Mouse embryos generated from frozen-thawed oocytes can successfully survive a second cryopreservation

BACKGROUND: To determine whether mouse embryos generated from frozen-thawed oocytes can successfully survive a second cryopreservation. METHODS: Immature C57BL6*BALB/c female mice underwent superovulation and the collected oocytes were divided into three groups. Group A oocytes (n = 107) underwent IVF. Group B oocytes (n = 167) underwent IVF and embryos generated were then cryopreserved. Group C oocytes (n = 94) were cryopreserved, thawed and underwent IVF. Two-four-cell stage embryos were re-cryopreserved and thawed. Embryos from all groups were then cultured to the blastocyst stage. RESULTS: Cleavage rates to the 2-4-cell stage were 78, 71 and 46% for groups A, B and C respectively. Blastulation rates from 2-4 cell-stage embryos were 37/83 (45%), 27/118 (23%) and 8/35 (23%) for groups A, B and C respectively. Development to blastocysts was observed in 37/107 oocytes (35%), 27/167 oocytes (16%) and only 8/94 oocytes (9%) for groups A, B and C respectively. CONCLUSION: Oocyte cryopreservation results in reduced fertilization rates. Embryo cryopreservation reduces blastulation rates by half regardless of whether the oocytes were fertilized fresh or frozen-thawed. Nevertheless, embryos generated from cryopreserved oocytes can survive cryopreservation and develop to the blastocyst stage at rates comparable with embryos obtained from fresh oocytes.     

The developmental potential of mouse oocytes matured in serum-free culture conditions

Studies were undertaken to identify serum-free conditions forthe maturation of mouse oocytes in vitro. Oocytes were recoveredfrom the antral follicles of juvenile mice 48 h after injectionwith gonadotrophin and allowed to resume meiosis in modifiedHam's F-10 (mHF-10) medium unsupplemented or supplemented withbovine serum albumin (BSA), fetal calf serum, human pre-ovulatoryserum, human follicular fluid or EDTA. They were inseminated14–16 h later, scored for polar body extrusion after 4–6h with spermatozoa, and transferred to protein-free mHF-10 forfurther development. In-vivo matured ova were inseminated andcultured in parallel as controls. Fertilization and developmentwere scored as two cells 24 h after insemination and blastocysts4 days following insemination respectively. Surprisingly, 41%of oocytes cultured in unsupplemented mHF-10 completed meiosisI, and of those, 50% fertilized; serum supplementation did notimprove maturation or fertilization rates. Although the additionof human follicular fluid to the mHF-10 improved meiosis (69%)and fertilization (68% of eggs with polar bodies) to levelscomparable with the in-vivo control eggs (79 and 66% respectively),BSA supplementation was equally beneficial. Blastocyst developmentvaried, but within each maturation/ fertilization group, thedevelopment from in-vitro matured eggs was comparable with embryosfrom in-vivo matured eggs. In addition, two out of eight 4-cellembryos from oocytes cultured in mHF-10 with BSA and EDTA gaverise to apparently normal pups following transfer to pseudopregnantrecipients. Thus, gonadotrophin-stimulated mouse oocytes cancomplete meiosis and fertilize in culture in the absence ofserum or follicular fluid. Oocytes cultured overnight in mHF-10,supplemented with EDTA and BSA, complete meiosis I, fertilizeand develop to blastocysts at rates comparable with eggs maturedin vivo. Serum-deprived oocytes have the potential to give riseto live offspring.     

Fertilization and early embryology: Cryopreservation of immature mouse oocytes

Mouse oocytes enclosed in cumulus cells were isolated from antralfollicles at the germinal vesicle (GV) stage. They were storedin straws at – 196°C by a conventional mouse embryofreezing method using dimethylsulphoxide (1.5 M) as the cryoprotectant.Overall survival assessed after removal of the cumulus cellswas 93% (299/320). A significantly greater proportion of freshoocytes remained arrested at the GV stage during culture (11versus 1%), but the rate of maturation to metaphase II was notsignificantly different between frozen and fresh oocytes (83versus 74%). The rate of fertilization in vitro was similarfor frozen and fresh oocytes matured in vitro (70 versus 81%)but significantly less than with mature ovulated oocytes (96%).Fertilization of frozen and fresh oocytes arrested after germinalvesicle breakdown was similar (77 versus 95%. No evidence ofparthenogenetic activation was found in the different groupsafter overnight incubation of metaphase II oocytes. Implantationwas similar for embryos derived from fresh and frozen GV-stageoocytes matured in vitro and mature ovulated oocytes, but theloss of embryos after implantation was significantly higherin the in-vitro matured groups (frozen, 40% and fresh, 46% versus24%). The overall survival of oocytes frozen at the GV stagewas 27%. This compares favourably with the estimated overallsurvival of mature oocytes cryopreserved by a similar procedure.We conclude that the increased post-implantation loss is dueto suboptimal conditions for maturation in vitro rather thanfreezing injury.     

Use of fetal bovine serum to protect against zona hardening during preparation of mouse oocytes for cryopreservation.

Addition of 20% fetal bovine serum (FBS) to media used for cryopreservation does not reduce the premature release of cortical granules but does prevent their action on the zona pellucida and thereby prevents zona hardening. In this paper, it is shown that the washing period required for removal of FBS is less than 12 min for cumulus-free oocytes and between 150 and 170 min for cumulus-intact oocytes. When these washing periods are observed after exposure of oocytes to the cryoprotectant dimethylsulphoxide (DMSO; 1.5 M) in the presence of 20% FBS, a subsequent exposure to calcium ionophore A23187 or a second exposure to 1.5 M DMSO both lead to zona hardening. This result suggests that sufficient cortical granules remain to elicit a block to polyspermy at fertilization. Oocytes, which had been exposed to DMSO and FBS and then washed free of both, were fertilized in vitro; the incidence of polyspermy was not found to be elevated over the level found in controls.     

Effects of sucrose concentration on the developmental potential of human frozen-thawed oocytes at different stages of maturity

BACKGROUND: Success of human oocyte cryopreservation depends on multiple cryobiological factors that could influence the developmental potential of the oocytes. The objective of this study was to examine the effects of different sucrose concentrations on the developmental potential of human frozen-thawed oocytes at different maturity stages. METHODS: A total of 355 oocytes collected from small follicles were randomly divided into three groups and two groups (B and C) were cryopreserved using slow-freezing method. Group A included 131 oocytes at different maturity stages without freezing. Another 119 oocytes in Group B were cryopreserved with 0.1 M sucrose and 105 oocytes in Group C with 0.2 M sucrose concentration. RESULTS: The post-thaw survival rate of the oocytes and the cleavage rate in Group C were significantly higher than that of Group B (P<0.05). For immature metaphase I (MI) stage oocytes, a significant difference was found in the maturation rate between Group C and Group B (P<0.05). The maturation rate for the GV oocytes in Groups A and C was significantly higher than Group B (P<0.01). CONCLUSIONS: The results suggested that sucrose concentration of 0.2 M in the cryoprotectant solution is more suitable for human oocyte cryopreservation.     

Cytoskeletal organization and zona sensitivity to digestion by chymotrypsin of frozen--thawed mouse oocytes

Mouse oocyte—cumulus masses were added to 1.5 dimethylsulphoxide (DMSO) + 20% fetal bovine serum (FBS) that had beenprecooled at +4°C, were frozen by slow cooling to an intermediatetemperature of –60°C before being plunged into liquidnitrogen at –196°C, subjected to a controlled thaw,expelled into 1.5 M DMSO + FBS at 4°C, and then washed inmedium + FBS at 37°C. Of 7733 oocytes treated, 78.4% wereviable (controls; no treatment: 94.2% of 2764 oocytes; cryoprotectantonly: 92.2% of 2991 oocytes). The oocyte losses were not dueto complete loss of all oocytes from some straws or mice, sinceanalysis of individual straws containing oocytes from a singlemouse revealed considerable inter-straw/mouse variation. Amongstsurviving oocytes, no significant differences between frozenand control oocytes in spindle, chromosomal or microfilamentorganization were recorded. Two significant differences wereobserved: (i) fewer frozen—thawed oocytes had zonae resistantto chymotrypsin digestion, and (ii) spindle organization incontrol oocytes, but not frozen—thawed oocytes, was improvedby 3 h incubation at 37°C. More of the abnormal than thenormal frozen—thawed and control oocytes were surroundedby zonae which were resistant to digestion by chymotrypsin.     

Cryopreservation of metaphase II human oocytes effects mitochondrial membrane potential: implications for developmental competence

BACKGROUND: Current outcome results with embryos derived from thawed MII human oocyes are significantly lower than with embryos cryopreserved at the pronuclear stage. Here, we investigated whether freezing-thawing was associated with changes in oocyte mitochondrial polarity (DeltaPsim) that could influence competence by altering ATP levels or the ability of the cytoplasm to regulate intracellular Ca2+. METHODS: Fresh and thawed uninseminated and unfertilized MII oocytes were stained with the DeltaPsim-specific probe JC-1 to detect clusters of high-polarized mitochondria (J-aggregate positive) and with the Ca2+- specific probe Fluo-4 to measure changes in intracellular levels of this cation. ATP content per oocyte was measured directly and cortical granules were visualized with a cortical granule-specific probe. RESULTS: A significant difference between fresh and thawed MII oocytes existed for pericortical J-aggregate fluorescence and for the ability of the cytoplasm to increase free Ca2+ in response to ionophore exposure. No significant difference in ATP contents was measured and cryopreservation was not associated with an apparent release of cortical granules. CONCLUSION: Irreversible loss of high DeltaPsim in thawed oocytes may be associated with defects in Ca2+ signalling after insemination and could have downstream consequences for normal embryogenesis.     

Epidermal growth factor enhances preimplantation developmental competence of maturing mouse oocytes

The objective of this study was to determine whether epidermal growth factor (EGF) promotes nuclear and cytoplasmic maturation of mouse oocytes grown in vivo or in vitro. In-vivo-grown oocytes were isolated at the germinal vesicle (GV) stage from gonadotrophin-primed (PR) or -unprimed (UPR) 22-day-old mice before in-vitro maturation (IVM). In-vitro-grown (IVG) oocytes were isolated from preantral follicles of 12-day-old mice and grown in vitro without gonadotrophins for 10 days before maturation (IVG/IVM oocytes). IVM and IVG/IVM oocytes were matured in medium supplemented with either EGF (10 ng/ml), follicle stimulating hormone (FSH) (100 ng/ml), EGF plus FSH, or with neither ligand (control). When oocyte-cumulus cell complexes were isolated from PR and UPR mice, IVM with EGF (10 ng/ml), alone or in combination with FSH (100 ng/ml), increased (P < 0.05) the incidence of nuclear maturation to metaphase II. Cytoplasmic maturation of oocytes from PR females, manifested as increased frequency of cleavage to the 2-cell stage and development to the blastocyst stage, was also enhanced with EGF (P < 0.05). Moreover, EGF increased the number of cells per blastocyst, but only in the absence of FSH (P < 0.01). In contrast, EGF, FSH, or EGF plus FSH did not affect the percentage of oocytes from UPR mice completing preimplantation development, but did increase the number of cells per blastocyst. These ligands also increased the proportion of IVG oocytes reaching metaphase II (53-57%) compared with controls (25%; P < 0.05). EGF alone or in combination with FSH increased (P < 0.05) the frequency of blastocyst formation (23% and 28%, respectively) compared with controls (13%). EGF treatment of maturing IVG oocytes produced blastocysts with more cells than other IVG groups (P < 0.05). It is concluded that gonadotrophins in vivo increase the sensitivity or responsiveness of cumulus cell-enclosed oocytes to EGF, thereby promoting both nuclear and cytoplasmic maturation. However, oocyte-granulosa cell complexes grown in vitro become responsive to EGF without gonadotrophin treatment. Thus, nuclear and cytoplasmic maturation of IVG oocytes is promoted by EGF treatment during meiotic maturation.     

Rescue of oocytes from antral follicles of cryopreserved mouse ovaries: competence to undergo maturation, embryogenesis, and development to term

Only primordial and primary follicles of frozen-thawed mouse ovaries survive after grafting to the ovarian bursa; large secondary follicles and antral follicles together with the oocytes contained in them degenerate. This study was undertaken to determine whether fully grown oocytes isolated from the antral follicles of frozen-thawed mouse ovaries are viable and can be rescued to undergo maturation, fertilization, and embryo development in vitro. Ovaries were cryopreserved after removal from 22-day-old (C57BL/6J x SJL/J)F(1) mice, with or without prior priming with equine chorionic gonadotrophin, and fresh non-frozen ovaries were used as controls. Only cumulus cell-denuded oocytes were recovered from frozen unprimed ovaries while both cumulus cell-enclosed and denuded oocytes were retrieved from frozen primed ovaries. Oocytes from both groups of frozen-thawed ovaries were able to undergo maturation, fertilization, and development to the blastocyst stage in vitro, though at lower percentages than oocytes from control unfrozen ovaries. Moreover, 19% of 2-cell stage embryos derived from frozen-thawed primed ovaries, compared with 42% of embryos derived from control primed ovaries, developed to term after transfer to pseudopregnant foster mothers (not significantly different). Therefore, fully grown oocytes in antral follicles survive the cryopreservation protocol, as demonstrated by maturation, fertilization and embryo development in vitro, and development to term after embryo transfer.     

Assessment of ultrarapid and slow freezing procedures for 1-cell and 4-cell mouse embryos

Three cryopreservation procedures were assessed for the freezingof mouse 1-cell and 4-cell embryos: the slow freezing protocolswith dimethylsulphoxide (DMSO, A) and propanediol (PROH, B)and the ultrarapid procedure with DMSO (C), which was describedby the Monash University group [A.Trounson et al. (1987) Fertil.Steril., 48, 843–850]. The evaluation of the differentprocedures included survival after freezing and thawing, furtherdevelopment after in-vitro culture to blastocysts and the abilityto implant and to form living fetuses after transfer of earlyblastocysts to pseudopregnant mice. In-vitro development waslower in all frozen embryos than in the unfrozen controls. ProceduresA and B induced comparable results and were significantly betterthan ultrarapid freezing. When unfrozen blastocysts were transferredto pseudopregnant mice, 64% of them implanted in the uterinewall and 59% developed to living fetuses. For zygotes the percentagesof implantation sites and living fetuses were 47 and 33% forA, 52 and 44% for B, and 29 and 17% for C, respectively. When4-cell embryos were cryopreserved, these results were 54 and46% for A, 60 and 53% for B and 37 and 23% for C, respectively.In the ultrarapid procedure we also looked at the influenceof the freezing solution. To set up a control, mouse zygotesand 4-cell embryos were exposed to DMSO as in the ultrarapidprocedure except that they were not frozen; the survival andblastocyst formation was not different from controls, but fewerliving fetuses were born (31% for zygotes and 41% for 4-cellembryos versus 67% in the controls). This study demonstratedthat slow freezing with 1.5 M DMSO or 1.5 M PROH yielded betterresults than the ultrarapid method with 3.5 M DMSO.     

Assessment of the integrity of human oocytes retrieved from cryopreserved ovarian tissue after xenotransplantation

BACKGROUND: Previous studies showed that immature oocytes stored in ovarian tissue could develop to the mature stage after transplantation. However, the quality and competency of the oocytes developed in xenografted ovarian tissue have never been investigated. As a pilot study to investigate this uncharted issue, we evaluated microtubule organization and chromatin configuration of human oocytes harvested from xenografted frozen-thawed ovarian tissue. METHODS: Frozen-thawed human ovarian tissue was transplanted into severe combined immunodeficient mice. All animals were stimulated with gonadotrophin from 20 weeks after transplantation. Grafts were recovered 36 h after hCG administration. The oocytes were retrieved from the antral follicles (>2 mm diameter), cultured in vitro, stained for microtubule and chromatin localization. RESULTS: Five oocytes from 21 female mice and seven oocytes from nine male mice were retrieved. Immunocytochemical examinations of these oocytes after in vitro maturation revealed only two developed to the metaphase II stage. Most oocytes were between prophase and metaphase with abnormal microtubule organization and chromatin configuration. CONCLUSIONS: Immature oocytes in stored human ovarian tissue can grow to maturity in host animals after xenotransplantation. Retrieval of oocytes from the xenograft can be carried out and is reproducible. However, many oocytes, grown in host animals and further matured in vitro, showed aberrant microtubule organization and chromatin patterns.     

Fertilization and early empryology: Use of fetal bovine serum substitutes for the protection of the mouse zona pellucida against hardening during cryoprotectant addition

The addition of 20% fetal bovine serum (FBS) to media used formouse oocyte cryopreservation prevents hardening of the zonapellucida that otherwise can occur due to premature releaseof cortical granule contents (George et al., Hum. Reprod., 7,401–412, 1992). Protection of human oocytes would ideallybe achieved by using a human macromolecular source or a moredefined bovine source than total FBS. Here we investigate whetherFBS can be replaced by human serum, human cord serum, humanserum albumin or fetuin, the major protein component of FBS.Only fetuin was found to be effective.     

Nuclear competence for maturation and pronuclear formation in mouse oocytes

BACKGROUND: In response to gonadotrophins, a fully grown mouse oocyte matures to the metaphase of the second meiotic division and becomes competent for the development of female and male pronuclei after fertilization. The present study was carried out to clarify when during the growth period an oocyte nucleus acquires the ability to promote pronuclei formation after fertilization. METHODS: Fully grown germinal vesicle (GV) oocytes were enucleated and fused with nuclei from growing oocytes from 1-20 day old mice by standard nuclear transfer technique. The reconstructed oocytes were matured and fertilized in vitro, and pronuclear formation was assessed. RESULTS: The oocytes whose nuclei were exchanged for those of the non-growing-stage oocytes matured to the metaphase of the second meiotic stage, but no normal female pronuclei were formed. Female pronuclei first formed in 27% of the oocytes reconstituted with the nuclei of oocytes from 8 day old pups after fertilization. Recondensed sperm chromatin was detected in 27% of the oocytes reconstructed with oocyte nuclei from 8 day old mice, and a male pronucleus was first formed in 6% of the oocytes that had been reconstructed with the nuclei of oocytes from 15 day old mice. The sizes of the female and male pronuclei increased with oocyte donor age, and reached normal size when the oocytes from 15 and 20 day old mice respectively were used. An electron microscopic study using oocytes that had received the oocyte nuclei of 8 day old mice confirmed these results. CONCLUSION: The factors required for pronuclear formation are derived from fully grown GV oocytes, and the transformation from decondensed sperm chromatin to a recondensed male pronucleus is governed by GV-derived factors.     

Recovery, survival and functional evaluation by transplantation of frozen-thawed mouse germ cells

BACKGROUND: Establishing a successful method for testicular stem cell transplantation of frozen-thawed testicular cells would be of immense benefit to boys with childhood cancer undergoing a sterilizing treatment. In this study, we evaluated different cryopreservation protocols in a mouse model by means of testicular germ cell transplantation (TGCT), in order to establish an optimal freezing protocol. METHODS AND RESULTS: In a first series of experiments, we compared an uncontrolled protocol with 1.5 mol/l dimethyl sulphoxide (DMSO) versus a controlled long protocol (cooling to -80 degrees C) and observed a better viability with the latter protocol (36% versus 48%, P < 0.05). We then compared survival after two thawing methods (37 degrees C water versus ice water) in either a DMSO- or an ethylene glycol (EG)-based protocol, and found no difference. In order to evaluate the functional capacity of the cryopreserved testicular suspension, TGCT was performed with both fresh and frozen-thawed suspensions. In 90% of the successfully injected testes, spermatogenesis was reinitiated using fresh suspensions. In contrast, this figure was only 12.5 and 22.7% after cryopreservation, for the short controlled EG protocol and the uncontrolled DMSO protocol, respectively. CONCLUSION: Reinitiation of spermatogenesis is possible after cryopreservation of testicular germ cell suspensions. Although cell survival was acceptable, our results after TGCT show that our protocols need further improvement.     

Effect of 1,2-propanediol and dimethylsulphoxide on the meiotic spindle of the mouse oocyte

At ovulation, the mouse oocyte is arrested at metaphase of thesecond meiotic division. Since microtubules are thermo-and chemosensitivestructures, the effects of 1.5 M dlmethylsulphoxide and 1.5M 1,2-propanediol were studied at room temperature on the morphologyof the meiotic spindle. Oocytes incubated at 37°C or atroom temperature served to estimate the effect of temperaturein the experiment. The meiotic spindle was visualized by immunogold-silverstaining of microtubules. In the control group at 37°C,88% of oocytes had normal spindles. After incubation at roomtemperature for the same time, 89% of oocytes showed abnormalspindles. In the oocytes exposed to dimethyl sulphoxide or 1,2-propanediolat room temperature a protective effect on spindle morphologycould be recognized. Subsequent incubation at 37°C resultedin partial restoration of the observed abnormalities after coolingto room temperature and after exposure to dhnethytsulphoxide.incubation at 37°C after exposure to 1,2-propanediol atroom temperature induced spindle absence in the majority ofoocytes. Although this latter condition allowed fertilizationwithout increased incidence of ploidy abnormalities, a rolefor 1 as an activating agent is hypothesized.     

The cortical reaction and development of activation competence in mammalian oocytes

Blocks to polyspermic fertilization are necessary to preventthe incorporation of two sperm nuclei into a zygote's genome,which would result in abnormal development. Many mammalian eggsutilize both an extracellular zona pellucida block to polyspermyand a plasma membrane block. Although little is known aboutthe plasma membrane block in mammals, fertilization resultsin zona glycoprotein modifications caused by enzymes releasedby the egg and its cortical granules (CG). This article reviewsother recent investigations demonstrating that the oocyte'sability to cause CG release and the block to polyspermy developsnear the time of ovulation. The development of normal 'activationcompetence' is likely to involve preovulatory changes in theoocyte's ability to signal the release of intracellular calciumas well as to respond to this calcium increase, resulting inCG exocytosis. Because normal activatin competence appears tohave a brief temporal window after oocyte meiotic maturationis resumed and since the oocytes are collected at various stagesin assisted reproductive procedures, these studies are relevantto optimizing clinical success.     

Cryopreservation of mouse and human oocytes using 1, 2-propanediol and the configuration of the meiotic spindle

Human and mouse oocytes were cryopreserved by a slow freeze,rapid thaw method, using propanediol (PROH) as the cryoprotectant.A simulated cryopreservation was also included in the studyto detect the level of damage attributable to the PROH alone.Comparison of the mouse and human oocytes cryopreserved by thesame method showed opposing results, with a poor morphologicalsurvival rate of 4% observed for mouse oocytes and a subsequentnormal fertilization rate of 0%. In 171 cryopreserved humanoocytes a higher survival rate of 64% was achieved, and thisshowed more similarity to the mouse pronuclear oocytes survivalof 53%. A comparison of human oocytes, cryopreserved withinthe cumulus and denuded of cumulus and corona prior to cryopreservation,demonstrated a higher survival rate in the denuded oocytes of69% compared to 48%. A delay prior to cryopreservation of 1or 2 days had no effect on the immediate survival of oocytes,but culture for a further 24 h after thawing reduced survival,with the day 1 oocytes exhibiting the most dramatic reductionin survival (28%). Using a lectin binding method, abundant corticalgranules were observed in all cryopreserved oocytes analysed.The meiotic spindle and chromosomes were examined in cryopreservedoocytes using fluorescence microscopy and 60% of the survivingoocytes had a normal spindle and chromosome configuration.     

Permeability characteristics of human oocytes in the presence of the cryoprotectant dimethylsulphoxide.

Equilibration of oocytes with cryoprotectants is a prerequisite of low temperature storage. However, cryoprotectant exposure may induce damage via osmotic stress. Knowledge of cell membrane permeability characteristics and their temperature dependence would facilitate the design of cryopreservation protocols in which osmotic stress is minimized and the incidence of intracellular freezing is reduced. To obtain such data, the volume change of donated human oocytes following exposure to cryoprotectant was measured at a variety of temperatures. After removal of cumulus cells, each oocyte was placed in a 5 microl droplet of phosphate-buffered medium. The oocyte was held in position by suction generated using a fine pipette and perfused with 1 ml 1.5 mol/l dimethylsulphoxide (DMSO) at 30, 24 or 10 degrees C. The volume of the oocyte before, during and after perfusion was recorded by videomicroscopy. Oocyte volume was calculated from radius measurements and the Kedem-Katchalsky (K-K) passive coupled transport coefficients, namely L(p) (hydraulic permeability), P(DMSO) (permeability to DMSO) and sigma (reflection coefficient) were derived. The resulting coefficients were L(p) = 1. 65 +/- 0.15, 0.70 +/- 0.06 and 0.28 +/- 0.04 microm/min.atm; P(DMSO) = 0.79 +/- 0.10, 0.25 +/- 0.04 and 0.06 +/- 0.01 microm/s and sigma = 0.97 +/- 0.01, 0.94 +/- 0.03 and 0.96 +/- 0.01 at 30, 24 and 10 degrees C respectively. The activation energy for L(p) was 14.70 and for P(DMSO) was 20.82 kcal/mol. The permeability parameters of human oocytes are higher than those of murine oocytes, suggesting that they require a shorter period of exposure to DMSO with concomitantly reduced toxic effects.     

Cryoprotection of human oocytes: inappropriate exposure to DMSO reduces fertilization rates

Human oocytes were exposed to the cryoprotectant dimethyl-sulphoxide (DMSO) at either 4 or 37 degrees C. Subsequent fertilization of these oocytes showed that exposure to DMSO at 37 degrees C was associated with a greatly reduced fertilization rate when compared to untreated control oocytes, whereas no such reduction was seen in oocytes exposed to DMSO at 4 degrees C. The significance of these results for the potential cryopreservation of human oocytes is discussed.     

Germinal vesicle transfer between fresh and cryopreserved immature mouse oocytes.

BACKGROUND: We assessed the maturational competence and the chromosomal pattern of mouse oocytes reconstructed by germinal vesicle (GV) transfer technique using nuclear and/or cytoplasmic components from cryopreserved GV stage oocytes. METHODS: From 657 GV oocytes (326 fresh and 331 frozen/thawed), four groups of reconstructed oocytes were obtained by micromanipulation and electrofusion: fresh GV-fresh cytoplast (FF), thawed GV-thawed cytoplast (TT), fresh GV-thawed cytoplast (FT), thawed GV-fresh cytoplast (TF). All reconstructed oocytes were cultured in vitro to metaphase II. RESULTS: Survival rate after manipulation and electrofusion, as well as progression to metaphase II, did not differ significantly among the four groups. Comparing reconstructed oocytes with fresh and thawed control pools, the only difference was a slightly but significantly higher maturation rate in the TT pool versus matched controls (P < 0.01). Cytogenetic analysis of 25 reconstructed oocytes showed the expected number of 20 chromosomes in 88% of them. CONCLUSIONS: We conclude that both nuclear and cytoplasmic components derived from cryopreserved immature oocytes are suitable for GV transfer procedure, and generate chromosomally normal oocytes able to progress to metaphase II in vitro. The possibility of using cryostored immature oocytes as a source of nuclei and cytoplasm could help in applying GV transfer procedure, both in research and clinical settings.