Physiology and endocrinology symposium: Influence of cattle genotype (Bos indicus vs. Bos taurus) on oocyte and preimplantation embryo resistance to increased temperature

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

From cell death to embryo arrest: Mathematical models of human preimplantation embryo development

Human preimplantation embryos exhibit high levels of apoptotic cells and high rates of developmental arrest during the first week in vitro. The relation between the two is unclear and difficult to determine by conventional experimental approaches, partly because of limited numbers of embryos. We apply a mixture of experiment and mathematical modeling to show that observed levels of cell death can be reconciled with the high levels of embryo arrest seen in the human only if the developmental competence of embryos is already established at the zygote stage, and environmental factors merely modulate this. This suggests that research on improving in vitro fertilization success rates should move from its current concentration on optimizing culture media to focus more on the generation of a healthy zygote and on understanding the mechanisms that cause chromosomal and other abnormalities during early cleavage stages.

The preimplantation embryo:handle with care

The past decade has seen considerable advances in our understanding of intrinsic developmental mechanisms associated with gametogenesis and embryogenesis and accompanying applications in the fields of reproductive medicine, embryonic stem cell biology, and nuclear reprogramming. However, a new focus has recently emerged concerning the homeostatic regulation of embryonic cells, how this is set, and how it may influence the longitudinal progression and optimization of the developmental program and indeed the phenotype of the offspring. Attention has been drawn to the preimplantation stage of development as a sensitive "window" when in vitro and in vivo manipulations, such as culture conditions or maternal diet, may have critical consequences. In this article, we review how changes in environmental conditions, mediated via a range of epigenetic, cellular, and metabolic mechanisms in the preimplantation embryo, may alter the pattern of cell division, gene expression, morphology, and potential. We consider how fetal and postnatal phenotype may become susceptible to the plasticity of the preimplantation embryo and the risks for adult health and physiology. Copyright © 2008 by Thieme Medical Publishers, Inc.

Dynamics of DNA Methylation during Early Development of the Preimplantation Bovine Embryo

There is species divergence in control of DNA methylation during preimplantation development. The exact pattern of methylation in the bovine embryo has not been established nor has its regulation by gender or maternal signals that regulate development such as colony stimulating factor 2 (CSF2). Using immunofluorescent labeling with anti-5-methylcytosine and embryos produced with X-chromosome sorted sperm, it was demonstrated that methylation decreased from the 2-cell stage to the 6-8 cell stage and then increased thereafter up to the blastocyst stage. In a second experiment, embryos of specific genders were produced by fertilization with X- or Y-sorted sperm. The developmental pattern was similar to the first experiment, but there was stage × gender interaction. Methylation was greater for females at the 8-cell stage but greater for males at the blastocyst stage. Treatment with CSF2 had no effect on labeling for DNA methylation in blastocysts. Methylation was lower for inner cell mass cells (i.e., cells that did not label with anti-CDX2) than for trophectoderm (CDX2-positive). The possible role for DNMT3B in developmental changes in methylation was evaluated by determining gene expression and degree of methylation. Steady-state mRNA for DNMT3B decreased from the 2-cell stage to a nadir for D 5 embryos >16 cells and then increased at the blastocyst stage. High resolution melting analysis was used to assess methylation of a CpG rich region in an intronic region of DNMT3B. Methylation percent decreased between the 6-8 cell and the blastocyst stage but there was no difference in methylation between ICM and TE. Results indicate that DNA methylation undergoes dynamic changes during the preimplantation period in a manner that is dependent upon gender and cell lineage. Developmental changes in expression of DNMT3B are indicative of a possible role in changes in methylation. Moreover, DNMT3B itself appears to be under epigenetic control by methylation. © 2013 Dobbs et al.

HLA-G expression during preimplantation human embryo development.

HLA-G is a nonclassical class I major histocompatibility complex molecule with a restricted pattern of expression that includes the placental extravillus cytotrophoblast cells in direct contact with maternal tissues. Circumstantial evidence suggests that HLA-G may play a role in protection of the semiallogeneic human fetus. We examined whether HLA-G is expressed during the critical period of preimplantation human development and whether expression of this molecule could be correlated with the cleavage rate of embryos. Using reverse transcription PCR on surplus human embryos and unfertilized oocytes from patients undergoing in vitro fertilization we detected HLA-G heavy chain mRNA in 40% of 148 of blastocysts tested. The presence of HLA-G mRNA was also detected in unfertilized oocytes and in early embryos, but not in control cumulus oophorus cells. beta 2-Microglobulin mRNA was also found in those embryos expressing HLA-G. In concordance with our mRNA data, a similar proportion of embryos stained positive for HLA-G utilizing a specific monoclonal antibody. Interestingly, expression of HLA-G mRNA was associated with an increased cleavage rate, as compared to embryos lacking HLA-G transcript. Thus, HLA-G could be a functional homologue of the mouse Qa-2 antigen, which has been implicated in differences in the rate of preimplantation embryo development. To our knowledge, the presence of HLA-G mRNA and protein in human preimplantation embryos and oocytes has not been reported previously. The correlation of HLA-G mRNA expression with cleavage rate suggests that this molecule may play an important role in human pre-embryo development.

The preimplantation mouse embryo is a target for cannabinoid ligand-receptor signaling.

Using a reverse transcription-coupled PCR, we demonstrated that both brain and spleen type cannabinoid receptor (CB1-R and CB2-R, respectively) mRNAs are expressed in the preimplantation mouse embryo. The CB1-R mRNA expression was coincident with the activation of the embryonic genome late in the two-cell stage, whereas the CB2-R mRNA was present from the one-cell through the blastocyst stages. The major psychoactive component of marijuana (-)-delta-9-tetrahydrocannabinol [(-)-THC] inhibited forskolin-stimulated cAMP generation in the blastocyst, and this inhibition was prevented by pertussis toxin. However, the inactive cannabinoid cannabidiol (CBD) failed to influence this response. These results suggest that cannabinoid receptors in the embryo are coupled to inhibitory guanine nucleotide binding proteins. Further, the oviduct and uterus exhibited the enzymatic capacity to synthesize the putative endogenous cannabinoid ligand arachidonylethanolamide (anandamide). Synthetic and natural cannabinoid agonists [WIN 55,212-2, CP 55,940, (-)-THC, and anandamide], but not CBD or arachidonic acid, arrested the development of two-cell embryos primarily between the four-cell and eight-cell stages in vitro in a dose-dependent manner. Anandamide also interfered with the development of eight-cell embryos to blastocysts in culture. The autoradiographic studies readily detected binding of [3H]anandamide in embryos at all stages of development. Positive signals were present in one-cell embryos and all blastomeres of two-cell through four-cell embryos. However, most of the binding sites in eight-cell embryos and morulae were present in the outer cells. In the blastocyst, these signals were primarily localized in the mural trophectoderm with low levels of signals in the polar trophectoderm, while little or no signals were noted in inner cell mass cells.These results establish that the preimplantation mouse embryo is a target for cannabinoid ligands. Consequently, many of the adverse effects of cannabinoids observed during pregnancy could be mediated via these cannabinoid receptors. Although the physiological significance of the cannabinoid ligand-receptor signaling in normal preimplantation embryo development is not yet clear, the regulation of embryonic cAMP and/or Ca2+ levels via this signaling pathway may be important for normal embryonic development and/or implantation.

Insulin-like growth factor-1 protects preimplantation embryos from anti-developmental actions of menadione

Menadione is a naphthoquinone used as a vitamin K source in animal feed that can generate reactive oxygen species (ROS) and cause apoptosis. Here, we examined whether menadione reduces development of preimplantation bovine embryos in a ROS-dependent process and tested the hypothesis that actions of menadione would be reduced by insulin-like growth factor-1 (IGF-1). Menadione caused a concentration-dependent decrease in the proportion of embryos that became blastocysts. All concentrations tested (1, 2.5, and 5.0 mu M) inhibited development. Treatment with 100 ng/ml IGF-1 reduced the magnitude of the anti-developmental effects of the two lowest menadione concentrations. Menadione also caused a concentration-dependent increase in the percent of cells positive for the TUNEL reaction. The response was lower for IGF-1-treated embryos. The effects of menadione were mediated by ROS because (1) the anti-developmental effect of menadione was blocked by the antioxidants dithiothreitol and Trolox and (2) menadione caused an increase in ROS generation. Treatment with IGF-1 did not reduce ROS formation in menadione-treated embryos. In conclusion, concentrations of menadione as low as 1.0 mu M can compromise development of bovine preimplantation embryos to the blastocyst stage of development in a ROS-dependent mechanism. Anti-developmental actions of menadione can be blocked by IGF-1 through effects downstream of ROS generation.

Comparison of the efficacy of two commercially available media for culturing one-cell embryos in the in vitro fertilization mouse model

Objective: To examine the effects of two commercial media on the development of mouse ova fertilized in vitro to the blastocyst stage. Design: Animal model. Setting: Academic institution. Animal(s): Eight-week old, superovulated mice. Intervention(s): One-cell embryos cultured in vitro up to the blastocyst stage in potassium-enriched simplex optimized medium (KSOM) or G1/G2 medium. Main Outcome Measure(s): Blastocyst and hatching rates, total cell number count, and proportion of allocation of cells to the inner cell mass (ICM) and trophectoderm (TE). Result(s): The percentage of zygotes that developed to the blastocyst stage 96 and 120 hours after insemination was statistically significantly higher in the KSOM group. The percentage of blastocysts that partially or completely hatched by day 5 of culture was 84% and 71% for the KSOM and G1/G2 groups, respectively, showing a statistically significant difference between the groups. The mean number of ICM cells was 11.7 +/- 4.0 and 9.2 +/- 5.2 for the zygotes cultured in KSOM and G1/G2 media, respectively, revealing a statistically significantly higher cell number in the ICM of blastocysts derived from culture in KSOM medium. The ICM/TE ratio in the blastocysts cultured in KSOM or G1/G2 media was similar in both groups. Conclusion(s): Commercially available KSOM medium is superior to sequential G1/G2 media for culturing one-cell embryos up to the blastocyst stage in the mouse IVF model.

Avaliação da fertilidade e de possíveis defeitos estruturais externos sobre a prole de ratos machos expostos cronicamente ao arsênio

Arsenic is an environmental pollutant that influences the male reproductive function, affecting the spermatogenesis process in the testis and causing alterations in the sperm. The objective of this work was evaluate the reproductive efficiency in adult male rats exposed to arsenic and the possible male-mediated structural malformations. The animal were treated with sodium arsenato (3,6 mg/Kg, i.p.), twice a weak, for 15, 30, 45, 60 or 75 days whereas control received only distilled water. Results have showed a reduced pregnancy rate associated to a decrease in the fertility potencial and to a increase in the preimplantation loss. There was a decrease in the fetal weights, as showed by its average. Malformations rate, postimplantation loss and resorptions were unaffect by treatment. These results suggest that chronic exposure to arsenic provokes reduction on fertility of male rats. Further studies are required to clarify the arsenic effects on male reproductive system and male-mediated effects on progeny.

HDAC inhibition decreases XIST expression on female IVP bovine blastocysts

During initial development, both X chromosomes are active in females, and one of them must be silenced at the appropriate time in order to dosage compensate their gene expression levels to male counterparts. Silencing involves epigenetic mechanisms, including histone deacetylation. Major X chromosome inactivation (XCI) in bovine occurs between hatching and implantation, although in vitro culture conditions might disrupt the silencing process, increasing or decreasing X-linked gene expression. In this study, we aimed to address the roles of histone deacetylase inhibition by trichostatin A (TSA) on female preimplantation development.We tested the hypothesis that by enhancing histone acetylation, TSA would increase the percentage of embryos achieving 16-cell stage, reducing percentage of embryos blocked at 8-cell stage, and interfere with XCI in IVF embryos. We noticed that after TSA treatment, acetylation levels in individual blastomeres of 8-16 cell embryos were increased twofold on treated embryos, and the samewas detected for blastocysts. Changes among blastomere levels within the same embryo were diminished on TSA group, as low-acetylated blastomeres were no longer detected. The percentage of embryos that reached the 5th cleavage cycle 118 h after IVF, analyzed by Hoechst staining, remained unaltered after TSA treatment. Then, we assessed XIST and G6PD expression in individual female bovine blastocysts by quantitative real-time PCR. Even though G6PD expression remained unaltered after TSA exposure, XIST expression was eightfold decreased, and we also detected a major decrease in the percentage of blastocysts expressing detectable XIST levels after TSA treatment. Based on these results, we conclude that HDAC is involved on XCI process in bovine embryos, and its inhibition might delay X chromosome silencing and attenuate aberrant XIST expression described for IVF embryos. © 2013 Society for Reproduction and Fertility.

Canonical WNT signaling regulates development of bovine embryos to the blastocyst stage

Objectives were to evaluate the role of canonical WNT signaling in development of the preimplantation embryo. Signaling was activated with 2-Amino-4-(3,4-(methylenedioxy)benzylamino)-6-(3-methoxyphenyl)pyrimidine (AMBMP) and inhibited with Dickkopf-related protein 1 (DKK1). Treatment of bovine embryos with AMBMP at day 5 after insemination decreased development to the blastocyst stage at day 7 and reduced numbers of trophectoderm and inner cell mass cells. At high concentrations, AMBMP caused disorganization of the inner cell mass. DKK1 blocked actions of AMBMP but did not affect development in the absence of AMBMP. Examination of gene expression in day 6 morulae by microarray revealed expression of 16 WNT genes and other genes involved in WNT signaling; differences in relative expression were confirmed by PCR for 7 genes. In conclusion, the preimplantation embryo possesses a functional WNT signaling system and activation of the canonical pathway can inhibit embryonic development.

A influência do sistema nitrérgico no cultivo in vitro de embriões bovinos

O Óxido Nítrico (NO-) é uma molécula de sinalização celular que regula o desenvolvimento embrionário pré-implantacional. Nós investigamos o papel do NO- no cultivo de embriões bovinos produzidos in vitro, através do uso de N-Nitro-L-Arginina Metil Ester (L-NAME), um inibidor da produção de NO-, e L-arginina (ARG), um precursor de NO-, em diferentes períodos de cultivo (ativação do genoma embrionário e compactação). Foram avaliados seus efeitos sobre as taxas de desenvolvimento, cinética do desenvolvimento, qualidade embrionária, e expressão gênica. Os embriões foram produzidos por maturação e fertilização in vitro de oócitos aspirados de ovários provenientes de abatedouro frigorífico. No experimento 1, as taxas de desenvolvimento foram avaliadas em SOFaa na presença de L-NAME em diferentes períodos: do 1º ao 4º dia de cultivo (LN1-4), do 4º ao 8º (LN4-8) e do 1º ao 8º dia de cultivo (LN1-8). A inibição foi prejudicial a partir do 4º dia de cultivo (grupos LN4-8 e LN1-8), ao reduzir as taxas de eclosão (17,3%±13,44 e 13,7%± 14,51, respectivamente, p<0,05). Entretanto, o efeito mais negativo ocorreu do 1º ao 8º dia (LN1-8) em que a taxa de blastocisto foi significativamente menor comparada ao controle (29,4%±3,72 vs 47,8%±11,34, respectivamente, p<0,05). Por isso no experimento 2, a ARG (1, 10 e 50mM) foi adicionada desde o 1º dia de cultivo. As taxas de blastocisto usando 1 e 10mM de ARG foram similares ao controle (48%±13,03 e 34,2%±3,92 vs 49,4%±4,82, respectivamente, p>0,05), mas 50mM prejudicou a taxa de desenvolvimento embrionário (10,7%±7,24, p<0,001). No experimento 3, ARG a 1mM foi adicionada do 5º ao 8º dia de cultivo. Foram observadas taxas de desenvolvimento similares ao grupo GLN (somente com glutamina). Mas comparada ao controle (sem ambos os aminoácidos), rendeu melhores taxa de eclosão (54,8%±6,9 vs 41,4%±11,47, respectivamente, p<0,05) e qualidade embrionária (84,8%±2,63 vs 52%±8,62, respectivamente, p<0,05), mas não de taxa de blastocisto (49,4%±6,5 vs 49,4%±4,8, respectivamente, p>0,05). Neste período a produção de NO- foi positivamente correlacionada com a taxa de eclosão (R²=96,4%, p<0,001) e a qualidade embrionária (R²=75,5%, p<0,05). Adicionalmente, embriões foram cultivados na presença de L-NAME e ARG simultaneamente (grupo ARG/LN), do 5º ao 8º dia de cultivo, e os transcritos de OCT-4 e INT-t foram quantificados por PCR tempo real. Foi encontrada expressão similar de OCT-4 (p>0,05), mas redução de 1,8x e 1,5x de INT-t em relação aos grupos controles ARG e GLUT (p<0,05), respectivamente. Esses dados fornecem evidências da contribuição do NO-, principalmente no período entre os estágios de mórula e blastocisto, para a melhoria da eclosão e qualidade embrionária. A produção de NO- é requerida para o desenvolvimento pré-implantacional de embriões bovinos produzidos in vitro, e pode ser mediada pela suplementação do meio de cultivo com ARG.

Short and long-term repercussions of the experimental diabetes in embryofetal development

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Differential gene expression and developmental competence in in vitro produced bovine embryos

The embryonic developmental block occurs at the 8-cell stage in cattle and is characterized by a lengthening of the cell cycle and an increased number of embryos that stop development. The maternal-embryonic transition arises at the same stage resulting in the transcription of many genes. Gene expression studies during this stage may contribute to the understanding of the physiological mechanisms involved in the maternal-embryonic transition. Herein we identified genes differentially expressed between embryos with high or low developmental competence to reach the blastocyst stage using differential display PCR. Embryos were analysed according to developmental kinetics: fast cleavage embryos showing 8 cells at 48 h post insemination (hpi) with high potential of development (F8), and embryos with slow cleavage presenting 4 cells at 48 hpi (54) and 8 cells at 90 hpi (S8), both with reduced rates of development to blastocyst. The fluorescence DDPCR method was applied and allowed the recovery of 176 differentially expressed bands with similar proportion between high and low development potential groups (52% to F8 and 48% in S4 and S8 groups). A total of 27 isolated fragments were cloned and sequenced, confirming the expected primer sequences and allowing the identification of 27 gene transcripts. PI3KCA and ITM2B were chosen for relative quantification of mRNA using real-time PCR and showed a kinetic and a time-related pattern of expression respectively. The observed results suggest the existence of two different embryonic genome activation mechanisms: fast-developing embryos activate genes related to embryonic development, and slow-developing embryos activate genes related to cellular survival and/or death.