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Laboratory of Fish Reproductive Physiology (T.M.), Faculty of Agriculture, Ehime University, Matsuyama 790-8566, Japan; "Time’s Arrow and Biosignaling" PRESTO Japan Science and Technology Agency (T.M., C.I.M.), Kawaguchi 332-0012, Japan; and Faculty of Fisheries (T.O., K.Y.), Hokkaido University, Hakodate 041-8611, Japan
Address all correspondence and requests for reprints to: Takeshi Miura, Laboratory of Fish Reproductive Physiology, Faculty of Agriculture, Ehime University, 3-5-7 Tarumi, Matsuyama 790-8566, Japan. E-mail: miutake{at}agr.ehimeu.ac.jp.
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Introduction |
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In germ cell development or spermatogenesis, estrogen has various stimulatory roles, one of the most important being the positive regulation of mitosis of gonocytes or spermatogonia in the early stage of this process (8, 9). However, the mechanisms underlying this regulation are not clear.
The male Japanese eel provides an excellent system for studying the regulation of spermatogenesis because spermatogonial stem cells are the only type of germ cell that it produces in fresh water conditions, and it is the only vertebrate studied to date in which all stages of spermatogenesis can be induced by artificial treatment with exogenous gonadotropin or androgen both in vivo and in vitro (10). Using the Japanese eel, we previously determined that estradiol-17ß (E2) has the following function in spermatogenesis.
Spermatogenesis begins with the mitotic proliferation of spermatogonia and then proceeds through two meiotic divisions followed by spermiogenesis, during which the haploid spermatids develop into spermatozoa. Spermatozoa then undergo maturation, acquiring the ability to fertilize eggs. Spermatogonial mitosis can be subdivided into two processes: spermatogonial stem cell renewal and spermatogonial proliferation toward meiosis (11). In the Japanese eel, estrogen induces only the renewal of spermatogonial stem cells, whereas other processes, including spermatogonial proliferation toward meiosis, are not induced by estrogen stimulation (12). In the present study, we used cDNA cloning to identify a factor that regulates spermatogonial stem cell renewal after estrogen stimulation and subsequently clarified its functions.
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Materials and Methods |
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Testicular organ culture techniques
Testicular organ culture techniques were performed as described previously (13), with minor modifications. In brief, freshly removed eel testes were cut into pieces of 1 x 1 x 0.5 mm3 and placed on floats of 1.5% agarose covered with a nitrocellulose membrane in 24-well plastic tissue culture dishes. The agarose disks had been floated in basal medium for 18 h before the start of testicular culture. For culture, testicular explants were maintained in 1 ml of basal medium with or without 1 µg/ml of E2 or 11-ketotestosterone (11-KT) for 6 d. The testicular fragments were then collected, and their poly (A)+ RNA was extracted using a Fast Track kit (Invitrogen, Carlsbad, CA).
cDNA subtraction
Subtractive cDNA libraries were constructed using poly(A)+ RNA extracted from testicular fragments cultured with (E+) or without (E-) E2. First-strand cDNA was synthesized from 5 µg of each poly(A)+ RNA using reverse transcriptase and an oligo (dT) primer. Second-strand cDNA was synthesized from the first strand with RNase H and Escherichia coli DNA polymerase, and the strands were ligated with bacteriophage T4 polynucleotide kinase and E. coli DNA ligase. After digestion with MboI, the cDNA fragments generated from each poly(A)+ RNA were subtracted from each other by a representational difference analysis (RDA) technique (14). After three cycles of subtractive enrichment, enriched up-regulated E+ and down-regulated E- cDNA fragments were packaged into 
zap II phage particles.
We predicted that the subtracted cDNA libraries would not include full-length cDNA clones, owing to digestion of the cDNA fragments by MboI. To obtain full-length cDNA clones, we therefore generated conventional cDNA libraries, using the same poly(A)+ RNA that was used for cDNA subtraction, containing full-length cDNA clones. Nondigested cDNA was size fractionated on a Sepharose CL4B column with a lower cut-off value of 500 bp and inserted into zap II arms after the addition of EcoRI adapters.
Screening and cloning
Enriched E+ and E- cDNAs were labeled using the Klenow fragment of E. coli DNA polymerase I and a linker primer with [
-32P]dCTP. Approximately 104 plaques from each subtractive library were blotted onto two nylon membranes (High Bond N+, Amersham, Piscataway, NJ). Radiolabeled E+ and E- cDNA probes were hybridized separately to duplicate copies of the library for 18 h at 65 C in hybridization solution [5x Denhart’s solution supplement with 6x standard sodium citrate, 0.1% sodium dodecyl sulfate (SDS), and 100 µg/ml of denatured, fragmented herring sperm DNA]. Only the clones that hybridized to the E+ or E- cDNA probe were collected and purified, and plasmids were excision rescued according to the manufacturer’s instructions (Stratagene, La Jolla, CA).
These partial cDNA fragments were labeled using the Random Primer Plus extension kit (Invitrogen) with [-32P]dCTP. Approximately 105 plaques from intact cDNA libraries were also blotted onto nylon membranes and hybridized with 1 x 107 cpm of the E+ and E- probes for 18 h at 65 C. These membranes were washed twice with 1x standard sodium citrate/1% (wt/vol) SDS at 65 C for 1 h.
The cDNA inserts from positively hybridized clones were collected and purified, and plasmids were excision rescued as above. Sequence determination was performed on an ABI Prism 310 DNA sequencer using the BigDye Terminator cycle sequencing FS ready reaction kit (Applied Biosystems, Foster City, CA). Sequence analysis and comparison were carried out using DNASIS software (Hitachi, Yokohama, Japan). Homology searching against the amino acid sequence deduced from the obtained cDNAs was done using the Fasta Sequence Similarity Search protein query (Genome Net, Kyoto, Japan).
RT-PCR
Poly(A)+ RNA was extracted from eel testes after hCG injection as described above. After DNase I treatment, 0.5 µg of poly(A)+RNA was transcribed by Superscript II (Invitrogen) using an oligo (dT) primer. The resulting cDNA was amplified by PCR with eel spermatogenesis-related substances 34 (eSRS34)-specific primers (sense primer: 5'-GGCCGATAAGGTGCTGTA-3'; antisense primer: 5'-GTCATGCCATCTACCAA-3') and eel elongation factor 1 (EF-1) primers (sense primer: 5'-GTTCTTCATGAGGTAGTCGG-3'; antisense primer: 5'-TTGCTGTCTCCAGCTACGTT-3'). The PCR cycling parameters were as follows: 35 cycles of 94 C for 30 sec, 55 C for 30 sec, and 72 C for 1 min.
The PCR products were resolved by electrophoresis on a 2% agarose gel, which was then stained with ethidium bromide
Production of polyclonal antibody
A PCR product encoding amino acids 8–456 of eSRS34 was inserted into the BamHI site of the pQE-30 expression vector (QIAGEN, Santa Clarita, CA) with a 6x histidine tag positioned upstream of the initiation codon. Host bacteria carrying the recombinant plasmids were grown at 37 C until log phase, and then 1 mM isopropyl ß-D-thiogalactoside was added to induce protein expression. After 4 h of induction, the bacteria were harvested and homogenized in lysis buffer (8 M urea, 0.1 M sodium phosphate monobasic, 0.01 M Tris-HCl, pH 8.0). Recombinant protein was purified by Ni-NTA agarose affinity chromatography (QIAGEN).
Female rabbits were immunized with 1 mg of purified recombinant protein mixed with Freund’s complete adjuvant. The rabbits received four immunizations at 2-wk intervals by sc injection, and sera were collected after the fourth injection. The diluted antiserum (1/1000) strongly reacted with purified antigen, as assessed by Western blot.
IgG from normal rabbit serum and immunized rabbit serum was purified by mixing pooled serum (15 ml) with the same volume of 80% saturated ammonium sulfate (SAS) in 0.01 M PBS, pH 7.0 (40% SAS-serum). The precipitate was then collected, dissolved in 2 ml PBS, and purified by ion-exchange chromatography on diethylaminoethyl-cellulose (DE52, Whatman, London, UK) column (1 x 15 cm; bed volume approximately 10 ml) equilibrated with 0.0175 M phosphate buffer (PB) (pH 6.8). The 40% SAS-serum supernatant was dialyzed against the starting buffer and loaded onto the column. Fractions including the purified IgG were collected and the absorbance was measured at 280 nm.
SDS-PAGE and Western blot analysis
Fresh testicular samples were homogenized in physiological saline solution for eel (15) at 4 C and then mixed with an equal volume of nonreducing [125 mM Tris-HCl, 4% (wt/vol) SDS, 20% (vol/vol) glycerol, and 0.05% (wt/vol) bromophenol blue] or reducing [nonreducing sample buffer plus 10% (vol/vol) 2-mercaptoethanol] sample buffer. The mixture was heated in a boiling water bath for 5 min and centrifuged at 9000 x g for 5 min to obtain the soluble tissue extract fraction. Three micrograms of sample total protein were separated by SDS-PAGE on 10% gels and transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA). After blotting, the membranes were shaken for 30 min in 5% skimmed milk in 20 mM Tris-HCl (pH 7.5) containing 0.5 M NaCl [Tris-buffered saline (TBS)] to block nonspecific binding sites. The blocked membranes were immersed overnight in 5% skimmed milk containing the primary antibody (diluted 1:1000) against eSRS34. After being washed twice with TBS containing 0.025% Tween 20 and then with TBS, the membranes were incubated with horseradish peroxidase-conjugated goat antirabbit IgG (diluted 1:1000 in TBS, Bio-Rad Laboratories, Hercules, CA) for 2 h at room temperature. The membranes were then washed, and horseradish peroxidase activity was visualized using a freshly prepared solution of 0.06% 4-chloro-1-naphthol in TBS plus 0.06% H2O2. To demonstrate the specificity of the eSRS34 antibody, the Western blot analysis was also carried out without the primary antibody as a negative control.
Immunohistochemistry
The eel testicular fragments were fixed in 4% paraformaldehyde in 0.1 M PB (pH 7.2) at 4 C for 18 h, embedded in paraffin wax, and cut into 5-µm serial sections. Sections were deparaffinized in xylene and hydrated in a graded ethanol series. Immunohistochemical analysis was performed using an ABC-AP-alkaline phosphatase substrate kit I (Red) (Vector Laboratories, Burlingame, CA), and sections were counterstained with Delafield’s hematoxylin.
Production of recombinant eSRS34
Recombinant eSRS34 (r-eSRS34) was constructed using a Bac-To-Bac baculovirus expression system (Invitrogen). The PCR product encoding nucleic acids 82–1560 of the esrs34E-10–5 clone (from 20 bases before the open reading frame to the glutamate codon before the stop codon) with a six-histidine tag at its C terminus was ligated into the pFast Bac 1 vector to produce a recombinant baculovirus containing the eSRS34 sequence. This recombinant baculovirus was used to infect Sf21 insect cells, and the conditioned medium was assayed by Western blot analysis to check production and secretion of eSRS34 protein.
Secreted eSRS34 was purified by Ni-NTA agarose affinity chromatography (QIAGEN), followed by ion exchange chromatography on CM-Sepharose Fast Flow column (Pharmacia, Buckinghamshire, UK) using a stepwise gradient of 0 to 1 M sodium chloride. The eluted protein was verified by Western blotting using the anti-eSRS34 antibody. Purified r-eSRS34 was visualized as a single band by SDS-PAGE under reduced conditions with silver staining.
Effect of the anti-eSRS34 antibody on the testicular germ cell/somatic cell coculture system
Testicular germ cell/somatic cell coculture techniques were performed as described by Miura et al. (16). In brief, germ cells and somatic cells containing mainly Sertoli cells isolated from eel testis were collected by centrifugation, and the cell pellets were incubated for 24 h at 20 C. The pellets were divided into seven groups, each consisting of five pellets. One group was analyzed immediately to obtain an initial control value; three groups were cultured with 1 ml of basal medium containing 50 µg anti-eSRS34 IgG with or without 100 pg/ml of E2 or 10 ng/ml of 11-KT for 15 d, and, as a control, three groups were treated with an equal amount of normal rabbit IgG instead of anti-eSRS34 IgG. Proliferating germ cells were detected by 5-bromo-2-deoxyuridine (BrdU) incorporation.
Detection of proliferating germ cells
To detect proliferation, germ cells were labeled with BrdU according to the manufacturer’s instructions (Amersham Bioscience, Buckinghamshire, UK). In brief, BrdU (1 µl/well) was added to the testicular fragments for the last 12 h of culture. After culture, samples were fixed in Bouin’s solution, and 5-µm-thick sections embedded in paraffin wax were cut and stained immunohistochemically and then counterstained with Delafield’s hematoxylin. The number of immunolabeled germ cells was counted and is expressed as a percentage of total number of germ cells.
Statistics
Results are expressed as means ± SEs (SEM). Data analysis was carried out by the Sceirer, Ray, and Hare extension of the Kruskal-Wallis test (a two-way ANOVA design for ranked data), followed by post hoc Benferroni adjustment. P < 0.05 per number of comparisons was considered to be significant.
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zap II arm. Fragments that hybridized to the same full-length cDNA clones were likely to be derived from the same mRNA. By this analysis, we identified seven and two cDNA clones that were, respectively, up-regulated and down-regulated after E2 treatment.
11-KT, which is an androgen of teleosts, induces complete spermatogenesis, including spermatogonial proliferation toward meiosis (13). Thus, if the genes identified in the screen are associated with spermatogonial mitotic proliferation, then their expression will be also regulated by 11-KT stimulation; however, if they are associated only with the regulation of spermatogonial stem cell renewal, then their expression will be altered only by E2. Therefore, Northern blot analysis was used to examine the effects of E2 and 11-KT on the expression of these genes in an eel testicular organ culture system. Among the cDNA clones, one clone was up-regulated only by stimulation with E2 (Fig. 1
). Using Northern blot analysis, the transcripts hybridizing to this cDNA clone represented two populations, i.e. a major transcript of 2.2 kb and a minor transcript of 4.5 kb. The mRNA expression of the other eight clones was regulated by treatment with both steroids.
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Characterization of eSRS34
esrs34e10–5, the longest cDNA clone of eSRS34, has a 1711-bp insert (DDBJ/EMBL/Gene Bank accession no. AB097149). The sequence contains a long open reading frame encoding 456 amino acids. Database searches showed that the predicted amino acid sequence of eSRS34 shares 53.9% similarity with human platelet-derived endothelial cell growth factor (PD-ECGF) (Fig. 2). The predicted sequence has several features of PD-ECGF, including a recognition motif for thymidine and pyrimidine nucleoside phosphorylases located between residues Ser119 (S) and Glu134 (17) as well as seven conserved cysteine residues, which indicates that the protein contains at least one free thiol group (18). Furthermore, as in human PD-ECGF, there are no typical signal peptides in the N-terminal region (19).
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). We found that eSRS34 mRNA is expressed continuously in testis during spermatogenesis.
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). Testicular eSRS34 protein was detected as a band of 53 kDa at every stage of spermatogenesis and as an additional band of 45 kDa at 15 and 18 d after hCG injection, the point at which spermatid and spermatozoa appeared in testis.
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). In contrast, the preimmune serum used as a negative control did not react to any of the samples (Fig. 5B).
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). Testicular fragments were cultured with increasing concentrations of r-eSRS34 (1, 10, 100, 1000 pg/ml) or with 10 ng/ml 11-KT or 100 pg/ml E2 as a positive control for 15 d. We then monitored the proliferation of spermatogonia by exposing the testicular tissues to BrdU to check for replicating DNA (Fig. 6). BrdU incorporation into germ cells was determined, and the results were expressed as a percentage of the total number of germ cells (BrdU index). Before cultivation, all germ cells in the eel testis were spermatogonial stem cells, and the BrdU index was 17.3 ± 1.4% on stem cell renewal. The addition of r-eSRS34 to the culture medium induced spermatogonial mitosis in a dose-dependent manner, with a peak (39.2 ± 0.8%) at 100 pg/ml. However, this treatment could not induce further spermatogenesis after meiosis. Significantly, in the control without any supplement, the BrdU index did not change from its initial value. The positive controls, 11-KT and E2, both stimulated DNA replication (BrdU index = 45.2 ± 4.2% and 37.0 ± 1.5%, respectively). In testes treated with E2, however, the germ cells were all spermatogonial stem cells, whereas proliferated spermatogonia and spermatocytes were observed in the 11-KT-treated testes.
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). After cultivation, spermatogonial proliferation was monitored by the BrdU index. In positive controls, 11-KT treatment alone significantly stimulated the proliferation of spermatogonia in control cell pellets cultured with normal rabbit IgG, and as a result late type B spermatogonia (proliferated spermatogonia) were detected in these pellets. Although E2 also stimulated spermatogonial mitosis, it did not induce the appearance of late type B spermatogonia, and it induced only spermatogonial stem cell renewal. These observations verified that our experimental system functioned normally. The subsequent addition of the anti-eSRS34 antibody significantly reduced both basal and E2-induced spermatogonial mitoses. However, this treatment did not reduce the spermatogonial proliferation toward meiosis that was induced by 11-KT stimulation.
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Discussion |
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Of these clones, the testicular expression of six of the up-regulated and all of the down-regulated clones was also regulated by 11-KT stimulation. 11-KT is a spermatogenesis-promoting androgen, and it induces spermatogonial proliferation toward meiosis, meiosis, and spermiogenesis in vitro (13, 20, 21). We therefore consider that these eight clones, which are regulated by both androgen and estrogen, are not only associated with spermatogonial stem cell renewal but also with spermatogonial proliferation toward meiosis and subsequent stages.
As a result of gene expression screening, we succeeded in identifying eSRS34, the expression of which was up-regulated in testes only by E2 stimulation. The transcription and translation of eSRS34 were detected in testes at every experimental stage; such expression parallels spermatogonial stem cell renewal, which occurs continuously throughout spermatogenesis (12). E2 existed in serum during spermatogenesis constantly (12). Furthermore, eSRS34 protein was expressed in Sertoli cells, in which the receptor for E2 is expressed (12). Taken together, these findings suggest that eSRS34 fulfills the criteria defined for a key factor regulating spermatogonial stem cell renewal regulated by E2.
The esrs34e10–5 clone was isolated from a normal cDNA library constructed from nondigested cDNA fragments inserted into a zapII vector. Northern analysis indicated that the transcripts of eSRS34 were composed of two populations, corresponding to a major population of 2.2 kb and a minor population of 4.5 kb in length. It is assumed that esrs34e10–5, the longest clone isolated in this experiment, represents a transcript of 2.2 kb in size.
A homology search of the predicted amino acid sequence showed that eSRS34 shares comparatively high similarity with PD-ECGF. Furthermore, eSRS34 contains seven conserved cysteine residues as well as the recognition motif for thymidine and pyrimidine nucleoside phosphorylase, which are key features of PD-ECGF (17, 18).
PD-ECGF stimulates endothelial cell growth and chemotaxis in vitro and angiogenesis in vivo (22). PD-ECGF has been also demonstrated to be identical to thymidine phosphorylase (dThdPase), an enzyme involved in pyrimidine nucleotide metabolism (17, 23, 24, 25, 26, 27, 28). To date, however, there is no evidence for a relationship between PD-ECGF/dThdPase and gametogenesis.
Similar to PD-ECGF/dThdPase, eSRS34 does not contain typical signal peptides sequences in its N-terminal region (22). In this study, however, recombinant eSRS34 protein was secreted from Sf21 cells infected with baculovirus, and an anti-eSRS34 antibody could prevent its action. These results suggest that eSRS34 is secreted from Sertoli cells. Basic fibroblast growth factor and IL-1 also lack N-terminal signal peptides (29, 30). These factors are secreted from producer cells via a mechanism of exocytosis that is independent of the endoplasmic reticulum-Golgi apparatus pathway (31, 32). In addition, it has been reported that PD-ECGF/dThdPase is secreted from some cancer cell lines in vitro (33). These findings support the idea that eSRS34 is secreted from Sertoli cells.
In Western blot analysis, testicular eSRS34 exhibits two forms with different molecular mass: one of 53 kDa and another of 45 kDa. It is not clear how these differences in mass may relate to differences in protein structure between the two forms in this experiment. The 53-kDa form was continuously expressed during all stages of maturation when spermatogonial stem cell renewal was progressing. In contrast, the 45-kDa form was expressed in testes only at 15 and 18 d after hCG injection, the stage at which spermatids and spermatozoa appear in eel testes (15). It is therefore possible that the 53-kDa form is related to the regulation of spermatogonial stem cell renewal. Although from its expression pattern it seems likely that the 45-kDa form may function in later stages of spermatogenesis, including spermiogenesis and/or sperm maturation other than spermatogonial stem cell renewal, the role of this molecule is not clear from this study.
Using r-eSRS34 protein produced from the baculovirus expression system, we investigated the effect of eSRS34 on spermatogenesis. Adding r-eSRS34 to the culture medium induced spermatogonial mitosis, with peak induction at 100 pg/ml. Because this treatment could not induce the appearance of proliferated spermatogonia (late type B spermatogonia) and/or more differentiated stages of germ cell after meiosis, r-eSRS34 seems to be specifically associated with the regulation of spermatogonial stem cell renewal, similar to E2 treatment.
Removing eSRS34 secreted from Sertoli cells by specific antibody binding reduced both basal and E2-induced spermatogonial mitosis in a testicular germ cell/somatic cell coculture system. By contrast, this treatment did not reduce spermatogonial proliferation induced by 11-KT, a spermatogenesis-inducing steroid. Thus, these results also indicate that eSRS34 induces only spermatogonial stem cell renewal and not spermatogonial proliferation toward meiosis.
We propose that eSRS34 should be called spermatogonial stem cell renewal factor for the following four reasons: 1) eSRS34 is expressed only in the Sertoli cells surrounding spermatogonial stem cells; 2) transcription of eSRS34 mRNA is up-regulated by E2, a steroid that promotes spermatogonial stem cell renewal; 3) r-eSRS34 induces spermatogonial stem cell renewal; and 4) removing naturally occurring eSRS34 protein from testis by a specific antibody prevents only spermatogonial stem cell renewal proliferation induced by E2 stimulation.
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Acknowledgments |
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Footnotes |
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Abbreviations: BrdU, 5-Bromo-2-deoxyuridine; dThdPase, thymidine phosphorylase; E2, estradiol-17ß; EF-1, elongation factor 1; eSRS34, eel spermatogenesis-related substances 34; hCG, human chorionic gonadotropin; 11-KT, 11-ketotestosterone; PB, phosphate buffer; PD-ECGF, platelet-derived endothelial cell growth factor; RDA, representational difference analysis; r-eSRS34, recombinant eSRS34; SAS, saturated ammonium sulfate; SDS, sodium dodecyl sulfate; TBS, Tris-buffered saline.
Received June 26, 2003.
Accepted for publication August 13, 2003.
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knockout mice. J Androl 21:107–121[Abstract]
and ß. Science 286:2328–2331 knockout mouse. Ann Endocrinol 60:143–148[Medline]
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