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Testis |
a Departments of Endocrinology, Faculty of Biology,
b Cell Biology, University Medical Center Utrecht, 3548 CH Utrecht, The Netherlands
c Department of Anatomy, Institute of Biomedicine, University of Turku, FIN-20520 Turku, Finland
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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developmental biology, spermatogenesis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In the present study, a method is developed in which highly purified, bovine type A spermatogonia, including spermatogonial stem cells together with contaminating testicular somatic cells, Sertoli cells, and myoid cells, can be successfully cultured for a long period of time, during which spermatogonia proliferate and colonies of spermatogonia are formed. The efficiency of various culture media, temperatures, and of the addition of serum was tested. In addition, the presence of spermatogonial stem cells in the culture was tested by transplanting the cultured cells into recipient mice testes. The differentiation of the germ cells in the culture was determined by ultrastructural analysis and specific biochemical markers for advanced germ cells.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Testes from 5-mo-old calves were collected from the slaughterhouse, and following enzymatic digestions and purification steps, 65–87% pure type A spermatogonia were obtained as described elsewhere [6]. To investigate the effect of the culture conditions (medium, temperature, and serum) on survival and proliferation, cells (5 x 104) were cultured in 96-well plates containing 200 µl of minimal essential medium (MEM) or KSOM (potassium-rich medium prepared according to the simplex optimization method) medium supplemented with 0%, 2.5%, 5%, or 10% fetal calf serum (FCS) for 2 wk. Medium was changed two times per week. Cells were cultured at 32°C or 37°C in a humidified atmosphere with 5% CO2. Viability and proliferation were assessed at the onset of the culture and at Days 7 and 14. The viability of cells during isolation and purification steps and during short-term culture was determined using the live/dead kit (Molecular Probes, Eugene, OR). In long-term culture, a colorimetric assay was used to quantify proliferative activity based on the cleavage of the tetrazolium salt 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate (WST-1) by mitochondrial dehydrogenases in viable cells (Boehringer, Mannheim, Germany).
To study the effect of cell density on survival of spermatogonia during culture, cells (75–600 x 103 cells/ml) were cultured in 24-well plates containing MEM supplemented with 2.5% FCS. At the onset of culture and at Day 7, viability was assessed using the live/dead kit. In addition, to study colony formation in long-term culture, the cells (250 x 103 per 24-well plate; i.e., 
104 cells/well) were cultured in MEM supplemented with 2.5% FCS for 2–4 wk. The numbers and sizes of spermatogonial colonies were evaluated using Dolichos Biflorus Agglutinin (DBA; E.Y. Laboratories, San Mateo, CA) immunolabeling.
Evaluation of Spermatogonial Proliferation and Differentiation During the First Month of Culture
Cultured cells were studied using an inverted light microscope (TE 200; Nikon, Tokyo, Japan) equipped with phase-contrast and Hoffmann (comparable to Nomarski) lenses. To identify spermatogonia during culture, the lectin DBA was used as a specific marker for type A spermatogonia [7]. In addition, to determine spermatogonial proliferation, 5-bromodeoxyuridine (BrdU; Sigma, St. Louis, MO) and DBA double-fluorescence labeling was used. For BrdU incorporation, cells were cultured in eight-well, glass-chamber slides, and at Days 4, 7, and 14, BrdU was added to a final concentration of 0.3 mg/ml. Cells were then cultured for another 2 h, fixed in Bouin solution for 15 min at room temperature, and kept in 70% ethanol until immunostaining. The BrdU immunolabeling was performed as described previously [8] with some modifications. After BrdU labeling and rinsing in PBS, the slides were incubated in DBA-fluorescein isothiocyanate (E.Y. Laboratories, San Mateo, CA) in PBS (1:100 [v/v]) for 1 h at 37°C. After long rinsing in PBS, they were mounted in Vectashield (Vector Laboratories, Burlingame, CA), sealed with nail polish, and evaluated under a Nikon (Tokyo, Japan) inverted light microscope equipped with an epifluorescence mercury lamp. To assess the proliferative activity of the spermatogonia within colonies, slides were studied under a confocal laser-scanning microscope (Leica TCS NT, Heidelberg Germany) equipped with a 63x oil-immersion objective. A 75-mW ArKr laser for dual excitation (488 and 568 nm), tuned by an acousta optical transfer filter, was used for visualization of the dual staining.
The differentiation stage of the spermatogonia during the first month of culture was studied using c-kit immunolabeling. At the beginning (Day 4) and at the end of the culture (4 wk), the cells were fixed in Bouin solution and preserved in 70% ethanol, and immunohistochemical localization of c-kit was performed using rabbit polyclonal anti c-kit (c19; Santa Cruz, Heerhugowaard, The Netherlands) according to the protocol described by Schrans-Stassen et al. [4].
Evaluation of Spermatogonial Differentiation after Long-Term Culture
To study differentiation of germ cells in long-term culture, in some experiments cells were cultured for as long as 150 days. Every 3–4 wk during this time, cells were removed from the culture dish by enzymatic digestion, a sample was taken for histological and immunohistological examination, and the rest of the cells were cultured on a new plate. During this period, some cellular structures were released from the plate into the medium. To avoid losing them with each medium exchange, before each refreshment the cells in the medium were pelleted, and a smear was prepared for histological and immunohistological investigations. In addition, samples were collected for protein and RNA extractions, and for ultrastructural studies, some cells were fixed within the plate after 100 days of culture.
Expression of Meiotic and Postmeiotic Markers
Protein expression of meiotic marker SCP3 Using an antibody against SCP3 [9], immunohistochemical analysis was performed on the cells after long-term culture and on cells isolated from an adult bull as a positive control [10].
Protein extracts of cells collected after 60 and 100 days of culture together with protein extracts from an adult bull testis and a mixture of isolated spermatocytes and spermatids (as positive controls) and those obtained from purified type A spermatogonia used for culture (as negative controls) were used for Western blot analysis. Total protein lysates were prepared by homogenizing the testes in a Polytron device (Janke & Kunkel GmbH, Staufen, Germany) in ice-cold RIPA buffer (PBS with 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM PMSF, 10 µg/ml of leupeptin, 10 µg/ml of aprotinin, 2 mM Na3VO4, 1 mM Na2MoD4, and 10 mM NaF). For protein extraction from the cultured cells, after removal from the culture plates and centrifugation the pellet was lysed in ice-cold RIPA. Lysates were sonicated on ice for 30 min and cleared by centrifugation for 30 min at 20 000 x g. Protein levels were measured using BCA analysis (Pierce Chemical Co., Rockford, IL). Proteins were separated using SDS-PAGE and blotted onto a polyvinylidene difluoride membrane (Millipore, Bedford, MA). Western blots were blocked using Blotto-A containing 5% Protifar (Nutricia, Zoetermeer, The Netherlands) in Tris-buffered saline (TBS; 10 mM Tris-HCl and 150 mM NaCl, pH 8.0), including 0.05% Tween-20 (TBT), and were washed in TBT in between each step. The first antibody was diluted 1:2000 (v/v) in Blotto-A. After incubation with secondary antibody conjugated to horseradish peroxidase (DAKO A/S, Glostrup, Denmark) diluted 1:5000 (v/v) in Blotto-A, the antigens were visualized using chemiluminescence (ECL; Amersham Pharmacia Biotech Benelux, Roosendaal, The Netherlands) and exposure to x-ray film (RX; Fuji Photo Film Co., Ltd., Tokyo, Japan).
Messenger RNA expression of acrosin and voltage anion-dependent channel Total RNA was extracted according to Rneasy Mini Handbook (Westburg b.v., Leusden, The Netherlands). Before reverse-transcription reaction, 10 µl of the RNA sample were incubated for 5 min at 70°C, vortexed for 5 sec, and chilled on ice. Reverse transcription was performed in a total volume of 20 µl containing 10 µl of sample RNA, 4 µl of 5x reverse transcriptase buffer (Gibco BRL, Breda, The Netherlands), 8 U of RNAsin (Promega, Madison, WI), 150 U of superscript II reverse transcriptase (Gibco BRL), 0.5 mg of Oligo (dt)12–18 primer, and final concentrations of 50 mM dithiothreitol (DTT) and 1 mM of each dNTP. The mixture was incubated for 1 h at 42°C, for 5 min at 95°C, and stored at -20°C. Primer design for acrosin was based on the bovine acrosin cDNA sequence as described by Adham et al [11], and for the voltage anion-dependent channel (VADC-2), primer design was based on human cDNA sequence as described by Blachly-Dyson et al. [12]. The cDNA samples from cultured spermatogonia after 60, 110, and 135 days of culture and samples of total adult testes (as positive control) as well as those obtained from purified type A spermatogonia used for culture (as negative controls) were amplified using primers for acrosin, VADC-2, and ß-actin as the housekeeping gene [13], and polymerase chain reaction (PCR) was performed as described before [14]. Ten microliters of the PCR product were resolved by 1% agarose gel containing 0.4 µg/ml of ethidium bromide. A 100-base pair (bp) ladder (Gibco BRL) was included as a reference for fragment size. An image of the gel was taken using a CCD camera (Appligene; B & L System, Zoetermeer, The Netherlands) and stored in digitized form.
Protein Expression of Outer Dense Fiber after Long-Term Culture
To study outer dense fiber (ODF-2) protein expression in adult bull testis, sections fixed in Bouin solution and embedded in paraffin were used. Immunohistochemistry was performed, incubating the sections overnight with polyclonal rabbit anti-ODF-2 (kindly donated by Dr. F. van de Hoorn, University of Calgary, Canada) antibody diluted in PBS-BSA-c (acetylated bovine serum albuin; 1:200 [v/v]). Subsequently, the slides were incubated with biotinylated goat anti-rabbit antibody diluted in PBS-BSA-c (1:100 [v/v]) for 1 h. After rinsing in PBS, the slides were incubated in avidin-biotin complex for 60 min and then for 20 min in TBS containing 25 mg of 3,3-diaminobenzidine tetrahydrochloride and 15 µl of 3.5% H2O2.
Protein extracts of the cells after 60 and 100 days of culture together with extracts from an adult bull testes and a mixture of isolated spermatocytes and spermatids (positive controls) and those obtained from purified type A spermatogonia used for culture (negative controls) were used for Western blot analysis with anti-ODF-2 antibody (1:500).
Electron Microscopy
Cell cultures were fixed in the culture dish with 2% paraformaldehyde and 0.2% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) for 2 h at room temperature. The cells were rinsed in 0.1 M cacodylate buffer (pH 7.4) and postfixed for 60 min in 1% OsO4 in 0.1 M cacodylate buffer (pH 7.4) and embedded in EPON 812, which was cured at 63°C. An area of interest on the culture dish was selected under a stereomicroscope and cut out. Ultrathin sections were prepared using a Reichert Ultracut E (Leica, Vienna, Austria) and poststained with uranyl acetate and lead citrate. Micrographs were taken at a magnification of 4000x on a JEOL 1200 EX electron microscope (JEOL, Tokyo, Japan).
Evaluation of Stem Cell Activity of Cultured Spermatogonia by Transplantation
Every week during the first month of culture, cells were collected from the culture plate using trypsin (0.05%) and EDTA (0.54 mM) treatment for approximately 15 min at 37°C. After repeated pipetting to break cell connections, trypsin was inactivated by addition of 10% FCS, and the cells were resuspended in MEM/BSA/DNase at a concentration of 20 x 106 cells/ml and kept on ice until transplantation. Ten adult NMRI nude mice (nu/nu; Harlan, Horst, The Netherlands) were used as recipients. The mice were housed with a photoperiod of 12L:12D at a constant temperature and provided with food and water ad libitum. The mice were given a fractionated dose of 1.5 and 12 Gy of x-rays (200 kV, 20 mA, 0.5 mm Cu filter; Philips, Eindhoven, Netherlands) with an interval of 24 h. This dose removes virtually all endogenous spermatogenesis and does not have any apparent harmful effect on the supporting Sertoli cells [15]. One month after irradiation, testes of recipient mice were exteriorized through a midline abdominal incision, and donor cells (25 µl containing 5 x 105 cells) were injected through a micropipette (Clark Electromedical Instruments, Reading, U.K.) via the efferent ducts into the rete testis as described by Ogawa et al [16]. The contralateral testis was used as the negative control. At 2–3 mo after transplantation, half of the testes was fixed in Bouin solution and used for DBA immunohistochemistry. The other half was used for DNA extraction and PCR analysis with specific primers for bovine DNA to detect bovine cells in the recipient mouse testes [17]. The experimental protocol of this study followed the Guidelines for the Care and Use of Laboratory Animals and was approved by the animal care and use committee of the Utrecht University.
Statistical Analysis
Results are presented as the mean ± SEM, and statistical analysis was performed by Student t-test. Differences were considered to be significant at P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Generally, culture of spermatogonia in MEM resulted in more proliferation than culture in KSOM. Addition of serum to both media significantly (P < 0.01) enhanced viability and cell numbers (Fig. 1). Furthermore, culture at 37°C resulted in more cells than culture at 32°C (data not shown). Although more serum resulted in more cells, the proportion of DBA-positive cells (i.e., spermatogonia) became less when more than 2.5% serum was added, indicating enhanced proliferation of somatic cells (Fig. 2). During the first week of culture in MEM supplemented with 2.5% FCS, the number of DBA-positive cells decreased, and approximately 50% of the cells survived. From Day 8 of culture onward, the number of spermatogonia increased continuously, and after 2 wk, approximately 2-fold more spermatogonia were found than at the onset of culture (Fig. 3). Furthermore, after 7 days of culture, no significant effect of cell density on viability of the spermatogonia was observed as measured by the live/dead kit.
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Spermatogonial Proliferation and Differentiation During the First Month of Culture
At the onset of culture, spermatogonia were round cells with a spherical nucleus and one to three dense, spherical nucleoli of approximately 1–3 µm in diameter occupying a central or slightly eccentric position in the nuclei; a high nucleus:cytoplasm ratio; and many cytoplasmic inclusions, mostly concentrated at one side of the cell (Fig. 4B). After a few days, the somatic cells constituted a confluent monolayer, and spermatogonia were observed on top of it. Somatic cells in the monolayer were large cells and positively stained with the Sertoli cell marker vimentin (Fig. 5K). Gradually, the number of spermatogonia increased, and after 1 wk, pairs and chains of spermatogonia were observed (Fig. 4, C–E). During the second and third week of culture, the number of spermatogonia increased further, and colonies were formed. Morphologically, two types of colonies could be distinguished: a round type and a radial type. Round colonies were larger and much less frequent than radial colonies (Table 1) and contained morphologically homogeneous cells with two or three small nucleoli (Fig. 4, F and G). In contrast, most of the cells within radial colonies contained one large, round nucleolus in their nuclei and were often seen to be interconnected through cytoplasmic bridges. However, single germ cells were also found in the radial colonies with one to three nucleoli in the nuclei, resembling the cells constituting the round colonies (Fig. 4, H and I). At 37°C, more and larger spermatogonial colonies were found, and a confluent monolayer of somatic cells was formed more rapidly, than at 32°C.
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In the testes of 5-mo-old calves (positive control), DBA exclusively stained type A spermatogonia (Fig. 5A). Shortly after the onset of culture, DBA-positive cells (i.e., spermatogonia) were found among the somatic cells (Fig. 5B). Whereas some DBA-positive cells were located in between the somatic cells, a few had cytoplasmic extrusions toward the somatic cells. After a few days, pairs and chains of DBA-positive cells could be found (Fig. 5C), which were more frequent at 2 wk. Both round (Fig. 5, D and E) and radial (Fig. 5, G and H) colonies stained positive for the spermatogonial marker DBA.
More than 80% of the isolated spermatogonia showed c-kit immunoreactivity (data not shown). After 4 wk of culture, almost all of the cells in the round colonies were c-kit negative (Fig. 5F), whereas most of the cells in the radial colonies were c-kit positive. Moreover, in some of the radial colonies, a central area was observed that contained dense and small cells, which were strongly positive for c-kit (Fig. 5I).
Coimmunolocalization of DBA-BrdU showed that after 2 wk of culture, approximately 30% of the cells were positive for DBA, and approximately 6–8% were positive for both DBA and BrdU (Fig. 5J). This indicates that approximately 20% of the type A spermatogonia in culture were in the S phase of the cell cycle. Coimmunolocalization of vimentin and BrdU showed the presence of BrdU-labeled cells among the Sertoli cells (Fig. 5K).
Spermatogonial Differentiation in Long-Term Culture
After the first passage (second month of culture), cells proliferated, and new colonies formed. Cells with the morphology of type A spermatogonia were numerous (Fig. 6A). In addition, some cells with the appearance of type B spermatogonia (Fig. 6B) and some similar to spermatocytes were observed (Fig. 6C). Particularly at 37°C, multinuclear structures were observed in the culture medium. These structures of variable size consisted of many nuclei without apparent cytoplasmic membranes (Fig. 6D). Similar structures were found in the cell suspensions isolated from the bull testis. The nuclei of these cells contained three nucleoli resembling those in round spermatids. After the second passage (third month of the culture), small round cells containing three nucleoli were frequently found together with other cells attached to the culture plate. In some of these cells, a small vesicle close to the nucleus resembling an acrosomal vesicle was found (Fig. 6E); however, no further acrosomal development was observed. Staining with periodic acid-Schiff (PAS)-hematoxylin revealed that many of these cells contained PAS-positive structures (Fig. 6F). In addition, some elongated cells were found mostly detached from the other cells floating in the culture medium (Fig. 6G). In some cells, a clear, tail-like structure was formed (Fig. 6, H and I); however, only occasionally was nuclear condensation observed (Fig. 6I).
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At the ultrastructural level, on the top and in between the somatic cell monolayer different cell types were observed (Fig. 7). Cells with large nuclei and little or no heterochromatin, resembling type A spermatogonia, were observed as well as cells with a round to slightly ovoid nucleus containing a moderate amount of heterochromatin along the nuclear rim, resembling intermediate or type B spermatogonia. Occasionally, cells with a complete flagellum structure containing a flagellar axoneme at the cell surface and two centrioles were found. These cells mostly contained numerous vesicles close to the Golgi complex, resembling proacrosomal vesicles, some of the cells containing proacrosomal granules. However, no complete acrosome structure was found.
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Expression of Meiotic and Postmeiotic Markers
A component of the synaptonemal complex, SCP3 is specifically expressed during meiosis in spermatocytes from the leptotene to the pachytene stage and has a molecular mass of 30–33 kDa in rodents and bovine species [18–20]. Immunolocalization of the SCP3 protein in a cell suspension from adult bull testis showed spotted, dotted lines and a linear localization in the nuclei of leptotene, zygotene, and early pachytene spermatocytes, respectively (Fig. 8, A and B). In cultured germ cells, the majority of SCP3-positive cells had a spotted localization similar to that in leptotene spermatocytes (Fig. 8C). Occasionally, a linear localization of SCP3 staining was found in condensed chromatin, resembling that of late pachytene spermatocytes (Fig. 8D). In addition, a band of the expected size of 33 kDa was found in Western blots of the cultured cells both after 60 and 100 days of culture as well as in protein extracts of an adult bull testis and isolated spermatocytes, which were used as positive controls. No similar band was found in the protein extracts obtained from purified type A spermatogonia used for culture (Fig. 9).
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Outer dense fibers are structural elements in the mammalian sperm tail, and ODF-2, with a molecular mass of 84 kDa, is the major protein of these fibers and is synthesized and assembled in elongating spermatids especially during step 16 of spermiogenesis [21]. Immunolocalization of the ODF-2 protein in the adult bull testis showed that it is exclusively expressed in the sperm tail (Fig. 8, E and F). In addition, a clear band of the expected size of 84 kDa was found in Western blots of the cultured cells both after 60 and 100 days of culture as well as in the positive controls. No similar band was found in the protein extracts of purified type A spermatogonia used for culture (Fig. 9).
Both acrosin and VADC-2 mRNAs were expressed in meiotic and postmeiotic germ cells (i.e., spermatocytes and spermatids) [22, 23]. The PCR analysis of the cDNA samples generated from the cells cultured for 60, 110, and 135 days as well as from adult testes demonstrated the presence of specific bands with the expected size of 280 bp for VADC-2 in these samples. However, a PCR product with the expected size of 241 bp for acrosin was only found in 110- and 135-day-old cultured spermatogonia and the positive control, not in 60-day-old cultured spermatogonia. All samples also showed a clear band for ß-actin (data not shown). No PCR products for acrosin or VADC-2 were detected in the samples generated from purified type A spermatogonia used for culture or when a water control was used as template for the PCR (Fig. 10).
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Stem Cell Activity of Cultured Spermatogonia
To investigate whether spermatogonial stem cells were in the culture and whether these cells remained functional during 1 mo of culture, every week cells were transplanted into recipient immunodeficient mouse testes. Three months after transplantation of either fresh or cultured spermatogonia, groups of bovine type A spermatogonia, as detected with DBA staining, were found in the tubule cross-sections of all recipient testes (Fig. 5L). No DBA-positive cells were found in the contralateral control testes. In addition, a PCR product with the expected size of 107 bp was detected in samples generated from mouse testes transplanted with cultured spermatogonia and from the bovine cells used as the positive control. No PCR products were detected when mouse cells or water were used as template for the PCR (Fig. 11).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Several culture variables were tested. First, it appeared that in the absence of serum, the viability of cultured spermatogonia dropped quickly (data not shown), and the cells died after a few days. However, addition of only 2.5% serum maintained both survival and proliferation. Addition of more serum (up to 10%) resulted in more proliferation, but this proliferation favored somatic cells more than spermatogonia. Furthermore, the proliferative activity and differentiation of the cultured spermatogonia were enhanced by culturing at 37°C compared to culturing at 32°C. Because the intratesticular temperature in this species is 34.5°C [24], some experiments were carried out at this temperature; however, preliminary results showed that at 34.5°C, colony formation was less than at 37°C. At 37°C, a confluent monolayer of testicular somatic cells, mostly consisting of Sertoli cells, was formed the quickest, which may have improved spermatogonial survival and proliferation. Comparing our results with those of van der Wee et al. [3] regarding culture of spermatogonia from prepubertal mice that were cocultured with an adult Sertoli cell feeder layer, we speculate that the success in culture might be caused by the presence of actively proliferating Sertoli cells in the monolayer, because cells were obtained from an immature calf testis. In other studies, feeder layers of nondividing Sertoli cells were used [3]. In our long-term culture system, the cultured cells had to be replated once a month because of increasing cell density, and the Sertoli cells, which kept proliferating, each time formed a new monolayer. The latter may well be essential.
Substitution of MEM with KSOM, a potassium-enriched medium, which has been shown to be superior for the culture of porcine [1] and mouse (unpublished results) spermatogonia, did not improve the viability nor the proliferation of bovine spermatogonia. This indicates important species differences in the needs of germ cells during culture. Taken together, these results indicate that culture in MEM containing 2.5% serum at 37°C provides an optimal condition for survival, proliferation, and differentiation of bovine type A spermatogonia.
After 1 mo of culture, two different types of colonies of type A spermatogonia could be discerned: one with a round appearance and one with a radial appearance. Round colonies were observed in which the spermatogonia did not show any formation of intercellular bridges and were negative for the c-kit receptor. Hence, the round colonies likely were composed of single type A spermatogonia that proliferate but apparently do not produce differentiating daughter cells. Alternatively, the round colonies could arise from gonocytes still present at 5 mo. However, because late gonocytes express c-kit receptor in the mouse [25] and in the bovine (unpublished results) but the round colonies do not express c-kit receptor, this possibility seems to be less likely. In contrast, radial colonies were abundant, many of the composing cells were connected through intercellular bridges, and most of the cells were c-kit positive.
An important question is whether spermatogonial stem cells stay present in our culture system. In rodents and the ram, cell kinetic and morphological studies have indicated that spermatogonial stem cells are single type A spermatogonia and that the first step toward differentiation involves incomplete cytokinesis at mitosis and formation of intercellular bridges. Although for the bull it has been suggested that pairs and chains of type A spermatogonia also have stem cell capacity [26–28], agreement exists that single type A spermatogonia are stem cells. The round colonies consist solely of single type A spermatogonia and may be colonies of spermatogonial stem cells that proliferate but are unable to take the first differentiation step. The radial colonies consist mostly of pairs and chains of type A spermatogonia but also, for a small part, single type A spermatogonia. Hence, on the basis of the single-cell criterion, both colonies contain spermatogonial stem cells. Another argument in favor of the presence of active stem cells in the culture is that after the monthly replating of the culture and concomitant dilution of the cells, new radial and round colonies keep being formed. The most obvious explanation of the ongoing formation of colonies is that after each replating, they are formed by the stem cells present in the cell suspension. Finally, after transplantation of cell suspensions of long-term cultures, extensive and progressive "repopulation" of recipient mouse testes by bovine spermatogonia has been found. As observed in most heterologous repopulation attempts, no differentiation of the spermatogonia into more advanced germ cells was seen. However, only spermatogonial stem cells are supposed to be able to migrate to the basal membrane of the recipient mouse seminiferous tubules and produce progeny [29], and the latter was clearly seen. Taken together, we conclude that in our culture system, spermatogonial stem cells remain present and that these cells proliferate and produce new stem cells as well as differentiating cells.
After 100 days of culture, cells with the appearance of spermatocytes and spermatids were observed. Because in vitro, in the absence of a normal seminiferous epithelium, the morphological identification of these cells could not be conclusive, we checked some molecular characteristics of the cells. Both in Western blot analysis and immunohistochemical experiments, expression of the spermatocyte marker SCP3 was found, indicating the presence of spermatocytes among the cultured cells. In addition, other cells expressed the ODF-2 protein, which is first expressed in elongated spermatids [30]. Finally, some cultured cells expressed the mRNAs of acrosin and VADC-2, which are specific for spermatocytes and spermatids [22, 23]. Clearly, our culture system allows at least some of the cells to develop all the way to elongating spermatids. Recently, the in vitro production of haploid germ cells by bovine gonocytes has been reported [31]. However, this culture system was not characterized in terms of proliferative activity and long-term behavior of the cultured germ cells. In a very recent study, human type A spermatogonia obtained from azoospermic patients were cultured on a Sertoli cell feeder layer. Here too, cells with the morphology of elongated spermatids were formed [32].
Hence, in our culture system, pairs and chains of spermatogonia keep being formed, even after replating the cultured cells as a single-cell suspension. An early differentiation step of spermatogonia, in which they acquire the c-kit protein, could clearly be demonstrated to occur. However, further differentiation of the cells was difficult to follow, because this seemed to occur in cell nests that were no longer firmly attached to the feeder layer. Also, these nests were disturbed during the replating procedure. Nevertheless, cells that at the light- and electron-microscopical level resembled spermatocytes and spermatids were seen, and the development of these cell types during culture could be proven by the expression of specific markers. However, many abnormalities, including incomplete or delayed nuclear condensation, cytoplasmic elongation, and acrosome formation, were observed. Similarly, at the molecular level, the expression of acrosin mRNA was also delayed. These observations are most likely caused by in vitro conditions. Therefore, more work needs to be done before this culture system can be used to examine the further differentiation of type A spermatogonia in vitro. Sex chromosome abnormalities were found in the in vitro-produced elongated spermatids in the study by Sousa et al. [32].
In conclusion, for the first time to our knowledge, a culture system has been developed in which type A spermatogonia, including spermatogonial stem cells, survive, proliferate, and form large colonies. In most of these colonies, groups of differentiating cells are formed that ultimately can acquire characteristics of spermatids. This novel culture system can now be used to study spermatogenesis in vitro, particularly factors that regulate differentiation of spermatogonial stem cells.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: D.G. de Rooij, Department of Endocrinology, Faculty of Biology, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands. FAX: 0031 30 2532837; d.g.derooij{at}bio.uu.nl
Received: 26 February 2002.
First decision: 13 March 2002.
Accepted: 16 July 2002.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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J.-B. Stukenborg, J. Wistuba, C. M. Luetjens, M. A. Elhija, M. Huleihel, E. Lunenfeld, J. Gromoll, E. Nieschlag, and S. Schlatt Coculture of Spermatogonia With Somatic Cells in a Novel Three-Dimensional Soft-Agar-Culture-System J Androl, May 1, 2008; 29(3): 312 - 329. [Abstract] [Full Text] [PDF]
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M. Kanatsu-Shinohara, T. Muneto, J. Lee, M. Takenaka, S. Chuma, N. Nakatsuji, T. Horiuchi, and T. Shinohara Long-Term Culture of Male Germline Stem Cells From Hamster Testes Biol Reprod, April 1, 2008; 78(4): 611 - 617. [Abstract] [Full Text] [PDF]
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 J.P. Milazzo, L. Vaudreuil, B. Cauliez, E. Gruel, L. Masse, N. Mousset-Simeon, B. Mace, and N. Rives Comparison of conditions for cryopreservation of testicular tissue from immature mice Hum. Reprod., January 1, 2008; 23(1): 17 - 28. [Abstract] [Full Text] [PDF]
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S. Goel, M. Sugimoto, N. Minami, M. Yamada, S. Kume, and H. Imai Identification, Isolation, and In Vitro Culture of Porcine Gonocytes Biol Reprod, July 1, 2007; 77(1): 127 - 137. [Abstract] [Full Text] [PDF]
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H. van den Berg, S. Repping, and F. van der Veen Parental desire and acceptability of spermatogonial stem cell cryopreservation in boys with cancer Hum. Reprod., February 1, 2007; 22(2): 594 - 597. [Abstract] [Full Text] [PDF]
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 F. K. Hamra, K. M. Chapman, D. Nguyen, and D. L. Garbers Identification of Neuregulin as a Factor Required for Formation of Aligned Spermatogonia J. Biol. Chem., January 5, 2007; 282(1): 721 - 730. [Abstract] [Full Text] [PDF]
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J. G. Jung, Y. M. Lee, T. S. Park, S. H. Park, J. M. Lim, and J. Y. Han Identification, Culture, and Characterization of Germline Stem Cell-Like Cells in Chicken Testes Biol Reprod, January 1, 2007; 76(1): 173 - 182. [Abstract] [Full Text] [PDF]
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E. Goossens, V. Frederickx, G. de Block, A. van Steirteghem, and H. Tournaye Evaluation of in vivo conception after testicular stem cell transplantation in a mouse model shows altered post-implantation development Hum. Reprod., August 1, 2006; 21(8): 2057 - 2060. [Abstract] [Full Text] [PDF]
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E. Goossens, V. Frederickx, G. De Block, A. Van Steirteghem, and H. Tournaye Blastocyst development after assisted reproduction using spermatozoa obtained after testicular stem cell transplantation in mice Hum. Reprod., July 1, 2006; 21(7): 1759 - 1764. [Abstract] [Full Text] [PDF]
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Y. Kim, V. Selvaraj, I. Dobrinski, H. Lee, M. C. Mcentee, and A. J. Travis Recipient Preparation and Mixed Germ Cell Isolation for Spermatogonial Stem Cell Transplantation in Domestic Cats J Androl, March 1, 2006; 27(2): 248 - 256. [Abstract] [Full Text] [PDF]
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M. Kanatsu-Shinohara, K. Inoue, J. Lee, H. Miki, N. Ogonuki, S. Toyokuni, A. Ogura, and T. Shinohara Anchorage-Independent Growth of Mouse Male Germline Stem Cells In Vitro Biol Reprod, March 1, 2006; 74(3): 522 - 529. [Abstract] [Full Text] [PDF]
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 F. K. Hamra, K. M. Chapman, D. M. Nguyen, A. A. Williams-Stephens, R. E. Hammer, and D. L. Garbers Self renewal, expansion, and transfection of rat spermatogonial stem cells in culture PNAS, November 29, 2005; 102(48): 17430 - 17435. [Abstract] [Full Text] [PDF]
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S. A. Beall, K. Boekelheide, and K. J. Johnson Hybrid GPCR/Cadherin (Celsr) Proteins in Rat Testis Are Expressed With Cell Type Specificity and Exhibit Differential Sertoli Cell-Germ Cell Adhesion Activity J Androl, July 1, 2005; 26(4): 529 - 538. [Abstract] [Full Text] [PDF]
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 N. Sofikitis, E. Pappas, A. Kawatani, D. Baltogiannis, D. Loutradis, N. Kanakas, D. Giannakis, F. Dimitriadis, K. Tsoukanelis, I. Georgiou, et al. Efforts to create an artificial testis: culture systems of male germ cells under biochemical conditions resembling the seminiferous tubular biochemical environment Hum. Reprod. Update, May 1, 2005; 11(3): 229 - 259. [Abstract] [Full Text] [PDF]
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M. Kanatsu-Shinohara, S. Toyokuni, and T. Shinohara Transgenic Mice Produced by Retroviral Transduction of Male Germ Line Stem Cells In Vivo Biol Reprod, October 1, 2004; 71(4): 1202 - 1207. [Abstract] [Full Text] [PDF]
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J. M. Oatley, J. J. Reeves, and D. J. McLean Biological Activity of Cryopreserved Bovine Spermatogonial Stem Cells During In Vitro Culture Biol Reprod, September 1, 2004; 71(3): 942 - 947. [Abstract] [Full Text] [PDF]
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 K. J. Bodensteiner, H. R. Sawyer, C. L. Moeller, C. M. Kane, K.-Y. F. Pau, G. R. Klinefelter, and D. N. R. Veeramachaneni Chronic Exposure to Dibromoacetic Acid, a Water Disinfection Byproduct, Diminishes Primordial Follicle Populations in the Rabbit Toxicol. Sci., July 1, 2004; 80(1): 83 - 91. [Abstract] [Full Text] [PDF]
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M. Kanatsu-Shinohara, T. Morimoto, S. Toyokuni, and T. Shinohara Regulation of Mouse Spermatogonial Stem Cell Self-Renewing Divisionby the Pituitary Gland Biol Reprod, June 1, 2004; 70(6): 1731 - 1737. [Abstract] [Full Text] [PDF]
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Y. Hong, T. Liu, H. Zhao, H. Xu, W. Wang, R. Liu, T. Chen, J. Deng, and J. Gui Establishment of a normal medakafish spermatogonial cell line capable of sperm production in vitro PNAS, May 25, 2004; 101(21): 8011 - 8016. [Abstract] [Full Text] [PDF]
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M. Kanatsu-Shinohara, N. Ogonuki, K. Inoue, A. Ogura, S. Toyokuni, and T. Shinohara Restoration of fertility in infertile mice by transplantation of cryopreserved male germline stem cells Hum. Reprod., December 1, 2003; 18(12): 2660 - 2667. [Abstract] [Full Text] [PDF]
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M. Kanatsu-Shinohara, N. Ogonuki, K. Inoue, H. Miki, A. Ogura, S. Toyokuni, and T. Shinohara Long-Term Proliferation in Culture and Germline Transmission of Mouse Male Germline Stem Cells Biol Reprod, August 1, 2003; 69(2): 612 - 616. [Abstract] [Full Text] [PDF]
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