Sutovsky P, Ramalho-Santos J, Moreno R D, Oko R, Hewitson L, Schatten G
Departments of Obstetrics and Gynecology, and Cell and Developmental Biology, Oregon Health Sciences University, and the Oregon Regional Primate Research Center, Beaverton, OR 97006, USA.
Hum Reprod. 1999 Sep;14(9):2301-12. doi: 10.1093/humrep/14.9.2301.
The selection of individual round spermatids for round spermatid injection (ROSI), a prerequisite for the successful application of this infertility treatment, has been hampered by the ambiguous definition of a round spermatid and the lack of specific vital and non-vital markers. Using cells from rhesus monkey and bull, we describe a non-invasive method for the on-stage selection of individual round spermatids for ROSI, based on the polarized patterns of mitochondria, visualized in live round spermatid cells by epifluorescence microscopy after incubation with MitoTracker(TM), a vital, mitochondrion-specific fluorescent probe. The correct identification of live round spermatid was confirmed by the presence of the acrosomal granule or acrosomal cap in parallel observations by Nomarski differential interference contrast microscopy. The existence of mitochondrial polarization was first established by the labelling of MitoTracker-tagged round spermatids with spermatid-specific antibodies against proteins of nascent sperm accessory structures combined with antibodies against a nuclear pore complex component, known to disappear at the round spermatid stage. Using an inverted microscope equipped with epifluorescence, the round spermatids can be individually selected from a heterogeneous population of testicular cells labelled with MitoTracker dyes. A major advantage of this approach is that the dyes are incorporated into the paternal mitochondria, destined for rapid elimination after fertilization. In addition, the relatively high excitation and emission wavelengths of MitoTracker dyes are less harmful to DNA after their photon excitation. Before the appropriate clinical testing is conducted, the MitoTracker-based round spermatid selection may be instrumental in the training of clinical staff.
单精子卵胞浆内注射（ROSI）中单个圆形精子细胞的选择是这种不育治疗成功应用的前提条件，但圆形精子细胞定义模糊以及缺乏特异性的活细胞和死细胞标记物阻碍了这一过程。我们利用恒河猴和公牛的细胞，描述了一种用于ROSI的单个圆形精子细胞在位选择的非侵入性方法，该方法基于线粒体的极化模式，在用线粒体特异性活性荧光探针MitoTracker(TM)孵育后，通过落射荧光显微镜在活的圆形精子细胞中观察到这种模式。通过诺马斯基微分干涉相差显微镜平行观察到顶体颗粒或顶体帽的存在，从而证实了活圆形精子细胞的正确识别。线粒体极化的存在首先通过用针对新生精子附属结构蛋白的精子细胞特异性抗体标记MitoTracker标记的圆形精子细胞，并结合针对已知在圆形精子细胞阶段消失的核孔复合体成分的抗体来确定。使用配备落射荧光的倒置显微镜，可以从用MitoTracker染料标记的异质睾丸细胞群体中单独选择圆形精子细胞。这种方法的一个主要优点是，这些染料被整合到父本线粒体中，受精后会迅速消除。此外，MitoTracker染料相对较高的激发和发射波长在光子激发后对DNA的危害较小。在进行适当的临床试验之前，基于MitoTracker的圆形精子细胞选择可能有助于临床工作人员的培训。
