Denuding and centrifugation of maturing bovine oocytes alters oocyte spindle integrity and the ability of cytoplasm to support parthenogenetic and nuclear transfer embryo development

The effects of cumulus cell removal and centrifugation of maturing bovine oocytes on nuclear maturation and subsequent embryo development after parthenogenetic activation and nuclear transfer were examined. Removal of cumulus cells at 4, 8, and 15 hr after in vitro maturation (IVM) or the centrifugation of denuded oocytes had no effect on maturation rates. Oocytes treated at 0 hr of IVM had a lower expulsion rate (50%) of the first polar body (PB1). The removal of cumulus cells and centrifugation affected the pattern of spindle microtubule distribution and division of chromosomes. There were almost no spindle microtubules allocated to PB1 and the spindles were swollen in anaphase I and telophase I oocytes. Approximately 20% of PB1 oocytes contained tripolar or multipolar spindles. After activation, oocytes denuded with or without centrifugation at 8 hr of IVM resulted in the lowest rate of development (3.0%). Denuded oocytes at 4, 15, and 24 hr of IVM with centrifugation or not resulted in similar blastocyst development rates (9.6%-13.2%). However, centrifugation of oocytes denuded at the beginning of IVM resulted in lower blastocyst development rate (8.1%, P < 0.05) than the noncentrifuged oocytes (17.3%). After nuclear transfer, the blastocyst development rates of oocytes denuded and centrifuged at 0, 4, and 8 hr of IVM were not different when compared to the same patch of noncentrifuged oocytes. However, oocytes denuded and centrifuged at 15 hr of IVM resulted in lower (P < 0.05) blastocyst development rates than the noncentrifuged oocytes. The results of this study suggest that removal of cumulus cells and centrifugation of denuded oocytes affect the spindle pattern. Embryo development of denuded and centrifuged oocytes may differ depending on the time of removal of cumulus cells.     

Effect of a serum extender containing growth factors on development of IVM and IVF bovine embryos

The present experiments were conducted to determine if supplementation of the culture medium with a serum extender containing growth factors would increase development of bovine embryos into morulae or blastocysts, following in vitro maturation (IVM) and in vitro fertilization (IVF). In Experiment 1, bovine zygotes were cultured in CR1 medium supplemented with 0, 0.01, 0.1, 1 or 10% serum extender. In Experiment 2, bovine zygotes were cultured in the presence of cumulus cells in CR1 medium supplemented with 0, 0.01, 0.1, 1 or 10% serum extender. In Experiment 3, bovine oocytes were matured in Medium 199 supplemented with 0, 0.01, 0.1, 1 or 10% serum extender. In Experiment 4, oocytes were matured in Medium 199 with 10% fetal bovine serum (FBS) or 5% FBS with serum extender. Following maturation, zygotes were cultured in CR1 medium with 10% FBS or 5 % FBS and serum extender. In all 4 experiments, the embryos were cultured in vitro until Day 7 after IVF, and development to the morula or blastocyst stage was assessed. The findings of the first 2 experiments showed that the serum extender did not directly influence embryo development but did stimulate development when cumulus cells were included in the culture system. The remaining 2 experiments showed that the serum extender did influence development through its interactions with cumulus cells during maturation and/or culture. These findings suggest that although growth factors or other products do not directly stimulate bovine embryo development their effects may be mediated through secondary cell systems.     

Improvement of in vitro co-culture systems for bovine embryos using a low concentration of carbon dioxide and medium supplemented with beta-mercaptoethanol

Two experiments were conducted to investigate the effect of carbon dioxide (CO2) gas atmosphere and beta-mercaptoethanol on the development of bovine embryos in an in vitro co-culture system. In Experiment 1, in vitro-matured bovine oocytes were inseminated and then co-cultured with cumulus cells in culture medium (CM; 25 mM HEPES buffered TCM-199 supplemented with 5% superovulated cow serum and 0.5 mM sodium pyruvate). Oocytes matured and fertilized in 2 or 5% CO2 in air exhibited similar cleavage rates, but the proportion of embryos that developed to the blastocyst stage was higher for embryos co-cultured in 2 versus 5% CO2 in air. In Experiment two, 4- to 8-cell embryos produced under the condition of 2% CO2 in air were co-cultured with cumulus cells in CM supplemented with various levels of beta-mercaptoethanol (0, 5, 10, 50 microM). The percentage of embryos that developed to the blastocyst stage in CM with 10 microM beta-mercaptoethanol was higher (P<0.05) than that of embryos co-cultured with 0 or 50 microM beta-mercaptoethanol. These results indicate that cumulus cell co-culture in an atmosphere of 2% CO2 in air has a marked stimulatory effect on in vitro development of bovine embryos and that addition of beta-mercaptoethanol to the co-culture medium 2 d after insemination improved the in vitro development of bovine 4- to 8-cell embryos to the blastocyst stage.     

Development, chromosomal composition, and cell allocation of bovine cloned blastocyst derived from chemically assisted enucleation and cultured in conditioned media

Treatment of in vitro matured bovine oocytes with colcemid results in a membrane protrusion that contains maternal chromosomes, which can be easily removed by aspiration. Four experiments were designed to evaluate the overall and temporal effects of conditioned medium (CM) by bovine cumulus cells on development of nuclear transfer (NT) bovine embryos and to examine the chromosomal composition and allocation of inner cell mass (ICM) and trophectoderm (TE) of the subsequent blastocysts. The nuclear transfer embryos were cultured in various CR1aa media conditioned by preculture with bovine cumulus cells. Development to the blastocyst stage in BSA-containing CM (BCM) and serum-containing CM (SCM) were similar to co-culture group (24-30%). The 24 hr-conditioned BCM yielded higher blastocyst development than 48 and 72 hr-conditioned BCM. Temporary exposure of embryos to BCM and SCM followed by CR1aa was also studied. Morula and blastocyst development were not different among the groups cultured in BCM for 72, 96, and 168 hr, but were significantly higher (P < 0.01) than groups exposed to BCM for 24 and 48 hr, respectively. Blastocyst development in SCM for 24 hr (29%), 96 hr (25%), and 168 hr (27%) were much higher (P < 0.05) than those in SCM for 48 hr (12%) and 72 hr (10%). The analyses of chromosomal composition of the resulting blastocysts indicate approximately 80% of the blastocysts cultured in CR1aa with co-culture or groups initially exposed to BCM for 24 hr followed by culture in CR1aa were diploid. However, the incidence of diploidy were only 36-60% in SCM-cultured groups and groups cultured in BCM beyond 48 hr. Conditioned media did not affect the allocation of ICM and TE in the blastocyst. No difference was found in the ratio of inner cell mass to total cells in co-culture, BCM or SCM groups (0.424, 0.441, and 0.473, respectively). In conclusion, bovine cumulus cell-CM and CR1aa with co-culture supported comparable development and blastocyst ICM:total cell ratio of bovine NT embryos. However, CM affected the blastocyst chromosomal composition and induced higher mixploidy.     

Cumulus cell function during bovine oocyte maturation,fertilization, and embryo development in vitro

Several contemporary micromanipulation techniques, such as sperm microinjection, nuclear transfer, and gene transfer by pronuclear injection, require removal of cumulus cells from oocytes or zygotes at various stages. In humans, the cumulus cells are often removed after 15–18 hr of sperm-oocyte coincubation to assist the identification of the fertilization status. This study was designed to evaluate the function of cumulus cells during oocyte maturation, fertilization, and in vitro development in cattle. Cumulus cells were removed before and after maturation and after fertilization for 0,7,20, and 48 hr. The cumulus-free oocytes or embryos were cultured either alone or on cumulus cell monolayers prepared on the day of maturation culture. Percentages of oocyte maturation, fertilization, and development to cleavage, morula, and blastocyst stages and to expanding or hatched blastocysts were recorded for statistical analysis by categorical data modeling (CATMOD) procedures. Cumulus cells removed before maturation significantly reduced the rate of oocyte maturation (4–26% vs. 93–96%), fertilization (0–9% vs. 91–92%), and in vitro development at all stages evaluated. Cumulus cells removed immediately prior to in vitro fertilization (IVF) or 7 hr after IVF reduced the rates of fertilization (58–60% and 71%, respectively, vs. 91–92% for controls), cleavage development (40–47% and 53–54% vs. 74–78% for controls), and morula plus blastocyst development (15% and 24% vs. 45%, P < 0.05). Cumulus cell co-culture started at various stages had no effect on fertilization and cleavage development but significantly improved rates of embryo development to morula or blastocyst stages (P < 0.05). Cumulus cell removal at 20 hr after IVF resulted in similar development to controls (P > 0.05) at all stages tested in this study. The intact state of surrounding cumulus cells of oocytes or embryos appears to be beneficial before or shortly after insemination (at or before 7 hr of IVF) but not essential at 20 hr after IVF. © 1995 Wiley-Liss, Inc.     

Glucose requirement at different developmental stages of in vitro fertilized bovine embryos cultured in semi-defined medium

Three experiments were conducted in which 2-cell bovine embryos were prepared from oocytes, obtained from abattoir ovaries, by in-vitro maturation for 22 to 24 hours, followed by exposure to spermatozoa for 8 hours and culture for 40 hours within the cumulus. The cumulus cells were then removed, and the cleaved embryos were cultured for a further 120 hours or longer, in the presence or absence of glucose, pyruvate and lactate. Very few embryos developed in the complete absence of energy substrates. Lactate and pyruvate, alone or combined, supported development to the 8-cell stage, but pyruvate was required to support development to the morula stage (Experiment 1). When present throughout culture or when added at 48 or 96 hours postinsemination, 5.56 mM glucose was detrimental to development (Experiments 1 and 2). However, when added at 120 hours postinsemination, 5.56 mM glucose improved development to the blastocyst and expanded blastocyst stages, compared with no glucose or 11.12 mM glucose (Experiment 3).     

Pregnancies after co-culture of cumulus cells with bovine embryos derived from in-vitro fertilization of in-vitro matured follicular oocytes

Bovine follicular oocytes surrounded by cumulus cells for more than one-third of their surface were matured, fertilized and developed in vitro utilizing a co-culture system with bovine cumulus cells. Embryos developed into blastocysts were non-surgically transferred to the uteri of cows at Day 6, 7 or 8 (Day 0 = oestrus). Out of 6 recipient cows (19 blastocysts transferred), 3 became pregnant. One of the 3 pregnant cows carried twins. The results of this study demonstrated the viability of embryos obtained from in-vitro maturation of bovine oocytes followed by in-vitro fertilization and culture to the blastocyst stage in vitro.     

Gas6在猪卵母细胞及早期胚胎中表达模式与功能的初步研究

该研究通过免疫组化和QRT-PCR等方法系统分析了Gas6（growth arrest-special gene 6）基因在猪卵泡发生及早期胚胎发育过程中的表达规律,并提出了一种改良猪卵母细胞体外成熟系统的方法。研究结果显示,Gas6基因表达于猪卵巢中的卵母细胞细胞核及其周围的卵丘细胞,在卵母细胞体外成熟及早期胚胎发育过程中始终有表达,且在囊胚中表达最高。Gas6 mRNA在卵母细胞成熟过程中始终存在,但在孤雌激活后迅速消失,直到发育到囊胚时再次出现。在猪卵母细胞体外培养系统中添加不同浓度的Gas6重组蛋白培养卵母细胞,发现添加Gas6重组蛋白对卵母细胞的极体率无显著影响;但是当添加浓度为100 ng/mL时,培养的卵母细胞孤雌激活后分裂率、囊胚率及囊胚细胞数都显著增高。Gas6可能是通过改善卵母细胞细胞质的成熟质量提高卵母细胞的发育潜能,从而获得了更多、更好的胚胎。     

Optimization of parthenogenetic activation protocol in porcine

The effects of the electrical field strengths, number of pulses, and post-activation media on chromatin conformation and parthenogenetic development were studied to optimize the activation protocol for porcine nuclear transfer. In experiment 1, electrical field strengths were examined. Oocytes were subjected to square direct current pulses at output voltages of 1.2, 1.7, 2.2, and 2.7 kV/cm for 1 x 30 microsec. The voltage resulting from experiment 1 was 2.2 kV/cm, in which 50.0% of activated oocytes developed to blastocysts in vitro. In experiment 2, the influence of 1, 2, and 3 pulses on blastocyst development was tested using field strengths and post-activation medium described in experiment 1. Oocytes activated by a single 30 microsec pulse of 2.2 kV/cm DC yielded a higher blastocyst rate (56.3%) than oocytes activated by 2 or 3 pulses (<42.5%). In experiment 3 and 4, we investigated the effects of cytochalasin B (CB), cycloheximide (CH), and CB + CH on nuclear development stages and parthenogenetic development following a single 30 microsec pulse of 2.2 kV/cm DC. The percentage of activated oocytes was not different among CB (93.3%), CB + CH (98.3%), control (80.0%), and CH (80.0%) groups 12 hr after activation. Treatment with CB (57.5%) or CB + CH (53.8%) enhanced the blastocyst rate compared with other groups, CH (23.8%) treated- and control group (18.8%). The results demonstrated that a single 30 microsec pulse of 2.2 kV/cm DC followed by culturing in post-activation medium with CB for 5 hr were effective parameters for parthenogenetic activation and blastocyst formation of in vitro matured porcine oocytes which suggests that a single calcium rise is sufficient to activate pig oocytes and to achieve high rate of blastocyst development.     

Effects of sodium pyruvate in nonserum maturation medium on maturation, fertilization, and subsequent development of bovine oocytes with or without cumulus cells

The present study was conducted to determine the effects of cumulus cells and sodium pyruvate during in vitro maturation of bovine oocytes on maturation, fertilization, and subsequent development. Cumulus-enclosed oocytes (CEOs) and cumulus-denuded oocytes (CDOs) were cultured for 24 h in polyvinylpyrrolidone-Hepes-tissue culture medium 199 with or without sodium pyruvate. Oocytes were fertilized in vitro and then cultured in CR1aa for 10 days. Before in vitro fertilization, the glutathione (GSH) content of some oocytes was measured. Maturation and normal fertilization rates of CDOs cultured with sodium pyruvate and CEOs were higher than that of CDOs cultured without sodium pyruvate. The CEOs showed significantly higher rates of development to the blastocyst stage than CDOs. The GSH contents of oocytes significantly decreased in CDOs after maturation culture, but the GSH contents of oocytes in CEOs remained at the same level as oocytes before culture. These results indicate that sodium pyruvate promotes nuclear maturation of bovine CDOs and that a continuing presence of cumulus cells during maturation is important for subsequent development of zygotes to the blastocyst stage. However, blastocysts produced from CDOs in the presence of sodium pyruvate showed a developmental competence to be normal calves, but it is not known if CDOs cultured without sodium pyruvate also were capable of developing into calves.     

Effect of culture media on in vitro development of cloned mouse embryos

The effect of simple and sequential embryo culture media on the preimplantation development of mouse nuclear transfer (NT) embryos reconstructed with cumulus cell nuclei using a mechanical NT technique was studied. Blastocyst formation rate was evaluated using CZB medium and the sequential media G1/G2 and KSOM/G2. Arrested two- and three-cell NT embryos were Hoechst-stained to check for nuclear abnormalities. Nonmanipulated and sham-manipulated parthenogenetic embryos served as controls for, respectively, the medium and the handling technique. Rates of blastocyst formation for medium and handling control embryos were similar in CZB (58% and 61%), in G1/G2 (94% and 85%), and in KSOM/G2 (88% and 84%). Development of NT embryos was significantly impaired from the two-cell stage onwards, reaching the blastocyst stage at a rate of 5% in CZB, 14% in G1/G2, and 28% in KSOM/G2. Arrested two- and three-cell stage NT embryos showed a high rate of binucleation. These data demonstrate not only that NT embryos are more sensitive to in vitro culture conditions than parthenogenetic control embryos but also that selection of culture media can influence the preimplantation development of NT embryos.     

Effects of co-culture and embryo number on the in vitro development of bovine embryos

It is generally accepted that culturing embryos in groups or with somatic cells improves both the yield and quality of the blastocysts obtained. The aims of this study were 1) to compare the yield and quality of the embryos obtained after culture in several number conditions and in several culture systems and 2) to assess the effect of co-culture started at various stages of embryo development. Under cell-free culture conditions (modified synthetic oviduct fluid [mSOF] supplemented with 10% fetal calf serum [FCS] 48 h post insemination, the rate of Day 10 blastocysts was lower when embryos were cultured in small groups (1 to 6 per drop) than in large groups (4 versus 23% ; P < 0.01). There was no group effect when embryos were co-cultured either with Buffalo rat liver (BRL) cells in TCM 199, or in a culture system allowing the progressive development of cumulus cells in mSOF, even if co-culture started at 66 or 114 h post insemination. However, embryos cultured singly had lower cell numbers than embryos cultured in large groups when co-culture started at 114 h post insemination. This suggests that 1) somatic cells improve the development of singly cultured bovine embryos up to the blastocyst stage after the 9-16 cell stage; 2) co-culture affects blastocyst cell number of singly cultured embryos by acting roughly between the 5-8 and the 9-16 cell stage; and 3) cooperation between embryos could replace the effect of co-culture either on the yield of blastocysts or on blastocyst cell number. Blastocysts appeared significantly earlier in co-culture with cumulus cells in mSOF than in co-culture with BRL cells in TCM 199 (detection of the blastocysts: 7.3 +/- 0.1 d post insemination with cumulus cells versus 8.1 +/- 0.1 d with BRL cells; P < 0.001) and had a significant higher number of cells (143 +/- 9 versus 85 +/- 11; P < 0.001). This system thus seems suitable for the culture of small numbers of embryos resulting from in vitro maturation and fertilization of oocytes from individual donor cows.     

Bovine parthenogenetic blastocysts following in vitro maturation and oocyte activation with ethanol

The appropriate in vitro bovine oocyte maturation and ethanol activation conditions for preimplantation bovine embryo parthenogenetic development to the blastocyst stage were investigated. A 7% ethanol concentration significantly enhanced (P<0.05) the proportion of activated, in vitro-matured bovine oocytes (7% ethanol, 83.4 +/- 3.2% versus 0% ethanol, 63.9 +/- 2.0%). The proportion of activated oocytes was significantly higher (P<0.05) by treatment with 7% ethanol for a minimum of 2 minutes (2 minutes, 89.8 +/- 4.0% versus 0.5 minutes 63.4 +/- 4.9%). Oocyte maturation for periods ranging from 30, 34, 38 and 44 hours resulted in a significant increase (P<0.05) in the proportion of activated oocytes, and in oocytes displaying 2 or 3 pronuclei versus oocytes matured for 26 hours. The proportion of cleaved, activated oocytes (2-cell stage), 4 -cell stage and parthenogenetic morula/blastocysts was significantly higher (P<0.05) within the 34-hour oocyte maturation treatment group. Although the 44-hour oocyte maturation treatment group displayed the highest proportion of activated oocytes with 2 pronuclei, it did not display the highest cleavage frequency, possibly due to the effects of postovulatory aging. Several morphologically normal parthenogenetic bovine blastocysts developed from oocytes that were in vitro matured for 34 hours. The ability to produce such parthenogenetic embryos will eventually facilitate investigation into the role(s) of the maternal and paternal genomes during bovine early development.     

In vitro fertilization and development of bovine oocytes matured in serum-free medium

This study was undertaken to investigate the effects of supplementation of serum (fetal calf serum), gonadotropins (LH, FSH, prolactin) and estradiol-17 beta (E2) to culture medium during in vitro maturation of bovine cumulus oocyte complexes on subsequent fertilization and development to the blastocyst stage in vitro. Serum supplementation during bovine oocyte maturation was not required but hormonal supplementation, gonadotropins (LH + FSH) and E2, enhanced the fertilizability and developmental ability of bovine oocytes matured in vitro. The addition of prolactin to maturation medium containing LH, FSH, and E2 did not further enhance frequencies of fertilization and development.     

Effects of oocyte quality, oxygen tension, embryo density, cumulus cells and energy substrates on cleavage and morula/blastocyst formation of bovine embryos

Various factors, such as quality of the oocyte, oxygen tension, embryo density, and kind of energy substrate during in vitro production of embryos may affect the rate of preimplantation embryo development. In the present study we used 12553 bovine oocytes aspirated from slaughterhouse ovaries to evaluate various culture conditions that would increase in vitro production of advanced stages of preimplantation embryos. The morphological quality of the oocyte based on the compactness and number of layers of cumulus cells had significant positive effects on the rates of in vitro maturation, fertilization and development to the morula and blastocyst stages. None of the corona-enclosed or nude oocytes progressed beyond the 8- to 16-cell stage. The level of oxygen (5 or 20%) did not affect the proportion of one-cell embryos undergoing cleavage or progressing to morula and blastocyst stages. The rate of development of one-cell embryos originating from inferior quality oocytes was significantly improved when cultured in groups of 40 instead of 20 embryos per 0.5 mL medium. In the presence of cumulus cells, glucose had beneficial effects on in vitro maturation and subsequent development of IVM-IVF zygotes. The presence of serum improved the rate of in vitro development of one-cell embryos. Minimum Essential Medium supplemented with energy substrates according to the findings of metabolic studies was less effective in supporting in vitro maturation and subsequent development than TCM-199. In conclusion, morphological grading of immature oocytes is an appropriate selection criterion for their developmental ability. Embryo yields from low quality oocytes can be increased by culturing them in large groups. Serum is not essential for in vitro generation of embryos but its addition improves rates of success.     

Nuclear transfer in cattle: Effect of nuclear donor cells,cytoplast age,co-culture,and embryo transfer

There are many factors affecting the efficiency of nuclear transfer technology. Some are evaluated here using our novel approach by enucleating oocytes at 20–22 hr after in vitro maturation (IVM), culturing the enucleated oocytes (cytoplasts) for 8–10 hr or 18–20 hr to gain activation competence and then conducting nuclear transfer. In the first experiment, we demonstrated that cumulus cell (CC) monolayer can support some cloned embryos to develop into morulae or blastocysts. Co-culture with CC and bovine oviduct epithelial cell (BOEC) monolayers resulted in no differences (P 0.05) in supporting the development of cloned embryos (Experiment 2). When in vitro matured oocytes were enucleated at 22 hr after IVM followed by nuclear transfer 18–20 hr later, cleavage and morula or blastocyst development of the cloned embryos were similar to those resulting from the enucleated oocytes which had been matured in vivo (Experiment 3). Frozen embryos as nuclear donor cells worked equally well as fresh embryos for cloning in embryo development which was superior to IVF embryos (Experiment 4). However, fresh embryos resulted in a higher proportion (P < 0.05) of blastomere recovery than did frozen or IVF ambryos. Finally, embryo transfer of cloned embryos from our procedure produced a viable calf, demonstrating the commercial value of this novel approach of the technology. © 1993 Wiley-Liss, Inc.     

Development of in vitro fertilized oocytes from pregnant and nonpregnant cows in oviductal epithelial and cumulus cell co-culture systems

The objectives of this study were 1) to measure cleavage, blastocyst formation, and blastocyst hatching after in vitro maturation (IVM), fertilization (IVF) and culture (IVC) of oocytes aspirated from pregnant versus nonpregnant cows, and 2) to compare embryo development in co-culture with bovine oviductal epithelial cells versus cumulus cells. No differences in cleavage (38 versus 40%), blastocyst formation (13 versus 13%), or blastocyst hatching (53 versus 51%) were observed for in vitro-matured, fertilized, and cultured oocytes from pregnant versus nonpregnant cows, respectively (P > 0.05), indicating that nonpregnant and early-pregnant cows are equally acceptable donors of oocytes for IVM/IVF/IVC procedures. Cleavage (36 versus 40%), blastocyst formation (11 versus 12%), and blastocyst hatching (50 versus 55%) were not different for embryos co-cultured with oviductal epithelial cells versus cumulus cells (P > 0.05). Thus, equivalent embryo development can be obtained with co-culture systems commonly used for in vitro-derived bovine embryos. These results help to define variables that affect comparison of results across laboratories and that are relevant to the practical application of IVM/IVF/IVC procedures to cattle.     

Effects of growth factors FGF2 and IGF2 on the development of parthenogenetic mouse embryos in utero and in vitro

We studied the effects of fibroblast growth factor 2 (FGF2) and insulin-like growth factor 2 (IGF2) on the development of parthenogenetic mouse embryos (CBA x C57BK/6)F1. The parthenogenetic embryos were treated in vitro during the preimplantation period and, at the blastocyst stage, transplanted into the uterus of pseudopregnant females. The addition of FGF2 at an optimal dose (2.5 ng/ml) to the culture medium increased twofold the number of embryos developed in utero to the somite stages as compared to the control: 18 and 43%, respectively. The parthenogenetic embryos (18-21 somites), treated and nontreated with FGF2 during the preimplantation period, were explanted for further development in vitro and treated with IGF2 at 2.5 micrograms/ml. As a result, many more parthenogenetic embryos (> 87%) of both groups developed in vitro to the stage of 30 or more somites as compared to the control (59%). The treatment of the parthenogenetic embryos with FGF2 alone at the preimplantation stages did not improve their development in vitro at the postimplantation stages. The results we obtained suggest that the treatment of parthenogenetic embryos in vitro with FGF2 during the preimplantation period increased twofold the number of somite embryos in utero, while their subsequent treatment in vitro with IGF2 leads to a significant prolongation of their development, as compared to the control.     

Effect of RU486 on different stages of mouse preimplantation embryos in vitro

17 beta-Hydroxy-11 beta(4-dimethylaminophenyl)-17 alpha-(1-propynyl)estra-4, 9-dien-3-one (RU486) inhibited the in vitro development of different stages of mouse preimplantation embryos under study. Two-celled embryos, morulae, and early blastocysts were obtained from B6D2F1 mice. The embryos were grown in Ham F-10 nutrient mixture (with glutamine) supplemented with sodium bicarbonate (2.1 g/L), calcium lactate (282 mg/L), and bovine serum albumin (fraction V, 3 mg/mL) at 37 degrees C in a humidified incubator supplied with 5% CO2 in air. RU486 was added to the culture medium at concentrations of 1, 5, 10, and 20 micrograms/mL. Culture medium with 0.05% ethanol served as the control. In vitro growth of embryos was assessed by the following criteria: (i) two-celled stage embryo development to blastocyst stage after 72 h, (ii) morula stage grown to blastocyst stage after 24 h, and (iii) early blastocyst stage development to hatching blastocyst after 12 h, in culture. RU486 inhibited the in vitro development of two-celled embryos, morulae, and early blastocysts at concentrations of 5, 10, and 20 micrograms/mL culture medium (p less than 0.001). The inhibitory effect of RU486 at these concentrations on the development of all the stages of embryos under study was irreversible. However, RU486 did not affect embryo development at 1 microgram/mL culture medium. The study indicates the direct adverse effect of RU486 at 5 micrograms/mL and higher concentrations in culture medium on the development of mouse preimplantation embryos in vitro, and it encourages its further investigation as a postcoital contraceptive in animal models and humans.