Byoung-Chul Yang

Comparison of maturation, fertilization, development, and gene expression of mouse oocytes grown in vitro and in vivo.

AbstractPurpose: To investigate the difference of in vitro and in vivo grown oocytes, we compared maturation, fertilization, development, and maternal gene expression from both in vitro and in vivo grown mouse oocytes. Methods: The preantral follicles isolated from 12-day-old mice were cultured on Transwell-COL membrane inserts. After culture, maturation, fertilization, and developmental rates were assessed. RT-PCR (reverse transcription—polymerase chain reaction) was performed to examine the expression of β-actin, GDF-9, and IGF-II in matured oocytes. Results: No difference in the nuclear maturation was detected between in vitro and in vivo grown oocytes, but the mean oocyte diameter of the in vitro group was smaller than that of the in vivo group. The fertilization rate was significantly lower in the in vitro group than in the in vivo group (p < 0.05). The capacities of in vitro grown oocyte to cleave and develop to blastocysts were significantly lower than those of the in vivo grown oocytes (p < 0.001). Moreover, blastocyst of in vitro group had fewer total cells than those of in vivo group (p < 0.05). In regards to the expression of genes in mature oocytes, growth differentiation factor-9 (GDF-9) expression was similar between the two groups, but β-actin was significantly reduced in the in vitro group compared to the in vivo group. Particularly, the expression of insulin-like growth factor II (IGF-II) was not found in the in vitro grown oocytes. Conclusions: These results showed that in vitro grown oocytes did not have the same developmental capacity as in vivo grown oocytes. We assume that the aberrant expression of maternal-derived genes in the in vitro grown oocytes may cause the poor embryo viability.

Length difference between equine ZFX and ZFY genes and its application for molecular sex determination

PurposeWe analyzed the sex chromosome-encoding ZFX-ZFY genes and tested molecular sexing using the amplification patterns of intron 9 of ZFX-ZFY in the horse.Methods and resultsThe amplification of the ZFX-ZFY produced two distinct patterns, reflecting sexual dimorphism based on a length difference between the X and Y chromosomes. The amplification products from foals showed two distinct bands: one was common to all foals and mares, indicating that this band was amplified from ZFX, while the other was specific to some foals, indicating that it was from ZFY. The result based on the PCR assay was identical to the results of amplification of the Y chromosome-specific SRY gene and those of investigations of the phenotypic gender in three different horse populations.ConclusionWe suggest that this PCR strategy for determining sexes by comparing the amplification patterns of ZFX-ZFY genes is a convenient and precise method for discriminating sexes in horses.

Quantitative analysis of sperm mRNA in the pig: relationship with early embryo development and capacitation

Although it is well known that mRNA is present in mammalian spermatozoa, the relevance of mRNA to capacitation and early embryo development in the pig remains unclear. In the present study, we investigated differences in the abundance of selected mRNAs coding for MYC, CYP19, ADAM2, PRM1 and PRM2 in purified porcine spermatozoa depending on embryo cleavage rate and capacitation (n=20 semen samples). Semen samples were used in IVF procedures, with subsequent embryo development classified into one of two groups based on cleavage rate (i.e. high (>75%) and low (<75%) cleavage groups) and mRNA abundance in purified spermatozoa compared between these two groups. In addition, mRNA abundance was compared between capacitated and non-capacitated spermatozoa. Comparison of mRNA levels between porcine spermatozoa revealed that the abundance of MYC, CYP19, ADAM2, PRM1 and PRM2 mRNA was significantly greater in the high cleavage group (n=10 high cleavage group semen samples) than in the low cleavage group (n=10; P<0.05). Significant downregulation of MYC mRNA was observed in capacitated spermatozoa (n=12; P<0.05). The results of the present study suggest that the amount of specific mRNAs could be used for estimating the quality of spermatozoa in the pig.

Characteristics of Korean native, Hanwoo, calves produced by transfer of in vitro produced embryos

The main objectives of this investigation were to monitor the birth weight of calves and gestation length following artificial insemination (AI) and transfer of in vivo or in vitro produced Korean native, Hanwoo embryos. Embryos produced in vivo were recovered from uterine flushings of superovulated cows 7 days after AI. Those embryos produced in vitro were co-cultured with cumulus cells for 7-8 days after in vitro fertilization. The birth weights of calves following the transfer of in vitro produced (IVP) embryos were heavier than calves from both of AI- and in vivo-derived embryo transferred calves in both sexes (29.6, 24.1 and 25.2kg, respectively, P<0.05). The IVP calves also had a longer gestation length (293.9, 285.8 and 283.8 days, respectively, P<0.05).

In vitro and in vivo genotoxic effects of somatic cell nuclear transfer cloned cattle meat.

Although the nutritional composition and health status after consumption of the meat and milk derived from both conventionally bred (normal) and somatic cell nuclear transferred (cloned) animals and their progeny are not different, little is known about their food safeties like genetic toxicity. This study is performed to examine both in vitro (bacterial mutation and chromosome aberration) and in vivo (micronucleus) genotoxicity studies of cloned cattle meat. The concentrations of both normal and cloned cattle meat extracts (0-10×) were tested to five strains of bacteria (Salmonella typhimurium: TA98, TA100, TA1535, and TA1537; Escherichia coli: WP2uvrA) for bacterial mutation and to Chinese hamster lung (CHL/IU) cells for chromosome aberration, respectively. For micronucleus test, ICR mice were divided into five dietary groups: commercial pellets (control), pellets containing 5% (N-5) and 10% (N-10) normal cattle meat, and pellets containing 5% (C-5) and 10% (C-10) cloned cattle meat. No test substance-related genotoxicity was noted in the five bacterial strains, CHL/IU cells, or mouse bone marrow cells, suggesting that the cloned cattle meat potentially may be safe in terms of mutagenic hazards. Thus, it can be postulated that the cloned cattle meat do not induce any harmful genotoxic effects in vitro and in vivo.

Human extracellular superoxide dismutase (EC-SOD) expression in transgenic chicken.

Extracellular superoxide dismutase (EC-SOD) is a metalloprotein and functions as an antioxidant enzyme. In this study, we used lentiviral vectors to generate transgenic chickens that express the human EC-SOD gene. The recombinant lentiviruses were injected into the subgerminal cavity of freshly laid eggs. Subsequently, the embryos were incubated to hatch using phases II and III of the surrogate shell ex vivo culture system. Of 158 injected embryos, 16 chicks (G0) hatched and were screened for the hEC-SOD by PCR. Only 1 chick was identified as a transgenic bird containing the transgene in its germline. This founder (G0) bird was mated with wild-type hens to produce transgenic progeny, and 2 transgenic chicks (G1) were produced. In the generated transgenic hens (G2), the hEC-SOD protein was expressed in the egg white and showed antioxidant activity. These results highlight the potential of the chicken for production of biologically active proteins in egg white. [BMB Reports 2013; 46(8): 404-409]

Effects of cloned-cattle meat diet on reproductive parameters in pregnant rabbits

In this paper, we report on the effects of a diet containing cloned-cattle meat on the reproductive parameters in pregnant rabbits. The artificially inseminated rabbits (gestation day 0) were fed a diet containing 5% or 10% of normal or cloned-cattle meat during the gestation period. Rabbits fed commercial pellet (no additional supplementations) were used as the control. Supplementation of cloned-cattle meat diets did not have any toxicologically significant effects on reproductive performance in dams (body weight, clinical signs, organ weight, and cesarean section analysis). And it also did not affect on fetal development (body and placental weight, and external, visceral and skeletal findings) compared to the controls. The only difference was a food consumption in the first week of gestation for all meat-based diet groups (p<0.05, 0.01, and 0.001, respectively). Our results collectively suggest that there are no obvious differences in reproductive parameters in pregnant rabbits fed cloned-cattle meat.

Delayed parturition in cloned calves associated with persistently elevated placentomal TGF-β1 expression

The objective of this study was to investigate hormonal and TGF-beta(1) characterizations of delayed parturition in the SCNT recipients (Korean native beef cattle: Hanwoo). The SCNT blastocysts produced by Hanwoo fetal fibroblast cells were transferred into the synchronized Hanwoo recipients. The artificially inseminated Hanwoo recipients (AI-R) were used as control. All AI-R were labored by natural delivery. The SCNT recipients (SCNT-R) with no signs of delivery were operated by Caesarean section. The blood and placentomes were collected during parturition. The weight of placentomes in SCNT-R (n=12, 301+/-41.22 g) was significantly higher than that of AI-R (n=10, 204.8+/-24.89 g) (p<0.05). There were significantly lower E2 (p<0.05) or higher P4 (p<0.01) and TGF-beta(1) (p<0.01) levels in the SCNT-R compared to that of AI-R, respectively. The SCNT-R showed a higher placentomal TGF-beta(1) protein level compared to that of AI-R (p<0.01). Interestingly, the TGF-beta(1) protein level in SCNT-R with normal delivery was dramatically decreased as same as AI-R, but it was highly maintained in C-sec at days 250 of pregnancy in AI-R. These results suggest that delayed parturition in clone calving may be associated with persistence of elevated TGF-beta-1 expression in late pregnancy.

A diet of somatic cell nuclear transfer cloned-cattle meat produced no toxic effects on behavioral or reproductive characteristics of F1 rats derived from dams fed on cloned-cattle meat

BACKGROUND The composition and nutritional value of meat and milk derived from cloned animals and their progeny has not been demonstrated to be different from normal animals, but possible food consumption risks that might arise from unidentified hazards remain. In this study, we investigated the effects of somatic cell nuclear transfer cloned-cattle meat diet on the behavioral and reproductive characteristics of F1 rats derived from dams that were also fed on cloned-cattle meat. METHODS AND RESULTS F1 rats were divided into five diet groups with their dams: commercial pellets (control), pellets containing 5% (N-5) and 10% (N-10) of normal-cattle meat, and diets containing 5% (C-5) and 10% (C-10) of cloned-cattle meat. In most cases, the cloned-cattle meat diet did not affect body weight and food consumption in both male and female F1 rats during 11 weeks, except for significantly higher body weight in both N-5 and N-10 (3-5 weeks, p<0.05 or p<0.01) and significantly higher food consumption in the both normal- and cloned-cattle meat groups (7-9 weeks, p<0.05 or p<0.01), as compared with the controls, respectively. We detected no signs of test substance-related toxicities on organ weights and behavioral characteristics (sensory reflex, motor function, and spatial learning and memory tests). Reproductive functions did not significantly differ among all examined rats (mating, fertility, and implantation). CONCLUSIONS These behavioral and reproductive toxicity results suggest that there are no obvious food safety concerns related to cloned-cattle meat in these parameters.

Enhanced biological effects of Phe140Asn, a novel human granulocyte colony-stimulating factor mutant, on HL60 cells.

Granulocyte colony-stimulating factor (G-CSF) is a cytokine secreted by stromal cells and plays a role in the differentiation of bone marrow stem cells and proliferation of neutrophils. Therefore, G-CSF is widely used to reduce the risk of serious infection in immunocompromised patients; however, its use in such patients is limited because of its non-persistent biological activity. We created an N-linked glycosylated form of this cytokine, hG-CSF (Phe140Asn), to assess its biological activity in the promyelocyte cell line HL60. Enhanced biological effects were identified by analyzing the JAK2/STAT3/survivin pathway in HL60 cells. In addition, mutant hG-CSF (Phe140Asn) was observed to have enhanced chemoattractant effects and improved differentiation efficiency in HL60 cells. These results suggest that the addition of N-linked glycosylation was successful in improving the biological activity of hG-CSF. Furthermore, the mutated product appears to be a feasible therapy for patients with neutropenia.