15 research outputs found
Evolutionary analysis of genes coding for Cysteine-RIch Secretory Proteins (CRISPs) in mammals
Cysteine-RIch Secretory Proteins (CRISP) are expressed in the reproductive tract of mammalian males and are involved in fertilization and related processes. Due to their important role in sperm performance and sperm-egg interaction, these genes are likely to be exposed to strong selective pressures, including postcopulatory sexual selection and/or male-female coevolution. We here perform a comparative evolutionary analysis of Crisp genes in mammals. Currently, the nomenclature of CRISP genes is confusing, as a consequence of discrepancies between assignments of orthologs, particularly due to numbering of CRISP genes. This may generate problems when performing comparative evolutionary analyses of mammalian clades and species. To avoid such problems, we first carried out a study of possible orthologous relationships and putative origins of the known CRISP gene sequences. Furthermore, and with the aim to facilitate analyses, we here propose a different nomenclature for CRISP genes (EVAC1-4, "EVolutionarily-analyzed CRISP") to be used in an evolutionary context.Fil: Arévalo, Lena. Consejo Superior de Investigaciones Científicas. Museo Nacional de Ciencias Naturales; España. Universitat Bonn; AlemaniaFil: Brukman, Nicolás Gastón. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Cuasnicu, Patricia Sara. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Roldan, Eduardo R. S.. Consejo Superior de Investigaciones Científicas. Museo Nacional de Ciencias Naturales; Españ
DETERMINACIÒN DE TIMOL Y CARVACROL EN HOJAS DE ORÉGANO POR HPLC FL
Oregano (Origanum vulgare L.) is an aromatic native plant originated from the Mediterranean region and it's traditionally grown in the south of Peru. The crop has adapted well over 2600 m.a.s.l. Bellow this altitude, the concentration of essential oils (thymol and carvacrol) decreases. The development of this method is essential to provide tools for decision making to identify regions where one or more ecotypes can potentially grow and produce high quality of essential oils contents of thymol and carvacrol. As part of the activities, it was necessary to develop a protocol to quantify HPLC for the oregano crop. The proposed method uses a HPLC with a fluorescence detector, isocratic mobile phase of acetonitrile and water (ACN): H O (50:50), a column Purospher® STAR rp-18e (4.6 x 2 150mm, 5µm), flow 1 ml/minute, injection volume of 20 µLand run time of 15 minutes. The present method has a linearity greater than 0.999, precision ≤2.27, 2.4 intra-day, and ≤2.47 and 1.94 for daily measurements for carvacrol and thymol respectively. Detection limits (DL) and quantification limits (QL) were 0.0007 to 0.002 mg/Lfor, thymol and 0.002 to 0.005 mg/L for carvacrol with a recovery of 98.68% and 90.95% respectively. Results show that the protocol is an appropriate method to quantify carvacrol and thymol via HPLC in oregano leaves.El orégano (Origanum vulgare L.) es una planta aromática originaria del mediterráneo; tradicionalmente es cultivada en la zona sur del Perú. Se adapta muy bien a los valles interandinos sobre los 2600 m.s.n.m. Debajo de esa altitud, la concentración de aceites esenciales (timol y carvacrol) disminuye. El desarrollo del presente método tiene por finalidad dotar de herramientas para tomar decisiones sobre la identificación de regiones donde uno o más ecotipos pueden desarrollarse potencialmente con respecto a la calidad de sus aceites esenciales en función al contenido de timol y carvacrol. Como parte de las actividades del proyecto fue necesario el desarrollo de un protocolo para la cuantificación por HPLC para el cultivo de orégano. El método propuesto consiste de un sistema HPLC con detector de Fluorescencia, fase móvil isocrática de acetonitrilo y agua (ACN):H O (50:50), columna Purospher® STAR rp-18e (4,6 2 x 150 mm, 5 ìm), flujo de 1 mL/min, volumen de inyección de 20 ìLy un tiempo de corrida de 15 minutos. El método ha presentado una linealidad mayor a 0,999, precisión ≤ 2,27 y 2,4 intra día, y ≤2,47 y 1,94 inter diario para el carvacrol y timol, respectivamente. Los límites de detección determinado LOD y límite de cuantificación LOQ fue de 0,0007 - 0,002 mg/L para el timol y 0,002 - 0,005 mg/L para el carvacrol, con una recuperación de 98,68 % y 90,95 %, respectivamente. Los resultados muestran que el protocolo desarrollado es un método adecuado para la determinación de timol y carvacrol en hojas de orégano
SPAG17 Mediates Nuclear Translocation of Protamines During Spermiogenesis
Protamines (PRM1 and PRM2) are small, arginine-rich, nuclear proteins that replace histones in the final stages of spermiogenesis, ensuring chromatin compaction and nuclear remodeling. Defects in protamination lead to increased DNA fragmentation and reduced male fertility. Since efficient sperm production requires the translocation of protamines from the cytoplasm to the nucleus, we investigated whether SPAG17, a protein crucial for intracellular protein trafficking during spermiogenesis, participates in protamine transport. Initially, we assessed the protein-protein interaction between SPAG17 and protamines using proximity ligation assays, revealing a significant interaction originating in the cytoplasm and persisting within the nucleus. Subsequently, immunoprecipitation and mass spectrometry (IP/MS) assays validated this initial observation. Sperm and spermatids fro
El acuerdo de paz de 2017 entre el gobierno y las FARC: un modelo de justicia transicional en Colombia
Máster Oficial en Estudios Avanzados en Derechos Humanos. Curso 2017/2018Presidente: Carlos Fernández Liesa ; Secretaria: Amparo Alcoceba Gallego ; Vocal: Félix Vacas Fernánde
A High Quality Genome for Mus spicilegus, a Close Relative of House Mice with Unique Social and Ecological Adaptations
Genomic data for the closest relatives of house mice (Mus musculus species complex) are surprisingly limited. Here, we present the first complete genome for a behaviorally and ecologically unique member of the sister clade to house mice, the mound-building mouse, Mus spicilegus. Using read cloud sequencing and de novo assembly we produced a 2.50 Gbp genome with a scaffold N50 of 2.27 Mbp. We constructed >25 000 gene models, of which the majority had high homology to other Mus species. To evaluate the utility of the M. spicilegus genome for behavioral and ecological genomics, we extracted 196 vomeronasal receptor (VR) sequences from our genome and analyzed phylogenetic relationships between M. spicilegus VRs and orthologs from M. musculus and the Algerian mouse, M. spretus. While most M. spicilegus VRs clustered with orthologs in M. musculus and M. spretus, 10 VRs with evidence of rapid divergence in M. spicilegus are strong candidate modulators of species-specific chemical communication. A high quality assembly and genome for M. spicilegus will help to resolve discordant ancestry patterns in house mouse genomes, and will provide an essential foundation for genetic dissection of phenotypes that distinguish commensal from non-commensal species, and the social and ecological characteristics that make M. spicilegus unique
Sexual selection towards a protamine expression ratio optimum in two rodent groups?
Post-copulatory sexual selection is thought to influence the evolution of genes involved in reproduction. However, the detection of straightforward effects has been proven difficult due to the complexity and diversity of reproductive landscapes found in different taxa. Here, we compare the possible effect of relative testes mass as a sperm competition proxy on protamine genotype (protamine 1/protamine 2 ratio) and the link to sperm head phenotype in two rodent groups, mice, and voles. In mice, protamine expression ratios were found to increase from low values toward a 1:1 ratio in a positive association with testes mass, and relative sperm head area. In contrast, in voles, decreasing protamine expression ratios were found in species with larger testes but, surprisingly, they range from high values, again toward a 1:1 ratio, and showing a negative correlation with relative sperm head area. Altogether, we found differences in the way protamines seem to be selected and involved in adaptations of the sperm head in voles and mice. However, sexual selection driven by sperm competition seems to exhibit a common evolutionary pattern in both groups toward an equilibrium in the expression of the two protamines.Fil: Arévalo, Lena. Universitat Bonn; Alemania. Consejo Superior de Investigaciones Científicas. Museo Nacional de Ciencias Naturales; EspañaFil: Tourmente, Maximiliano. Consejo Superior de Investigaciones Científicas. Museo Nacional de Ciencias Naturales; España. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones Biológicas y Tecnológicas. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas, Físicas y Naturales. Instituto de Investigaciones Biológicas y Tecnológicas; Argentina. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas, Físicas y Naturales. Centro de Biología Celular y Molecular; ArgentinaFil: Varea Sánchez, María. Consejo Superior de Investigaciones Científicas. Museo Nacional de Ciencias Naturales; EspañaFil: Ortiz García, Daniel. Consejo Superior de Investigaciones Científicas. Museo Nacional de Ciencias Naturales; EspañaFil: Roldan, Eduardo R. S.. Consejo Superior de Investigaciones Científicas. Museo Nacional de Ciencias Naturales; Españ
Transcriptional CDK Inhibitors as Potential Treatment Option for Testicular Germ Cell Tumors
Type II testicular germ cell tumors (TGCT) are the most frequently diagnosed solid malignancy in young men. Up to 15% of patients with metastatic non-seminomas show cisplatin resistance and a very poor survival rate due to lacking treatment options. Transcriptional cyclin-dependent kinases (CDK) have been shown to be effective targets in the treatment of different types of cancer. Here, we investigated the effects of the CDK inhibitors dinaciclib, flavopiridol, YKL-5-124, THZ1, NVP2, SY0351 and THZ531. An XTT viability assay revealed a strong cytotoxic impact of CDK7/12/13 inhibitor SY0351 and CDK9 inhibitor NVP2 on the TGCT wild-type cell lines (2102EP, NCCIT, TCam2) and the cisplatin-resistant cell lines (2102EP-R, NCCIT-R). The CDK7 inhibitor YKL-5-124 showed a strong impact on 2102EP, 2102EP-R, NCCIT and NCCIT-R cell lines, leaving the MPAF control cell line mostly unaffected. FACS-based analysis revealed mild effects on the cell cycle of 2102EP and TCam2 cells after SY0351, YKL-5-124 or NVP2 treatment. Molecular analysis showed a cell-line-specific response for SY0351 and NVP2 inhibition while YKL-5-124 induced similar molecular changes in 2102EP, TCam2 and MPAF cells. Thus, after TGCT subtype determination, CDK inhibitors might be a potential alternative for optimized and individualized therapy independent of chemotherapy sensitivity
Loss of the cleaved-protamine 2 domain leads to incomplete histone-to-protamine exchange and infertility in mice.
Protamines are unique sperm-specific proteins that package and protect paternal chromatin until fertilization. A subset of mammalian species expresses two protamines (PRM1 and PRM2), while in others PRM1 is sufficient for sperm chromatin packaging. Alterations of the species-specific ratio between PRM1 and PRM2 are associated with infertility. Unlike PRM1, PRM2 is generated as a precursor protein consisting of a highly conserved N-terminal domain, termed cleaved PRM2 (cP2), which is consecutively trimmed off during chromatin condensation. The carboxyterminal part, called mature PRM2 (mP2), interacts with DNA and together with PRM1, mediates chromatin-hypercondensation. The removal of the cP2 domain is believed to be imperative for proper chromatin condensation, yet, the role of cP2 is not yet understood. We generated mice lacking the cP2 domain while the mP2 is still expressed. We show that the cP2 domain is indispensable for complete sperm chromatin protamination and male mouse fertility. cP2 deficient sperm show incomplete protamine incorporation and a severely altered protamine ratio, retention of transition proteins and aberrant retention of the testis specific histone variant H2A.L.2. During epididymal transit, cP2 deficient sperm seem to undergo ROS mediated degradation leading to complete DNA fragmentation. The cP2 domain therefore seems to be a key aspect in the complex crosstalk between histones, transition proteins and protamines during sperm chromatin condensation. Overall, we present the first step towards understanding the role of the cP2 domain in paternal chromatin packaging and open up avenues for further research
SPAG17 mediates nuclear translocation of protamines during spermiogenesis
Protamines (PRM1 and PRM2) are small, arginine-rich, nuclear proteins that replace histones in the final stages of spermiogenesis, ensuring chromatin compaction and nuclear remodeling. Defects in protamination lead to increased DNA fragmentation and reduced male fertility. Since efficient sperm production requires the translocation of protamines from the cytoplasm to the nucleus, we investigated whether SPAG17, a protein crucial for intracellular protein trafficking during spermiogenesis, participates in protamine transport. Initially, we assessed the protein-protein interaction between SPAG17 and protamines using proximity ligation assays, revealing a significant interaction originating in the cytoplasm and persisting within the nucleus. Subsequently, immunoprecipitation and mass spectrometry (IP/MS) assays validated this initial observation. Sperm and spermatids from Spag17 knockout mice exhibited abnormal protamination, as revealed by chromomycin A3 staining, suggesting defects in protamine content. However, no differences were observed in the expression of Prm1 and Prm2 mRNA or in protein levels between testes of wild-type and Spag17 knockout mice. Conversely, immunofluorescence studies conducted on isolated mouse spermatids unveiled reduced nuclear/cytoplasm ratios of protamines in Spag17 knockout spermatids compared to wild-type controls, implying transport defects of protamines into the spermatid nucleus. In alignment with these findings, in vitro experiments involving somatic cells, including mouse embryonic fibroblasts, exhibited compromised nuclear translocation of PRM1 and PRM2 in the absence of SPAG17. Collectively, our results present compelling evidence that SPAG17 facilitates the transport of protamines from the cytoplasm to the nucleus
DataSheet1_SPAG17 mediates nuclear translocation of protamines during spermiogenesis.pdf
Protamines (PRM1 and PRM2) are small, arginine-rich, nuclear proteins that replace histones in the final stages of spermiogenesis, ensuring chromatin compaction and nuclear remodeling. Defects in protamination lead to increased DNA fragmentation and reduced male fertility. Since efficient sperm production requires the translocation of protamines from the cytoplasm to the nucleus, we investigated whether SPAG17, a protein crucial for intracellular protein trafficking during spermiogenesis, participates in protamine transport. Initially, we assessed the protein-protein interaction between SPAG17 and protamines using proximity ligation assays, revealing a significant interaction originating in the cytoplasm and persisting within the nucleus. Subsequently, immunoprecipitation and mass spectrometry (IP/MS) assays validated this initial observation. Sperm and spermatids from Spag17 knockout mice exhibited abnormal protamination, as revealed by chromomycin A3 staining, suggesting defects in protamine content. However, no differences were observed in the expression of Prm1 and Prm2 mRNA or in protein levels between testes of wild-type and Spag17 knockout mice. Conversely, immunofluorescence studies conducted on isolated mouse spermatids unveiled reduced nuclear/cytoplasm ratios of protamines in Spag17 knockout spermatids compared to wild-type controls, implying transport defects of protamines into the spermatid nucleus. In alignment with these findings, in vitro experiments involving somatic cells, including mouse embryonic fibroblasts, exhibited compromised nuclear translocation of PRM1 and PRM2 in the absence of SPAG17. Collectively, our results present compelling evidence that SPAG17 facilitates the transport of protamines from the cytoplasm to the nucleus.</p