published_or_final_version / abstract / Zoology / Doctoral / Doctor of Philosophy

published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

Determining the genetic basis of reproductive isolation is a fundamental goal in evolutionary biology. Intrinsic reproductive isolation often arises due to epistasis between divergent interacting genes. The rapid evolution of hybrid male sterility is known to have several causes, including the exposure of recessive X-linked incompatibilities in males and the rapid evolution of male reproductive traits. Despite these insights, little is known about the genetics of reproductive isolation during the early stages of speciation. This deficiency inspired parallel studies on the molecular evolution of male reproduction in house mice and the genetic basis of hybrid male sterility between two mouse species, Mus domesticus and M. musculus. Evolutionary analysis of 946 genes showed that the intensity of positive selection varies across sperm development and acts primarily on phenotypes that develop late in spermatogenesis (Appendix A). Several reciprocal crosses between wild-derived strains of M. musculus and M. domesticus were used to examine F1 hybrid male sterility (Appendix B). These crosses revealed hybrid male sterility linked to the M. musculus X chromosome and a novel sterility polymorphism within M. musculus. A large introgression experiment was used to further dissect the genetic basis of X-linked incompatibilities between M. musculus and M. domesticus (Appendix C). Introgression of the M. musculus X chromosome into a M. domesticus genetic background produced male sterility and involved a minimum of four factors. No sterility factors were uncovered on the M. domesticus X chromosome. These data demonstrate the complex genetic basis of hybrid sterility in mice and provide numerous X-linked candidate sterility genes. The molecular evolution of five rapidly evolving candidate genes was examined using population and phylogenetic sampling in Mus (Appendix D). Four of these loci showed evidence of positive natural selection. One locus, 4933436I01Rik, showed divergent protein evolution between M. domesticus and M. musculus and was one of a handful of testis-expressed genes within a narrow interval involved in hybrid male sterility. In summary, these data demonstrate that hybrid male sterility has a complex genetic basis between two closely related species of house mice and provide a foundation for the identification of specific mutations that isolate these species.

The role of male germ cell death in spermatogenesis is an important one as it removes dysfunctional or genetically damaged germ cells and is necessary to maintain an optimal germ cell to Sertoli cell ratio. The formation of the bloodtestis barrier requires the elimination of excessive germ cells and a surge of germ cell apoptosis occurs prior to puberty regulating the ratio of germ cells to Sertoli cells. The aim of this study was to evaluate the presence of four apoptotic markers on sperm from patients with various grades of fertility using flow cytometry. Furthermore, any correlations between the apoptotic marker assays and the standard semen analysis results were identified. This study compares early and late parameters of apoptosis with morphological features in spermatozoa in the same samples. The three sample groups were identified as: teratozoospermic [G-pattern] (n=26), teratozoospermic [P-pattern] (n=98) and oligoteratozoospermic [Ppattern] (n=36). Standard semen analysis was conducted on the semen samples according to the WHO guidelines. Four apoptotic marker assays using flow cytometry was applied in this study to examine the apoptotic alterations in ejaculate sperm. These assays included the Annexin-V staining for the determination of phosphatidylserine exposure, APO-Direct to identify DNA fragmentation, caspase-3 to detect expression of this active protease during early apoptosis and Fas expression. For the Annexin-V and caspase-3 assays, statistically significant differences (P&lt / 0.05) were evident between the three groups. No significant differences (P&gt / 0.05) were found between the groups with respect to the APO-Direct assay. A significant difference (P&lt / 0.05) was found when comparing the teratozoospermic [G-pattern] group and the oligoteratozoospermic [P-pattern] group for the Fas assay. A strong positive correlation was evident between the Fas and the caspase-3 assays in the teratozoospermic [G-pattern] group. For the teratozoospermic [P-pattern group] the following positive correlations existed between the APO-Direct and the Fas assays, APO-Direct and caspase-3 assays and between caspase-3 and Fas assays. The only strong positive correlation was between the caspase-3 and APO-Direct assays in the oligoteratozoospermic [P-pattern] group. The presence of spermatozoa showing microscopic features resembling apoptosis has been identified in ten human ejaculate samples per sample group. Electron microscopy was used to identify morphological features of apoptosis in these human sperm samples. Classical apoptosis as observed in diploid cells could be identified in sperm and these included: loose fibrillarmicrogranular chromatin network, presence of vacuoles in the nuclear chromatin, membranous bodies within the vacuoles of the chromatin, partially disrupted nuclear membranes, plasma membrane protuberances and apoptotic bodies containing cytoplasmic vacuoles and dense masses. This study has confirmed that semen samples with abnormal semen parameters exhibit the presence of apoptotic markers in sperm. The identification of apoptotic markers on the sperm suggests that abnormalities occur during their developmental process, however, the exact mechanism thereof remains unclear. These findings may suggest that certain apoptotic markers may be an indicator of abnormal sperm function and possibly indicative of male infertility.

A infertilidade é uma doença do sistema reprodutivo considerada um problema de saúde pública global, afetando aproximadamente 15% dos casais e 7% dos homens. Falência espermatogênica primária é definida como incapacidade dos testículos produzirem espermatozoides, apesar do suporte hormonal adequado. Esse estudo é o primeiro a comparar dois grupos de pacientes com falência espermatogênica primária por meio da técnica array-CGH+SNP, utilizando a plataforma 4X180 (Agilent®). Adicionalmente, foram comparados os resultados obtidos por essa técnica com bancos de dados genômicos de expressão. Foram analisados vinte e três pacientes com falência espermatogênica primária, sendo sete com oligozoospermia extrema e dezesseis com azoospermia não obstrutiva, assim como seis homens comprovadamente férteis (grupo controle). A análise genômica foi realizada pelo software Nexus 8.0 e as análises de bancos de dados genômicos de expressão por meio do software Nexus Expression 3.0 com nível de significância de 5%. Os primeiros resultados evidenciaram diferenças de número e de tamanho das alterações genômicas (CNVs e LOHs) entre os dois grupos da amostra e o grupo controle, sendo a diferença do número de CNVs de perdas do grupo com oligozoospermia significativamente maior do que no grupo controle. O grupo de pacientes com azoospermia apresentou alterações genômicas de maior tamanho, entre 552pb a 11.3MB, quando comparado aos homens oligozoospérmicos e ao grupo controle. A análise descritiva permitiu identificação de um gene candidato a fenótipo de falência espermatogênica primária na região 12q21.1-q21.2, o gene GLIPR1L1. Por meio da análise individualizada, identificamos dois genes candidatos ao fenótipo de infertilidade, o SHH, localizado na região 7q21.3, e o TMEM184A, localizado na região 7p22.3. Foram também descritas oito novas regiões de CNVs e LOHs nos dois grupos da amostra, direcionando novos estudos para correlação entre genótipo e fenótipo. / Infertility is a disease of the reproductive system and a global public health problem affecting approximately 15% of couples and 7% of men. Primary spermatogenic failure is defined as the inability of the testes to produce sperm although there is adequate hormonal support. This study is the first to compare two groups of patients with primary spermatogenic failure using array-CGH + SNP technique with the 4X180 (Agilent®) platform. Also, we compared the results obtained by this technique with genomic expression databases. Twenty-three patients with primary spermatogenic failure were analyzed, seven with extreme oligozoospermia and sixteen with nonobstructive azoospermia. We used six proven fertile men as a control group. Genomic analysis was performed by Nexus 8.0 software and analysis of genomic databases of expression was performed using the Nexus Expression 3.0 software with 5% significance level. Initial results showed differences in both the number and size of genomic alterations (CNVs and LOHs) between the two sampled infertility groups and the control group. The main difference was that the number of overall CNVs losses which was significantly higher in the oligozoospermia group than in the control group. The group of patients with azoospermia showed larger genomic alterations, between 552bp and 11.3MB size, compared to oligozoospermic men and the control group. A descriptive analysis was used to identify a candidate gene, GLIPR1L1, for the primary spermatogenic failure phenotype in the region 12q21.1-q21.2. Through individualized analysis, we identified two other candidate genes that may have a relationship with the infertility phenotype, SHH, located in the 7q21.3 region, and TMEM184A, located on the 7p22.3 region. Eight new areas of CNV and LOHs have also been described in the two sample groups, for more extensive genotype and phenotype correlative studies.

Les microtubules sont des éléments du cytosquelette, composées d'hétérodimères de tubuline de type α et β. Ils jouent un rôle important dans plusieurs processus cellulaires, dont le transport cytoplasmique, la mobilité et la division cellulaire. Cependant, les mécanismes par lesquels les microtubules sont adaptés à ces rôles très différents restent largement méconnus.Les modifications post-traductionnelles de la tubuline pourraient contribuer à la diversité de fonction des microtubules. Parmi celles-ci, la polyglutamylation pourrait jouer un rôle important en changeant d'une manière importante les propriétés des microtubules, et les adaptant ainsi à leurs différents rôles. La polyglutamylation correspond à l'addition de longues chaînes latérales d'acides glutamiques aux extrémités C-terminales des tubulines α et β. Ces régions sont des sites connues d'interactions de la tubuline avec ses protéines associées (MAP) et les moteurs moléculaires. Au cours de mes travaux, j'ai étudié le rôle de polyglutamylation de la tubuline dans le développement des spermatozoïdes. En utilisant la souris et la drosophile comme organismes modèles, j'ai montré que le changement de niveau de polyglutamylation de la tubuline dans les spermatozoïdes, soit par une régulation positive soit par régulation négative, provoque des anomalies structurales des spermatozoïdes et entraîne une stérilité des mâles. J'ai également étudié la rôle de la katanine, un enzyme coupant les microtubules, effecteur potentiel de la polyglutamylation. J'ai montré qu'en absence de la katanine, la production des cellules germinales est gravement compromise chez les mâles, provoquant également une stérilité. Pris ensemble, ces résultats démontrent que la régulation de polyglutamylation de la tubuline est indispensable pour le développement correct des spermatozoïdes et que son effet pourrait être médié par la katanine, enzyme dont l'activité pourrait dépendre de la polyglutamylation de la tubuline.Durant mes travaux, j'ai également développé une nouvelle technique de production des microtubules différentiellement glutamylés. En utilisant comme matière primaire de la tubuline de cerveau porcin, hautement polyglutamylée, j'ai réalisé une déglutamylation produisant ainsi une tubuline déglutamylée. En utilisant les deux types de microtubules (avec ou sans polyglutamylation), il est possible de tester si les interactions entre les microtubules et les MAP dépendent de la polyglutamylation de la tubuline. / Microtubules are essential cytoskeletal elements composed of α- and β-tubulin heterodimers. They are involved in a number of cellular processes, including intracellular transport, cell motility and cell division. However, how microtubules can adapt to all these different functions remains largely unknown. One of the mechanism, which could contribute to microtubule diversity are posttranslational modifications of tubulin. Among tubulin modifications polyglutamylation has a high potential for changing microtubule properties and thus adapting them to different roles. It consists of addition of long glutamate side chains to multiple glutamate residues located in the C-terminal tail of both α- and β-tubulin, which are known as interaction sites for many microtubule associated proteins (MAPs) and molecular motors. In my studies I focused on the role of polyglutamylation in sperm development. Using mice and Drosophila as model systems, I showed that changing the levels of this modification, either by up- or downregulation, results in the assembly of structurally abnormal sperm and causes male sterility. In addition, I also addressed the role of one of the potential effectors of polyglutamylation, a microtubule-severing enzyme called katanin. I demonstrated that in the absence of katanin the production of male germ cells is severely compromised leading to male sterility. Taken together my data suggest that proper balance of tubulin polyglutamylation is essential for sperm development and that its effects may be mediated by katanin whose activity has been proposed to be dependent on tubulin polyglutamylation. Moreover, during my PhD project I developed a method for production of differentially glutamylated microtubules. Using porcine brain tubulin, which is known to be highly glutamylated, as a starting material I perform deglutamylation to obtain the non-glutamylated version of it. Obtaining these two types of tubulin allows now to directly testing whether the interactions between microtubules and the MAPs of interests are dependent on tubulin polyglutamyaltion.

As dificuldades técnicas atuais para o manejo e controle de pragas que atacam as lavouras em nosso país, vêm incentivando a busca por métodos de controle alternativos e/ou complementares aos convencionais, destacando-se o potencial dos inseticidas botânicos. O Neem é uma planta nativa da Índia pertencente à família Meliaceae, conhecida popularmente como Nim ou Neem e tem sido usada por séculos no Oriente como: planta medicinal no tratamento de inflamações, infecções virais, hipertensão e febre, planta sombreadora, repelente, material para construção, combustível, lubrificante, adubo e mais recentemente como praguicida natural. Mesmo alavancada pela produção orgânica de alimentos, o uso de bioinseticidas ainda carece de mais pesquisas acerca de cada formulação principalmente sua ação sobre os organismos, sendo assim esta pesquisa visou avaliar os efeitos do extrato aquoso das folhas de Neem (Azadiractha indica) em ratos Wistar machos em idade reprodutiva, objetivando avaliar quais possíveis danos induzidos a exposição por oito dias ao extrato da planta pode resultar e também avaliar sua capacidade contraceptiva trazendo respostas importantes afim de se conhecer esta importante alternativa ao uso de agrotóxicos sintéticos. Para este estudo foram utilizados 20 ratos Wistar que foram divididos em 4 grupos compostos por 5 animais cada, sendo: G1 - 10.000ppm, G2 - 7.500ppm, G3 - 5.000 ppm e G4 - controle com água destilada. O volume administrado foi padronizado em 1 ml para cada animal e a administração deu-se por gavagem, por oito dias. Para se estudar as reações ocasionadas pelo tratamento realizamos nos animais após a administração das diferentes doses do composto o reconhecimento de sinais clínicos de toxicidade, a investigação das alterações fisiológicas, análise da qualidade do sêmen e avaliação macro e microscópicas dos órgãos alvo fígado, rins e testículos. Nossos resultados apontam que o extrato aquoso de Neem administrado por oito dias, via oral, nas doses empregadas apresentou baixa toxicidade, uma vez que não foi revelada nenhuma alteração nos quesitos avaliados, contudo apresentou resultados que levam a considerar sua capacidade contraceptiva, por terem alterando de forma negativa a motilidade e viabilidade espermática nas maiores doses administradas. / The current technical difficulties for the management and control of pests that attack the crops in our country have been encouraging the search for alternative and / or complementary control methods to conventional ones, highlighting the potential of the botanical insecticides. Neem is a plant native to India belonging to the family Meliaceae, popularly known as Nim or Neem and has been used for centuries in the East as: medicinal plant (in the treatment of inflammation, viral infections, hypertension and fever), shading plant, repellent, building material, fuel, lubricant, fertilizer and more recently as natural pesticide. Even though the use of bio-insecticides is still underpinned by organic food production, there is still a need for more research on each formulation, especially its action on organisms. This research aimed to evaluate the effects of the aqueous extract of Neem leaves (Azadiractha indica) on Wistar rats males of reproductive age, aiming to evaluate which possible induced damages the exposure for eight days to the extract of the plant can result and also to evaluate its contraceptive capacity bringing important answers in order to know this important alternative to the use of synthetic agrochemicals. For this study 20 Wistar rats were divided into 4 groups composed of 5 animals each, being: G1 - 10.000ppm, G2 - 7,500ppm, G3 - 5,000 ppm and G4 - control with distilled water. The volume administered was standardized in 1 ml for each animal and administration was given by gavage for eight days. In order to study the reactions caused by the treatment, the clinical signs of toxicity were confirmed in the animals after administration of the different doses of the compound, investigation of physiological changes after administration of the extract, analysis of the semen quality and macro and microscopic evaluation of the organs target liver, kidneys and testicles. After analysis of the results obtained it can be considered that the aqueous extract of Neem administered for eight days, orally, in the doses used presented low toxicity, since no changes were revealed in the evaluated items, however presented results that lead to consider its contraceptive capacity, because it has negatively altered motility and sperm viability at the highest doses administered.

Orientador: Rosicleire Veríssimo Silveira / Banca: Cristiele da Silva Ribeiro / Banca: George Shigueti Yasui / Resumo: Em teleósteos, a utilização de transplante de espermatogônias tronco está criando um cenário novo e promissor na área de biotecnologia e produção em aquicultura, possibilitando a preservação de espécies em perigo de extinção. Esta técnica consiste na remoção de células tronco espermatogoniais do testículo de um animal doador e a sua transferência para o testículo de um receptor. Para garantir a eficiência desta técnica o receptor deve ter sua espermatogênese endógena suprimida, pois a eficiência do transplante depende do sucesso na competição entre células germinativas transplantadas e células germinativas endógenas nos túbulos seminíferos. Este estudo teve por objetivo promover experimentalmente a depleção da espermatogênese endógena de Devario aequipinnatus, por meio da droga quimioterápica Myleran (Busulfan) e sua associação com a temperatura de 30°C. Foram definidas quatro fases de maturação gonadal para o ciclo testicular de D. aequipinnatus: Maturação Inicial, Maturação Intermediária, Maturação Final e Regressão, essas definições tiveram como base as alterações do epitélio germinativo testicular associado aos estágios das células germinativas presentes. Para depleção foram testadas duas dosagens, no T15/18 foram aplicadas duas injeções, via intracelomática, com intervalo de 10 dias entre as mesmas, sendo a primeira dose de 15 mg kg -1 Myleran (Busulfan) e a segunda com 18 mg kg -1 de Myleran. No T40 foi aplicado somente uma injeção contendo 40 mg kg -1 de Myleran (Busulfan). Como grupo controle foi utilizado uma unidade experimental sem a inclusão de qualquer tipo de droga, em que os animais permaneceram sob as mesmas condições de temperatura, alimentação e fotoperíodo oferecidas para os animais utilizados dos tratamentos. O T15/18 e o T40 apresentaram uma redução significativa na quantidade de Espermatócitos e consequentemente um aumento na quantidade de lúmen... / Abstract: In teleosts, the use of spermatogonial stem transplant is creating a new and promising scenario in the area of biotechnology and aquaculture production, enabling the preservation of species in danger of extinction. This technique involves the removal of spermatogonial stem cells of the testis of a donor animal and its transfer to the testicle of a receiver. To ensure the efficiency of this technique the receiver shall have its endogenous spermatogenesis deleted because the transplant efficiency depends on the success in the competition between transplanted germ cells and endogenous germ cells in the seminiferous tubules. This study aimed to experimentally promote depletion of endogenous spermatogenesis Devario aequipinnatus through the chemotherapy drug Myleran (Busulfan) and its association with the temperature 30 ° C. Maturation of four phases have been defined for testicular cycle D. aequipinnatus: Maturation Home, Intermediate maturation, maturation and Final Regression, these definitions were based on the changes of the testicular germinal epithelium associated with the stages of germ cells present. For depletion two doses were tested in the T15 / 18 were applied two injections via intracelomic with an interval of 10 days between them, the first dose of 15 mg kg-1 Myleran (Busulfan) and second with 18 mg kg- 1 Myleran. T40 was applied only in an injection containing 40 mg kg-1 Myleran (Busulfan). The control group was used an experimental unit without the inclusion of any type of drug, where the animals were kept under the same conditions of temperature, photoperiod and food offered to the animals used treatments. The T15 / 18 and T40 showed a significant reduction in the number of spermatocytes and consequently an increase in the amount tubular lumen in the testes of the animals examined. Three days after application of Busulfan for both single dose and double dose of Busulfan for an increase in pyknosis in spermatogonia ... / Mestre

Utilizaram-se 27 ratos Wistar, machos com 98 dias de idade, originados do Biotério da FMVZ-USP, com o objetivo de avaliar os possíveis efeitos do diabetes mellitus, sobre a função reprodutiva relacionada ao macho.Os animais foram divididos em três grupos, grupo A (GA) constituído de 10 animais sadios, grupo B (GB) constituído de oito ratos Wistar, com diabetes mellitus induzida quimicamente através da administração intraperitonial de estreptozotocina (65mg/Kg) e grupo C (GC) constituído por nove animais com diabetes mellitus induzida quimicamente pela administração intraperitonial de estreptozotocina (65mg/Kg), associada a insulinoterapia (3UI/rato por dia). Após quatro dias da administração da droga (GB e GC), os animais pertencentes ao grupo C (GC) receberam insulinoterapia (três IU/rato/dia) durante 42 dias. No final do experimento (46 dias após a administração da estreptozotocina), os animais foram sacrificados e foram observados os seguintes resultados; O diabetes mellitus leva ao aumento da glicemia, diminuição do peso corpóreo, diminuição do peso e tamanho testicular, diminuição das concentrações séricas de testosterona e FSH. Porém através da avaliação dos níveis séricos de inibina B, foi constatado que o diabetes não promove nem redução, nem aumento das concentrações séricas desse hormônio. Conclui-se também que a reposição exógena de insulina foi capaz de impedir a diminuição do peso corporal, redução do peso testicular, diminuição dos níveis de testosterona e FSH, porém apesar da insulinoterapia ter impedido a diminuição do peso corpóreo, não foi capaz de proporcionar o mesmo desenvolvimento corporal observado nos animais controle. / The objective of the present experiment was to evaluate the possible effect of diabetes mellitus, on the reproductive function in male rats. Towards this end, 27 male Wistar rats (98 days old), housed at the FMVZ-USP animal holding facility, were randomly assigned into three groups: Group A (GA) consisting of 10 healthy animals; Group B(GB) consisting of eight Wistar rats, with diabetes mellitus chemically induced through the intraperitoneal administration of estreptozotocin (65mg/Kg); and C group (GC) constituted by nine animals with diabetes mellitus chemically induced by the intraperitoneal administration of estreptozotocin (65mg/Kg), associate to an insulin treatment (3IU/rat per day for 42 days) that begun 4 days after the streptozotocin administration. Animals were euthanized 46 days after the administration of the estreptozotocin and the following results were obtained: diabetes mellitus led to an increase of the glicemia, reduction of the corporeal weight, decreased testicular wheight, serum concentrations of testosterone and FSH. No effect of diabeted were found for the serum levels of inhibin B. Results suggested that the exogen replacement of insulin was capable of hindering the reduction on corporal weight, reduction of testicular weight and reduction of the testosterone levels and FSH. On the other hand despite the fact that insulin treatament was capable of avoiding the reduction on bofy weight, it was not capable to provide similar body development observed in the normal animals.