| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
From the Laboratory of Reproductive Medicine, Nanjing Medical University, Nanjing, People's Republic of China.
| Correspondence to: Dr Jiahao Sha, Laboratory of Reproductive Medicine, Nanjing Medical University, Nanjing, 210029, People's Republic of China (e-mail: shajh{at}njmu.edu.cn). |
| Received for publication July 7, 2004; accepted for publication September 30, 2004. |
 |
Abstract |
|---|
 Top Abstract Materials and MethodsResultsDiscussionReferences |
|---|
Key words: Alternative splicing, spermatogenesis, regulatory mechanism, protein diversity, spliceoforms
The testis is a rich source for identifying gene regulation mechanisms because germ cell expansion and differentiation requires many cellular changes and regulatory steps. In developing germ cells, the lengths of mRNA transcripts often vary as the cells mature, reflecting on-going regulatory changes (Eddy, 2002); alternative splicing is thought to be an ideal mechanism for controlling gene expression at transcription level in these tissues (Walker et al, 1999). Recently, a genome-wide analysis of expressed sequence tags (ESTs) showed that the testis has the greatest enrichment of tissue-specific splicing (Xu et al, 2002), which could be involved in various functions, including spermatogenesis and steroidogenesis (Carreau et al, 2001; Hammes et al, 2001; Venables, 2002). Although recent studies have collected alternative splicing information from annotated resources such as the alternative splicing annotation project (ASAP; http://www.bioinformatics.ucla.edu/ASAP/) and EST databases (Gelfand et al, 1999; Hu et al, 2001; Lee et al, 2003), there is a paucity of large-scale information on development-related alternatively spliced mRNAs in the human testis.
Here, we used a human testis complementary DNA (cDNA) microarray hybridized with 33P-labeled human adult testis, fetal testis, and adult spermatozoa cDNA probes to identify novel testicular development-related alternatively spliced mRNAs. Sixty-seven Unigene clusters showed a total of 74 novel alternative spliceoforms, allowing us to investigate the relationship between alternative splicing and developmental state. Together, our results illustrate the significance of alternative splicing in human testis development and spermatogenesis.
|
Materials and Methods |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Screening of Genes Related to Testis Development
In previous studies, a human testis cDNA microarray was constructed in our
laboratory (Sha et al, 2002;
Wang et al, 2004). For
identification of full-length cDNAs, we employed the Human Testis Large Insert
Phage cDNA Library (HL5503U; Clontech, Mississauga, Ontario, Canada);
this library has an average insert size of greater than 3.0 kilobases (kb) and
an insert size range between 0.8 and 7.0 kb. Genes related to testis
development and spermatogenesis were identified by hybridization of adult
testis, fetal testis, and adult spermatozoa probes onto cDNA microarrays
containing 9216 human clones (2 dots for each clone were spotted). In brief,
after subtraction of the background from an area in which no clone was
spotted, clones with an intensity density greater than 10 were considered
positive signals, whereas if the standard difference of the signal intensity
of the paired dots was greater than 0.3, the result was not analyzed. The
hybridization intensity of correspondent dots in adult and fetus were
compared. If the difference in values of spot intensity in adult and fetus was
more than twofold higher or lower, the correspondent genes were considered
differentially expressed. Genes expressed at a higher level in adult
spermatozoa were considered potentially related to spermatogenesis
(Sha et al, 2002; Wang et al, 2004).
Identification and Characterization of Novel Alternative Splices in Human Testis
All differentially expressed cDNA plasmids were proliferated, extracted,
and purified. MegaBACE 1000 (Amersham Pharmacia, Stockholm, Sweden) was used
to sequence the full insert length. The National Center for Biotechnology
Information (NCBI) BLAST-nr tool was used to search for sequence homologies,
and 3 types of genes were identified: known genes, novel genes, and novel
alternative spliceoforms. Here, we focus on the last type. The NCBI Human
Genome database
(http://www.ncbi.nlm.nih.gov/genome/guide/human/)
was searched for exon/intron information for each novel spliceoform, and the
NCBI LocusLink database
(http://www.ncbi.nlm.nih.gov/LocusLink)
was examined for splice site information. Finally, the SMART
(http://smart.embl-heidelberg.de/)
and protein family (PFAM;
http://www.sanger.ac.uk/Software/Pfam/)
databases of protein domain sequences were used to assess the effects of the
observed alternative splices on the predicted protein products.
Tissue Distribution of Novel Spliceoforms
Multiple tissue cDNA panels (including brain, heart, kidney, liver, lung,
pancreas, placenta, and skeletal muscle) were purchased from Clontech
(K1420-1). Testis cDNA was amplified by reverse transcription (RT)-PCR from
normal human adult testis with the use of specific primers (AY009109 sense:
5'-CAG GAA CAG TGA CAG TGT AGG ACG-3' and antisense: 5'-TGT
GGA GAC GCT CTT GCT G-3'; AF327561 sense: 5'-TGT TAA AGA ACA GAG
AAG TGA TCC T-3' and antisense: 5'-GTG TTT ATA TTG CTT CAG GGC TTG
T-3'; AY014284 sense: 5'-TGA CAA TCT CCA GGA ACA GT-3' and
antisense: 5'-TCT AGA TTC TGC ATG GAG AT-3'; AF332009 sense:
5'-TAA ATT GTG GGA CTC AAA GGA GAC-3' and antisense: 5'-GCC
AAG GTT GAT TCT GAA GGC CA-3') designed to span at least 1 intron to
guard against genomic contamination. Amplification of the glucose-3-phosphate
dehydrogenase (G3PDH) housekeeping gene was used as the positive control. The
PCR mixtures (20 µL) contained 2 µL of cDNA template, 2 µL of
10x reaction buffer, 1.5 µL of MgCl2 (25 mM), 1.5 µL of
desoxynucleotide triphosphate (dNTP; 2 mM), 0.1 µL of Taq DNA
polymerase (5 U/µL), 10.9 µL of distilled water, and 1 µL of each
primer (5 pmol/µL). PCR was performed with a PTC100 thermocycler (MJ
Research Inc, Watertown, Mass) with an initial denaturation at 94°C for 5
minutes, followed by 35 cycles of 94°C for 30 seconds, 55°C for 30
seconds, and 72°C for 1 minute, with a final extension for 7 minutes at
72°C. The PCR products were separated by electrophoresis and analyzed.
NYD-SP20 and CRISP2: Developmental Stage-Dependent Expression of Novel Spliceoforms
Template cDNAs were RT-PCR amplified from human fetal, adult, and elderly
testis tissues and spermatozoa. Gene NYD-SP20 (Hs.351068), which had
4 spliceoforms (a, AF367472; b, AY032684; c, AY035867; d, AY035868), was
amplified by primer pair P1, which amplified all 4 spliceoforms (sense:
5'-GCC TAA GTG AAA ATT CAG CTC G-3' and antisense: 5'-CAT
CTC TGT AGC TCC AAC CAC C-3'); primer pair P2, which amplified
spliceoforms c and d (sense: 5'-AGT TGT GTG TCG TGT TG-3' and
antisense: 5'-CAC TGT TTT CAT TAC AGG AGC-3'); and primer pair P3,
which amplified spliceoforms a and b (sense: 5'-ACG GGG CCG CCT GGA
CGA-3' and antisense: 5'-CAT CTC TGT AGC TCC AAC CAC C-3').
One set of primers was used to amplify gene CRISP2 (cysteine-rich
secretory protein 2, Hs.2042; sense: 5'-TCT GGT TAC TGT GCT GCT
TCC-3' and antisense: 5'-TCG TTG TTA CCT CTC TGC TCC-3').
PCR was performed as described previously.
|
Results |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
|
|
|
Types of Novel Spliceoforms and Their Corresponding Open Reading Frame Changes
There are several types of alternative RNA processing. According to the
classification of Lou and Gagel
(2001), 3 types of alternative
splicing exist: alternative exon (including cassette exon and mutually
exclusive exon), alternative intron, and alternative splice site (including a
5' splice site and 3' splice site). All novel alternative
splicings were classified on the basis of the described method. Although
alternative splicing can remove or retain intronic sequences, this form of
splice was not observed in this work. Interestingly, we found that 74.63% of
spliceoform employed both alternative transcription then alternative splicing
because they used different promoters to initiate transcription. Among them,
70% used a downstream promoter (Table
2). We also separated the spliceoforms into 2 groups on the basis
of whether or not they showed open reading frame (ORF) changes
(Table 2) and found that 57
(83.58%) of the spliceoforms changed the predicted ORFs.
Tissue Distribution of Novel Alternative Spliceoforms
After bioinformatic analysis, several novel alternative spliceoforms were
considered to have important functions because the ORFs were changed. Previous
studies had identified tissue distribution of 10 spliceoforms of various genes
via RT-PCR, 7 of which were uniquely expressed in human adult testis
(Cheng et al, 2002; Xiao et al, 2002;
Yin et al, 2002;
Zhou et al, 2002;
Zhu et al, 2002;
Fang et al, 2004;
Huang et al, 2004;
Zhu et al, 2004; Hu et al, 2004;
Zheng et al, 2004). Here, we
randomly chose another 4 novel alternative spliceoforms (accession numbers
AF332009, AY009109, AF327561, and AY014284) and examined their tissue
expression profiles with 9 tissues. PCR followed by electrophoresis showed
that all 4 were highly expressed in human adult testis and 1 (AF332009) was
widely expressed, whereas 3 (AY009109, AF327561, and AY014284) were uniquely
expressed in adult testis (Figure
2).
|
NYD-SP20 and TPX1: Developmental Stage-Dependent Expression of Novel Spliceoforms
As shown in Figure 3,
NYD-SP20 was found to have 4 novel alternatively spliced forms (a, b,
c, and d), and CRISP2 was found to have a known spliced form (a,
NM_003296) and 3 novel alternatively spliced forms (b, AY289796; c, AY292862;
d, AY292863). All NYD-SP20 spliceoforms were expressed in human adult
testis and human spermatozoa, whereas human fetal and elderly testis tissues
expressed only spliceoforms b, c, and d
(Figure 4A through D). All
forms of CRISP2 were highly expressed in human adult testis and
spermatozoa but were not detected in fetal and elderly testis tissues
(Figure 4E and F).
|
|
|
Discussion |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Although spermatogenesis is the fundamental function of the testis (Eddy, 2002), it is not an autonomous process. It is subject to secondary regulation by endocrine cues transmitted indirectly through surrounding somatic cells. Both germ cells and testis somatic cells, such as Leydig cells and Sertoli cells, are important to testis development (Jegou and Sharpe, 1993; Griswold, 1998). We found that Unigene cluster Hs.408096 (FXR1P) had 2 alternatively spliced forms: known variant FXR1P-a and novel variant FXR1P-b (Table 1). Both of them were highly expressed in human adult testis, but FXR1P-a was highly expressed in human spermatozoa, whereas FXR1P-b was not (Figure 1). FXR1P encodes an RNA binding protein containing 2 KH (hnRNP K-protein homology) domains and 1 RGG amino acid repeat motif box; it associates with polyribosomes, predominantly with 60S large ribosomal subunits. Huot et al (2001) investigated the mouse homolog of FXR1P-a by immunohistologic staining of mouse testes at different developmental stages. Low-level immunoreactivity was observed in testis sections from postnatal day 6 animals. On day 14, increased staining was observed predominantly in areas containing primary spermatocytes at the early pachytene stage. Finally, in postnatal day 20 and adult testes, positive cytoplasmic signals were observed in almost all testicular cells, ranging from pachytenes to round spermatids. Surprisingly, in testes from adult mice, strong staining was also present in the luminal centers, which contained dispersed bundles of flagella from mature spermatids. Integrating this information with our data, we propose that FXR1P-a is highly expressed in human spermatogenic cells. In contrast, FXR1P-b, not detected in human spermatozoa, seems to be expressed in human testicular somatic cells, Sertoli cells, or Leydig cells. These transcripts might influence germ cell development through converging signal transduction pathways, perhaps causing post-translational modifications of transcription factors or other proteins, or both, that regulate alternative transcript processing in spermatogenic cells (Eddy, 1998).
The identified novel alternatively spliced forms were classified into 3 groups according to their splicing types: alternative exon (including cassette exon and mutually exclusive exon), alternative intron, and alternative splice site (Table 2). We did not find an alternative intron in our report. Interestingly, we identified that 74.63% of spliceoforms employed both alternative transcription then alternative splicing because they used different promoters to initiate transcription. Among them, 70% used a downstream promoter (Table 2). This is an interesting phenomenon that has been seen in previous cases, such as the genes for angiotensin-converting enzyme (ACE; Howard et al, 1990) and calcium+2/calmodulin-dependent kinase IV (CaMKIV; Means et al, 1991), both of which use a downstream promoter to produce truncated, enzymatically distinct proteins specific to male germ cells. Together, these data suggest that the complex genetics of the testis might not come from transcription of new, testis-specific genes, but rather from transcription of somatic genes from different, germ cell-specific downstream promoters that then experience alternative splicing to produce various proteins specific to male germ cells.
Spermatogenesis occurs in successive mitotic, meiotic, and postmeiotic phases, so the genes expressed during these processes must encode proteins specific to the different phases of germ cell development. Alternative splicing of pre-mRNA is important for the production of functionally diverse proteins (Lopez, 1998), and alternative transcripts can benefit male germ cells at least 4 ways. First, domain addition or replacement can expand protein function and allow addition of germ cell-specific functions. Second, spliceoform-associated changes in protein sequences can alter intracellular protein distributions. Third, long poly(A) tails, which are often found on transcripts in postmeiotic germ cells, can cause a delay of several days between transcription and translation. Fourth, alternate transcripts can arise with the use of a promoter within an intron, resulting in a protein that differs functionally in somatic cells vs male germ cells (Eddy, 2002). In support, we observed that 83.58% of the alternatively spliced transcripts had changes in their predicted ORFs. For example, the gene for testis calpastatin (accession number AF327443) showed alternative splicing in our microarray experiments. Calpain and calpastatin are involved in numerous membrane fusion events that are important in the testicular acrosome reaction. Sequence comparisons between somatic and testis calpastatin revealed 2 single amino acid changes and large-scale deletions both of domain L and of 13 amino acids between inhibitory domains I and II. However, these changes were not found to alter inhibitory activities (Takano et al, 1993), suggesting that the alternatively spliced form might have alterations in its 3-dimensional structure, inhibitory activity, subcellular location, or a combination of these factors, allowing it to influence the acrosome reaction (Zhu et al, 2002). Besides this splice, we also discovered that other novel alternative splices of various genes (accession numbers AF333336, AF327560, AF353720, AY026505, AY359880, AY313777, AY186741, AY303972, AF361054) change their predicted ORF and might be related to spermatogenesis (Cheng et al, 2002; Xiao et al, 2002; Yin et al, 2002; Zhou et al, 2002; Fang et al, 2004; Huang et al, 2004; Zhu et al, 2004; Hu et al, 2004; Zheng et al, 2004).
We further focused our attention on NYD-SP20, a novel Unigene cluster that showed 4 novel alternative spliceoforms in our tested microarrays. All of the spliceoforms differed in exon 1, and spliceoform b lacked exon 3, thus generating a shortened predicted protein product (Figure 3A). All 4 spliceoforms were expressed in human adult testis and spermatozoa, whereas only 3 were detected in embryonic and elderly testis tissues. Another examined gene, CRISP2, had 4 alternative spliceoforms in the microarrays, 3 of which were novel and 1 of which (spliceoform c) had a longer exon 8, thus encoding a longer predicted protein product (Figure 3B). All 4 CRISP2 spliceoforms were expressed in adult testis and spermatozoa but were not detected in embryonic or elderly testis samples. Developmentally, spermatogenesis does not occur in male embryos. In adults, spermatogenic cells undergo successive mitotic, meiotic, and postmeiotic phases and then form mature sperm. In elderly males, spermatogenesis weakens and the quantity and quality of sperm decreases (Bellve et al, 1977; Eddy, 2002). We found that 3 spliceoforms of NYD-SP20 (b, c, and d) were present in adult testis tissues and spermatozoa, but not in embryonic and elderly testes samples. In addition, the previously known alternatively spliced form of CRISP2 (a) was located mainly in the testis, and the mouse homolog was identified in round spermatids (Kratzschmar et al, 1996). These observations suggest that all forms of CRISP2 could be located in germ cells and highly related to spermatogenesis. Additionally, 3 spliceoforms of NYD-SP20 (a, c, and d) encode a different protein than form b, and 3 forms of CRISP2 (a, b, and d) encode a different protein than form c. Taken together, these results suggest that alternative splicing could be an important mechanism for proper testis development and function, allowing genetic diversity from an array of normally somatic gene products.
We herein identified many novel development-related alternative splicings in the testis and propose that these play a role in the complicated process of testis development and spermatogenesis. Further work will be necessary to determine the regulation mechanism of alternative splicing and the function of these proteins in human testis development.
|
Footnotes |
|---|
|
References |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Bellve AR, Cavicchia JC, Millette CF, O'Brien DA, Bhatnagar YM, Dym
M. Spermatogenic cells of the prepuberal mouse. Isolation and morphological
characterization. J Cell Biol. 1977; 74: 68
-85.
Boue S, Letunic I, Bork P. Alternative splicing and evolution. Bioessays. 2003; 25: 1031 -1034.[Medline]
Carreau S, Bourguiba S, Lambard S, Galeraud-Denis I, Genissel C,
Bili
ska B, Benahmed M, Levallet J. Aromatase expression in male germ
cells. J Steroid Biochem Mol Biol. 2001; 79: 203
-208.[Medline]
Cheng LJ, Zhou ZM, Li JM, et al. Expression of a novel HsMCAK mRNA splice variant, tsMCAK gene in human testis. Life Sci. 2002; 71: 2741 -2757.[Medline]
Copland JA, Davies PJ, Shipley GL, Wood CG, Luxon BA, Urban RJ. The use of DNA microarrays to assess clinical samples: the transition from bedside to bench to bedside. Recent Prog Horm Res. 2003; 58: b25 -b53.
Eddy EM. Regulation of gene expression during spermatogenesis. Semin Cell Dev Biol. 1998; 9: 451 -457.[Medline]
Eddy EM. Male germ cell gene expression. Recent Prog
Horm Res. 2002;57: 103
-128.
Fang X, Zhou ZM, Li JM, et al. Expression of a novel pyridoxal kinase mRNA splice variant, PKH-T, in human testis. Asia J Androl. 2004;6: 83 -91.
Gelfand MS, Dubchak I, Dralyuk I, Zorn M. ASDB: database of
alternatively spliced genes. Nucleic Acids Res. 1999; 27: 301
-302.
Griswold MD. The central role of Sertoli cells in spermatogenesis. Semin Cell Dev Biol. 1998; 9: 411 -416.[Medline]
Hammes A, Guo JK, Lutsch G, Leheste JR, Landrock D, Ziegler U, Gubler MC, Schedl A. Two splice variants of the Wilms' tumor 1 gene have distinct functions during sex determination and nephron formation. Cell. 2001;106: 319 -329.[Medline]
Howard TE, Shai S-Y, Langford KG, Martin BM, Bernstein KE.
Transcription of testicular angiotensin-converting enzyme (ACE) is initiated
within the 12th intron of the somatic ACE gene. Mol Cell
Biol. 1990;10: 4294
-4302.
Hu GH, Madore SJ, Moldover B, Jatkoe T, Balaban D, Thomas J, Wang
Y. Predicting splice variant from DNA chip expression data. Genome
Res. 2001;11: 1237
-1245.
Hu YQ, Zhou ZM, Huang XY, Xu M, Lu L, Xu ZY, Li JM, Sha JH. Expression of a novel DnaJA1 alternative splicing in human testis and sperm. Int J Androl. 2004; 2: 343 -349.
Huang XY, Wang H, Xu M, Lu L, Xu ZY, Li JM, Zhou ZM, Sha JH.
Expression of a novel RAD23B mRNA splice variant in the human testis.
J Androl. 2004;25: 363
-368.
Huot M-E, Mazroui R, Leclerc P, Khandjian EW. Developmental
expression of the fragile X-related 1 proteins in mouse testis: association
with microtubule elements. Hum Mol Genet. 2001; 10: 2803
-2811.
Jegou B, Sharpe RM. Paracrine mechanism in testicular control. In: de Kretser DM, ed. Molecular Biology of the Male Reproductive System. San Diego, Calif: Academic Press; 1993: 271 -310.
Kratzschmar J, Haendler B, Eberspaecher U, Roosterman D, Donner P, Schleuning WD. The human cysteine-rich secretory protein (CRISP) family. Primary structure and tissue distribution of CRISP-1, CRISP-2 and CRISP-3. Eur J Biochem. 1996; 236: 827 -836.[Medline]
Lee C, Atanelov L, Modrek B, Xing Y. ASAP: the alternative splicing
annotation project. Nucleic Acids Res. 2003; 31: 101
-105.
Lockhart DJ, Winzeler EA. Genomics, gene expression and DNA arrays. Nature. 2000;405: 827 -836.[Medline]
Lopez AJ. Alternative splicing of pre-mRNA: development consequences and mechanisms of regulation. Ann Rev Genet. 1998;32: 279 -305.[Medline]
Lou H, Gagel RF. Alternative ribonucleic acid processing in
endocrine systems. Endocr Rev. 2001; 22: 205
-225.
Maniatis T, Tasic B. Alternative pre-mRNA splicing and proteome expansion in metazoans. Nature. 2002; 418: 236 -243.[Medline]
Means AR, Cruzalegui F, LeMagueresse B, Needleman DS, Slaughter GR,
Ono T. A novel calcium+2/calmodulin-dependent protein kinase and a
male germ cell-specific calmodulin-binding protein are derived from the same
gene. Mol Cell Biol. 1991; 11: 3960
-3971.
Sha JH, Zhou ZM, Li JM, et al. Identification of testis development
and spermatogenesis-related genes in human and mouse testes using cDNA arrays.
Mol Hum Reprod. 2002; 8: 511
-517.
Smith L, Greenfield A. DNA microarrays and development.
Hum Mol Genet. 2003; 12: 1
-8.
Takano E, Nosaka T, Lee WJ, et al. Molecular diversity of calpastatin in human erythroid cells. Arch Biochem Biophys. 1993;303: 349 -354.[Medline]
Venables JP. Alternative splicing in the testes. Curr Opin Genet Dev. 2002;12: 615 -619.[Medline]
Walker WH, Delfino FJ, Habener JF. RNA processing and the control of spermatogenesis. Front Horm Res. 1999; 25: 34 -58.[Medline]
Wang H, Zhou ZM, Min Xu, Li JM, Xiao JH, Xu ZY, Sha JH. A spermatogenesis-related gene expression profile in human spermatozoa and its potential clinical application. J Mol Med. 2004; 82: 317 -324.[Medline]
World Health Organization. WHO Laboratory Manual for the Examination of Human Semen and Semen-Cervical Mucus Interactions. 4th ed. Cambridge, United Kingdom: Cambridge University Press; 1999 .
Xiao JH, Yin LL, Li JM, Zhu H, Zhou ZM, Zhao BG, Sha JH. Molecular cloning, identification and characteristics of NYD-SP9: gene coding protein kinase presumably involved in spermatogenesis. Chinese Sci Bull. 2002;47: 896 -901.
Xu Q, Modrek B, Lee C. Genome-wide detection of tissue-specific
alternative splicing in the human transcriptome. Nucleic Acids
Res. 2002;30: 3754
-3766.
Yin LL, Li JM, Zhu H, Cheng LJ, Wang YQ, Zhou ZM, Sha JH. Identification and characterization of a gene coding a novel spliceoform of DEAD-box preteen. Reprod Fertil Dev. 2002; 14: 185 -189.[Medline]
Zheng Y, Zhou ZM, Yin LL, Li JM, Sha JH. Molecular cloning and characterization of a novel splicing variant of PIASx1. Acta Pharmacol Sin. 2004; 25: 1058 -1064.[Medline]
Zhou ZM, Sha JH, Li JM, et al. Expression of a novel reticulon-like gene in human testis. Reproduction. 2002; 123: 227 -234.[Abstract]
Zhu H, Zhou ZM, Huo R, et al. Identification and characteristics of a novel E1 like gene, nUBE1L, in human testis. Acta Biochimica Et Biophysica Sin. 2004;36: 227 -234.
Zhu H, Zhou ZM, Li JM, Zhu H, Cheng LJ, Shan YX, Yin LL, Sha JH. Cloning and characterization of a novel spliceoform calpastatin in human adult testis. Acta Pharmacol Sin. 2002; 23: 150 -154.
This article has been cited by other articles:
|
|
 |
|
  R. B. Parmigiani, F. Bettoni, M. D. Vibranovski, M. H. Lopes, W. K. Martins, I. W. Cunha, F. A. Soares, A. J. G. Simpson, S. J. de Souza, and A. A. Camargo Characterization of a cancer/testis (CT) antigen gene family capable of eliciting humoral response in cancer patients PNAS, November 28, 2006; 103(48): 18066 - 18071. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
