RNA Binding Protein Ptbp2 Is Essential for Male Germ Cell Development

Abstract

RNA binding proteins (RBPs) are increasingly recognized as essential factors in tissue development and homeostasis. The polypyrimidine tract binding (PTB) protein family of RBPs are important posttranscriptional regulators of gene expression. In the nervous system, the function and importance of PTB protein 2 (Ptbp2) as a key alternative splicing regulator is well established. Ptbp2 is also abundantly expressed during spermatogenesis, but its role in this developmental program has not been explored. Additionally, the importance of alternative splicing regulation in spermatogenesis is unclear. Here, we demonstrate that Ptbp2 is essential for spermatogenesis. We also describe an improved dual fluorescence flow cytometry strategy to discriminate, quantify, and collect germ cells in different stages of development. Using this approach, in combination with traditional histological methods, we show that Ptbp2 ablation results in germ cell loss due to increased apoptosis of meiotic spermatocytes and postmeiotic arrest of spermatid differentiation. Furthermore, we show that Ptbp2 is required for alternative splicing regulation in the testis, as in brain. Strikingly, not all of the alternatively spliced RNAs examined were sensitive to Ptbp2 loss in both tissues. Collectively, the data provide evidence for an important role for alternative splicing regulation in germ cell development and a central role for Ptbp2 in this process.

ACKNOWLEDGMENTS

We are grateful to the following individuals for their contributions to this work: Douglas Black (UCLA) for providing mice with the Ptbp2^flox allele; Nancy Edgehouse and Jenifer Mikulan at the CWRU Histology Core for tissue processing and embedding; Adam Kresak at the CWRU Tissue Resources Core for assistance with photomicroscopy; Robert Darnell for providing anti-Ptbp2 antibody; and Christopher Geyer, Timothy Nilsen, and Jo Ann Wise for comments on the manuscript.

This work was supported by NIH grant GM107331 to D.D.L.