Degradation of securin in mouse and pig oocytes is dependent on ubiquitin-proteasome pathway and is required for proteolysis of the cohesion subunit, Rec8, at the metaphase-to-anaphase transition.
Although securin/separase/cohesion pathway was reported to regulate chromosome segregation during meiotic metaphase-to-anaphase transition, little biochemical evidence was provided. We recently found that oocytes could not progress beyond meiotic metaphase when ubiquitin-proteasome pathway was inhibited, but the mechanisms remain unclear. In the present study, we investigated the quantity of securin and Rec8 protein and the localization of securin, a cohesion subunit, during oocyte meiosis providing data in support of the hypothesis that the effect of ubiquitin-proteasome pathway on metaphase-to-anaphase transition was mediated by regulating securin and Rec8 degradation in mouse and pig oocytes. In germinal vesicle-stage oocytes, immunostaining of securin was mainly localized in the germinal vesicle. Shortly after germinal vesicle breakdown, immunoreactive securin accumulated around the condensed chromosomes at prometaphase I. At metaphase I and metaphase II, when chromosomes were organized at the equatorial plate, immunoreactive securin was concentrated around the aligned chromosomes, putatively associated with the position of the metaphase spindle. The accumulation of securin could not be detected at anaphase I and anaphase II. In both mouse and pig oocytes, Western blot analysis showed that securin protein was low at germinal vesicle stage, reached the highest level at metaphase I, while decreased at anaphase I. Securin was increased again at metaphase II, while it was decreased at anaphase II. Rec8 protein was present in germinal vesicle-stage oocytes and remained until metaphase I, while it was decreased at anaphase I. Like securin, Rec8 was increased at metaphase II, while it was decreased again at anaphase II. The inhibition of the ubiquitin-proteasome pathway inhibited the decrease in securin and Rec8 at metaphase-to-anaphase transitions in both mouse and pig oocytes. Microinjection of securin antibody into MII-arrested oocytes leads to the degradation of Rec8. In conclusion, these results suggest that the proteolysis of securin is dependent on ubiquitin-proteasome pathway and is necessary for the degradation of Rec8 during meiotic metaphase-to-anaphase transitions in mouse and pig oocytes.
Huo
Li-Jun
LJ
State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100080, China.
Zhong
Zhi-Sheng
ZS
Liang
Cheng-Guang
CG
Wang
Qiang
Q
Yin
Shen
S
Ai
Jun-Shu
JS
Yu
Ling-Zhu
LZ
Chen
Da-Yuan
DY
Schatten
Heide
H
Sun
Qing-Yuan
QY
D000705:Anaphase / Q000502:physiology; D000818:Animals; D000906:Antibodies; D015153:Blotting, Western; D002352:Carrier Proteins / Q000032:analysis / Q000378:metabolism*; D018929:Cell Culture Techniques; D018797:Cell Cycle Proteins; D005260:Female; D008540:Meiosis; D008677:Metaphase / Q000502:physiology; D051379:Mice; D009687:Nuclear Proteins / Q000378:metabolism*; D009865:Oocytes / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex / Q000502:physiology*; D064249:Securin; D013552:Swine; D025801:Ubiquitin / Q000502:physiology*
D000906:Antibodies; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D009687:Nuclear Proteins; D010750:Phosphoproteins; C465863:Rec8 protein, mouse; D064249:Securin; D025801:Ubiquitin; D046988:Proteasome Endopeptidase Complex