Initial germ cell to somatic cell ratio impacts the efficiency of SSC expansion <i>in vitro</i>

<p>Spermatogonial Stem Cell (SSC) expansion <i>in vitro</i> remains a major challenge in efforts to preserve fertility among pubertal cancer survivor boys. The current study focused on innovative approaches to optimize SSC expansion. Six- to eight-week-old CD-1 murine testicular samples were harvested by mechanical and enzymatic digestion. Cell suspensions were incubated for differential plating (DP). After DP, we established two experiments comparing single vs. repetitive DP (S-DP and R-DP, respectively) until passage 2 (P2) completion. Each experiment included a set of cultures consisting of 5 floating-to-attached cell ratios (5, 10, 15, 20, and 25) and control cultures containing floating cells only. We found similar cell and colony count drops during P0 in both S- and R-DP. During P2, counts increased in S-DP in middle ratios (10, 15, and especially 20) relative to low and high ratios (5 and 25, respectively). Counts dropped extensively in R-DP after passage 2. The superiority of intermediate ratios was demonstrated by enrichment of GFRα1 by qPCR. The optimal ratio of 20 in S-DP contained significantly increased proportions of GFRα1-positive cells (25.8±5.8%) as measured by flow cytometry compared to after DP (1.9±0.7%, <i>p</i><0.0001), as well as positive immunostaining for GFRα1 and UTF1, with rare Sox9-positive cells. This is the first report of the impact of initial floating-to-attached cell ratios on SSC proliferation <i>in vitro</i>.</p> <p><b>Abbreviations</b>: SSC: spermatogonial stem cells; DP: differential plating; NOA: non-obstructive azoospermia; MACS: magnetic-activated cells sorting; FACS: fluorescence-activated cells sorting </p

CDC28 phosphorylates Cac1p and regulates the association of chromatin assembly factor i with chromatin

<div><p>Chromatin Assembly Factor I (CAF-I) plays a key role in the replication-coupled assembly of nucleosomes. It is expected that its function is linked to the regulation of the cell cycle, but little detail is available. Current models suggest that CAF-I is recruited to replication forks and to chromatin via an interaction between its Cac1p subunit and the replication sliding clamp, PCNA, and that this interaction is stimulated by the kinase <i>CDC7</i>. Here we show that another kinase, <i>CDC28</i>, phosphorylates Cac1p on serines 94 and 515 in early S phase and regulates its association with chromatin, but not its association with PCNA. Mutations in the Cac1p-phosphorylation sites of <i>CDC28</i> but not of <i>CDC7</i> substantially reduce the <i>in vivo</i> phosphorylation of Cac1p. However, mutations in the putative <i>CDC7</i> target sites on Cac1p reduce its stability. The association of CAF-I with chromatin is impaired in a <i>cdc28–1</i> mutant and to a lesser extent in a <i>cdc7–1</i> mutant. In addition, mutations in the Cac1p-phosphorylation sites by both <i>CDC28</i> and <i>CDC7</i> reduce gene silencing at the telomeres. We propose that this phosphorylation represents a regulatory step in the recruitment of CAF-I to chromatin in early S phase that is distinct from the association of CAF-I with PCNA. Hence, we implicate <i>CDC28</i> in the regulation of chromatin reassembly during DNA replication. These findings provide novel mechanistic insights on the links between cell-cycle regulation, DNA replication and chromatin reassembly.</p></div