Immature Mouse Oocytes Research Articles

Artificial expression of aquaporin-3 improves the survival of mouse oocytes after cryopreservation.

Successful cryopreservation of mammalian cells requires rapid transport of water and cryoprotective solutes across the plasma membrane. Aquaporin-3 is known as a water/solute channel that can transport water and neutral solutes such as glycerol. In this study we examined whether artificial expression of aquaporin-3 in mouse oocytes can improve water and glycerol permeability and oocyte survival after cryopreservation. Immature mouse oocytes were injected with aquaporin-3 cRNA and were cultured for 12 h. Then the hydraulic conductivity (L(P)) and glycerol permeability (P(GLY)) of matured oocytes were determined from the relative volume changes in 10% glycerol in PB1 medium at 25 degrees C. Mean +/- SD values of L(P) and P(GLY) of cRNA-injected oocytes (3.09 +/- 1.22 micro m min(-1) atm(-1) and 3.69 +/- 1.47 x 10(-3) cm/min, respectively; numbers of oocytes = 25) were significantly higher than those of noninjected oocytes (0.83 +/- 0.02 micro m min(-1) atm(-1) and 0.07 +/- 0.02 x 10(-3) cm/min, respectively; n = 13) and water-injected oocytes (0.87 +/- 0.10 micro m min(-1) atm(-1) and 0.08 +/- 0.02 x 10(-3) cm/min, respectively; n = 20). After cryopreservation in a glycerol-based solution, 74% of cRNA-injected oocytes (n = 27) survived as assessed by their morphological appearance, whereas none of the water-injected oocytes survived (n = 10). When cRNA-injected oocytes that survived cryopreservation were inseminated in vitro, the penetration rate was 40% (n = 48) and the cleavage rate was 31% (n = 70), showing that oocytes retain their ability to be fertilized. This is the first report to show that artificial expression of a water/solute channel in a cell improves its survival after cryopreservation. This approach may enable cryopreservation of cells that have been difficult to cryopreserve.

Neurotrophin-4/5 and neurotrophin-3 are present within the human ovarian follicle but appear to have different paracrine/autocrine functions.

Neurotrophins are a family of soluble polypeptide growth factors widely recognized for their role in the mammalian nervous system. We first reported the unique presence of one neurotrophin, brain-derived neurotrophic factor (BDNF), in the follicular fluid of the human ovarian follicle. The BDNF receptor, Trk B, was identified in mouse oocytes, and BDNF accelerated first polar body extrusion in vitro. In the present study, we examined human follicular fluid and mouse immature oocytes to determine whether another Trk B ligand, neurotrophin-4/5 (NT-4/5), is present within the ovarian follicle and if so, whether it demonstrates activity similar to that of BDNF. We also examined whether a non-Trk B neurotrophin ligand, neurotrophin-3 (NT-3), is present within the follicle and has a possible role in oocyte maturation. NT-4/5 and NT-3 were noted to be present in all human follicular fluid samples aspirated from follicles of women undergoing in vitro fertilization. NT-4/5, but not NT-3, significantly promoted mouse oocyte polar body extrusion. Trk C receptors were not noted to be present in mouse oocytes. This study demonstrates for the first time that NT-4/5 and NT-3 are present in the follicular fluid of the human ovary. These data suggest that NT-4/5, like BDNF, promotes oocyte nuclear maturation. In contrast, NT-3 does not promote oocyte maturation but may contribute to follicle-oocyte maturation by mechanisms not yet identified.

TACC3 expression and localization in the murine egg and ovary.

A protein spot cored from a silver-stained two dimensional (2D) gel of germinal vesicle stage immature mouse oocytes was identified as Transforming Acidic Coiled Coil containing protein (TACC3) by tandem mass spectrometry. PCR amplification revealed two alternatively spliced forms, Tacc3a and Tacc3b, in mouse ovarian cDNA libraries. TACC3a encoded a 630 aa protein with a predicted mass of 70 kDa. It contained seven 24 aa repeats at the N-terminus and two coiled-coil domains at the C-terminus. TACC3b encoded a 426 aa protein with a predicted mass of 49 kDa also containing two coiled coil domains, but lacking the 168 aa repeat region. In addition to homology to the TACC family members, murine TACC3 also showed 35.7% identity to the Xenopus protein, Maskin, a cytoplasmic polyadenylation element binding protein (CPEB)-associated factor. Northern blot analysis demonstrated that TACC3a is abundantly expressed in adult testis and spleen and is moderately expressed in the ovary, heart, and lung, suggesting a wide tissue distribution. Both myc-tagged TACC3a and TACC3b targeted to the cytoplasm of transiently transfected CV-1 cells. In situ hybridization of mouse ovarian tissue sections displayed abundant expression of TACC3 specifically in the cytoplasm of growing oocytes, but not in primordial or atretic follicles. This pattern of expression suggests that TACC3 is expressed in ovarian cells undergoing active growth and development.

Vitrification of ICR mouse immature oocytes in a straw to prevent the risk of liquid nitrogen contamination during storage

Objective: Selection of an oocyte cryopreservation method is a prerequisite factor for developing an effective bank system. In previous study, we had examined the effect of vitrification with ethylene glycol (EG)-based cryoprotectants on mouse oocytes in grid, and found higher post-thawed survival and subsequent embryo development than conventional slow freezing methods. The disadvantage of vitrification by using grids is that oocytes with the vitrification solution directly contact with liquid nitrogen during cooling or storage. To protect the risk of LN2 contamination, this study was conducted to develop effective vitrification method by using straws.

Somatic and embryonic cell nucleus transfer into intact and enucleated immature mouse oocytes.

The aim of our study was to evaluate the possibility of embryonic or somatic cell haploidization after fusion with intact or enucleated immature oocytes which were subsequently cultured in vitro. Embryonic or somatic cell nuclei do not undergo premature chromosome condensation when fused to intact or enucleated immature oocytes whose maturation is prevented by dibutyryl cyclic AMP (dbcAMP). The presence of dbcAMP permits, however, the completion of DNA replication in somatic cell nuclei. The chromosomes condensed when the reconstructed cells were released from the dbcAMP block. When somatic or embryonic nuclei were introduced into intact immature meiotically competent oocytes and subsequently cultured their chromosomes assembled on a common spindle with meiotic chromosomes and proceeded through the meiotic-like division, judged according to the presence of the first polar body extruded. When embryonic cell nuclei were introduced into cytoplasts obtained from immature meiotically competent oocytes, polar bodies were extruded in about 75% of reconstructed cells but the metaphase plates were abnormal in almost all cases. When somatic cell nuclei were inserted into the above cytoplasts, polar bodies were extruded only very exceptionally and in these cells chromosomes were arranged in abortive metaphase plates. Our results suggest that somatic cell nuclei are unable to proceed through the reduction division (haploidization) when introduced into an immature oocyte meiotic cytoplasm.

Nuclear transfer can accelerate nuclear maturation of immature mouse oocytes derived from preantral follicles.

Objective: It has been demonstrated that transferring nuclei of GV oocytes to fully competent cytoplasts can induce meioses. The object of this study was to test whether transferring nuclei taken from immature oocytes derived from preantral follicles and transferred into cytoplasts can induce a similar pattern of meioses and participate in embryogenesis.

Normal live birth produced from preovulatory mouse oocytes reconstructed by germinal vesicle transfer.

Objective: The germinal vesicle (GV) of an immature mouse oocyte can undergo maturation to metaphase II (MII) in heterocytoplasm of same developmental stage and, upon artificial activation, transform into a haploid pronucleus (PN). In this study, we have coupled GV transfer with MII spindle and PN transfer to assess the developmental competence of the transferred GV to support embryonic growth to term.

Germinal Vesicle Transfer Between Fresh and Cryopreserved Immature Mouse Oocytes

Objective: Germinal vesicle (GV) transfer offers the potential to assess and rescue genomic maturation in mammalian oocytes. However, as a clinical or research procedure, this technique presents a major logistic problem to ensure that germinal vesicle oocytes are obtained in the same cell cycle stage. This problem could be eliminated if cryostored, immature oocytes are shown to be appropriate for use as GV donors in the transfer procedure. Design: Genomic maturation, as judged by first polar body extrusion, was assessed in mouse oocytes reconstructed by GV transfer using nuclear and/or cytoplasmic components from cryopreserved oocytes. Materials and Methods: Female mice (CD-1 strain) were treated with PMSG (10 IU ip). The mice were killed at 36–48 h post injection and the eggs harvested by follicular puncture. Oocytes were frozen in propanediol-sucrose using routine slow embryo freezing and rapid thawing protocols. GV transfer was performed to exchange nuclei between fresh and frozen oocytes; the transfer and electrofusion protocols have been described previously (Liu et al, Human Reproduction 14:2357, 1999). Throughout these procedures, the oocytes were treated with isobutyl-methylxanthine to block meiotic progression. Following electrofusion the eggs were cultured in HTF containing 10% fetal bovine serum and monitored between 16–20 h for first polar body extrusion. Staining with Hoechst 3342 revealed DNA in both oocyte and polar body. Results: legendPreparationlegendThe survival rate for the freeze-thaw procedures was 76% (679/890).Electrofusion rateMaturation rateFresh GV-fresh cytoplasm41% (55/135)80% (44/55)Frozen GV-frozen cytoplasm41% (57/138)91% (52/57)Fresh GV-frozen cytoplasm51% (73/143)86% (63/73)Frozen GV-fresh cytoplasm40% (49/124)78% (38/49)Fresh control (no micromanipulation)–77% (167/218)Fresh control (electrofusion pulses only)–86% (36/42)Frozen control (no micromanipulation)–79% (216/275)legend The survival rate for the freeze-thaw procedures was 76% (679/890). Open table in a new tab Conclusions: Immature oocytes reconstructed by GV transfer with nuclei or cytoplasm from frozen eggs show comparable electrofusion and maturation rates as non-manipulated fresh oocytes. These results suggest cryobanked immature oocytes may be suitable for use in research or clinical GV transfer procedures.

Na+-Ca2+ exchange in mouse oocytes: modifications in the regulation of intracellular free Ca2+ during oocyte maturation

Increases in intracellular free Ca(2+)+ concentration (Ca(2+)+ oscillations) occur during meiotic maturation and fertilization of mammalian oocytes but little is known about the mechanisms of Ca(2+) homeostasis in these cells. Cells extrude Ca(2+) from the cytosol using two main transport processes, the Ca(2+)-ATPase and the Na(+)-Ca(2+) exchanger. The aim of this study was to determine whether Na(+)-Ca(2+) exchange activity is present in immature and mature mouse oocytes. Na(+)-Ca(2+) exchange can be revealed by altering the Na(+) concentration gradient across the plasma membrane and recording intracellular free Ca(2+) concentrations using Ca(2+)-sensitive fluorescent dyes. Depletion of extracellular Na(+) caused an immediate increase in Ca(2+) concentration in immature oocytes and a delayed increase in mature oocytes. The Na(+) ionophore, monensin, caused an increase in intracellular Ca(2+) in immature oocytes similar to that induced by Na(+)-depleted medium. In mature oocytes, monensin had no effect on intracellular Ca(2+) but the time taken for Ca(2+) to reach a peak value on removal of extracellular Na(+) was significantly decreased. Finally, addition of Ca(2+) to immature oocytes incubated in Ca(2+)-free medium caused an increase in the concentration of intracellular Ca(2+) that was dependent upon the presence of extracellular Na(+). This effect was not seen in mature oocytes. The data show that Na(+)-Ca(2+) exchange occurs in immature and mature mouse oocytes and that Ca(2+) homeostasis in immature oocytes is more sensitive to manipulations that activate Na(+)-Ca(2+) exchange.

Na+-Ca2+ exchange in mouse oocytes: modifications in the regulation of intracellular free Ca2+ during oocyte maturation.

Increases in intracellular free Ca(2+)+ concentration (Ca(2+)+ oscillations) occur during meiotic maturation and fertilization of mammalian oocytes but little is known about the mechanisms of Ca(2+) homeostasis in these cells. Cells extrude Ca(2+) from the cytosol using two main transport processes, the Ca(2+)-ATPase and the Na(+)-Ca(2+) exchanger. The aim of this study was to determine whether Na(+)-Ca(2+) exchange activity is present in immature and mature mouse oocytes. Na(+)-Ca(2+) exchange can be revealed by altering the Na(+) concentration gradient across the plasma membrane and recording intracellular free Ca(2+) concentrations using Ca(2+)-sensitive fluorescent dyes. Depletion of extracellular Na(+) caused an immediate increase in Ca(2+) concentration in immature oocytes and a delayed increase in mature oocytes. The Na(+) ionophore, monensin, caused an increase in intracellular Ca(2+) in immature oocytes similar to that induced by Na(+)-depleted medium. In mature oocytes, monensin had no effect on intracellular Ca(2+) but the time taken for Ca(2+) to reach a peak value on removal of extracellular Na(+) was significantly decreased. Finally, addition of Ca(2+) to immature oocytes incubated in Ca(2+)-free medium caused an increase in the concentration of intracellular Ca(2+) that was dependent upon the presence of extracellular Na(+). This effect was not seen in mature oocytes. The data show that Na(+)-Ca(2+) exchange occurs in immature and mature mouse oocytes and that Ca(2+) homeostasis in immature oocytes is more sensitive to manipulations that activate Na(+)-Ca(2+) exchange.

Meiosis-activating sterol-mediated resumption of meiosis in mouse oocytes in vitro is influenced by protein synthesis inhibition and cholera toxin.

To explore the possible signaling pathways of meiosis-activating sterol (MAS)-induced oocyte maturation and to elucidate whether the MAS pathway involves transcription or translation, arrested immature mouse oocytes were cultured with either the protein synthesis inhibitor cycloheximide or the heteronuclear RNA inhibitors alpha-amanitin or actinomycin D, respectively. Moreover, the possible involvement of a G protein-coupled receptor mechanism in MAS-mediated oocyte maturation was explored by influencing oocyte maturation with cholera toxin (CT). MAS-induced oocyte maturation was completely blocked by the addition of 50 microg/ml cycloheximide 4 h before the addition of MAS. Simultaneous addition of MAS and the protein synthesis inhibitor also significantly reduced the meiotic resumption compared to that in MAS-treated controls. In contrast, neither of the treatment regimens to inhibit transcription of DNA to RNA was observed to have any effect on the MAS-induced resumption of meiosis. CT was observed to inhibit MAS-induced, but not spontaneous, oocyte maturation in vitro, suggesting a putative involvement of G protein-coupled receptor mechanism in the MAS mode of action. In conclusion, protein synthesis was found to be an essential requirement for maintaining the oocytes' responsiveness to MAS-induced resumption of meiosis, in contrast to transcription.

A reliable technique of nuclear transplantation for immature mammalian oocytes.

Transplanting a germinal vesicle (GV) to another enucleated oocyte provides a possible way to avoid age-related aneuploidy in metaphase II (MII) oocytes from older women. This study was conducted to examine the efficiency of each step of nuclear transplantation as reflected in the survival and maturation capacity of immature mouse oocytes subjected to this procedure. GV stage oocytes were retrieved from unstimulated ovaries. A GV removed with a small amount of cytoplasm (karyoplast) was transferred subzonally into a previously enucleated oocyte, which was then exposed to direct current to promote fusion. Such reconstituted oocytes were placed in culture to allow maturation, and some that had extruded a first polar body were fixed and processed for chromosome analysis. Each step of nuclear transplantation - survival, enucleation, grafting, and reconstitution - was successful in >90%, with the overall efficiency of reconstitution being 80%. The observation of normal karyotypes confirmed that the procedure did not increase chromosomal aneuploidy. An electrolytic medium, revealed to be superior for the reconstitution procedure, also allowed haploidization of the transplanted nucleus. These findings suggest that this technique can be applied to study the effects of a 'younger' woman's ooplasm on the disjunction of an 'older' woman's chromosomes during meiosis I.

Sugars Exert a Major Influence on the Vitrification Properties of Ethylene Glycol-Based Solutions and Have Low Toxicity to Embryos and Oocytes

A systematic approach was taken to assess the vitrification properties of ethylene glycol-based solutions supplemented with carbohydrates. Solutions were prepared by weight (gravimetrically) using ethylene glycol as the cryoprotectant, 0.9% NaCl in water, and six different sugars:d-glucose,d(−)-fructose,d-sorbitol, sucrose,d(+)-trehalose, and raffinose. Sugars were added on a molal basis (0.1, 0.5, and 1m). Characteristics of the solutions were measured during warming by differential scanning calorimetry using a cooling rate of 100°C/min and a warming rate of 10°C/min. In the absence of carbohydrates a 59 wt% EG-saline solution formed a stable glass. When EG was replaced by an equimolal concentration of glucose, fructose, or sorbitol (monosaccharides) at 0.1, 0.5, or 1.0mthere was no change in the total solute concentration at which vitrification occurred, but the glass transition (Tg) occurred at a higher temperature than in EG-saline alone. When EG was replaced by an equimolal concentration of sucrose or trehalose (disaccharides) both the Tg and the lowest total solute concentration required for vitrification became progressively higher as the molecular weight, or the ratio of sugar to EG in the solutions, increased. At the highest tested disaccharide concentration (1m) vitrification was achieved at a total solute concentration of 65 wt% (sucrose) and 67 wt% (trehalose). The polysaccharide raffinose significantly modified the vitrification properties of ethylene glycol solutions. When 0.5 or 0.1mraffinose replaced EG on an equimolal basis the glass transition point was raised more than with either the monosaccharides or the disaccharides. Raffinose allowed vitrification at a total solute concentration of 67 wt% (0.5m) and 63 wt% (0.1m). The maturation of immature mouse oocytes, and the development of embryos in media containing 5–7 mM of any sugar was comparable to controls, indicating that they are not toxic. Exposure of freshly collected GV or MII oocytes to sugar concentrations between 0.5 and 1.0 M, for up to 10 min had no significant effect on the proportion which subsequently formed two cells. We conclude that added sugars do contribute to a solutions overall vitrification properties, and their properties should be taken into consideration when vitrification solutions are being designed or modified.

Vitrification of mouse germinal vesicle oocytes: effect of treatment temperature and egg yolk on chromatin and spindle normality and cumulus integrity.

The success rates for cryopreservation of immature oocytes from several species including human remain low, in contrast to major improvements with mature oocytes. In this study, a new approach has been developed using a short exposure ultra-rapid freezing protocol, examining the effect of temperature and egg yolk (two factors which may be expected to influence membrane flexibility) on the cryostability of immature mouse oocytes and cumulus complexes. These two factors were tested in various patterns for their cryoprotective effect using ethylene glycol as the principal cryoprotectant. The results showed that 37 degrees C pre- and post-freeze exposure significantly improved both survival and normal spindle configuration after in-vitro maturation. Egg yolk was found to produce further beneficial effects on both the oocyte and cumulus cell integrity, with the best effects being obtained at 37 degrees C with inclusion of egg yolk both before and after the freezing. This protocol produced > 80% normal survival post-thaw with intact and attached cumulus complex, 84% maturation rate and 45% normal metaphase configuration. In summary, a unique combination of high survival and meiotic normality together with good preservation of the attached cumulus cell mass has been achieved using a simple new vitrification procedure.

Nuclear calcium release by InsP3-receptor channels plays a role in meiosis reinitiation in the mouse oocyte

Our purpose was to investigate the presence of nuclear specific elements of the phosphoinositide pathway, and the link between nuclear calcium events and the first step of meiosis resumption, i.e. germinal vesicle breakdown (GVB) in mouse immature oocytes. Using confocal laser scanning microscopy, we analyzed the effects of nuclear microinjection of inositol 1,4,5-trisphosphate (InsP3), heparin and anti-InsP3 receptor monoclonal antibodies on both spontaneous nuclear and cytoplasmic calcium oscillations, as well as the effects of these components on the GVB. First we observed that nuclear Ca 2+ events were dependent upon both nucleoplasmic and cytoplasmic InsP3 levels, highlighting a cross-talk between the GV and the cytoplasm concerning the Ca 2+/InsP3 pathway. We demonstrated also that: 1) type 1 InsP3 receptors were localized at the nuclear membrane level while type 3 were absent from the nucleus; 2) calcium release from nuclear stores was mediated by type 1 rather than type 3 InsP3 receptor associated channels; 3) the anti-InsP3 R-1 mAB microinjected into the nucleus inhibited the GVB. These results demonstrate that reinitiation of meiosis requires an increase in nuclear phosphoinositide dependent Ca 2+. Thus, the role of nuclear Ca 2+ homeostasis is discussed with particular emphasis on nuclear envelope dynamics.

Differential effects of cryopreservation on nuclear or cytoplasmic maturation in vitro in immature mouse oocytes from stimulated ovaries.

The aim of this study was to develop a maturation protocol for immature oocytes and assess the protocol with cryopreserved oocytes. Nuclear maturation (mature spindle and aligned chromosomes) occurred irrespective of the treatment regime: 71-89% of oocytes matured in vitro had a normal spindle and chromosomes compared with 87% matured in vivo. Fertilization rates were not significantly different from those of in-vivo matured oocytes. Of the maturation treatment regimes investigated, the initial treatment producing best development to blastocyst (cytoplasmic maturation) involved a 2 h incubation in standard maturation medium (SMM) containing 7.5 IU follicle stimulating hormone (FSH) followed by 14 h in SMM plus 7.5 IU FSH:luteinizing hormone with follicular cells [62% (range 49-69)]. The addition of 1 ng/ml epidermal growth factor (EGF) in this protocol resulted in development [75% (range 71-81)] that was not significantly different from in-vivo matured oocytes [82% (range 73-90)]. Exposure of the oocytes to 1.5 M dimethylsulphoxide (DMSO) did not affect fertilization or development rates. Following a slow-cool/thaw freezing regime, 81% (range 74-89) of the oocytes were morphologically normal, i.e. had a spherical shape with an intact zona and oolemma; they had, however, lost their previously attached cumulus and corona cells. Maturation of frozen-thawed oocytes in the presence of EGF gave good fertilization rates but poor development rates [80% (range 77-86) and 37% (range 33-40) respectively]. In conclusion, the best maturation, both nuclear and cytoplasmic, occurred in the presence of a combination of gonadotrophins, EGF and follicular cells. Oocytes cryopreserved using a slow-cool/thaw regime can be matured to produce blastocysts after in-vitro fertilization.

Induction of DNA replication in germinal vesicles and in nuclei formed in maturing mouse oocytes by 6-DMAP treatment.

Immature mouse oocytes (germinal vesicle stage, GV), oocytes at different stages during maturation (prometaphase to anaphase I) and matured oocytes (metaphase II arrested) were cultured in 6-dimethylaminopurine (6-DMAP)-supplemented medium also containing bromodeoxyuridine for the assessment of DNA replication in these cells. Immature oocytes remained arrested at the GV stage and DNA replication was never detected in them. On the other hand, oocytes at the prometaphase to anaphase-telophase I stages responded to 6-DMAP treatment by forming nuclei which synthesised DNA. Mature (metaphase II) oocytes did not respond to 6-DMAP and their chromatin remained condensed. DNA synthesis could even be induced in GV-staged oocytes, but only when they were fused to freshly activated oocytes and incubated in 6-DMAP-supplemented medium.

Unique protein kinase C profile in mouse oocytes: lack of calcium-dependent conventional isoforms suggested by rtPCR and Western blotting

rtPCR and Western blotting were used to determine which members of the PKC family are present in both immature and mature mouse oocytes. Using isoform-specific PCR primers and antibodies PKC- δ and - λ were detected while such techniques failed to observe the conventional isoforms of PKC- α, - β, - γ. This isoform profile was confirmed using an alternative PCR strategy, which allowed discrimination of PCR products derived from conventional and novel PKC isoforms. In addition PKC- ϵ, - η, - θ and - ζ were not detected by rtPCR. These results suggest that the predominant isoforms in oocytes are PKC- δ and - λ.

R-163. A chromosomal and spindle study of cryopreserved immature mouse oocytes

R-163. A chromosomal and spindle study of cryopreserved immature mouse oocytes

Cryopreserved immature mouse oocytes: a chromosomal and spindle study.

Cryopreservation of human oocytes might provide an alternative approach to freezing supernumerary embryos obtained during IVF. This process, performed on immature denuded prophase 1 mouse oocytes, was investigated. We first investigated the capacity of frozen, immature, murine oocytes to continue in vitro maturation after thawing. We then evaluated the risk to offspring from chromosomal damage by cytogenetical and cytological (spindle) analysis. Finally, we attempted to determine the reasons for and the stage of maturation failure. A total of 700 immature oocytes was frozen, 629 (90%) were recovered intact after thawing, and 53% extruded the first polar body, versus 74% for the control group. Freezing was not accompanied by an increase in aneuploidy in maturing oocytes (18 and 15% for thawed and control oocytes, respectively). Consequently, the first meiotic division occurred normally, without an increase in nondisjunction. Spindle analysis demonstrated only a few abnormalities (15 and 2% for thawed and control oocytes, respectively) incompatible with further development. Oocytes arrested during in vitro maturation were mainly at the metaphase I stage (64 and 76% for thawed and control oocytes, respectively). Whereas 17% of thawed oocytes were blocked before the formation of the first meiotic spindle, this never occurred in the control group. Immature murine oocytes can withstand cryo-preservation, which is encouraging for future human application of this technique.