RESUMEN

Hyaluronidases (hyases) are a family of enzymes that catalyse the breakdown of hyaluronic acid (HA), which is abundant in the extracellular matrix. Two unlinked gene clusters encode these six proteins: three each in the somatic (or ubiquitous) acid-active subgroup and the neutral-active germ-cell subgroup. This review analyses the data on the expression and role of hyases in gamete biology and fertilization, using electronic databases until October 2010. Evidence indicates that hyases are membrane proteins with multifunctional essential, enzymatic and non-enzymatic, roles (cumulus penetration, zona binding and HA receptor) in fertilization. While sperm adhesion molecule-1 (SPAM1), which has neutral and acidic (bimodal) activity, is the widely conserved mammalian sperm hyase, it co-exists with an acidic hyase in murine and human spermatozoa. Thus, sperm function depends on the concerted activity of both germ cell and 'somatic' hyases. Some hyases are in low abundance in the ovary, somatic testicular cells, the male accessory organs and the male and female genital tracts where they are secreted and acquired by spermatozoa. The latter opens up the possibility of treating hyase-deficient spermatozoa via assisted reproductive technology. The findings challenge the existing classification of hyases, and support the notion that hyase activities are polygenic traits controlled by as many as five hyase genes in mice. Multiple sperm hyases may function cooperatively in a quantitative system and/or serve redundant roles. Unsolved problems include functional redundancy, which can be addressed by double gene-knockouts, and identifying the murine hyase(s) involved in zona binding or whether this role shows species specificity.

Asunto(s)

Fertilización/fisiología , Hialuronoglucosaminidasa/fisiología , Espermatozoides/fisiología , Animales , Moléculas de Adhesión Celular/biosíntesis , Moléculas de Adhesión Celular/metabolismo , Membrana Celular/metabolismo , Femenino , Células Germinativas/metabolismo , Humanos , Ácido Hialurónico/metabolismo , Hialuronoglucosaminidasa/biosíntesis , Hialuronoglucosaminidasa/metabolismo , Masculino , Ratones , Ovario/enzimología , Cabeza del Espermatozoide/enzimología , Interacciones Espermatozoide-Óvulo/fisiología , Espermatozoides/enzimología , Testículo/enzimología

Asunto(s)

Cromosomas/genética , Hibridación Fluorescente in Situ , Conejos/genética , Receptores de Hormona Paratiroidea/genética , Animales , Bandeo Cromosómico , Cromosomas Humanos Par 3/genética , Humanos , Mapeo Físico de Cromosoma , Receptor de Hormona Paratiroídea Tipo 1

RESUMEN

The sperm adhesion molecule 1 (SPAM1 or PH-20) is an important sperm surface protein with a hyaluronidase activity and bifunctional roles in mammalian fertilization. Recently we reported that in the mouse, Spam1 is synthesized independently in the testis and the epididymis, where it is found in membranous vesicles in the principal cells of the epithelium in all three regions. Here we used mouse epididymal luminal fluid and cultured epididymal epithelial cells to demonstrate that epididymal Spam1 may be a secretory protein. Using a dual environment culture chamber system in which corpus or cauda epithelial cells are cocultured with their corresponding epididymal fibroblasts in medium supplemented with androgens and epidermal growth factor, we show that in 2- to 6-day cultures Spam1 can be detected immunocytochemically in the epithelial cells. The protein was also detected by Western blot analysis in extracts of the cultured cells and in their serum-free conditioned medium, as well as in luminal fluid from fresh caput, corpus, and caudal epididymis. Importantly, it was shown to have hyaluronidase activity, using hyaluronic acid substrate gel electrophoresis, and to be expressed in greater quantities in the corpus compared with the cauda and caput. The results not only confirm our previous finding that Spam1 is synthesized in the epididymis, but extend them by showing that it is released in the luminal fluid where it may effect posttesticular maturation and function of sperm. Results from transcript analysis indicate that epididymal and testicular Spam1 are under different transcriptional regulation.

Asunto(s)

Moléculas de Adhesión Celular/metabolismo , Epidídimo/metabolismo , Andrógenos/administración & dosificación , Animales , Western Blotting , Moléculas de Adhesión Celular/biosíntesis , Células Cultivadas , Técnicas de Cocultivo , Medios de Cultivo , Factor de Crecimiento Epidérmico/administración & dosificación , Células Epiteliales/fisiología , Fibroblastos/metabolismo , Hialuronoglucosaminidasa/metabolismo , Immunoblotting , Queratinas/análisis , Masculino , Ratones , Ratones Endogámicos ICR , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transcripción Genética

RESUMEN

Gametic equality is thought to exist, despite haploid gene action in mammalian spermiogenesis, because of product sharing via the intercellular bridges of conjoined spermatids. However, mice carrying different t-alleles have been known to produce functionally different sperm, leading to transmission ratio distortion (TRD), whose mechanism is unknown. The reduced Spam1 mRNA levels, previously shown to be associated with TRD and reduced fertility in mice carrying the Rb(6.16) or the Rb(6.15) Robertsonian translocation, are reflected in the levels of its encoded membrane protein (Spam1) and its accompanying insoluble hyaluronidase activity. Studies of the temporal expression pattern of Spam1 reveal that it is haploid expressed, with both the RNA and protein first appearing on Day 21.5. RNA fluorescence in situ hybridization and immunocytochemistry show both the mRNA and the protein to be compartmentalized. Compartmentalization of the mRNA along with its immediate translation and insertion of the protein in the plasma membrane suggests the nonsharing of Spam1 transcripts among spermatids, resulting in functionally different sperm in males with different Spam1 alleles. Evidence for biochemically different sperm in these heterozygous males was revealed by flow cytometry and confocal microscopy. Our findings support the notion that the Spam1 antigen is not shared, and we may have uncovered a mechanism for TRD.

Asunto(s)

Moléculas de Adhesión Celular/genética , Expresión Génica , Espermátides/química , Animales , Membrana Celular/química , Femenino , Citometría de Flujo , Técnica del Anticuerpo Fluorescente Indirecta , Haploidia , Hialuronoglucosaminidasa/metabolismo , Inmunohistoquímica , Hibridación Fluorescente in Situ , Infertilidad Masculina/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos ICR , Microscopía Confocal , ARN Mensajero/análisis , Translocación Genética

RESUMEN

In mice bearing the Rb(6.16) or Rb(6.15) Robertsonian translocation (Rb), sperm dysfunction associated with the Rbs has been shown to lead to transmission ratio distortions (TRDs) in heterozygotes. The severity of the TRDs is directly related to the severity in the alteration of expression of the gene for the Sperm Adhesion Molecule 1 (Spam1), which maps to proximal mouse Chromosome 6 (Chr 6) near the translocation junction and encodes a sperm antigen with hyaluronidase activity. Here we demonstrate that there is a significantly reduced fertility in the Rb homozygotes (P < 0.001), based on litter size; and that with the Sperm Select Penetration assay Rb-bearing sperm have significantly decreased (P < 0.02-0.001) rates of penetration of hyaluronic acid. Catalytic kinetics studies indicate that reduced Spam1 (PH-20) hyaluronidase activity in the Rb(6.15) mice results from a qualitative defect, while for Rb(6.16) with the greater TRD both a qualitative and a quantitative deficiency (confirmed by Western analysis) of Spam1 exist. Six point mutations were shown to be clustered in the Spam1 hyaluronic acid-binding domain in Rb(6.15). For Rb(6.16) which has a gross genomic alteration at the Spam1 locus, 11 point mutations are scattered in the 5' and 3' UTRs and the coding region, where one leads to the replacement of a conserved residue. Entrapment of spontaneous Spam1 mutations, owing to recombination suppression near the Rb junctions, is proposed as the major underlying defect of the sperm dysfunction.

Asunto(s)

Moléculas de Adhesión Celular/genética , Interacciones Espermatozoide-Óvulo/genética , Translocación Genética , Animales , Análisis Mutacional de ADN , ADN Complementario/química , ADN Complementario/genética , Femenino , Hialuronoglucosaminidasa/metabolismo , Tamaño de la Camada/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Mutación , Mutación Puntual , Recuento de Espermatozoides , Espermatozoides/enzimología

RESUMEN

The gene for the sperm adhesion molecule 1 (PH-20), SPAM1, has been known to be testis-specific and exclusively haploid expressed. We show that in mice, the 2 common isoforms of the protein (Spam1) observed in sperm are also present in the caput, corpus, and cauda epididymides. Both qualitative and quantitative variation of expression of the protein were observed in epididymis with the highest expression detected in the corpus. The endogenous production of enzymatically active (via hyaluronidase) Spam1 by epididymal cells is supported by the detection of steady-state Spam1 epididymal messenger RNA in both wild type and germ cell-deficient mice. In situ transcript hybridization shows the transcript to be localized to the principal cells of the epithelium. The protein was similarly immunolocalized to these cells, predominantly in vesicles near the apical region. The results suggest a mechanism for transportation of Spam1 from the epididymal epithelium to sperm during their transit and storage in the cauda. None of the current categories of spermatogenic-expressed genes shows the dual transcription pattern (haploid testicular/diploid epididymal) observed for Spam1. The work also confirms and extends the finding that Spam1 is expressed in the kidney.

Asunto(s)

Moléculas de Adhesión Celular/genética , Epidídimo/fisiología , Regulación de la Expresión Génica , Espermatogénesis/genética , Animales , Moléculas de Adhesión Celular/análisis , Moléculas de Adhesión Celular/fisiología , Cruzamientos Genéticos , Epidídimo/citología , Femenino , Hialuronoglucosaminidasa/genética , Hibridación in Situ , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos ICR , ARN Mensajero/análisis , Espermatozoides/fisiología , Testículo/fisiología , Transcripción Genética

RESUMEN

We previously have identified murine heparin/heparan sulfate-interacting protein (HIP) identical to mouse ribosomal protein L29 that is, like its human orthologue, distinctively expressed both on the cell surface and intracellularly in different adult tissues and cell types. In the present study, we show that mouse HIP/RPL29 is encoded by a single mRNA and that it is expressed to different extents in most of the tissues of the developing embryo without restriction to a specific cell type. We isolated the single-copy gene coding for murine Hip/Rpl29 among a large number of pseudogenes, established its structure, and assigned its location to distal chromosome 9. Similar to other ribosomal protein promoters, the promoter of Hip/Rpl29 is rich in polypyrimidine tracts, contains binding motifs for ubiquitously expressed transcription factors, and lacks a TATA box. Progressive 5' deletion analyses identified a strong enhancer region that includes CT-rich sequences and a potential consensus binding site for NF-kappaB. These data will provide valuable tools to progress the understanding of HIP/RPL29 function as a ribosomal protein and/or as a regulator of growth and cell adhesion through interaction with heparan sulfate proteoglycans.

Asunto(s)

Proteínas Ribosómicas/genética , Animales , Secuencia de Bases , Línea Celular , Bandeo Cromosómico , Mapeo Cromosómico , Clonación Molecular , ADN/química , ADN/genética , Embrión de Mamíferos/metabolismo , Femenino , Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Genes/genética , Hibridación in Situ , Hibridación Fluorescente in Situ , Luciferasas/genética , Luciferasas/metabolismo , Masculino , Ratones , Ratones Endogámicos , Datos de Secuencia Molecular , Regiones Promotoras Genéticas/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Análisis de Secuencia de ADN

Asunto(s)

Mapeo Cromosómico , Proteínas del Huevo/genética , Glicoproteínas de Membrana/genética , Ratones Endogámicos C57BL/genética , Muridae/genética , Receptores de Superficie Celular , Animales , Cruzamientos Genéticos , Hibridación Fluorescente in Situ , Cariotipificación , Ratones , Zona Pelúcida/fisiología , Glicoproteínas de la Zona Pelúcida

RESUMEN

Diverse cellular functions are regulated by the calcium-sensing receptor, encoded by the CASR gene, which plays an important role in calcium homeostasis. Here we provide the sequence for exon VII of the rabbit CASR gene and show that it is 91% identical to the human gene at the nucleotide level, and 95% identical at the amino acid level. The gene was mapped by fluorescence in situ hybridization, using a cosmid isolated from a genomic library, to chromosome 14q11 and this was confirmed independently by PCR amplification of flow sorted chromosomes. In addition, the cosmid detected sites with lower frequencies on four other chromosomes: 3q, 5p, 8p, and 13p. Two of these sites (5p and 13p) were also detected by a related but unidentical cosmid, and map to regions that are homologous to the mouse calcium-sensing receptor related sequences (Casr-rs); suggesting that they may represent CASR-related sequences in the rabbit. The data support the presence of a family of genes related to the calcium-sensing receptor in the G-protein coupled receptor (GPCR) superfamily, as well as extend the existing knowledge of homology for several human and rabbit chromosomes.

Asunto(s)

Mapeo Cromosómico , Conejos/genética , Receptores de Superficie Celular/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Calcio/metabolismo , Cósmidos , Exones , Femenino , Biblioteca Genómica , Humanos , Hibridación Fluorescente in Situ , Riñón/metabolismo , Masculino , Ratones , Datos de Secuencia Molecular , Familia de Multigenes , Receptores Sensibles al Calcio , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico

RESUMEN

The Sperm Adhesion Molecule 1 (SPAM1), also known as PH-20, is a sperm membrane-bound protein that has been shown to have bifunctional roles in fertilization. It is encoded by a gene that is widely conserved in mammalian species, underscoring its importance in the fertilization process. Here we determined the genomic structure of the murine homologue, Spam1, using PCR analysis, and studied its transcriptional regulation. The gene covers approximately 10.5 kb of genomic DNA, is encoded by four exons, and the splice site consensus sequence is maintained in all intron-exon junctions, similar to that reported for the human homologue. With primer extension analysis, two transcription initiation sites were detected. One was assigned to the residue C and the other (a minor site) to the residue G, at positions 1 and -56, respectively. These are at 313 and 369 nucleotides upstream of the translation initiation codon, ATG. In about 770 bp upstream region of Spam1 that has been cloned and sequenced, multiple transcription factor binding sites including a CRE (cAMP-responsive element) were found. We specifically studied the function of the eight nucleotide CRE sequence (TGATGTCA) at -57 (or -62 depending on the strain of mice) of the promoter region. It can bind to the transcription factor CREM (cAMP-responsive element modulator) in gel mobility shift assays using mouse testis nuclear extract, and the binding can be inhibited by a 28 bp oligonucleotide containing the CRE sequence. Similar binding and inhibition assays using rat nuclear extract suggest the existence of a rat CRE sequence and the involvement of CREM in rat Spam1 expression. In vitro transcription assays suggest that CRE is necessary for the transcriptional activity of the murine promoter, and Northern analysis shows that Spam1 transcripts are absent in CREM-knockout mice. The results strongly suggest that the murine Spam1 expression is under the control of CREM, and that this transcriptional regulator for Spam1 might be conserved in other mammals, at least in the rat.

Asunto(s)

Moléculas de Adhesión Celular/genética , AMP Cíclico/farmacología , Proteínas de Unión al ADN/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Regiones Promotoras Genéticas , Proteínas Represoras , Animales , Secuencia de Bases , Clonación Molecular , AMP Cíclico/genética , Modulador del Elemento de Respuesta al AMP Cíclico , Proteínas de Unión al ADN/genética , Exones , Hialuronoglucosaminidasa , Ratones , Datos de Secuencia Molecular , Proteínas Nucleares/análisis , Reacción en Cadena de la Polimerasa , Empalme del ARN/genética , ARN Mensajero/genética , Ratas , Transcripción Genética

RESUMEN

Indirect immunofluorescence of mouse caput and caudal sperm shows distinctly different distributions of Spaml protein, which is associated with structural and functional differences of the molecule. Spam1 is uniformly distributed over the surface of the head of caput sperm while in caudal sperm, light and confocal microscopy demonstrate that it is localized to the anterior and posterior regions. The hyaluronidase activity of Spaml in acrosome-intact caput sperm was significantly lower (4.3-fold; P < 0.0001) than that of caudal sperm. The increase in enzymatic activity in caudal sperm is accompanied by a reduction in the molecular weight (MW): in extracts from caput sperm there was a major band at approximately 74 kDa and a minor band at approximately 67 kDa; while for the cauda there was a major band at approximately 67 kDa and minor bands at approximately 70 and -56 kDa. Additionally, the bands from caput sperm were 4.9 to 7.7-fold less intense than those from caudal sperm. This decreased affinity for the polyclonal anti-Spaml suggests the presence of different surface characteristics of the molecule from the two epididymal regions. Computer analysis of the protein structure from Spam1 cDNA sequence reveals four putative N-linked glycosylation sites, and enzymatic deglycosylation suggests that all sites are functional. After endoglycosidase activity of extracts from caput and caudal sperm, both show a major band with a MW of approximately 56 kDa, the size of the membrane-anchored polypeptide backbone. Based on the difference in size and intensity of the Spaml bands and hyaluronidase activities from caput and caudal sperm, the data suggest that the activation of Spaml during epididymal maturation is regulated by deglycosylation.

Asunto(s)

Moléculas de Adhesión Celular/metabolismo , Epidídimo/fisiología , Hialuronoglucosaminidasa/metabolismo , Glicoproteínas de Membrana/metabolismo , Oligosacáridos/metabolismo , Procesamiento Proteico-Postraduccional , Acrosoma , Secuencia de Aminoácidos , Animales , Moléculas de Adhesión Celular/genética , Epidídimo/citología , Glicosilación , Hialuronoglucosaminidasa/genética , Concentración de Iones de Hidrógeno , Isoenzimas/metabolismo , Masculino , Glicoproteínas de Membrana/genética , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Peso Molecular , Espermatozoides/metabolismo

RESUMEN

Stannin is a protein that has been localized to trimethyltin-sensitive cell populations, and evidence suggests it plays a role in the toxic effects of organotins. In this study, we have isolated a mouse stannin genomic clone and have characterized the gene's intron-exon organization, promoter region, and chromosomal location. We have also isolated a partial human stannin cDNA clone and analyzed the open reading frame. The mouse genomic clone spans approximately 19 kb and consists of one intron and two exons. The splice site consensus sequence was maintained at all intron-exon junctions. Promoter analysis suggests that two putative promoter sites exist, each containing multiple regulatory elements and transcription factor-binding sites. Fluorescence in situ hybridization analysis localized stannin to mouse Chromosome (Chr) 16 at band A2. This region is homologous to the proximal region of human Chr 16 (16p13) to which stannin has been previously mapped. Sequence analysis revealed that the 264-bp open reading frame was identical between rat and mouse. The human sequence was 98% identical, with two amino acid substitutions near the c-terminal end of the peptide. These data suggest that stannin is highly conserved between species, and its unusual pattern of cellular expression may, in part, be explained via cell-specific promoters.

Asunto(s)

Mapeo Cromosómico , Neuropéptidos/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Cromosomas Humanos Par 16 , Clonación Molecular , Secuencia Conservada , ADN , Exones , Biblioteca de Genes , Humanos , Intrones , Masculino , Ratones , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Regiones Promotoras Genéticas , Empalme del ARN , Transcripción Genética

RESUMEN

Rat embryonic d 14 (E14) mesencephalic cells, 2.5% of which are glioblasts, were incubated in medium containing 10% of fetal bovine serum for 12 h and subsequently expanded in a serum-free medium using basic fibroblast growth factor (bFGF) as the mitogen. On a single occasion, after more than 15 d in culture, several islets of proliferating, glial-like cells were observed in one dish. The cells, when isolated and passaged, proliferated rapidly in either a serum-free or serum-containing growth medium. Subsequent immunocytochemical analysis showed that they stained positive for GFAP and vimentin, and negative for A2B5, O4, GalC, and MAP2. Serum-free conditioned medium (CM) prepared from these cells caused a fivefold increase in survival and promoted neuritic expansion of E14 mesencephalic dopaminergic neurons in culture. These actions are similar to those exerted by CM derived from primary, mesencephalic type-1 astrocytes. The pattern of expression of the region-selective genes; wnt-1, en-1, sis showed that 70% of the cells were heteroploid, and of these, 50% were tetraploid. No apparent decline in proliferative capacity has been observed after 25 passages. The properties of this cell line, named ventral mesencephalic cell line one (VMCL1), are consistent with those of an immortalized, type-1 astrocyte. The mesencephalic origin of the cell line, and the pattern and potency of the neurotrophic activity exerted by the CM, strongly suggest that the neurotrophic factor(s) identified are novel, and will likely be strong candidates with clinical utility for the treatment of Parkinson's disease.

Asunto(s)

Astrocitos/metabolismo , Línea Celular , Dopamina/fisiología , Mesencéfalo/citología , Proteínas del Tejido Nervioso/análisis , Neuronas/efectos de los fármacos , Animales , Astrocitos/citología , Tamaño de la Célula , Supervivencia Celular/efectos de los fármacos , Medios de Cultivo Condicionados , Medio de Cultivo Libre de Suero , Relación Dosis-Respuesta a Droga , Feto , Expresión Génica , Inmunohistoquímica , Factores de Crecimiento Nervioso , Proteínas del Tejido Nervioso/metabolismo , Proteínas del Tejido Nervioso/farmacología , Neuritas/efectos de los fármacos , Neuritas/metabolismo , Neuronas/citología , Ratas , Ratas Sprague-Dawley , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa

RESUMEN

Recently we mapped the murine Spam1 gene to the proximal region of chromosome 6 (MMU 6). Based on the map location and physiological characteristics of its encoded sperm antigen, the gene is an attractive candidate for the sperm dysfunction seen in Rb(6.16) translocation heterozygotes and the reduced fertility of homozygotes. We have analyzed the expression of Spam1 mRNA in normal and Rb(6.16) mice. The expression of Spam1 mRNA was found to be: 1) tissue specific; it is expressed exclusively in testis; and 2) developmentally regulated, with a haploid expression. Notably, the steady-state mRNA level of Spam1 in Rb(6.16) homozygotes was 25-30% of that in chromosomally normal consomic mice and those homozygous for Rb(2.8) (7.18). In Rb(6.16) and Rb(6.15) heterozygotes the levels were 61% and 66% of the normal. Studies are currently under way to determine the protein levels and gene structure of Spam1, to detect the underlying cause of the mRNA reduction associated with these translocations.

Asunto(s)

Moléculas de Adhesión Celular/genética , ARN/metabolismo , Ribosomas/metabolismo , Animales , Northern Blotting , Bandeo Cromosómico , Mapeo Cromosómico , Hialuronoglucosaminidasa , Masculino , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Testículo/química

RESUMEN

We have determined the chromosomal localization of the murine gene encoding the 68-kDa sperm adhesion molecule 1, Spam1 or Ph-20. Using two independent approaches, fluorescence in situ hybridization (FISH) and interspecific backcross analysis we show the Spam1 maps to proximal mouse Chromosome (Chr) 6. This map position is within the conserved linkage group corresponding to human Chr 7q, where the human homolog, SPAM 1, has been shown to map previously. Genetic mapping shows the gene to be very closely linked to Met, one of the most proximal loci on MMU 6. It thus places the gene near the centromere and the junction of the Rb(6.16)24Lub and Rb(6.15)1Ald translocations. The essential role of the Spam1 sperm antigen in mouse sperm-egg interactions and its gene location provide strong support for its candidacy as the gene involved in the dysfunction of mouse sperm bearing the Rb(6.16)24Lub or Rb(6.15)1Ald translocation.

Asunto(s)

Moléculas de Adhesión Celular/genética , Mapeo Cromosómico , Espermatozoides/fisiología , Animales , Secuencia de Bases , Cromosomas , ADN Complementario , Femenino , Ligamiento Genético , Heterocigoto , Humanos , Hialuronoglucosaminidasa , Masculino , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Interacciones Espermatozoide-Óvulo , Translocación Genética

RESUMEN

Segregants of the Rb(11.14) translocation in 17 heterozygous male mice were analyzed in G- and C-banded first-cleavage metaphases after in vivo (n = 440) and in vitro (n = 267) fertilization. Mating intervals of 3 and 14 d provided unaged and physiologically aged sperm for the oocytes, which were from chromosomally normal females. Significantly more normal than balanced sperm segregants were seen in both the in vivo fertilized (P < 0.01) and in vitro fertilized (P < 0.001) study groups. The distortion was elevated in vitro (P < 0.02), unaccompanied by a sex-ratio distortion, and had a segregant distribution that was independent of sperm age; thus it differs from that seen for other translocations. The findings argue for chromosome-specific effects of Robertsonian translocations on sperm function. A significantly (P < 0.05) increased hyperhaploidy rate (unrelated to the translocation) supports the sperm-aging hypothesis.

Asunto(s)

Translocación Genética , Animales , Bandeo Cromosómico , Femenino , Fertilización , Fertilización In Vitro , Heterocigoto , Masculino , Ratones , Ratones Mutantes , Edad Paterna , Razón de Masculinidad , Espermatozoides , Cigoto

RESUMEN

We have determined the chromosomal localization of the murine gene encoding a 75-kDa inositol polyphosphate-5-phosphatase (Inpp5b). Using two independent approaches, fluorescence in situ hybridization and interspecific backcross analysis, we show that Inpp5b maps to distal mouse Chromosome 4. This map position is within the conserved linkage group corresponding to the short arm of human Chromosome 1, where the human homologue, INPP5B, has been shown to map previously. The position of Inpp5b on mouse Chromosome 4 is in the vicinity of the mouse developmental mutation dysgenetic lens (dyl). However, using a genetic approach, we show that Inpp5b maps distal to dyl on mouse Chromosome 4.

Asunto(s)

Mapeo Cromosómico , Genes , Enfermedades del Cristalino/genética , Ratones/genética , Monoéster Fosfórico Hidrolasas/genética , Animales , Secuencia de Bases , Cruzamientos Genéticos , Femenino , Humanos , Hibridación Fluorescente in Situ , Inositol Polifosfato 5-Fosfatasas , Masculino , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Ratones Mutantes , Datos de Secuencia Molecular , Muridae/genética , Especificidad de la Especie

RESUMEN

Previously it has been shown that male mice heterozygous for the Rb(6.16) translocation show a transmission distortion of the segregants in resulting zygotes, particularly for sexually active animals (Aranha and Martin-DeLeon, 1991). Sperm bearing the translocation have an impaired fertilizing ability, compared to chromosomally normal sperm, and this dysfunction is potentiated in X-bearing cells leading to a sex ratio distortion. Since earlier data suggested that a deficiency of Chromosome 6 was associated with sperm dysfunction (Zackowski and Martin-DeLeon, 1989), the present study was designed to determine the uniqueness of the coupling of the distortions for the Rb(6.16) translocation and the effect of 0 and 2 copies of Chr 6 on sperm function. First cleavage metaphases, resulting from chromosomally normal females that were mated to sexually active or rested Rb(6.15) or Rb(2.8) heterozygous males, were sequentially G- and C-banded. For unaged sperm from sexually active Rb(6.15) males, cytogenetic analysis showed that the ratio of normal (90) to balanced (37) zygotes deviated significantly (P < 0.001) from 1:1, as did the sex ratio, 46X:81Y (P < 0.01). The latter distortion was related to a deficiency of X-bearing sperm with the translocation (8X:29Y). Both segregation and sex ratio distortions disappeared for aged sperm (69:50, 51X:68Y) and a contingency table showed that segregant type was related to sperm age (P < 0.05). For the Rb(2.8) the ratio of normal to balanced zygotes (65:46) was not significantly different from 1:1 for unaged sperm, and although the ratio from aged sperm was 62:39, there was no relationship between segregant type and sperm age (P > 0.05). There was also no sex ratio distortion for either unaged or aged sperm (P > 0.05). To generate zygotes with sperm complements containing 0 and 2 copies of Chr 6, males doubly heterozygous for Rb(6.16) and Rb(6.15) were mated to chromosomally normal females. No zygotes were recovered and the males were shown histologically to have azoospermia and spermatogenic arrest. The results support the conclusion that the presence of Chr 6 in Rb translocations specifically affects segregant and sex ratio distributions. They also indicate, that as the common arm in compound heterozygotes, this chromosome grossly affects spermatogenesis.

Asunto(s)

Heterocigoto , Translocación Genética , Animales , Femenino , Cariotipificación , Masculino , Ratones , Ratones Endogámicos ICR , Testículo/patología

RESUMEN

The segregation products in zygotes of females heterozygous for the mouse Rb(6.16) translocation were studied. Of 191 first-cleavage metaphase zygotes recovered from 16 females mated to chromosomally normal males, 132 were cytogenetically analyzed after sequential G- and C-banding. There were no products of adjacent segregation, since the six chromosomal imbalances were unrelated to the translocation. Alternate segregation was seen in 126 (95.5%) of the zygotes, with 85 being chromosomally normal and 41 carrying the translocation. These results indicate preferential alternate segregation and a significant deviation from the Mendelian 1:1 ratio (P < 0.005) for reciprocal alternate segregants, as seen earlier for males with this translocation. However, the approximately 2:1 ratio for normal-to-balanced segregants in females is lower than the ratios consistently seen for males. This supports the notion that there are different underlying causes of the distortion in the sexes.

Asunto(s)

Mapeo Cromosómico , Tamización de Portadores Genéticos , Ratones/genética , Translocación Genética , Cigoto/fisiología , Animales , Bandeo Cromosómico , Femenino , Fertilización , Masculino , Ratones Endogámicos ICR , Óvulo/citología , Cigoto/citología

RESUMEN

The segregation products of the mouse Rb-(6.16)24 Lub male translocation carrier were analyzed at first cleavage metaphase to determine whether the proportion of normal, balanced, and unbalanced sperm segregants differ in fertilizations occurring in vivo and in vitro. From 34 males, the sperm genomes in 268 first-cleavage mouse embryos were analyzed cytogenetically: 137 and 131 following in vivo and in vitro fertilization, respectively. Both systems demonstrated a preponderance of alternate (67.2% and 54.2%) as compared to adjacent segregation (10.2% and 13.7% as estimated). A contingency table showed that the distribution of reciprocal alternate segregants differed significantly between the two fertilization environments (chi 2 = 20.64, P < 0.0005). Whereas chromosomally normal sperm were 3.6 times more likely than the balanced reciprocals to fertilize in vivo (78.3% normal: 21.7% balanced), 1:1 ratios were recovered following in vitro fertilization (43.7% normal: 56.3% balanced). The data also showed an excess of Y-bearing sperm with the translocation in both in vivo and in vitro fertilization groups. In the latter these segregants were 3 times more likely than X-bearing ones to effect fertilization. These data suggest a phenotypic disadvantage of translocation-X-bearing sperm, possibly mediated through altered haploid gene expression on chromosome 6 and gene expression on the Y. The results show clear evidence for prezygotic selection in vivo and indicate that the environment in which fertilization occurs significantly affects the transmission frequency of this specific translocation.

Asunto(s)

Fertilización In Vitro , Fertilización/genética , Espermatozoides/fisiología , Translocación Genética/genética , Animales , Femenino , Masculino , Ratones , Razón de Masculinidad