Thesis (Ph. D.)--University of Missouri-Columbia, 2006. / The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Vita. Includes bibliographical references.

Thesis (Ph. D.)--University of Hong Kong, 2007. / Title proper from title frame. Also available in printed format.

Thesis (Ph. D.)--University of Missouri--Columbia, 2001. / Typescript. Vita. Includes bibliographical references (leaves 217-230). Also available on the Internet.

Thesis (Ph. D.)--University of Hong Kong, 2000. / Includes bibliographical references (leaves 97-146).

Thesis (Ph.D.) -- University of Texas Southwestern Medical Center at Dallas, 2005. / Not embargoed. Vita. References located at the end of each chapter.

In this study, for the first time we used CRISPR/Cas9 gene editing technology in sturgeons i.e., sterlets (Acipenser ruthenus). The sequences of sgRNA and primers were designed based on published dnd1 sterlet sequence. Each pair of sgRNA oligos after ligation ready duplex DNA fragment was cloned into vector pX330-U6-Chimeric_BB-CBh-hSpCas9 backbone and thereafter the transformation to competent cells Escherichia coli DH5 was done. The plasmid carried sgRNA was extracted for downstream applications. We diluted extracted plasmid with 10% of 2 M KCl and injection into animal pole of fertilized eggs of sterlets at one to four-cell-stage, 4 hours post fertilization (hpf). At the same time, second microinjection with 2.5% FITC-biotin-dextrans was injected into vegetal pole for labelling PGCs. In the control groups, the eggs were only injected by 2.5% FITC into vegetal pole. PGCs of sterlet were visualized and photographed using a uorescent stereo microscope Leica M165 FC. To confirm the presence or deletion/insertion occurring in the target gene, we used MCE-202 MultiNA microchip electrophoresis system for DNA analysis, in which the targeted gene after amplifying by PCR was analyzed. Mutations in both treated and control embryos of sterlet were further assessed by Sanger sequencing of the PCR product. In present study, we successfully established basic protocols such as preparation of competent cells, construction of vector carrying sgRNA and its transformation into competent cells to carry out the CRISPR/Cas9 technology in sturgeons. Less number of PGCs was observed in embryos that were treated with CRISPR/Cas9; however, sequencing did not provide us a reliable evidence for mutation of the targeted gene probably due to an unspecific PCR. Therefore, more authentication of dnd1 knockout should be done in future by more specific PCR and repeated sequencing.

No description available.

O transplante de células germinativas tem sido uma importante abordagem experimental para o estudo da preservação genética de espécies ameaçadas de extinção ou economicamente importantes. A técnica consiste na remoção das células germinativas indiferenciadas das gônadas do animal doador e na transferência das mesmas para a gônada de um indivíduo receptor. A fim de aumentar a eficiência da técnica, a identificação prévia das células germinativas a serem transplantadas torna-se preferível, visto que a cavidade que as receberá apresenta tamanho limitado. Sendo assim, é importante o desenvolvimento de marcadores moleculares que identifiquem precisamente as células a serem transplantadas na cavidade do individuo receptor, e os genes mais utilizados para esta finalidade são o dead end e o gene vasa, os quais são expressos apenas nas células da linhagem germinativa. Devido à importância do Colossoma macropomum (tambaqui) para a economia brasileira, esta espécie foi escolhida como uma espécie modelo de preservação para este estudo. Através do isolamento e sequenciamento dos genes dead end e vasa, desenvolvemos sondas de hibridização capazes de reconhecer as células onde estes genes são expressos e estudar o seu padrão de expressão nas gônadas. Ambos os genes apresentaram intensa expressão nos oócitos pré-vitelogênicos e fraca expressão em algumas espermatogônias. Pela primeira vez na literatura, diferentes isoformas causadas por splicing alternativo foram identificadas no gene dead end. A quantificação da expressão temporal dos diferentes transcritos mostrou que o padrão de expressão da sequência completa do gene teve uma tendência distintiva comparada ao padrão dos transcritos curtos, sugerindo que as diferentes isoformas desempenham papéis específicos e importantes para o desenvolvimento da linha germinativa nesta espécie. / Germ cell transplantation has been an important experimental approach to the study of the genetic preservation of endangered or economically important species. The technique consists in removing undifferentiated germ cells from the donor animal's gonads and transferring them to the gonad of a recipient individual. In order to increase the efficiency of the technique, the prior identification of the germ cells to be transplanted becomes preferable, since the receiving cavity presents limited size. Therefore, it is important to develop molecular markers to precisely identify the cells to be implanted in the recipient cavity, and the genes most used for this purpose are the dead end and the vasa genes, which are expressed only in germline cells. Due to the importance of Colossoma macropomum (tambaqui) for the Brazilian economy, this species was chosen as a model species for preservation in this study. By isolating and sequencing the dead end and vasa genes, we developed hybridization probes capable of recognizing the cells where these genes are expressed and better studying their expression pattern in the gonads. Both genes presented intense expression in pre-vitellogenic oocytes and poor expression in some spermatogonia. For the first time in the literature, different isoforms caused by alternative splicing were identified in the dead end gene. Quantification of the temporal expression of the different transcripts showed that the expression pattern of the full-length sequence had a distinctive tendency compared to the short transcripts pattern, suggesting that the different isoforms play specific roles for the germline development in this species.

Cryopreservation and transplantation of germ cells in fish provides a suitable tool for preserving genetic information. By method of surrogate reproduction, the offspring with characters of the chosen donor can be obtained. In this case of our commercially important species common carp. However, for the successful cryopreservation of the germ cells, a suitable protocol for each species must be established. Several cryoprotectants were tested. The best of them, Me2SO, regarding the viability of spermatogonia, was tested for its different concentrations depending also on the rate of freezing. Further testing, related to the effect of tissue size, incubation time and added sugar, was performed. The result of the assay identified best cryomedium composed of 2.5M dimethylsulfoxide, added sugar of 0.3M glucose, 1.5% BSA and 25nM Hepes dissolved in PBS. The most suitable size of tissue was 100 mg, incubation time was 30 min and coolig rate was -1 ° C/min. This protocol ensures the highest viability rate of cryopreserved spermatogonia of common carp. The second part of the work was to verify the success of the transplantation of cryopreserved and fresh spermatogonia into a suitably chosen recipient, the goldfish, which shares similar reproductive characteristics with carp, but also offers reduction of space requirements or resistance to koi-herpes virus. The transplanted germ cells colonized the germ line and started gametogenesis in 42.5% (cryopreserved spermatogonia) and 52.5% (fresh spermatogonia) goldfish recipients, which demonstrated that the transplantation of cryopreserved spermatogonia of common carp can be successfully achieved.

Submitted by ANGEL ANDRÉS ARIAS VIGOYA null (lakotah1@gmail.com) on 2017-01-25T17:53:42Z No. of bitstreams: 1 TESE VERSAO DEFINITIVA.pdf: 6803879 bytes, checksum: 2803b2bfb33863aee3dc638d6b31456b (MD5) / Approved for entry into archive by LUIZA DE MENEZES ROMANETTO (luizamenezes@reitoria.unesp.br) on 2017-01-27T13:30:27Z (GMT) No. of bitstreams: 1 ariasvigoya_aa_dr_jabo.pdf: 6803879 bytes, checksum: 2803b2bfb33863aee3dc638d6b31456b (MD5) / Made available in DSpace on 2017-01-27T13:30:27Z (GMT). No. of bitstreams: 1 ariasvigoya_aa_dr_jabo.pdf: 6803879 bytes, checksum: 2803b2bfb33863aee3dc638d6b31456b (MD5) Previous issue date: 2016-12-08 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Nos vertebrados, a espermatogênese é um processo de desenvolvimento celular altamente conservado e organizado, no qual uma pequena população de espermatogônias tronco produz continuamente milhões de espermatozóides, os quais são os responsáveis pela propagação do genótipo dos machos. Este processo é sustentado pelas espermatogônias tronco através da sua capacidade de auto-renovação e diferenciação. Vários aspectos relacionados com a regulação da atividade e fisiologia das espermatogônias tronco são ainda desconhecidos nos peixes teleósteos. Portanto, a técnica de transplante de células germinativas torna-se uma poderosa abordagem para melhorar o conhecimento da biologia das espermatogônias tronco e da espermatogênese. Resumidamente, a técnica envolve a transferência de células germinativas isoladas de um doador fértil para os testículos de um receptor estéril, e as células transplantadas são capazes de restaurar a gametogênese do receptor. Neste contexto, realizamos uma caracterização morfológica e estereológica dos diferentes tipos de células encontradas na espermatogênese da carpa comum (Cyprinus carpio). Também caracterizamos fenotipicamente as potenciais espermatogônias tronco. Portanto, descrevemos cinco tipos espermatogôniais na carpa comum: espermatogônias indiferenciadas do tipo A (Aund* - Aund), espermatogônias diferenciadas do tipo A (Adiff) e espermatogônias do tipo B (inicial e final). Nesta espécie, o processo espermatogênico durou aproximadamente uma semana. Nossos resultados demonstraram que a população espermatogonial expressa as proteínas c-Kit, Gfrα-1 e POU2. Neste estudo, também foi padronizada a técnica de transplante de espermatogônias na carpa comum. Assim, células germinativas isoladas de kinguios sexualmente maduros foram transplantadas através da papila urogenital de carpas macho quimicamente esterilizadas. As células germinativas derivadas dos doadores foram capazes de colonizar e se desenvolver nos testículos dos receptores. Em geral, nossos resultados reforçam a compreensão da biologia das células germinativas, em especial das potenciais células tronco. Além disso, o transplante, padronizado aqui, é uma abordagem com implicações significativas para a conservação e manejo das espécies valiosas e/ou ameaçadas de extinção. / In vertebrates, spermatogenesis is a highly conserved and organized developmental process, in which a small population of spermatogonial stem cells (SSC) continuosly produce millions of spermatozoa, which are responsible for spreading the male genotype. Likewise other stem cells, SSC are capable of either self-renew or differentiate. Several aspects related to the regulation of SSC activity and physiology are still unknown in teleost fish. Thus, the germ cell transplantation technique becomes a powerful approach to improve the knowledge of SSC biology and spermatogenesis. Briefly, the technique involves the transfer of germ cells isolated from a fertile donor into the testes of a sterile recipient, and transplanted donor germ cells are able to restore the recipient gametogenesis. Taking advantage of this background, we performed morphological and stereological characterization of the different cell types found in the common carp (Cyprinus carpio) spermatogenesis. We also characterized the putative SSC candidates by morphology. Therefore, we described five spermatogonial types in common carp: type A undifferentiated spermatogonia (Aund* - Aund), type A differentiated spermatogonia (Adiff) and type B spermatogonia (early and late). In this species, the spermatogenic process lasted approximately one week. Our findings demonstrated that the spermatogonial population expressed the c-Kit, Gfrα1 and POU2 proteins. Donor germ cells isolated from goldfish were transplanted non-surgically through urogenital papilla into the sexually mature cytoablated common carp recipients. Donor transplanted germ cells were able to colonize and develop in recipients’ testes. Overall, our results strengthens the knowledge of germ cell biology, focusing on stem cells. Finally, transplantation, standardized here, is an approach with significant implications for the conservation and management of endangered and valuable fish species. / CAPES: 15213-12-9