Yes / In each generation, the germline is tasked with producing somatic lineages that form the body, and segregating a population of cells for gametogenesis. During animal development, when do cells of the germline irreversibly commit to producing gametes? Integrating findings from diverse species, we conclude that the final commitment of the germline to gametogenesis - the process of germ cell determination - occurs after primordial germ cells (PGCs) colonize the gonads. Combining this understanding with medical findings, we present a model whereby germ cell tumors arise from cells that failed to undertake germ cell determination, regardless of their having colonized the gonads. We propose that the diversity of cell types present in these tumors reflects the broad developmental potential of migratory PGCs. / This work was supported by the Howard Hughes Medical Institute where D.C.P. is an Investigator, and the Frontier Research Program from the Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology. P.K.N. is a recipient of the Hope Funds for Cancer Research Fellowship (HFCR-15-06-06) and an Early Career Fellowship from the National Health and Medical Research Council, Australia (GNT1053776).

Introdução: a alta prevalência do câncer e o aumento significativo da sobrevivência em longo prazo geraram interesse quanto à preservação da fertilidade em mulheres jovens expostas a quimioterapia e radioterapia. Neste sentido estudos de congelamento de tecido ovariano para posterior transplante, abriram uma nova perspectiva de aplicação no tratamento e prevenção da infertilidade feminina. Objetivos: comparar dois protocolos de congelamento de tecido ovariano, um lento não automatizado e um por vitrificação, com o intuito de avaliar a viabilidade dos tecidos para posterior transplante autólogo. Método: Foram utilizadas 30 camundongos fêmea CF1 com aproximadamente 8 semanas e pesando 29,29g±2,9. •Os ovários extraídos foram vitrificados ou congelados, mantidos em nitrogênio líquido por 30 dias e descongelados. Após o descongelamento, o ovário esquerdo foi destinado às análises histológicas e caracterização por imuno histoquímica para o marcador mouse vasa homologue (MVH) e o ovário direito foi utilizado para os testes de viabilidade celular com exclusão por azul de trypan. Resultados: Nas análises de Hematoxilina e Eosina (HE) foram contados folículos primordiais, primários, pré-antrais e antrais. Não houve diferença significativa na proporção de folículos primordiais, primários e pré-antrais após descongelamento entre os grupos testados. A contagem de folículos antrais foi significativamente maior no grupo de vitrificação (p = 0,004). No ensaio de imunohistoquímica para o marcador MVH, folículos MVH + e MVH- foram contados e comparados com o número total de folículos. O grupo congelamento lento apresentou maior número de células não marcadas (p = 0,012). Conclusão: Embora ambos os protocolos tenham apresentado resultados semelhantes na análise histológica das contagens foliculares, o protocolo de vitrificação foi significativamente melhor para preservar a população de células tronco ovarianas. / Introduction: The high prevalence of cancer and the significant increase in long-term survival have generated interest as the preservation of fertility in young women exposed to chemotherapy and radiotherapy. Experimental techniques have been tried in an attempt to reverse the ovarian failure induced by these treatments. In this regard studies of ovarian tissue freezing for subsequent transplantation disclose a new application perspective in the treatment and prevention of female infertility. Objective: two ovarian tissue freezing protocols were tested, a non-automated slow-freezing and by vitrification, in order to assess the viability of the tissues for subsequent autologous transplantation. Methods: as ovaries donors, were used 30 female CF1 mice approximately 8 weeks and weighing 29,29g±2,9. • The ovaries were vitrified or frozen, stored in liquid nitrogen for 30 days and thawed. After thawing, the left ovary was intended for histological and immunohistochemical characterization by histochemical marker for MVH and right ovary was used for the tests with cell viability by trypan blue exclusion. Results: In HE slides was counting primordial, primary, pre antral and antral follicles. No significant difference was found in the proportion of high-quality primordial, primary and pre antral follicles after thawing/warming in the slow-freezing and vitrification group, respectively. The antral follicle counting was significant higher in vitrification group (p=0,004). In immunohistochemistry assay for MVH Antibody , MVH+ and MVH- follicles were counted and compared with the total number of follicles and slow freeze group had a higher number of not marked cells (p=0,012). Conclusion: Although both protocols showed similar results in the histological analysis for follicular counts, the vitrification protocol was significantly better for preserve the ovarian stem cell population.

The studies in this thesis examined piglet gonocyte identification, isolation, purification, preservation and potential for initiation of spermatogenesis after transplantation into irradiated recipient testes. As a first step, we characterized a previously non-described auto-fluorescence in the piglet testis tissue. This auto-fluorescence mainly originated from granules among the testis interstitial cells, and we found that its interference with immuno-fluorescence can be overcome using Sudan black staining. We also showed that porcine gonocytes can be specifically labelled with the lectin Dolichos biflorus agglutinin (DBA). To optimize gonocyte isolation, we found that ~9-fold more live cells could be harvested by enzymatic digestion of testis tissues than with mechanical methods. However, the proportion of gonocytes (~7%) did not differ between the mechanical and enzymatic methods of testis cell isolation. We then developed a novel three-step strategy for isolation of gonocytes by combining enzymatic digestion and vortexing, resulting in a gonocyte proportion of ~40% (~5-fold more than that from conventional methods). For short-term preservation of testis cells, we found that the survival of testis cells under hypothermic conditions was dependent on the cell type, and affected by storage duration, temperature and medium used. More than 80% of live testis cells survived the 6-day hypothermic preservation period in 20% FBS-L15, without visible changes to the cell culture potential or gonocyte proportion. In another experiment where testis tissues were maintained under hypothermic conditions, we found that ~25% of testis cells could survive for 6 days if preserved in HypoThermosol-FRS solution (HTS-FRS), without morphological changes. To purify gonocytes, we showed that centrifugation of testis cells using 17% Nycodenz can lead to precipitation of gonocytes in pellets (with a purity of > 80%). We also found that pre-coating tissue culture plates with both fibronectin and poly-D-lysine can result in the negative selection of gonocytes (with a purity of up to 85%). We subsequently showed that further purification of gonocytes (to > 90%) could be achieved by combining the two latter approaches. To prepare recipients for germ cell transplantation, we used local irradiation of piglet testes which reduced testis growth, decreased seminiferous tubule diameters and completely eliminated spermatogenesis at 4 months post-irradiation. Compared with the absence of endogenous spermatogenesis in the control testes, spermatogenesis up to elongating spermatids was observed in the irradiated testes after gonocyte transplantation. In summary, we investigated several critical elements in the study and manipulation of gonocytes in a large animal model.

The studies in this thesis examined piglet gonocyte identification, isolation, purification, preservation and potential for initiation of spermatogenesis after transplantation into irradiated recipient testes. As a first step, we characterized a previously non-described auto-fluorescence in the piglet testis tissue. This auto-fluorescence mainly originated from granules among the testis interstitial cells, and we found that its interference with immuno-fluorescence can be overcome using Sudan black staining. We also showed that porcine gonocytes can be specifically labelled with the lectin Dolichos biflorus agglutinin (DBA). To optimize gonocyte isolation, we found that ~9-fold more live cells could be harvested by enzymatic digestion of testis tissues than with mechanical methods. However, the proportion of gonocytes (~7%) did not differ between the mechanical and enzymatic methods of testis cell isolation. We then developed a novel three-step strategy for isolation of gonocytes by combining enzymatic digestion and vortexing, resulting in a gonocyte proportion of ~40% (~5-fold more than that from conventional methods). For short-term preservation of testis cells, we found that the survival of testis cells under hypothermic conditions was dependent on the cell type, and affected by storage duration, temperature and medium used. More than 80% of live testis cells survived the 6-day hypothermic preservation period in 20% FBS-L15, without visible changes to the cell culture potential or gonocyte proportion. In another experiment where testis tissues were maintained under hypothermic conditions, we found that ~25% of testis cells could survive for 6 days if preserved in HypoThermosol-FRS solution (HTS-FRS), without morphological changes. To purify gonocytes, we showed that centrifugation of testis cells using 17% Nycodenz can lead to precipitation of gonocytes in pellets (with a purity of > 80%). We also found that pre-coating tissue culture plates with both fibronectin and poly-D-lysine can result in the negative selection of gonocytes (with a purity of up to 85%). We subsequently showed that further purification of gonocytes (to > 90%) could be achieved by combining the two latter approaches. To prepare recipients for germ cell transplantation, we used local irradiation of piglet testes which reduced testis growth, decreased seminiferous tubule diameters and completely eliminated spermatogenesis at 4 months post-irradiation. Compared with the absence of endogenous spermatogenesis in the control testes, spermatogenesis up to elongating spermatids was observed in the irradiated testes after gonocyte transplantation. In summary, we investigated several critical elements in the study and manipulation of gonocytes in a large animal model.

No description available.

No description available.

Introdução: a alta prevalência do câncer e o aumento significativo da sobrevivência em longo prazo geraram interesse quanto à preservação da fertilidade em mulheres jovens expostas a quimioterapia e radioterapia. Neste sentido estudos de congelamento de tecido ovariano para posterior transplante, abriram uma nova perspectiva de aplicação no tratamento e prevenção da infertilidade feminina. Objetivos: comparar dois protocolos de congelamento de tecido ovariano, um lento não automatizado e um por vitrificação, com o intuito de avaliar a viabilidade dos tecidos para posterior transplante autólogo. Método: Foram utilizadas 30 camundongos fêmea CF1 com aproximadamente 8 semanas e pesando 29,29g±2,9. •Os ovários extraídos foram vitrificados ou congelados, mantidos em nitrogênio líquido por 30 dias e descongelados. Após o descongelamento, o ovário esquerdo foi destinado às análises histológicas e caracterização por imuno histoquímica para o marcador mouse vasa homologue (MVH) e o ovário direito foi utilizado para os testes de viabilidade celular com exclusão por azul de trypan. Resultados: Nas análises de Hematoxilina e Eosina (HE) foram contados folículos primordiais, primários, pré-antrais e antrais. Não houve diferença significativa na proporção de folículos primordiais, primários e pré-antrais após descongelamento entre os grupos testados. A contagem de folículos antrais foi significativamente maior no grupo de vitrificação (p = 0,004). No ensaio de imunohistoquímica para o marcador MVH, folículos MVH + e MVH- foram contados e comparados com o número total de folículos. O grupo congelamento lento apresentou maior número de células não marcadas (p = 0,012). Conclusão: Embora ambos os protocolos tenham apresentado resultados semelhantes na análise histológica das contagens foliculares, o protocolo de vitrificação foi significativamente melhor para preservar a população de células tronco ovarianas. / Introduction: The high prevalence of cancer and the significant increase in long-term survival have generated interest as the preservation of fertility in young women exposed to chemotherapy and radiotherapy. Experimental techniques have been tried in an attempt to reverse the ovarian failure induced by these treatments. In this regard studies of ovarian tissue freezing for subsequent transplantation disclose a new application perspective in the treatment and prevention of female infertility. Objective: two ovarian tissue freezing protocols were tested, a non-automated slow-freezing and by vitrification, in order to assess the viability of the tissues for subsequent autologous transplantation. Methods: as ovaries donors, were used 30 female CF1 mice approximately 8 weeks and weighing 29,29g±2,9. • The ovaries were vitrified or frozen, stored in liquid nitrogen for 30 days and thawed. After thawing, the left ovary was intended for histological and immunohistochemical characterization by histochemical marker for MVH and right ovary was used for the tests with cell viability by trypan blue exclusion. Results: In HE slides was counting primordial, primary, pre antral and antral follicles. No significant difference was found in the proportion of high-quality primordial, primary and pre antral follicles after thawing/warming in the slow-freezing and vitrification group, respectively. The antral follicle counting was significant higher in vitrification group (p=0,004). In immunohistochemistry assay for MVH Antibody , MVH+ and MVH- follicles were counted and compared with the total number of follicles and slow freeze group had a higher number of not marked cells (p=0,012). Conclusion: Although both protocols showed similar results in the histological analysis for follicular counts, the vitrification protocol was significantly better for preserve the ovarian stem cell population.

Introdução: a alta prevalência do câncer e o aumento significativo da sobrevivência em longo prazo geraram interesse quanto à preservação da fertilidade em mulheres jovens expostas a quimioterapia e radioterapia. Neste sentido estudos de congelamento de tecido ovariano para posterior transplante, abriram uma nova perspectiva de aplicação no tratamento e prevenção da infertilidade feminina. Objetivos: comparar dois protocolos de congelamento de tecido ovariano, um lento não automatizado e um por vitrificação, com o intuito de avaliar a viabilidade dos tecidos para posterior transplante autólogo. Método: Foram utilizadas 30 camundongos fêmea CF1 com aproximadamente 8 semanas e pesando 29,29g±2,9. •Os ovários extraídos foram vitrificados ou congelados, mantidos em nitrogênio líquido por 30 dias e descongelados. Após o descongelamento, o ovário esquerdo foi destinado às análises histológicas e caracterização por imuno histoquímica para o marcador mouse vasa homologue (MVH) e o ovário direito foi utilizado para os testes de viabilidade celular com exclusão por azul de trypan. Resultados: Nas análises de Hematoxilina e Eosina (HE) foram contados folículos primordiais, primários, pré-antrais e antrais. Não houve diferença significativa na proporção de folículos primordiais, primários e pré-antrais após descongelamento entre os grupos testados. A contagem de folículos antrais foi significativamente maior no grupo de vitrificação (p = 0,004). No ensaio de imunohistoquímica para o marcador MVH, folículos MVH + e MVH- foram contados e comparados com o número total de folículos. O grupo congelamento lento apresentou maior número de células não marcadas (p = 0,012). Conclusão: Embora ambos os protocolos tenham apresentado resultados semelhantes na análise histológica das contagens foliculares, o protocolo de vitrificação foi significativamente melhor para preservar a população de células tronco ovarianas. / Introduction: The high prevalence of cancer and the significant increase in long-term survival have generated interest as the preservation of fertility in young women exposed to chemotherapy and radiotherapy. Experimental techniques have been tried in an attempt to reverse the ovarian failure induced by these treatments. In this regard studies of ovarian tissue freezing for subsequent transplantation disclose a new application perspective in the treatment and prevention of female infertility. Objective: two ovarian tissue freezing protocols were tested, a non-automated slow-freezing and by vitrification, in order to assess the viability of the tissues for subsequent autologous transplantation. Methods: as ovaries donors, were used 30 female CF1 mice approximately 8 weeks and weighing 29,29g±2,9. • The ovaries were vitrified or frozen, stored in liquid nitrogen for 30 days and thawed. After thawing, the left ovary was intended for histological and immunohistochemical characterization by histochemical marker for MVH and right ovary was used for the tests with cell viability by trypan blue exclusion. Results: In HE slides was counting primordial, primary, pre antral and antral follicles. No significant difference was found in the proportion of high-quality primordial, primary and pre antral follicles after thawing/warming in the slow-freezing and vitrification group, respectively. The antral follicle counting was significant higher in vitrification group (p=0,004). In immunohistochemistry assay for MVH Antibody , MVH+ and MVH- follicles were counted and compared with the total number of follicles and slow freeze group had a higher number of not marked cells (p=0,012). Conclusion: Although both protocols showed similar results in the histological analysis for follicular counts, the vitrification protocol was significantly better for preserve the ovarian stem cell population.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21663号 / 医博第4469号 / 新制||医||1035(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 篠原 隆司, 教授 万代 昌紀, 教授 近藤 玄 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医科学) / 甲第20285号 / 医科博第76号 / 新制||医科||5(附属図書館) / 京都大学大学院医学研究科医科学専攻 / (主査)教授 篠原 隆司, 教授 浅野 雅秀, 教授 近藤 玄 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM