Expression of a novel beta adaptin subunit mRNA splice variant in human testes

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Identification and characterization of the BGR-like gene with a potential role in human testicular development/spermatogenesis

Aim: To investigate the roles of the BGR-like gene in testicular development/spermatogenesis. Methods: A human testis cDNA microarray was hybridized with probes from human adult testes and embryo testes. The differentially expressed clones were sequenced and analyzed. Expression of the BGR-like gene was analyzed by reverse transcription-polymerase chain reaction (RT-PCR). Results: A new gene exhibiting 50-fold difference in expression level between adult and fetal human testes was cloned and named the BGR-like gene. The cDNA consisted of 2500 nucleotides and had an open reading frame of 1437 nucleotides encoding a putative protein of 497 amino acid residues. Homologous comparison showed that the BGR-like gene was a new alternative splicing variant of the BGR gene and had sequence homology with the bubblegum gene of human, mouse, rat and Drosophilia. Protein motif analysis of the BGR-like gene revealed that it contained a conserved adenosine monophosphate (AMP)-binding domain and a fatty acyl-CoA synthetase signature motif which existed in all acyl-CoA synthetases. The BGR-like gene transcript was imperceptibly expressed in human fetal testes, highly in human adult testes and moderately in elderly testes and human Leydig cells. RT-PCR-based tissue distribution experiments showed that the BGR-like gene was exclusively expressed in testes and was a testes-specific isoform of the BGR gene. A BGR-like gene transcript was not detected in some azoospermic testes. Conclusion: The BGR-like gene may play an important role in spermatogenesis/testicular development and may be correlated with male infertility.

Expression of a novel bHLH-Zip gene in human testis

<abstract>Aim: To identify specifically expressed genes in the adult and fetal testes. Methods: A human testis cDNA microarray was established. Then the mRNA of adult and fetal testis was purified and probes were prepared by a reverse transcription reaction with the testis mRNA as template. The microarray was hybridized with probes of adult and fetal testes. The nucleic sequences of differentially expressed genes were determined and homologies were searched in the databases of the GenBank. Results: When hybridized with adult or fetal testis probes, the positive clones were 96.8 % and 95.4 %, respectively. Among these genes, one was a new testis-specific gene, which was named TSP1. TSP1 was highly expressed in human adult testis. The cDNA of TSP1 was 1,484 bp in length. The cDNA sequence of this clone was deposited in the Genbank (AF333098). TSP1 was also determined as Interim Gen Symbol (Unigene, No. Hs.98266). Protein analysis showed that TSP1 contained two functional domains: an N-terminal basic helix-loop-helix (bHLH) and a C-terminal leucine zipper (Zip). Homologous analysis showed that the 430 amino acid sequences deduced from the 1,293 bp open reading frame (ORF) had a homology with the human gene FLJ2509 (AK098575). TSP1 had also a sequence homology with Spz 1 protein of mouse. Expression profiles showed that TSP1 was specifically and strongly expressed in the testis. Conclusion: TSP1 is a gene highly expressed in adult testis. It may play an important role in spermatogenesis in the humans.

Expression of a novel pyridoxal kinase mRNA splice variant,PKH-T, in human testis

Aim: To identify the genes specifically expressed in human adult and fetal testes and spermatozoa. Methods: A human testis cDNA microarray was established. Then mRNAs of human adult and fetal testis and spermatozoa were purified and probes were prepared by a reverse transcription reaction with mRNA as the template. The microarray was hybridized with probes of adult and fetal testes and spermatozoa. The nucleic acid sequences of differentially expressed genes were determined and homologies were searched in the databases of GenBank. Results: A novel human testis-specific gene, PKH-T, was identified by hybridizing adult and fetal testis and spermatozoa probes with a human testis cDNA microarray. The cDNA of PKH-T was 1 069 bp in length. The cDNA sequence of this clone was deposited in the Genbank (AY303972) and PKH-T was also determined as Interim GenSymbol (Unigene, HS.38041). PKH-T contained most PKH conserved motif. The 239 amino acid sequences deduced from the 719 bp open reading frame (ORF) had a homology with the gene PKH (U89606). PKH-T was specifically and strongly expressed in the testis. Comparison of the differential expressions of PKH and PKH-T in testes of different develop-mental stages indicated that PKH-T was expressed in the adult testis and spermatozoa, while PKH, in the adult, fetal and aged testes. PKH-T had no expression in the testis of Sertoli cell only and partially spermatogenic arrest patients. Conclusion: PKH-T is a gene highly expressed in adult human testis and spermatozoa. It may play an important role in spermatogenesis and could be related to male infertility. (Asian J Androl 2004 Jun; 6: 83-91)

Identification and characterization of cul-3b,a novel hominine CUL-3 transcript variant

Aim:To identify genes related to the human testis development by substrate hybridization technique.Methods:A human testis cDNA microarray was constructed and hybridized with probes prepared from human adult and fetal testes and spermatozoa mRNAs by reverse transcription reactions.The differentially expressed genes were sequenced. And a newly identified cullin-3 (CUL-3) transcript variant (designated cul-3b) was bio-informatically analyzed with an online GenBank database.Multi-tissue reverse transcription polymerase chain reaction (RT-PCR) was used to deter- mine the tissue expression profile of cul-3b.Results:Cul-3b,a novel CUL-3 transcript variant,was identified.The expression level of cul-3b in adult testes was 3.79-fold higher than that in fetal ones.Cul-3b differed from cul-3 (including NM_003590 and AY337761) in the opening reading frame and had three internal ribosomal entry sites (IRESes) in the 5'-UTR.These led to a 24 amino acid (aa) truncation at N-terminus of CUL-3b as compared with CUL-3 and a more motivated expression pattern of cul-3b under some strict circumstances.Additionally,cul-3b expressed ubiquitously in human tissues according to multi-tissue RT-PCR.Conclusion:Cul-3b is a novel transcript variant of CUL-3,which may be important not only for the development of human testis but also for that of other organs.