A typical clinical cis-diamminedichloroplatinum (II) (cisplatin) dosing regimen consists of repeated cycles of five to seven daily low dose treatments followed by a one to two week recovery period. While effective, this dosing structure results in a prolonged, and sometimes permanent, infertility in men. Undifferentiated spermatogonia, including spermatogonial stem cells (SSCs), are theoretically capable of repopulating the seminiferous tubules after exposure has ceased. It is proposed that an altered spermatogonial environment during recovery from the initial treatment cycle may drive an increase in SSC mitotic cell activity, rendering the SSC pool increasingly susceptible to cisplatin-induced cell death from subsequent cycles. The undifferentiated spermatogonia population and niche of the adult mouse (C57/BL/6J) were examined during the recovery period of a clinically-relevant course of one and two cycles of 2.5 mg/kg/d of intraperitoneal cisplatin and were compared to mice receiving an equivalent cumulative dose in a single cycle (5.0 mg/kg/d) and vehicle treated controls. Histological examination of the testicular epithelium revealed an increase in the disorganization of spermatogenesis correlating with the number of exposure cycles. Quantification of TUNEL positive cells showed an increase in apoptotic germ cells early in the recovery period in mice exposed to cisplatin compared to control animals. Immunohistochemical (IHC) examination of Foxo1 (undifferentiated spermatogonia marker) showed an increase in the undifferentiated spermatogonia population late in the recovery period in mice exposed to one cycle of 2.5 mg/kg/d, but not following two cycles of 2.5 mg/kg/d. Analysis of BrdU incorporation after dosing indicated a decrease in mitotic activity of early germ cells immediately after cisplatin exposure followed by a return to basal levels by the conclusion of the initial recovery period. No such rebound was observed during the second recovery period. IHC investigation of glial cell line-derived neurotrophic factor (GDNF), a recognized SSC niche factor, revealed an increase in production along the basal Sertoli cell membrane throughout the recovery period in all treatment groups. Taken together, these data establish that the impact of cisplatin exposure on the functional stem cell pool and niche correlates with: (1) the number of dosing cycles; (2) mitotic activity of early germ cells; and (3) alterations in the basal Sertoli cell GDNF expression levels after cisplatin-induced testicular injury. / text

Les Chondrichtyens sont des espèces d’intérêt de par leur position phylogénétique à la base des Vertébrés. Au cours de cette thèse réalisée sur un petit requin, la petite roussette, l’étude des protéines Nanos, essentielles pour le maintien de la lignée germinale chez les Ostéichtyens, a mis en évidence la présence de deux protéines Nanos1 (1A et 1B) chez les Chondrichtyens, résultant d’une duplication du gène en amont des Gnathostomes. Chaque paralogue a révélé des profils d’expression spécifiques suggérant une spécification génique. De plus, la niche des spermatogonies souches (SSCs) a été mieux caractérisée et de nouveaux marqueurs de pluripotence tels que SSEA4 et Sox2 ont été détectés dans les SSCs potentielles chez cette espèce. Une culture primaire enrichie en spermatogonies de la zone germinative a montré une hétérogénéité d’expression des facteurs de SSC analysés (GFRα1, SSEA4, POU2, Nanos1A, Nanos1B, c-Kit), suggérant une hétérogénéité des cellules souches et/ou des progéniteurs. Afin de valider le caractère souche de ces cellules en culture, un test fonctionnel de transplantation a été initié. Le développement de cette technologie, pour la première fois chez un Chondrichtyen, ouvre de nouvelles perspectives en termes de préservation de ces espèces. Enfin, la recherche de facteurs à potentiel thérapeutique au niveau testiculaire chez la petite roussette a permis l’identification d’un peptide capable de réguler la glycémie et l’insulinémie de souris présentant un diabète de type 2, mais son mode d’action reste à explorer. L’ensemble des résultats confirme l’intérêt de la petite roussette pour l’étude évolutive de la niche germinale, essentielle au maintien de la gamétogenèse, et la recherche de molécules à potentiel thérapeutique. / Chondrichthyes are species of interest because of their phylogenetic position at the base of the Vertebrates. In this thesis, based on a small shark, the small spotted dogfish, the study of Nanos proteins, essential for the maintenance of the germ line in Osteichthyes, showed the presence of two Nanos1 proteins (1A and 1B) in Chondrichthyes, resulting from a gene duplication upstream of the Gnathostomata. Each paralog revealed specific expression profiles suggesting a gene specification. In addition, the spermatogonial stem cells (SSCs) niche was better characterized and new pluripotency markers such as SSEA4 and Sox2 were detected in potential SSCs in this species. A primary culture of the germinative zone enriched in spermatogonia showed a heterogeneity of expression of the analysed SSC factors (GFRα1, SSEA4, POU2, Nanos1A, Nanos1B, c-Kit), suggesting heterogeneity of stem cells and / or progenitors. In order to validate the stemness potential of these cells in culture, a functional transplantation test was initiated. The development of this technology, for the first time in a Chondrichthyes, opens new perspectives in terms of preservation of these species. Finally, the search of potential therapeutic factors in the dogfish testis led to the identification of a peptide able to regulate blood glucose and insulin levels in mice presenting type 2 diabetes, but its mode of action remains to be explored. All the results confirm the interest of the small spotted dogfish for the evolutionary study of the germinal niche, essential to the maintenance of gametogenesis, and the search for molecules with therapeutic potential.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23086号 / 医博第4713号 / 新制||医||1050(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 斎藤 通紀, 教授 藤田 恭之, 教授 近藤 玄 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Yes / The spermatogonial stem cells (SSCs) are the only germline stem cells in adults that are responsible for the transmission of genetic information from mammals to the next generation. SSCs play a very important role in the maintenance of progression of spermatogenesis and help provide an understanding of the reproductive biology of future gametes and a strategy for diagnosis and treatment of infertility and male reproductive toxicity. Androgens/oestrogens are very important for the suitable maintenance of male germ cells. There is also evidence confirming the damaging effects of oestrogen-like compounds on male reproductive health. We investigated the effects in vitro, of diethylstilbestrol (DES) on mouse spermatogonial stem cells separated using Staput unit-gravity velocity sedimentation, evaluating any DNA damage using the Comet assay and apoptotic cells in the TUNEL assay. Immunocytochemistry assays showed that the purity of isolated mouse spermatogonial cells was 90%, and the viability of these isolated cells was over 96%. Intracellular superoxide anion production (O2−) in SSCs was detected using p-Nitro Blue Tetrazolium (NBT) assay. The viability of cells after DES treatment was examined in the CCK8 (cell counting kit-8) cytotoxicity assay. The results showed that DES-induced DNA damage causes an increase in intracellular superoxide anions which are reduced by the flavonoid, quercetin. Investigating the molecular mechanisms and biology of SSCs provides a better understanding of spermatogonial stem cell regulation in the testis.

A espermatogênese em mamíferos é um processo sustentado pela auto-renovação e diferenciação de células-tronco espermatogoniais (SSCs). O estudo destas células oferece um excelente modelo para o melhor entendimento da biologia das células-tronco adultas e dos mecanismos que controlam as funções das SSCs. Além do potencial biomédico para estudos sobre infertilidade em diferentes espécies, as SSC possuem uma aplicação promissora na biotecnologia para a produção de animais transgênicos. Assim, o objetivo deste trabalho foi responder à pergunta: "SSCs bovinas LacZ+ podem integrar-se aos túbulos seminíferos de bezerros pré-púberes da raça Nelore após transplante autólogo?" Para isso, bezerros Nelore de 5 meses de idade (n=16) foram submetidos a uma orquiectomia unilateral para o isolamento de células espermatogoniais por digestão enzimática. Após o plaqueamento diferencial, as células foram transduzidas com um vetor lentiviral contendo a sequencia do gene marcador LacZ. Para isso, os animais foram aleatoriamente alocados em um dos quatro grupos experimentais: LacZ+/PKH26+, LacZ+/PKH26-, LacZ-/PKH26+, LacZ-/PKH26-. Após 60 h do início do cultivo in vitro, as células espermatogoniais foram transplantadas autologamente para o mediastino do testículo remanescente por injeção guiada por ultrassonografia. O testículo transplantado foi removido cirurgicamente após 45 dias e amostras de tecido foram submetidas a reação com x-gal para verificação da integração de células espermatogoniais transgênicas aos túbulos seminíferos. Células espermatogoniais foram isoladas e cultivadas in vitro com sucesso. Contudo, não foi possível obter uma população pura de SSCs por plaqueamento diferencial. Embora tenha sido eleito o transplante de células espermatogoniais e não de SSCs somente, sabe-se que também foram transplantadas SSCs, pois a caracterização das células isoladas demonstrou a expressão dos marcadores de SSCs ITGA6, GFRa-1, PGP 9.5 e afinidade pela lectina DBA. Crioseções de amostras de tecido testicular coradas com x-gal permitiram a observação de células transgênicas em 8 de 8 animais que receberam células LacZ+. Contudo, todas as células transgênicas observadas estavam situadas no interstício. Concluindo, não foi possível observar a integração das células transgênicas transplantadas aos túbulos seminíferos do testículo receptor após 45 dias do transplante autólogo utilizando a técnica de injeção intratesticular de células espermatogoniais LacZ+ no mediastino de bezerros pré-púberes da raça Nelore. / Mammalian spermatogenesis is sustained by self renewal and differentiation of spermatogonial stem cells (SSCs). The study of these cells provides a model to better understand adult stem cell biology and the mechanisms that control SSC functions. Besides the biomedical potential to perform studies of infertility in many species, SSCs hold a promising biotechnological application at animal transgenesis. In this manner, the goal of this study was to answer the question: "Can LacZ+ bovine SSCs be integrated into seminiferous tubule of prepubertal Nelore bulls subjected to autologous transplantation?" Hence, 5 months old bulls (n=16) were hemicastrated and spermatogonial cells were isolated by a two step enzymatic digestion procedure. After differential plating, cells were transduced with a lentivirus vector carrying the LacZ reporter gene sequence. Animals were randomly allocated in four experimental groups: LacZ+/PKH26+, LacZ+/PKH26-, LacZ-/PKH26+, LacZ-/PKH26-. After 60 h of the onset of in vitro culture, spermatogonial cells were autologously transplanted to the remaining testes by an ultrasound guided needle injection at the testis mediastinum. The transplanted testes were surgically removed after 45 days and testicular tissue samples were subjected to x-gal staining to assess the integration of transgenic spermatogonial cells to seminiferous tubule. Spermatogonial cells were successfully isolated and in vitro cultured. However, it was not possible to obtain a SSC enriched population of cells by differential plating. Although it was decided by the transplant of spermatogonial cells instead of pure SSCs only, it was detected the expression of SSC marker genes ITGA6, PGP9.5, GFR-1 and the affinity for DBA by the isolated cells. Cryosections of x-gal stained testicular tissue samples allowed the observation of transgenic cells in 8 out of 8 animals that received LacZ+ cells. However, all transgenic cells observed were located at the interstitial space. In conclusion, it was not possible to observe the integration of the transplanted transgenic cells into seminiferous tubule of prepubertal Nelore bulls subjected to autologous transplantation using an ultrasound guided needle injection at the testis mediastinum, after 45 days of transplant.

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.

A espermatogênese em mamíferos é um processo sustentado pela auto-renovação e diferenciação de células-tronco espermatogoniais (SSCs). O estudo destas células oferece um excelente modelo para o melhor entendimento da biologia das células-tronco adultas e dos mecanismos que controlam as funções das SSCs. Além do potencial biomédico para estudos sobre infertilidade em diferentes espécies, as SSC possuem uma aplicação promissora na biotecnologia para a produção de animais transgênicos. Assim, o objetivo deste trabalho foi responder à pergunta: "SSCs bovinas LacZ+ podem integrar-se aos túbulos seminíferos de bezerros pré-púberes da raça Nelore após transplante autólogo?" Para isso, bezerros Nelore de 5 meses de idade (n=16) foram submetidos a uma orquiectomia unilateral para o isolamento de células espermatogoniais por digestão enzimática. Após o plaqueamento diferencial, as células foram transduzidas com um vetor lentiviral contendo a sequencia do gene marcador LacZ. Para isso, os animais foram aleatoriamente alocados em um dos quatro grupos experimentais: LacZ+/PKH26+, LacZ+/PKH26-, LacZ-/PKH26+, LacZ-/PKH26-. Após 60 h do início do cultivo in vitro, as células espermatogoniais foram transplantadas autologamente para o mediastino do testículo remanescente por injeção guiada por ultrassonografia. O testículo transplantado foi removido cirurgicamente após 45 dias e amostras de tecido foram submetidas a reação com x-gal para verificação da integração de células espermatogoniais transgênicas aos túbulos seminíferos. Células espermatogoniais foram isoladas e cultivadas in vitro com sucesso. Contudo, não foi possível obter uma população pura de SSCs por plaqueamento diferencial. Embora tenha sido eleito o transplante de células espermatogoniais e não de SSCs somente, sabe-se que também foram transplantadas SSCs, pois a caracterização das células isoladas demonstrou a expressão dos marcadores de SSCs ITGA6, GFRa-1, PGP 9.5 e afinidade pela lectina DBA. Crioseções de amostras de tecido testicular coradas com x-gal permitiram a observação de células transgênicas em 8 de 8 animais que receberam células LacZ+. Contudo, todas as células transgênicas observadas estavam situadas no interstício. Concluindo, não foi possível observar a integração das células transgênicas transplantadas aos túbulos seminíferos do testículo receptor após 45 dias do transplante autólogo utilizando a técnica de injeção intratesticular de células espermatogoniais LacZ+ no mediastino de bezerros pré-púberes da raça Nelore. / Mammalian spermatogenesis is sustained by self renewal and differentiation of spermatogonial stem cells (SSCs). The study of these cells provides a model to better understand adult stem cell biology and the mechanisms that control SSC functions. Besides the biomedical potential to perform studies of infertility in many species, SSCs hold a promising biotechnological application at animal transgenesis. In this manner, the goal of this study was to answer the question: "Can LacZ+ bovine SSCs be integrated into seminiferous tubule of prepubertal Nelore bulls subjected to autologous transplantation?" Hence, 5 months old bulls (n=16) were hemicastrated and spermatogonial cells were isolated by a two step enzymatic digestion procedure. After differential plating, cells were transduced with a lentivirus vector carrying the LacZ reporter gene sequence. Animals were randomly allocated in four experimental groups: LacZ+/PKH26+, LacZ+/PKH26-, LacZ-/PKH26+, LacZ-/PKH26-. After 60 h of the onset of in vitro culture, spermatogonial cells were autologously transplanted to the remaining testes by an ultrasound guided needle injection at the testis mediastinum. The transplanted testes were surgically removed after 45 days and testicular tissue samples were subjected to x-gal staining to assess the integration of transgenic spermatogonial cells to seminiferous tubule. Spermatogonial cells were successfully isolated and in vitro cultured. However, it was not possible to obtain a SSC enriched population of cells by differential plating. Although it was decided by the transplant of spermatogonial cells instead of pure SSCs only, it was detected the expression of SSC marker genes ITGA6, PGP9.5, GFR-1 and the affinity for DBA by the isolated cells. Cryosections of x-gal stained testicular tissue samples allowed the observation of transgenic cells in 8 out of 8 animals that received LacZ+ cells. However, all transgenic cells observed were located at the interstitial space. In conclusion, it was not possible to observe the integration of the transplanted transgenic cells into seminiferous tubule of prepubertal Nelore bulls subjected to autologous transplantation using an ultrasound guided needle injection at the testis mediastinum, after 45 days of transplant.

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

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第21166号 / 農博第2292号 / 新制||農||1061(附属図書館) / 学位論文||H30||N5140(農学部図書室) / 京都大学大学院農学研究科応用生物科学専攻 / (主査)教授 今井 裕, 教授 久米 新一, 准教授 南 直治郎 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM