AIMTo determine the possible roles of the t-complex testis expressed gene 5 (Tctex5) on sperm functions, the full-length sequence of mRNA was studied and compared in the testis between the normal wild-type and the sterile t-haplotype mutant mice.

METHODSWe applied rapid amplification of cDNA ends, Northern blot and reverse transcription polymerase chain reaction to analyze the full length of Tctex5 mRNAs isolated from testes of the wild-type and the t-haplotype mice. Reverse transcription polymerase chain reaction was used to semi-quantitatively compare expression of Tctex5 transcripts in the 16 tissues and 9.5 day stage embryos in the wild-type mice. E-translation was applied to estimate the amino acid sequences.

RESULTSOne long and one short transcript of Tctex5 mRNA were discovered in mouse testis of wild-type (Tctex5(long-+) and Tctex5(short-+)) and t-haplotype (Tctex5(long-t) and Tctex5(short-t)) mice, respectively. Being enhanced only in the testis, Tctex5(long-t) had 17 point mutations and one 15-bp-deletion in the exon 1 region, comparing with the Tctex5(long-+), whereas the Tctex5(short-t) was similar to the Tctex5(short-+). The short isoforms of Tctex5 mRNAs in the two models encoded exactly the same peptides, but the long isoforms did not. The estimated peptide encoded by Tctex5(long-t) had significant mutations on putative sites of phosphorylation and PP1 binding.

CONCLUSIONWe established that mutations that occur in the Tctex5 long transcript of the t-haplotype mice are important for normal sperm function, whereas the short transcript of Tctex5 might have a conserved function among different tissues.

Animals ; Gene Expression ; Haplotypes ; Infertility, Male ; Male ; Mice ; Microtubule-Associated Proteins ; chemistry ; genetics ; Mutation ; Nuclear Proteins ; chemistry ; genetics ; Protein Phosphatase 1 ; Sequence Analysis, Protein ; Spermatozoa ; metabolism ; Testis ; metabolism ; t-Complex Genome Region

OBJECTIVETo identify a novel isoform of hTCP11 gene and investigate its expression and alternative splicing.

METHODSAccording to the sequence of human ESTs which are highly homologous to hTCP11a, primers for PCR were synthesized. Then, the amplified fragments were cloned and sequenced; some methods including BLAST, ClustalW and RT-PCR were used for genomic analysis, study of alternative splicing and gene expression among multiple tissues and different testis tissues.

RESULTSA novel isoform of hTCP11 gene was isolated. It encodes a 440 amino acid protein that is highly homologous to the mouse 566 amino acid protein which is important to sperm function because it encodes the receptor for fertilization promoting peptide (FPP). Among TCP11a, TCP11b and TCP11c, the complicated alternative splicing was found. RT-PCR analysis of RNA extracted from human tissues revealed that the gene is only expressed in fertile adult testes, but not in azoospermic patient testes, fetal testes or other human tissues.

CONCLUSIONOur results along with the mouse Tcp-11 function suggest that the isoforms of TCP11 gene play important roles in sperm function and fertility.

Adult ; Alternative Splicing ; Amino Acid Sequence ; Animals ; Base Sequence ; Cloning, Molecular ; DNA-Binding Proteins ; biosynthesis ; genetics ; Gene Expression ; Humans ; Male ; Membrane Proteins ; Mice ; Microtubule-Associated Proteins ; genetics ; Molecular Sequence Data ; Nuclear Proteins ; genetics ; Protein Isoforms ; Sequence Homology ; t-Complex Genome Region