Expression of connexins in human preimplantation embryos in vitro

Intercellular communication via gap junctions is required to coordinate developmental processes in the mammalian embryo. We have investigated if the connexin (Cx) isoforms known to form gap junctions in rodent preimplantation embryos are also expressed in human embryos, with the aim of identifying species differences in communication patterns in early development. Using a combination of polyA PCR and immunocytochemistry we have assessed the expression of Cx26, Cx31, Cx32, Cx40, Cx43 and Cx45 which are thought to be important in early rodent embryos. The results demonstrate that Cx31 and Cx43 are the main connexin isoforms expressed in human preimplantation embryos and that these isoforms are co-expressed in the blastocyst. Cx45 protein is expressed in the blastocyst but the protein may be translated from a generally low level of transcripts: which could only be detected in the PN to 4-cell embryos. Interestingly, Cx40, which is expressed by the extravillous trophoblast in the early human placenta, was not found to be expressed in the blastocyst trophectoderm from which this tissue develops. All of the connexin isoforms in human preimplantation embryos are also found in rodents pointing to a common regulation of these connexins in development of rodent and human early embryos and perhaps other species

Cattle Embryo Growth Development and Viabilty.

End of Project ReportA major problem for the cattle breeding industry is the high rate of early embryo loss which compromises reproductive efficiency and genetic improvement, resulting in serious financial loss to farmers. An important part of the Teagasc research programme in this area is the investigation of basic parameters of cattle embryo growth, development and viability during the critical period when most of the embryo loss occurs. We have now characterised this period of embryo development and to our knowledge, this is the first report describing the morphology, growth rate, protein content and metabolic activity of cattle embryos during this period. The main results are summarised here and detailed results have been published in the papers listed at the end of this report. Embryo growth rate and protein content increased exponentially between days 8 and 13 after fertilisation. Furthermore, there was a high rate of protein synthetic activity, energy and amino acid metabolism and signal transduction activity, all reaching a peak between days 8 and 13 after fertilisation. Because of the high rate of metabolic activity evident during this time it is likely that the embryos are very susceptible to environmental changes that have the potential to interfere with normal developmental mechanisms. The results arising from this project suggest that the critical period of early embryo loss in cattle may now be narrowed to a time window of day 8 to 13 rather than day 8 to 16 as presumed up to now. The main results are summarised.European Union 4th Framework Programme (Contract CT-95-0190)

Energy metabolism in pig oocytes and early embryos

Pig oocytes and embryos differ from those of other species in having a large quantity of endogenous lipid, a potential role for which has yet to be identified. In the present study, the hypothesis that endogenous triglyceride acts as a metabolic substrate during in vitro maturation and early embryo development was tested. Embryos were produced by in vitro fertilization (IVF) of in vitro-matured, abattoir-derived immature oocytes, cultured in medium NCSU23 up to the blastocyst stage. The triglyceride content of single oocytes and embryos was measured throughout development. Oxygen and glucose consumption and the formation of lactate were measured non-invasively over the same period, enabling total ATP production to be calculated. The triglyceride content of oocytes before maturation (135+/-4.9 ng) decreased by 13 ng (P<0.05) during in vitro maturation, but there was no apparent change in triglyceride content during embryo development (117.68 ng). Oxygen consumption was low throughout embryo cleavage before reaching a peak at the blastocyst stage (P<0.01), a pattern similar to that seen in other mammals studied. Glucose consumption and lactate production were also at a maximum at the blastocyst stage (P<0.05). These data indicate that pig oocytes may use endogenous triglyceride as an energy source during in vitro maturation and that most (91-97%) of the ATP produced during embryo development comes from oxidative phosphorylation. The high exogenous glucose concentration in NCSU23 (5.5 mmol l(-1)) may be needed to form pyruvate, which in turn, produces oxaloacetate, which is required to prime the tricarboxylic acid cycle. However, the reason for the high lipid content in early pig embryos remains to be elucidated

Can Embryo metabolism be used for selecting bovine embryos before transfer?

International audienc

Glucose utilization by components of the mouse conceptus during early embryogenesis

Mouse conceptuses were collected from female mice between day 6.5 and day 9.5 of pregnancy, dissected into their component parts and incubated for 2.5 h at 37 degrees C in droplets of Hepes-buffered medium containing 1 or 5 mmol glucose l-1 supplemented with 0.33 mmol pyruvate l-1 plus either 1 or 5 mmol (DL) lactate l-1 under oil. Glucose disappearance and lactate appearance were measured enzymatically at the end of incubation. High glucose concentration doubled utilization of this substrate per microgram of embryonic protein, but the change in lactate concentration had no effect on glucose turnover. Over the whole period of development studied, tissue from the ectoplacental cone exhibited the lowest rate of glucose turnover of all tissues isolated. At day 8.5, there was little difference between yolk sac and embryonic tissues, but by day 9.5, the yolk sac had a higher rate of utilization of glucose than did embryonic tissues. By this time, the embryonic tissues had started to show some metabolic differentiation, with head and visceral tissue exhibiting 20% higher turnover of glucose than that of body tissue. Overall, the rate of glucose utilization fell as development progressed and the estimate of the relative rate of glucose utilization on day 9.5 was half the value on day 6.5

Effect of inhibiting nitric oxide production on mouse preimplantation embryo development and metabolism

Nitric oxide (NO) is a free radical that functions as a cell signaling molecule but at high concentrations can be toxic. It is formed from arginine, which is consumed by the mouse blastocyst, but its effect on early embryo development has been little studied. In this study, the role of NO in mouse preimplantation development has been examined in terms of developmental rate and oxidative metabolism. Zygotes were cultured in one of four media; potassium simplex optimization medium (KSOM), KSOM with amino acids (KSOMaa), KSOM without glutamine (KSOM-glut), or KSOM with 0.5 mM arginine (KSOMarg) ± L-NAME (a specific inhibitor of NO production). End points were Day 4 blastocyst rates, cell counts determined using bisbenzimide and oxygen consumption. In KSOM and KSOM-glut, the blastocyst rate was decreased by 1 mM L-NAME from 50.2% ± 3.1% and 37.4% ± 4.5% to 6% ± 3% and 0%, respectively. In KSOMaa, cavitation rates were unaltered but the blastocysts contained fewer cells (P &lt; 0.001). Blastocysts cultured in KSOM and KSOM-glut consumed significantly more oxygen than those cultured in KSOMaa (P &lt; 0.001 and P &lt; 0.05, respectively). However, the addition of 0.1 mM or 1 mM L-NAME to KSOMaa significantly increased the amount of oxygen consumed (P &lt; 0.05 and P &lt; 0.001, respectively). The data suggest a physiological role for NO in mouse preimplantation metabolism and development. One possibility is that NO may limit oxygen consumption at the blastocyst stage at the level of mitochondrial cytochrome c oxidase

Pyruvate uptake during early cleavage predicts blastocyst development

International audienc

The delivery of PEBBLE nanosensors to measure the intracellular environment

Cellular introduction of PEBBLEs (photonic explorers for bioanalysis with biologically localized embedding) has been investigated by a wide variety of methods in a range of cell types. These methods include surface functionalization with CPPs (cell-penetrating peptides), pinocytosis, commercial lipid transfection agents, cytochalasin D, picoinjection, and Gene gun bombardment. This paper will overview several of the most popular methods used for the introduction of PEBBLE nanosensors to the cellular environment and discuss the efficacy of the techniques

Substrate utilization and maturation of cumulus cell-enclosed mouse oocytes: evidence that pyruvate oxidation does not mediate meiotic induction

This study was performed to address the possible role of pyruvate in meiotic induction in mouse oocytes. Cumulus cell-enclosed oocytes from primed, immature mice were cultured in 7.5 μl microdrops under oil for 9 or 18 h in medium containing 4 mmol hypoxanthine l−1 plus 0.23 mmol pyruvate l−1, l mmol pyruvate l−1, or 1 mmol pyruvate l−1 plus 5.5 mmol glucose l−1. When compared with cultures containing 0.23 mmol pyruvate l−1, 1 mmol pyruvate l−1 induced germinal vesicle breakdown, and this was preceded by an increase in pyruvate utilization. Addition of glucose prevented both the increase in pyruvate consumption and the meiotic induction. When different combinations of pyruvate and glucose were tested on oocyte maturation in microdrop cultures, a high concentration of pyruvate or glucose alone was stimulatory to maturation. Addition of the complementary energy substrate prevented the induction of germinal vesicle breakdown and reduced the amount of substrate consumption. During spontaneous maturation in vitro, oocyte–cumulus cell complexes consumed glucose for the first 3 h; however, during the second 3 h period, which followed germinal vesicle breakdown, glucose consumption decreased and net pyruvate utilization was initiated. Treatment of hypoxanthine-arrested oocytes with dichloroacetate, an activator of pyruvate dehydrogenase, stimulated pyruvate consumption but had no effect on germinal vesicle breakdown. Although FSH stimulates meiotic resumption, no changes in pyruvate consumption were observed in response to this gonadotrophin. Measurement of oxygen consumption by hypoxanthine-treated complexes revealed no effect of high concentrations of pyruvate on respiration, and FSH treatment resulted in a suppression of oxygen utilization. These data indicate that, in mouse oocyte–cumulus cell complexes, pyruvate and glucose can each modulate metabolism of the other substrate, and this can significantly influence meiotic maturation of the oocyte. In addition, augmentation of pyruvate oxidation does not appear to play a mediating role in meiotic induction triggered by energy substrate manipulation or gonadotrophin treatment