The effects of roscovitine on cumulus cell apoptosis and the developmental competence of domestic cat oocytes.
ABSTRACT The developmental competence of cat oocytes matured in vitro is relatively poor when compared with that of in vivo oocytes. The study aimed to investigate the effect of roscovitine on the developmental competence of cat Felis catus oocytes matured in vitro. Cumulus-oocyte complexes (COCs) were classified as Grade I and II to III. Groups of COCs were cultured in 0, 12.5, 25, 50, 100, and 200 microM roscovitine for 24h and were either fixed to assess the stages of nuclear maturation (Experiment 1) or additionally matured in vitro for 24h before fixation (Experiment 2). In Experiment 3, cumulus cells from the COCs treated with roscovitine were examined for apoptosis. Experiment 4 examined the developmental competence of cat oocytes after roscovitine treatment and in vitro fertilization in terms of cleavage and morula and blastocyst formation rates. Roscovitine reversibly arrested cat oocytes at an immature stage in a dose-dependent manner. Roscovitine at 12.5 and 25 microM demonstrated less efficiency compared with that of other doses. However, higher doses of roscovitine induced cumulus cell apoptosis and resulted in a high number of degenerated oocytes after in vitro maturation. Roscovitine at 12.5 and 25 microM were therefore used to evaluate their effect on embryo development. Pretreatment with 12.5 and 25 microM roscovitine prior to in vitro maturation decreased the developmental competence of cat oocytes compared with that of non-roscovitine-treated controls. In conclusion, roscovitine reversibly maintained cat oocytes at the germinal vesicle stage without detrimental effect on nuclear maturation. However, it negatively affected cumulus cell viability and developmental competence.
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ABSTRACT: Objective To determine the effects of insulin-like growth factor-1 (IGF-1) and the mRNA expression of IGF-1 receptor (IGF-1R) during the in vitro development of cat embryos cultured in groups versus singly. Methods Cumulus-oocyte complexes (COCs) were matured and fertilized in vitro with frozen-thawed semen. Cleaved embryos (48 h post-fertilization) were randomly assigned to one of the following treatments: 1) group embryo culture without IGF-1 (10 embryos per 50 μl droplet), 2) single-embryo culture without IGF-1, and 3) to 6) single-embryo culture (50 μl droplet per embryo) supplemented with different concentrations of IGF-1 (5, 25, 50 and 100 ng/ml, respectively). During in vitro culture, the embryos were analyzed for development to the morula, blastocyst and hatching blastocyst stage. Relative mRNA expression of IGF-1R was also examined by qPCR at the morula and blastocyst stages. In addition, the mRNA expression of IGF-1R in morula-stage embryos treated with IGF-1 was determined. The influence of IGF-1 to preimplantation embryo development was then explored by co-incubation with 0.5 μM IGF-1R inhibitor (Picropodophyllin; PPP). Results Group embryo culture led to a significantly higher blastocyst development rate compared with single-embryo culture (P < 0.05). The poor development of singly cultured embryos coincided with the significantly lower IGF-1R expression in morulae than in group-cultured morulae. IGF-1 (25 or 50 ng/ml) supplementation significantly improved the blastocyst formation rate of single embryos to a level similar to group culture by promoting the morula-to-blastocyst transition. IGF-1 supplementation (25 or 50 ng/ml) of singly cultured embryos upregulated the expression of IGF-1R mRNA in morula-stage embryos to the same level as that observed in group-cultured embryos (without IGF-1). The beneficial effects of IGF-1 on singly cultured embryo was (P < 0.05) suppressed by PPP even in the group culture embryo without growth factor supplementation. Conclusion IGF-1 supplementation improves the developmental competence of feline embryos cultured individually and also increases IGF-1R gene expression to levels similar to group-cultured embryos.Growth hormone & IGF research: official journal of the Growth Hormone Research Society and the International IGF Research Society 04/2014; 24(2-3). DOI:10.1016/j.ghir.2014.03.002 · 1.33 Impact Factor
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ABSTRACT: It is believed the temporary meiosis arrest with roscovitine or cycloheximide may improve the in vitro developmental competence of oocytes in different animal species. However, little is known about the effects of these inhibitors on ultrastructure of ovines cumulus–oocyte complexes (COCs). The aim of this study was to evaluate the progression of cytoplasmic maturation and the ultrastructural changes in sheep COCs exposed to roscovitine or cycloheximide, at acceptable concentrations. COCs were in vitro cultured for 24 h in maturation medium (control group) containing 100 μM roscovitine or 1 μg/mL cycloheximide (treatment groups). After this time, some COCs were cultured for further 22 h in inhibitor-free medium. The ultrastructure organization of COCs was evaluated by transmission electron microscopy before (immature group) and after in vitro culture for 24 and 46 h. As expected, signs of immaturity and maturity were observed in immature and control groups, respectively. In treatment with roscovitine, there were cumulus cells degeneration, swelling of mitochondrias, few cortical granules and many vesicles with electron-dense material. However, in cycloheximide treatment there were not signs of degeneration or cellular senescence. Metabolic units and mitochondrial pleomorphism were found in all experimental groups. These evidences demonstrate that roscovitine promoted irreversible ultrastructural changes while cycloheximide did not affect the cytoplasmic maturation. However, the implications on embryo development are still unclear.Small Ruminant Research 01/2013; 109(s 2–3):156–162. DOI:10.1016/j.smallrumres.2012.07.006 · 1.10 Impact Factor
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ABSTRACT: The aim of this study is to produce live kittens from oocytes fertilized by intracytoplasmic sperm injection (ICSI) with frozen/thawed testicular spermatozoa. Spermatozoa were collected from thawed testicular tissue and subsequently injected into in vitro matured cat oocytes. At 24 h post-ICSI, presumptive zygotes/cleaved embryos were treated with 10 μm forskolin for 24 h to reduce intracellular lipid content of embryos (delipidation). At 48 h after oocyte injection, cleaved embryos (2- to 8-cell stage) were frozen in 10% (v/v) ethylene glycol-based medium by a slow controlled rate method and stored in liquid nitrogen. To evaluate in vitro and in vivo developmental competence, frozen embryos were thawed and then cultured for 6 days (n = 155) or cultured for 2 h before transferred (n = 209) to hormonal (equine chorionic gonadotropin/hCG)-treated cat recipients. Cleavage frequency at day 2 after ICSI with frozen/thawed testicular spermatozoa was ~30%. The percentages of frozen/thawed embryos that developed to morula and blastocyst stage (on day 3 and day 6 of in vitro culture, respectively) were significantly lower than that of fresh ICSI embryos (22.6 vs 45.2% and 21.3 vs 38.7%, respectively; p < 0.05). However, no difference was found in the number of blastomeres between frozen/thawed (242.5 ± 43.1) and fresh (320.2 ± 28.1) blastocysts. Three of seven cat recipients were pregnant and one pregnant cat delivered two healthy kittens. This is the first report of the birth of kittens after the transfer of frozen-thawed embryos produced by ICSI with frozen/thawed testicular sperm.Reproduction in Domestic Animals 12/2012; 47 Suppl 6:305-8. DOI:10.1111/rda.12072 · 1.18 Impact Factor