Accumulation of Securin on Spindle During Female Meiosis I

Abstract

Chromosome segregation during female meiosis is frequently incorrect with severe consequences including termination of further development or severe disorders, such as Down syndrome. Accurate chromosome segregation requires tight control of a protease called separase, which facilitates the separation of sister chromatids by cohesin cleavage. There are several control mechanisms in place, including the binding of specific protein inhibitor securin, phosphorylation by cyclin-dependent kinase 1 (CDK1), and complex with SGO2 and MAD2 proteins. All these mechanisms restrict the activation of separase for the time when all chromosomes are properly attached to the spindle. In our study, we focused on securin and compared the expression profile of endogenous protein with exogenous securin, which is widely used to study chromosome segregation. We also compared the dynamics of securin proteolysis in meiosis I and meiosis II. Our study revealed that the expression of both endogenous and exogenous securin in oocytes is compartmentalized and that this protein accumulates on the spindle during meiosis I. We believe that this might have a direct impact on the regulation of separase activity in the vicinity of the chromosomes.

Early onset of APC/C activity renders SAC inefficient in mouse embryos

“…on the protection of animals against cruelty and was approved by the Central Commission for Animal Welfare, under approval ID 37/2021. The methods for the collection of oocytes and embryos were described previously ( Pauerova et al, 2021 ). In brief, to collect embryos, mice were stimulated with 5 IU of pregnant mare serum gonadotropin (PMSG; BioVendor, Brno, Czech Republic), followed by stimulation with 5 IU of human chorionic gonadotropin (hCG; Merck, Darmstadt, Germany) 44–48 h later, and mated with BDF1 males.…”

“…The microinjection of mouse oocytes and embryos was performed in M2 media (Merck), as previously described ( Pauerova et al, 2021 ). The cRNAs used were as follows: securin-CFP, securin-EGFP, securin-DK EGFP, a fragment of the first 90 aa of cyclin B1 (1–90 cyB1) fused to Venus, tubulin-CFP, tubulin-EGFP, Mad1-Venus, histone H2B-mCherry, and histone H2B-mPlum.…”

“…The immunofluorescence protocol was described by Pauerova et al (2021) . In brief, oocytes and embryos were harvested after the previous culture.…”

“…Fiji (ImageJ, National Institutes of Health, Bethesda, MA, USA), LAS AF ( http://www.leica-microsystems.com ), and Imaris software ( www.bitplane.com ) was used for image analysis. For the quantification of the securin and 1–90 cyclin B1 signal the expression level was normalized to the level at GVBD or NEBD, as previously ( Pauerova et al, 2021 ). To quantify the Mad1 signal on the kinetochores, the CREST signal was used to define the ROI.…”