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,
From the * Section on Developmental Genomics,
Laboratory of Clinical Genomics, National Institute of Child Health and Human
Development, National Institutes of Health, Bethesda, Maryland; the
Departments of Cell Biology and
Pediatrics, Georgetown University, Washington,
DC; and the Department of Pathology, Loyola
University Medical Center, Maywood, Illinois.
| Correspondence to: Dr Wai-Yee Chan, Section on Developmental Genomics, Laboratory of Clinical Genomics, National Institute of Child Health and Human Development, National Institutes of Health, 49 Convent Dr, Room 2A08, MSC 4429, Bethesda, MD 20892-4429 (e-mail: chanwy{at}mail.nih.gov). |
| Received for publication April 28, 2003; accepted for publication July 11, 2003. |
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Key words: Type A spermatogonia, pachytene spermatocytes, round spermatids, spermatogenesis, complementary DNA microarray, quantitative polymerase chain reaction
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Materials and Methods |
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Hybridization of cDNA Microarray and Data Analysis
Radiolabeled cDNA probes were prepared from each type of germ cell by
reverse-transcribing 1 µg of total RNA in the presence of
oligo-deoxythymidine primers and 10 µL of
[
-33P]deoxycytidine triphosphate (10 mCi/mL, 3000 Ci/mmol,
Amersham Pharmacia, Piscataway, NJ). Mouse GeneFilters microarrays (GF400,
Release I) containing 5184 mouse sequence-verified cDNA elements, each of them
comprising of a 
1-kb fragment from the 3' end of the corresponding
gene, were purchased from Research Genetics (Huntsville, Ala). Microarray
hybridizations were performed according to manufacturer's instruction. Two
microarrays were hybridized with probes generated from 2 separate preparations
of germ cells of each stage. Two extra microarrays were hybridized in the same
way with probes from a reference cell line C418. After washing, the hybridized
microarrays were exposed to a phosphor screen for 5 hours and scanned for
signals with a Storm 840 scanner (Molecular Dynamics, Piscataway, NJ) at a
resolution of 50 µm. Images were analyzed by IPLab/ArraySuite v2.0
(NGHRI/NIH) as described previously (Su et
al, 2000). In the preliminary selection, only genes giving signal
intensities less than 2-fold variation between duplicate experiments were
considered to be expressed in that particular cell type. The expression level
of selected genes was compared to that of C418 cells to obtain a reference
ratio to eliminate experimental variation. The genes were required to exhibit
comparable changes in signal ratio in the duplicate experiments. Genes showing
2-fold or greater difference in signal ratio in any 2 stages were considered
to be differentially expressing. Gene identities and GenBank accession
identifications were extracted from the mouse GeneFilters database (version
gf400a; available at
ftp://ftp.resgen.com/pub/genefilters/gf400a_final_data_070300.txt).
Unigene assignment of selected genes was finalized based on Mouse Unigene
cluster Build #118 (December 4, 2002) from NCBI. Biological functions of gene
products were queried against LocusLink of NCBI
(http://www.ncbi.nlm.nih.gov/LocusLink),
Mouse Genome Informatics of the Jackson Laboratory
(http://www.informatics.jax.org),
and GeneCards of the Weizmann Institute of Science
(http://bioinfo.weizmann.ac.il/).
Quantitative Polymerase Chain Reaction
Equal amounts of total RNA from different stages of germ cells were reverse
transcribed to prepare the first-strand cDNA samples for QPCR analyses.
Gene-specific primers (Table 1)
were designed by Primer Express Version 2.0 (Applied Biosystems, Foster City,
Calif) according to the sequence information provided for the cDNAs on the
microarray. QPCR was carried out with the 7900 HTS Sequence Detection System
(Applied Biosystems) and SYBR Green I chemistry according to manufacturer's
instruction. To compare the expression level of each gene among the different
cell types, a standard curve was first generated by plotting the threshold
cycle (CT) values of a series of fixed amount of RdSd cDNAs
(arbitrarily assigned 0.1x, 1x, and 10x) against these
amounts of cDNAs. The CT values for a gene in SgA or PcSc were
fitted onto the standard curve to obtain the respective expression levels. A
smaller CT value indicates a higher expression level, and vice
versa. Genes showing CT values of 40 or higher were considered to
be nonexpressing. The abundance of 18S rRNA in each cell type also was
monitored and the gene expression levels were normalized to that of 18S rRNA.
Genes showing 2-fold or greater difference in expression level in any 2 stages
were considered to be differentially expressing.
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Results and Discussion |
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TopAbstractMaterials and MethodsResults and DiscussionReferences |
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Based on the microarray signals, 79 differentially expressing genes (including known genes, uncharacterized transcripts, and ESTs) were identified, which exhibited 11 expression patterns as a function of the 3 stages of spermatogenesis (Table 5). The expression patterns of all 79 genes were verified by QPCR. When verifying gene expressions in PcSc and RdSd, only 37 genes (47%) were found to demonstrate concordant changes in expression between the microarray and QPCR experiments. Among them, 20 genes showed a 2-fold or greater difference in expression level between PcSc and RdSd. The expression levels of these genes, plus several known genes showing concordant change in expression but with less than a 2-fold difference in QPCR, in SgA, were examined. According to our selection scheme, a total of 23 genes were confirmed to be differentially expressed in the 3 stages of germ cells, with one half of them (12) representing known genes and the remaining 11 genes being ESTs or uncharacterized transcripts (Table 6). The high percentage of ESTs and uncharacterized transcripts identified corroborates the fact that little is known about gene expression in germ cells.
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We speculate the transition of cells throughout spermatogenesis to be the result of a programmed change in gene expression at different stages of differentiation. Up-regulation of a gene at a particular stage would suggest requirement at that period of the gene expression, as well as the inverse corollary. A comparison of the expression patterns at different stages should provide insight into the potential roles these genes would play during spermatogenesis. The expression of the 23 genes could be clustered into 5 changing patterns (group I through V; Figure). Group I genes (n = 7) displayed very low or no expression in SgA, a gradual increase in PcSc, and a maximal increase in RdSd. These genes may be specific for meiotic or postmeiotic functions or cellular activities in a more differentiated state. Four of the genes in this group are known genes, and the remaining 3 are ESTs and a gene encoding a hypothetical protein (Table 6). In the mouse, glucokinase (glycerol kinase) activity-related sequence 1 (Gk-rs1) is an autosomal intronless retrotransposed element from the X-linked glycerol kinase and expressed only in the testis (Pan et al, 1999). Nothing is known about its biological function because it lacks glycerol kinase activity. From our data, the total absence in SgA suggests that Gk-rs1 is involved in activities of postmitotic germ cells. Within this group there is a novel gene Smaf1 that has uncharacterized biological function.
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Two immunocyte-specific genes, namely complement receptor 2
(Cr2/CD21) and T-cell receptor CD3 eta precursor
(CD3
), also were identified. Cr2 is a receptor for C3d,g
complement fragment-tagged immune complexes. The receptor is expressed mainly
on follicular dendritic and B cell surfaces and was found to enhance B-cell
activation and differentiation by lowering the signal threshold for activation
(Prechl and Erdei, 2000).
CD3 is one of the noncovalently associated subunits of the
T-cell receptor (TCR) complexes. It has been shown to participate in the
assembly and cell surface expression of TCR complexes and transduction of
signal from TCR that leads to intrathymic T-cell differentiation
(Bauer et al, 1991;
Malissen et al, 1993). Because
cross-contamination by B or T cells is very unlikely in our germ cell
isolation procedure, the expression of Cr2 and CD3 in
male germ cells strongly suggests their involvement in spermatogenesis and the
immunocyte-specific expression is the result of exclusion of testis tissues in
previous studies. As both gene products were expressed on cell surface and
involved in signaling processes, we speculate a similar mode of action for Cr2
and CD3 in germ cells by regulating or transducing signals for cellular
differentiation from the extracellular environment.
Group II genes (n = 8) expressed at a lower level in SgA; after reaching a maximum in PcSc, the expression level declined in RdSd. Such expression pattern implies that the gene activities are more important to meiotic germ cells, as the expression level dropped beyond this stage, possibly involved in meiosis or the maintenance of the tetraploid state. Three known genes are in this group.
Mitochondrial elongation factor G (Gfm) catalyzes the A-to-P site translocation of peptidyl-tRNA after peptide bond formation in protein biosynthesis (Gao et al, 2001). Its preferential expression in PcSc suggests a more demanding need of the germ cell for protein synthesis at this stage, presumably to support cellular events at the tetraploid state or the 2 rounds of meiosis.
Regulator of G-protein signaling 2 (Rgs2) belongs to a family of
proteins that regulate G-protein signaling by accelerating hydrolysis of
guanosine triphosphate bound to activated G subunits, thus limiting the
duration of signaling (Kehrl and
Sinnarajah, 2002). Rgs2 also was involved in cellular
differentiation (Imagawa et al,
1999). Specifically it was up-regulated during early stages of
differentiation but down-regulated thereafter. We did not observe a concordant
expression pattern of Rgs2 in germ cells; this may be attributed to
the difference in cellular contexts. The increased expression of Rgs2
in PcSc suggests the occurrence of more active transmembrane signaling events
during this stage.
The large conductance calcium-activated potassium channel (BK or MaxiK) is
a member of the Shaker-related 6 transmembrane domain potassium channel family
that is activated by voltage and calcium. BK channel is composed of a
pore-forming subunit and a modulatory transmembrane ß subunit.
The tissue specificity of ß subunits confers different physiological
properties to the channels, for example, ß4 subunit (encoded by
KCNMB4), which is highly expressed in brain and testis, could enhance
the opening of BK channel at high [Ca2+]
(Brenner et al, 2000). Despite
the subunit gene being absent on the microarray, the detection of
Kcnmb4 in germ cells indicates the presence of functional and
ß4 BK channels. In fact, both - and ß4-subunits were found to
be active in human testes (Behrens et al,
2000; Brenner et al,
2000). The augmented expression of Kcnmb4 in PcSc
suggests more active modulation of the BK channel during this stage. In
neurons, BK channels were associated with calcium channels
(Marrion and Tavalin, 1998). We postulate in male germ cells, the Ca2+ influx resulting from
signaling events would activate BK channels to open to allow entry of
K+ that triggers downstream biochemical responses. Interestingly,
heterologously expressed and ß4 BK channels could be activated by
17ß-estradiol (Behrens et al,
2000). This finding suggests that sex steroids may act on
and ß4 BK channels in germ cells. It would be tempting to investigate the
interplay between sex steroids and and ß4 BK channels in
modulating germ cell physiology in vivo.
The expression level of group III genes (n = 2) was maintained in SgA and
PcSc but dropped in RdSd, suggesting the encoded functions were less essential
to the postmeiotic germ cells. Only 1 known gene is present in this group.
Trim10 (also known as Herf1) was reported to be involved in
erythroid differentiation (Harada et al,
1999). The higher expression of Trim10 in SgA and PcSc
suggests its involvement in differentiation in the earlier stages of
spermatogenesis. Similar to Cr2 and CD3,
Trim10 was previously not known to be active in testis.
Group IV genes (n = 5) had the highest expression level in SgA, the lowest in PcSc, and more elevated in RdSd, but less than in SgA. These genes are likely to mediate functions specific to spermatogonia. The up-regulation in RdSd may reflect the requirement of the gene products at this stage (eg, for subsequent sperm maturation) or result from relaxation of restraints imposed by X-chromosome inactivation process (McCarrey et al, 2002). Four known genes and 1 uncharacterized transcript are in this group.
Cartilage-associated protein (Crtap) was first identified to exhibit a developmentally regulated expression pattern in chick chondrocytes (Castagnola et al, 1997). Crtap was implied to function in the differentiation process, because its expression was up-regulated when chondrocytes differentiated. Examination of our data demonstrated a similar expression pattern of Crtap, in which the more differentiated RdSd showed a higher expression than PcSc. However, the less differentiated SgA showed the highest expression level. This observation suggests an alternate function for Crtap in SgA or Crtap may play a totally different role in germ cells than in chick chondrocytes. Because little is known about Crtap, the significance of this gene in germ cell development will be revealed by direct manipulation of its expression.
Prolyl oligopeptidase (Pop) is a widely distributed serine endopeptidase catalyzing the hydrolysis of the carboxyl side of proline residues in peptides. Pop was found to involve in peptide hormone maturation and degradation, cellular differentiation (Kimura et al, 1999), and recently meiosis of spermatocytes and differentiation of spermatids in mice (Kimura et al, 2002). In the latter study, Pop showed the highest expression level in 2-week-old mouse testes that contained differentiated cell species from SgA to PcSc; the expression decreased successively until 8 weeks of age when all stages of spermatogenesis were present. In situ hybridization revealed Pop mRNA in all germ cells in 2-week-old testes, but only in RdSd in 8-week-old testes. Because the PcSc and RdSd used in our study were isolated from animals at a similar age (60 days) as in study of Kimura et al (2002) (8 weeks), we expected a similar result. Indeed, we observed a higher expression of Pop in RdSd. In contrast, we did not see a total absence of Pop expression in PcSc. We also found that SgA displayed the highest level of Pop expression, which was not addressed by Kimura et al (2002). Thus, rather than being involved in meiosis, we believe Pop plays a more important role in SgA mitotic functions.
Among the genes in this group are two X-linked genes, namely B-cell receptor-associated protein 31 (Bap31) and centrin 2 (Cetn2). Bap31 associates with membrane immunoglobulin D on mature B cells and is highly enriched in endoplasmic reticulum (Ng et al, 1997). It also regulates apoptosis by processing procaspase-8L to modulate caspase activation (Breckenridge et al, 2002). In fact, Bap31 is a preferred substrate of caspase-8, and its cleavage product p20 contributes directly to apoptosis progression (Nguyen et al, 2000). Cetn2 is a centriole protein within the centrosome. In addition to its involvement in cytokinesis and cell cycle progression, Cetn2 is essential to centriole duplication and the proper progression of mitosis; its loss leads to aberrant mitosis and cell death (Salisbury et al, 2002). Both genes were preferentially expressed in SgA, suggestive of a role for Bap31 and Cetn2 in regulating spermatogonial apoptosis and proper maintenance of mitosis, respectively. As germ cells differentiate, the expression of Bap31 and Cetn2 dropped dramatically in PcSc, a phenomenon attributable to X-chromosome inactivation. This functionally elusive inactivation process takes place in PcSc, which results in a transient repression of gene transcription from the single X chromosome in male germ cells (McCarrey et al, 2002). The down-regulation of Cetn2 coincides with an up-regulation of its autosomal retrotransposon Cetn1 that may compensate for its repression and allow completion of meiosis (Hart et al, 1999). This mechanism implies an indispensable role of the Cetn family in spermatogenesis. To date no retrotransposon of Bap31 has been identified. The repression likely reflects a cessation of the need for apoptosis in PcSc. For some X-linked genes the inactivation process persists beyond the pachytene stage, but other X-linked genes would be reactivated in post-meiotic RdSd, for example, Ube1x (Odorisio et al, 1996) and Pgk-1 (Kumari et al, 1996), or activated for the first time at this stage, for example, Mage (McCarrey et al, 2002). From our data, both Bap31 and Cetn2 became reactivated in postmeiotic RdSd, indicating that their functions were required in this stage.
Only 1 EST was classified into group V. The limited sequence information made us unable to speculate on the function of the encoded protein. This EST reached the highest expression level in SgA but it dropped drastically in PcSc and RdSd, implying that the translated product is important to cellular activities in spermatogonia, possibly related to mitosis, and is less important in the following stages. The full characterization of the ESTs and uncharacterized transcripts identified in the 5 groups should permit speculation about their roles in spermatogenesis.
A search of the GenBank cDNA database identified 7 of the 23 genes not
known to be expressed in the testis (Table
6). In addition to the discovery of novel testicular transcripts,
the identification of 3 known genes (Cr2, CD3, and
Trim10) suggests a more general biological function of them in
diverse cellular contexts than previously postulated. Of the known genes
identified, a high proportion are implicated to be functional in the process
of signaling (4 of 12) and cellular differentiation (3 of 12;
Table 6), suggesting these as
the major biological activities in spermatogenesis. Nevertheless, the small
number of genes identified limits the generality of this speculation. A more
detailed study is required to elucidate the biochemistry of male germ
cells.
Extensive analysis of gene expression in germ cells is limited by the small number of germ cells at earlier stages of development and the availability of efficient germ cell isolation method. Nevertheless, recent advances in molecular biotechnology have led to multiple studies of testicular gene expression on a larger scale, for example, identification of novel genes at particular stages of spermatogenesis by differential display (Anway et al, 2003), cDNA subtraction (Wang et al, 2001; Fujii et al, 2002), and microarray analysis of testes in mutant animals enriched for specific types of germ cells (Tanaka et al, 2002). Gene profiling experiments were reported on the spermatozoal mRNA profiles of healthy fertile men (Ostermeier et al, 2002) and the pattern of testicular gene expression in neonatal and mature animals (Sha et al, 2002). However, the conclusions that may be drawn from these experiments are limited to single stages of germ cells only. We adopted the use of 3 different developmental stages of germ cells isolated from mice to examine the changes in gene expression patterns during spermatogenesis. Although one may challenge the potential risk of altering the properties, and consequently the gene expression, of germ cells upon isolation because the cells are not maintained in their natural microenvironment, the use of whole testes from animals of different ages would yield results that may be difficult to interpret because the identified transcripts can be contributed by a single or multiple cell types. Also, one cannot eliminate the interference from other testicular somatic cells whose gene expression patterns can be changing over time.
Just before submission of this manuscript, a report on gene expression profiling of various stages of mouse germ cells with a 1176 cDNA microarray was published (Yu et al, 2003). In that report, radioactive signals of the genes from one stage were compared with those of the neighboring stage to identify differentially expressed genes. From our experience and that of others (Piétu et al, 1996; Eickhoff et al, 1999), radioactive signals generated from membrane-based microarrays are not totally reliable because of various experimental variances. This is illustrated by the low level of concordance between the membrane hybridization and QPCR results in this report. Conclusions based solely on radioactive signals are highly prone to errors. In contrast, we used microarray analysis as a tool to screen for differentially expressed genes. The leads identified were confirmed by QPCR. Only genes showing consistent changes in both experiments were considered to be truly differentially expressed during spermatogenesis. To our knowledge, our report is the most comprehensive comparison of the changes in gene expression patterns during spermatogenesis. In our study, the transcriptomes of germ cells were partially characterized because of the incomplete representation of genes on the microarray. A more detailed analysis by more powerful tools such as SAGE is required to identify the molecular signature of male germ cells. We reported the differential expression patterns of a set of genes and transcripts at different stages of spermatogenesis. The specific gene/transcript expression patterns strongly suggest specialized functions for the encoded products during male germ cell development, and identify targets for manipulation to unravel the molecular mechanism of spermatogenesis.
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