Abnormal changes in mitochondria, lipid droplets, ATP and glutathione content, and Ca(2+) release after electro-activation contribute to poor developmental competence of porcine oocyte during in vitro ageing.

The present study aims to investigate major changes in porcine oocytes during ageing in vitro. After the oocytes were cultured for 44, 56, 68 and 80 h, changes to porcine oocytes in ultrastructure, mitochondrial distribution, glutathione (GSH) and ATP content, Ca(2+) release patterns and developmental competence after electro-activation were observed. Mitochondria were evenly distributed in oocytes at 44 h, aggregated in clusters or in peripheral cytoplasm at 68 h and dimly dispersed throughout ooplasm at 80 h. Mitochondrial shape during ageing was also observed by transmission electron microscopy (TEM) at the same time intervals. Most mitochondria were spherical at 44 h, and became elongated when the culture time was extended to 68 h and 80 h. Moreover, mitochondrial clustering became increasingly loose from 56 h. Lipid droplets in oocytes appeared prominent and electron-dense at 44 h, but electron density was lost at 56 h. Lipid droplets were solidified as of 68 h. There was an age-dependent decrease in ATP content per oocyte. Glutathione content per oocyte decreased significantly and remained lower after 56 h. Amplitudes of [Ca(2+)] rise decreased dramatically following 56 h, and the time required for [Ca(2+)] to plateau became shorter after electro-activation with prolonged culture time. Cleavage and blastocyst rates of aged oocytes progressively decreased, while the fragmentation rate gradually increased after electro-activation. It is concluded that abnormal changes in mitochondria, lipid droplets, Ca(2+) release after electro-activation, and ATP and GSH content in oocytes during ageing may result in poor developmental competence of parthenotes.

Effects of SOF and CR1 media on developmental competence and cell apoptosis of ovine in vitro fertilization embryos.

The present study was to investigate effects of synthetic oviductal fluid (SOF) and Charles Rosenkrans medium (CR1) culture systems on developmental competence and cell apoptosis of ovine in vitro fertilization (IVF) embryos. Ovine presumptive IVF zygotes were cultured in the following six media: (1) SOF supplemented with amino acids (SOFaa) and 8 mg/ml bovine serum albumin (BSA) for 9 days (SOFaaBSA); (2) SOFaa supplemented with 10% fetal bovine serum (FBS) for 9 days (SOFaaFBS); (3) SOFaaBSA for first 3 days and then SOFaaFBS for later 6 days (SOFaaBSA-FBS); (4) CR1 supplemented with amino acids (CR1aa) and 8 mg/ml BSA for 9 days (CR1aaBSA); (5) CR1aa supplemented with 10% FBS for 9 days (CR1aaFBS); (6) CR1aaBSA for first 3 days and then CR1aaFBS for later 6 days (CR1aaBSA-FBS). The rates of blastocyst and hatched blastocyst in group 1, group 3 and group 6 were not different (P>0.05), but were greater than in other three groups (P<0.05). In SOF and CR1 cultural system, SOFaaBSA and CR1aaBSA-FBS provided the highest blastocyst rates respectively. Both numbers of total cell and trophectoderm (TE) in expanded or hatched blastocyst from SOFaaBSA were significantly higher than CR1aaBSA-FBS (P<0.05). However, the inner cell mass (ICM) cell number and ratio of ICM to TE cell in expanded or hatched blastocysts were not different between two groups (P>0.05). The apoptotic signals were firstly observed at 8-cell stage in two groups and became stronger and stronger with the development of embryos. Rates of embryos with apoptotic signals in group 6 at morula or blastocyst were greater than in group 1 (P<0.05). The apoptotic nuclei numbers of morula or blastocyst in group 6 were also significantly higher than group 1 (P<0.05). It is concluded that CR1aaBSA-FBS can support in vitro development of ovine IVF embryos, but SOFaaBSA is more suitable.

Factors affecting the efficiency of producing porcine embryos expressing enhanced green fluorescence protein by ICSI-mediated gene transfer method.

This study aims to investigate factors that affect the efficiency of blastocyst development and enhanced green fluorescence protein (EGFP) expression in porcine embryos following intracytoplasmic sperm injection (ICSI)-mediated DNA transfer. Frozen-thawed dead spermatozoa were exposed to different concentrations (0.01 microg/mL, 0.05 microg/mL or 0.1 microg/mL) of EGFP DNA solution, and then microinjected into in vitro matured oocytes. The optimal concentration for EGFP expression of resultant embryos was 0.05 microg/mL. When oocytes were microinjected on a warm stage at 30 degrees C, the percentage of EGFP-expressing embryos was higher than that at 38.5 degrees C (40.1% vs. 20.9%, P<0.01). The efficiency of EGFP expression in embryos following ICSI using linear EGFP DNA-exposed spermatozoa was higher than using circular DNA (40.8% vs. 28.2%, P<0.05). ICSI oocytes treated with 6-DMAP after electro-activation had a higher percentage of embryos expressing EGFP than those not treated (52.5% vs. 26.3%, P<0.01). However, neither incubation temperatures of spermatozoa and DNA (4 degrees C, 24 degrees C or 39 degrees C) nor BSA addition to the incubation medium affected the efficiency of producing EGFP-expressing embryos. Furthermore, treatment with DNase I after preincubation of sperm and DNA prevented the embryos from expressing EGFP. The EGFP expression of ICSI oocytes was affected neither by intracytoplasmic injection using sperm heads or whole spermatozoa, nor by washing of the sperm after preincubation. The above-mentioned factors did not affect embryonic developmental competence, apart from 6-DMAP treatment after electro-activation. In conclusion, most exogenous DNA molecules were tightly bound on the membranes of sperm head after incubation of DNA and sperm, and the temperature during ICSI, 6-DMAP treatment, exogenous DNA concentrations and constructs could significantly affect EGFP expression in porcine embryos following ICSI-mediated DNA transfer.