RESUMEN
Follicle cultures reproduce in vitro the functional features observed in vivo. In a search for an ideal model, we cultured bovine antral follicle wall sections (FWS) in a serum-free defined medium (DM) known to induce 17ß-estradiol (E2) production, and in a nondefined medium (NDM) containing serum. Follicles were sectioned and cultured in NDM or DM for 24 or 48 h. Morphological features were determined by light microscopy. Gene expression of steroidogenic enzymes and follicle-stimulating hormone (FSH) receptor were determined by RT-PCR; progesterone (P4) and E2 concentrations in the media were measured by radioimmunoassay. DM, but not NDM, maintained an FWS morphology in vitro that was similar to fresh tissue. DM also induced an increase in the expression of all steroidogenic enzymes, except FSH receptor, but NDM did not. In both DM and NDM, there was a gradual increase in P4 throughout the culture period; however, P4 concentration was significantly higher in NDM. In both media, E2 concentration was increased at 24 h, followed by a decrease at 48 h. The E2:P4 ratio was higher in DM than in NDM. These results suggest that DM maintains morphological structure, upregulates the expression of steroidogenic enzyme genes, and maintains steroid production with a high E2:P4 ratio in FWS cultures.
Asunto(s)
Medios de Cultivo/farmacología , Estradiol/farmacología , Folículo Ovárico/efectos de los fármacos , Progesterona/farmacología , Técnicas de Cultivo de Tejidos , Análisis de Varianza , Animales , Aromatasa/genética , Bovinos , Enzima de Desdoblamiento de la Cadena Lateral del Colesterol/genética , Medio de Cultivo Libre de Suero , Femenino , Expresión Génica , Folículo Ovárico/anatomía & histología , Fosfoproteínas/genética , Progesterona Reductasa/genética , Receptores de HFE/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Esteroide 17-alfa-Hidroxilasa/genéticaRESUMEN
Follicle cultures reproduce in vitro the functional features observed in vivo. In a search for an ideal model, we cultured bovine antral follicle wall sections (FWS) in a serum-free defined medium (DM) known to induce 17β-estradiol (E2) production, and in a nondefined medium (NDM) containing serum. Follicles were sectioned and cultured in NDM or DM for 24 or 48 h. Morphological features were determined by light microscopy. Gene expression of steroidogenic enzymes and follicle-stimulating hormone (FSH) receptor were determined by RT-PCR; progesterone (P4) and E2 concentrations in the media were measured by radioimmunoassay. DM, but not NDM, maintained an FWS morphology in vitro that was similar to fresh tissue. DM also induced an increase in the expression of all steroidogenic enzymes, except FSH receptor, but NDM did not. In both DM and NDM, there was a gradual increase in P4 throughout the culture period; however, P4 concentration was significantly higher in NDM. In both media, E2 concentration was increased at 24 h, followed by a decrease at 48 h. The E2:P4 ratio was higher in DM than in NDM. These results suggest that DM maintains morphological structure, upregulates the expression of steroidogenic enzyme genes, and maintains steroid production with a high E2:P4 ratio in FWS cultures.
Asunto(s)
Animales , Bovinos , Femenino , Medios de Cultivo/farmacología , Estradiol/farmacología , Folículo Ovárico/efectos de los fármacos , Progesterona/farmacología , Técnicas de Cultivo de Tejidos , Análisis de Varianza , Aromatasa/genética , Medio de Cultivo Libre de Suero , Enzima de Desdoblamiento de la Cadena Lateral del Colesterol/genética , Expresión Génica , Folículo Ovárico/anatomía & histología , Fosfoproteínas/genética , Progesterona Reductasa/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Receptores de HFE/genética , /genéticaRESUMEN
The objective was to study the effect of a defined culture system, on nuclear and cytoplasmic maturation of bovine oocytes, using the two-step procedure of IVM to detect possible inhibition and subsequent resumption of meiosis arrest. In the first step, called the prematuration period (PMP), COCs were cultured in T1-non-defined medium (NDM), or T2-defined medium (DM), both for 24 h. In step 2, called the resumption period (RP), COCs were cultured in: NDM (T1); DM + NDM (T3); or DM+DM (T4) for 24 h in each medium. The NDM was composed of TCM-199 supplemented with FCS and FSH. The DM was composed of alpha-MEM supplemented with PVA, insulin, IGF-1, androstenedione, nonessential amino acids, transferrin, and sodium selenium. Oocytes from T2 had a lower (P < 0.05) rate of nuclear maturation (19.8%) than T1 oocytes (83.2%). Also, T2 COCs appeared to be in the process of cytoplasmic maturation, according to the distribution of organelles assessed by transmission electron microscopy (MET). These COCs had characteristics previously described as mature: erect microvilli on the plasmembrane, presence of cortical/evenly distributed mitochondria throughout the ooplasm, and presence of 50% aligned/cluster cortical granules. Immature characteristics such as small PvS, compact cumulus cells, and presence of 50% cortical granule clusters were also observed. The T1 COCs had only characteristics of maturation (P < 0.05). In step 2 (RP), meiosis arrest induced by DM was resumed after an additional 24 h of culture in NDM (T3) with 79.2% mature COCs, whereas in T4, meiosis arrest was maintained, resulting in almost 70% immature COCs (P < 0.05). At the end of RP, T3 COCs had the mature characteristics of mitochondria spread throughout the cytoplasm (P < 0.05), cumulus expansion, and alignment of cortical granules, whereas the T4 group had both immature and mature characteristics. We inferred that DM can be used in lieu of meiosis inhibitors and furthermore, it can provide extra time to study nuclear and cytoplasmic maturation synchrony of IVM.
Asunto(s)
Bovinos , Técnicas de Cultivo de Célula , Medios de Cultivo/química , Meiosis/efectos de los fármacos , Oocitos/efectos de los fármacos , Animales , Medios de Cultivo/farmacología , Citoplasma/fisiología , Estradiol/metabolismo , Femenino , Oocitos/citología , Oocitos/crecimiento & desarrollo , Alcohol Polivinílico/farmacología , Factores de TiempoRESUMEN
BACKGROUND: The incidence of occupational injuries among children and adolescents is not well known in Brazil. This study aims to identify occurrences and characteristics of occupational injuries in children and adolescents at the two major public emergency hospitals in Aracaju, Brazil. METHODS: In a cross sectional study, all children and adolescents (5-17 years) attending the services because of accidents of external cause in October-November 2006 and May-June 2007 were evaluated. The patients and their responsibles were interviewed to obtain information about the injury, family and patient characteristics and relation to work in the last 7 days. Data were analysed in number and percentages using descriptive statistics and chi-squared and Fisher's exact tests. RESULTS: There were 917 patients with injuries of external cause in the period studied. Seventy-one (8%) had worked in the last 7 days and in 40 (4%) the injury was related to work, with 2 (0.21%) deaths. Occupational injuries occurred more frequently in boys (95%); in the 14- to 17-year-old age group (77%); in agricultural (46%) and commercial activities (18%); to help with family budget (54%); without any safety training (73.5%) and not using personal protective equipment (94%). Main mechanisms of injuries were transportation incidents (42.5%) and contact with tools and equipment (27.5%). Injuries were mostly fractures (37.5%) and lacerations (22.5%). Upper extremities were injured in 49% of cases and the lower extremities in 26.5%. Comparison between the two groups (accident related or not related to labour) shows that there were significant differences for gender, age group, the main mechanism of injury and the part of the body injured, but not for the nature of injury. CONCLUSION: Working children and adolescents have been injured during their occupational activities and work-related injuries represent 4% of injuries of external causes in the emergency departments of Aracaju with 5% lethality.