Nam-Hyung Kim

Effect of antifreeze glycoprotein 8 supplementation during vitrification on the developmental competence of bovine oocytes

The purpose of this study was to investigate the effect of antifreeze glycoprotein 8 (AFGP8) supplementation during vitrification on the survival, fertilization, and embryonic development of bovine oocytes and the underlying molecular mechanism(s). Survival, fertilization, early embryonic development, apoptosis, DNA double-strand breaks, reactive oxygen species levels, meiotic cytoskeleton assembly, chromosome alignment, and energy status of mitochondria were measured in the present experiments. Compared with that in the nonsupplemented group; survival, monospermy, blastocyst formation rates, and blastomere counts were significantly higher in the AFGP8-supplemented animals. Oocytes of the latter group also presented fewer double-strand breaks and lower cathepsin B and caspase activities. Rates of normal spindle organization and chromosome alignment, actin filament impairment, and mitochondrial distribution were significantly higher in the AFGP8-supplemented group. In addition, intracellular reactive oxygen species levels significantly decreased in the AFGP8-supplemented groups, maintaining a higher ΔΨm than that in the nonsupplemented group. Taken together, these results indicated that supplementation with AFGP8 during vitrification has a protective effect on bovine oocytes against chilling injury.

Melatonin enhances the developmental competence of porcine somatic cell nuclear transfer embryos by preventing DNA damage induced by oxidative stress

Melatonin has antioxidant and scavenger effects in the cellular antioxidant system. This research investigated the protective effects and underlying mechanisms of melatonin action in porcine somatic cell nuclear transfer (SCNT) embryos. The results suggested that the developmental competence of porcine SCNT embryos was considerably enhanced after melatonin treatment. In addition, melatonin attenuated the increase in reactive oxygen species levels induced by oxidative stress, the decrease in glutathione levels, and the mitochondrial dysfunction. Importantly, melatonin inhibited phospho-histone H2A.X (γH2A.X) expression and comet tail formation, suggesting that γH2A.X prevents oxidative stress-induced DNA damage. The expression of genes involved in homologous recombination and non-homologous end-joining pathways for the repair of double-stranded breaks (DSB) was reduced upon melatonin treatment in porcine SCNT embryos at day 5 of development under oxidative stress condition. These results indicated that melatonin promoted porcine SCNT embryo development by preventing oxidative stress-induced DNA damage via quenching of free radical formation. Our results revealed a previously unrecognized regulatory effect of melatonin in response to oxidative stress and DNA damage. This evidence provides a novel mechanism for the improvement in SCNT embryo development associated with exposure to melatonin.

Progesterone influences cytoplasmic maturation in porcine oocytes developing in vitro

Progesterone (P4), an ovarian steroid hormone, is an important regulator of female reproduction. In this study, we explored the influence of progesterone on porcine oocyte nuclear maturation and cytoplasmic maturation and development in vitro. We found that the presence of P4 during oocyte maturation did not inhibit polar body extrusions but significantly increased glutathione and decreased reactive oxygen species (ROS) levels relative to that in control groups. The incidence of parthenogenetically activated oocytes that could develop to the blastocyst stage was higher (p < 0.05) when oocytes were exposed to P4 as compared to that in the controls. Cell numbers were increased in the P4-treated groups. Further, the P4-specific inhibitor mifepristone (RU486) prevented porcine oocyte maturation, as represented by the reduced incidence (p < 0.05) of oocyte first polar body extrusions. RU486 affected maturation promoting factor (MPF) activity and maternal mRNA polyadenylation status. In general, these data show that P4 influences the cytoplasmic maturation of porcine oocytes, at least partially, by decreasing their polyadenylation, thereby altering maternal gene expression.

Effects of antifreeze glycoprotein 8 (AFGP8) supplementation during vitrification on the in vitro developmental capacity of expanded bovine blastocysts

Cryopreservation is an effective method for the long-term storage of valuable germplasm in the field of reproductive research. The present study examined the developmental capacity of post-thaw bovine blastocysts during vitrification after supplementation with antifreeze glycoprotein 8 (AFGP8). Survival and re-expansion rates in culture during the 12h after thawing were significantly higher in the AFGP8-treated than untreated group. In addition, blastocysts from the AFGP8-treated group exhibited lower rates of apoptosis. Real-time reverse transcription-polymerase chain reaction analysis showed that the expression of the Bcl-2 gene, coding for an anti-apoptotic protein, was increased significantly, whereas the expression of the pro-apoptotic gene Bax was decreased significantly in the AFGP8-treated group. The cellular proliferation rate and mitochondrial membrane potential were significantly higher in post-thaw re-expanded blastocysts from the AFGP8-treated compared with untreated group. In addition, outgrowth potential in post-thaw blastocysts in re-expanded blastocysts after vitrification was significantly increased in the AFGP8-treated compared with untreated group. Together, these results are the first to demonstrate that the addition of AFGP8 during vitrification can help protect bovine blastocysts against chill-induced injury.

Toxic effects of atrazine on porcine oocytes and possible mechanisms of action

Because atrazine is a widely used herbicide, its adverse effects on the reproductive system have been extensively researched. In this study, we investigated the effects of atrazine exposure on porcine oocyte maturation and the possible mechanisms. Our results showed that the rates of oocyte maturation significantly decreased after treatment with 200 μM atrazine in vitro. Atrazine treatment resulted in abnormal spindle morphology but did not affect actin distribution. Atrazine exposure not only triggered a DNA damage response but also decreased MPF levels in porcine oocytes. Our results also revealed that atrazine worsened porcine oocyte quality by causing excessive accumulation of superoxide radicals, increasing cathepsin B activity, and decreasing the GSH level and mitochondrial membrane potential. Furthermore, atrazine decreased developmental competence of porcine oocytes up to the blastocyst stage and changed some properties: cell numbers, apoptosis, and related gene expression levels. Collectively, our results indicate that porcine oocyte maturation is defective after atrazine treatment at least through disruption of spindle morphology, MPF activity, and mitochondrial function and via induction of DNA damage, which probably reduces developmental competence.

Autophagy is required for proper meiosis of porcine oocytes maturing in vitro

Autophagy is an essential cellular mechanism that degrades cytoplasmic proteins and organelles to recycle their components; however, the contribution of autophagy during meiosis has not been studied in porcine oocytes maturing in vitro. In this study, we observed that the autophagy-related gene, LC3, was expressed in porcine oocytes during maturation for 44 h in vitro. Knockdown of the autophagy-related gene, BECN1, reduced both BECN1 and LC3 protein expression levels. Moreover, BECN1 knockdown and treatment with the autophagy inhibitor, LY294002, during maturation of porcine oocytes in vitro impaired polar body extrusion, disturbed mitochondrial function, triggered the DNA damage response, and induced early apoptosis in porcine oocytes. Autophagy inhibition during oocyte maturation also impaired the further developmental potential of porcine oocytes. These results indicate that autophagy is required for the in vitro maturation of porcine oocytes.

171 The effect of trichostatin a on the development and the global gene expression patterns in mouse embryos developing in vitro

Trichostatin A (TSA) is an inhibitor of histone deacetylase and is able to alter gene expression patterns by interfering with the removal of acetyl groups from histones. The aim of this study was to determine the effect of TSA treatment on the development and gene expression patterns of mouse zygotes developing in vitro. The addition of 100 nm TSA to the culture medium did not affect the cleavage of mouse embryos (TSA treatment, 148/150 (99%) v. control, 107/107 (100%)); however, embryos that were treated with TSA arrested at the 2-cell stage (145/148, 98%). We estimated the number of nuclei in control and TSA-treated embryos by propidium iodide staining, taking into account the presence of any cells with two or more nuclei. At 62–63 h post-hCG stimulation, control zygotes had developed to the 4-cell stage and exhibited one nucleus in each blastomere, indicative of normal development. In contrast, we observed tetraploid nuclei in at least one blastomere in 20.8% (11/53) of the embryos that had been treated with TSA. At 28–29 h post-hCG stimulation (metaphase of the 1-cell stage), there was no difference in the mitotic index (as determined by analyzing the microtubule configuration) in the TSA group compared to the control group. At the 2-cell stage, however, we did not observe mitotic spindles and metaphase chromatin in embryos in the TSA treatment group compared to the controls. Interestingly, when embryos were cultured in TSA-free medium from 35 h post-hCG stimulation (S- or early G2-phase of the 2-cell stage) onward, almost all of them (47/50) developed to the blastocyst stage. In contrast, when embryos were cultured in TSA-free medium from 42 h post-hCG stimulation (middle G2-phase of the 2-cell stage) onward, they did not develop to the 4-cell stage. We used Illumina microarray technology to analyze the gene expression profiles in control and TSA-treated late 2-cell-stage embryos. Applied Biosystems Expression System software was used to extract assay signals and assay signal-to-noise ratio values from the microarray images. Our data showed that 897 genes were significantly (P < 0.05; 2-sample t-test) up- or down-regulated by TSA treatment compared to controls. Analysis using the PANTHER classification system (https://panther.appliedbiosystems.com) revealed that the 575 genes that were differentially expressed in the TSA group compared to the control were classified as being associated with putative biological processes or molecular function. Overall, in terms of putative biological processes, more nucleoside, nucleotide, and nucleic acid metabolism, protein metabolism and modification, signal transduction, developmental process, and cell cycle genes were differentially expressed between the TSA and control groups. In terms of putative molecular function, more nucleic acid-binding transcription factor and transferase genes were differentially expressed between the groups. The results collectively suggest that inhibition of histone acetylation in mouse embryos affects gene expression profiles at the time of zygotic genome activation, and this subsequently affects further development.

265 DICER GENE EXPRESSION IN PRE-IMPLANTATION MOUSE EMBRYOS: IMPLICATION OF TRANSCRIPTION REGULATION AT THE BLASTOCYST STAGE

Dicer is an RNAse III enzyme that is related to the generation of the microRNAs involved in the gene silencing pathway. In order to obtain insight into the role of Dicer in early embryo development, we first evaluated its gene expression levels in mouse oocytes and embryos during in vitro development. The relative abundance of Dicer1 transcripts was established by real-time RT-PCR using the 2-ddCt method. H2a was applied as an internal standard to normalize the real-time RT-PCR reaction efficiency and quantify Dicer1 mRNA. Relatively high expression levels of mRNA in germinal vesicle-stage oocytes steadily decreased up to the 2-cell stage embryo, and then expression remained during morulae and blastocyst formation. Protein synthesis of Dicer was also observed in the mouse oocytes and early embryos. Specific silencing of mRNA expression and protein synthesis by RNA interference (siRNA) did not inhibit developmental events up to the blastocyst (BL) stage. However, Dicer1 siRNA reduced (P <0.05) total nuclei numbers in the BL-stage embryos (Dicer1: 77.2±4.2 vs. control: 62.7±3.1). Real-time RT-PCR also confirmed that, following Dicer1 siRNA microinjection into zygotes, transcription levels of several non-target genes, Cdc42, Cdh1, Dbc2, ILK, Tuba1, Plat, and Tie1, were not changed in blastocyst-stage embryos. However, selected transcription factors, Pou5f1 (P <0.01), Nanog (P <0.005), and Sox2 (P <0.01), in blastocysts were significantly down-regulated. Additionally, POU5F1 protein synthesis was also reduced. Using Applied Biosystem microarray technology, we compared gene expression profiles in control and Dicer1 siRNA microinjected blastocysts. This technique confirmed that 397 or 737 of 16354 genes were up- or down-regulated, respectively, following siRNA microinjection (P <0.05), including 52 transcription factors. The results suggest that expression of Dicer regulates gene expression at the blastocyst-stage embryo for cell fate, possibly by the transcription control.

299 LEPTIN ACCELERATES PRONUCLEAR FORMATION FOLLOWING INTRACYTOPLASMIC SPERM INJECTION OF PORCINE OOCYTES: POSSIBLE ROLE OF MITOGEN-ACTIVATED PROTEIN KINASE INACTIVATION

Although evidence suggests that leptin modulates oocyte maturation in vitro, few studies have examined the direct effect of leptin on cytoplasmic maturation and pronuclear formation following fertilization of porcine oocytes.The aim of this studywas to determine microtubule and microfilament assembly following oocyte maturation, and to assess blastocyst formation, mitogen-activated protein kinase (MAPK) activity, and pronuclear formation following parthenogenetic stimuli or intracytoplasmic sperm injection (ICSI) of leptin-treated oocytes.A symmetrical shape of the meiotic spindle was considered to be the normal microtubule assembly, and a clear, thick microfilament area containing a second metaphase chromatin was considered to be morphologically normal. Significant differences were determined using Tukeys multiple range test with P < 0.05 being considered significant. Addition of 10 ng mL-1 of leptin to the in vitro maturation (IVM) medium significantly increased the percentage of zygotes that developed to the blastocyst stage (26.1 ± 2.1%; P < 0.05) compared with that of the control (0 ng mL-1: 16.3 ± 2.8%) or 1 ng mL-1 (18.6 ± 2.6%) leptin treatment groups following parthenogenetic activation. After IVM for 44 h, the percentage of oocytes with normal microtubules was higher in the leptin (10 ng mL-1) group than in the control group (89.1 ± 4.3% vs. 78.5 ± 4.0%, respectively; P < 0.05). However, the percentage of normal microfilament assembly was similar in the leptin-treated and control groups (85.6 ± 4.3% and 82.9 ± 5.0%, respectively). In oocytes matured in vitro in the presence of 10 ng mL-1 of leptin and subsequently induced by parthenogenetic activation via chemical stimulation, there was a significant increase in the formation of 2 pronuclei (2PN: 52.1 ± 5.2%; P < 0.05) at 6 h, compared with the control non-leptin-treated oocytes (40.7 ± 4.0%). Similarly, addition of 10 ng mL-1 of leptin to the IVM medium also increased 2PN formation at the same time point following ICSI (leptin treatment: 47.9 ± 4.0; control: 36.2 ± 3.8%; P < 0.05). The addition of 10 ng mL-1 of leptin to the IVM medium decreased the level of phospho-ERK1/2 at 6 and 9 h and was lower in the leptin-treated group compared with the control non-leptin-treated group (P < 0.01). These results suggest that exogenous leptin enhances cytoplasmic maturation and pronuclear formation following fertilization, possibly via the MAPK pathway.

252 DIFFERENCES IN GENE EXPRESSION PROFILES IN MOUSE FERTILIZED BLASTOCYSTS AND DIPLOID BLASTOCYST STAGE PARTHENOTES

Following parthenogenetic activation, in the absence of a male contribution, oocytes progress into early gestation. To gain insight into the role of the paternal genome during pre-implantation development, we used microarray to compare gene expression profiles in pre-implantation embryos following fertilization and parthenogenetic activation. Fertilized embryos and oocytes were collected from superovulated C57BL/6J female mice. The oocytes were activated with 50 ¼M calcium ionophore A23187 for 5 min. After 5 h of culture in M16 medium with 7.5 ¼g/mL cytochalasin B, oocytes with one polar body and two pronuclei were used in this experiment. The activated oocytes and zygotes were cultured in M16 to the blatocyst stage. Messenger RNA from 50 blastocysts was extracted by means of the Dynabeads mRNA Direct Kit (Dynal, Oslo, Norway), and then linearly amplified for two rounds using the RiboAmp HS RNA Amplification Kit (Arcturus Bioscience, Inc., Mountain View, CA, USA). A set of cRNA targets from the embryos was assembled into a hybridization reaction on the Applied Biosystems 1700 chemiluminescent microarray analyzer (Jung Hwa Scientific Co., Ltd., Seoul, Korea). Each set was repeated three times. All of the correlation coefficients were above 0.9 for experiment replications. Differences in microarray intensities were normalized and grouped by using the Avadis Prophetic 3.3 version, and categories are based on the PANTHER classification system. According to the cDNA microarray data, we additionally categorized genes into transcription- and developmental process-related genes and compared them in both fertilized and parthenogenetically activated blastocysts. Five transcription-related genes (Goosecoid, transcription factor 1, LIM domain, Spi-C transcription factor, and hypoxia inducible factor 3) and seven developmental process related genes (metaxin 1, serine/threonine kinase 22, stromal antigen, butyrophilin, anti-Mullerian hormone type 2 receptor, prolactin-like protein C2, and otoconin 90) were identified in the fertilized blastocysts compared to the blastocyst-stage parthenotes. In contrast, seven transcription- (Amnionless, EHOX-like, calcium signal transducer 2, nuclear receptor 0B, transcription factor CP2, Iroquois related homeobox 3, and zinc finger protein 3) and eight developmental process-related genes (prion protein dublet, X-linked lymphocyte-regulated 3a, muscleblind-like 3, stathmin-like 2, SRY-box-containing gene 7, ephrin B1, muscleblind-like 3, and Iroquois-related homeobox 3) were expressed at a higher level in parthenotes than in fertilized blastocysts. These genes were selected, and their expression levels confirmed, by real-time quantitative RT-PCR. The results indicate that diploid parthenotes at the blastocyst stage may lack or over express genes related to transcription and development processes which possibly result in fetal lethality. Further studies are required to determine whether aberrant gene expression in parthenotes is due to lack of paternal contribution. This work was funded by a grant from the National Research Laboratory Program in Korea.