Abstract
Background
Preferentially expressed antigen in melanoma (PRAME) is a cancer/testis antigen (CTA) that is predominantly expressed in normal gametogenic tissues and a variety of tumors. Members of the PRAME gene family encode leucine-rich repeat (LRR) proteins that provide a versatile structural framework for the formation of protein–protein interactions. As a nuclear receptor transcriptional regulator, PRAME has been extensively studied in cancer biology and is believed to play a role in cancer cell proliferation by suppressing retinoic acid (RA) signaling. The role of the PRAME gene family in germline development and spermatogenesis has been recently confirmed by a gene knockout approach. To further understand how PRAME proteins are involved in germ cell development at a subcellular level, we have conducted a systematic immunogold electron microscopy (IEM) analysis on testis sections of adult mice with gene-specific antibodies from two members of the mouse Prame gene family: Pramel1 and Pramex1. Pramel1 is autosomal, while Pramex1 is X-linked, both genes are exclusively expressed in the testis.
Results
Our IEM data revealed that both PRAMEL1 and PRAMEX1 proteins were localized in various cell organelles in different development stages of spermatogenic cells, including the nucleus, rER, Golgi, mitochondria, germ granules [intermitochondrial cement (IMC) and chromatoid body (CB)], centrioles, manchette, and flagellum. Unlike other germ cell-specific makers, such as DDX4, whose proteins are evenly distributed in the expressed-organelle(s), both PRAMEL1 and PRAMEX1 proteins tend to aggregate together to form clusters of protein complexes. These complexes were highly enriched in the nucleus and cytoplasm (especially in germ granules) of spermatocytes and spermatids. Furthermore, dynamic distribution of the PRAMEL1 protein complexes were observed in the microtubule-based organelles, such as acroplaxome, manchette, and flagellum, as well as in the nuclear envelope and nuclear pore. Dual staining with PRAMEL1 and KIF17B antibodies further revealed that the PRAMEL1 and KIF17B proteins were co-localized in germ granules.
Conclusion
Our IEM data suggest that the PRAMEL1 and PRAMEX1 proteins are not only involved in transcriptional regulation in the nucleus, but may also participate in nucleocytoplasmic transport, and in the formation and function of germ cell-specific organelles during spermatogenesis.
Protein components of ribonucleoprotein granules from Drosophila germ cells oligomerize and show distinct spatial organization during germline development
