Mouse Ova Research Articles

A Helminth-Induced Mucosal Th2 Response Alters Nonresponsiveness to Oral Administration of a Soluble Antigen

A fascinating feature of the intestinal mucosal immune system is its ability to guard against invasion by pathogens while avoiding a response to the many potential Ags present in food. The phenomenon of systemic tolerance after oral administration of protein Ags is well documented, but the cellular and molecular basis for the observed nonresponsiveness is not fully understood. To gain insight into the role of the mucosal microenvironment in the induction of orally induced nonresponsiveness, we attempted to induce tolerance to OVA in mice primed for a Th2-biased mucosal immune response by infection with the nematode parasite Heligmosomoides polygyrus. We found that oral tolerance for Th1-type responses to OVA is maintained when OVA is fed during the peak of the mucosal immune response to H. polygyrus. Tolerance for Th2 cytokine responses or a Th2-dependent isotype of IgG is not induced in this Th2-biased mucosal environment. Treatment of infected mice with rIL-12 to reverse the Th2 polarity of the parasite-specific immune response restores tolerance of both Th1 and Th2 responses to OVA. We conclude that the polarized Th2 response induced by this enteric infection plays a central role in determining whether or not systemic tolerance is induced. Our results imply that attempts to use oral administration of Ag to suppress systemic immune responses will be influenced strongly by the presence of mucosal infection.

Transgenic expression of a CD46 (membrane cofactor protein) minigene: studies of xenotransplantation and measles virus infection.

CD46 (membrane cofactor protein) is a human cell-surface regulator of activated complement and a receptor for the measles virus. A CD46 transgenic mouse line with an expression pattern similar to that of human tissues has been produced, to develop an animal model of (i) the control of complement activation by complement regulators in hyperacute rejection of xenografts, and (ii) measles virus infection. The mouse line was made using a CD46 minigene that includes promoter sequence and the first two introns of genomic CD46, which was coinjected into mouse ova with chicken lysozyme matrix attachment region DNA. A high level of CD46 expression in homozygotic transgenic mice was obtained with spleen cells having approximately 75% of the level found on human peripheral blood mononuclear cells. CD46 was detected in all tissues examined by immunohistochemistry, radioimmunoassay and Western blotting, showing that these mice were suitable for transplantation and measles virus infection studies. It also indicated that the transgene included the important regulatory elements of the CD46 promoter. Transgenic spleen cells were significantly protected in vitro from human complement activated by either the classical or alternative pathways and from alternative pathway rat complement. Furthermore, transgenic mouse hearts transplanted to rats regulated complement deposition in an in vivo model of antibody-dependent hyperacute xenograft rejection. Similar to human lymphocytes, transgenic lymphoblasts could be infected in vitro with measles virus; infected cells expressed viral proteins and produced infectious viral particles. The data demonstrate the suitability of this minigene for obtaining high-level CD46 expression sufficient for enhanced resistance of transgenic cells to complement attack and for obtaining wide tissue distribution of CD46, analogous to human tissues and, therefore, useful for comparative studies.

Adherence of human spermatozoa to trypsin-modified hamster ova.

Human spermatozoa do not attach to the zona pellucida of other species because of their high specificity compared to that of other animals in recognizing the zona pellucida. This study describes the recognition mechanism of human spermatozoa through trypsin treatment of the ovum of human and other species. This research has revealed two findings: (1) The "lattice-like" structure, the surface of the zona pellucida of the hamster ovum, was lost and became a uniformly coarse and flat structure. More than 200 human spermatozoa attached to it. The number of attaching spermatozoa increased significantly along with the prolongation of the incubation period and in direct dependence on the spermatozoa concentration. (2) Human spermatozoa did not attach to the zona pellucida of mouse, rat, and human ovum. This result confirmed that human spermatozoa attach to hamster ovum if modified with trypsin.

Maternal and paternal genomes function independently in mouse ova in establishing expression of the imprinted genes Snrpn and Igf2r: no evidence for allelic trans-sensing and counting mechanisms.

It has often been suggested that the parental-specific expression of mammalian imprinted genes might be dependent on maternal-paternal intergenomic or interallelic interactions. Using quantitative allele-specific RT-PCR single nucleotide primer extension assays developed for two imprinted genes, Snrpn and Igf2r, we demonstrate: (i) No role for maternal-paternal allelic interactions: the modes of parental-specific expression of Snrpn and Igf2r in normal ova were unchanged in gynogenetic and androgenetic ova; the latter contain two maternal and two paternal genomes respectively, and cannot undergo maternal-paternal interactions. (ii) No role for allelic counting or exclusion mechanisms: in individual blastomeres of androgenetic ova, both paternal Snrpn alleles were active (Snrpn was not expressed in gynogenetic ova), and in individual gynogenetic and androgenetic blastomeres, both maternal and paternal Igf2r alleles, respectively, were active. (iii) No role for ploidy: the mode of parental-specific expression of Snrpn and Igf2r in normal diploid ova was unchanged in individual blastomeres of triploid and tetraploid ova. Thus, the maternal and paternal genomes function independently in establishing the pre-implantation mode of parental-specific expression of Snrpn and Igf2r, with no role for trans-allelic/genomic interaction phenomena. In addition, the results show that inactive and biallelic modes of expression of imprinted genes are potential mechanisms for the death of gynogenones and androgenones at the peri-implantation stage.

Parthenogenic activation of pig oocytes by protein kinase inhibition.

Previous studies have shown that protein kinase stimulators can induce the release of the metaphase II arrest in mouse ova. The present report is about the role of protein kinase in parthenogenic activation of pig oocytes, which was studied using a broad-spectrum protein kinase inhibitor. Metaphase II oocytes were obtained via in vitro maturation. Two sources of H7 [1-(5-isoquinolinesulfonyl)-2-methylpiperazine, HCl] were tested--Sigma (H7s) and Calbiochem (H7c). Both were found to be equally effective in promoting release of the metaphase II block. Activation, release of the metaphase II arrest, and progression to the first interphase could be induced at the highest percentage with an exposure to 2.0 mM H7s for 80 min (68.1%). In another experiment, H7c resulted in 69.5% activation, while iso-H7 [1-(5-isoquinolinesulfonyl)-3-methylpiperazine, HCl] at a similar concentration and exposure duration resulted in 25.7% activation. H7s and H7c were more effective than iso-H7 in inducing the appearance of a 22-kDa protein that is associated with normal fertilization in the pig. In contrast, although pronuclei could form and the protein profiles were indicative of activation, cortical granule exocytosis did not occur, and oocytes failed to develop to the blastocyst stage after H7 treatment. In contrast to the H7-treated oocytes, electrostimulation resulted in pronuclear formation, the appearance of the 22-kDa protein, release of cortical granules, and development to the blastocyst stage. These data demonstrate that broad-spectrum inhibitors of protein kinase are unable to induce all the events associated with normal fertilization.

Age-Related Changes of Lectin Bindings on the Cell Surface of Unfertilized Mouse Ova.

Lectin bindings on the surface of unfertilized mouse ova immediately after ovulation were histochemically examined, and were compared among 30-day-old, 60- to 90-day-old, 180- to 210-day-old and 270-day-old mice. The bindings of PNA, GS-I, DBA, SBA, BPA, MPA, GS-II, WGA and Con A were observed from a weak to an intense degree on the cell surface of ova in mice from different age groups, but those of UEA-I and LPA were not. Although the strength in bindings of PNA, DBA, SBA, GS-II, BPA, WGA and Con A did not differ among the age groups, the strength of GS-I and MPA bindings intensified in 270-day-old mice. From the results, it may be said that the glycoconjugates on the cell surface of mouse ova immediately after ovulation contain galactose, N-acetylgalactosamine, N-acetylglucosamine and mannose, and that the amount of galactose and N-acetylgalactosamine increases as the animals get older.

Apoptosis in the Degeneration Process of Unferilized Mouse Ova.

In mammals, ova which are not fertilized undergo degeneration. Thus, it seems that the ovum is programmed to die unless fertilization and embryogenesis occur, but little is known about the mechanism of such degeneration. To investigate this process, we observed the morphological changes of cultured unfertilized ova and stained DNA fragmentation by the modified TUNEL method (treating floating samples in liquid reagents) to detect apoptosis. Ova were collected from the oviducts of superovulated mice and cultured for observation. The number of morphologically abnormal ova with shrinkage of the ooplasm and cytoplasmic fragmentation, which are typical features of apoptosis, showed a significant gradual increase from 24 to 32 hr (p < 0.05) and an abrupt increase from 40 hr of incubation (p < 0.001). DNA fragmentation, which is one of the biological changes seen in apoptosis, was observed in the ooplasm of both intact and abnormal ova, cells with cytoplasmic fragmentation, and in the first polar body at the time of ovum collection as well as after incubation. These findings demonstrate that apoptosis is related to the process of degeneration in the mouse ovum and first polar body.

Transgene detection during early murine embryonic development after pronuclear microinjection

The polymerase chain reaction (PCR) technique was used to detect a whey acidic protein (WAP) gene and transgene presence in mouse ova cultured to various stages of development after pronuclear microinjection at the one-cell stage. The PCR technique detected an endogenous 442 bp WAP DNA sequence in 78% of one-cell, 88% of two-cell and 94% of four-cell ova, and in 95% of morulae and 97% of blastocysts. The heterologous WAP-human protein C transgene was detected in 88% of one-cell, 88% of two-cell and 44% of four-cell ova, and in 40% of morulae and 29% of blastocysts. For comparison, the integration frequency for transgenic mouse production using the same DNA construct was 22%. After five days of in vitro culture, embryos that were either developmentally arrested or fragmented were tested for the presence of the transgene. The injected construct was detected in 83% of arrested one-cell, 85% of arrested two-cell, and 85% of fragmented ova. In culture, only 28% of zygotes microinjected with DNA developed to the blastocysts stage compared to 74% of noninjected zygotes, while 63% of zygotes developed to the blastocyst stage after injection of buffer alone. Pronuclear injection of the transgene at concentrations of 1.5, 15 and 50 micrograms ml-1 resulted in 28, 11 and 9% development to blastocysts and 29, 86 and 88% transgene detection, respectively. Transgene detection was 85, 96 and 97% in degenerate embryos at the respective doses of DNA. These data show that pronuclear microinjection of the transgene is detrimental to subsequent embryonic development.(ABSTRACT TRUNCATED AT 250 WORDS)

Indirect effects of oral tolerance in mice.

Anti-DNP antibody formation resulting from intraperitoneal (i.p.) immunization with DNP-KLH may be blocked by simultaneous (i.p.) injection of DNP-Ova or native Ova in mice orally tolerant to Ova, but not in normal mice. In Ova-tolerant mice the inhibition of anti-DNP antibody formation also occurred when DNP-Ova and DNP-KLH were given by separate routes of immunization: subcutaneous (s.c.) and i.p. A second exposure to Ova by gastric intubation (gavage) or intravenous administration simultaneously with i.p. immunization with DNP-KLH failed to inhibit anti-DNP antibody formation. There was inhibition of responses to DNP-KLH i.p. by DNP-Ova given 24 h before, but not 24 h after, and in the Ova-tolerant mice, addition of DNP-Ova only to the primary immunization with DNP-KLH inhibited secondary and tertiary responses to DNP-KLH in the absence of further exposures to DNP-Ova. These results suggest that the indirect effects of parenteral exposure of tolerant mice to the tolerated immunogen may inhibit unrelated immune responses. This inhibition is not due to 'innocent bystanding' suppression, i.e., to inhibitory cytokines provided locally by specific suppressor lymphocytes; it may derive from more durable perturbations of immune system.

Effects of low molecular weight oviductal factors on the development of mouse one-cell embryos in vitro.

The relationship between the oviduct and embryo development in the mouse was investigated and the period at which the influence of oviduct can be concerned in the development of mouse embryos in vitro was identified. In addition, the relative molecular weight of oviductal factors that promote embryo development was demonstrated. Mouse zygotes developed to the blastocyst stage when co-cultured with ampulla. The period of embryo co-culture significantly affected the further development of the embryos. Fewer one-cell embryos co-cultured with dissected ampullae for less than 24 h developed to blastocysts than those co-cultured for more than 28 h (P < 0.001). A high percentage of embryos co-cultured with ampullae after 24 h of culture in vitro developed to the blastocyst stage, which suggests that the influences of ampulla on the development of mouse embryos are restricted to a specific period at the two-cell stage (about 55-56 h after hCG injection) in vitro. Mouse ova that were cultured in media conditioned by ampullae could also develop to the blastocyst stage. The fractionated medium that contained low molecular weight fractions was more effective (P < 0.001) on the development of embryos to the blastocyst stage than that containing high molecular weight fractions. These results suggest that the low molecular weight oviductal factors play an important role in the development of mouse embryos at a certain critical age in vitro.

Subzonal insemination with a single spermatozoon using manipulation assisted sperm adhesion onto the ooplasmic membrane in mouse ova.

A simple and successful method of microinjection of a single spermatozoon under the zona pellucida of a mouse oocyte has been developed. A characteristic of this method is that the tip of the sperm injection needle pierces the zona pellucida without touching the ooplasmic membrane. All the ova (277) used for this series of experiments had normal morphology after the injection procedure. Spermatozoa preincubated in culture medium for capacitation and those treated with ionophore A23187 for induction of acrosome reaction were used. In combination with some of these injections, a manipulation assisting the adhesion of the sperm head onto the ooplasmic membrane was employed. The fertilization rate (67.3%) of the ova injected with the ionophore-treated sperm using the sperm-adhesion treatment was significantly higher (P less than 0.005) than that obtained by the injection of the preincubated sperm without applying the adhesion treatment (23.6%). All three of the recipients that received the 24 fertilized ova became pregnant and gave birth to 11 offspring (45.8%). The inseminations performed with the sperm-adhesion treatment using the immotile sperm from the preincubated population and/or those from the ionophore-treated population did not result in fertilization in any case. These results suggest that the fertilization rate of subzonal insemination with motile ionophore-treated sperm can be improved by applying the sperm-adhesion treatment and that sperm motility might be involved in the establishment of fertilization, even after the adhesion of the sperm head with the mouse ovum membrane.

Metallothionein gene expression and metal regulation during preimplantation mouse embryo development (MT mRNA during early development)

In order to provide information concerning gene expression and regulation in the preimplantation mammalian embryo, and to explore the roles of metallothionein (MT) during this period of development, the constitutive and metal-induced MT mRNA levels in mouse ova, preimplantation embryos, and oviducts were determined. These results were correlated with the effects of transient exposure to high levels of metals (zinz (Zn) or cadmium (Cd)) on the continued development of preimplantation embryos into blastocysts in culture. RNA from preimplantation mouse embryos at different stages of development (Days 1 through 4 of gestation; D1 = vaginal plug) was analyzed using the reverse transcriptase-polymerase chain reaction (RT-PCR) to specifically amplify MT-I and MT-II mRNA transcripts. MT-I mRNA in ova, preimplantation embryos, and oviducts was detected using in situ hybridization. This mRNA in the oviduct was also analyzed by Northern blotting. The results establish that the mouse MT genes are coordinately and constitutively expressed at low basal levels in ova and preimplantation mouse embryos. In unfertilized (ova), fertilized (one-cell) eggs, and two-cell embryos, the MT-I gene was not detectably responsive to metal ions, whereas in later cleavage stage embryos (four- and eight-cell) the MT-I gene was detectably responsive to metals in some blastomeres of some of the embryos. In contrast, after the third cleavage this gene was highly metal-inducible in essentially all cells of the embryo (morula/blastocyst). Surprisingly, the appearance of metal responsiveness of the MT genes during development correlated with decreased Zn toxicity and increased Cd toxicity; two-cell embryos were Zn-sensitive and Cd-resistant, whereas eight-cell and older embryos were Zn-resistant and Cd-sensitive. In the oviduct, MT-I mRNA was not abundant in total RNA, but was detected specifically in the epithelial cells of the isthmus region and was elevated in these cells on D3 and D4 of gestation. In the oviduct, only isthmus epithelial cells responded to metals (Zn or Cd) by increased accumulation of this mRNA. These studies suggest that preimplantation mouse embryo develops the capacity to respond to metals in the environmental milieu by induction of MT gene expression at about the third cleavage. Whether the lack of responsiveness of these genes before this stage reflects transcriptional repression or attenuated metal ion influx and/or enhanced efflux remains to be determined. Sensitivity and resistance of preimplantation embryos to acute metal toxicity involve mechanisms other than MT gene expression in preimplantation mouse embryos. The cell-specific expression of MT genes in the isthmus epithelia of the oviduct suggests that these cells play a role in metal ion homeostasis on D3 and D4 of pregnancy, perhaps by creating a local environment rich in the essential metals Zn and copper and/or by protecting from the toxic effects of Cd.

Development Potential of Mouse Embryos Conceived in Vitro and Cultured in Ethylenediaminetetraacetic Acid with or without Amino Acids or Serum1

Mouse ova were inseminated in vitro in modified Earle's balanced salts solution (EBSS) supplemented with 10 or 100 microM EDTA and 4 mg/ml BSA. After 4 h exposure to sperm, the ova were transferred to five different culture conditions based on albumin-free EBSS supplemented with 10 microM EDTA minus or plus amino acids, or with 100 microM EDTA minus or plus amino acids, or with human cord serum. After 44 h of culture, four-cell embryos from each culture group were transferred in cohorts of five into the left oviduct of pseudopregnant recipients (13-16 per culture condition). Two-cell embryos developed in vivo were similarly transferred to a separate group of recipients to serve as controls. The pregnancy rates following transfer of embryos cultured in 10 microM EDTA minus or plus amino acids or in 100 microM EDTA plus amino acids (38%, 43%, and 50%, respectively) were not significantly different from those of the in vivo control group (43%). The pregnancy rates following transfer of embryos cultured in 100 microM EDTA plus amino acids (21%) or plus cord serum (8%) were significantly lower (p less than 0.01) than those of the other groups. The overall yield of fetuses from total embryos transferred was significantly higher (p less than 0.01) for the groups developed in 100 microM EDTA plus amino acids (29%) and in vivo (26%) compared with embryos developed in 10 or 100 microM EDTA with no amino acids, 10 microM EDTA plus amino acids, or 100 microM EDTA plus cord serum (15%, 15%, 9%, and 3%, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Experimental distribution of permeability parameters of mouse ova and their behavior during freezing

Experimental distribution of permeability parameters of mouse ova and their behavior during freezing

The role of antigen-presenting B cells in T cell priming in vivo. Studies of B cell-deficient mice.

Mice rendered B cell deficient by treatment with rabbit anti-mouse IgM (anti-mu) antibodies from birth fail to respond when primed with soluble protein antigens in CFA, as measured by T cell proliferation when challenged with antigen in vitro. The role of B cells in T cell priming in vivo was examined by adoptively transferring hapten-specific B cells into anti-mu mice, followed by immunization with haptenated Ag in CFA. The T cell proliferative response to OVA of anti-mu BALB/c mice was partially restored by the administration of TNP or FITC-specific B cells and immunization with TNP-OVA or FITC-OVA, respectively. This reconstitution was Ag-specific, inasmuch as hapten-binding B cells restored the T cell responses to OVA in mice immunized with the same hapten coupled to OVA. The mechanism of B cell reconstitution of T cell priming in anti-mu mice was addressed using parental to F1 B cell transfers. The Ia restriction pattern of the activated T cells from these mice indicated that both direct presentation of Ag by transferred B cells and antibody-mediated enhancement of Ag presentation by non-B, host Ag-presenting cells occurred. Thus, Ag-specific B lymphocytes play a critical role in priming of T cells in vivo.

Rate of gonadotrophin-induced abnormalities in mouse ova is related to the site of hormone administration.

Adult female mice, regardless of the stage of the oestrous cycle, were superovulated with PMSG and hCG. Ovulated oocytes were recovered 20-22 h after hCG and fertilized ova 72-74 h after hCG. Compared with the controls, the gonadotrophin treatment increased the mating rate of the females, and the incidence of abnormal ova. Regardless of the site of gonadotrophin injections, the numbers of ova were equal, but the proportion of abnormal eggs in mice injected intraperitoneally was significantly higher than in mice injected subcutaneously.

Estimation and manipulation of glutathione levels in prepuberal mouse ovaries and ova:Relevance to sperm nucleus transformation in the fertilized egg

AbstractGlutathione (GSH), the major low‐molecular‐weight thiol in mammalian cells, is believed to be a necessary factor for the transformation of the disulfide‐stabilized sperm nucleus into the male pronucleus after fertilization. Its concentration in mouse ova, isolated from the ampulla of the oviduct after hormone‐induced superovulation of 3–4‐week‐old mice, has been determined by an enzymic cycling microassay. The level found was 1.80 pmol per ovum. Mean ovum diameter was estimated as 71–72 μm, indicating a GSH concentration of 9–10 mM in the mouse egg. Administration of L‐buthionine S, R‐sulfoximine, an inhibitor of GSH biosynthesis, during the 2 days preceding ovulation, reduced ovum GSH content below 0.20 pmol (&lt;1.0 mM). The mean GSH concentration of the hormone‐stimulated ovaries was reduced from 3.2 mM to 0.2 mM under these conditions.It has also been demonstrated that measurement and manipulation of ovum and ovarian levels of GSH can aid in studying its function in ovaries, ova, and early embryos. Hormone‐induced superovulation was achieved in BSO‐treated prepuberal C57B1/6 X SJL mice whose ovaries contained less than 10% of control levels of GSH. Over 50% of the isolated ova were fertilized in vitro. However, abnormal one‐cell embryos resulted in which the maternally derived pronucleus coexisted with an unchanged sperm nucleus, thus confirming that adequate levels of GSH are necessary for initiating transformation of the fertilizing sperm nucleus.

Introduction to recombinant DNA.

This paper describes the current state of knowledge of methods for analysing gene structure and localization. Illustrations are given of the preparation of complementary DNA libraries and their screening by positive-negative selection, the use of synthetic oligodeoxynucleotides and the use of antibodies. Analysis of the EGF precursor is used as an example to show its close relationship to plasma membrane receptor and its homology with the LDL receptor. Analysis of cloned genome DNA by use of bacteriophage lambda or cosmids gives useful information about gene regulation and evolution. Mutations by frame shift, point or missence mutations are discussed with reference to the LDL receptor and the apolipoproteins. The techniques of gene mapping by rat-human cell hybridization and hybridization in situ are illustrated, again with reference to genes coding for enzymes of cholesterol metabolism, the apolipoproteins and insulin-like growth factors. Finally the potential of in vitro mutagenesis and the injection of cloned DNA into the fertilized mouse ovum are discussed.

In vitro fertilization and development of mouse ova in protein-free medium.

Mouse ova were fertilized and developed in vitro in protein-free media. Superovulated ova from (JCL-ICR strain) female mice were fertilized in vitro by epidi-dymal sperm of the same strain and cultured for 114 h under 5% CO2 in air and the sub sequent development of the fertilized ova was examined.In the separate experiment, 2-cell embryos were flushed out of the oviducts of super-ovulated JCL-ICR mice 48 h after hCG injection and mating. The 2-cell embryos were cultured for 68 h and the subsequent development of the embryos was examined.Fertilization rates judged from the second polar body extrusion at 6 h after insemina-tion were 28.3 (45/159), 26.8 (71/265) and 62.5% (60/96) in protein-free medium containing 0, 50 and 100 μm EDTA, respectively. The development rates of these fertilized ova to bla-stocyst stage were 0 (0/45), 53.5 (38/71) and 76.7% (46/60) after 114 h of culture in protein-free medium containing 0, 50 and 100 μm EDTA, respectively. The development rate of the late 2-cell embryos collected from the oviducts to blastocyst stage was 95.7% (45/47) after 68 h of culture in protein-free medium. From these findings, it is suggested that mouse ova can be fertilized and be developed in protein-free media.