Dissociation of oocyte nuclear and cytoplasmic maturation by the addition of insulin in cultured bovine antral follicles

Abstract

Follicles of 4-8 mm in diameter were dissected from ovaries and cultured in Waymouth culture medium in the presence or absence of insulin (5 mg/ml) at 39 8C in a humidified atmosphere of 45% O 2 , 5% CO 2 and 50% N 2 for 24 h. Following follicle culture, the oocytes were collected and examined for developmental potential, total protein profile and ultrastructural aspects. Oocytes aspirated directly from follicles of the same size were used as controls. Addition of insulin to the follicle culture medium significantly reduced expression of the low molecular weight insulin-like growth factor-binding proteins (IGFBPs) in the follicular fluid, and significantly reduced the cleavage rate of subsequently matured and fertilised oocytes (0.52 vs 0.61). However, there were no differences in the proportion of cleaved embryos which developed to the blastocyst stage (0.30 vs 0.28), nor embryo quality as assessed by total cell number (137 6 8.53 vs124.6 6 6.95). The total protein profiles of immature oocytes recovered after 24 h of follicle culture were compared by PAGE. There were marked differences between the two groups, unmatured oocytes recovered from the insulin-positive follicle group showed a protein pattern similar to that of matured oocytes. In addition, examination of ultrastructural features by transmission electron microscopy indicated that oocytes from follicles cultured in the presence of insulin undergo many of the cytoplasmic changes associated with oocyte maturation. In conclusion, follicle culture in the presence of insulin is beneficial for follicular survival and significantly reduces cleavage but has no detrimental effects on the development of cultured embryos. However, many of the cytoplasmic changes associated with oocyte maturation occur prior to the induction of nuclear maturation.

Effect of dietary-induced changes in plasma insulin concentrations during the early post partum period on pregnancy rate in dairy cows

“…As outlined in the introduction, feeding cows diets designed to increase plasma insulin has been found to have beneficial effects on follicular development, leading to earlier resumption of ovarian cyclic activity (Gong et al 2002a), but can have negative effects on oocyte developmental competence (Fouladi-Nashta et al 2005). High insulin also has adverse effects on nuclear maturation of oocytes after follicle culture (Fouladi-Nashta & Campbell 2006). The results of this experiment support the concept that responses to nutrition can vary at different stages of the reproductive cycle.…”

Impact of linoleic acid on bovine oocyte maturation and embryo development

“…For experiments designed to assess the effects of LA in maturation medium on the developmental potential of oocytes, in vitro-matured oocytes (in the presence of 0, 50 or 100 mM LA) were fertilized with frozen semen (gift from Genus ABS, Nantwich Cheshire, UK) from a single bull as previously described (Fouladi-Nashta & Campbell 2006). Briefly, motile sperm were selected by swim up for 45 min in calcium-free medium followed by centrifugation at 300 g at room temperature and re-suspension of the pellet in fertilization medium (TALP supplemented with 0.6% (w/v) fatty acid-free BSA, 1 mg/ ml heparin, 50 ng/ml adrenaline and 50 ng/ml hypotaurine).…”

Oocyte quality in lactating dairy cows fed on high levels of n-3 and n-6 fatty acids

“…All oocytes were cultured for 24 h in 35 mm culture dishes (Nunc, Roskilde, Denmark) containing 10 ml maturation medium per oocyte (TCM199 supplemented with 10 mg FSH/ml (follitropin; Bioniche Animal Health, Bellevile, Ontario, Canada), 10 mg LH/ml (leutropin; Bioniche Animal Health) 1 mg estradiol/ml, 50 mg gentamicin/ml, and 10% HIFCS under mineral oil in a humidified atmosphere of 5% CO 2 in air at 39 8C. In vitromatured oocytes were fertilized with frozen sperm from a single bull, as described previously (Fouladi-Nashta & Campbell 2006). Briefly, motile sperm were prepared after 45 min of swim up in calcium-free medium followed by centrifugation at 300 g at room temperature and re-suspension of the pellet in fertilization medium.…”