RESUMEN
Shadoo and PrP belongs to the same protein family, whose biological function remains poorly understood. Previous experiments reported potential functional redundancies or antagonisms between these two proteins, depending on the tissue analysed. While knockdown experiments suggested the requirement of Shadoo in the absence of PrP during early mouse embryogenesis, knockout ones, on the contrary, highlighted little impact, if any, of the double-knockout of these two loci. In the present study, we reinvestigated the phenotype associated with the concomitant knockout of these two genes using newly produced FVB/N Sprn knockout mice. In this genetic background, the combined two genes' knockout induces intra-uterine growth retardations, likely resulting from placental failures highlighted by transcriptomic analyses that revealed potential redundant or antagonist roles of these two proteins in different developmental-related pathways. It also induced an increased perinatal-lethality and ascertained the role of these two loci in the lactation process.
Asunto(s)
Proteínas del Tejido Nervioso/metabolismo , Proteínas Priónicas/metabolismo , Reproducción/fisiología , Animales , Animales Recién Nacidos/crecimiento & desarrollo , Desarrollo Embrionario , Femenino , Proteínas Ligadas a GPI , Genes Letales , Lactancia/genética , Lactancia/fisiología , Masculino , Ratones , Ratones Noqueados , Ratones Transgénicos , Proteínas del Tejido Nervioso/deficiencia , Proteínas del Tejido Nervioso/genética , Fenotipo , Placentación , Embarazo , Proteínas Priónicas/deficiencia , Proteínas Priónicas/genética , Reproducción/genética , TranscriptomaRESUMEN
Neuropathies are neurodegenerative diseases affecting humans and other mammals. Many genetic causes have been identified so far, including mutations of genes encoding proteins involved in mitochondrial dynamics. Recently, the "Turning calves syndrome", a novel sensorimotor polyneuropathy was described in the French Rouge-des-Prés cattle breed. In the present study, we determined that this hereditary disease resulted from a single nucleotide substitution in SLC25A46, a gene encoding a protein of the mitochondrial carrier family. This mutation caused an apparent damaging amino-acid substitution. To better understand the function of this protein, we knocked out the Slc25a46 gene in a mouse model. This alteration affected not only the nervous system but also altered general metabolism, resulting in premature mortality. Based on optic microscopy examination, electron microscopy and on biochemical, metabolic and proteomic analyses, we showed that the Slc25a46 disruption caused a fusion/fission imbalance and an abnormal mitochondrial architecture that disturbed mitochondrial metabolism. These data extended the range of phenotypes associated with Slc25a46 dysfunction. Moreover, this Slc25a46 knock-out mouse model should be useful to further elucidate the role of SLC25A46 in mitochondrial dynamics.
Asunto(s)
Dinámicas Mitocondriales/genética , Proteínas Mitocondriales/genética , Proteínas de Transporte de Fosfato/genética , Polineuropatías/genética , Proteómica , Sustitución de Aminoácidos/genética , Animales , Bovinos , Humanos , Ratones , Mitocondrias/genética , Mitocondrias/patología , Mutación , Fenotipo , Polineuropatías/patología , Polineuropatías/veterinariaRESUMEN
The insulin family of growth factors (insulin, IGF1, and IGF2) are critical in sex determination, adrenal differentiation, and testicular function. Notably, the IGF system has been reported to mediate the proliferation of steroidogenic cells. However, the precise role and contribution of the membrane receptors mediating those effects, namely, insulin receptor (INSR) and type-I insulin-like growth factor receptor (IGF1R), have not, to our knowledge, been investigated. We show here that specific deletion of both Insr and Igf1r in steroidogenic cells in mice leads to severe alterations of adrenocortical and testicular development. Double-mutant mice display drastic size reduction of both adrenocortex and testes, with impaired corticosterone, testosterone, and sperm production. Detailed developmental analysis of the testes revealed that fetal Leydig cell (LC) function is normal, but there is a failure of adult LC maturation and steroidogenic function associated with accumulation of progenitor LCs (PLCs). Cell-lineage tracing revealed PLC enrichment is secondary to Insr and Igf1r deletion in differentiated adult LCs, suggesting a feedback mechanism between cells at different steps of differentiation. Taken together, these data reveal the cell-autonomous and nonautonomous roles of the IGF system for proper development and maintenance of steroidogenic lineages.-Neirijnck, Y., Calvel, P., Kilcoyne, K. R., Kühne, F., Stévant, I., Griffeth, R. J., Pitetti, J.-L., Andric, S. A., Hu, M.-C., Pralong, F., Smith, L. B., Nef, S. Insulin and IGF1 receptors are essential for the development and steroidogenic function of adult Leydig cells.
Asunto(s)
Diferenciación Celular , Células Intersticiales del Testículo/metabolismo , Receptor de Insulina/metabolismo , Receptores de Somatomedina/metabolismo , Células Madre/metabolismo , Corteza Suprarrenal/citología , Corteza Suprarrenal/metabolismo , Animales , Corticosterona/genética , Corticosterona/metabolismo , Células Intersticiales del Testículo/citología , Masculino , Ratones , Ratones Noqueados , Receptor de Insulina/genética , Receptores de Somatomedina/genética , Células Madre/citología , Testosterona/genética , Testosterona/metabolismoRESUMEN
Cell lineages of the early human gonad commit to one of the two mutually antagonistic organogenetic fates, the testis or the ovary. Some individuals with a 46,XX karyotype develop testes or ovotestes (testicular or ovotesticular disorder of sex development; TDSD/OTDSD), due to the presence of the testis-determining gene, SRY Other rare complex syndromic forms of TDSD/OTDSD are associated with mutations in pro-ovarian genes that repress testis development (e.g. WNT4); however, the genetic cause of the more common non-syndromic forms is unknown. Steroidogenic factor-1 (known as NR5A1) is a key regulator of reproductive development and function. Loss-of-function changes in NR5A1 in 46,XY individuals are associated with a spectrum of phenotypes in humans ranging from a lack of testis formation to male infertility. Mutations in NR5A1 in 46,XX women are associated with primary ovarian insufficiency, which includes a lack of ovary formation, primary and secondary amenorrhoea as well as early menopause. Here, we show that a specific recurrent heterozygous missense mutation (p.Arg92Trp) in the accessory DNA-binding region of NR5A1 is associated with variable degree of testis development in 46,XX children and adults from four unrelated families. Remarkably, in one family a sibling raised as a girl and carrying this NR5A1 mutation was found to have a 46,XY karyotype with partial testicular dysgenesis. These unique findings highlight how a specific variant in a developmental transcription factor can switch organ fate from the ovary to testis in mammals and represents the first missense mutation causing isolated, non-syndromic 46,XX testicular/ovotesticular DSD in humans.
Asunto(s)
Proteínas de Unión al ADN/genética , Trastorno del Desarrollo Sexual 46,XY/genética , Insuficiencia Ovárica Primaria/genética , Desarrollo Sexual/genética , Factor Esteroidogénico 1/genética , Adulto , Síndrome de Resistencia Androgénica/genética , Síndrome de Resistencia Androgénica/patología , Linaje de la Célula/genética , Niño , Trastorno del Desarrollo Sexual 46,XY/patología , Femenino , Gónadas/crecimiento & desarrollo , Gónadas/patología , Humanos , Cariotipo , Masculino , Mutación Missense , Ovario/crecimiento & desarrollo , Ovario/patología , Linaje , Insuficiencia Ovárica Primaria/patología , Procesos de Determinación del Sexo , Testículo/crecimiento & desarrollo , Testículo/patologíaRESUMEN
The Hedgehog (Hh) family of secreted proteins act as morphogens to control embryonic patterning and development in a variety of organ systems. Post-translational covalent attachment of cholesterol and palmitate to Hh proteins are critical for multimerization and long range signaling potency. However, the biological impact of lipid modifications on Hh ligand distribution and signal reception in humans remains unclear. In the present study, we report a unique case of autosomal recessive syndromic 46,XY Disorder of Sex Development (DSD) with testicular dysgenesis and chondrodysplasia resulting from a homozygous G287V missense mutation in the hedgehog acyl-transferase (HHAT) gene. This mutation occurred in the conserved membrane bound O-acyltransferase (MBOAT) domain and experimentally disrupted the ability of HHAT to palmitoylate Hh proteins such as DHH and SHH. Consistent with the patient phenotype, HHAT was found to be expressed in the somatic cells of both XX and XY gonads at the time of sex determination, and Hhat loss of function in mice recapitulates most of the testicular, skeletal, neuronal and growth defects observed in humans. In the developing testis, HHAT is not required for Sertoli cell commitment but plays a role in proper testis cord formation and the differentiation of fetal Leydig cells. Altogether, these results shed new light on the mechanisms of action of Hh proteins. Furthermore, they provide the first clinical evidence of the essential role played by lipid modification of Hh proteins in human testicular organogenesis and embryonic development.
Asunto(s)
Aciltransferasas/genética , Trastorno del Desarrollo Sexual 46,XY/genética , Proteínas Hedgehog/metabolismo , Lipoilación/genética , Mutación Missense , Transducción de Señal/genética , Aciltransferasas/química , Aciltransferasas/metabolismo , Secuencia de Aminoácidos , Animales , Femenino , Homocigoto , Humanos , Masculino , Ratones , Datos de Secuencia Molecular , Linaje , Homología de Secuencia de Aminoácido , Testículo/embriologíaRESUMEN
Mouse sex determination provides an attractive model to study how regulatory genetic networks and signaling pathways control cell specification and cell fate decisions. This study characterizes in detail the essential role played by the insulin receptor (INSR) and the IGF type I receptor (IGF1R) in adrenogenital development and primary sex determination. Constitutive ablation of insulin/IGF signaling pathway led to reduced proliferation rate of somatic progenitor cells in both XX and XY gonads prior to sex determination together with the downregulation of hundreds of genes associated with the adrenal, testicular, and ovarian genetic programs. These findings indicate that prior to sex determination somatic progenitors in Insr;Igf1r mutant gonads are not lineage primed and thus incapable of upregulating/repressing the male and female genetic programs required for cell fate restriction. In consequence, embryos lacking functional insulin/IGF signaling exhibit (i) complete agenesis of the adrenal cortex, (ii) embryonic XY gonadal sex reversal, with a delay of Sry upregulation and the subsequent failure of the testicular genetic program, and (iii) a delay in ovarian differentiation so that Insr;Igf1r mutant gonads, irrespective of genetic sex, remained in an extended undifferentiated state, before the ovarian differentiation program ultimately is initiated at around E16.5.
Asunto(s)
Gónadas , Insulina , Receptor IGF Tipo 1 , Receptor de Insulina , Procesos de Determinación del Sexo/genética , Corteza Suprarrenal/crecimiento & desarrollo , Corteza Suprarrenal/patología , Glándulas Suprarrenales/crecimiento & desarrollo , Glándulas Suprarrenales/metabolismo , Animales , Diferenciación Celular/genética , Linaje de la Célula , Proliferación Celular , Trastornos del Desarrollo Sexual/genética , Femenino , Gónadas/crecimiento & desarrollo , Gónadas/metabolismo , Gónadas/patología , Humanos , Insulina/genética , Insulina/metabolismo , Masculino , Ratones , Ovario/crecimiento & desarrollo , Ovario/metabolismo , Receptor IGF Tipo 1/genética , Receptor IGF Tipo 1/metabolismo , Receptor de Insulina/genética , Receptor de Insulina/metabolismo , Cromosomas Sexuales , Transducción de Señal , Testículo/crecimiento & desarrollo , Testículo/metabolismoRESUMEN
Azoospermia (the complete absence of spermatozoa in the semen) is a common cause of male infertility. The etiology of azoospermia is poorly understood. Whole-genome analysis of azoospermic men has identified a number of candidate genes, such as the X-linked testis-expressed 11 (TEX11) gene. Using a comparative genomic hybridization array, an exonic deletion (exons 10-12) of TEX11 had previously been identified in two non-apparent azoospermic patients. However, the putative impact of this genetic alteration on spermatogenesis and the azoospermia phenotype had not been validated functionally. We therefore used a CRISPR/Cas9 system to generate a mouse model (Tex11Ex9-11del/Y) with a partial TEX11 deletion that mimicked the human mutation. Surprisingly, the mutant male Tex11Ex9-11del/Y mice were fertile. The sperm concentration, motility, and morphology were normal. Similarly, the mutant mouse line's testis transcriptome was normal, and the expression of spermatogenesis genes was not altered. These results suggest that the mouse equivalent of the partial deletion observed in two infertile male with azoospermia has no impact on spermatogenesis or fertility in mice, at least of a FVB/N genetic background and until 10 months of age. Mimicking a human mutation does not necessarily lead to the same human phenotype in mice, highlighting significant differences species.
Asunto(s)
Azoospermia , Meiosis , Espermatogénesis , Animales , Masculino , Ratones , Espermatogénesis/genética , Meiosis/genética , Azoospermia/genética , Azoospermia/patología , Infertilidad Masculina/genética , Eliminación de Secuencia , Humanos , Testículo/metabolismo , Testículo/patología , Sistemas CRISPR-CasRESUMEN
The continuous production of spermatozoa is a tightly regulated biological process, both at the transcriptional and post-transcriptional levels. Recently, different classes of small non-coding RNAs have emerged as important regulators of spermatogenesis. Available molecular and genetic data, although still fragmented, underscore their crucial role in regulating the fine tuning of gene expression required for testicular function. Here, we review the latest advances accomplished in this domain, spanning from the biogenesis of these small non-coding RNAs to their roles in male reproductive function.
Asunto(s)
ARN Pequeño no Traducido/fisiología , Espermatogénesis/genética , Animales , Linaje de la Célula/genética , Linaje de la Célula/fisiología , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/metabolismo , ARN Helicasas DEAD-box/fisiología , Células Germinativas/metabolismo , Células Germinativas/fisiología , Humanos , Masculino , Modelos Biológicos , ARN Pequeño no Traducido/clasificación , ARN Pequeño no Traducido/genética , ARN Pequeño no Traducido/metabolismo , Reproducción/genética , Reproducción/fisiología , Ribonucleasa III/genética , Ribonucleasa III/metabolismo , Ribonucleasa III/fisiología , Espermatogénesis/fisiologíaRESUMEN
Disorders/differences of sex development (DSD) cause profound psychological and reproductive consequences for the affected individuals, however, most are still unexplained at the molecular level. Here, we present a novel gene, 3-hydroxy-3-methylglutaryl coenzyme A synthase 2 (HMGCS2), encoding a metabolic enzyme in the liver important for energy production from fatty acids, that shows an unusual expression pattern in developing fetal mouse gonads. Shortly after gonadal sex determination it is up-regulated in the developing testes following a very similar spatial and temporal pattern as the male-determining gene Sry in Sertoli cells before switching to ovarian enriched expression. To test if Hmgcs2 is important for gonad development in mammals, we pursued two lines of investigations. Firstly, we generated Hmgcs2-null mice using CRISPR/Cas9 and found that these mice had gonads that developed normally even on a sensitized background. Secondly, we screened 46,XY DSD patients with gonadal dysgenesis and identified two unrelated patients with a deletion and a deleterious missense variant in HMGCS2 respectively. However, both variants were heterozygous, suggesting that HMGCS2 might not be the causative gene. Analysis of a larger number of patients in the future might shed more light into the possible association of HMGCS2 with human gonadal development.
Asunto(s)
Trastornos del Desarrollo Sexual/genética , Disgenesia Gonadal/genética , Gónadas/crecimiento & desarrollo , Hidroximetilglutaril-CoA Sintasa/genética , Adolescente , Animales , Trastornos del Desarrollo Sexual/patología , Femenino , Regulación del Desarrollo de la Expresión Génica/genética , Disgenesia Gonadal/patología , Gónadas/patología , Heterocigoto , Humanos , Masculino , Ratones , Mutación Missense/genética , Ovario/crecimiento & desarrollo , Ovario/patología , Células de Sertoli/metabolismo , Proteína de la Región Y Determinante del Sexo/genética , Testículo/crecimiento & desarrollo , Testículo/patologíaRESUMEN
Shadoo belongs to the prion protein family, an evolutionary conserved and extensively studied family due to the implication of PrP in Transmissible Spongiform Encephalopathies. However, the biological function of these genes remains poorly understood. While Sprn-knockdown experiments suggested an involvement of Shadoo during mouse embryonic development, Sprn-knockout experiments in 129Pas/C57BL/6J or 129Pas/FVB/NCr mice did not confirm it. In the present study, we analyzed the impact of Sprn gene invalidation in a pure FVB/NJ genetic background, using a zinc finger nuclease approach. The in-depth analysis of the derived knockout transgenic mice revealed a significant increase in embryonic lethality at early post-implantation stages, a growth retardation of young Sprn-knockout pups fed by wild type mice and a lactation defect of Sprn-knockout females. Histological and transcriptional analyses of knockout E7.5 embryos, E14.5 placentas and G7.5 mammary glands revealed specific roles of the Shadoo protein in mouse early embryogenesis, tissue development and differentiation with a potential antagonist action between PrP and Shadoo. This study thus highlights the entanglement between the proteins of the prion family.
Asunto(s)
Diferenciación Celular/genética , Desarrollo Embrionario/genética , Proteínas del Tejido Nervioso/genética , Proteínas Priónicas/genética , Animales , Proteínas Ligadas a GPI , Humanos , Glándulas Mamarias Animales/crecimiento & desarrollo , Glándulas Mamarias Animales/metabolismo , Ratones , Ratones Noqueados , Células Madre Embrionarias de Ratones/metabolismo , Organogénesis/genética , Enfermedades por Prión/genética , Enfermedades por Prión/patologíaRESUMEN
Identification of novel genes involved in sexual development is crucial for understanding disorders of sex development (DSD). Here, we propose a member of the START domain family, the X chromosome STARD8, as a DSD candidate gene. We have identified a missense mutation of this gene in 2 sisters with 46,XY gonadal dysgenesis, inherited from their heterozygous mother. Gonadal tissue of one of the sisters contained Leydig cells overloaded with cholesterol droplets, i.e., structures previously identified in 46,XY DSD patients carrying mutations in the STAR gene encoding another START domain family member, which is crucial for steroidogenesis. Based on the phenotypes of our patients, we propose a dual role of STARD8 in sexual development, namely in testes determination and testosterone synthesis. However, further studies are needed to confirm the involvement of STARD8 in sexual development.
Asunto(s)
Cromosomas Humanos X/genética , Proteínas Activadoras de GTPasa/genética , Disgenesia Gonadal 46 XY/genética , Mutación/genética , Procesos de Determinación del Sexo/genética , Hermanos , Adolescente , Secuencia de Bases , Femenino , Gónadas/patología , Humanos , Lactante , FenotipoRESUMEN
Sertoli cells (SCs), the only somatic cells within seminiferous tubules, associate intimately with developing germ cells. They not only provide physical and nutritional support but also secrete factors essential to the complex developmental processes of germ cell proliferation and differentiation. The SC transcriptome must therefore adapt rapidly during the different stages of spermatogenesis. We report comprehensive genome-wide expression profiles of pure populations of SCs isolated at 5 distinct stages of the first wave of mouse spermatogenesis, using RNA sequencing technology. We were able to reconstruct about 13 901 high-confidence, nonredundant coding and noncoding transcripts, characterized by complex alternative splicing patterns with more than 45% comprising novel isoforms of known genes. Interestingly, roughly one-fifth (2939) of these genes exhibited a dynamic expression profile reflecting the evolving role of SCs during the progression of spermatogenesis, with stage-specific expression of genes involved in biological processes such as cell cycle regulation, metabolism and energy production, retinoic acid synthesis, and blood-testis barrier biogenesis. Finally, regulatory network analysis identified the transcription factors endothelial PAS domain-containing protein 1 (EPAS1/Hif2α), aryl hydrocarbon receptor nuclear translocator (ARNT/Hif1ß), and signal transducer and activator of transcription 1 (STAT1) as potential master regulators driving the SC transcriptional program. Our results highlight the plastic transcriptional landscape of SCs during the progression of spermatogenesis and provide valuable resources to better understand SC function and spermatogenesis and its related disorders, such as male infertility.
Asunto(s)
Túbulos Seminíferos/metabolismo , Células de Sertoli/metabolismo , Espermatogénesis/genética , Transcriptoma , Animales , Perfilación de la Expresión Génica , Masculino , Ratones , Ratones TransgénicosRESUMEN
We report the case of a female patient suffering from a 46,XY disorder of sexual development (DSD) with complete gonadal dysgenesis and Wiedemann-Steiner Syndrome (WDSTS). The coexistence of these 2 conditions has not yet been reported. Using whole exome sequencing and comparative genome hybridization array, we identified a de novo MLL/KMT2A gene nonsense mutation which explains the WDSTS phenotype. In addition, we discovered novel genetic variants, which could explain the testicular dysgenesis observed in the patient, a maternally inherited 167-kb duplication of DAAM2 and MOCS1 genes and a de novo LRRC33/NRROS gene mutation. These genes, some of which are expressed during mouse gonadal development, could be considered as potentially new candidate genes for DSD.
Asunto(s)
Trastorno del Desarrollo Sexual 46,XY/genética , Disgenesia Gonadal 46 XY/genética , N-Metiltransferasa de Histona-Lisina/genética , Proteína de la Leucemia Mieloide-Linfoide/genética , Codón sin Sentido , Anomalías Craneofaciales/genética , Discapacidades del Desarrollo/genética , Femenino , Estudios de Seguimiento , Genitales/patología , Humanos , Recién Nacido , Síndromes de Malabsorción/genética , Masculino , Linaje , SíndromeRESUMEN
Testis size and sperm production are directly correlated to the total number of adult Sertoli cells (SCs). Although the establishment of an adequate number of SCs is crucial for future male fertility, the identification and characterization of the factors regulating SC survival, proliferation, and maturation remain incomplete. To investigate whether the IGF system is required for germ cell (GC) and SC development and function, we inactivated the insulin receptor (Insr), the IGF1 receptor (Igf1r), or both receptors specifically in the GC lineage or in SCs. Whereas ablation of insulin/IGF signaling appears dispensable for GCs and spermatogenesis, adult testes of mice lacking both Insr and Igf1r in SCs (SC-Insr;Igf1r) displayed a 75% reduction in testis size and daily sperm production as a result of a reduced proliferation rate of immature SCs during the late fetal and early neonatal testicular period. In addition, in vivo analyses revealed that FSH requires the insulin/IGF signaling pathway to mediate its proliferative effects on immature SCs. Collectively, these results emphasize the essential role played by growth factors of the insulin family in regulating the final number of SCs, testis size, and daily sperm output. They also indicate that the insulin/IGF signaling pathway is required for FSH-mediated SC proliferation.
Asunto(s)
Hormona Folículo Estimulante/metabolismo , Receptor de Insulina/metabolismo , Células de Sertoli/citología , Células de Sertoli/metabolismo , Animales , Recuento de Células , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Proliferación Celular , Forma de la Célula/efectos de los fármacos , Femenino , Feto/citología , Feto/embriología , Perfilación de la Expresión Génica , Células Germinativas/citología , Células Germinativas/efectos de los fármacos , Células Germinativas/metabolismo , Humanos , Células Intersticiales del Testículo/citología , Células Intersticiales del Testículo/efectos de los fármacos , Células Intersticiales del Testículo/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Mutación/genética , Tamaño de los Órganos/efectos de los fármacos , Tamaño de los Órganos/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptor IGF Tipo 1/genética , Receptor IGF Tipo 1/metabolismo , Receptor de Insulina/genética , Túbulos Seminíferos/citología , Túbulos Seminíferos/efectos de los fármacos , Túbulos Seminíferos/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Espermatogénesis/efectos de los fármacos , Espermatogénesis/genética , Espermatozoides/citología , Espermatozoides/efectos de los fármacos , Espermatozoides/metabolismo , Hormonas Tiroideas/farmacologíaRESUMEN
Spermatogenesis is a highly sophisticated process involved in the transmission of genetic heritage. It includes halving ploidy, repackaging of the chromatin for transport, and the equipment of developing spermatids and eventually spermatozoa with the advanced apparatus (e.g., tightly packed mitochondrial sheat in the mid piece, elongating of the tail, reduction of cytoplasmic volume) to elicit motility once they reach the epididymis. Mammalian spermatogenesis is divided into three phases. In the first the primitive germ cells or spermatogonia undergo a series of mitotic divisions. In the second the spermatocytes undergo two consecutive divisions in meiosis to produce haploid spermatids. In the third the spermatids differentiate into spermatozoa in a process called spermiogenesis. Paracrine, autocrine, juxtacrine, and endocrine pathways all contribute to the regulation of the process. The array of structural elements and chemical factors modulating somatic and germ cell activity is such that the network linking the various cellular activities during spermatogenesis is unimaginably complex. Over the past two decades, advances in genomics have greatly improved our knowledge of spermatogenesis, by identifying numerous genes essential for the development of functional male gametes. Large-scale analyses of testicular function have deepened our insight into normal and pathological spermatogenesis. Progress in genome sequencing and microarray technology have been exploited for genome-wide expression studies, leading to the identification of hundreds of genes differentially expressed within the testis. However, although proteomics has now come of age, the proteomics-based investigation of spermatogenesis remains in its infancy. Here, we review the state-of-the-art of large-scale proteomic analyses of spermatogenesis, from germ cell development during sex determination to spermatogenesis in the adult. Indeed, a few laboratories have undertaken differential protein profiling expression studies and/or systematic analyses of testicular proteomes in entire organs or isolated cells from various species. We consider the pros and cons of proteomics for studying the testicular germ cell gene expression program. Finally, we address the use of protein datasets, through integrative genomics (i.e., combining genomics, transcriptomics, and proteomics), bioinformatics, and modelling.
Asunto(s)
Proteómica , Espermatogénesis , Animales , Diferenciación Celular , Perfilación de la Expresión Génica , Humanos , Masculino , Meiosis , Mitosis , Espermátides/citología , Espermatocitos/citología , Espermatogénesis/genética , Espermatogénesis/fisiología , Espermatogonias/citología , Espermatozoides/citología , Espermatozoides/fisiología , Testículo/citología , Testículo/embriología , Testículo/metabolismoRESUMEN
Sperm are, arguably, the most differentiated cells produced within the body of any given species. This is owing to the fact that spermatogenesis is an intricate and highly specialized process evolved to suit the individual particularities of each sexual species. Despite a vast diversity in method, the aim of spermatogenesis is always the same, the idealized transmission of genetic patrimony. Towards this goal certain requirements must always be met, such as a relative twofold reduction in ploidy, repackaging of the chromatin for transport and specialized enhancements for cell motility, recognition and fusion. In the past 20 years, the study of molecular networks coordinating male germ cell development, particularly in mammals, has become more and more facilitated thanks to large-scale analyses of genome expression. Such postgenomic endeavors have generated landscapes of data for both fundamental and clinical reproductive biology. Continuous, large-scale integration analyses of these datasets are undertaken which provide access to very precise information on a myriad of biomolecules. This review presents commonly used transcriptomic and proteomic workflows applied to various testicular germ cell studies. We will also provide a general overview of the technical possibilities available to reproductive genomic biologists, noting the advantages and drawbacks of each technique.
Asunto(s)
Espermatogénesis/fisiología , Testículo/fisiología , Animales , Perfilación de la Expresión Génica/métodos , Masculino , Proteómica/métodos , Espermatogénesis/genética , Testículo/metabolismoRESUMEN
In a recent proteomic study of rat spermatogenesis, we identified CLPH (for Casein-Like PHosphoprotein), a new testis-specific protein expressed exclusively in postmeiotic germ cells. In situ hybridization showed that the CLPH transcript was mainly present in round spermatids, whereas the protein was specifically detected by immunohistochemistry in elongated spermatids and in residual bodies. Electron microscopy showed the protein to be mostly cytoplasmic, but also frequently associated with the mitochondrial inner membrane during the last steps of spermatid differentiation. The Clph gene was found to be present solely in mammalian genomes, in a chromosomal region syntenic to the mammalian cluster of secretory calcium-binding phosphoprotein (SCPP) genes. CLPH has several distinctive properties in common with SCPPs: calcium overlay experiments showed that CLPH was a calcium-binding protein, whereas trypsin digestion assay, circular dichroism and fluorescence experiments demonstrated its intrinsically disordered structure. We also showed that CLPH was phosphorylated in vitro and in vivo by casein kinase 2, an enzyme critical for spermatid elongation. Given the specific and strong production of CLPH during rat spermiogenesis, together with the particular biochemical properties of this protein, we suggest that CLPH is involved in the extremely complex structural rearrangements occurring in haploid germ cells during spermiogenesis.
Asunto(s)
Caseínas/metabolismo , Espermátides/metabolismo , Espermatogénesis , Secuencia de Aminoácidos , Animales , Quinasa de la Caseína II/metabolismo , Caseínas/química , Caseínas/genética , Simulación por Computador , Electroforesis en Gel Bidimensional , Humanos , Hibridación in Situ , Masculino , Espectrometría de Masas , Meiosis , Datos de Secuencia Molecular , Fosforilación , Pliegue de Proteína , Proteómica , Ratas , Ratas Sprague-Dawley , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Testículo/citología , Testículo/metabolismoRESUMEN
The molecular mechanisms underlying normal and pathological spermatogenesis remain poorly understood. We compared protein concentrations in different germ cell types to identify those proteins specifically or preferentially expressed at each stage of rat spermatogenesis. Crude cytosolic protein extracts and reversed-phase HPLC prefractionated cytosolic extracts from spermatogonia, pachytene spermatocytes, and early spermatids were subjected to two-dimensional difference gel electrophoresis (2-D DIGE). By comparing gels and carrying out statistical analyses, we were able to identify 1274 protein spots with relative abundances differing significantly between the three cell types. We found that 265 of these spots displaying highly differential expression (ratio > or = 2.5 between two cell types), identified by mass fingerprinting, corresponded to 123 nonredundant proteins. The proteins clustered into three clades, corresponding to mitotic, meiotic, and post-meiotic cell types. The differentially expressed proteins identified by 2-D DIGE were confirmed and validated by western blotting and immunohistochemistry, in the few cases in which antibodies were available. 2-D DIGE appears a relevant proteomics approach for studying rat germ cell differentiation, allowing the establishment of the precise expression profiles for a relatively large number of proteins during normal spermatogenesis.