Animal Physiology and Reproduction Division, ICAR-Central Institute for Research On Goats, Makhdoom, Farah, 281 122, Mathura, Uttar Pradesh, India.
Animal Health Division, ICAR-Central Institute for Research On Goats, Makhdoom, Farah, 281 122, Mathura, Uttar Pradesh, India.
Cell Stress Chaperones. 2021 Nov;26(6):989-1000. doi: 10.1007/s12192-021-01236-y. Epub 2021 Sep 22.
The present study aims to evaluate culture temperature-dependent variation in survival, growth characteristics and expression of stress, pluripotency, apoptosis, and adhesion markers in enriched caprine male germline stem cells (cmGSCs). For this, testes from pre-pubertal bucks (4-5 months; n = 4) were used to isolated cells by a two-step enzymatic digestion method. After enrichment of cmGSCs by multiple methods (differential platting, Percoll density gradient centrifugation, and MACS), viability of CD90 cells was assessed before co-cultured onto the Sertoli cell feeder layer at different temperatures (35.5, 37.0, 38.5, and 40.0 °C). The culture characteristics of cells were compared with MTT assay (viability); cluster-forming activity assay, SA-β1-gal assay (senescence), BrdU assay (proliferation), and transcript expression analyses by qRT-PCR. Moreover, the co-localization of pluripotency markers (UCHL-1, PLZF, and DBA) was examined by a double-immunofluorescence method. The cells grown at 37.0 °C showed faster proliferation with a significantly (p < 0.05) higher number of viable cells and greater number of cell clusters, besides higher expression of pluripotency markers. The transcript expression of HSPs (more noticeably HSP72 than HSP73), anti-oxidative enzymes (GPx and CuZnSOD), and adhesion molecule (β1-integrin) was significantly (p < 0.05) downregulated when grown at 35.0, 38.5, or 40.0 °C compared with 37.0 °C. The expression of pluripotency-specific transcripts was significantly (p < 0.05) lower in cmGSCs grown at the culture temperature lower (35.5 °C) or higher (38.5 °C and 40.0 °C) than 37.0 °C. Overall, the culture temperature significantly affects the proliferation, growth characteristics, and expression of heat stress, pluripotency, and adhesion-specific markers in pre-pubertal cmGSCs. These results provide an insight to develop strategies for the improved cultivation and downstream applications of cmGSCs.
本研究旨在评估培养温度依赖性对富集山羊雄性生殖干细胞（cmGSCs）存活、生长特性和应激、多能性、细胞凋亡、黏附标志物表达的影响。为此，使用来自青春期前小山羊（4-5 月龄；n=4）的睾丸通过两步酶消化法分离细胞。通过多种方法（差 plating、Percoll 密度梯度离心和 MACS）富集 cmGSCs 后，在不同温度（35.5、37.0、38.5 和 40.0°C）下将 CD90 细胞共培养到支持细胞饲养层上之前，评估细胞活力。通过 MTT 分析（活力）、集落形成活性分析、SA-β1-半乳糖苷测定（衰老）、BrdU 测定（增殖）和 qRT-PCR 进行转录表达分析比较细胞培养特性。此外，通过双免疫荧光法检测多能性标志物（UCHL-1、PLZF 和 DBA）的共定位。在 37.0°C 下生长的细胞增殖更快，活细胞数量和细胞集落数量显著增加（p<0.05），多能性标志物表达更高。与 37.0°C 相比，在 35.0°C、38.5°C 或 40.0°C 下生长时，应激相关 HSPs（HSP72 比 HSP73 更明显）、抗氧化酶（GPx 和 CuZnSOD）和黏附分子（β1-整合素）的转录表达显著下调（p<0.05）。与 37.0°C 相比，在培养温度较低（35.5°C）或较高（38.5°C 和 40.0°C）时，cmGSCs 的多能性特异性转录表达显著降低（p<0.05）。总之，培养温度显著影响青春期前 cmGSCs 的增殖、生长特性和热应激、多能性和黏附特异性标志物的表达。这些结果为开发 cmGSCs 的改良培养和下游应用策略提供了参考。
