[show abstract][hide abstract] ABSTRACT: The present study was conducted to generate transgenic pigs coexpressing human CD55, CD59, and H-transferase (HT) using an IRES-mediated polycistronic vector. The study focused on hyperacute rejection (HAR) when considering clinical xenotransplantation as an alternative source for human organ transplants. In total, 35 transgenic cloned piglets were produced by somatic cell nuclear transfer (SCNT) and were confirmed for genomic integration of the transgenes from umbilical cord samples by PCR analysis. Eighteen swine umbilical vein endothelial cells (SUVEC) were isolated from umbilical cord veins freshly obtained from the piglets. We observed a higher expression of transgenes in the transgenic SUVEC (Tg SUVEC) compared with the human umbilical vein endothelial cells (HUVEC). Among these genes, HT and hCD59 were expressed at a higher level in the tested Tg organs compared with non-Tg control organs, but there was no difference in hCD55 expression between them. The transgenes in various organs of the Tg clones revealed organ-specific and spatial expression patterns. Using from 0 to 50% human serum solutions, we performed human complement-mediated cytolysis assays. The results showed that, overall, the Tg SUVEC tested had greater survival rates than did the non-Tg SUVEC, and the Tg SUVEC with higher HT expression levels tended to have more down-regulated α-Gal epitope expression, resulting in greater protection against cytotoxicity. By contrast, several Tg SUVEC with low CD55 expression exhibited a decreased resistance response to cytolysis. These results indicated that the levels of HT expression were inversely correlated with the levels of α-Gal epitope expression and that the combined expression of hCD55, hCD59, and HT proteins in SUVECs markedly enhances a protective response to human serum-mediated cytolysis. Taken together, these results suggest that combining a polycistronic vector system with SCNT methods provides a fast and efficient alternative for the generation of transgenic large animals with multiple genetic modifications.
PLoS ONE 01/2013; 8(5):e63241. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: To determine the presence of sexual dimorphic transcription and how in vitro culture environments influence X-linked gene transcription patterns in preimplantation embryos, we analyzed mRNA expression levels in in vivo-derived, in vitro-fertilized (IVF), and cloned porcine blastocysts. Our results clearly show that sex-biased expression occurred between female and male in vivo blastocysts in X-linked genes. The expression levels of XIST, G6PD, HPRT1, PGK1, and BEX1 were significantly higher in female than in male blastocysts, but ZXDA displayed higher levels in male than in female blastocysts. Although we found aberrant expression patterns for several genes in IVF and cloned blastocysts, similar sex-biased expression patterns (on average) were observed between the sexes. The transcript levels of BEX1 and XIST were upregulated and PGK1 was downregulated in both IVF and cloned blastocysts compared with in vivo counterparts. Moreover, a remarkable degree of expression heterogeneity was observed among individual cloned embryos (the level of heterogeneity was similar in both sexes) but only a small proportion of female IVF embryos exhibited variability, indicating that this phenomenon may be primarily caused by faulty reprogramming by the somatic cell nuclear transfer (SCNT) process rather than in vitro conditions. Aberrant expression patterns in cloned embryos of both sexes were not ameliorated by treatment with Scriptaid as a potent HDACi, although the blastocyst rate increased remarkably after this treatment. Taken together, these results indicate that female and male porcine blastocysts produced in vivo and in vitro transcriptional sexual dimorphisms in the selected X-linked genes and compensation of X-linked gene dosage may not occur at the blastocyst stage. Moreover, altered X-linked gene expression frequently occurred in porcine IVF and cloned embryos, indicating that X-linked gene regulation is susceptible to in vitro culture and the SCNT process, which may eventually lead to problems with embryonic or placental defects.
PLoS ONE 12/2012; 7(12):e51398. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: Human interferon α (IFN-α) and erythropoietin (EPO) have been used for a variety of purposes in clinical medicine. Human IFN-α has been used to treat several types of viral infection and cancer, as well as renal anemia, via stimulation of erythrocyte formation in the bone marrow. Transgenic cattle are excellent candidates for pharmaceutical production for humans due to their ability to produce recombinant proteins in milk. The purpose of the present study was to generate bovine transgenic fibroblasts capable of producing recombinant human IFN-α and EPO proteins in transgenic cattle milk. First, we analyzed the promoter activities of various bovine milk protein genes in HC11 mouse mammary epithelial cells. The bovine milk protein gene promoters were cloned into the Luc gene in a promoter-less pGL3-Basic vector. Presence of the αS1-casein promoter (-175 to +796 nt) resulted in an up to 16-fold increase in luciferase activity compared with that of the promoter-less construct. In addition, the human IFN-α and EPO genes were identified as significantly overexpressed in HC11 cells compared with the promoter-less construct. Together, the present results demonstrate that the construct with the αS1-casein promoter may induce secretion of recombinant human IFN-α and EPO into bovine milk. Furthermore, we generated transgenic fibroblasts expressing human IFN-α and EPO cDNA controlled by the αS1-casein promoter and two screening markers, enhanced green fluorescent protein and neomycin resistance. These transgenic fibroblasts may be a source of somatic cells for generating transgenic cattle that produce recombinant human IFN-α and EPO proteins during lactation.
Molecular Medicine Reports 11/2012; · 1.17 Impact Factor
[show abstract][hide abstract] ABSTRACT: Type 2 diabetes mellitus (T2DM) is one of the most common complex metabolic disorders in humans, and is characterized by hyperglycemia and metabolic alterations. In T2DM, fasting hyperglycemia is attributed to excessive hepatic glucose production, and increased gluconeogenesis has been ascribed to increased transcriptional expression of phosphoenolpyruvate carboxykinase (PEPCK). In this study, we analyzed porcine PEPCK promoter activities to generate liver-specific expression vectors. We generated miniature pig fibroblasts overexpressing PEPCK via transgenes to provide an animal model of human T2DM. Various regions of the promoter showed high levels of activity in the presence of glucocorticoids, a PEPCK gene inducer, in liver cells compared to a positive control promoter. The selected promoter region for a liver-specific expression system was adopted based on the current targeting vector containing two selection markers, green fluorescence protein and a neomycin-resistance gene. The linearized vector was introduced into pig fibroblasts which facilitated liver-specific PEPCK overexpression and screening according to the two selection markers. In the present study, we used a liposome-mediated transfection protocol rather than a virus-mediated gene delivery system, since the virus may cause side effects. Following transfection, 46 colonies out of 33 transfection trials had positively integrated the overexpression vector, indicating that a relatively high transfection efficiency rate was obtained by the liposomal-mediated system. Thus, we recommend the optimal liver-specific expression system for safe and effective transfection of pig cells. We plan to use these cells for somatic cell nuclear transfer to produce piglets that overexpress liver-specific PEPCK as an animal model for human T2DM.
Molecular Medicine Reports 07/2012; 6(1):45-50. · 1.17 Impact Factor
[show abstract][hide abstract] ABSTRACT: Animals cloned by somatic cell nuclear transfer (SCNT) sometimes have abnormalities that result in large offspring syndrome or early death during gestation due to respiratory and metabolic defects. We cloned pigs using two sources of donor cells and observed phenotypic anomalies in three pigs cloned from one type of cell, s-pig fetal fibroblasts. These animals had many wrinkles on their faces and bodies and looked older than age-matched normal pigs. We performed the present study to examine whether the wrinkled phenotype in the cloned pigs was due to senescence, a genetic problem with donor specificity, or epigenetic problems with reprogramming. To address this issue, we investigated biomarkers of senescence, including telomere length and the expression of senescence-associated β-galactosidase (SA-β-gal), glyceraldehyde phosphate dehydrogenase (GAPDH) and β-actin. We also assessed the methylation status of euchromatic PRE-1 repetitive sequences and centromeric satellite DNA, and measured the mRNA levels of six imprinted genes, Copg2, Mest, Igf2R, GNAS, SNRPN and Ube3a. The telomeres of the wrinkled cloned pigs were much shorter than those of the normal cloned pigs and age-matched normal pigs. In the wrinkled cloned pigs, SA-β-gal activity was detected and GAPDH and β-actin were repressed. The mRNA levels of Mest, GNAS and Ube3a were reduced in the wrinkled cloned pigs, although there was no difference between the normal cloned pigs and normal controls. This gene expression analysis indicates that the wrinkled abnormality of our pigs originates from genetic abnormalities in the donor cells used for SCNT.
International Journal of Molecular Medicine 05/2012; 30(2):383-91. · 1.96 Impact Factor
[show abstract][hide abstract] ABSTRACT: Dogs are useful models for studying human metabolic diseases such as type 2 diabetes mellitus due to similarities in physiology, anatomy and life styles with humans. Somatic cell nuclear transfer (SCNT) facilitates the production of transgenic dogs. In this study, we generated transgenic dogs expressing the phosphoenolpyruvate carboxykinase (PEPCK) gene, which is closely involved in the pathogenesis of type 2 diabetes mellitus. In addition, we assessed the cloning efficiency associated with adult or fetal (cloned or natural mating) fibroblasts as a nuclear source. Cloning efficiency was determined by the fusion, pregnancy and cloning rates. The fusion rates were significantly high for fibroblasts from cloned fetuses, but the pregnancy and cloning rates were relatively high for cells from normal fetuses. Based on these data, fetal fibroblasts were selected as the nuclear donor for SCNT and genetically engineered to overexpress the PEPCK gene and dual selection marker genes controlled by the PEPCK promoter. The transgenic cells were introduced into oocytes and transferred into five recipient dogs, resulting in two pregnancies. Finally, three puppies were born and confirmed by microsatellite analysis to be genetically identical to the donor. One puppy successfully overexpressed PEPCK mRNA and protein in the liver. This canine disease model may be useful for studying the pathogenesis and/or therapeutic targets of type 2 diabetes mellitus.
International Journal of Molecular Medicine 05/2012; 30(2):321-9. · 1.96 Impact Factor
[show abstract][hide abstract] ABSTRACT: Diabetes mellitus is a metabolic disease caused by impaired insulin secretion from the pancreatic β cells and increased insulin resistance in peripheral tissues. Recently, the overexpression of inducible cyclic AMP (cAMP) early repressor (ICER) Iγ in rodent pancreatic β cells was found to induce insulin deficiency and glucagon overproduction similar to that found in human diabetes mellitus. ICER Iγ with only a DNA binding domain interrupts the transcriptional regulation of the cAMP responsive element-binding protein (CREB) target genes. Based on this information, we hypothesized that the overexpression of ICER Iγ, the most powerful competitor to CREB, could be useful for generating a pig model of diabetes. First, we evaluated the promoter activities of the human insulin gene for the β cell-specific overexpression of ICER Iγ in the pig pancreas. The maximum promoter activity region [-1,431 nucleotides (nt) to +1 nt, +1 = the transcriptional start site] of the insulin gene presented an activity level 3-fold higher than a promoterless construct. Second, ICER Iγ overexpression controlled by this promoter region significantly blocked the glucose-mediated insulin transcription, such as that regulated by the viral promoter in the pancreatic β cell line, MIN6. This suggests that the human insulin promoter may facilitate the overexpression of ICER Iγ in porcine pancreatic β cells. In addition, the overexpression of ICER Iγ in porcine β cells may induce human-like type 1 diabetes mellitus in pigs. In the present study, we generated transgenic fibroblasts containing ICER Iγ cDNA controlled by the human insulin promoter, as well as two screening markers, the green fluorescence protein and the neomycin resistance gene. These fibroblasts may provide a source for somatic cell nuclear transfer to generate a pig model that mimics human diabetes mellitus.
Molecular Medicine Reports 05/2012; 6(1):239-45. · 1.17 Impact Factor
[show abstract][hide abstract] ABSTRACT: To artificially activate embryos in somatic cell nuclear transfer (SCNT), chemical treatment with ionomycin has been used to induce transient levels of Ca(2+) and initiate reprogramming of embryos. Ca(2+) oscillation occurs naturally several times after fertilization (several times with 15- to 30-min intervals). This indicates how essential additional Ca(2+) influx is for successful reprogramming of embryos. Hence, in this report, the experimental design was aimed at improving the developmental efficiency of cloned embryos by repetitive Ca(2+) transients rather than the commonly used ionomycin treatment (4 min). To determine optimal Ca(2+) inflow conditions, we performed three different repetitive ionomycin (10 µM) treatments in reconstructed embryos: Group 1 (4-min ionomycin treatment, once), Group 2 (30-sec treatment, 4 times, 15-min intervals) and Group 3 (1-min treatment, 4 times, 15-min intervals). Pronuclear formation rates were checked to assess the effects of repetitive ionomycin treatment on reprogramming of cloned embryos. Cleavage rates were investigated on day 2, and the formation rates of blastocysts (BLs) were examined on day 7 to demonstrate the positive effect of repeated ionomycin treatment. In Group 3, a significant increase in BL formation was observed [47/200 (23.50%), 44/197 (22.33%) and 69/195 (35.38%) in Groups 1, 2 and 3, respectively]. Culturing embryos with different ionomycin treatments caused no significant difference among the groups in terms of the total cell number of BLs (164.3, 158.5 and 145.1, respectively). Additionally, expression of the anti-apoptotic Bcl-2 gene and MnSOD increased significantly in Group 3, whereas the expression of the pro-apoptotic Bax decreased statistically. In conclusion, the present study demonstrated that repeated ionomycin treatment is an improved activation method that can increase the developmental competence of SCNT embryos by decreasing the incidence of apoptosis.
Journal of Reproduction and Development 12/2011; 58(1):132-9. · 1.76 Impact Factor
[show abstract][hide abstract] ABSTRACT: Several mammals, including dogs, have been successfully cloned using somatic cell nuclear transfer (SCNT), but the efficiency of generating normal, live offspring is relatively low. Although the high failure rate has been attributed to incomplete reprogramming of the somatic nuclei during the cloning process, the exact cause is not fully known. To elucidate the cause of death in cloned offspring, 12 deceased offspring cloned by SCNT were necropsied. The clones were either stillborn just prior to delivery or died with dyspnea shortly after birth. On gross examination, defects in the anterior abdominal wall and increased heart and liver sizes were found. Notably, a significant increase in muscle mass and macroglossia lesions were observed in deceased SCNT-cloned dogs. Interestingly, the expression of myostatin, a negative regulator of muscle growth during embryogenesis, was down-regulated at the mRNA level in tongues and skeletal muscles of SCNT-cloned dogs compared with a normal dog. Results of the present study suggest that decreased expression of myostatin in SCNT-cloned dogs may be involved in morphological abnormalities such as increased muscle mass and macroglossia, which may contribute to impaired fetal development and poor survival rates.
Molecular Reproduction and Development 05/2011; 78(5):337-46. · 2.81 Impact Factor
[show abstract][hide abstract] ABSTRACT: Relaxin, a member of the insulin superfamily, has diverse functions in both reproductive and nonreproductive tissues. The aim of the present study was to evaluate the effects of recombinant relaxin on the in vitro maturation of porcine oocytes and their subsequent embryonic development following in vitro fertilization. Three concentrations of relaxin (1, 10, and 100 ng/ml) were used in the in vitro maturation (IVM) medium [TCM supplemented with 10% (v/v) porcine follicular fluid, 10 ng/ml of epidermal growth factor, 4 IU/ml of pregnant mare serum gonadotropin, and (only for the first 22 hr) 4 IU/ml of human chorionic gonadotropin]. Relaxin was used during the entire IVM period. Nuclear maturation of oocytes was examined under ultraviolet light following staining with bisbenzimide (Hoechst 33342) for 5 min and mounted on a glass slide. The glutathione (GSH) content in oocytes, an important indicator of cytoplasmic maturity, was measured using a micro-glutathione assay. Cryopreserved boar semen was used for in vitro fertilization. Embryos were cultured in modified NCSU-23 medium supplemented with 0.5 mM pyruvate and 5 mM lactate. Although nuclear maturation of oocytes did not vary, the GSH content in oocytes was significantly higher when cultured with 1 ng/ml (7.9 pmol/oocyte) and 10 ng/ml (8.47 pmol/oocyte) compared to a control group. However, no additional beneficial effect was observed when 100 ng/ml of relaxin was added to the IVM medium. A significantly higher rate of blastocyst formation was observed with 10 ng of relaxin (32.4%) compared to the control (14.4%) or 100 ng of relaxin (21.4%). No difference between 1 ng and 10 ng was observed in terms of the blastocyst production rate. The inner cell mass cell numbers in relaxin-treated groups were significantly higher than control, and trophectoderm cell number was the highest in the 10 ng relaxin group. Relaxin (10 ng/ml) can be supplemented in IVM medium to support the maturation of porcine oocytes.
Journal of Veterinary Medical Science 12/2009; 72(3):333-7. · 0.88 Impact Factor
[show abstract][hide abstract] ABSTRACT: The present study was undertaken to evaluate two activation methods for somatic cell nuclear transfer (SCNT), namely, fusion and simultaneous activation (FSA, fusion medium contains calcium), versus fusion followed by chemical activation (F+CA, fusion medium does not contain calcium), and to evaluate the effects of parity of recipient dogs on the success of SCNT. Oocytes retrieved from outbred dogs were reconstructed with adult somatic cells collected from an 11-year-old female dog named Missy. In the FSA method, oocytes were fused and activated at the same time using two DC pulses of 1.75 kV/cm for 15 microsec. In the F+CA method, oocytes were fused with two DC pulses of 1.75 kV/cm for 15 microsec, and then activated 1 h after fusion by 10 microM calcium ionophore for 4 m and cultured for 4 h in 1.9 mM 6-dimethylaminopurine for postactivation. Activation method had a significant impact on the production efficiency of cloned dogs. There was a significant difference in full-term pregnancy rate and percentage of live puppies between the two methods (6.3% and 38.5% for FSA and F+CA, respectively). In our study, four out of five live offspring produced by F+CA survived versus FSA, which did not result in any surviving puppies. Overall, as few as 14 dogs and 54 reconstructed embryos were needed to produce a cloned puppy. In addition, the parity of recipient bitches had no effect on the success of SCNT in canine species. Both the nullipara and multipara bitches produced live puppies following SCNT-ET.
Cloning and Stem Cells 03/2009; 11(1):123-30. · 2.66 Impact Factor
[show abstract][hide abstract] ABSTRACT: The present study was undertaken to evaluate two enucleation methods for somatic cell nuclear transfer (SCNT), and to standardize the optimum number of embryos for transfer to each recipient for canines. Oocytes retrieved from outbreed dogs were reconstructed with adult somatic cells from a male Beagle dog. A total of 134 or 267 oocytes were enucleated either by aspiration or squeezing method, fused with two DC pulses of 1.75 kV/cm for 15 micros electrical stimulation, chemically activated after 1h of fusion using 10 microM calcium ionophore for 4 min and cultured 4h in 1.9 mM 6-dimethylaminopurine. Finally, 103 or 214 embryos for aspiration or squeezing method were transferred to 6 or 11 naturally synchronized recipients, respectively. A total of 53, 317 and 342 embryos were transferred to 7, 17 and 12 recipients for the group of 4-10, 11-25 and 26-40 embryos, respectively. There was no difference between fusion rate (76.87% vs. 80.15%), full term pregnancy rate (16.66% vs. 27.27%) and percent of live puppies born (0.97% vs. 1.87%) for aspiration and squeezing method (P>0.05). Production efficiency of cloned dogs was significantly affected by the number of embryos transferred to each recipient. No pregnancy was established for the group of 4-10 embryos (n=7) and 26-40 embryos (n=12) while pregnancy was detected in 23.53% recipients received a group of 11-25 embryos (n=17). Among them, five (1.76%) live puppies were born (P<0.05). These data show an increase in the overall efficiency of SCNT in canine species.
[show abstract][hide abstract] ABSTRACT: To improve the efficiency of somatic cell nuclear transfer (SCNT) in dogs, we evaluated whether or not the interval between fusion and activation affects the success rate of SCNT. Oocytes retrieved from outbred dogs were reconstructed with adult somatic cells from a male or female Golden Retriever. In total, 151 and 225 reconstructed oocytes were transferred to 9 and 14 naturally synchronized surrogates for male and female donor cells, respectively. Chromosomal morphology was evaluated in 12 oocytes held for an interval of 2 hr between fusion and activation and 14 oocytes held for an interval of 4 hr. Three hundred seventy-six and 288 embryos were transferred to 23 and 16 surrogates for the 2 and 4 hr interval groups, respectively. Both the male (two pregnant surrogates gave birth to three puppies) and female (one pregnant surrogate gave birth to one puppy) donor cells gave birth to live puppies (P > 0.05). In the 2 hr group, significantly more reconstructed oocytes showed condensed, metaphase-like chromosomes compared to the 4 hr group (P < 0.05). A significantly higher pregnancy rate and a greater number of live born puppies were observed in the 2 hr group (13.0% and 1.1%, respectively) compared to the 4 hr group (0%) (P < 0.05). In total, three surrogate dogs carried pregnancies to term and four puppies were born. These results demonstrate that decreasing the interval between fusion and activation increases the success rate of clone production and pregnancy. These results may increase the overall efficiency of SCNT in the canine family.
Molecular Reproduction and Development 10/2008; 76(5):483-9. · 2.81 Impact Factor
[show abstract][hide abstract] ABSTRACT: In the present study, we investigated the effect of melatonin on the preimplantation development of porcine parthenogenetic and somatic cell nuclear transfer (SCNT) embryos. Parthenogenetic embryos were cultured in mNCSU-23 supplemented with various concentrations of melatonin for 7 days. The results revealed that 100 pM was the optimal concentration, which resulted in significantly increased cleavage and blastocyst formation rates. Additionally, 100 pM melatonin provided the highest increase in total cell number of blastocysts. Therefore, the subsequent experiments were performed with 100 pM melatonin. ROS level in 2-8 cell stage embryos in the presence or absence of melatonin was evaluated. Embryos cultured with melatonin showed significantly decreased ROS. Blastocysts cultured with melatonin for 7 days were analyzed by the TUNEL assay. It was observed that melatonin not only increased (P < 0.05) the total cell number but also decreased (P < 0.05) the rate of apoptotic nuclei. Blastocysts cultured with melatonin were assessed for the expression of apoptosis-related genes Bcl-xl and Bax, and of pluripotency marker gene Oct-4 by real-time quantitative PCR. Analysis of data showed that the expression of Bcl-xl was higher (1.7-fold) compared to the control while the expression of Bax was significantly decreased relative to the control (0.7-fold) (P < 0.05). Moreover, the expression of Oct-4 was 1.7-fold higher than the control. These results indicated that melatonin had beneficial effects on the development of porcine parthenogenetic embryos. Based on the findings of parthenogenetic embryos, we investigated the effect of melatonin on the development of porcine SCNT embryos. The results also demonstrated increased cleavage and blastocyst formation rates, and the total cell numbers in blastocysts were significantly higher when the embryos were cultured with melatonin. Therefore, these data suggested that melatonin may have important implications for improving porcine preimplantation SCNT embryo development.
Molecular Reproduction and Development 07/2008; 75(7):1127-35. · 2.81 Impact Factor
[show abstract][hide abstract] ABSTRACT: Insulin-transferrin-selenium (ITS) together has been used in different in vitro maturation system to support in vitro maturation of oocytes. The present study was designed to evaluate the effects of ITS in defined (0.1% PVA) and porcine follicular fluid (10% pFF) supplemented IVM media on the developmental competence of porcine oocytes. Three combinations of ITS, 10 mg/L insulin (Ins), 5.5mg/L transferrin (Tf) and 5 microg/L selenium (Se), 20mg/L Ins, 11 mg/L Tf and 10 microg/L Se, and 30 mg/L Ins, 16.5 mg/L TF and 15 microg/L Se, were used. The data were analyzed by one-way ANOVA and Tukey was used as the post hoc test. Both in the defined and pFF supplemented media, higher concentration of intracellular glutathione was observed in presence of ITS (4.6-4.8, and 6.9-7.1 picomole/oocyte for defined and pFF groups, respectively) compared to the respective control (2.1 and 4.3 picomole/oocyte for defined and pFF group, respectively). ITS decreased polyspermy and increased male pronucleus formation in both the defined and pFF supplemented medium. There was no difference in different treatment groups. The highest frequency of blastocyst formation rate and number of cells in blastocyst following IVF and SCNT was observed in pFF+ITS group (p<0.05). In conclusion, ITS addition during IVM improved the developmental competence of porcine oocytes in both the defined and pFF supplemented groups. Thus, we recommended to supplement porcine IVM medium with 10 microg/mL insulin, 5.5 microg/mL transferrin and 5 microg/mL selenium.
[show abstract][hide abstract] ABSTRACT: During follicular development, the importance of the interaction between oocytes and somatic cells is well-known for maturation oocyte. Growth differentiation factor-9 (gdf-9) is thought to play an important role in such process. In this study, we characterized porcine gdf-9 gene and investigate its expression during development. Also we determined its expression by different hormones during in vitro oocyte maturation. Overall length of pig gdf-9 cDNA was cloned and its presumptive protein sequence of isolated cDNA has high homology to human or mouse GDF-9 sequences. The gdf-9 transcript was expressed in all stage of preimplantation embryos and fetuses, and was also expressed in the heart, brain, cumulus cells, spleen, ovary, and testis. Porcine GDF-9 protein was specifically localized in in vitro matured oocyte and oocytes from adult ovary. In order to investigate the potential regulator of gdf-9 gene expression, expression level of gdf-9 gene was determined using serum-free in vitro maturation (IVM) system. Supplementing with human chorionic gonadotrophin (hCG), pregnant mare serum gonadotrophin (PMSG), gonadotropin-releasing hormone (GnRH), follicular stimulating hormone, luteinizing hormone (LH), or epidermal growth factor (EGF) did not affect level of gdf-9 gene expression during ooycte maturation. In conclusion, porcine gdf-9 gene is expressed in the porcine reproductive organ, especially in oocytes and embryos, and its expression is decreased during IVM.
Molecular Reproduction and Development 06/2008; 75(5):707-14. · 2.81 Impact Factor
[show abstract][hide abstract] ABSTRACT: The collection of in vivo matured canine oocytes relies on the accurate prediction of ovulation. The present study was designed to develop a protocol for the recovery of in vivo matured canine oocytes based on once daily measurements of serum progesterone (P(4)) concentrations. Blood samples (2 mL) were collected every day at 0900 h, and P(4) concentrations were analyzed using a DSL-3900 ACTIVE Progesterone Coated-Tube Radioimmunoassay Kit. The average number of oocytes at the metaphase II (M II) stage was significantly higher at or after 72 h (6.7 to 7.5) compared to 56 h (1.7) following ovulation. The highest numbers of corpora lutea, and therefore the highest numbers of oocytes, were recovered from bitches with initial ovulatory P(4) concentrations ranging from 6.0 to 8.0 ng/ mL (12.2 and 11.4, respectively) compared to from 4.0 to 4.9 ng/ mL (9.6 and 8.8, respectively; p < 0.05). The average number of M II oocytes recovered at 84 h from bitches with initial ovulatory P(4) levels of 5.0 to 5.9 ng/mL (7.7) was higher compared to bitches with P(4) levels of 4.0 to 4.9 ng/ mL (3.5) and 6.0 to 8.0 ng/ mL (4.8; p < 0.05). When oocyte recovery time was adjusted for initial ovulatory P(4) concentration, no significant difference in recovery rates or oocyte quality were observed. In conclusion, once daily measurements of P(4) can be used to predict ovulation in bitches, and oocyte recovery time should be adjusted for initial ovulatory serum P(4) concentrations.
Cloning and Stem Cells 06/2008; 10(3):403-8. · 2.66 Impact Factor
[show abstract][hide abstract] ABSTRACT: The restricted supply of oocytes in the domestic dog limits the development of reproductive biotechnologies in this species. Inter-species somatic cell nuclear transfer could be an alternative for cloning animals whose oocytes are difficult to obtain. In this study, the possibility of cloning dog embryos using pig oocytes was investigated by evaluating nuclear remodeling. Chromatin remodeling, assessed by premature chromosome condensation, pseudo-pronuclei formation, DNA methylation and histone acetylation, along with the developmental ability was compared between intra- and inter-species cloned embryos. The incidence of premature chromosome condensation was significantly higher in intra-species cloned embryos relative to inter-species cloned embryos (87.2% vs. 61.7%; P<0.05), but comparable pseudo-pronuclei formation was observed in both (85.3% vs. 75.8%). None of the inter-species cloned embryos developed beyond the 8-cell stage while 18.3% of intra-species cloned embryos developed to the blastocyst stage. The relative level of both DNA methylation and histone acetylation was similar between intra- and inter-species cloned embryos at all times examined. These results suggest that although partial chromatin remodeling occurs, further investigation is needed to be able to use pig oocytes as recipient oocytes in dog cloning.
[show abstract][hide abstract] ABSTRACT: Various thiol compounds are known to improve cytoplasmic and/or nuclear maturation of oocytes in vitro. The present study examined the effects of two thiol compounds, cysteine (0.1, 0.5, and 1.0 mM) and cysteamine (50, 100, and 200 microM), on cytoplasmic and nuclear maturation of canine oocytes. Oocytes collected from different reproductive stages were cultured in TCM-199 supplemented with 10% fetal bovine serum, 2.2 mg/ml sodium carbonate, 2.0 microg/ml estrogen, 0.5 microg/ml FSH, 0.03 IU/ml hCG, and 1% penicillin-streptomycin solution for 72 h. Data were analyzed by two-way ANOVA after arcscine transformation and protected by Bonferroni post hoc test. The effects of cysteine and cysteamine on canine IVM were varied depending on the reproductive stage of oocyte donor bitches. In the follicular stage, significantly more oocytes reached the metaphase II (M II) stage when cultured with 0.5 or 1.0 mM cysteine (16.7% and 16.9%, respectively) compared to the control (6.2%). In the follicular stage, cysteamine increased oocyte maturation rate upto the M II stage (15.1% to 17.0%) compared to the control (4.4%). Both the 0.5 mM cysteine and 100 microM cysteamine, alone or together, increased the intracellular GSH level of canine oocytes compared to the control. Irrespective of reproductive stage, no further beneficial effects on nuclear or cytoplasmic maturation were observed when 0.5 mM cysteine and 100 microM cysteamine were supplemented together. In conclusion, addition of 0.5 mM cysteine and 100 microM cysteamine to the maturation medium improved IVM of canine oocytes.
Molecular Reproduction and Development 10/2007; 74(9):1213-20. · 2.81 Impact Factor