Culture of bovine embryos to the blastocyst stage using Buffalo rat liver (BRL) cells

A comparison was made between the development of in vitro matured and fertilized bovine oocytes in co-culture with bovine oviduct epithelial (BOE) cells or with Buffalo rat liver (BRL) cells. Both cell types supported development from the 1-cell to the blastocyst stage with equal efficiencies (4.4% for BRL cells, 4.0% for BOE cells). Medium conditioned by either cell type supported development to the blastocyst stage as efficiently as co-cultures (6.4 and 7.3% blastocysts for BOE and BRL conditioned medium, respectively). A higher percentage of blastocyst development was found when embryos were cultured closely apposed in small drops of BRL-conditioned medium compared with larger volumes (20.5 versus 7.0%). The ability of BRL-conditioned medium to support embryonic development was dependent on the duration of the conditioning period (optimum 24 to 48 h), and was not lost when the medium was stored at -20 degrees C for extended periods. The effects were independent of the conditions used to promote maturation in vitro and the procedure for fertilization. With 2 different methods to produce embryos in culture, both the BRL cell co-culture and BRL-conditioned medium in microdrops supported embryo development to the blastocyst stage. The use of the BRL cell line reduces the variability associated with primary BOE cell cultures.     

Differential preimplantation development of rhesus monkey embryos in serum-supplemented media

Several media, some augmented with amino acids, have been formulated recently, based on simplex optimization, to support the preimplantation development of mouse embryos. For the highly limited studies on preimplantation development of nonhuman primate embryos, a complex medium (CMRL-1066) has been employed. Our objective was to compare the developmental ability of rhesus monkey embryos in a simple medium containing amino acids, KSOM/AA, with the complex media used previously. Zygotes (99) were recovered following in vitro fertilization (IVF) from six monkeys, allocated to either CMRL or KSOM/AA both containing 10% fetal calf serum (FCS), and monitored daily until reaching the expanded or hatched blastocyst stage. The distribution of cells between the inner cell mass (ICM) and trophectoderm was determined at the end of culture by differential nuclear staining. Although a greater number of embryos cultured in KSOM/AA vs. CMRL developed to the morula stage (80%) and beyond (66% to expanded blastocyst), the differences were not significant. Such embryos in KSOM/AA did, however, develop at a significantly faster rate, on average, reaching the expanded blastocyst stage 26 hr earlier than did embryos cultured in CMRL. KSOM/AA embryos hatched in less time and had a higher percentage (43 vs. 34) of cells allocated to the ICM. These results indicate that a simple medium, KSOM/AA, in the presence of serum, supports the development of rhesus monkey embryos at high efficiency and at a faster rate than that observed for embryos cultured in the complex medium, CMRL-1066. © 1996 Wiley-Liss, Inc.     

In vitro culture of bovine IVM-IVF embryos: Cooperative interaction among embryos and the role of growth factors

The objective of this study was to determine whether there is a cooperative interaction among bovine embryos during in vitro culture. Furthermore, culture medium was supplemented with the growth factors, epidermal growth factor (EGF) and transforming growth factor-beta1 (TGF-beta1), to determine if these factors had a stimulatory effect on bovine embryo development similar to that seen in mouse development. In vitro matured - in vitro fertilized bovine embryos (2- to 8-cell) were cultured singly and in groups of five in 25 mul of medium (CR1 + amino acids + fatty acid-free bovine serum albumin) with or without EGF and TGF-beta1. Bovine embryos cultured in groups had a significantly higher rate of development to the blastocyst stage than embryos cultured singly. Neither EGF (10 ng/ml) nor TGF-beta1 (2 ng/ml) affected blastocyst development, hatching or the cell number of the embryos cultured in groups. Epidermal growth factor stimulated hatching of embryos cultured singly from the 8-cell stage, but did not significantly affect blastocyst development.     

In vitro development of bovine embryos in Buffalo rat liver- or bovine oviduct-conditioned medium

A culture system for bovine embryos was developed using Buffalo rat liver cell (BRL) line-conditioned medium without serum. Zygotes, obtained by in vitro maturation and fertilization of oocytes, were cultured either in unconditioned medium (TCM 199 or DMEM/F12) or in the same medium conditioned by bovine oviduct or BRL cells. No serum was added during conditioning or during embryo culture. The DMEM/F12 medium was superior to TCM 199 for development of bovine embryos to the 5 to 8-cell stage: on average between 50 and 57% of the embryos reached this stage after 2 d of culture in DMEM/F12 or in conditioned medium, while 36% reached this stage in TCM 199. Further development to the blastocyst stage was enhanced by conditioning. The highest percentage of blastocysts was achieved in DMEM/F12 medium conditioned with BRL cells (30%). The yield of blastocysts was similar in TCM 199 and in DMEM/F12 media conditioned with bovine oviduct cells (22 versus 20%), but after conditioning with BRL cells, DMEM/F12 medium yielded a higher percentage of blastocysts than TCM 199 (30 versus 18%). This might be explained by the fact that viability of BRL cells was better in DMEM/F12 medium than in TCM 199 when serum was omitted. Blastocysts produced in BRL-conditioned medium had a higher number of cells than blastocysts obtained in bovine oviduct-conditioned medium, and their transfer to recipients led to pregnancies and birth of calves. In conclusion, culture of bovine embryos in DMEM/F12 medium conditioned with BRL cells without serum led to the development of good-quality blastocysts and is thus a promising method for producing embryos for the study of potential embryotrophic factors. The use of rat liver cell lines guarantees against bovine viruses and allows for better production of embryos.     

Immunohistochemical demonstration of prostaglandin E-2 in preimplantation mouse embryos

An antiserum to prostaglandin (PG) E-2 and indirect immunofluorescence were used to demonstrate immunohistochemically the presence of PGE-2 in preimplantation mouse embryos. Fluorescence was observed in the cytoplasm of unfertilized 1-cell embryos to the blastocyst stage. The strongest fluorescence was detected at the 8-cell and morula stages. In embryos cultured from the 2-cell stage on, the fluorescence was observed in the cytoplasm of 4-cell embryos to the blastocyst stage. No differences were observed in the intensity and the distribution of the fluorescence between embryos in vivo and those in vitro. However, when blastocysts were cultured in a medium containing 100 microM-indomethacin, the fluorescence was diminished markedly. We therefore suggest that preimplanted mouse embryos contain PGE-2 during their early developmental stages and that the embryos synthesize the PGE-2.     

The development of preimplantation mouse parthenogenones in vitro in absence of glucose: Influence of the maternally inherited components

Mouse preimplantation embryo development is characterized by a switch from a dependence on the tricarboxylic acid cycle pre-compaction to a metabolism based on glycolysis post-compaction. In view of this, the role of glucose in embryo culture medium has come under increased analysis and has lead to improved development of outbred mouse embryos in glucose free medium. Another type of embryo that has proven difficult to culture is the parthenogenetic (PN) mouse embryo. With this in mind we have investigated the effect of glucose deprivation on PN embryo development in vitro. Haploid and diploid PN embryos were grown in medium M16 with or without glucose (M16-G) and development, glycolytic rate, and methionine incorporation rates assessed. Haploid PN and normal embryo development to the blastocyst stage did not differ in either M16 or M16-G. In contrast, although diploid PN embryos formed blastocysts in M16 (28.3%), they had difficulty in undergoing the morula/blastocyst transition in M16-G (7.6%). There was no significant difference in mean cell numbers of haploid PN, diploid PN and normal embryos cultured in M16 and M16-G at the morula and blastocyst stage. Transfer of diploid PN embryos from M16-G to M16 at the four- to eight-cell stage dramatically increased blastocyst development. At the morula stage diploid PN embryos grown in M16-G exhibited a higher glucose metabolism and protein synthesis compared to those grown in M16 and to haploid PN embryos. Difficulties of diploid PN embryos in undergoing the morula/blastocyst transition in absence of glucose infer the existence of a link between the maternally inherited components and the preimplantation embryos dependence on glucose. © 1996 Wiley-Liss, Inc.     

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.     

The influence of insulin on the in vitro development of mouse and bovine embryos

To further investigate the role of insulin during preimplantation embryo development, we compared the effects of insulin on the development of mouse and bovine preimplantation embryos and on cell proliferation during culture in vitro in simplex media. The influence of insulin on the development of mouse zygotes was determined during cultivation in mSOF medium, alone or supplemented with glucose. Similarly, the effects of insulin on the bovine preimplantation embryo development were studied in mSOF medium. The addition of insulin into mSOF medium enhanced significantly the number of cells per mouse blastocyst. Moreover, when mSOF medium was supplemented with insulin and 0.2 mmol x l(-1) glucose, the percentage of hatched blastocysts and the mean cell number of mouse blastocysts were significantly higher. Insulin had no significant effect on the development of bovine embryos, produced by in vitro fertilization of in vitro matured oocytes. Neither the rates of developing embryos nor the mean number of cells in blastocysts were different in comparison with control embryos. Our results suggest that the in vitro development of mouse embryos could be enhanced by the addition of insulin to the culture medium and is further improved by the addition of glucose. In contrast to this our results indicate that insulin has no detectable beneficial effect on the preimplantation development of bovine embryos in mSOF medium.     

Effects of interferon and encephalomyocarditis virus on in vitro development of preimplantation mouse embryos with and without the zona pellucida

Development of preimplantation mouse embryos, with or without the zona pellucida, in the presence of interferon (IFN) and mouse encephalomyocarditis (EMC) virus was studied using the in vitro culture method. The embryos (2- to 8-cell stages) were obtained from superovulated mice and cultured in modified Witten's medium under paraffin oil in 5% CO2 in air at 37 degrees C. Removal of the zona pellucida does not affect the subsequent development of the embryos: 90% of embryos with and 87% of embryos without the zona pellucida reached the morula-early blastocyst stages. Mouse IFN (10(4) units/ml) had no inhibitory effect on the developmental ability of the preimplantation embryos with or without the zona pellucida: 88 and 89% of the embryos in each group, respectively, reached the morula-early blastocyst stages. The preimplantation mouse embryos were sensitive to the embryotoxic effect of EMC virus: at a multiplicity of 20 infection particles per embryo the development of 43% of embryos was inhibited. The zona pellucida had no significant protective effect: Its removal changed only slightly the susceptibility of the preimplantation embryos to this virus. Pretreatment of embryos with IFN did not protect them from the embryotoxic effect of EMC virus. This work indicates that preimplantation mouse embryos appear to be resistant for both the antiviral and antiproliferative activities of IFN.     

Culture of one-cell bovine embryos in explanted mouse oviduct and bovine oviductal epithelial cells

One-cell bovine embryos fertilized in vivo were cultured in TCM-199 and bovine oviductal epithelial cells, in TCM-199, or in explanted immature mouse oviducts supported by TCM-199 to compare development to the blastocyst stage. The morphological stage of development and cell number were determined following 144 hours of culture. Of the embryos that cleaved at least once, 52.6, 30.4 and 0.0% developed to the morula/blastocyst stage after culture in oviductal epithelial cells, in TCM-199 alone, or in explanted mouse oviducts, respectively. The mean total cell number for embryos cultured in oviductal epithelial cells (24.5) was higher than for embryos cultured in TCM-199 (12.8) or in explanted mouse oviducts (5.9; P<0.05). The mean cell number of embryos cultured in TCM-199 or in explanted mouse oviducts did not differ. The explanted immature mouse oviduct supported by TCM-199 did not provide an environment adequate for development of one-cell bovine embryos to the blastocyst stage. Development of one-cell bovine embryos was best supported by co-culture with oviductal epithelial cells in TCM-199 medium.     

Influence of short-term maternal zinc deficiency on the in vitro development of preimplantation mouse embryos

In this study, we evaluated the use of mouse preimplantation embryos as a model to study zinc deficiency-induced abnormal development. In Experiment 1, the effect of culture medium Zn concentrations on blastocyst development was studied. Preimplantation embryos (2 and 4 cells) obtained from superovulated females developed normally in media containing 0.7-30 microM Zn for up to 72 hr; higher levels of medium Zn resulted in abnormal development. In Experiment 2A, females were fed diets containing 50 (+Zn) or 0.4 (-Zn) micrograms Zn/g (760 vs 6 nmol/g, respectively) from 1 day before to 1 day after mating (3 days total). Preimplantation embryos were removed from the dams and cultured for 72 hr in 0.7 microM Zn medium. Embryos from the -Zn dams were morphologically normal at time zero; however, over the 72-hr period, these embryos tended to develop at a slower rate than controls, although compaction and cavitation frequency were similar. By the end of the 72-hr culture period, embryos from -Zn dams had significantly fewer cells than did embryos from control dams. In Experiment 2B, an extended period of maternal Zn deprivation (6 days) was used to investigate the potential for further impairment of in vitro preimplantation embryo development observed in Experiment 2A. Results from this experiment were consistent with those from Experiment 2A, in addition to providing evidence that the developmental progress of embryos obtained from mice fed Zn-deficient diets for 6 days was significantly impaired. In Experiment 3, the potential for supplemental Zn in culture medium to overcome the impairment in development due to maternal Zn deficiency was investigated. Embryos from female mice subjected to the same dietary regimen described in Experiment 2A were cultured to the blastocyst stage in medium containing Zn at a concentration of either 0.7 or 7.7 microM. Medium Zn supplementation did not improve development of embryos from dams fed Zn-deficient diets. In summary, embryos from mice fed -Zn diets for a 3- or 6-day period encompassing oocyte maturation and fertilization exhibited impaired development in vitro. This impairment was not overcome by medium Zn supplementation.     

Enhanced in vitro growth of murine fibroblast cells and preimplantation embryos cultured in medium supplemented with an amphipathic peptide.

Preliminary studies on the proliferative effects of lytic peptides were carried out using NIH 3T3 murine fibroblast cells and human lymphocytes. Cells were cultured in various concentrations of three different amphipathic peptides (SB-37, Shiva-1, and Vishnu), and enhanced proliferation was determined by uptake of 3H-thymidine with treated cells compared with control cultures. Enhanced proliferation of 3T3 cells was observed in cultures containing 50 microM or less SB-37. The primary study consisted of 263 four-cell- to eight-cell-stage mouse embryos from naturally bred mice and incubated in Whitten's medium containing 0.2, 1, or 10 microM of the amino terminus of an amphipathic cecropin B analog (Vishnu) or in Whitten's medium alone. Embryos were cultured to the hatched blastocyst stage, and effect of treatment was determined by the rate of growth to that stage of development. Statistical analysis revealed that culture in all three levels of Vishnu significantly accelerated in vitro growth of these stages of preimplantation embryos compared with controls. These results indicate that Vishnu promotes increased cleavage rates of embryos in vitro. A growth factor receptor clustering mechanism of action is proposed. This peptide may have some potential as an embryo culture medium additive to enhance in vitro growth rate.     

Glutamine uptake and utilization by preimplantation mouse embryos in CZB medium

At least 71% of CF1 x B6SJLF1/J embryos developed from the 1-cell stage to the blastocyst stage in an optimum glutamine concentration of 1 mM, as long as glucose was present after the first 48 h of culture. Blastocysts raised under these conditions had significantly more cells than did blastocysts raised in CZB medium alone (glutamine present, glucose absent). Embryos raised in vivo accumulated 170-200 fmol glutamine/embryo/h at the unfertilized egg and 1-cell stages with a decline to 145 fmol/embryo/h at the 2-cell stage, followed by sharp increases to 400 and 850 fmol/embryo/h at the 8-cell and blastocyst stages. The presence or absence of glucose in the labelling medium had no effect on glutamine uptake by these embryos. Embryos raised in vitro accumulated 2-3 times more glutamine at stages comparable to those of embryos raised in vivo. In all cases in which 1-cell to blastocyst development in vitro was successful, glucose was present in the culture medium and the incremental uptake of glutamine between the 8-cell stage and the blastocyst stage was approximately 2-fold. This was also the increment for in-vivo raised embryos. When glucose was not present after the first 48 h, the 8-cell to blastocyst glutamine increment was not significant, and development into blastocysts was reduced. The results also show that glutamine can be used as an energy source for the generation of CO2 through the TCA cycle by all stages of preimplantation mouse development, whether raised in vivo or in vitro from the 1-cell stage. Two-cell embryos raised in vivo converted as much as 70% of the glutamine uptake into CO2, consistent with an important role for glutamine in the very earliest stages of preimplantation development. Cultured blastocysts appeared to convert less glutamine and the presence of glucose in the culture medium seemed to inhibit this conversion.     

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.     

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.     

Interleukin-1 beta induces autophagy of mouse preimplantation embryos and improves blastocyst quality

Autophagy is one of the basic cellular mechanism during preimplantation development of mammalian embryos, and it plays crucial role in several physiological processes. It is induced by interleukin (IL)-1β in mammalian cells. Our present study shows that IL-1β is important for autophagy activation in embryo development. Our in vitro culture system analysis shows effect of IL-1β in medium on the development of mouse embryos and it was found to be concentration dependent. A preimplantation embryo culture using medium containing IL-1β did not improve cleavage and blastocyst development rates of mouse embryos; however, blastocyst quality was significantly improved by increasing total cell number, especially in supplementary 20 ng/mL IL-1β. Furthermore, autophagy activation mainly occurs in 2 to 4 cell embryo and blastocyst, 20 ng/mL IL-1β into culture medium can effectively enhance levels of messenger RNA and protein of autophagy-related-factors in 2 to 4 cell embryos and blastocyst, while these factors reduce in VGX-1027 (IL-1β inhibitor) groups that also reduce the quality of blastocyst. Effects of IL-1β on the development of embryo reduced in 20 ng/mL IL-1β supplemented group when 5 mM 3-methyladenine (3-MA) was also added, which used to inhibit autophagy activation in endogenous PtdIns3Ks signal pathway. Our current results show that exogenous IL-1β can effectively induce autophagy in mouse embryos at stages of 2 to 8 cell and blastocyst, that also help to improve the quality of blastocyst.