Beall Stephanie A, Boekelheide Kim, Johnson Kamin J
Department of Pathology and Laboratory Medicine, Brown University, Providence, Rhode Island, USA.
J Androl. 2005 Jul-Aug;26(4):529-38. doi: 10.2164/jandrol.05003.
Spermatogenesis requires Sertoli cell-germ cell adhesion for germ cell survival and maturation. Cadherins are a diverse superfamily of adhesion proteins; structurally unique members of this superfamily (celsr cadherins) are hybrid molecules containing extracellular cadherin repeats connected to a G protein-coupled receptor transmembrane motif. Here we demonstrate postnatal testicular mRNA expression of the 3 celsr paralogs (celsr1, celsr2, and celsr3), protein localization of celsr2 and celsr3, and functional analysis of celsr2 adhesion activity in primary Sertoli cell-germ cell co-cultures. Evaluation of celsr mRNA levels during a postnatal time course indicated that celsr1 and celsr2 were Sertoli cell and/or early-stage germ cell products, whereas celsr3 was expressed in later-stage germ cells. Cell type-specific expression was verified using the Sertoli cell line 93RS2, where celsr1 and celsr2 mRNA, but not celsr3, were detected. Immunostaining of testicular cryosections resulted in celsr2 protein localization to a spokelike pattern in the basal seminiferous epithelium and punctate figures in the apical epithelium, consistent with both Sertoli cell and germ cell expression. Celsr3 localized to punctate structures in the adluminal epithelium from postnatal day 40, consistent with elongate spermatid expression. The subcellular localization of celsr2 was examined further to define its localization in Sertoli cells and germ cells. Celsr2 localized to the Golgi complex in Sertoli cells and germ cells. In addition, germ cell celsr2 localized to a rab7-positive structure, which may be an endocytic compartment. Neither celsr2 nor celsr3 immunostaining was present at classic cadherin-based adhesion junctions. Nonetheless, the addition of a recombinant celsr2 protein fragment consisting of extracellular cadherin domains 4 through 8 to Sertoli cell-germ cell co-cultures resulted in germ cell detachment from Sertoli cells. Collectively, these data indicate that celsr cadherins have a cell type-specific expression pattern, and celsr2 may mediate Sertoli cell-germ cell adhesion outside of classic cadherin-based adhesion junctions.
精子发生需要支持细胞与生殖细胞的黏附，以确保生殖细胞的存活和成熟。钙黏蛋白是一类多样的黏附蛋白超家族；该超家族结构独特的成员（celsr钙黏蛋白）是杂合分子，包含与G蛋白偶联受体跨膜基序相连的细胞外钙黏蛋白重复序列。在此，我们展示了3种celsr旁系同源物（celsr1、celsr2和celsr3）在出生后睾丸中的mRNA表达、celsr2和celsr3的蛋白定位，以及在原代支持细胞 - 生殖细胞共培养中对celsr2黏附活性的功能分析。对出生后不同时间点的celsr mRNA水平评估表明，celsr1和celsr2是支持细胞和/或早期生殖细胞的产物，而celsr3在后期生殖细胞中表达。使用支持细胞系93RS2验证了细胞类型特异性表达，在该细胞系中检测到了celsr1和celsr2的mRNA，但未检测到celsr3的mRNA。对睾丸冰冻切片进行免疫染色，结果显示celsr2蛋白在基底生精上皮呈辐条状定位，在顶端上皮呈点状分布，这与支持细胞和生殖细胞的表达均相符。从出生后第40天起，celsr3定位于近腔上皮的点状结构，与伸长型精子细胞的表达一致。进一步研究了celsr2的亚细胞定位，以确定其在支持细胞和生殖细胞中的定位。celsr2定位于支持细胞和生殖细胞的高尔基体复合体。此外，生殖细胞中的celsr2定位于一种rab7阳性结构，这可能是一个内吞区室。在基于经典钙黏蛋白的黏附连接中均未出现celsr2和celsr3的免疫染色。尽管如此，将由细胞外钙黏蛋白结构域4至8组成的重组celsr2蛋白片段添加到支持细胞 - 生殖细胞共培养体系中，导致生殖细胞与支持细胞分离。总体而言，这些数据表明celsr钙黏蛋白具有细胞类型特异性表达模式，并且celsr2可能在基于经典钙黏蛋白的黏附连接之外介导支持细胞与生殖细胞的黏附。
