Alternative splicing of premessenger RNA is an important regulatory mechanism that increases the diversity of proteins transcribed from a single gene.This is particularly important in the testis because germ cell expansion and differentiation require many cellular changes and regulatory steps.To investigate novel developmentrelated alternative splicings in the human testis, complementary DNA microarray studies were conducted with the use of probes from human fetal testes,adult testes,and human spermatozoa.Of a total of 386 Unigene clusters found to be related to the development of the testis,67 clusters showed a total of 74 novel alternative spliceoforms. Developmental stage–dependent expression was also performed for a novel Unigene, NYD-SP20 (Hs.351068), which had 4 possible novel spliceoforms and another Unigene,CRISP2 (cysteine-rich secretory protein 2,Hs.2042),which had 3 possible novel spliceoforms.These results indicate that alternative splicing plays an important role in the complicated processes of testis development and spermatogenesis.

A gene coding a novel isoform of carbamyl phosphate synthetase I (CPS1) was cloned from a human testicular library.As shown by cDNA microarray hybridization, this gene was expressed at a higher level in human adult testes than in fetal testes. The full length of its cDNA was 3831 bp, with a 3149 bp open reading frame, encoding a 1050-amino-acid protein. The cDNA sequence was deposited in the GenBank (AY317138). Sequence analysis showed that it was homologous to the human CPS1 gene.The putative protein contained functional domains composing the intact large subunit of carbamoyl phosphate synthetase,thus indicated it has the capability of arginine biosynthesis.A multiple tissue expression profile showed high expression of this gene in human testis,suggesting the novel alternative splicing form of CPS1 may be correlated with human spermatogenesis.

Identification of genes specifically expressed in adult and fetal testis is important in furthering our understanding of testis development and function. In this study,a novel human transcript, designated human testis cAMP-responsive element-binding protein (htCREB), was identified by hybridization of adult and fetal human testis cDNA probes with a human cDNA microarray containing 9216 clones.The htCREB transcript (GenBank Accession no. AY347527) was expressed at 2.35-fold higher levels in adult human testes than in fetal testes.Sequence and ntBLAST analyses against the human genome database indicated that htCREB was a novel splice variant of human CREB.RT-PCR-based tissue distribution experiments demonstrated that the htCREB transcript was highly expressed in adult human testis and in healthy sperm, but not in testes from patients with Sertoli cell-only syndrome. Taken together, these results suggest that the htCREB transcript is chiefly expressed in germ cells and is most likely involved in spermatogenesis.

A novel human testis-specific gene,NYD-SP12,was identified by hybridizing human adult or fetal testes cDNA samples with a human cDNA microarray containing 9216 clones.mRNA expression level of NYD-SP12 was 30-fold higher in human adult testes than fetal testes.Similarly,semi-quantitative RT

By hybridizing human adult testis cDNA microarrays with human adult and embryo testis cDNA probes,a novel human testis gene NYD-SP16 was dentified. NYD-SP16 expression was 6.44-fold higher in adult testis than in fetal testis.NYD-SP16 contains 1595 base pairs (bp) and a 762-bp open reading frame encoding a 254-amino acid protein with 73% amino acid sequence identity with the mouse testis homologous protein.The NYD-SP16 gene was localized to human chromosome 5q14.The deduced structure of the NYD-SP16 protein contains one transmembrane domain, which was confirmed by GFP/NYD-SP16 fusion protein expression in the cytomembrane of the transfected human choriocarcinoma JAR cells,suggesting that it is a transmembrane protein. Multiple tissue distribution indicated that NYD-SP16 mRNA is highly expressed in the testes and pancreas, with little or no expression elsewhere. Further analysis of abnormal expression in infertile male patients revealed complete absence of NYD-SP16 in the testes of patients with Sertoli-cellonly syndrome and variable expression in patients with spermatogenic arrest.Homologous gene expression in mouse testis was confirmed in spermatogenic cells by in situ hybridization.The results of cDNA microarray,in situ hybridization,and quantitative polymerase chain reaction in mouse testis of different stages indicated that NYD-SP16 expression is developmentally regulated.These results suggest that the putative NYD-SP16 protein may play an important role in testicular development/spermatogenesis and may be an important factor in male infertility.

We have constructed cDNA microarrays from the human testis large insert cDNA library, containing 9216 genes,together with several housekeeping genes. The cDNA microarrays were used to identify gene expression differences between human fetal and adult testes. Of >8700 hybridized clones, 731 exhibited significant differential expression characteristics.About 7500 genes were identified when the same cDNA microarrays were used for hybridization with cDNA probes from mouse testis,with 256 genes having significant differential expression between the age of 1-4 weeks. Among these genes,101 were identified as critically related to testis development and possibly to spermatogenesis since they were found in both human and mouse testes, and expressed differentially at different stages of testis development. Of the 101 development-related genes,59 full-length cDNAs have been sequenced previously, while the full-length cDNAs of the other 42 genes have not been published. We have obtained 11 full-length sequences of the 42 genes and deposited them in the GenBank. The conserved testis development-related genes found in both human and mouse testes may include genes that are likely to be involved in testicular functions, especially spermatogenesis,thus providing a basis for further functional characterization of the genes in mouse models.

Using cDNA microarray hybridization from a human testicular cDNA library, a novel gene exhibiting 30-fold difference in expression level between adult and embryo human testes was cloned and named NYDSP6,which was 1858 bp in length with 87% nucleotide identity with the mouse homologue sequence.The deduced protein structure of NYD-SP6 was found to contain two plant homeodomain (PHD) finger domains,believed to be involved in activating transcriptional regulation.Tissue distribution analysis using Northern blot indicated that the NYD-SP6 gene was expressed in a wide range of tissues,with a high expression level in the testis.Its expression in human and mouse testes by in situ hybridization was confined to Sertoli cells and the expression was developmentally regulated as demonstrated by cDNA microarray,in situ hybridization,and semiquantitative PCR in mouse testes.GFP/NYD-SP6 protein was predominantly localized in the nucleus of the transfected CHO cells,indicating its role in transcriptional regulation.In contrast,the N-terminal truncated NYD-SP6 (tNYDSP6) localized in the nuclear envelope,indicating this region function as a nuclear localization signal.Further Northern blot analysis of gene expression in patients with spermatogenesis arrest revealed that NYDSP6 was absent in one patient whose spermatogenesis was blocked at the stage of spermatocytes.Taken together,these results suggest that the putative protein of NYD-SP6 may play an important role in stimulating transcription involved in testicular development and/or spermatogenesis.

Identification of genes that are specifically expressed in the adult testis or the fetal testis is important for the study of genes related to the development of the testis.In this study,a human testis cDNA microarray was established.PCR products of 9216 clones from a human testis cDNA library were dotted on a nylon membrane;mRNA from adult and fetal testes were purified and probes were prepared by a reverse transcription reaction with testis mRNA as template.The microarray was hybridized with probes of adult and fetal testes,and 96.8 and 95.4% of clones were positive,respectively.In total,731 clones were differentially expressed:592 were highly expressed in adult testis and 139 were highly expressed in fetal testis.Among these genes,a new reticulon (Rtn)-like gene was detected and named Rtn-T.Rtn-T was highly expressed in adult human testis. The cDNA of Rtn-T contains 3491 bp and the putative protein had 968 amino acids.This protein is homologous to the six known members of the Rtn family (KIAA0886,Rtn xL,reticulon 4a,Nogo-A, Nogo-A short form, and brain my043) but was different at the 5’end.All homologues originate from one gene,and result from both different promotor regions and different splicing. Rtn-T lacks the first exon and contains a second exon that is lacking in the other homologues.Rtn-T is shorter than KIAA0886,Rtn xL,reticulon 4a and Nogo-A,but longer than the Nogo-A short form and brain my043.Sequence analysis showed that Rtn-T protein has two hydrophobic regions that may be branespanning domains.Expression profiles showed that Rtn-T is specifically and strongly expressed in testis.The results of the present study indicate that the Rtn-T gene is differentially expressed in adult and fetal testes and encodes a membrane protein that may have a function in testis development.