Jung-Kai Tseng

Sonic hedgehog promotes porcine oocyte maturation and early embryo development

In the present study, we investigated the effects of the Sonic hedgehog (Shh) protein on porcine oocyte maturation and early embryo development. Immunohistochemistry showed activation of Shh signalling in cumulus-oocyte complexes (COCs), as reflected by Patched (Ptc), Smoothened (Smo) and Gli1 expression in oocytes, cumulus cells and granulosa cells, particularly those of small follicles (<2 mm in diameter). Western blot analysis showed Smo expression in COCs and in denuded oocytes derived from small and medium (3-7 mm)-sized follicles. Small follicles contained the highest concentration of Shh in follicular fluid compared with medium-sized and large (>7 mm in diameter) follicles. Supplementation with Shh (0.5 or 1 microg mL(-1)) enhanced oocyte maturation compared with the control group (92.4% and 90.4% v. 81.9%, respectively; P < 0.05). This effect was reversed by the simultaneous addition of cyclopamine (1-2 microm), an Shh inhibitor. Similar to intact COCs, denuded COCs showed enhanced maturation following Shh supplementation. Furthermore, cyclin B1 content, extracellular signal-regulated kinase 1/2 phosphorylation, intracellular calcium release, blastocyst rate and total cell numbers were greater (P < 0.05) in oocytes matured in the presence of 0.5 and 1 microg mL(-1) Shh compared with control oocytes. The findings of the present study provide the first evidence that the Shh signalling pathway is active, or at least partially activated, in the porcine ovary and is likely to promote oocyte cytoplasmic and nuclear maturation, as well as subsequent in vitro development, although the underlying mechanisms remain to be elucidated.

Characterising visual deficits in children of an urban elementary school in Taiwan

Purpose:  The aim was to screen children from Grades 1 to 6 in an urban elementary school in Central Taiwan for visual deficits and associated parameters and, as an extension, to examine the acceptance of cycloplegic therapy as well as the lag in optimal vision correction.

Calcium Release and Development of Heat-Shocked Porcine Oocytes after Nucleus-Ooplasm Reconstruction

We determined the effect of heat shock (HS) on the alterations of development and calcium releasing capacity of nuclear-ooplasmic reconstructed porcine oocytes stimulated by thimerosal. The non-HS (39 degrees C) and the HS2h (41.5 degrees C for 2 h) matured oocytes were enucleated and their spindles/chromosomes were exchanged between these two groups followed by parthenogenetic activation. In the Control group (Csp-Coop), the non-HS spindle (Csp) was transferred to the non-HS ooplasm (Coop). Blastocyst and cleavage rates were higher in both Csp-HSoop (non-HS spindle transferred to the HS ooplasm) and HSsp-Coop (HS spindle transferred to non-HS ooplasm) reconstructed oocytes, but no difference was detected in the average cell number per blastocyst. However, intracellular calcium concentrations ([Ca(2+)](i)) generally declined (p < 0.05) in the reconstructed HS oocytes, with a greater blastocyst rate after parthenogenetic activation. In the present study, time for the completion of spindle transfer in these oocytes was 1-2 h, during which some physiological remodeling or adaptation might have been occurred in the oocytes. Therefore, changes in heat-shock protein70 (HSP70) expression and developmental competence of the HS2h oocytes with 1 or 2 h of recovery time under normal culture temperature (39 degrees C) were examined. The results showed that the expression of HSP70 in the HS2h oocytes was higher (p < 0.05) than those had recovery incubation for 1 h (HC1h) after HS, but the cleavage and blastocyst rates were greater (p < 0.05) in the HC1h group. We demonstrated that a recovery period prior to activation of porcine oocytes and reconstructed oocytes is beneficial to further development. Heat shock to either the karyoplast or the ooplasm enhances embryonic development but reduces intracellular calcium release in the cloned porcine oocytes.

357 CALCIUM RELEASE INDUCED BY THIMEROSAL AND INOSITOL 1,4,5-TRIPHOSPHATE IN HEAT-SHOCKED PORCINE OOCYTES

Elevated ambient temperature has been known to be deleterious to the developmental competence of mammalian oocytes and embryos, although the mechanism is still unclear. The objective of this study was to determine the effect of heat shock (HS) on the alteration of intracellular calcium concentrations ([Ca2+]i) of matured pig oocytes by two different calcium releasing agents. Porcine cumulus–oocyte complexes were aspirated from the follicles (3–6 mm) and subjected to standard in vitro maturation procedure for 42 h. Matured oocytes were then randomly allocated to different heat treatments at 41.5°C for 0 (Control, C0h), 1 (HS1h), 2 (HS2h), or 4 h (HS4h). An additional control group was cultured for 4 h without heat shock (C4h). Oocytes were incubated with 2 µM fura-2 acetoxymethyl ester (AM) and 0.02% pluronic F-127 in Ca2+-free PBS (40 min) following heat shock, and then washed with Ca2+-free PBS (30 min) for detection of [Ca2+]i. Fluorescent images were captured with alternative excitation wavelengths at 340/380 nm by a rotating chopper disk equipped with an Axon imaging system. Data from both experiments were analyzed by ANOVA using the General Linear Model (GLM) of the SAS (SAS Institute, Inc., Cary, NC, USA). In Experiment 1, matured oocytes were activated by 200 mM thimerosal (10 min) following heat treatment. The maximal [Ca2+]i in the HS2h group was the highest among all treatment groups. The lowest maximal peak of [Ca2+]i was observed in the HS4h group, but it was still higher than that in the C4h group (P < 0.05). The total amount of Ca2+ release represented by the total area of the peaks in C4h was lower than in any other groups except HS4h (P < 0.05). In Experiment 2, each matured oocyte was injected with approximately 10 pL of inositol 1,4,5-triphosphate (IP3, 0.5 mM); the Ca2+ transient was recorded as described in the previous experiment. The maximal value of [Ca2+]i in the C4h group was still the lowest among the heat-shocked and C0h groups (P < 0.05). The total Ca2+ release in the HS2h group was the highest among all treatment groups, but only significantly higher than the HS1h and C4h groups (P < 0.05). A similar pattern of Ca2+ release in HS-oocytes was induced by thimerosal and IP3 stimulations. These results indicate that Ca2+ releasing capacity of matured pig oocytes is enhanced by a shorter duration of heat shock, but declines after prolonged exposure of heat shock and/or in vitro culture. The differential Ca2+ releasing capacity of heat-shocked oocytes prior to fertilization revealed physiological changes of pig oocytes after heat shock. This finding provides further insight for the low fertilization and developmental competence that occurs in farm species during hot seasons.