The sensitivity of whole,half and quarter sea urchin embryos to cytotoxic neuropharmacological drugs

1. Embryos of sea urchins Echinocardium cordatum, Echinus melo and E. esculentus are supersensitive to the same cytotoxic neurochemicals as Arbacia lixula embryos studied earlier.2. White half and clear quarter embryos of A. lixula have no obvious supersensitivity to neurochemicals.3. Supersensitivity is restored in granular quarter embryos up to the initial level, and it does not disappear in the red halves and in corresponding quarter embryos.4. There is a non-obligatory coupling between the supersensitivity receptors and cell division processes.     

Differential integrin expression in pre-implantation embryos developing under in vivo and in vitro conditions

Implantation failure is a major problem in human assisted reproduction, which persists regardless the optimization of endometrial receptivity and selection of genetically and morphologically healthy embryos. Since embryo-endometrium interaction depends on cell junctional, cell adhesion and cell-substratum adhesion molecules, the present study inquired whether in vitro growing murine embryos display similar to the in vivo growing embryos patterns of adhesion molecules. To this extend aVb3 expression and distribution in zygotes and 2-cell stage embryos were studied. The results demonstrated that only the in vivo growing embryos displayed specifically polarized aVb3 distribution, indicating their potential successful interaction with endometrium. Based on previous studies showing that L-carnitine (L-Cn) could affect embryonic development, it was demonstrated that the addition of L-Cn to the culture medium, could lead the in vitro growing embryos to acquire aVb3 expression and distribution similar to the in vivo growing embryos. Visualization of the effect of L-Cn using third harmonic generation imaging showed decreased lipid droplet levels in 2-cell-stage embryos, observation that correlates with an active energetic state of the growing embryos. Thus, the application of L-Cn to the culture medium could assist pre-implantation-state embryos to acquire aVb3 expression and distribution similar to the in vivo developing conditions.     

Expression analysis of the NLRP gene family suggests a role in human preimplantation development

Background

The NLRP (Nucleotide-binding oligomerization domain, Leucine rich Repeat and Pyrin domain containing) family, also referred to as NALP family, is well known for its roles in apoptosis and inflammation. Several NLRPs have been indicated as being involved in reproduction as well.

Methodology

We studied, using the unique human gametes and embryo materials, the expression of the NLRP family in human gametes and preimplantation embryos at different developmental stages, and compared the expression levels between normal and abnormal embryos using real-time PCR.

Principal Findings

Among 14 members of the NLRP family, twelve were detected in human oocytes and preimplantation embryos, whereas seven were detected in spermatozoa. Eight NLRPs (NLRP4, 5, 8, 9, 11, 12, 13, and 14) showed a similar expression pattern: their expression levels were high in oocytes and then decreased progressively in embryos, resulting in a very low level in day 5 embryos. However, NLRP2 and NLRP7 showed a different expression pattern: their expression decreased from oocytes to the lowest level by day 3, but increased again by day 5. The expression levels of NLRP5, 9, and 12 were lower in day 1 abnormal embryos but higher in day3 and day5 arrested embryos, when compared with normal embryos at the same stages. NLRP7 was down-regulated in day 1 and day 5 abnormal embryos but over-expressed in day3 arrested embryos.

Conclusions

According to our results, different NLRPs possibly work in a stage-dependent manner during human preimplantation development.     

小鼠孤雌胚与体内正常胚H3K27三甲基化模式的差异

为了考察小鼠(Mus musculus)孤雌激活胚胎H3K27三甲基化模式与体内正常胚胎之间的差异,以及曲古抑菌素A(TSA)对孤雌胚H3K27三甲基化水平的影响,探究表观遗传修饰对孤雌胚胎发育的作用。首先,用H3K27me3特异性抗体对MⅡ期卵母细胞染色,利用激光共聚焦对其荧光强度进行检测,结果发现该时期的甲基化荧光强度相对较低。接着,采用同样的方法对小鼠孤雌胚胎和体内正常胚胎植入前各时期的H3K27me3模式进行比较,结果显示,从2-细胞到囊胚期孤雌组呈现逐渐升高的趋势,与体内组变化趋势完全相反,且总体平均荧光强度较体内组普遍偏低。孤雌胚胎经TSA处理后,处理组和未处理组在前三个时期虽然没有显著性差异(P0.05),但是处理之后的H3K27三甲基化水平有所提高,囊胚期与未处理组相比有显著性差异(P0.05)。以上结果表明,小鼠孤雌胚胎的H3K27三甲基化模式与体内胚胎之间存在着巨大的差异,这可能是造成孤雌胚胎发育能力差的重要原因之一。TSA处理对H3K27me3模式造成了一定的影响,使体外培养环境有所改善,这可能对提高孤雌胚胎发育能力具有一定的意义。     

Genome editing with RNA-guided Cas9 nuclease in Zebrafish embryos

Recent advances with the type II clustered regularly interspaced short palindromic repeats (CRISPR) system promise an improved approach to genome editing. However, the applicability and efficiency of this system in model organisms, such as zebrafish, are little studied. Here, we report that RNA-guided Cas9 nuclease efficiently facilitates genome editing in both mammalian cells and zebrafish embryos in a simple and robust manner. Over 35% of site-specific somatic mutations were found when specific Cas/gRNA was used to target either etsrp, gata4 or gata5 in zebrafish embryos in vivo. The Cas9/gRNA efficiently induced biallelic conversion of etsrp or gata5 in the resulting somatic cells, recapitulating their respective vessel phenotypes in etsrp^y11 mutant embryos or cardia bifida phenotypes in fau^tm236a mutant embryos. Finally, we successfully achieved site-specific insertion of mloxP sequence induced by Cas9/gRNA system in zebrafish embryos. These results demonstrate that the Cas9/gRNA system has the potential of becoming a simple, robust and efficient reverse genetic tool for zebrafish and other model organisms. Together with other genome-engineering technologies, the Cas9 system is promising for applications in biology, agriculture, environmental studies and medicine.     

Sequence of Reactivation of Ribonucleic Acid Synthesis during Early Germination of the Maize Embryo

The onset of RNA synthesis in primary roots of germinating Zea mays embryos was studied. Incubation of excised embryos in the radioactive precursor solution was performed after different germination periods.     

The rate of oxygen consumption during embryogenesis of Lymnaea stagnalis (Gastropoda)

We studied the rate of oxygen consumption by the Lymnaea stagnalis embryos. The rate of oxygen consumption increased consistently during embryogenesis. The volume specific rate of oxygen consumption increased initially from the early cleavage stages until the gastrula stage and then decreased gradually to the eclosion of snails. There are three periods in embryogenesis of L. stagnalis, which differ in the coefficients of allometric dependence between the rate of oxygen consumption and volume of embryos: (1) early embryogenesis, when the increase in the rate of oxygen consumption is not accompanied by the growth of volume of the embryos; (2) larval period (trochophore and veliger stages; exponential coefficient k = 0.514), and (3) postlarval period (exponential coefficient k = 0.206).     

Expression of Maternally and Embryonically Derived Hypoxanthine Phosphoribosyl Transferase (Hprt) Activity in Mouse Eggs and Early Embryos

X-chromosome activity in early mouse development has been studied by a gene dosage method that involves measuring the activity level of the X-linked enzyme hypoxanthine phosphoribosyl transferase (HPRT) in single eggs and embryos from XO females and from females heterozygous for In(X)1H, a paracentric inversion of the X chromosome. The HPRT activity in oocytes increased threefold over a 24-hr period beginning after ovulation. Afterward, the activity plateaued in unfertilized eggs but continued to increase for at least 66 hr in presumed OY embryos. Both before and after ovulation, the level of activity in unfertilized eggs from In(X)/X females was twice that from XO females, and the distributions of activity in eggs for both sets of females remained unimodal. Beginning with the two-cell stage, distributions of activity for embryos from In(X)/X females were trimodal, which is evidence for embryonic activity. It is proposed that activation of a maternal mRNA or proenzyme is responsible for the HPRT activity increase in oocytes and early embryos and is supplemented by dosage-dependent activity of the embryonic Hprt gene as early as the two-cell stage.     

The reduced folate carrier-1 (RFC1 696T>C) polymorphism is associated with spontaneously aborted embryos in Koreans

The major cause of early spontaneous abortion is believed to be chromosomal abnormality. However, the genetic etiologies of spontaneous abortion are still unknown. A central feature of fetal development is widespread rapid cell division. Due to its role in DNA synthesis, the need for folate increases during periods of rapid fetal growth. Folate transport across cell membranes is mediated by reduced folate carrier-1 (RFC1). Variants within SLC19A1 may influence folate and homocysteine concentrations. The aim of this study was to investigate the association of RFC1 mutations with spontaneous abortion in aborted embryos. We studied 115 spontaneously aborted embryos at <20 weeks of gestational age, 102 child controls, and 353 adult controls. The genotype frequencies of RFC1 polymorphisms, 80A>G and 696T>C, in spontaneously aborted embryos were measured. The RFC1 696T>C mutation was significantly increased in spontaneously aborted embryos compared to child controls. Further studies will be required to examine the functional significance of the RFC1 696T>C polymorphism.     

Specific control of messenger translation in Drosophila oocytes and embryos

The distribution of messenger RNA between polysomes and mRNP in oocytes and embryos of Drosophila melanogaster has been studied by in vitro translational analysis. Poly(A)⁺ RNA was purified from polysomes or mRNA from mature oocytes and young embryos. The messenger populations were translated in vitro and the peptides synthesized were separated by two-dimensional electrophoresis. Analysis of the 2D gel patterns enabled the detection of three peptides coded by messengers present predominantly in the mRNA pools of mature oocytes. When DNA-binding peptides were selected from the in vitro translation products, they showed, after separation by two-dimensional electrophoresis, less than 100 spots. The analysis of the 2D gels indicated that three DNA-binding peptides are coded by messengers present only in the mRNP of the oocytes. These messengers are later found in the polysomal fraction of embryos.     

NDR Kinases Are Essential for Somitogenesis and Cardiac Looping during Mouse Embryonic Development

Studies of mammalian tissue culture cells indicate that the conserved and distinct NDR isoforms, NDR1 and NDR2, play essential cell biological roles. However, mice lacking either Ndr1 or Ndr2 alone develop normally. Here, we studied the physiological consequences of inactivating both NDR1 and NDR2 in mice, showing that the lack of both Ndr1/Ndr2 (called Ndr1/2-double null mutants) causes embryonic lethality. In support of compensatory roles for NDR1 and NDR2, total protein and activating phosphorylation levels of the remaining NDR isoform were elevated in mice lacking either Ndr1 or Ndr2. Mice retaining one single wild-type Ndr allele were viable and fertile. Ndr1/2-double null embryos displayed multiple phenotypes causing a developmental delay from embryonic day E8.5 onwards. While NDR kinases are not required for notochord formation, the somites of Ndr1/2-double null embryos were smaller, irregularly shaped and unevenly spaced along the anterior-posterior axis. Genes implicated in somitogenesis were down-regulated and the normally symmetric expression of Lunatic fringe, a component of the Notch pathway, showed a left-right bias in the last forming somite in 50% of all Ndr1/2-double null embryos. In addition, Ndr1/2-double null embryos developed a heart defect that manifests itself as pericardial edemas, obstructed heart tubes and arrest of cardiac looping. The resulting cardiac insufficiency is the likely cause of the lethality of Ndr1/2-double null embryos around E10. Taken together, we show that NDR kinases compensate for each other in vivo in mouse embryos, explaining why mice deficient for either Ndr1 or Ndr2 are viable. Ndr1/2-double null embryos show defects in somitogenesis and cardiac looping, which reveals their essential functions and shows that the NDR kinases are critically required during the early phase of organogenesis.     

The cell cycle and retinal histogenesis fidget mutant mice.

The autoradiographic method using [³H]thymidine has shown that the autosomal recessive mutant gene fidget (gene symbol fi) prolonging the presynthetic period of the cell cycle in the retinal anlage in homozygotes retards the transition of retinal cells to the differentiated state. Some retinal cells of normal embryos (+/+) start their transition to the differentiated state on the 11th day of embryogenesis, while in $\text{fi}\text{fi}$ embryos this process starts only on the 12th day. An active transition of retinal cells to the differentiated state especially in the peripheral zone of the mutant retina takes place 2 days later as compared to normal embryos. The number of differentiating cells in the retina of mutants at the stages of development studied is considerably lower as compared to the norm. The analysis of the cell cycle parameters in 15-day embryos has shown that in the mutants the retina is less mature as compared to +/+ embryos. The sequence of transition of various cell types to the differentiated state in the retina of $\text{fi}\text{fi}$ embryos is the same as in the norm. Gene fidget seems to interfere with proliferative rather than critical (quantal) cell cycles in the developing mouse retina.     

Inbreeding effects on in vitro embryo production traits in Guzerá cattle

Inbreeding has been associated with the impairment of reproductive performance in many cattle breeds. Although the usage of reproductive biotechnologies has been increasing in bovine populations, not much attention has been given to the impact of inbreeding over cow’s performance on artificial reproduction. The objective of this study was to estimate the impact of inbreeding on in vitro embryo production in a Guzerá breed population. The inbreeding coefficient (F), calculated as half of the co-ancestry of the individual’s parents, was used as an estimate of inbreeding. The inbreeding coefficients of the donor, sire (used on in vitro fertilization) and of the embryos were included, separately, in the proposed models either as classificatory or continuous variables (linear and quadratic effects). The percentage of non-inbred individuals (or embryos) and mean F of donors, embryos and sires were 29.38%; 35.76%; 42.86% and 1.98±2.68; 1.32±3.13; 2.08±2.79, respectively. Two different models were considered, one for oocyte production traits and other for embryo production traits. The increase of F of the donor significantly (P<0.05) impaired the number of viable oocytes (N_OV), number of grade I oocytes (N_GI) and number of cleaved embryos (N_CLV). Moreover, the donor’s F influenced the percentage of grade I oocytes (P_GI), percentage of viable embryos (P_EMB) and percentage of cleaved embryos that turned into embryos (P_CXE). No significant (P>0.05) effects were observed for the sire (father of the embryos) inbreeding coefficient over the traits analysed. Embryo’s F influenced (P<0.05) the number of viable embryos (N_EMB), percentage of viable embryos (P_EMB) and percentage of cleaved embryos that turn into embryos (P_CXE). Results suggested that an increase in the inbreeding coefficient might impair the embryos ability to survive through challenges imposed by the in vitro environment. Submitting highly inbred Guzerá female donors to in vitro embryo production may, in the long-term, have negative implications on the number of embryos obtained per cow and increase the relative costs of the improvement programmes based on this technology. High levels of inbreeding should be avoided when selecting Guzerá female donors and planning in vitro fertilization mating.