Analysis of HLA-G in maternal plasma, follicular fluid, and preimplantation embryos reveal an asymmetric pattern of expression

Soluble HLA-G (sHLA-G) secretion by human preimplantation embryos in culture has been associated with successful embryo development, and therefore has potential to serve as a noninvasive marker of embryo viability. We have examined the spatial and temporal expression of HLA-G in embryos of varying developmental competence and the role of maternal factors in human embryonic HLA-G expression. Embryos that reached blastocyst stage on day 5 showed a higher frequency of sHLA-G secretion than those at morula or arrested stages (p < 0.05). There was no significant difference in sHLA-G secretion between normal embryos and those diagnosed as chromosomally abnormal by preimplantation genetic diagnosis. HLA-G detected in maternal plasma and follicular fluid did not appear to correlate with HLA-G expressed in the embryo or embryo supernatants. Confocal microscopy analysis indicated that HLA-G protein expression in embryos was not homogeneous; mostly, it was confined to blastocysts localized on trophectoderm and trophectoderm projections. Single-particle fluorescent imaging analysis of HLA-G on the cell surface of JEG-3 cells showed that HLA-G particles were mostly monomeric, but dimeric and higher order oligomers were also observed. These results suggest that HLA-G play an important role in preimplantation embryo development. However, the observed expression of HLA-G in arrested and chromosomally abnormal embryos indicates that HLA-G testing should be used with caution and in conjunction with conventional methods of embryo screening and selection.     

Stage-specific response of preimplantation mouse embryos to W-7, a calmodulin antagonist

Involvement of calmodulin-dependent processes in preimplantation development of mouse embryos was studied with the use of N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), a specific antagonist of calmodulin. At 25 microM, W-7 interfered with compaction of eight-cell embryos, caused decompaction of compacted eight-cell embryos, inhibited cavitation of late morulae, and caused collapse and degeneration of blastocysts. These effects of W-7 appear to be due to specific inhibition of calmodulin-dependent processes, because W-5, a less active analogue of W-7, was less effective in interfering with development; at 25 microM, W-5 had only a slight effect on compaction and had no effect on blastocyst formation, maintenance of blastocoels, or post-blastocyst development. In addition to the developmental effects just described, W-7 inhibited cell proliferation in four-cell embryos and reduced cell numbers of morulae after treatment at the two- to eight-cell stages. There was a marked increase in embryos' sensitivity to W-7 at the late morula stage, and the sensitivity increased further as embryos developed into blastocysts; the effects of W-7 were largely reversible after treatment at the two-cell through the compacted eight-cell stages, but not after treatment at the late morula or blastocyst stage. At the blastocyst stage, inner cell mass cells appeared to be slightly more resistant to W-7 than trophectoderm cells. This differential sensitivity became more pronounced at the late blastocyst stage: after 3.5-4-h exposure of late blastocysts to 25 microM W-7, all trophectoderm cells degenerated but most of the inner cell masses survived. From these results it appears that calmodulin-dependent processes are involved in development of mouse embryos at all of the preimplantation stages examined.     

Substrate utilization in porcine embryos cultured in NCSU23 and G1.2/G2.2 sequential culture media

Embryo metabolism is an indicator of viability and, therefore, efficiency of the culture medium. Currently, little is known regarding porcine embryo metabolism. The objective of our study was to evaluate glucose and pyruvate uptake and lactate production in porcine embryos cultured in two different media systems. Oocytes were matured and fertilized according to standard protocols. Embryos were allocated randomly into two culture treatments, NCSU23 medium or G1.2/G2.2 sequential culture media 6-8 h post-insemination (hpi). Embryo substrate utilization was measured at the two-cell (24-30 hpi), 8-cell (80 hpi), morula (120 hpi), and blastocyst (144 hpi) stages using ultramicrofluorimetry. Glucose uptake was higher (P < 0.05) in two-cell embryos cultured in G1.2 than in NCSU23 medium (4.54 +/- 0.71, 2.16 +/- 0.87 pmol/embryo/h, respectively). Embryos cultured in G1.2/G2.2 produced significantly more lactate than those in NCSU23 at the eight-cell stage (9.41 +/- 0.71, 4.42 +/- 0.95 pmol/embryo/hr, respectively) as well as the morula stage (11.03 +/- 2.31, 6.29 +/- 0.77 pmol/embryo/hr, respectively). Pyruvate uptake was higher (P < 0.05) in morula cultured in G1.2/G2.2 versus NCSU23 (22.59 +/- 3.92, 11.29 +/- 1.57 pmol/embryo/h, respectively). Lactate production was greater (P < 0.05) in blastocysts cultured in G1.2/G2.2 (38.13 +/- 15.94 pmol/embryo/h) than blastocysts cultured in NCSU23 (8.46 +/- 2.38 pmol/embryo/h). Pyruvate uptake was also greater in blastocysts cultured in G1.2/G2.2 (24.3 +/- 11.04) than those in NCSU23 (11.30 +/- 2.70). When cultured in NCSU23 medium, two- and eight-cell embryos utilized less glucose than morulae and blastocysts, and two-cell embryos produced less lactate than blastocysts (P < 0.05). In G1.2/G2.2 media, two-cells took up less pyruvate than morulae or blastocysts, while blastocysts produced more lactate and utilized more glucose than two-cell, eight-cell and morula stage embryos (P < 0.05). As in other species, glycolysis appears to be the primary metabolic pathway in post-compaction stage porcine embryos. Culture medium composition affects not only substrate uptake, but also metabolic pathways by which these substrates are utilized in porcine embryos at several developmental stages.     

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.     

Production of monozygotic twin calves using the blastomere separation technique and Well of the Well culture system

The present study was conducted to establish a simple and efficient method of producing monozygotic twin calves using the blastomere separation technique. To produce monozygotic twin embryos from zona-free two- and eight-cell embryos, blastomeres were separated mechanically by pipetting to form two demi-embryos; each single blastomere from the two-cell embryo and tetra-blastomeres from the eight-cell embryo were cultured in vitro using the Well of the Well culture system (WOW). This culture system supported the successful arrangement of blastomeres, resulting in their subsequent aggregation to form a demi-embryo developing to the blastocyst stage without a zona pellucida. There was no significant difference in the development to the blastocyst stage between blastomeres separated from eight-cell (72.0%) and two-cell (62.0%) embryos. The production rates of the monozygotic pair blastocysts and transferable paired blastocysts for demi-embryos obtained from eight-cell embryos (64.0 and 45.0%, respectively) were higher than those for demi-embryos obtained from two-cell embryos (49.0 and 31.0%, P<0.05). The separated demi-embryos obtained from eight-cell embryos produced by IVM/IVF of oocytes collected by ovum pick-up (OPU) from elite cows and cultured in wells tended to have a higher pregnancy rate (78.9% vs. 57.1%) and similar monozygotic twinning rate (40.0% vs. 33.3%) compared with monozygotic twin blastocysts obtained by the conventional bisection of in vivo derived blastocysts. In conclusion, producing twins by separation of blastomeres in OPU-IVF embryos, followed by the WOW culture system, yielded viable monozygotic demi-embryos, resulting in high rates of pregnancy and twinning rates after embryo transfer.     

Inhibition of endoplasmic reticulum stress improves mouse embryo development

X-box binding protein-1 (XBP-1) is an important regulator of a subset of genes during endoplasmic reticulum (ER) stress. In the current study, we analyzed endogenous XBP-1 expression and localization, with a view to determining the effects of ER stress on the developmental competency of preimplantation embryos in mice. Fluorescence staining revealed that functional XBP-1 is localized on mature oocyte spindles and abundant in the nucleus at the germinal vesicle (GV) stage. However, in preimplantation embryos, XBP-1 was solely detected in the cytoplasm at the one-cell stage. The density of XBP-1 was higher in the nucleus than the cytoplasm at the two-cell, four-cell, eight-cell, morula, and blastocyst stages. Furthermore, RT-PCR analysis confirmed active XBP-1 mRNA splicing at all preimplantation embryo stages, except the one-cell stage. Tunicamycin (TM), an ER stress inducer used as a positive control, promoted an increase in the density of nuclear XBP-1 at the one-cell and two-cell stages. Similarly, culture medium supplemented with 25 mM sorbitol displayed a remarkable increase active XBP-1 expression in the nuclei of 1-cell and 2-cell embryos. Conversely, high concentrations of TM or sorbitol led to reduced nuclear XBP-1 density and significant ER stress-induced apoptosis. Tauroursodeoxycholic acid (TUDCA), a known inhibitor of ER stress, improved the rate of two-cell embryo development to blastocysts by attenuating the expression of active XBP-1 protein in the nucleus at the two-cell stage. Our data collectively suggest that endogenous XBP-1 plays a role in normal preimplantation embryonic development. Moreover, XBP-1 splicing is activated to generate a functional form in mouse preimplantation embryos during culture stress. TUDCA inhibits hyperosmolar-induced ER stress as well as ER stress-induced apoptosis during mouse preimplantation embryo development.     

Protein databases for compacted eight-cell and blastocyst-stage mouse embryos

High-resolution two-dimensional sodium dodecyl sulfate-polyacrylamide (2D-SDS) gel electrophoresis combined with computerized analysis of gel images was used to construct and analyze protein databases for two stages of preimplantation mouse embryogenesis, the compacted eight-cell stage and the fully expanded blastocyst stage. These stages were chosen for their ease in identification of multiple synchronous embryos. Synchronous cohorts of 30–50 embryos were labelled with L-[³⁵S]methionine for 2 hr. The embryos were then lysed in 30 μl hot SDS sample buffer, and the lysates were stored at ?80°C until the gels were run. Five replicates were run for eight-cell embryos, and four for blastocyst-stage embryos. The samples were processed for 2D gel electrophoresis and fluorography; multiple exposures were made. Gel images were analyzed using the PDQUEST system, and databases were constructed. Analysis of the databases for both developmental stages showed high reproducibility of protein spots in multiple gel images. Of 1,674 total spots in eight-cell embryo standards, >79% of spots had a percentage error (S.E.M./average) <50%, and >45% had a percentage error <30%. Similarly, of 1,653 total spots in blastocyst-stage embryo standards, 74% of spots had a percentage error <50%, and approximately 47% of spots had a percentage error <30%. Forty-three spots (approximately 3% of the total spots) were found to be detected only in the eight-cell stage, while 75 spots were detected solely in the blastocyst stage. Sixty-nine proteins showed a greater than threefold increase in isotope incorporation from the eight-cell to the blastocyst stage, with a percentage error <50% in both the eight-cell and the blastocyst stages. In contrast, 41 of the proteins showed a decrease during this period. Analysis of the protein databases described in this study has allowed us to document the overall quantitative changes in proteins from the compacted eight-cell stage to the blastocyst stage of mouse preimplantation development. These databases provide a valuable tool for further detailed quantitative analysis of specific proteins associated with developmental events. In addition they will permit analysis of the effects of environmental factors, such as growth factors, on early embryo development. © 1994 Wiley-Liss, Inc.     

Perturbations in mouse embryo development and viability caused by ammonium are more severe after exposure at the cleavage stages

The presence of ammonium in culture medium has a detrimental effect on embryo physiology and biochemistry; however, the stage at which the embryo is most sensitive to this effect is unknown. The aim of this study was to determine the exact stage at which the embryo is most vulnerable to ammonium by exposing the preimplantation embryo to 300 muM ammonium either at the precompaction stage (between the zygote and two-cell or the two-cell to eight-cell) or at the postcompaction stage (between the eight-cell and blastocyst). This study determined that exposure of embryos to ammonium at the precompaction stage from either the zygote to two-cell stage or from the two-cell to the eight-cell stage did not affect the rate of development to the blastocyst stage; however, the resultant blastocysts had decreased cell numbers and inner cell mass cells. Furthermore, these blastocysts had increased levels of cellular apoptosis and perturbed levels of Slc2a3 expression and glucose uptake. Transfer of these blastocysts revealed that, while implantation was not affected, the number of fetuses was reduced by culture with ammonium at the precompaction stage and fetal development was delayed, as observed by reduced crown-rump length and maturity. In contrast, the later stage embryo was more resistant to the negative effects of ammonium, with only Slc2a3 expression and fetal maturity affected. This raises the possibility that the later stage embryo is more able to protect itself from in vitro-derived stress and that the majority of in vitro-induced damage to mouse embryos is inflicted at the early stages of development.     

Functions of the IGFs in early mammalian development

The identification of growth factors and/or receptors produced by mammalian embryos or present in the maternal reproductive tract is of basic interest, as well as having practical application. Early studies established that receptors binding insulin and the insulin-like growth factors (IGFs) are expressed by preimplantation mouse embryos. These studies have been confirmed at the molecular level using RT-PCR techniques. In addition, high resolution electron microscopy has shown that insulin is internalized by the cells of the blastocyst stage mouse embryo, and that immunologically intact insulin is detectable in the cells of the trophectoderm and inner cell mass. Similar studies with gold labelled IGF-I have shown that this ligand is also bound and internalized by mouse blastocysts. However, although all blastocysts express receptors that bind IGF-I on the basolateral cell surface of the trophectoderm, only 30% exhibit apically located receptors. In order to elucidate the functions of IGFs in early mouse development, we are in the process of constructing protein databases for embryos at the eight-cell and blastocyst stage. By the use of the database, it should prove possible to elucidate targets of growth factor action. © 1993 Wiley-Liss, Inc.     

Development of interspecies cloned embryos in yak and dog

Interspecies nuclear transfer (NT) could be an alternative to replicate animals when supply of recipient oocytes is limited or in vitro embryo production systems are incomplete. In the present study, embryonic development was assessed following interspecies NT of donor cumulus cells derived from yak and dog into the recipient ooplasm of domestic cow. The percentages of fusion and subsequent embryo development to the eight-cell stage of interspecies NT embryos were comparable to those of intraspecies NT embryos (cow-cow NT embryos). The percentage of development to blastocysts was significantly lower (p < 0.05) in yak-cow NT embryos than that in cow-cow NT embryos (10.9% vs. 39.8%). In dog-cow NT embryos, only one embryo (0.4%) developed to the blastocyst stage. These results indicate that interspecies NT embryos possess equally developmental competence to the eight-cell stage as intraspecies NT embryos, but the development to blastocysts is very low when dog somatic cells are used as the donor nuclei.