Interaction between embryos and culture conditions during in vitro development of bovine early embryos

Various factors such as embryo density and substances in the medium can influence embryo development in vitro. These factors and the embryos probably interact with each other, however the interactions are not fully understood. To investigate the interactions, we examined the effects of the number of embryos, drop size, oxygen concentration and glucose and inorganic phosphate in the medium during protein-free culture of bovine IVM/IVF embryos. In Experiment 1, different numbers of embryos were cultured in a 50 microl drop of medium. The frequencies of blastocyst development in the groups with 25, 50 and 100 embryos per drop were higher than in the other groups. One, five and 25 embryos were cultured in different drop sizes (Experiment 2), a 50 microl drop of medium at different O2 concentrations (Experiment 3) and a 50 microl drop of medium excluding glucose and/or inorganic phosphate (Experiment 4). In Experiment 2, the size of the medium drops did not improve blastocyst development. In Experiment 3, the highest frequency of blastocyst development for one, five and 25 embryos per drop was obtained at 1, 2.5 and 5% O2, respectively. In Experiment 4, blastocyst development for one and five embryos per drop were improved in the medium excluded inorganic phosphate. These results indicate that there is a cooperative interaction among embryos during culture and that this interaction may be mediated by reduction of toxic factors in the medium. At low embryo density, reduced oxygen concentration or the exclusion of inorganic phosphate enhanced blastocyst development.     

Effects of various electric fields on the fusion and in vitro development of mouse two-cell embryos

This study was undertaken to examine the effects of various electric fields such as alternating current (a.c.) voltage, fusion pulse strength, pulse duration, pulse number and electrode geometry on blastomere fusion and developmental rates of mouse two-cell embryos. The a.c. voltages (6 and 12 V/mm) did not affect the fusion and developmental rates. High fusion and developmental rates were obtained when pulse strengths of 1.0 to 2.5 kV/cm, pulse durations of 30 to 90 mu sec and pulse numbers of 1 to 6 were applied using a wire chamber. Comparison of electrode geometries showed that fusion rates were similarly high (93 to 98%) when pulse strengths of 1.0 to 2.5 kV/cm were applied, regardless of the electrode geometry. However, significantly lower developmental rates were observed in a rectangular chamber compared with those in a wire chamber, except when the pulse strength was 1.0 kV/cm. It was further observed that in a rectangular chamber, the developmental rate decreased with increasing pulse strength from 1.0 to 2.0 and 2.5 kV/cm. The results of this study indicate that by using a wire chamber, electric fields can be successfully applied across a relatively wide range of pulse strength, duration and number to provide sufficiently high fusion and subsequent developmental rates. The fusion conditions did, however, vary with chambers of different electrode geometries.     

Proembryo culture: in vitro development of early globular-stage zygotic embryos from Brassica juncea

We have systematically investigated the nutritional requirements for in vitro culture of zygotic proembryos of Brassica juncea. Normal embryo development in vitro was achieved in a new embryo culture medium (ECM) which contains mineral salts, sugars, amino acids, organic acids and coconut water. The culture system is comprised of two agar layers, with the top layer containing a higher osmolality than the bottom layer. Proembryos were embedded in the top layer in which the osmotic pressure decreased gradually during culture because of the diffusion of osmotically active compounds into the bottom layer. Using such a double-layer culture system and the ECM, proembryos as small as 35 μm (8–36 cells) could be cultured and developed into normal, mature embryos with an efficiency of at least 75%. In contrast to previous findings, we found that the removal of the suspensor had only a small effect on the development of embryos 55 μm or smaller, but no effect on larger proembryos. We expect this system to be very useful for investigations of the mechanism of plant embryogenesis.     

Hypoxanthine phosphoribosyltransferase activity in bovine embryos during the early embryonic development

The activity of hypoxanthine phosphoribosyltransferase (HPRT) was determined in the bovine embryo during early embryonic development. Microassay, using [(3)H] hypoxanthine, was improved to measure enzyme activity in the embryonic extract. This activity depended on the reaction time and the concentration of phosphorybosyl pyrophosphate (PRPP) in a reaction. mixture. Maximum activity was obtained at 4 hours of reaction time and at a concentration of 1 mM PRPP, but was much lower than the activity recorded in the mouse embryo. During early embryonic development, HPRT activity rapidly increased beyond the 8-cell stage. When distributions and activities of HPRT, adenine phosphorybosyltransferase (APRT), and the ratio of HPRT: APRT were examined in individual blastocysts, HPRT activity was broadly distributed, but it did not clearly show the bimodal distribution expected. Six of demi-embryos with high or low HPRT:APRT ratios were transferred to recipient cows from which 2 calves were obtained. Both offspring were of the sex predicted by the HPRT: APRT ratio. These results indicate that HPRT activity of bovine preimplantation embryos can be microassayed using radiolabeled hypoxanthine, and this assay could provide an alternative method for embryo sexing.     

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.     

Synchronous division of mouse two-cell embryos with nocodazole in vitro.

Mouse two-cell embryos were cultured in a medium supplemented with nocodazole or colcemid for 12.5-14.5 h in vitro, and development after elimination of these drugs was examined. All embryos cultured with nocodazole stopped at the metaphase of the second cell cycle. When nocodazole was removed, almost all embryos divided to the normal four-cell stage within 1 h and then developed into blastocysts (98%). The proportion of embryos that developed into young after transfer to recipients was not significantly different from the control (35 versus 36%), but the developmental ability of the embryos treated with colcemid was reduced, especially after transfer to recipients.     

Whole-embryo culture of Drosophila : development of embryonic tissues in vitro

Summary We have devised techniques to culture whole, dissected embryos of Drosophila melanogaster. We examine multiple aspects of the morphological and physiological development of the epidermis, musculature, nervous system, and internal organs in this cultured preparation, and show that in vitro development closely parallels normal embryogenesis. These techniques permit a wide range of experimental manipulations during embryogenesis and allow us to extend observations through late embryonic stages, after cuticle deposition. Applications of this technique are presented.     

Development of hamster two-cell embryos in the isolated mouse oviduct in organ culture system

Hamster early two-cell embryos developed to the expanded blastocyst stage within the isolated mouse ampulla maintained in organ culture system. Mouse ampullae isolated at different times after treating the mice with human chorionic gonadotropin (hCG) (0–72 h) or pregnant mare's serum gonadotropin (PMSG) (30–32 h) were flushed with culture medium, and hamster early two-cell embryos were introduced into these ampullae. Mouse ampullae isolated at 14–32 h after hCG injection were more favorable for the development of the embryos than those isolated at 70–72 h. When mouse ampullae were isolated 30–32 h after hCG or PMSG treatment, 39% of the cultured eggs developed, some of them to the expanded blastocyst stage after additional culture for 65–70 h. These results indicate that unknown oviductal factors stimulate the development of hamster early two-cell embryos, and these factors are under the control of hCG or PMSG. In addition, these factors are common to the mouse and hamster.     

Early embryonic development and in vitro culture of in vivo produced embryos in the farmed European polecat (Mustela putorius)

Early embryonic development and in vitro culture of in vivo produced embryos in the farmed European polecat (Mustela putorius) was investigated as a part of an ex situ conservation program of the endangered European mink (Mustela lutreola), using the European polecat as a model species. The oestrus cycles of 34 yearling polecat females were monitored by visual examination of the vulval swelling and, to induce ovulation, the females were mated once daily on two consecutive days. Sixteen yearling males were used for mating. The females were humanely killed 3-14 days after the first mating and the uteri and oviducts were collected for embryo recovery. Uterine and oviductal flushings yielded a total number of 295 embryos, representing developmental stages from the 1-cell stage to large expanded and hatched blastocysts. On Day 3 after the first mating, only 1-16-cell stage embryos were recovered. Between Days 4 and 6 after the first mating, 1-16-cell stage embryos and morulae were found. The first blastocysts were recovered on Day 7 after the first mating. The first implanted blastocysts were detected on Day 11 after the first mating. A total number of 85 embryos were in vitro cultured after recovery. Blastocyst production rates for in vitro cultured 1-16-cell stage embryos and for morulae/compact morulae were 68 and 84%, respectively. For all cultured embryos, the hatching rate was 15%. The in vitro culture requirements for the preimplantation embryos of the farmed European polecat remain to be determined before further utilization of the technique.     

In vitro formation of tetraploid rat blastocysts after fusion of two-cell embryos

Gene targeting technology is not available in the rat which is an animal model of major importance, e.g., in cardiovascular research. This is due to the fact that the rat embryonic stem cell (ESC)-like cells established by several groups do not form germ-line chimeras when injected into blastocysts. In the mouse, the aggregation of ESC with tetraploid embryos has allowed the generation of animals completely derived from these cells. However, aggregation of rat ESC-like cells with tetraploid rat embryos has not yet been attempted to evaluate their developmental capacity. Therefore, we established a method to produce tetraploid rat embryos by fusion at the two-cell stage. Chemical fusion by polyethylene glycol (PEG) was shown to be less efficient (56.3% fused embryos) than electrofusion (96.1% fused embryos). The rate of development of fused embryos to blastocysts was independent of the fusion method and similar to the rate of control embryos. However, this rate was lower when the embryos had been cultured from the zygote state before fusion (14-20%) compared to freshly isolated two-cell embryos (41-63%). Alike for the mouse, blastocysts derived from fused two-cell rat embryos contained about half the number of cells as control blastocysts and were homogeneously tetraploid with no evidence of mosaicism. This method may be useful for the establishment of gene-targeting technology in the rat.     

Effects of superovulation, culture and microinjection on development of rabbit embryos in vitro

Factors influencing the developmental potential of cultured rabbit zygotes and their ability to incorporate and integrate the WAP-hPC (human protein C) gene were investigated. Rabbit zygotes (n = 1053) were recovered from both superovulated and nontreated New Zealand White females. The hormonal treatment of rabbit donors resulted in a doubling of the number of recovered ova per donor when compared with the nontreated group (18 vs 9 ova). However, the quality of recovered zygotes (presence of both pronuclei) was significantly better in the nontreated group (99 vs 88%, Experiment 1). The effect of various culture media on the development of rabbit zygotes in vitro was evaluated after incubation under CO2-free conditions (Experiment 2). In serum-free, growth factor-supplemented medium (BSEITS, DME/F12, 1.5% BSA, EGF, insulin, transferrin and sodium selenite) the percentage of morula/blastocyst stage embryos was significantly higher (88%) than in DME/FCS, (DME/F12, 10% fetal calf serum, 59%) or the control group (DME/F12, 1.5% BSA, 25%). In Experiment 3, zygotes were microinjected with the WAP-hPC gene and were examined after 72 h of culture. Zygote cleavage and the percentage of morula/blastocyst stage intact embryos were higher (79 and 58%, respectively) than in microinjected embryos (31.0 and 21.5%, respectively). Summarized data of the PCR assay of microinjected zygotes demonstrated positive signals for the integration of the WAP-hPC gene in 6.6% (34 of 515) of all the microinjected zygotes.     

Full-term development of mouse blastomere nuclei transplanted into enucleated two-cell embryos

The nuclei from four- and eight-cell mouse embryos were transplanted into enucleated two-cell embryos. It was found that such embryos not only developed to the blastocyst stage in vitro (72% and 35%), but also developed to full term (22% and 8%) after transfer to recipient mice. However, development of embryos which contained nuclei from the inner cell mass was not observed. Since the development of enucleated zygotes which contain advanced nuclei is limited (the present study; McGrath and Solter: Science, 226:1317-1319, '84; Robl, Gilligan, Critser, and First: Biol. Reprod., 34:733-739, '86), it appears that cytoplasmic factors are important for the development of nuclei from advanced cells.     

Electrofusion of two-cell bovine embryos for the production of tetraploid blastocysts in vitro

Tetraploid bovine blastocysts were produced experimentally by electrofusion of in vitro matured and fertilized, zona-enclosed two-cell embryos (33-35 hr after initiation of sperm-egg incubation) using three fusion protocols. Field strengths of 1.0, 1.4, and 2.4 kV/cm were tested and the rate of fusion, subsequent cleavage, and blastocyst development were measured for each. High rates of fusion (76.5% +/- 2.8%), cleavage (72.5% +/- 7.4%) and blastocyst development (56.1% +/- 6.4%) were achieved with the application of 1. 4 kV/cm as a single 100-microseconds pulse. Embryos were scored 30 and 60 min after stimulation for fusion. No time effect for fusion, cleavage, or blastocyst development was observed. Chromosome preparations of day 7 blastocysts revealed 12.5% of fused embryos were tetraploid. This is a significant increase from that found in nonfused embryos where spontaneous tetraploidy did not occur. An electrical stimulus of 1.0 kV/cm applied as two 50-microseconds pulses produced significantly less one-cell embryos (64.2% +/- 3.0%) compared to 1.4 kV/cm while cleavage (79.9% +/- 3.4) and blastocyst development (44.6% +/- 4.0%) were not different from that for unexposed control embryos (89.5% +/- 2.3% and 57.2% +/- 3.2%, respectively). Embryos fused at 2.4 kV/cm applied as a single 30-microseconds pulse (69.7% +/- 5.7%) showed significantly lower cleavage (72.1% +/- 3.7%) and blastocyst rates (40.2% +/- 4.6%) compared to the unexposed control.     

Chromosomal control of early embryonic development in mice I. Experiments on embryos with autosomal monosomy

The effects of autosomal monosomy on early embryonic development were studied in mice with Robertsonian and reciprocal (non-Robertsonian) translocations. It was found that monosomy for autosomes 1, 2, 3, 5, 6, 16, 17 and 19 had substantially different effects on preimplantation development and survival of mouse embryos. Monosomy for autosomes 1, 3, 6, 16 and 19 does not affect cleavage, compaction and blastulation and in some cases is compatible with implantation. Most of these embryos, however, die as early blastocysts (Ms 3, 6 or 19) and some of them are eliminated at early postimplantation stages (Ms 1 or 16). The embryos with monosomy for autosomes 2, 5 or 17 can be identified during cleavage owing to the reduced blastomere number and pathological changes in the nuclei. Most of these monosomies do not survive beyond the morula stage. The results indicate that differential genetic activity of autosomes in mice becomes already evident in very early embryonic development. A hypothetical mechanism for homologous autosome activation at the onset of embryonic development in mice is suggested.     

Collection of two-cell pig embryos and their culture in media containing different protein components

Eggs were flushed from the oviducts of slaughtered gilts that had been inseminated after synchronization of estrus and ovulation with pregnant mare serum gonadotrophin (PMSG) and human chorionic gonadotrophin (HCG). Of 347 eggs collected at a recovery rate of 73.8%, 41.8% had not cleaved by 32 h after expected ovulation time. Of those cleaved, 86.9% were at the two-cell stage.Two-cell embryos were cultured in Dulbecco's medium containing either no protein or 20% of one of the following: lyophilized bovine serum albumin, lyophilized fetal calf serum or heat-inactivated serum from slaughter heifers, rabbits or barrows. Dulbecco's medium without protein did not support further development of embryos. Addition of heat-inactivated blood serum from slaughter heifers, rabbits or barrows resulted in development rates similar to those obtained by using commercially available products. Optimal embryonic development rates of 54.5% were obtained with addition of heat-inactivated bovine serum. Of the two-cell embryos only three (2.1%) developed past the four-cell stage in these culture media.     

Insulin-related peptides and the two-cell block to development in pre-implantation mouse embryos

Abstract. In many mammalian embryos development in vitro is arrested after the first zygotic division, a phenomenon known as the two-cell block. In the mouse several strains exhibit a two-cell block to further development and it was the purpose of this investigation to determine whether the inability of embryos to progress through the block was due to lack of insulin or insulin-like growth factors (IGFs) in the medium. Several factors have been implicated in the two-cell block, amongst which oxidative stress, glucose and missing maternal factors have been examined to date. Because of their anabolic and anti-apoptotic properties, IGFs are good candidates for such missing maternal factors. Using MF1 strain mice and M16 medium we have examined the effects of IGF-I, II and insulin on the two-cell block. No effects were discernable at concentrations known to support development of non-blocking embryos and we conclude that the IGFs and insulin may be excluded as critical factors in the two-cell block under the culture conditions used.