犬卵母細(xì)胞體外成熟和冷凍保存基礎(chǔ)研究.pdf

1、The influence of 9-cis-retinoic acid on nuclear and cytoplasmicmaturation and gene expression in canine oocytes duringin vitro maturationRetinoidshave important roles in the regulation ofoocyte nuclearandcytoplasmic matu

2、ration.The present study investigated the effects of a retinoidmetabolite onnuclear maturation，cytoplasmicmaturation,and gene expression incanineoocytesduringin vitro maturation IVM.COCs were harvested from ovaries by sl

3、icing.Only oocytes that were >120 μmin diameter,with a homogeneous dark cytoplasm and three or more layers of compact cumulus cellswere used.Varying concentrationsof 9-cis-RA(0，5，50,and 500 nM) were included in thematura

4、tionmedium,and the following were measured:(i)oocyte nuclear maturationafterculture for 48 h; (ii)cytoplasmicgranular migration by labeling of oocytes with fluorescein isothiocyanate labeled lectins;and(iii)relative expr

5、essionof genes related to apoptosis(BAX and Bcl2)in cumulus cells detached from oocytes，by semi-quantitativereverse transcriptase-polymerase chain reaction.After48h culturewith IVM,the highestpercentageof oocytes that ha

6、d developed to the MII stagewere in the5 nM 9-cis-RAtreatment group (18.3±2.5％; P < 0.05).Completegranular migration was observed in oocytes matured with5 nM 9-cis-RA，consistent with a commensurate gain in developmental

7、competence.Treatment with 5 nM 9-cis-RA had no effect on Bcl2gene expression，but down-regulatedBAXexpression.In conclusion,since5 nM9-cis-RAwas beneficial tonuclear and cytoplasmic maturation of canine oocytes,we inferre

8、d an important role for 9-cis-RAduring IVM.Influenceof ovarian cortex cellsco-culture during in vitro maturation on meiotic resumption of canine oocytesThe present studywas carried out to evaluate the effects ofCOCCsmono

9、layersco-culture on themeioticresumption competence ofcanine immatureoocytes during IVM.COCs were harvested from ovaries by slicingandevaluatedunderstereomicroscope.Only oocytes that were larger than 120μmin diameter,wit

10、h a homogeneous dark cytoplasm andmore three layers of compact cumulus cells were usedandrandomlyallocated intotwotreatmentgroups and cultured for 48h with the followingculture systems: Control group(COCswere cultured in

11、 maturation medium); Co-culturegroup (COCswere co-culturedwithCOCCs monolayers). The COCCs wascultured inDMEMwithouthormone until the confluence,and then cultured inmaturation mediumsupplemented with hormone for 12 hbefo

12、re the oocytes added.After cultured in vitro for 48 h，meiosis process of the oocytes wasexaminedbyHoechst33342 stainingunderinverted fluorescence microscope.The proportion ofoocytes meiotic resumption(GVBD to MII)cu

13、ltured in co-culturegroup (58.2%)was higher than for oocytes cultured incontrol group.More GVBDdeveloped in the co-culture group than that in the control group; however，the percentage of meioticprogression toMII stage sh

14、owed no significant difference between the treat group and control group.These results indicate that co-culturewith COCCsmonolayersduringIVMcould positivelyinfluencethe meiotic resumptioncompetence of canineimmatureoocyt

15、es.Canine oocyte collection during cryopreservation of the ovarian tissuesThe aim ofthepresent study was toevaluatetheeffectof canine ovarian tissues cryopreservation onthe survival ofoocytesfromvitrified-warmed ovarian

16、tissues.The ovariantissueswere harvested frombitchovariesandcut intosmall fragments using a scalpel blade and thenvitrified using solid-surface vitrification. Oocyteswerealso collected and vitrified following the procedu

17、re forovarian tissues. After storage in liquid nitrogenand thawing,the vitrifiedoocytesand theoocytes invitrifiedovarian tissueswere recovered and thenthe viability of the oocyteswere examinedby PIstainingunder inverted

18、fluorescence microscope. Although oocytes survivalrate in vitrified oocytes groupwashigher than oocytescollected from vitrified ovarian tissues group，nostatisticallydifference inviability was observedbetweenthetwogroups(

19、22.7%versus25.3%).These results demonstrate that cryopreserved canine ovarian tissuescouldpreserve canine oocytes and providean alternative technology forcryopreserved canine oocytesforcanineassisted reproductive program