Department of Animal Nutrition and Feed Science, College of Animal Science and Technology, Huazhong Agricultural University, Wuhan, 430070, China.
State Key Laboratory of Agricultural Microbiology, Department of Chemistry, College of Science, Huazhong Agricultural University, Wuhan, 430070, China.
Anal Chim Acta. 2023 May 15;1255:341054. doi: 10.1016/j.aca.2023.341054. Epub 2023 Mar 7.
Mineral elements play an important role in the spermatogenesis, maturation, and fertilization of sperm. It is of great scientific significance to study the role of mineral elements in spermatogenesis by accurately measuring the content of elements in different spermatogenic cells and analyzing the distribution pattern of elements in spermatogenesis. Here, time-resolved inductively coupled plasma mass spectrometry (ICP-MS) was used to analyze the content and distribution patterns of mineral elements in spermatogenic cells of different types at the single cell level. Firstly, spermatogonia, spermatocytes, round spermatids and elongating spermatids were successfully isolated from testis of mice of different weeks of age by differential adherent method and discontinuous bovine serum albumin (BSA) density gradient method. Then, signal profiles and elemental distributions of Mg, P, Cr, Mn, Fe and Zn in spermatogenic cells were obtained with dwell time at 0.1 ms. Based on the results of acid digestion, we derived a formula to calculate element content in single cell from peak area for each element, and the feasibility and universality of the formula in the quantitative detection of single cell elements were verified by sperm samples to a certain extent. The detection results of element content in single cell showed that the content of P in elongating spermatids was significantly higher than that in spermatogonia, spermatocytes and round spermatids (P < 0.01), and the distribution range was wider. However, the Cr and Fe content of elongating spermatids was lower than that of spermatogonia, spermatocytes and round spermatids (P < 0.05). When spermatogonia developed into round spermatids, the contents of Mn and Zn in single cell increased significantly (P < 0.05), then decreased to the lowest in elongating spermatids. In addition, the significant decrease of Cr, Mn, Fe and Zn content in elongating spermatids also be visually observed from the center of the fitting curve of the element signal intensity distribution moving to the left. This study provides an elemental view of the changes in elemental content at various stages of spermatogenesis at the single-cell level. Time-resolved ICP-MS is used to detect mineral elements content and distribution patterns in spermatogenic cells of testis, which is helpful to better explore the stages and modes of action of various elements in spermatogenesis, and provide direct evidence for revealing the effects of element content changes on spermatogenesis and semen quality regulation.
矿物质元素在精子发生、成熟和受精过程中发挥着重要作用。通过准确测量不同生精细胞中元素的含量，并分析元素在精子发生过程中的分布模式，研究矿物质元素在精子发生中的作用具有重要的科学意义。本研究采用时间分辨电感耦合等离子体质谱法（ICP-MS），在单细胞水平上分析了不同类型生精细胞中矿物质元素的含量和分布模式。首先，通过差速贴壁法和不连续牛血清白蛋白（BSA）密度梯度法，从不同周龄小鼠睾丸中成功分离出精原细胞、精母细胞、圆形精子细胞和伸长精子细胞。然后，采用 dwell time 为 0.1ms 的方法，获得了精原细胞中 Mg、P、Cr、Mn、Fe 和 Zn 的信号轮廓和元素分布。基于酸消解的结果，我们推导出了一个公式，用于根据每个元素的峰面积计算单细胞中元素的含量，并且通过对精子样本的检测，在一定程度上验证了该公式在单细胞元素定量检测中的可行性和通用性。单细胞元素含量检测结果表明，伸长精子细胞中 P 的含量明显高于精原细胞、精母细胞和圆形精子细胞（P<0.01），分布范围也较宽。然而，伸长精子细胞中的 Cr 和 Fe 含量低于精原细胞、精母细胞和圆形精子细胞（P<0.05）。当精原细胞发育为圆形精子细胞时，细胞内 Mn 和 Zn 的含量显著增加（P<0.05），然后在伸长精子细胞中降至最低。此外，从元素信号强度分布拟合曲线的中心向左移动，也可以直观地观察到伸长精子细胞中 Cr、Mn、Fe 和 Zn 含量的显著下降。本研究在单细胞水平上提供了精子发生各个阶段元素含量变化的元素视图。时间分辨 ICP-MS 用于检测睾丸生精细胞中的矿物质元素含量和分布模式，有助于更好地探索各种元素在精子发生中的作用阶段和作用方式，为揭示元素含量变化对精子发生和精液质量调节的影响提供直接证据。
