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fbtwitterlinkedinvimeoflicker grey 14rssslideshare1
Moawad, Adel Reda
Languages: English
Types: Unknown
Subjects:
Oocyte cryopreservation represents one of the most recent developments in the field of reproductive technologies. However, despite of significant progress, the efficiency of oocyte cryopreservation is still very low. Cryopreservation of mature metaphase II (MIl) oocytes has been reported to induce disorganization of the meiotic spindle and chromosome damage. However, cryopreservation of immature oocyte at germinal vesicle (GV) stage may provide an alternative which avoids these problems. Slow freezing protocols have more recently been replaced by vitrification approaches. In this thesis, recovery, viability and subsequent developmental potential following in vitro fertilisation (IVF), parthenogenetic activation or somatic cell nuclear transfer (SCNT) of ovine oocytes vitrified at GV stage and matured in vitro were studied. Solid surface vitrification (SSV) and cryoloop technologies share the advantages of using a containerless system and small volumes of solution (less than t J.ll) which favours rapid cooling. Maturation, fertilisation, cleavage and blastocyst development were significantly decreased in SSV vitrified oocytes as compared to controls. Following cryoloop\ud vitrification, frequencies of in vitro maturation (43.4 vs 63.2%), oocytes with normal spindle and chromosome configuration (50.0 vs 70.4%) and fertilisation (54.0 vs 74. t %) did not differ significantly between vitrified and control oocytes. Numbers of cleaved embryos that developed to the blastocyst stage following IVM/IVF/IVC did not differ significantly between vitrified and control groups (29.4 vs 45.1 %). In vitro matured ovine oocytes vitrified at GV stage using cryoloop were activated by two different protocols (I) a combination of calcium ionophore (A 23187), cycloheximide and cytochalasin 13 (CA+CHX/CI3), (2) strontium and CB (Sr/Cll). No blastocysts developed in vitrified oocytes activated by CA+CHX/CB; however, 3.8% were obtained following Sr/CI3 activation. Developmental competence of ovinc oocytes vitrified at GV stage and used as cytoplast recipients for SCNT was evaluated. Although the frequencies of cleaved embryos were significantly decreased in vitrified oocytes as compared to control, development to morula and blastocyst stage embryos was not significantly different. No significant differences were observed in total cell numbers, number of apoptotic nuclei as detected by Hoechst and TUNEL assay and proportions of diploid embryos in day 7 blastocysts produced following IVF or seNT of vitrified oocytes as compared to control. Pre-treatment of ovine GV-oocytes with cytochalasin 13 (7.5 J.lglml for 60 min) or demecolcine (0.1 flg/ml for 20 min) prior to vitrification improved frequencies of maturation, fertilisation and subsequent development following IVF or parthenogenetic activation. Caffeine treatment during IVM (10 mM for 6 h) increased the frequencies of blastocyst development in vitrified/thawed GV ovine oocytes.\ud Taken together, these studies suggest that, ovine oocytes vitrified at GV stage can be matured, fertilised and develop in vitro to blastocyst stage embryos. Cryoloop vitrification resulted in higher maturation, fertilisation and subsequent development as compared to SSV. Strontium can be used effectively for parthenogenetic activation of vitrified/thawed ovine GV oocytes. Ovine oocytes vitrified at GV stage can be used effectively as cytoplast recipients for SCNT.
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    • CIIAPTER 3................................. ... ............ ............ ............
    • 3.1 INTRODUCTION.............................................................
    • 3.2 MATERIALS AND METHODS.................. .................. .........
    • 3.3 RESULTS....................................................................... 3.3.1 Effects of different cryoprotectants on oocyte viability........ .... 3.3.2 Effects of different vitrification solutions on cumulus cell expansion following IVM............................................ .... 3.3.3 Effects of different cryoprotectants on meIotIc maturation following IVM......... ............ ...... ...... ............ ............. 3.4 DISSCUSION..................................................................
    • CIIAPTER 4.........................................................................
    • IN VITRO FERTILISATION OF OVINE OOCYTES VITRIFIED BY SOLID SURFACE VITRIFICATION (SSV) AT GERMINAL VESICLE STAGE............... ...... ......... ...... ... .......................... 4.1 INTRODUCTION............................................................. 4.2 MATERIALS AND METHODS............................................ 4.3 RESULTS...................................................................... 4.3.1 Recovery rates and oocyte viability following SSV............... 4.3.2 Cumulus cell expansion and meiotic maturation following IVM 4.3.3 Frequencies of fertilisation and polyspermy following IVF...... 4.3.4 Kinetics of first cleavage following IVF............................. 4.3.5 In vitro embryo development following IVF........................ 4.4 DISCUSSION..................................................................
    • CIIAPTER 5........................................................ ......................
    • N, FERTILISATION AND SUBSEQUENT DEVELOPMENT......... 5.1 INTRODUCTION............................................................. 5.2 MATERIALS AND METHODS...... ......... ......... ...... .............. 5.3 RESULTS....................................................................... 5.3.1 Recovery rates and oocyte survival.................................. 5.3.2Cumulus cell expansion and nuclear maturation following IVM. 5.3.3Spindle and chromosome organization.................. ............. 5.3.4 In vitro fertilisation and polyspermy................................. 5.3.5 In vitro embryo development following IVM/IVF...... ........... 5.3.6 Total cell numbers and apoptotic nuclei of in vitro produced blastocysts............................................................... 5.3.7 Chromosomal analysis of in vitro produced embryos...... ........ 5.4 Discussion........................ .......................................
    • CIIAPTER 6............ ............ ......... ......... ......... ......... ...... .......
    • 6.1 INTRODUCTION........................................................... ..
    • 6.2 MATERIALS AND METHODS...... ...... ...... ......... .................
    • 6.3 RESULTS....................................................................... 6.3.1 MPF/MAPK activities.................. ............... ...... ........... 6.3.2 Frequencies of cleavage following parthenogenetic activation.. 6.3.3 In vitro embryo development following parthenogenetic activation... .. . ... . .. . .... . ... .. . . .. . . .. .. .. . . .. . . . . . . .. . . .. . .. . . . . .... 6.3.4 Apoptosis in blastocysts produced following parthenogenetic activation........................................................... ... 6.3.5 Frequencies of enucleation and fusion....................... 6.3.6 Cleavage and subsequent development ofSCNT embryos....... 6.3.7 Total cell numbers and apoptosis in blastocysts produced by SCNT...................................................................... 6.3.8 Karyotyping ofSCNT embryos...... ............ ... ....... ........... 6.4 DISCUSSION.....................................................................
    • CIIAPTER 7 ....................................................................... II
    • ON VIABILITY AND SUBSEQUENT DEVELOPMENT................... 7.1 INTRODUCTION............................................................. 7.2 MATERIALS AND METHODS........................................... 7.3 RESULTS....................................................................... 7.3.1 Effect of CB pre-treatment on viability of vitrified COCs....... 7.3.2 Effect ofCB treatment on fertilisation events following IYF... 7.3.3Effect of CB treatment on in vitro embryo development following IYF........................ .................................
    • 7.4 DISCUSSION..................................................................
    • CIIAPTER 8............ ... ............ ........................ .....................
    • GERMINAL VESICLE (GV) STAGE........................................ 8.1 INTRODUCTION............................................................ 8.2 MATERIALS AND METHODS........................................... 8.3 RESULTS...................................................................... 8.3.1 Effect of caffeine treatment on MPF/MAP kinases activities. 8.3.2Effect of caffeine treatment on spindle and chromatin configuration......................................................... 8.3.3 Effect of caffeine treatment on frequency of cleavage following IYF........................................................ 8.3,4 Effect of caffeine treatment on subsequent development following IYF............ ...... ............ .................. ........ 8.4 DISCUSSION.................................................................
    • CIIAPTER 9........................................................................
    • STAGE.................................................................................. 9.1 INTRODUCTION............................................................... 9.2 MATERIALS AND METHODS............................................. 9.3 RESULTS......... .................. ......... ................................. ... 9.3.1 Effect of Deme pre-treatment on viability of vitrified oocytes.... 9.3.2 Timing of first polar body (PBI) extrusion........................... 9.3.3 Effect of Deme pre-treatment on spindle and chromosome organization of ovine oocytes vitrified at GV stage............ ... 9.3.4 Effect of Deme pre-treatment on in vitro embryo development following IVMIIVF/IVC of ovine oocytes vitrified at GV stage.. 9.3.5 Effect of Deme pre-treatment on parthenogenetic development of ovine oocytes vitrified at GV stage.....................................
    • 9.4 DISCUSSION....................................................................
    • Figure 1.2 Schematic representation of an oocyte/embryo (circle) during slow freezing, conventional vitrification, and ultrarapid vitrification.. ....
    • Figure 2.1 Vitrification of COCs using SSV.......................................
    • Figure 2.2 Cryoloop vitrification of immature ovine oocytes ...............
    • Figure 2.3 Production of oocyte somatic cell couplets from ovine oocyte vitrified at GV-stage... ......... ... ...... ............ ... ... ...............
    • Figure 3.1 Effects of exposure of immature ovine oocytes to different vitrification solutions on survival rates.........................
    • Figure 3.2 Cumulus cell expansion following IVM of ovine oocytes exposed to different vitrification solutions at GV-stage... ......... ............
    • Figure 3.3 Nuclear maturation of ovine oocytes...... ............ ...... .........
    • Figure 4.1 Recovery rates of ovine oocytes vitrified at GV-stage by Solid Surface Vitrification (SSV)............... ...... ... ...... .... ..... ......
    • Figure 4.2 Viability of ovine oocytes vitrified at GV-stage by Solid Surface Vitrification (SSV)....... ........... ............ ... ...... ...... ... ....
    • Figure 4.3 Degenerated oocyte recovered following SSV of ovine COCs....
    • Figure 4.4 Cumulus cell expansion following IVM of ovine oocytes vitrified at GV-stage using SSV .................. ...... ...... ......
    • Figure 4.5 Oocytes with expanded cumulus cells.................................
    • Figure 4.6 Meiotic maturation of ovine oocytes.............................. ...
    • Figure 4.7 Fertilisation events in ovine oocytes.................................
    • Figure4.8 Developmental stages of ovine embryos produced by IVF of ovine oocytes vitrified at GV-stage using SSV......... ......... ......
    • Figure 5.1 Recovery rates of ovine oocytes vitrified at GV-stage using the cryoloop ............................. ...................................................
    • Figure 5.2 Survival of ovine oocytes vitrified at GV-stage by the cryoloop.. ...
    • Figure 5.3 Damaged oocytes recovered following cryoloop vitrification
    • Figure 5.4 Cumulus cell expansion following IVM of ovine oocytes vitrified at GV-stage using cryoloop ..............................................
    • Figure 5.5 Oocytes with expanded cumulus cells................................ ...
    • Figure 5.6 Meiotic maturation of ovine oocytes .................................
    • Figure5.7 Examples of spindle and chromosome organization in ovine oocytes .................. ... ............... ............ ...... ......... .......
    • Figure 5.8 Fertilisation status of ovine oocytes following IVF................. ...
    • Figure 5.9 Developmental stages of ovine embryos produced by IVF of vitrified/ thawed GV-stage oocytes...... ......... ...... ... ................
    • Figure 5.10 Total cell numbers and apoptotic nuclei in day 7 embryos determined by Hoechst and TUNEL assay...... ...... ...................
    • Figure 5.11 Chromosomal analysis of day 7 in vitro produced blastocysts.. ....
    • Figure 6.1 Maturation promoting factor (MPF) and mitogen-activated protein kinase (MPAK) activities ....................................................
    • Figure 6.2 Representative blastocyst stage embryos stained with Hoechst and TUNEL assay...............................................................
    • Figure 6.3 Enucleation of ovine oocytes vitrified at GV-stage...... ...... .........
    • Figure 6.4 Day 7 blastocysts produced by SCNT................................. ...
    • Figure 7.1 Effects of CB pre-treatment on viability of vitrified/thawed ovine GV oocytes....................................................................
    • Figure 7.2 In vitro fertilisation of ovine oocytes...... ..............................
    • Figure 7.3 Day 7 blastocysts produced by IVF of ovine oocytes vitrified at GV stage and pre-treated with CB............ .... .... .... ..... .... .. .. ..
    • Figure 7.4 Total cell numbers in day 7 in vitro produced blastocysts............
    • Figure 8.1 Activation of MPF.........................................................
    • Figure 8.2 Effects of caffeine treatment on maturation promoting factor (MPF) and mitogen-activated protein kinase (MPAK) activities......
    • Figure 8.3 Spindle and chromatin configuration..................................
    • Figure 8.4 Day 7 ovine blastocysts produced by IVF...............................
    • Figure 9.1 Effect of demecolcine (Dcme) pre-treatment on viability of ovine oocytes vitrified at GV-stage................................................
    • Figure 9.2 Effect of demecolcine (Deme) pre-treatment on timing of first polar body extrusion (PBI) in ovine oocytcs vitrified at GV -stage....
    • Figure 9.3 Ovine oocyte showing first polar body (PBI) following 24 h IVM..
    • Figure 9.4 Spindle and chromatin configuration of ovine oocytes following 24IVM...........................................................................
    • Figure 9.5 Day 7 in vitro produced blastocysts......................................
    • Figure 10.1 The best conditions used for vitrification of immature ovine oocytes applied in this thesis...... ..........................................
    • Table 1.1 Mammalian species yielding normal offspring following the transfer of cryopreserved embryos to foster mother .............................. .
    • Table 1.2 Applications of oocyte cryopreservation ................................ .
    • Table 1.3 Comparison of the three approaches for cryopreservation of oocytes
    • Table 1.4 A comparison of vitrification with slow-cooling procedures ..........
    • Table 1.5Variables in vitrification that can profoundly influence its effectiveness ................................................................. .
    • Table 3.1 Effects of exposure of immature ovine oocytes to different vitrification solutions on survival rates .................................. ..
    • Table 3.2Effect of exposure to different vitrification solutions on subsequent nuclear maturation of immature ovine oocytes ........................ .
    • Table 4.1 Recovery rates and viability of ovine oocytes vitri fied at GV-stage bY S0 II'd Surf:ace VI"tnfiIcatl.On (SS V) .................................... .
    • Table 4.2 Nuclear maturation of ovine oocytes vitrified at GV-stage by SSV.
    • Table 4.3 Frequencies of in vitro fertilisation and polyspermy following IVMIIVF of ovine oocytes vitrified at GV-stage by SSV ..............
    • Table 4.4 First cleavage following IVM/IVF of ovine oocytes vitrified at GV stage .......................................................................... ..
    • Table 4.5 In vitro embryo development following IVF of vitrified/thawed ovine COCs using SSV .................................................... ..
    • Table 5.1 Recovery rates and viability of ovine oocytes vitrified at GVstage using cryoloop ..................................................... ..
    • Table 5.2 Nuclear maturation of ovine oocytes vitrified at GV-stage using cryoloop .................................................................... .
    • Table 5.3 Effect of vitrification of immature ovine oocytes on spindle and chromosome configuration following IVM ...................... ..
    • Table 5.4 In vitro fertilisation of ovine oocytes vitrified at GV-stage using cryoloop .................................................................. .
    • Table 5.5 Effects of cryoloop vitrification on survival rates after IVF ...... ..
    • Table 5.6 Developmental potential of ovine oocytes vitrified at GV-stage using cryoloop ............................................................. .
    • Table 5.7 Total cell numbers and apoptotic nuclei in day 7 blastocyst embryos ........................... · .. ··· .. · .. · ... · ... ····· .. ·· .... · ........ .
    • Table 5.8 Chromosomal composition of day 7 blastocysts produced by IVF of ovine oocytes vitri fied at GV-stage ................................ .
    • Table 6.1 Cleavage rates folIowing parthenogenetic activation of ovine oocytes vitrified at GV stage ............................................ .
    • Table 6.2 In vitro embryo development following parthenogenetic activation of ovine oocytes vitrified at GV stage .................................. ..
    • Table 6.3 Numbers of apoptotic nuclei and apoptotic index in day 7 blastocysts produced by Sr/CD parthenogenetic activation ......... EO
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