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research-article |
Department of Animal Sciences and Center for Reproductive Biology, Washington State University, Pullman, Washington 99164
ABSTRACT
The purpose of this study was to identify factors that contribute to bovine testis development and donor age-dependent differences in the abilities of bovine ectopic testis tissue grafts to produce elongated spermatids. We used real-time RT-PCR and microarrays to evaluate and to identify the expression of genes that are involved in Sertoli and germ cell development in bovine testis tissues. Testis tissues were obtained from 2-, 4-, and 8-wk-old bull calves and were grafted immediately. Grafted bovine testis tissue was removed from mice, RNA was isolated from the grafts, and real-time RT-PCR was used to evaluate gene expression during the grafting period. In addition, the gene expression in the donor tissue was analyzed using Affymetrix Bovine GeneChips, to identify differentially expressed genes. Examination of the testis tissue grafts indicated that Sertoli cell-specific gene expression was lower in 8-wk donor tissue grafts compared to the donors of other ages. Furthermore, the expression of KIT, which is a germ cell-specific gene, was low in testis tissue grafts. Microarray analysis of the donor tissue showed that several genes that are involved in angiogenesis or tissue growth were differentially expressed in 2-, 4-, and 8-wk-old bovine testes. The levels of expression of the genes for angiogenin, transgelin, thrombomodulin, early growth response 1, insulin-like growth factor 2, and insulin-like growth factor-binding protein 3 were lower in testis tissues from older animals. Using these data, it will be possible in the future to manipulate the testis xenograft microenvironment so as to improve the efficiency of sperm production within the graft.
angiogenesis,, developmental biology, gene regulation, growth, microarray, spermatogenesis
Spermatogenesis is a complex process that occurs in the testis of the male. This process is dependent upon successful testis development and includes mitosis of diploid germ cells, meiosis, and morphological differentiation of postmeiotic germ cells. Few techniques exist to study spermatogenesis from early postnatal gonadal development through to the production of elongated spermatids. Development of novel tools to study testis function would dramatically increase the potential to understand fully this complex process.
Ectopic testis tissue xenografting is a technique in which small pieces of testis tissue are grafted under the dorsal skin of immunodeficient mice [1]. This technique has been used to generate sperm in grafted testis tissue from a variety of species, including mice, pigs, bulls, monkeys, and cats [1–8], and provides a unique tool to investigate the basic mechanisms of testis development and sperm production. Testis tissue can be treated prior to grafting or during the grafting period [5], and the dorsal location of the graft sites allows for increased replication and access to the testis tissue for removal and manipulation during the grafting period. Thus, factors that regulate testis function in these animals can be studied by manipulating the mouse host environment. Additional applications of ectopic testis tissue grafting include the generation of genetically modified sperm for the production of transgenic animals [2] and the preservation of the germ line of valuable livestock, endangered species, and prepubertal human cancer patients. For these applications to be successful, a significant percentage of seminiferous tubules within the grafts must undergo complete germ cell differentiation.
Several factors necessary for successful sperm production in xenografted testis tissue have been identified. Prepubertal donor tissue has the highest rate of survival on mice, and castrated, immunocompromised recipients provide the most suitable host environment for the development of prepubertal testis tissue [1, 3–7]. Prepubertal donor tissue has the greatest potential to mature and undergo complete germ cell differentiation.
Bovine testis tissue grafted onto the backs of castrated immunodeficient nude mice has the ability to grow, differentiate, and undergo complete germ cell development [2–6]. Furthermore, different aged bovine donor testis tissues have different potentials for development and germ cell differentiation when xenografted onto mice. Moreover, 8-wk-old donor tissue has the greatest potential to produce elongating spermatids, while 1- and 2-wk-old donor tissues have the greatest growth potential. Growth and germ cell differentiation are limited in 4-wk-old donor testis tissue grafts [3–4]. There could be several reasons for the differential development of bovine testis tissue when grafted. Differential gene expression (and subsequent protein expression) in the donor testis tissue prior to grafting could alter the initial ability of the grafted tissue to be accepted by the host environment. In fact, Rathi and colleagues [6] have demonstrated that the low efficiency of spermatogenesis in bovine ectopic xenografts is due in part to an initial loss of germ cells. In addition, grafted tissues from different aged donors may have differential gene and protein expression patterns during the grafting period, resulting in a differential developmental pattern dependent on donor age.
The present study aimed to identify genes that are differentially expressed in different aged donor testis tissues prior to grafting and during the grafting period. To accomplish this objective, two techniques were utilized. Affymetrix Bovine Microarray GeneChips were used to analyze the transcriptomes of 2-, 4-, and 8-wk-old bull testes prior to grafting, in order to identify differentially expressed genes. A second objective was to determine if genes critical for testis development and spermatogenesis are differentially expressed during the grafting periods of testis tissue grafts from 2-, 4-, and 8-wk-old donor bull calves. Our hypothesis was that different aged donor tissues both prior to and during the grafting period would have differential gene expression. Identification of differentially expressed genes in either the donor tissue or the grafts themselves could provide new strategies to manipulate the graft and/or the host to improve the efficiency of sperm production in bovine ectopic testis xenografts.
All reagents, unless stated otherwise, were purchased from Sigma. Donor bulls (Angus cross) were maintained at Washington State University. Recipient immunodeficient nude mice (Taconic; CrTac:NCR-Fox1<nu>) were raised under normal conditions on a 14L:10D cycle and a standard rodent chow was provided ad libitum. Immunodeficiency in this mouse strain is due to homozygosity of the autosomal recessive nude gene (nu/nu), which results in an abnormal thymus. All animal procedures were approved by the Washington State University Animal Care and Use Committee.
Testis tissue samples were obtained from at least three bull calves of each age (2, 4, and 8 wk) using standard castration procedures, and immediately placed in Hanks balanced salt solution (HBSS) on ice. In the laboratory, the tunica albuginea was removed from the testes and the parenchymal tissue was dissected away, cut into 3–5 mg (2–3 mm) pieces and returned to HBSS on ice until the time of grafting. At the time of castration, additional tissue samples were preserved in Trizol for RNA isolation and in Bouins fixative for preservation for histological analysis of the donor tissue. The tissues were left in Bouins for 24 h at 4°C, followed by dehydration and storage in 70% ethanol.
Ectopic Testis Tissue Xenografting
The grafting procedure was performed as previously described [2–5]. Testis tissues from three 2-, 4-, and 8-wk-old bull calves were grafted onto mice such that the grafts could be evaluated at 4, 8, and 16 wk of tissue age. Four pieces of testis tissue were ectopically grafted onto each mouse at distinct sites. Two-week-old donor tissue was grafted onto 27 recipient mice (three mice from each bull were killed at 2, 6, and 14 wk postgrafting), 4-wk donor testis tissue was grafted onto 18 recipient mice (three mice from each bull were killed at 4 and 12 wk postgrafting), and 8-wk donor tissue was grafted onto nine recipient mice (three mice from each bull were killed 8 wk postgrafting). This experimental design allowed for the comparison of different aged donor tissues at specific developmental testis time-points.
Recipient mice were killed between 2 and 14 wk after grafting by CO2 inhalation and cervical dislocation. The donor grafts were removed and weighed. Three randomly selected grafts were pooled and homogenized in Trizol. Total RNA was isolated from the grafts and age-matched bovine testis tissue. Total RNA was resuspended in RNase-free water (Ambion), and five µg of total RNA was reverse-transcribed into cDNA using oligo(dT) priming and M-MLV reverse transcriptase (Invitrogen). Complementary DNA synthesis was confirmed using PCR for the ß-actin gene (ACTB).
Complementary DNA samples from the grafts from three to four recipient mice and cDNA samples from 2-, 4-, 8-, and 16-wk-old bull calves (3–4 samples per age) were examined for the expression of genes important for testis development and germ cell differentiation using real-time RT-PCR. The primers were designed using PrimerExpress (Applied Biosystems) and obtained from Operon Biotechnologies or Invitrogen (Table 1). The PCR reaction mixture contained 200 ng cDNA, 20 pmol of each primer, 12.5 µl iQ TM Supermix (Bio-Rad), made up to a volume of 25 µl with water. SYBR Green I (Molecular Probes) was used to detect amplimer production. PCR reactions were conducted in an iCycler iQ TM Real-Time PCR Detection System (Bio-Rad) and consisted of 40 cycles of 95°C for 30 sec, 58°C for 30 sec, and 72°C for 30 sec. Samples were run in duplicate. Relative gene expression was determined based on the expression of ribosomal protein S2 (RPS2) as the baseline using the Q-Gene method [9] and standardized to the 2-wk bull calf expression level.
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Histology and Immunohistochemistry
Grafts placed in Bouins fixative were washed in xylene, blocked in paraffin, and sectioned at 8-µm thickness. Slides were deparaffinized, rehydrated, and stained with hematoxylin and eosin to evaluate gem cell differentiation. The grafts were evaluated using light microscopy and digital images were captured with a Leica DFC 280 Camera and a Leica DME compound microscope (Leica Microsystems Imaging Solutions) at 400x magnification.
Immunohistochemistry was used for localization of proteins involved in testis development and germ cell maturation. The procedure was conducted using goat anti-rabbit or rabbit anti-mouse IgG Histostain SP AEC Kits (Zymed/Invitrogen) according to the manufacturers directions. Antigen retrieval was conducted by boiling in sodium citrate (pH 6.0), and primary antibodies (Santa Cruz Biotechnology) were used at a 1:100 to 1:350 dilutions depending on the specific primary antibody. Slides were counterstained with hematoxylin and digital images were captured as described.
Ten micrograms of total RNA from 2-, 4-, and 8-wk-old bull testes (two samples per age) were used for microarray analysis. Biotinylated cRNA was created from total RNA using oligo(dT) primer with the T7 promoter in a reverse-transcriptase reaction. Complementary RNA was utilized for in vitro transcription using the MEGAScript Kit (Ambion) with biotinylated cytosine and uridine triphosphate. The labeled cRNA was fragmented and hybridized to Affymetrix GeneChip Bovine Genome arrays (Affymetrix). The arrays were stained in accordance to the manufacturers protocol using the Affymetrix GeneChip Fluidics Station 400 and scanned using a Gene/Array Scanner 2500A (Agilent). All reactions and microarray hybridizations were performed in the Laboratory for Biotechnology and Bioanalysis I (LBBI) at Washington State University.
Absolute and Statistical Analyses
The real-time RT-PCR data were analyzed using the SAS software with the Proc GLM function. Raw relative gene expression values, as determined using the Q-Gene method [9], were utilized for the statistical analysis. Differences in gene expressions over time were determined using Duncans test for significance and were considered significant at P 
0.05. In all figures, the data are normalized to the levels in 2-wk bull tissues, and are presented as the mean ± SEM.
The microarray output was analyzed using the GeneChip Operating Software (GCOS; Affymetrix), and all probe sets on each array were scaled to a mean target signal intensity of 125. Using GCOS, an absolute analysis of the GeneChip data was conducted to assess the relative abundance of the 
24 000 transcripts based on signal and detection (present, absent or marginal). Absolute analysis from GCOS was imported into GeneSpring 7.2 (Silicon Genetics), and normalized using the default normalization methods, which include setting the signal values at less than 0.1 up to 0.1, total chip normalization to the 50th percentile, and normalization of each gene to the median. Data restrictions and analytical tools in GeneSpring were applied to identify noteworthy patterns of gene expression. Differentially expressed transcripts were identified using a one-way ANOVA parametric test with variances not assumed to be equal, a P-value cutoff of 0.05, and a Benjamini and Hochberg false discovery rate multiple testing correction. This analysis was applied to all time-points and assumed that all transcripts were represented on the arrays. Expression restrictions were applied to transcripts that were expressed in a significant manner. In addition to being significantly expressed, each transcript had to have a signal value of at least 100 at one of the three time-points, the range of replicates could not exceed 1, and differences in gene expression between two of the three time-points had to be at least two-fold. Transcripts that satisfied these restrictions were considered for further analysis.
Clustering analysis was utilized to characterize further the expression pattern of genes that satisfied the described restrictions. Expression patterns were identified using unsupervised cluster analysis within the set of differentially expressed transcripts. In this analysis, a hierarchical clustering algorithm utilizing a smooth correlation with the default parameters was used to isolate distinct, nonrepetitive patterns of expression within the time course. A nonphylogenetic gene tree produced for this analysis illustrates the major expression patterns within the differentially expressed transcripts. The cluster analysis was further used for ontological clustering of differentially expressed transcripts using GeneSpring.
Gene Expression and Histological Analyses of Grafted Testis Tissues
The expression of genes that are important for testis development and spermatogenesis was determined. The genes analyzed included those for androgen receptor (exon 8; AR), KIT, clusterin (CLU), fibroblast growth factor 2 (FGF2), follicle-stimulating hormone receptor (FSHR), GATA-binding protein 4 (GATA4), glial cell line-derived neurotrophic growth factor (GDNF), GDNF family receptor 
1 (GFRA1), hypoxia-inducible factor 1 (HIF1A), insulin-like growth factor 1 (IGF1), inhibin (INHA), KIT ligand (KITLG), nuclear receptor subfamily 4 group A member 1 (NR4A1), and vascular endothelial growth factor (VEGFA).
The interaction between KITLG produced by the Sertoli cells and its receptor KIT on germ cells is important for germ cell differentiation [10]. The levels of KIT and KITLG mRNAs were higher in 16-wk-old bull testis tissues than in 2-, 4-, and 8-wk-old bull testis tissues (Table 2). Testis graft expression of KIT mRNA was significantly lower in all testis grafts compared to bull testis tissues. Furthermore, 2-wk donor tissue grafts had higher levels of KIT gene expression than 8-wk donor tissue grafts at a tissue age of 16 wk (Fig. 1A). The expression patterns of KIT protein mirrored the transcript levels (Fig. 2, A–C). In contrast to the expression of KIT, expression of KITLG mRNA was higher in 2-wk donor testis grafts than in age-matched bull tissues and all other grafted tissues at 4, 8, and 16 wk of tissue age (Fig. 1B). However, KITLG gene expression in 4-wk donor tissue grafts was not different from that in bull tissues. In addition, 8-wk donor testis grafts had lower KITLG mRNA levels than all the other different aged donor grafts at 16 wk. The expression of KITLG protein was lower in all grafts compared to age-matched bull tissue (Fig. 2, D–F). These data indicate that although KIT and KITLG levels are increasing in the developing bull testis, the graft expression of KIT is dramatically reduced, whereas KITLG expression is dependent on the age of the donor of the graft.
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Clusterin and GATA4 are expressed by Sertoli cells in the testis [11, 12]. The expression of GATA4 mRNA was higher in 16-wk bull testis tissue than in 2-, 4-, and 8-wk-old bull testis tissues, and the expression of CLU mRNA was higher in 16-wk bull testis tissue than in 2- and 4-wk-old bull testes (Table 2). The expression pattern of GATA4 protein (Fig. 3) was similar to that of GATA4 mRNA (described below), although no difference was observed for the location or intensity of CLU protein production between testis grafts and bull testes (data not shown). No differences in GATA4 protein, GATA4 mRNA or CLU mRNA expression were apparent between the 2- or 4-wk-old donor testis tissue grafts compared to bulls at tissue age of 4 or 8 wk (Fig. 4, A and B). In contrast, 16-wk-old testis grafts from 8-wk-old donors had lower GATA4 and CLU gene expression levels than all other 16-wk-old testis tissues (Fig. 4). Furthermore, the 2-wk donor testis grafts had higher CLU gene expression at 16 wk than did the 16-wk bull testis (Fig. 4).
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GDNF and GFRA1 are important for the regulation of spermatogonial stem cell self-renewal [13, 14]. There was no difference in the expression patterns of GDNF or GFRA1 mRNA during bovine testis development (Table 2). However, GDNF mRNA expression was lower in all testis grafts at 16 wk compared to 16-wk bull testis tissue, regardless of donor age (Fig. 5). Furthermore, 4-wk donor tissue grafts had lower GDNF mRNA expression than 2-wk donor tissue grafts at 16 wk. No difference was observed in GFRA1 mRNA expression between testis grafts and bull testis tissue (data not shown).
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FGF2 has been proposed to be involved in the modulation of spermatogenesis via regulation of FSHR numbers and Sertoli cell secretions [15, 16]. Expression of FGF2 mRNA was higher in 16-wk bull testis tissue than in 2-, 4-, and 8-wk-old bull testis tissues (Table 2). The 8-wk donor testis tissue grafts had significantly lower FGF2 gene expression at 16 wk than age-matched control testis tissues (Fig. 6). No other differences in FGF2 expression between testis grafts and age-matched control tissues were observed.
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VEGFA stimulates sperm production in bovine testis grafts [5] and HIF1A has been linked to the presence of VEGFA in many cells [17]. The expression levels of both HIF1A and VEGFA mRNA increased over time in developing bull testes (Table 2). Expression of HIF1A and VEGFA mRNAs was higher in 2-wk donor testis grafts at 4 wk of age than in 4-wk bull testis tissue. The 4-wk donor testis grafts had higher HIF1A and VEGFA mRNA expression levels at 16 wk than all the 16-wk-old bull testis tissues examined (Fig. 7).
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Time-dependent developmental expression differences in different aged bull testis tissues were observed for the expression of NR4A1 and IGF1 mRNA. However, no developmental differences were observed for the expression of INHA, FSHR or AR mRNAs (Table 2). No differences were observed for the expression levels of AR, INHA-FSHR, NR4A1 or IGF1 between grafts and age-matched bull testis tissues (data not shown). In addition, no differences were observed for the levels or locations of NR4A1 or AR protein expression in grafted testis tissue compared to age-matched bull tissues (data not shown). In contrast, FSHR immunohistochemistry indicated that testis grafts had decreased protein expression compared to age-matched testis tissue, although no difference was observed in the cellular location of FSHR (data not shown).
Microarray Analysis and Evaluation of Gene Expression
A genome-wide analysis of the developing bovine testis transcriptome was conducted using Affymetrix Bovine Microarray GeneChips. The microarray data have been deposited in the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO) database (www.ncbi.nlm.nih.gov/geo; series GEO series # GSE5970). The average percentages of transcripts present on the GeneChips that were expressed in 2-, 4-, and 8-wk-old bull testis tissues during these times were 53.8%, 54.4%, and 59.4 %, respectively.
Using the restrictions described in Materials and Methods, a group of approximately 200 transcripts was identified for further analysis. These transcripts represented approximately 0.8% of the transcripts on the GeneChip and approximately 1.5% of the transcripts expressed in the testis during this time period.
Microarray and Cluster Analyses
Cluster analysis was used to characterize unique patterns of expression of the 200 differentially expressed transcripts in 2-, 4-, and 8-wk-old bovine testis tissues. A hierarchical clustering algorithm (within GeneSpring) was utilized to generate a gene tree representative of the major patterns of developmental expression (Fig. 8). Three major patterns of gene expression were identified using this analysis. These unique patterns included increasing expression from 2 to 8 wk (Fig. 8A), decreasing expression from 2 to 8 wk (Fig. 8B), and relatively constant expression at 2 wk and 8 wk with decreased expression at 4 wk (Fig. 8C). Most of the genes were either gradually increasing or decreasing over time.
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Further ontological clustering of genes with GeneSpring revealed several different expression patterns of gene families during the 2–8-wk developmental period (Fig. 9). Genes involved in differentiated cell activity (catalytic activity, signal transduction, and cellular maintenance) appeared to increase during the developmental period while genes involved in proliferation and differentiation (cell communication and nucleic acid binding) appeared to decline as the testis aged.
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Several genes were identified that may be responsible for the observed differences in the developmental potential of testis tissues from 2-, 4-, and 8-wk-old donor bulls grafted onto the backs of immunodeficient mice. These genes were all present, had a signal of at least 100 for one of the three time-points, and had a change of two-fold or greater in expression between at least two of the time-points. Candidate genes included genes involved in angiogenesis (angiogenin [ANG], transgelin 2 [TAGLN2], and thrombomodulin [THBD]) and genes involved in growth (early growth response 1 [EGR1], insulin-like growth factor 2 [IGF2], and insulin-like growth factor-binding protein 3 [IGFBP3]). The expression patterns of these genes were confirmed by real-time RT-PCR (Table 1, Fig. 10).
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Bovine testis tissue grafted onto the back of castrated, immunodeficient mice undergoes complete germ cell differentiation [2–6]. Previously, grafted testis tissue function was evaluated based on the production of testosterone and the presence of elongating spermatids in the seminiferous tubules of the grafts. Under optimal conditions, approximately 10% of all seminiferous tubules in bovine testis xenografts produce sperm, with variation depending on bull calf donor age [3–4]. The endpoint of this analysis does not provide information about the somatic and germ cell differentiation leading to the development of spermatogenesis in grafted testis tissue. Many proteins that are essential for the establishment of the seminiferous tubules and for qualitative and quantitative spermatogenesis have been identified from natural mutants or with the use of targeted gene disruption in mice. The experiments presented in the current study generate several valuable datasets, which will lead to a better understanding of the development of bovine spermatogenesis and ectopic testis tissue xenografting.
The bovine testis reaches functional maturity at approximately 32 wk of age. Prior to 16 wk of age, the testis is undergoing few cellular changes, whereas after this time-point, Sertoli and germ cells begin to proliferate and differentiate [18]. The expression patterns of the genes presented in the current study further support this developmental time-line, in that the vast majority of significant increases in gene expression occur between 8 and 16 wk of age. This is most pronounced by genes that reflect Sertoli cell function, such as CLU, GATA4, FGF2, and NR4A1 [11, 12, 15, 16, 19]. Furthermore, the lack of increases in the expression levels of AR, FSHR, GDNF, and INHA indicates that these cells are still functionally immature. Moreover, the increased expression levels of HIF1A and VEGFA indicate that the testis may be undergoing structural organization at this time-point. An increase in KIT and KITLG expression at 16 wk with no difference in GDNF and GFRA1 expression may indicate that early spermatogenesis is differentially regulated in the bull compared to the rodent.
We hypothesized that differences in gene expression in testis tissues prior to or after grafting are responsible for the differences observed in sperm production in grafts from different aged donors. In the present study, evaluation of the expression patterns of genes critical for cell growth, angiogenesis, and testis function indicates that bovine testis tissue grafts express many of the same genes in a similar manner as age-matched bovine testis tissue. However, several genes associated with testis function were differentially expressed between grafted testis tissues and bovine testes. In addition, the expression levels of genes associated with blood vessel growth were higher in bovine testes that originated from 8-wk-old bull calves, which produce more sperm than other donors of other ages.
As previously reported [3–4], 2-wk donor tissues grow substantially more during the grafting period compared to tissues from other donors. However, 8-wk donor tissue has a better potential to complete spermatogenesis. In addition, grafted testis tissue androgen production is similar regardless of calf donor age, which suggests that differences in sperm production between grafted testis tissues are due to differences in the abilities of the cells in the seminiferous tubules to grow and mature. However, previous analyses of androgen production have been performed in animals with mature grafts, and it is possible that different aged donor tissues may respond differently to the mouse endocrine environment, resulting in differential growth during the early grafting period. The lack of differential expression of AR or FSHR in the present study suggests that the androgen environment is not a factor in differential graft development.
HIF1A and VEGFA are important for the initiation of angiogenesis in hypoxic tissues and for the subsequent migration and proliferation of vascular endothelial cells [17]. VEGFA is expressed in adult testis tissue in humans, although no active angiogenesis is occurring in this tissue [20]. HIF1A is expressed by spermatocytes and Sertoli cells, and has been shown to locate to the midpiece of spermatozoa in the testis and epididymis [21]. In grafted testis tissue, HIF1A and VEGFA mRNA were differentially expressed in 2-wk donor tissue at 2 wk postgrafting, but their levels were similar to the endogenous control levels by 8 wk and 16 wk of testis age. This expression pattern suggests that angiogenesis in 2-wk donor tissue grafts is complete within 6 wk of grafting. More rapid angiogenesis in 2-wk donor testis tissues may be responsible for increased graft growth, resulting in functional testis tissue later in testis graft development [3–4]. Interestingly, analysis of VEGFA protein in grafted tissues indicates that protein expression was decreased in the grafts, even though transcription was not decreased, which indicates that VEGFA mRNA turnover or translation may undergo complex regulation in the testis. VEGFA is expressed in Sertoli cells [20], and bovine testis tissue treated with VEGFA at the time of xenografting has significantly more tubules that contain elongating spermatids than the controls. However, this increase occurs without a significant increase in blood vessel growth into the tissue, which suggests that VEGFA regulates somatic or germ cell differentiation in the bovine testis [5].
The expression of FGF2 mRNA was lower in 8-wk donor tissue 8 wk after grafting (tissue age of 16 wk) compared to age-matched bull testis tissue. This factor is important for the proliferation of germ cells and Sertoli cells [14, 16, 22]. Reduced production of FGF2 in 8-wk donor tissue may indicate that Sertoli cell proliferation is impeded, resulting in fewer Sertoli cells per seminiferous tubule. One characteristic of bovine testis tissue grafts is that they contain large seminiferous tubules that are devoid of germ cells. These tubules probably do not support germ cell differentiation due to an increase in the number of Sertoli cells, which results in an increase in intratubular pressure. Therefore, testis grafts from 8-wk bull calves may have a higher percentage of tubules that contain differentiating germ cells due to decreased Sertoli cell proliferation, as suggested by lower FGF2 expression. This conclusion is supported by the expression of GATA4 and CLU in Sertoli cells of grafts. These data indicate that there are fewer Sertoli cells in these grafts, resulting in a seminiferous tubule environment that is better suited for germ cell differentiation, than in grafts that originate from younger calves.
Processes that are essential for adult spermatogenesis include spermatogonial stem cell self-renewal and the differentiation of spermatogonia into more advanced types of germ cells. Spermatogonial stem cell (SSC) self-renewal is regulated by the interaction between Sertoli cell-produced GDNF and the GFRA1/RET receptor complex, which is located on spermatogonia [13–14]. The expression of GDNF mRNA was significantly lower in grafted tissue at 16 wk of age than in 16-wk bull tissue, regardless of donor age. The spermatogonial stem cell self-renewal pathway in grafted testis tissue may be less efficient than in normal bull testis. While GDNF regulation of bovine SSCs has been reported [23], the regulation of GDNF expression in vivo has not been investigated in cattle. Xenografting of bovine testis tissue may provide a novel approach to determine the regulation and effect of GDNF on the stem cell pool during testis development.
Differentiating spermatogonia are first present in bull testes by 12 wk of age [18]. The initiation of germ cell differentiation is due in part to the interaction between KITLG and KIT. The 2-wk grafted bovine testis tissue had significantly higher KITLG expression at all testis tissue ages examined. In contrast to 2-wk donor tissue, 4-wk donor tissue was not different from age-matched bull tissue at any age, and 8-wk donor tissue had significantly lower expression of KITLG compared to all other 16-wk-old testis tissues examined. Differential expression of KITLG in different aged donor testis tissue indicates a differential ability of Sertoli cells to produce and or secrete sufficient amounts of KITLG to stimulate germ cell differentiation. These differences may be due to different rates of differentiation of testis tissues from different aged animals after grafting.
Analyses of gene expression in grafted testis tissue indicate that factors involved in tissue survival, somatic cell function, and germ cell differentiation are involved in the capacity of testis tissue to produce sperm following grafting. To investigate intrinsic differences in testis tissues that may have impacts on tissue survival and sperm production, we investigated gene expression in testis tissues from 2-, 4- and 8-wk-old bull calves using Affymetrix Bovine Microarray GeneChips. Previous studies have successfully utilized microarray technologies to identify genes that are important in mammalian testis biology [24–28]. Based on the data from the real-time RT-PCR analysis of gene expression in testis tissues following grafting, analysis of the GeneChip data focused on the identification of differentially expressed genes that are involved in angiogenesis and growth.
The ANG, THBD, and TRGLN2 genes were more highly expressed in the testes from 2- or 4-wk-old calves compared to samples from 8-wk-old calves. These factors are involved in new blood vessel formation, prevention of clot formation, and cytoskeleton formation [29–31]. Higher expression of these angiogenic factors at 2 wk of age supports the hypothesis that the larger grafts from younger donors are due to an increase in the angiogenic potential of the younger tissues.
Microarray analysis of the 2-, 4-, and 8-wk-old bull testes also indicated that EGR1, IGF2 and IGFBP3 were differentially regulated in bovine testis development. The expression of all three genes decreased from 2 to 8 wk of age. These factors are involved in a myriad of functions, including lymphocyte mitosis, growth factor regulation, apoptosis, angiogenesis, memory, gonadotropin production (EGR1 [32–37]), fetal growth hormone activity, muscle growth, fat deposition, placental growth, parthenogenesis (IGF2 [38–42]), and IGF regulation (IGFBP3 [43–44]). Known roles for these genes in the testis include regulation of sex determination (EGR1, [45]), growth (IGF2 [46–47]), and IGF1 regulation (IGFBP3 [48]).
Different aged prepubertal neonatal bovine testis tissues have different potentials for development when ectopically grafted onto recipient mice. Testis tissues from 8-wk-old donors provide the best materials for the production of elongating spermatids (compared to 2- and 4-wk-old donors). In the present study, several cohorts of genes involved in Sertoli cell proliferation and function, angiogenesis, and germ cell function were found to be differentially expressed. We attribute the higher levels of sperm production in bovine testis xenografts from different age bull calf donors to differences in Sertoli cell proliferation and differentiation and blood vessel growth. A successful graft must have adequate angiogenesis for functional Sertoli cells, which in turn are necessary for functional germ cells. The experiments presented in the current study generate several valuable datasets that will be useful for future studies of bovine spermatogenesis and may lead to improvements in bovine ectopic testis tissue xenografting. Future experiments include analyses of differential gene expression between bovine testis tissue and testis xenografts within specific cell types and the treatment of xenografts with factors (e.g., GDNF, angiogenic promoters, TSH) that may improve the efficiency of spermatogenesis within the graft. Elucidation of the differentially expressed factors will provide novel methods to improve ectopic testis tissue grafting, as well as shed light on the roles of genes that are important for successful bovine testis development.
ACKNOWLEDGMENTS
The authors thank Nada Cummings, Craig Blakesley, So Nagaoka, and Liang-Yu Chen for assistance with the grafting technique, and Michelle Schmidt for assistance with the microarray analysis. The authors also thank the past and present members of the McLean laboratory for laboratory assistance and critical evaluation of the manuscript.
FOOTNOTES
1The microarray data have been deposited in the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO) database (series no. GSE5970). 
Correspondence: 2FAX: 509 335 4246; e-mail: dmclean{at}wsu.edu
Received: 18 October 2006.
First decision: 14 November 2006.
Accepted: 21 February 2007.
REFERENCES
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