Loss Of Developmental Competence Research Articles

Non-linear hierarchy of the quorum sensing signalling pathway in bloodstream form African trypanosomes.

Trypanosoma brucei, the agents of African trypanosomiasis, undergo density-dependent differentiation in the mammalian bloodstream to prepare for transmission by tsetse flies. This involves the generation of cell-cycle arrested, quiescent, stumpy forms from proliferative slender forms. The signalling pathway responsible for the quorum sensing response has been catalogued using a genome-wide selective screen, providing a compendium of signalling protein kinases phosphatases, RNA binding proteins and hypothetical proteins. However, the ordering of these components is unknown. To piece together these components to provide a description of how stumpy formation arises we have used an extragenic suppression approach. This exploited a combinatorial gene knockout and overexpression strategy to assess whether the loss of developmental competence in null mutants of pathway components could be compensated by ectopic expression of other components. We have created null mutants for three genes in the stumpy induction factor signalling pathway (RBP7, YAK, MEKK1) and evaluated complementation by expression of RBP7, NEK17, PP1-6, or inducible gene silencing of the proposed differentiation inhibitor TbTOR4. This indicated that the signalling pathway is non-linear. Phosphoproteomic analysis focused on one pathway component, a putative MEKK, identified molecules with altered expression and phosphorylation profiles in MEKK1 null mutants, including another component in the pathway, NEK17. Our data provide a first molecular dissection of multiple components in a signal transduction cascade in trypanosomes.

Prediction of blastocyst development and implantation potential in utero based on the third cleavage and compaction times in mouse pre-implantation embryos.

Cytokinesis and cell division during pre-implantation embryonic development occur as an orchestrated spatiotemporal program. Cleavage, compaction, and blastulation in pre-implantation embryos are essential for successful implantation and pregnancy. Their alteration is associated with chromosomal imbalance and loss of developmental competence. In this study, we evaluated the time of cleavage and compaction as predictors for in vitro pre- and peri-implantation development and in utero implantation potential by time-lapse monitoring. Mouse 2-cell embryos were collected on 1.5 days post coitum (dpc) and were individually cultured to the outgrowth (OG) stage (7.5 dpc). Developmental stages were classified as 3-cell, 4-cell, 8-cell, morula, blastocyst, and OG. Cut-off times for successful blastocyst development were determined by receiver operating characteristic curve analysis. When cut-off times were set as 9 h for the third cleavage from the 2- to 4-cell stage, and 40 h for compaction from the 2-cell to morula stage, blastocyst and OG development rates, respectively, were significantly higher (P < 0.0001). Embryos were grouped according to the above cut-off time and transferred to the contralateral uterine horn on 3.5 dpc. Implantation rates in utero on 5.5 dpc were significantly higher in early third cleaved (≤ 9 h from 2- to 4-cell) and early compacted embryos (≤ 40 h from 2-cell to morula) than those in delayed embryos (P < 0.05). Therefore, the time of the third cleavage from 2- to the 4-cell stage and compaction from 2-cell to morula stage may be a useful morphokinetic parameter for predicting developmental potential, including successful implantation and pregnancy in human in vitro fertilization-embryo transfer programs.

153 ZINC INSUFFICIENCY DURING PORCINE OOCYTES IN VITRO MATURATION CAUSED MEIOTIC BLOCK AND DEVELOPMENTAL FAILURE

The objective was to investigate the effects of zinc (Zn) insufficiency during in vitro maturation (IVM) of porcine oocytes. Zinc insufficiency was induced by treatment of Zn chelator, N,N,N′,N′-tetrakis-(2-pyridylmethyl)-ethylendiamine (TPEN). In experiment 1, we investigated the effect of duration of Zn insufficiency in IVM on oocytes maturation and subsequent embryonic development after parthenogenetic activation (PA). First, 10 μM TPEN was added to the IVM medium for 0, 7, 15, or 22 h. After TPEN treatment, 10 μM Zn were supplemented on IVM medium except in the 0 h group. Reductions in the nuclear maturation rates were dependent on TPEN duration. The 0-h-treated oocytes showed 83.9 ± 3.9% metaphase II (MII) rate; the 7-h-treated oocytes had significantly lower MII rate (44.8 ± 3.0%) than 0-h-treated oocytes. The majority of 15- and 22-h-treated oocytes were arrested at metaphase I (MI rate: 98.0 ± 1.0 and 97.2 ± 1.7%, MII rate: 0 and 0%, respectively). Embryonic developmental competence was similar to maturation results. Reduction in cleavage and blastocyst (BL) rates were also dependent on duration of TPEN treatment (cleavage rate: 65.3 ± 1.4, 42.6 ± 4.8, 2.6 ± 0.1, and 3.0 ± 1.6%; BL formation rate: 29.3 ± 2.8, 9.2 ± 1.5, 0, and 0% for 0, 7, 15, and 22 h). Total cell number of BL was also significantly different. Total cell number of BL in the 0-h-treated group (51.4 ± 4.5) was significantly higher than that in the 7-h-treated group (23.2 ± 1.6). In experiment 2, to confirm that the Zn insufficiency caused oocyte immaturities and loss of developmental competence in TPEN-treated oocytes, we investigated nuclear maturation and subsequent embryonic development following 3 groups: (1) non treatment (control); (2) 10 μM TPEN treatment during 22 h of IVM; (3) 10 μM TPEN + 10 μM Zn treatment during 22 h of IVM. Only TPEN-treated oocytes and TPEN+Zn-treated oocytes showed contrasting results. Oocyte maturation rates and subsequent embryonic development competence in TPEN with Zn-treated oocytes were similar to control (MII rate: 93.0 ± 1.2 and 92.7 ± 1.8%, BL formation rate: 42.0 ± 6.7 and 40.0 ± 7.5% for TPEN+Zn-treated oocytes and control). These results were significantly different compared with only TPEN-treated oocytes’ results (MII rate: 0.61 ± 0.61%, BL formation rate: 0%). In conclusion, Zn is an essential element for successful oocyte maturation and embryo development in porcine. Zinc insufficiency caused meiotic block and had lasting effects on early embryo development. This work was supported, in part, by a grant from the Next-Generation BioGreen 21 Program (No. PJ00956901), Rural Development Administration, and the National Research Foundation of Korea Grant funded by the Korean Government (NRF-2012R1A1A4A01004885, NRF-2013R1A2A2A04008751), Republic of Korea.

156 PRETREATMENT WITH THE ANTIOXIDANT EPIGALLOCATECHIN GALLATE (EGCG) COUNTERACTS THE NEGATIVE EFFECTS OF MATERNAL HYPERTHERMIA ON EMBRYONIC DEVELOPMENT IN MICE

Hyperthermia-induced oxidative stress is one of the suggested mechanisms underlying the loss of developmental competence in heat-stressed embryos. The objective of the present study was to determine whether pretreatment with the antioxidant epigallocatechin gallate (EGCG) would counteract the negative effects of maternal hyperthermia on oocyte competence and improve subsequent embryonic development. Exp. 1 examined the effect of pretreatment with EGCG on ex vivo embryonic development under normal culture conditions (KSOM, 37°C, 5% CO2, 95% RH in air). Female mice (CB6F1) were synchronized (PMSG + hCG) and injected with 0.4 mL EGCG (100 mg/kg body weight) or with saline. Both EGCG- and saline-treated females were paired with stud males overnight. Mated mice were sacrificed and putative zygotes recovered and cultured in vitro. Cleavage and blastocyst formation rates were recorded on Days 1 and 5 post-fertilization, respectively. The percentage of putative zygotes that cleaved into the two-cell stage did not differ between the groups; however, blastocyst formation rate was higher (P &lt; 0.05) in the EGCG group (85 ± 2%) than in the saline group (75 ± 2%). In Exp. 2 (a 2 × 2 factorial study), both EGCG- and saline-treated mice were exposed to normo-thermal conditions (NT; 22°C, 45% RH) or heat stress (HS; 40°C, 70% RH) for 1.5 to 2 h, the latter to induce a rise of 2°C in body temperature. Synchronized mice were paired with stud males overnight and mated mice were then sacrificed. Putative zygotes were recovered and cultured in vitro as described above. The number of putative zygotes recovered, cleavage and blastocyst formation rates and percentage of hatched blastocysts are presented in Table 1. Blastocyst formation rate was higher (P &lt; 0.05) in the HS-EGCG group than in the HS-saline group. In addition, HS-EGCG embryos exhibited developmental competence similar to that of embryos from both NT groups. In summary, pretreatment with EGCG improved developmental competence under the described culture conditions. Furthermore, pretreatment with the antioxidant EGCG counteracted the negative effects of maternal hyperthermia. Further studies are warranted to evaluate the effect of pretreatment with EGCG on embryo quality. Table 1. Effect of pretreatment with EGCG on developmental competence of heat stressed oocytes

Mammalian Oocyte Maturation and Microtubule-Associated Proteins Dynamics

Mammalian Oocyte Maturation and Microtubule-Associated Proteins Dynamics

Redox status of the oviduct and CDC2 activity in 2-cell stage embryos in heat-stressed mice.

Mammalian preimplantation embryos are vulnerable to heat stress. However, the mechanisms by which maternal heat stress compromises embryonic development are unclear. We hypothesized that the loss of developmental competence in maternally heat-stressed embryos results from enhanced oxidative stress in the oviducts. In experiment 1, oviducts and zygotes were collected from mice that were heat-stressed at 35 degrees C and 60% relative humidity for 12 h on the day of pregnancy as well as from control mice. The zygotes were cultured for 84 h to assess their development, and the H(2)O(2) level, glutathione concentration, and free radical scavenging activity (FRSA) were measured in the oviduct. In experiment 2, zygotes were cultured for 22 h to reach the late G(2) phase in the 2-cell stage, and Cdc2 activity was assessed using immunoblotting. A high percentage (87.6%) of control embryos developed to morulae or blastocysts, whereas the majority (67.4%) of the heat-stressed group arrested at the 2-cell stage. Although heat stress did not alter the FRSA or glutathione concentration in the oviducts, the H(2)O(2) level (P < 0.01) and its ratio to the FRSA (P < 0.05) significantly increased in the heat-stressed group. The Cdc2 activation at the 2-cell stage, as shown by the ratio of the dephosphorylated form to the phosphorylated form, was evident in control embryos but absent in heat-stressed embryos, and the level was similar to that in embryos blocked at the 2-cell stage (positive control). These results indicate that maternal heat stress enhances oxidative stress in the oviducts and that loss of developmental competence in maternally heat-stressed embryos correlates with a defect in Cdc2 activity at the 2-cell stage.

Developmental competence and oxidative state of mouse zygotes heat-stressed maternally or in vitro.

Mammalian preimplantation embryos are sensitive to maternal and direct heat stress. However, the mechanisms by which heat stress affects early embryonic development in vivo or in vitro are unknown. This study examined whether heat-stress-induced loss of developmental competence in mouse embryos was mediated by physiological changes in the maternal environment or by high temperatures alone. After fertilization, zygotes at the same stage were heat-stressed at 39.5 degrees C for 12 h either maternally (measured by maternal rectal temperature) or directly in culture. Zygotes in each group were cultured at 37.5 degrees C for a further 84 h to assess their developmental ability. Neither type of heat stress affected the first cleavage rate. However, the proportion of embryos that developed to morulae or blastocysts was significantly lower in the maternally heat-stressed group, but not in the directly heat-stressed group. Moreover, maternal heat stress significantly reduced intracellular glutathione concentrations and enhanced hydrogen peroxide concentrations in both zygotes and two-cell embryos that were recovered immediately after heat stress or 12 h later, respectively. In contrast, direct heat stress had little effect on concentrations of glutathione or hydrogen peroxide in cultured early embryos. These results demonstrate that maternal heat stress at the zygote stage reduces the developmental ability of mouse embryos via physiological changes in the maternal environment that lead to an increase in intracellular oxidative stress on the embryo.

Maturation of human oocytes in vitro and their developmental competence.

Complete maturation of oocytes is essential for the developmental competence of embryos. Any interventions in the growth phase of the oocyte and the follicle in the ovary will affect oocyte maturation, fertilization and subsequent embryo development. Oocyte size is associated with maturation and embryo development in most species examined and this may indicate that a certain size is necessary to initiate the molecular cascade of normal nuclear and cytoplasmic maturation. The minimum size of follicle required for developmental competence in humans is 5-7 mm in diameter. Maturation in vitro can be accomplished in humans, but is associated with a loss of developmental competence unless the oocyte is near completion of its preovulatory growth phase. This loss of developmental competence is associated with the absence of specific proteins in oocytes cultured to metaphase II in vitro. The composition of culture medium used successfully for maturation of human oocytes is surprisingly similar to that originally developed for maturation of oocytes in follicle culture in vitro. The presence of follicle support cells in culture is necessary for the gonadotrophin-mediated response required to mature oocytes in vitro. Gonadotrophin concentration and the sequence of FSH and FSH-LH exposure may be important for human oocytes, particularly those not exposed to the gonadotrophin surge in vivo. More research is needed to describe the molecular and cellular events, the presence of checkpoints and the role of gene expression, translation and protein uptake on completing oocyte maturation in vitro and in vivo. In the meantime, there are very clear applications for maturing oocytes in human reproductive medicine and the success rates achieved in some of these special applications are clinically valuable.

Maturation of human oocytes in vitro and their developmental competence

Complete maturation of oocytes is essential for the developmental competence of embryos. Any interventions in the growth phase of the oocyte and the follicle in the ovary will affect oocyte maturation, fertilization and subsequent embryo development. Oocyte size is associated with maturation and embryo development in most species examined and this may indicate that a certain size is necessary to initiate the molecular cascade of normal nuclear and cytoplasmic maturation. The minimum size of follicle required for developmental competence in humans is 5-7 mm in diameter. Maturation in vitro can be accomplished in humans, but is associated with a loss of developmental competence unless the oocyte is near completion of its preovulatory growth phase. This loss of developmental competence is associated with the absence of specific proteins in oocytes cultured to metaphase II in vitro. The composition of culture medium used successfully for maturation of human oocytes is surprisingly similar to that originally developed for maturation of oocytes in follicle culture in vitro. The presence of follicle support cells in culture is necessary for the gonadotrophin-mediated response required to mature oocytes in vitro. Gonadotrophin concentration and the sequence of FSH and FSH-LH exposure may be important for human oocytes, particularly those not exposed to the gonadotrophin surge in vivo. More research is needed to describe the molecular and cellular events, the presence of checkpoints and the role of gene expression, translation and protein uptake on completing oocyte maturation in vitro and in vivo. In the meantime, there are very clear applications for maturing oocytes in human reproductive medicine and the success rates achieved in some of these special applications are clinically valuable.

Egg culture: the foundation.

Ralph Brinster began his classic work on egg culture more than 35 years ago. His interest in mammalian egg culture had developed, in part, as a consequence of his experiences with animal breeding and reproduction that he gained while growing up on a farm. Ralph decided early in his career that an in vitro approach to culturing eggs would provide a powerful tool with which to study the development of these cells. Beginning at the close of the 19th century, a number of investigators had performed in vitro studies on egg culture and the related area of egg transfer; however, the ability to recover and transplant eggs had reached a much higher level of perfection than had culture. Eggs of many species could be successfully transferred, but there was no reliable technique for egg culture. In 1963, Ralph reported a method for culturing eggs in microdrops of medium under oil (Brinster, 1963), which has become universally used. Two years later, he identified pyruvate as the central and essential energy source for early stages of mouse eggs (Brinster, 1965b). These two developments revolutionized in vitro studies of mammalian eggs and issued in an era of intense research activity concerning egg culture and egg manipulation. Effective formulations of culture media could now be developed to allow routine in vitro maintenance of eggs, and important parameters for these recipes were soon determined. It was quickly established that the requirement for pyruvate as an energy source exists at ovulation in many species and is already present in germ cells of the mouse fetus. The metabolic activity of the fertilized mouse egg was shown to be low and comparable to bone; however, four days later, at the blastocyst stage of development, the metabolic activity was comparable to brain. Thus, a foundation of understanding about the biology of early mammalian eggs was established between 1960 and 1970, and subsequent studies have broadened this understanding. However, the greatest impact of a simple, reliable egg culture method has been to provide the ability to perform complicated manipulative procedures on preimplantation stages of mammalian embryos. In no area has this been more important than in development of transgenic animals. All methods for generating germ line genetic modifications rely on the ability to maintain and manipulate eggs and early developmental stages in vitro without loss of developmental competence. The importance of efficient egg culture to manipulation and transgenesis is fundamental and enabling.