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Testis |
a Murdoch Childrens Research Institute, Royal Children's Hospital, Parkville, Victoria 3052, Australia
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2) but had no effect on spermatogonia-derived colony size and number. For testis somatic cell underlays, there was a profound inhibition of all colony types, and immunohistochemical staining of testis cell underlays showed inhibin/activinßA subunit expression. This finding suggests that negative regulation of germ cell proliferation is mediated by inhibin. Addition of activin A to these cultures resulted in significant recovery (P = 0.046) of gonocyte-derived colony numbers but not spermatogonia-derived colonies, which may reflect the functional regulation by these factors observed in vivo. This proliferation assay also highlights many similarities in the regulation of gonocyte and spermatogonia proliferation in vitro, suggesting that proliferation potential is not noticeably affected by the transition of gonocytes to spermatogonia. For example, the average colony cloning efficiency was 80% for gonocytes and 76% for spermatogonia. This technology forms a basis for optimizing growth of neonatal germ cells for applications such as introduction of genetic material into the germ line to produce transgenic mice and to explore gene therapy.
activin, developmental biology, gamete biology, spermatogenesis, testis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Our in vitro culture system for gonocytes has been extremely useful for directly testing the effect of purified growth factors but has also been useful for understanding the regulatory control of germ cells by somatic cells [8, 9]. At birth, gonocytes are easily recognized in testis by their large size in comparison with Sertoli cells and other somatic cells. The methodology for gonocyte-derived colony formation and the clonogenic assay system has been described by Hasthorpe et al. [8]. Previously, cocultures of gonocytes and Sertoli cells were used to show survival effects of added growth factors on germ cells [10]. The difficulty with the coculture system was the complex interaction of germ cells with somatic cells and the lack of demonstrable cell division in vitro. To date attempts to culture spermatogonia in vitro have been unsuccessful because of difficulties such as purity of the testis population and lack of knowledge of proliferation and differentiation regulation in vitro. In the present study, spermatogonia from Day 15 testes, in which an expanded and enriched spermatogonia population occurs, were micromanipulated and selected by their large relative cell size.
Previous experiments have shown that testis cell underlays exert a pronounced inhibitory action on gonocyte-derived colony formation [9], and our study shows spermatogonia were similarly affected. The inhibin/activin family is a known gonadal regulatory system [11–14] in which activin and inhibin have antagonistic actions. Rat gonocytes contain the activin ßA subunit mRNA and protein and presumably regulate their own maturation by production of endogenous activin A [15]. Activin binds to either a constitutively expressed and active activin type II receptor or to one of four isoforms of the activin type IIB receptor. Following type II receptor binding to activin A, the type I receptor is recruited [16]. In many tissues, activin signaling is antagonized by the inhibin heterodimer. Follistatin also is an activin-binding protein in the testis and can antagonize all the known actions of activin [17]. Local regulation of activin by inhibin, which is produced in Sertoli cells, appears to be an important component of spermatogenic development [18]. In the present study, we extended cloning of germ cells to in vitro culture of spermatogonia and further analyzed the effect of somatic cell regulation on germ cell proliferation in vitro.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Inbred HSD OLA:ICR Swiss mice were used throughout, and the day of birth was denoted as Day 1. Testes were dissected from 
10 1-day-old mice for gonocyte cultures, and testes from 5 15-day-old mice were used for spermatogonial cell cloning. Testes were stored in PBS, and enzymatic digestion of testes involved a 20-min incubation at 37°C with a mixture containing hyaluronidase (100 µg/ml), trypsin (100 µg/ml), collagenase (100 µg/ml), and DNase 1 (5 µg/ml). All enzymes were purchased from Sigma Chemical Co. (St. Louis, MO). After washing, the testes were gently disrupted on ice using a dounce homogenizer with Iscove modified Dulbecco medium (IMDM; Gibco BRL, Life Technologies, Grand Island, NY) containing 1% (w/v) BSA (fraction V; Sigma).
Germ cell cloning medium contained IMDM, nucleosides [19], nonessential amino acids (Gibco BRL), 10-4 M ß-mercaptoethanol (Sigma), and 20% fetal calf serum with growth factors added as indicated. Microtiter wells were incubated with collagen IV (10 µg/cm; Collaborative Research Products, Becton Dickinson, Bedford, MA) or laminin (5 µg/cm; Boehringer-Mannheim, Mannheim, Germany) for 1 h. Plates were then washed and used for cloning experiments.
In some experiments, the following agents were added to the culture medium: platelet-derived growth factor bb (PDGF, 10 ng/ml; Sigma), recombinant rat stem cell factor (SCF, 100 ng/ml; Amgen, Thousand Oaks, CA), human platelet-derived transforming growth factor ß (TGFß, 25 µg/ml; ICN Pharmaceuticals, Costa Mesa, CA), recombinant human activin A, inhibin A, and inhibin B (100 ng/ml; R & D Systems, Minneapolis, MN), and recombinant human follistatin 300 (100 and 400 ng/ml; R & D Systems).
Adherent testis cell underlays were made by preparing a single-cell suspension of testis as described above. Microtiter wells were coated with collagen IV (1 µg/well; Collaborative Research), and 104 cells were plated into each well. Two days later, when confluent, the cells were treated with mitomycin C for 2 h at 37°C in the above medium. After incubation, the medium was aspirated, the cells were washed twice, fresh medium was added, and the underlays were kept at 37°C in a humidified atmosphere for experiments within 7 days. Fresh testis cells (Tsc, 104 cells/well) depleted of germ cells were plated simultaneously with individual micromanipulated gonocytes.
A drawn-out Pasteur pipette (tip diameter approximately 250 µm) was used to deposit germ cells, which were viewed under phase-contrast microscopy and placed into 60-mm-diameter tissue culture-treated Petri dishes. Single germ cells were transferred to individual wells of a 96-well coated microtiter plate containing 150 µl of culture medium with additives (Fig. 1).
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Assay of germ cell-derived colonies was carried out after 4–5 days or 5–7 days of culture for gonocytes and spermatogonia, respectively. The percentage of wells containing a colony was determined using a Leica inverted-phase microscope, and results from three or more experiments were used to calculate mean and SEM. In the case of Figure 9, factorial analysis was carried out using ANOVA. The proportion of wells in which colonies developed was transformed to the log scale, and the differences were determined for the various groups, gonocyte alone (G), inhibin A (InA) + G, and testis cell underlay (TUL) + G, and for activin A present or absent. Analysis of frequency of colonies was performed on data for the frequency of colonies of different sizes in different treatment groups using the chi-square test (see Figs. 3 and 7). In other cases, the t-test was applied to determine the significance of differences between control and test groups.
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Immunohistochemical Staining
Immunohistochemical staining for the activin/inhibin ßA subunit was performed with the E4 monoclonal antibody (Oxford Bio-Innovation Ltd., Oxfordshire, UK) or the 69403.11 antibody (R & D Systems). Cultured testis cells were fixed in 2% paraformaldehyde, and Day 1 normal testis was fixed in 10% neutral formalin. Testis sections or testis cells cultured on glass slides were stained using the CSA system (DAKO Corp., Carpinteria, CA) with primary antibodies and incubated for 30 min.
Paraffin-embedded sections of testes from normal newborn, Day 5, and Day 15 mice were prepared and stained with germ cell nuclear antigen (GCNA) according to the method of Enders and May [10]. For immunofluorescent staining of colony cells, at 5 days of culture colonies were pooled into a 5-ml tube (Falcon Plastics, Becton Dickinson), fixed in a 1% paraformaldehyde (w/v) solution for 10 min, washed, and resuspended in 0.1% Triton X-100 (v/v) in PBS. The GCNA-1 antibody (a gift from Dr. G. Enders, Kansas City, KS) hybridoma supernatant was not diluted, and cells were incubated for 30 min on ice and washed with 0.1% Triton X-100 in PBS. Anti-mouse F(ab')2-fluorescein isothiocyanate conjugate was added for 30 min at 4°C, and the cells were observed under a fluorescence microscope. The relative size estimate of germ cells and somatic cells was determined using cytospin (Shandon Inc., Pittsburgh, PA) preparations. The preparations were fixed for GCNA staining as described above, incubated with GCNA-1 antibody hybridoma supernatant for 30 min at room temperature, processed for horseradish peroxidase staining using sheep anti-rat IgM-biotin (Biosource International, Camarillo, CA) and streptavidin-horseradish peroxidase (Silenus, Melbourne, Australia), and counterstained with hematoxalin. The diameters of 500 GCNA-positive and 500 GCNA-negative cells were determined using the Scion Image for Windows program. The mean and SD of these measurements were calculated as 10 ± 1.6 µm and 6 ± 1.5 µm, respectively.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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At birth (Day 1), the gonocyte is located centrally in the seminiferous tubule (Fig. 2a) and migrates over the next 3 days to the base of the tubule. Type A spermatogonia are found lining the seminiferous tubule (Fig. 2b), forming an annular ring by Day 15 (Fig. 2c), and intermediate and type B spermatogonia and spermatocytes are also present in Day 15 testis tubules. The distinctive advantages of studying germ cells from the gonocyte and spermatogonia stage is their ease of identification by size, relative to somatic cells. In a single-cell suspension of Day 15 testis, germ cells have a cell diameter 1.6 times that of GCNA-negative cells (somatic cells) of the testis.
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Clonogenicity of Gonocytes and Spermatogonia
Gonocyte-derived colonies were of various sizes, from 4 to >256 cells/colony after 4–5 days of culture (Figs. 3 and 4). Addition of various growth factors to gonocyte culture medium produced quite similar colony numbers and colony size distribution profiles, except in the case of PDGF, which had an increased colony size. There were fewer colonies with >16–64 cells when compared with control cultures, but this reduction in number of colonies was offset by a higher number of PDGF colonies with >64–128 cells. Chi-square analysis confirmed the significance of this difference and showed strong evidence of differences in distribution of colony size between PDGF treatment and controls (P < 0.001). The differentiation status of these colony cells is not known, but the presence of large and smaller cells suggests that these cells were later-stage germ cells. The lack of stage-specific differentiation markers precludes positive identification. GCNA was expressed in colony cells, confirming their germ cell origin (Fig. 4f, inset). Although all colonies were derived from gonocytes, there was a broad size range, indicating varying potential for proliferative expansion, with a modal size of >16–64 cells/colony (Fig. 3). The cloning efficiency of gonocytes was approximately 80% and was achieved without addition of specific mitogenic growth factors.
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Spermatogonia-derived colonies were counted after 5–7 days of culture and were very similar in appearance to gonocyte-derived colonies (Figs. 4 and 5). Experiments comparing different extracellular matrix coatings of culture wells indicated that the spermatogonia-derived colony incidence was the same whether wells were coated with laminin (56% ± 13%) or collagen IV (56% ± 6%). Colony size peaked at >32–64 cells/colony for laminin and >16–32 cells/colony for collagen IV. However, there was a higher incidence of larger colonies (>128–256 and >256 cells/colony) with collagen IV, which was used to coat wells for subsequent experiments.
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The percentage of colony formation (percentage of wells with a colony) for spermatogonia cultures with PDGF and/or SCF was comparable, and the cloning efficiency ranged from 63% ± 10% to 85% ± 8% (Fig. 6). Colony size distribution showed a higher frequency of colonies with >5–16 and >64–128 cells/colony with PDGF as compared with controls, which peaked at >16–32 cells/colony (Fig. 7), but chi-square analysis showed no significant difference between all treatment groups and controls (P = 0.07).
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Somatic Cell, Activin, and Inhibin Regulation of Gonocyte Proliferation
Experiments with established testis cell underlays and cloned gonocytes revealed a dramatic and significant decrease (P = 0.0003) in gonocyte-derived colony frequency when compared with gonocytes only (Fig. 8a). This effect was specific to testis cells; underlays from an irrelevant cell line, A549, did not have a significant inhibitory effect (66% ± 19% compared with 73% ± 13% colonies with no underlay present). When gonocytes were cloned with a testis single-cell suspension, a significant reduction in colony number also occurred (P = 0.03) (Fig. 8b). With addition of activin A to gonocyte cultures having a testis underlay, colony formation was increased to 53% ± 12% as compared with 20% ± 8%, and an ANOVA revealed a significant interaction between TUL + G and the presence of activin A (P = 0.046), indicating that there was a postitive effect of activin A in the TUL + G + AA group. Other groups with added inhibin A and/or activin A did not show any significant interactons (P 0.1) (Fig. 9). Anti-activin A (ßA subunit; 5 µg/ml) monoclonal antibody did not reduce gonocyte-derived colony numbers (data not shown), and follistatin, which binds activin, also did not alter colony formation when 100 and 400 ng/ml was added to cultures (data not shown).
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Immunohistochemical staining of testis underlays was done with two different anti-human activin/inhibin ßA subunit antibodies; the E4 antibody was raised against a synthetic peptide, and the 69403.11 antibody was prepared against the mature recombinant human activin A molecule. Both ßA subunit antibodies revealed the presence of the ßA subunit in the cytoplasm of adherent cells (Fig. 10), indicating the presence of the inhibin or activin protein in somatic cells. Staining in Figure 10B appears to be around and over the nucleus, although the appearance of nuclear staining may be due to the overlying cytoplasm in these whole cell preparations grown on slides in vitro.
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Activin, Somatic Cell, and Inhibin Regulation of Spermatogonia Proliferation
Testis cell underlays made with 1-day-old mouse testis cells showed significant inhibition of spermatogonia colony formation (P 
0.015), similar to that observed with gonocyte cultures (Fig. 11). Activin A produced an nonsignificant increase in spermatogonia-derived colony number (P = 0.32) in the presence of a testis cell underlay. When spermatogonia cultures were exposed directly to activin A, follistatin, inhibin A, or inhibin B, there was no significant effect on spermatogonia-derived colony formation (P 0.16) (Fig. 12).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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6 and ß1 integrin [24] on type A spermatogonia. We used identical culture conditions for spermatogonia and gonocytes, which suggests that spermatogonia colonies may be largely derived from type A spermatogonia for which the gonocyte is the immediate precursor. Collagen IV matrix coating resulting in the same cloning efficiency for spermatogonia as did laminin matrix coating of culture wells, but Shinohara et al. [24] found that spermatogonia were more enriched for transplantation when testis cells were selected on the basis of their adherence to laminin rather than to collagen IV or fibronectin. Laminin adherence may be more selective for transplantation of type A spermatogonia, but it is not a requirement for initiation of proliferation in culture. Germ cell development in the testis has been classified into functional temporal stages with the amplifying compartment, which includes up to type B spermatogonia with a flexible number of cell divisions, and the differentiating compartment of spermatocytes, which are only capable of two divisions [5, 25]. Spermatocytes in Day 15 testis would at most have the potential to form a four-cell colony in the proliferation assay described here. Colonies containing four cells or less were therefore not included in the analysis involving Day 15 testis (Fig. 7) so that only spermatogonial cell colonies from the amplifying compartment were assayed.
The early stages of spermatogenesis involve complex regulation and cell-cell interactions. In vivo and coculture studies have been informative [26–28], but germ cells still need to be studied in isolation. Efforts have therefore been directed to establishing germ cell lines from neonatal and older mice to study meiosis and early stages of spermatogenic differentiation. Immortalized germ cell lines have been generated using SV-40 large T antigen, and these lines include GC-1spg cells with pachytene spermatocyte characteristics and GC-2spd(ts) and GC-3spc(ts) cells, which have round spermatid features [29, 30]. More recently, rat spermatogonial lines have been established using SV-40 large T antigen, and these cell lines have stem cell, proliferating, and differentiating spermatogonial cell properties [31]. These cells can be transplanted and can colonize seminiferous tubules but do not differentiate past the spermatogonial stage, replicating without differentiation in vivo [31]. This lack of normal differentiation capabilities limits the usefulness of these cell lines. Our approach has been to grow normal gonocytes and spermatogonia in vitro and then compare characteristics at these two stages of differentiation both in vivo and in vitro and explore any morphological differences in proliferation in vitro [8].
Differentiated and proliferative type A spermatogonia express c-kit and are sensitive to inhibition by the antibody to the c-kit receptor (ACK2) at 5 days of age in vivo, whereas spermatogonia of 2-day-old mice, which are negative for c-kit protein, are not affected [22]. The majority of gonocytes in mice express c-kit mRNA but fail to respond to SCF in vitro [8], indicating that the receptor is not functional at that stage, possibly because of an uncoupling of the receptor and intracellular signaling [32]. Evidence from our studies suggests that SCF has a role in spermatogenesis once spermatogonial differentiation occurs, as has also been reported by Packer et al. [33]. Our findings indicate that spermatogonial colony-forming cells do not respond to soluble SCF in vitro and that more highly differentiated spermatogonia are the target cell population for SCF. The effect of membrane-bound SCF cannot be studied in this system because underlays of STO cells produce an inhibitory response [9].
The PDGF receptor is expressed on gonocytes and Sertoli cells during the first 5 days after birth in the rat [34], and PDGF potentiates the proliferative ability of gonocytes in mice, resulting in a shift to larger colony size [8]. A comparable observation has been reported with purified rat gonocytes in vitro, which showed elevation of bromodeoxyuridine incorporation in the presence of PDGF [34]. Spermatogonial colony number was not altered by PDGF, and PDGF produced a smaller modal colony size compared with control cultures. PDGF appears to affect gonocyte but not 15-day spermatogonia proliferation, which may reflect the existing functional regulation of germ cell production in vivo [35].
Inhibins and activins are feedback regulators for the release of the pituitary hormone FSH, but they also have wide-ranging effects on gonadal and nongonadal tissues. Evidence from in vitro culture of testis fragments and cocultures have shown that FSH and activin appear to stimulate Sertoli cell proliferation during early postnatal testis development [15, 26, 36]. FSH acts indirectly to induce Sertoli cell expression of follistatin and inhibin [37, 38], which in turn regulates both germ cells and Sertoli cells. Meehan et al. [15] proposed that germ cells regulate their own maturation initially through endogenous activin A production and then follistatin neutralizes activin action, thus leading the way for the onset of spermatogenesis. In our culture system, activin addition overrides the antagonistic effect of somatic testis cells, which produce the inhibin ßA subunit, and increases gonocyte colony formation by more than 2-fold. However, very little effect on spermatogonia was measured. This finding is in agreement with those of Meehan et al. [15], who demonstrated that activin A caused a decrease in the spermatogonia:Sertoli cell ratio whereas the gonocyte:Sertoli cell ratio was elevated because of an increase in gonocyte number in the presence of activin.
We developed a novel approach to cloning germ line cells in vitro. The optimal culture conditions are essentially identical for gonocytes and spermatogonia, both requiring fetal calf serum and ß-mercaptoethanol in the culture medium. Growth factors had a minor effect when compared with the strong inhibition of colony formation in the presence of testis cell underlays. The way is now clear for characterizing colony cells functionally using transplantation into the testis tubules and assay for stem cell repopulating ability. Characteristics such as proliferative potential and self renewal await investigation, as does in vitro manipulation of these germ line cells for transgenic technology and new therapies. This culture system provides a model for directly studying and manipulating differentiation and for investigating the signals required to induce meiosis in the germ line.
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FOOTNOTES |
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2 Correspondence: S. Hasthorpe, Germ Cell Research, Murdoch Childrens Research Institute, Royal Children's Hospital, Flemington Road, Parkville, Victoria 3052, Australia. FAX: 613 03 9345 6668; hasthors{at}cryptic.rch.unimelb.edu.au
Received: 17 June 2002.
First decision: 15 July 2002.
Accepted: 28 October 2002.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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6-Integrin are surface markers on mouse spermatogonial stem cells. Proc Natl Acad Sci U S A 1999 96:5504-5509This article has been cited by other articles:
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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, K. Inoue, N. Ogonuki, H. Miki, S. Yoshida, S. Toyokuni, J. Lee, A. Ogura, and T. Shinohara Leukemia Inhibitory Factor Enhances Formation of Germ Cell Colonies in Neonatal Mouse Testis Culture Biol Reprod, January 1, 2007; 76(1): 55 - 62. [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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F. W. Atchison and A. R. Means Spermatogonial Depletion in Adult Pin1-Deficient Mice Biol Reprod, December 1, 2003; 69(6): 1989 - 1997. [Abstract] [Full Text] [PDF]
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; SCF + PDGF, 
TGFß, 
; no added growth factors, 
