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 M roscovitine or 1 g/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.

Temporary meiosis arrest with cyclin-dependent kinases inhibitors has been proposed in order to improve the quality of in vitro matured oocytes. In sheep, however, this phenomenon has been rarely investigated. Therefore, the present study aimed to evaluate the effect of different incubation times with roscovitine on nuclear maturation and cumulus cell expansion of sheep cumulus-oocyte complexes (COCs). For this, COCs were cultured for 0, 6, 12 or 20 h in basic maturation medium (Control) containing 75 μM roscovitine (Rosco). After, they were in vitro matured (IVM) for 18 h in the presence of luteinizing hormone (LH) and follicle-stimulating hormone (FSH). At the end of each treatment, cumulus cell expansion and nuclear maturation were assessed under a stereomicroscope and by Hoechst 33342 staining, respectively. In the Control and Rosco groups, the absence of cumulus cell expansion prevailed at 0, 6, 12 and 20 h. After IVM for 18 h, total cumulus cell expansion in the Rosco treatments was dependent on the exposure time to roscovitine. A significantly high percentage of oocytes treated with roscovitine for 6 h (87%), 12 h or 20 h (65%) were arrested at the germinal vesicle (GV) stage. In contrast, 23% GVBD, 54% metaphase I (MI) and 61% MII oocytes were observed in the Control groups at 6, 12 and 20 h, respectively. In all treatments, a significant percentage of oocytes reached MII after IVM for 18 h. Therefore, roscovitine reversibly arrested the meiosis of sheep oocytes during different culture times with the maximal efficiency of meiotic inhibition reached at 6 h. In addition, reversibility of its inhibitory action on cumulus cells was exposure-time dependent.

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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.

Apis melífera L. propolis is a resinous and balsamic material whose biological effects are related to its chemical composition. This chemical composition is greatly influenced by seasonality, so propolis from different seasons and regions has a different chemical composition. The increasing need for natural options to control fungi that cause damage to food crops makes propolis an alternative that deserves more research. In this context, the aim of this study was to evaluate the effect of seasonality on the antifungal potential of propolis collected in Campo Grande, Mato Grosso do Sul, Brazil, on the fungus Lasiodiplodia theobromae . Effects of different concentrations of ethanolic extracts of green propolis on the mycelial growth of the pathogen were evaluated. Concentrations of flavonoids and phenolic compounds in the samples were also determined by spectrophotometric methods. Results showed that the propolis extracts have a different chemical composition, potential fungi static effects on the tested fungus, and that there is interference of seasonality on the mycelial growth of the fungus, pointing to the concentration of 1250µg 100mL-1of the samples collected in the summer, in a first moment, as the most efficient.

The majority of mammalian oocytes destined for in vitro maturation (IVM) have not undergone all molecular and structural changes necessary for competence acquisition to support the fertilization and early embryogenesis. In this context, different methods able to provide a transient arrest of meiosis resumption have been tested in order to improve the in vitro developmental potential of oocytes. Based on that, our study aimed to evaluate the effect of temporary meiosis inhibition using roscovitine on gene expression in sheep oocytes and cumulus cells. For this, cumulus-oocyte complexes (COCs) were cultured for 6 h in modified TCM199 medium with (Rosco) and without (Control) 75 M roscovitine. Subsequently, they were in vitro matured for a further 18 h in inhibitor-free TCM199 medium supplemented with gonadotropins. At 0, 6 and 24 h of culture, nuclear status of oocytes and expression of selected genes were evaluated by Hoescht staining and qRT-PCR, respectively. The analysis of oocyte chromatin organization revealed that roscovitine efficiently inhibited the meiosis of sheep oocytes for 6 h and its action was completely reversed after 18 h of in vitro maturation in inhibitor-free medium. Besides, no detrimental effect on cumulus expansion was observed. The expression profile of most investigated genes in cumulus cells (PTX3, GREM1, GLUT1, PTGS2, ALK5, ALK6) and oocytes (ZAR1, NLRP5, SOD1, BMP15, GDF9) was similar between Control and Rosco treatments and the ratio BCL2/BAX was maintained in both cell types even in the presence of roscovitine. These results indicate that reversible meiotic arrest promoted by roscovitine, at the concentration and exposure time studied, neither impaired nor improved the expression of investigated genes in sheep oocytes and cumulus cells. Moreover, the efficiency of temporary meiotic arrest and the absence of deleterious effect on COCs suggest that roscovitine provides a useful method for transportation or manipulation of sheep oocytes at onset of maturation. However, further investigations are necessary to evaluate the benefits of roscovitine treatment on in vitro development of sheep embryos and its effects on cellular ultrastructure.

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RESUMOO presente estudo descreve uma técnica para obtenção de fragmentos luteais empregando-se a colpotomia. Os animais foram submetidos à anestesia epidural e no fórnix vaginal e, após dez minutos, realizou-se uma incisão no fundo vaginal com lâmina de bisturi, bem como procedeu-se à dissecção do tecido, até que fossem possíveis o acesso à cavidade pélvica e a tração dos ovários para o interior vaginal. Realizou-se, então, a colheita de biopsia luteínica, com o auxílio de uma pinça do tipo Yomann. Sinais de dor e estresse foram observados apenas em duas colheitas durante a incisão no fundo vaginal, a tração do ovário ou durante a biopsia luteínica. Contudo, esses sinais foram observados em dez colheitas durante a dissecção da parede vaginal e peritôneo. Registrou-se a ocorrência de ataxia em 38,81% colheitas, a qual esteve relacionada, normalmente, a um procedimento mais longo. As ataxias podem ser divididas em leve (15/26), moderada (6/26) e severa (5/26). A avaliação da presença de adesões ovarianas ipsilaterais à incisão realizou-se apenas até a quarta colheita, sendo notadas em dezesseis colheitas. O protocolo empregado mostrou-se um método seguro e eficiente na obtenção de fragmentos luteais. A baixa incidência de aderências permite o uso consecutivo das fêmeas sem interferência nas ovulações e colheitas subsequentes.