Testis-specific Exon Research Articles

The catalytic function of hormone-sensitive lipase is essential for fertility in male mice.

In male mice, deficiency of hormone sensitive lipase (HSL, Lipe gene, E.C.3.1.1.3) causes deficient spermatogenesis, azoospermia, and infertility. Postmeiotic germ cells express a specific HSL isoform that includes a 313 amino acid N-terminus encoded by a testis-specific exon (exon T1). The remainder of testicular HSL is identical to adipocyte HSL. The amino acid sequence of the testis-specific exon is poorly conserved, showing only a 46% amino acid identity with orthologous human and rat sequences, compared with 87% over the remainder of the HSL coding sequence, providing no evidence in favor of a vital functional role for the testis-specific N-terminus of HSL. However, exon T1 is important for Lipe transcription; in mouse testicular mRNA, we identified 3 major Lipe transcription start sites, finding numerous testicular transcription factor binding motifs upstream of the transcription start site. We directly explored two possible mechanisms for the infertility of HSL-deficient mice, using mice that expressed mutant HSL transgenes only in postmeiotic germ cells on a HSL-deficient background. One transgene expressed human HSL lacking enzyme activity but containing the testis-specific N-terminus (HSL-/-muttg mice). The other transgene expressed catalytically inactive HSL with the testis-specific N-terminal peptide (HSL-/-atg mice). HSL-/-muttg mice were infertile, with abnormal histology of the seminiferous epithelium and absence of spermatozoa in the epididymal lumen. In contrast, HSL-/-atg mice had normal fertility and normal testicular morphology. In conclusion, whereas the catalytic function of HSL is necessary for spermatogenesis in mice, the presence of the N-terminal testis-specific fragment is not essential.

Molecular design of a splicing switch responsive to the RNA binding protein Tra2

Tra2β regulates a number of splicing switches including activation of the human testis-specific exon HIPK3-T in the Homeodomain Interacting Protein Kinase 3 gene. By testing HIPK3-T exons of different intrinsic strengths, we found Tra2β most efficiently activated splicing inclusion of intrinsically weak exons, although these were spliced at a lower overall level. Both the RRM and N-terminal RS-rich region of Tra2β were required for splicing activation. Bioinformatic searches for splicing enhancers and repressors mapped four physically distinct exonic splicing enhancers (ESEs) within HIPK3-T, each containing the known Tra2β AGAA-rich binding site. Surprisingly disruption of each single ESE prevented Tra2β-mediated activation, although single mutated exons could still bind Tra2β protein by gel shifts and functional splicing analyses. Titration experiments indicate an additive model of HIPK3-T splicing activation, requiring availability of an array of four distinct ESEs to enable splicing activation. To enable this efficient Tra2β-mediated splicing switch to operate, a closely adjacent downstream and potentially competitive stronger 5′-splice site is actively repressed. Our data indicate that a novel arrangement of multiple mono-specific AGAA-rich ESEs coupled to a weak 5′-splice site functions as a responsive gauge. This gauge monitors changes in the specific nuclear concentration of the RNA binding protein Tra2β, and co-ordinately regulates HIPK3-T exon splicing inclusion.

Characterization of the testis‐specific promoter region in the human pituitary adenylate cyclase‐activating polypeptide (PACAP) gene

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a pleiotropic neuropeptide localized in the testis at concentration comparable to that found in the brain, suggesting involvement in spermatogenesis. In this study, we identified the human PACAP testis-specific exon (TSE) 10.9 kb upstream from the translational start site and found that the testis-specific transcript of the human PACAP gene was found to be spliced from the TSE into a region of intron 2 without a frameshift. The resulting PACAP precursor has no signal peptide, suggesting that PACAP functions physiologically in an intracrine manner in the testis. The 5'-flanking region of the TSE contains an 80-bp fragment with potent promoter activity in testicular F9 cell. Electrophoresis mobility shift assays showed that proteins from the F9 nuclear extract interacted specifically with the 80-bp fragment. DNA affinity chromatography allowed isolation of the specific proteins bound to the 80-bp fragment, two of which were identified as Poly (ADP-ribose) polymerase-1 (PARP-1) and TIA-1-related protein (TIAR) by mass spectrometry. By using their siRNAs, the depletion of their proteins in F9 cells affected the potent promoter activity of the 80-bp fragment, suggesting that they might be involved in the testis-specific gene expression of PACAP.

The germ cell nuclear proteins hnRNP G-T and RBMY activate a testis-specific exon.

The human testis has almost as high a frequency of alternative splicing events as brain. While not as extensively studied as brain, a few candidate testis-specific splicing regulator proteins have been identified, including the nuclear RNA binding proteins RBMY and hnRNP G-T, which are germ cell-specific versions of the somatically expressed hnRNP G protein and are highly conserved in mammals. The splicing activator protein Tra2β is also highly expressed in the testis and physically interacts with these hnRNP G family proteins. In this study, we identified a novel testis-specific cassette exon TLE4-T within intron 6 of the human transducing-like enhancer of split 4 (TLE4) gene which makes a more transcriptionally repressive TLE4 protein isoform. TLE4-T splicing is normally repressed in somatic cells because of a weak 5′ splice site and surrounding splicing-repressive intronic regions. TLE4-T RNA pulls down Tra2β and hnRNP G proteins which activate its inclusion. The germ cell-specific RBMY and hnRNP G-T proteins were more efficient in stimulating TLE4-T incorporation than somatically expressed hnRNP G protein. Tra2b bound moderately to TLE4-T RNA, but more strongly to upstream sites to potently activate an alternative 3′ splice site normally weakly selected in the testis. Co-expression of Tra2β with either hnRNP G-T or RBMY re-established the normal testis physiological splicing pattern of this exon. Although they can directly bind pre-mRNA sequences around the TLE4-T exon, RBMY and hnRNP G-T function as efficient germ cell-specific splicing co-activators of TLE4-T. Our study indicates a delicate balance between the activity of positive and negative splicing regulators combinatorially controls physiological splicing inclusion of exon TLE4-T and leads to modulation of signalling pathways in the testis. In addition, we identified a high-affinity binding site for hnRNP G-T protein, showing it is also a sequence-specific RNA binding protein.

Alternative 5′-untranslated first exons of the mouse Cyp19A1 (aromatase) gene

The human aromatase gene ( CYP19A1) has eleven tissue-specific untranslated first exons, while only three have been described in the mouse Cyp19A1 namely brain-, ovary- and testis-specific exons 1. The present study aims to elucidate the complete structure of the mouse Cyp19A1 gene. We detected aromatase transcripts in mouse bone, aorta, hypothalamus, adipose, gonads and placenta, but not nulliparous mammary fat pad. BestFit algorithm analysis against the human CYP19A1 has identified ten putative first exons upstream of mouse Cyp19A1. Based on these putative sequences, we were able to design specific primers for RT-PCR and detected for the first time, the presence of exons I.4 and I.3 in murine fat and gonads, respectively. These are novel 5′UTRs of mouse Cyp19A1. Using RT-PCR and 5′ RACE, we confirmed the expression of exon 1f in the hypothalamus and proximal exon P2 in the ovary. The testis-specific exon 1 begins 217 bp further upstream than previously reported. Putative exons 2a, I.5, I.7, I.6 and I.2 were not detected in mouse tissues. Therefore, we showed that mouse Cyp19A1 contains more tissue-specific first exons than previously thought and displays a similar genomic organization to human CYP19A1.

Cloning of chicken lanosterol 14α-demethylase (CYP51) cDNA: Discovery of a testis-specific CYP51 transcript

A lanosterol 14alpha-demethylase (CYP51) cDNA, which consisted of a 1419 bp open reading frame encoding 472 amino acids and a 918 bp 3'-untranslated region, was isolated from the chicken testis. The sequence corresponding to exon 1 of this cDNA was completely different from those of CYP51 cDNAs in other tissues, including the liver. The expression level of the CYP51 gene with the testis-specific exon 1 was much higher in mature (2-year-old) male chickens than in immature (5-week-old) chickens. In addition, a CYP51 transcript common to several tissues, including the liver, adrenal gland, cerebellum, cerebrum, lobus opticus, kidney, lung, heart, muscle, spleen, small intestine and ovary, was also detected in the testis by RT-PCR. Furthermore, testis-specific shorter transcripts, which have been observed in mammals such as humans, rats and pigs, were not detected in the chicken testis. The results of this study demonstrate for the first time the presence of a chicken testis-specific CYP51 transcript and its sexual maturation-related expression, although its biological significance remains unclear.

Characterization of a Novel Testicular Form of Human Hormone-Sensitive Lipase

Hormone-sensitive lipase (HSL) is an esterase and lipase, which are essential for spermatogenesis. Two HSL mRNAs are expressed in human testis. A long form is encoded by a testis-specific exon and nine exons common to testis and adipocyte HSL. Here we show that the short-form 3.3-kb mRNA possesses a unique 5′ end that is transcribed from a novel testis-specific exon. The corresponding protein is similar to the 775-amino-acid-long adipocyte HSL. Immunohistochemistry experiments on human testis sections revealed that the long form is strictly expressed in haploid germ cells whereas the short form is expressed in interstitial and tubular somatic cells as well as premeiotic germ cells.

Genomic structure and chromosomal localization of the human hepatocyte growth factor activator inhibitor type 1 and 2 genes.

Hepatocyte growth factor activator inhibitor type 1 (HAI-1) and type 2 (HAI-2) are recently discovered Kunitz-type serine protease inhibitors which can be purified and cloned from human stomach cancer cell line MKN45 as specific inhibitors against hepatocyte growth factor activator (HGFA). HAI-2 was identical with the protein originally reported as placental bikunin. Both proteins contain two Kunitz inhibitor domains (KDs), of which the first domain (KD1) is mainly responsible for the inhibitory activity against HGFA, and are expressed ubiquitously in various tissues. In this study, we cloned the genes coding for these two structurally similar proteins by screening of human genomic bacterial artificial chromosome (BAC) library and their genomic structures were compared. HAI-1 and -2 genes consist of 11 and 8 exons spanning 12 kbp and 12.5 kbp, respectively. Three exons were inserted between KD1 and KD2 of each gene, of which the middle one was the low-density lipoprotein (LDL) receptor-like domain (HAI-1) and the testis specific exon (HAI-2). Apparently homologous regions between HAI-1 and -2 were not found in 5'-flanking region and neither TATA nor CAAT box was present. The genes were mapped to chromosome 15q15 (HAI-1) and 19q13.11 (HAI-2). These results suggested that although HAI-1 and -2 genes might be derived from same ancestor gene, they acquired distinctive in vivo roles during their evolution.

Pituitary Adenylate Cyclase-Activating Polypeptide Gene Expression Regulated by a Testis-Specific Promoter in Germ Cells during Spermatogenesis

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a member of the glucagon-related family of hormones that is widely expressed in various tissues. The PACAP messenger RNA (mRNA) and protein is expressed at high levels in the germ cells of the testis, where it locally activates cAMP-coupled receptors located in the somatic Sertoli cells. The PACAP mRNA expressed specifically in the testis is shorter than the mRNA expressed in hypothalamus and includes 127 nucleotides of novel sequence at the 5′-end, suggesting a different start site of transcription in the testes and the utilization of a tissue-specific promoter. Here we present evidence that a single PACAP gene uses a testis-specific promoter to express a mRNA containing a unique exon located 13.5 kb upstream from the first coding exon. As determined by RT-PCR analysis of testis mRNA, the expression of the first testis-specific exon is relatively specific for the testis, as no PACAP mRNA containing the testis-specific first exon was detected in hypothalamic mRNAs. The promoter for the testis-specific PACAP gene was cloned, and a start site for transcription was mapped by primer extension. The testis-specific promoter sequence directs germ cell-specific expression upon transfection of promoter-transcriptional reporter plasmids to populations of testicular cells in vitro and upon expression of a promoter-reporter transgene in mice. Analyses of PACAP gene expression during the spermatogenic cycle, accomplished by RT-PCR of segments of isolated seminiferous tubules, identified intense expression in the postmeiotic round spermatids during developmental stages I–VIII. These observations establish the existence of a specialized PACAP gene promoter whose activity is highly regulated during the spermatogenic cycle.

Pituitary adenylate cyclase-activating polypeptide gene expression regulated by a testis-specific promoter in germ cells during spermatogenesis.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a member of the glucagon-related family of hormones that is widely expressed in various tissues. The PACAP messenger RNA (mRNA) and protein is expressed at high levels in the germ cells of the testis, where it locally activates cAMP-coupled receptors located in the somatic Sertoli cells. The PACAP mRNA expressed specifically in the testis is shorter than the mRNA expressed in hypothalamus and includes 127 nucleotides of novel sequence at the 5'-end, suggesting a different start site of transcription in the testes and the utilization of a tissue-specific promoter. Here we present evidence that a single PACAP gene uses a testis-specific promoter to express a mRNA containing a unique exon located 13.5 kb upstream from the first coding exon. As determined by RT-PCR analysis of testis mRNA, the expression of the first testis-specific exon is relatively specific for the testis, as no PACAP mRNA containing the testis-specific first exon was detected in hypothalamic mRNAs. The promoter for the testis-specific PACAP gene was cloned, and a start site for transcription was mapped by primer extension. The testis-specific promoter sequence directs germ cell-specific expression upon transfection of promoter-transcriptional reporter plasmids to populations of testicular cells in vitro and upon expression of a promoter-reporter transgene in mice. Analyses of PACAP gene expression during the spermatogenic cycle, accomplished by RT-PCR of segments of isolated seminiferous tubules, identified intense expression in the postmeiotic round spermatids during developmental stages I-VIII. These observations establish the existence of a specialized PACAP gene promoter whose activity is highly regulated during the spermatogenic cycle.

Testis Expression of Hormone-sensitive Lipase Is Conferred by a Specific Promoter That Contains Four Regions Binding Testicular Nuclear Proteins

The testicular isoform of hormone-sensitive lipase (HSLtes) is encoded by a testis-specific exon and 9 exons common to the testis and adipocyte isoforms. In mouse, HSLtes mRNA appeared during spermiogenesis in round spermatids. Two constructs containing 1.4 and 0.5 kilobase pairs (kb) of the human HSLtes gene 5'-flanking region cloned upstream of the chloramphenicol acetyltransferase gene were microinjected into mouse oocytes. Analyses of enzyme activity in male and female transgenic mice showed that 0.5 kb of the HSLtes promoter was sufficient to direct expression only in testis. Cell transfection experiments showed that CREMtau, a testis-specific transcriptional activator, does not transactivate the HSLtes promoter. Using gel retardation assays, four testis-specific binding regions (TSBR) were identified using testis and liver nuclear extracts. The testis-specific protein binding on TSBR4 was selectively competed by a probe containing a SRY/Sox protein DNA recognition site. Sox5 and Sox6 which are expressed in post-meiotic germ cells bound TSBR4. Mutation of the AACAAAG motif in TSBR4 abolished the binding. Moreover, binding of the high mobility group domain of Sox5 induced a bend within TSBR4. Together, our results showed that 0.5 kb of the human HSLtes promoter bind Sox proteins and contain cis-acting elements essential for the testis specificity of HSL.

Alternative splicing of exons in the alpha1 subunit of the rat testis L-type voltage-dependent calcium channel generates germ line-specific dihydropyridine binding sites.

Cell-specific isoforms of the alpha1 subunit of the L-type voltage-dependent calcium channel (VDCC) have unique pharmacological reactivities. Prior sequence analysis of nucleotide bases 3908-6077 of the VDCC alpha1 subunit expressed in rat testis differed from cardiac sequences only in a 84 base pair region corresponding to exons 31/32 encoding a putative dihydropyridine binding region. We now report that sequence analysis of bases 3048-3936 identifies a second difference between the rat testis and rat cardiac alpha1 sequence in a 60 base pair region corresponding to exons 21/22 and encoding another putative dihydropyridine binding site. Variable VDCC exons 21/22 and 31/32 and their linking introns were sequenced using genomic DNA from rat lung as template, providing evidence that the rat testis and cardiac alpha1 isoforms are products of the same gene. Reverse transcription in-situ polymerase chain reaction (PCR) with frozen sections of rat testis was carried out with primers identifying the testis-specific exon 32 of the VDCC alpha1 subunit. PCR products were confined to seminiferous tubules and were associated with the germ cell lineage from Type A spermatogonia to mature spermatozoa. Close coupling of testis alpha1 VDCC gene transcription and translation was established by in-situ immunolabelling of serial frozen sections with a monoclonal antibody (IIF7) directed against epitopes on rabbit skeletal muscle L-type VDCC alpha1. Western blot analysis of rat proteins extracted from heart, skeletal muscle, testis and spermatozoa which were reactive with the IIF7 antibody detected primarily 175-220 kDa proteins in the size range of VDCC. These data unequivocally demonstrate that an L-type VDCC is expressed in rat testis and that VDCC isoforms from rat testis and heart differ in deduced amino acid composition in and around potential binding sites for calcium channel blocking drugs such as the dihydropyridines.