Developmental Capability Research Articles

198 CILOSTAMIDE SUSTAINS GAP JUNCTION-MEDIATED COMMUNICATION AND CHROMATIN REMODELLING IN PIG OOCYTES

In the pig, the efficiency of in vitro embryo production procedures is still limited. It has been suggested that prematuration treatments could improve the developmental capability of oocytes. In particular, recent studies conducted in the bovine (Luciano, 2011, BOR, in press) indicate that the prolongation of a patent bidirectional crosstalk between the oocyte and the surrounding cumulus cells, together with the maintenance of a proper level of cAMP during the prematuration culture, could be beneficial to oocytes that have not yet acquired full meiotic and developmental capability. The aim of the present study was to assess the effect of treatment with cilostamide, an inhibitor of the phosphodiesterase 3 (PDE3), which degrades cAMP, on the functional status of gap junction-mediated communication (GJC) in pig cumulus–oocyte complexes (COC). Moreover, since chromatin configuration represents a marker of oocyte differentiation and competence, the effect of cilostamide on the process of chromatin remodeling was also evaluated during the culture period. To this aim, COC were collected from 3- to 6-mm antral follicles and cultured for up to 24 h in defined culture medium supplemented with 0.1 IU mL–1 of FSH in the presence or absence of 1 μM cilostamide. The GJC functionality was assessed by Lucifer Yellow fluorescent dye microinjection at the time of collection (0 h) and after 12, 18, or 24 h of culture. Chromatin configuration was evaluated by fluorescence microscopy after removal of cumulus cells and DNA staining with Hoechst and oocytes were classified according to Bui et al. (2004 BOR 70, 1843–1851) as SC, (with stringy chromatin within the germinal vesicle), GVI (with chromatin condensed in a rim around the nucleolus), GVII-IV (where the beginning of formation of chromatin strands is typical), ProMI (prometaphase I) and MI (metaphase I). The administration of cilostamide sustained functional coupling for up to 24 h of culture as the percentage of COC with open GJC was significantly higher when compared with the control group (62.2% vs 30%; P < 0.05) and not significantly different from the time 0 h (80%). The maintenance of the coupling during the culture period was accompanied by a delay of the meiotic resumption as only 26.3% of cilostamide-treated oocytes underwent germinal-vesicle breakdown and reached ProMI stage compared to the control group (62.1%; P < 0.05). Moreover the transition towards advanced stages of differentiation, as judged by the chromatin configuration, was slowed down in the presence of cilostamide. In conclusion, our study indicates that the maintenance of elevated cAMP levels through the inhibition of PDE3 sustains a functional bidirectional communication between the oocyte and cumulus cells and delays meiotic resumption in the pig oocyte. This could be a useful approach for the development of prematuration treatments aimed at improving the embryonic developmental potential of pig oocytes. Experiments are in progress in our laboratories to confirm this hypothesis. This study has been supported by EU FP6 grant n LSHB-CT-2006-037377 (Xenome) EU FP7- n°223485 (Plurisys).

Electrical Activation Enhances Pre-Implantation Embryo Development Following Sperm Injection into <i>In Vitro</i> Matured Pig Oocytes

The objective of this study was to evaluate the effect of electrical stimulation (EST) on pronuclear formation, chromosomal constitution, and developmental capability among in vitro matured pig oocytes following intracytoplasmic sperm injection (ICSI). After ICSI, the oocytes were randomly distributed and cultured into 3 groups: the EST activated ICSI group, non-activation ICSI group, and in vitro fertilization (IVF) group. The proportion of oocytes in which 2 pronuclei were formed in ICSI groups was significantly higher in the former groups than in the IVF group (96.2 and 93.5 vs. 64.5%, respectively, P<0.05). The cleavage rate was significantly higher in EST activated ICSI group (78.6%) than in the IVF and non-activated ICSI groups (51.8 and 46.0%, respectively, P<0.05), as was the proportion of oocytes that developed to the blastocyst stage at day 7 (18.9 vs. 11.6 and 9.1%, respectively, P<0.05). Diploid blastocysts were observed in 52.4, 63.0, and 65.2% of oocytes in the IVF, activated, and non-activated ICSI groups, respectively. Eight out of 23 gilts (34.8%) were confirmed to be pregnant in activated ICSI groups, but none of these pregnancies were carried to term. These results show that oocyte activation after ICSI is effective in elevating the cleavage rate and blastocyst development, while ensuring normal chromosome composition. Further research is needed to determine the pregnancy maintenance requirements for ICSI-embryos in pigs.

Maternal topoisomerase II alpha, not topoisomerase II beta, enables embryonic development of zebrafish top2a -/- mutants

BackgroundGenetic alterations in human topoisomerase II alpha (TOP2A) are linked to cancer susceptibility. TOP2A decatenates chromosomes and thus is necessary for multiple aspects of cell division including DNA replication, chromosome condensation and segregation. Topoisomerase II alpha is also required for embryonic development in mammals, as mouse Top2a knockouts result in embryonic lethality as early as the 4-8 cell stage. The purpose of this study was to determine whether the extended developmental capability of zebrafish top2a mutants arises from maternal expression of top2a or compensation from its top2b paralogue.ResultsHere, we describe bloody minded (blm), a novel mutant of zebrafish top2a. In contrast to mouse Top2a nulls, zebrafish top2a mutants survive to larval stages (4-5 day post fertilization). Developmental analyses demonstrate abundant expression of maternal top2a but not top2b. Inhibition or poisoning of maternal topoisomerase II delays embryonic development by extending the cell cycle M-phase. Zygotic top2a and top2b are co-expressed in the zebrafish CNS, but endogenous or ectopic top2b RNA appear unable to prevent the blm phenotype.ConclusionsWe conclude that maternal top2a enables zebrafish development before the mid-zygotic transition (MZT) and that zebrafish top2a and top2b are not functionally redundant during development after activation of the zygotic genome.

Linking Service-Dominant Logic and Strategic Business Practice

Service-dominant (S-D) logic can function as a strategic business logic that portrays creating superior value in conjunction with—rather than for—customers as a source of competitive advantage for organizations. From this perspective, strategy is about making choices in terms of how to best facilitate and enhance value cocreation for mutual and long-term betterment. While the literature has pointed out the managerial merits of cocreating value, less is known about the organizational capabilities necessary to execute S-D logic in practice. This article devises an S-D orientation, specified as a portfolio of six strategic capabilities, namely individuated, relational, ethical, empowered, developmental, and concerted interaction capability. In combination, these six strategic capabilities constitute a cocreation capability. The authors develop the conceptual model of S-D orientation through (a) an in-depth literature review and (b) input from 21 expert academics. Conceptualizing S-D orientation provides a foundation for bridging S-D logic and strategy research with a more general framework, and for guiding much-needed empirical research that will inform managers. From a managerial point of view, S-D orientation provides a holistic approach to align the organization with its value network partners. The article also sets out an agenda for future research.

Gap Junction-Mediated Communications Regulate Chromatin Remodeling During Bovine Oocyte Growth and Differentiation Through cAMP-Dependent Mechanism(s)1

Oocyte development is characterized by impressive changes in chromatin structure and function in the germinal vesicle (GV) that are crucial in conferring to the oocyte meiotic and developmental competence. During oogenesis, oocyte and follicular cells communicate by paracrine and junctional mechanisms. In cow, cumulus-enclosed oocytes (CEOs) isolated from early antral follicles have uncondensed chromatin (GV0), functionally open gap junction (GJ)-mediated communications, and limited meiotic competence. The aim of the present study was to analyze the role of GJ communications on the chromatin remodeling process during the specific phase of folliculogenesis that coincides with the transcriptional silencing and the sequential acquisition of meiotic and developmental capability. CEOs were cultured in a follicle-stimulating hormone-based culture system that sustained GJ coupling and promoted oocyte growth and transition from GV0 to higher stages of condensation. When GJ functionality was experimentally interrupted, chromatin rapidly condensed, and RNA synthesis suddenly ceased. These effects were prevented by the addition of cilostamide, a phosphodiesterase 3 inhibitor, indicating that the action of GJ-mediated communication on chromatin structure and function is mediated by cAMP. Prolonging GJ coupling during oocyte culture before in vitro maturation enhanced the ability of early antral oocytes to undergo meiosis and early embryonic development. Altogether, the evidence suggests that GJ-mediated communication between germinal and somatic compartments plays a fundamental role in the regulation of chromatin remodeling and transcription, which in turn are related to competence acquisition.

Establishing Naming in Typically Developing Two-Year-Old Children as a Function of Multiple Exemplar Speaker and Listener Experiences

Naming is a verbal developmental capability and cusp that allows children to acquire listener and speaker functions without direct instruction (e.g., incidental learning of words for objects). We screened 19 typically developing 2- and 3-year-old children for the presence of Naming for 3-dimensional objects. All 9 3-year-olds had Naming, and 8 of 10 2-year-olds lacked Naming. For the 2-year-old children who lacked Naming, we used multiple-probe designs (2 groups of 4 children) to test the effect of multiple exemplar instruction (MEI) across speaker and listener responses on the emergence of Naming. Prior to the MEI, the children could not emit untaught listener or speaker responses following match-to-sample instruction with novel stimuli, during which they had heard the experimenter tact the stimuli. After MEI with a different set of novel stimuli, the children emitted listener and speaker responses when probed with the original stimuli, in the absence of any further instruction with those stimuli. Seven of 8 children acquired the speaker and listener responses of Naming at 83% to 100% accuracy. We discuss the basic and applied science implications.

Histone Deacetylase 1 Down-Regulation on Developmental Capability and Histone Acetylation in Bovine Oocytes and Parthenogenetic Embryos

Histone deacetylase 1 (HDAC1) is one of the most conserved enzymes present in the nuclei of cells, including bovine oocytes and pre-implantation embryos. However, the biological functions of HDAC1 in supporting the growth and development of bovine oocytes and embryos are still not fully elucidates. In this study, three siRNAs (si299, si672, and si1272) targeting to HDAC1 mRNA sequence were designed. After transfection into bovine fibroblast cells, si299, the most effective one in HDAC1 knock-down, was selected. The selected siRNA was microinjected into bovine germinal vesicle (GV) stage oocytes to determine the functions of HDAC1 in the maturation of bovine oocytes. Finally, the siRNA was microinjected into mature oocytes, which were then parthenogenetically activated and cultured in vitro until the blastocyst stage. The rates of cleavage, blastocyst development and acetylation of lysine 14 of H3 (H3K14) state were checked. The results suggest that HDAC1 knock-down in oocytes did not influence the rates of maturation or cleavage of parthenogenetic embryos. However, the rates of blastocyst decreased after siRNA microinjection. Furthermore, histone H3K14 acetylation level increased after siRNA microinjection into parthenogenetic embryos.

164 EPIGENETIC ANALYSIS OF GENOMIC DNA IN PREPUBERAL AND ADULT BOVINE OOCYTES

The use of oocytes obtained from prepuberal cattle shortens the generation interval by producing descendants of genetically valuable animals before achieving actual cultivation maturity. However, several studies proved that oocytes derived from prepuberal animals differ significantly from oocytes of adult animals with regard to their developmental capability and therefore reproductive potential. Epigenetic events are taken into consideration as a possible reason for this phenomenon. Particularly DNA methylation, allele specific gene expression in a parent-of-origin-specific manner (imprinting), and certain histone modifications, like acetylations, carboxylations, and phosphorylations, play an important role. This project aims to gain knowledge about the mechanisms involved in attaining of the full developmental potential of bovine oocytes. Using immature and in vitro matured oocytes of prepuberal and adult cattle, a comparative study was conducted by measuring mRNA expression of 4 developmentally relevant, but non-imprinted genes (GDF9, GLUT1, PRDX1, and ZAR1) as well as the general DNA methylation status, performed by bisulfite sequencing of 2 satellite sequences [bovine testis satellite I DNA segment 2 (BTSS2) and Bos taurus α satellite I DNA (BTS)]. After various pretreatments, immature bovine oocytes were collected from prepuberal calves [6–9 months, either left untreated (Ca1) or treated with FSH (Ca2) or FSH+IGF1 (Ca3) or FSH+IGFK (Ca4)] and adult animals [≥2nd lactation, either left untreated (Ad1) or treated with FSH (Ad2)] using the Ovum-pick-up (OPU) technique. The Ad1 group was considered the control group. First results of the qPCR analyses of immature oocytes show differences between treatment groups for GLUT1, PRDX1, and ZAR1 transcripts. Compared with Ad1, GLUT1 expression increased in Ad2 [fold change (FC) 2.2], Ca1 (FC 2.0), Ca2 (FC 1.8), and Ca3 (FC 1.4). The genes PRDX1 and ZAR1 were reduced in all groups by 0.02 to 0.07 in comparison with Ad1. The GDF9 showed generally a very low expression. The methylation analysis shows for BTSS2 and BTS significant differences before and after in vitro maturation in the groups Ad1 (BTSS2: 49.6 v. 64.9%), Ad2 (BTS: 76.7 v. 52.5%), Ca1 (BTSS2: 74.6 v. 53.3%), Ca2 (BTS: 72.8 v. 57.8%) and Ca3 (BTSS2: 60.6 v. 71.7%). Currently, the first experiment and statistical analysis are under way. The preliminary data confirm differences in gene expression between prepuberal and adult animals, and demonstrates the dependence of the methylation pattern on age and maturation status. These results contribute to a better understanding of the developmental potential of prepuberal oocytes in order to optimize their use for in vitro production of embryos. This work was supported by the H. Wilhelm Schaumann Foundation, Hamburg.

96 THE EFFECT OF CHEMICAL DELIPIDATION ON CRYOPRESERVABILITY OF CAT EMBRYOS

Embryo cryopreservation is a desired technique for long-term preservation of embryos. However, the success rate of cryopreserved in vitro produced cat embryos is currently poor. Until recently, the mechanism underlying the cause of cryoinjury that occurs during cooling and cryopreservation is not well understood. This study aimed to examine the effect of chemical delipidation (forskolin) before cryopreservation of 4- to 8-cell cat embryos on post-thaw embryo survival and in vitro developmental capability. A total of 333 cumulus oocyte complexes (COCs) were matured and fertilized in vitro. At 24 h post-IVF, the presumptive embryos were randomly assigned into one of the following groups: 1) non-frozen control (n = 63); 2) forskolin treatment without freezing (n = 52); 3) freezing without forskolin (n = 77); and 4) freezing after forskolin treatment (n = 89). The embryos were cryopreserved using a programmable controlled-rate freezer. After freezing and thawing, the embryos were subsequently cultured in vitro for a further 6 days. The development competence was assessed by morula and blastocyst rates on Days 5 and 8 of their development, respectively. Percentages of cleaved embryos on Day 2 (IVF = Day 0) did not significantly differ among groups, indicating that there was no adverse effect of forskolin on cleavage rates. Furthermore, blastocyst formation rates of cat embryos treated with forskolin (53.5 ± 3.1) did not significantly differ when compared with non-treated controls (54 ± 9.3). Forskolin-treated embryos survived after cryopreservation at a higher rate than non-forskolin treatment, in terms of survival (93.1 ± 2.6 v. 88.2 ± 1.4), morula (56.9 ± 7.6 v. 40.8 ± 5.7), and blastocyst formation (47.6 ± 6.4 v. 35.6 ± 3.6) rates. It is concluded that partial delipidation of cat embryos before cryopreservation improves the cryopreservability of cat embryos. This study demonstrates that intracellular lipid has an impact on cryopreservability of cat embryos. Further study is required to examine in utero development of these delipidated embryos after embryo transfer. This study was financially supported by the Zoo organisation of Thailand and CHE-TRF Senior Research Scholars RTA-5080010.

Modeling the development toward the knowledge economy: a national capability approach

PurposeThis paper aims to model the national development of the knowledge economy and argue its policy implications.Design/methodology/approachThe methodology is a deductive research approach. First, the paper examines fundamental epistemological assumptions of the literature on the knowledge economy to identify major views on the knowledge economy. Second, it synthesizes relevant studies of the knowledge economy to develop key concepts to be used in the theoretical model of the knowledge economy.FindingsThe paper argues that among three views of the knowledge economy (i.e., knowledge‐as‐asset, knowledge‐as‐relation, and knowledge‐as‐capability views), the knowledge‐as‐capability view is the most appropriate to explain the knowledge economy. However, this view is still evolving to explain the knowledge economy. Only a few studies have discussed national capabilities, but they omitted many points of capability as an aspect of knowledge. Although many studies have discussed organizational capabilities and provided some insight, these ideas are not directly applicable at the national level.Practical implicationsThis paper suggests that to develop a knowledge economy a national government should be concerned about the balanced development of the whole system of the economy, while paying due attention to knowledge‐related activities.Originality/valueThis paper proposes a theoretical model of the knowledge economy, using original concepts of three types of national basic capability, i.e., epistemic capability, economic capability, and institutional capability and national developmental capability as the meta‐capability of leveraging, orchestrating, and restructuring those basic capabilities.

Non-invasive real-time spindle visualization under DIC optics and its predictability of oocyte development

OBJECTIVE: The presence of meiotic spindle predicts the fertilizability and developmental capability of ICSI oocytes. Real-time spindle imaging is believed to require a designated polarized microscope. Here, we attempted to identify meiotic spindles in living oocytes under one of the conventional settings for ICSI, and assessed the developmental ability of oocytes depending on the spindle detection. DESIGN: Meiotic spindle was visualized in living oocytes prior to ICSI. Fertilization and embryonic cleavage were compared between oocytes with or without spindles. MATERIALS AND METHODS: In this study, 103 infertile couples undergoing 115 ICSI cycles were involved with written informed consent. Visualization of the spindle was performed immediately before ICSI on a glass-bottomed dish. Oocytes with the first polar body (PBI) were examined at 400 or 600x magnification under an inverted microscope equipped with differential interference contrast optics and a heated stage. Embryological outcome were compared between the two groups with and without spindle. RESULTS: Total of 427 oocytes with PBI were assessed and subjected to ICSI. Spindle was distinctly identified as a barrel shaped bipolar array of filaments and occasionally even with chromosomes aligned on the equatorial plane, and detected in 30 seconds. The spindle was visualized in 82.0% of oocytes (350/427). The fertilization rate was 65.4% of spindle positive oocytes and similar to that negative (66.2%). However, the incidence of normally fertilized oocytes developed to good quality embryos on day 3 as well as to the blastocyst stage on day 5 was significantly higher in the positive than the negative group (55.5 vs. 31.4%, 61.8 vs. 36.1%). CONCLUSIONS: It was possible to detect the spindles with an expected incidence. The spindle visualization enabled safer injections. The presence of the spindle was associated with higher developmental potential. This system is immediately applicable to the current conventional ICSI setting without introducing expensive devices.

Effects of chemical activation and season on birth efficiency of cloned pigs

The effects of chemical activation on birth efficiency of cloned pigs were studied by investigating the developmental process from porcine oocyte activation to birth of cloned pigs. Three different activation methods were used: (i) Electroporation (Ele); (ii) Ele followed by incubation with 6-dimethylaminopurine (6-DMAP); and (iii) Ele followed by a treatment with cycloheximide (CHX). In experiment 1, the rates of cleavage, developmental rates and cell number of porcine parthenogenetic (PA) embryos were investigated in the three treatment groups. In experiment 2, NT embryos produced by the three different activation treatments were compared for the rates of cleavage, development and cell number. Finally, the effects of Ele and Ele+CHX activation methods on birth efficiency of cloned pigs were compared. The activated oocytes treated by combination activation generally showed a higher (P<0.05) blastocyst rate and produced more expanded blastocysts than oocytes activated with Ele. The rates of cleavage and total cell number of parthenotes were not significantly different. Parthenogenetic embryos activated with 6-DMAP developed into blastocyst and expanded blastocyst stages at a significantly (P<0.05) higher rate than those treated with Ele, but the developmental capability was dramatically decreased in NT embryos. With the CHX activation method, the NT embryo blastocyst rate was substantially (P<0.05) increased although the production of expanded blastocysts was not significantly different from that by the other two methods. The birth rate of cloned pigs increased in the CHX group, though the rate was not significantly different from Ele. The effects of season on developmental rate of the porcine PA embryos and birth rate of cloned pigs were also examined in our study. Porcine oocytes collected in the spring had higher developmental capabilities than those collected in the winter. However, no difference in birth rate of the cloned pigs was found between the oocytes collected in the two seasons. The results obtained from PA and NT embryos, following different activation methods, were inconsistent, suggesting that activation mechanisms are dissimilar in PA and NT embryos. Although the chemical activation in our study leads to an elevation of the blastocyst rate, it does not improve the oocyte's molecular programming and so does not significantly improve the efficiency of producing cloned pig births.

Effect of different cryopreservation protocols on cytoskeleton and gap junction mediated communication integrity in feline germinal vesicle stage oocytes

Oocyte cryopreservation in carnivores can significantly improve assisted reproductive technologies in animal breeding and preservation programs for endangered species. However, the cooling process severely affects the integrity and the survival of the oocyte after thawing and may irreversibly compromise its subsequent developmental capability. In the present study, two different methods of oocyte cryopreservation, slow freezing and vitrification, were evaluated in order to assess which of them proved more suitable for preserving the functional coupling with cumulus cells as well as nuclear and cytoplasmic competence after warming of immature feline oocytes. From a total of 422 cumulus enclosed oocytes (COCs) obtained from queens after ovariectomy, 137 were stored by vitrification in open pulled straws, 147 by slow freezing and 138 untreated oocytes were used as controls. Immediately after collection and then after warming, functional coupling was assessed by lucifer yellow injection and groups of fresh and cryopreserved oocytes were fixed to analyze tubulin and actin distribution, and chromatin organization. Finally, COCs cryopreserved with both treatments were matured in vitro after warming. In most cases, oocytes cryopreserved by slow freezing showed a cytoskeletal distribution similar to control oocytes, while the process of vitrification induced a loss of organization of cytoskeletal elements. The slow freezing protocol ensured a significantly higher percentage of COCs with functionally open and partially open communications (37.2 vs. 19.0) and higher maturational capability (32.5 vs. 14.1) compared to vitrified oocytes. We conclude that although both protocols impaired intercellular junctions, slow freezing represents a suitable method of GV stage cat oocytes banking since it more efficiently preserves the functional coupling with cumulus cells after thawing as well as nuclear and cytoplasmic competence. Further studies are needed to technically overcome the damage induced by the cryopreservation procedures on immature mammalian oocytes.

The apical region of developing tooth root constitutes a complex and maintains the ability to generate root and periodontium‐like tissues

Mammalian tooth root development is a long-term process during which root elongates along the apical direction and is accompanied with the formation of periodontium. Considering the heterogeneous apical region of developing root as a functional entity, we observed and characterized the developing apical complex of rat molar root. The aim of the present study was to investigate the characteristics and developmental capability of developing apical complex in situ and in vitro. Histological analysis of rat developing apical complex was performed using hematoxylin and eosin staining and immunohistochemical staining. Cell counting, bromodeoxyuridine incorporation, flow cytometry assays and western blot analyses were performed to assess the proliferation potential of developing apical complex cells in vitro, and its mineralization potential was investigated by alkaline phosphatase activity, alizarin red staining and reverse transcription-polymerase chain reaction analysis. In vivo transplantation of both developing apical complex tissues and cells were used to characterize the differentiation capacity of developing apical complex cells. Dental pulp cells were used as a control in this study. Isolated developing apical complex maintained the developmental capability to form tooth root/periodontal complex ectopically. Developing apical complex cells exhibited relatively higher proliferation and mineralization potential compared with dental pulp cells in culture. When cultured developing apical complex cells were putatively depleted of Hertwig's epithelial root sheath cells, only osteodentin-like tissues and fibrous connective tissues formed in cell-scaffold explants. The sustainable development ability of developing apical complex qualifies it as the growth center of tooth root and as a promising candidate source of cells for tooth root and periodontal regeneration.

Effects of conditioning reinforcement for print stimuli on match-to-sample responding in preschoolers.

Introduction One of the major challenges in the fields of education, psychology, and speech therapy is to develop science based research procedures to teach children with language disabilities how to acquire repertoires for learning more complex verbal skills. Keohane, Delgado, and Greer (2008) argue that the foundations for the development of early verbal language in children consists of child's observing responses such as looking, listening, tasting, smelling and touching. When observing responses such as looking and making sustained eye contact with adults and other visual stimuli do not function as conditioned reinforcement for children, these children fail to develop correspondence between what they hear and see concurrently. Moreover, children that lack these conditioned reinforcers for observing responses are likely to find it difficult, if not impossible, to learn more complex skills such as language acquisition, generalized matching and becoming more aware of different stimuli in their environment. Children with disabilities often have difficulty attending and discriminating among the relevant properties of stimuli to master both basic and generalized matching. Greer and Ross (2008) based on Skinner's (1957) theory of verbal behavior identified this deficit (lack of observing/attention to instructional) stimuli as developmental capability or Drawing on Ruiz and Baer's (1997) treatment of the acquisition of critical repertoires as behavioral developmental cusps, Greer and Ross summarized body of research that they argue identify several verbal developmental They suggest that these building blocks make it possible for children to learn listener and speaker responses and the joining of listener and speaker repertoires that make progressively more complex verbal behavior possible. Ruiz and Baer characterized developmental cusp as a special instance of behavior, change crucial to what can come next.... (p. 533). Several years earlier Staats (1968, p. 267) suggested something like behavioral developmental cusps. Several verbal developmental cusps and stages have been identified in the research literature and described as verbal developmental capabilities. The very earliest foundations for verbal development include cusps that consist of conditioned reinforcement for observing responses (Dinsmoor, 1985; Donahoe & Palmer, 2004; Greer & Ross, 2008) Recently, Keohane, Greer, & Ackerman (2006), tested the effects of conditioning sustained eye contact with three-dimensional (3D) objects (Visual Tracking Protocol) on the acquisition of matching responses for participant for whom looking at or observing 3D instructional stimuli on table that were not reinforcers for visual observing responses. They found that when 3D stimuli were paired with unconditioned reinforcers (e.g. food) until the 3D stimuli alone reinforced observing, the participants' mean-learn-units-to criteria dramatically decreased for learning match-to-sample instruction, and also his attending skills increased. The stimuli were conditioned as reinforcers for observing after the stimulusstimulus paring, which resulted in significant decreases in the numbers of learn units the participants required to master visually related instructional material. The significant acceleration in rate of learning for these participants suggest the role of conditioned reinforcement for observing responses as developmental cusp, since the participants could learn discriminations they could not learn prior to intervention. The newly conditioned reinforcers for observing responses constituted acquisition of developmental cusp. Keohane, Delgado, and Greer (2008), surmised that observing responses in children are tied to and are result of conditioned reinforcement for observing. When child is missing this capability, experimentally based conjugate reinforcement procedures or stimulus-stimulus pairing procedure can be used to induce conditioned reinforcement for observing responses for print and 3D instructional stimuli. …

Developmental competence of gametes reconstructed by germinal vesicle transplantation from fresh and cryopreserved bovine oocytes

To evaluate the use of fresh or frozen bovine oocytes as an animal model for reconstructing artificial gametes by germinal vesicle transplantation (GVT), to study nucleocytoplasmic interaction and define clinical procedures for ooplasm donation in humans. Prospective experimental study. University-based experimental laboratory. Bovine oocytes from slaughterhouse ovaries. A total of 446 gametes were reconstructed from fresh immature oocytes; nuclear and cytoplasmic competencies were analyzed through the assessment of meiotic progression and cytoskeleton reorganization; embryonic developmental capability was evaluated after parthenogenetic activation of metaphase II (MII) reconstructed oocytes. Furthermore, the distribution of mitochondria in karyoplast and cytoplast in grafted oocytes was studied. Finally, meiotic and developmental competencies were determined in 199 gametes reconstructed from vitrified immature oocytes. Maturational and developmental rate of reconstructed oocytes, cytoskeleton organization, and mitochondrial distribution. Gametes reconstructed from either fresh or cryopreserved immature oocytes showed similar meiotic competence (41.6% vs. 37.7%, respectively). All reconstituted oocytes that reached MII displayed a normal distribution of cytoskeletal elements. Embryonic developmental capability was higher in oocytes derived from fresh than from cryopreserved gametes (30.8% vs. 8.1%, respectively). Finally, oocyte centrifugation was effective in obtaining karyoplasts with <5% of mitochondria. Cows can provide a suitable organism model to develop GVT technique in both research and clinical settings as well as in fertility preservation programs.

Establishment and Characterization of a Pluripotent Stem Cell line Derived from Human Amniotic Membranes and Initiation of Germ Layers in vitro

Pluripotent stem cells are proposed to be used in regenerative therapy and may exist in the human amniotic membrane. The present article is aimed at establishing a pluripotent stem cell line from human placenta. HAM-1 (stem cell line derived from human amniotic membranes) was established by the colonial cloning technique using aMEM culture medium containing 10 ng/ml of EGF, 10 ng/ml of hLIF and 10% fetal bovine serum. HAM-1 cells appeared to maintain a normal karyotype indefinitely in vitro and expressed markers characteristic of stem cells from mice and human, namely alkaline phosphatase. Also, these cells contributed to the formation of chimeric mouse embryoid bodies and gave rise to cells of all germ layers in vitro. This study demonstrates that human amniotic membranes derived stem cells have a wide developmental capability and might be utilized to regenerate different types of cells or tissues for transplantation therapy.

The vertical distribution, density and the development of the tick Ixodes ricinus in mountain areas influenced by climate changes (The Krkonoše Mts., Czech Republic)

The altitudinal distribution and density of host-seeking Ixodes ricinus ticks were investigated by flagging in 2002–2006 along two vertical transects in the Krkonoše Mts. Comparison with historical data revealed that the I. ricinus upper distribution limit shifted from the former 700–800 m limit at least up to 1100 m above sea level (a.s.l.) during the last two decades. The tick thus has newly colonised the whole montane belt, covered predominantly with Norway spruce forests. The density of host-seeking nymphs decreased with increasing altitude: In the established mixed forests located at 600–800 m a.s.l., it reached the mean value of 31 nymphs per hour of flagging (with a maximum density of 125 nymphs per hour). The mean density attained 7.1 nymphs per hour (maximum 66) in 870–1020 m and 2 nymphs per hour (maximum 6.3) in the newly colonised areas at 1080–1100 m a.s.l. Regular records of host-seeking I. ricinus larvae and nymphs up to 1020 m a.s.l., together with rather regular records of nymphs and presence of single host-seeking adults up to 1100 m a.s.l. indicated viability of the newly established local tick populations. The present empirical upper limit was experimentally confirmed by the investigation of the developmental capability of eggs, fed larvae and nymphs along a vertical transect (650–1550 m a.s.l.) in 2004–2006. The experimental results demonstrated a strong relationship between tick developmental success and duration on the one hand and altitude on the other. They showed that climatic conditions up to 1100 m a.s.l. are at present suitable for the development of all tick stages. However, it also revealed that ticks were able to complete their development up to at least 1250 m a.s.l. in the unusually warm summer and autumn 2006 (the mean temperature for June–October 2006 exceeded the 1960–1990 mean value by 1.8 °C). The causes of tick expansion could be found when analysing long-term (1961–2005) climatic data from the Krkonoše Mts., which showed a systematic and significant rise of the mean annual air temperature by 1.4 °C at 1000 m a.s.l. The most pronounced temperature increase (mean monthly temperature by 1.5–3.5 °C at 1000 m a.s.l.) was determined in May–August, which is crucial for host finding and development of I. ricinus. Abundance of the newly settled local tick populations at higher altitudes may increase in the future after their closer establishment in local mountain ecosystems.

Factors affecting the production of potential transgenic pigs by DNA microinjection; a six-year retrospective study

Transgenesis is a complex process. Among the most important factors affecting the production of potential transgenic pigs using standard DNA microinjection into zygote pronuclei are the type of promoter used to express the gene construct and the method of zygote transfer used for transformation. The transformation of zygotes using different gene constructs showed that the promoter type infl uenced the developmental capability of the transformed zygotes, and this affected the percentage of born piglets. The transfer of transformed zygotes into a single oviduct resulted in a higher percentage of potential transgenic piglets born, this fi gure was almost doubled compared with transfer into both oviducts. Recently, research into the production of transgenic pigs has shown that factors connected with reproduction physiology and embryological factors such as the number of zygotes transferred after transformation and the season of the year have a signifi cant infl uence but are not critical to the effi ciency of production of transgenic pigs. The best results were obtained when less than 20 transformed zygotes per recipient were transplanted and the autumn season is the time when the environmental factors are optimal for production of potential transgenic pigs.

Early pregnancy loss in sows after low dose, deep uterine artificial insemination with sex-sorted, frozen–thawed sperm

Recent developments in reproductive technologies have enabled the production of piglets of a predetermined sex via non-surgical, low dose artificial insemination. The practical application of sex-sorting technology to the pig is made challenging by the large numbers of sperm required for successful insemination of sows. One way of overcoming the time required for sex-sorting may be to create a bank of cryopreserved, sex-sorted sperm, thus making available appropriate doses as sows require insemination. To date, little success has been achieved with non-surgical inseminations of sex-sorted boar sperm. This study attempted to achieve litters of a predetermined sex after a double insemination of sows with 160 × 10 6 sex-sorted, frozen–thawed sperm. Sows were synchronised and sperm were non-surgically inseminated into the proximal third of the uterine horn at 36 and 42 h after hCG administration. Sows inseminated with sex-sorted sperm achieved similar pregnancy rates to those receiving an equal dose of unsorted, frozen–thawed sperm. However, all sows conceiving after insemination with sex-sorted sperm returned to oestrus within 57 days of insemination. This was a higher rate of pregnancy loss than observed for sows inseminated with unsorted sperm (37.5%; P = 0.031). A combination of low sperm numbers and potentially compromised developmental capability of embryos derived from sex-sorted sperm may have resulted in this early stage loss of pregnancy.