Aging Results in Molecular Changes in an Enriched Population of Undifferentiated Rat Spermatogonia1

A strong correlation exists between increasing paternal age and a decline in reproductive function. Testis aging is associated with testicular atrophy, increased DNA damage, and de novo mutations. It is unclear whether these problems arise from the spermatogonial stem cells (SSCs), a buildup of anomalies as older germ cells progress through spermatogenesis, or both. We hypothesize that with the continual divisions of SSCs that maintain the germ cell population, an alteration of these cells occurs over time. To test this, we utilized young (4-mo-old) and aged (18- and 21-mo-old) transgenic rats that express GFP in germ cells only. We first examined the number and activity of SSCs from the different age groups by transplantation. Aged rats had numerically fewer SSCs than young rats (<50%; not significant) despite the lack of testicular atrophy, and 21-mo-old rats show a significant reduction in colony length, suggesting that the quality of SSCs also deteriorates. To evaluate any molecular changes occurring in the early cells of spermatogenesis with age, we isolated an SSC-enriched population of CD9-positive (CD9(+)) cells using fluorescence-activated cell sorting (confirmed by transplantation studies) and extracted RNA for microarray analysis. In the aged CD9(+) cells, 60 transcripts were upregulated and more than 500 downregulated compared to the young cells. An altered expression was found for transcripts involved in mitosis and in DNA damage response. These results suggest molecular alterations in the SSC-enriched population of aged CD9(+) cells, implying that reproductive aging originates in the undifferentiated cells of spermatogenesis.