Use of Low-Salt Culture Medium for in Vitro Maturation of Porcine Oocytes is Associated with Elevated Oocyte Glutathione Levels and Enhanced Male Pronuclear Formation after in Vitro Fertilization1

Abstract

The effects of sodium chloride (NaCI) in Whitten's medium on intracellular glutathione concentration and on cytoplasmic maturation, as determined by monospermic penetration and male pronuclear formation of porcine oocytes, were examined. Porcine cumulus-oocyte complexes were cultured for 20 h in BSA-free Whitten's medium containing different NaCl concentrations (44.50, 68.49, 92.40, 116.40, or 140.35 mM) and supplemented with 10% porcine follicular fluid and hormonal supplements; the complexes were then cultured without hormonal supplements for an additional 20-h period. The mean width of the perivitelline space of oocytes was increased with decreased concentration of NaCI in the culture medium. Intracellular glutathione concentration was elevated in oocytes cultured in medium with lower NaCI concentrations. After co-culture with spermatozoa for 6 h and culture in modified Whitten's medium for an additional 6 h, there were no differences in maturation and penetration rates among experimental groups. However, the rate of male pronuclear formation was higher in oocytes matured in media with the lower NaCI concentrations. In addition, the rates of monospermic penetration and male pronuclear formation were higher in oocytes matured in medium containing 44.50 mM NaC1 (59.3 ± 8.1 and 70.9 + 2.0%, respectively) than in medium containing 68.49 mM NaCI (39.4 ± 5.5 and 57.1 4.5%, respectively). These data indicated that decreasing NaCI concentration in maturation medium for porcine oocytes below the customary level improved the quality of the matured oocytes as reflected in higher intracellular glutathione content, wider perivitelline space, higher monospermic penetration rate, and increased frequency of male pronuclear formation.

Developmental competence of prepubertal goat oocytes selected with brilliant cresyl blue and matured with cysteamine supplementation

“…With BCB+ oocytes, the difference between the percentage of cleaved embryos (45.3%) and the percentage of 2PN oocytes (39.7%) was reduced which suggests a lower number of haploid embryos obtained with BCB+ oocytes matured with cysteamine. Several authors have found an effect of intracytoplasmic GSH levels on MPN formation [15,16,28,31,33,34]. In our study we observed significantly higher percentages of MPN in oocytes matured with cysteamine compared to the control group (80.3 vs. 61.5%, respectively).…”

Effects of heat stress during in vitro maturation on cytoplasmic versus nuclear components of mouse oocytes

“…Intracellular content of GSH was measured as described by Funahashi et al (1994). Cumulus-free oocytes were washed three times in M2.…”

Low Salt Maturation Medium Enhances the Histone H1 Kinase Activity of Porcine Oocytes at the End of In Vitro Maturation.

“…As described in our previous report [12], the maturation medium used was BSA-free Whitten's Medium containing various concentrations (44.50, 68.49, 92.40, 116.40 and 140.35 mM) of NaCl supplemented with 10% (v/v) porcine follicular fluid and hormonal supplements (10 IU eCG/ml, 10 IU hCG/ml and 1 µg estradiol/ml). Mean osmolarity of the maturation medium containing various concentrations of NaCl (44.50, 68.49, 92.40, 116.40 and 140.35 mM) was 214.5 ± 0.7, 254.5 ± 0.5, 295.0 ± 0.4, 333.5 ± 0.7 and 373.3 ± 0.3 mOsM, respectively.…”

“…Further, the histone H1 kinase activity is correlated with oocyte glutathione content which, in turn, also affects male pronuclear development [12,13]. The present studies were undertaken to determine; 1) the effects of sodium chloride concentration in maturation medium on histone H1 kinase activity at the end of in vitro maturation, and 2) the correlation of histone H1 kinase activity and oocyte glutathione content at the end of culture for maturation.…”