Confocal fluorescence microscopy in a murine model of microdissection testicular sperm extraction to improve sperm retrieval.
Confocal fluorescence microscopy in a murine model of microdissection testicular sperm extraction to improve sperm retrieval.
复制标题
共聚焦荧光显微镜在小鼠睾丸精子提取显微解剖模型中的应用，以改善精子检索。
DOI：
10.1016/j.juro.2011.12.053
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发表时间：
2012
期刊：
影响因子：
--
通讯作者：
Lysiak,JeffreyJ
中科院分区：
文献类型：
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作者：
Smith,RyanP;Lowe,GregJ;Kavoussi,ParvizK;Steers,WilliamD;Costabile,RaymondA;Herr,JohnC;Shetty,Jagathpala;Lysiak,JeffreyJ
目的显微解剖睾丸精子提取显着提高非梗阻性无精症男性的精子回收率。然而，定位精子焦点可能非常耗时，而且并不总是成功。光纤共焦荧光显微镜具有快速体内检测生精小管中荧光标记精子的优点。 材料和方法在建立光纤共焦荧光显微镜体内识别抗体标记精子的可行性后，C57/B6小鼠腹腔注射白消安诱导无精子症。在大约16周的精子发生重建过程中，将小鼠麻醉并通过低中线切口递送睾丸。将异硫氰酸荧光素标记的顶体内蛋白 Hs-14 抗体逆行注射到单个小鼠睾丸网中。使用光纤共焦荧光显微镜对睾丸进行体内成像并检测精子灶。切除各自的生精小管并准备挤压用于免疫荧光显微镜检查。结果通过体内光纤共聚焦荧光显微镜鉴定注射荧光标记抗体的睾丸中的精子灶。每只小鼠的对侧对照睾丸未显示特异性信号。切除的小管的免疫荧光显微镜提供了标记精子存在的形态学证实，而对照中不存在。研究的可行性部分和非梗阻性无精子症的白消安模型中的结果是一致的。结论在无精子小鼠模型中显微切割睾丸精子提取过程中，光纤共聚焦荧光显微镜在体内识别荧光标记的精子是可行的。转化为临床环境可以减少手术时间并提高精子收获率。
PurposeMicrodissection testicular sperm extraction markedly improves the sperm retrieval rates in men with nonobstructive azoospermia. However, localizing sperm foci can be time-consuming and it is not always successful. Fiberoptic confocal fluorescent microscopy offers the advantage of rapid in vivo detection of fluorescently labeled sperm in the seminiferous tubules.Materials and MethodsAfter establishing the feasibility of fiberoptic confocal fluorescent microscopy to identify antibody labeled sperm in vivo C57/B6 mice underwent intraperitoneal injection of busulfan to induce azoospermia. During spermatogenesis reestablishment at approximately 16 weeks the mice were anesthetized and the testes were delivered through a low midline incision. Fluorescein isothiocyanate labeled antibody to intra-acrosomal protein Hs-14 was injected retrograde into a single murine rete testis. The testes were imaged in vivo with fiberoptic confocal fluorescent microscopy and sperm foci were detected. The respective seminiferous tubules were excised and squash prepared for immunofluorescence microscopy.ResultsSperm foci were identified in the testis injected with fluorescently tagged antibody by in vivo fiberoptic confocal fluorescence microscopy. The contralateral control testis of each mouse showed no specific signal. Immunofluorescence microscopy of the excised tubules provided morphological confirmation of the presence of labeled sperm with an absence in controls. Findings were consistent in the feasibility portion of the study and in the busulfan model of nonobstructive azoospermia.ConclusionsFiberoptic confocal fluorescent microscopy was feasible during microdissection testicular sperm extraction in an azoospermic mouse model to identify fluorescently labeled sperm in vivo. Translation to the clinical setting could decrease operative time and improve the sperm harvest rate.