Our purpose was to determine the effects on mammary gland development in prepubertal heifers given the anti-estrogen tamoxifen. Sixteen Holstein calves were randomly assigned to one of two treatment groups: tamoxifen-injected (TAM) or control (CON). Calves were subcutaneously injected daily from 28 to 120 days of age with 0.3 mg/kg tamoxifen or carrier. At 120 days calves were euthanized and udders removed. Weight of trimmed parenchymal tissue (left rear quarter) was dramatically lower in TAM calves than in CON calves (p < 0.0003; 16.1 vs. 34.8 g). Parenchymal samples from three regions of the left rear quarter (lower, middle and outer regions) were processed for immunohistochemical staining for Estrogen Receptor α and Progesterone Receptor, myoepithelial cells, and label retaining cells. Overall, the proportion of neither ER nor PR labeled cells was impacted by TAM treatment. However, imaging analysis indicated a markedly higher intensity of ER expression in CON calves. TAM caused an increase in myoepithelial cell differentiation similar to what is seen in ovariectomy. We were able to effectively use a new technique of multispectral imaging to identify label retaining cells, which led to the discovery of an increase in the percentage of label retaining cells in TAM compared to CON. While treatment with the anti-estrogen tamoxifen reduced mammary parenchymal mass similarly to OVX, the mechanism(s) involved appear to differ. This suggests that the impacts of ovariectomy are only partially explained by the absence of estrogen. / Master of Science

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20235号 / 医博第4194号 / 新制||医||1019(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 別所 和久, 教授 鈴木 茂彦, 教授 渡邊 直樹 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Le thymus subit un vieillissement précoce, appelé involution thymique, qui cause une perte de fonction du thymus avec l’âge. À ce jour, les mécanismes de renouvellement des cellules épithéliales thymiques (TECs) sont encore mal compris, c’est pourquoi nous avons voulu identifier les cellules souches de l’épithélium thymique. Comme les cellules souches sont quiescentes dans plusieurs tissus, les objectifs de notre étude étaient de déterminer si l’épithélium thymique contenait des cellules quiescentes et d’étudier la cinétique de prolifération des TECs chez les souris jeunes et adultes. Pour ce faire, nous avons utilisé une souris transgénique (H2B-GFP Tet-On) nous permettant d’identifier les cellules ne se divisant pas sur une longue période de temps (LRC, label-retaining cells¬). Nous avons d’abord montré que les TECs proliféraient plus rapidement chez les femelles que les mâles. De plus, nous avons trouvé plusieurs différences entre l’épithélium thymique post-natal et adulte : (1) les TECs corticales (cTECs) et médullaires (mTECs) ont un taux de prolifération similaire chez les jeunes souris, mais chez l’adulte, les cTECs prolifèrent plus lentement que les mTECs; (2) les TECs prolifèrent plus rapidement chez les souris jeunes que adultes; (3) des LRC sont détectées chez l’adulte, mais pas chez les jeunes souris. Les LRC, retrouvées dans le compartiment cTEC, sous-expriment des gènes associés à la sénescence et surexpriment des gènes importants pour le développement et le renouvellement des TECs. Ces résultats montrent que ces cellules sont quiescentes et suggèrent qu’elles pourraient bel et bien être les progéniteurs thymiques responsables du renouvellement des TECs adultes. / The thymus undergoes a rapid degeneration with age termed thymic involution that causes a loss of function of the thymus with age. To this day, mechanisms of thymic maintenance are still unknown. This is why we aimed to identify thymic epithelial stem cells. Since stem cells are quiescent in many tissues in adults, our main objectives were to determine whether the thymic epithelium contains quiescent cells and study the proliferation kinetics of thymic epithelial cells in neonatal and adult mice. To this end, we used the transgenic mouse model H2B-GFP Tet-On, a label-retaining assay allowing us to identify cells that have not divided for a prolonged period of time, which are called label-retaining cells (LRC). First, we showed that in the adult thymus, females’ thymic epithelial cells (TECs) proliferated more actively than males’ TECs. We observed three main differences between neonatal and adult thymi: (1) cTECs and mTECs have similar proliferation rates in young, but mTECs cycled more actively in adult mice; (2) neonatal TECs have a higher turnover rate than adult’s TECs, and (3) we were able to detect LRC in adult mice, but not in neonatal mice. These LRC are contained in the cTEC compartment and express very low levels of senescence-associated proteins and show a high expression of genes important for thymic development and. These results show that the LRC identified in adult thymi are not senescent cells and therefore might represent the elusive thymic progenitor cells responsible for thymic maintenance and regeneration in adult mice.