RESUMEN
Over the past few years, significant progress has been made in characterizing the expression and localization of proteins that act as scaffolds for cAMP-dependent protein kinase (PK-A). These A-kinase anchor proteins (AKAPs) tether PK-A to intracellular organelles and structures, sequestering the kinase near its physiological substrates. The compartmentalization of distinct pockets of PK-A activity serves to provide spatial regulation of this signaling pathway. In addition, other signaling proteins bind to AKAPs, as do some newly described proteins of unknown function, suggesting that proteins of various pathways are anchored through AKAPs.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Proteínas Portadoras/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Sistema Endocrino/metabolismo , Proteínas de la Membrana/metabolismo , Oocitos/metabolismo , Reproducción/fisiología , Espermatozoides/metabolismo , Proteínas de Anclaje a la Quinasa A , Animales , Femenino , Humanos , MasculinoRESUMEN
Using reverse transcription-polymerase chain reaction (RT-PCR) with degenerate oligonucleotides corresponding to two highly conserved motifs within the protein kinase family of catalytic domains, we isolated a PCR fragment encoding a novel member of the testis-specific serine/threonine kinases (STK) from mouse male mixed germ cell mRNA. This PCR fragment recognized a 1020-bp transcript in male germ cells by northern blot analysis and was used to clone a full-length cDNA from a mouse mixed germ cell cDNA library. This cDNA has an open reading frame of 804 bases encoding a protein of 268 amino acids. This novel gene is almost identical to Stk22c, encoding a recently described testis-specific protein kinase, except for base-pair deletions that result in a shift in the coding region and an alteration of 22 amino acids (residues 109-131). Due to its homology with Stk22c, we have called this protein kinase gene Stk22d. Northern blot analysis revealed that this protein kinase is developmentally expressed in testicular germ cells and is not present in brain, ovary, kidney, liver, or early embryonic cells. We then cloned the human homologue of this protein kinase gene (STK22C) and found it to be expressed exclusively in the testis. Fluorescence in situ hybridization with both the human and mouse cDNA clones revealed syntenic localization on chromosomes 1p34-p35 and 4E1, respectively.
Asunto(s)
Proteínas Serina-Treonina Quinasas/genética , Espermatozoides/enzimología , Testículo/enzimología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , Mapeo Cromosómico , Cromosomas Humanos Par 1/genética , Clonación Molecular , ADN Complementario , Femenino , Expresión Génica , Biblioteca de Genes , Humanos , Hibridación Fluorescente in Situ , Masculino , Ratones , Datos de Secuencia Molecular , Especificidad de Órganos , Proteínas Serina-Treonina Quinasas/química , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Ácido NucleicoRESUMEN
OBJECTIVE: To determine whether mutations in the pro-hAKAP82 gene and the resulting pro-hAKAP82 and hAKAP82 proteins were associated with the infertility seen in a patient with stump tail sperm. DESIGN: Case report. SETTING: Academic research and teaching environment, tertiary care hospital. PATIENT(S): A single, infertile Caucasian male diagnosed with essentially 100% stump tail sperm. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Electrophoresis, silver staining, and immunoblotting of patient and control sperm proteins; RII (type II regulatory subunit of protein kinase-A) overlay assay of patient and control sperm proteins, partial DNA sequence analysis of patient's pro-hAKAP82 gene; indirect immunofluorescence and immunogold electron microscopy of patient and control sperm. RESULTS(S): No significant abnormalities in the size or amount of pro-hAKAP82 and hAKAP82 or in the ability of these proteins to bind the regulatory subunit of protein kinase-A were identified in the patient's sperm. Partial sequence analysis of the patient's pro-hAKAP82 gene was identical to the published normal sequence. Indirect immunofluorescence and immunoelectron microscopy of sperm localized pro-hAKAP82/hAKAP82 to the sperm flagellum and demonstrated that the proteins were present in a disorganized, amorphous region, which apparently represented the fibrous sheath. CONCLUSION(S): These results suggest that, although pro-hAKAP82 and hAKAP82 localize to the correct structural component of the flagellum and are not directly responsible for the stump tail phenotype, they are unable to assemble normally into the fibrous sheath. Although this study did not identify abnormalities in the pro-hAKAP82 gene or its resulting proteins in a patient with stump tail sperm, several regions of the gene and protein remain to be examined.
Asunto(s)
Infertilidad Masculina/genética , Precursores de Proteínas/genética , Proteínas/genética , Proteínas de Plasma Seminal , Espermatozoides/anomalías , Proteínas de Anclaje a la Quinasa A , Adulto , Proteína Quinasa Tipo II Dependiente de AMP Cíclico , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Técnica del Anticuerpo Fluorescente Indirecta , Humanos , Infertilidad Masculina/metabolismo , Masculino , Microscopía Inmunoelectrónica , Peso Molecular , Fenotipo , Precursores de Proteínas/química , Proteínas/química , Análisis de Secuencia de ADNRESUMEN
Dysplasia of the fibrous sheath (DFS) is characterized by male infertility, asthenozoospermia, and morphologically abnormal flagella that possess a severely malformed fibrous sheath. In many cases, DFS is familial, suggesting a genetic component. Human AKAP4 and AKAP3 are structural proteins of the fibrous sheath that also function to anchor protein kinase A to this structure via the regulatory subunit of the kinase. We hypothesized that defects in either AKAP4 or AKAP3 might cause DFS. No quantitative or qualitative differences between patients with DFS and normal controls were detected when sperm proteins were analyzed by either silver staining or immunoblot analysis using antibodies raised against AKAP4 and AKAP3. Additionally, AKAP4 and AKAP3 from DFS sperm retained the ability to bind the regulatory subunit of protein kinase A. Localization at the light and electron microscopic levels showed that AKAP3 and AKAP4 localized correctly to the FS of the amorphous flagellum in DFS sperm. Partial sequence analysis of the AKAP4 and AKAP3 genes in patients with DFS did not identify any significant alterations in potential AKAP4/AKAP3 binding regions, suggesting that the two proteins interact normally in DFS sperm. Our results did not find evidence to support the hypothesis that mutations in either gene are responsible for DFS in humans.
Asunto(s)
Proteínas Portadoras/genética , Enfermedades de los Genitales Masculinos/genética , Espermatozoides/metabolismo , Adulto , Secuencia de Bases , Cartilla de ADN , Electroforesis en Gel de Poliacrilamida , Humanos , Inmunohistoquímica , Focalización Isoeléctrica , Masculino , Microscopía Inmunoelectrónica , Reacción en Cadena de la PolimerasaRESUMEN
In somatic cells, caveolin-1 plays several roles in membrane dynamics, including organization of detergent-insoluble lipid rafts, trafficking of cholesterol, and anchoring of signaling molecules. Events in sperm capacitation and fertilization require similar cellular functions, suggesting a possible role for caveolin-1 in spermatozoa. Immunoblot analysis demonstrated that caveolin-1 was indeed present in developing mouse male germ cells and both mouse and guinea pig spermatozoa. In mature spermatozoa, caveolin-1 was enriched in a Triton X-100-insoluble membrane fraction, as well as in membrane subdomains separable by means of their light buoyant densities through sucrose density gradient centrifugation. These data indicated the presence of membrane rafts enriched in caveolin-1 in spermatozoa. Indirect immunofluorescence analysis revealed caveolin-1 in the regions of the acrosome and flagellum in sperm of both species. Confocal immunofluorescence analysis of developing mouse male germ cells demonstrated partial co-localization with a marker for the acrosome. Furthermore, syntaxin-2, a protein involved in acrosomal exocytosis, was present in both raft and nonraft fractions in mature sperm. Together, these data indicated that sperm membranes possess distinct raft subdomains, and that caveolin-1 localized to regions appropriate for involvement with acrosomal biogenesis and exocytosis, as well as signaling pathways regulating such processes as capacitation and flagellar motility.
Asunto(s)
Caveolinas/metabolismo , Microdominios de Membrana/metabolismo , Espermatozoides/metabolismo , Acrosoma/metabolismo , Animales , Caveolina 1 , Técnica del Anticuerpo Fluorescente Indirecta , Cobayas , Immunoblotting , Masculino , Ratones , Microscopía Confocal , Transducción de Señal , Capacitación Espermática , Motilidad Espermática , Espermatogénesis , Espermatozoides/ultraestructuraRESUMEN
Spermatozoa are highly polarized cells with specific metabolic pathways compartmentalized in different regions. Previously, we hypothesized that glycolysis is organized in the fibrous sheath of the flagellum to provide ATP to dynein ATPases that generate motility and to protein kinases that regulate motility. Although a recent report suggested that glucose is not essential for murine sperm capacitation, we demonstrated that glucose (but not lactate or pyruvate) was necessary and sufficient to support the protein tyrosine phosphorylation events associated with capacitation. The effect of glucose on this signaling pathway was downstream of cAMP, and appeared to arise indirectly as a consequence of metabolism as opposed to a direct signaling effect. Moreover, the phosphorylation events were not affected by uncouplers of oxidative respiration, inhibitors of electron transfer, or by a lack of substrates for oxidative respiration in the medium. Further experiments aimed at identifying potential regulators of sperm glycolysis focused on a germ cell-specific isoform of hexokinase, HK1-SC, which localizes to the fibrous sheath. HK1-SC activity and biochemical localization did not change during sperm capacitation, suggesting that glycolysis in sperm is regulated either at the level of substrate availability or by downstream enzymes. These data support the hypothesis that ATP specifically produced by a compartmentalized glycolytic pathway in the principal piece of the flagellum, as opposed to ATP generated by mitochondria in the mid-piece, is strictly required for protein tyrosine phosphorylation events that take place during sperm capacitation. The relationship between these pathways suggests that spermatozoa offer a model system for the study of integration of compartmentalized metabolic and signaling pathways.
Asunto(s)
Transducción de Señal , Capacitación Espermática , Espermatozoides/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Diferenciación Celular , AMP Cíclico/metabolismo , Relación Dosis-Respuesta a Droga , Glucosa/metabolismo , Glucólisis , Hexoquinasa/química , Immunoblotting , Cinética , Ácido Láctico/farmacología , Masculino , Ratones , Fosforilación/efectos de los fármacos , Isoformas de Proteínas , Ácido Pirúvico/farmacología , Espectrofotometría , Capacitación Espermática/efectos de los fármacos , Factores de Tiempo , Tirosina/metabolismoRESUMEN
Although three germ cell-specific transcripts of type 1 hexokinase exist in murine male germ cells, only one form, HK1-sc, is found at the protein level. This single isoform localizes to three distinct structures in mouse spermatozoa: the membranes of the head, the mitochondria in the midpiece, and the fibrous sheath in the flagellum (Travis, A. J., Foster, J. A., Rosenbaum, N. A., Visconti, P. E., Gerton, G. L., Kopf, G. S., and Moss, S. B. (1998) Mol. Biol. Cell 9, 263-276). The mechanism by which one protein is targeted to multiple sites within this highly polarized cell poses important questions of protein targeting. Because the study of protein targeting in germ cells is hampered by the lack of established cell lines in culture, constructs containing different domains of the germ cell-specific hexokinase transcripts were linked to a green fluorescent protein and transfected into hexokinase-deficient M+R42 cells. Constructs containing a nonhydrophobic, germ cell-specific domain, present at the amino terminus of the HK1-SC protein, were targeted to the endoplasmic reticulum and the plasma membrane. Mutational analysis of this domain demonstrated that a complex motif, PKIRPPLTE (with essential residues italicized), represented a novel endoplasmic reticulum-targeting motif. Constructs based on another germ cell-specific hexokinase transcript, HK1-sa, demonstrated the specific proteolytic removal of an amino-terminal domain, resulting in a protein product identical to HK1-SC. Such processing might constitute a regulatory mechanism governing the spatial and/or temporal expression of the protein.
Asunto(s)
Membrana Celular/enzimología , Retículo Endoplásmico/enzimología , Hexoquinasa/metabolismo , Espermatozoides/enzimología , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Transporte Biológico , Línea Celular , Proteínas Fluorescentes Verdes , Hexoquinasa/química , Hexoquinasa/genética , Hidrólisis , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Proteínas Luminiscentes/genética , Masculino , Ratones , Microscopía Confocal , Mitocondrias/enzimología , Datos de Secuencia Molecular , Mutación , Procesamiento Proteico-Postraduccional , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Homología de Secuencia de Aminoácido , Factores de Tiempo , TransfecciónRESUMEN
Sperm motility is regulated by the cAMP-dependent protein kinase (protein kinase-A)-mediated phosphorylation of a group of largely unidentified flagellar proteins. Human AKAP82 (hAKAP82) and its precursor protein, pro-hAKAP82, are members of the A-kinase anchor protein (AKAP) family. These proteins tether protein kinase-A to the fibrous sheath of human spermatozoa and presumably localize the activity of the kinase near specific targets in the sperm flagellum. In this way, pro-hAKAP82 and hAKAP82 may be involved in regulating sperm motility. Similar to its homologues in other species, pro-hAKAP82 is proteolytically processed to hAKAP82. However, the amount of processing of pro-hAKAP82 in human spermatozoa is less than the amount of processing of the precursor in other species. We postulated that this lower extent of processing may be related to lower percentages of human sperm motility. In addition, both pro-hAKAP82 and hAKAP82 are tyrosine phosphorylated in a capacitation-dependent manner. Since capacitation is associated with hyperactivated motility, we postulated that tyrosine phosphorylation of pro-hAKAP82/hAKAP82 is associated with changes in motility. However, using a combination of immunofluorescence and immunoblotting approaches, we found no evidence for an association between either processing or tyrosine phosphorylation of pro-hAKAP82/hAKAP82 and significant differences in motility in spermatozoa from normal men.
Asunto(s)
Precursores de Proteínas/metabolismo , Proteínas/metabolismo , Proteínas de Plasma Seminal , Motilidad Espermática/fisiología , Tirosina/metabolismo , Proteínas de Anclaje a la Quinasa A , Secuencia de Aminoácidos , Humanos , Masculino , Datos de Secuencia Molecular , Fosforilación , Procesamiento Proteico-Postraduccional , Valores de Referencia , Capacitación Espermática , Cola del Espermatozoide/metabolismoRESUMEN
Protein kinase A regulates sperm motility through the cAMP-dependent phosphorylation of proteins. One mechanism to direct the activity of the kinase is to localize it near its protein substrates through the use of anchoring proteins. A-Kinase anchoring proteins (AKAPs) act by binding the type II regulatory subunit of protein kinase A and tethering it to a cellular organelle or cytoskeletal element. We showed previously that mAKAP82, the major protein of the fibrous sheath of the mouse sperm flagellum, is an AKAP. The available evidence indicates that protein kinase A is compartmentalized to the fibrous sheath by binding mAKAP82. To characterize AKAP82 in bovine sperm, a testicular cDNA library was constructed and used to isolate a clone encoding bAKAP82, the bovine homologue. Sequence analysis showed that the primary structure of bAKAP82 was highly conserved. In particular, the amino acid sequence corresponding to the region of mAKAP82 responsible for binding the regulatory subunit of protein kinase A was identical in the bull. Bovine AKAP82 was present in both epididymal and ejaculated sperm and was localized to the entire principal piece of the flagellum, the region in which the fibrous sheath is located. Finally, bAKAP82 bound the regulatory subunit of protein kinase A. These data support the idea that bAKAP82 functions as an anchoring protein for the subcellular localization of protein kinase A in the flagellum.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Proteínas/química , Proteínas de Plasma Seminal , Cola del Espermatozoide/química , Proteínas de Anclaje a la Quinasa A , Secuencia de Aminoácidos , Animales , Proteínas Portadoras/química , Bovinos , Subunidad RIIalfa de la Proteína Quinasa Dependiente de AMP Cíclico , Proteína Quinasa Tipo II Dependiente de AMP Cíclico , Epidídimo/química , Masculino , Ratones , Datos de Secuencia Molecular , Proteínas/metabolismo , Homología de Secuencia de Aminoácido , Cola del Espermatozoide/enzimologíaRESUMEN
The imprinted mouse H19 gene is hypomethylated on the expressed maternal allele and hypermethylated on the silent paternal allele. A 2-kb region of differential methylation located from -2 to -4 kb relative to the H19 transcriptional start site has been proposed to act as the imprinting mark since hypermethylation in this region is inherited from sperm and retained on the paternal allele throughout development. Here, we describe a temporal analysis of the methylation patterns at the H19 locus during postnatal male germ cell development. The 2-kb region is methylated on the paternal allele throughout spermatogenesis, suggesting that methylation is acquired in this region prior to the resumption of mitosis in postnatal male mice. Likewise, more than half of the maternal alleles are hypermethylated prior to the resumption of mitosis. However, the remaining maternal alleles are not hypermethylated until the completion of meiosis I, indicating that de novo methylation in this region is a continuous process. Sequences proximal to the H19 promoter, which are methylated in spermatozoa and on the paternal allele in somatic cells, are differentially methylated in diploid, mitotic spermatogonia. The maternal allele becomes hypermethylated in this region during meiotic prophase. Thus, the parental H19 alleles acquire methylation differentially in the male germline.
Asunto(s)
Impresión Genómica , Proteínas Musculares/genética , ARN no Traducido , Espermatogénesis/genética , Alelos , Animales , Southern Blotting , ADN/efectos de los fármacos , ADN/genética , ADN/metabolismo , Femenino , Regulación del Desarrollo de la Expresión Génica , Masculino , Metilación , Ratones , Ratones Endogámicos C57BL , Proteínas Musculares/metabolismo , Mutagénesis , Regiones Promotoras Genéticas , ARN Largo no Codificante , Espermatozoides/citología , Espermatozoides/metabolismo , Sulfitos/farmacologíaRESUMEN
One approach to studying the mechanisms governing sperm motility is to permeabilize sperm and examine the regulation of motility by manipulating the intracellular milieu of the cell. The most common method of sperm permeabilization, detergent treatment, has the disadvantage that the membranes and many proteins are extracted from the cell. To avoid this problem, we have developed a method that uses streptolysin O to create stable pores within the plasma membrane while leaving internal membranes intact. Sperm were permeabilized, preincubated, and then treated with 0.6 U/ml of streptolysin O. Permeabilization was assessed by fluorescent dye technologies and endogenous protein phosphorylation using exogenously added [gamma-32P]ATP. Streptolysin O-induced permeabilization rendered the sperm immotile, and the effect was Ca2+-dependent. When the cells were treated simultaneously with a medium containing ATP, streptolysin O-treated sperm maintained flagellar movement. These results demonstrate that the streptolysin O permeabilization model system is a useful experimental method for studying the mechanisms that regulate sperm motility since it allows the flagellar apparatus to be exposed to various exogenously added molecules.
Asunto(s)
Permeabilidad de la Membrana Celular/efectos de los fármacos , Motilidad Espermática/efectos de los fármacos , Estreptolisinas/farmacología , Adenosina Trifosfato/farmacología , Animales , Proteínas Bacterianas , Calcio/farmacología , Colorantes Fluorescentes , Masculino , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Microscopía Fluorescente , Fosfoproteínas/metabolismo , Radioisótopos de Fósforo/metabolismo , Fosforilación , Motilidad Espermática/fisiología , Cola del Espermatozoide/fisiologíaRESUMEN
Mammalian sperm motility is regulated by a cascade of cAMP-dependent protein phosphorylation events mediated by protein kinase A. A-kinase anchor proteins (AKAPs) direct protein kinase A activity by tethering the enzyme near its physiological substrates. We have characterized a major human sperm fibrous sheath AKAP, hAKAP82, and its precursor, pro-hAKAP82, the homologues of the mouse fibrous sheath proteins mAKAP82 and pro-mAKAP82. The cDNA sequence of pro-hAKAP82 was highly homologous to the mouse sequence, and the functional domains of the pro-hAKAP82 protein, the protein kinase A binding, and the pro-hAKAP82/hAKAP82 cleavage sites were identical to those of the mouse protein. The genomic organization of mouse pro-AKAP82 was determined. Alternative splicing occurred in both the mouse and human pro-AKAP82 genes that resulted in at least two distinct transcripts and possibly two different proteins. Compared with pro-mAKAP82, considerably less pro-hAKAP82 was processed to hAKAP82 in human sperm. Although pro-mAKAP82 localizes only to the proximal portion of the principal piece of the flagellum, pro-hAKAP82 localized to the entire length of the principal piece. The pro-hAKAP82 gene mapped to human chromosome Xp11.2, indicating that defects in this gene are maternally inherited. These studies suggest several roles for hAKAP82 in sperm motility, including the regulation of signal transduction pathways.
Asunto(s)
Empalme Alternativo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Precursores de Proteínas/genética , Proteínas/genética , Proteínas de Plasma Seminal , Cola del Espermatozoide/metabolismo , Cromosoma X , Proteínas de Anclaje a la Quinasa A , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Mapeo Cromosómico , Femenino , Biblioteca de Genes , Impresión Genómica , Humanos , Hibridación Fluorescente in Situ , Cariotipificación , Sustancias Macromoleculares , Masculino , Ratones , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Precursores de Proteínas/metabolismo , Proteínas/metabolismo , Alineación de Secuencia , Homología de Secuencia de Ácido Nucleico , Cola del Espermatozoide/ultraestructura , Testículo/metabolismoRESUMEN
Basonuclin was first described as a human keratinocyte zinc finger protein present in the nuclei of proliferative basal keratinocytes in the epidermis. It disappears from keratinocytes that have lost their proliferative ability and have entered terminal differentiation. We now report that basonuclin is present also in the germ cells of the mouse testis and ovary. Immunocytochemical staining detected basonuclin in the nuclei of spermatogonia and spermatocytes at various developmental stages. During spermiogenesis, it relocated from the nucleus to the midpiece of the flagellum of the spermatozoa. In the ovary, basonuclin was found mainly in the nuclei of developing oocytes. The dual presence of basonuclin in differentiated spermatozoa and oocytes suggests that it may play a role in their differentiation and the early development of an embryo.
Asunto(s)
Núcleo Celular/metabolismo , Células Germinativas/metabolismo , Proteínas/metabolismo , Espermatogénesis/fisiología , Espermatozoides/metabolismo , Dedos de Zinc , Animales , Núcleo Celular/ultraestructura , Proteínas de Unión al ADN , Femenino , Técnica del Anticuerpo Fluorescente Indirecta , Células Germinativas/ultraestructura , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Oocitos/metabolismo , Ovario/citología , Ovario/metabolismo , Fosfoproteínas , Reacción en Cadena de la Polimerasa , Espermatozoides/ultraestructura , Testículo/citología , Testículo/metabolismo , Factores de TranscripciónRESUMEN
Multiple isoforms of type 1 hexokinase (HK1) are transcribed during spermatogenesis in the mouse, including at least three that are presumably germ cell specific: HK1-sa, HK1-sb, and HK1-sc. Each of these predicted proteins contains a common, germ cell-specific sequence that replaces the porin-binding domain found in somatic HK1. Although HK1 protein is present in mature sperm and is tyrosine phosphorylated, it is not known whether the various potential isoforms are differentially translated and localized within the developing germ cells and mature sperm. Using antipeptide antisera against unique regions of HK1-sa and HK1-sb, it was demonstrated that these isoforms were not found in pachytene spermatocytes, round spermatids, condensing spermatids, or sperm, suggesting that HK1-sa and HK1-sb are not translated during spermatogenesis. Immunoreactivity was detected in protein from round spermatids, condensing spermatids, and mature sperm using an antipeptide antiserum against the common, germ cell-specific region, suggesting that HK1-sc was the only germ cell-specific isoform present in these cells. Two-dimensional SDS-PAGE suggested that all of the sperm HK1-sc was tyrosine phosphorylated, and that the somatic HK1 isoform was not present. Immunoelectron microscopy revealed that HK1-sc was associated with the mitochondria and with the fibrous sheath of the flagellum and was found in discrete clusters in the region of the membranes of the sperm head. The unusual distribution of HK1-sc in sperm suggests novel functions, such as extramitochondrial energy production, and also demonstrates that a hexokinase without a classical porin-binding domain can localize to mitochondria.
Asunto(s)
Hexoquinasa/análisis , Isoenzimas/análisis , Mitocondrias/enzimología , Porinas/metabolismo , Espermatozoides/enzimología , Secuencia de Aminoácidos , Animales , Especificidad de Anticuerpos , Hexoquinasa/química , Hexoquinasa/genética , Hexoquinasa/metabolismo , Punto Isoeléctrico , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Masculino , Ratones , Datos de Secuencia Molecular , Peso Molecular , Especificidad de Órganos , Fosforilación , Solubilidad , Cabeza del Espermatozoide/enzimología , Espermatogénesis/fisiología , Tirosina/metabolismoRESUMEN
The assembly of the mammalian sperm flagellum is a complex developmental event requiring the sequential activation of genes encoding the component parts and the coordinated assembly of these proteins during the differentiation of the haploid spermatid. In this study, the mechanism underlying the assembly of the fibrous sheath surrounding the axoneme was examined. The subject of the study was the major fibrous sheath protein of the mouse sperm flagellum, AKAP82, a member of the A Kinase Anchor Protein (AKAP) family of polypeptides that bind the regulatory (RII) subunit of protein kinase A (PK-A). Immunoelectron microscopy demonstrated that AKAP82 is present throughout the transverse ribs and longitudinal columns of the fibrous sheath. Since AKAP82 is initially synthesized as a precursor (pro-AKAP82) during spermiogenesis, an antiserum was raised against a peptide from the processed region of pro-AKAP82 (M(r) 97,000). In immunoblotting experiments, the antibody detected pro-AKAP82 in condensing spermatids but not in epididymal sperm. In addition, two other immunoreactive proteins of M(r) 109,000 (p109) and M(r) 26,000 (p26, representing the "pro" domain of the precursor) were present in epididymal sperm. Alkaline phosphatase treatment of epididymal sperm proteins demonstrated that p109 was a phosphorylated form of pro-AKAP82 that remained in sperm. By immunofluorescence, pro-AKAP82 was localized to the entire length of the principal piece in testicular sperm, while in epididymal sperm p109 and p26 were present only in the proximal portion of the principal piece. Pro-AKAP82 was solubilized when germ cells were extracted with Triton X-100. However, in sperm, both AKAP82 and p109 were almost totally resistant to these extraction conditions and remained in the particulate fraction even after extraction with Triton and dithiothreitol. Similar to pro-AKAP82, the RII subunit of PK-A was present in the Triton X-100-soluble fraction of developing germ cells. In sperm, much of the RII also became particulate, consistent with the hypothesis that AKAP82 anchors RII in the flagellum. These data indicate that pro-AKAP82 is synthesized in the cell body, transported down the axoneme to its site of assembly in the fibrous sheath, and then proteolytically clipped to form mature AKAP82.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Proteínas/metabolismo , Proteínas de Plasma Seminal , Cola del Espermatozoide/metabolismo , Animales , Western Blotting , Sueros Inmunes/inmunología , Masculino , Ratones , Peso Molecular , Fragmentos de Péptidos/inmunología , Fosforilación , Precursores de Proteínas/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas/inmunología , Solubilidad , Espermátides/metabolismoRESUMEN
The molecular basis of mammalian sperm capacitation, defined as those biochemical and functional changes that render the sperm competent to fertilize the egg, is poorly understood. This extratesticular maturational process is accompanied by the activation of a unique signal transduction pathway involving the cAMP-dependent up-regulation of protein tyrosine phosphorylation presumably through the activation of protein kinase A (PK-A). We demonstrate in this report that capacitation of cauda epididymal mouse sperm in vitro was accompanied by a time-dependent increase in PK-A activity. This increase in PK-A activity did not occur in a medium that does not support capacitation. While PK-A catalytic and RI/RII regulatory subunits, as well as PK-A enzyme activity, were found in both the Triton X-100-soluble and -insoluble fractions of the sperm, the increase in PK-A activity accompanying capacitation was associated with enzyme activity found in the soluble fraction. Moreover, the regulatory and catalytic subunits of PK-A were observed by indirect immunofluorescence to be present throughout the head, midpiece, and principal piece of the sperm. Thus, PK-A appears to be functional in multiple compartments of this highly differentiated cell. A fraction of the Triton X-100-insoluble PK-A is presumably tethered by AKAP82, the major protein of the fibrous sheath of the sperm flagellum which anchors and compartmentalizes PK-A to the cytoskeleton via the RII subunit of PK-A. Using various recombinant truncated AKAP82 constructs in a gel overlay assay, the RII subunit-binding domain of this protein was mapped to a 57-amino-acid residue region at its N-terminus. Computer analysis revealed a 14-amino-acid region that resembled the RII-binding domains of other A Kinase Anchor Proteins. A synthetic peptide corresponding to this domain inhibited AKAP82-RII binding in a gel overlay assay, providing further support that AKAP82 is an anchoring protein for the subcellular localization of PK-A in the mouse sperm fibrous sheath. This work, along with previous findings that cAMP is a key intermediary second messenger in regulating protein tyrosine phosphorylation and capacitation, further supports the importance of PK-A in these processes and necessitates a further understanding of the contribution of both the soluble and insoluble forms of PK-A, as well as AKAP82, to sperm function.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Proteínas/metabolismo , Proteínas de Plasma Seminal , Capacitación Espermática/fisiología , Cola del Espermatozoide/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Compartimento Celular , AMP Cíclico/fisiología , Técnica del Anticuerpo Fluorescente Indirecta , Masculino , Ratones , Datos de Secuencia Molecular , Fragmentos de Péptidos/metabolismo , Sistemas de Mensajero Secundario/fisiología , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Solubilidad , Cabeza del Espermatozoide/metabolismoRESUMEN
Signal transduction pathways regulate various aspects of mammalian sperm function. When human sperm were incubated in a medium supporting capacitation, proteins became tyrosine-phosphorylated in a time-dependent manner. This phosphorylation was inhibited by genistein, a protein tyrosine kinase inhibitor. Phosphorylation was also reduced when sperm were incubated either in the presence of increasing concentrations of extracellular Ca2+ or in a medium containing the Ca2+ ionophore A23187. This Ca2+-induced dephosphorylation was calmodulin-dependent, suggesting that calcineurin was involved. In this regard, the calcineurin inhibitor deltamethrin inhibited the Ca2+ ionophore-induced dephosphorylation. A limited number of Mr 80,000-105,000 polypeptides were the most prominent phosphotyrosine-containing proteins present in human sperm. Unlike mouse sperm, which contains a tyrosine-phosphorylated isoform of hexokinase, a phosphotyrosine-containing hexokinase in human sperm was not detected. Most of the tyrosine-phosphorylated proteins were Triton X-100-insoluble and were localized to the principal piece of the flagellum, the region where the cytoskeletal fibrous sheath is found. Prominent phosphotyrosine-containing proteins of Mr 82,000 and 97,000 were identified as the human homologues of mouse sperm AKAP82, the major fibrous sheath protein, and pro-AKAP82, its precursor polypeptide, respectively. These proteins are A Kinase Anchor Proteins, polypeptides that sequester protein kinase A to subcellular locations. Taken together, these results suggest that protein tyrosine phosphorylation may be part of a signal transduction cascade(s) regulating events pertaining to capacitation and/or motility in mammalian sperm and that an interrelationship between tyrosine kinase and cAMP signaling pathways exists in these cells.
Asunto(s)
Calcio/metabolismo , Calmodulina/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Fosfotirosina/metabolismo , Proteínas/metabolismo , Proteínas de Plasma Seminal , Espermatozoides/metabolismo , Alcaloides , Animales , Benzofenantridinas , Calcimicina/farmacología , Inhibidores Enzimáticos/farmacología , Genisteína , Humanos , Isoflavonas/farmacología , Masculino , Ratones , Fenantridinas/farmacología , Fosforilación , Fosfotirosina/análisis , Inhibidores de Proteínas Quinasas , Espermatozoides/efectos de los fármacos , Estaurosporina/farmacologíaRESUMEN
A number of cell-cycle checkpoint genes have been shown to play important roles in meiosis. We have characterized the human and mouse counterpart of the Schizosaccharomyces pombe Rad3 protein, named Atr (for ataxia-telangiectasia- and rad3-related), and the protein that is mutated in ataxia-telangiectasia, Atm. We demonstrate that ATR mRNA and protein are expressed in human and mouse testis. More detailed analysis of specific cells in seminiferous tubules shows localization of Atr to the nuclei of cells in the process of meiosis I. Using immunoprecipitation and immunoblot analysis, we show that Atr and Atm proteins are approximately 300 and 350 kD relative molecular mass, respectively, and further demonstrate that both proteins have associated protein kinase activity. Further, we demonstrate that Atr and Atm interact directly with meiotic chromosomes and show complementary localization patterns on synapsing chromosomes. Atr is found at sites along unpaired or asynapsed chromosomal axes, whereas Atm is found along synapsed chromosomal axes. This is the first demonstration of a nuclear association of Atr and Atm proteins with meiotic chromosomes and suggests a direct role for these proteins in recognizing and responding to DNA strand interruptions that occur during meiotic recombination.
Asunto(s)
Proteínas de Ciclo Celular/análisis , Cromosomas/química , Meiosis/fisiología , Proteínas Quinasas/química , Proteínas Serina-Treonina Quinasas , Proteínas/análisis , Animales , Proteínas de la Ataxia Telangiectasia Mutada , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Línea Celular , Núcleo Celular/química , Cromatina/química , Cromosomas/metabolismo , Proteínas de Unión al ADN , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Peso Molecular , Profase , Proteínas Quinasas/análisis , Proteínas Quinasas/metabolismo , Proteínas/química , Proteínas/metabolismo , ARN Mensajero/análisis , Túbulos Seminíferos/química , Espermatozoides/química , Testículo/química , Proteínas Supresoras de TumorRESUMEN
Mouse sperm possess a phosphotyrosine-containing hexokinase type 1 (HK1) that is associated with the plasma membrane fraction of these cells (Kalab et al., 1994; J. Biol Chem 269:3810-3817). This apparent plasma membrane association appears unique, since somatic HK1 is normally cytoplasmic or bound to the outer mitochondrial membrane via contact sites with a voltage-dependent anion channel (porin) through a porin-binding domain. In male germ cells, three cDNA clones have been described that encode unique HK1 isoforms (HK1-sa, HK1-sb, HK1-sc) that do not contain porin binding domains (Mori et al., 1993: Biol Reprod 49:191-203). This suggests that these proteins might not be localized to the outer mitochondrial membrane and could have alternative functions in germ cells and/or sperm. We demonstrate in the mouse that male germ cells and sperm could potentially express four HK1 isoforms (HK1-sa, HK1-sb, HK1-sc, and the somatic HK1). At the protein level, at least one of the HK1 isoforms becomes phosphorylated on tyrosine residues during spermatogenesis. Treatment of sperm membrane fractions to dissociate the phosphotyrosine-containing HK1 (pY-mHK1) yields results demonstrating that pY-mHK1 has properties of an integral membrane protein. Indirect immunofluorescence using a monoclonal antibody to HK1 demonstrates specific staining both in the head and tail regions of sperm. Surface biotinylation of intact sperm followed by precipitation with either polyclonal HK1 antiserum or with avidin-Sepharose suggests that pY-mHK1 possesses an extracellular domain. These results suggest that mouse sperm contain at least one HK1 isoform that is present on the sperm head, has an extracellular domain, and behaves as an integral membrane protein.