Johns Hopkins University老師趙玉蓮博士合影
我的海報2研究主題
生殖保存是一項有前瞻性的醫學應用，特別是年輕的癌症患者及瀕臨絕種的生物，若能適當的保存其性腺，生殖能力是可以延續的。玻璃化的冷凍為冷醫學最有前途的研究。異體性腺移植能恢復性腺衰竭後的功能，移植胚胎細胞至同種成熟生物體的對應器官將保留最大的分化及發育潛能，尤其降低免疫排斥性與致畸率。新生精子在異體卵巢移植睪丸後產生精子將對精子生成利於研究及臨床應用。本計劃以基因轉殖鼠冷光活體追蹤生殖保存與延續的動物模式。
1. 異體卵巢移植恢復卵巢衰竭的功能，但缺乏免疫排斥性的研究，基因轉殖鼠FVB/N-Tg(PolII-luc)Ltc帶有冷光基因片段運用在性腺的移植做為捐贈者，移植到基因相同的 FVB/N 野生型鼠或基因不同的Balb/c 野生型鼠, 基因不同且免疫缺乏的NOD-SCID鼠及應用抗排斥藥物於Balb/c 野生型的個體，當排斥發生，移植物會定性與定量的表現冷光的強弱及結合傳統光學染色及荷爾蒙的分析，血中及組織CD4/CD8的表現。
2. 玻璃化的冷凍其高濃度的冷凍保護劑較具生物毒性，我們也運用生物冷光在傳統的光學濾泡觀察及傳統光學染色之外於基因轉殖鼠的性腺以玻璃化的冷凍(vitrification)於後於體外培養盤定性與定量快速測量移植前存活的濾泡或性腺組織，以及測試冷凍保護劑及方式及濃度不同的生物毒性的前驅實驗，進而觀察移植。
我的海報1研究主題
具有功能之雄性生殖細胞的生產是依賴於生殖幹細胞(精原幹細胞, Spermatogonial stem cells-SSC)的連續活性，在雄性出生後，其精原幹細胞能自體更新且生產大量已分化的生殖細胞，此已分化的生殖細胞會形成精子並將遺傳訊息傳衍至下一代。移植分析系統可用於明確地識別雄性的生殖細胞，並允許此生殖細胞進行體外培養、冷凍保存及基因修飾。此外，移植分析系統能確認有關幹細胞自體更新的環境與因素、精子形成作用與精子的產生。這些關於生殖幹細胞的知識有很大的實用價值，例如，能以冷凍保存生殖幹細胞的方式保護癌症治療後的男孩之生殖系。本研究之目的在於應用移植分析系統使精原幹細胞能行體外培養、冷凍保存並恢復受損的受孕力。
一：在成熟小鼠的睪丸中，SSCs的濃度為每三千至四千顆細胞中才僅有一顆SSC，且幹細胞上並沒有形態或生化上的標識存在。我們將應用小鼠的SSCs表面抗原(MHC class I- Thy-1lo/+ c-Kit- αv-integrin-/dim α6-integrin+)以提高取得SSCs的數目。
二：成功的SSCs冷凍保存與培養對移植SSCs保存生殖力來說是個重要的因素。我們將評估使用多樣的冷凍保存與培養技術對SSCs的存活與功能之影響，以找到理想的SSCs冷凍保存與培養方式。
三：我們將從具有生殖能力的成熟小鼠或初生小鼠上取得SSCs，隨後移植至無生殖能力之成熟小鼠的曲細精管中，並評估上述兩種小鼠所提供的SSCs對於重建精子形成作用與修復生殖力之影響。這項移植分析系統可被使用來研究SSCs的生物學，並且能使支持小鼠SSCs的體外自體更新與增生之外部因子被確認。
Lobby of Orange Convention Center, Orlando, FL
這次美國生殖學會年會陳啟煌發表之兩作品 (Oct.15-19, 2011)於ORANGE COUNTY CONVENTION CENTER ORLANDO, FLORIDA
[P-330] TRACKING THE DEVELOPMENT POTENTIAL AND THE ONSET INHIBITION OF PRIMORDIAL FOLLICLE OF THE CRYOPRESERVED MURINE OVARY WITH BIOLUMINESCENT IMAGING (BLI) IN VIVO.
C.-H. Chen, C.-R. Tzeng, C.-W. Wang, M.-I. Hsu Center for Reproductive Medicine & Sciences, Taipie Medical University Hospital, Taipei, Taiwan; Department of Obstetrics & Gynecology, Wan Fang Hospital, Taipei Medical University, Taipei, Taiwan
OBJECTIVE: The effect of cryopreservation of ovarian tissue on the development of primordial follicles remains to be elucidated.
DESIGN: Transgenic mouse study.
MATERIALS AND METHODS: The ovarian tissues of donor FVB/N-Tg(PolII–Luc)Ltc transgenic mice (n=15), five-week-old, with a luciferase transgene as the reporter, were transplanted into inbred strain of age-matched FVB/NJNarl wild-type mice as recipient (n=15). Before transplantation, donor ovaries were slowly cryopreserved using a modification of the method of Gosden. All the recipient underwent oophorectomy. Fresh grafts were used as control group. In vivo BLI which expedites the fast-throughput screening of grafts, in a longitudinal study to monitor transplanted ovarian grafts every other day until 10 day after transplantation using LIVINGIMAGE v2.50 software (Xenogen). Genes for GDF-9, p53, AMH and GAPDH were quantified by real-time PCR. Ovarian tissue were analyzed by histology. The student's test was used for statistical analysis and P<0.05 was considered significant.
RESULTS: Follicle count in experimental group revealed significant lower than that of fresh group (p=0.006, p=0.003, p=0.012 and p=0.008, respectively. The trend of GDF-9, p53 and AMH by time course revealed significant higher in fresh group than experimental group by time course. The in vivo BLI also demonstrated poor ovarian reserve with fluctuate estrous cycle in cryopreserved ovary in contrast to fresh control after 10-day follow-up.
CONCLUSION: This study suggests that ovarian cryopreservation using slow-freezing inhibits early development of primordial follicles and long term compromised ovarian reserve, which were caused by follicle loss and cryoinjury of ovarian stroma. This study also delivers the promising tool of luciferase systems widely used in the field of genetic engineering as reporter genes. Luciferase systems have also been harnessed for reproductive biomedical research using bioluminescence imaging.
Supported by: NSC99-2314-B-016-013-MY3, Taiwan, ROC
Wednesday, October 19, 2011 7:00 AM
Poster Session II: Fertility Preservation
[P-333] BIOLUMINESCENCE IMAGING (BLI) AS A TOOL TO EVALUATE GERM CELL IN VITRO AND TRANSPLANTATION IN VIVO AS FERTILITY PRESERVATION OF PREPUBERTAL MALE MICE.
C.-H. Chen, C.-R. Tzeng, C.-W. Wang, M.-I. Hsu Center for Reproductive Medicine & Sciences, Taipei Medical University Hospital, Taipei, Taiwan; Department of Obstetrics & Gynecology, Wan Fang Hospital, Taipei Medical University, Taipei, Taiwan
OBJECTIVE: Fertility preservation for infertile male mice is feasible after spermatogonial germinal cells transplantation. BLI was used as a tool to evaluate the efficiency of germ cell transplantation in vitro and in vivo.
DESIGN: Transgenic mouse model.
MATERIALS AND METHODS: A. Donor cell preparation
Cells for transplantation are obtained from FVB/N-Tg (PolII-luc) Ltc transgenic mouse testes of mice 3-week-old after birth by a two-step enzymatic digestion protocol.
B. Transplantation of SCC into recipient mouse
Donor cells are harvested from the testes of fertile donor mice that express a reporter transgene, and a single cell suspension of the cells is microinjected into seminiferous tubules of 8 weeks FVB/NJNarl wild type recipient infertile adult mice after busulfan-induced testis failure.
C. In Vitro BLI
Isolated germinal cells were suspended in Eppendorf tubes, followed by adding d-luciferin in excess (0.15 mg/ml). Bioluminescence was imaged using the IVIS 50 imaging system.
D. In Vivo BLI
BLI was obtained accordingly. 3-day-old newborn mice born from wild-type recipient-mated wild-type female mice were injected with luciferin (150 mg/kg IP) 10 min before imaging. Luminescence was quantified using Living Image software.
D. Fertility assay
Mating the recipient male to a wild-type female, then progeny is produced by nature fertility 4-5 month after FVB/N-Tg (PolII-luc) SCC transplantation.
RESULTS: Wild-type female mice were mated with the wild-type infertile male recipients 4–5 months after germinal cell transplantation. Live FVB/N-Tg (PolII-luc) Ltc transgenic pups were born with a mean of 11 ± 0.7 pups per litter.
CONCLUSION: Spermatogonial transplantation could be used to restore male fertility preservation. This study demonstrates this small transgenic animal model and technique. BLI at a quantity and quality basis is a useful tool to evaluate germ cell in vitro and transplantation in vivo to demonstrate the efficiency and success of transplantation.
Supported by: NSC 99-2314-B-038-033-MY3, Taiwan, ROC
Wednesday, October 19, 2011 7:00 AM
Poster Session II: Fertility Preservation
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