RESUMEN
Y-box proteins are single-strand DNA- and RNA-binding proteins distinguished by a conserved cold shock domain (CSD) and a variable C-terminal domain organized into alternating short modules rich in basic or acidic amino acids. A huge literature depicts Y-box proteins as highly abundant, staggeringly versatile proteins that interact with all mRNAs and function in most forms of mRNA-specific regulation. The mechanisms by which Y-box proteins recognize mRNAs are unclear, because their CSDs bind a jumble of diverse elements, and the basic modules in the C-terminal domain are considered to bind nonspecifically to phosphates in the RNA backbone. A survey of vertebrate Y-box proteins clarifies the confusing names for Y-box proteins, their domains, and RNA-binding motifs, and identifies several novel conserved sequences: first, the CSD is flanked by linkers that extend its binding surface or regulate co-operative binding of the CSD and N-terminal and C-terminal domains to proteins and RNA. Second, the basic modules in the C-terminal domain are bona fide arginine-rich motifs (ARMs), because arginine is the predominant amino acid and comprises 99% of basic residues. Third, conserved differences in AA (amino acid) sequences between isoforms probably affect RNA-binding specificity. C-terminal ARMs connect with many studies, demonstrating that ARMs avidly bind sites containing specific RNA structures. ARMs crystallize insights into the under-appreciated contributions of the C-terminal domain to site-specific binding by Y-box proteins and difficulties in identifying site-specific binding by the C-terminal domain. Validated structural biology techniques are available to elucidate the mechanisms by which YBXprot (Y-box element-binding protein) CSDs and ARMs identify targets.
Asunto(s)
Proteínas de Unión al ADN , Proteínas de Unión al ARN , ARN , Secuencias de Aminoácidos , Animales , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Humanos , Dominios Proteicos , ARN/química , ARN/genética , ARN/metabolismo , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismoRESUMEN
Gordon Dixon's pioneering work on the replacement of histones by protamines during spermatogenesis inspired research as recombinant DNA became widely used to analyze gene expression in mammalian spermatogenic cells. The impact of recombinant DNA began immediately with the identification of mouse protamine 1 as a haploid-expressed mRNA, resolving a decades-long controversy whether gene expression in haploid spermatogenic cells distorts transmission of alleles to progeny. Numerous insights into the biology of spermatogenesis followed as the sequences of many mRNAs revealed that the patterns of gene expression in spermatogenic cells are astonishingly different from those in other cells in the mammalian body. Studies of these phenomena have generated fundamental insights across reproductive, molecular and evolutionary biology. Abbreviations: PRM1: protamine 1; PRM2: protamine 2; TCE: translation control element.
Asunto(s)
ADN Recombinante , Regulación de la Expresión Génica , Expresión Génica , Protaminas/metabolismo , Espermatogénesis , Animales , Ensamble y Desensamble de Cromatina , Masculino , Proteínas de Unión al ARN/metabolismo , Espermátides/metabolismoRESUMEN
Many mRNAs encoding proteins needed for the construction of the specialized organelles of spermatozoa are stored as translationally repressed, free messenger ribonucleoproteins in round spermatids, to be actively translated in elongating and elongated spermatids. The factors that repress translation in round spermatids, however, have been elusive. Two lines of evidence implicate the highly abundant and well-known translational repressor, Y-box protein 2 (YBX2), as a critical factor: First, protamine 1 (Prm1) and sperm-mitochondria cysteine-rich protein (Smcp) mRNAs are prematurely recruited onto polysomes in Ybx2-knockout mouse round spermatids. Second, mutations in 3' untranslated region (3'UTR) cis-elements that abrogate YBX2 binding activate translation of Prm1 and Smcp mRNAs in round spermatids of transgenic mice. The abundance of YBX2 and its affinity for variable sequences, however, raise questions of how YBX2 targets specific mRNAs for repression. Mutations to the Prm1 and Smcp mRNAs in transgenic mice reveal that strong repression in round spermatids requires YBX2 binding sites located near the 3' ends of their 3'UTRs as locating the same sites in upstream positions produce negligible repression. This location-dependence implies that the assembly of repressive complexes is nucleated by adjacent cis-elements that enable cooperative interactions of YBX2 with co-factors. The available data suggest that, in vertebrates, YBX2 has the important role of coordinating the storage of translationally repressed mRNAs in round spermatids by inhibiting translational activity and the degradation of transcripts via translation-dependent deadenylation. These insights should facilitiate future experiments designed to unravel how YBX2 targets mRNAs for repression in round spermatids and how mutations in the YBX2 gene cause infertility in humans. Mol. Reprod. Dev. 83: 190-207, 2016. © 2016 Wiley Periodicals, Inc.
Asunto(s)
Biosíntesis de Proteínas/fisiología , Estabilidad del ARN/fisiología , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Espermátides/metabolismo , Animales , Humanos , Masculino , Ratones , Ratones Noqueados , ARN Mensajero/genética , Proteínas de Unión al ARN/genética , Espermátides/citologíaRESUMEN
The protamine 1 (Prm1) and sperm mitochondria-associated, cysteine-rich protein (Smcp) mRNAs exemplify a widespread pattern of mRNA-specific regulation of mRNA translation in post-meiotic spermatogenic cells, spermatids. Both mRNAs are transcribed and initially stored in free-mRNPs in early spermatids, and translated on polysomes in late spermatids. In this study, we demonstrate that the 5' and 3'-UTRs and the 3' terminus of the Smcp 3'-UTR are required for normal repression of the Smcp mRNA in transgenic mice. RNA affinity chromatography and mass spectrometry sequencing identified Y-box protein 2 (YBX2/MSY2) as the major protein that interacts with the 3' terminus of the Smcp 3'-UTR and a Y-box recognition sequence, GCCACCU, in the translation control element that is necessary for Prm1 mRNA repression. Depletion of YBX2 in Ybx2-null mice prematurely activates Prm1 and Smcp mRNA translation in early spermatids. Fluorescent in situ hybridization reveals that the Smcp intron, the Smcp mRNA, and both Smcp-Gfp transgenic mRNAs are strongly concentrated in the chromatoid body, and that theYbx2-null mutation does not eliminate the Smcp mRNA from the chromatoid body. This and previous findings suggest that the Smcp pre-mRNA is spliced and associates with YBX2 in the chromatoid body, and that repressed free-mRNPs are stored in the general cytoplasm. As YBX2 is the predominant protein in testis free-mRNPs, it likely represses many mRNAs in early spermatids. The mechanisms by which YBX2 represses the Smcp and Prm1 mRNAs are relevant to reproductive medicine because mutations in the human YBX2 gene correlate with abnormal protamine expression and male infertility.
Asunto(s)
Regulación de la Expresión Génica , ARN Mensajero/genética , Proteínas de Unión al ARN/fisiología , Selenoproteínas/genética , Espermátides/metabolismo , Transcripción Genética , Regiones no Traducidas 3' , Animales , Secuencia de Bases , Western Blotting , ATPasas Transportadoras de Calcio/genética , ATPasas Transportadoras de Calcio/metabolismo , Células Cultivadas , Humanos , Hibridación Fluorescente in Situ , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Datos de Secuencia Molecular , ARN Mensajero/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Selenoproteínas/metabolismo , Homología de Secuencia de Ácido Nucleico , Espermátides/citologíaRESUMEN
mRNA-specific regulation of translational activity plays major roles in directing the development of meiotic and haploid spermatogenic cells in mammals. Although many RNA-binding proteins (RBPs) have been implicated in normal translational control and sperm development, little is known about the keystone of the mechanisms: the interactions of RBPs and microRNAs with cis-elements in mRNA targets. The problems in connecting factors and elements with translational control originate in the enormous complexity of post-transcriptional regulation in mammalian cells. This creates confusion as to whether factors have direct or indirect and large or small effects on the translation of specific mRNAs. This review argues that gene knockouts, heterologous systems, and overexpression of factors cannot provide convincing answers to these questions. As a result, the mechanisms involving well-studied mRNAs (Ddx4/Mvh, Prm1, Prm2, and Sycp3) and factors (DICER1, CPEB1, DAZL, DDX4/MVH, DDX25/GRTH, translin, and ELAV1/HuR) are incompletely understood. By comparison, mutations in elements can be used to define the importance of specific pathways in regulating individual mRNAs. However, few elements have been studied, because the only reliable system to analyze mutations in elements, transgenic mice, is considered impractical. This review describes advances that may facilitate identification of the direct targets of RBPs and analysis of mutations in cis-elements. The importance of upstream reading frames in the developmental regulation of mRNA translation in spermatogenic cells is also documented.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Espermatogénesis , Espermatozoides/metabolismo , Regiones no Traducidas 3' , Regiones no Traducidas 5' , Animales , Proteínas de Unión al ADN/metabolismo , Técnicas de Inactivación de Genes , Genes mos , Haploidia , Humanos , Masculino , Meiosis , MicroARNs/metabolismo , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Mutación , Sistemas de Lectura Abierta , Protaminas/genética , Protaminas/metabolismo , ARN Interferente Pequeño/metabolismo , Proteínas de Unión al ARN/metabolismoRESUMEN
To facilitate identifying translational control elements by studies of mutations in transgenic mice, a database of orthologous 5' and 3' ends of 12 messenger RNA (mRNA) species from 13 to 23 mammals that undergo delayed translational activation in spermatids was constructed for the Acev2, Akap3, Akap4v2, Gapdhs, Odf1, Prm1, Prm2, Prm3, Smcp, Spata18, Tnp1, and Tnp2 mRNAs. This database, available here, was searched for conserved sequences in conserved positions and known translational control elements. Numerous potential mRNA-specific elements were identified, including upstream open reading frames, conserved sequences upstream and downstream of the poly(A) signal, and noncanonical and multiple poly(A) signals. RNA electrophoresis mobility shift assays demonstrate that Y-box proteins bind 30 of the 36 permutations of the degenerate Y-box recognition sequence (YRS), [UAC][CA]CA[UC]C[ACU], and this information was used to identify hundreds of YRSs in the untranslated region (UTR) database. Collectively, these findings suggest that the distal ends of both UTRs are particularly well conserved, implying that translation of each mRNA is regulated by mechanisms involving the poly(A) binding protein and the closed loop. In addition, the 5' flanking regions of all 12 genes have conserved, gene-specific sequences and configurations of elements that resemble the binding site of the testis-specific isoform of cyclic AMP response element modulator, and all 12 genes lack retrogene paralogues, demonstrating the efficacy of mechanisms that limit the proliferation of retroposons in the male germ line. This study illustrates the power of comparative genomics in identifying novel hypothetical regulatory elements for analysis with biochemical and in vivo genetic approaches.
Asunto(s)
Regiones no Traducidas 3' , Regiones no Traducidas 5' , Hibridación Genómica Comparativa , ARN Mensajero/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos , Espermátides/metabolismo , Animales , Secuencia de Bases , Sitios de Unión , Secuencia Conservada , Bases de Datos Genéticas , Ensayo de Cambio de Movilidad Electroforética , Regulación de la Expresión Génica , Humanos , Masculino , Ratones , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Proteínas de Unión a Poli(A)/metabolismo , Biosíntesis de Proteínas , Proteínas de Unión al ARN/metabolismo , Ratas , Sitio de Iniciación de la TranscripciónRESUMEN
The chromatoid body is a dynamic organelle that is thought to coordinate the cytoplasmic regulation of mRNA translation and degradation in mammalian spermatids. The chromatoid body is also postulated to function in repression of mRNA translation by sequestering dormant mRNAs where they are inaccessible to the translational apparatus. This review finds no convincing evidence that dormant mRNAs are localized exclusively in the chromatoid body. This discrepancy can be explained by two hypotheses. First, experimental artifacts, possibly related to peculiarities of the structure and function of the chromatoid body, preclude obtaining an accurate indication of mRNA localization. Second, mRNA is not stored in the chromatoid body, because, like perinuclear P granules in Caenorhabditis elegans, the chromatoid body functions as a center for mRNP remodeling and export to other cytoplasmic sites.
Asunto(s)
ARN Mensajero Almacenado/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Espermátides/metabolismo , Animales , Transporte Biológico/fisiología , Humanos , Masculino , Mamíferos/genética , Mamíferos/metabolismo , Biosíntesis de Proteínas/fisiología , Interferencia de ARN/fisiología , Procesamiento Postranscripcional del ARN/fisiología , Espermátides/ultraestructuraRESUMEN
BACKGROUND: Developmental and global regulation of mRNA translation plays a major role in regulating gene expression in mammalian spermatogenic cells. Sucrose gradients are widely used to analyze mRNA translation. Unfortunately, the information from sucrose gradient experiments is often compromised by the absence of quantification and absorbance tracings, and confusion about the basic properties of sucrose gradients. METHODS: The Additional Materials contain detailed protocols for the preparation and analysis of sucrose and Nycodenz gradients, obtaining absorbance tracings of sucrose gradients, aligning tracings and fractions, and extraction of equal proportions of RNA from all fractions. RESULTS: The techniques described here have produced consistent measurements despite changes in personnel and minor variations in RNA extraction, gradient analysis, and mRNA quantification, and describes for the first time potential problems in using gradients to analyze mRNA translation in purified spermatogenic cells. CONCLUSIONS: Accurate quantification of the proportion of polysomal mRNA is useful in comparing translational activity at different developmental stages, different mRNAs, different techniques and different laboratories. The techniques described here are sufficiently accurate to elucidate the contributions of multiple regulatory elements of variable strength in regulating translation of the sperm mitochondria associated cysteine-rich protein (Smcp) mRNA in transgenic mice.
Asunto(s)
Centrifugación por Gradiente de Densidad/métodos , Biosíntesis de Proteínas , ARN Mensajero/genética , Animales , Expresión Génica , Yohexol , Masculino , Ratones , Ratones Transgénicos , ARN Mensajero/metabolismo , Selenoproteínas/biosíntesis , Selenoproteínas/genética , Espermatozoides/metabolismo , SacarosaRESUMEN
The sperm mitochondria-associated cysteine-rich protein (Smcp) mRNA is transcribed in step 3 spermatids, and is stored in free mRNPs until translation begins â¼6 days later in step 11. To identify sequences that control the timing of Smcp mRNA translation, mutations in both UTRs were analyzed in transgenic mice using green fluorescent protein (GFP), squashes of seminiferous tubules, and quantification of polysomal loading in adult and 21 dpp testes in sucrose and Nycodenz gradients. GFP fluorescence is first detected in step 9 spermatids in lines harboring a transgene containing the Gfp 5' UTR and Smcp 3' UTR. Unexpectedly, this mRNA is stored in large, inactive mRNPs in early spermatids that sediment with polysomes in sucrose gradients, but equilibrate with the density of free mRNPs in Nycodenz gradients. Randomization of the segment 6-38 nt upstream of the first Smcp poly(A) signal results in early detection of GFP, a small increase in polysomal loading in 21 dpp testis, inactivation of the formation of heavy mRNPs, and loss of binding of a Y-box protein. GFP is first detected in step 5 spermatids in a transgene containing the Smcp 5' UTR and Gfp 3' UTR. Mutations in the start codons in the upstream reading frames eliminate translational delay by the Smcp 5' UTR. Collectively, these findings demonstrate that Smcp mRNA translation is regulated by multiple elements in the 5' UTR and 3' UTR. In addition, differences in regulation between Smcp-Gfp mRNAs containing one Smcp UTR and the natural Smcp mRNA suggest that interactions between the Smcp 5' UTR and 3' UTR may be required for regulation of the Smcp mRNA.
Asunto(s)
Biosíntesis de Proteínas/genética , Secuencias Reguladoras de Ácido Ribonucleico/fisiología , Ribonucleoproteínas/metabolismo , Selenoproteínas/genética , Espermátides/metabolismo , Regiones no Traducidas 3'/genética , Regiones no Traducidas 5'/genética , Animales , Secuencia de Bases , Análisis Mutacional de ADN , Regulación hacia Abajo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Datos de Secuencia Molecular , Interferencia de ARN/fisiología , Ribonucleoproteínas/fisiología , Homología de Secuencia de Ácido NucleicoRESUMEN
The comparative genomics of the Odf1, Prm1, Prm2, Tnp1, and Tnp2 genes in 13-21 diverse mammalian species reveals striking similarities and differences in the sequences that probably function in the transcriptional and translational regulation of gene expression in haploid spermatogenic cells, spermatids. The 5' flanking regions contain putative TATA boxes and cAMP-response elements (CREs), but the TATA boxes and CREs exhibit gene-specific sequences, and an overwhelming majority of CREs differ from the consensus sequence. The 5' and 3' UTRs contain highly conserved gene-specific sequences including canonical and noncanonical poly(A) signals and a suboptimal context for the Tnp2 translation initiation codon. The conservation of the 5' UTR is unexpected because mRNA translation in spermatids is thought to be regulated primarily by the 3' UTR. Finally, all of the genes contain a single intron, implying that retroposons are rarely created from mRNAs that are expressed in spermatids.
Asunto(s)
Proteínas Cromosómicas no Histona/genética , Regulación del Desarrollo de la Expresión Génica , Genoma , Proteínas de Choque Térmico/genética , Protaminas/genética , Elementos Reguladores de la Transcripción , Espermátides/metabolismo , Región de Flanqueo 3'/genética , Región de Flanqueo 5'/genética , Animales , Secuencia de Bases , Bovinos , Secuencia Conservada , Perros , Dosificación de Gen , Genómica , Cobayas , Humanos , Masculino , Ratones , Datos de Secuencia Molecular , PoliadenilaciónRESUMEN
The protamine gene cluster containing the Prm1, Prm2, Prm3, and Tnp2 genes is present in humans, mice, and rats. The Prm1, Prm2, and Tnp2 genes have been extensively studied, but almost nothing is known about the function and regulation of the Prm3 gene. Here we demonstrate that an intronless Prm3 gene encoding a distinctive small acidic protein is present in 13 species from seven orders of mammals. We also demonstrate that the Prm3 gene has not generated retroposons, which supports the contention that genes that are expressed in meiotic and haploid spermatogenic cells do not generate retroposons. The Prm3 mRNA is first detected in early round spermatids, while the PRM3 protein is first detected in late spermatids. Thus, translation of the Prm3 mRNA is developmentally delayed similar to the Prm1, Prm2, and Tnp2 mRNAs. In contrast to PRM1, PRM2, and TNP2, PRM3 is an acidic protein that is localized in the cytoplasm of elongated spermatids and transfected NIH-3T3 cells. To elucidate the function of PRM3, the Prm3 gene was disrupted by homologous recombination. Sperm from Prm3(-/-) males exhibited reductions in motility, but the fertility of Prm3(-/-) and Prm3(+/+) males was similar in matings of one male and one female. We have developed a competition test in which a mutant male has to compete with a rival wild-type male to fertilize a female; the implications of these results are also discussed.
Asunto(s)
Protaminas/genética , Protaminas/metabolismo , Motilidad Espermática/genética , Motilidad Espermática/fisiología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Secuencia Conservada , Cartilla de ADN/genética , Femenino , Fertilidad/genética , Fertilidad/fisiología , Expresión Génica , Masculino , Mamíferos/genética , Ratones , Ratones Noqueados , Datos de Secuencia Molecular , Familia de Multigenes , ARN Mensajero/genética , ARN Mensajero/metabolismo , Retroelementos , Homología de Secuencia de Aminoácido , Espermatogénesis/genética , Espermatogénesis/fisiologíaRESUMEN
The Smcp mRNA encoding the sperm mitochondria-associated cysteine-rich protein is translationally repressed in round spermatids and translationally active in elongated spermatids. The patterns of transcription and translation of fusions of the Smcp promoter, the green fluorescent protein coding region (Gfp) and various combination of the Smcp and Gfp 5' UTR and 3' UTR have been studied in transgenic mice. 518 nt of Smcp 5' flanking region and 8 nt of 5' UTR drive transcription of mRNAs containing the Gfp coding region in early round spermatids at the same transcription start site as the natural Smcp gene. Transcripts containing both the Gfp 5' and 3' UTRs are translationally active in step 2 spermatids as detected by GFP fluorescence in squashes of living seminiferous tubules from adult testes, and the presence of polysomal mRNAs in sucrose gradient analyses of testes from 21-day-old prepubertal mice, which contain early round spermatids and lack elongated spermatids. By comparison, expression of GFP is delayed until steps 5 and 10, respectively, in transcripts containing the Smcp 5' UTR and Gfp 3' UTR and the Gfp 5' UTR and the Smcp 3' UTR. Sucrose gradient analysis of 21-day-old testes demonstrates that transcripts containing the Smcp 3' UTR exhibit a bimodal distribution in free-mRNPs and polysomes, indicating that the 3' UTR blocks the expression of GFP after the transcripts have entered the elongation phase, a mechanism that may involve microRNAs. The Smcp 5' UTR reduces the levels and size of polysomes in adult testis. In addition, the natural Smcp mRNA contains a positive control element that counteracts the inhibition of translation by the Smcp 5' UTR in adult testis, and the Smcp 3' UTR strongly localizes GFP fluorescence in step 10 spermatids.
Asunto(s)
Selenoproteínas/genética , Espermátides/fisiología , Regiones no Traducidas 3' , Regiones no Traducidas 5' , Animales , Northern Blotting , Centrifugación por Gradiente de Densidad , Regulación de la Expresión Génica , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Masculino , Ratones , Ratones Transgénicos , Biosíntesis de Proteínas , ARN Mensajero , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Selenoproteínas/metabolismo , Sacarosa , Testículo/fisiología , Sitio de Iniciación de la TranscripciónRESUMEN
The sperm mitochondrial cysteine-rich protein (SMCP) is a rapidly evolving cysteine- and proline-rich protein that is localized in the mitochondrial capsule and enhances sperm motility. The sequences of the SMCP protein, gene, and mRNA in a variety of mammals have been compared to understand their evolution and regulation. SMCP can now be reliably identified by its tripartite structure including a short amino-terminal segment; a central segment containing short tandem repeats rich in cysteine, proline, glutamine, and lysine; and a C-terminal segment containing no repeats, few cysteines, and a C-terminal lysine. The SMCP gene is located in the epidermal differentiation complex (EDC), a large gene cluster that functions in forming epithelial barriers. Similarities in chromosomal location, molecular function, intron-exon structure, and protein organization argue that SMCP originated from an EDC gene and acquired spermatogenic cell-specific transcriptional and translational regulation and a novel cellular function in sperm motility. The SMCP 5' UTR and 3' UTR contain conserved elements and uORFs that may function in cytoplasmic regulation of gene expression, and the levels of SMCP mRNA in human are much lower than in other mammals, a feature of male-biased expression. The evolution of SMCP has been accompanied by changes in the sequence, number, and length of repeat units, including three alleles in dogs. The major proteins associated with the mitochondrial capsule, SMCP and phospholipid hydroperoxide glutathione peroxidase, provide outstanding examples of changes in cellular function driven by selective pressures on sperm motility, an important determinant of male reproductive success.
Asunto(s)
Genómica/métodos , Selenoproteínas/genética , Regiones no Traducidas 3'/genética , Regiones no Traducidas 5'/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , Bovinos , Mapeo Cromosómico , Cromosomas Humanos Par 1/genética , Cricetinae , Bases de Datos de Ácidos Nucleicos , Perros , Epitelio/metabolismo , Femenino , Perfilación de la Expresión Génica , Humanos , Masculino , Mesocricetus , Ratones , Datos de Secuencia Molecular , Peromyscus , Seudogenes/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico , Testículo/metabolismo , Sitio de Iniciación de la TranscripciónRESUMEN
Triple knockout mice were used to investigate the interactions of five genes that were expressed in meiotic and haploid spermatogenic cells in mice, transition protein 2 (Tnp2), proacrosin (Acr), histone H1.1 (H1.1), histone H1t (H1t), and sperm mitochondria-associated cysteine-rich protein (Smcp). TNP2 functions in the replacement of histones and the initial condensation of the spermatid nucleus. The linker histone subtypes H1.1 and H1t are expressed at high levels in meiotic and early haploid cells. ACR, a protease that is stored as a proenzyme in the acrosome, is activated during the acrosome reaction and functions in binding of sperm to the zona pellucida. SMCP is a structural protein in the outer membranes of sperm mitochondria that functions in motility. Previous work demonstrates that homozygous knockout mice lacking each of these proteins individually exhibit no defect in fertility on mixed genetic backgrounds. In contrast, the present study demonstrates that five triple knockout lines, Acr/H1.1/Smcp, Acr/Tnp2/Smcp, Tnp2/H1.1/Smcp, Acr/H1t/Smcp, Tnp2/H1t/Smcp, exhibit drastic reductions in fertility on mixed genetic backgrounds. Analysis of fertility parameters reveal that the decreased fertility is due to line-dependent defects in sperm motility in vitro correlated with reduced migration in the female reproductive tract, and decreased fertilization due to defects in adhesion of sperm to the zona pellucida, the membrane surrounding the egg. It was also found that triple knockout males, that are hemizygous for one locus and homozygous for two other loci, are as subfertile as homozygous triple knockout males, a phenomenon known as haploinsufficiency. These findings demonstrate that male fertility involves synergistic interactions of genes that function in sperm motility and sperm-egg adhesion during fertilization.
Asunto(s)
Fertilización/genética , Infertilidad Masculina/genética , Motilidad Espermática/genética , Espermatozoides/metabolismo , Reacción Acrosómica/genética , Reacción Acrosómica/fisiología , Animales , Epidídimo/citología , Fertilización/fisiología , Infertilidad Masculina/metabolismo , Masculino , Ratones , Ratones Noqueados , Motilidad Espermática/fisiologíaRESUMEN
This review proposes that the peculiar patterns of gene expression in spermatogenic cells are the consequence of powerful evolutionary forces known as sexual selection. Sexual selection is generally characterized by intense competition of males for females, an enormous variety of the strategies to maximize male reproductive success, exaggerated male traits at all levels of biological organization, co-evolution of sexual traits in males and females, and conflict between the sexual advantage of the male trait and the reproductive fitness of females and the individual fitness of both sexes. In addition, spermatogenesis is afflicted by selfish genes that promote their transmission to progeny while causing deleterious effects. Sexual selection, selfish genes, and genetic conflict provide compelling explanations for many atypical features of gene expression in spermatogenic cells including the gross overexpression of certain mRNAs, transcripts encoding truncated proteins that cannot carry out basic functions of the proteins encoded by the same genes in somatic cells, the large number of gene families containing paralogous genes encoding spermatogenic cell-specific isoforms, the large number of testis-cancer-associated genes that are expressed only in spermatogenic cells and malignant cells, and the overbearing role of Sertoli cells in regulating the number and quality of spermatozoa.
Asunto(s)
Expresión Génica , Secuencias Repetitivas de Ácidos Nucleicos , Selección Genética , Espermatogénesis , Animales , Evolución Biológica , División Celular , Copulación , Femenino , Humanos , Masculino , ARN Mensajero/análisis , Cromosomas Sexuales , Proteína de Unión a TATA-Box/genéticaRESUMEN
Phospholipid hydroperoxide glutathione peroxidase (PHGPx) is a selenoprotein belonging to the family of glutathione peroxidases and has been implicated in antioxidative defense and spermatogenesis. PHGPx accounts for almost the entire selenium content of mammalian testis. In an attempt to verify the expression pattern of PHGPx, testes of mouse mutants with arrest at different stages of germ cell development and testes of mice at different ages were subjected to immunostaining with a monoclonal anti-PHGPx antibody. PHGPx was detected in Leydig cells of testes in all developmental stages. In the seminiferous tubuli, the PHGPx staining was first observed in testes of 21-day-old mice which is correlated with the appearance of the first spermatids. This result was confirmed when the testes of mutant mice with defined arrest of germ cell development were used. An immunostaining was observed in the seminiferous tubuli of olt/olt and qk/qk mice which show an arrest at spermatid differentiation. In Western blot analysis of proteins extracted from testes of mutant mice and from developing testes, two signals at 19- and 22-kDa were observed which confirm the existence of two PHGPx forms in testicular cells. In mouse spermatozoa, a subcellular localization of PHGPx and sperm mitochondria-associated cysteine-rich protein (SMCP) was demonstrated, indicating the localization of PHGPx in mitochondria of spermatozoa midpiece. For verifying the midpiece localization of PHGPx in other species, spermatozoa of Drosophila melanogaster, frog, fish, cock, mouse, rat, pig, bull, and human were used in immunostaining using anti-PHGPx antibody. A localization of PHGPx was found in the midpiece of spermatozoa in all species examined. In electronmicroscopical analysis, PHGPx signals were found in the mitochondria of midpiece. These results indicate a conserved crucial role of PHGPx during sperm function and male fertility.
Asunto(s)
Glutatión Peroxidasa/metabolismo , Espermatozoides/metabolismo , Espermatozoides/ultraestructura , Testículo , Animales , Evolución Biológica , Peces , Expresión Génica , Cabras , Humanos , Masculino , Ratones , Fosfolípido Hidroperóxido Glutatión Peroxidasa , Ratas , Porcinos , Testículo/citología , Testículo/crecimiento & desarrollo , Testículo/metabolismoRESUMEN
The patterns of transcription and translation of the ribosomal protein L32 (Rpl32) mRNA differ greatly in adult testis and somatic tissues. Northern blots reveal that the levels of Rpl32 mRNA are four- to five-fold higher in prepubertal and adult testes, and purified pachytene spermatocytes and round spermatids than in a variety of nongrowing adult somatic tissues. 5' RACE demonstrates that transcription in 8-day prepubertal testis, which lacks meiotic and haploid cells, strongly prefers the same start site in the 5' terminal oligopyrimidine tract (5' TOP) that is used is somatic cells. The 5' TOP is a cis element that inhibits translation of many mRNAs in nongrowing somatic cells. Although the sizes of deadenylated Rpl32 mRNAs are indistinguishable in somatic and spermatogenic cells, transcription initiates at 11 sites over a 31-nt segment in adult testis and approximately 62% of Rpl32 mRNAs lack a 5' TOP. In agreement with previous studies, low levels of cycloheximide increase the proportions and sizes of polysomes in absorbance profiles, and increase the proportions and sizes of polysomes translating four 5' TOP mRNA species including the Rpl32 mRNA in 8-day seminiferous tubules. In contrast, cycloheximide has little or no effect on the absorbance profiles and distribution of Rpl32 mRNA and 5' TOP mRNAs in adult seminiferous tubules. The failure of cycloheximide to increase the size of polysomes in adult seminiferous tubules implies a block in the pathway by which ribosomes are recruited onto translationally active mRNAs.
Asunto(s)
ARN Mensajero/metabolismo , Proteínas Ribosómicas/genética , Espermatocitos/metabolismo , Espermatogénesis/genética , Testículo/metabolismo , Envejecimiento/genética , Envejecimiento/metabolismo , Animales , Codón de Terminación/genética , Cicloheximida/farmacología , Regulación del Desarrollo de la Expresión Génica/genética , Masculino , Ratones , Polirribosomas/efectos de los fármacos , Polirribosomas/genética , Polirribosomas/metabolismo , Biosíntesis de Proteínas/genética , Inhibidores de la Síntesis de la Proteína/farmacología , Secuencia de Oligopirimidina en la Región 5' Terminal del ARN/genética , ARN Mensajero/genética , Espermátides/citología , Espermátides/metabolismo , Espermatocitos/citología , Testículo/citología , Transcripción Genética/genéticaRESUMEN
The sperm mitochondria-associated cysteine-rich protein (SMCP) is a cysteine- and proline-rich structural protein that is closely associated with the keratinous capsules of sperm mitochondria in the mitochondrial sheath surrounding the outer dense fibers and axoneme. To investigate the function of SMCP, we generated mice with a targeted disruption of the gene Smcp by homologous recombination. Homozygous mutant males on a mixed genetic background (C57BL/6J x 129/Sv) are fully fertile, while they are infertile on the 129/Sv background, although spermatogenesis and mating are normal. Homozygous Smcp(-/-) female mice are fertile on both genetic backgrounds. Electron microscopical examination demonstrated normal structures of sperm head, mitochondria, and tail. In vivo experiments with sperm of Smcp(-/-) 129/Sv mice revealed that the migration of spermatozoa from the uterus into the oviduct is reduced. This result is supported by the observation that sperm motility as determined by the computer-assisted semen analysis system (CASA) is significantly affected as compared to wild-type spermatozoa. In vitro fertilization assays showed that Smcp-deficient spermatozoa are able to bind to the oocyte but that the number of fertilized eggs is reduced by more than threefold relative to the wild-type control. However, removal of the zona pellucida resulted in an unaffected sperm-egg fusion which was monitored by the presence of pronuclei and generation of blastocyts. These results indicate that the infertility of the male Smcp(-/-) mice on the 129/Sv background is due to reduced motility of the spermatozoa and decreased capability of the spermatozoa to penetrate oocytes.