Fertilization and development of the human oocyte following exposure to cryoprotectants, low temperatures and cryopreservation: a comparison of two techniques.

Both glycerol and dimethyl sulphoxide (DMSO) equilibration prior to cryopreservation produced adequate rates of survival and development to the hatching blastocyst stage for mouse oocytes using the two chosen cooling and warming regimes. Successful fertilization of human oocytes and further development of the embryos produced, was achieved following equilibration with DMSO at 0 degrees C. Although fertilization of fresh human ova previously exposed to glycerol was recorded, all oocytes with two pronuclei failed to continue to undergo cell division by 48 h in culture. Following cryopreservation, human oocytes were successfully inseminated after equilibration with both glycerol and DMSO. However, cell division to the 2-cell stage was recorded in only one oocyte which had undergone exposure to DMSO, freezing and thawing. No cell divisions were recorded after glycerol cryopreservation, or even after simple exposure to glycerol. Therefore it appears that DMSO and slow cooling may be the best protocol, although further evaluation will be necessary.     

Osmotic responses and tolerance limits to changes in external osmolalities, and oolemma permeability characteristics, of human in vitro matured MII oocytes

BACKGROUND: Oocyte cryopreservation remains a realistic objective, provided that more systematic approaches are applied, such as thorough analysis of the oocyte oolemma permeability to water and diverse cryoprotectants. METHODS: We prospectively investigated volume changes over time at different temperatures (30 degrees C, 22 degrees C and 8 degrees C) of human metaphase II (MII) oocytes (obtained in stimulated ICSI cycles and matured in vitro from the germinal vesicle stage) when exposed to changes in external osmolality. We also investigated human in vitro matured (IVM) oocytes membrane permeability characteristics at 22 degrees C to 1,2-propanediol (PG) and dimethylsulphoxide (DMSO) and at 30 degrees C, 22 degrees C and 8 degrees C to ethylene glycol (EG), and calculated corresponding oocyte oolemma permeability coefficients (Lp and Pcpa). Furthermore, we investigated the osmotic tolerance limits of IVM oocytes exposed to changes in external osmolality as assessed by their developmental competence during the course of 72 h after ICSI. RESULTS: The results of our studies describe human oocyte membrane permeability coefficients for EG at 30 degrees C (2.85+/-0.15x10(-3) cm/min), 22 degrees C (1.17+/-0.60x10(-3) cm/min) and 8 degrees C (0.37+/-0.15x10(-3) cm/min). Furthermore, at 22 degrees C the EG oolemma permeability coefficient was lower than that of PG and DMSO (1.17+/-0.60x10(-3) cm/min versus 2.15+/-0.70x10(-3) and 1.56+/-0.38x10(-3) cm/min, respectively). Our results also indicate, that human IVM MII oocytes tolerated exposure to solutions in the range of 39-2264 mOsmol/kg H2O as assessed by the oocytes' developmental competence after exposure. CONCLUSIONS: The results of the present study may contribute to a better understanding of the biology and cryobiology of human oocytes, and to the design of better and more robust cryopreservation (freezing or vitrification) protocols.     

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.     

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.     

Temporary developmental arrest after storage of fertilized mouse oocytes at 4 degrees C: effects on embryonic development, maternal mRNA processing and cell cycle

The aim of this study was to examine whether fertilized mouse oocytes can survive after short-term incubation (for 6-48 h) at 4 degrees C. When fertilized oocytes of ICR and C57BL/6 (B6) strain were incubated at 4 degrees C and returned to normal culture conditions (37 degrees C), development of these 4 degrees C-treated embryos for up to 12 h (for ICR) to blastocyst stage did not differ from that of untreated oocytes. Even 4 degrees C-treated embryos for 48 h developed to blastocysts at relatively good rates (33.3% for ICR and 50.8% for B6). The in vivo development of 4 degrees C-treated embryos for 12, 24 and 36 h to fetal stage was similar to that of untreated ones. BrdU labelling assay revealed temporary cessation of DNA replication in 4 degrees C-treated fertilized oocytes. Post-fertilization events including cytoplasmic polyadenylation of maternal mRNAs, mRNA degradation of a cell cycle-related gene and elevated mRNA expression of zygotic gene activation-related genes were temporarily suppressed in 4 degrees C-treated embryos. These findings indicate that 4 degrees C-treatment of fertilized murine oocytes results in temporary cessation of molecular events. We also show that 4 degrees C-treated fertilized oocytes for 12 h can be used for preparation of transgenic mice.     

In vitro maturation of mouse germinal vesicle-stage oocytes following cooling, exposure to cryoprotectants and ultrarapid freezing: limited effect on the morphology of the second meiotic spindle.

Cryopreservation of germinal vesicle (GV)-stage mouse oocytes results in a developmental block. As an approach to explain the failure in development, we have investigated the morphology of the second meiotic spindle after in-vitro maturation. Fully grown GV-stage mouse oocytes were collected from the ovaries of primed mice and kept in meiotic arrest with dibutyryl cyclic AMP. These oocytes were submitted to different variables of cryopreservation: (i) cooling to 22 degrees C or 0 degrees C; (ii) exposure to 1.5 M 1,2-propanediol at 22 degrees C or 0 degrees C; (iii) exposure to 1.5 M dimethylsulphoxide (DMSO) at 22 degrees C or 0 degrees C; (iv) ultrarapid freezing with 3.5 M DMSO/0.5 M sucrose; (v) exposure to a sucrose dehydration series according to the ultrarapid freezing protocol. The morphology of the second meiotic spindle was evaluated 16 h after release from meiotic arrest. We were able to demonstrate that following cooling, exposure to cryoprotectants or ultrarapid freezing of GV-stage mouse oocytes, a normal barrel-shaped spindle with the chromosomes in midplane position is found in 79-94% of oocytes except for two conditions with great exposure to dehydration stress. Exposure to DMSO at 0 degrees C or exposure to a sucrose dehydration series resulted in significantly lower percentages of barrel-shaped spindles, respectively 64% and 62%. The effect on spindle morphology has to be put into perspective, however, since the observed abnormalities were changes of spindle shape, such as elongation or reduction, which are assumed to be restorable, and since no polar organizational defects were found.(ABSTRACT TRUNCATED AT 250 WORDS)     

Limited recovery of meiotic spindles in living human oocytes after cooling-rewarming observed using polarized light microscopy.

BACKGROUND: Spindles are formed from microtubules and are exquisitely sensitive to changes in temperature. An orientation-independent polarized light microscope, the Polscope, can be used to image spindles in living oocytes allowing analysis of spindle kinetics in the living state. This study examined the effects of cooling on spindle disassembly in living human oocytes and spindle recovery after rewarming. METHODS: Oocytes were imaged continuously with the Polscope during cooling and rewarming. The quantity of microtubules in the spindles was measured by its birefringence using the Polscope. RESULTS: Spindles had completely disassembled by 5 min after cooling and recovered by 20 min after rewarming to 37 degrees C if rewarming started soon after the oocyte's temperature dropped to room temperature. However, when oocytes were cooled and kept at 33, 28 or 25 degrees C for 10 min and then warmed, it was found that warming allowed 5/5, 2/5 and 0/5 oocytes of the spindles to recover respectively. CONCLUSIONS: These results indicate that human meiotic spindles are exquisitely sensitive to alterations in temperature. The maintenance of temperature at 37 degrees C during in-vitro manipulation is important for spindle integrity and, therefore, is likely to be important for normal fertilization and subsequent embryo development.     

The effects of cooling human oocytes

Preovulatory human oocytes were cooled to 0°C at 1°C/minwith or without the cryoprotectant dimetbyl suiphoxide (DMSO),to assess the effects of cooling on the meiotic spindles andon oocyte structure. Batches of oocytes, cultured for 3–9h, were held at 0°C for 20 or 60 min and then fixed fortransmission electron microscopy (TEM) either at 0 or 8°C.Control oocytes were not cooled and were fixed at 22 or 37°Cfor comparison. TEM revealed that 80% of the oocytes were atmetaphase II, while 20% were at metaphase I and most had resumedmeiosis recently. Control oocytes had more or less barrel-shapedmeiotic spindles composed of microtubules (MT), some associatedwith chromosomes at kinetochores. Both metaphase I and II spindleswere disassembled when cooled and fixed at 0°C, with orwithout DMSO, due to extensive depolymerization of MT. The fewMT that survived were found at the poles or were bundled togetheror were associated with chromosomes. Kinetochores were not prominent.Some oocytes cooled with DMSO and fixed at 0 or 8°C showedevidence of MT, but the spindles were still disorganized andwere abnormal In structure. Chromosomes tended to dump togetheror were dislocated in the cortical ooplasm in cooled oocytes,but widespread scattering was not observed. This was particularlyevident in the absence of DMSO. Elements of the endoplasmicreticulum, Golgi, mitochondria and the cytosol were also adverselyaffected In some of the cooled oocytes and their surroundingcumulus cells. The results show that meiotic spindles are verysensitive to simple cooling and that DMSO does not provide substantialstabilization of the meiotic spindle even at 0/C. The findingsare discussed with reference to recent work on frozen humanand mouse oocytes.     

Comparative results on survival of human and animal eggs using different cryoprotectants and freeze-thawing regimens. II. Human

A total of 269 human pronuclear embryos and 84 oocytes weresubjected to four different protocols of cryoprotectant equilibration,washing out and freeze-thawing. The morphological survival,rate of development, fertilization in vitro and overall survivalrate were estimated in the groups of fresh, aged oocytes, diploidand multipronuclear embryos used. With some restrictions, theconclusion can be drawn that slow, low intermediary temperature1, 2-propanediol (1, 2-PROH) and 1, 2-PROH/dimethyl sulphoxide(DMSO) systems are superior to the rapid, high intermediarytemperature 1, 2-PROH and the traditionally used DMSO systems.The best success rates were reached with the combination ofcryoprotectants in the low intermediary temperature group. Thirty-threepatients had 37 transfers of cryopreserved pronuclear embryos(n= 103), resulting in eight pregnancies (24.2% per transfer)thus doubling the pregnancy rate in the stimulation cycles forthe same period (21% per transfer). Thawing at a rate of 50?C/minis not incompatible with the survival of slowly frozen humanoocytes and pronuclear embryos cooled to an intermediary temperatureof –70?C using the DMSO system.     

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.     

Cryopreservation of human and rabbit oocytes and one-cell embryos: a comparison of DMSO and propanediol

The aim of this study was to improve the cryopreservation ofhuman oocytes and pronuclear embryos. One-step and multiple-stepaddition of dimethyl sulphoxide (DMSO) and 1, 2-propanediol(PROH) and three different freezing protocols with intermediatetemperatures of –35, –70 and –110°C wereinvestigated. This work was performed using rabbit oocytes aswell as human oocytes and one-cell embryos from the routineIVF programme. Also, human polyploid pronucleate oocytes wereused in controlled prospective studies of morphological intactnessand development in vitro. Rabbit oocytes survived best (113/126)when PROH was added in one step and controlled freezing stoppedat –110°C. But the development was better (141/187)if DMSO was added in multiple steps and the oocytes were cooledto –70°C before being plunged into liquid nitrogen.The mode of addition of the cryoprotectant influenced developmentonly if slow freezing was stopped at –35°C (51 versus34%). Using PROH, the development after thawing was also betterif cooling was stopped at –35°C (51 versus 37%) andDMSO was superior to PROH when the oocytes were cooled slowlyto –110°C (66 versus 37%). In the human, significantlymore pronucleated than unfertilized oocytes developed afterfreezing (92 versus 50%). The best results were achieved withpronuclear embryos using 1.5 M PROH and cooling to –110°C,when 91.7% of the surviving oocytes developed further. Thisis a marked improvement of the development rate and comparableto embryo freezing     

IVF within microfluidic channels requires lower total numbers and lower concentrations of sperm

BACKGROUND: Microfluidic technology has been utilized in numerous biological applications specifically for miniaturization and simplification of laboratory techniques. We sought to apply microfluidic technology to murine IVF. METHODS: Microfluidic devices measuring 500 microm wide, 180 microm deep, and 2.25 cm in length were designed and fabricated using poly(dimethylsiloxane) (PDMS). Controls were standard centre-well culture dishes with 500 microl of media, half of which also contained PDMS as a material control. Denuded mouse oocytes were placed into microchannels or centre-well dish controls in groups of 10, then co-incubated overnight with epididymal mouse sperm at various concentrations. Fertilization was assessed and Fisher's exact test was used for statistical analysis (P < 0.05 significant). RESULTS: Fertilization rates between the two control groups (42%, no PDMS; 41%, with PDMS; not significant) were similar. Fertilization rates for denuded oocytes at standard mouse insemination sperm concentration (1 degrees 10(6) sperm/ml) was poorer in microchannels (12%) than controls (43%; P < 0.001). As insemination concentrations decreased, fertilization rates improved in microchannels with a plateau between 8 degrees 10(4) and 2 degrees 10(4) sperm/ml (4000-1000 total sperm). At these concentrations, combined fertilization rate for denuded oocytes was significantly higher in microchannels than centre-well dishes (27 versus 10%, respectively; P < 0.001), and was not significantly different from corresponding controls with a sperm concentration of 1 degrees 10(6) (37%; P = 0.06). CONCLUSIONS: Murine IVF can be conducted successfully within microfluidic channels. Lower total numbers and concentrations of sperm are required. Microfluidic devices may ultimately be useful in clinical IVF.     

Fertilization and embryonic development of human oocytes after cooling.

Injury to living cells resulting from rapid cooling to temperatures at or near 0 degrees C has long been recognized, and the phenomenon, which is termed 'cold shock', has been known to occur in some mammalian gametes. Although human embryos have been successfully stored at low temperatures, cryopreservation of the human oocyte is proving to be more difficult. Whether or not this lack of success is a direct result of cellular injury brought about by 'cold shock' is the purpose of the current investigation. Human oocytes were cooled, in the absence of cryoprotectants, at two different cooling rates (-3 degrees C/min and -1000+ degrees C/min) to a temperature of 0 degrees C and rewarmed prior to insemination. In both cases fertilization after cooling was similar to the rates achieved in a routine in-vitro fertilization and embryo transfer procedure. After cooling at -3 degrees C/min, the rate of fertilization was 19/22 (86%) and after cooling at -1000+ degrees C/min, 9/9 (100%), with non-cooled control rates of 62/87 (71%) and 35/50 (70%) respectively. Fertilized oocytes from both groups were successfully cultured for a further 24 h before termination of the experiment.     

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.     

Relationship between meiotic spindle location with regard to the polar body position and oocyte developmental potential after ICSI

BACKGROUND: The recent development of a computer-assisted polarization microscopy system (Polscope) with which the meiotic spindle can be visualized in living oocytes on the basis of its birefringence permits analysis of the meiotic spindles of oocytes subjected to ICSI. Previous studies have shown that the meiotic spindle is not always aligned with the first polar body (PB) in metaphase II human oocytes prepared for ICSI. In the present study, the relationship between the degree of meiotic spindle deviation from the first PB location and ICSI outcome was analysed. METHODS: Oocytes were divided into four groups according to the angle of meiotic spindle deviation from the PB position. The angle of deviation was 0-5 degrees, 6-45 degrees, 46-90 degrees and >90 degrees for groups I to IV respectively. RESULTS: The rates of normal [2 pronuclei (PN)] and abnormal (1PN or >2PN) fertilization did not differ between groups I, II and III. However, the rate of normal fertilization was lower among oocytes in which the meiotic spindle deviation angle was >90 degrees; this led to an increased proportion of tripronucleated zygotes that failed to extrude the second PB. When embryos developed from normally fertilized oocytes were evaluated on day 3 after ICSI, no relationship was found between the angle of meiotic spindle deviation and embryo quality. The meiotic spindle was not detected in only 9% of oocytes, and these showed a higher incidence of fertilization and cleavage abnormalities than did oocytes in which the spindle was detected. When oocytes at metaphase I after cumulus oophorus and corona radiata removal were matured in vitro, the meiotic spindle was detected in 53.8% of those that reached metaphase II. In these in-vitro-matured oocytes the meiotic spindle was always aligned with the first PB, suggesting that misalignment seen in those oocytes matured in vivo resulted from PB displacement during manipulations for cumulus and corona removal. CONCLUSION: High degrees of misalignment between the meiotic spindle and the first PB predict an increased risk of fertilization abnormalities. However, when normal fertilization had occurred, the cleavage potential of embryos developing from such oocytes was not impaired. These findings facilitate the selection of oocytes for ICSI in situations when the creation of supernumerary embryos is to be avoided.     

The influence of cooling on the properties of the zona pellucida of the mouse oocyte

Mouse oocytes were exposed to a variety of cooling regimes priorto insemination in vitro. Exposure to 4°C, but not to 25°C,was associated with a reduced fertilization rate, but developmentto the blastocyst stage of those oocytes that fertilized wasnot consistently different from that of non-cooled controls.The reduced fertilization rate seems to result from an effectof cooling on the zona pellucida, since it was not observedif the zona was removed prior to insemination, and since coolingrendered the zona pellucida resistant to the action of chymotrypsin.Using chymotrypsin resistance as an assay, the nature of thecooling-induced effect on the zona was investigated. It is suggestedthat rapid cooling to 4°C may promote release of corticalgranules and a premature zona reaction.     

Cryoloop vitrification of rabbit oocytes

BACKGROUND: Vitrification is assumed to be a promising method to cryopreserve human oocytes but still needs optimization. In this study, rabbit oocytes (fertilized by ICSI) were vitrified with cryoloops, and the effect of three different cryopreservation protocols on spindle configuration and embryo quality was assessed. METHODS: Metaphase II rabbit oocytes were randomly assigned to one of four groups: (i) control; (ii) E40 [40% ethylene glycol (EG)]; (iii) ED20 [20% EG + 20% dimethylsulphoxide (DMSO)]; and (iv) ED20 + M (20% EG + 20% DMSO + vitrification machine). After warming, one part of each group was fertilized by ICSI to examine the fertilization and embryo cleavage ability, and the others were immunostained for tubulin and chromatin before visualization using confocal microscopy. RESULTS: The survival rates after warming were 79.1, 83.1 and 82.3%, respectively. In protocols E40 and ED20, the spindles were severely injured and the embryo quality not good compared with those in the ED20 + M group. CONCLUSIONS: The fastest cooling rate in combination with EG and DMSO as cryoprotectants had the fewest adverse effects on the spindle configuration of rabbit oocytes and embryo development.     

Cryopreservation of pronucleate mouse ova: slow versus ultrarapid freezing

The usefulness of ultrarapid freezing of mouse pronucleate ova was investigated in comparison with slow, programmed freezing. Pronucleate mouse ova were frozen using an ultrarapid method in either 3.5 M dimethylsulphoxide (DMSO), 3.5 M propanediol (PROH) or a 1:1 mixture of both. After a brief exposure to the cryoprotectant, they were plunged into liquid nitrogen. Thawing was done at 37 degrees C and the cryoprotectant was rapidly diluted in a sucrose solution. Pronucleate ova were slowly cooled in a biological freezer using 1.5 M PROH as cryoprotectant. Thawing was done at room temperature and PROH was removed by multi-step dilutions. As control groups, pronucleate ova were either given no treatment, or exposed to PROH or DMSO without freezing and cultured in vitro. Ultrarapid freezing using 3.5 M DMSO as cryoprotectant resulted in rates of survival, cleavage and post-thaw development to blastocysts of 85, 89 and 37%, respectively. PROH as cryoprotectant, however, was inadequate in the ultrarapid freezing protocol and the combination of DMSO and PROH showed no further improvement. Slow, programmed freezing (using PROH) compared to ultrarapid freezing (using DMSO) resulted in similar rates of survival, cleavage and development of 80, 84, 20% and 92, 81 and 23% respectively. In conclusion, DMSO is a better cryoprotectant than PROH for ultrarapid freezing of pronucleate mouse ova, and ultrarapid freezing with 3.5 M DMSO is as effective as slow freezing with 1.5 M PROH, as evaluated by their subsequent development in vitro.     

Recombinant human albumin supports hamster in-vitro fertilization

BACKGROUND: Serum albumin is normally required to support sperm capacitation and IVF, but its mechanism of action is not well understood. Commercial serum albumin preparations are contaminated with a variety of other proteins and compounds, and their biological activity is variable. Recombinant human albumin (rHA) might replace serum albumin for IVF. METHODS: rHA was examined for its ability to capacitate hamster spermatozoa and to support fertilization in vitro. A standardized hamster IVF system was used to compare the capacitation-supporting activities of rHA and two commercial preparations of bovine serum albumin (BSA) in a chemically defined culture medium. Epididymal spermatozoa were incubated for 4 h at 37 degrees C under 5% CO(2) in air in either the basic medium containing rHA, one of the two BSA preparations or no protein, and then cultured in the same medium with ovulated oocytes for another 4 h. The experiment was replicated five times. RESULTS: Spermatozoa incubated in protein-free medium fertilized only one oocyte (2% of total), significantly less than any of the other three treatment conditions (P < 0.01); spermatozoa incubated in medium containing rHA or BSA fertilized 86-93% of oocytes. There were no differences between the three albumin-containing treatment groups. CONCLUSION: rHA is equivalent to commercial serum albumin preparations in its ability to support sperm capacitation and fertilization in this test system. This finding has considerable practical implications for human IVF and may also help efforts to elucidate the mechanism of sperm capacitation.     

Diffusion and distribution of dimethyl sulphoxide in the isolated guinea-pig taenia coli

1. The diffusion of the cryoprotective non-electrolyte dimethyl sulphoxide (DMSO) in the isolated guinea-pig taenia coli at 37, 25 and 0 degrees C has been studied using [(35)S]DMSO.2. Within 1 hr after immersion at 37 degrees C in Krebs solution containing 20% (w/v) DMSO and trace amounts of [(35)S]DMSO, the non-electrolyte was distributed uniformly throughout a volume equivalent to the total initial water content of the muscle.3. The kinetics of efflux of [(35)S]DMSO from muscles at constant volume were analysed on the basis of two models: one incorporated radial diffusion in extracellular fluid with simultaneous permeation into the cells, the other involved only radial diffusion in homogeneous cylinders of tissue having no internal barriers to diffusion; the former was found to give a better representation of the efflux kinetics.4. If it was assumed that the rate of diffusion of DMSO in the extracellular space of taenia coli was the same as that in the bathing medium, the values of the extracellular space and the permeability of smooth muscle to DMSO, obtained from the analysis of the efflux kinetics, were 454 +/- 19 ml./kg and 2.36 +/- 0.05 x 10(-6) cm sec(-1) at 37 degrees C.5. The activation energy for the transfer of DMSO across the surface of the cell was estimated to be 6.0 kcal/mole at 37 degrees C, 6.6 kcal/mole at 25 degrees C and 11.6 kcal/mole at 0 degrees C, indicating either that the equivalent pore radius of the cells decreased with temperature or that the cell permeability represented the sum of two fluxes, one through the aqueous pores of the cell and the other through the lipid phase of the cell membrane, each with a different energy of activation.6. A net flux of water across the surface of the cells, superimposed on the efflux of DMSO, markedly affected the rate of diffusion of the non-electrolyte out of the whole tissue; however, it was considered that an analysis of the efflux kinetics was not possible under these conditions.7. These results provide a basis for methods which will be used to investigate the possibility of preserving tissue in unfrozen aqueous media at sub-zero temperatures.