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BOR - Papers in Press, published online ahead of print October 23, 2002.
Biol Reprod 2002, 10.1095/biolreprod.102.005132
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BIOLOGY OF REPRODUCTION 68, 860–866 (2003)
DOI: 10.1095/biolreprod.102.005132
© 2003 by the Society for the Study of Reproduction, Inc.

Reproductive Technology

Determinants of Retroviral-Mediated Gene Delivery to Mouse Spermatogonia1

Maria P. De Miguela, and Peter J. Donovan2,a

a Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania 19107


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Spermatogonia represent a new route to transgenesis in mice and potentially in some commercially important domesticated animals. In addition, these cells are also a potential target for viral integration in patients receiving somatic cell gene therapy. But the factors influencing retroviral transduction into spermatogonia are not well understood. Because retroviral transduction is affected in part by the proliferative status of the host cell, we developed an improved cell culture system in which spermatogonia survive and proliferate for several days. We used this system to test the ability of a variety of murine and avian retroviruses to infect spermatogonia. We investigated the factors influencing retroviral transduction of spermatogonia, including the proliferative status of the infected cell, the type of viral envelope, the type of retroviral long terminal repeat, and the method of viral delivery. Here we show that many of the widely used retroviral vector systems can be used to successfully transduce spermatogonia at high efficiency. Moreover, we show that retroviral delivery of MDM2, the major downregulator of p53, promotes spermatogonial survival in culture, suggesting that p53 plays a role in regulating spermatogonial apoptosis induced by growth factor deprivation. These results further demonstrate the usefulness of this novel system of targeting substances of interest to the testis. These data have important implications for improving animal transgenesis and for understanding the risks associated with somatic cell gene therapy.

gametogenesis, Sertoli cells, spermatogenesis, testis


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Type A spermatogonia are a heterogeneous population of cells containing a small percentage of spermatogonial stem cells that have self-renewal capability. But the vast majority of A spermatogonia are destined to become spermatozoa and lack self-renewal capability. To date, these two populations of A spermatogonia cannot be distinguished morphologically or biochemically. Spermatogonial stem cells can be identified functionally by transplantation from one animal to another and can colonize the recipient gonad and generate mature sperm. Therefore, such transplanted spermatogonia represent a potentially new route to transgenesis in mice and other commercially important species [1]. Exogenous DNA can be introduced into transplanted spermatogonial cells using a variety of techniques including lipofection, electroporation, and viral transduction. Understanding the factors that affect transduction of spermatogonia with DNA should facilitate the goal of widespread application of this technology. Moreover, studies on viral infection of spermatogonia could also provide insights into the possible hazards that viral vectors present when used in somatic cell gene therapy. Potentially, viral vectors introduced into the tissues or bloodstream of a patient could find their way into the testis and into spermatogonial stem cells. Integration of the viral genome into the DNA of spermatogonia could generate potentially undesirable gene mutations that will be passed on to successive generations.

DNA can be introduced into spermatogonia either in vitro using transfection techniques such as lipofection, which affects only transient gene expression [2] or in vivo via whole testis electroporation, which has very low efficiency [3, 4]. Adenoviruses or adeno-associated viruses (AAV-2) introduced into testes in vivo fail to transduce spermatogonial cells [5, 6]. Attempts to introduce retroviruses into spermatogonia in vitro have been moderately successful, although the efficiency of infection is low [7, 8]. This is likely because retroviral integration into the host cell DNA requires cells to go through M-phase [9] and the techniques for culturing spermatogonia are still poor. Few of the cells are able to divide or even survive in culture. This may reflect the in vivo situation where only a small percentage of spermatogonia proliferate [10]. Spermatogonia naturally undergo extensive apoptosis in vivo [11–13]. This fact is paralleled in the primary culture systems developed so far. Apoptotic regulators such as p53 may play an important role in regulating male germ cell survival [14]. In fact, Trp53 knockout mice testes contain 40% higher numbers of type A spermatogonia than wild-type mice [15].

In previous studies, we and others have begun to identify the molecules implicated in the regulation of in vitro spermatogonial survival and proliferation. Among other growth factors, leukemia inhibitory factor (LIF; [16]) and oncostatin M (OSM; [17, 18]) have been shown to improve both survival and proliferation of spermatogonia in coculture with Sertoli cells. Proliferation of spermatogonia in vitro is also improved by addition of vitamin A [19, 20] or epidermal growth factor (EGF; [21]). In addition, the cAMP agonist, forskolin, has been demonstrated to improve in vitro proliferation of a variety of cell types, including primordial germ cells (PGCs), the embryonic precursors of spermatogonia [22]. However, none of these factors alone is enough to promote the long-term survival or stimulate high proliferative activity of spermatogonia (for review, see [23]). Establishment of specific culture conditions for spermatogonia that promote high levels of cell survival and stimulate proliferation will benefit subsequent efforts in retroviral transduction of this cell type.

In addition to improving conditions for spermatogonial proliferation, manipulation of different viral elements could improve efficiency of retroviral transduction of spermatogonia. Viral envelope proteins that bind to specific receptors on the host cell can regulate retroviral entry into cells. For example, the 10A1 amphotropic viral envelope not only binds to the typical amphotropic cell surface retroviral receptor but also to the gibbon ape leukemia virus receptor. Theoretically, therefore, viruses coated with the 10A1 amphotropic envelope have an increased chance of viral-cell surface receptor recognition and exhibit a broader host range. Therefore, judicious packaging of viruses in different envelopes can improve the efficiency of retroviral transduction. Moreover, expression of a specific retroviral receptor in a cell type can dramatically improve the efficiency of retroviral delivery. For example, the avian leukosis viruses (ALVs) can infect avian cells but are incapable of infecting mammalian cells because mammalian cells do not express the ALV receptor [24]. Expression of the cloned ALV receptor, tva, in mammalian cell types allows them to be infected with ALVs and facilitates use of ALV-based gene delivery systems [25]. Finally, dramatic improvements in retroviral transduction of cells can be achieved by spinning viral supernatants onto the cells, a method known as spinoculation [26].

Once retroviral entry and integration have occurred, expression of viral genes in cells is driven by the promoter in the retroviral long terminal repeat (LTR). Pluripotent stem cells possess mechanisms to prevent viral transduction by expressing specific proteins that bind the murine leukemia viral (MLV) LTR and prevent retroviral gene transcription [27]. This effect could explain the low efficiency of spermatogonial transduction achieved in previous studies [7, 8]. In embryonal carcinoma cells [27], embryonal stem cells [28], and PGCs [29], the use of a mutated MLV LTR, termed the murine stem cell virus (MSCV) LTR, which cannot be inactivated by stem cells, dramatically improves transduction efficiency. Similarly, the ALV LTR is not recognized by mammalian cells and thus escapes the mechanisms that inhibit retroviral transduction [24].

The factors affecting viral infection of mammalian spermatogonia are ill defined. The goals of the present study were first to establish cell culture conditions in which spermatogonia would both survive and proliferate efficiently in order to allow retroviral transduction. Second, we sought to characterize the parameters that affect both retroviral infection of and retroviral-mediated gene expression in spermatogonia. Third, we sought to test this retroviral transduction system by infecting spermatogonia with a retrovirus encoding a molecule with therapeutic potential to demonstrate the functional utility of this system. These studies should aid in the development of conditions for routinely manipulating gene expression in male germline stem cells and also improve our understanding of the risks associated with viral-based somatic cell gene therapy.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Mice

B6D2F1 mice were obtained from Jackson Laboratories (Bar Harbor, ME). In experiments using ALV-derived vectors, transgenic mice expressing the receptor for the subgroup A ALVs (tva; [30]) under the control of the constitutive chicken ß-actin promoter (ß-AKE) [31] were used. Most tissues in ß-AKE mice are susceptible to ALV infection, including the germline, as the provirus is transmitted as a transgene. Mice were used at 3 days postpartum (dpp). All animals were maintained and used under protocols approved by the Institutional Animal Care and Use Committee of Thomas Jefferson University according to the Guide for Care and Use of Laboratory Animals (National Research Council publication copyright 1996, National Academy of Science).

Cell Isolation and Culture

A detailed procedure for spermatogonial isolation and culture has been described previously [32]. Cultures were derived from mice at 3 dpp, at which time the only germ cells present in the seminiferous tubules are A spermatogonia. Type A spermatogonia and supporting Sertoli cells were isolated by sequential collagenase/trypsin digestion and differential unit gravity sedimentation. The cultures therefore consisted of type A spermatogonia and supporting Sertoli cells. Culture media consisted of Dulbecco modified Eagle medium/F-12 with 15 mM HEPES buffer (Life Technologies, Carlsbad, CA) supplemented with 0.1% Fraction V BSA (Sigma, St. Louis, MO), 200 ng/ml retinol acetate (Sigma), 5 U/ml penicillin/streptomycin (Life Technologies), 2 mM L-glutamine (Life Technologies), 1 mM Na-pyruvate (Sigma), and 5 µg/ml insulin/transferrin (Sigma). To increase the survival and proliferation rate of spermatogonia, media was supplemented with different combinations of growth factors. The concentrations of growth factors were 100 µm forskolin (Sigma), 1000 U/ml mLIF (Life Technologies), 10 ng/ml mOSM (Sigma), and 10 ng/ml mEGF (Life Technologies).

BrdU and Immunohistochemistry

Spermatogonia were identified in the spermatogonia-Sertoli cell cocultures by indirect immunohistochemistry using an antibody to the cytoplasmic germ cell marker vasa [33] and either a biotinylated or a rhodamine-conjugated goat anti-rabbit antibody (Vector Labs [Burlingame, CA] and Roche [Indianapolis, IN], respectively). Labeled cells were identified either by rhodamine fluorescence or with an alkaline phosphatase-streptavidin conjugate developed with Fast Red (Sigma). To determine the survival rate of spermatogonia, the number of vasa-positive cells was counted in a randomly selected 12 mm2 area. To determine the proliferative rate of all cell types, the cultures were incubated with BrdU for 1 h. Anti-BrdU immunohistochemistry was performed following the manufacturer's instructions (Amersham, Piscataway, NJ). Cells incorporating BrdU were identified by the presence of a black nuclear precipitate. The Sertoli cell proliferation rate was quantified by counting BrdU-labeled Sertoli cells in an area of 4 mm2 that was selected at random in each culture. For spermatogonia, at least 150 germ cells were identified and counted in each culture. Data presented are expressed as the mean percentage of BrdU positive versus total number of cells of one representative experiment. Experiments were repeated twice, each experiment comprising at least triplicate cultures.

Retroviral Vectors

All reporter vectors carried the enhanced green fluorescent protein (GFP) as a reporter gene. The wild-type MLV-GFP vector LGIN was purchased from Clontech (Palo Alto, CA). The MSCV-GFP vector MGIN has been described elsewhere [34]. The ALV-GFP vector RCASBP(A) has also been described elsewhere [24, 35]. The RCASBP(A)-MDM2 vector was a gift from W.P. Hively, E. Holland, and H. Varmus and was derived by subcloning the EcoRI fragment of mouse MDM2 cDNA into the YAP6 shuttle vector and then the Pac1 fragment ligated into RCASBP(A)-X cloning vector.

Production and Titer of Retrovirus Stocks

For MGIN and LGIN retroviral production, PA317 amphotropic packaging cells (a 293T derivative) were used [34]. For MGIN 10A1 retroviral production, PT67 packaging cells expressing the 10A1 envelope were used [36]. For RCASBP(A)-GFP retroviral production, the avian fibroblast cell line DF-1 was used [31]. Retroviral production was performed according to the procedure described by Miller et al. [37]. The end-point titers of viral stocks were measured in NIH3T3 cells based on GFP fluorescence and also when possible based on neomycin resistance. Titers were adjusted to 5 x 105 colony-forming units for all viral supernatants used.

Infection

Infection of spermatogonia and Sertoli cells with the different viral supernatants was performed the day after plating. When using MLV and MSCV, polybrene (5 µg/ml) was added to the viral supernatant, as it has been reported to increase infection efficiency [26]. Cultures were infected daily with viral supernatant mixed 1:1 with medium during the experimental time period (repeated infection method) or undiluted supernatant was centrifuged onto the cultures at 1800 x g for 2 h on the first day of culture (spinoculation method) [26, 34].

Assessment of Expression of Introduced DNA

To specifically identify spermatogonia in the postnatal cultures, anti-vasa immunohistochemistry was performed [33] using a secondary antibody coupled with rhodamine isothiocyanite. An area of 4 mm2 was selected at random for each culture and time point to determine infection efficiency of Sertoli cells. Transduced green cells were identified and counted under a fluorescence microscope (Nikon). For spermatogonia, at least 100 germ cells were identified and counted in each culture. The percentage of infected cells was determined after 3, 5, or 7 days of culture. Data presented are expressed as the mean percentage of transduced versus total number of cells of at least four pooled experiments, each experiment comprising at least triplicate cultures.

Retroviral-Mediated Manipulation of Spermatogonial Growth

In the functional experiments, cultures were established in the same way as described above and infected with the ALV-MDM2 viral vector by spinoculation on the first day of culture. Cultures were treated with growth factors during 4 days to promote proliferation and allow retroviral integration. The cultures were then subjected to growth-factor deprivation for 24 h prior to fixation in order to induce spermatogonial death. Spermatogonia were identified by indirect immunohistochemistry using an antibody to the cytoplasmic germ cell marker vasa as described above and counted in a randomly selected 6 mm2 area. Experiments were repeated three times, and each experiment consisted of triplicate cultures. Statistical significance was assessed by the Student t-test.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Establishment of Culture Conditions Permissive of Retroviral Transduction of Spermatogonia

Gene transfer by retroviruses occurs only in cells that are actively replicating at the time of infection [9]. In vivo, spermatogonia proliferation is low both at 3 dpp and in the adult testis [10, 38]. Even when spermatogonia are cultured with Sertoli cells, they proliferate very poorly in medium lacking growth factors [39] and could not be transduced with retroviruses (not shown). In order to improve both survival and proliferation of spermatogonia in culture, we tested different combinations of growth factors, including forskolin, LIF, EGF, and OSM, which we previously found to improve either survival or proliferation of spermatogonia in the same culture conditions (see Introduction; for a review, see [23]). Proliferation rates were measured by BrdU incorporation. Spermatogonia were isolated at 3 dpp because, at this stage of mouse development, the seminiferous tubules contain the highest percentage of spermatogonial stem cells. Spermatogonia were identified in the spermatogonia-Sertoli cell cocultures by anti-vasa staining (Fig. 1, A–C). Using every growth factor combination, 80% of the spermatogonia plated on Day 1 survived the first 5 days in culture (not shown). However, after 7 days in culture, spermatogonia survived only in cultures supplemented with both OSM and EGF together (Fig. 1, B and C). In cultures lacking OSM, there was a dramatic decrease in spermatogonial survival at Day 7, most likely because of the requirement for OSM for long-term spermatogonial survival. In spermatogonial cultures supplemented with both OSM and EGF together, spermatogonial survival was further enhanced by the addition of LIF. With this growth factor combination (forskolin, OSM, EGF, and LIF), 45% of the plated spermatogonia survived until Day 7. Spermatogonial proliferation was high in all cultures supplemented with OSM, but only in cultures also supplemented with LIF was proliferation steady during the 7-day time course of the experiments (Fig. 2A). The most effective combination of growth factors for stimulating survival and proliferation of spermatogonia was found to be forskolin, LIF, EGF, and OSM (Fig. 2A). Sertoli cells survived equally independently of growth factor conditions (not shown). Interestingly, the higher proliferative activity achieved by spermatogonia was paralleled by lower Sertoli cell proliferation (Fig. 2B). This mirrors the in vivo situation, where Sertoli cell proliferation declines after birth, at the same time as spermatogonia start proliferating [10]. In this 7-day period, we also observed that Sertoli cells became aggregated and, in the presence of EGF, formed cord-like structures (Fig. 1B). The lower proliferative activity of Sertoli cells was accomplished by the same combination of forskolin, LIF, EGF, and OSM (Fig. 2B). Therefore, all subsequent experiments were performed using the above described growth factors combination.



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  		FIG. 1. A) Sertoli cell-A spermatogonia cocultures were incubated for 1 h with BrdU to assess proliferation rates under different growth factors combinations (statistical data shown in Fig. 2). Anti-BrdU was developed with DAB/Cl2Ni that produces a black precipitate in the cells nuclei. Spermatogonia are distinguished from Sertoli cells by anti-vasa immunohistochemistry developed with Fast Red that produces a red precipitate in the spermatogonia cytoplasm. Proliferating spermatogonia (black arrowheads) as well as nonproliferating spermatogonia (white arrowheads), together with proliferating Sertoli cells (black arrows) and nonproliferating Sertoli cells (white arrows) can be recognized. Bar = 95 µm. B) A phase-contrast image of a Sertoli cell-A spermatogonia coculture after 7 days of culture. By this stage of culture, Sertoli cells have begun to aggregate into cord-like structures and germ cells can be seen growing on top of the Sertoli cells. C) Anti-vasa immunocytochemistry of the culture shown in B. Labeled cells were identified with a rabbit anti-vasa antibody and a goat anti-rabbit immunoglobulin antiserum coupled to rhodamine isothiocyanite. Germ cells are stained red. Bar = 95 µm. D, E) Fluorescence photomicrographs of GFP expression in retrovirally infected cultures. Sertoli cell-A spermatogonia cocultures were infected with the MSCV-GFP vector for 8 days. D) Transduced cells are recognized under fluorescent light using GFP excitation and emission filters. E) Expression of GFP in spermatogonia. The same field as D, in which two spermatogonia can be recognized by staining for the vasa antigen with a rabbit antibody and a rhodamine-conjugated anti-rabbit antibody as in C. Bar = 34 µm



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  		FIG. 2. Cell proliferation in Sertoli cell-A spermatogonia cocultures under different growth factors combination conditions. To determine the proliferative rate of both cell types, the cultures were incubated with BrdU for 1 h at 1, 2, 3, 5, or 7 days of culture. Anti-BrdU immunohistochemistry was performed, and the percentage of BrdU-positive cells by comparison with the total number of cells was determined. A) Percentage of spermatogonia in S-phase. B) Percentage of Sertoli cells in S-phase. F, Forskolin; L, LIF; E, EGF; O, OSM. Data represent the mean ± SEM of one representative experiment. The experiments were repeated at least twice, each experiment comprising at least triplicate cultures

Efficiency of Spermatogonia and Sertoli Cells Retroviral Transduction Depends on Their Proliferation Rate

We tested the ability of several retroviral vectors to infect spermatogonia and to drive gene expression. The vectors used included those based on the MLV, the MSCV, and the ALV. All vectors carried the enhanced GFP reporter gene. Gene transduction was assessed by monitoring GFP expression. Spermatogonia were identified in the spermatogonia-Sertoli cell cocultures by anti-vasa staining. We found that, under the previously identified culture conditions, spermatogonia were infected with all retrovirus types (Figs. 1, D and E, and 3A). Infected spermatogonia were detectable 24 h after infection (not shown), but the number of transduced spermatogonia increased afterward, reaching a maximum at Day 7 of culture (Fig. 3A).



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  		FIG. 3. Retroviral transduction efficiencies achieved in A spermatogonia (A) and Sertoli cells (B). The percentage of transduced cells was determined after 7 days of culture by comparing the number of GFP-positive cells with the total number of cells. The data are expressed as the mean percent of transduced cells of all pooled experiments. The experiments were repeated at least four times and each experiment comprised at least triplicate cultures. ALV, Avian leukosis virus; MLV, murine leukemia virus; MSCV, murine stem cell virus; MSCV 10A1, murine stem cell virus coated with the 10A1 envelope; repeat, repeated infection method; spin, spinoculation method

In general, spermatogonia were more efficiently transduced than the accompanying Sertoli cells (compare Fig. 3, A and B), which correlates with the differences in proliferation rates of the two cell types in our culture conditions (compare Fig. 2, A and B). The exception was seen using viruses coated with the 10A1 envelope, which transduced Sertoli cells very effectively (Fig. 3B).

Several Retroviral Vector Elements Determine Transduction Efficiency

The ability of retroviral vectors to transduce cells depends on many factors, including the retroviral LTR, the retroviral envelope, and the method of infection. The highest efficiency of spermatogonial transduction (approximately 50% transduced spermatogonia) was achieved using the ALV vector, either by repeated infection or spinoculation (Fig. 3A), followed by the amphotropic MLV vector (37% transduced spermatogonia). Surprisingly, the mutated LTR of the MSCV vector did not improve transduction efficiency in spermatogonia (Fig. 3A). Neither the use of the 101A envelope nor the spinoculation method improved the efficiency of spermatogonial transduction (Fig. 3A). These data contrast with the results obtained in the accompanying Sertoli cells, which were more efficiently transduced using viruses coated with the 10A1 envelope than with the amphotropic envelope, suggesting that, in contrast with spermatogonia, Sertoli cell plasma membranes display a high number of gibbon ape leukemia virus receptors. Sertoli cells were also more effectively transduced by the spinoculation method than by the repeated infection method (Fig. 3B), in contrast with spermatogonia (Fig. 3A).

Retroviral-Mediated Expression of MDM2 Improves Spermatogonial Survival in Culture

In order to demonstrate the functional usefulness of this retroviral delivery system, we sought to investigate the possible role of the p53 tumor suppressor protein in the control of spermatogonial apoptosis in vitro. We infected spermatogonia cultures with either an ALV vector expressing murine double minute clone 2 (MDM2), the main down-regulator of p53, or an ALV vector expressing GFP as a control. Spermatogonia were infected on the first day of culture and then cultured for 4 days in growth factors to allow for retroviral integration and retroviral-mediated gene expression. Cultures were then deprived of growth factors for 24 h to induce spermatogonial cell death. Expression of MDM2 resulted in a dramatic increase in spermatogonial cell numbers by comparison with the control (GFP) virus (Fig. 4). These data suggest that p53 is acting to induce apoptosis in cultured spermatogonia and that MDM2 can prevent the spermatogonial apoptosis induced by growth factors depletion. These data demonstrate the usefulness of this retroviral-mediated gene delivery system for studying the biology of spermatogonia.



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  		FIG. 4. Effect of MDM2 expression on spermatogonial survival. Spermatogonia cultures were infected with either an ALV vector expressing MDM2 or an ALV vector expressing GFP as a control. Spermatogonia were infected on the first day of culture and then cultured for 4 days in growth factors to allow for retroviral integration and retroviral-mediated gene expression. Cultures were then deprived of growth factors for 24 h to induce spermatogonial cell death. Expression of MDM2 resulted in a dramatic increase in spermatogonial cell numbers by comparison with the control (GFP) virus. Data represent the mean ± SEM of one representative experiment. The experiments were repeated three times, each experiment comprised triplicate cultures


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Understanding the factors that affect the ability of spermatogonia to be transduced with retroviruses could aid in both the development of techniques for animal transgenesis and in our knowledge of risks associated with somatic cell gene therapy. To address the susceptibility of mouse spermatogonia to retroviral infection, we developed a culture system for mouse spermatogonia in which they both survive and proliferate. Successful gene transduction with retroviruses requires viral integration, which in turn requires cells to transit through the M-phase of the cell cycle [9]. Therefore, we tested various growth factors for their ability to stimulate proliferation of spermatogonia in culture. These studies have been performed in a heterogeneous population of undifferentiated A spermatogonia, some of which are spermatogonial stem cells. We found that addition of a combination of growth factors, including LIF, OSM, and EGF as well as the cAMP agonist, forskolin, improved survival and proliferation of spermatogonia cocultured with Sertoli cells. Our data show that approximately 40–50% of spermatogonia can be successfully infected with both MLV and ALV-derived vectors, and represent a significant improvement over previously published reports [7, 8]. Because retroviral integration requires cells to transit through the cell cycle, these data suggest that 40–50% of cells are dividing in culture. These data are in agreement with data obtained from BrdU pulsing experiments, from which we estimated that approximately 40% of spermatogonia were proliferating in culture (Fig. 2A). These observations demonstrate that, in the presence of a combination of growth factors, spermatogonia can be stimulated to proliferate more than they would in vivo [40]. Taken together, these results also demonstrate that, in the cell culture conditions described here, all of the spermatogonia that are capable of being transduced are being transduced.

A variety of retroviral elements affect the ability of cells to be successfully transduced with retroviruses, including the retroviral envelope, the type of retroviral LTR, and the method of retroviral delivery. We found that few of these factors affected gene transduction in spermatogonia. Viruses with an amphotropic envelope were more effective than those with a 10A1 envelope in infecting spermatogonia, suggesting that spermatogonia have either few or no gibbon ape leukemia virus receptors on their cell surface. We also found that spermatogonia derived from mice expressing the ALV receptor were highly susceptible to infection with ALV. This is probably related to high levels of expression of the ALV receptor gene in these cells. Viruses containing the wild-type MLV LTR were more effective in driving gene expression in spermatogonia than those containing the mutant MSCV LTR, suggesting that spermatogonia lack the mechanisms for silencing retroviruses that exist in embryonal carcinoma cells [27], embryonal stem cells [28], and PGCs [29]. Previously, we found that retroviral transduction of PGCs, the cells from which spermatogonia are derived, is more effective following spinoculation of virus onto cells [29], as is seen with hematopoietic stem cells [26, 34]. Spinoculation is thought to increase the chances for interaction between viral envelope proteins and cell surface receptors, perhaps by overcoming diffusion forces that maintain viral particles in suspension [41]. However, we found that spermatogonia were equally infected by daily repeated infection of cultures throughout the time course of the experiments or spinoculation on the first day of culture. These results may reflect the steady proliferative activity of spermatogonia throughout the time course of the experiments. The fact that spermatogonial transduction does not require extraordinary methods of retroviral delivery opens up the possibility of simple methods of retroviral delivery in the in vivo situation. Our data indicate that many of the most commonly used retroviral vector types can be used to infect and transduce mouse spermatogonia. Moreover, by selective expression of the ALV receptor, it should be possible to target ALV infection to specific cell types, thereby overcoming some of the problems of lack of cell specificity of gene expression associated with MLV-based vectors. We are currently generating mice in which the ALV receptor is expressed exclusively in the germ cell lineage from the promoter of the vasa gene [42] through a knock-in strategy.

The ease with which retroviruses can be used to transduce spermatogonia contrasts with that seen for other types of viruses, including adenovirus and adeno-associated virus (AAV-2). Adenoviruses do not infect spermatogonia when introduced intravenously [43] or into the seminiferous tubule [5]. In a previous study [6], we found that AAV-2 is incapable of effecting gene transduction in spermatogonia either in vivo or in vitro using the same culture conditions described here. These findings suggest that adenoviruses and AAV-2 may be a relatively safe alternative for human somatic cell gene therapy.

We also show here that expression of MDM2, a protein that functions normally to downregulate p53 protein [44], promotes spermatogonia survival in culture. These data agree with previous studies showing that Trp53 knockout mice testes contain higher numbers of type A spermatogonia than wild-type mice [15]. In a recent study, Trp53 deficiency partially rescued male fertility in KitW-v mice [45]. Our data showing that MDM2 expression from a retroviral vector promotes spermatogonia survival in culture agrees with these studies [15, 45] and supports the idea that p53 plays a role in regulating spermatogonial apoptosis induced by growth factors deprivation. Our results further demonstrate the usefulness of this novel system of targeting substances of interest to spermatogonia.

In conclusion, we report here the successful culture of spermatogonia from neonatal mice and stimulation of proliferation with a combination of growth factors. These culture conditions enable high efficiency viral transduction of spermatogonia. Based on comparing cell proliferation data with viral infection data, we estimate that possibly all spermatogonia that are capable of being infected in these cultures are in fact infected. We found that many types of retroviral vectors could be used to transduce spermatogonia. Moreover, we have shown that retroviral transduction can be used in functional assays to identify the molecules controlling spermatogonial growth. These studies should aid in the development of conditions for routinely manipulating gene expression in germline stem cells. The application of retroviral-mediated gene transduction to analysis of spermatogenesis could yield important information about the molecular mechanisms regulating spermatogenesis in mammals as well as improving methods for animal transgenesis.


   ACKNOWLEDGMENTS
 
We are grateful to M.J. Federspiel for the ALV-GFP vector, L. Cheng for the MLV and MSCV vectors, W.P. Hively, E.C. Holland, and H.E. Varmus for the ALV-MDM2 vector, S. Hughes for the ß-AKE mice, and T. Noce for the anti-vasa antibody. We are also indebted to L. Lock for helpful comments on the manuscript.


   FOOTNOTES
 
1 This work was supported by institutional funds from Thomas Jefferson University and NCI Cancer Core grant P30CA56036 to the Kimmel Cancer Center. Back

2 Correspondence: Peter J. Donovan, Kimmel Cancer Center, Thomas Jefferson University, Bluemle Life Sciences Building, Room 706, 233 South 10th Street, Philadelphia, PA 19107. FAX: 215 923 4153; e-mail: pdonovan{at}lac.jci.tju.edu Back

Received: 1 March 2002.

First decision: 2 April 2002.

Accepted: 19 September 2002.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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