The 'Developmental Origins' Hypothesis: relevance to the obstetrician and gynecologist.

The recognition of 'fetal origins of adult disease' has placed new responsibilities on the obstetrician, as antenatal care is no longer simply about ensuring good perinatal outcomes, but also needs to plan for optimal long-term health for mother and baby. Recently, it has become clear that the intrauterine environment has a broad and long-lasting impact, influencing fetal and childhood growth and development as well as future cardiovascular health, non-communicable disease risk and fertility. This article looks specifically at the importance of the developmental origins of ovarian reserve and ageing, the role of the placenta and maternal nutrition before and during pregnancy. It also reviews recent insights in developmental medicine of relevance to the obstetrician, and outlines emerging evidence supporting a proactive clinical approach to optimizing periconceptional as well as antenatal care aimed to protect newborns against long-term disease susceptibility.

Embryonic genotype and inbreeding affect preimplantation development in cattle.

Infertility in cattle herds is a growing problem with multifactorial causes. Embryonic genotype and level of inbreeding are among the many factors that can play a role on reproductive efficiency. To investigate this issue, we produced purebred and crossbred bovine embryos by in vitro techniques from Holstein oocytes and Holstein or Brown Swiss semen and analyzed several cellular and molecular features. In the first experiment, purebred and crossbred embryos, obtained from abattoir oocytes, were analyzed for cleavage, development to morula/blastocyst stages, amino acid metabolism and gene expression of developmentally important genes. The results indicated significant differences in the percentage of compacted morulae, in the expression of three genes at the blastocyst stage (MNSOD, GP130 and FGF4) and in the utilization of serine, asparagine, methionine and tryptophan in day 6 embryos. In the second experiment, bovine oocytes were collected by ovum pick up from ten Holstein donors and fertilized with the semen of the respective Holstein sires or with Brown Swiss semen. The derived embryos were grown in vitro up to day 7, and were then transferred to synchronized recipients and recovered on day 12. We found that purebred/inbred embryos had lower blastocyst rate on days 7-8, were smaller on day 12 and had lower expression of the trophoblast gene PLAC8. Overall, these results indicate reduced and delayed development of purebred embryos compared with crossbred embryos. In conclusion, this study provides evidence that embryo genotype and high inbreeding can affect amino acid metabolism, gene expression, preimplantation development and therefore fertility in cattle.

Expression of genes involved in early cell fate decisions in human embryos and their regulation by growth factors.

Little is understood about the regulation of gene expression in human preimplantation embryos. We set out to examine the expression in human preimplantation embryos of a number of genes known to be critical for early development of the murine embryo. The expression profile of these genes was analysed throughout preimplantation development and in response to growth factor (GF) stimulation. Developmental expression of a number of genes was similar to that seen in murine embryos (OCT3B/4, CDX2, NANOG). However, GATA6 is expressed throughout preimplantation development in the human. Embryos were cultured in IGF-I, leukaemia inhibitory factor (LIF) or heparin-binding EGF-like growth factor (HBEGF), all of which are known to stimulate the development of human embryos. Our data show that culture in HBEGF and LIF appears to facilitate human embryo expression of a number of genes: ERBB4 (LIF) and LIFR and DSC2 (HBEGF) while in the presence of HBEGF no blastocysts expressed EOMES and when cultured with LIF only two out of nine blastocysts expressed TBN. These data improve our knowledge of the similarities between human and murine embryos and the influence of GFs on human embryo gene expression. Results from this study will improve the understanding of cell fate decisions in early human embryos, which has important implications for both IVF treatment and the derivation of human embryonic stem cells.

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.

Identification of viable embryos in IVF by non-invasive measurement of amino acid turnover.

BACKGROUND: IVF is limited by low success rates and an unacceptably high multiple pregnancy rate. These outcomes would be improved significantly if a single embryo of high viability could be replaced in each treatment cycle, but widespread acceptance of such a policy is hindered by the lack of predictive factors for embryo selection. We have conducted a retrospective clinical study of a novel non-invasive method of embryo selection based on the depletion/appearance of amino acids in the culture medium. METHODS: Fifty-three cycles of IVF treatment using ICSI were studied. Embryos were cultured for 24 h in 4 microl drops of medium containing a physiological mixture of 18 amino acids. The spent medium was analysed for amino acid content by high performance liquid chromatography. RESULTS: The turnover of three amino acids, Asn, Gly and Leu, was significantly correlated with a clinical pregnancy and live birth. These correlations were independent of known predictors, such as female age, basal levels of FSH, embryo cell number and embryo morphological grade. CONCLUSIONS: Non-invasive assay of amino acid turnover has the potential to improve significantly the prospective selection of the most viable embryos, or single embryo, for replacement in an IVF cycle.

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 < 0.001). Blastocysts cultured in KSOM and KSOM-glut consumed significantly more oxygen than those cultured in KSOMaa (P < 0.001 and P < 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 < 0.05 and P < 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.

Functional significance of gap junctional coupling in preimplantation development.

Gap junctional intercellular coupling allows cells to share low molecular weight metabolites and second messengers, thus facilitating homeostatic and developmental processes. Gap junctions make their appearance very early in rodent development, during compaction in the eight-cell stage. Surprisingly, preimplantation mouse embryos lacking the gap junction protein connexin 43 develop normally and establish full-term pregnancies despite severely reduced gap junctional coupling. It was suggested that this might be explained by the presence of at least five additional connexins known to be expressed in blastocysts. In the present study, we set out to clarify the number of connexins present in preimplantation rodent embryos and the role of gap junctional coupling, if any, in blastocyst development. We provide evidence from reverse transcription-polymerase chain reaction analysis that the genes encoding 3 additional connexins (connexin 30 or beta6, connexin 36 or alpha9, and connexin 57 or alpha10) are also transcribed in preimplantation mouse embryos. Furthermore, we show that multiple connexins are expressed in rat preimplantation embryos, indicating that multiplicity of connexin expression may be a common feature of early mammalian embryogenesis. We could detect no up-regulation of any of 3 coexpressed connexins examined in mouse embryos lacking connexin 43. Impaired intercellular coupling caused either by the loss of connexin 43 or by treatment of cultured embryos with the gap junctional coupling blocker 18alpha-glycyrrhetinic acid (AGA) had no discernable effect on either apoptosis or glucose utilization, parameters known to be affected by gap junctional coupling in other contexts. These results, taken together with the reported inability of AGA to perturb blastocyst formation, imply that gap junctional coupling is not essential during this developmental period. We propose that connexin expression and the assembly of multiple types of gap junction channels in preimplantation embryos facilitates the diversification of communication pathways that will appear during postimplantation development. New evidence of this diversification is presented using rat blastocyst outgrowths.

Doubly mutant mice, deficient in connexin32 and -43, show normal prenatal development of organs where the two gap junction proteins are expressed in the same cells.

The connexins are a family of proteins that form the intercellular membrane channels of gap junctions. Genes encoding 13 different rodent connexins have been cloned and characterized to date. Connexins vary both in their distribution among adult cell types and in the properties of the channels that they form. In order to explore the functional significance of connexin diversity, several mouse connexin-encoding genes have been disrupted by homologous recombination in embryonic stem cells. Although those experiments have illuminated specific physiological roles for individual connexins, the results have also raised the possibility that connexins may functionally compensate for one another in cells where they are coexpressed. In the present study, we have tested this hypothesis by interbreeding mice carrying null mutations in the genes (Gjb1 and Gja1) encoding connexin32 (beta 1 connexin) and connexin43 (alpha 1 connexin), respectively. We found that fetuses lacking both connexins survive to term but, as expected, the pups die soon thereafter from the cardiac abnormality caused by the absence of connexin43. A survey of the major organ systems of the doubly mutant fetuses, including the thyroid gland, developing teeth, and limbs where these two connexins are coexpressed, failed to reveal any morphological abnormalities not already seen in connexin43 deficient fetuses. Furthermore, the production of thyroxine by doubly mutant thyroids was confirmed by immunocytochemistry. We conclude that, at least as far as the prenatal period is concerned, the normal development of those three organs in fetuses lacking connexin43 cannot simply be explained by the additional presence of connexin32 and vice-versa. Either gap junctional coupling is dispensable in embryonic and fetal cells in which these two connexins are coexpressed, or coupling is provided by yet another connexin when both are absent.

Normal development of preimplantation mouse embryos deficient in gap junctional coupling.

The connexin multigene family (13 characterized members in rodents) encodes the subunits of gap junction channels. Gap junctional intercellular coupling, established during compaction of the preimplantation mouse embryo, is assumed to be necessary for development of the blastocyst. One member of the connexin family, connexin43, has been shown to contribute to the gap junctions that form during compaction, yet embryos homozygous for a connexin43 null mutation develop normally, at least until implantation. We show that this can be explained by contributions from one or more additional connexin genes that are normally expressed along with connexin43 in preimplantation development. Immunogold electron microscopy confirmed that roughly 30% of gap junctions in compacted morulae contain little or no connexin43 and therefore are likely to be composed of another connexin(s). Confocal immunofluorescence microscopy was then used to demonstrate that connexin45 is also assembled into membrane plaques, beginning at the time of compaction. Correspondingly, embryos homozygous for the connexin43 null mutation were found to retain the capacity for cell-to-cell transfer of fluorescent dye (dye coupling), but at a severely reduced level and with altered permeability characteristics. Whereas mutant morulae showed no evidence of dye coupling when tested with 6-carboxyfluorescein, dye coupling could be demonstrated using 2',7'-dichlorofluorescein, revealing permeability characteristics previously established for connexin45 channels. We conclude that preimplantation development in the mouse can proceed normally even though both the extent and nature of gap junctional coupling have been perturbed. Despite the distinctive properties of connexin43 channels, their role in preimplantation development can be fulfilled by one or more other types of gap junction channels.

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 microliters microdrops under oil for 9 or 18 h in medium containing 4 mmol hypoxanthine l-1 plus 0.23 mmol pyruvate l-1, 1 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 the 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.

Expression and activity of hexokinase in the early mouse embryo.

The maximal activity and Michaelis constant, KM, of hexokinase have been measured in the peri-implantation mouse embryo using an ultramicrofluorescence technique. In addition, transcript detection of the predominant isoenzyme hexokinase I has been determined in single preimplantation mouse embryos at successive stages of development using reverse transcriptase-mediated cDNA amplification. Maximal hexokinase activity decreased dramatically peri-implantation, from 0.97 +/- 0.19 nmol/microgram protein/h at the blastocyst stage to 0.31 +/- 0.05 nmol/microgram protein/h on day 6.5. The KM remained relatively low and constant over this period (0.23-0.39 mM), indicating the absence of the hexokinase type IV isoenzyme. The pattern of hexokinase activity resembled that of glucose consumption suggesting a possible regulatory role for the enzyme during this period of development. Hexokinase I mRNA was detected in the oocyte and all preimplantation stages of development. The blastocyst polymerase chain reaction (PCR) product, when cloned and sequenced was found to be 98% homologous with mouse tumour hexokinase I. Taken together, these data suggest that the hexokinase gene is not under transcriptional control during early mouse embryo development but plays a significant role in the regulation of glucose consumption. A role for hexokinase in the phosphate-induced inhibition of early embryo development is also proposed.

Oxygen consumption and energy metabolism of the early mouse embryo.

Oxygen consumption of preimplantation and early postimplantation mouse embryos has been measured using a novel noninvasive ultramicrofluorescence technique, based on an oil-soluble, nontoxic quaternary benzoid compound pyrene, whose fluorescence is quenched in the presence of oxygen. Pyruvate and glucose consumption, lactate production, and glycogen formation from glucose were also measured. Preimplantation mouse embryos of the strain CBA/Ca x C57BL/6 were cultured in groups of 10-30 in 2 microliters of modified M2 medium containing 1 mmol l-1 glucose, 0 mmol l-1 lactate, and 0.33 mmol l-1 pyruvate, for between 4-6 hr. Day 6.5 and 7.5 embryos were cultured singly in 40 microliters M2 medium for between 2-3 hr. Oxygen consumption was detected at all stages of development, including, for the first time, in the early postimplantation embryo. Consumption remained relatively constant from zygote to morula stages before increasing in the blastocyst and day 6.5-7.5 stages. When expressed as QO2 (microliters/mg dry weight/hr), oxygen consumption was relatively constant from the one-cell to morula stages before increasing sharply at the blastocyst stage and declining to preblastocyst levels on days 6.5 and 7.5. Pyruvate was consumed during preimplantation stages, with glucose uptake undetectable until the blastocyst stage. Glucose was the main substrate consumed by the 6.5 and 7.5 day embryo. The proportions of glucose accounted for by lactate appearance were 81%, 86%, and 119% at blastocyst, day 6.5, and day 7.5 stages, respectively. The equivalent figures for glucose incorporated into glycogen were 10.36%, 0.21%, and 0.19%, respectively. The data are consistent with a switch from a metabolism dependent on aerobic respiration during early preimplantation stages to one dependent on both oxidative phosphorylation and aerobic glycolysis at the blastocyst stage, a pattern which is maintained on days 6.5 and 7.5. Our technique for measuring oxygen consumption may have diagnostic potential for selecting viable embryos for transfer following assisted conception techniques in man and domestic animals.

Oxygen uptake and carbohydrate metabolism by in vitro derived bovine embryos.

The consumption of oxygen, uptake of pyruvate and glucose and production of lactate were determined for groups of bovine embryos produced in vitro from the one-cell to the blastocyst stage (day 0-6 of culture). Measurements were made in Hepes-buffered synthetic oviduct fluid medium supplemented with 1.0 mmol pyruvate l-1, 10 mmol D,L-lactate l-1 and 1.5 mmol glucose l-1 and also 3 mg BSA ml-1 and, from day 5 of development, 10% (v/v) fetal calf serum. The amount of ATP production was determined from oxygen consumption and the proportion of glucose taken up that could be accounted for by lactate production. The data revealed that oxygen consumption was relatively constant from days 0-4 of culture (0.24-0.27 nl per embryo h-1), but increased with the initiation of compaction (0.39 nl per embryo h-1) and continued to increase with the formation and expansion of the blastocoel (0.9 nl per embryo h-1). Both pyruvate and glucose uptake followed similar patterns. Furthermore, when plotted against oxygen consumption, both pyruvate and glucose uptake increased significantly (P < 0.001) in a linear relationship (R2 = 0.61 and 0.49, respectively). Lactate production also increased with development and accounted for 40% of glucose uptake at day 0 of culture (putative zygotes), increasing to 70% by day 2 (eight-cell stage) and 100% of glucose uptake from day 4 of culture onwards. ATP production followed a similar pattern to that of oxygen consumption (60-85 pmol per embryo h-1 from day 0 to day 4) increasing with compaction (124 pmol per embryo h-1) and blastulation (221 pmol per embryo h-1). For precompaction stages, 93-96% of ATP production was derived from oxidative phosphorylation, decreasing to 82% with compaction. ATP produced by oxidative phosphorylation could be accounted for by the uptake of pyruvate, suggesting that bovine embryos produced in vitro utilize little endogenous substrates when appropriate exogenous substrates are present in the culture medium. The data revealed that bovine embryos were dependent on oxidative phosphorylation for energy (ATP) production at all stages of pre-elongation development, with perhaps a shift in dependence towards glycolysis in conjunction with compaction. It follows that oxidizable substrates, such as pyruvate and certain amino acids, are preferred in embryo culture medium during development in vitro.