Chromosome number and development of artificial mouse oocytes and zygotes

BACKGROUND: Infertility due to the absence of gametes is one of the last frontiers in reproductive medicine. Sperm or oocyte donation is currently the only treatment option but this approach lacks the genetic contribution of both partners. Artificial production of gametes through haploidization may offer an alternative strategy. The aim of this study was to evaluate the efficiency of producing artificial oocytes and zygotes with correct chromosome number. METHODS AND RESULTS: Somatic cumulus cell nuclei were injected into non-enucleated oocytes to produce artificial zygotes and into enucleated mature mouse oocytes to produce artificial oocytes. The expected chromosome number of artificial zygotes and oocytes is 40 and 20 chromosomes respectively. Fertilization and developmental potential of artificial zygotes and oocytes inseminated by IVF or ICSI were investigated. The expected chromosome numbers were found in 12% of artificial zygotes and 15% of artificial oocytes. Varying the time interval between injection of the somatic nucleus and activation (3, 5, 8 h) tended to increase the efficiency up to 18 and 23% for zygotes and oocytes respectively. Two-cell formation rates were 90% for artificial zygotes and 37% for artificial oocytes after IVF and 53% for artificial oocytes after ICSI. Blastocyst formation rates were 15, 8 and 9% respectively. CONCLUSIONS: Chromosome number analysis shows that the efficiency of obtaining artificial zygotes and oocytes with correct chromosome number was low and that developmental potential was severely hampered. These observations question the possibility of obtaining chromosomally normal embryos from artificial oocytes or zygotes.     

The fertilization and development of mouse oocytes following cortical granule discharge in the presence of a protease inhibitor.

The effect of leupeptin, a serine protease inhibitor, on the fertilization and development potential of oocytes stimulated to undergo cortical granule exocytosis has been investigated. An in-vitro bioassay system was used in which mouse oocytes were exposed to calcium ionophore, A23187, in the presence and absence of leupeptin, before their fertilization and development to the blastocyst stage was assessed. We have demonstrated that the presence of leupeptin in the incubation medium, at concentrations of 1 micrograms/ml and 10 micrograms/ml during the first 10 min of cortical granule exocytosis, reversed the ionophore-induced decrease in the capacity of oocytes to fertilize and develop to blastocysts. The induction of exocytosis of cortical granules by calcium ionophore was confirmed using fluorescence microscopy. Using this technique, we also confirmed that leupeptin did not inhibit ionophore-induced cortical granule exocytosis, thus supporting the contention that leupeptin acted upon released cortical exudate. It was concluded that leupeptin acted by inhibiting proteases released into the perivitelline space during the early stages of cortical granule exocytosis. Based on these results it was proposed that leupeptin could be used to prevent premature loss of fertility of human oocytes which are inadvertently activated under in-vitro conditions.     

Fertilization and early embryology: Osmotic and physiological responses of mouse zygotes and human oocytes to mono- and disaccharides

To survive cryopreservation, oocytes, zygotes and embryos musttolerate a sequence of volumetric contractions and expansions.These result as an egg or an embryo is exposed to a permeatingcryoprotective additive, then to an increase followed by a decreasein the osmolality of its extracellular milieu as water freezesduring cooling and then melts during warming, and finally tothe dilution of the cryoprotective additive solution. Measurementsof the extent to which mouse zygotes and human oocytes undergoosmotic contraction have been made by exposing them to solutionsof monosaccharides (fructose, galactose, glucose) or disaccharides(maltose, sucrose, trehalose), ranging in concentration from0.25 to 1.50 M. Mouse zygotes and human oocytes exhibit verysimilar responses to these solutions. Their volumes contractlinearly as a function of 1/(osmolality) of the solutions, yieldingestimates of non-osmotic volumes of 13–23%. Mouse zygotesexposed to 1.5 M concentrations of these solutions for 10 minlose 85% of their cell water. Yet >75% of treated zygotesdevelop into hatching blastocysts. Human oocytes also appearto survive such extreme dehydration, based on a vital dye assay.These results suggest that solutions of various non-permeatingsaccharides can serve as osmotic buffers for the recovery ofcryopreserved oocytes, zygotes and embryos.     

Regulation of neurite outgrowth mediated by neuronal calcium sensor-1 and inositol 1,4,5-trisphosphate receptor in nerve growth cones

Calcium acts as an important second messenger in the intracellular signal pathways in a variety of cell functions. Strictly controlled intracellular calcium is required for proper neurite outgrowth of developing neurons. However, the molecular mechanisms of this process are still largely unknown. Neuronal calcium sensor-1 (NCS-1) is a high-affinity and low-capacity calcium binding protein, which is specifically expressed in the nervous system. NCS-1 was distributed throughout the entire region of growth cones located at a distal tip of neurite in cultured chick dorsal root ganglion neurons. In the central domain of the growth cone, however, NCS-1 was distributed in a clustered specific pattern and co-localized with the type 1 inositol 1,4,5-trisphosphate receptor (InsP₃R1). The pharmacological inhibition of InsP₃ receptors decreased the clustered specific distribution of NCS-1 in the growth cones and inhibited neurite outgrowth but did not change the growth cone morphology. The acute and localized loss of NCS-1 function in the growth cone induced by chromophore-assisted laser inactivation (CALI) resulted in the growth arrest of neurites and lamellipodial and filopodial retractions. These findings suggest that NCS-1 is involved in the regulation of both neurite outgrowth and growth cone morphology. In addition, NCS-1 is functionally linked to InsP₃R1, which may play an important role in the regulation of neurite outgrowth.     

Fertilization, embryonic development and implantation of mouse oocytes with one or two laser-drilled holes in the zona, and inseminated at different sperm concentrations

The zonae pellucidae of mouse oocytes were photo-ablated byan ultraviolet laser (337.1 nm) to create one or two 10 µmholes. The pulse energy used was 3 µJ/s, with a frequencyof 10 pulses/s. These laser zona-drilled (LZD) oocytes withone hole (LZD1) or two holes (LZD2) and the zona-intact controlswere inseminated with spermatozoa at standard concentrationsof 5x104, 5x105, 1x106 and 2xl06/ml. Fertilization was significantlyimproved at all sperm concentrations in LZD1 and LZD2 oocytesas compared to the controls. However, there was no significantdifference in fertilization rates between LZD1 and LZD2. LZD2produced significantly higher numbers of blastocysts at day5. Hatching was significantly enhanced in the presence of eitherone or two holes in the zona. Poly-ploidy was generally absent,except in LZD2 oocytes (1%) inseminated at higher sperm concentrations.Differential cell counts of expanded LZD blastocysts were similarto those of the controls. Significantly fewer LZD2 blastocystsimplanted and produced viable fetuses than LZD1 and controlblastocysts. Morphological abnormalities of the fetuses wereabsent in all three groups. Laser zona-drilling using the ultravioletlaser was shown to be fast, efficient and safe.     

Fertilization and development of mouse oocytes cryopreserved using a theoretically optimized protocol

Rational design of a cryopreservation protocol was demonstratedby using theoretical models of the cryopreservation processto develop an optimal freezing protocol for mouse oocytes. Acoupled mechanistic model of the processes of freeze-inducedcell dehydration and intracellular ice formation was developed,and cryomicroscopical measurements of intracellular ice formationkinetics were used to determine biophysical parameters requiredby the model, and to test model predictions of the freezingbehaviour of mouse oocytes. A simple phenomenological modelfor oocyte damage resulting from exposure to concentrated electrolyteand cryoprotectant solutions during cryopreservation was obtainedby defining a cost function equal to the duration of the freezingprotocol. A two-step freezing protocol was theoretically optimizedby using a sequential simplex algorithm to minimize the costfunction, subject to the constraint that the predicted probabilityof intracellular ice formation remain below 5%, yielding a putativeoptimum at the cooling rate B = 0.59°C/min, and plunge temperatureTp = –67°C. By systematically varying B and Tp aboutthese values in experiments with mouse oocytes cryopreservedin 1.5 M dimethyl sulphoxide, the maximal recovery of intactoocytes with a normal morphology (82%) was obtained for B =0.59°C/min and Tp = –80°C. Further evaluationof the fertilizability and developmental capacity of oocytescryopreserved using the optimized protocol yielded cleavageto the 2-cell stage in 65% of oocytes inseminated, and blastocystformation in 50% of these 2-cell embryos.     

Fertilization and early embryology: Temporal effects of ouabain on in-vitro development of mouse zygotes

Ouabain is a specific inhibitor of Na⁺-K⁺-ATPase, an enzymewhich controls the intracellular Na⁺ and K⁺ levels. In thisstudy, in-vitro fertilized zygotes from a hybrid mouse strainwere used to examine the temporal effects of 50 µM ouabainon embryonic development in vitro during the preimplantationperiod. A higher incidence of blastocyst formation at the endof the culture period was found when embryos were cultured inthe presence of ouabain from 22 to 46 h post-insemination, orany other period that included this time period. When zygotesfrom randomly bred mice were used, inhibition of Na⁺-K⁺-ATPasewith ouabain clearly promoted development through the 2-cellblock in vitro. As Na⁺-K⁺-ATPase is the most important regulatorof intracellular electrolyte concentrations in mammalian cells,these results suggest that an ionic imbalance exists in embryoscultured in conventional media which can be positively influencedby inhibiting this enzyme.     

Endothelin-1 induces intracellular calcium rise and inositol 1,4,5-trisphosphate formation in cultured rat and human glioma cells

The effects of endothelin-1 (ET) on the signal transduction in rat and human glioma cell line cells have been investigated. ET was found to initiate the increase of intracellular calcium ([Ca2+]i) levels in both C6 and A-172 cells, which was concurrent with the formation of inositol 1,4,5-trisphosphate (IP3(1,4,5)). In the presence of [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA) in the incubation media, the duration of the intracellular calcium response was reduced, indicating that the ET-induced increase of intracellular calcium in glioma cells may be mediated by a dual mechanism, intracellular calcium mobilization and influx of extracellular calcium. These results suggest that ET may also act as a neuropeptide in the central nervous system.     

Comparison of ethylene glycol, 1,2-propanediol and glycerol for cryopreservation of slow-cooled mouse zygotes, 4-cell embryos and blastocysts

The aim of the study was to analyse the toxicity, the osmolar and cryoprotective activity of ethylene glycol (ETG) in terms of survival rate (SR), cleavage rate (CR) and expanded blastocysts percentage (EBP) of mouse embryos. Early mouse embryos and blastocysts were slowly cooled with ETG, 1,2-propanediol (PROH) or glycerol, and thawed. The Van t'Hoff curve for 1.5 mol/l ETG showed recovery of initial volume within 4 min. No differences were observed in CR and EBP of ETG-exposed compared with non-exposed mouse zygotes. The SR of zygotes frozen with PROH was significantly better than with ETG (92% and 60% respectively; P < 0.01), and a significantly better EBP was achieved for blastocysts frozen with glycerol compared with ETG (75% and 50% respectively; P < 0.05). For 4-cell stage embryos, no differences were observed in SR and EBP between ETG and PROH. Higher EBP was observed for 4-cell stage embryos (53%) frozen with ETG compared with pronucleate stage (19%) and blastocysts (48%). Low toxicity, good SR and EBP were observed for mouse embryos frozen with ETG, the best results being obtained at the 4-cell stage. At other embryonic stages, PROH and glycerol respectively seemed to provide better results.     

Strontium promotes calcium oscillations in mouse meiotic oocytes and early embryos through InsP3 receptors, and requires activation of phospholipase and the synergistic action of InsP3

BACKGROUND: Sr2+ is the most efficient agent for mouse oocyte activation and functions by inducing Ca2+ oscillations. However, its specific mechanism of action remains unknown. Here we investigated the specificity and possible mechanism of Sr2+-induced Ca2+ oscillations in mouse oocytes and early embryos. METHODS: Ca2+ oscillations in oocytes and embryos were measured by ratiometric fluorescence imaging using fura-2AM. The role of phospholipase C (PLC) and inositol trisphosphate (InsP3) receptors in Sr2+-induced Ca2+ oscillations was examined by selective inhibitors. RESULTS: Sr2+ can induce Ca2+ oscillations in both immature and mature oocytes, and in early embryos. A cell cycle stage-dependent phenomenon to Sr2+ stimulation was observed in 1-cell embryos. By using a low molecular weight heparin to antagonize the function of InsP3 receptors, we were able to show that InsP3 receptors are essential for Sr2+-induced Ca2+ oscillations. Treating metaphase II (MII) oocytes with the PLC inhibitor, U73122, abolished Sr2+-induced increases in Ca2+. This inhibitory effect of U73122 could be rescued by microinjection of InsP3, indicating that Sr2+-induced Ca2+ oscillations require the synergistic action of InsP3. CONCLUSIONS: Sr2+-induced calcium oscillations in mouse oocytes and early embryos are mediated through InsP3 receptors, and require PLC activation and the synergistic action of InsP3.     

Fertilization and embryo development in a mouse ICSI model using human and mouse sperm after immobilization in polyvinylpyrrolidone

BACKGROUND: For human ICSI, sperm are normally immobilized immediately prior to injection. However, there are some situations when only sperm of questionable viability are available. There are few evaluations of fertilization or developmental problems in human or animal models using sperm having known intervals between immobilization and injection. METHODS: Immobilized human sperm were maintained for 1-24 h in 10% polyvinylpyrrolidone (PVP) before injection into mouse oocytes. Mouse sperm heads were similarly maintained in either PVP or a high potassium-containing 'nucleus isolation medium' (NIM) before ICSI and embryo development to the blastocyst stage. RESULTS: Immobilized human sperm activated mouse oocytes comparably to controls even 24 h after immobilization. However, mouse sperm heads showed a decrease in activating ability 6 h after isolation, either in PVP or NIM. A significant reduction in blastocyst development occurred if mouse sperm heads were maintained for even 1 h in PVP. After 6 h, no blastocysts formed, with arrest occurring at the morula stage. NIM provided partial protection for up to 3 h. CONCLUSIONS: Immobilized human sperm maintained oocyte activating activity for 24 h. However, mouse sperm are susceptible to alterations that affect both fertilization and development.     

Propanediol-induced alterations in membrane integrity, metabolism and developmental potential of mouse zygotes

The effects of 1, 2-propanediol (PROH) on embryonic development,membrane integrity and metabolism on B₆D₂F₁ mouse zygotes inthe pronuclear stage were evaluated. In both the control andthe group treated with 1.5 M PROH, 78% of the zygotes developedinto 2-cell embryos. With 3 M PROH, the proportion of 2-cellembryos was only 7% (P < 0.05). In a second series of experiments,pronuclear mouse eggs were incubated in either fluorescein diacetate(FDA) or 5µM Acridine Orange (AO) then transferred toPROH. FDA-induced fluorescence, which is maintained until thecell membrane is damaged, was retained in 100% of the controland 98% of the zygotes treated with 1.5 M PROH. Exposure to3.0 and 6.0M PROH reduced the percentage of zygotes with FDA-inducedfluorescence to 81% (P < 0.05) and 5% (P < 0.05) respectively.AO fluoresces yellow-green within the physiological pH range(7.4). After AO exposure, 95% of the control zygotes and 95%of the zygotes exposed to 1.5 M PROH possessed yellow–greenfluorescence, indicating a normal cellular pH. Treatment with3.0 and 6.0 M PROH caused a shift in the fluorescence such that93% (P < 0.05) and 100%(P < 0.05) of the zygotes respectivelyno longer fluoresced yellow-green, indicating a lower pH. Theseresults demonstrate that a 20-min exposure to 1.5 M PROH doesnot affect embryonic development, while PROH at 3.0 M inhibitsembryonic development. Treatment with 3 M PROH causes both cellmembrane damage and pH changes, which are in turn associatedwith a decrease in embryonic development.     

Fertilization and early embryology: The effect of pentoxifylline on mouse in-vitro fertilization and early embryonic development

This study was designed to assess the effect of pentoxifylline(PTX) on fertilization and early embryonic development in themouse. Oocytes from superovulated B6CBA female mice were inseminatedin vitro with spermatozoa from B6CBA males incubated with PTXaccording to different protocols, i.e. (i) 3.6 and 7.2 mM PTXwashed out prior to insemination, (ii) 3.6 and 7.2 mM PTX dilutedsix times in the insemination medium and (iii) PTX present at3.6 and 7.2 mM in the insemination medium. After inseminationand washing, fertilization was assessed by the presence of 2-cellstage embryos. These were further cultured up to the blastocystor egg-cylinder stage to asses embryonic development. Parthenogeneticactivation was evaluated by exposing post-ovulatory oocytesto 3.6 and 7.2 mM PTX. If spermatozoa were washed free fromPTX before insemination, no effect on either fertilization orsubsequent development was found. If PTX was not washed out,fertilization was reduced significantly, yet development offertilized oocytes was unaffected. If insemination was performedin the presence of PTX both fertilization and development wereimpaired. Parthenogenetic activation was not increased by PTXexposure. We conclude that if used in in-vitro fertilization,exposure of oocytes and/or zygotes to PTX has to be avoidedby washing out the compound thoroughly to prevent adverse effectson early embryonic development.     

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.     

Effect of the number of inseminated spermatozoa on subsequent human and mouse embryonic development in vitro.

It has been shown, in both human and mouse in-vitro fertilization (IVF), that an excess number of spermatozoa in the insemination medium leads to reduced fertilization rates. In this study, we evaluated human embryonic development after dividing the oocytes of each of 62 IVF attempts into two groups on the basis of insemination with two widely used concentrations (50,000 and 100,000 spermatozoa/ml). The embryonic growth was retarded in the group inseminated with 100,000 spermatozoa/ml: significantly fewer fast developing embryos (4-cell and 5- to 8-cell stages) were found (53.4% in the 100,000/ml group and 65.5% in the 50,000 group; P less than 0.05). In two experimental series, mouse embryonic development was evaluated in the presence of 0, 50,000, 100,000 and 500,000 spermatozoa per ml. In the first series, the spermatozoa were present during 5-20 h after insemination, while in the second series, the spermatozoa were present during the whole culture period of 120 h. The development of mouse embryos was impaired when 500,000/ml spermatozoa were present during the whole culture period. In contrast with human IVF results, the presence of up to 500,000 spermatozoa during the first 20 h after insemination did not have any significant detrimental effect on blastocyst formation in the mouse.     

Impact of hyperglycemia on early embryo development and embryopathy: in vitro experiments using a mouse model

BACKGROUND: The aim of this study is to model the processes of early embryopathy seen in human pregnancy complicated by maternal hyperglycemia secondary to maternal diabetes using a mouse embryo culture system. METHODS: Female mice were superovulated and mated in pairs. Two-cell embryos were harvested from the oviducts and cultured in vitro in KSOM medium (synthetic oviductal medium enriched with potassium) supplemented with 0.2, 5.56, 15.56 or 25.56 mM d-glucose. Cell proliferation, differentiation and apoptosis were assessed. Experiments were performed in constant, embryos exposed to a particular concentration of glucose (0.2, 5.56, 15.56 or 25.56 mM) from harvest to either Day 5 post fertilization (pf) or Day 8 pf, and fluctuating, embryos exposed to alternate high 25.56 mM and normal 5.56 mM concentrations of glucose between harvest and Day 5 pf, glycemic culture. RESULTS: Expected levels of blastocyst formation and hatching were seen at 0.2 and 5.56 mM concentrations of glucose but both were impaired at higher concentrations (chi(2), P < 0.005; P < 0.001). Total cell numbers (P < 0.002) and cell allocation to the inner cell mass (P < 0.01) were reduced, but with no evidence of enhanced apoptosis in the hyperglycemic cultures. Variation in hyperglycemic exposure of the embryos on Days 2, 3 and 4 showed no adverse effects of hyperglycemia up to 24 h, but 48 and 72 h exposures were equally embryopathic (P < 0.01). CONCLUSIONS: Hyperglycemic exposure for >24 h is toxic to early embryo development. These findings may explain the lower than expected implantation rates and higher than expected rates of congenital abnormality and early pregnancy loss seen in patients with diabetes, particularly those with poor diabetic control.     

The influence of in-vitro culture versus stimulated and untreated oviductal environment on mouse embryo development and implantation.

A prospective randomised study was performed to evaluate stimulated versus natural oviductal environment in comparison with in-vitro culture for the developmental capacity of mouse embryos. Therefore, embryos of superovulated F1 hybrid CBAxC57Bl females were collected at 17, 22, 41 and 46 h after human chorionic gonadotrophin treatment and randomly divided into five groups. They were either transferred immediately to untreated pseudopregnant females, cultured in vitro for 5, 24 or 29 h before transfer, or cultured in vitro for 96 h to blastocysts. The transfers resulted in an impaired implantation (P < 0.001) and a lower numbers of living fetuses (P < 0.001) when embryos had been exposed longer to the stimulated oviductal environment. Similar results were obtained after a longer period of in-vitro culture (P < 0.05). However when embryos were flushed earlier from the superovulated mice and cultured longer in-vitro until the transfer was performed, the implantation rate was improved (P < 0.01). Blastocyst development, however, was better (P < 0.001) when embryos were flushed later. In conclusion, the stimulated oviductal environment impairs the developmental capacity of embryos in comparison with untreated pseudopregnant females. In-vitro culture is also suboptimal but better than the stimulated oviductal environment.     

The effects of the sperm motility activators 2-deoxyadenosine and pentoxifylline used for sperm micro-injection on mouse and human embryo development

The sperm motility stimulants 2-deoxyadenosine (DOA) and pentoxifylline(PTF), used to improve the success of insemination and spermmicro-injection for low motility sperm samples, were studiedfor their effects on the developmental capacity of mouse andhuman oocytes. When human oocytes were micro-injected with spermatozoain 3 mM DOA 80% of them became blocked at the 1-cell pronuclearstage. However, when spermatozoa in 3 mM PTF were used for micro-injectionor when spermatozoa were washed to remove DOA before micro-injectiononly a few oocytes (9–10%) were blocked. Pregnancies occurredin five of 14 patients into whom cleaving embryos from all threetreatments had been transferred, indicating that once cleavagewas initiated, development of embryos occurred at expected rates.Exposure of mouse oocytes to DOA for a short period during insemination(4–6 h) or a longer period during the pronuclear cellcycle (18 – 20 h) significantly reduced cleavage beyondthe 2-cell stage, resulting in a dramatic reduction in blastocystformation. PTF did not significantly reduce mouse embryo development.Similar results were obtained for oocytes inseminated in vitroor micro-injected with a spermatozoon into the perivitellinespace. Neither DOA nor PTF increased fertilization of mouseoocytes. PTF reduced fertilization, particularly in cumulus-enclosedoocytes and oocytes micro-injected with spermatozoa in PTF.We conclude that DOA is a potent inhibitor of embryo developmentand oocytes should not be exposed to DOA. Exposure of oocytesto PTF had little effect on their subsequent development buttreatment of cauda epididymal mouse spermatozoa can reduce theirfertilizing capacity.     

Fetal development after transfer is increased by replacing protein with the glycosaminoglycan hyaluronan for mouse embryo culture and transfer.

The effect of macromolecules on mouse embryo development and viability after culture in sequential media was investigated. It was found that high rates of viable blastocysts could be obtained in the absence of any macromolecule. Blastocyst cell numbers were increased when bovine serum albumin was present in the culture medium, although this benefit was not manifest after blastocyst transfer. Rather, the highest rates of implantation and fetal development after blastocyst transfer were observed when hyaluronan was the macromolecule in the culture media. Subsequent analysis revealed that the beneficial effects of hyaluronan were due to its presence in the transfer medium. As the highest cell numbers and hatching rates obtained in this study occurred when both serum albumin and hyaluronan were present in the same medium, it is proposed that embryo culture media should contain both serum albumin and hyaluronan, while the transfer medium need only contain hyaluronan.     

Fertilization and early embryology: Effects of embryo density and co-culture of unfertilized oocytes on embryonic development of in-vitro fertilized mouse embryos

We have evaluated the effects of embryo density and the co-cultureof unfertilized (degenerating) oocytes on the development ofin-vitro fertilized (IVF) mouse embryos. In experiment 1, groupsof one, five, 10 or 20 zygotes were cultured in 20 µldrops of modified human tubal fluid (HTF) medium for 168 h at38.7°C in 5% CO₂ and 95% air. As the embryo density increased,significantly (P < 0.05) higher rates of embryos reachedhatched blastocyst stage. In addition, the time required forhatching after IVF was significantly (P < 0.05) shortenedby the increase in embryo density. In experiment 2, 10 IVF zygoteswere cultured with or without 10 unfertilized (degenerating)oocytes in 20 µl drops of HTF medium. The rates of IVFembryos that developed to morula, blastocyst, expanded blastocystand hatched blastocyst stages were decreased significantly (P< 0.01) by culturing embryos with unfertilized oocytes comparedwith culturing embryos alone. In experiment 3, groups of oneor 10 IVF zygotes or 10 IVF zygotes plus 10 unfertilized oocyteswere cultured in 20 µl drops of HTF medium and the numberof cells per blastocyst was examined at 120 h after IVF. Increasingembryo density resulted in a significant (P < 0.05) increasein the number of cells per blastocyst. In contrast, the cellnumber of IVF embryos that developed to blastocyst decreasedsignificantly (P < 0.05) when they were cultured with unfertilizedoocytes. The results suggest that in-vitro development of IVFmouse embryos is enhanced by increasing embryo density and isimpaired by co-culture with unfertilized (degenerating) oocytes.