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Le parvovirus adéno-associé (AAV) possède un génome à ADN linéaire simple brin de 4,7kb encadré par deux séquences palindromiques inversées et identiques de 145 nucléotides appelées ITRs, requises en cis pour la réplication et l’encapsidation de l’ADN viral. Dans un AAV recombinant (rAAV), la totalité de la partie codante du génome viral est remplacée par une cassette d’expression et seuls les ITRs sont conservés. Le rAAV constitue un outil de choix pour le transfert de gènes dans diverses applications thérapeutiques. Cependant, dans bon nombre d’entre elles, il est nécessaire de pouvoir moduler l’expression du transgène quantitativement et au cours du temps. Plusieurs systèmes de régulation ont été décrits dont le système d’activation (Tet-On) de l’expression du transgène par la tétracycline et ses analogues (ex : la doxycycline). Le transfert et l’activation de l’expression du transgène par la doxycycline (Dox) nécessite deux vecteurs d’expression, un premier vecteur dans lequel le transactivateur (rtTA) est exprimé à partir d’un promoteur constitutif et un second qui porte le gène d’intérêt sous le contrôle du promoteur tétracycline (Ptet). Le Ptet est constitué du promoteur minimal du cytomégalovirus humain (PhCMVmini) placé en aval d’une répétition de séquences dites "opérateurs" (tetO). En présence de la Dox, le rtTA change de conformation, se lie au tetO et active la transcription du gène d’intérêt à partir du PhCMVmini. Pour le transfert de gène in vivo, il est cependant préférable de disposer d’un vecteur portant les deux cassettes d’expression au sein d’une seule construction (rAAV unique). Toutefois, les ITRs d’AAV d’une part et les séquences "enhancers" du promoteur utilisé pour exprimer le rtTA d’autre part, interfèrent avec le Ptet donnant lieu à une expression du gène d’intérêt à l’état non induit et par conséquent à un faible facteur d’induction. Nous décrivons dans ce travail un vecteur rAAV unique dont l’expression du transgène est activée par la tétracycline après transfert dans le cerveau de rat. En effet, nous avons développé un vecteur autorégulable dans lequel les deux cassettes d’expression sont placées en orientations opposées et la transcription du transgène et du rtTA est initiée à partir d’un promoteur tétracycline bidirectionnel (Ptet-bidi) et terminée par les signaux de polyadénylation bidirectionnels de SV40. Placées à côté de chaque ITR, ces dernières séquences pourraient également servir à arrêter la trancription à partir des ITRs d’AAV en absence de l’inducteur. Les performances de notre vecteur portant le gène rapporteur egfp (rAAV-ptetbidi-EGFP) ont été établies dans diverses lignées cellulaires immortalisées, dans des cultures primaires de cellules de Schwann ainsi que dans le cerveau du rat et des facteurs d’induction allant de 20 à 100 fois ont été observés. Nous avons également évalué la capacité de la minocycline (Mino), un antibiotique de la famille des tétracyclines utilisé pour ses propriétés anti-inflammatoires dans le cerveau, à induire l’expression du transgène à partir du Ptet dans une lignée de cellules U87-MG exprimant de façon stable le plasmide ptetbidi-EGFP. Quoique l’induction maximale de l’expression du transgène par la Mino nécessite des doses plus élevées et un temps plus long de traitement comparée à la Dox, elle apparaît moins toxique à des doses effectrices. Nous avons également évalué la réversibilité du système. Les résultats montrent une extinction plus rapide dans des cellules induites par la Mino comparée à celle obtenue dans des cellules induites par la Dox. Cependant, la cinétique d’induction du rAAV-ptetbidi-EGFP était lente et le niveau basal d’expression était encore élevé. De plus, à l’état induit, le nombre de cellules transduites par ce vecteur in vitro et in vivo reste inférieur à celui obtenu avec un vecteur équivalent portant le transgène sous le contrôle d’un promoteur constitutif. Nous avons réussi à améliorer l’inductibilité de notre vecteur portant le gène rapporteur egfp ou le gène thérapeutique hgdnf codant pour un facteur neurotrophique ayant un effet neuroprotecteur sur les neurones dopaminergiques mais également des effets non désirés : i) en plaçant, en aval du rtTA, le WPRE, une séquence de régulation post-transcriptionnelle d’origine virale permettant l’accumulation du transactivateur à concentration plus élevée dans les cellules transduites. Il en résulte un démarrage plus rapide et un niveau plus élevé de l’expression du transgène ainsi qu’une augmentation du nombre de cellules transduites dans le striatum de rat en réponse à la Dox; ii) en remplaçant le rtTA par le rtTA2SM2 moins toxique, plus stable et ayant une meilleure affinité de liaison au tetO. L’utilisation du rtTA2SM2 permet une réduction du niveau basal d’expression du transgène et son induction à plus faible dose d’inducteur. La version améliorée de notre vecteur a été ensuite encapsidée dans le sérotype 1 d’AAV, qui, injecté dans le striatum de rat, permet d’améliorer le volume de transduction et d’augmenter le nombre de cellules "GFP-positives" transduites comparé au sérotype 2 couramment utilisé. Un facteur d’induction de l’ordre de 10 fois a été également obtenu au moyen d’un rAAV1-ptet-bidi-hGDNF avec une quantité de GDNF exprimée à l’état induit dans la gamme des concentrations neuroprotectrices (100 pg/mg de tissu).

Submitted by Cristiane Chim (cristiane.chim@ucpel.edu.br) on 2017-11-27T11:42:50Z No. of bitstreams: 1 FERNANDA BURLANI NEVES.pdf: 1796364 bytes, checksum: 1cd868160548173be0f57cea7319fb27 (MD5) / Made available in DSpace on 2017-11-27T11:42:50Z (GMT). No. of bitstreams: 1 FERNANDA BURLANI NEVES.pdf: 1796364 bytes, checksum: 1cd868160548173be0f57cea7319fb27 (MD5) Previous issue date: 2015-02-27 / The purpose of this study was to contribute to the knowledege of women’s health in perimenopause phase and analyse the symptons related to this period with women’s quality of life. This period is characterized by a critical transition where several disturbances occurs due to endocrinous, psichological and biological alterations. This alterations generally are marked by periods of depression, anxiety and hormone levels alterations, particularly on strogens levels. Firstly it was conducted a bibliographic research with the objective of verify the effect of physical activity (PA) on psichological symptons of menopause period. After that, a transversal study was realized to evaluate BDNF, NGF e GDNF levels and the relation of these seric proteins levels with quality of life in climateric women. It was applied a questionnaire to identify socioeconomic, demographic and health behavior matters of these women. To evaluate quality of life it was administered Women’s Health Questionnaire (WHQ) and blood sample was colected from all sample. Statistical analyses were conducted using SPSS 21 and GraphPad Prism 6.0. Non-parametric test was performed and all values were presented as mean ± standart deviation. Spearman coeficent was used to analyse correlation between BDNF, GDNF, NGF and classifications of WHQ. p < 0,05 was cosidered significant. About serum NGF, the association between neurotrophic factor and WHQ presented a correlation between NGF and anxiety domain (r = 0,373, p ≤ 0,05) and attractiveness domain (r = 0,344, p ≤ 0,05). The literature review pointed that PA could promote reduction of stress and depressive symptons and better quality of life. Correlations between seric levels of BDNF, GDNF, NGF reveals that women who presented higher scores in anxiety and attractiveness domains had higher seric level of NGF in comparison with those who presented lower scores in these refered domains. Results suggests that this neutrophin could perform a relevant function related to behaviour during perimenopause period. Thus, more studies are needed to elucidate this question with te aim of reach clearly understanding of facts related to this period. The perspectives of this study are evaluate psichological and hormonal aspects by the effect of anaerobic ans aerobic training in cronic and acute phases, analyse cognitive function, control hormonal replacement, increase the number of participants and idealize PA as non-pharmacological treatment in perimenopause and all phases of women’s life. / Este estudo tem como objetivo contribuir para o conhecimento da saúde das mulheres na fase da perimenopausa e ainda analisar os sintomas referentes a esse período na qualidade de vida dessas mulheres. Esse período é caracterizado por uma crítica transição na qual a mulher lida com diversos transtornos consequentes de alterações endócrinas, psicológicas e biológicas que variam de acordo com as características da personalidade de cada mulher. As alterações geralmente são marcadas por períodos de depressão, ansiedade e alterações nos níveis hormonais, particularmente nos níveis de estrogênio. Inicialmente realizou-se uma pesquisa Bibliográfica com objetivo de verificar o efeito da Atividade Física (AF) nos sintomas psicológicos no período da menopausa, após desenvolveu-se um estudo transversal com objetivo de avaliar os níveis de BDNF, NGF e GDNF em mulheres climatéricas e a relação desses níveis séricos destas proteínas com a qualidade de vida de mulheres que estão passando por essa fase vital. Primeiramente foi aplicado um instrumento para identificar o nível socioeconômico e condições demográficas e questões de saúde comportamental. Para determinar a qualidade de vida foi usado Women’s Health Questionnaire (WHQ) e foi coletado amostra sanguínea de todas as participantes. Para as análises estatísticas foram realizadas utilizando SPSS 21 e GraphPad Prism 6.0. Foi realizado teste não paramétrico e todos os valores foram apresentados como média ± desvio padrão. O Coeficiente de correlação de Spearman foi utilizado para analisar a correlação entre BDNF, GDNF, NGF e as pontuações do WHQ. Os valores de p < 0,05 foram considerados estatisticamente significativos. Em relação aos níveis de NGF no soro, a associação entre fator neurotrófico e os domínios WHQ apresentou uma correlação entre os níveis de NGF com domínio ansiedade (r = 0,373, p ≤ 0,05) e domínio de atratividade (r = 0,344, p ≤ 0,05). Foi possível verificar na revisão de literatura que a AF pode promover a redução da ansiedade, do estresse e dos sintomas depressivos e a melhora da qualidade de vida. Na correlação entre os níveis séricos do BDNF, GDNF, NGF com o WHQ foi observado que as mulheres com escores mais altos em domínios de ansiedade e atratividade apresentam o maior nível sérico de NGF em comparação com aquelas com pontuações mais baixas nestes referidos domínios. Estes resultados sugerem que esta neurotrofina pode desempenhar um papel relevante na comportamento durante a perimenopausa. Deste modo, mais pesquisas são necessárias para elucidar essa questão com a intenção de alcançar claramente a compreensão dos fatos envolvidos neste período. Como perspectivas deste estudo pode-se promover a avaliação dos objetivos estudados sob o efeito de um treinamento físico aeróbio e anaeróbio na fase aguda e crônica, avaliar a função cognitiva, controlar o uso da reposição hormonal, aumentar o número das participantes e ainda realizar nas diferentes fases da vida, para de fato propor a Atividade Física como um plano de tratamento efetivo não farmacológico na perimenopausa e em outras fases da vida da mulher.

<p>The symptoms of Parkinson's disease occur due to degeneration of dopamine neurons in substantia nigra. It has been demonstrated that glial cell line-derived neurotrophic factor (GDNF) is a potent neurotrophic factor when it comes to protect and enhance survival of dopamine neurons in animal models of Parkinson's disease. The aim of this study was to evaluate short- and long-term effects of GDNF on survival and nerve fiber outgrowth of dopamine cells and astrocytic migration in mouse fetal ventral mesencephalic (VM) tissue. Primary tissue cultures were made of mouse fetal VM tissue and evaluated at 7 and 21 days in vitro (DIV) in terms of dopaminergic nerve fiber outgrowth and astrocytic migration when developed with GDNF present, partially, or completely absent. The results revealed that VM tissue cultured in the absence of GDNF did not exhibit any significant differences in migration of astrocytes or dopaminergic nerve fiber outgrowth neither after 7 DIV nor after 21 DIV, when compared with tissue cultured with GDNF present. Migration of astrocytes and dopaminergic nerve fiber outgrowth reached longer distances when tissue was left to develop for 21 DIV in comparison with 7 DIV. In order to study the long-term effects of GDNF, mouse fetal dopaminergic tissue was transplanted into the ventricles of adult mice and evaluated after 6 months. No surviving dopamine neurons were present in the absence of GDNF. In contrast dopamine neurons developed with GDNF did survive, indicating that GDNF is an essential neurotrophic factor when it comes to long-term dopamine cell survival. More cases have to be assessed in the future in order to strengthen the findings. Thus, transplanted dopamine neurons will be assessed after 3 and 12 months in order to map out when dopamine neurons deprived of GDNF undergo degeneration.</p>

Parkinson’s disease is a disorder mainly characterized by progressive degeneration of dopamine producing neurons in the substantia nigra of the midbrain. The most commonly used treatment strategy is to pharmacologically restore the lost function by the administration of the dopaminergic precursor L-DOPA. Another treatment strategy is to replace the degenerated neurons with immature fetal ventral mesencephalic tissue, or ultimately stem cell-derived tissue. Grafting trials have, however, revealed poor reinnervation capacity of the grafts, leaving much of the striata dopamine-denervated. An additional drawback is the upcoming of dyskinesia (involuntary movements), a phenomenon also observed during L-DOPA treatment of Parkinson’s disease patients. Attempts to characterize nerve fiber formation from dopamine neurons have demonstrated that the nerve fibers are formed in two morphologically diverse outgrowth patterns, one early outgrowth seen in the absence of astrocytes and one later appearing outgrowth seen in co-existence with astrocytes. The overall objective of this thesis has been to study the dopaminergic outgrowth including guidance of nerve fiber formation, and to look into the mechanisms of L-DOPA-induced dyskinesia. The first paper in this thesis characterizes the different outgrowth patterns described above and their relation to different glial cells. The study demonstrated the two different outgrowth patterns to be a general phenomenon, applying not only to dopamine neurons. Attempts of characterization revealed no difference of origin in terms of dopaminergic subpopulations, i.e. A9 or A10, between the outgrowth patterns. Furthermore, the “roller-drum” technique was found optimal for studying the dual outgrowth sequences. The second and the third paper also utilized the “roller-drum” technique in order to promote both patterns of neuronal fiber formation. The effects of glial cell line-derived neurotrophic factor (GDNF) on the formation of dopamine nerve fibers, was investigated. Cultures prepared from gdnf knockout mice revealed that dopaminergic neurons survive and form nerve fiber outgrowth in the absence of GDNF. The dopaminergic nerve fibers exhibited an outgrowth pattern consistent with that previous observed in rat. GDNF was found to exert effect on the glial-associated outgrowth whereas the non-glial-associated was not affected. Astrocytic proliferation was inhibited using cytosine β-D-arabinofuranoside, resulting in reduced glial-associated outgrowth. The non-glial-associated dopaminergic outgrowth was on the other hand promoted, and was retained over longer time in culture. Furthermore, the non-glial-associated nerve fibers were found to target the fetal frontal cortex. Different developmental stages were shown to promote and affect the outgrowths differently. Taken together, these data indicate and state the importance of astrocytes and growth factors for neuronal nerve fiber formation and guidance. It also stresses the importance of fetal donor age at the time for transplantation. The fourth and fifth studies focus on L-DOPA dynamics and utilize in vivo chronoamperometry. In study four, 6-OHDA dopamine-depleted rats were exposed to chronic L-DOPA treatment and then rated as dyskinetic or non-dyskinetic. The electrochemical recordings demonstrated reduced KCl-evoked release in the intact striatum after chronic L-DOPA treatment. Time for maximal dopamine concentration after L-DOPA administration was found to be shorter in dyskinetic animals than in non-dyskinetic animals. The serotonergic nerve fiber content in the striatum was evaluated and brains from dyskinetic animals were found to exhibit significantly higher nerve fiber density compared to non-dyskinetic animals. Furthermore, the mechanisms behind the conversion of L-DOPA to dopamine in 6-OHDA dopamine-depleted rats were studied. Local administration of L-DOPA in the striatum increased the KCl-evoked dopamine release in the intact striatum. Acute application of L-DOPA resulted sometimes in a rapid conversion to dopamine, probably without vesicle packaging. This type of direct conversion is presumably occurring in non-neuronal tissue. Furthermore, KCl-evoked dopamine releases were present upon local application of L-DOPA in the dopamine-depleted striatum, suggesting that the conversion to dopamine took place elsewhere, than in dopaminergic nerve fibers. In conclusion, these studies state the importance of astrocytes for neuronal nerve fiber formation and elucidate the complexity of L-DOPA conversion in the brain.

La maladie de Parkinson est une maladie neurodégénérative caractérisée par la mort progressive des neurones dopaminergiques de la substance noire (SN). Cette mort neuronale entraîne un déficit en dopamine (DA) dans le striatum, responsable de la survenue des symptômes. <br />Dans la perspective d'un traitement visant à protéger les neurones de la SN, de nombreuses études ont été centrées sur l'utilisation du glial cell-line derived neurotrophic factor (GDNF), facteur neurotrophique et neuroprotecteur des neurones dopaminergiques. Son efficacité thérapeutique a été démontrée par de nombreuses études dans des modèles animaux de la MP et lors d'essais cliniques. <br />Depuis une dizaine d'années, de nombreux travaux ont démontré l'efficacité de la thérapie génique (TG) comme méthode d'administration du GDNF et un effort particulier est entrepris pour optimiser cette approche. Cependant, malgré des résultats très encourageants obtenus dans les modèles murins et de primates de la MP, il s'est révélé que l'utilisation des vecteurs viraux se confrontait à une limite majeure. En effet, le ciblage préférentiellement neuronal de ces vecteurs entraîne la dispersion de la protéine dans la voie striatonigrale provoquant ainsi la formation massive de collatérales dans des zones non désirées. <br />Afin d'éviter la dispersion du GDNF, nous avons étudié la restriction de sa localisation au striatum en ciblant préférentiellement les astrocytes striataux via un vecteur lentiviral.<br /> Une première partie du travail porte sur la caractérisation d'un vecteur pseudotypé par l'enveloppe Mokola. Chez la souris, l'enveloppe permet un ciblage spécifique des astrocytes quel que soit le promoteur utilisé, contrairement aux rat et primate, chez lesquels un ciblage préférentiellement astrocytaire est obtenu en combinaison avec le promoteur CMV. <br />Dans une seconde partie, le vecteur pseudotypé par Mokola a été utilisé pour étudier l'effet d'une expression restreinte de GDNF aux astrocytes striataux sur la protection de la voie nigrostriée. L'injection d'un vecteur permettant la synthèse de GDNF (Mok-GDNF) dans un modèle de rat de la MP permet une forte et stable synthèse de GDNF pendant au moins un an. Bien que le GDNF soit très majoritairement retrouvé au niveau du striatum et du globus pallidus, un très faible transport du GDNF dans les zones de projection est détecté. Cette dispersion résiduelle ne permet pas la génération de collatérales ectopiques. L'injection du vecteur Mok-GDNF permet une protection du système dopaminergique de la voie nigrostriée ainsi que le rétablissement d'un turn-over de la DA normal, traduit par une restauration complète du comportement moteur. Ces résultats indiquent que l'utilisation du vecteur pseudotypé par Mokola pour exprimer le GDNF se révèle optimale pour la TG de protection de la MP.

The symptoms of Parkinson's disease occur due to degeneration of dopamine neurons in substantia nigra. It has been demonstrated that glial cell line-derived neurotrophic factor (GDNF) is a potent neurotrophic factor when it comes to protect and enhance survival of dopamine neurons in animal models of Parkinson's disease. The aim of this study was to evaluate short- and long-term effects of GDNF on survival and nerve fiber outgrowth of dopamine cells and astrocytic migration in mouse fetal ventral mesencephalic (VM) tissue. Primary tissue cultures were made of mouse fetal VM tissue and evaluated at 7 and 21 days in vitro (DIV) in terms of dopaminergic nerve fiber outgrowth and astrocytic migration when developed with GDNF present, partially, or completely absent. The results revealed that VM tissue cultured in the absence of GDNF did not exhibit any significant differences in migration of astrocytes or dopaminergic nerve fiber outgrowth neither after 7 DIV nor after 21 DIV, when compared with tissue cultured with GDNF present. Migration of astrocytes and dopaminergic nerve fiber outgrowth reached longer distances when tissue was left to develop for 21 DIV in comparison with 7 DIV. In order to study the long-term effects of GDNF, mouse fetal dopaminergic tissue was transplanted into the ventricles of adult mice and evaluated after 6 months. No surviving dopamine neurons were present in the absence of GDNF. In contrast dopamine neurons developed with GDNF did survive, indicating that GDNF is an essential neurotrophic factor when it comes to long-term dopamine cell survival. More cases have to be assessed in the future in order to strengthen the findings. Thus, transplanted dopamine neurons will be assessed after 3 and 12 months in order to map out when dopamine neurons deprived of GDNF undergo degeneration.

Normal intestinal functions are coordinated by enteric neurons within the enteric nervous system (ENS). In the embryonic and neonatal gut, enteric neuron survival is dependent on the expression of glial cell line-derived neurotrophic factor (GDNF) from its targets of innervation - the intestinal smooth muscle cells (ISMC). In the inflamed adult intestine, enteric neuron loss is immediately followed by ISMC proliferation, resulting in severe disruption of normal intestinal functions. Although GDNF can support the survival of postnatal enteric neurons, whether adult ISMC can secrete GDNF and support neuron survival is unclear. Results from qPCR analysis showed that freshly isolated adult ISMC have acquired the ability to express GDNF at the onset of proliferation, in vitro. Western blot analysis indicates that GDNF continues to be upregulated in ISMC at Passage 2 (P2), but its expression is decreased after long periods of proliferation at Passage 10 (P10). A neuron survival bioassay suggests that GDNF expression is correlated with enteric neuron survival. Results showed that P2 ISMC or conditioned media (CM) - but not P10 ISMC and CM, significantly increased enteric neuron survival. In subsequent experiments, the RET tyrosine kinase inhibitor vandetanib was used to block GDNF receptor-ligand interactions, and anti-GDNF neutralizing antibody was used to sequester soluble GDNF within the culture media. Both methods were successful at decreasing myenteric neuron survival. Furthermore, abolishing GDNF expression in P2 ISMC with GDNF siRNA also resulted in a decreased myenteric neuron survival. The above observations suggest that ISMC-derived GDNF is important in supporting myenteric neuron survival in vitro. / Thesis (Master, Neuroscience Studies) -- Queen's University, 2012-09-28 09:43:23.968

Abstract The embryonic urogenital system (UGS) generates the metanephric kidney, gonad and the adrenal gland. It is well known that the development of the UGS is regulated by sequential and reciprocal epithelial and mesenchymal tissue interactions but the secreted mediators involved are still poorly known. The action of such inductive signals is typically regulated by specific antagonists. The Sprouty (Spry) proteins compose one family of cytoplasmic regulators that typically repress the function of the receptor tyrosine kinase (RTK) signal transduction pathways. The DAN/Cerberus (Cer) family that encodes secreted proteins bind and antagonize the Bmp, Wnt and Nodal signals. In this study the roles of Spry and Cer1 was addressed during mouse UGS development by targeted expression of SPROUTY2 (SPRY2) and Cer1 in the ureteric bud and Wolffian duct under the Pax2 promoter. Changes induced in the UGS assembly process were analyzed in detail to reveal the normal developmental roles of these proteins. SPRY2 expression led to either complete lack of the kidney, reduction in the kidney size or formation of unilateral kidney with reduced size. The SPRY2 mediated reduction in kidney size was accompanied by inhibition of expression of genes that are known to regulate kidney development. The results indicated that the Spry may take part in kidney development by coordinating the reciprocal cell signaling between the ureteric bud, the mesenchymal cells and stromal cells. In addition to the kidney, the gain of SPRY2 function revealed an important role in the control of male gonadogenesis. SPRY2 over expression in the Wolffian duct malformed the Wolffian duct derivatives, diminished the number of seminiferous tubules and the amount of the interstitial tissue associated with reduced mesonephric cell migration to the testis. Exogenous FGF9 rescued mesonephric cell migration inhibited by SPRY2. It was concluded that Spry protein contribute to male sexual organogenesis by antagonizing Fgf9 signaling. When the Cer1 gene was over expressed in the ureteric bud this lead unexpectedly to increased kidney size. The Cer1 mediated promotion of kidney size was demonstrated to involve enhanced ureteric bud morphogenesis. At the molecular level Cer1 protein function lead to inhibition of Bmp4 gene expression and concurrent upregulation of Gdnf and Wnt11 expression. Notably, excess BMP4 reduced the Cer1 stimulated ureteric bud branching and downregulated normally expression of Gdnf and Wnt11 in the embryonic kidney. Based on the presented data it is proposed that the establishment of mammalian organ size is under the control of both systemic and the intrinsic factors. Together the work demonstrates significant roles for the proteins that typically inhibit growth factor signaling or signal transduction. Hence organogenesis is controlled by coordination between positive and negative growth factor regulator signals.

In our Laboratory of Developmental Biology there was established a long term culture derived from Xenopus tropicalis testes. It contains pre-Sertoli cells mostly. They compose a feeder layer allowing cultivation of stem cells, revealing the morphology of spermatogonial stem cells. This diploma thesis was focused on a preparation of two growth factors, FGF2 (fibroblast growth factor 2) and GDNF (glial cell line-derived neurotrophic factor), with the subsequent characterization of their influence at cell culture mentioned above. Factors were selected on the basis of the microenvironmental niche theory, according which FGF2 and GDNF are the most important factors for spermatogonial stem cells proliferation and self-renewal. FGF2 recombinant factor was gained using the expression plasmid pET-15b. Its characterization in the testicular culture brought surprising result. Even a low concentration of FGF2 factor (2.5ng/ml) caused cell detaching and dying. Similar result was previously shown in differentiating osteoblast culture only. More experiments need to be done to prove if apoptose take place and why do testicular cells act this way. Key words: Xenopus tropicalis, FGF2, GDNF, SSC, pre-Seroli cells