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Gamete Biology |
Department of Cell Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100005, China
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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gametogenesis, sperm, spermatogenesis, testis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Most cells in our bodies contain the same genome, but not all the genes are used in each cell. Some genes are turned on (or expressed when needed), and many genes are used to specify features unique to each type of cells. To know how genes are controlled to express at the exact time and which genes function uniquely in special cells, an important step is defining gene expression profiles—that is, comparing patterns of expression in different tissues and at different developmental stages in both normal and disease states. This can be accomplished using reverse transcription-polymerase chain reaction (RT-PCR), RNase protection assays, or Northern blot analysis, but these methods focus on only a few genes at a time. A more promising approach for analyzing multiple genes simultaneously is the hybridization of entire cDNA populations to nucleic acid arrays, a method that has been adopted for high-throughput analysis of gene expression [4–7]. It allows rapid detection of the gene expression profiles of hundreds to thousands of genes simultaneously. This technology, otherwise known as cDNA microarray, offers tremendous potential for characterizing gene expression patterns during normal biological or disease processes as well as for identifying differentially expressed genes that may play an integral role in these processes. In this regard, using this approach has resulted in the identification and cloning of genes with potential relevance for growth control and terminal differentiation in human melanoma cells [8], ovarian carcinomas [9], renal cell carcinoma [10], breast cancer [11], embryonic stem cells [12], folliculogenesis [13], and follicular lymphoma [14]. In addition, cDNA microarrays have been used to study the temporal program of gene expression in human fibroblasts in response to serum [15] and in spermatogenic cells in response to stress [16] and to hyperthermia [17].
In a similar vein, the cDNA microarray technology can be applied to gain a comprehensive view of gene expression involved in spermatogenesis with the purpose of studying the mechanisms and regulation of spermatogenesis at the genetic level. In the present study, we first isolated six spermatogenic cell types from mouse testis. We then used cDNA microarray technology to identify spermatogenesis-related as well as differentially expressed genes among differently developed stages of germ cells and to further elucidate their functions and relationships in spermatogenesis.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Male and female Balb/c mice (age, 80–100 days) were obtained from Laboratorial Animals Center of Beijing University (Beijing, China) and maintained in a temperature- and humidity-controlled room on a 12L:12D photoperiod. The mice had free access to food and water. Female mice were naturally mated and observed at 12-h intervals near the end of pregnancy to record the time at which parturition occurred. The day of birth was designated as Day 0. Litter size was adjusted to a maximum of 10 by removing the appropriate number of female pups. All the measures taken for the mice were in accordance with approved guidelines (Guideline for the Care and Use of Laboratory Animals) established by the Chinese Council on Animal Care.
Isolation of Spermatogenic Cell Types
The procedure for the isolation of spermatogenic cells was based on that previously described by Bellvé et al. [18] with a modification. Based on the majority of germ cell types, different ages of male Balb/c mice were used for the isolation of differently developed stages of spermatogenic cell types: 6 days of age for primitive type A spermatogonia, 9 days for type B spermatogonia, 14 days for preleptotene spermatocytes, 21 days for pachytene spermatocytes, 35 days for round spermatids, and 60 days for elongating spermatids. The number of mice used for cell separation was 25, 25, 20, 15, 10, and 6, respectively.
Briefly, mice were anesthetized with CO2 and then killed by cervical dislocation. The testes were removed and decapsulated. The tubulous tissue was cut into small pieces and incubated in 5 ml of PBS containing 0.5 mg/ml of collagenase (Sigma, St. Louis, MO) with continuous agitation at 33°C for 15 min. The dispersed seminiferous cords and cells were allowed to sediment for 5 min, and then the supernatant was decanted. The pellet was resuspended in 5 ml of PBS containing 0.5 mg/ml of trypsin (Sigma) and 1 µ/ml of DNase (Promega, Madison, WI) and incubated under the same conditions as above for 15 min. The tissue was dissociated to disperse seminiferous cells by pipetting gently with a Pasteur pipette, and the cell suspension was centrifuged at 80 x g for 10 min. The pellet was then washed twice with PBS, filtered through a filter cloth (200 mesh), and resuspended in 20 ml of PBS solution containing 0.5% BSA.
Cell numbers were counted using a hemocytometer. A total of 108 cells was bottom-loaded into a cell-separator apparatus and followed by a 2% to 4% BSA gradient in RPMI medium 1640 (Gibco, Grand Island, NY). After 3 h of velocity sedimentation at unit gravity, the cell fractions (10 ml/fraction) were collected from the bottom of the separator apparatus at a rate of 5 ml/min. The cell type and purity in each fraction were assessed using light microscopy based on their diameters and morphological characteristics. Only fractions with expected cell type and high purity (>85%) were pooled together. The average purity for each cell type was as follows: primitive type A spermatogonia, 94%; type B spermatogonia, 90%; preleptotene spermatocytes, 88%; pachytene spermatocytes, 95%; round spermatids, 96%; and elongating spermatids, 92%.
cDNA Array Hybridization Procedures
RNA extraction Total RNA was extracted using Trizol Reagent (Gibco) according to the manufacturer's protocol, followed by treatment with RNase-free DNase (Promega) at 37°C for 20 min to avoid contamination of genomic DNA. The RNA quality and concentration were assessed using agarose gel electrophoresis and spectrophotometric reading.
Probe preparation
Five micrograms of total RNA were reverse transcribed using reagents provided in the Atlas cDNA Expression Array Kit (Clontech, Palo Alto, CA) and radiolabeled with [
32P]dATP (10 µCi/µl; Amersham Pharmacia Biotech, Beijing, China). The labeled cDNAs were purified from unincorporated 32P-labeled nucleotides by Chroma Spin-200 columns (Clontech), and the radioactivity of the probes was counted using a scintillation counter.
Hybridization The array membranes (Atlas Mouse 1.2 cDNA Expression Arrays; Clontech) that contained 1176 genes were prehybridized for 30 min at 68°C in ExpressHyb hybridization solution containing 100 µg/ml of salmon testis DNA. The denatured 32P-labeled cDNA was added to ExpressHyb hybridization solution (Clontech) at a final concentration of 1 x 106 cpm/ml, and the array membranes were hybridized with the labeled cDNA overnight at 68°C. The next day, the membranes were washed three times for 30 min with prewarmed (68°C) washing solution 1 (2x SSC [1x SSC: 0.15 M sodium chloride and 0.015 M sodium citrate] and 1% SDS) and once for 30 min with prewarmed (68°C) washing solution 2 (0.1x SSC and 0.5% SDS) with continuous agitation at 68°C. After a final wash with 2x SSC at room temperature, the membranes were immediately wrapped with plastic film to keeping them from drying and exposed to x-ray film (XK-1; Kodak, Shantou, China) for 12 h at -80°C.
Analysis of Gene Expression
The films were scanned, and the images were quantitated and analyzed with AtlasImage 2.01 software (Clontech). Expression data from replicate membranes were averaged and normalized. Adjusted intensity equals the intensity of each gene minus the background value. The genes with an adjusted intensity less than 2-fold the background value were not detected, nor were those that had opposite changes in replicate pairs. The signal ratio of each given gene between two compared array membranes was calculated. In the present study, the ratio threshold was set at 2.0. Only those genes that showed an increase or decrease of 2.0-fold or greater were considered to be differentially expressed.
RT-PCR Analysis
Expression of some prominently changed genes in type B spermatogonia and spermatids was further examined by RT-PCR to confirm the array results. First-strand cDNA was synthesized from total RNA using Moloney murine leukemia virus reverse transcriptase at 42°C for 1 h. The PCRs were performed with first-strand cDNA under conditions of 30 sec at 94°C, 30 sec at 55°C (varied with different genes), and 1 min at 72°C each cycle. Thirty-two cycles (25 cycles for glyceraldehyde phosphate dehydrogenase [GAPDH]) were done and followed by extension for 10 min at 72°C. The primers for PCR are listed in Table 1.
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The RT-PCR products were subjected to electrophoresis in 1% agarose gels. To ensure that equal amounts of reverse-transcribed cDNA were applied to the PCR reaction, GAPDH (a housekeeping gene) was also included in the PCR.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To characterize genes that may be associated with the initial differentiation decisions and mechanisms directing mammalian spermatogenesis, we examined the gene expression profiles of different stages of spermatogenic cells using the Atlas Mouse 1.2 Array. The Atlas Mouse 1.2 Array includes 1176 cDNAs, nine housekeeping control cDNAs, and negative controls on a single nylon membrane. The cDNAs immobilized on the membrane have been specially prepared to minimize the problem of nonspecific hybridization. Each cDNA fragment is 200 to 600 base pairs and amplified from a region of the mRNA that lacks the poly-A tail, repetitive elements, or other highly homologous sequences. Gene location and information are available from Clontech (http://atlasinfo.clontech.com/).
The images of the Atlas array membranes hybridized to cDNAs from different types of spermatogenic cells are shown in Figure 1. Of the 1176 genes on the array, 260 were detected during spermatogenesis in at least one of the six cell types. Their cellular distribution was as follows: 181 genes expressed in primitive type A spermatogonia, 256 in type B spermatogonia, 221 in preleptotene primary spermatocytes, 160 in pachytene primary spermatocytes, 141 in round spermatids, and 126 in elongating spermatids. Of the total number of genes detected during spermatogenesis, 46% (120 genes) were detected in all six cell types. Interestingly, some stage-specific genes were observed. For example, nine genes expressed uniquely in type B spermatogonia, including connexin 26 (CXN26), cyclin D1, semaphorin B, neuroendocrine convertase 2 (NEC 2), interleukin 5-receptor subunit (IL-5R), fyn proto-oncogene, corticotropin-releasing hormone receptor 2, cathepsin C, and meiotic recombination 11 homologue A. Four genes expressed only in round spermatids, including low-affinity immunoglobulin G Fc receptor II beta (FCGR2B), heat shock 86-kDa protein 1 (HSP86-1), defender against cell death 1 protein (DAD1), and mitogen-activated protein kinase 14 (MAPK14).
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Identification of Differentially Expressed Genes
To identify genes that differentially expressed during spermatogenesis, we compared the gene expression profiles of 1176 genes among different stages of spermatogenic cells during spermatogenesis. Differentially expressed genes between one stage and the neighboring stage of spermatogenic cells are listed in the tables.
Of the total 260 genes detected during spermatogenesis, 64 were differentially expressed from primitive type A spermatogonia to type B spermatogonia. The expression of 2 genes was down-regulated and of 62 genes up-regulated (Table 2), including 17 exclusively expressed in type B cells compared to primitive type A spermatogonia. Among up-regulated genes, several were observed to have a dramatic increase in type B spermatogonia as compared to primitive type A spermatogonia, such as cyclin E1 (increased by 10-fold), cyclin F (increased by 10-fold), nucleoside diphosphate kinase B (NDK B; increased by 9-fold), and vascular endothelial growth factor precursor (VEGF; increased by 7-fold). In contrast, two genes decreased from primitive type A spermatogonia to type B spermatogonia: glutathione S-transferase 5 (GST-5; decreased by 63%) and follistatin precursor (FST; 50%). The results also showed that 20 genes expressed strongly in both primitive type A spermatogonia and type B spermatogonia, then dramatically decreased to near-background level in the following stages (Table 3).
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From type B spermatogonia to preleptotene primary spermatocytes, 38 genes were up-regulated (Table 4) and 51 down-regulated (Table 5). The majority of the genes remained unchanged in their expression intensities.
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A little change of gene expression occurred from preleptotene spermatocytes to pachytene spermatocytes. Most of the differentially expressed genes in this differentiation step were down-regulated (35 genes). Only three genes were up-regulated: sine oculis-related homeobox protein 2 homologue (SIX2), single-mined 2 transcription factor (SIM2), and oncostatin M (OSM).
As compared to pachytene spermatocytes, the expression of most genes was down-regulated in both round and elongating spermatids (Table 6), but expression of a few genes was up-regulated in these two types of spermatids. Among the expressed genes, 67 down-regulated and 7 up-regulated genes were detected from pachytene spermatocytes to round spermatids. Of them, the expression of 20 genes decreased more than 10-fold. The seven up-regulated genes in round spermatids were FCGR2B, LIM domain-binding 2 (LIM2), mothers against decapentaplegic homologue 1 (MADH1), HSP86-1, DAD1, glial cell line-derived neurotropic factor (GDNF), and MAPK14. Three up-regulated genes were detected in elongating spermatids: suppressor of cytokines signaling protein 1 (SOCS-1), CEK7 ligand (CEK7-L)/ephrin A2 (EFNA2), and ERA-1 protein (ERA-1). These differentially expressed genes can be classified into several categories according to their general functions: basic transcription factors, cyclins, cytoskeleton proteins, and growth factors and their receptors.
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RT-PCR Analysis
Although differences in gene expression during spermatogenesis were observed with the cDNA array, it was essential to verify these results using other methods. We monitored the mRNA level of selected genes noted to be differentially expressed in the Atlas arrays by RT-PCR. We selected seven genes up-regulated prominently from primitive type A spermatogonia to type B spermatogonia, seven genes up-regulated in round and elongating spermatids, and one gene that gradually decreased during spermatogenesis to reach an undetectable level in elongating spermatids to perform RT-PCR analysis. Agarose electrophoresis graphs of the RT-PCR results are shown in Figures 2 through 4. The E2F transcription factor 3 (E2F-3), VEGF, Cek 5 receptor protein tyrosine kinase ligand (CEK5-R), factor associated with N-smase activation (FAN), potassium voltage-gated channel (PVGC), G1/S-specific cyclin E1 (CCNE1) and signal transducing adapter molecule (STAM) were expressed at low or undetectable levels in primitive type A spermatogonia and up-regulated strongly in type B spermatogonia (Fig. 2). Expression of FCGR2B, LIM 2, MADH 1, SOCS-1, CEK7-L, and ERA-1 was up-regulated from pachytene spermatocytes to round and elongating spermatids (Fig.3). The gene CEK5-R was highly expressed in the early stage of spermatogenesis (type B spermatogonia), then gradually down-regulated in the middle stage (preleptotene spermatocytes, pachytene spermatocytes, and round spermatids), and was undetectable in the late stage (elongating spermatids) (Fig. 4). These results are identical to the data from the cDNA array study. However, the gene MAPK14 did not show a remarkable change in expression from pachytene spermatocytes to round and elongating spermatids (Fig. 3), which was not in accordance with the cDNA array results, in which the expression was up-regulated.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Gene Expression in the Early Stages of Spermatogenesis
Most reports regarding gene expression during spermatogenesis have concentrated on the late stages. Gene expression patterns in spermatogonia or early spermatocytes have seldom been reported. The stage of preleptotene primary spermatocytes is often considered to be the point of entry into meiosis, but it should be noted that type B spermatogonia have already embarked on a one-way pathway leading to the meiotic prophase. That is to say, the type B stage is an important step as preparation for meiosis. The present results show that transcriptional activity of many genes in primitive type A spermatogonia is low, whereas nearly 30% (62 genes) of the detected genes are up-regulated in expression from primitive type A to type B spermatogonia, including cyclins, growth factors, transcription factors, and oncogenes. From them, seven genes were selected to be further confirmed by RT-PCR, and the PCR results of these seven genes were completely in accord with the microarray results. Thirty-eight genes had expressions that increased from type B spermatogonia to preleptotene primary spermatocytes. We also found 20 genes that only expressed in both primitive type A and type B spermatogonia and then dramatically decreased to near-background level after entering the meiosis phase. We hypothesize that, perhaps, they do not function during the late stage of spermatogenesis or that other germ cell-specific genes are expressed to compensate for the absence of the silenced or inactivated genes. The present results indicate that most detected genes (256 of 260) express in type B spermatogonia and that a number of detected genes have the highest expression level in type B spermatogonia and preleptotene primary spermatocytes, supporting our speculation that a series of specific events during spermatogenesis, such as meiosis and condensation of the nucleus, are determined at the early stage of spermatogenesis. The initiation or up-regulation of specific gene expression in spermatogonia and early primary spermatocytes is responsible for the further differentiation and meiosis of these cells.
The expression of cyclins during spermatogenesis is interesting. The G2/mitotic-specific cyclin A1 and G1-specific cyclin C are both expressed consistently at a high level in all germ cells, whereas the S/G2/M-specific cyclin F, G1/S-specific cyclin D1, and G1/S-specific cyclin E1 are only expressed in spermatogonia, and up-regulated markedly from primitive type A to type B spermatogonia. Equally, as a member of the G1/S-specific cyclin family, cyclin D3 is undetectable in spermatogonia but is expressed strongly in spermatocytes and spermatids. This differential expression during spermatogenesis may be indicative of a different susceptibility of germ cells to such agents. More interestingly, gene E2F, which is related to the function of cyclin D1 and cyclin E1 [19], playing regulatory roles in the G1/S-phase transition of the cell cycle, is also up-regulated from primitive type A to type B spermatogonia, similar to the up-regulation of cyclins D1 and E1. This may reflect the increase in the proliferative capacity of type B spermatogonia. Thus, it is possible that in spermatogonia, E2F is a transcriptional activator of both cyclin E1 and cyclin D1, which in turn cause growth and proliferation of type B spermatogonia. In a similar vein, cyclin D1 has been shown to activate the gene HSP70 [20], which is a spermatogenic cell-specific stress gene. The gene HSP70 is not found on the Atlas arrays used in the present study. However, another member of the same gene family, HSP105, which is contained on the arrays and has the same regulatory motif with HSP70, was observed to up-regulate in type B spermatogonia.
A number of growth factors and receptors have been shown to play essential roles in spermatogenesis. The present study showed that the expression of transforming growth factor ß1 (TGFß1) was undetectable in primitive type A spermatogonia and weakly expressed in type B spermatogonia, whereas no expression signal of TGFß2 was observed in both primitive type A spermatogonia and type B spermatogonia. This corresponds to previous reports that TGFß2 is expressed only in the late spermatogenetic phase. Another previous study [21] reported that targeted inactivation of genes for several TGFß superfamily members expressed in the testis did not cause primary defects in spermatogenesis, suggesting that they did not have a key role in this process. Whether the TGF family plays a role in spermatogenesis remains to be clarified. In the insulin-like growth factor (IGF) family, we found that IGF-1 expressed at a low level in primitive type A and type B spermatogonia, whereas IGF-2 had a strong expression signal in both. In contrast, IGF-1 receptor (IGFR-I) expressed strongly in the two cell types, whereas IGF-2 receptor (IGFR-II) was undetectable. Previous studies showed that the loss of IGFR-II results in overaccumulation of circulating levels of IGF-2 [22]. One of the receptors for IGF-2, IGFR-II, exerts its function of limiting the biological effects of IGF-2. This suggests that the inactivation of IGFR-II is in coincidence with the strong expression of IGF-2 in the present study. We infer that IGF-2 may play a more important role than IGF-1 during spermatogenesis. Moreover, some genes for adapters and death receptor-associated proteins, such as STAM and FAN, were strongly up-regulated in type B spermatogonia. This finding supports those of previous reports that programmed cell death or apoptosis plays an important role in establishing the adult population of spermatogonia of proper composition and size [23]. Among genes related to DNA replication, DNA polymerase 
and DNA polymerase were expressed in type B spermatogonia. These two kinds of DNA polymerases participate in synthesis of DNA during the early phase of meiosis and play roles in DNA replication of spermatogonia as well [24].
When spermatogenic cells differentiate into the preleptotene spermatocyte stage, the process of meiosis begins. The activities of most genes are maintained unchangeable from preleptotene to pachytene spermatocytes, whereas three genes are up-regulated, including SIX2, SIM2, and OSM. Both SIX2 and SIM2 have been found to behave as transcriptional repressors in mammals [25, 26]. Up-regulation of the transcriptional repression genes in pachytene spermatocytes may be partly responsible for the gene transcription inactivation in spermatids. Other studies have reported that OSM is expressed in the rat testis and likely plays a role at the start of rat spermatogenesis [27].
Gene Expression in the Late Stages of Spermatogenesis
In the present study, we found that the number of expressed genes and their expression level remarkably decreased from pachytene spermatocytes to round spermatids and elongating spermatids. These results are in agreement with the previous concept that histone-to-protamine transition and the ensuing chromatin compaction in condensing spermatids render sperm gene transcription virtually inactive. For example, we found that gene CEK5-R had the highest expression level in type B spermatogonia and was down-regulated from spermatocytes to spermatids. This was also confirmed by RT-PCR analysis. It is interesting that seven genes were up-regulated as pachytene spermatocytes differentiated to round spermatids and that three genes were up-regulated during the transition from round spermatids to elongating spermatids. These genes may be involved in the induction of spermiogenesis. The up-regulated genes in round spermatids are FCGR2B, LIM2, MADH1, HSP86-1, DAD1, GDNF, and MAPK14.
The role of the heat shock protein (HSP) gene in spermatogenesis has been studied. It has been reported [28] that HSP86 is mainly expressed in spermatogenic cells and is up-regulated along with germ cell differentiation during development of the testis. The present results correspond to those of this previous report, suggesting that the method used in the present study is reliable. Furthermore, our results indicate that the HSP86 gene has the highest expression level in round spermatids. A recent report [29] demonstrated that GDNF expression could be detected in Leydig and Sertoli cells. However, the high-level expression of GDNF was only detected in round spermatids and some types of spermatocytes, suggesting that GDNF may play essential roles in spermatogenesis and testis maturation. We also found three up-regulated genes in elongating spermatids. To confirm the results of cDNA microarrays, seven genes, which were up-regulated in round and elongating spermatids in the present study and were not reported as expressing in spermatogenesis previously, were selected and further analyzed by RT-PCR. The results show that the expression patterns of six genes selected are consistent with those from cDNA arrays. This suggests that the cDNA array results are stable and reliable. Nevertheless, it is necessary to confirm these results using other methods. It is important that most of the differentially expressed genes that we found have not yet been reported. These results could be helpful for finding more spermatogenesis-related, especially spermiogenesis-related, genes. Further studies on the functions of these differentially expressed genes may provide insight regarding the molecular mechanisms of spermatogenesis.
Conclusions
We have demonstrated the utility of the cDNA microarray in analyzing gene expression changes at different stages of spermatogenic cells during spermatogenesis. For a number of genes that have been shown previously to be developmentally related in spermatogenic cells, the data obtained by this approach are comparable to those obtained by other methods. Importantly, additional genes with specific expression in spermatogenic cells as identified by Atlas arrays may be differentially regulated in male germ cells. Furthermore, genes and gene networks identified as being significant by microarrays provide important leads for pursuing a more complete understanding of spermatogenetic mechanisms.
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FOOTNOTES |
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2 Correspondence: Daishu Han, Department of Cell Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences & Peking Union Medical College, 5 Dong Dan San Tiao, Beijing 100005, China. FAX: 86 10 65296466; daishu{at}public.bta.net.cn
Received: 1 November 2002.
First decision: 3 December 2002.
Accepted: 3 February 2003.
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