Down-regulation of Flt-1 gene expression by the proteasome inhibitor MG262.

The mechanisms involved in the anti-angiogenic actions of the proteasome inhibitors are poorly understood. Here, we report that the gene expression of the VEGF receptor Flt-1 (vascular endothelial growth factor receptor 1) was down-regulated by the reversible proteasome inhibitor MG262 in explant cultures of the developing chicken pecten oculi, a vascular organ consisting of endothelial cells, pericytes, and macrophages. In addition, the inhibitor prevented the induction of Flt-1 by lipopolysaccharide (LPS) in macrophages and down-regulated the expression of Flt-1 after LPS induction. Flt-1 gene expression was also down regulated by MG262 in cultures of human microvascular endothelial cells. Interestingly, a transcript of Flt-1, coding for a soluble form of the receptor (sFlt-1) with anti-angiogenic properties, was not down-regulated in the same extent. Only a small decrease in the expression of VEGF and Ang-2 was detected in the pecten oculi upon inhibition of the proteasome, while no major changes were observed in the expression of other angiogenic molecules, such as KDR or Ang-1. Since recent experiments have demonstrated the importance of anti-Flt-1 therapy in the inhibition of tumor angiogenesis, retinal angiogenesis, arthritis, and atherosclerosis (Luttun et al. [2002]: Nat Med 8:831-840), our observation on down-regulation of Flt-1 in microvascular endothelial cells and macrophages by MG262 supports the postulated role of the proteasome inhibitors as potential candidates for therapeutic modulation of angiogenesis and inflammation.

Constitutive and heat-shock induced expression of Hsp70 mRNA during chicken testicular development and regression.

The constitutive and heat shock induced expression of Hsp70 mRNA was investigated in normal adult chicken testis and in adult testis after testicular regression induced by diethylstilbestrol (DES) treatment. In addition to the canonical form of Hsp70 mRNA, we have detected transcripts with an extended 5'UTR and transcripts containing, in the 5'UTR, sequences of the 18S ribosomal RNA. Hsp70 was expressed in unstressed male gonads in adult and regressed testis, being the expression much lower in regressed testis. Upon heat shock at 44 degrees C or 46 degrees C, Hsp70 was highly induced in both tissues. However, when testicular seminiferous tubules were incubated at the chicken internal temperature of 39 degrees C, no induction of Hsp70 was observed in mature testis, while the expression markedly increased in regressed testis. Induction at 39 degrees C was completely inhibited in the presence of 6 mM aspirin. Aspirin in the range 3-10 mM decreases the expression of Hsp70 in unstressed and stressed testicular cells, in striking contrast with the effect observed in other tissues as liver. These data suggest that the expression of Hsp70 is regulated in a specific manner in chicken testis and particularly in the male gonad undergoing regression.

Genomic structure and alternative splicing of chicken angiopoietin-2.

Angiopoietin-1 (Ang-1) prevents endothelial cell apoptosis and promotes blood vessel stability, while angiopoietin-2 (Ang-2), a natural antagonist of Ang-1, disrupts blood vessel structure and induces apoptosis. We have sequenced the chicken angiopoietin-2 gene that spans about 46 kb of DNA and is split in 9 exons by 8 introns. Alternative splicing of the gene gives rise to three different species of Ang-2 mRNAs: Ang-2A, Ang-2B, and Ang-2C. The three mRNA isoforms, also present in humans, codify for proteins with an identical fibrinogen-like C-terminal domain and a different coiled-coil N-terminal domain. Ang-2A and particularly Ang-2C are expressed in immature testis and regressed adult testis undergoing vascular remodeling, while their expression is barely detectable in quiescent adult testis. Conversely, Ang-2B is only detectable in adult testis. The new isoforms with truncated amino-terminal domains may modulate the Tie2 receptor during vascular angiogenesis and regression.

Expression of lactate dehydrogenases A and B during chicken spermatogenesis: characterization of testis specific transcripts.

The substrates required for glycolysis change markedly at successive stages of spermatogenesis suggesting a considerable plasticity in the expression of glycolytic enzymes. Lactate dehydrogenase (LDH) isoenzymes, LDH-A and LDH-B, are expressed in premeiotic, meiotic cells, and early spermatids, both in avian and mammalian spermatogenesis. Highly polyadenylated forms, particularly of LDH-A, were detected in chicken testis. While mammals and columbid birds express the testis specific LDH-C gene in meiotic and postmeiotic cells, several LDH-B testis specific transcripts were detected in the corresponding cells during chicken spermatogenesis. These testis specific transcripts and the mRNA of mammalian LDH-C show several properties in common, such as temporal correlation of expression, mRNA stability, and repression of premature translation. These observations suggest that the testis specific transcripts could perform during chicken spermatogenesis the functions of the LDH-C mRNA in mammalian testis.

Characterization of a novel form of angiopoietin-2 (Ang-2B) and expression of VEGF and angiopoietin-2 during chicken testicular development and regression.

The complex process of angiogenesis is controlled by the vascular endothelial growth factor (VEGF) and its receptors and by the recently isolated angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2) that signal through the transmembrane endothelial receptor tyrosine kinase Tie2. We report here the characterization of a novel form of Ang-2 (Ang-2B) with a truncated amino-terminal domain resulting from an alternative splicing of the gene. While previous reports have found the expression of Ang-2 limited to the embryo, female reproductive organs, and tumor tissues, we have observed striking changes in Ang-2 expression during chicken testicular development and regression. The expression of Ang-2 and VEGF is abundant in prepuberal testis and low in quiescent adult testis. Testicular regression is accompanied by high expression of Ang-2 and very low expression of VEGF. These observations are in accordance with the proposal that Ang-2 induces angiogenesis in the presence of VEGF and vascular regression in its absence.

Novel transcripts of carbonic anhydrase II in mouse and human testis.

Intracellular and extracellular sources of bicarbonate are essential for sperm motility, sperm binding to the zona pellucida and the acrosome reaction. Carbonic anhydrase II, catalysing the synthesis of bicarbonate within spermatozoa, must play a significant role in these mechanisms. We report here the expression of carbonic anhydrase II during mouse spermatogenesis and the primary structure of testicular transcripts coding for carbonic anhydrase II isolated from adult mouse and human testes. The mouse carbonic anhydrase II (Car2) mRNA displays a 5' untranslated region (UTR) larger than the corresponding somatic sequence. The additional 5' sequence contains the 'TATA box' used in somatic tissues and other promoter sequences, suggesting the use of testis-specific promoters further upstream with read-through of downstream promoters. The 3'UTR of the Car2 mRNA is shorter in mature testicular cells than in somatic cells. Polysomal gradient analysis of carbonic anhydrase II transcripts isolated from adult mouse testis and kidney revealed different translation potential: most of the testicular transcripts were present in the non-polysomal fractions, whereas a considerable fraction of kidney transcripts were polysome-associated. These results suggest that specific transcriptional and post-transcriptional mechanisms regulate the expression of carbonic anhydrase II during mammalian spermatogenesis.

Marked differences between avian and mammalian testicular cells in the heat shock induction and polyadenylation of Hsp70 and ubiquitin transcripts.

Mammalian male germ cells undergo apoptosis at the body's internal temperature of 37 degrees C. Birds, however, are unique among homeothermic animals in developing spermatogenesis at the elevated avian internal body temperature of 40-41 degrees C. To shed light on the mechanisms that maintain an efficient avian spermatogenesis at elevated temperatures we compared, in mouse and chicken testicular cells, the expression of genes that are essential for stress resistance: Hsp70 and ubiquitin. While the expression of Hsp70 and ubiquitin did not change upon heat shock in mouse testicular cells, both the amount and polyadenylation of Hsp70 and ubiquitin transcripts increased when male germ cells from adult chicken testis were exposed to elevated temperatures.

Several novel transcripts of glyceraldehyde-3-phosphate dehydrogenase expressed in adult chicken testis.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), in addition to being a classic glycolytic enzyme, is a multifunctional protein involved in relevant cell functions such as DNA replication, DNA repair, translational control of gene expression, and apoptosis. Although the multifunctional nature of GAPDH suggests versatility in the mechanisms regulating its expression, no major qualitative changes and few quantitative changes in the GAPDH transcripts have been reported. While studying the expression of GAPDH during spermatogenesis, we detected alternative initiations to TATA box and alternative splicings in the 5' region of the pre-mRNA, resulting in at least six different types of mRNAs. The amount and the polyadenylation of the GAPDH transcripts increased in mature testis in relation to immature testis and further increased when cell suspensions from mature testis were exposed to heat shock. These results suggest that alternative initiation, alternative splicing, and polyadenylation could provide the necessary versatility to the regulation of the expression of this multifunctional protein during spermatogenesis.

Four isoforms of the signal-transduction and RNA-binding protein QKI expressed during chicken spermatogenesis.

Genes expressed during spermatogenesis undergo alternative initiation and alternative splicing and may be under the control of a coordinated mechanism of RNA processing. A family of proteins that combine features of signal-transduction and RNA-binding molecules could be instrumental in this process. We have characterized a cDNA from adult chicken testis that codifies a highly conserved member of the STAR protein family, the orthologue of the mouse quaking gene qki. The predicted chicken protein differs only in four amino acids from the corresponding mouse protein. Messages of 7, 6, and 5 kb are expressed differentially during chicken spermatogenesis. The 5-kb message, the predominant form in adult testis, presents heterogeneity in the coding region, showing insertions of 51 and 75 bp and a deletion of 24 bp, which gives rise to four possible isoforms of the protein.

Characterization and expression of two chicken cDNAs encoding ubiquitin fused to ribosomal proteins of 52 and 80 amino acids.

We have determined the complete nucleotide sequence of two chicken cDNAs, Ub-t52 and Ub-t80, encoding ubiquitin fused to ribosomal proteins of 52 and 80 amino acids. The deduced amino acid sequences of the ribosomal proteins are identical or very similar to the homologous human and rat proteins and to the corresponding proteins of other species. Unexpectedly, the ubiquitin moiety of the Ub-t52 protein showed two amino acid substitutions: serine-20 has been replaced by asparagine and serine-57 by alanine. Ubiquitin is a protein strongly conserved during evolution, with no changes in sequence previously reported in vertebrates. Ub-t52 and Ub-t80 are highly expressed in early embryogenesis and during postmitotic stages of spermatogenesis, in parallel with the expression of the polyubiquitin gene UbII. Whereas the 5' untranslated regions (5'UTRs) of the chicken polyubiquitin mRNAs showed marked differences in mature testes in relation to somatic tissues, no differences were observed in the 5'UTRs of the ubiquitin-ribosomal protein mRNAs. These mRNAs possess a 5'-terminal oligopyrimidine tract that could be used as a mechanism to postpone translation during postmitotic stages of spermatogenesis, as has been proposed in quiescent cells.

Differential expression of bcl-2 and bcl-x during chicken spermatogenesis.

We report the isolation and characterization of a chicken testis bcl-XL cDNA coding for a long bcl-x protein with a hydrophobic tail, and the expression of bcl-2 and bcl-x during chicken spermatogenesis. Bcl-2 is highly expressed in embryonic and immature testes enriched in spermatogonia and barely detectable in mature testes, where most of the cells are meiotic and postmeiotic. Bcl-x is expressed in both mature and immature testes, but in a lesser amount in mature testes. Differential expression of bcl-2 and bcl-x during spermatogenesis is consistent with the reported different susceptibility to apoptosis of spermatogonia, and meiotic and postmeiotic cells.

Heat-shock inducible polyubiquitin gene UbI undergoes alternative initiation and alternative splicing in mature chicken testes.

Ubiquitin, a heat-shock protein highly expressed during spermatogenesis, plays an essential role in the differentiation of the germinal cells, particularly in the structural changes of chromatin taking place at the end of the process. To shed light on the mechanisms that modulate transcriptional activity of the heat-shock inducible polyubiquitin gene UbI during spermatogenesis and stabilize the message when transcription is not longer active, we have compared the characteristics of UbI transcripts in mature and immature testes and somatic cells. In mature chicken testes, transcription starts at a site placed closer to the heat-shock promoters than in somatic tissues. This site is upstream from the TATA box used in somatic cells. In addition, UbI transcript undergoes an alternative splicing that produces a longer 5' untranslated region in mature testis. These findings may provide a basis for the observed increase in expression of UbI in mature chicken testes and for the stability of the message when transcription ceases at the end of spermatogenesis.

Alternative transcriptional initiation and alternative use of polyadenylation signals in the alphaB-crystallin gene expressed in different chicken tissues.

Overexpression of alphaB-crystallin is associated with numerous neurodegenerative diseases and abnormal cell growth patterns. To study the mechanisms involved in the control of the transcriptional activity of the gene we have characterized its expression in different chicken tissues. The sequence of the alphaB-crystallin cDNA isolated from chicken testis and 6-day-old chick embryo is highly homologous to the duck alphaB-crystallin cDNA and differs from the previously reported chicken lens alphaB-crystallin cDNA in the 5' untranslated region (5'-UTR) and in one amino acid of the coding sequence. Four forms of the alphaB-crystallin cDNA detected in chicken testes arise from the use of alternative transcription initiation sites and alternative polyadenylation signals. The two principal hybridizing bands found in lens and embryonic tissues possess a short 5'-UTR and differ in the length of the 3'-UTR. Forms with longer 5'-UTR are present in testis, muscle, and heart. The use of different start sites and polyadenylation signals could modulate transcriptional activity and the stability of the messages. The expression of the alphaB-crystallin gene decreases from day 6 to day 8 of chick embryogenesis, in parallel with the expression of the polyubiquitin gene UbII.

Study of the 3'-ApoB minisatellite performed by PCR in the population of Catalonia (northeast Spain).

Allele and genotype frequencies for the 3'-ApoB locus were determined in a population sample from Catalonia (northeast Spain) using PCR amplification and nonradioactive detection. In a total of 308 unrelated individuals, 16 alleles and 50 genotypes were observed. The 3'-ApoB locus demonstrated a heterozygosity of 80%. The distribution of genotypes is in agreement with expected values according to the Hardy-Weinberg equilibrium.

Suitability of the YNZ22 (D17S5) VNTR polymorphism for legal medicine investigations in the population of Catalonia (Spain).

Allele and phenotype frequencies for the YNZ22 locus were determined in a population sample from Catalonia (Spain) using the polymerase chain reaction (PCR). In 311 unrelated individuals, 14 alleles and 56 phenotypes were observed. No deviation from Hardy-Weinberg equilibrium was found. The observed heterozygosity was 81.35%. The YNZ22 polymorphism is useful for paternity testing with a CE value of 70% and an Essen-Möller value of 9.35 (log.).

A novel carbonic anhydrase II mRNA isolated from mature chicken testis displays a TATA box and other promoter sequences in a leader 5' untranslated region not present in somatic tissues.

The primary structure of a novel carbonic anhydrase II-encoding cDNA clone (CAII) isolated from a chicken testis cDNA library is presented. The size of the CAII mRNA obtained from meiotic and haploid chicken testis cells is larger than the corresponding mRNA from immature testis and somatic tissues. The nucleotide sequence of the chicken testis CAII clone revealed a protein-coding region identical to the published sequence of CAII mRNA from erythroid cells. However, the 5' untranslated region (UTR) of the testis CAII mRNA is larger than the corresponding somatic sequence. The 5' UTR contains a leader sequence not present in the CAII mRNA isolated from erythroid cells or chick retina. The additional 5' UTR of the mRNA displays a TATA box, located 23-30 bp upstream from the cap site of the CAII mRNA transcribed in erythroid cells, and several G+C-rich boxes. Our results suggest that the use of a testis-specific promoter would result in the incorporation of somatic promoter sequences into the 5' UTR of the testis message.

Intron and intronless transcription of the chicken polyubiquitin gene UbII.

We have previously reported that the chicken polyubiquitin gene UbII is preferentially expressed during spermatogenesis and we show here that UbII is the predominant polyubiquitin gene expressed in early embryogenesis. Two main initiation sites were detected. Transcription from the initiation site used in early embryos results in the presence of an intron in the 5'-untranslated region of the transcripts as has been reported for other polyubiquitin messages. In mature testis, however, the use of a different initiation site, located within the intron, produces intronless transcripts. Distinct promoter sequences, present in each initiation site, may regulate the differential expression observed in this gene.