The effects of roscovitine on cumulus cell apoptosis and the developmental competence of domestic cat oocytes

Department of Obstetrics, Gynaecology and Reproduction, Faculty of Veterinary Science, Chulalongkorn University, Bangkok, Thailand.
Theriogenology (Impact Factor: 1.8). 11/2009; 73(2):199-207. DOI: 10.1016/j.theriogenology.2009.08.013
Source: PubMed


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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    • "According to Lagutina et al. (2002), bovine oocytes pre-matured with this inhibitor reached the MII stage about 4 h earlier than untreated oocytes. It occurs probably due to accumulation of developmentally relevant factors during the meiosis block and suggests that meiotic step may be shorter (Han et al., 2006; Sananmuang et al., 2010). Based on that, it was established that 18 h of culture with gonadotropins could be sufficient for the oocyte to complete the meiosis, as already reported by Máximo et al. (2012) in sheep. "
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    ABSTRACT: Attempts to maintain the meiotic arrest under suitable culture conditions have been performed in order to improve the quality of oocytes in vitro matured. So, our study aimed to evaluate the potential of roscovitine, an inhibitor of cyclin-dependent kinases, to reversibly arrest the meiosis of sheep oocytes in medium supplemented with gonadotropins and/or serum. Cumulus-oocyte complexes (COCs) were treated, for 20 h, with 75 μM roscovitine (Rosco) in basic maturation medium (Basic) containing 10% fetal bovine serum (FBS) and 0.1 IU/mL FSH plus 0.1 IU/mL LH (Enriched). After this, COCs were in vitro matured (IVM) for a further 18 h in the presence of gonadotropins and serum. At 20 and 38 h of culture, cumulus expansion and nuclear maturation were assessed under stereomicroscope and by Hoechst 33342 staining, respectively. Our findings demonstrate that total cumulus expansion occurred only in medium containing gonadotropins and serum. In the presence of roscovitine, however, the cumulus expansion was inhibited in a not fully reversible manner, independently of medium composition. The GV rate in the Rosco FBS group (64.6%) was significantly higher than that of Rosco Enriched (51.8%); however, both did not differ from that observed in the Rosco Basic (56.2%). In all roscovitine treatments, the meiotic arrest was reversible after the additional culture for 18 h in inhibitor-free medium. Besides, significant proportion of oocytes from Basic, FBS and Enriched groups reached the MII stage after 20 and 38 h of culture. Therefore, we can infer that (i) gonadotropins and serum did not affect the reversible meiotic arrest promoted by roscovitine in sheep oocytes; (ii) the maximum efficiency of meiosis inhibition occurred in serum-supplemented medium; (iii) roscovitine action on cumulus cells was not fully reversible independently of medium composition.
    Small Ruminant Research 03/2015; 126. DOI:10.1016/j.smallrumres.2015.02.022 · 1.13 Impact Factor
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    • "By competing with ATP for the binding site p34 cdc2 , roscovitine prevents the MPF activation (Meijer et al. 1997). The reversible meiotic arrest using this inhibitor has been reported in several species as bovine (Mermillod et al. 2000), goat (Han et al. 2006), pig (Romar and Funahashi 2006), cat (Sananmuang et al. 2010) and horse (Consiglio et al. 2010). In sheep, however, few similar studies have been conducted. "
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    ABSTRACT: Inhibitors of cyclin-dependent kinases, as roscovitine, have been used to prevent the spontaneous resumption of meiosis in vitro and to improve the oocyte developmental competence. In this study, the interference of oil overlay on the reversible arrest capacity of roscovitine in sheep oocytes as well as its effects on cumulus expansion was evaluated. For this, cumulus-oocyte complexes (COCs) were cultured for 20 h in TCM 199 with 10% foetal bovine serum (Control) containing 75 μm roscovitine (Rosco). Subsequently, they were in vitro matured (IVM) for further 18 h in inhibitor-free medium with LH and FSH. The culture was performed in Petri dishes under mineral oil (+) or in 96 well plates without oil overlay (-) at 38.5°C and 5% CO2 . At 20 and 38 h, the cumulus expansion and nuclear maturation were evaluated under stereomicroscope and by Hoechst 33342 staining, respectively. No group presented cumulus expansion at 20 h. After additional culture with gonadotrophins, a significant rate of COCs from both Control groups (+/-) exhibited total expansion while in both Rosco groups (+/-) the partial expansion prevailed. Among the oocytes treated with roscovitine, 65.2% were kept at GV in the absence of oil overlay while 40.6% of them reached MII under oil cover (p < 0.05). This meiotic arrest was reversible, and proper meiosis progression also occurred in the Control groups (+/-). So, the culture system without oil overlay improved the meiotic inhibition promoted by roscovitine without affecting the cumulus expansion rate or the subsequent meiosis progression. © 2015 Blackwell Verlag GmbH.
    Reproduction in Domestic Animals 02/2015; 50(3). DOI:10.1111/rda.12506 · 1.52 Impact Factor
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    • "In vitro oocyte maturation (IVM) and fertilization (IVF) were performed as previously described by Sananmuang et al. [33]. For IVM, groups of 25–40 COCs were cultured in 500 μl of IVM medium (NaHCO 3 buffered M199 with 1.0 mM sodium pyruvate, 2.0 mM L-glutamine, 100 IU/ml penicillin, 50 μg/ml gentamicin, 4 mg/ml BSA and 0.05 IU/ml recombinant human follicle-stimulating hormone (rhFSH; Organon, Bangkok, Thailand) and 25 ng/ml EGF). "
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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 · 1.41 Impact Factor
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