Protamines: Selection and function in mammals

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 23-11-2015Los protaminas son proteínas básicas localizadas en el nucleo espermático y tienen un papel crucial en la reorganización y protección del genoma paterno que tiene lugar en las fases finales de la formación de los espermatozoides. Las alteraciones en la expresión génica de las protaminas tienen un impacto importante sobre la forma de la célula espermática, la fertilidad masculina y la supervivencia embrionaria. Existe gran variabilidad en los fenotipos de los espermatozoides de mamíferos. Se piensa que esta diversidad se ha producido principalmente como resultado de la selección sexual postcópula (es decir, la competición espermática y la elección críptica de la hembra). Para comprender cómo se ha desarrollado esta diversidad, es importante entender los patrones selectivos comunes entre especies y aquellos que son diferentes, así como los cambios en los genes que son clave para la formación y función de los espermatozoides. Se ha propuesto que la evolución de los genes de las protaminas se produce bajo la influencia de la competición espermática. Esta tesis doctoral se ha centrado por lo tanto en un estudio evolutivo de relaciones genotipo-fenotipo en mamíferos, examinando la evolución de la secuencia y la regulación de las protaminas y los cambios en el fenotipo de las cabezas de los espermatozoides. Se realizaron análisis de genes de protaminas y la evolución de las secuencias reguladoras, así como de la expresión génica comparada con el fin de mostrar la importancia de la función de las protaminas en el tamaño y la forma de la cabeza espermática. Se examinaron especies que exhiben diferentes niveles de competición espermática como modelos para estudiar las relaciones entre la competición espermática y el fenotipo de la cabeza de los espermatozoides. Los resultados de este estudio son importantes para conocer mejor la compleja interacción entre las fuerzas selectivas que potencialmente pueden actuar sobre las secuencias de ADN y el fenotipo celular. Las secuencias del gen y de los dominios reguladores se obtuvieron mediante técnicas estandar de PCR así como PCR con desplazamiento sobre el gen. Se empleó PCR cuantitativa para obtener información sobre expresión génica. Los estudios de asociación genotipo-fenotipo se desarrollaron calculando el "ratio dN/dS desde base al extremo" (ratio de tasas de substituciones no sinónimas / sinónimas) para conocer las tasas evolutivas, así como análisis filogenéticos generalizados de cuadrados mínimos (PGLS) para comparar datos genéticos y morfométricos. Los resultados de este estudio han revelado un patrón sorprendentemente complejo de restricción selectiva, así como el efecto de la competición espermática sobre la divergencia de la secuencia de protaminas y de niveles de expresión. Más aún, estos resultados representan una evidencia sólida del efecto de las protaminas sobre la forma y tamaño de la cabeza de los espermatozoidesProtamines are basic sperm nuclear proteins with a crucial role in the reorganization and protection of the paternal genome that takes place during the final stages of sperm formation. Alterations in protamine gene expression have a major impact on shape of the sperm cell, male fertility and embryo survival. There is great variability in mammalian sperm phenotypes and it is thought that such diversity has been brought about largely by postcopulatory sexual selection (i.e., sperm competition and cryptic female choice). To understand how this diversity is established it is important to examine both common and differing selective patterns across species, along with changes in genes crucial to sperm formation and function. It has been proposed that the evolution of protamine genes takes place under the influence of sperm competition. This doctoral thesis thus focuses on an evolutionary genotype-phenotype study in mammals, examining sequence and regulatory evolution of protamines and accompanying changes in sperm head phenotype. Analyses were undertaken of protamine gene and regulatory sequence evolution as well as comparative gene expression aiming to show the importance of protamine function on sperm head size and shape. Species exhibiting different levels of sperm competition served as models to assess relationships between sperm competition and sperm head phenotype brought about by selective pressures acting on these proteins. The results presented here are important for a better understanding of the complex interplay between selective forces potentially acting on DNA sequences and cell phenotype. Gene and regulatory sequences were obtained by standard PCR techniques as well as gene walking PCR. Quantitative PCR was used to obtain gene expression data. Genotype-phenotype association studies were developed using root-to-tip dN/dS (nonsynonymous / synonymous substitutions rate ratio) to account for evolutionary rates and phylogenetic generalized least squares analyses to compare genetic and morphometric data. The results of this study revealed a surprisingly complex pattern of selective constraints and sperm competition acting on protamine sequence divergence and expression levels and, moreover, they provide compelling evidence for the effect of protamines on sperm head shape and sizeThe work presented here was funded by the Spanish Ministry of Economy and Competitiveness (Grants No. CGL 2011-26341 and CGL 2012-37423). The doctoral candidate holds a studentship from the Spanish Research Council (JAEpre-CSIC) cofinanced by the European Social Fund (ESF

Sexual selection of protamine 1 in mammals

Protamines have a crucial role in male fertility. They are involved in sperm chromatin packaging and influence the shape of the sperm head and, hence, are important for sperm performance. Protamine structure is basic with numerous arginine-rich DNA-binding domains. Postcopulatory sexual selection is thought to play an important role in protamine sequence evolution and expression. Here, we analyze patterns of evolution and sexual selection (in the form of sperm competition) acting on protamine 1 gene sequence in 237 mammalian species. We assessed common patterns as well as differences between the major mammalian subclasses (Eutheria, Metatheria) and clades. We found that a high arginine content in protamine 1 associates with a lower sperm head width, which may have an impact on sperm swimming velocity. Increase in arginine content in protamine 1 across mammals appears to take place in a way consistent with sexual selection. In metatherians, increase in sequence length correlates with sexual selection. Differences in selective pressures on sequences and codon sites were observed between mammalian clades. Our study revealed a complex evolutionary pattern of protamine 1, with different selective constraints, and effects of sexual selection, between mammalian groups. In contrast, the effect of arginine content on head shape, and the possible involvement of sperm competition, was identified across all mammals.This work was supported by funds from the Spanish Ministry of Economy and Competitiveness (grant number CGL2011-26341).Peer reviewe

Schulentscheidungsprozesse und Schulzufriedenheit in Familien mit einem sprachauffälligen Kind

Die Einleitung des Verfahrens zur Feststellung eines sonderpädagogischen Förderbedarfs (AO-SF-Verfahren) forciert bei den betroffenen Eltern eine (erneute) intensive Auseinandersetzung mit der schulischen Zukunft ihres Kindes. Gleichzeitig wird auf politischer Ebene im Zuge der Diskussion um die bildungspolitische Umsetzung der UN-Behindertenrechtskonvention zunehmend das Wahlrecht der Eltern thematisiert. Bislang fehlt in der Diskussion um die optimalen Bedingungen schulischer Förderung sprachauffälliger Kinder jedoch die wissenschaftliche Evidenz. Die Studie soll ein erster Schritt sein, diese Wissenslücke zu schließen. So wurden das AO-SF-Verfahren und die aktuelle Beschulungssituation Gegenstand einer Elternbefragung. Dabei wurde untersucht, wie zufrieden Eltern mit der Förderschule ihres Kindes sowie rückblickend mit dem AO-SF-Verfahren sind und welche wesentlichen Einflussfaktoren auf die Zufriedenheit identifiziert werden können. Weiterhin wurde erfasst, wie die Eltern die Zukunftschancen ihrer Kinder einschätzen und von welchen Faktoren diese Einschätzung beeinflusst wird. Mit Hilfe eines ad hoc Fragebogens wurde der Entscheidungsfindungs¬prozess von Eltern sprach-auffälliger Kinder sowie die Zufriedenheit mit der Förderschule erfasst. Insgesamt haben sich 408 Eltern sprachauffälliger Kinder im Grundschulalter aus 22 Förderschulen mit dem Förderschwerpunkt Sprache in NRW an der Studie beteiligt. Die Zufriedenheit der Eltern sowohl mit dem Verlauf des Schulentscheidungsverfahrens (AO-SF-Verfahren) als auch mit der Schule ihres Kindes insgesamt ist sehr hoch. Die meisten Eltern fühlten sich maßgeblich an der Entscheidung über die Schulform beteiligt und sind dementsprechend mit der Entscheidung der Schul-aufsichtsbehörde einverstanden. Eltern, die jedoch ihren eigenen Einfluss auf die Entscheidung eher gering einschätzen, sind mit dem Verlauf des AO-SF-Verfahrens sowie mit der Schule ihres Kindes deutlich unzufriedener. Darüber hinaus konnten relevante Faktoren identifiziert werden, anhand derer sich die Zufriedenheit der Eltern sowie ihre Prognosen für die Zukunft ihres Kindes vorhersagen lassen. Die hohe Zufriedenheit von Eltern mit den Förderschulen mit dem Förderschwerpunkt Sprache in NRW wirkt sich positiv auf das psycho-soziale Gesamtgefüge aus, in dem die Kinder gefördert werden. Gleichwohl ist weitere empirische Forschung dringend notwendig, um diejenigen Elemente der schulischen Förderung identifizieren zu können, die einen maßgeblichen sprachlichen Förderbeitrag leisten

Evolution of protamine genes and changes in sperm head phenotype in rodents

Little is known about the genetic basis of evolutionary changes in sperm phenotype. Postcopulatory sexual selection is associated with differences in protamine gene sequences and promoters and is a powerful force acting on sperm form and function, although links between protamine evolution and sperm phenotype are scarce. Protamines are involved in sperm chromatin condensation, and protamine deficiency negatively affects sperm morphology and male fertility, thus suggesting that they are important for sperm design and function. We examined changes in protamine genes and sperm phenotype in rodents to understand the role of sexual selection on protamine evolution and sperm design. We performed a genotype-phenotype association study using root-to-tip dN/dS (nonsynonymous/ synonymous substitutions rate ratio) to account for evolutionary rates and phylogenetic generalized least squares analyses to compare genetic and morphometric data. Evolutionary rates of protamine 1 and the protamine 2 domain cleaved off during chromatin condensation correlated with head size and elongation. Protamine 1 exhibited restricted positive selection on some functional sites, which seemed sufficient to preserve its role in head design. The cleaved-protamine 2, whose relaxation is halted by sexual selection, seems to ensure small, elongated heads that would make sperm more competitive. No association existed between mature-protamine 2 and head phenotype, suggesting little involvement during chromatin condensation and a likely role maintaining the condensed state. Our results suggest that evolutionary changes in protamines could be related to complex developmental modifications in the sperm head. This represents an important step toward understanding the role of changes in gene coding sequences in the divergence of germ cell phenotype. © 2014 by the Society for the Study of Reproduction, Inc.Peer Reviewe

Sexual selection on protamine and transition nuclear protein expression in mouse species

Post-copulatory sexual selection in the form of sperm competition is known to influence the evolution of male reproductive proteins in mammals. The relationship between sperm competition and regulatory evolution, however, remains to be explored. Protamines and transition nuclear proteins are involved in the condensation of sperm chromatin and are expected to affect the shape of the sperm head. A hydrodynamically efficient head allows for fast swimming velocity and, therefore, more competitive sperm. Previous comparative studies in rodents have documented a significant association between the level of sperm competition (as measured by relative testes mass) and DNA sequence evolution in both the coding and promoter sequences of protamine 2. Here,we investigate the influence of sexual selection on protamine and transition nuclear protein mRNA expression in the testes of eight mouse species that differ widely in levels of sperm competition.We also examined the relationship between relative gene expression levels and sperm head shape, assessed using geometric morphometrics. We found that species with higher levels of sperm competition express less protamine 2 in relation to protamine 1 and transition nuclear proteins. Moreover, therewas a significant association between relative protamine 2 expression and sperm head shape. Reduction in the relative abundance of protamine 2 may increase the competitive ability of sperm in mice, possibly by affecting sperm head shape. Changes in gene regulatory sequences thus seem to be the basis of the evolutionary response to sexual selection in these proteins. © 2014 The Author(s) Published by the Royal Society. All rights reserved.This work was supported by the Spanish Ministry of Economy and Competitiveness (grant no. CGL2011-26341)Peer Reviewe

Sexual Selection Halts the Relaxation of Protamine 2 among Rodents

Sexual selection has been proposed as the driving force promoting the rapid evolutionary changes observed in some reproductive genes including protamines. We test this hypothesis in a group of rodents which show marked differences in the intensity of sexual selection. Levels of sperm competition were not associated with the evolutionary rates of protamine 1 but, contrary to expectations, were negatively related to the evolutionary rate of cleaved- and mature-protamine 2. Since both domains were found to be under relaxation, our findings reveal an unforeseen role of sexual selection: to halt the degree of degeneration that proteins within families may experience due to functional redundancy. The degree of relaxation of protamine 2 in this group of rodents is such that in some species it has become dysfunctional and it is not expressed in mature spermatozoa. In contrast, protamine 1 is functionally conserved but shows directed positive selection on specific sites which are functionally relevant such as DNA-anchoring domains and phosphorylation sites. We conclude that in rodents protamine 2 is under relaxation and that sexual selection removes deleterious mutations among species with high levels of sperm competition to maintain the protein functional and the spermatozoa competitive

Proteins involved in motility and sperm-egg interaction evolve more rapidly in mouse spermatozoa

Proteomic studies of spermatozoa have identified a large catalog of integral sperm proteins. Rapid evolution of these proteins may underlie adaptive changes of sperm traits involved in different events leading to fertilization, although the selective forces underlying such rapid evolution are not well understood. A variety of selective forces may differentially affect several steps ending in fertilization, thus resulting in a compartmentalized adaptation of sperm proteins. Here we analyzed the evolution of genes associated to various events in the sperm's life, from sperm formation to sperm-egg interaction. Evolutionary analyses were performed on gene sequences from 17 mouse strains whose genomes have been sequenced. Four of these are derived from wild Mus musculus, M. domesticus, M. castaneus and M. spretus. We found a higher proportion of genes exhibiting a signature of positive selection among those related to sperm motility and sperm-egg interaction. Furthermore, sperm proteins involved in sperm-egg interaction exhibited accelerated evolution in comparison to those involved in other events. Thus, we identified a large set of candidate proteins for future comparative analyses of genotype-phenotype associations in spermatozoa of species subjected to different sexual selection pressures. Adaptive evolution of proteins involved in motility could be driven by sperm competition, since this selective force is known to increase the proportion of motile sperm and their swimming velocity. On the other hand, sperm proteins involved in gamete interaction could be coevolving with their egg partners through episodes of sexual selection or sexual conflict resulting in species-specific sperm-egg interactions and barriers preventing interspecies fertilization. © 2014 Vicens et al.This work was funded by the Spanish Ministry of Economy and Competitiveness (grant CGL2011-26341 to E.R.S.R. and postgraduate scholarship BES- 2009-029239 to A.V.), and by a postgraduate scholarship from the Consejo Superior de Investigaciones Cientı´ficas (JAE-Predoc) co-funded by the European Social Fund (to L.L.)Peer Reviewe

Entwicklung von Unterrichtsmaterial zum Uhrenlernen und erste Evaluation im Multiple Baseline Design

Das Lesen der Uhr und ein fortgeschrittenes Zeitverständnis sind einerseits Kompeten zen, die höchst relevant für die spätere gesellschaftliche Partizipation sind, andererseits stellen sie für viele Lernende eine große Herausforderung dar. Auch aus der Sicht von Lehrkräften besteht hier ein großer Bedarf an didaktischen Konzepten und Materialien zur Erarbeitung dieses Lernbereiches in inklusiven Lernsettings. Im Rahmen des Projekts wurden Unterrichtsmaterialien für das Lernen im Lernbereich Zeit entwickelt. Diese Materialien wurden anschließend hinsichtlich ihrer Effektivität und Einsetzbarkeit im Rahmen eines Multiple Baseline Designs erprobt. An der Evaluationsstudie beteiligten sich fünf Mädchen und eine erwachsene Frau. Für die Messung des Lernfortschritts wur den 60 Testaufgaben entwickelt. Bei allen Teilnehmenden zeigen sich Verbesserungen, wobei die Effektstärken variieren. Insgesamt führte die Förderung mit den entwickelten Materialien zu einer Verbesserung der Kompetenzen im Uhrlesen und der Zeitwahr nehmung. Durch die Modularisierung ist das Material beliebig einsetzbar und auch für unterschiedliche Lernniveaus geeignet. Das gesamte Lernmaterial wird unter offener Li zenz als Open Educational Resource verfügbar gemacht