Macromolecular source as dependent on osmotic pressure and water source: effects on bovine in vitro embryo development and quality

This study evaluated the protective effect of protein, as dependent on osmolarity, and the quality of water sources used to prepare embryo culture media. In Experiment 1, two concentrations of NaCl were used to obtain culture media with normal (280 mOSM) and low (245 mOSM) osmolarity, each supplemented with either bovine serum albumin (BSA) or polyvinyl alcohol (PVA). Low osmolarity improved blastocyst rates in the presence of BSA (P < 0.01) and tended to do it in medium containing PVA (P < 0.07). Furthermore, low osmolarity allowed PVA to increase inner cell mass (ICM) numbers and ICM/total cell rate (P < 0.05), while trophectoderm (TE) and total cell counts tended to decrease (P < 0.08). In Experiment 2, culture media were prepared with two water sources (Milli-Q and Sigma-W3500-) in combination with BSA or PVA. Both water sources yielded similar embryo development rates, but in the presence of BSA, Milli-Q water produced embryos with increased ICM/total cells rates (P < 0.05). On the contrary, Sigma water tended to increase trophectoderm cell counts (P < 0.08). In conclusion, the present study showed that low osmolarity is beneficial to embryo development and combinations of macromolecule and osmolarity influence trophectoderm differentiation. Both Milli-Q and Sigma supported embryo development at comparable rates, although in the presence of BSA, blastocysts obtained in the medium prepared with Milli-Q water had superior quality in terms of ICM/total cells rates.     

Maintenance of the inner cell mass in human blastocysts from fragmented embryos

The degree of fragmentation during early cleavage is universally used as an indicator of embryo quality during human in vitro fertilization treatment. Extensive fragmentation has been associated with reduced blastocyst formation and implantation. We examined the relationship between early fragmentation and subsequent allocation of cells to the trophectoderm and inner cell mass in the human blastocyst. We retrospectively analyzed data from 363 monospermic human embryos that exhibited varying degrees of fragmentation on Day 2. Embryos were cultured from Day 2 to Day 6 in Earle balanced salt solution with 1 mM glucose and human serum albumin. Rates of development and blastocyst formation were measured. The number of cells in the trophectoderm and inner cell mass and the incidence of apoptosis were assessed following differential labeling with polynucleotide-specific fluorochromes. Increasing fragmentation resulted in reduced blastocyst formation and lower blastocyst cell numbers. For minimal and moderate levels of fragmentation, the reduction in cell numbers was confined largely to the trophectoderm and a steady number of inner cell mass cells was maintained. However, with extensive fragmentation of more than 25%, cell numbers in both lineages were reduced in the few embryos that formed blastocysts. Apoptotic nuclei were present in both the trophectoderm and inner cell mass, with the lowest incidence in blastocysts that had developed from embryos with minor (5-10%) fragmentation. Paradoxically, higher levels of apoptosis were seen in embryos of excellent morphology, suggesting a possible role in regulation of cell number.     

The effects of cell size and ploidy on cell allocation in mouse chimaeric blastocysts

In a previous study of mouse tetraploid<-->diploid chimaeric blastocysts, tetraploid cells were found to be more abundant in the trophectoderm than the inner cell mass (ICM) and more abundant in the mural trophectoderm than the polar trophectoderm. This non-random allocation of tetraploid cells to different regions of the chimaeric blastocyst may contribute to the restricted tissue distribution seen in post-implantation stage tetraploid<-->diploid chimaeras. However, the tetraploid and diploid embryos that were aggregated together differed in several respects: the tetraploid embryos had fewer cells and these cells were bigger and differed in ploidy. Each of these factors might underlie a non-random allocation of tetraploid cells to the chimaeric blastocyst. A combination of micromanipulation and electrofusion was used to produce two series of chimaeras that distinguished between the effects of cell size and ploidy on the allocation of cells to different tissues in chimaeric blastocysts. When aggregated cells differed in cell size but not ploidy, the derivatives of the larger cell contributed significantly more to the mural trophectoderm and polar trophectoderm than the ICM. When aggregated cells differed in ploidy but not cell size, the tetraploid cells contributed significantly more to the mural trophectoderm than the ICM. In both experiments the contributions to the polar trophectoderm tended to be intermediate between those of the mural trophectoderm and ICM. These experiments show that both the larger size and increased ploidy of tetraploid cells could have contributed to the non-random cell distribution that was observed in a previous study of tetraploid<-->diploid chimaeric blastocysts.     

Differential effect of hexoses on hamster embryo development in culture

The effects of glucose, fructose, and galactose on hamster embryo development in the absence of phosphate were studied in culture. One- and two-cell embryos were cultured to the blastocyst stage in HECM-9 medium without hexose or in medium with increasing concentrations of hexoses. Embryo development, cell number, and cell allocation were assessed in blastocysts. Blastocyst viability was determined by transfer to pseudopregnant recipients. Although 0.25 mM fructose increased mean cell number, low glucose concentrations had no stimulatory effect on development to blastocyst. Both galactose and 5.0 mM glucose were detrimental to embryos. Addition of 0.5 mM glucose increased implantation and fetal viability as compared with controls. Compared with 0.5 mM glucose, treatment with 0.25 mM fructose gave similar implantation and fetal viability, whereas 5.0 mM glucose tended to decrease implantation and significantly decreased fetal development. These data demonstrate that morphology is a poor indicator of embryo viability and that exposure of preimplantation embryos to glucose or fructose is important for embryo viability post-transfer. Although no difference in blastocyst viability was detected between embryos cultured with 0.25 mM fructose and those cultured with 0.5 mM glucose, increased cell numbers obtained with fructose suggest that fructose may be more appropriate than glucose for inclusion in culture medium.     

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.     

The effect of in vitro treatment of bovine embryos with IGF-1 on subsequent development in utero to Day 14 of gestation

Culture of bovine embryos with insulin-like growth factor-1 (IGF-1) can improve development to the blastocyst stage and embryo survival following transfer to heat-stressed, lactating dairy cows. Two experiments were conducted to determine whether IGF-1 could improve embryo survival and development at Day 14 after ovulation. In Experiment 1, non-lactating Holstein cows (n=58) were selected as recipients following synchronization for timed-embryo transfer. Embryos were produced in vitro and cultured with or without 100ng/mL IGF-1. At Day 7 after expected ovulation (Day 0), groups of 7-12 embryos were randomly transferred to each recipient. Embryos were recovered at Day 14. Embryo length and the presence or absence of an embryonic disc was recorded. Recovered embryos were cultured individually for 24h to determine interferon-tau (IFN-tau) secretion. There was no effect of IGF-1 on embryo recovery rate, embryo length or IFN-tau secretion. In Experiment 2, non-lactating (n=56) and lactating (n=35) Holstein cows were selected as recipients following synchronization for timed-embryo transfer. Embryos were produced as described in Experiment 1. At Day 7 after expected ovulation (Day 0), a single embryo was randomly transferred to each recipient. Embryos were recovered at Day 14. Embryo length and IFN-tau secretion were determined as in Experiment 1. Recovery rate at Day 14 tended (P=0.1) to be higher for recipients that received IGF-1 treated embryos compared to control embryos (43.2% versus 26.1%, respectively). There was no effect of IGF-1 on embryo length or IFN-tau secretion. In conclusion, results suggest that exposure to IGF-1 through Days 7-8 of development does not enhance capacity of embryos to prevent luteolysis. Results of the single embryo-transfer experiment suggested that IGF-1 treatment might affect embryo survival post-transfer as early as Day 14 after ovulation. Further experimentation is warranted to verify this finding.     

Cell allocation in half- and quadruple-sized preimplantation mouse embryos

The size of preimplantation mouse embryos was experimentally manipulated in order to examine the consequences for the allocation of cells to the two primary tissues, trophectoderm and inner cell mass (ICM). Half embryos were produced by the mechanical lysis of one cell at the two-cell stage and quadruple embryos by the aggregation of four whole eight-cell embryos. Such procedures are shown not only to alter the absolute number of cells that are assigned to the trophectoderm and ICM, but also to disturb significantly the proportions of these two tissues in the blastocyst. The proportion of trophectoderm is directly related to the surface area of the morula, as is predicted by a purely epigenetic scheme for cell allocation.     

Trichostatin A-treated eight-cell bovine embryos had increased histone acetylation and gene expression, with increased cell numbers at the blastocyst stage

The objective was to determine the effects of trichostatin A (TSA), a potent histone deacetylase inhibitor, on eight-cell bovine embryos. That treatment increased histone acetylation was confirmed by immunostaining with anti-AcH4K5 and AcH4K8 antibodies. Embryos treated with TSA (100 nM) for various intervals (4, 8, and 12 h) developed to the blastocyst stage as frequently as untreated embryos (average development rate, 49.5%). Treatment with TSA for 12 h increased (P < 0.05) the numbers of inner cell mass (ICM) cells and total cells (TC), as well as the ICM/TC ratio in the blastocyst, but the number of cells in the trophectoderm decreased (P < 0.05). Treated embryos had increased relative abundance (RA) of OCT3/4 and E-CADHERIN mRNA relative to controls at the morula stage (P < 0.05), however, the RA of CDX2 mRNA was unchanged. In conclusion, TSA-treated eight-cell stage embryos had increased histone acetylation and gene expression, which increased ICM and TC numbers and the ICM/TC ratio, but significantly decreased the number of cells in the trophectoderm of resulting blastocysts.     

Fibroblast growth factor requirements for in vitro development of bovine embryos

The overall goal was to describe the importance of fibroblast growth factors (FGFs) during development of the bovine embryo. An inhibitor of FGF receptor kinase activity (SU5402) was used to examine whether FGF signaling is required for embryo development. Addition of 20 μM SU5402 on Day 0 (Day of IVF) reduced (P = 0.04) the percentage of oocytes becoming blastocysts on Day 7 compared to controls (5.9 ± 2.1 vs 16.9 ± 2.4; average ± SEM). Also, Day-8 blastocysts placed into individual culture drops of medium containing SU5402 tended to have decreased (P = 0.08) blastomere numbers at Day 11 (211.1 ± 27.5 vs 297.8 ± 25.0). A second series of studies determined if supplemental FGF2 enhances development in vitro. There was no effect of FGF2 on cleavage or blastocyst development rates when 5 or 100 ng/mL FGF2 was provided immediately after fertilization. Also, FGF2 supplementation beginning on Day 5 post-fertilization did not significantly affect blastocyst rates or the number of trophoblast and inner cell mass cells. However, addition of 500 ng/mL FGF2 at both Day 0 and Day 4 increased (P = 0.03) the percentage of oocytes that became blastocysts on Day 7 compared with controls (27.4 ± 1.3 vs 19.7 ± 1.3). In a final study, the thermal-protective ability of FGF2 was examined by adding FGF2 1 h before exposing Day 5 embryos to heat shock. Addition of FGF2 did not significantly influence embryo thermal-tolerance. In conclusion, FGF receptor activation was important for optimal blastocyst formation and FGF2 supplementation increased bovine blastocyst formation when provided at high concentrations.     

Probing morphological,genetic and metabolomic changes of in vitro embryo development in a microfluidic device

Assisted reproduction technologies for clinical and research purposes rely on a brief in vitro embryo culture which, despite decades of progress, remain suboptimal in comparison to the physiological environment. One promising tool to improve this technique is the development of bespoke microfluidic chambers. Here we present and validate a new microfluidic device in polydimethylsiloxane (PDMS) for the culture of early mouse embryos. Device material and design resulted embryo compatible and elicit minimal stress. Blastocyst formation, hatching, attachment and outgrowth formation on fibronectin-coated devices were similar to traditional microdrop methods. Total blastocyst cell number and allocation to the trophectoderm and inner cell mass lineages were unaffected. The devices were designed for culture of 10–12 embryos. Development rates, mitochondrial polarization and metabolic turnover of key energy substrates glucose, pyruvate and lactate were consistent with groups of 10 embryos in microdrop controls. Increasing group size to 40 embryos per device was associated with increased variation in development rates and altered metabolism. Device culture did not perturb blastocyst gene expression but did elicit changes in embryo metabolome, which can be ascribed to substrate leaching from PDMS and warrant further investigation.     

Insulin-like growth factor-I promotes resistance of bovine preimplantation embryos to heat shock through actions independent of its anti-apoptotic actions requiring PI3K signaling

For the bovine preimplantation embryo, insulin-like growth factor-I (IGF-I) is a survival factor that blocks the induction of apoptosis and reduces the decrease in development caused by heat shock. The first objective was to determine the signaling pathways whereby IGF-I acts to increase embryo cell number while inhibiting heat-shock induced apoptosis. Exposure of embryos to heat shock reduced cell number and increased percent apoptosis, but IGF-I increased cell number and blocked induction of apoptosis caused by heat shock. Actions of IGF-I to increase cell number were blocked by treatment with the mitogen activated protein kinase kinase (MAPKK) inhibitor PD 98059 whereas the phosphatidylinositol 3-kinase (PI3K) inhibitor LY 294002 had no effect. Conversely, LY 294002 but not PD 98059 blocked actions of IGF-I to inhibit induction of apoptosis caused by heat shock. The second objective was to determine whether IGF-I blocks effects of heat shock on development to the blastocyst stage by preventing apoptosis. Culture of embryos with IGF-I was effective in blocking the reduction in blastocyst development caused by heat shock-this action occurred even in the presence of LY 294002. Addition of another inhibitor of apoptosis, the caspase-3 inhibitor z-DEVD-fmk, did not mimic the protective effects of IGF-I on blastocyst development. Surprisingly, IGF-I was not effective in blocking the reduction in blastocyst development caused by heat shock when cultured with z-DEVD-fmk. In conclusion, the anti-apoptotic actions of IGF-I require PI3K signaling while actions to promote proliferation require MAPKK signaling. Moreover, actions of IGF-I to allow heat-shocked embryos to continue development to the blastocyst stage are independent of its anti-apoptotic effects.