Ohta Hiroshi, Sakaide Yuko, Wakayama Teruhiko
Laboratory for Genomic Reprogramming, Center for Developmental Biology, RIKEN, Chuo-ku, Kobe 650-0047, Japan.
Hum Reprod. 2007 Jun;22(6):1612-6. doi: 10.1093/humrep/dem064. Epub 2007 Apr 11.
Although ICSI is a useful technique, low elongated spermatid numbers frequently causes technical difficulties, especially in the case of azoospermic patients. Enrichment of elongated spermatids from the testis prior to ICSI may solve this problem.
To determine whether elongated spermatids had a characteristic phenotype suitable for purification, testicular cells prepared from 25-day-old mice (from spermatogonia to round spermatids) and adult mice (from spermatogonia to elongated spermatids) were compared by flow cytometry. After flow-cytometric cell sorting (FCS) based on their side (SSC) and forward scatter (FSC), purity of the elongated spermatids in the fractionated population was microscopically examined, and functional ability of purified elongated spermatids was assessed by ICSI.
Elongated spermatids in testicular cells showed characteristic SSC and FSC phenotypes. In the purified population, approximately 70-80% of the cells were morphologically determined as elongated spermatids, in contrast to only 10% before sorting. Using ICSI, purified elongated spermatids supported full-term development similar to that of unsorted elongated spermatids. Furthermore, we succeeded in enriching the elongated spermatids from the infertile testis model by approximately 10-fold.
Elongated spermatids with normal developmental ability can be efficiently purified by FCS based on SSC and FSC characteristics.
尽管卵胞浆内单精子注射（ICSI）是一项有用的技术，但长形精子细胞数量少常常导致技术难题，尤其是在无精子症患者中。在ICSI之前从睾丸中富集长形精子细胞可能解决这一问题。
为了确定长形精子细胞是否具有适合纯化的特征性表型，通过流式细胞术比较了从25日龄小鼠（从精原细胞到圆形精子细胞）和成年小鼠（从精原细胞到长形精子细胞）制备的睾丸细胞。基于侧向散射（SSC）和前向散射（FSC）进行流式细胞分选（FCS）后，显微镜检查分选群体中长形精子细胞的纯度，并通过ICSI评估纯化的长形精子细胞的功能能力。
睾丸细胞中的长形精子细胞表现出特征性的SSC和FSC表型。在纯化群体中，约70%-80%的细胞在形态上被确定为长形精子细胞，相比之下分选前仅为10%。使用ICSI，纯化的长形精子细胞支持与未分选的长形精子细胞相似的足月发育。此外，我们成功地将不育睾丸模型中的长形精子细胞富集了约10倍。
具有正常发育能力的长形精子细胞可基于SSC和FSC特征通过FCS有效纯化。
