Interactions between mouse ZP2 glycoprotein and proacrosin; a mechanism for secondary binding of sperm to the zona pellucida during fertilization.

The mouse zona pellucida glycoprotein, mZP2, is thought to be the secondary receptor on eggs for retention of acrosome-reacted sperm during fertilization. Here, we present evidence that one of its complementary binding proteins on sperm is proacrosin/acrosin. mZP2 binds to proacrosin null sperm considerably less effectively than to wild-type sperm. Binding is mediated by a strong ionic interaction between polysulphate groups on mZP2 and basic residues on an internal proacrosin peptide. The stereochemistry of both sulphate groups and basic amino acids determines the specificity of binding. Structurally relevant sulphated polymers and suramin, a polysulphonated anticancer drug, compete with mZP2 for complementary binding sites on proacrosin/acrosin in solid-phase binding assays. The same competitors also displace attached sperm from the zona pellucida of eggs in an in vitro fertilization system. This combination of genetic, biochemical and functional data supports the hypothesis that mZP2-proacrosin interactions are important for retention of acrosome-reacted sperm on the egg surface during fertilization. Safe mimetics of suramin have potential as non-steroidal antifertility agents.

Assessment of promoter elements of the germ cell-specific proacrosin gene.

The testis-specific proacrosin gene encodes for a fertilization-promoting protein. In mouse and rat it is first transcribed in late pachytene spermatocytes and revealed to be translationally regulated. Former proacrosin promoter studies demonstrated that elements necessary for conducting a stage and temporal-specific expression of the gene are located within 0.9 kb upstream of the translational start codon. In the present study we analyzed putative cis-acting elements located in this promoter region for their specific binding properties to nuclear factors assumed to be involved in proacrosin gene regulation. Supplement of specific antibodies in electrophoretic mobility shift assays (EMSA) revealed that two Y-box proteins and the transcription factors CREM and YY1 interact with proacrosin promoter elements. The Y-box proteins, antigenically related to the frog Y-box proteins FRGY1 and FRGY2, bound to the Y-box (55-66 bp upstream of the ATG initiation codon) in brain and testis nuclear extracts, respectively. CREM bound to three elements (30-37, 252-259, and 717-724 bp upstream of ATG). The ubiquitous transcription factor YY1 bound to a conserved element in the central proacrosin promoter (457-473 bp upstream of ATG) and showed almost germ cell-specific truncates in EMSA. These results suggest that the identified factors are involved in proacrosin gene regulation.

Developmental stage- and germ cell-regulated expression of a calcium-binding protein mRNA in mouse Sertoli cells.

There is considerable evidence that germ cells, mainly spermatocytes and spermatids, contribute to the regulation of Sertoli cell activity. We developed an in vitro system to investigate the genes involved in Sertoli cell-germ cell interaction in the mouse by using the differential mRNA display technique. One of the isolated differentially expressed genes, named calgizzarin, belongs to the family of S100 calcium-binding proteins and shows a decreased expression in Sertoli cell-germ cell cocultures compared to cultured Sertoli cells alone. Calgizzarin is expressed in all adult tissues examined, including testis and ovary; however, a high mRNA level for calgizzarin in mouse testis is maintained until day 15 of postnatal development and then declines dramatically, whereas the expression pattern in the ovary remains constantly high. Furthermore, Northern blot studies on testicular RNA from different mouse mutants with defects in spermatogenesis revealed that high levels of calgizzarin transcripts can only be detected in testes of mouse mutants with either no germ cells or primary spermatocytes, but only weak signals for calgizzarin are observed in testes of mutants containing spermatids. In addition, using both RT-PCR analysis and whole-mount in situ hybridization on dissected gonads it was demonstrated that mouse calgizzarin expression starts at 13.5 dpc in the prenatal male gonad and at 16.5 dpc in the embryonic ovary, respectively. The mouse calgizzarin gene was localized on mouse chromosome 5, region E-F. Taken together, our results indicate that calgizzarin expression could be repressed by factors originated from pachytene spermatocytes and/or spermatids.

Molecular cloning of a SALL1-related pseudogene and mapping to chromosome Xp11.2.

SALL1 and SALL2 have been identified as two human homologs of the region-specific homeotic gene spalt (sal) of Drosophila, which encodes a zinc finger protein of characteristic structure. SALL1 has recently been found to be mutated in patients with Townes-Brocks syndrome (TBS, OMIM No. 107480). Here we report the isolation and mapping of another sal-like human gene, named SALL1P, on chromosome Xp11.2. This intronless gene closely resembles SALL1 but displays several mutations, suggesting that SALL1P represents a sal-related pseudogene. The high similarity of SALL1P to SALL1 is of considerable importance for mutation analysis of SALL1 in TBS.

Yeast one-hybrid assay identifies YY1 as a binding factor for a proacrosin promoter element.

The proacrosin gene is specifically expressed in the testis and encodes an acrosomal enzyme. Previously, footprint analyses have shown binding of nuclear extracts from testis and brain to a highly conserved 17 bp motif (F1 element: 5'-AACTTCAAAATGGCTCC/T-3') located in the proacrosin promoter. By using this DNA-element as a target in a yeast one-hybrid assay, a cDNA fragment coding for the C-terminal part of the transcription factor YY1 was isolated. The binding of YY1 to this F1 element was confirmed by immunocompetition in EMSA. Because putative YY1 binding sites were also found in the promoters of other testis-specific genes, the YY1 transcription factor could play an important role in testicular gene expression.

Subtype selectivity of the novel nonpeptide neuropeptide Y Y1 receptor antagonist BIBO 3304 and its effect on feeding in rodents.

1. The novel Y1-selective argininamide derivative BIBO 3304 ((R)-N-[[4-(aminocarbonylaminomethyl)-phenyl]methyl]-N2-(diphen ylacetyl)-argininamide trifluoroacetate) has been synthesized and was examined for its subtype selectivity, its in vitro antagonistic properties and its food intake inhibitory properties. 2. BIBO 3304 displayed subnanomolar affinity for both the human and the rat Y1 receptor (IC50 values 0.38+/-0.06 nM and 0.72+/-0.42 nM, respectively). The inactive enantiomer of BIBO 3304 (BIBO 3457) had low affinity for both the human and rat Y1 receptor subtype (IC50> 1000 nM). BIBO 3304 showed low affinity for the human Y2 receptor, human and rat Y4 receptor as well as for the human and rat Y5 receptor (IC50 values > 1000 nM). 3. 30 microg BIBO 3304 administered into the paraventricular nucleus inhibited the feeding response induced by 1 microg NPY as well as the hyperphagia induced by a 24 h fast implying a role for Y1 receptors in NPY mediated feeding. The inactive enantiomer had no effect. 4. BIBO 3304 inhibits neither the galanin nor the noradrenaline induced orexigenic response. but it blocked feeding behaviour elicited by both [Leu31, Pro24]NPY and NPY (3 36) suggesting an interplay between different NPY receptor subtypes in feeding behavior. 5. The present study reveals that BIBO 3304 is a subtype selective nonpeptide antagonist with subnanomolar affinity for the Y1 receptor subtype that significantly inhibits food intake induced by application of NPY or by fasting.

Mutations in the SALL1 putative transcription factor gene cause Townes-Brocks syndrome.

Townes-Brocks syndrome (TBS, OMIM #107480) is a rare autosomal-dominant malformation syndrome with a combination of anal, renal, limb and ear anomalies. Cytogenetic findings suggested that the gene mutated in TBS maps to chromosome 16q12.1, where SALL1 (previously known as HSAL1), a human homologue of spalt (sal), is located. SAL is a developmental regulator in Drosophila melanogaster and is conserved throughout evolution. No phenotype has yet been attributed to mutations in vertebrate sal-like genes. The expression patterns of sal-like genes in mouse, Xenopus and the fish Medaka, and the finding that Medaka sal is regulated by Sonic hedgehog (Shh; ref. 11), prompted us to examine SALL1 as a TBS candidate gene. Here we demonstrate that SALL1 mutations cause TBS in a family with vertical transmission of TBS and in an unrelated family with a sporadic case of TBS. Both mutations are predicted to result in a prematurely terminated SALL1 protein lacking all putative DNA binding domains. TBS therefore represents another human developmental disorder caused by mutations in a putative C2H2 zinc-finger transcription factor.

Five novel mutations in the L1CAM gene in families with X linked hydrocephalus.

Five novel mutations have been identified in the gene encoding L1CAM, a neural cell adhesion protein, in families with X linked hydrocephalus (XHC). Interestingly, all five mutations are in the evolutionarily highly conserved Ig-like domains of the protein. The two frameshift mutations (52insC and 955delG) and the nonsense mutation (Trp276Ter) most probably result in functional null alleles and complete absence of L1CAM at the cell surface. The two missense mutations (Tyr194Cys and Pro240Leu) may considerably alter the structure of the L1CAM protein. These data provide convincing evidence that XHC is genetically extremely heterogeneous.

Cloning, expression, and chromosomal localization of the rat mitochondrial capsule selenoprotein gene (MCS): the reading frame does not contain potential UGA selenocysteine codons.

The mitochondrial capsule selenoprotein (MCS) is a selenium-containing polypeptide. It is one of three proteins that are important for the maintenance and stabilization of the crescent structure of the sperm mitochondria. In this paper, we report the isolation and characterization of the rat MCS cDNA and gene. The cDNA contains a reading frame for a 145-amino-acid protein and it lacks the UGA codons, which have been found in the reading frame of the mouse MCS cDNA and have been presumed to encode the selenocysteine in the amino terminal of the deduced mouse amino acid sequence. The deduced amino acid sequence of the rat and mouse MCS shows a high level of homology (79%). The rat MCS gene contains two exons; the intron sequence interrupts the 5' untranslated sequence at the same position as in the mouse MCS gene. The transcription start site is located 184 bp upstream of the translation start site. Alignment of the 5'-flanking regions of the mouse and rat genes reveals that the first 400 nucleotides upstream of the transcription start site exhibit an overall sequence similarity of 73%. This conserved region contains no TATA or CAAT box motifs. Northern blot analysis indicates that the MCS mRNA is detectable only in the testis after day 30 of postnatal development. Moreover, in situ hybridization revealed that the rat MCS gene is mainly expressed in round spermatids. From the analysis of mouse-rat cell hybrids that segregate rat chromosomes, the MCS gene was assigned to rat chromosome 2.

Kinetic analysis of thyrotropin-releasing hormone binding in the central nervous system: evidence for receptor desensitization.

To investigate the mechanism(s) of experimentally and clinically observed refractoriness of spinal lower motor neurons (LMNs) to the excitatory effects of high-dose TRH, we examined the kinetics of dissociation of [3H]TRH from its CNS-receptor. At 23 degrees C, the receptor was rapidly (40 min) and completely converted from a form with fast dissociation kinetics (complex I; t1/2 20-30 min) to one from which the peptide dissociated much more slowly (complex II; t1/2 greater than 120 min). This conversion required the presence of added agonist ([3H]TRH) and was not prevented by the GTP-analog Gpp(NH)p. We suggest that complexes I and II may respectively represent active and inactive (desensitized) forms of the TRH-receptor and that TRH-induced I to II conversion of the receptor is responsible for refractoriness of LMNs to the drug.

bc1-Complex from beef heart. One-step purification by hydroxyapatite chromatography in Triton X-100, polypeptide pattern and respiratory chain characteristics.

A new simple method for the purification of the bc1-complex has been developed. The polypeptide composition of the complex was analysed by dodecyl sulfate-polyacrylamide gel electrophoresis. The content of chain components and phospholipids was determined. The b-type cytochromes were further characterized by their absorbance spectra and midpoint potentials. (1) Starting from a Triton X-100 extract of submitochondrial particles supplemented with antimycin, the bc1-complex is purified by adsorption chromatography on hydroxyapatite with citrate as specific eluant. (2) The complex splits in dodecyl sulfate into five main polypeptides with apparent molecular weight of 47, 44, 31, 11 and less than 10 kdalton. (3) The purified complex has a heme-b content of 8.0 mumol/g protein and a cytochrome c1 content of 3.8 mumol/g protein. (4) The cytochromes show the typical absorbance spectra of cytochromes b-562 and b-565 and are present in approximately equal amounts with midpoint potentials of Em7 = + 100 mV and Em7 = + mV respectively. Carbon monoxide does not bind to the cytochromes. (5) The nonheme iron protein content of the complex is diminished to 0.6 mumol/g protein. (6) The use of the nonionic surfactant Triton X-100 leads to a complete loss of lipids and ubiquinone of the bc1-complex. (7) The complex contains no succinate dehydrogenase as indicated by the absence of the 69 kdalton subunit in the dodecyl sulfate gel electrophoresis. In addition, it lacks an ubiquinone cytochrome c reductase activity and other electron transferring activities. This may be inferred from an inhibition by antimycin and depletion of ubiquinone and phospholipids. The highly purified and relative stable complex can be prepared giving 50% yield and may be suitable for protein chemistry studies.