Characterization and population dynamics of germ cells in adult macaque testicular cultures

BackgroundFrom a biological and clinical perspective, it is imperative to establish primate spermatogonial cultures. Due to limited availability of human testicular tissues, the macaque (Macaca fascicularis) was employed as non-human primate model. The aim of this study was to characterize the expression of somatic as well as germ cell markers in testicular tissues and to establish macaque testicular primary cell cultures.Materials and methodsCharacterization of macaque testicular cell population was performed by immunohistochemical analyses for somatic cell markers (SOX9, VIM, SMA) as well as for germ cell markers (UTF1, MAGEA4, VASA). Testicular cells from adult macaque testes (n = 4) were isolated and cultured for 21 days using three stem cell culture media (SSC, PS and SM). An extended marker gene panel (SOX9, VIM, ACTA2; UTF1, FGFR3, MAGEA4, BOLL, DDX4) was then employed to assess the changes in gene expression levels and throughout the in vitro culture period. Dynamics of the spermatogonial population was further investigated by quantitative analysis of immunofluorescence-labeled MAGEA4-positive cells (n = 3).ResultsRNA expression analyses of cell cultures revealed that parallel to decreasing SOX9-expressing Sertoli cells, maintenance of VIM and ACTA2-expressing somatic cells was observed. Expression levels of germ cell marker genes UTF1, FGFR3 and MAGEA4 were maintained until day 14 in SSC and SM media. Findings from MAGEA4 immunofluorescence staining corroborate mRNA expression profiling and substantiate the overall maintenance of MAGEA4-positive pre- and early meiotic germ cells until day 14.ConclusionsOur findings demonstrate maintenance of macaque germ cell subpopulations in vitro. This study provides novel perspective and proof that macaques could be used as a research model for establishing in vitro germ cell-somatic cell cultures, to identify ideal culture conditions for long-term maintenance of primate germ cell subpopulation in vitro.研究背景 从生物学与临床医学视角出发，建立灵长类精原细胞培养体系至关重要。由于人类睾丸组织可获取量有限，本研究选用食蟹猴（Macaca fascicularis）作为非人灵长类模型。本研究旨在鉴定睾丸组织中体细胞与生殖细胞标志物的表达特征，并建立食蟹猴睾丸原代细胞培养体系。 材料与方法 本研究通过免疫组织化学分析，检测了体细胞标志物（SOX9、VIM、SMA）与生殖细胞标志物（UTF1、MAGEA4、VASA），以鉴定食蟹猴睾丸细胞群特征。从4只成年食蟹猴睾丸中分离睾丸细胞，采用SSC、PS、SM三种干细胞培养基进行21天体外培养。随后通过扩展的标志物基因组合（SOX9、VIM、ACTA2；UTF1、FGFR3、MAGEA4、BOLL、DDX4），评估体外培养过程中基因表达水平的变化。进一步通过对免疫荧光标记的MAGEA4阳性细胞进行定量分析（n=3），探究精原细胞群的动态变化。 结果 细胞培养的RNA表达分析显示，随着表达SOX9的支持细胞数量减少，表达VIM与ACTA2的体细胞得以维持。在SSC和SM培养基中，生殖细胞标志物基因UTF1、FGFR3及MAGEA4的表达水平可维持至第14天。MAGEA4免疫荧光染色结果印证了mRNA表达谱分析结果，证实直至第14天，表达MAGEA4的减数分裂前及早期减数分裂生殖细胞群整体得以维持。 结论 本研究结果证实，食蟹猴生殖细胞亚群可在体外维持存活。本研究为以食蟹猴作为研究模型建立体外生殖细胞-体细胞共培养体系提供了新的思路与实验依据，旨在筛选出可长期维持灵长类生殖细胞亚群体外存活的最优培养条件。