Sexual Medicine and Andrology Unit, Department of Experimental, Clinical and Biomedical Sciences, Center of Excellence DeNothe, University of Florence, Florence, Italy.
Andrology. 2014 May;2(3):394-401. doi: 10.1111/j.2047-2927.2014.00208.x. Epub 2014 Apr 3.
Human semen is a complex biological matrix. It contains mature spermatozoa, immature germ cells, residual apoptotic bodies and, in some cases, epithelial cells and leucocytes. Hence, one of the challenges in applying flow cytometry in spermatology is the correct recognition of spermatozoa and their separation from signals of other semen cells/elements. In this study, we show that semen spermatozoa are included in a well-defined, flame-shaped FSC/SSC region (FR), by demonstrating that the count of the spermatozoa contained in such region overlaps that obtained by microscopy in the same samples. In FR, nuclear staining of semen samples reveals three different populations: unstained, brighter and dimmer. Unstained elements were previously characterized as apoptotic bodies of testis origin and the brighter elements represent the majority of semen spermatozoa, whereas the composition and the origin of the population with a lower nuclear staining is less clear, albeit we have previously shown that all the elements constituting it are positive for TUNEL. In this study, we sorted all the elements contained in FR region and demonstrated that the dimmer elements are spermatozoa. To further characterize dimmer spermatozoa, we evaluated apoptotic caspases and chromatin immaturity, the latter detected by aniline blue (AB) and chromomycin A (CMA3) staining. We found that caspases were much more expressed in the dimmer spermatozoa (71.4 ± 18.8%) than in the brighter (46.7 ± 15.1%), whereas similar amounts of spermatozoa with chromatin immaturity were found in both populations (brighter, AB: 48.2 ± 19.5%; CMA3: 48.5 ± 20.4% and dimmer, AB: 43.4 ± 19.8%; CMA3: 36.1 ± 18.0%). Hence, the role of apoptosis in generating dimmer spermatozoa and their DNA fragmentation appears clear, whereas the involvement of defects during the chromatin packaging remains elusive.
人类精液是一种复杂的生物基质。它包含成熟的精子、未成熟的生殖细胞、残留的凋亡小体，在某些情况下，还包含上皮细胞和白细胞。因此，在应用流式细胞术于精子研究中面临的挑战之一，是正确识别精子并将其与其他精液细胞/成分的信号区分开来。在本研究中，我们通过证明包含在该区域中的精子计数与在相同样本中通过显微镜获得的计数重叠，表明精液精子包含在一个定义明确的火焰状 FSC/SSC 区域（FR）中。在 FR 中，对精液样本进行核染色显示出三个不同的群体：未染色、更亮和更暗。未染色的元素先前被表征为睾丸来源的凋亡小体，而更亮的元素代表了大多数精液精子，而核染色较低的群体的组成和来源则不太清楚，尽管我们之前已经表明，构成该群体的所有元素均对 TUNEL 呈阳性。在本研究中，我们对 FR 区域中包含的所有元素进行了分选，并证明更暗的元素是精子。为了进一步表征更暗的精子，我们评估了凋亡半胱天冬酶和染色质不成熟，后者通过吖啶蓝（AB）和色霉素 A3（CMA3）染色检测。我们发现，更暗的精子中半胱天冬酶的表达要高得多（71.4 ± 18.8%），而在更亮的精子中（46.7 ± 15.1%）则要低得多，而在两个群体中都发现了具有染色质不成熟的相似数量的精子（更亮的，AB：48.2 ± 19.5%；CMA3：48.5 ± 20.4%和更暗的，AB：43.4 ± 19.8%；CMA3：36.1 ± 18.0%）。因此，凋亡在产生更暗的精子及其 DNA 碎片化中的作用是明确的，而在染色质包装过程中出现缺陷的参与仍然难以捉摸。
