Cell-type specific inactivation of sumoylation during mouse spermatogenesis - Abstract. Previous studies from our laboratory have obtained evidence implicating sumoylation in
regulation of different events during spermatognenesis. SUMO is abundantly expressed in germ cells from
different species, and the localization pattern suggests diverse and potentially multiple roles of sumoylation
in testicular function. We have also performed a broad-spectrum cell-specific identification of SUMO targets
in purified mouse germ cells. Numerous proteins with important roles during mitosis, meiosis and spermatid
differentiation have been identified. Level of sumoylation correlated with the proliferation activity of germ
cells. Notably, in mouse spermatocytes, inhibition of global sumoylation with the inhibitor Ginkgolic acid
(GA) arrested cells at the G2/M1 checkpoint in vitro due to the inability of spermatocytes to condense
chromatin and disassemble their synaptonemal complexes (SC). Our preliminary data show that Polo
kinase1 (PLK1) and Aurora B (AURKB) are activated upon G2/M meiotic transition, but significantly
inhibited upon inhibition of sumoylation. Since PLK1and AURKB are responsible for the disassembly of the
SC and chromatin condensation, respectively, these kinases are "at the right place, at the right time" to at
least, in part, explain the meiotic arrest. However, definite evidence regarding the importance of
sumoylation and its targets in germ cells in vivo is lacking because of the embryonic lethality of mice with
inactivated sumoylation machinery. The specific aims of the current application will be: 1) To perform a cell-
specific inactivation of sumoylation and characterize the consequences of this inactivation in mitotic
spermatogonia, meiotic spermatocytes and differentiating spermatids in vivo using a UBA2 (SUMO-
activating enzyme) loxP mouse model that we possess as well as commercially available mouse models
expressing Cre recombinase in the corresponding germ cells; 2) To dissect the molecular events regulated
by sumoylation in spermatocytes through analysis of the activity, interacting partners and phosphorylation
status of known downstream targets of PLK1 and AURKB upon inhibition of sumoylation in vivo and in vitro.
Together, the proposed research and follow-up studies will advance knowledge across the field of germ cell
biology by the characterization of post-translational modification networks necessary for the progression
throughout spermatogenesis. A significant part of the proposed project will be conducted by undergraduate
students who will have the opportunity to use state-of-the-art equipment, learn hypothesis generation and
practice basic and advanced biomedical techniques. They will be involved in germ cell isolation, preparation
of cell lysates, western blot, PCR, microscopy, and bioinformatics analyses. Opportunities to participate in
research early in their careers will give students a better understanding of a career in science and will
motivate them to pursue their education in STEM fields.
