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Stage-Specific Expression of Genes Associated with Rat Spermatogenesis: Characterization by Laser-Capture Microdissection and Real-Time Polymerase Chain Reaction¹

^a Prince Henry's Institute of Medical Research, Clayton, Victoria, Australia 3168 ^b Department of Anatomy and Cell Biology, Monash University, Clayton, Victoria, Australia 3800

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Spermatogenesis in the rat consists of 14 unique morphologic cellular associations between Sertoli cells and developing germ cells within the seminiferous epithelium. The complexity of the cellular associations leads to difficulty in the isolation of individual cells at a defined stage of development for the study of their unique patterns of gene or protein expression. Thus, laser-capture microdissection is an ideal technique to permit such analysis. This study used laser-capture microdissection and real-time reverse transcription-polymerase chain reaction (RT-PCR) to quantitate the stage-specific expression of a series of genes of functional significance in hormonal regulation and cell-cell interactions in spermatogenesis, including cathepsin-L, CREM-, transition protein-1, androgen receptor, ß1-integrin, N-cadherin, and hypoxanthine phosphoribosyltransferase (HPRT). Frozen sections (10 µm) were obtained from normal adult rat testes. Laser-capture microdissection (LCM) was used to capture all cells in cross-sections of seminiferous tubules that were grouped into stages I–V, VII–VIII, and IX–XIII. Transition protein-1 expression was lowest during stages I–V and increased 5.9-fold during stages VII–VIII and IX–XIII (P < 0.01). Cathepsin-L expression was highest during stages I–V and VII–VIII, falling 4.9-fold during stages IX–XIII (P < 0.05). Similarly, CREM- expression was highest during stages I–V and VII–VIII, falling 1.6-fold during stages IX–XIII (P < 0.05). A novel CREM- isoform lacking the phosphorylation domain was also characterized but was not stage-specific. ß1-Integrin, N-cadherin, and androgen receptor expression did not change between the spermatogenic stages examined. HPRT housekeeper expression was lowest during stages I–V but increased 1.5-fold during stages VII–VIII and IX–XIII (P < 0.05). This study is the first to apply LCM and real-time RT-PCR analysis to quantitate stage-specific changes in the expression of multiple genes in the seminiferous epithelium.

gene regulation, Sertoli cells, spermatid, spermatogenesis

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Spermatogenesis is the process of germ cell development whereby diploid germ cells proliferate and differentiate into haploid spermatozoa [1]. The spermatogenic process takes place within the seminiferous tubules of the testis. The seminiferous epithelium is comprised of the somatic Sertoli cells together with closely associated germ cells. Each Sertoli cell is in contact with germ cells at various phases of development, from immature spermatogonia through to mature elongated spermatids. Spermatogenesis occurs in a cyclic manner known as the spermatogenic cycle (see de Kretser et al. [2] for review), which can be divided into 14 stages (I–XIV) on the basis of distinct associations of germ cells [3]. These 14 stages follow one another sequentially along the tubule, and a stage XIV tubule becomes a stage I tubule at the beginning of the next cycle.

Spermatogenesis is known to be regulated by the hormones FSH and testosterone (T) [4, 5]. It is well known that hormones regulate spermatogenesis in a stage-specific manner, e.g., androgens exert their effects on spermatogenesis primarily during stages VII–VIII [6, 7], while FSH receptors are highest in Sertoli cells during stages XII–II [8].

Spermatogenesis can be disrupted at various stages in conditions of infertility. Such conditions can have a genetic basis [9, 10], can also be due to defects in reproductive hormone production or action [11], or alternatively, be induced by exposure to environmental or chemical toxicants [12, 13]. Germ cell development is often arrested at discrete stages of spermatogenesis in conditions of infertility, e.g., arrested at the point of spermatogonial or postmeiotic germ cell development [14, 15]; however, the mechanism(s) for these arrests remain unknown.

Proteins are known to be expressed in a stage-specific and cell-specific manner in the seminiferous epithelium. However, due to the complexity of the epithelium, it is very difficult to isolate pure cell populations at discrete stages of development in order to analyze stage- and cell-specific gene and protein expression. Previous studies investigating gene expression in the seminiferous epithelium have therefore been restricted to using whole-tissue approaches such as in situ hybridization [16, 17], immunocytochemistry [18, 19], or dissection of seminiferous tubule segments in which stages are subsequently defined by transillumination microscopy and morphologic analysis [8, 20]. These approaches are not readily quantifiable. Alternatively, in vitro techniques in which particular cell types (e.g., Sertoli cells, pachytene spermatocytes, round spermatids) purified from all stages of spermatogenesis have been used in semiquantitative gene expression analysis [21]. The major disadvantages of this latter approach are in obtaining pure cell populations, particularly from adult animals, and in the inability to separate cell types from particular stages.

Recent advances in methodology have now made it possible to quantitate gene expression in highly purified cell populations derived from complex tissues. Laser-capture microdissection (LCM) is a novel technique that allows for the procurement of individual cells or cell groups directly from tissue sections using cell morphology as the means of selection [22–25]. When coupled with real-time reverse transcriptase-polymerase chain reaction (RT-PCR) for quantitation of gene expression, use of LCM circumvents the difficulties of cell isolation encountered with previous techniques [22]. For example, LCM has been used to isolate samples for gene expression analysis in kidney glomeruli [26] and neuronal subtypes [27] and also to analyze different stages of breast cancer progression [28] and protein production in colon cancer [29]. However, LCM and quantitative real-time RT-PCR have not been applied to the study of gene expression in the seminiferous epithelium.

The aim of this study was to show that changes in testicular gene expression over the spermatogenic cycle in the normal rat can be quantified using LCM and real-time RT-PCR. A series of genes previously shown to be stage specific were studied, including cathepsin-L [30, 31], transition protein-1 (TP-1) [17, 32], cyclic-AMP response element modulator- (CREM-) [33], and androgen receptor (AR) [16, 34, 35]. In addition, the stage-specificity of two cell adhesion molecules, N-cadherin and ß1-integrin, for which available evidence suggests regulation by FSH and T [36–38] was investigated.

	MATERIALS AND METHODS

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Tissue Preparation for Laser-Capture Microdissection

Adult male (100–150 days old) Sprague-Dawley outbred rats were killed by CO₂ asphyxiation. All animal work was approved by the Monash Medical Centre Animal Ethics Committee. Testes were excised, frozen in isopentane over liquid N₂, and stored at -80°C. Tissue sections (10 µm thick) were collected at -20°C onto autoclaved glass slides (uncharged and uncoated; Objektträger, HD Scientific, Melbourne, Australia), which were wiped with 100% ethanol and cooled to -20°C. Sections were bonded to the slide by gentle warming of the slide undersurface and then refreezing at -20°C. Sections were then fixed in cold (0°C) acetone for 1 min, stained with 4',6-diamidino-2-phenylindole dihydrochloride (DAPI; Molecular Probes, Eugene, OR) (100 nM in 70% ethanol, 5 min), and dehydrated in 95% ethanol (1 wash, 30 sec), 100% ethanol (three washes), and xylene (two washes, 5 min each). Sections were then allowed to air dry and were stored at room temperature in a desiccator. It was noted that sections could be stored in this way for up to 14 days without any loss in RNA yield (data not shown). We compared the effect of tissue fixation (acetone, ethanol, 3.7% formaldehyde, Bouin) and staining on RNA recovery from tissue sections. Optimal morphology in conjunction with highest total RNA yield was obtained with acetone fixation, whereas other fixatives either yielded poorer tissue morphology (ethanol, formaldehyde) or prevented the recovery of total RNA (Bouin) (formaldehyde not assessed). In addition, any form of aqueous processing of frozen sections (e.g., hematoxylin and eosin staining) resulted in poor morphology and was not investigated further. Nuclear staining with DAPI was possible because this compound is soluble in ethanol, and its use did not alter the yield of total RNA (data not shown).

Laser-Capture Microdissection

Spermatogenic stages were morphologically identified by a combination of light and fluorescence microscopy using the fluorescent stain DAPI to label cell nuclei. Tubules at stages I–V were characterized by the presence of compact elongate spermatid heads embedded deep within the seminiferous epithelium and extensive elongate spermatid cytoplasm in the tubule lumen. Stage VII–VIII tubules were characterized by the alignment of elongate spermatids adjacent to the tubule lumen prior to spermiation, while stage IX–XIII tubules were recognized by the presence of spermatids undergoing nuclear compaction in the absence of elongate spermatids. Tubules at stage VI or XIV of spermatogenesis were not captured because they represent transitional stages that were difficult to categorize unambiguously.

Samples containing 24 tubule cross-sections of one of the three stage groupings described above were collected by LCM for RT-PCR analysis. The entire sampling scheme was repeated three times from the same testis (n = 3). LCM was performed using a PixCell II laser capture microdissection microscope (Arcturus Engineering, Mountain View, CA), equipped with a fluorescence light source. Each section was pretreated with a PrepStrip tissue preparation strip (Arcturus) to remove loose debris and to flatten the tissue. Sections were then visualized using a 20x objective, and capture was performed using a 30-µm diameter laser spot size set at 20–30 mW with a pulse duration of 5 msec. Cells were captured using CapSure LCM caps (Arcturus) and stored in a desiccator prior to extraction of total RNA.

Isolation of Total RNA

Total RNA was isolated from each LCM sample using the TRIzol method (Gibco BRL, Rockville, MD). LCM caps were initially incubated with TRIzol reagent with added glycogen (250 ng/µl) for 60 min at room temperature, after which the manufacturer's protocol was followed. RNA samples were then resuspended in 30 µl diethyl pyrocarbonate-treated H₂O, treated for the removal of any contaminating DNA using the DNAse-free kit (Ambion, Austin, TX), and stored at -80°C. A comparison of total RNA yields extracted from frozen tissue sections using TRIzol reagent or RNeasy (Qiagen, Hilden, Germany) demonstrated that 493 ± 77 ng/tissue section (mean ± SD) was obtained with TRIzol reagent, while 352 ± 52 ng/tissue section was obtained using RNeasy.

RNA concentrations were determined using the Ribogreen fluorescence RNA assay (Molecular Probes) with an Escherichia coli ribosomal RNA preparation of known concentration as standard (Molecular Probes) and Ribogreen reagent at a final dilution of 1:500. A standard curve of 0–80 ng/well in a 96-well flat-bottomed plate was employed. This assay gave an intraassay CV, as defined by the repeated analysis of a single RNA sample, of 3.3% (n = 16) and an interassay variation of 10.3% (n = 14).

Reverse Transcription

Reverse transcription was performed on 8 ng total RNA/sample using Sensiscript RT (1 µl/reaction; Qiagen) with 2 µg random hexamer primers (Amersham Pharmacia Biotech Inc., Piscataway, NJ), RNAsin (1 U/reaction) in a final volume of 20 µl according to the manufacturer's instructions. For each stage grouping, the absence of contaminating genomic DNA in cDNA samples was confirmed using reactions in which the RT enzyme was omitted.

Real-Time PCR Analysis

Quantitative real-time PCR analysis was performed using the Roche LightCycler (Roche, Mannheim, Germany) and the FastStart DNA Master SYBR-green 1 system (Roche). Oligonucleotide primer sequences for cathepsin-L, transition protein 1, ß1-integrin, androgen receptor, cyclic AMP-response element modulator- (CREM-), and hypoxanthine phosphoribosyltransferase (HPRT) were obtained either from published sources or designed using the Oligo program (version 6; Molecular Biology Insights, Cascade, CO) (Table 1). For PCR analysis, sample cDNA was diluted 1:5- to 1:24-fold, and PCR reaction conditions, including Mg²⁺ concentration, primer concentration, anneal time, and extension time, were optimized for each primer pair as summarized in Table 2. For all PCR analyses, standard curves were generated using dilutions of an adult rat testicular cDNA preparation of arbitrary unitage. PCR of all standards and samples was performed using duplicate reactions for approximately 40–45 cycles, after which a melting curve analysis was performed to monitor PCR product purity (see Table 2). In initial experiments, PCR product identities were verified by agarose gel electrophoresis and DNA sequencing.

Statistical Analysis

Data are represented as mean ± SD, with n = 3 samples in each group. Statistical analyses were performed using GB Stat (Dynamic Systems Inc., Silver Spring, MD), and all data were log transformed prior to analysis. Initially, homogeneity of variance was assessed for all groups. Homogeneous groups were assessed using one-way ANOVA, followed by the Neuman-Keuls post hoc multiple group comparisons test. Nonhomogeneous groups were assessed using the nonparametric Kruskal-Wallis test, followed by the Neuman-Keuls analog post hoc multiple group comparisons test.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Whole seminiferous tubule cross-sections of defined spermatogenic stages were successfully captured, as shown in Figure 1, A–C. For the purpose of this study, spermatogenic stages were pooled into three groups, these being stages I–V, VII–VIII, and IX–XIII. These stage groupings were chosen because they represent times in the spermatogenic cycle when distinct developmental events occur (e.g., release of mature elongate spermatids), during which it is anticipated that discrete molecular mechanisms are functional. Identification of these stages was as outlined in Materials and Methods and is shown in Figure 1, D–I.

View larger version (169K): [in this window] [in a new window]   FIG. 1. A–C) Laser-capture microdissection of rat seminiferous tubules. Rat testis cross-sections were cut on a cryostat (10 µm thickness) and processed for LCM as described in Materials and Methods. A) Micrograph of intact testis section viewed by light microscopy prior to LCM. B) Isolated tubule cross-section on the LCM cap following LCM. C) Remaining tissue section. Bar = 20 µm. D–I) Examples of spermatogenic stages collected by LCM for mRNA expression analysis. D–F) Images viewed by light microscopy. G–I) The same sections viewed by UV showing cell nuclei as indicated by the fluorescent stain DAPI. Seminiferous tubules at the following stages of spermatogenesis were grouped: (D, G) stages I–V, (E, H) stages VII–VIII, (F, I) stages IX–XIII. Short arrows indicate compact elongate spermatid heads embedded within the seminiferous epithelium; asterisks indicate elongating spermatid cytoplasm; arrowheads indicate elongate spermatids adjacent to the tubule lumen prior to spermiation, x indicates absence of elongate spermatids; long arrows indicate compacting heads of elongate spermatids. Bar = 20 µm

Gene expression in the defined spermatogenic stages was performed using real-time RT-PCR. All PCR reactions amplified the expected gene products, as assessed by PCR product size (see Fig. 2) and by DNA sequencing (data not shown). In addition, PCR product purity was routinely monitored on the LightCycler by determination of the PCR product melting temperature (see Table 2). The presence of a single melting-curve peak at the expected temperature indicated the formation of a single PCR product of expected size.

View larger version (65K): [in this window] [in a new window]   FIG. 2. Agarose gel electrophoresis of PCR products. Following real-time PCR amplification, PCR products were retrieved from the LightCycler capillaries, electrophoresed on a 2.0% agarose gel containing EtBr, and visualized under UV light. PCR product sizes are indicated in parentheses. A minus RT control with HPRT primers (-RT HPRT) was also performed, and similar blank lanes were obtained for all stage groupings (not shown). A 50-bp DNA ladder was included to indicate PCR product size (base pairs)

In initial experiments optimizing the RT reaction, it was established that the relationship between the amount of total RNA applied to each reaction and the amount of cDNA produced was linear from 0 to 16 ng RNA/reaction (Fig. 3A). Furthermore, increasing the concentration of random hexamer primers used to prime the reverse transcription reaction above that recommended by the manufacturer (6.4 ng/reaction) in the presence of a constant amount of total RNA (8 ng/reaction) increased the amount of cDNA produced. This effect was maximal at >1000 ng random hexamers per reaction (Fig. 3B). Finally, the parallelism of sample cDNA dilutions with the rat testicular cDNA standard was assessed for all genes, a representative result of which is shown in Figure 3C. It was observed that sample cDNA diluted in parallel with the standard over a 16-fold range, thus verifying that PCR analysis was quantitative.

View larger version (12K): [in this window] [in a new window]   FIG. 3. Validation of the reverse transcription-real time PCR process. A) Correlation between total RNA concentration applied to the reverse-transcriptase (RT) reaction (x-axis) and yield of cDNA as measured by real-time PCR for TP-1 (y-axis). The RT enzyme was primed with 6.4 ng random hexamers/reaction, as recommended by the manufacturer. B) Correlation between primer (random hexamer) concentration applied to the RT reaction (x-axis) and yield of cDNA as measured by real-time PCR for TP-1 (y-axis). A constant amount of total RNA (8 ng) was used in each reaction. C) Parallelism of dose-response curves for real-time PCR analysis. The testicular cDNA standard was diluted over a 16-fold range, while two testicular cDNA samples were diluted over a 4-fold range and subjected to PCR amplification for cathepsin-L. All data are expressed in terms of arbitrary units (mean ± SD, n = 3 samples)

Once the application of quantitative RT-PCR had been validated as described above, the stage-specific expression of TP-1, cathepsin-L, N-cadherin, ß1-integrin, AR, and HPRT in the pools of spermatogenic stages captured was determined using real-time RT-PCR (see Fig. 4). TP-1 expression (Fig. 4A) was lowest during stages I–V and increased 5.9-fold during stages VII–VIII and IX–XIII (P < 0.01). Cathepsin-L expression (Fig. 4B) was highest during stages I–V and VII–VIII, falling 4.9-fold during stages IX–XIII (P < 0.05). The expression levels of N-cadherin (Fig. 4C), ß1-integrin (Fig. 4D), and AR (Fig. 4E) did not change between the spermatogenic stages examined. Expression of the HPRT housekeeper gene (Fig. 4F) varied significantly over the spermatogenic stages examined and was lowest during stages I–V but increased 1.5-fold during stages VII–VIII and IX–XIII (P < 0.05). Given that HPRT expression during spermatogenesis was variable, it was not used to normalize quantitative PCR data.

View larger version (28K): [in this window] [in a new window]   FIG. 4. Stage-specific expression of genes during spermatogenesis. Gene expression in tubule cross-sections of defined spermatogenic stages (I–V, VII–VIII, IX–XIII) isolated by laser capture microdissection was assessed by real-time PCR. Data is presented as mean ± SD for n = 3 separate samples per stage grouping and is expressed in terms of arbitrary units per ng total RNA in the reverse transcription reaction. Significant differences between groups are denoted by letters: a and b, P < 0.01; c and d, P < 0.05

In addition to the genes described above, the stage-specific expression of CREM- was analyzed (see Fig. 5). CREM- expression was highest during stages I–V and VII–VIII, falling 1.6-fold during stages IX–XIII (P < 0.05). Following quantitative PCR, the PCR products were separated by agarose gel electrophoresis, revealing the expected 446-base pair (bp) PCR product, along with a smaller 208-bp PCR product (Fig. 5B). Sequence analysis of the two CREM- PCR products revealed that they represent splicing isoforms of the CREM- gene (see Fig. 5C), where the phosphorylation domain (P-Box) located between the CREM--specific glutamine-rich domains (labeled I and II) is alternatively spliced. In the larger 446-bp PCR product, the P-Box is spliced in, whereas in the smaller 208-bp PCR product, the P-Box is spliced out. The existence of a CREM- isoform containing both glutamine-rich domains but lacking the P-Box has not been previously described. In order to quantitate the relative expression levels of the two CREM- isoforms, the PCR products generated in Figure 5A were separated by gel electrophoresis, and the two bands were analyzed using densitometry (see Fig. 5D). Expression of the 446-bp PCR product was highest during stages I–V and VII–VIII, falling 1.6-fold during stages IX–XIII (P < 0.05). Expression of the 208-bp PCR product was lower than the 446-bp PCR product and did not vary over the spermatogenic cycle.

View larger version (17K): [in this window] [in a new window]   FIG. 5. Stage-specific expression of CREM- during spermatogenesis. A) CREM- expression in tubule cross sections of defined spermatogenic stages, as determined by real-time PCR. (Note: PCR was halted during the log-linear phase of PCR amplification following 33 cycles to facilitate analysis by densitometry in D). B) Agarose gel electrophoresis of the two CREM- real-time PCR products (446 and 208 bp, respectively) and a minus RT control. C) Schematic representation of the rat CREM- gene, showing location of two glutamine-rich exons (I, II), phosphorylation domain (P-Box), and DNA-binding domain. The location of the forward and reverse CREM- PCR primers used is indicated, together with the 446- and 208-bp products observed and sequenced after real-time PCR. D) Samples quantitated in A were separated by gel electrophoresis, and the stage-specific expression of the two PCR products was further analyzed by densitometry. Data is mean ± SD for n = 3 separate samples. Significant differences between groups are denoted by letters: a and b, P < 0.05

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
This study has shown that changes in gene expression associated with the stages of rat spermatogenesis can be quantified using LCM and real-time RT-PCR. The mRNAs for cathepsin-L, transition protein-1, CREM-, and the putative housekeeping gene HPRT were expressed at varying levels during spermatogenesis, while the mRNAs for ß1-integrin, N-cadherin, and androgen receptor did not change. Furthermore, a novel CREM- splice isoform lacking the phosphorylation domain was identified in the rat seminiferous epithelium and was found to be present at equal levels during all stages of spermatogenesis. The combination of these techniques thus allows a sensitive analysis of stage-specific gene expression previously unavailable in the study of spermatogenesis.

In order to evaluate the applicability of LCM-RT-PCR for testicular gene expression, a number of candidate genes of known stage specificity were selected, including cathepsin-L, TP-1, and androgen receptor. Cathepsin-L is a cysteine protease expressed by Sertoli cells [39] and Leydig cells [40] and is thought to be involved in the turnover of Sertoli cell-elongate spermatid and Sertoli-Sertoli cell junctions [40]. Previous data by Northern blot analysis [30] and in situ hybridization [31] has shown that cathepsin-L mRNA is undetectable at stage II and is greatest during stages VI–VII, after which it is down-regulated. Similarly, cathepsin-L immunostaining is present during stages V–VIII in Sertoli cells, being strongest during stages VI–VII [41], and is not found in other stages. This pattern of mRNA expression was confirmed and for the first time quantitated in the current study, in which cathepsin-L mRNA was observed at highest levels in I–V and VII–VIII stage groupings but decreased in stages IX–XIII.

TP-1 is a haploid germ cell-specific DNA-binding protein involved in the compaction of chromatin required for spermatid head compaction during steps 7–10 [32]. Previous studies by in situ hybridization have shown that TP-1 mRNA expression begins during stage VII, is highest during stages VIII–XIII, and decreases during stage XIV [17, 32]. A similar trend was observed in the current study in which greatest TP-1 mRNA expression was observed across stages VII–XIII, with a 5.9-fold decrease in stages I–V. Immunocytochemical data has demonstrated that TP-1 protein is progressively increased during stages XI through XIII, decreasing thereafter, becoming absent by stage IV [42, 43].

Testicular CREM- is an FSH-dependent germ cell transcription factor essential for spermiogenesis, which is the process of germ cell maturation from round to elongate spermatids [44, 45] (for a review, see Fimia et al. [46]). CREM- mRNA is synthesized in pachytene spermatocytes [47] under the control of FSH [48]. CREM- protein is synthesized and activated by phosphorylation predominantly in the postmeiotic round spermatids [33, 49] and is involved in regulating the expression of downstream target genes such as the transition and protamine nucleoproteins [44]. Although CREM- protein has been reported to be highest during stages IV–VII of spermatogenesis in the rat [49], stage specificity of CREM- mRNA was not apparent [50] using in situ hybridization techniques. The ability of the current study to identify a 1.6-fold decline in CREM- expression during stages IX–XIII likely indicates the increased sensitivity gained by using a quantitative PCR-based technique to examine gene expression.

Multiple CREM- isoforms arising from alternative exon splicing have previously been identified [51, 52]. In the testis, exons that have been alternatively spliced out of full-length CREM- consist of one of the glutamine-rich domains either alone or in combination with the phosphorylation domain [52]. CREM- isoforms lacking the glutamine-rich and phosphorylation domains inhibit gene transcription, being able to bind cyclic-AMP response elements, however not being activated by phosphorylation [52]. The CREM- isoform identified in this study is different from those identified by [52] in that the phosphorylation domain is spliced out while both glutamine-rich domains are present. We hypothesize that this novel CREM- isoform would also be a repressor of gene transcription in the testis.

The actions of T on spermatogenesis are mediated via the androgen receptor, which is found in Sertoli cells, peritubular myoid cells, and Leydig cells but is not believed to be present in germ cells [35, 53]. Within the seminiferous epithelium, Sertoli cell AR mRNA levels by in situ hybridization are low during stages I–II, increase during stages IV–V, are maximal during stages VII–VIII, and drop to basal levels during stages IX–XI [16]. Similarly, AR protein exhibits a stage-dependent expression by immunohistochemistry, being most predominant in Sertoli cell nuclei during stages IV–VII [35]. In contrast, this study surprisingly found no evidence for stage-specific expression of AR mRNA expression in whole cross-sections of seminiferous tubules. It is expected that this is most likely due to the use of whole seminiferous tubule cross-sections, which contained not only Sertoli cells and germ cells but also peritubular myoid cells, which line the periphery of seminiferous tubules. Peritubular myoid cells have been shown to express AR mRNA at all stages of spermatogenesis [35], which could potentially mask any stage-specific expression of Sertoli cell AR mRNA as seen in previous studies. Alternatively, stage-specific differences in AR expression could have been masked in our experiments by the stage groupings used, which may also have contributed to the large errors observed with this primer set in comparison with the other genes analyzed. Hence, a more appropriate approach to quantitate AR gene expression would be to isolate more discrete spermatogenic stages and/or to specifically ablate peritubular cells surrounding seminiferous tubules prior to laser capture. The use of the PALM microbeam LCM system would likely be more appropriate for the latter experiment [54].

Integrins are dimeric transmembrane proteins involved in cell-cell and cell-matrix adhesion along with cell signaling and are comprised of one and one ß chain (for reviews, see Hynes [55] and van der Flier and Sonnenberg [56]). Many subunits of each and ß integrin subunit have been described, with specific combinations of the two subunits producing the specificity of integrin interactions [55, 56]. In the rat testis, the ß1-integrin subunit protein has been localized by immunohistochemistry to regions of round spermatid-Sertoli cell contact from stage VII onward and Sertoli-Sertoli cell contacts [18, 57, 58] as well as to peritubular cells [18, 57]. ß1-Integrin mRNA has been shown to be expressed in the rat [59] and marmoset testis [60]; however, no evidence for stage specificity has been reported. The current study has shown that ß1-integrin mRNA is expressed at equal levels during all stages of rat spermatogenesis.

The cadherins are a family of transmembrane proteins involved in homophilic calcium-dependent cell-cell adhesion (for a review, see Potter et al. [61]). Many cadherin members are expressed in the testis [62]; however, of the classic cadherins (being N-, E-, and P-cadherin), only N-cadherin is expressed at significant levels in the adult rat testis [63]. N-cadherin protein is found at sites of Sertoli-Sertoli and Sertoli-germ cell contact [19]. It is known that both Sertoli cells [36, 64] and germ cells [64] isolated from 20-day-old rats synthesize N-cadherin mRNA; however, examination of the stage-specific expression of N-cadherin mRNA in the adult rat testis has not previously been examined. This study has shown that N-cadherin mRNA is produced during all stages of spermatogenesis and that the levels of N-cadherin mRNA do not change over the spermatogenic cycle. In confirmation, cadherin protein, detected using an antibody that recognizes all classical cadherins, has been localized by immunohistochemistry to sites of Sertoli-Sertoli cell contact at all stages of spermatogenesis [58, 65].

In this study, we have established that stage-specific gene expression can successfully be quantified in the rat testis. However, a number of limitations regarding the applicability of LCM need to be considered. While spermatogenic stages were pooled into three groups, it would be of preference to study changes in gene expression at each individual stage. This was not possible because the fixation method used did not provide sufficiently defined tissue morphology to allow each individual stage to be conclusively identified. The use of immunostaining techniques to assist in stage identification has previously been suggested [24] but was not used in this study because attempts to treat frozen tissue sections with aqueous solutions were detrimental to morphology. Although it is possible to examine gene expression in testicular cells captured by LCM from testis tissue exhibiting better morphology, e.g., following Bouin immersion fixation and wax embedding [66], in our hands, these steps reduced the recovery of RNA to levels below those required for quantitative analysis of multiple gene products. Improved fixation techniques compatible with RNA isolation and perhaps the use of tissues embedded in a low melting-temperature wax [24] will therefore be required to allow gene expression studies in defined spermatogenic stages or single-cell populations.

When quantifying gene expression with RT-PCR techniques, data need to be normalized to a reference. The most common method used is to normalize data to the expression of a housekeeper gene; however, numerous examples now exist to show that this method can be problematic, as reviewed by Bustin [67] and Thellin et al. [68]. This was confirmed in the present study, in which it was found that the expression of HPRT was not constant between the stage groupings analyzed. For this reason, data were normalized to total RNA as recommended elsewhere [67].

In summary, this study has validated a method to isolate seminiferous tubule cross-sections containing Sertoli cells, germ cells, and peritubular cells from whole sections of testis tissue using LCM, with quantitation of gene expression using real-time RT-PCR. This approach provides the foundations to not only further our understanding of gene expression associated with spermatogenesis in the normal animal but also to identify changes in gene expression associated with male infertility or animal models in which hormones (e.g., FSH, testosterone) have been selectively suppressed and replaced [7, 69]. Furthermore, the analysis of genes in individual cell types (e.g., round spermatids) at specific stages of spermatogenesis should now be possible. Studies of this nature are currently being undertaken within this laboratory.

	ACKNOWLEDGMENTS

 
The authors would like to thank Drs. D.M. Robertson and R.I. McLachlan for helpful discussions during the course of this work.

	FOOTNOTES

 
First decision: 18 March 2002.

¹ National Health and Medical Research Council of Australia Program Grant Regkey No. 983212.

² Correspondence: Peter Stanton, Prince Henry's Institute of Medical Research, P.O. Box 5152, Clayton 3168, VIC, Australia. FAX: 61 3 9594 6125; peter.stanton{at}med.monash.edu.au

Accepted: April 24, 2002.

Received: February 22, 2002.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

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