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PiRNAs and their Piwi family protein partners are part of a germline specific epigenetic regulatory mechanism essential for proper spermatogenesis, silencing of transposable elements, and maintaining germline genome integrity, yet their role in the response of the male germline to genotoxic stress is unknown. Ionizing radiation (IR) is known to cause transgenerational genome instability that is linked to carcinogenesis. Although the molecular etiology of IR-induced transgenerational genomic instability is not fully understood, it is believed to be an epigenetically mediated phenomenon. IR-induced alterations in the expression pattern of key regulatory proteins involved in the piRNA pathway essential for paternal germline genome stability may be directly involved in producing epigenetic alterations that can impact future generations. Here we show whole body and localized X-irradiation leads to significant altered expression of proteins that are necessary for, and intimately involved in, the proper functioning of the germline specific piRNA pathway in mice and rats. In addition we found that IR-induced alterations to piRNA pathway protein levels were time and dose dependent. / ix, 123 leaves : ill. (some col.) ; 29 cm

In a 4-cell C. elegans embryo the ventral blastomere EMS requires polarity signaling from its posterior sister cell, P2. This signaling event enables EMS to orient its division spindle along the anterior-posterior (A/P) axis and to specify the endoderm fate of its posterior daughter cell, E. Wnt pathway components have been implicated in mediating P2/EMS signaling. However, no single mutants or various mutant combinations of the Wnt pathway components disrupt EMS polarity completely. Here we describe the identification of a pathway that is defined by two tyrosine kinase related proteins, SRC-1 and MES-1, which function in parallel with Wnt signaling to specify endoderm and to orient the division axis of EMS. We show that SRC-1, a C. elegans homolog of c-Src, functions downstream of MES-1 to specifically enhance phosphotyrosine accumulation at the P2/EMS junction in order to control cell fate and mitotic spindle orientation in both the P2 and EMS cells. In the canonical Wnt pathway, GSK-3 is conserved across species and acts as a negative regulator. However, in C. elegans we find that GSK-3 functions in a positive manner and in parallel with other components in the Wnt pathway to specify endoderm during embryogenesis. In addition, we also show that GSK-3 regulates C. elegans germline development, a function of GSK-3 that is not associated with Wnt signaling. It is required for the differentiation of somatic gonadal cells as well as the regulation of meiotic cell cycle in germ cells. Our results indicate that GSK-3 modulates multiple signaling pathways to regulate both embryogenesis and germline development in C. elegans.

The studies presented in this thesis addressed mainly two aspects of Piwi-interacting RNA (piRNA) biology in the Drosophilagermline. We investigated the role of the piRNA pathway in embryonic axis specification. piRNAs mediate silencing of retrotransposons and the Stellate locus. Mutations in the Drosophila piRNA pathway genes armitage and aubergine disrupt embryonic axis specification, triggering defects in microtubule polarization and asymmetric localization of mRNA and protein determinants in the developing oocyte. Mutations in the ATR/Chk2 DNA damage signal transduction pathway dramatically suppress these axis specification defects, but do not restore retrotransposon or Stellatesilencing. Furthermore, piRNA pathway mutations lead to germline-specific accumulation of γ-H2Av foci characteristic of DNA damage. We conclude that piRNA based gene silencing is not required for axis specification, and that the critical developmental function for this pathway is to suppress DNA damage signaling in the germline. We have also identified a new member of the piRNA pathway. We show that mutations in rhino, which encodes a rapidly evolving Heterochromatin Protein 1 (HP1) chromo box protein, lead to germline specific DNA break accumulation, trigger Chk2 kinase dependent defects in axis specification, and disrupt germline localization of Piwi proteins. Mutations in rhino and the piRNA pathway gene armitage disrupt silencing of all major transposon families, but do not alter expression of euchromatic or heterochromatic protein coding genes. Deep sequencing studies show that rhino mutations significantly reduce or eliminate anti-sense piRNAs derived from the majority of transposable elements in the Drosophila genome, and lead to a dramatic reduction in piRNAs derived from major piRNA production clusters on chromosomes 2R and 4. Rhino protein localizes to distinct nuclear foci, and associates with the chromosome 2R and 4 clusters by chromatin immunoprecipitation. The Rhino HP1 homologue is therefore required for piRNA biogenesis, transposon silencing, and maintenance of germline genome integrity.

Orientador: Mary Anne Heidi Dolder / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-17T22:42:21Z (GMT). No. of bitstreams: 1 Gomes_MarcosdeLuccaMoreira_D.pdf: 17022356 bytes, checksum: 8f52bd976bb7a3f25e260b1c7c06dc69 (MD5) Previous issue date: 2011 / Resumo: A espécie Heteropterys aphrodisiaca é amplamente utilizada como afrodisíaca. Estudos mostram o potencial efeito estimulante sobre o tamanho das vesículas seminais e de populações de células de Leydig em ratos, ambos andrógenos dependentes. Neste trabalho foi proposto o tratamento com a infusão de H. aphrodisiaca aliado ao exercício de resistência física com o intuito de observar seus efeitos nos testículos de ratos. Foram utilizados ratos Wistar (90 dias) divididos em 4 grupos (n=10, cada): controles - água, sedentário e com treinamento físico; tratados com H. aphrodisiaca (104mg/kg/dia) sedentário e com treinamento físico. O experimento teve duração total de 56 dias. Foi realizada dosagem de testosterona plasmática e avaliados parâmetros morfométricos e estereológicos do parênquima testicular. No epitélio seminífero, foram contados os números de células germinativas e somáticas, calculando-se o rendimento da espermatogênese, índices mitótico e meiótico e eficiência e capacidade de células de Sertoli. Foi realizado Western Blotting para quantificação da concentração de receptores de andrógeno, e TUNEL para determinação de porcentagem de células apoptóticas. A concentração de testosterona circulante foi significativamente maior nos animais tratados e sedentários. Os diâmetros médios tubulares dos animais treinados foram maiores quando comparados aos dos animais sedentários. Não ocorreram variações significativas quanto ao volume do tecido intersticial, na proporção de macrófagos, espaço linfático e vasos sanguíneos. Entretanto, ocorreram diminuições significativas na proporção de tecido conjuntivo em ambos os grupos que receberam a infusão vegetal. Houve aumentos significativos do diâmetro e volume nucleares das células de Leydig dos animais treinados. Houve diminuição significativa do número de células de Leydig por grama de parênquima testicular nos animais controle/treinados quando comparados aos outros grupos experimentais. O grupo tratado/treinado apresentou o maior índice de mitoses espermatogoniais, sendo significativamente maior que a média do grupo sedentário/tratado. As curvas de rendimento e índice mitótico possuem mesmo padrão de distribuição, entretanto, para o índice meiótico, o grupo sedentário/tratado mostrou as maiores médias quando comparado aos dois grupos controle, sedentário e treinado. Não houve alteração de índices apoptóticos tampouco na concentração de receptores androgênicos entre os grupos experimentais. O protocolo de treinamento aplicado no presente experimento não acarretou em danos no parênquima testicular, como tem sido afirmado em outros experimentos envolvendo exercício aeróbico. Contudo, a infusão de nó-de-cachorro parece influenciar tanto no estímulo de células de Leydig, com aumento do volume celular, quanto no comportamento espermatogonial, induzindo mitoses e aumentando o rendimento geral do processo espermatogênico / Abstract: The species Heteropterys aphrodisiaca is traditionally used as an aphrodisiac. Some studies showed its stimulating potential of seminal vesicle and Leydig cell population growth, both androgen dependent. In this study, we proposed to observe the effects of H. aphrodisiaca infusion treatment combined with endurance exercise protocol. For this investigation, 40 adult Wistar rats (90 days) were used (4 groups, n = 10 each): two controls receiving water, sedentary and trained, and two treated with H. aphrodisiaca (104mg/kg/dia), sedentary and trained. The experiment lasted for 56 days. Plasma testosterone concentration was measured by radioimmunoassay. Morphometrical and stereological parameters were evaluated from the testicular parenchyma. The numbers of somatic and germ cells were estimated by counting the seminiferous epithelium cell population. Also, the spermatogenic yield, meiotic and mitotic index, efficiency and capacity of Sertoli cells were estimated. Western blotting was performed to quantify the concentration of androgen receptors, and TUNEL technique to determine the percentage of apoptotic cells within the seminiferous tubules. Control/trained animals showed a significant decrease in body mass gain compared to sedentary animals. The testosterone concentrations were significantly higher in treated/sedentary animals. The mean tubular diameters of trained animals were higher when compared to sedentary animals. There were no significant differences regarding the volume of interstitial tissue, the proportion and volume of macrophages, lymphatic space and blood vessels. However, the treated/sedentary group showed lower connective tissue content compared with the control/sedentary, as well as the diminished volume of Leydig cells, compared with the treated/trained group. There was a significant increase of both nuclear diameter and volume of Leydig cells in trained animals. On the other hand, there was a significant decrease of Leydig cell number per gram of testicular parenchyma in control/trained animals. Considering the spermatogenesis dynamics, both treated groups showed the highest spermatogenic yield, in relation to the control/trained group. The treated/trained group showed the highest index of spermatogonial mitosis - significantly higher than the sedentary/treated one. Spermatogenic yield and mitotic index showed the same distribution pattern when plotted on graphs; however, the meiotic index was significantly higher only in the sedentary/treated group. The apoptotic indexes, as well as the androgen receptor concentrations were not affected by the protocols employed. Treatment with the plant infusion did not lead to an increase of the parameters analyzed. However, the infusion seems to stimulate either the Leydig cell (increase of cell volume) or the spermatogonial behavior, inducing more mitosis and increasing the spermatogenic yield / Doutorado / Biologia Celular / Doutor em Biologia Celular e Estrutural

L’infertilité représente un problème majeur de santé publique en concernant 10 à 15% des couples en âge de procréer. Un facteur masculin est responsable de l’infertilité du couple dans près de la moitié des cas. Pour environ 30% d'entre eux, l'étiologie reste inexpliquée. Le premier axe du travail a concerné l’étude moléculaire d’une cohorte de patients infertiles (azoospermie non-obstructive/cryptozoospermie ou désordre du développement sexuel ou DSD) pour lesquels les analyses du caryotype standard et/ou des microdélétions des régions AZF par PCR n’ont pas permis d’expliquer le phénotype. L'impact des variations de nombre de copies de l'ADN (CNV) détectées par l'hybridation génomique comparative sur puce à ADN est peu documenté. Un design personnalisé de puce à ADN de format 400K, pangénomique et enrichi sur un large panel de 445 gènes liés à l'infertilité et à un DSD a été développé. Cette puce a permis l’identification de 171 CNV d’intérêt. Ces résultats soulignent l’intérêt de ce design comme outil diagnostic dans le cadre du bilan de l’infertilité masculine. Le second axe du travail a été de modéliser l’infertilité masculine in vitro dans un contexte d’anomalie génétique. Des cellules souches pluripotentes induites humaines (hiPS) ont été générées à partir d’érythroblastes de deux patients infertiles porteurs d’un remaniement chromosomique complexe ou d’un caryotype 46,XX-SRY négatif avec mutation du gène de l’AMH. Dans un deuxième temps, la fonctionnalité des lignées de cellules hiPS générées a été testée par différenciation in vitro en cellules germinales primordiales (CGP). Elles expriment les marqueurs clés du stade CGP dont SOX17, le déterminant germinal le plus précoce des CGP. Les perspectives de ce travail seront de poursuivre la différenciation germinale vers des stades plus matures et ainsi de pouvoir étudier le processus méiotique dans un contexte d’anomalie génétique. / Infertility represents a major public health problem and concerns 10 to 15% of couples in the general population. A male factor is responsible for the infertility of the couple in about half of all cases. In approximately 30% of them, the etiology remains unexplained.The first working axis concerned the molecular study of a cohort of infertile patients (nonobstructiveazoospermia/ cryptozoospermia and disorder of the sex development or DSD) for whom analyses of standard karyotype and/or microdeletions of AZF regions were not able to explain the phenotype. The impact of copy number variations of DNA (CNVs) detected by comparative genomic hybridization (CGH-array) is poorly documented. A custom design 400K micoarray, genome-wide and enriched on a wide panel of 445 genes linked with infertility and DSD has been achieved. This array allowed the identification of 171 CNVs of interest.These results underline the potential of this design for diagnosis of male infertility. The second objective of this work was the in vitro modelisation of male infertility in a context of genetic abnormality. For that purpose, human induced pluripotent stem cells (hiPSCs) were generated from erythroblasts by means of not integrative Sendaï virus, in two patients carrying genetic abnormalities (complex chromosomal rearrangement and 46,XX-SRY negative karyotype associated with AMH gene mutation). Secondly, functionality of hiPSCs generated was tested by germ cells in vitro differentiation. Primordial germ cell (PGC) stage was successfully obtained. Cells expressed key PGC markers such as SOX17. The perspectives of this work will be to continuethe germinal differentiation towards more mature stages and so to be able studying the meiotic process in a context of genetic abnormality.

During each life cycle, gametes must preserve and pass on both genetic and epigenetic information, making the germline both immortal and totipotent. In the male germline the dramatic morphological transformation of a germ cell through meiosis, into a sperm competent for fertilization, while retaining this information is an incredible example of cellular differentiation. This process of spermatogenesis is inherently thermosensitive in numerous metazoa ranging from worms to man. Here, I describe the role of two redundant AGO-class paralogs, ALG-3/4, and their small RNA cofactors, in promoting thermotolerant male fertility in Caenorhabditis elegans. alg-3/4 double mutants exhibit temperature dependent sterility resulting from defective spermiogenesis, the postmeiotic differentiation of haploid spermatids into spermatozoa competent for fertilization. The essential Argonaute CSR-1 functions with ALG-3/4 to positively regulate target genes required for spermiogenesis by promoting transcription via a small RNA positive feedback loop. Our findings suggest that ALG-3/4 functions during spermatogenesis to amplify a small-RNA signal loaded into CSR-1 to maintain transcriptionally active chromatin at genes required for spermiogenesis and to provide an epigenetic memory of male-specific gene expression. CSR-1, which is abundant in mature sperm, appears to transmit this memory to offspring. Surprisingly, in addition to small RNAs targeting male-specific genes, we show that males also harbor an extensive repertoire of CSR-1 small RNAs targeting oogenesis-specific mRNAs. The ALG-3/4 small RNA pathway also initiates silencing small RNA signals loaded into WAGO vii Argonautes, which function to posttranscripitonally silence their target mRNAs. Silencing WAGO/small RNA-complexes are present in sperm and presumably transmitted to offspring upon fertilization. Together these findings suggest that C. elegans sperm transmit not only the genome but also epigenetic activating and silencing signals in the form of Argonaute/small-RNA complexes, constituting a memory of gene expression in preceding generations.

In the preceding chapters, I have discussed my doctoral research on studying the siRNA loading pathway in Drosophila using both biochemical and genetic approaches. We established a gel shift system to identify the intermediate complexes formed during siRNA loading. We detected at least three complexes, named complex B, RISC loading complex (RLC) and RISC. Using kinetic modeling, we determined that the siRNA enters complex B and RLC early during assembly when it remains double-stranded, and then matures in RISC to generate Argonaute bearing only the single-stranded guide. We further characterized the three complexes. We showed that complex B comprises Dcr-1 and Loqs, while both RLC and RISC contain Dcr-2 and R2D2. Our study suggests that the Dcr-2/R2D2 heterodimer plays a central role in RISC assembly. We observed that Dcr-1/Loqs, which function together to process pre-miRNA into mature miRNA, were also involved in siRNA loading. This was surprising, because it has been proposed that the RNAi pathway and miRNA pathway are separate and parallel, with each using a unique set of proteins to produce small RNAs, to assemble functional RNA-guided enzyme complexes, and to regulate target mRNAs. We further examined the molecular function of Dcr-1/Loqs in RNAi pathway. Our data suggest that, in vivo and in vitro, the Dcr-1/Loqs complex binds to siRNA. In vitro, the binding of the Dcr-1/Loqs complex to siRNA is the earliest detectable step in siRNA-triggered Ago2-RISC assembly. Futhermore, the binding of Dcr-1/Loqs to siRNA appears to facilitate dsRNA dicing by Dcr-2/R2D2, because the dicing activity is much lower in loqslysate than in wild type. Long inverted repeat (IR) triggered white silencing in fly eyes is an example of endogenous RNAi. Consistent with our finding that Dcr-1/Loqs function to load siRNA, less white siRNA accumulates in loqs mutant eyes compared to wild type. As a result, loqs mutants are partially defective in IR trigged whitesilencing. Our data suggest considerable functional and genetic overlap between the miRNA and siRNA pathways, with the two sharing key components previously thought to be confined to just one of the two pathways. Based on our study on siRNA loading pathway, we also elucidated the molecular function of Armitage (Armi) protein in RNAi. We showed that armi is required for RNAi. Lysates from armi mutant ovaries are defective for RNAi in vitro. Native gel analysis of protein-siRNA complexes suggests that armi mutants support early steps in the RNAi pathway, i.e., the formation of complex B and RLC, but are defective in the production of the RISC.

Transposon silencing is required to maintain genome stability. The non-coding piRNAs effectively suppress of transposon activity during germline development. In the Drosophila female germline, long precursors of piRNAs are transcribed from discrete heterochromatic clusters and then processed into primary piRNAs in the perinuclear nuage. However, the detailed mechanism of piRNA biogenesis, specifically how the nuclear and cytoplasmic processes are connected, is not well understood. The nuclear DEAD box protein UAP56 has been previously implicated in protein-coding gene transcript splicing and export. I have identified a novel function of UAP56 in piRNA biogenesis. In Drosophila egg chambers, UAP56 co-localizes with the cluster-associated HP1 variant Rhino. Nuage is a germline-specific perinuclear structure rich in piRNA biogenesis proteins, including Vasa, a DEAD box with an established role in piRNA production. Vasa-containing nuage granules localize directly across the nuclear envelope from cluster foci containing UAP56 and Rhino, and cluster transcripts immunoprecipitate with both Vasa and UAP56. Significantly, a charge-substitution mutation that alters a conserved surface residue in UAP56 disrupts co-localization with Rhino, germline piRNA production, transposon silencing, and perinuclear localization of Vasa. I therefore propose that UAP56 and Vasa function in a piRNA-processing compartment that spans the nuclear envelope.