Genes Associated with Establishment of the Spermatogenic Lineage in Rats.

Primordial germ cells (PGCs) in midgestation gonads become gonocytes in late gestation and perinatal testes, which give rise to spermatogonial stem cells (SSCs) in the adult testis. Previous studies in mice and our current studies in rats indicate that these stages of germline development are accompanied by changes in developmental potential (pluripotent vs. germline restriction). In rats, we found that pluripotency (teratoma production) was exhibited by germ cells from mid-gestation testes (12.5 or 15.5 days postcoitum; dpc), but not late gestation or postnatal testes (18.5dpc or 8.5 days post partum; dpp). Spermatogenic activity (spermatogonial stem cell transplantation) was exhibited by germ cells from 18.5 dpc and 8.5 dpp gonads but was not observed in earlier stages (12.5 and 15.5 dpc). These biological assays indicate that a pluripotent to spermatogenic transition occurs on about day 18.5 of rat germline development. To begin elucidating the molecular mechanisms controlling this transition, we prospectively isolated germ cells from each developmental stage using a phase contrast microscope, micromanipulator and pulled-glass capillary pipette. The transcriptome of germ cells from each time point was amplified and subjected to microarray analysis. We identified 237 genes with an expression profile that correlated with loss of pluripotency in germ cells (15.5dpc > 8.5dpp), including Sox2, Dppa3 (Stella), and Ifitm1 (Fragilis). An additional 281 genes exhibited an expression pattern that correlated with gain of spermatogenic activity (8.5dpp > 15.5dpc), including Zbtb16 (Plzf) and Gfralpha1, consensus markers of SSCs. These results validated the experimental design and suggested that this design might reveal genes that are up-regulated or down-regulated coincident with the transition in germ cell developmental potential on 18.5 dpp. We identified 75 down-regulated genes (15.5dpc > 18.5 dpc < 8.5 dpp) and 152 up-regulated genes (15.5 dpc < 18.5 dpc > 8.5 dpp). Functional annotation using Ingenuity Pathway Analysis indicated that cell cycle control genes were down-regulated on 18.5 dpc, including Mki67 (Ki67) and Aurkb (Aurora kinase B). Similar annotation indicated that genes associated with cell migration and reproductive system development were upregulated at 18.5dpc, including Pard3, Igtb2 (beta2-Integrin), and Piwil2 (Mili). Immunohistochemical staining confirmed the expression patterns revealed by microarray and co-staining with DDX4 (VASA) demonstrated that candidate gene expression corresponded with germ cells in the developing rat testis. Ongoing studies are designed to determine whether these genes are functionally required to establish the spermatogenic lineage in late gestation.

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