Scaldaferri Maria-Lucia, Fera Stefania, Grisanti Laura, Sanchez Massimo, Stefanini Mario, De Felici Massimo, Vicini Elena
Department of Public Health and Cell Biology, University of Rome Tor Vergata, Rome, Italy.
Int J Dev Biol. 2011;55(2):209-14. doi: 10.1387/ijdb.092977ms.
In mammals, the stem cells of spermatogenesis are derived from an embryonic cell population called primordial germ cells (PGCs). Spermatogonial stem cells displaying the “side population” (SP) phenotype have been identified in the immature and adult mouse testis, but noting is known about the expression of the SP phenotype during prenatal development of germ cells. The SP phenotype, defined as the ability of cells to efflux fluorescent dyes such as Hoechst, is common to several stem/progenitor cell types. In the present study, we analyzed and characterized the Hoechst SP via cytofluorimetric analysis of disaggregated gonads at different time points during embryonic development in mice. To directly test the hypothesis that the SP phenotype is a feature of germ cell lineage, experiments were performed on transgenic animals expressing enhanced green fluorescent protein (EGFP) under the control of the Oct4 promoter, to identify early germ cells up to PGCs. We found that prenatal gonads contain a fraction of SP cells at each stage analyzed, and the percentage of cells in the SP fraction decreases as development proceeds. Surprisingly, more than 50% of the PGCs displayed the SP phenotype at 11.5 dpc (days post coitum). The percentage of germ cells with the SP phenotype decreased steadily with development, to less than 1% at 18.5 dpc. Cytofluorimetric analysis along with immunocytochemistry performed on sorted cells indicated that the SP fraction of prenatal gonads, as in the adult testis, was heterogeneous, being composed of both somatic and germ cells. Both cell types expressed the ABC transporters Abcg2, Abcb1a, Abcb1b and Abcc1. These findings provide evidence that the SP phenotype is a common feature of PGCs and identifies a subpopulation of fetal testis cells including prospermatogonia whose differentiation fate remains to be investigated.
在哺乳动物中，精子发生的干细胞源自一种称为原始生殖细胞（PGCs）的胚胎细胞群体。在未成熟和成年小鼠睾丸中已鉴定出具有“侧群”（SP）表型的精原干细胞，但对于生殖细胞产前发育过程中SP表型的表达情况却一无所知。SP表型定义为细胞外排荧光染料（如Hoechst）的能力，在几种干/祖细胞类型中很常见。在本研究中，我们通过对小鼠胚胎发育不同时间点解离的性腺进行细胞荧光分析，对Hoechst SP进行了分析和表征。为了直接验证SP表型是生殖细胞谱系特征这一假设，我们对在Oct4启动子控制下表达增强型绿色荧光蛋白（EGFP）的转基因动物进行了实验，以鉴定直至PGCs的早期生殖细胞。我们发现，在所分析的每个阶段，产前性腺都含有一部分SP细胞，并且随着发育的进行，SP部分中的细胞百分比会降低。令人惊讶的是，在妊娠11.5天（受孕后天数）时，超过50%的PGCs表现出SP表型。具有SP表型的生殖细胞百分比随着发育而稳步下降，在妊娠18.5天时降至不到1%。对分选细胞进行的细胞荧光分析以及免疫细胞化学表明，与成年睾丸一样，产前性腺的SP部分是异质的，由体细胞和生殖细胞组成。两种细胞类型均表达ABC转运蛋白Abcg2、Abcb1a、Abcb1b和Abcc1。这些发现提供了证据，表明SP表型是PGCs的共同特征，并鉴定出胎儿睾丸细胞的一个亚群，包括其分化命运仍有待研究的精原细胞。
