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Research Article |
Department of Animal Sciences and Center for Reproductive Biology, Washington State University, Pullman, Washington 99164
ABSTRACT
Bovine ectopic testis tissue grafting is a technique that can be used to study bovine spermatogenesis and for the production of germ cells for a variety of applications. Approximately 10% of seminiferous tubule cross sections in testis grafts contain spermatids, providing a unique tool to investigate what regulates germ cell differentiation. We hypothesized that manipulation of testis tissue grafts would increase the percentage of seminiferous tubule cross sections undergoing complete germ cell differentiation. To test this hypothesis, bovine testis tissue was treated with vascular endothelial growth factor (VEGF) at the time of grafting or explant cultured for 1 wk prior to grafting. For the VEGF experiment, 8-wk donor tissue and graft sites were treated with 1 µg of VEGF in order to increase angiogenesis at the graft site. For the testis tissue culture experiment, 4-wk-old donor testis was cultured for 1 wk prior to grafting to stimulate spermatogonial stem cell proliferation. Testis tissue grafts were removed from the mice 24 wk after grafting. VEGF treatment increased graft weight and the percentage of seminiferous tubule cross sections with elongating spermatids at the time of graft removal. Cultured testis tissue grafts were smaller and had fewer seminiferous tubules per graft. However, there was no difference in the percentage of seminiferous tubule cross sections that contained any germ cell type between groups. These data indicate for the first time that bovine testis tissue can be manipulated to better support germ cell differentiation in grafted tissue.
gametogenesis, growth factors, sperm, spermatogenesis, testis
INTRODUCTION
Ectopic testis tissue grafting is a technique in which small pieces of prepubertal donor testis tissue are grafted subcutaneously on the backs of immunodeficient mice. The donor testis grafts grow, mature, and undergo complete germ cell differentiation characterized by the presence of elongating spermatids in the graft. Testis grafts that contain elongating spermatids have been demonstrated using testis tissue from a variety of donor species, including pigs, goats, mice, monkeys, hamsters, bulls, and cats [1–6]. Haploid germ cells have been isolated from mouse testis allografts and successfully used with intracytoplasmic sperm injection (ICSI) to generate normal embryos and ultimately progeny [7]. This technique could potentially be used as a tool to investigate basic mechanisms regulating spermatogenesis in multiple species and to generate genetically modified sperm for the production of transgenic animals [4].
Research with bovine ectopic testis tissue xenografting has demonstrated that prepubertal bull testis tissue will grow, differentiate, and undergo complete germ cell differentiation when grafted onto castrated immunodeficient mice [4, 5]. In addition, it was demonstrated that testis tissue from 8-wk-old bull calf donors had the greatest potential for growth, development, and subsequent sperm production compared with testis tissue from 2-, 4-, 12-, and 16-wk-old animals when grafted for 24 wk [5]. Published reports indicate that between 5% and 10% of seminiferous tubule cross sections in grafted bovine testis tissue generate elongating spermatids 24 wk after grafting [4, 5]. The low percentage of seminiferous tubules with elongating spermatids provides a unique model to study factors needed for quantitative spermatogenesis. Two potential ways to treat testis tissue to increase the percentage of seminiferous tubule cross sections with complete germ cell differentiation after grafting are to stimulate angiogenesis into the testis tissue and to increase the initial population of spermatogonial stem cells in the testis tissue before grafting.
Vascular endothelial growth factor (VEGF) is a potent angiogenic factor that stimulates blood vessel development and migration [8]. Previous research has utilized exogenous VEGF to increase vascular development into ischemic tissues [9], and anti-VEGF treatments have been shown to be effective as potential cancer treatments by limiting blood vessel growth into tumors [10]. We hypothesized that treatment of ectopic testis tissue graft sites with VEGF could potentially increase the rate and extent of angiogenesis into the grafts and ultimately increase the percentage of seminiferous tubule cross sections that undergo complete germ cell differentiation.
The spermatogonial stem cell (SSC) provides the basis for continual sperm production throughout the life of a male [11]. Spermatogonial stem cells can undergo mitotic divisions without differentiation in a process of stem cell self-renewal. Alternatively, SSCs can undergo mitotic divisions and differentiate into A spermatogonia. This process of continual self-renewal and differentiation is highly regulated, and disruption of this mechanism results in altered numbers of sperm. For example, male mice treated with the chemo-toxin busulfan are sterile due to complete loss of spermatogenesis resulting from death of dividing SSCs. Testes in busulfan-treated mice can support spermatogenesis when SSCs from wild-type mice are transplanted into the testes. Recently, Oatley and colleagues [12] described an approach that results in an increase in the number of SSCs in pieces of 4-wk-old bull testis tissue during 1 wk of tissue culture. We hypothesized that tissue culture to increase the number of SSCs in testis tissue prior to ectopic testis xenografting would result in a higher percentage of seminiferous tubule cross sections with elongating spermatids at the time of graft removal.
The objective of the following research was to manipulate bovine testis tissue prior to xenografting in order to increase the percentage of seminiferous tubule cross sections that had undergone complete germ cell differentiation at the time of graft removal. To achieve this objective, two experiments were conducted. In the first experiment, grafts were treated with exogenous VEGF at the time of grafting to increase angiogenesis. For the second experiment, donor testis tissue was maintained in tissue culture for 1 wk prior to grafting to increase the initial population of SSCs in the testis tissue.
MATERIALS AND METHODS
Materials and Animals
All reagents were purchased from Sigma (www.sigmaaldrich.com) unless otherwise stated. Recipient immunodeficient NCr nude mice (Taconic, http://www.taconic.com; CrTac:NCR-Fox1<nu>) were raised under normal conditions and were fed a standard rodent chow ad libitum. Immunodeficiency in this mouse strain stems from an abnormal thymus due to homozygosity of the autosomal recessive nude gene (nu/nu). In order to evaluate migration of cells from the recipient animals into the donor testis tissue, the immunodeficient NCr nude mice were crossed with a transgenic mouse line ROSA26 (B6, 129 TgR(ROSA26)26Sor) originally purchased from Jackson Laboratory (www.jax.org). The ROSA26 mice express the Escherichia coli ß-galactosidase gene. Immunodeficient nude progeny from this cross that were positive for the ß-galactosidase transgene as indicated by staining with 5-bromo-4-chloro-3-indoyl ß-D-galactoside (X-gal) were termed "Ronu" and were used as graft recipients [4]. Angus cross bull calves from the Washington State University Beef Center were castrated at 4 or 8 wk of age. The Washington State University Animal Care and Use Committee approved all animal procedures.
Tissue Collection
Testis tissue samples were obtained from three 4-wk-old bull calves and five 8-wk-old bull calves using standard castration protocols. One testis from each donor was immediately placed in Hanks Balanced Salt Solution (HBSS) on ice. In the laboratory, the tunica was removed from the testis and parenchymal tissue was cut into 3–5 mg (2–3 mm) pieces and returned to HBSS on ice until the time of grafting. The second testis from each animal was prepared in the same way except that testis tissue was placed in Bouins fixative for 4 h at 4°C followed by dehydration and storage in 70% ethanol.
Ectopic Testis Tissue Xenografting
Immediately after donor testis tissue dissection, four pieces of testis tissue were ectopically grafted onto castrated immunodeficient nude mice. Briefly, mice were anesthetized with ketamine (0.1 mg/kg body weight [BW]) and xylazine (0.5 mg/kg BW) in sterile physiological saline. A ventral medial incision was made in the abdomen and the testes were removed and the peritoneum and skin sutured closed using absorbable suture (Ethicon, http://www.novartis.com). After castration, mice were placed in ventral recumbence, and four incisions were made in the skin on the back of the mice. Donor testis tissue was inserted at each site and the incisions were sutured closed. The mice were allowed to recover and were returned to their cages.
Tissue Treatments
Vascular Endothelial Growth Factor Donor testis from five 8-wk-old bulls was used in this experiment. After the testis tissue grafts were inserted and the dorsal sites were sutured closed, graft sites were treated with either 10 µl of carrier (1X PBS) or 10 µl of 100 µg/ml (1 µg per graft site) recombinant mouse VEGF 164 (R & D Systems, rndsystems.com) by inserting the tip of a 10 µl pipette between sutures and injecting the treatment. From the five donor bulls, nine mice (36 grafts) were treated with control and nine mice (36 grafts) were treated with VEGF. Four additional Ronu mice were grafted with 8-wk donor tissue for macroscopic evaluation of tissue angiogenesis.
Pregraft Culture Donor testis tissue from three 4-wk-old bulls was used in this experiment. On the day of collection, four pieces of testis tissue were ectopically grafted onto four castrated immunodeficient mice per donor as previously described. The remainder of the tissue was cultured for 1 wk as previously described [12] in order to increase the population of spermatogonial stem cells in the tissue. Briefly, testis tissue pieces were cultured on 0.45-µm pore membranes (Millipore, http://www.millipore.com) floating in a single well of a six-well culture plate. Ten tissue pieces were placed on a membrane, and one plate was used for each donor (60 pieces per donor). Cultures were maintained for 1 wk in Dulbecco Modified Eagle Medium (DMEM) containing 10% fetal bovine serum, 30 mg/ml penicillin, and 50 mg/ml streptomycin at 32°C in an atmosphere of 5% CO2. Medium was changed every 2 days. After 1 wk in culture the tissue was grafted onto 4 castrated immunodeficient mice per donor.
Histological Analysis of Donor Grafts
Grafts were recovered at 24 wk after grafting for both the culture and the VEGF treatments. Mice were killed by CO2 inhalation and cervical dislocation. Testis tissue grafts were removed, weighed, and fixed in Bouin fixative at 4°C for 4 h prepared for histology and sectioned at 8 µm. Slides were deparaffinized, rehydrated, stained with hematoxylin and eosin, and evaluated using light microscopy with digital images captured using a Leica DFC 280 camera and a Leica DME compound microscope (Leica Microsystems Imaging Solutions Ltd., http://www.leica-microsystems.com) at 400x magnification.
Seminiferous tubule cross sections were visually evaluated to determine the average number of seminiferous tubule cross sections with spermatogonia, meiotic germ cells, elongating spermatids, or no germ cells. Spermatogonia were identified by the presence of condensed nuclei and cellular basal location within the seminiferous tubule. Meiotic germ cells were identified by the presence of diffuse nuclear staining. Elongating spermatids were identified by the presence of highly condensed elongating nuclei. The percentage was determined by dividing the total number of seminiferous tubule cross sections within a testis tissue graft cross section containing a germ cell type in all grafts on a given mouse by the total number of seminiferous tubule cross sections in the same graft cross sections from that mouse. Seminiferous tubule diameter was determined for all round tubules within a testis tissue graft section and averaged per mouse. Testis tissue grafts were analyzed per mouse to prevent unequal weighting of grafts due to the range in the number of seminiferous tubule cross sections per graft.
Cross sections of testis tissue grafts treated with VEGF or PBS were also probed for the presence of von Willebrand factor (VWF). After rehydration the slides were boiled in sodium citrate (pH 6.0), and exogenous peroxidase activity was blocked using 3% H2O2 (v/v) in methanol followed by rinsing in PBS. Nonspecific antibody binding was blocked by incubation of samples with nonimmune rabbit serum for 15 min. The primary antibody, VWF (rabbit anti-human VWF; Santa Cruz Biotechnology, http://www.scbt.com) diluted to 1:200, was applied to samples and incubated at 4°C overnight in a humidified chamber. Samples were then washed in PBS three times for 2 min and biotinylated secondary antibody (rabbit anti-goat IgG; Histostain SP AEC Kit, Invitrogen, http://www.invitrogen.com) was added and incubated for 10 min at room temperature, followed by washing with PBS. HRP-streptavidin was added to the sections and allowed to incubate at room temperature for 10 min, followed by washing in PBS. Samples were developed with aminoethyl carbazole (AEC) for 10 min, counterstained with hematoxylin, mounted with GVA mounting reagent (Invitrogen), and viewed using light microscopy. Digital images were captured with a Leica DFC 280 camera and a Leica DME compound microscope at x400 magnification.
Recipient Mouse Analysis
At the time of killing, blood was collected from the recipient mice by cardiac puncture. Serum was isolated from the blood and assayed for testosterone as an indicator of testis tissue graft viability. Testosterone concentrations were determined by RIA using a commercial kit (DSL-400; Diagnostic Systems Laboratory Inc., http://www.dslabs.com). Vesicular glands were also removed and weighed as an indicator of the bioactivity of testosterone produced by testis tissue grafts.
Statistical Analysis
All seminiferous tubules from the largest center cross section of each graft were evaluated for germ cell proportions. Germ cell proportions and tubule diameter were averaged per mouse because of the variability in the number of seminiferous tubule cross sections per testis tissue graft. All data were analyzed using the SAS system software with the Proc GLM function. Differences between means for testis tissue graft size, percentage of seminiferous tubule cross sections with germ cell types per testis tissue graft per mouse, mouse vesicular gland size, and mouse serum testosterone concentration were determined using the Duncan test for significance. Differences between treatments were considered significant at P 
0.05. Data are presented as the mean ± SEM.
RESULTS
VEGF Experiment
Testis Tissue Graft Weight, Seminiferous Tubule Diameter, Serum Testosterone Concentration, and Vesicular Gland Weight In order to evaluate the effect of vascular endothelial growth factor on testis graft function, bovine testis tissue was xenografted on the backs of castrated immunodeficient nude mice followed by treatment with VEGF or vehicle. Eight-week-old donor testis tissue was recovered 24 wk after grafting [5] and evaluated to determine if somatic and germ cell differentiation took place in the testis tissue and if treatment improved any of these parameters. Eight-week-old donor testis tissue has been previously demonstrated to be the optimal age for complete germ cell differentiation when tissue was grafted for 24 wk [5]. In the present experiment, a total of 86.1% of the testis tissue grafts were recovered from control mice recipients, and 64.5% of recovered grafts were functional (56% of total grafted). Functional testis tissue grafts were defined as grafts with active spermatogenesis recovered from mice with at least 1 ng/ml serum testosterone and/or vesicular glands weighing 100 mg or more. Approximately 69% of the testis tissue grafts treated with VEGF were recovered, with 64% of the recovered grafts being functional (44% of total grafted; Table 1).
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Testis tissue graft weight was measured at the time of removal to determine if VEGF had an effect on grafted testis tissue growth. VEGF-treated testis tissue grafts were significantly (P < 0.05) larger than control grafts (Fig. 1A). To rule out the possibility that VEGF-treated mice received larger pieces of testis tissue at the time of grafting, the numbers of seminiferous tubule cross sections per testis tissue graft were counted. There was no significant difference (P 
0.05) between the number of seminiferous tubule cross sections in VEGF and control treated testis tissue grafts (Fig. 1B). Seminiferous tubule diameter was also measured to determine the developmental potential of testis tissue grafts that were treated with VEGF at the time of grafting. There was no significant difference (P 0.05) in seminiferous tubule diameter (Fig. 1C) between treatments.
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Recipient mouse serum testosterone levels were determined to evaluate the ability of Leydig cells within the testis tissue grafts to proliferate, differentiate, and ultimately produce testosterone. Serum testosterone concentrations in mice with testis tissue grafts treated with VEGF were not different (P 0.05) compared to mice receiving control treatment (Fig. 2A). Recipient mouse vesicular glands were weighed to assess the bioactivity of the testosterone produced by the testis tissue grafts. No significant difference (P 0.05) was observed in vesicular gland weights between VEGF and control treatment (Fig. 2B).
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Testis Tissue Graft Histological Analysis Histological evaluation of donor tissue and grafted testis tissue was conducted to determine whether VEGF treatment had any effect on the ability of the testis tissue grafts to support germ cell differentiation and undergo spermatogenesis. All seminiferous tubule cross sections from the middle of each testis tissue graft were evaluated for the presence of spermatogonia, meiotic germ cells, or spermatids.
Evaluation of 8-wk bull donor tissue revealed seminiferous tubule cross sections containing only spermatogonia and Sertoli cells (Fig. 3A). Regardless of treatment, grafted testis tissue increased in size and supported germ cell differentiation at the time of graft removal. Evaluation of functional testis tissue grafts indicated that 19 of 20 supported complete germ cell differentiation (Fig. 3, B and C). The extent of germ cell differentiation in testis tissue grafts was evaluated to determine if VEGF treatment had an effect on the proportion of tubules undergoing spermatogenesis. No significant difference (P 0.05) in the percentage of tubule cross sections with spermatogonia, meiotic germ cells, or no germ cells was observed between VEGF-treated and control testis tissue grafts. However, VEGF-treated testis tissue grafts had a significantly higher (P < 0.05) percentage of seminiferous tubule cross sections with elongating spermatids than control treated grafts (Fig. 4, A and B).
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Angiogenesis is important for testis tissue graft survival. This is supported by blood vessel growth into large functional testis tissue grafts (Fig. 5A). Furthermore, evaluation of angiogenesis into grafts on Ronu mice indicates that blood vessels appear to grow from the mouse recipient into the testis graft (Fig. 5, B and C). To determine if VEGF treatment had any effect on angiogenesis into donor testis tissue, graft cross sections were probed using an antibody for the endothelial cell marker VWF to count the number of blood vessels in each testis tissue graft [13]. In both control and VEGF-treated grafts the vascular endothelial cells were positive for VWF as expected, however Sertoli cells also expressed VWF, which has previously been undemonstrated (Fig. 3D). Functional testis tissue grafts had significantly (P < 0.05) more blood vessels than nonfunctional grafts (Fig. 6A); however, VEGF-treated testis tissue grafts did not have significantly more blood vessels than controls (P = 0.15; Figure 6B).
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Testis Tissue Culture Experiment
Graft Weight, Seminiferous Tubule Diameter, Serum Testosterone Concentration, and Vesicular Gland Weight Previous research demonstrates that short-term explant culture of 4-wk-old bovine testis tissue for 1 wk results in an increase in the population of spermatogonial stem cells in the cultured tissue [12]. Bovine testis tissue from 4-wk-old bull calves was cultured for 7 days prior to grafting to determine if the increase in spermatogonial stem cells [12] in this tissue would increase the number of germ cells that differentiate into elongating spermatids within the grafted tissue. Testis tissue was either grafted on castrated nude mice on the day of castration (control) or maintained in vitro for 7 days prior to grafting (treatment). Approximately 64% of the testis tissue grafts from control mice were recovered and 82.6% of recovered grafts were functional (53% of total grafted). Culture of testis tissue prior to grafting did not affect tissue survival, as indicated by recovery of 70.5% of the cultured grafts. Additionally, 90.3% of the recovered grafts were functional (64% of total grafted; Table 1). Testis tissue graft weight was measured at the time of removal to determine if pregraft culturing had an effect on grafted testis tissue growth. Cultured testis tissue grafts were significantly (P < 0.05) smaller than control grafts (Fig. 7A), and control grafts had significantly more (P < 0.05) seminiferous tubule cross sections per graft than the cultured grafts (Fig. 7B).
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Seminiferous tubule diameter was also measured to determine the developmental potential of testis tissue grafts that were cultured prior to grafting, and there was no difference (P 0.05) in seminiferous tubule diameter between groups (Fig. 7C). Serum testosterone concentrations in mice with cultured testis tissue grafts were not different (P 0.05) compared to controls (Fig. 8A). Recipient mouse vesicular glands were weighed to assess the bioactivity of the testosterone produced by the testis tissue grafts. In contrast to the serum testosterone data, mice that received cultured testis tissue grafts had significantly (P < 0.05) smaller vesicular glands than control mice (Fig. 8B).
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Testis Tissue Graft Histological Analysis To determine if culturing of testis tissue prior to grafting had an effect on spermatogenesis, histological evaluation of donor tissue and grafted tissue was conducted. All seminiferous tubules in a cross section from the middle of each graft were evaluated for the presence of spermatogonia, meiotic germ cells, or spermatids. The largest graft cross section was utilized for this histological analysis.
Evaluation of 4-wk bull donor tissue revealed seminiferous tubules containing gonocytes and Sertoli cells (Fig. 9A). Cultured testis tissue was very similar in appearance to precultured donor tissue (Fig. 9B). Regardless of treatment, testis tissue grafted on mice increased in size and supported germ cell differentiation. All evaluated recipient mice had at least one graft with elongating spermatids (Fig. 9, C and D). The extent of germ cell differentiation in testis tissue grafts was evaluated to determine if culturing of testis tissue prior to grafting had any long-term effect on the proportion of tubules undergoing germ cell differentiation. No significant difference (P 0.05) in the percentage of seminiferous tubule cross sections with spermatogonia, meiotic germ cells, spermatids, or no germ cells was observed between control and cultured testis tissue grafts (Fig. 10).
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DISCUSSION
The objective of this research was to manipulate bovine testis tissue prior to xenografting in order to increase the percentage of seminiferous tubule cross sections that had undergone complete germ cell differentiation at the time of graft removal. To complete this objective, two approaches were used. The first approach was to improve the initial rate and extent of angiogenesis into the grafted tissue by treatment with VEGF. The second approach was to increase the initial population of spermatogonial stem cells within the grafted tissue by culturing the tissue prior to grafting [12].
Evaluation of testis tissue grafts revealed a significant difference in the number of blood vessels between functional and nonfunctional grafts, indicating an essential role of angiogenesis for the survival, development, and eventual production of elongating spermatids in grafted testis tissue. Treatment of testis tissue graft sites with VEGF resulted in an increase in graft size and an increase in the percentage of seminiferous tubules with elongating spermatids. No difference was observed in the number or diameter of the seminiferous tubules within treated and nontreated testis tissue grafts. This indicates that the initial graft size was consistent across recipients. Additionally, control testis tissue graft size was similar to previously reported weights of 8-wk donor testis grafted for 24 wk (33 mg vs. 
40mg; [5]). Although not significant, there was a numerical increase in the seminiferous tubule diameter and the number of seminiferous tubule cross sections per graft in the VEGF-treated grafts that combined could account for the increase in graft size in the treatment group. Serum testosterone concentration and vesicular gland weights were not different between treatments, indicating that the testis tissue grafts had the same steroidogenic activity regardless of size.
VEGF-treated testis tissue grafts had a higher percentage of seminiferous tubule cross sections with elongating spermatids than the control grafts. This percentage (over 14%) is higher than any previously reported for bovine testis tissue xenografts. Interestingly, no difference was observed in the proportion of seminiferous tubule cross sections with spermatogonia, meiotic cells, or Sertoli cells only between treated and control groups. Indeed, the values for percent with meiotic cells presented here are consistent with previously reported data [5]. These data indicate that VEGF treatment at the time of grafting indirectly increases survival of germ cells in the transition from meiosis to spermiogenesis. However, the total number of spermatogonia or meiotic cells in the seminiferous tubules of each testis tissue graft was not determined, so we cannot rule out the possibility that there are more total germ cells. Therefore, it is possible that VEGF treatment at the time of grafting increased germ cell survival by acting directly on germ cells. Another possible explanation for the increase in graft size and the percentage of tubules with elongating spermatids is that VEGF increased the amount of vascular flow to the grafts. The number of blood vessels in grafts was determined using immunohistochemistry with an antibody to a factor (VWF) specific for endothelial cells. There was a trend for the VEGF-treated grafts to have more blood vessels than the control mice; however, a significant difference was not detected (P = 0.15).
The increase in the percentage of seminiferous tubule cross sections undergoing complete germ cell differentiation in bovine testis grafts following a single treatment of VEGF without a significant increase in blood vessel growth is intriguing in regard to how VEGF may influence somatic and germ cell differentiation in the seminiferous tubules. VEGF has been shown to be produced in the testis [14] and is induced by hCG [15]. The fact that VEGF is actively produced in the testis is surprising, because there is no active angiogenesis in the adult testis. In the human testis, VEGF and its receptors, VEGFR-1 and VEGFR-2, are localized to both the Sertoli and Leydig cells. Additionally, VEGFR-1 and VEGFR-2 are also found on the testicular capillary endothelial cells [14] and germ cells [16]. Interestingly, transgenic mice that overexpress VEGF in the testis are infertile [16]. Different VEGF receptors are also differentially expressed on developing germ cells. VEGFR-2 is present on spermatogonia, whereas VEGFR-1 is present on spermatids [17]. These data indicate that VEGF may have non-endothelial cell targets. It has been hypothesized that VEGF may regulate spermatogonial proliferation. If this is true, the increase in the percentage of seminiferous tubule cross sections with elongating spermatids could be due to an initial increase in spermatogonia in the grafted testis tissue. The use of the Ronu mice indicates that some blood vessels in testis grafts are of recipient mouse origin. Further evaluation of grafted testis tissue with these recipient animals will allow for a more detailed analysis of angiogenesis and cell migration into the grafts. The results of the VEGF treatment experiment indicate that it is possible to manipulate bovine testis tissue prior to xenografting to increase the percentage of seminiferous tubule cross sections undergoing complete germ cell differentiation within the graft.
Culturing small pieces of bovine testis tissue has been demonstrated to nearly double the population of SSCs in the testis tissue [12]. We hypothesized that this increase of SSCs in testis tissue prior to grafting would result in more seminiferous tubules with complete germ cell differentiation in grafted testis tissue over time. Pregraft testis tissue culture resulted in grafts that were significantly smaller, with fewer seminiferous tubules than the controls. It is highly unlikely that a difference in the number of seminiferous tubules existed prior to culture. All testis tissue (control and culture) was treated identically and randomly selected for culture or immediate grafting (control). Control testis tissue graft weight in this study was very similar to previously reported data for 4-wk donor testis grafted for 24 wk [4]. These data initially indicate that culturing prior to grafting caused a degeneration or loss of seminiferous tubules in the cultured tissue. It is highly unlikely that some seminiferous tubules dissociated from the testis tissue and were lost during the culture period. However, it is possible that some of the seminiferous tubules degenerated to an unrecoverable state and were overgrown by interstitial cells during the grafting period. It is also possible that the smaller number of seminiferous tubule cross sections in a cross section of testis graft is due to decreased lateral growth of cultured seminiferous tubules, resulting in shorter tubules and thus fewer cross sections. Interestingly, the tubules that survived the culturing period were relatively similar to the control tubules. No difference was present in seminiferous tubule diameter or the percentage of seminiferous tubules with germ cells, meiotic cells, elongating spermatids, or Sertoli cells only between control and treatment groups. An interesting result was that the recipient mice grafted with cultured testis tissue had significantly smaller vesicular gland weights without a difference in serum testosterone compared to the controls. This suggests that cultured testis tissue took longer to establish and produce adequate testosterone to maintain vesicular gland growth. These data suggest that although culturing of testis tissue has a negative impact on graft growth, overall germ cell differentiation is unaffected. Thus, culturing of testis tissue in various treatments and culture media could serve as a useful technique to study bovine spermatogenesis.
Donor tissue of different ages was utilized for the two different experiments presented. This is due to the optimal age for ectopic testis tissue grafting and the donor age used for bovine testis tissue explant culture identified in previous experiments [4, 5, 12]. It is likely that similar results would be observed if the donor ages utilized in these studies were reversed. However, developmental differences between 4- and 8-wk-old testis tissue could alter the ability of the tissue to respond to VEGF or explant culture.
Ectopic bovine testis xenografting is a useful technique to investigate basic mechanisms regulating bovine spermatogenesis. The present research indicates for the first time that treating grafts with exogenous factors can positively influence the efficiency of spermatogenesis in testis tissue grafts. Furthermore, although culturing testis tissue prior to grafting negatively influences testis tissue graft growth, germ cell differentiation in the grafts remains relatively unaffected. These data provide the avenue for further research on the process of bovine spermatogenesis using ectopic testis xenografting.
ACKNOWLEDGMENTS
The authors would like to thank current and past members of the McLean Laboratory for laboratory assistance and critical evaluation of the manuscript. The authors would also like to thank Andrianna Oliver and Jon Oatley for their assistance in the grafting procedure.
FOOTNOTES
1 Correspondence: FAX: 509 335 4246; dmclean{at}wsu.edu 
Received: 29 November 2005.
First decision: 24 January 2006.
Accepted: 24 March 2006.
REFERENCES
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  L. Arregui, R. Rathi, S. O Megee, A. Honaramooz, M. Gomendio, E. R S Roldan, and I. Dobrinski Xenografting of sheep testis tissue and isolated cells as a model for preservation of genetic material from endangered ungulates Reproduction, July 1, 2008; 136(1): 85 - 93. [Abstract] [Full Text] [PDF]
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 J. A. Schmidt, J. M. d. Avila, and D. J. McLean Analysis of Gene Expression in Bovine Testis Tissue Prior to Ectopic Testis Tissue Xenografting and During the Grafting Period Biol Reprod, June 1, 2007; 76(6): 1071 - 1080. [Abstract] [Full Text] [PDF]
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