Effect of insulin,transferrin and selenium and epidermal growth factor on development of buffalo oocytes to the blastocyst stage in vitro in serum-free,semidefined media

The in vitro development of buffalo oocytes up to the blastocyst stage was studied in serum-free, semidefined media containing bovine serum albumin, follicle-stimulating hormone (FSH), insulin, transferrin and selenium (ITS) and epidermal growth factor (EGF). In experiment 1, oocytes aspirated from abattoir-derived ovaries were cultured in eight serum-free, semidefined culture media containing different combinations of these four factors. In experiment 2, the maturation of buffalo oocytes and the development of the embryos were compared in a complex co-culture system and in the serum-free, semidefined media. Supplementation with FSH and EGF significantly (P < 0.05) increased the maturation rates of buffalo oocytes, and the yield of blastocysts was higher (P < 0.05) in media containing EGF and ITS. The yield of blastocysts was lower in the serum-free semidefined media (P < 0.05) than in the complex co-culture system.     

Influence of cysteine in conjunction with growth factors on the development of in vitro-produced bovine embryos

Cysteine supplementation to in vitro maturation (IVM) media of bovine oocytes increases cellular glutathione production. Beneficial effects of growth factors for improving the rate of blastocyst development have been reported, but combined effects are unknown. This study was conducted to determine the additive effect of cysteine with epidermal growth factor (EGF) and/or insulin-like growth factor-I (IGF-I) on embryo development. Bovine oocytes from slaughterhouse ovaries were matured in TCM-199 (control), with or without the addition of 0.6 mm cysteine (C) at 0 or 12 h of maturation. After in vitro fertilization, embryos were allocated to culture treatments containing synthetic oviductal fluid medium. Culture treatments included fetal calf serum (FCS, 4%) alone; IGF-I (100 ng/ml); EGF (10 ng/ml); and IGF-I + EGF (100 + 10 ng/ml). Although rates for blastocysts development were not different among treatments, an increased proportion of embryos attaining morula formation was achieved when cysteine was added to the maturation media (12 h C IGF-I + EGF, 41.4%; 0 h C EGF, 40.0%) as compared to control (FCS: 34.6%). When cysteine treatments were combined, percent cleavage was greater for IGF-I + EGF (70.8%) compared to FCS (61.2%). The abundance of mRNA from the apoptotic genes, Bax and Bcl-2, and the oxidative stress genes, copper (Cu)-zinc (Zn) superoxide dismutase (SOD) and manganese (Mn) SOD in embryos was assessed. No treatment effect was observed on the expression of these genes. In conclusion, supplementation of cysteine during IVM of oocytes, in conjunction with growth factors could effectively be used as a replacement for FCS.     

Leucemia inhibitory factor; investigating the time‐dependent effect on viability of vitrified bovine embryos

Leucemia inhibitory factor (LIF) is involved in various reproductive processes, including sperm development, regulation of ovulation, as well as blastocyst formation, hatching and implantation in embryos. Moreover, LIF has also been shown significantly to enhance the blastocyst formation rates of bovine embryos, a finding that remains controversial. Our purpose was to investigate time‐dependent effect of LIF on bovine embryo culture, especially in terms of addition timing. Presumptive zygotes were cultured in five different groups. In this study, 100 ng/ml LIF was added to the culture medium were as follows; control: SOF alone, group A: at day 0 (fertilization day), group B: at day 4 post‐insemination (p.i.), group C: at day 4 to 7 (p.i. before vitrification) and group D: at day 8 (p.i. after thawing). Addition of LIF to the culture medium at day 4 significantly increased the percentage of blastocyst rate when compared day 0, day 4 at 6/7 and control group (41.8% versus 24.3%, 19.7%, 34.6%). In conclusion, the addition of LIF only on day 4 (p.i.) to the culture medium was found to be beneficial for bovine embryonic development based on several measures, including blastocysts rate, re‐expansion rate and cellular cryotolerance after vitrification.     

Lipid-rich bovine serum albumin improves the viability and hatching ability of porcine blastocysts produced in vitro

The effects of lipid-rich bovine serum albumin (LR-BSA) on the development of porcine blastocysts produced in vitro were examined. Addition of 0.5 to 5 mg/ml LR-BSA to porcine blastocyst medium (PBM) from Day 5 (Day 0 = in vitro fertilization) significantly increased the hatching rates of blastocysts on Day 7 and the total cell numbers in Day-7 blastocysts. When Day-5 blastocysts were cultured with PBM alone, PBM containing LR-BSA, recombinant human serum albumin or fatty acid-free BSA, addition of LR-BSA significantly enhanced hatching rates and the cell number in blastocysts that survived compared with other treatments. The diameter, ATP content and numbers of both inner cell mass and total cells in Day-6 and Day-7 blastocysts cultured with PBM containing LR-BSA were significantly higher than in blastocysts cultured with PBM alone, whereas LR-BSA had no effect on mitochondrial membrane potential. The mRNA levels of enzymes involved in fatty acid metabolism and β-oxidation (ACSL1, ACSL3, CPT1, CPT2 and KAT) in Day-7 blastocysts were significantly upregulated by the addition of LR-BSA. The results indicated that LR-BSA enhanced hatching ability and quality of porcine blastocysts produced in vitro, as determined by ATP content, blastocyst diameter and expression levels of the specific genes, suggesting that the stimulatory effects of LR-BSA arise from lipids bound to albumin.     

Melatonin Supplementation During In Vitro Maturation and Development Supports the Development of Porcine Embryos

Melatonin has been reported to improve the in vitro development of embryos in some species. This study was conducted to investigate the effect of melatonin supplementation during in vitro maturation (IVM) and development culture on the development and quality of porcine embryos. In the first experiment, when the in vitro fertilized embryos were cultured with different concentrations of melatonin (0, 10, 25 and 50 ng/ml) for 8 days, the blastocyst formation rate of embryos cultured with 25 ng/ml melatonin (10.7%) was significantly increased (p < 0.05) compared to the control embryos cultured without melatonin (4.2%). The proportion of DNA‐fragmented nuclei in blastocysts derived from embryos cultured with 50 ng/ml melatonin was significantly lower (p < 0.05) than that of embryos cultured without melatonin (2.1% vs 7.2%). In the second experiment, when oocytes were cultured in the maturation medium supplemented with different concentrations of melatonin (0, 10, 25 and 50 ng/ml), fertilized and then cultured with 25 ng/ml melatonin for 8 days, there were no significant differences in the rates of cleavage and blastocyst formation among the groups. However, the proportions (2.7–5.4%) of DNA‐fragmented nuclei in blastocysts derived from oocytes matured with melatonin were significantly decreased (p < 0.05) compared to those (8.9%) from oocytes matured without melatonin, irrespective of the concentration of melatonin. Our results suggest that supplementation of the culture media with melatonin (25 ng/ml) during IVM and development has beneficial effects on the developmental competence and quality of porcine embryos.     

Normal calves produced after transfer of embryos cultured in a chemically defined medium supplemented with epidermal growth factor and insulin-like growth factor I following ovum pick up and in vitro fertilization in Japanese black cows

The objective of this study was to examine whether high concentrations of epidermal growth factor (EGF) and/or insulin-like growth factor I (IGF-I) would have a beneficial effect on bovine embryo development in vitro and to obtain normal calves by using an ovum pick up method and embryo culture in a chemically defined medium. When compared with controls, EGF (100 or 200 ng/ml) or IGF-I (50 or 100 ng/ml) significantly increased the rate of embryos that developed into blastocysts during an 8-day culture after the in vitro fertilization of oocytes obtained from ovaries from a slaughterhouse. IGF-I induced a dose-dependent increase in cell number in both the inner cell mass and the trophectoderm, whereas EGF stimulated proliferation only in the inner cell mass. A combination of EGF (100 ng/ml) and IGF-I (50 ng/ml) produced an additive effect, and embryos developed into blastocysts at a comparatively high rate (27.9%) compared with controls (12.0%). A similar rate of development was achieved using a combination of EGF and IGF-I in the culture of embryos following ovum pick up by ultrasound-guided transvaginal follicular aspiration and in vitro fertilization, and 5 blastocysts that developed after the culture were transferred into uteri; two embryos implanted, and normal calves were born. These results suggest that the combined use of EGF and IGF-I makes bovine embryo culture in a chemically defined medium a practical and useful procedure for producing blastocysts, and its application to embryo culture following ovum pick up and in vitro fertilization could be useful for producing normal calves.     

Effect of ovine granulocyte-macrophage colony-stimulating factor on bovine in vitro embryo development and blastocyst interferon-τ secretion

This experiment examined the effects of including recombinant ovine granulocyte-macrophage colony-stimulating factor (GMCSF) in in vitro culture on secretion of interferon-τ (IFNT) by bovine blastocysts. At 32 h post-insemination (p.i.), cleaved bovine zygotes were selected and incubated with or without GMCSF for either 48 h only (between 32 and 80 h p.i., Early) or until day 9 p.i. (Throughout). Concentrations of GMCSF (ng/ml) examined were as follows: Experiment 1: 2, 5, 10 and 50 (Early only); Experiment 2: 50 (Early and Throughout); Experiment 3: 2 and 10 (Early and Throughout). In none of the experiments did GMCSF have an effect (p > 0.05) on the numbers of blastocysts formed or blastocyst characteristics as assessed by cell number, proportion of apoptotic cells or oxidation of pyruvate. When GMCSF was included in culture medium between 32 and 80 h p.i. (Early), IFNT concentrations were lower (in media drops recovered after culture of groups of embryos for 48 h between days 7 and 9 p.i. and normalized by the numbers of blastocysts developing within each drop) compared to no inclusion of GMCSF or GMCSF present Throughout culture (Experiment 2, p > 0.05; Experiment 3, p = 0.038). IFNT was present in media drops in which groups of embryos had been incubated between days 7 and 9 p.i. but in which no blastocysts had developed. Experimental treatment did not influence (p > 0.05) IFNT secretion by blastocysts incubated individually for 24 h. However, during the 24-h individual culture, blastocysts recovered on day 7 secreted less IFNT than blastocysts recovered on day 8 (mean ± SE; 15 ± 1.3 v 30 ± 3.6 pg/ml; p < 0.001). In conclusion, in contrast to previous studies in the ovine, GMCSF did not increase IFNT secretion but in agreement with the ovine did not affect bovine blastocyst development.     

Effects of purple sweet potato anthocyanins on development and intracellular redox status of bovine preimplantation embryos exposed to heat shock

The development of cleavage stage preimplantation embryos is disrupted by exposure to heat shock, such as high temperatures in the summer season. In this study, we investigated whether addition of anthocyanins, which are strong scavengers of reactive oxygen species (ROS), improves development and intracellular redox status of heat-exposed bovine preimplantation embryos by reduction of heat shock-derived oxidative stress. After in vitro fertilization (IVF), embryos were cultured at 38.5 C through Day 8 (Day 0=day of IVF) with 0, 0.1, 1 and 10 microg/ml anthocyanins (non-heat-shocked group). On Day 2, embryos were cultured at 41.5 C for 6 h with 0, 0.1, 1 and 10 microg/ml anthocyanins followed by culture at 38.5 C until Day 8 (HS group). After exposure to heat shock, the intracellular ROS and glutathione (GSH) contents of individual embryos were measured in the non-heat-shocked and HS groups using fluorescent probes. On Day 8, the blastocysts formation rates of the embryos and total cell numbers of blastocysts were evaluated. Embryos exposed to heat shock without anthocyanins showed a significant decrease in blastocyst formation rate and GSH content (P<0.05) and an increase in intracellular ROS (P<0.05) compared with non-heat-shocked embryos. In contrast, addition of 0.1 microg/ml anthocyanins significantly (P<0.05) improved the blastocyst formation rate of the heat-shocked embryos. Addition of any dose of anthocyanins produced a significant decrease in the ROS levels (P<0.05) and tended to increase the GSH levels under heat-shock conditions. However, addition of higher concentrations (1 and 10 microg/ml) of anthocyanins to the culture media under heat shock did not improve the development of embryos. These results indicate that anthocyanins maintain the intracellular redox balance of heat-shocked bovine embryos by reducing intracellular oxidative stress and increasing the GSH levels. Thus, alterations of the redox state using natural antioxidative polyphenols is a useful approach for reducing heat shock-derived oxidative stress.     

Effects of ghrelin on in vitro development of porcine in vitro fertilized and parthenogenetic embryos

The objective of the present study was to investigate the effect of addition of ghrelin to in vitro culture medium on preimplantation development of porcine in vitro fertilized and parthenogenetic embryos. In Experiment 1, we sought to compare the in vitro developmental competence of IVF and parthenogenetic embryos. No significant (P<0.05) differences were detected for cleavage rate or blastocyst rate between the in vitro fertilization (IVF)- and parthenogenetic activation-derived embryos. In Experiment 2, parthenogenetic embryos were cultured in Porcine Zygote Medium-3 containing various concentrations of ghrelin. The blastocyst rate was remarkably (P<0.05) increased when 5 ng/ml (PA-5) and 500 ng/ml (PA-500) of ghrelin was added to in vitro culture medium compared with the other groups. Total cell number per blastocyst was slightly promoted in the ghrelin treatment groups compared with the controls. However, the ratio of inner cell mass (ICM) cell number/total cell number was significantly reduced in the PA-50 group compared with the controls (P<0.05). In Experiment 3, we cultured in vitro fertilized embryos in Porcine Zygote Medium-3 supplemented with ghrelin at different dosages. The rate of blastocyst formation was markedly (P<0.05) elevated when 500 ng/ml ghrelin was added to culture medium (IVF-500) compared with the controls. Increased total cell numbers (P<0.05) were observed when in vitro fertilized embryos were cultured in IVF-50 and IVF-500 compared with the controls. However, the ratio of ICM cell number/total cell number was decreased in the ghrelin treatment groups compared with the controls (P<0.05). Taken together, the results suggest that ghrelin can enhance blastocyst formation of porcine in vitro fertilized and parthenogenetic embryos while exerting a negative effect on the structural integrity of the blastocysts.     

提高猪囊胚后期贴壁能力的优化培养体系

为了提高猪孤雌囊胚贴壁率,试验从饲养层及培养液两方面研究猪孤雌囊胚贴壁能力;用小鼠、猪和牛的胎儿成纤维细胞制作饲养层,分别添加DMEM、NCSU-23、DMEM/NCSU-23培养液,探讨猪孤雌囊胚在3种饲养层上的发育效果。结果表明,BEF饲养层能更好地促进猪孤雌囊胚贴壁生长,其囊胚贴壁率为33.67%,与MEF饲养层组的囊胚贴壁率(19.08%)之间差异显著(P0.05),与PEF饲养层组之间囊胚贴壁率差异不显著(P0.05),MEF饲养层组和PEF饲养层组之间囊胚贴壁率差异不显著(P0.05);在BEF牛胎儿成纤维细胞饲养层组,用猪胚胎培养液NCSU-23培养猪孤雌囊胚后,囊胚贴壁率(22.53%)显著高于DMEM培养液组(10.41%)和DMEM/NCSU-23培养液半量混合组(12.05%)(P0.05),DMEM培养液组和DMEM/NCSU-23培养液半量混合组之间差异不显著(P0.05)。牛胎儿成纤维细胞饲养层和猪胚胎培养液NCSU-23能更好地促进猪孤雌囊胚后期贴壁。     

Effect of nerve growth factor (NGF) on the development of preimplantation rabbit embryos <Emphasis Type="Italic">in vitro</Emphasis>

This study aimed to investigate the effect of nerve growth factor (NGF) on the development of preimplantation rabbit embryos in vitro. Zygotes were collected from superovulated New Zealand rabbits 19 h after injection of hCG and immediately mating and cultured in TCM-199 plus fatty-acid free BSA with different concentrations of NGF. Zygotes not treated with NGF served as control. At 24 h, 48 h, 72 h and 96 h of the culture, the numbers of the early cleavage stage, morulae, blastocysts and hatching blastocysts were determined. The intrazonal diameter of the blastocyst and the total cell numbers per blastocyst were measured after 96 h of culture. The results showed: (1) NGF at 100 ng/mL and 1000 ng/mL could improve the numbers of the hatching blastocysts which developed compared to the control treatment (p < 0.05); (2) All concentrations of NGF increased the total cell numbers in the blastocysts compared to the control treatment (p < 0.05); (3) NGF had no significant effect on the blastocyst intrazonal diameter of the blastocysts at 96 h of culture (p = 0.493); (4) The proportion in the early cleavage stage at 24 h of culture (p = 0.635), of morulae at 48 h of culture (p = 0.812) and of blastocysts at 72 h of culture (p = 0.812) in all treatments were not significantly different.     

Effect of phosphonoformic acid in the development of bovine embryos in vitro

This research evaluated the ability of phosphonoformic acid to inhibit bovine herpesvirus 1 (BHV-1) in cumulus cells commonly used in co-culture with bovine in vitro-produced embryos. At 200 and 400 microg/ml, phosphonoformic acid inhibited 4 logs of BHV-1. Subsequently, phosphonoformic acid (200 and 400 microg/ml) added to both in vitro fertilization and culture medium resulted in a decrease in the proportion of developed blastocysts, and the number of cells per blastocyst was lower in the treated embryos. Therefore, while phosphonoformic acid can effectively inhibit replication of BHV-1 in co-culture cells, it also inhibits development of in vitro-produced bovine embryos.     

In vitro development of IVF-derived bovine embryos following cytoplasmic microinjection for the episomal expression of the IGF2 gene

Important genomic imprinting changes usually occur following the in vitro production (IVP) of bovine embryos, especially in the imprinting pattern of components of the IGF system. This study aimed to evaluate the effects of a transient episomal overexpression of the IGF2 gene in bovine IVP embryos following embryo cytoplasmic microinjection (CMI) at the 1-cell stage on embryo survival, early and late developmental kinetics and morphological quality up to Day 7 of development. Selected cumulus–oocyte complexes (COCs) were matured and fertilized in vitro and subsequently segregated into six experimental groups: non-CMI control group and five CMI groups at increasing doses (0, 10, 20, 40 and 80 ng/μl) of a GFP vector built for the episomal expression of bovine IGF2. Zygote CMI was effective in delivering the expression vector into the ooplasm, irrespective of the groups, with 58% of positive GFP fluorescence in Day 7 blastocysts. Considering developmental rates and late embryo kinetics, the 10-ng/μl CMI vector dose promoted a lower blastocyst rate (10.4%), but for blastocysts at more advanced stages of development (93.0% blastocysts and expanded blastocysts), and higher number of cells (116.0 ± 3.0) than non-CMI controls (23.3%, 75.0% and 75.0 ± 6.8 were obtained, respectively). In conclusion, CMI at the 1-cell stage did not compromise subsequent in vitro development of surviving embryos, with the 10-ng/μl group demonstrating a possible growth-promoting effect of the IGF2 gene on embryo development, from the 1-cell to the blastocyst stage.     

Effect of Human Menopausal Gonadotropin,17β-estradiol and Epidermal Growth Factor on the Quality of in vitro Maturation Bovine Oocytes

For optimizing in vitro maturation system of bovine oocytes,we firstly examined the influence of four different hormonal regimes(FSH+LH,HMG,FSH+LH+E₂ and HMG+E₂) on oocyte maturation rates.Then we studied the effects of epidermal growth factor (EGF) in the above defined medium on bovine oocyte maturation,in vitro development and quality of parthenogenetic embryos.The cell apoptotic index of parthenogenetic blastocysts was detected by TUNEL.No significant difference was observed in maturation rates in four groups supplemented with different hormones.However,human menopausal gonadotropin (HMG) provided steady maturation results in replicates.Maturation of oocytes was promoted by supplementation with 17β-estradiol (E₂).Combination of HMG and E₂ gave rise to steady and efficient mature results.The presence of EGF at 30 ng/mL concentration significantly increased maturation rate and blastocyst rate and reduced apoptotic cells in parthenogenetic blastocysts.Therefore,the optimal oocyte maturation solution could be supplemented with 0.075 IU/mL HMG,1 μg/mL E₂ and 30 ng/mL EGF.     

Effects of Acetoacetate on in vitro Development of Bovine Embryos in Medium Containing Citrate and Myo-inositol

This study investigated bovine embryo development in vitro in the presence of acetoacetate in serum-free medium. In vitro -matured and fertilized oocytes from ovaries of slaughtered cows were cultured in synthetic oviduct fluid (SOF) containing citrate, myo-inositol, lactate and pyruvate. In the medium with acetoacetate this compound replaced both lactate and pyruvate as energy sources. Three experiments were carried out: (1) to test development in medium with acetoacetate and bovine serum albumin; (2) to analyse the effects of acetoacetate that were dependent upon citrate and myo-inositol; and (3) to determine the effects of acetoacetate in the presence of serum. Blastocyst development was recorded at day 8 and the number of cells of expanded blastocysts obtained were counted. Blastocysts development was reduced in medium with 1.8, 3.6 or 7.2 m M acetoacetate in comparison with the control with or without lactate and pyruvate. The detrimental effect of acetoacetate was independent of the presence of citrate and myo-inositol, but serum added to culture medium protected against this effect. Citrate and myo-inositol did not improve blastocyst formation. Morphological quality and cell number of blastocysts were similar between groups.     

In vitro culture conditions using chemically defined media for in vitro matured and intracytoplasmically inseminated porcine oocytes

The present study investigated in vitro culture methods [droplet and Well of the Well (WOW)] using semi-defined and defined media [modified porcine zygote medium (mPZM)] and the additional effects of insulin on in vitro matured and intracytoplasmically inseminated porcine oocytes. In Experiment 1, in vitro matured and intracytoplasmically inseminated porcine oocytes were cultured for 6 days in the following four groups: 1) mPZM-3 (containing bovine serum albumin) + droplet (30 mul), 2) mPZM-3 + WOW, 3) mPZM-4 (containing polyvinyl alcohol) + droplet, and 4) mPZM-4+ WOW. The culture media (mPZM-3 and mPZM-4) and methods (droplet and WOW) did not significantly affect the cleavage rate, but the blastocyst rate of the oocytes cultured in mPZM-3 was significantly (P<0.01) higher than that of mPZM-4 (20.1 and 9.4%, respectively). The blastocyst rates as percentages of the cleaved oocytes (51.8 and 16.9%) and the hatched blastocyst rate as percentages of the number of blastocysts (12.3 and 2.2%) were also significantly (P<0.01) higher in mPZM-3 compared with those in mPZM-4. There was significant interaction (P<0.05) between the two main factors; the effects of the culture media and methods on the rate of hatched blasyocysts as percentages of the blastocysts produced and, the hatched blastocyst rate (20.3%) as percentages of the number of blastocysts produced in mPZM-3 were significantly (P<0.05) higher than in the other groups. In Experiment 2, the additional effects of insulin (100 ng/ml) in mPZM-3 and mPZM-4 media was investigated in the WOW culture system. Insulin addition did not improve cleavage, blastocyst formation, or the number of cells in blastocysts. However, as in Experiment 1, mPZM-3 resulted in a significantly higher blastocyst rate as percentages of the cleaved oocytes than mPZM-4 (33.9 and 18.4%). These results indicate that a chemically defined medium (mPZM-4) needs to be improved to provide more suitable culture conditions for in vitro development of in vitro matured and intracytoplasmically inseminated porcine oocytes. However, the WOW system may be a useful IVC method for blastocyst development of in vitro matured porcine oocytes following ICSI when a semi-defined medium (mPZM-3) is used.     

人尿促性腺激素、雌二醇及表皮生长因子对牛卵母细胞体外成熟质量的影响

试验旨在研究不同激素配比及表皮生长因子(EGF)浓度对牛卵母细胞体外成熟及卵母细胞质量的影响。将随机分组的卵丘-卵母细胞复合体于添加FSH+LH、HMG、FSH+LH+E₂、HMG+E₂ 4种不同激素组合配比的成熟基础液中培养,对比其体外成熟率,比较了EGF对牛卵母细胞体外成熟率和孤雌胚胎体外发育的影响,并采用TUNEL法检测添加不同浓度EGF的牛孤雌激活囊胚细胞凋亡情况。结果表明,添加HMG的成熟试验结果稳定,E₂对牛卵母细胞成熟有一定的促进作用,HMG+E₂联合使用可以得到高效稳定的成熟结果;在此基础上,在成熟液中添加30 ng/mL EGF对牛卵母细胞的成熟质量、胚胎发育及降低胚胎细胞凋亡都有明显的促进作用。因此,在体外成熟培养液中添加0.075 IU/mL HMG、1 μg/mL E₂和30 ng/mL EGF对牛卵母细胞的成熟和质量较为有益。     

Expression levels of mRNA-encoding PDGF receptors in goat ovaries and the influence of PDGF on the in vitro development of caprine pre-antral follicles

The aims of this study were to investigate the expression levels of mRNA for platelet-derived growth factor (PDGF) receptors (PDGFR-α and -β) in caprine follicles at different developmental stages and to evaluate the influence of PDGF on the in vitro development of pre-antral follicles. For this, goat primordial, primary and secondary follicles, as well as small (1-3 mm) and large (3-6 mm) antral follicles, were obtained, and PDGFR-α and -β mRNA levels were quantified by real-time PCR. Furthermore, pre-antral follicles (≥ 200 μm) were isolated from goat ovaries and cultured for 18 days in α- minimum essential medium supplemented with PDGF at 50 or 100 ng/ml, containing or not FSH. Real-time PCR showed highest PDGFR-α mRNA levels in secondary follicles, while PDGFR-β mRNA levels were highest in primary follicles onwards. Both receptors showed higher mRNA levels in granulosa/theca cells from small and large antral follicles than in their corresponding cumulus-oocyte complexes. In culture, the percentage of antrum formation was significantly higher in 100 ng/ml PDGF compared with the same PDGF concentration associated with FSH. After 18 days, PDGF in both concentrations associated with FSH promoted follicular growth significantly higher than the control. Moreover, the addition of FSH to 50 ng/ml PDGF positively influenced the follicular growth when compared with the same PDGF concentration in the absence of FSH. In conclusion, PDGF is important for early goat folliculogenesis, because the presence of PDGFR-α and -β mRNA was detected in all follicular categories, and PDGF associated with FSH stimulated the growth of goat pre-antral follicles isolated and cultured in vitro.     

注入嵌合法制作黄牛和水牛种间嵌合胚的初步研究

采用注入嵌合法初步建立了一套黄牛和水牛种间嵌合的程序与方法。采用机械剥离法或免疫外科法分离胚胎内细胞团(ICM),然后注入到已去除ICM的受体囊胚中构建形成水牛和黄牛的嵌合胚。结果发现,在用免疫外科法分离ICM时,抗血清的灭活温度从57℃升至63.5℃,ICM的获得数显著升高(0%vs100%,P<0.01),如若在分离培养液中添加6%的胎牛血清(FCS),ICM的获得数大大降低(97.6%vs0%,P<0.01)。采用免疫外科法分离得到的黄牛ICM进行水牛囊胚的ICM置换重组,重组胚的存活率与机械剥离法得到的ICM无显著差异(91.4%vs87.5%,P>0.05);但囊胚孵化率则显著提高(80%vs43.8%;P<0.05)。以上结果表明,⑴水牛和黄牛胚胎通过ICM置换获得的种间嵌合胚胎能继续发育;⑵用于黄牛ICM分离的兔抗牛抗血清需在63.5℃灭活30min,且分离需在无血清的培养液中进行;⑶通过分离ICM置换进行胚胎嵌合时,免疫外科法优于显微手术法。     

The role of insulin, glucagon, dexamethasone, and leptin in the regulation of ketogenesis and glycogen storage in primary cultures of porcine hepatocytes prepared from 60 kg pigs

A study was conducted to elucidate hormonal control of ketogenesis and glycogen deposition in primary cultures of porcine hepatocytes. Hepatocytes were isolated from pigs (54-68 kg) by collagenase perfusion and seeded into collagen-coated T-25 flasks. Monolayers were established in medium containing fetal bovine serum for 1 day and switched to a serum-free medium for the remainder of the culture period. Hepatocytes were maintained in DMEM/M199 containing 1% DMSO, dexamethasone (10(-6) or 10(-7) M), linoleic acid (3.4 x 10(-5) M), and carnitine (10(-3) M) for 3 days. On the first day of serum-free culture, insulin was added at 1 or 100 ng/ml and glucagon was added at 0, 1, or 100 ng/ml. Recombinant human leptin (200 ng/ml) was added during the final 24 h; medium and all cells were harvested on the third day. Concentrations of acetoacetate and beta-hydroxybutyrate (ketone bodies) in media and glycogen deposition in the cellular compartment were determined. Ketogenesis was highly stimulated by glucagon (1 and 100 ng/ml) and inhibited by insulin. In contrast, glycogen deposition was stimulated by insulin and attenuated by glucagon; high insulin was also associated with a reduction in the ketone body ratio (acetoacetate:beta-hydroxybutyrate). High levels of dexamethasone stimulated ketogenesis, but inhibited glycogen deposition at low insulin. Culture of cells with leptin for 24 h, over the range of insulin, glucagon, and dexamethasone concentrations had no effect on either glycogen deposition or ketogenesis. These data suggest that while adult porcine hepatocytes are indeed sensitive to hormonal manipulation, leptin has no direct influence on hepatic energy metabolism in swine.