Preimplantation Embryogenesis Research Articles

Mitochondrial inhibition during preimplantation embryogenesis shifts the transcriptional profile of fetal mouse brain

Environmental stress results in perturbations to mitochondrial function in the preimplantation embryo and hinders subsequent embryo and possibly offspring development. Global gene expression in fetal mouse brain was investigated following targeted mitochondrial inhibition by amino-oxyacetate (AOA) from the 2-cell to the blastocyst stage. Blastocysts were transferred to pseudopregnant recipients and RNA extracted from Day 18 fetal brains for microarray interrogation. Exposure to 5 μM AOA during preimplantation embryo development induced differential expression of 166 genes (>1.25 fold) in the fetal brain, relative to control medium-cultured embryos. Altered expression pathways included carbohydrate metabolism, neurological development, cellular proliferation and death, DNA replication, recombination and repair. Of 28 genes exhibiting the greatest change in expression, qPCR confirmed that 16 were significantly altered. Targeted qPCR assessment of a further 20 genes associated with methylation, acetylation and mitochondrial dysfunction revealed that three were significantly altered (Immp1l, Nars2, Sat2) and Dmap1 exhibited a sex-specific response to AOA exposure. Only 2/48 genes had significantly altered expression by qPCR (Nola3, Timm8b) in fetal brains exposed to 50 μM AOA embryo culture, excluding an AOA dose-dependent response. It was concluded that perturbation of mitochondrial function induced by 5 μM AOA during preimplantation embryo development alters gene expression in the neonatal brain in a manner that suggests that proper brain development may be compromised.

Effect of vitrification and thawing on human oocyte ATP concentration

Vitrification/thawing has a significant negative impact on oocyte ATP concentration. However, incubating oocytes up to 180 minutes after thawing allowed oocytes to regain some ATP level.

Aneuploid Human Oocytes and Embryos Possess Significantly Reduced Quantities of Telomere DNA.

Aneuploidy represents the most prevalent form of genetic instability found in human embryos and is the leading cause of miscarriage and developmental delay in newborns. Although maternal age is a well known risk factor, the underlying molecular etiology of human aneuploidy is unknown. Since telomere DNA content is associated with genomic instability and is naturally reset during mammalian preimplantation embryonic development, telomere DNA quantity may play a role in the development of aneuploidy commonly found in human embryos. To begin to characterize the potential association of telomere biology with development of human aneuploidy, we have characterized relative telomere DNA quantity at various stages of human preimplantation embryogenesis. First, we developed a method capable of accurate quantitation of telomere DNA from single cells. Whole genome amplification (WGA) of picogram amounts of DNA from cells with previously established telomere DNA quantities provided results equivalent to those obtained using large quantities of purified total DNA (Pearson correlation coefficient=0.97). This technology was then applied to human oocytes and embryos. Excess WGA DNA was available for telomere DNA analysis of oocyte and embryo biopsies as a result of previously performing SNP microarray based 24 chromosome aneuploidy screening on human in vitro fertilization derived samples. Paired analysis of aneuploid and euploid polar bodies and embryonic cells from within the same patients' oocytes and embryos and within the same treatment cycle provided the greatest control over critical variables including maternal age. Embryo fragmentation rates, which were previously shown to correlate with telomere DNA length, weren't significantly different among aneuploid (9.2%) and euploid (9.7%) samples (P=0.75). Results demonstrated, for the first time, that aneuploid human polar bodies possess significantly less telomere DNA than euploid polar bodies (-3.07 fold, P=0.016). Since the first and second polar bodies are extruded upon completion of meiosis I and II, respectively, and reflect the chromosomal complement of the remaining oocyte, abnormalities observed in polar bodies are indicative of development of aneuploidy during meiosis of the oocyte. Therefore, the present results indicate that oocytes with reduced telomere DNA quantity are prone to development of aneuploidy during meiosis. We also show that aneuploid embryonic cells possess significantly less telomere DNA than euploid embryonic cells at the cleavage stage (-2.60 fold, P=0.002) but not at the blastocyst stage (-1.18 fold, P=0.340). These results indicate that reduced telomere length is associated with aneuploidy prior to embryonic genome activation between the cleavage and blastocyst stages of development. This further suggests that aneuploidy is not associated with failed resetting of telomere DNA length but is instead associated with failed telomere maintenance in the maternal genome during oogenesis and early embryo development. (platform)

Maternal phosphatidylinositol 3‐kinase signalling is crucial for embryonic genome activation and preimplantation embryogenesis

Maternal effect factors derived from oocytes are important for sustaining early embryonic development before the major wave of embryonic genome activation (EGA). In this study, we report a two-cell-stage arrest of embryos lacking maternal 3-phosphoinositide-dependent protein kinase 1 as a result of suppressed EGA. Concurrent deletion of maternal Pten completely rescued the suppressed EGA and embryonic progression through restored AKT signalling, which fully restored the fertility of double-mutant females. Our study identifies maternal phosphatidylinositol 3-kinase signalling as a new maternal effect factor that regulates EGA and preimplantation embryogenesis in mice.

Telomere DNA is significantly reduced in aneuploid human oocytes and embryos

OBJECTIVE: Since telomere DNA (telDNA) content is associated with genomic instability and is naturally reset during mammalian preimplantation embryogenesis, we tested the hypothesis that telomere DNA quantity is associated with the development of aneuploidy. DESIGN: Paired analysis of telDNA quantity in aneuploid and euploid human polar bodies and embryo biopsies. MATERIALS AND METHODS: A telDNA real-time PCR method was developed to evaluate single cells after whole genome amplification (WGA). Results from picogram amounts of DNA were compared to results obtained using large quantities of purified total DNA from the same cell lines. WGA DNA was available for analysis from oocyte (n=18) and embryo (n=44) biopsies that had undergone microarray based 24 chromosome aneuploidy screening. Paired analysis of sibling aneuploid and euploid polar bodies and embryonic cells from within the same patients' oocytes and embryos and within the same treatment cycle provided the greatest control over critical variables including maternal age. RESULTS: WGA of picograms of DNA from cells provided results equivalent to those obtained using large quantities of purified total DNA (Pearson R2=0.97). Embryo fragmentation rates, which were previously shown to correlate with telDNA length, weren't significantly different among aneuploid (9.2%) and euploid (9.7%) samples (P=0.75). Aneuploid human polar bodies displayed significantly less telDNA than euploid polar bodies (-3.07 fold, P=0.016). Aneuploid embryonic cells displayed significantly less telDNA than euploid embryonic cells at the cleavage stage (-2.60 fold, P=0.002) but not at the blast stage (-1.18 fold, P=0.340). CONCLUSION: These results indicate that reduced telomere length is associated with aneuploidy prior to embryonic genome activation. This further suggests that aneuploidy is not associated with resetting of telomere DNA length but is instead associated with telomere maintenance in the maternal genome during oogenesis and early embryo development.

Review: Role of tubal environment in preimplantation embryogenesis: application to co-culture assays

The culture of early preimplantation stage embryo is still delicate and the metabolic pathways of embryos are not completely understood. Embryo needs are evolutionary during the preimplantation development, consequently it is difficult to meet embryo needs in vitro. Culture conditions have to respect several physical and chemical equilibria: such as redox potential, pH, osmotic pressure, metabolic flux of energetic compounds, endogenous pools of amino acids and transcripts, etc. Embryo culture media are generally supplemented with amino acids, glucose, other energetic metabolites and antioxidant compounds, vitamin, and growth factors etc. Furthermore autocrine and paracrine regulation of embryo development probably exist. In fact embryo culture conditions have to be as non-toxic as possible. Various types of co-culture systems have been devised to overcome these problems. Complex interrelations exist between embryos and co-cultured cells. The beneficial effects of co-cultured cells may be due to continuous modifications of the culture medium, i.e. the elimination of toxic compounds and/or the supply of embryotrophic factors.

Stem cell factor/c-Kit signaling in in vitro cultures supports early mouse embryonic development by accelerating proliferation via a mechanism involving Akt-downstream genes

stem cell factor (SCF)/c-Kit regulates the proliferation and survival of germ cells or stem cells; however, little is known about the role of SCF/c-Kit in pre-implantation embryo development. using exogenous SCF supplementation and c-Kit siRNA injection, we investigated the role and mechanism of SCF/c-Kit in pre-implantation mouse embryos. addition of soluble SCF to the culture medium improved blastocyst formation. c-Kit gene silencing reduced the rate of blastocyst formation and delayed embryonic development. The number of proliferating cells in c-Kit gene-silenced blastocysts decreased, whereas the number of apoptotic cells in blastocysts obtained from both experimental and the control groups was not affected. RT-PCR, immunostaining and western blotting revealed that proliferation-related Akt downstream targets were substantially affected by c-Kit gene silencing. SCF/c-Kit signaling through Akt downstream targets is likely involved in mediating the cleavage and proliferation of blastomeres during mouse pre-implantation embryogenesis.

Characterization of Metastasis Associated Protein 3 (MTA3) in Mouse Oocytes and Pre-Implantation Embryos.

Metastasis associated protein 3 (MTA3) is a constituent of the Mi-2/nucleosome remodeling and deacetylase (Mi-2/NuRD) complex that regulates gene expression by altering chromatin structure, e.g., histone deacetylation. The biological function of MTA3 within the Mi-2/NuRD complex is unknown. An initial computational analysis of expressed sequence tags in the GenBank database revealed very high relative expression of MTA3 in mouse oocytes, fertilized eggs and blastocysts when compared to all other screened tissues. Because global chromatin reorganization is a critical aspect of preimplantation embryogenesis, we initiated studies to characterize MTA3 expression at this time of development. Immunohistochemical analysis of postnatal ovaries revealed that MTA3 is highly expressed in the germinal vesicle (GV) region of oocytes throughout postnatal development and in the nucleus of granulosa cells at all ovarian follicle stages. Surprisingly, we also found very high MTA3 expression in the cortical region of oocytes at all stages of follicular development. Confocal microscopic analysis of oocytes confirmed that MTA3 was present in the GV but that the perinuclear and cortical localization was much greater. During the progression of preimplantation development we observed nuclear localization that was most pronounced in the morula and blastocyst stages; however, the majority of MTA3 was in the cytoplasm where it localized to clusters of increasing size. Western blot analysis demonstrated that MTA3 protein expression in whole ovarian extracts gradually increased and reached its maximal level on postnatal day 19. MTA3 was also detected by immunoblotting of isolated GV oocytes and ovulated eggs. Although the amount of protein did not change, there was a shift in apparent size of MTA3 that suggested a post-translational protein modification occurs during oocyte maturation. Sequence analysis of isolated mRNA revealed that the predominant MTA3 variant in granulosa cells has 1679 nucleotides, whereas the predominant variant present in eggs and blastocysts has 1682 nucleotides because there is a 3 nucleotide insertion encoding a serine residue. This difference in the MTA3 protein sequence and its predominant expression in the cytoplasmic compartment suggest that it has functions in oocytes and preimplantation embryos in addition to supporting Mi-2/NuRD chromatin remodeling. (poster)

91 INHIBITION OF TELOMERASE REVERSE TRANSCRIPTASE (TERT) INDUCES EMBRYO ARREST IN BOVINE PRE-IMPLANTATION EMBRYOS

Previous studies from our laboratory have detected telomerase activity in embryos from all stages of early bovine development. However, the regulation of the telomerase subunits remains poorly understood. The objective of this study was to characterize the expression and function of the bovine telomerase reverse transcriptase (bTERT) subunit during bovine pre-implantation embryogenesis. Using RT-PCR and immunofluorescence staining procedures (n = 20 embryos at timed stages of development; r = 3), we demonstrate that mRNA transcripts and protein for bTERT were detected in pre-implantation bovine embryos from 1-cell to the blastocyst stage. The specificity of bTERT PCR products was confirmed by sequencing and exhibited 94% sequence homology to the human hTERT cDNA sequence. In immature oocytes, the bTERT protein was localized within the germinal vesicle and after 18 to 20 h of in vitro maturation, bTERT was observed as doublet foci co-localized with the condensed metaphase II meiotic stage chromosomes. Post-fertilization, the expression of bTERT was observed within the pronuclei. Through the initial cleavage stages, the expression of bTERT was variable, with some blastomeres showing punctate staining, whereas others exhibited a more-uniform staining pattern within the cytoplasm. By the 8- to 16-cell stage, the embryos demonstrated a peri-nuclear presence of bTERT. However, while in morulae and blastocysts, bTERT was localized to the nuclei as demonstrated by co-localization with 4′,6-diamidino-2-phenylindole (DAPI) staining. Treatment of zygotes with Telomerase Inhibitor III (telomere mimic that inhibits telomerase activity; Calbiochem, San Diego, CA) significantly induced embryos to permanently arrest in a senescence-like state at the 2- to 4-cell cleavage stage compared with nontreated controls. These arrested embryos showed abnormal nuclear phenotypes (elongated chromosomes, anaphase bridging) and phosphorylated histone γ-H2A.X foci suggesting a critical role(s) of TERT in chromosomal segregation and telomere integrity in early embryos. The translocation of bTERT protein from the cytoplasm to the nucleus in morulae supports the known telomere elongation event, whereas low levels of TERT at the 2- to 4-cell stage are associated with the previously documented state of permanent embryo arrest. Future studies would be directed toward understanding the relationship between TERT expression, telomere dysfunction, and permanent embryo arrest. Operating grants from the Canadian Institutes of Health Research (CIHR) to D.H.B. and W.A.K. supported this research.

Maternal depletion of NLRP5 blocks early embryogenesis in rhesus macaque monkeys (Macaca mulatta)

Genetic defects in the oocyte can lead to female infertility due to failures in early embryogenesis. NLRP5 (NLR family, Pyrin domain containing 5) is a maternal effect factor required for early embryo development beyond the 2-cell stage in mice. The NLRP5 gene is conserved in mammals. Applying RNA interference (RNAi) technique in the macaque oocyte, we studied the in vitro effect of NLRP5 in preimplantation embryo development in rhesus macaques. Macaque MII oocytes were collected and subjected to NLRP5 long double-stranded (ds) RNA microinjection, followed by intracytoplasmic sperm injection and in vitro embryo culture. NLRP5 mRNA and protein expression were examined, and the progress of preimplantation embryo development was monitored until the blastocyst stage. While 46.7 and 41.4% of embryos derived from uninjected and EGFP (Enhanced Green Fluorescence Protein) dsRNA-injected oocytes (control) reached the blastocyst stage within 10 days of culture, respectively, only 7.0% of the NLRP5 knockdown embryos developed to term. The majority of NLRP5 knockdown embryos were arrested between the 8-cell and the 16-cell stages. The work provides the first evidence that long dsRNA-mediated RNAi can be applied to specific gene silencing in rhesus macaque oocytes, and demonstrates that maternal NLRP5 plays an important role in preimplantation embryogenesis in non-human primates.

The hyaluronan binding assay HBA™: a novel and statistically significant predictor of clinical pregnancy (CP) in in vitro fertilization (IVF) and assisted reproductive technology (ART)

OBJECTIVE: Established andrology assays assist in directing the clinician towards the selection of the optimal ART for each couple facing infertility. The HBA assay assesses the ability of the seminal population to bind to an immobilized substrate of hyaluronan. H-bound (HB) sperm carry reduced levels of chromosomal aberrations and enhanced levels of developmental maturity. This relationship led to the current study examining sperm binding potential, descriptive parameters of the male and female partners and their individual contribution to IVF outcome. The study sought to explore the potential association between the HBA index, morphology (M), total number of motile sperm (MS), ART treatment, age, follicle stimulating hormone (FSH) level, number of dominant follicles (DF) and the likelihood of CP.DESIGN: A retrospective analysis of 253 infertility couples examined each parameter as to its contribution to a positive CP. Data collection followed an IRB-approved protocol. The sample consisted of patients requiring intracytoplasmic sperm injection (ICSI) (n=84), IVF (n=93) and intrauterine insemination (IUI) (n=76).MATERIALS AND METHODS: The HBA score, M, MS, ART treatment, age, FSH and DF were the independent variables (IV) recorded for each patient. The HBA score was calculated as # bound MS/# total MS. With CP as the dependent variable, all IV were entered into a logistic regression model (LRM). Descriptive analyses included ANOVA and Pearson's χ 2 (chi square) using SPSS 15.0.RESULTS: Overall, 11.8% of IUI, 68% of IVF and 72.6% of ICSI patients demonstrated a CP. There was a statistically significant difference in mean HBA scores of IUI and IVF patients (79.87, 78.58, respectively (resp.)) as compared to ICSI patients (62.26, p<0.001). The LRM showed that the HBA score, female age and ART method had a statistically significant role in contributing to CP (p=.003, p=.001, p<.001, resp.). Male age, MS, M and FSH level showed no statistical significance (p>0.05) in contributing to CP.CONCLUSIONS: The use of the HBA index in the evaluation of the infertile couple merits strong consideration in the selection of the most appropriate ART to achieve a CP. The statistically significant value of the HBA index in predicting CP suggests the influence HB-sperm exert on preimplantation embryogenesis. Use of refined criteria for the HBA index may better direct the ART technologies selected for infertile patients thus optimizing CP and improving patient care. OBJECTIVE: Established andrology assays assist in directing the clinician towards the selection of the optimal ART for each couple facing infertility. The HBA assay assesses the ability of the seminal population to bind to an immobilized substrate of hyaluronan. H-bound (HB) sperm carry reduced levels of chromosomal aberrations and enhanced levels of developmental maturity. This relationship led to the current study examining sperm binding potential, descriptive parameters of the male and female partners and their individual contribution to IVF outcome. The study sought to explore the potential association between the HBA index, morphology (M), total number of motile sperm (MS), ART treatment, age, follicle stimulating hormone (FSH) level, number of dominant follicles (DF) and the likelihood of CP. DESIGN: A retrospective analysis of 253 infertility couples examined each parameter as to its contribution to a positive CP. Data collection followed an IRB-approved protocol. The sample consisted of patients requiring intracytoplasmic sperm injection (ICSI) (n=84), IVF (n=93) and intrauterine insemination (IUI) (n=76). MATERIALS AND METHODS: The HBA score, M, MS, ART treatment, age, FSH and DF were the independent variables (IV) recorded for each patient. The HBA score was calculated as # bound MS/# total MS. With CP as the dependent variable, all IV were entered into a logistic regression model (LRM). Descriptive analyses included ANOVA and Pearson's χ 2 (chi square) using SPSS 15.0. RESULTS: Overall, 11.8% of IUI, 68% of IVF and 72.6% of ICSI patients demonstrated a CP. There was a statistically significant difference in mean HBA scores of IUI and IVF patients (79.87, 78.58, respectively (resp.)) as compared to ICSI patients (62.26, p<0.001). The LRM showed that the HBA score, female age and ART method had a statistically significant role in contributing to CP (p=.003, p=.001, p<.001, resp.). Male age, MS, M and FSH level showed no statistical significance (p>0.05) in contributing to CP. CONCLUSIONS: The use of the HBA index in the evaluation of the infertile couple merits strong consideration in the selection of the most appropriate ART to achieve a CP. The statistically significant value of the HBA index in predicting CP suggests the influence HB-sperm exert on preimplantation embryogenesis. Use of refined criteria for the HBA index may better direct the ART technologies selected for infertile patients thus optimizing CP and improving patient care.

Identification of oocyte‐selective NLRP genes in rhesus macaque monkeys (Macaca mulatta)

Oocyte-selective genes control multiple aspects of female gamete development and preimplantation embryogenesis. Several key oocyte-selective factors have been identified in mice recently; however, these factors are not well documented in more advanced species such as nonhuman primates. One of such oocyte-selective factors is NLRP5 (NLR family, Pyrin domain containing 5), also known as Maternal Antigen That Embryos Require (MATER), which is required for preimplantation embryo development beyond the 2-cell stage in mice. Human NLRP family contains 14 members. We identified 14 NLRP gene homologues and examined their spatial and temporal expression in rhesus macaque monkeys (Macaca mulatta). While all 14 NLRP genes are detectable in the macaque gonad, eight of them (NLRP2, 4, 5, 8, 9, 11, 13, and 14) are specifically or preferentially expressed in the ovary. In situ hybridization elucidated a specific oocyte expression pattern of the eight NLRP genes within the ovary. During the oocyte-to-embryo transition, seven of these oocyte-selective NLRP transcripts (excluding NLPR2) are enriched in maturing oocytes and early preimplantation embryos but diminish upon embryo genome activation, indicating an exclusive maternal origin of these transcripts. Though functionally unknown, the spatial and temporal distribution of these oocyte-selective NLRP genes implies important roles of the NLRP family in oogenesis and early embryo development in nonhuman primates.

Histone Deacetylase 1 Regulates Gene Expression During Mouse Preimplantation Embryogenesis.

Superimposed on the activation of embryonic genome in the preimplantation mouse embryo is the formation of a transcriptionally repressive state during the late two-cell stage. Several lines of evidence suggest that histone deacetylases (HDAC) may underlie the development of the transcriptionally repressive state during zygotic gene activation (ZGA) by mediating changes in the chromatin structure. In this study, we investigated expression and possible functions of Class I HDACs HDAC1, HDAC2, and HDAC3 during mouse preimplantation embryogenesis. We show that HDAC1 is likely a major deacetylase and its expression inversely correlates with changes of histone H4K5 acetylation state. RNAi-mediated reduction of HDAC1 leads to hyperacetylation of histone H4 and inhibited the incidence of development to the blastocyst stage even though expression of Hdac2 and Hdac3 is significantly induced in Hdac1-suppresssed embryos. RNAi-mediated reduction of HDAC2 has no noticeable effect on preimplantation development, suggesting that individual HDACs have distinct functions during preimplantation development. We also demonstrate that HDAC1 knockdown results in elevated levels of expression of a subset of genes that are normally repressed following genome activation and that this expression correlates with hyperacetylation of histone H4. Results of these experiments suggest that HDAC1 is involved in the development of a transcriptionally repressive state that initiates in 2-cell embryos.

On the Horizon: Nonsteroidal Contraceptive Targets in the Female Ovary and Reproductive Tract.

Half of all unintended pregnancies in the United States occur among contraceptive users. There is an urgent need for non-coitally related methods that are free of undesirable hormone-related side effects to control fertility. Novel contraceptive agents could be targeted to specific sites in the female reproductive tract to alter gamete (inhibit oocyte maturation, fertilization), ovarian (suppress follicle rupture, oocyte release) and uterine (prevent implantation) function to prevent pregnancy. During the gonadotropin surge, cAMP levels rise in granulosa cells and decrease in the oocyte prior to the resumption of meiosis. Hydrolysis of cAMP is achieved intracellularly via phosphodiesterases (PDE). PDE4 is expressed in granulosa cells, while PDE3 exists in the oocyte. This compartmentalization suggests a novel contraceptive strategy wherein selective inhibition of PDE3 maintains elevated cAMP levels and prevents meiotic maturation of the oocyte without affecting follicle rupture, a concept demonstrated in rodents and recently applied to primates. Immature, GV-intact macaque oocytes obtained from large pre-ovulatory follicles during gonadotropin-stimultated cycles in the absence of an ovulatory hCG bolus were exposed in vitro to various doses of PDE3 inhibitors or a selective PDE4 inhibitor. Resumption of meiosis was specifically blocked by PDE3 inhibitors, but not the PDE4 inhibitor. To determine whether PDE3 inhibitors prevent oocyte maturation in vivo, oocytes were collected after an ovulatory stimulus (hCG) from macaques either receiving no further treatment (control) or the PDE3 inhibitor, ORG 9935 (NV Organon) during gonadotropin-stimulated and natural menstrual cycles. In gondaotropin-stimulated cycles, 85% of oocytes from controls were mature compared to 13% from treated animals. In natural cycles, all oocytes from controls resumed meiosis, whereas 83% from treated animals were GV-intact. Although 60% of GV oocytes from treated animals progressed to metaphase I during culture, none fertilized in vitro compared with 67% fertilization in controls. Thus, the ability of PDE3 inhibitors to block primate oocyte maturation in vitro and in vivo suggests a potential clinical use as contraceptives in women. Very low doses of the antiprogestin, ZK 137 316 (Schering AG), permit ovarian and menstrual cyclicity when administered daily for up to 6 months in rhesus monkeys, but disrupt endometrial growth. This antiprogestin regimen was 100% effective in preventing pregnancy in female macaques during cohabitation with males. The contraceptive efficacy and reversiblility with respect to subsequent fertility was tested in macaques receiving vehicle or antiprogestin daily for 1 year. Antiprogestin-treated females cohabitating with males did not become pregnant. After 1 year, live offspring were born in 80% and 60% of females previously treated with vehicle or antiprogestin, respectively. Antiprogestin exposure during follicular development in macaques altered oocyte functions necessary for normal preimplantation embryogenesis that may contribute to pregnancy prevention. Thus, chronic, low dose antiprogestin permits continued ovarian cyclicity and prevents pregnancy in primates. The rapid reversal of anti-fertility effects and live births after antiprogestin treatment increases confidence in the feasibility of this regimen as a potential contraceptive in women. HD18185, HD031398, RR00163, HD01243-03 (JJ), HD042710 (JJ), HD31633 (MZ)

The environmental toxicant 2,3,7,8-tetrachlorodibenzo-p-dioxin disrupts morphogenesis of the rat pre-implantation embryo

BackgroundEnvironmental toxicants, whose actions are often mediated through the aryl hydrocarbon receptor (AhR) pathway, pose risks to the health and well-being of exposed species, including humans. Of particular concern are exposures during the earliest stages of development that while failing to abrogate embryogenesis, may have long term effects on newborns or adults. The purpose of this study was to evaluate the effect of maternal exposure to the AhR-specific ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on the development of rat pre-implantation embryos with respect to nuclear and cytoskeletal architecture and cell lineage allocation.ResultsWe performed a systematic 3 dimensional (3D) confocal microscopy analysis of rat pre-implantation embryos following maternal exposure to environmentally relevant doses of TCDD. Both chronic (50 ng/kg/wk for 3 months) and acute (50 ng/kg and 1 μg/kg at proestrus) maternal TCDD exposure disrupted morphogenesis at the compaction stage (8–16 cell), with defects including monopolar spindle formation, f-actin capping and fragmentation due to aberrant cytokinesis. Additionally, the size, shape and position of nuclei were modified in compaction stage pre-implantation embryos collected from treated animals. Notably, maternal TCDD exposure did not compromise survival to blastocyst, which with the exception of nuclear shape, were morphologically similar to control blastocysts.ConclusionWe have identified the compaction stage of pre-implantation embryogenesis as critically sensitive to the effects of TCDD, while survival to the blastocyst stage is not compromised. To the best of our knowledge this is the first in vivo study to demonstrate a critical window of pre-implantation mammalian development that is vulnerable to disruption by an AhR ligand at environmentally relevant doses.

Putative Role of Hyaluronan and Its Related Genes,HAS2andRHAMM, in Human Early Preimplantation Embryogenesis and Embryonic Stem Cell Characterization

Human embryonic stem cells (hESC) promise tremendous potential as a developmental and cell therapeutic tool. The combined effort of stimulatory and inhibitory signals regulating gene expression, which drives the tissue differentiation and morphogenetic processes during early embryogenesis, is still very poorly understood. With the scarcity of availability of human embryos for research, hESC can be used as an alternative source to study the early human embryogenesis. Hyaluronan (HA), a simple hydrating sugar, is present abundantly in the female reproductive tract during fertilization, embryo growth, and implantation and plays an important role in early development of the mammalian embryo. HA and its binding protein RHAMM regulate various cellular and hydrodynamic processes from cell migration, proliferation, and signaling to regulation of gene expression, cell differentiation, morphogenesis, and metastasis via both extracellular and intracellular pathways. In this study, we show for the first time that HA synthase gene HAS2 and its binding receptor RHAMM are differentially expressed during all stages of preimplantation human embryos and hESC. RHAMM expression is significantly downregulated during differentiation of hESC, in contrast to HAS2, which is significantly upregulated. Most importantly, RHAMM knockdown results in downregulation of several pluripotency markers in hESC, induction of early extraembryonic lineages, loss of cell viability, and changes in hESC cycle. These data therefore highlight an important role for RHAMM in maintenance of hESC pluripotency, viability, and cell cycle control. Interestingly, HAS2 knockdown results in suppression of hESC differentiation without affecting hESC pluripotency. This suggests an intrinsic role for HAS2 in hESC differentiation process. In accordance with this, addition of exogenous HA to the differentiation medium enhances hESC differentiation to mesodermal and cardiac lineages. Disclosure of potential conflicts of interest is found at the end of this article.

Dissection of the high velocity air control (HVAC) system serving the in vitro fertilization (IVF) laboratory: the impact of ultraviolet-c band (UVC) irradiation on clinical pregnancy rates (CPR)

Successful preimplantation embryogenesis (pe) is critically dependent upon the culture environment and changing organic chemistry of the ambient air (aa) provided by the IVF laboratory. The HVAC system serving the IVF laboratory plays a key role in controlling aa quality. Its design and maintenance is critical as it can serve as both the source of and perfect conduit for the spread of embryotoxic bioaersols and microorganisms. Placement of UVC light sources within the HVAC system will kill pathogens threatening the aa.

PICSI™ vs. ICSI: statistically significant improvement in clinical outcomes in 240 in vitro fertilization (IVF) patients

The in vitro selection of sperm for intracytoplasmic sperm injection (ICSI) is critical and directly influences the paternal contribution to preimplantation embryogenesis. Hyaluronan (H), a major constituent of the cumulus matrix, may play a critical role in the selection of functionally competent sperm during in vivo fertilization. H-bound (HB) sperm carry increased levels of developmental maturity and sperm chromatin integrity. The relationship between HB-sperm and potentially enhanced levels of functional competence led to the current study examining the use of HB-sperm in the treatment of ICSI patients.

Dynamic changes in the localization of five members of the methyl binding domain (MBD) gene family during murine and bovine preimplantation embryo development

There are five methyl binding domain (MBD) proteins characterized by a methyl CpG-binding domain. Four MBD proteins (MeCP2 and MBDs 1-3) are linked to transcriptional repression and one (MBD4), to DNA repair. During preimplantation development, the embryo undergoes global demethylation following fertilization and selective remethylation following the maternal to zygotic transition (MZT). This study characterized changes in MBD mRNA expression and protein localization during both murine and bovine preimplantation development. These species were selected because they undergo MZT at different developmental stages. Gene expression profiling during preimplantation development detected the presence of all MBDs examined, although stage and species-specific differences were observed. MBD2 was not expressed in murine or bovine oocytes and MeCP2 was not detected in murine blastocysts, subcellular protein localization was found to vary at time points critical in development. Most MBDs showed species-specificity in localization patterns and differences were found between individual MBDs. MBD1 localization is consistent with a novel role during MZT for both species. MBD3, known to play a crucial role in murine embryogenesis, was highly localized to the nucleus before and after, but not during the MZT in the bovine. MBD2, MBD4, and MeCP2 show varying patterns of localization which indicate possible roles in the early cleavage stages and in inner cell mass differentiation. Further experiments are currently underway to define discreet functional roles for specific MBDs during bovine preimplantation embryogenesis.

The kinesin related motor protein, Eg5, is essential for maintenance of pre-implantation embryogenesis

Eg5 is a plus end directed kinesin related motor protein (KRP) previously shown to be involved in the assembly and maintenance of the mitotic spindle. KRPs are molecular motors capable of generating forces upon microtubules (MTs) in dividing cells and driving structural rearrangements necessary in the developing spindle. In vitro experiments demonstrate that loss of Eg5 results in cell cycle arrest and defective centrosome separation resulting in the development of monopolar spindles. Here we describe mice with a genetrap insertion in Eg5. Heterozygous mutant mice appear phenotypically normal. In contrast, embryos homozygous for the Eg5 null allele recovered at embryonic days 2.5–3.5 display signs of a proliferation defect as reduced cell numbers and failure of compaction and progression to the blastocyst stage was observed. These data, in conjunction with previous in vitro data, suggest that loss of Eg5 results in abnormal spindle structure, cell cycle arrest and thereby reduced cell proliferation of early cleavage pre-implantation embryos. These observations further support the conclusion that Eg5 is essential for cell division early in mouse development, and that maternal contribution may sustain the embryo through the maternal to zygotic transition at which point supplies of functional Eg5 are exhausted, preventing further cell cleavage.