A melatonina é um antioxidante muito eficaz e protege as células contra o estresse oxidativo causado pelas espécies reativas, e indiretamente, modula a expressão de genes associados ao ciclo celular, metabolismo oxidativo e apoptose celular. Deste modo, a suplementação da melatonina ao meio de maturação in vitro, a torna uma alternativa para promover melhorias na viabilidade das células germinativas e embrionárias. O estudo 1 avaliou o efeito da adição de melatonina ao meio de maturação por meio da maturação nuclear (progressão meiótica) e citoplasmática (migração de grânulos corticais) e dos níveis de ROS em oócitos suínos maturados in vitro. A suplementação do meio de maturação com melatonina estimulou a progressão da meiose, a migração de grânulos corticais e reduziu os níveis intracelulares de ROS nos oócitos. O estudo 2 avaliou o efeito da melatonina na expressão de genes antioxidantes (Catalase, SOD1, SOD2 e GPX) envolvidos na proteção celular de oócitos e células do cumulus. A adição da melatonina ao meio de maturação influenciou positivamente a expressão dos genes antioxidantes nos oócitos e células do cumulus. O estudo 3 avaliou o efeito da melatonina nos processos biológicos por meio do perfil de trascriptomas, via RNA-Seq, em células do cumulus oriundas de oócitos suínos maturados in vitro. A partir da análise de expressão diferencial foi possível identificar que a adição da melatonina ao meio de maturação influenciou 80 genes associados a nove processos biológicos (ciclo celular; proteólise; organização de citoesqueleto; via energética; adesão e transporte celular; via de sinalização; fator de transcrição; metabolismo oxidativo e apoptose; e componente celular). A suplementação do meio de maturação com melatonina potencialmente influencia a viabilidade e o funcionamento celular, uma vez que modulou a expressão de genes associados à processos fisiológicos essenciais, tais como: divisão celular, metabolismo energético e oxidativo, vias de sinalização e apoptose. O estudo 4, avaliou o efeito da melatonina sobre os genes associados à viabilidade oocitária e o subsequente desenvolvimento embrionário por meio do perfil de trascriptomas, via RNA-Seq, de células do cumulus oriundas de oócitos suínos. A adição da melatonina ao meio de maturação influenciou 59 genes associados a nove funções biológicas (expansão do cumulus, comunicação em COCs, maturação nuclear, maturação citoplasmática, reparo e integridade do DNA, viabilidade oocitária, esteroidogênese, fertilização e embriogênese). Em conclusão, a suplementação do meio de maturação com melatonina influencia positivamente a maturação oócitária, reduz os níveis intracelulares de ROS, aumenta a expressão de genes antioxidantes, em adição, interfere no transcriptoma de um número expressivo de genes associados à aquisição da competência oócitária, da viabilidade embrionária e desenvolvimento subsequente. / Melatonin is a very effective antioxidant and protects cells against oxidative stress caused by reactive species, and indirectly modulates expression of genes associated with cell cycle, oxidative metabolism, and apoptosis. Thus, melatonin supplementation to in vitro maturation media becomes an alternative to improve the viability of germ and embryonic cells. The first study assessed the effects of adding melatonin to the maturation medium on nuclear (meiotic progression) and cytoplasmic (cortical granules migration) maturation and ROS levels in in vitro matured porcine cumulus-oocyte complexes (COCs). Melatonin supplementation stimulated meiosis progression and cortical granules migration, and reduced intracellular ROS levels in oocytes. The second study evaluated the effects of melatonin on the expression of antioxidant genes (Catalase, SOD1, SOD2, and GPX) involved in cellular protection in oocytes and cumulus cells. The addition of melatonin to the maturation medium positivity influence the expression of antioxidant genes in oocytes and cumulus cells. The third study evaluated the effect of melatonin on genes associated with biological processes through transcriptomic profile via RNA-Seq in cumulus cells derived from porcine COCs in vitro matured. Melatonin addition to maturation medium differentially affected expression of 80 genes associated with nine biological processes (cell cycle, proteolysis, cytoskeletal organization, energy pathaway, cell adhesion and transport, signalling pathway, transcription factor, oxidative metabolism and apoptosis, and cell components). The fourth study assessed the effect of melatonin on genes associated with oocyte viability and subsequent embryo development through transcriptomic profile via RNA-Seq in cumulus cells derived from porcine COCs. Melatonin in the maturation medium affected 59 genes associated with nine biological functions related with oocyte viability and embryo development (cumulus expansion, communication between cumulus cells and oocytes, nuclear maturation, cytoplasmic maturation, DNA repair and integrity, oocyte viability, steroidogenesis, fertilization and embryogenesis). In conclusion, supplementation of melatonin to maturation environment influences oocyte nuclear and cytoplasmic maturation, reduces intracellular ROS levels, positivity influence the expression of antioxidant and also interferes in the transcriptome of a significant number of genes associated with oocyte competence acquisition, embryo viability and subsequent development.

66 Abstract Legal issues of gametes in relation to assisted reproduction The thesis deals with the issue of assisted reproduction with a specific focus on gametes and some issues that are associated with them. In order to better understand contradictory opinions and views on a particular issue, the Czech legislation is compared with the British legislation. Then, in each individual case, it is assessed which country has dealt with the problem better, using a comparative method. The introduction of the thesis deals with assisted reproduction in general, especially from the point of view of definition of the concepts and historical development of this issue. Subsequently, the basic legal framework for assisted reproduction is defined both in the Czech Republic and the United Kingdom. Briefly, there are also summarized the most basic requirements for gametes donors in both countries. In terms of specific problems related to gametes, much of the work focuses on the anonymity of sperm donors, in particular summarizing the most important arguments of its opponents and subsequently refuting these arguments. The conclusion of this chapter submits why, in my view, the anonymity of sperm donors should be maintained. The next part is devoted to financial rewards for donation of gametes, their admissibility and amount....

Diss. (sammanfattning). Uppsala : Sveriges lantbruksuniversitet, 2006. / Härtill 4 uppsatser.

A melatonina é um antioxidante muito eficaz e protege as células contra o estresse oxidativo causado pelas espécies reativas, e indiretamente, modula a expressão de genes associados ao ciclo celular, metabolismo oxidativo e apoptose celular. Deste modo, a suplementação da melatonina ao meio de maturação in vitro, a torna uma alternativa para promover melhorias na viabilidade das células germinativas e embrionárias. O estudo 1 avaliou o efeito da adição de melatonina ao meio de maturação por meio da maturação nuclear (progressão meiótica) e citoplasmática (migração de grânulos corticais) e dos níveis de ROS em oócitos suínos maturados in vitro. A suplementação do meio de maturação com melatonina estimulou a progressão da meiose, a migração de grânulos corticais e reduziu os níveis intracelulares de ROS nos oócitos. O estudo 2 avaliou o efeito da melatonina na expressão de genes antioxidantes (Catalase, SOD1, SOD2 e GPX) envolvidos na proteção celular de oócitos e células do cumulus. A adição da melatonina ao meio de maturação influenciou positivamente a expressão dos genes antioxidantes nos oócitos e células do cumulus. O estudo 3 avaliou o efeito da melatonina nos processos biológicos por meio do perfil de trascriptomas, via RNA-Seq, em células do cumulus oriundas de oócitos suínos maturados in vitro. A partir da análise de expressão diferencial foi possível identificar que a adição da melatonina ao meio de maturação influenciou 80 genes associados a nove processos biológicos (ciclo celular; proteólise; organização de citoesqueleto; via energética; adesão e transporte celular; via de sinalização; fator de transcrição; metabolismo oxidativo e apoptose; e componente celular). A suplementação do meio de maturação com melatonina potencialmente influencia a viabilidade e o funcionamento celular, uma vez que modulou a expressão de genes associados à processos fisiológicos essenciais, tais como: divisão celular, metabolismo energético e oxidativo, vias de sinalização e apoptose. O estudo 4, avaliou o efeito da melatonina sobre os genes associados à viabilidade oocitária e o subsequente desenvolvimento embrionário por meio do perfil de trascriptomas, via RNA-Seq, de células do cumulus oriundas de oócitos suínos. A adição da melatonina ao meio de maturação influenciou 59 genes associados a nove funções biológicas (expansão do cumulus, comunicação em COCs, maturação nuclear, maturação citoplasmática, reparo e integridade do DNA, viabilidade oocitária, esteroidogênese, fertilização e embriogênese). Em conclusão, a suplementação do meio de maturação com melatonina influencia positivamente a maturação oócitária, reduz os níveis intracelulares de ROS, aumenta a expressão de genes antioxidantes, em adição, interfere no transcriptoma de um número expressivo de genes associados à aquisição da competência oócitária, da viabilidade embrionária e desenvolvimento subsequente. / Melatonin is a very effective antioxidant and protects cells against oxidative stress caused by reactive species, and indirectly modulates expression of genes associated with cell cycle, oxidative metabolism, and apoptosis. Thus, melatonin supplementation to in vitro maturation media becomes an alternative to improve the viability of germ and embryonic cells. The first study assessed the effects of adding melatonin to the maturation medium on nuclear (meiotic progression) and cytoplasmic (cortical granules migration) maturation and ROS levels in in vitro matured porcine cumulus-oocyte complexes (COCs). Melatonin supplementation stimulated meiosis progression and cortical granules migration, and reduced intracellular ROS levels in oocytes. The second study evaluated the effects of melatonin on the expression of antioxidant genes (Catalase, SOD1, SOD2, and GPX) involved in cellular protection in oocytes and cumulus cells. The addition of melatonin to the maturation medium positivity influence the expression of antioxidant genes in oocytes and cumulus cells. The third study evaluated the effect of melatonin on genes associated with biological processes through transcriptomic profile via RNA-Seq in cumulus cells derived from porcine COCs in vitro matured. Melatonin addition to maturation medium differentially affected expression of 80 genes associated with nine biological processes (cell cycle, proteolysis, cytoskeletal organization, energy pathaway, cell adhesion and transport, signalling pathway, transcription factor, oxidative metabolism and apoptosis, and cell components). The fourth study assessed the effect of melatonin on genes associated with oocyte viability and subsequent embryo development through transcriptomic profile via RNA-Seq in cumulus cells derived from porcine COCs. Melatonin in the maturation medium affected 59 genes associated with nine biological functions related with oocyte viability and embryo development (cumulus expansion, communication between cumulus cells and oocytes, nuclear maturation, cytoplasmic maturation, DNA repair and integrity, oocyte viability, steroidogenesis, fertilization and embryogenesis). In conclusion, supplementation of melatonin to maturation environment influences oocyte nuclear and cytoplasmic maturation, reduces intracellular ROS levels, positivity influence the expression of antioxidant and also interferes in the transcriptome of a significant number of genes associated with oocyte competence acquisition, embryo viability and subsequent development.

Citrus is the main fruit crop in the world and Spain is the 6th producer and the major exporter for the fresh fruit market. Seedlessness is one of the most important fruit quality traits for this market since consumers do not accept seedy fruits. Recovery of triploid hybrids has become an important breeding strategy to develop new seedless citrus varieties and several of them have been already released from citrus breeding programs worldwide. Despite the undisputable importance of polyploidy in plant species, their genetics are much less well known than those of their diploid counterparts. Citrus triploid hybrids can be routinely recovered from sexual polyploidization (2x ¿ 2x) or interploid crosses (2x ¿ 4x and 4x ¿ 2x). In 2x ¿ 2x sexual crosses, spontaneous triploid hybrids arise from the union of an unreduced (2n) megagametophyte with haploid pollen. In the case of interploid sexual crosses (2x ¿ 4x and 4x ¿ 2x), triploid hybrids result from the fecundation of a diploid gamete arising from the tetraploid parent and a haploid gamete arising from the diploid parent. The genetic and phenotypic structures of triploid populations greatly depend on the parental heterozygosity restitution (HR) in the diploid gamete at each locus, which is mainly affected by the triploid recovery strategy. In 2x ¿ 2x crosses, HR depends on the underlying mechanism leading to the unreduced gamete formation, which are genetically equivalent to First Division Restitution (FDR) or Second Division Restitution (SDR) mechanisms. Moreover, under each restitution mechanism, HR also depends on the locus-centromere genetic distance. In the case of interploid crosses, parental heterozygosity restitution from tetraploid parents depends on the double reduction frequency. In citrus, the unreduced gamete formation mechanism is still controversial; FDR has been the mechanism proposed for sweet orange, whereas SDR has been proposed for clementine. On the other hand, inferring the allelic configurations of genetic markers is a main challenge in polyploidy crops to infer genotypic and gametic structures with the objective to analyze meiosis and inheritance mechanisms. According to this scientific context, the objectives of the thesis where: (i) to develop a new approach for allele dosis assignation when using co-dominant markers, (ii) to implement and apply methods for the analysis of 2n gametes origin and locate centromeres, and (iii) to take advantage of this knowledge to locate a major gene of resistance to Alternaria Brown Spot (ABS) which is a major constraint for triploid mandarin breeding. For microsatellite (SSR) markers, we have demonstrated that triploid progeny genotyping can be successfully performed using the microsatellite allele-counting peak ratio (MAC-PR) method. However, SSR analysis remains relatively costly and time consuming compared with actual SNP genotyping methods. Moreover, with the increasing availability of EST databases and whole genome sequences, SNPs have become the most abundant and powerful polymorphic markers that can be selected along the entire genome. In this thesis, a new method based on competitive allele-specific PCR has been developed to assign SNP allele dosage in an accurate, simple, and cost effective way. Combining the MAC-PR and the new developed SNP genotyping methods offers the possibility to utilize a broad range of molecular markers in genotyping triploid genotypes. Both methods have been used in further works included in this thesis. SDR has been demonstrated as the mechanism underlying unreduced gamete production in `Fortune¿ mandarin by genotyping triploid progenies with SSR markers. In addition, a new method to locate the centromere, based on the best fit between observed heterozygosity restitution within a linkage group and theoretical functions under either partial or no chiasma interference hypotheses has been developed and successfully applied. To expand the knowledge of the mechanism underlying unreduced gamete formation to other citrus genotypes besides clementines and `Fortune¿ mandarin, a maximum likelihood method based on parental heterozygosity restitution of centromeric loci was developed and successfully applied in sixteen mandarin cultivars. The new method developed in the study allows inferring the restitution mechanism both at population level and even at individual level. Maternal origin of 2n gametes was confirmed for all triploid hybrids and SDR was proposed as the restitution mechanism for all analyzed progenies. The information acquired from the mode of heterozygosity restitution in citrus was useful to determine the genetic and phenotypic structures of new triploid populations arising from different breeding strategies. We studied these structures for the resistance to Alternaria brown spot (ABS), a serious fungal disease producing necrotic lesions on fruits and young leaves in susceptible citrus genotypes. In the present work, different approaches were combined taking advantage of the particular genetic structures of 2n gametes resulting from SDR to map a genome region linked to ABS resistance in triploid citrus progeny. The monolocus dominant inheritance of the susceptibility, proposed on the basis of diploid population studies, was corroborated in triploid progeny. A 3.3 Mb genomic region linked to ABS resistance was located near the centromere on chromosome III, which includes clusters of resistance genes. SSR and SNP markers were developed for an efficient early selection of ABS resistant hybrids and they are currently used in our breeding program to perform marker assisted selection. The knowledge obtained in this thesis on the mechanism of sexual polyploidization and inheritance of concrete traits in citrus will allow implementing much more efficient triploid breeding programs on the basis of current and future needs. Indeed, applied outcomes of this PhD are already routinely used in the IVIA triploid breeding program. / Cuenca Ibáñez, J. (2013). Mechanisms of sexual polyploidization and inheritance in triploid citrus populations [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/34671 / TESIS / Premios Extraordinarios de tesis doctorales

No description available.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniv. / Härtill 5 uppsatser. På titels. felaktigt: 1997.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / O estudo trata da induÃÃo anestÃsica, caracterizaÃÃo do lÃquido seminal, refrigeraÃÃo e congelaÃÃo do sÃmen de tilÃpia-do-Nilo, Oreochromis niloticus. No primeiro capÃtulo foram testados os anestÃsicos naturais mentol e eugenol em espÃcimes adultos objetivando determinar a concentraÃÃo ideal para manejo de tilÃpia-do-Nilo. No segundo capÃtulo, o lÃquido seminal da tilÃpia-do-Nilo foi caracterizado de acordo com os seguintes parÃmetros: volume coletado, que revelou a viabilidade de coleta em volumes suficientes, sem a necessidade de sacrificar o peixe; concentraÃÃo espermÃtica, que mostrou relevante variaÃÃo entre espÃcimes; pH e osmolaridade, que ratificam as informaÃÃes presentes na literatura; tempo de motilidade, que comprovou ser o espermatozoide de tilÃpia diferenciado quanto à energia acumulada quando comparado a outras espÃcies de Ãgua doce; e morfometria da cabeÃa do espermatozoide por meio das seguintes medidas: comprimento, largura, perÃmetro e Ãrea. TambÃm foram registrados quatro parÃmetros derivados do formato da cabeÃa: elipticidade, rugosidade, elongaÃÃo e regularidade. No terceiro capÃtulo foi avaliada a viabilidade do sÃmen de tilÃpia-do-Nilo submetido a diferentes diluentes e refrigerado a 4ÂC. A avaliaÃÃo foi realizada por meio da taxa de motilidade espermÃtica determinada pelo sistema CASA (Computer Assisted Sperm Analysis). Neste, o ACP-104, diluente à base de Ãgua de coco em pÃ, ofereceu melhor taxa de motilidade dos espermatozoides por atà 72 horas de estocagem. Esta resposta mostrou que o ACP-104 à um diluente promissor para o sÃmen da referida espÃcie. No quarto capÃtulo foi avaliado o efeito de diferentes criodiluentes sobre do sÃmen de tilÃpia-do-Nilo pÃs-descongelaÃÃo, utilizando o software (Sperm Class Analyser, SCA). Os resultados de motilidade espermÃtica e de parÃmetros de velocidade do espermatozoide revelaram que o ACP-104 combinado ao dimetilsulfÃxido apresenta-se como um criodiluente promissor para a criopreservaÃÃo do sÃmen de tilÃpia-do-Nilo em longo prazo. / The present study deals with anesthetic induction, characterization of sperm fluid, cooling and freezing of sperm of the Nile tilapia Oreochromis niloticus. In the first chapter the natural anesthetics menthol and eugenol were tested in adult specimens aiming to determine the ideal concentration for handling the Nile tilapia. In the second chapter the sperm fluid of the Nile tilapia was characterized according to the following parameters: collected volume, which showed the feasibility of collect in sufficient volume without the need of sacrificing the fish; sperm concentration, which showed significant variation between specimens; pH and osmolarity, which confirm the information of the literature; sperm motility time, which proved that the spermatozoa of tilapia are differentiated concerning accumulated energy when compared to other freshwater species; and sperm head morphometry, characterized by size (length, width, perimeter and area) and head shape (ellipticity, rugosity, elongation and regularity). In the third chapter we evaluated the feasibility of preservation of tilapia sperm using different extenders and cooled at 4ÂC. The sperm motility rate was evaluated with the aid of the Computer-assisted Sperm Analyzer (CASA) System. In this, the ACP-104, media based on powdered coconut water, showed best sperm motility rate for up to 72 hours after storage. In the fourth chapter was evaluated the effects of different cryoprotectants on the post-thaw Nile tilapia sperm, using the software Sperm Class Analyzer (SCA). The results of the sperm motility rate and sperm velocity parameters revealed that ACP-104 in combination with dimethylsulfoxide is a promising cryodiluent for Nile tilapia sperm in long-term conservation.

Contact zones of cytotypes harbour a unique opportunity to study the dynamics and evolution of mixed-ploidy systems. Butomus umbellatus (flowering rush) is one of a few species in which diploid and triploid cytotype is present in nature. The first known mixed-ploidy populations of B. umbellatus were discovered in south-eastern Slovakia during the previous studies. Common occurrence of the species in an area with substantially natural dynamics of wetland ecosystems represents a unique model system for unbiased comparisons of traits and genetic diversity between diploid and triploid individuals along with their dispersal capabilities on a landscape level. The main aim of of this thesis is to uncover the truth behind evolutionary processes that are taking place in the contact zone of diploid and triplioid cytotype of B. umbellatus in south-eastern Slovakia. Using flow cytometry and molecular methods (microsatellites) we will test whether this is a primary or secondary contact zone and assess the level of genetic variability between individuals and populations of both cytotypes. Using the analysis od microsatellite loci we discovered that the contact zone of cytotype is of both primary and secondary character and gene flow between cytotypes was also detected. These results were supported by high production and...

Dans l’histoire évolutive des végétaux, la polyploïdisation a été un phénomène récurrent qui a façonné les génomes, aurait contribué à l’avènement de grandes étapes évolutives et aurait favorisé la survie de nombreuses lignées lors de crises écologiques majeures. Le principal mécanisme d’apparition d’espèces polyploïdes est la polyploïdisation sexuelle, qui implique la formation de gamètes 2n résultant de modifications de la division méiotique. Récemment plusieurs mutants produisant des taux élevés de gamètes 2n ont été identifiés chez A. thaliana. Chez cette espèce, la perte de fonction du gène AtPS1 conduit à la mise en place de fuseaux parallèles en méiose II et celle du gène AtCYCA1;2/TAM à l’omission de la seconde division méiotique. L’objectif de cette thèse a été de déterminer des facteurs et mécanismes responsables de la formation de gamètes 2n, en utilisant le rosier comme modèle végétal. Ces travaux ont permis : (i) d’identifier un facteur abiotique, la température élevée, comme inducteur de production de forts taux de gamètes 2n, (ii) de montrer que la fenêtre de sensibilité à ce facteur est restreinte à la méiose et (iii) de révéler que ces gamètes 2n produits sont principalement issus de la mise en place de fuseaux parallèles en méiose II. Afin de déterminer les mécanismes moléculaires à l’origine de leur formation, deux gènes candidats, RhPS1 et RhCYCA1 ont été identifiés chez Rosa. L’analyse de leur expression a révélé : (i) en condition non inductible, leur forte expression dans les étamines au stade méiose et (ii) la répression rapide de leurs niveaux de transcrits en condition d’induction de gamètes 2n. La fonction méiotique du gène RhPS1 a été validée par complémentation du mutant atps1-1 d’A. thaliana et par l’obtention d’une lignée rosier transgénique p35S::ARNi-RhPS1. Compte tenu de ces résultats, l’étude de la polyploïdisation et de ses mécanismes peut désormais être replacée dans le contexte actuel de changement climatique. / In the evolutionary history of plants, polyploidization has been a recurring phenomenon that has shaped the genomes, might have contributed to the occurrence of major evolutionary step and might have facilitated the survival of many plant families during major ecological crises. The main mechanism of polyploidization is sexual polyploidization, which involves the formation of 2n gametes resulting from meiotic division changes. Recently, mutants highly producing 2n gametes have been isolated in A. thaliana. Loss of AtPS1 gene function leads to parallel spindles orientation in meiosis II and loss of AtCYCA1;2/TAM gene function leads to the omission of the second meiotic division. The aim of this PhD project was to identify factors and mechanisms responsible for the 2n gametes formation, using Rosa as a model. This work permitted to: (i) discover an abiotic factor, high temperature, that can induce a high production of 2n gametes, (ii) show that the sensitivity window to this factor is narrow and restricted to meiosis and (iii) reveal that 2n gamete production in inductive condition, results from parallel spindle orientation in meiosis II. To determine molecular mechanisms responsible for their formation, two candidate genes, RhPS1 and RhCYCA1 were identified in Rosa. Analysis of their expression revealed: (i) their high expression level in stamens at meiosis stage in non-inductive condition and (ii) the rapid repression of their transcript levels under inductive condition. Meiotic gene function of RhPS1 was validated by complementation of atps1-1 mutant and by generating a rose transgenic line p35S:: RNAi-RhPS1. According to these results, polyploidization and its mechanisms can now be replaced in the context of the current climate.