyltriethoxy-silane (Sigma Chemical Co., St. Louis, MO), and then allowed to air-dry. Following sectioning, the coverslips were used immediately or, in some cases, were stored at –80°C before labeling. The sections were washed three times in PBS, and then incubated in 1% BSA in PBS for 1 h at room temperature to block nonspecific protein-binding sites. The sections were incubated with the TES101 mAb (0.3–8 µg/ml) for 60 min at room temperature or overnight at 4°C, and then washed at least four times in PBS. The sections were subsequently incubated for 1.5 h at room temperature with either Alexa Fluor 488- or 594-labeled goat anti-mouse immunoglobulin G (IgG) (5–10 µg/ml; Molecular Probes, Eugene, OR), and subsequently washed three times in PBS. The sections were counterstained with Hoechst 33342 or 4',6-diamidino-2-phenylindole dihydrochloride (DAPI; Molecular Probes), washed five times in PBS, and then mounted in ProLong antiphotobleaching medium (Molecular Probes) on glass microscope slides. The sections were examined with a Provis AX80TR microscope (Olympus, Tokyo, Japan) or a BX60 microscope (Olympus) equipped with the Spot RT SE6 CCD camera (Diagnostic Instruments, Sterling Heights, MI). The sex of the gonads of embryos at 12 dpc was determined by PCR analysis of a gene in the sex-determining region of the Y chromosome (Sry) in liver samples from the same embryos. Control sections received the same treatment, with the exception that the primary antibody was either omitted or replaced with purified nonimmune mouse IgG.
Some fresh tissue samples were embedded in O.C.T. compound and flash-frozen in liquid nitrogen. These sections were cut using the cryostat, mounted on coverslips, and then fixed in acetone for 10 min at 4°C. These sections were immunolabeled in the same manner as described above.
Reverse Transcription-Polymerase Chain Reaction
Total RNA was extracted from gonadal tissues using the Isogen reagent (Nippon Gene, Tokyo, Japan) according to the manufacturer's instructions, and cDNA was synthesized using 500 ng of total RNA, MultiScribe reverse transcriptase (Applied Biosystems, Foster City, CA), and oligo(dT) primer (Invitrogen, Carlsbad, CA). The primer sequences (forward and reverse, respectively) for Tex101 (MGI representative transcript sequence NM_019981), GFR
-1 (also known as Gfra1; NM_010279), c-kit (also known as Kit; NM_021099), and ß-actin (also known as Actb; NM_ 007393) were as follows: Tex101 (544-base pair [bp] PCR product), 5'-TAGACCGTTCCCAGGTCTTG-3' and 5'-AGCACTGAGTTGTGCCATTG-3'; GFR-1 (264-bp PCR product) 5'-CTAGCCACTCTGTACTTCGT-3' and 5'-GCTTGCAGCGGCAGTTGTAGA-3'; c-kit (MGI primer accession number 2652151; 765-bp PCR product), 5'-TGTCTCTCCAGTTTCCCTGC-3' and 5'-TTCAGGGACTCATGGGCTCA-3'; ß-actin (UniSTS accession number 273493; 454-bp PCR product), 5'-ATGGGTCAGAAGGACTCCTA-3' and 5'-TTGATGTCACGCACGATTTC-3'.
The primer pair for Tex101 was designed using the Primer3 software (http://frodo.wi.mit.edu/primer3/primer3_code.html); the forward primer was located in exon 2 and the reverse primer in exon 5, to eliminate genomic DNA amplification. PCR was performed using ExTaq DNA polymerase (TaKaRa Bio, Shiga, Japan), and 1/20th volumes of the RT reactions as templates, with a reaction volume of 20 µl in the TaKaRa PCR Thermal Cycler Dice Gradient. The following PCR conditions were used: initial denaturation for 2 min at 94°C, followed by 30 cycles for 30 sec at 94°C, 30 sec at 60°C, and 1 min at 72°C, with a final extension step for 5 min at 72°C. The PCR products were electrophoresed on 2% agarose gels and stained with SYBR Green I (Molecular Probes).
In Situ Hybridization
For ISH, cryostat sections of mouse testes were prepared as described above, except that the sections were mounted on glass microscope slides (No. 1 thickness; Matsunami). Probes were designed using the Genetyx software version 6.03 (Genetyx, Tokyo, Japan) as described previously [14]. Two sense and two antisense probes were constructed: one for the N-terminal region of the Tex101 cDNA sequence (antisense, 5'-CGGCCTGGATCGTCTTCCAGACTCAGGG-3'; sense, 5'-CCCTGAGTCTGGAAGACGATCCAGGCCG-3'); and one for the C-terminal region (antisense, 5'-CCGCCTCTCCTCCTTGAGAAACACAGCTCTTACTGGCC-3'; sense, 5'-GGCCAGTAAGAGCTGTGTTTCTCAAGGAGAGGCGG-3'). The oligonucleotide probes were biotinylated at the 5' end (TaKaRa Bio).
The sections were treated with 1 µg/ml proteinase K (Merck, Darmstadt, Germany) for 10 min, and then postfixed in 0.1 M phosphate buffer (pH 7.6) that contained 4% paraformaldehyde. The sections were preincubated in the hybridization solution (see below) without oligonucleotide probe for 1 h at room temperature, and subsequently incubated in the hybridization solution in a moist chamber at 37°C overnight. The hybridization solution contained 50% deionized formamide, 10% dextran sulfate (Pharmacia Biotech, Uppsala, Sweden), 450 mM NaCl, 45 mM sodium citrate, 1x Denhardt solution (0.02% BSA [Sigma], 0.02% Ficoll 400 [Merck], and 0.02% polyvinylpyrrolidone [Wako, Osaka, Japan]), 100 µg/ ml salmon sperm DNA (Sigma), 125 µg/ml yeast RNA (Ambion, Austin, TX), and 1 µg/ml of each oligonucleotide probe. After hybridization, the solution was removed by washing with 2x SSC (1x SSC contains 150 mM NaCl and 15 mM sodium citrate) at 40°C. Posthybridization washes (40°C) were carried out twice in 1x SSC for 10 min and twice in 0.5x SSC for 10 min. Next, the specimens were rinsed in PBS. All the buffers were rendered RNase-free by exposing them to diethylpyrocarbonate at 37°C for 1 h, followed by autoclaving at 121°C for 40 min.
To visualize ISH signals, we employed tyramide signal amplification followed by fluorescein isothiocyanate-labeled streptavidin (FITC-SA) as described previously [15]. Briefly, the sections were incubated with horseradish peroxidase (HRP)-conjugated avidin-biotin complex (ABC; Vector laboratories, Burlingame, CA) for 30 min at room temperature. The sections were then incubated for 10 min at room temperature with biotin-XX tyramide (Molecular Probes)-PBS containing 0.01% H2O2 to increase the number of biotin sites through the enzymatic reaction of HRP. The biotin precipitate was amplified further by a second incubation with ABC for 30 min at room temperature, which was followed by incubation with FITC-SA (10 µg/ml; DAKO, Glostrup, Denmark) for 1 h at room temperature. The sections were then counterstained with Hoechst 33342 (Molecular Probes), and mounted in ProLong antiphotobleaching medium (Molecular Probes). Controls included the addition of the antisense probe to sections that were pretreated with 1 µg/ml RNase (Boehringer-Mannheim, Mannheim, Germany), and the addition of the sense probe to untreated sections.
Double-Staining for BrdU and TEX101
From 1 dpp through 12 dpp, male mice were injected i.p. with BrdU (1 µM/g body weight; Boehringer-Mannheim) in PBS. Two hours later, the testes were excised and fixed with 4% paraformaldehyde for 2 h at room temperature. Cryostat-cut sections were mounted on round glass coverslips, which were prepared as described above.
Immunohistochemistry using the TES101 mAb was carried out in the manner described above. Following the TEX101-labeling procedure, the sections on coverslips were temporarily mounted in Slowfade Antifade mounting medium (Molecular Probes) on a glass microscope slide. The sections were examined immediately by optical microscopy and images were collected. The temporary slide preparations were subsequently disassembled, and the sections on coverslips were washed in PBS with three changes for 9 min each. The sections were then immersed in 2 N HCl for 60 min to denature the genomic DNA. After rinsing, the sections were treated with 1% BSA and incubated with the FITC-conjugated anti-BrdU mouse monoclonal antibody (20 µg/ml; Boehringer-Mannheim) at 37°C for 60 min. The sections were washed with PBS for 15 min with five changes of the solution. Subsequently, the sections were incubated with Alexa 488-labeled goat anti-FITC antibody (5–10 µg/ml; Molecular Probes). The sections were washed five times in PBS, and then mounted in ProLong antiphotobleaching medium. The regions that were previously examined by fluorescence microscopy were relocated, and immunofluorescence micrographs were collected. Control sections from mice with or without BrdU injection received the same treatment, with the exception that the FITC-conjugated anti-BrdU was omitted or replaced with purified nonimmune mouse IgG. Sections of the small intestine of BrdU-injected mice were also used as positive control sections.
Double-Staining for TUNEL and TEX101
Nuclei with DNA fragmentation, which is a hallmark of apoptosis, were detected by TUNEL analysis, which was performed using a modification of the method described by Gavrieli et al. [16]. From 1 dpp through 21 dpp, the testes were excised and fixed with 4% paraformaldehyde for 2 h at room temperature. Cryostat sections were mounted on round glass coverslips and prepared as described above. TEX101 immunohistochemistry was carried out in the same manner as described above. Following the TEX101-labeling procedure, the sections were digested with 1 µg/ml proteinase K for 10 min at 37°C, and then washed three times with PBS for 5 min each. The reaction with terminal deoxynucleotidyltransferase (TdT) was conducted for 1 h at 37°C in a solution that contained TdT buffer (Boehringer-Mannheim), 1.5 mM CoCl2, 10 µM biotinylated UTP (Boehringer-Mannheim), and 0.15 U/µl TdT (Boehringer-Mannheim). The samples were washed with PBS for 15 min with five changes of the solution. TUNEL-positive sites were visualized by incubation with FITC-SA (10 µg/ml) for 1 h at room temperature. As a control, some sections were subjected to reaction either in the absence of TdT or where biotinylated UTP was replaced with TTP. The sections were then counterstained with Hoechst 33342 (Molecular Probes) and mounted in ProLong antiphotobleaching medium.
Western Blot Analysis
Mouse testes were homogenized in octyl-glucopyranoside-based lysate buffer (150 mM Na2HPO4 pH 7.4, 60 mM N-octyl-BD glucopyranoside [Nacalai Tesque, Kyoto, Japan], 10 mM D-gluconic acid lactone [Sigma], 20 mM EDTA [Dojindo, Kumamoto, Japan], 30 mM NaN3, 1.19 mg/ml PMSF [Sigma], 10 µg/ml aprotinin [Sigma], 100 µg/ml leupeptin [Sigma], and 20 µg/ml pepstatin A [Sigma]) using the Polytron PT1200C homogenizer (Kinematica AG, Lucerne, Switzerland) for 10 sec at 4°C. Following incubation for 20 min on ice, the debris was pelleted in the Iwaki CFM-200 centrifuge (Iwaki, Tokyo, Japan) at 8000 x g, and the lysates were stored at –80°C until used. The lysates were subjected to SDS-PAGE on 5%–20% gradient gels under nonreducing conditions. The proteins were transferred to polyvinylidene difluoride (PVDF) membranes (Bio-Rad, Hercules, CA), and subsequently probed with the TES101 mAb in 5% low-fat milk in 10 mM TBS that contained 0.1% Tween-20 (Sigma). The signal was detected by the addition of the LumiGLO chemiluminescent substrate reagent (KPL, Gaithersburg, MD) and visualized on BioMax Light film (Kodak, Rochester, NY).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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TEX101 expression during prespermatogenesis To determine the localization of TEX101-positive cells, we performed immunohistochemistry of the developing gonads of the mouse. PGCs had already reached the genital ridge at 12 dpc, and germ cells were identifiable by nuclear staining with Hoechst 33342 or DAPI. TES101 mAb-positive (TEX101-positive) staining was not present in the mouse testes at 12 dpc (data not shown). At 14 dpc, the seminiferous cords of the testis had almost formed. In the seminiferous cords, the germ cells, which are referred to as prospermatogonia [7, 17], occupied the centers of the cords. Immature Sertoli cells were located in the peripheral regions of the cords, whereas Leydig and other interstitial cells were located between the cords. Sections prepared from testes at 14 dpc showed that TEX101 was present on the cell surfaces of some prospermatogonia (Fig. 1A), which were distinguished from Sertoli cells by the shape and the size of the nucleus; the prospermatogonia were characterized by relatively large, spherical nuclei and a few prominent, spherical nucleoli. Positive staining was not observed in either the Sertoli cells or the interstitial cells (Fig. 1A). In the testis at 16 dpc, most of the prospermatogonia expressed TEX101 on their cell surfaces (Fig. 1C). After birth, TEX101-positive cells moved from the center region toward the basement membrane of the seminiferous tubule, and then formed an arrangement around the basement membrane. By 8 dpp, the prospermatogonia, which included the neonatal-type undifferentiated spermatogonia described by Dettin et al. [18] and Ohbo et al. [19], constitutively expressed TEX101, whereas the Sertoli cells and other cell types in the testis showed no reactivity to the TES101 mAb throughout the fetal and postnatal stages (Fig. 1, A–H). It should be noted that the TES101 mAb may be used to detect prospermatogonia in the fetal and early postnatal periods.
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TEX101 expression after initiation of spermatogenesis At 10–12 dpp, male germ cells were readily identified by Hoechst 33342 or DAPI staining as adult-type spermatogonia (i.e., type A, intermediate, and type B spermatogonia) rather than prospermatogonia. After this stage, TEX101 was no longer detectable in these types of spermatogonia (Fig. 1I). TEX101-positive cells were observed occasionally in testes at 10 dpp (data not shown); eventually these cells transformed into spermatogonia that lacked TEX101 expression. In testes at 12–14 dpp, spermatogenesis started, and leptotene and later-stage spermatocytes were found in the superficial portions of the epithelia of some seminiferous tubules. TEX101 was detectable on the surfaces of spermatocytes in the tubules (Fig. 1I).
From 12 dpp through 28 dpp, as the spermatogonia differentiated to produce spermatozoa, TEX101 was highly expressed on the plasma membranes of spermatocytes and spermatids, but not on those of spermatogonia, Sertoli cells, or interstitial cells, including Leydig cells (Fig. 1, I–N). At 28 dpp, cells at all stages of spermatogenesis, including spermatozoa, were observed. The sections of the 28 dpp testis essentially showed the same TEX101 immunostaining patterns as observed for each cell type of the mature testis, as reported previously [10].
Control sections for all age groups did not show any specific positive staining of testicular cells (data not shown). The results from the experiments are summarized in Table 1.
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TEX101 expression on female germ cells TEX101-positive staining was not observed for the 12 dpc mouse ovary (data not shown). In the 14 dpc ovary, there were no obvious cords surrounding the oogonia and primary oocytes. TEX101 appeared on the cell surfaces of the germ cells in the 14 dpc ovary (Fig. 2A); the morphology of the female germ cells was similar to that of prospermatogonia. By 18 dpc, the cortical region of the ovary had developed, and germ cells were grouped in clusters in the cortex. The germ cells at 18 dpc still expressed TEX101 on the cell surface (Fig. 2C). By around 4 dpp, the oocytes became enclosed by follicular cells, thereby forming the primordial follicle. Concomitantly, TEX101 disappeared from the primary oocytes of the primordial follicle (Fig. 2, D and E). From 6 dpp onward, TEX101 was no longer detectable on the oocytes (Fig. 2F).
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RT-PCR Detection of Tex101 Expression during Gonadal Development
To confirm that Tex101 mRNA was present in the developing gonads, we used RT-PCR to evaluate the expression of Tex101 transcripts in male and female gonads from 1 dpp to 28 dpp. In addition to the detection of Tex101 mRNA, the expression levels of GFR-1, which is a putative marker for undifferentiated germ cells [18, 20], as well as of c-kit, were examined in germ cells during the neonatal period.
Tex101 mRNA was detected in the male gonad at 1 dpp and increased thereafter, with peak expression on and after the initiation of spermatogenesis (Fig. 3A). In contrast, in the neonatal female gonad, the Tex101 mRNA expression pattern was strikingly different. The intensities of the PCR bands for Tex101 transcripts were significantly reduced at 4 dpp, and Tex101 mRNA was no longer detectable after 6 dpp (Fig. 3B). It should be noted that these RT-PCR results are consistent with the results obtained by immunohistochemistry (see Figs. 1 and 2). We also examined the expression of the GFR-1 and c-kit genes. In the neonatal testis, GFR-1 mRNA was detectable at 1 dpp, and was strongly down-regulated after 12 dpp (Fig. 3C). In the ovary, significant amounts of GFR-1 mRNA were not detected throughout the investigation period (Fig. 3D). In the male gonad, c-kit mRNA was up-regulated on and after 8 dpp (Fig. 3E). In the female gonad, c-kit mRNA was expressed strongly during the neonatal period (Fig. 3F). These expression patterns of GFR-1 and c-kit obtained by RT-PCR are in good agreement with earlier results from Northern blotting [20] and ISH [21].
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ISH Detection of Tex101 Transcripts During Testicular Development
To confirm that the Tex101 gene was expressed specifically in prospermatogonia, using ISH we examined the Tex101 mRNA expression levels in developing male gonads from 14 dpc to 28 dpp.
In the testis at 14 dpc, hybridization with antisense probes for Tex101 was observed for prospermatogonia, with no hybridization being observed for Sertoli cells and interstitial cells (Fig. 4A). This finding is consistent with the results of the immunohistochemical analysis using TES101 mAb, as mentioned above (see Fig. 1). Tex101 mRNA was detectable in prospermatogonia until around 6 dpp (Fig. 4B). However, the message was not detectable in 8–10 dpp prospermatogonia (Fig. 4C). In the 6–8 dpp testis, early (preleptotene) spermatocytes were formed, which showed expression of Tex101 (Fig. 4C). From 12 dpp onward, with the first wave of spermatogenesis, Tex101 transcripts were detected by ISH in both the spermatocytes and spermatids, while no Tex101 mRNA was found in the spermatogonia (Fig. 4D). Neither Sertoli cells nor interstitial cells (including Leydig cells) expressed histochemically detectable levels of Tex101 mRNA during the fetal and postnatal stages.
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Specific staining was absent both in sections that were hybridized with the sense probes and in sections that were pretreated with RNase followed by hybridization with the antisense probes (data not shown).
Relationship Between TEX101 Expression and Cellular Proliferation and Apoptosis During Testicular Development
The proliferation and degeneration of male germ cells are important events in prespermatogenesis. To provide additional characterization of TEX101-positive prospermatogonia in the developing testis, we examined the relationship between TEX101 expression levels and these events. To identify proliferating prospermatogonia, the in vivo BrdU-labeling method was used, in combination with immunohistochemistry with the TES101 mAb (Fig. 4). In addition, to examine the extent of prospermatogonium death, the TUNEL method was employed in combination with TEX101 immunohistochemistry (Fig. 4).
Fluorescence signaling indicating the uptake of BrdU was not detected in TEX101-positive prospermatogonia at 1–2 dpp (Fig. 4E). This was in contrast to Sertoli cells, which were labeled with BrdU at this stage (Fig. 4E). BrdU-labeled prospermatogonia appeared on 3–4 dpp. By 10 dpp, some of the BrdU-positive prospermatogonia were TEX101-positive, while others were TEX101-negative (Fig. 4, F and G). From 12 dpp onward, spermatogonia that were virtually identical to those in the adult testis were identified, and were labeled with BrdU (Fig. 4H). In the days of initiation of spermatogenesis, earlier stage spermatocytes that incorporated BrdU were sometimes encountered. Control experiments (with or without BrdU injection), in which preimmune antibody replaced the FITC-conjugated anti-BrdU mouse monoclonal antibody, or in which the primary antibody was omitted, did not give levels of fluorescence above the background (data not shown).
TUNEL staining revealed a lack of TUNEL-positive cells within a few days before and after birth (Fig. 4I). TUNEL-positive cells were detected in the 6 dpp mouse testes (Fig. 4J). At this stage, most if not all of the TUNEL-positive cells were TEX101-negative, and these cells were found predominantly in the inner part of the seminiferous tubules, away from the basement membranes. On the other hand, most of the TEX101-positive cells had already localized to the walls of the seminiferous tubules (Fig. 4J). The number of TUNEL-positive cells increased in the seminiferous tubules until 10–12 dpp (Fig. 4, K and L). When the control sections were incubated with reaction mixtures that lacked TdT, or that contained TTP in place of biotin-UTP, no positive nuclei were found (data not shown).
Measurement of TEX101 Expression by Western Blotting
Mouse adult testis samples probed with the TES101 mAb showed an intense single band with an apparent molecular mass of about 38 kDa under nonreducing conditions (Fig. 5, lane A). When an equal amount (9 µg) of total protein from a 6 dpp testis sample was applied, TEX101 immunoreactivity was abrogated (Fig. 5, lane B). However, low levels of TEX101 could be detected after the application of relatively large amounts (540 µg) of the 6 dpp testis sample (Fig. 5, lane C). The apparent molecular mass of TEX101 was slightly heavier in the 6 dpp testis than in the adult testis (Fig. 5, lanes A and C). This may be explained by the differences in the amounts of proteins in the loaded samples.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Prespermatogenesis begins once the PGCs reach the genital ridge in the male mouse. Differentiation from PGCs to type A spermatogonia occurs in the developing testis. In the pregonadal period of gametogenesis, several genes are temporally and spatially regulated in PGCs, and PGC-specific proteins have been documented (for a review see [22– 24]). However, the characteristics of germ cells during prespermatogenesis have not been revealed. Hilscher and collaborators histochemically classified rat prospermatogonia differentiation during prespermatogenesis into three stages [7, 25]: M-spermatogonia are present during the proliferation phase in the prenatal period; T1-prospermatognia appear during the mitotic arrest phase, around the time of birth; and T2-prospermatogonia are detected during the reproliferation phase in the neonatal period. There have been a few reports regarding changes in the expression levels of markers for prospermatogonia. Although Plzf, which is a transcriptional repressor, is expressed in neonatal prospermatogonia and in undifferentiated spermatogonia after the start of spermatogenesis [26], no prospermatogonium-specific cell surface antigen has been identified. Consequently, detailed studies on the reproductive biology of these important cells have been hindered. In this study, we clearly demonstrate that TEX101 is a specific marker for prospermatogonia.
We also attempted to characterize TEX101-positive prospermatogonia from the viewpoints of proliferation and cell death in the developing testis. BrdU-labeled prospermatogonia appeared after birth. Our results on BrdU-labeling are consistent with earlier findings, which suggested that reproliferation of prospermatogonia occurs after birth [6]. It seems that the TEX101 expression is independent of prospermatogonial proliferation. On the other hand, most of the TUNEL-positive cells were not prospermatogonia but preleptotene-like spermatocytes, which were localized away from the basement membranes of the seminiferous tubules. Our TUNEL results are in good agreement with those of Wang et al. [12]; early spermatocytes that occur during rat early spermatogenesis perish [25]. In addition, some researchers have reported prospermatogonium cell death during prespermatogenesis [11, 12, 27]. In this study, it was difficult to find cells that were both TEX101-positive and TUNEL-positive. TEX101-positive cells appear to represent the surviving germ cells that can transform into spermatogonia.
In a preliminary study from our laboratories, the 38 kDa band with reactivity for the TES101 mAb was first observed around 20 dpp by Western blot analysis [10]. On the other hand, in this study, the TEX101-reactive band was detectable in the 6 dpp testis (Fig. 5). The TEX101-positive cell population in the neonatal testis may be much smaller than that in the adult testis. We confirmed by RT-PCR and ISH that Tex101 mRNA was present in the prospermatogonia (Figs. 3 and 4).
Surprisingly, TEX101 appeared transiently in female germ cells, and only during oogenesis. Hilscher et al. have reported that the similarity, in terms of kinetics, between female and male rat gametogenesis continues until the beginning of folliculogenesis and spermatogenesis [7]. Our data provide evidence that TEX101 is a molecule with specificity for both female and male precursors of adult-type germ cells. The tyrosine-kinase c-kit receptor/stem cell factor system is essential for PGC migration toward the gonadal ridges, and their survival in the early embryonal gonads of both sexes (for review, see [23]). In the developing male mouse gonad, c-kit expression appears on differentiating type A1–A4 spermatogonia from 8 dpp onward [5, 21, 28]. In the adult testis, the c-kit protein is mainly expressed in type A1–A4 and type B spermatogonia, and in Leydig cells, and is down-regulated concomitant with the onset of meiosis [5, 29–31]. In the female, the c-kit protein is detected in oocytes within follicles from 1 dpp until ovulation, as well as in theca cells [21, 32]. Using RT-PCR, we observed similar c-kit mRNA expression patterns for male and female gonads during the early postnatal period (see Fig. 3). Taken together, the results presented in this study indicate that the expression patterns of TEX101 and c-kit receptor in germ cells are substantially in reciprocal manner during gametogenesis. We conclude that TEX101 may play an important physiological role in germ cell development.
Recently, Halova et al. [33] reported the novel lipid raft-associated and glycosylphosphatidylinositol-anchored glycoprotein TEC-21. This glycoprotein was originally found in the rat basophilic leukemia cell line RBL-2H3, and it belongs to the urokinase plasminogen activator receptor/Ly-6/snake neurotoxin family. TEC-21 shows 87% identity to TEX101 at the cDNA level (coding region) and 79% identity at the protein level, which strongly suggests that TEC-21 is a rat homologue of mouse TEX101. A homologous molecule has also been identified recently as a novel cancer/testis antigen in humans [34]. To examine the details of the physiological function or functions of TEX101 and its regulatory role in gametogenesis, we are currently undertaking further studies that include Tex101 gene suppression or protein suppression experiments (or both).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Yoshihiko Araki, Institute for Environmental and Gender-Specific Medicine, Juntendo University Graduate School of Medicine, 2-1-1 Tomioka, Urayasu-City, Chiba 279-0021, Japan. FAX 81 47 353 3178; yaraki{at}med.juntendo.ac.jp
3 These authors contributed equally to this work
Received: 5 December 2004.
First decision: 11 January 2005.
Accepted: 31 January 2005.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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receptor. Int Immunol 2002 14:213-223
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