Immortalization of Mouse Germ Line Stem Cells
http://www.100md.com
《干细胞学杂志》
a Department of Biology, University of Dayton, Dayton, Ohio, USA;
b Department of Cell Biology, Georgetown University Medical Center, Washington, DC, USA
Key Words. Type A spermatogonia ? SV40 ? Large T antigen ? Testis ? Germ line stem cell ? GFR-1 ? Glial cell line–derived neurotrophic factor (GDNF)
Correspondence: Marie-Claude Hofmann, Ph.D., Department of Biology, University of Dayton, 300 College Park, Dayton, OH 45469–2320. Telephone: 937-229-2894; Fax: 937-229-2021; e-mail: Marie-Claude.Hofmann@notes.udayton.edu
ABSTRACT
Stem cells are unique cell populations that are able to undergo both self-renewal and differentiation and are found in the embryo, as well as in the adult animal. In the early mammalian embryo, pluripotent embryonic stem cells are derived from the blastocyst stage and have the ability to form any fully differentiated cell of the body. As the embryo develops, stem cells become restricted in their ability to form different lineages (multipotent stem cells). Multipotent stem cells are also found in a wide variety of adult tissues such as bone marrow and brain. However, in the adult animal, the ability of certain stem cells to differentiate can be restricted to only one cell lineage (unipotent stem cells). Examples of mammalian unipotent stem cells include the stem cells residing in the gut epithelium, the skin, and the seminiferous epithelium of the testis.
A widely accepted model for spermatogonial development has been originally proposed by Huckins and Oakberg . In this scheme, the Asingle (As) spermatogonia are the putative stem cells. They can self-renew or differentiate into Apaired (Apr) spermatogonia that remain connected by an intercellular bridge. The Apr spermatogonia further divide to form chains of 4, 8, or 16 Aaligned (Aal) spermatogonia. Further, the Aal cells will differentiate into type A1 spermatogonia. The A1 spermatogonia resume division to form A2 to A4 spermatogonia. Next, A4 cells divide to form intermediate (In) spermatogonia, and In spermatogonia divide to produce type B spermatogonia. Finally, type B spermatogonia divide to form primary spermatocytes that will enter meiosis. Spermatogonial stem cells and their progeny are contained in the basal part of the germinal epithelium, in contact with the basement membrane, and they are in close association with the somatic Sertoli cells . Regulatory mechanisms mediated by growth factors produced by the Sertoli cells induce or inhibit the proliferation, differentiation, and further development of the germ cells .
Spermatogonial stem cells can generate spermatogenesis when transplanted into the seminiferous tubules of an infertile male . Currently, there is a limited understanding of the molecular mechanisms that control the development of these cells into mature sperm. Spermatogonial stem cells exhibit a distinct phenotype such as the high expression of ?-1 and -6 integrins and specific light-scattering properties . While the stem cell identity of the As spermatogonia has not yet been rigorously demonstrated, their morphology and location in the seminiferous epithelium make them good candidates for being stem cells . As spermatogonia express GFR-1, the receptor for glial cell line–derived neurotrophic factor (GDNF) . As the As differentiate into Apr and Aal spermatogonia, they start expressing the surface receptor c-kit, which binds to stem cell growth factor (SCF) produced by Sertoli cells .
The ability to isolate, culture, and manipulate the germ line stem cell in vitro would allow us to unravel the molecular mechanisms that drive the first steps of spermatogenesis and to characterize the signaling pathways that induce spermatogonial differentiation versus self-renewal. In turn, this could help us understand the origin of certain testicular neoplasias and the causes of male infertility. To look at these issues, an in vitro system in which these cells could be maintained in long-term cultures would be ideal. In the study reported here, we attempted to establish a mouse spermatogonial stem cell line using the large T antigen under the control of an inducible promoter. The resulting immortalized cells express detectable levels of protein and RNA specific for As and Apr spermatogonia such as the GFR-1 membrane receptor. Further analysis revealed the expression of additional markers accounting for a stem cell phenotype such as the genes oct-4, piwi12, and prame11 that have a role in stem cell maintenance and renewal in the germ line and other tissues.
MATERIALS AND METHODS
Cell Immortalization
In our immortalization strategy, the ecdysone receptor from Drosophila was expressed from a vector called pVgRxR . The ecdysone-responsive promoter, which drives the expression of the large T-antigen gene, is on a second vector called pIND-LTAg. Both vectors were stably cotransfected into freshly isolated type A spermatogonia that were induced to express the LTAg when treated with Ponasterone A, an analog of ecdysone. While the cells escaped the hormonal control after a finite number of generations and expressed the LTAg constitutively, their growth remained slow, with a doubling time of about 3 days. After several rounds of subcloning by limiting dilution, a clone was obtained that we called C18-4. The cells appeared round to polygonal, with a round nucleus, and a large nucleus-to-cytoplasm ratio in phase contrast microscopy (Fig. 1).
Figure 1. Morphology of the C18-4 cells in phase contrast microscopy. The cells are round to polygonal, with a round nucleus, and a large nucleus-to-cytoplasm ratio.
Immunocytochemistry
The purified C18-4 cells were stained by immunocytochemistry for the presence of the germ cell–specific Dazl protein using antisera nos. 149 and 150 . Figure 2 shows that Dazl is expressed in the nucleus when it is stained with antiserum no. 149 (Fig. 2A), and it is expressed in the nucleus and the cytoplasm when it is stained with antiserum no. 150 (Fig. 2C). The cells were also positive for GFR-1, the coreceptor of Ret, and a marker for germ line stem cells (Fig. 3A). Further, they stained positive for the Ret transmembrane receptor, a marker for spermatogonia and spermatocytes (Fig. 3C). The cell nuclei, in particular the nucleoli, stained positive for Oct-4, an early marker for the germ line (Fig. 3E). The expression of the c-kit receptor could not be revealed using immunocytochemistry, but it was detected by RT-PCR in early passages only (Fig. 4).
Figure 2. C18-4 cells, immunostained for the Dazl RNA-binding protein. (A): Staining for Dazl, a general marker for germ cells, revealed by immunocytochemistry using antiserum no. 149 and horseradish peroxidase. (B): Negative control without primary antibody. (C): Staining for Dazl, a general marker for germ cells, revealed by immunocytochemistry using antiserum no. 150 and horseradish peroxidase. (D): Negative control without primary antibody.
Figure 3. C18-4 cells, immunostained for GFR-1, the Ret transmembrane receptor, and the Oct-4 transcription factor. (A): Staining for GFR-1, revealed by immunocytochemistry and horseradish peroxidase (x100). (B): Negative control without primary antibody (x100). (C): Staining for Ret, revealed by immunocytochemistry and horseradish peroxidase (x100). (D): Negative control without primary antibody (x100). (E): Staining for Oct-4, revealed by immunocytochemistry and horseradish peroxidase (x40). (F): Negative control without primary antibody (x40).
Figure 4. c-kit gene expression in the C18-4 cells visualized by reverse transcription polymerase chain reaction. Lane A: size markers; lane B: adult testis; lane D: C18-4 cell line; lane F: freshly isolated Sertoli cell. Lanes C, E, and G: negative controls without reverse transcriptase.
Immunoprecipitation
The expression of GFR-1 and Dazl in the C18-4 cell line was confirmed by immunoprecipitation (Fig. 5). To increase the specificity of detection, GFR-1 was immunoprecipitated using a polyclonal antibody made in goat, then revealed after blotting using a polyclonal antibody made in rabbit. In Figure 5A, lane E, we show that the C18-4 cell line produces a protein exhibiting a molecular weight of 42 kDA that corresponds to the molecular weight of GFR-1. As positive controls, we used protein extracts from developing brain, kidney, and testis (lanes B–D, respectively). As negative controls, we used NIH 3T3 cells and the Sertoli cell line 15P1 (lanes F and G) .
Figure 5. Immunoprecipitation of GFR-1, Dazl, and Oct-4 using C18-4 protein extracts. (A): Immunoprecipitation of GFR-1. Lane A: molecular weight markers; lane B: adult kidney; lane C: developing brain (6-day-old); lane D: adult testis; lane E: C18-4 cell line; lane F: 15P1 Sertoli cell line; lane G: NIH 3T3 cell line. (B): Immunoprecipitation of Dazl. Lane A: molecular weight markers; lane B: adult testis; lane C: C18-4 cell line. (C): Immunoprecipitation of Oct-4. Lane A: C18-4 cell line; lane B: adult testis; lane C: molecular weight markers.
The Dazl protein was immunoprecipitated and revealed after blotting using antiserum no. 149 . As seen in Figure 5B, a strong band with a molecular weight of approximately 33 kDa was revealed in the testis extract, corresponding to the molecular weight of Dazl . The same band was observed using C18-4 extracts (Fig. 5B). Similarly, the Oct-4 protein was revealed after immunoprecipitation (Fig. 5C).
Finally, immunoprecipitation failed to detect the expression of GDNF in the purified cells, indicating that the C18-4 cells are not contaminated by Sertoli cells.
Reverse Transcriptase Polymerase Chain Reaction
Neither 3?HSD, a marker for Leydig cells, nor -actin, a marker for peritubular myoid cells, could be found by RT-PCR, further indicating that the cells are of germ cell origin. The absence of LDHC4 expression indicated that the cells are not spermatocytes.
To further confirm the spermatogonial origin of the C18-4 cell line, we investigated by RT-PCR the expression of 36 spermatogonial-specific genes, which were previously identified by Wang and colleagues in the 6-day-old mouse testis. There was a RT-PCR product for the housekeeping gene Gapd in both cell lines and in the neonatal testis samples, indicating that the RNA isolated contained intact transcripts (data not shown). RT-PCR further revealed that all 36 spermatogonial-specific genes tested were expressed in the 6-day-old testis, as expected. None of these genes were expressed in the SF7 Sertoli cell line, confirming the specificity of the transcripts. The expression of 10 spermatogonial-specific genes was detected in the C18-4 cell line. The results of these 10 are reported in Table 1.
Table 1. Spermatogonial genes expressed in the C18-4 cell line, arranged in alphabetical order; 6-day-old testes were used as positive control, and the Sertoli cell line SF7 as negative control
One of the genes expressed by the C18-4 cells was the Ott gene (Fig. 6A), which is transcribed in vivo in the germ cells of testis and ovary . The primers used in this study can detect the presence of two bands by RT-PCR, corresponding to two isoforms, with sizes of 551 and 503 bp, respectively . As shown in Figure 6A, two bands of the correct size are expressed by the C18-4 cells (lane B). However, only one band (551 bp) was detectable in the neonatal testis sample (lane D). Sertoli cells do not express the Ott gene (lane F). Figure 6B shows that the C18-4 cell line also expresses Sycp1, a gene coding for a protein of the synaptonemal complex, which is already expressed in spermatogonia . The band obtained is of 311 bp, as expected (lane B). The same band is visible for the 6-day-old testis sample (lane D), but not in Sertoli cells (lane F). Figure 6C represents the expression of the gene Fth117 that codes for a testis-specific ferritin-binding protein . Fth117 is expressed in the C18-4 cells, giving the expected band of 292 bp (lane B) by RT-PCR. Fth117 expression is found in the 6-day-old testis but is not found in Sertoli cells (lanes D and F, respectively). In addition to spermatogonial-specific genes with a known function, the C18-4 cell line expressed five novel genes that belong to the Tex (testis-expressed) gene family (Fig. 6D) . None of those genes were expressed by the Sertoli cell line.
Figure 6. Expression of spermatogonia-specific genes in the C18-4 cell line, visualized by RT-PCR. RNA samples were isolated from the C18-4 cells, from 6-day-old testes as positive control, and from the SF7 Sertoli cell line as negative control. After reverse transcription, the cDNA samples were amplified using the primers described by Wang et al. . (A): RT-PCR for Ott (PCR product = 551 and 503 bp). (B): RT-PCR for Sycp1 (PCR product = 311 bp). (C): RT-PCR for Fth117 (PCR product = 292 bp). (D): RT-PCR for Tex13 (PCR product = 220 bp), Tex14 (PCR product = 635 bp), Tex15 (PCR product = 411 bp), Tex16 (PCR product = 660 bp), and Tex19 (PCR product = 184 bp). (E): RT-PCR for Piwi1 (PCR product = 241 bp). (F): RT-PCR for Prame11 (PCR product = 448 bp). Lanes A: size markers (100 bp ladder); B: C18-4 cell line; C: negative control for C18-4; D: 6-day-old testis; E: negative control for 6-day-old testis; F: SF7 Sertoli cells; G: negative control for SF7 Sertoli cells. Abbreviation: RT-PCR, reverse transcriptase polymerase chain reaction.
Interestingly, the C18-4 spermatogonial cell line also shows the expression of the piwi12 (piwi-like 2) gene (Fig. 6E). Piwi belongs to a family of genes that is involved in stem cell maintenance and renewal in Drosophila . Two mouse homologues of piwi have recently been cloned: miwi (mouse piwi) and mili (miwi-like) . The primers used in this study are derived from the Drosophila sequence and give a band of 241 bp. As expected, Piwi12 was also detected in the neonatal testis but not in Sertoli cells. Additionally, the C18-4 cells express a PRAME-like gene (Fig. 6F). PRAME is a protein expressed in the testis, as well as in several carcinomas, in acute myeloid leukemias, and in CD34+ hematopoietic stem cells .
Influence of GDNF and SCF on the Behavior of the C18-4 Cells
To determine if the C18-4 cell line responds to growth factors, we cultivated the cells in the presence or absence of 100 ng/ml GDNF in the culture medium. When GDNF is present, the rate of proliferation of the cells increases significantly (p < .005) (Fig. 7A). No influence on cell morphology or expression of the c-kit receptor was observed. Interestingly, the growth effect of GDNF is detected only when the culture medium also contains 10% FCS. No change in the rate of proliferation can be detected when FCS is replaced by a defined serum (Nu serum), which contains a minimal amount of cytokines. Further, addition of SCF to the culture medium did not stimulate the proliferation of the cells (Fig. 7B). SCF did not induce the cells to differentiate since RT-PCR analysis did not reveal the expression of LDHC4, a germ cell–specific isozyme that is detected at the earliest in preleptotene spermatocytes .
Figure 7. Rates of proliferation of the C18-4 cells with GDNF or SCF. (A): In the presence of GDNF, the rate of proliferation of the C18-4 cells increases significantly in comparison with the control cultures. (B): No changes could be observed when the cells are cultured with SCF. Abbreviations: GDNF, glial cell line–derived neurotrophic factor; SCF, stem cell factor.
DISCUSSION
We thank Dr. Renee Reijo-Pera, Department of Obstetrics, Gynecology and Reproductive Sciences, University of California, San Francisco, for providing us with the Dazl no. 149 and 150 antibodies. We also thank Ms. Kathy van der Wee for valuable technical assistance and Dr. Kathy Beal for statistical analysis. This work was supported by the National Institutes of Health grant RO1-HD36483.
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b Department of Cell Biology, Georgetown University Medical Center, Washington, DC, USA
Key Words. Type A spermatogonia ? SV40 ? Large T antigen ? Testis ? Germ line stem cell ? GFR-1 ? Glial cell line–derived neurotrophic factor (GDNF)
Correspondence: Marie-Claude Hofmann, Ph.D., Department of Biology, University of Dayton, 300 College Park, Dayton, OH 45469–2320. Telephone: 937-229-2894; Fax: 937-229-2021; e-mail: Marie-Claude.Hofmann@notes.udayton.edu
ABSTRACT
Stem cells are unique cell populations that are able to undergo both self-renewal and differentiation and are found in the embryo, as well as in the adult animal. In the early mammalian embryo, pluripotent embryonic stem cells are derived from the blastocyst stage and have the ability to form any fully differentiated cell of the body. As the embryo develops, stem cells become restricted in their ability to form different lineages (multipotent stem cells). Multipotent stem cells are also found in a wide variety of adult tissues such as bone marrow and brain. However, in the adult animal, the ability of certain stem cells to differentiate can be restricted to only one cell lineage (unipotent stem cells). Examples of mammalian unipotent stem cells include the stem cells residing in the gut epithelium, the skin, and the seminiferous epithelium of the testis.
A widely accepted model for spermatogonial development has been originally proposed by Huckins and Oakberg . In this scheme, the Asingle (As) spermatogonia are the putative stem cells. They can self-renew or differentiate into Apaired (Apr) spermatogonia that remain connected by an intercellular bridge. The Apr spermatogonia further divide to form chains of 4, 8, or 16 Aaligned (Aal) spermatogonia. Further, the Aal cells will differentiate into type A1 spermatogonia. The A1 spermatogonia resume division to form A2 to A4 spermatogonia. Next, A4 cells divide to form intermediate (In) spermatogonia, and In spermatogonia divide to produce type B spermatogonia. Finally, type B spermatogonia divide to form primary spermatocytes that will enter meiosis. Spermatogonial stem cells and their progeny are contained in the basal part of the germinal epithelium, in contact with the basement membrane, and they are in close association with the somatic Sertoli cells . Regulatory mechanisms mediated by growth factors produced by the Sertoli cells induce or inhibit the proliferation, differentiation, and further development of the germ cells .
Spermatogonial stem cells can generate spermatogenesis when transplanted into the seminiferous tubules of an infertile male . Currently, there is a limited understanding of the molecular mechanisms that control the development of these cells into mature sperm. Spermatogonial stem cells exhibit a distinct phenotype such as the high expression of ?-1 and -6 integrins and specific light-scattering properties . While the stem cell identity of the As spermatogonia has not yet been rigorously demonstrated, their morphology and location in the seminiferous epithelium make them good candidates for being stem cells . As spermatogonia express GFR-1, the receptor for glial cell line–derived neurotrophic factor (GDNF) . As the As differentiate into Apr and Aal spermatogonia, they start expressing the surface receptor c-kit, which binds to stem cell growth factor (SCF) produced by Sertoli cells .
The ability to isolate, culture, and manipulate the germ line stem cell in vitro would allow us to unravel the molecular mechanisms that drive the first steps of spermatogenesis and to characterize the signaling pathways that induce spermatogonial differentiation versus self-renewal. In turn, this could help us understand the origin of certain testicular neoplasias and the causes of male infertility. To look at these issues, an in vitro system in which these cells could be maintained in long-term cultures would be ideal. In the study reported here, we attempted to establish a mouse spermatogonial stem cell line using the large T antigen under the control of an inducible promoter. The resulting immortalized cells express detectable levels of protein and RNA specific for As and Apr spermatogonia such as the GFR-1 membrane receptor. Further analysis revealed the expression of additional markers accounting for a stem cell phenotype such as the genes oct-4, piwi12, and prame11 that have a role in stem cell maintenance and renewal in the germ line and other tissues.
MATERIALS AND METHODS
Cell Immortalization
In our immortalization strategy, the ecdysone receptor from Drosophila was expressed from a vector called pVgRxR . The ecdysone-responsive promoter, which drives the expression of the large T-antigen gene, is on a second vector called pIND-LTAg. Both vectors were stably cotransfected into freshly isolated type A spermatogonia that were induced to express the LTAg when treated with Ponasterone A, an analog of ecdysone. While the cells escaped the hormonal control after a finite number of generations and expressed the LTAg constitutively, their growth remained slow, with a doubling time of about 3 days. After several rounds of subcloning by limiting dilution, a clone was obtained that we called C18-4. The cells appeared round to polygonal, with a round nucleus, and a large nucleus-to-cytoplasm ratio in phase contrast microscopy (Fig. 1).
Figure 1. Morphology of the C18-4 cells in phase contrast microscopy. The cells are round to polygonal, with a round nucleus, and a large nucleus-to-cytoplasm ratio.
Immunocytochemistry
The purified C18-4 cells were stained by immunocytochemistry for the presence of the germ cell–specific Dazl protein using antisera nos. 149 and 150 . Figure 2 shows that Dazl is expressed in the nucleus when it is stained with antiserum no. 149 (Fig. 2A), and it is expressed in the nucleus and the cytoplasm when it is stained with antiserum no. 150 (Fig. 2C). The cells were also positive for GFR-1, the coreceptor of Ret, and a marker for germ line stem cells (Fig. 3A). Further, they stained positive for the Ret transmembrane receptor, a marker for spermatogonia and spermatocytes (Fig. 3C). The cell nuclei, in particular the nucleoli, stained positive for Oct-4, an early marker for the germ line (Fig. 3E). The expression of the c-kit receptor could not be revealed using immunocytochemistry, but it was detected by RT-PCR in early passages only (Fig. 4).
Figure 2. C18-4 cells, immunostained for the Dazl RNA-binding protein. (A): Staining for Dazl, a general marker for germ cells, revealed by immunocytochemistry using antiserum no. 149 and horseradish peroxidase. (B): Negative control without primary antibody. (C): Staining for Dazl, a general marker for germ cells, revealed by immunocytochemistry using antiserum no. 150 and horseradish peroxidase. (D): Negative control without primary antibody.
Figure 3. C18-4 cells, immunostained for GFR-1, the Ret transmembrane receptor, and the Oct-4 transcription factor. (A): Staining for GFR-1, revealed by immunocytochemistry and horseradish peroxidase (x100). (B): Negative control without primary antibody (x100). (C): Staining for Ret, revealed by immunocytochemistry and horseradish peroxidase (x100). (D): Negative control without primary antibody (x100). (E): Staining for Oct-4, revealed by immunocytochemistry and horseradish peroxidase (x40). (F): Negative control without primary antibody (x40).
Figure 4. c-kit gene expression in the C18-4 cells visualized by reverse transcription polymerase chain reaction. Lane A: size markers; lane B: adult testis; lane D: C18-4 cell line; lane F: freshly isolated Sertoli cell. Lanes C, E, and G: negative controls without reverse transcriptase.
Immunoprecipitation
The expression of GFR-1 and Dazl in the C18-4 cell line was confirmed by immunoprecipitation (Fig. 5). To increase the specificity of detection, GFR-1 was immunoprecipitated using a polyclonal antibody made in goat, then revealed after blotting using a polyclonal antibody made in rabbit. In Figure 5A, lane E, we show that the C18-4 cell line produces a protein exhibiting a molecular weight of 42 kDA that corresponds to the molecular weight of GFR-1. As positive controls, we used protein extracts from developing brain, kidney, and testis (lanes B–D, respectively). As negative controls, we used NIH 3T3 cells and the Sertoli cell line 15P1 (lanes F and G) .
Figure 5. Immunoprecipitation of GFR-1, Dazl, and Oct-4 using C18-4 protein extracts. (A): Immunoprecipitation of GFR-1. Lane A: molecular weight markers; lane B: adult kidney; lane C: developing brain (6-day-old); lane D: adult testis; lane E: C18-4 cell line; lane F: 15P1 Sertoli cell line; lane G: NIH 3T3 cell line. (B): Immunoprecipitation of Dazl. Lane A: molecular weight markers; lane B: adult testis; lane C: C18-4 cell line. (C): Immunoprecipitation of Oct-4. Lane A: C18-4 cell line; lane B: adult testis; lane C: molecular weight markers.
The Dazl protein was immunoprecipitated and revealed after blotting using antiserum no. 149 . As seen in Figure 5B, a strong band with a molecular weight of approximately 33 kDa was revealed in the testis extract, corresponding to the molecular weight of Dazl . The same band was observed using C18-4 extracts (Fig. 5B). Similarly, the Oct-4 protein was revealed after immunoprecipitation (Fig. 5C).
Finally, immunoprecipitation failed to detect the expression of GDNF in the purified cells, indicating that the C18-4 cells are not contaminated by Sertoli cells.
Reverse Transcriptase Polymerase Chain Reaction
Neither 3?HSD, a marker for Leydig cells, nor -actin, a marker for peritubular myoid cells, could be found by RT-PCR, further indicating that the cells are of germ cell origin. The absence of LDHC4 expression indicated that the cells are not spermatocytes.
To further confirm the spermatogonial origin of the C18-4 cell line, we investigated by RT-PCR the expression of 36 spermatogonial-specific genes, which were previously identified by Wang and colleagues in the 6-day-old mouse testis. There was a RT-PCR product for the housekeeping gene Gapd in both cell lines and in the neonatal testis samples, indicating that the RNA isolated contained intact transcripts (data not shown). RT-PCR further revealed that all 36 spermatogonial-specific genes tested were expressed in the 6-day-old testis, as expected. None of these genes were expressed in the SF7 Sertoli cell line, confirming the specificity of the transcripts. The expression of 10 spermatogonial-specific genes was detected in the C18-4 cell line. The results of these 10 are reported in Table 1.
Table 1. Spermatogonial genes expressed in the C18-4 cell line, arranged in alphabetical order; 6-day-old testes were used as positive control, and the Sertoli cell line SF7 as negative control
One of the genes expressed by the C18-4 cells was the Ott gene (Fig. 6A), which is transcribed in vivo in the germ cells of testis and ovary . The primers used in this study can detect the presence of two bands by RT-PCR, corresponding to two isoforms, with sizes of 551 and 503 bp, respectively . As shown in Figure 6A, two bands of the correct size are expressed by the C18-4 cells (lane B). However, only one band (551 bp) was detectable in the neonatal testis sample (lane D). Sertoli cells do not express the Ott gene (lane F). Figure 6B shows that the C18-4 cell line also expresses Sycp1, a gene coding for a protein of the synaptonemal complex, which is already expressed in spermatogonia . The band obtained is of 311 bp, as expected (lane B). The same band is visible for the 6-day-old testis sample (lane D), but not in Sertoli cells (lane F). Figure 6C represents the expression of the gene Fth117 that codes for a testis-specific ferritin-binding protein . Fth117 is expressed in the C18-4 cells, giving the expected band of 292 bp (lane B) by RT-PCR. Fth117 expression is found in the 6-day-old testis but is not found in Sertoli cells (lanes D and F, respectively). In addition to spermatogonial-specific genes with a known function, the C18-4 cell line expressed five novel genes that belong to the Tex (testis-expressed) gene family (Fig. 6D) . None of those genes were expressed by the Sertoli cell line.
Figure 6. Expression of spermatogonia-specific genes in the C18-4 cell line, visualized by RT-PCR. RNA samples were isolated from the C18-4 cells, from 6-day-old testes as positive control, and from the SF7 Sertoli cell line as negative control. After reverse transcription, the cDNA samples were amplified using the primers described by Wang et al. . (A): RT-PCR for Ott (PCR product = 551 and 503 bp). (B): RT-PCR for Sycp1 (PCR product = 311 bp). (C): RT-PCR for Fth117 (PCR product = 292 bp). (D): RT-PCR for Tex13 (PCR product = 220 bp), Tex14 (PCR product = 635 bp), Tex15 (PCR product = 411 bp), Tex16 (PCR product = 660 bp), and Tex19 (PCR product = 184 bp). (E): RT-PCR for Piwi1 (PCR product = 241 bp). (F): RT-PCR for Prame11 (PCR product = 448 bp). Lanes A: size markers (100 bp ladder); B: C18-4 cell line; C: negative control for C18-4; D: 6-day-old testis; E: negative control for 6-day-old testis; F: SF7 Sertoli cells; G: negative control for SF7 Sertoli cells. Abbreviation: RT-PCR, reverse transcriptase polymerase chain reaction.
Interestingly, the C18-4 spermatogonial cell line also shows the expression of the piwi12 (piwi-like 2) gene (Fig. 6E). Piwi belongs to a family of genes that is involved in stem cell maintenance and renewal in Drosophila . Two mouse homologues of piwi have recently been cloned: miwi (mouse piwi) and mili (miwi-like) . The primers used in this study are derived from the Drosophila sequence and give a band of 241 bp. As expected, Piwi12 was also detected in the neonatal testis but not in Sertoli cells. Additionally, the C18-4 cells express a PRAME-like gene (Fig. 6F). PRAME is a protein expressed in the testis, as well as in several carcinomas, in acute myeloid leukemias, and in CD34+ hematopoietic stem cells .
Influence of GDNF and SCF on the Behavior of the C18-4 Cells
To determine if the C18-4 cell line responds to growth factors, we cultivated the cells in the presence or absence of 100 ng/ml GDNF in the culture medium. When GDNF is present, the rate of proliferation of the cells increases significantly (p < .005) (Fig. 7A). No influence on cell morphology or expression of the c-kit receptor was observed. Interestingly, the growth effect of GDNF is detected only when the culture medium also contains 10% FCS. No change in the rate of proliferation can be detected when FCS is replaced by a defined serum (Nu serum), which contains a minimal amount of cytokines. Further, addition of SCF to the culture medium did not stimulate the proliferation of the cells (Fig. 7B). SCF did not induce the cells to differentiate since RT-PCR analysis did not reveal the expression of LDHC4, a germ cell–specific isozyme that is detected at the earliest in preleptotene spermatocytes .
Figure 7. Rates of proliferation of the C18-4 cells with GDNF or SCF. (A): In the presence of GDNF, the rate of proliferation of the C18-4 cells increases significantly in comparison with the control cultures. (B): No changes could be observed when the cells are cultured with SCF. Abbreviations: GDNF, glial cell line–derived neurotrophic factor; SCF, stem cell factor.
DISCUSSION
We thank Dr. Renee Reijo-Pera, Department of Obstetrics, Gynecology and Reproductive Sciences, University of California, San Francisco, for providing us with the Dazl no. 149 and 150 antibodies. We also thank Ms. Kathy van der Wee for valuable technical assistance and Dr. Kathy Beal for statistical analysis. This work was supported by the National Institutes of Health grant RO1-HD36483.
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