Effects of synchronization of donor cell cycle on embryonic development and DNA synthesis in porcine nuclear transfer embryos

The relationship between donor cell cycle and the developmental ability of somatic cell nuclear transfer (SCNT) embryos has not fully been elucidated. Donor cells that are usually prepared by serum starvation or confluent-cell culture for SCNT represent a heterogeneous population that includes mainly G0 phase cells, other cells in different phases of the cell cycle and apoptotic cells. In this study, we compared the developmental ability of porcine SCNT embryos reconstructed from G0 phase cells (G0-SCNT embryos) and strictly synchronized-G1 phase cells (G1-SCNT embryos), and examined the developmental rates and timing of first DNA synthesis. The G0 phase cells were synchronized by confluent culture, and the G1 phase cells were prepared from actively dividing M phase cells. The G1-SCNT embryos showed a significantly higher (P<0.05) developmental rate to the blastocyst stage per cleaved embryo (59%) than the G0-SCNT embryos (43%). Moreover, initiation of first DNA synthesis and cleavage occurred significantly earlier in the G1-SCNT embryos than in the G0-SCNT embryos. Delay of initiation of first DNA synthesis in the SCNT embryos by aphidicolin resulted in decreased developmental rates to the blastocyst stage without any effect on cleavage rates. Our data demonstrates that synchronized-G1 phase cells can be used as donor cells for SCNT embryos and that earlier initiation of first DNA synthesis may be important for subsequent development of SCNT embryos. The SCNT system using G1-synchronized cells, in terms of their highly uniform and viable cell states, can be useful for studying the reprogramming processes and embryonic development of SCNT embryos.     

Role of the hyaluronan receptor CD44 during porcine oocyte maturation

Previous our studies have shown that CD44, the principal receptor for hyaluronan, is present on cumulus cells during oocyte maturation. Although hyaluronan-CD44 interaction has been implicated in cumulus expansion and/or oocyte maturation, the full significance of CD44 remains unknown. The objective of the present study was to further investigate the role of CD44 in cumulus expansion and oocyte maturation in pigs. We demonstrate here in that CD44 has a key role in oocyte maturation but not in cumulus expansion. Previous studies have reported the physiological significance of cumulus expansion in oocyte maturation. However, our results suggest that cumulus expansion is a necessary condition for oocyte maturation, but that it is not sufficient on its own. Furthermore, western blot analysis demonstrated that the CD44 of the in vitro-matured cumulus-oocyte complexes (COCs) had a larger molecular weight and more terminal sialic acid, which has been proven to inhibit the hyaluronan-binding ability of the receptor, than the CD44 of the in vivo-matured COCs, indicating that the hyaluronan-CD44 interactions during in vitro maturation might be insufficient compared with those in vivo. The insufficient interactions of hyaluronan-CD44 during in vitro maturation may cause the inferior capacity of fertilization and development of oocytes matured in vitro.     

Spindle formation and microtubule organization during first division in reconstructed rat embryos produced by somatic cell nuclear transfer

The present study was conducted to demonstrate the spindle formation and behavior of chromosomes and microtubules during first division in reconstructed rat embryos produced by somatic cell nuclear transfer (SCNT) with cumulus cell nuclei. To demonstrate the effect of oocyte aging after ovulation on the cleavage of SCNT embryos, micromanipulation was carried out 11, 15 and 18 h after injection of hCG. SCNT oocytes were activated by incubation in culture medium supplemented with 5 microM ionomycin for 5 min followed by treatment with 2 mM 6-dimethylaminopurine (6-DMAP) in mR1ECM for 2-3 h. For immunocytochemical observation, the SCNT embryos were incubated with monoclonal anti-alpha-tubulin antibody and then fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG. Cleavage rates were significantly higher for oocytes collected after 15 and 18 h rather than for those collected 11 h after injection of hCG (56 and 53%, respectively vs. 28%; P<0.05). Premature chromosome condensation occurred before activation of the SCNT oocytes, but adequate spindle formation was only rarely observed. The distribution of microtubules in SCNT embryos after activation was different from those of fertilized and parthenogenic oocytes, i.e., a dense microtubule organization shaped like a ring was observed. Eighteen to 20 h post-activation, most SCNT embryos were in the 2-cell stage, but no nucleoli were clearly visible, which was quite different from the fertilized oocytes. In addition, first division with and without small cellular bodies containing DNA was observed in the rat SCNT embryos in some cases. The present study suggests that reorganization of transferred nuclei in rat SCNT embryos may be inadequate in terms of formation of the mitotic assembly and nucleolar reorganization.     

A novel culture method of canine peripheral blood lymphocytes with concanavalin a and recombinant human interleukin-2 for adoptive immunotherapy

This study was designed to develop a novel culture method for the efficient proliferation of canine peripheral blood lymphocytes (cPBL) for adoptive immunotherapy. When cPBL were cultured in the presence of concanavalin A (Con A), proliferation of cPBL was induced and expression of interleukin-2 receptor (IL-2R) which enables to respond to exogenously added IL-2 was upregulated. And then, when cPBL were cultured with recombinant human interleukin-2 (rhIL-2) in addition to Con A, proliferation was accelerated and increased to about 10-fold after 1 week. The phenotypic analysis showed that the main population of the cultured cPBL was consisted of CD8+ positive lymphocytes. Among them, CD4+CD8+ double positive (DP) lymphocytes had significantly increased, and the ratio of CD4+ single positive (SP) lymphocytes to CD8+ SP lymphocytes (CD4+SP/CD8+SP) was decreased as compared to before culturing. To evaluate the cytotoxic activity of cPBL cultured with Con A and rhIL-2, furthermore, cytotoxic assay was carried out against xenogeneic melanoma cell line (MeWo), which resulted in MHC-unrestricted cytokilling. These results suggest that the culture method of cPBL by the use of Con A and rhIL-2 may be useful for generating lymphokine activated killer cells, and also this may be beneficial for adoptive immunotherapy of tumor-bearing dogs.     

Using midday stem water potential for scheduling deficit irrigation in mid–late maturing peach trees under Mediterranean conditions

Irrigation techniques that reduce water applications are increasingly applied in areas with scarce water resources. In this study, the effect of two regulated deficit irrigation (RDI) strategies on peach [Prunus persica (L.) Batsch cv. “Catherine”] performance was studied over three growing seasons. The experimental site was located in Murcia (SE Spain), a Mediterranean region. Two RDI strategies (restricting water applications at stage II of fruit development and postharvest) based on stem water potential (Ψ_s) thresholds (?1.5 and ?1.8 MPa during fruit growth and ?1.5 and ?2.0 MPa during postharvest) were compared to a fully irrigated control. Soil water content (θ_v), Ψ_s, gas exchange parameters, vegetative growth, crop load, yield and fruit quality were determined. RDI treatments showed significantly lower values of θ_v and Ψ_s than control trees when irrigation water was restricted, causing reductions in stomatal conductance and photosynthesis rates. Vegetative growth was reduced by RDI, as lower shoot lengths and pruning weights were observed under those treatments when compared to control. However, fruit size and yield were unaffected, and fruit quality was slightly improved by RDI. Water savings from 43 to 65 % were achieved depending on the year and the RDI strategy, and no negative carryover effect was detected during the study period. In conclusion, RDI strategies using Ψ_s thresholds for scheduling irrigation in mid–late maturing peach trees under Mediterranean conditions are viable options to save water without compromising yield and even improving fruit quality.     

Measured and estimated water use and crop coefficients of grapevines trained to overhead trellis systems in California’s San Joaquin Valley

The use of overhead trellis systems for the production of dry-on-vine (DOV) raisins and table grapes in California is expanding. Studies were conducted from 2006 to 2009 using Thompson Seedless grapevines grown in a weighing lysimeter trained to an overhead arbor trellis and farmed as DOV raisins for the first two years and for use as table grapes thereafter. Maximum canopy coverage for the two lysimeter vines across years was in excess of 80 %. Seasonal (15 March–31 October) evapotranspiration for the lysimeter vines (ET_Lys) was 952 mm in 2007 (farmed as DOV raisins) and 943 and 952 mm (when farmed as table grapes). The maximum crop coefficient (K _cLys) across all 4 years ranged from 1.3 to 1.4. These maximum values were similar to those estimated using the relationship where K _c is a function of the amount of shaded area measured beneath the canopy at solar noon (K _c = 0.017 × percent shaded area). Covering the lysimeter’s soil surface with plastic (and then removing it) numerous times during the 2009 growing season (1 June–14 September) reduced ET_Lys from an average of 6.4 to 5.6 mm day^?1 and the K _c from 1.07 to 0.93. A seasonal basal K _c (K _cb) was calculated for grapevines using an overhead trellis system with a 13 % reduction in the K _cLys across the growing season.     

Crop response of drought-tolerant and conventional maize hybrids in a semiarid environment

In the High Plains, corn (Zea mays L.) is an important commodity for livestock feed. However, limited water resources and drought conditions continue to hinder corn production. Drought-tolerant (DT) corn hybrids could help maintain high yields under water-limited conditions, though consistent response of such hybrids is unverified. In this two-year study, the effects of three irrigation treatments were investigated for a DT and conventional maize hybrid, Pioneer AQUAMax P0876HR and Pioneer 33Y75, respectively. In 2013, the drier of the 2 years, irrigation amounts and crop water use (ET_c) were greater for the conventional hybrid, but grain water use efficiency (WUE) and harvest index were significantly greater for the DT hybrid. In 2014, grain yields and WUE were not significantly different between hybrids. However, irrigation amounts, ET_c and biomass yields were greater for the conventional hybrid. Results from both years indicate that the DT hybrid required less water to maximize grain yield as compared to the conventional hybrid. Producing relatively high yields with reduced amounts of water may provide a means for producers to continue corn production in a semiarid environment with declining water supplies.