Cross-talk between sumoylation and phosphorylation in mouse spermatocytes

Abstract

The meiotic G2/M1 transition is mostly regulated by posttranslational modifications, however, the cross-talk between different posttranslational modifications is not well-understood, especially in spermatocytes. Sumoylation has emerged as a critical regulatory event in several developmental processes, including reproduction. In mouse oocytes, inhibition of sumoylation caused various meiotic defects and led to aneuploidy. However, the role of sumoylation in male reproduction has only begun to be elucidated. Given the important role of several SUMO targets (including kinases) in meiosis, in this study, the role of sumoylation was addressed by monitoring the G2/M1 transition in pachytene spermatocytes in vitro upon inhibition of sumoylation. Furthermore, to better understand the cross-talk between sumoylation and phosphorylation, the activity of several kinases implicated in meiotic progression was also assessed upon down-regulation of sumoylation. The results of the analysis demonstrate that inhibition of sumoylation with ginkgolic acid (GA) arrests the G2/M1 transition in mouse spermatocytes preventing chromosome condensation and disassembling of the synaptonemal complex. Our results revealed that the activity of PLK1 and the Aurora kinases increased during the G2/M1 meiotic transition, but was negatively regulated by the inhibition of sumoylation. In the same experiment, the activity of c-Abl, the ERKs, and AKT were not affected or increased after GA treatment. Both the AURKs and PLK1 appear to be "at the right place, at the right time" to at least, in part, explain the meiotic arrest obtained in the spermatocyte culture.

Keywords: AURKs; Ginkgolic acid; Meiosis; PLK1; Phosphorylation; Sumoylation.

Figure 1. Inhibition of sumoylation blocks the G2/M1 progression in spermatocytes

A highly purified population of spermatocytes (A) was treated with the phosphatase inhibitor okadaic acid (OA) with (B) and without (C) a preceding addition of 30 μM of a sumoylation inhibitor (ginkgolic acid, GA). Color-coding is indicated for each image group. Nuclei are stained by DAPI (blue). In a control culture, OA treatment induced massive H3Ser10 phosphorylation (which hallmarks chromosome condensation) and disassembly of the synaptonemal complex (B); In contrast, neither disassembly of the synaptonemal complex nor H3Ser10 phosphorylation were detectable in GA-treated spermatocytes (C).

Figure 2

A) Effect of increasing concentration of sumoylation inhibitor ginkgolic acid (GA) on disassembly of synaptonemal complex and appearance of H3Ser10 phosphorylation. Fifty cells were scored for each condition and the experiments were repeated three times. Data are presented and mean ± standard deviation. B₁) The effect of inactivation of sumoylation by ginkgolic acid (GA) on tyrosine phosphorylation in spermatocytes. Spermatocytes were treated with 30 μM of GA for 1 hour followed by a preparation of the whole cell lysate and western blot analysis with anti-SUMO or anti-phospho-tyrosine antibody. Anti-actin antibody was used to confirm equal loading. B₂) The effect of specific inactivation of sumoylation on tyrosine phosphorylation in GC-1 spermatogonia cell lines. Si-RNA mediated inactivation of UBC9 (SUMO-conjugating enzyme) decreased sumoylation and caused significant changes in tyrosine phosphorylation of the GC-1 spermatogonia cell line.

Figure 3. The effect of inhibition of sumoylation during the G2/M transition on the activity of several kinases implicated in the regulation of meiosis

A purified population of spermatocytes was treated with okadaic acid (+OA) with (+GA) and without (−GA) a preceding addition of 30 μM ginkgolic acid (GA). Specific antibodies (Table 1) against either activating (c-Abl, PLK1, AUR, ERK, AKT) or inhibitory (CDC2) phosphorylation on several kinases were used to examine changes in their activity upon addition of OA and inhibition of sumoylation.