Role of glucose in mouse preimplantation embryo development

Mouse preimplantation embryos consume pyruvate preferentially during the early developmental stages, before glucose becomes the predominant energy substrate in the blastocyst. To investigate the importance of the switch to glucose utilization at the later developmental stages, mouse embryos from F1 hybrid mice (CBA/Ca × C57BL/6) were cultured from the one-and two-cell stages (22 and 46 h post hCG, respectively) for 5 days in a modified medium, M16, containing 0.33 mM pyruvate and 5 or 23 mM D+L-lactate, in the presence and absence of 1 mM glucose (M16+G and M16-G, respectively). Nutrient uptakes were also determined over this time. Some embryos cultured in M16-G were transferred to M16+G at 94 or 118 h post hCG. Embryos cultured from the two-cell stage in M16+G exhibited the characteristic fall in pyruvate consumption between the morula and the blastocyst stage; those cultured from the two-cell stage in M16-G compensated for the lack of glucose by consuming increasing amounts of pyruvate, from 2.78 pmol/embryo/h at 58 h post hCG to 5.21 pmol/embryo/h at 154 h post hCG. However, the percentage of embryos developing to the blastocyst stage, the hatching rate, and blastocyst cell numbers (50.6 ± 2.5 [28] vs. 105 ± 3.8 [37]) were all lower in this group. When exposed to glucose at 94 or 118 h post hCG, embryos cultured from the two-cell stage in M16-G readily consumed glucose in preference to pyruvate, although the characteristic fall in pyruvate consumption was not observed. One-cell embryos cultured continuously in M16-G were only able to develop to the morula stage, after which time they degenerated. In these embryos pyruvate was readily consumed between 22 and 94 h post hCG, before falling from 2.77 pmol/embryo/h at 83 h post hCG to 0.045 pmol/embryo/h at 130 h post hCG. Transfer of these embryos to M16+G at 94 and 118 h post hCG did not support development to the hatching blastocyst stage. The results show that mouse preimplantation embryos from F1 hybrid mice (CBA/Ca × C57BL/6) need only be exposed to glucose for less than 24 h between 22 and 94 h post hCG in order to develop from the morula to the blastocyst stage in vitro. However, the exposure time needs to be increased to between 24 and 72 h in order that blastocyst cell numbers reach control levels. The importance of glucose before the morula stage may relate to the need to synthesize glycogen for later use. If the obligatory requirement for glucose is fulfilled, embryos are able to utilize pyruvate in the absence of glucose at the later stages of development. These results show that the mouse preimplantation embryo can, to some extent, adapt metabolically to changes in its external environment. © 1995 Wiley-Liss, Inc.     

Observations on in vitro development of bovine morulae in Ham's F-10 and Dulbecco's phosphate buffered saline supplemented with normal steer serum

This study was conducted to compare in vitro development of bovine morulae in Ham's F-10 and Dulbecco's phosphate buffered saline (D-PBS) media supplemented with 10% (v/v) normal steer serum. Fifty-three excellent and good embryos were obtained by superovulating 15 non-lactating Holstein cows. Embryos were placed randomly in culture with Ham's F-10 or D-PBS media and development was recorded at 12-h intervals for the duration of culture. All embryos reached early blastocyst, blastocyst and expanded blastocyst stage. Nineteen of 27 embryos (70.1%) cultured in Ham's F-10 developed to hatched blastocyst stage in contrast to three out of 26 in D-PBS (11.5%). The mean developmental scores at 24, 48, 72, 96 and 120 h of culture were significantly (P<0.001) higher for embryos cultured in Ham's F-10. Also, the mean times to reach early blastocyst (25.84 +/- 6.65 vs 46.67 +/- 9.99 h), blastocyst (44.57 +/- 11.45 vs 61.89 +/- 16.62 h) and expanded blastocyst stage (65.00 +/- 13.20 vs 73.41 +/- 15.80 h) were significantly (P<0.001) shorter for embryos cultured in Ham's F-10. No difference was observed in the mean time to reach hatching (90.00 +/- 10.85 vs 84.00 +/- 16.97 h) and hatched blastocyst stage (97.26 +/- 18.71 vs 96.00 +/- 0.00 h). The results obtained support the concept that Ham's F-10 and normal steer serum provide for optimal bovine embryo development and suggest that 10% normal steer serum could be used as a protein supplement with D-PBS for short term storage and culture of bovine embryos.     

In vitro embryo development and blastocyst hatching rates following vitrification of river buffalo embryos produced from oocytes recovered from slaughterhouse ovaries or live animals by ovum pick-up

The present study was undertaken to determine whether the source of oocytes (ovum pick up versus slaughterhouse ovaries) affected in vitro embryo production and embryo survival (as measured by blastocyst hatching rates) following vitrification in buffaloes (Bubalus bubalis). Oocytes recovered from live buffaloes (n=6) by ovum pick up (OPU) and by manual aspiration from slaughterhouse ovaries were in vitro matured, fertilized and cultured to blastocyst stage under same culture conditions. Vitrification of blastocysts was carried out in two steps at 24 degrees C. Embryos were equilibrated in 10% EG+10% DMSO+0.3 M sucrose in base medium for 4 min. Subsequently, the embryos were transferred into 25% EG+25% DMSO+0.3 M sucrose in base medium for 45 s and then the embryos were loaded into straws and immersed in liquid nitrogen. Following warming, blastocysts were cultured in vitro for 48 h to assess hatching. Oocytes derived from live animals by OPU resulted in a significantly higher blastocyst yield then those derived from slaughterhouse ovaries (30.6+/-4.3 versus 18.5+/-1.8). Blastocyst hatching rates following vitrification of buffalo embryos produced from the oocytes collected from live animals by OPU was significantly higher than the oocytes collected from slaughterhouse ovaries (52.8+/-4.2 versus 40.2+/-4.4). In conclusion, the present study showed that source of oocytes (OPU versus slaughterhouse ovaries) affects the in vitro embryo development and blastocyst hatching rates following vitrification of embryos in buffaloes.     

Buffalo rat liver cells produce factors that support preimplantation development of mouse embryos cultured in vitro

To examine the effects of buffalo rat liver (BRL) cells on the preimplantation development of mouse embryos in vitro, we first cultured two-cell mouse embryos alone in serum-free Dulbecco modified Eagle medium. As expected, the embryos did not develop to subsequent stages. However, when cocultured with BRL cells, the embryos developed to the blastocyst stage efficiently. Direct contact of embryos with BRL cells was not necessary for development: the medium conditioned by BRL cells contained soluble factors that supported the preimplantation development of mouse embryos. Embryos cultured with BRL-conditioned medium that was replaced at various intervals had a further increased rate of development to the blastocyst stage. This finding indicated that the activities of the factors were maintained only briefly. Seven proteins between 35 and 44 kDa that were detected in the medium were highly beneficial to the development of the embryos. Follistatin-related protein and pigment epithelium-derived factor are believed to be the factors supporting embryo development. The other five proteins also may improve the environment for the development of mouse embryos cultured in vitro.     

Effects of uranium poisoning on cultured preimplantation embryos

The toxic effect of uranium in cultured preimplantation embryos of the mouse is presented. Embryos were obtained from hybrid females CBA×C57 BL following induction of superovulation and were incubated in M16 cultured medium. Two different experiments were performed. In one, embryos in a one-cell stage were placed in culture media with final concentrations of uranyl nitrate of 104 and 208 μg/mL during 120 h in the same dish. In the other experiment, embryos in a one-cell stage were placed in culture medium with uranyl nitrate with final U concentrations of 26, 52, 104, and 208 μg/mL. At 24 h, those embryos which had reached the two-cell stage were transferred to another culture dish to which fresh solutions with uranyl nitrate were added. The percentage of embryos in two-cell stage, morula, early blastocyst, expanded blastocyst, and hatched blastocyst were recorded at 24, 72, 96 and 120 h of culture. The results obtained showed that concentrations as from 26 μg U/mL induced the delay of embryo development and the impairment of blastomere proliferation. The toxic effect of uranium increased in those experiments in which the embryos were transferred to a new medium. This embryo-culture system appears to be appropriate to evaluate the toxic effect of uranium on embryos removed from maternal influences and represents a suitable test system for environmental pollutants.     

Development of fertilized embryos transferred to oviducts of immature mice

The in-vitro culture of fertilized 1-cell mouse embryos to the blastocyst stage is associated with subsequent decreased viability. In this study, 1-cell embryos were cultured for 3 days in the reproductive tract of immature female mice as an alternative to in-vitro culture. Embryos that spent 3 days in immature females were developmentally more advanced, had higher cell numbers and better viability, as measured by development to mid-gestation, after transfer to pseudopregnant recipient females than did embryos maintained for the same period in culture. Embryos that developed in immature females had lower cell numbers but comparable rates of development and subsequent viability when compared with embryos transferred to synchronous pseudopregnant females for the same preimplantation period. The immature mouse oviduct is therefore a suitable alternative environment to in-vitro culture or a pseudopregnant host for complete preimplantation development and has the additional advantage that synchrony between embryo and temporary host is not necessary. This method will allow for evaluation of manipulation procedures while maintaining viability before the embryos are finally committed to a foster mother for development to term.     

Co-culture of day-5 to day-7 equine embryos in medium with oviductal tissue

Oviductal and uterine embryos were collected from mares at 5 to 7 days following ovulation 1) to evaluate the effects of oviductal tissue explants on in vitro growth and development of equine embryos and 2) to study the morphologic development of equine embryos in culture. Embryos were incubated for 5 days in a medium (control group) or in medium supplemented with oviductal tissue explants (co-culture group). Embryos were evaluated and the media changed daily. Following 5 days in culture, 10 10 (100%) control embryos and 27 29 (93%) co-cultured embryos had doubled in diameter. All embryos that were recovered as morulae developed to the blastocyst stage in culture. By 5 days in culture, 6 10 (60%) control embryos and 19 29 (66%) co-cultured embryos had reached the hatching blastocyst stage of development. By 3 days in culture, significantly more (P<0.05) control embryos versus co-cultured embryos had degenerated (4 10 vs 2 29 , respectively). By 5 days in culture, significantly more (P<0.01) control embryos versus co-cultured embryos had degenerated (6 10 vs. 3 29 , respectively). Embryos cultured with oviductal tissue were sustained longer than embryos cultured in medium alone. Hatching was characterized by the blastocyst squeezing through a small opening in the zona pellucida or by the zona pellucida thinning over approximately half of the blastocyst surface and subsequently disappearing entirely.     

Effects of amino acids and α-amanitin on bovine embryo development in a simple protein-Free medium

Five experiments, utilizing 3741 embryos produced in vitro, were designed to test the effects of Eagle's nonessential amino acids, and combinations of Eagle's essential amino acids and the RNA polymerase inhibitor α-amanitin on the development of preimplantation bovine embryos in a modified protein-free KSOM medium. Embryos were cultured in 5% O₂:5% CO₂:90% N₂ at 39°C for the first 40–44 hr in modified KSOM, and embryos with ≥4 cells were cultured in modified KSOM-PVA with different amino acids in experiments 1–4, and with the addition of α-amanitin in experiment 5. In experiment 1, addition of 0.5× of the essential amino acids, with different concentrations of nonessential amino acids significantly increased hatching of blastocysts and decreased blastocyst degeneration, but increasing the nonessential amino acids from 1× to 5×, did not stimulate embryo development. In experiments 2–4, increasing only the glycine concentration, or adding each of the 12 essential amino acids singly or several in combination to the medium containing nonessential amino acids, did not significantly improve embryo development. Taurine (0.4 mM) in the modified KSOM medium reduced blastocyst degeneration. In experiment 5, α-amanitin (20 μM) completely inhibited further embryo development when it was added at several stages from 4-cell embryos to morulae. The study with protein-free KSOM plus amino acids provided a completely defined simple medium for culturing bovine embryos, with evidence that continuous mRNA activity and presumed protein synthesis was obligatory to meet the complex and continuous requirements for proteins by the developing blastocyst. Mol. Reprod. Dev. 46:278–285, 1997. © 1997 Wiley-Liss, Inc.     

Expression and function of amphiregulin during murine preimplantation development

Amphiregulin (Ar) is an EGF receptor ligand that functions to modulate the growth of both normal and malignant epithelial cells. We asked whether mouse preimplantation embryos express Ar, and if so, what the function of Ar is during preimplantation development. We used RT-PCR to show expression of Ar mRNA in mouse blastocysts, and using a polyclonal anti-Ar antibody and indirect immunofluorescence, we detected the presence of Ar protein in morula- and blastocyst-stage embryos. Ar protein was present in both the cytoplasm and nucleus in both morulae- and blastocyst-stage embryos, which is similar to Ar distribution in other cell types. Embryos cultured in Ar developed into blastocysts more quickly and also exhibited increased cell numbers compared to control embryos. In addition, 4-cell stage embryos cultured in an antisense Ar phosphorothioate-modified oligodeoxynucleotide (S-oligo) for 48 hr exhibited slower rates of blastocyst formation and reduced embryo cell numbers compared to embryos exposed to a random control S-oligo. TGF-α significantly improved blastocyst formation, but not cell numbers, for embryos cultured in the antisense Ar S-oligo. From these observations, we propose that Ar may function as an autocrine growth factor for mouse preimplantation embryos by promoting blastocyst formation and embryo cell number. We also propose that blastocyst formation is stimulated by Ar and TGF-α, while Ar appears to exert a greater stimulatory effect on cell proliferation than does TGF-α in these embryos. Mol. Reprod. Dev. 47:271–283, 1997. © 1997 Wiley-Liss, Inc.     

Effect of endothelial cell growth supplement and fibroblastic growth factor on early mouse embryo development in vitro

This study was conducted to examine the effect of endothelial cell growth supplement (ECGS) and fibroblastic growth factor (FGF) on early mammalian embryo development in vitro. Two hundred mouse blastocysts were placed randomly in culture wells containing one of five treatments: 1) Ham's F-10, 2) Ham's F-10 + 10 mug ECGS, 3) Ham's F-10 + 10 ng ECGS, 4) Ham's F-10 + 100 ng FGF and 5) Ham's F-10 + 10 ng FGF. In all cases, media were supplemented with 10% (v/v) normal steer serum. Embryos were cultured at 37 C with an atmosphere of 5% O(2), 5% CO(2) and 90% N and development was recorded at 12 h intervals for the duration of culture. The percentage of embryos that developed to expanded blastocyst (94.8), hatching blastocyst (74.4) and hatched blastocyst (71.8) in Ham's F-10 media were not different from embryos cultured in all other treatments except Ham's F-10 + 10 mug ECGS. A decrease in the percentage of embryos reaching expanded blastocyst (44.7), hatching blastocyst (23.7) and hatched blastocyst (18.4) was observed in Ham's F-10 + 10 mug ECGS. Also, a significant (P<0.01) decrease in development scores at 24, 48 and 72 h was observed for embryos cultured in Ham's F-10 + 10 mug ECGS. No difference was observed in the mean time to reach different developmental stages among treatments. The data suggest that ECGS and FGF at the doses tested have no beneficial effect on early mouse embryo development in vitro and 10 mug of ECGS has inhibitory effect.     

Development of pig blastocysts in vitro is altered by serum, bovine serum albumin and amino acids and vitamins

We tested the effects of the amino acids and vitamins in minimum essential medium (MEM) and Eagle's medium (BME) on pig blastocyst development and nuclei number. Embryos were recovered either 5 or 6 d after first detected estrus and were cultured for 96 h in U-bottomed wells (0.2 ml). In Experiment 1, addition of MEM amino acids and vitamins to modified Krebs-Ringer bicarbonate (MKRB) medium containing either bovine serum albumin (BSA, 4 mg/ml) or lamb serum (10%, v/v) resulted in fewer (P<0.001) nuclei and smaller (P<0.05) embryo volumes at the end of culture as compared to embryos cultured in MKRB without MEM-supplements. Addition of MEM-amino acids without glutamine (Experiment II) depressed blastocyst volume and rate of hatching, but glutamine (2 mM) had no effect on embryo development. Dialysis (molecular weight > 12,000 retained) of fetal bovine serum (Experiment III) did not affect blastocyst expansion but reduced (P<0.05) the number of nuclei/blastocyst at the end of the culture. Embryos cultured in MKRB with dialyzed serum and the amino acids and vitamins in BME were smaller (P<0.05) and had fewer (P<0.05) nuclei than embryos cultured in MKRB with dialyzed serum but without the BME-supplements. We conclude that, under our culture conditions, MEM and BME amino acids and vitamins are detrimental to the development of early pig blastocysts and that this effect is not due to glutamine. Also, dialysis of fetal bovine serum removes some component(s) that are important for cell division by pig embryos, but it does not affect blastocyst expansion.     

Factors influencing the in vitro hatching of mouse blastocysts

The influence of bovine serum albumin (BSA) concentration on embryo hatching and the number of embryos cultured per drop of culture medium was examined in F1 (C57BL/6J × DBA/2J), C3HeB/FeJ strain and Line E mice. Embryos collected from F1 and Line E mice exhibited uniform hatching rates at BSA concentrations between 1 and 10 mg/ml, and embryo numbers ranging from 1 to 10 per 3 μ1 of culture medium. The hatching of C3HeB/FeJ blastocysts was greater at the higher concentrations of BSA and higher embryo densities. When the C3HeB/FeJ embryos were grown at high densities until morula and then cultured singly in fresh media they hatched at a low rate. However, when allowed to develop until the blastocyst stage before replotting, the embryos hatched at a high rate. C3HeB/FeJ embryos cultured singly until morula and then placed in groups of 10 hatched at a high rate. Single C3HeB/FeJ embryos, cultured in medium conditioned by the prior presence of embryos at high densities, hatched at a slightly higher frequency than those cultured in fresh medium. There was no tendency of embryos developing from the two-cell to the eight-cell stages to hatch when cultured in the presence of high densities of hatching blastocysts.     

Inhibition of DNA replication in preimplantation mouse embryos by aphidicolin

The regulation of trophectoderm differentiation in mouse embryos was studied by inhibiting DNA synthesis with aphidicolin, a specific inhibitor of DNA polymerase alpha. Embryos were exposed to aphidicolin (0.5 micrograms/ml) for 16 h at various preimplantation stages and scored for their ability to form a blastocyst and develop beyond the blastocyst stage. Embryos were most sensitive to aphidicolin at the late 4-cell stage and became progressively less sensitive as they developed. Aphidicolin inhibited blastocyst formation by 70%, 100%, 77%, and 24% after treatment at the 2-cell, 4-cell, noncompacted 8-cell, and compacted 8-cell stages, respectively. Although the inhibitory effect of aphidicolin on blastocyst formation decreased markedly as 8-cell embryos underwent compaction, developmental capacity beyond the blastocyst stage was poor after treatment of either noncompacted or compacted 8-cell embryos. Treatment at the morula and early blastocyst stages was less harmful to embryos than treatment at earlier stages but reduced the number of trophoblast outgrowths by interfering with hatching. Autoradiographic analysis showed that during aphidicolin treatment, incorporation of 3H-thymidine was inhibited over 90% at all stages examined, indicating an inhibition of DNA synthesis. Because inhibition of blastocyst formation by aphidicolin decreased at the compacted 8-cell stage, we suggest that approximately the first half of the fourth DNA replication cycle is critical for subsequent blastocyst formation. Furthermore, the poor further development of blastocysts formed after aphidicolin treatment of compacted 8-cell embryos suggests that the DNA replication requirements for initial trophectoderm differentiation are distinct from requirements for further development of blastocysts in vitro.     

Temporal effect of human oviductal cell and its derived embryotrophic factors on mouse embryo development.

Mouse embryos at different stages of development were cocultured with human oviduct cells or cultured in the presence of oviduct-derived embryotrophic factor-1, -2, and -3 (ETF-1, -2, and -3) for various amounts of time within the preimplantation period. Cocultures that included the period from 48 to 72 h post-hCG stimulated cell division and increased the cell numbers in the inner cell mass (ICM) of the exposed blastocyst. Exposure of embryos to oviductal cells from 96 to 120 h post-hCG increased the cell number in the trophectoderm (TE), blastocyst size, hatching rate, attachment, and in vitro spreading of the blastocyst. ETF-1 and ETF-2 affected embryos between 48 and 72 h post-hCG by increasing the number of cells in the ICM. In contrast, ETF-3 had a more profound effect on embryos that were exposed from 96 to 120 h post-hCG, where it mostly affected the development of TE cells, leading to higher hatching rate. Human oviductal cells improved mouse embryo development partly by the production of high molecular weight embryotrophic factors. These factors had differential effects on mouse embryo development.     

Improved development of rabbit one-cell embryos to the hatching blastocyst stage by culture in a defined, protein-free culture medium

In Exp. 1, Medium 199 and Medium RD (RPMI-1640 and Dulbecco's MEM, 1:1 v/v) were compared in a 2 x 2 factorial design by supplementing each with 15 mg bovine serum albumin (BSA)/ml of 1 mg polyvinyl alcohol (PVA)/ml. All media contained 5 micrograms insulin/ml, 5 micrograms transferrin/ml, 5 ng selenium/ml (ITS), and 10 ng epidermal growth factor (EGF)/ml. One-cell embryos were cultured at 39 degrees C with 5% CO2 in air for 65 h and then stained with Hoechst 33342 to determine blastomere number. Embryos in Medium 199 developed poorly (P less than 0.001) when PVA was used instead of BSA (30 vs 76 cells/embryo), but developed rapidly in Medium RD with PVA or BSA (118 and 121 cells). Similar results were obtained in Exp. 2 in BSA- and PVA-free medium. In Exp. 3, the development of 1-cell embryos after 65 h in unsupplemented (protein-free) Medium RD (68% blastocysts, 117 cells) did not differ (P greater than 0.37) from that obtained using Medium RD with insulin, ITS or EGF alone. Culture in protein-free Medium RD for 96 h resulted in 82% of the 1-cell embryos forming blastocysts and 40% hatching through the zona pellucida. In a preliminary test of viability, 1-cell embryos cultured in this medium for 48 or 65 h and transferred to synchronous recipients resulted in 5/18 (28%) and 3/24 (12%) Day-15 viable fetuses. Cell counts of approximately 120 per blastocyst after culturing 1-cell embryos for 65 h in Medium RD indicated that cell division was more rapid than that obtained with all other media tested previously in this laboratory. This is the first report of rabbit embryo development from the 1-cell to the hatching blastocyst stage in a defined protein-free culture medium.     

Effects of media and the presence of bovine oviduct epithelial cells during in vitro fertilization on fertilizability and developmental capacity of bovine oocytes

The effect of the presence of bovine oviduct epithelial cells (BOEC; Experiment 1) as well as the effects of media (Tyrode fertilization medium: TFM vs synthetic oviduct fluid: SOF), fertilization containers (drops in petri dish vs 96-wells), and the number of oocytes per drop and well (5 vs 10) for in vitro fertilization (Experiment 2) on the fertilizability and in vitro development of bovine oocytes were investigated. Immature oocytes with cumulus cells were cultured in TCM199 supplemented with 10% ECS and 2.5x10(6) granulosa cells for 24 hours at 39 degrees C under 5% CO(2) in air. In vitro fertilization was performed with frozen-thawed, heparin-treated spermatozoa (100 mug/ml, 15 minutes) and with BOEC (Experiment 1). In Experiment 2, in vitro fertilization was performed with two different media (TFM and SOF) and various conditions (culture dish and different number of oocytes). Cleavage, development to the blastocyst stage were evaluated on Day 2 and Day 7 after the start of culture. Effect of the presence of BOEC on fertilizability and developmental capacity (Experiment 1) was not significantly different. In Experiment 2, alterations in media, containers and number of oocytes during in vitro fertilization had no affect. The SOF medium showed results similar to those of TFM (normal fertilization rate: 63.2 vs 64%; cleavage: 69.3 vs 73.9%; development to the blastocyst stage: 14 vs 15%; and mean number of nuclei per blastocyst: 80.5 vs 86.6). The results indicate that the presence of BOEC during in vitro fertilization did not improve fertilizability, and that SOF as well as TFM medium can be utilized as a simple fertilization medium.