Unfertilized mouse oocytes were frozen by directly plunging them into liquid nitrogen vapour after equilibration in a freezing medium containing 3 mol ethylene glycol l⁻¹ with 0.25 mol sucrose or trehalose l⁻¹ for 5–40 min. After thawing and dilution of the cryoprotectant, oocytes of normal morphology were inseminated in vitro and the effect of equilibration period on the rates of fertilization and development in vitro was examined. Regardless of the equilibration in the freezing medium, no significant difference was observed on the fertilization rate of frozen–thawed oocytes. However, higher fertilization and higher normal fertilization rates were obtained with equilibration in 3 mol ethylene glycol l⁻¹ with either 0.25 mol sucrose l⁻¹ or trehalose for 20 and 40 min than with 5 and 10 min equilibration. Development rates to two-cell embryos and expanded blastocysts of in vitro fertilized frozen–thawed oocytes that were equilibrated in the freezing medium for 20 and 40 min were significantly higher (P < 0.05 or P < 0.01) than with 5 min equilibration. Development in vivo was assessed by transferring blastocysts derived from unfertilized oocytes frozen by the optimum treatment (20 min equilibration in the freezing medium before freezing) into the uterine horns of day 3 pseudopregnant female recipients. The development rate of frozen–thawed oocytes to the blastocyst stage after insemination in vitro was significantly lower than that of the non-frozen control (P < 0.001). However, transfer of the blastocysts derived from frozen-thawed oocytes to the uterine horns of the recipients resulted in fetal development and implantation rates similar to those of the control. The overall development rates to fetuses of blastocysts derived from in vitro fertilization of mouse oocytes frozen after 20 min equilibration in 3 mol ethylene glycol l⁻¹ with 0.25 mol sucrose l⁻¹ or trehalose were 20.3 and 22.5%, respectively.

Preantral follicles mechanically isolated from the ovaries of 12-day-old mice were exposed to 2 mol ethylene glycol l(-1) for 2 or 5 min and then to a vitrification solution containing 6 mol ethylene glycol l(-1) and 0.3 mol raffinose l(-1) for 0.5, 1.0 or 2.0 min before vitrification. The vitrified and fresh preantral follicles were treated with collagenase, and the oocyte-granulosa cell complexes (OGCs) obtained were cultured in vitro for 10 days in membrane inserts. Preantral follicles exposed to 2 mol ethylene glycol l(-1) for 5 min and then to the vitrification solution for 0.5 or 1.0 min showed the highest survival rates after warming. The follicular loss after warming was approximately 20%. After in vitro culture, the proportion of viable OGCs from the vitrified follicles was 10% lower than that of the fresh preantral follicles. There were no differences in the rates of maturation, fertilization and subsequent development to blastocysts between the oocytes derived from vitrified follicles and those derived from fresh preantral follicles; however, the developmental competence of the oocytes derived from both vitrified and fresh preantral follicles grown in vitro was lower than that of oocytes grown in vivo. One of the five recipient mice that received 20 blastocysts derived from vitrified preantral follicles gave birth to six live pups. The results of the present study demonstrate for the first time that mouse preantral follicles can be vitrified and that some of the embryos derived from vitrified preantral follicles can develop to live pups.

Summary. In Exp. 1 pig oocytes matured in vitro were used to evaluate the fertilizability in vitro of frozen epididymal (4 boars) and ejaculated (3 boars) spermatozoa that were preincubated in modified TCM-199 for 4 h at 37°C. The percentages of penetrated oocytes with the frozen epididymal spermatozoa were 0–40%. In contrast, none of the occytes were penetrated with the frozen ejaculated spermatozoa. In Exp. 2, oocytes matured in vivo were inseminated in vitro with the frozen epididymal spermatozoa that were known to penetrate oocytes matured in vitro. The penetration rate was 79% and the percentage of polyspermic oocytes was 57%. Culture for 30 h of oocytes matured in vivo and fertilized in vitro resulted in 51% (34/67) developing to the 2-cell stage. These embryos were transferred to 2 recipient gilts. One gilt became pregnant and a litter of 3 (1 live and 2 dead) was born. These results indicate that frozen epididymal spermatozoa can be used for in-vitro fertilization in the pig.

Keywords: in vitro; oocyte; fertilization; pig; embryo transfer

Summary. The effect of macrophages on proliferation of granulosa cells was examined in gonadotrophin-primed immature female rats. The mouse anti-rat macrophage monoclonal antibodies TRPM-2 and TRPM-3 were used and macrophages were observed in the granulosa layer and antrum of follicles and in corpora lutea and stroma around follicles. There was no difference in distribution between TRPM-2-positive cells and TRPM-3-positive cells. Macrophages with some cytoplasmic vacuoles of various sizes were also demonstrated in growing follicles. The average ratios of macrophages to granulosa cells in preantral, antral and mature follicles were 0·008, 0·007 and 0·002, respectively. Labelling with [³H]thymidine of granulosa cells cultured with peritoneal macrophages was significantly greater and the labelling index peaked to 25·0% when the ratio of macrophages to granulosa cells was 0·01, compared with the value of 14·2% when the granulosa cells were cultured alone. This ratio of macrophages to granulosa cells was similar to that in the preantral and antral follicles in vivo. These results suggest that macrophages participate in promoting proliferation of granulosa cells as local mediators in growing follicles.

Keywords: macrophage; granulosa cell proliferation; immunohistochemistry; culture; rat

Summary. Mouse blastocysts were exposed to solutions containing four concentrations (10, 20, 30 and 40% v/v) of six permeating cryoprotectants (glycerol, ethylene glycol, propylene glycol, dimethyl sulfoxide, 1,3-butanediol and 2,3-butanediol) in phosphate-buffered saline (PBS) with calf serum (CS) at room temperature (20–22°C). Blastocysts were exposed to these solutions for various periods, diluted into PBS plus CS with or without 1 mol trehalose l⁻¹ solution and their subsequent survival in vitro was examined. Two-way anova showed a significant interaction (P < 0·01) between cryoprotectant type, concentration of cryoprotectant and method of dilution. However, no significant interaction was observed between cryoprotectant type and duration of exposure. Results suggest that cryoprotectant-induced injury to nonfrozen blastocysts is variable and depends on the cryoprotectant used. On the basis of toxicity assays, ethylene glycol was the least harmful and was combined with dimethyl sulfoxide and 1,3-butanediol to produce a new vitrification solution. Mouse blastocysts were successfully cryopreserved using a vitrification solution (designated as VSv) consisting of 20% ethylene glycol, 20% dimethyl sulfoxide and 10% 1,3-butanediol (v/v). Embryos were equilibrated in two steps, first in an equilibration solution (designated as ESv: 10% ethylene glycol, 10% dimethyl sulfoxide and 5% 1,3-butanediol; v/v) and then to VSv or one-step in VSv at different exposure times at room temperature, and then vitrified by direct plunging into liquid nitrogen. High developmental rates were obtained in vitro when the embryos were exposed to ESv and VSv for 3 and 0.5 min, respectively (96·2%) or exposed to VSv for 0·5 min (95·4%). Prolonged exposure time proved detrimental to subsequent embryo development in vitro. When vitrified warmed embryos were transferred immediately to pseudopregnant recipients, the rate of development to normal fetuses did not significantly differ from that of the nonvitrified control (two-step, 54·2 and one-step, 45·0 versus 60·0%, P > 0·05).

These results suggest that the simple vitrification solution described in this study is effective for the cryopreservation of mouse blastocysts.

Keywords: mouse; blastocyst; vitrification; cryoprotectant; equilibration

In the process of nuclear transfer, heteroplasmic sources of mitochondrial DNA from a donor cell and a recipient oocyte are mixed in the cytoplasm of the reconstituted embryo. The distribution of mitochondrial DNA heteroplasmy in nuclear transfer bovine embryos and resultant offspring was investigated by measuring polymorphism in the displacement loop region of mitochondrial DNA using PCR-mediated single-strand conformation polymorphism. Most offspring (20 of 21 calves) from recipient oocytes of undefined mitochondrial DNA genotypes showed different genotypes from the mitochondrial DNA of donor cells. The single calf that was an exception showed heteroplasmy, including the donor mitochondrial DNA genotype. Six cloned calves were produced from oocytes of a defined mitochondrial DNA genotype. All of these clonal members and various tissues showed only the mitochondrial DNA genotype derived from the oocyte. The mitochondrial DNA from donor cells appeared to be eliminated during early embryonic development; it gradually decreased at the early cleavage stages and was hardly detectable by the blastocyst stage. These results indicate that the genotype of mitochondrial DNA from recipient oocytes may become the dominant category of mitochondrial DNA in calves resulting from nuclear transfer.

Syntaxin is an integral membrane protein that is involved in membrane fusion. The exocytosis of the contents of cortical granules, secretory vesicles located in the cortex of an egg, modify the extracellular environment to block additional spermatozoa from penetrating the newly fertilized egg. The aim of this study was to characterize syntaxin expression in mouse oocytes, and to determine the specific isoform that is expressed. Syntaxin was demonstrated in the mouse ovary and in mouse oocytes by both western blot and reverse transcription-polymerase chain reaction analyses. Syntaxin 4 was specifically expressed in metaphase II oocytes. Syntaxin was also immunolocalized within metaphase II oocytes and one-cell embryos with pronuclei using laser scanning confocal microscopy. In metaphase II oocytes, syntaxin was located on the plasma membrane and in the cortex, where cortical granules are present, but was not seen at sites free of cortical granules. In one-cell embryos, no cytoplasmic region was free of syntaxin immunoreactivity. Immunoelectron microscopy detected syntaxin on both the plasma membrane and the vesicle membranes in mouse metaphase II oocytes. In conclusion the results indicate that syntaxin 4 co-localizes with cortical granules and participates in membrane fusion and exocytosis during the cortical reaction.