Isolation of Sertoli cells from adult rat testes: An approach to ex vivo studies of Sertoli cell function
Isolation of Sertoli cells from adult rat testes: An approach to ex vivo studies of Sertoli cell function
复制标题
DOI：
10.1095/biolreprod.102.008045
复制
发表时间：
2003-03-01
影响因子：
3.6
通讯作者：
Zirkin, BR
中科院分区：
文献类型：
--
作者：
Anway, MD;Folmer, J;Zirkin, BR
许多已知的关于成人支持细胞的分子调控和功能已经被推断出来。在体外对未成熟的支持细胞进行研究。然而，成熟细胞和未成熟细胞在许多方面存在显著差异，而且，许多支持细胞的功能是由难以在体外复制的条件调节的。我们的目标是开发一种快速、数量和纯度分离支持细胞的方法，以便在分离细胞后立即评估支持细胞的功能。本文所述的分离过程需要少于4小时，并且不需要培养细胞。从一只4岁大的成年大鼠中，我们常规地每个睾丸获得7.0 +/- 0.4 X 10(6)个支持细胞，从一只21岁大的大鼠中，每个睾丸获得7.2 +/- 0.4 X 10(6)个支持细胞。通过塑料包埋细胞的形态学分析或酪氨酸-微管蛋白或波形蛋白染色确定的纯度平均为80%。的。污染物通常包括生殖细胞（10%）和肌样细胞（10%）。在Sertoli细胞制备中未检测到生殖细胞表达基因蛋白蛋白-2和半铁蛋白，但Sertoli细胞表达基因clusterin、cathepsin L和transferrin高表达。老龄大鼠分离的支持细胞中转铁蛋白mRNA水平高于年轻成年大鼠，这与之前对整个睾丸的分析一致；和组织蛋白酶L mRNA水平在精原上皮周期6 - 7期分离的Sertoli细胞中远高于其他阶段的表达，这也与之前对整个睾丸和分离的小管的分析一致。这些研究表明，新分离的细胞保留了分化的功能，因此可以通过分离支持细胞并立即评估其功能来评估成年支持细胞的体内功能。
Much of what is known about the molecular regulation and function of adult Sertoli cells has been inferred. from in vitro studies of immature Sertoli cells. However, adult and immature cells differ in significant ways and, moreover, many Sertoli cell functions are regulated by conditions that are difficult to replicate in vitro. Our objective was to develop a procedure to isolate Sertoli cells rapidly and in sufficient number and purity to make it possible to assess Sertoli cell function immediately after the isolation of the cells. The isolation procedure described herein takes less than 4 h and does not require culturing the cells. From a single 4-mo-old adult rat, we routinely obtain 7.0 +/- 0.4 X 10(6) Sertoli cells per testis, and from a 21-mo-old rat, 7.2 +/- 0.4 X 10(6) Sertoli cells per testis. The purity, determined by morphologic analyses of plastic-embedded cells or after staining for tyrosine-tubulin or vimentin, averaged 80%. The. contaminants typically included germ cells (10%) and myoid cells (10%). The germ cell-expressed genes protamine-2 and hemiferrin were not detected in the Sertoli cell preparations by Northern blot analyses, but the Sertoli cell-expressed genes clusterin, cathepsin L, and transferrin were highly expressed. Transferrin mRNA levels were greater in Sertoli cells isolated from aged than from young adult rats, consistent with previous analyses of whole testes; and cathepsin L mRNA levels were far more highly expressed in Sertoli cells isolated from stages VI-VII than from other stages of the cycle of the seminiferous epithelium, also consistent with previous analyses of whole testes and isolated tubules. These studies indicate that the freshly isolated cells retain differentiated function, and thus it should be possible to assess the in vivo function of adult Sertoli cells by isolating the Sertoli cells and immediately assessing their function.