The Study On The Roles And Regulatory Mechanisms Of Chromosome Cohesion Proteins Escol And Esco2 During Mammalian Oocyte Meiotic Maturation

Oocyte maturation disorder and aneuploidy in mammals are the major indicators of low quality of oocytes,severely impairing the livestock fertility and production.Oocyte maturation failure usually causes the defects of nuclear maturation and cytoplasmic maturation of oocytes,leading to the fertilization failure.Although aneuploid eggs could be fertilized,embryonic lethality,abortion and birth defects will occur subsequently.However,their mechanisms have not fully clarified and the regulators are less known.These molecules can be regarded as the evaluation indicators of oocyte quality,contributing to the screen of the female livestock with high fertility for production.As two critical events during oocyte meiotic maturation,normal spindle assembly and precise chromosome segregation are highly correlated to the oocyte quality.Chromosome cohesion proteins play key roles in cohesion establishment and maintenance and are the prerequisite for the accurate chromosome segregation.In addition,this protein family takes important part in the DNA damage repair,gene expression and chromatin structure maintenance.Although the functions and regulatory mechanisms of chromosome cohesion proteins have been extensively studied,their roles in oocyte meiosis have not clearly defined.Therefore,exploration and discovery of more regulators of oocyte maturation and oocyte quality provide the theoretical basis for improvement of livestock fertility and animal husbandry development.In budding yeast,acetyltransferase Ecol acetylates cohesin subunit Smc3 in S phase to establish the chromosome cohesion.However,in mammalian cells,it evolves into two acetyltransferases Escol and Esco2 to exert this function,ensuring the accurate chromosome cohesion and segregation.Although Escol and Esco2 have redundant roles in chromosome cohesion,they exert different functions in other biological processes through their distinct substrates.In this study,we used mouse and porcine oocytes as the model and applied the methods of targeted siRNA or morpholino-based gene silencing,microinjection of point mutation mRNA,immunofluorescence,immunoblotting as well as immunoprecipitation to investigate the functional mechanisms of Esco1 and Esco2 during mouse oocyte meiotic maturation and their conservation in porcine oocytes,which provides scientific evidence for defining chromosome cohesion proteins as the molecular indicators of oocyte quality.The study is divided into four parts.The specific research content and results are as follows:Experiment 1.The cohesion establishment factor Esco1 acetylates ?-tubulin to ensure proper spindle assembly in mouse oocyte meiosisIt has been reported that Esco1 plays important roles in chromatin structure maintenance and gene expression in addition to the chromosome cohesion.In the present study,we documented that Esco1 was expressed and localized to both the nucleus and cytoplasm during mouse oocyte meiotic maturation.Depletion of Esco1 by siRNA microinjection caused the meiotic progression arrest with a severe spindle abnormality and chromosome misalignment,which was coupled with a higher incidence of the erroneous kinetochore-microtubule attachments and activation of spindle assembly checkpoint.In addition,depletion of Esco1 led to the impaired microtubule stability shown by the weakened resistance ability to the microtubule depolymerizing drug nocodazole and the decreased level of acetylated ?-tubulin.Conversely,overexpression of Esco1 caused hyperacetylation of ?-tubulin and spindle defects.Moreover,we found that Esco1 bound to?-tubulin and was required for its acetylation.The reduced acetylation level of ?-tubulin in Esco1-depleted oocytes could be restored by the ectopic expression of exogenous wild-type Esco1 but not enzymatically dead Esco1-G768D.Purified wild-type Esco1 instead of mutant Esco1-G768D acetylated the synthesized peptide of a-tubulin in vitro.Collectively,our data assign a novel function to Esco1 as a microtubule regulator during oocyte meiotic maturation beyond its conventional role in chromosome cohesion.Experiment 2.Cohesin acetyltransferase Esco2 regulates SAC and kinetochore functions via maintaining H4K16 acetylation during mouse oocyte meiosisAs a critical regulator during chromosome cohesion establishment,mutations in Esco2 cause Roberts syndrome,a developmental disease characterized by severe prenatal retardation as well as limb and facial abnormalities.However,its exact roles during oocyte meiosis have not clearly defined.Here,we discovered that Esco2 localized to the chromosomes during oocyte meiotic maturation.Depletion of Esco2 by morpholino microinjection led to the precocious polar body extrusion,the escape of metaphase I arrest induced by nocodazole treatment and the loss of BubR1 from kinetochores,indicative of inactivated SAC.Furthermore,depletion of Esco2 caused a severely impaired spindle assembly and chromosome alignment,accompanied by the remarkably elevated incidence of defective kinetochore-microtubule attachments which consequently led to the generation of aneuploid eggs.Notably,we found that the involvement of Esco2 in SAC and kinetochore functions was mediated by its binding to histone H4 and acetylates H4 at Lys16 both in vivo and in vitro.Thus,our data assign a novel meiotic function to Esco2 beyond its role in the cohesion establishment during mouse oocyte meiosis.Experiment 3.Escol regulates spindle assembly to promote in vitro maturation in porcine oocytesOur study has uncovered the novel function of Escol to regulate tubulin acetylation and spindle assembly in mouse oocytes,but whether this function is conserved among species has not been known.Porcine oocytes were used as a model to investigate the conserved function of Esco1.Antibody stain and Escol-RFP mRNA microinjection showed that both endogenous and exogenous Esco1 had the spindle localization patterns.Depletion of Escol by siRNA microinjection caused the meiotic arrest of porcine oocytes by showing the reduced frequency of first polar body extrusion and SAC activation.Furthermore,depletion of Esco1 resulted in defective spindle/chromosome structure and weakened microtubule stability in porcine oocytes.Last,Escol bound to ?-tubulin and was required for its acetylation level to maintain the microtubule dynamics in porcine oocytes.Thus,our data reveal that Escol performs conserved functions in microtubule dynamics and spindle assembly in meiosis to promote oocyte maturation in mammals.Experiment 4.Esco2 modulates spindle assembly checkpoint to drive porcine oocyte meiotic maturationWe have validated that Esco2 maintains the euploidy of oocytes by regulating SAC and kinetochore functions as well as H4K16 acetylation during mouse oocyte meiosis.The present experiment further determined the role of Esco2 during porince oocyte maturation.Immunofluorescence result showed that Esco2 was distributed on the chromosomes at different development stages of porcine oocytes.Depletion of Esco2 by siRNA microinjection resulted in the accelerated meiotic progression by displaying the precocious polar body extrusion and inactivation of spindle assembly checkpoint.In addition,Esco2 was shown to be associated with histone H4 for the acetylation of H4K16 to modulate the kinetochore function in porcine oocytes.In conclusion,our data demonstrate that Esco2 exerts conserved functions in reguation of SAC and euploidy to drive the meiotic progression,ensuring the maturation and quality of oocytes.In summary,our study uncovered the novel and unique functions of cohesin acetyltransferases Esco1 and Esco2 during oocyte meiosis using mouse and porcine oocytes as the research objects.Taking advantage of mouse model,we performed mechnism-based research and found that Escol acetylates ?-tubulin to ensure proper spindle assembly and meiotic maturation in mouse oocytes.In addition,Esco2 maintains the euploidy by regulating SAC and kinetochore functions as well as H4K16 acetylation during mouse oocyte meiosis.More importantly,we verified the noncanonical roles of Esco1 and Esco2 in porince oocytes,indicating that their functions are highly conserved among specise.Together,our findings clarifed the molecular mechanisms about the role of chromosome cohesion proteins in oocyte maturation and oocyte quality control,which provides the theoretical evidence to define chromosome cohesion proteins as the molecular indicator of oocyte quality and offers the new strategies for improvement of livestock fertility.