College of Animal Science and Technology, Anhui Agricultural University, Hefei, Anhui, China.
College of Animal Science and Technology, Anhui Agricultural University, Hefei, Anhui, China; Anhui Provincial Laboratory of Local Livestock and Poultry Genetic Resource Conservation and Bio-Breeding, Hefei, Anhui, China.
Theriogenology. 2014 Mar 1;81(4):545-55. doi: 10.1016/j.theriogenology.2013.11.007. Epub 2013 Nov 19.
The genetic manipulation of spermatogonial stem cells (SSCs) can be used for the production of transgenic animals in a wide range of species. However, this technology is limited by the absence of an ideal culture system in which SSCs can be maintained and proliferated, especially in domestic animals like the goat. The aim of this study therefore was to investigate whether the addition of vitamin C (Vc) in cell culture influences the growth of caprine SSCs. Various concentrations of Vc (0, 5, 10, 25, 40, and 50 μg/mL(-1)) were added to SSC culture media, and their effect on morphology and alkaline phosphatase activity was studied. The number of caprine SSC colonies and area covered by them were measured at 10 days of culture. The expression of various germ cell and somatic cell markers such as VASA, integrins, Oct-4, GATA-4, α-SMA, vimentin, and Thy-1 was studied to identify the proliferated cells using immunostaining analyses. Further, the intracellular reactive oxygen species (ROS) level was measured at the 3rd, 6th, and 9th day after culture, and expression of Bax, Bcl-2, and P53, factors involved in the regulation of apoptosis, were analyzed on the 7th day after culture using reverse transcription polymerase chain reaction and quantitative real-time polymerase chain reaction. The results showed that the SSCs formed compact colonies and had unclear borders in the different Vc-supplemented groups at 10 days, and there were no major morphologic differences between the groups. The number and area of colonies were both the highest in the 40 μg/mL(-1) Vc group. Differential expression of markers for germ cells, undifferentiated spermatogonia, and testis somatic cells was observed. Cultured germ cell clumps were found to have alkaline phosphatase activity regardless of the Vc dose. The number of Thy-1- and Oct-4-positive cells was the most in the 40 μg/mL(-1) Vc group. Moreover, the level of ROS was dependent on the Vc dose and culture time. The Vc dose 40 μg/mL(-1) was found to be optimum with regard to decreasing ROS generation, and increasing the expression of the antiapoptotic gene Bcl-2 and decreasing the expression of the proapoptotic genes Bax and P53. In conclusion, the addition of 40 μg/mL(-1) Vc can maintain a certain physiological level of ROS, trigger the expression of the antiapoptosis gene Bcl-2, suppress the proapoptotic gene P53 and Bax pathway, and further promote the proliferation of caprine SSCs in vitro.
精原干细胞（SSC）的基因操作可用于在广泛的物种中生产转基因动物。然而，这项技术受到缺乏理想的培养体系的限制，在这种培养体系中，SSC 可以得到维持和增殖，特别是在家畜如山羊中。因此，本研究的目的是研究细胞培养中添加维生素 C（Vc）是否会影响山羊 SSC 的生长。将不同浓度的 Vc（0、5、10、25、40 和 50μg/mL（-1））添加到 SSC 培养基中，并研究其对形态和碱性磷酸酶活性的影响。在培养的第 10 天测量山羊 SSC 集落的数量和它们所覆盖的面积。使用免疫染色分析研究各种生殖细胞和体细胞标志物（如 VASA、整合素、Oct-4、GATA-4、α-SMA、波形蛋白和 Thy-1）的表达，以鉴定增殖细胞。此外，在培养第 3、6 和 9 天测量细胞内活性氧（ROS）水平，并在培养第 7 天使用逆转录聚合酶链反应和定量实时聚合酶链反应分析凋亡调节因子 Bax、Bcl-2 和 P53 的表达。结果表明，在不同的 Vc 补充组中，SSC 在第 10 天形成了紧密的集落，边界不清晰，各组之间没有明显的形态差异。在 40μg/mL（-1）Vc 组中，集落的数量和面积均最高。观察到未分化精原细胞和睾丸体细胞的生殖细胞、未分化的精原细胞和睾丸体细胞标志物的差异表达。无论 Vc 剂量如何，培养的生殖细胞团块均具有碱性磷酸酶活性。在 40μg/mL（-1）Vc 组中，Thy-1 和 Oct-4 阳性细胞的数量最多。此外，ROS 水平取决于 Vc 剂量和培养时间。发现 Vc 剂量 40μg/mL（-1）最有利于减少 ROS 的产生，增加抗凋亡基因 Bcl-2 的表达，减少促凋亡基因 Bax 和 P53 的表达。总之，添加 40μg/mL（-1）的 Vc 可以维持一定的生理 ROS 水平，触发抗凋亡基因 Bcl-2 的表达，抑制促凋亡基因 P53 和 Bax 途径，进一步促进山羊 SSC 在体外的增殖。
