Blastocyst production by in vitro maturation and development of porcine oocytes in defined media following intracytoplasmic sperm injection.

The present study was carried out to establish porcine defined IVP. In Experiments 1 and 2, we investigated the efficacy of additional 0.6 mM cystine and/or 100 microM cysteamine (Cys) to a defined TCM199 maturation medium with regard to the intracellular glutathione (GSH) concentration and the developmental competence of in vitro matured porcine oocytes following intracytoplasmic sperm injection (ICSI). The control medium was a modified TCM199 containing 0.05% (w/v) polyvinyl alcohol (PVA). Cys and/or cystine were added to the control medium. The control group and immature oocytes (presumptive germinal vesicle oocytes; GV) were prepared for GSH assay. In Experiment 3, the efficacy of epidermal growth factor (EGF) addition to a modified porcine zygote medium (mPZM) for in vitro culture (IVC) medium was investigated on embryonic development and the mean cell number of blastocysts following ICSI. As a positive or negative control, 0.3% BSA (mPZM-3) or 0.3% PVA (mPZM-4), respectively, was added to the base medium. The defined IVC medium was supplemented with 5 or 10 ng/ml EGF. In Experiment 1, no significant difference was found in the rates of cleavage (31.4-64.3%) and blastocyst formation (6.5-22.9%) among the treatment and control groups. The mean cell numbers per blastocyst ranged from 30 to 48 among the groups without significant differences. However, in Experiment 2, the intracellular GSH concentrations in the oocytes cultured in the medium supplemented with 100 microM Cys (9.6 pmol/oocyte) or Cys + cystine (9.9 pmol/oocyte) were significantly (p < 0.05) higher than the control (2.5 pmol/oocyte) and 0.6 mM cystine (6.5 pmol/oocyte) groups, but not different from the GV group (9.0 pmol/oocyte). The GSH concentration in the cystine group was also significantly (p < 0.05) higher than that in the control group, but not different from the GV group. In Experiment 3, the rates of cleavage and blastocyst formation and the mean cell numbers of blastocysts were not significantly different among the groups. However, the addition of 5 ng/ml EGF into the mPZM-4 resulted in a significantly (p < 0.05) higher blastocyst rate per cleaved embryo than the other two defined groups (mPZM-4 + 5 ng/ml: 48.6%, mPZM-4 and mPZM-4 +10 ng/ml: 23.4% and 23.1%, respectively). The present results indicate that the addition of Cys to a defined medium for in vitro maturation (IVM) of porcine oocytes increases intracellular GSH concentration. Further addition of cystine into the IVM medium containing 100 microM Cys is not necessary and TCM199 plus Cys (100 microM) could be used as a defined IVM medium for porcine oocytes. The addition of 5 ng/ml EGF to a defined IVC medium has enhanced subsequent development after ICSI. This study shows that porcine blastocysts can be produced by defined media throughout the steps of IVP (IVM, ICSI and IVC).

Kobayashi M ，Asakuma S ，Fukui Y 《ZYGOTE》

In vitro maturation of porcine oocytes using a defined medium and developmental capacity after intracytoplasmic sperm injection.

To establish a defined in vitro maturation culture system for porcine oocytes, we examined the effects of adding cysteine (Cys) and epidermal growth factor (EGF) to the maturation medium. Furthermore, to evaluate cytoplasmic maturation, we investigated GSH concentrations and embryo development after intracytoplasmic sperm injection (ICSI). The basic media for IVM were modified TCM199 containing 10% newborn calf serum (NBCS) or 0.1% polyvinyl alcohol (PVA), supplemented with amino acids. Adding EGF (10 ng/ml) or EGF + Cys (0.57 mM) to the defined medium (0.1% PVA + amino acids) increased (P < 0.05) the rate of nuclear maturation relative to the defined medium (without these additives). After ICSI, oocytes matured in a medium supplemented with NBCS, Cys and EGF had a higher (P < 0.05) rate of pronuclear formation rate than oocytes matured in the defined IVM medium. Although there was no significant difference in cleavage rates between NBCS- and PVA-containing media supplemented with both Cys and EGF, the rate of blastocyst development was lower (P < 0.05) in the defined medium than in the NBCS-containing medium. Intracellular GSH concentrations of oocytes matured in the NBCS- and PVA-containing media supplemented with both Cys and EGF were higher (P < 0.05) than in oocytes matured in PVA alone or in oocytes before maturation. Adding Cys and EGF to a defined medium for porcine IVM improved rates of nuclear maturation and cleaved oocytes following ICSI, probably due to increased GSH concentrations. Also, embryos derived from oocytes matured in the defined medium (with the addition of Cys and EGF) developed into blastocysts after ICSI.

Kishida R ，Lee ES ，Fukui Y 《THERIOGENOLOGY》

Cysteamine or beta-mercaptoethanol added to a defined maturation medium improves blastocyst formation of porcine oocytes after intracytoplasmic sperm injection.

The present study was carried out to investigate the effect of adding 100 microM cysteamine (Cys) or 100 microM beta-mercaptoethanol (beta-ME) to a defined maturation medium on in vitro maturation (IVM), and fertilization and developmental competence of in vitro matured porcine oocytes following intracytoplasmic sperm injection (ICSI). The two control media for IVM culture were modified TCM199 containing 10% (v/v) porcine follicular fluid (pFF) or 0.05% (w/v) polyvinyl alcohol (PVA), and Cys or beta-ME was supplemented to the PVA-control medium. There was no significant difference in the proportions of in vitro matured oocytes among the four treatment groups (94.5-98.4%). The percentages of pronuclear formation (51.0-64.2%) after ICSI were also not significantly different among the four groups. The cleavage rate (72.8%) in the oocytes treated with Cys showed no significant difference compared with those of the two control media containing pFF (72.2%) or PVA (61.5%), but was higher (P<0.05) than that in the oocytes treated with beta-ME (56.3%). However, the rates of blastocyst formation of Cys (36.7%), beta-ME (27.1%) and pFF (31.4%) were higher (P<0.05) than that using the control medium containing PVA (15.6%). The mean cell number of blastocysts ranged from 42 to 52 among the four groups, without significant differences. In conclusion, the addition of Cys or beta-ME to a defined maturation medium enhanced blastocyst formation after ICSI, to a level similar to that achieved by adding pFF.

Kobayashi M ，Lee ES ，Fukui Y 《THERIOGENOLOGY》

Metabolic requirements associated with GSH synthesis during in vitro maturation of cattle oocytes.

Glutathione (GSH) concentration increases in bovine oocytes during in vitro maturation (IVM). The constitutive amino acids involved in GSH synthesis are glycine (Gly), glutamate (Glu) and cysteine (Cys). The present study was conducted to investigate the effect of the availability of glucose, Cys, Gly and Glu on GSH synthesis during IVM. The effect of the amino acid serine (Ser) on intracellular reduced/oxidized glutathione (GSH/GSSG) content in both oocytes and cumulus cells was also studied. Cumulus-oocyte complexes (COC) of cattle obtained from ovaries collected from an abattoir were matured in synthetic oviduct fluid (SOF) medium containing 8 mg/ml bovine serum albumin-fatty acid-free (BSA-FAF), 10 microg/ml LH, 1 microg/ml porcine FSH (pFSH) and 1 microg/ml 17 beta-estradiol (17beta-E2). GSH/GSSG content was measured using a double-beam spectrophotometer. The COC were cultured in SOF supplemented with 1.5mM or 5.6mM glucose (Exp. 1); with or without Cys+Glu+Gly (Exp. 2); with the omission of one constitutive GSH amino acid (Exp. 3); with 0.6mM Cys or Cys+Ser (Exp. 4). The developmental capacity of oocytes matured in IVM medium supplemented with Cys and the cell number per blastocyst were determined (Exp. 5). The results reported here indicate (1) no differences in the intracellular GSH/GSSG content at any glucose concentrations. Also, cumulus cell number per COC did not differ either before or after IVM (Exp. 1). (2) Glutathione content in oocytes matured in SOF alone were significantly different from oocytes incubated with SOF supplemented with Cys+Glu+Gly (Exp. 2). (3) Addition of Cys to maturation medium, either with or without Gly and Glu supplementation resulted in an increase of GSH/GSSG content. However, when Cys was omitted from the IVM medium intracellular GSH in oocytes or cumulus cells was less but not significantly altered compared to SOF alone (Exp. 3). (4) Glutathione content in both oocytes and cumulus cells was significantly reduced by incubation with 5mM Ser (Exp.4). (5) There was a significant increase in cleavage and blastocyst rates when Cys was added to maturation medium. In contrast, the cleavage, morula and blastocyst rates were significantly different when 5mM Ser was added to maturation media. There was also a significant difference in mean cell number per blastocyst, obtained from oocytes matured with 5mM Ser (Exp. 5). This study provides evidence that optimal embryo development in vitro is partially dependent on the presence of precursor amino acids for intracellular GSH production. Moreover, the availability of Cys might be a critical factor for GSH synthesis during IVM in cattle oocytes. Greater Ser concentration in IVM medium altered "normal" intracellular GSH in both oocytes and cumulus cells with negative consequences for subsequent developmental capacity.

Furnus CC ，de Matos DG ，Picco S ，García PP ，Inda AM ，Mattioli G ，Errecalde AL ... -  《-》

Development and viability of pig oocytes matured in a protein-free medium containing epidermal growth factor.

This study examined the ability of epidermal growth factor (EGF) to improve the developmental competence of pig oocytes matured in a protein-free (PF) in vitro maturation (IVM) system. Oocyte maturation was done in one of three media: 1. PF-TCM: tissue culture medium (TCM) 199 + 0.1% polyvinylalcohol (PVA); 2. PF-TCM+EGF: PF-TCM + 10 ng/ml EGF; and 3. +ve CONT: North Carolina State University (NCSU) 23 medium + 10% porcine follicular fluid. All media contained 0.57 mM cysteine. Hormonal supplements, 0.5 microg/mL LH and 0.5 microg/mL FSH, were present only for the first half (20 to 22 h) of the culture period. After maturation, oocytes were co-incubated with frozen-thawed spermatozoa for 5 to 6 h and transferred to embryo culture medium, NCSU 23 containing 0.4% BSA, for 144 h. In Experiment 1, differences in cumulus expansion were observed for oocytes matured in +ve CONT (Category 4), PF-TCM (Category 2) and PF-TCM+EGF (Category 3). However, no significant differences in nuclear maturation to metaphase II stage were observed. In Experiment 2, no differences in fertilization parameters were observed. Significant (P < 0.01) differences in cleavage rates were observed among the three media for a proportion of the oocytes matured (52, 60 and 69% in PF-TCM, PF-TCM+EGF, and +ve CONT, respectively). Oocytes matured in PF-TCM showed the lowest (P < 0.01) blastocyst development (22%). However, the same rate of blastocyst development was obtained for +ve CONT (37%) and PF-TCM+EGF (37%). Blastocyst cell numbers were significantly higher when oocytes were matured in the presence of EGF (26 vs. 37 to 41). In Experiment 3, oocytes matured in PF-TCM+EGF had a significantly (P < 0.05) higher intracellular glutathione (GSH) concentration (5.9 vs. 11.4 pmol/oocyte) compared with PF-TCM. Twenty-two of 25 embryo transfer recipients became pregnant (Experiment 4). Four animals returned to estrus in within 60 days. Six pregnant animals slaughtered at 26 to 45 days had 43 fetuses (range: 4 to 12) and the remaining 12 animals farrowed 82 piglets (range: 3 to 12). These results indicate that EGF enhances the developmental competence of pig oocytes matured in a protein-free culture medium which is correlated with higher GSH level in oocytes. Birth of piglets indicate that embryos derived from oocytes matured in the presence of EGF are viable.

Abeydeera LR ，Wang WH ，Cantley TC ，Rieke A ，Murphy CN ，Prather RS ，Day BN ... -  《THERIOGENOLOGY》