HSFY (heat shock transcription factor, Y chromosome) has been mapped in the AZFb region of the Y chromosome, whose deletion results in severe male infertility. HSFY belongs to the heat shock factor family that has been shown to be implicated in spermatogenesis both in animals and humans. We report the characterization of the genomic structure, the number of copies on the Y chromosome and the expression of the gene. By comparison of data obtained from expression analysis and from GenBank cDNA sequences, seven exons were identified. Alternative splicing generates three different transcripts and proteins, each containing an HSF domain typical of HSF proteins. Two identical and functional full-length copies of HSFY map in palindrome P4 of AZFb, whereas four similar sequences mapping in two clusters in palindrome P1 of AZFc and P3 seem to represent pseudogenes. Sequences similar to few HSFY exons are also located in the short arm of chromosomes Y, X and 22. Expression analysis revealed that the three HSFY transcripts are differentially expressed, transcript 1 being present in many tissues including testis and ejaculated sperm, and transcripts 2 and 3 being testis-specific. These data suggest that HSFY could have an important role in human spermatogenesis.

Vascular endothelial growth factor (VEGF) is one of the key regulators of tumor neoangiogenesis. It acts through two types of high-affinity tyrosine kinase receptors (VEGF receptor-1 [VEGFR-1]/fms-related tyrosine kinase 1 [Flt-1] and VEGFR-2/kinase domain receptor [KDR]) expressed on endothelial cells. VEGFRs have also been detected on cancer cells, suggesting a possible autocrine effect of VEGF on their growth. We studied the expression of VEGF, VEGFR-1, and VEGFR-2 in human medulloblastoma cell lines (DAOY, D283Med, and D341Med) and investigated the possible autocrine mechanisms of VEGF on medulloblastoma cell proliferation. Reverse transcriptase PCR analysis showed the presence of VEGF and VEGFR mRNAs in all cell lines studied. Of the three VEGF isoforms, VEGF(121) and VEGF(189) were detected by Western blot analysis in all three medulloblastoma cell lines, whereas VEGF(165) was identified only in DAOY cells. Medulloblastoma cell lines expressed both VEGFR-1 and VEGFR-2. We also demonstrated expression of VEGF and its receptors in medulloblastoma tumor specimens. Exogenous VEGFR-2 inhibitor reduced the VEGF-dependent cell proliferation of DAOY and D283Med cells. In DAOY cells, VEGF(165) induced phosphorylation of VEGFR-2/KDR and of downstream proteins in the signal transduction pathway. These data suggest a possible autocrine role for VEGF in medulloblastoma growth. Targeting VEGF signaling may represent a new therapeutic option in the treatment of medulloblastoma.

During testicular development and maturation, extracellular matrix (ECM) remodelling is a fundamental process which requires the presence of several proteases and protease inhibitors. Among the proteases, a pivotal role has been proposed for matrix metalloproteases (MMPs) and their tissue inhibitors (TIMPs). Here we report an analysis of MMP-2, MT1-MMP and TIMP-2 expression by rat Sertoli cells in culture using RT-PCR and zymographic techniques. Stimulating Sertoli cells with follicle-stimulating hormone (FSH) in vitro induced evident changes in the level of their mRNA in a time-dependent manner. In the case of TIMP-2 and MT1-MMP, the respective transcripts were augmented up to three-fold after 24 h of treatment, and MMP-2 transcripts increased by four times in the same period. MMP-2 activity determined by gelatin zymography showed an increase in enzyme secretion after FSH stimulation. The results of this study suggest that: (i) at the surface of Sertoli cells pro-MMP-2 activation mediated by MT1-MMP may occur, involving TIMP-2 as a receptor; and (ii) the expression of these molecules is not constitutive in this cell type, but may be modulated by FSH and is therefore implicated in spermatogenesis.

PD1 is a novel protein particularly expressed at the testicular level. The relative cDNA sequences were cloned from human and rat testis libraries revealing an open reading frame for a protein of 520 and 511 amino acids respectively. The human PD1 amino acid sequence shows 85% identity with rat sequence suggesting that PD1 gene has been highly conserved during mammalian evolution. Immunohistochemical analysis showed that this protein is detected in the tubular compartment of the testis and, in particular, in the cytoplasm of the Sertoli cells. PD1 expression is not constitutive but seems to be under the influence of neighboring spermatogenic cells as demonstrated by its reduction in hypospermatogenesis with respect to normal spermatogenesis and a further reduction in Sertoli cell-only syndrome. During testicular development in the rat (from 2 to 45 days of age) the PD1 mRNA level became detectable at 14 days and then increased steadily with an advancement of age. These findings suggest that PD1 may play a role in the regulation of spermatogenesis and may be a potential candidate gene for defects of male fertility.

Spata2 (spermatogenesis-associated protein 2) was originally described as a novel gene involved in the spermatogenic process. In this study, we cloned a potential zebrafish spata2 orthologue. The consensus open reading frame (1650 bp) encodes a polypeptide of 550 amino acids which shares 37% identity with the human SPATA2. Bioinformatic analysis reveals a small pattern PW [KR] KE [YF][RK] which seems to be of particular interest in the light of its strong conservation between SPATA2 and the recently discovered TAMO protein of D. melanogaster. RT-PCR analysis in adult zebrafish tissues revealed that spata2 mRNA has a broad distribution. Whole-mount in situ hybridization demonstrated that spata2 transcripts are maternally derived and becomes strongly localized in the central nervous system at early developmental stages. From 5 dpf, spata2 expression becomes detectable in the gut and pronephric duct epithelium, suggesting a wide tissue function during vertebrate development. KEY WORDS: spata2, zebrafish (Danio rerio), whole-mount in situ hybridizationpd1 was initially identified and cloned as a novel human gene from testis cDNA library (Graziotto et al., 1999). The rat orthologue of human pd1 was further cloned and, according to the Gene Nomenclature Committee, was renamed spata2 (spermatogenesis-associated protein 2) gene on the basis of expression analysis performed on rat testis at different testicular developmental stages (Onisto et al., 2000). The analysis of the human and rat cDNA sequences disclosed an open reading frame for a protein of 520 and 511 amino acids respectively, with an overall identity of 85%. High expression spata2 levels have been detected in the brain, both in human and rodents, whereas less abundant transcripts were identified by virtue of RT-PCR experiments in skeletal muscle and kidneys (Onisto et al., 2001). Using a bioinformatic approach, it has been predicted that SPATA2 protein is highly hydrophilic, contains a cluster of 17 cysteins at its C-terminus and shares a significant similarity with the recently characterized Drosophila melanogaster TAMO protein at the N-terminus (Minakhina et al., 2003).The promoter region and the structural organization of the human spata2 gene were also determined showing that this gene is composed of three exons and two introns spanning a region of about 12.1 kb localized at 20q13.13 (Slongo et al., 2003). The transient expression of chimeric SPATA2-GFP in HL-TAT cells (Onisto et al., 2001) revealed that spata2 has a nuclear localization, but a clear function at the cellular level has Int. J. Dev. Biol. 51: 241-246 (2007) Abbreviations used in this paper: aa, aminoacid; BCIP, bromo-4-chloro-3-indolyl phosphate; bp, base pair; CNS, central nervous system; cDNA, complementary DNA; Dig, digoxygenin; dpf, days post fertilization; EDTA, ethylenediaminetetra-acetic acid; hpf, hours post fertilization; kb, kilobase; NBT, nitro blue tetrazolium; nt, nucleotide; PCR, polymerase chain reaction; RACE, rapid amplification of cDNA ends; RT-PCR, reverse tr...