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1.
Zygote ; 27(5): 315-320, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31412974

RESUMO

We compare the efficiency of mechanical or enzymatic methods, and their combination, for the isolation of ovarian preantral follicles (PFs) from collared peccaries. The ovaries from six females were subjected to the different methods investigated here. For the enzymatic method, ovary fragments were exposed to collagenase type IV in TCM-HEPES medium; the mechanical procedure was based on ovarian cortex dissociation by using a scalpel blade. The residual solution obtained after the mechanical isolation was subjected to the enzymatic procedure. The number of isolated PFs was quantified and classified as primordial, primary, or secondary; their viability was assessed using trypan blue dye assay. To confirm the results, PFs derived from the most efficient method were evaluated for integrity using scanning electron microscopy (SEM) and subjected to a 24 h in vitro culture for subsequent evaluation of viability by using fluorescent probes. A higher number of PFs (P < 0.05) was obtained from the enzymatic method (961.7 ± 132.9) in comparison with the mechanical method (434.3 ± 88.9), but no difference was observed between the two methods and their combination (743.2 ± 92.8). The trypan blue assay showed that the enzymatic method (98.7 ± 0.6%) provided the highest percentage of viable follicles (P < 0.05). Furthermore, SEM confirmed the ultrastructural integrity of the surface architecture of peccary PFs isolated by the enzymatic procedure; epifluorescence microscopy was used to confirm their viability (86.0%). In conclusion, we suggest that the enzymatic method investigated here is useful for the isolation of viable ovarian PFs from collared peccaries.


Assuntos
Artiodáctilos , Folículo Ovariano , Coleta de Tecidos e Órgãos/veterinária , Animais , Colagenases , Feminino , Corantes Fluorescentes , Microscopia Eletrônica de Varredura , Microscopia de Fluorescência , Técnicas de Cultura de Tecidos , Coleta de Tecidos e Órgãos/métodos
2.
Zygote ; 25(3): 341-357, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28669364

RESUMO

This study aims to investigate the effect 5-azacytidine (5-Aza) during induction of pluripotency in bovine fibroblasts and to evaluate the effects of BMP2, BMP4 or follicular fluid in the differentiation of reprogrammed fibroblasts in primordial germ cells and oocytes. It also analysis the mRNA levels for OCT4, NANOG, REX, SOX2, VASA, DAZL, cKIT, SCP3, ZPA and GDF9 after culturing 5-Aza treated fibroblasts in the different tested medium. Dermal fibroblasts were cultured and exposed to 0.5, 1.0 or 2.0 µM of 5-Aza for 18 h, 36 h or 72 h. Then, the cells were cultured in DMEM/F12 supplemented with 10 ng/ml BMP2, 10 ng/ml BMP4 or 5% follicular fluid. After culture, morphological characteristics, viability and gene expression were evaluated by qPCR. Treatment of skin fibroblasts with 2.0 µM 5-Aza for 72 h significantly increased expression of mRNAs for SOX2, OCT4, NANOG and REX. The culture in medium supplemented with BMP2, BMP4 or follicular fluid for 7 or 14 days induced formation of oocyte-like cells, as well as the expression of markers for germ cells and oocyte. In conclusion, treatment of bovine skin-derived fibroblasts with 2.0 µM 5-Aza for 72 h induces the expression of pluripotency factors. Culturing these cells in differentiation medium supplemented with BMP2, BMP4 or follicular fluid induces morphological changes and promotes expression of markers for germ cells, meiosis and oocyte.


Assuntos
Azacitidina/farmacologia , Diferenciação Celular/efeitos dos fármacos , Fibroblastos/citologia , Fibroblastos/fisiologia , Marcadores Genéticos/genética , Animais , Proteína Morfogenética Óssea 2/farmacologia , Proteína Morfogenética Óssea 4/farmacologia , Bovinos , Diferenciação Celular/genética , Meios de Cultura/química , Meios de Cultura/farmacologia , RNA Helicases DEAD-box/genética , Feminino , Fibroblastos/efeitos dos fármacos , Líquido Folicular/fisiologia , Células-Tronco Pluripotentes/fisiologia , Pele/citologia , Pele/embriologia , Glicoproteínas da Zona Pelúcida/genética
3.
Zygote ; 25(3): 256-264, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28300526

RESUMO

The aim of this study was to evaluate the effects of different concentrations of BMP4 on activation, development and mRNA expression of GDF9, BMP15, PCNA, Bax and Bcl2 in cultured bovine follicles enclosed in ovarian tissues. Ovarian tissue fragments were cultured for 6 days in α-MEM+ alone or supplemented with different concentrations of BMP4 (10, 50 or 100 ng/ml). Classical histology was performed to analyze follicle growth and morphology, while real-time PCR was used to analyze mRNA levels in fresh and cultured tissues. After 6 days, the culture of ovarian tissue in α-MEM+ alone or supplemented with 10, 50 or 100 ng/ml BMP4 promoted follicular activation. The different concentrations of BMP4 maintained the percentage of normal follicles similar to results of the control. The presence of 100 ng/ml BMP-4 in culture medium increased oocyte and follicular diameters of primary and secondary follicles when compared with those follicles from uncultured control or cultured in α-MEM+ alone (P < 0.05). The tissues cultured in the presence of increasing concentrations of BMP4 had an increase in mRNA expression of the tested genes, but despite this the differences were not statistically significant. In conclusion, 100 ng/ml BMP4 promotes an increase in diameters of follicles and oocytes of primary and secondary follicles after 6 days of in vitro culture.


Assuntos
Proteína Morfogenética Óssea 4/farmacologia , Folículo Ovariano/efeitos dos fármacos , Folículo Ovariano/fisiologia , Ovário/citologia , Técnicas de Cultura de Tecidos/métodos , Animais , Proteína Morfogenética Óssea 15/genética , Bovinos , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Fator 9 de Diferenciação de Crescimento/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteína X Associada a bcl-2/genética
4.
Zygote ; 23(1): 41-52, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23941689

RESUMO

The role of activin-A in follicular development and on the mRNA expression levels of different genes in goat secondary follicles was evaluated. Goat secondary follicles (≥ 150 µm) were cultured for 18 days under control conditions or with the addition of either 50 or 100 ng/ml activin-A (Experiment 1). The mRNA levels for the genes that code for activin-A, ActR-IA, ActR-IB, ActR-IIA, ActR-IIB, follicle stimulating hormone receptor (FSH-R) and P450 aromatase were measured in each condition (Experiment 2). We observed that after 6 days of culture, the antrum formation rate was higher in cultures with added activin-A than in the cultured control (P < 0.05). The addition of 50 ng/ml activin-A increased the follicular growth rate in the final third of the culture (days 12-18), resulting in a higher percentage of meiosis resumption (P < 0.05). On day 6, the addition of activin-A (50 ng/ml) increased the levels of ActR-IA mRNA compared with the cultured control (P < 0.05). After 18 days, the addition of 50 ng/ml activin-A significantly increased the levels of its own mRNA compared with the non-cultured control. Moreover, this treatment reduced the mRNA levels of P450 aromatase in comparison with the cultured control (P < 0.05). Higher levels of P450 aromatase mRNA were found for both activin-A treatments compared with the non-cultured control (P < 0.05). No difference in estradiol levels was detected among any of the tested treatments. In conclusion, the addition of activin-A to culture medium stimulated early antrum formation as well as an increase in the daily follicular growth rate and the percentage of meiosis resumption.


Assuntos
Ativinas/farmacologia , Folículo Ovariano/efeitos dos fármacos , Folículo Ovariano/fisiologia , Receptores de Ativinas Tipo I/genética , Receptores de Activinas Tipo II/genética , Ativinas/genética , Animais , Aromatase/genética , Células Cultivadas , Estradiol/análise , Estradiol/metabolismo , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Cabras , Técnicas de Maturação in Vitro de Oócitos/métodos , Folículo Ovariano/ultraestrutura , Receptores do FSH/genética
5.
Acta Cir Bras ; 29 Suppl 3: 22-7, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25351152

RESUMO

PURPOSE: To evaluate morphological and functional aspects of the ovarian graft in transplanted rats treated with NAC. METHODS: Female Wistar rats, virgin, 3 to 4 months old, weighing 200-250 grams were used in experiments. The rats have been kept in proper sanitary conditions, receiving food and water ad libitum. Five groups (n=10, each) were constituted: 4 groups treated subcutaneously with NAC, at doses of 150, 300, 600 and 1200 mg/kg (NAC150, NAC300, NAC600 and NAC1200, respectively), one hour of before the ovarian transplantation and control group (GTx) - treated with physiological solution and submitted to ovarian transplantation. The rats were anesthetized and submitted to autologous left ovarian transplantation, without anastomosis in retroperitoneum, and contralateral oophorectomy. During follow-up of 4 or 15 days, the estrous cycle was evaluated by vaginal smears to determine cycle regularity. At the end of 4th or 15th days, rats were re-anesthetized and blood and graft were obtained to estradiol analysis and morphological assessment. Data were analysed by One Way Analysis of Variance (ANOVA) or ANOVA on ranks complemented by Student-Newman-Keuls test. RESULTS: At 4th day, viable follicles in the graft did not altered by NAC treatments. The NAC300 and NAC600 groups showed increasing in follicle atresia (p=0.012) compared to GTx and NAC1200 group. At 15th day, 50% of GTx, NAC150, and NAC300 rats showed regular oestrous cycle; 83% of NAC600 and 100% of NAC1200 rats returned to regular cycle. NAC1200 group showed increasing in primordial follicle compared to GTx, NAC150 or NAC300 (p=0.011). NAC did not interfere in estradiol levels after 4 or 15 days of transplantation. CONCLUSION: In autologous ovarian transplantation, high dose of NAC promotes graft viability with recovery of estrous cycle.


Assuntos
Acetilcisteína/farmacologia , Ovário/transplante , Transplantes/efeitos dos fármacos , Acetilcisteína/administração & dosagem , Animais , Estradiol/sangue , Ciclo Estral/efeitos dos fármacos , Feminino , Modelos Animais , Folículo Ovariano/efeitos dos fármacos , Ovário/anatomia & histologia , Ovário/efeitos dos fármacos , Distribuição Aleatória , Ratos Wistar , Fatores de Tempo , Transplantes/fisiologia
6.
Acta cir. bras ; 29(supl.3): 22-27, 2014. tab, graf
Artigo em Inglês | LILACS | ID: lil-726248

RESUMO

PURPOSE: To evaluate morphological and functional aspects of the ovarian graft in transplanted rats treated with NAC. METHODS: Female Wistar rats, virgin, 3 to 4 months old, weighing 200-250 grams were used in experiments. The rats have been kept in proper sanitary conditions, receiving food and water ad libitum. Five groups (n=10, each) were constituted: 4 groups treated subcutaneously with NAC, at doses of 150, 300, 600 and 1200 mg/kg (NAC150, NAC300, NAC600 and NAC1200, respectively), one hour of before the ovarian transplantation and control group (GTx) - treated with physiological solution and submitted to ovarian transplantation. The rats were anesthetized and submitted to autologous left ovarian transplantation, without anastomosis in retroperitoneum, and contralateral oophorectomy. During follow-up of 4 or 15 days, the estrous cycle was evaluated by vaginal smears to determine cycle regularity. At the end of 4th or 15th days, rats were re-anesthetized and blood and graft were obtained to estradiol analysis and morphological assessment. Data were analysed by One Way Analysis of Variance (ANOVA) or ANOVA on ranks complemented by Student-Newman-Keuls test. RESULTS: At 4th day, viable follicles in the graft did not altered by NAC treatments. The NAC300 and NAC600 groups showed increasing in follicle atresia (p=0.012) compared to GTx and NAC1200 group. At 15th day, 50% of GTx, NAC150, and NAC300 rats showed regular oestrous cycle; 83% of NAC600 and 100% of NAC1200 rats returned to regular cycle. NAC1200 group showed increasing in primordial follicle compared to GTx, NAC150 or NAC300 (p=0.011). NAC did not interfere in estradiol levels after 4 or 15 days of transplantation. CONCLUSION: In autologous ovarian transplantation, high dose of NAC promotes graft viability with recovery of estrous cycle. .


Assuntos
Animais , Feminino , Acetilcisteína/farmacologia , Ovário/transplante , Transplantes/efeitos dos fármacos , Acetilcisteína/administração & dosagem , Estradiol/sangue , Ciclo Estral/efeitos dos fármacos , Modelos Animais , Folículo Ovariano/efeitos dos fármacos , Ovário/anatomia & histologia , Ovário/efeitos dos fármacos , Distribuição Aleatória , Ratos Wistar , Fatores de Tempo , Transplantes/fisiologia
7.
Homeopathy ; 102(1): 41-8, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23290878

RESUMO

OBJECTIVE: To evaluate the effect of dynamized follicle-stimulating hormone (FSH) on the survival, activation and growth of ovine preantral follicles (PFs) in vitro. METHODS: Ovarian fragments were cultured for 1 or 7 days in alpha minimum essential medium (α-MEM(+)) control in the absence or presence of alcohol (Al control) or FSH (6cH, 12cH and 30cH) added at intervals of 24 or 48 h. The ovarian fragments were processed, coded and analyzed by a blinded observer by classical histology (CH), fluorescence microscopy (FM) and transmission electron microscopy (TEM). RESULTS: After 7 days of culture, the group which to which FSH 6cH was added at 24 h intervals showed better rates of follicle survival and activation compared to α-MEM(+) control or Al control (p < 0.05). This group also showed higher follicle and oocyte growth than α-MEM(+) control (p < 0.05). FM and TEM techniques confirmed that FSH 6cH promoted viability and ultrastructural integrity of follicles after 7 days of culture. CONCLUSIONS: FSH 6cH (24 h) treatment maintained the viability, and promoted the activation and in vitro growth of ovine PFs.


Assuntos
Hormônio Foliculoestimulante/farmacologia , Folículo Ovariano/efeitos dos fármacos , Animais , Técnicas de Cultura de Células , Sobrevivência Celular , Feminino , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Oócitos/efeitos dos fármacos , Oócitos/crescimento & desenvolvimento , Oócitos/fisiologia , Folículo Ovariano/crescimento & desenvolvimento , Folículo Ovariano/fisiologia , Ovinos
8.
Ciênc. rural ; 43(1): 107-113, jan. 2013. ilus, tab
Artigo em Inglês | LILACS-Express | LILACS | ID: lil-659669

RESUMO

This study investigated the levels of messenger ribonucleic acids (mRNA) for inhibin-ßA subunit in goat primordial, primary and secondary follicles, as well as in cumulus-oocyte complexes (COCs) and mural granulosa / theca cells of antral follicles. The effects of activin-A (100ng mL-1) and/or follicle stimulating hormone (FSH, 50ng mL-1) on growth and expression of mRNA for activin-A and FSH receptor (FSH-R) in secondary follicles cultured for six days were evaluated. The data showed that the expression of inhibin-ßA is lower in secondary follicles than in primary follicles and is higher in large antral follicles than in small antral follicles. After culture, activin-A and/or FSH promoted growth of secondary follicles, while FSH increased the levels of mRNA for inhibin-ßA, and activin-A increased the levels of FSH-R mRNA. In conclusion, mRNA for inhibin-ßA is expressed at different levels in pre-antral and antral follicles and activin-A acts as a stimulator of the FSH-R expression in goat follicles. On its turn, the expression of inhibin-ßA is stimulated by FSH, which together with activin-A promotes secondary follicle growth in-vitro.


Este estudo investigou os níveis de ácidos ribonucleicos (RNAm) para a subunidade ßA da inibina em folículos primordiais, primários e secundários caprinos, bem como em complexos cumulus-oócitos (CCOs) e células da granulosa mural/teca de folículos antrais. Além disso, avaliaram-se os efeitos da ativina-A (100ng mL-1) e/ou hormônio folículo estimulante (FSH, 50ng mL-1) sobre o crescimento e a expressão do RNAm para inibina-ßA e receptores de FSH (FSH-R) em folículos secundários cultivados por seis dias. Os dados mostraram que a expressão de inibina-ßA é menor em folículos secundários do que em folículos primários e é maior em grandes folículos antrais que nos pequenos folículos antrais. Após o cultivo, ativina-A e/ou FSH promoveram o crescimento de folículos secundários. Enquanto o FSH aumentou os níveis de RNAm para inibina-ßA, a ativina-A aumentou os níveis de RNAm para FSH-R. Em conclusão, a inibina-ßA é expressa em diferentes níveis em folículos pré-antrais e antrais e a ativina-A atua como um estimulador da expressão de FSH-R em folículos caprinos. Por sua vez, a expressão de inibina-ßA é estimulada pelo FSH, que, juntamente com ativina, promove o crescimento de folículos secundários in vitro.

9.
Zygote ; 21(2): 125-8, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-22717039

RESUMO

Summary This study investigated the effect of three different culture media (α minimum essential medium (α-MEM), McCoy or TCM199 during the in vitro culture (IVC) of bovine isolated pre-antral follicles. Pre-antral follicles greater than 150 µm in size were isolated and cultured for 0 (control), 8 or 16 days in one of the abovementioned culture media. Follicles were evaluated for survival, growth and antrum formation at days 8 and 16. The results showed that TCM199 was the most suitable medium to preserve follicular viability and ultrastructure, resulting in the highest rates of antrum formation. In conclusion, TCM199 promotes the in vitro development of isolated pre-antral follicles without hampering follicular functionality by sustaining in vitro growth and antrum formation.


Assuntos
Sobrevivência Celular/efeitos dos fármacos , Meios de Cultura/farmacologia , Técnicas de Maturação in Vitro de Oócitos , Folículo Ovariano/citologia , Folículo Ovariano/efeitos dos fármacos , Animais , Bovinos , Células Cultivadas , Feminino , Compostos Orgânicos/farmacologia , Folículo Ovariano/ultraestrutura
10.
Braz. j. vet. res. anim. sci ; 43(2): 250-255, 2006. graf
Artigo em Português | LILACS | ID: lil-454662

RESUMO

O objetivo deste estudo foi avaliar foliculos pré-antrais (FOPA) ovinos isolados após sua exposição e criopreservação utilizando glicerol (GLI), etilenoglicol (EG), propanodiol (PROH) ou dimetilsulfóxido (DMSO) a 1,5 e 3,0 M. Cada par ovariano de 5 ovelhas sem raça definida foi coletado em abatedouro local e submetido ao isolamento folicular. Da suspensão obtida, uma aliquota foi imediatamente destinada à análise da viabilidade folicular com o auxílio do corante vital azul de trypan. O restante da suspensão foi dividida em 16 aliquotas de 0,9 mL, suspensas (v/v) em MEM+ com EG, DMSO, GLI ou PROH a 1,5 ou 3,0 M, para teste de toxicidade e criopreservação. Após o término de cada tratamento, a viabilidade folicular foi analisada e os FOPA considerados viáveis se não corados ou não viáveis, quando corados. A análise dos dados mostrou que após o teste de toxicidade e criopreservação, em todos os crioprotetores e em ambas as concentrações, a percentagem de FOPA viáveis foi significativamente reduzida quando comparada ao controle. No teste de toxicidade, quando os crioprotetores foram comparados entre si nas mesmas concentrações, foram observadas percentagens signifIcativamente menores de FOPA viáveis no PROH 3,0 M (38,9%), apresentando-se, portanto, mais tóxico quando comparado aos demais crioprotetores. Após criopreservação, obteve-se percentagens significativamente maiores de foliculos pré-antrais viáveis quando o EG e o DMSO foram utilizados. Em conclusão, FOPA ovinos isolados podem ser criopreservados com sucesso utilizando-se D MSO e EG a 1,5 e 3,0 M.


The aim of this study was to evaluate isolated sheep preantral follicles (PF) after exposure and cryopreservation using glycerol (GLI), ethylene glycol (EG), propanediol (PROH) or dimethyl sulfoxide (DMSO) at 1.5 and 3.0 M. Each ovarian pair from 5 mixed breed adult sheeps was obtained at a local slaughterhouse and submited to follicular isolation. From the obtained suspension, one aliquot was immediately analysed with trypan blue. The remaining suspension was divided in 16 aliquots of 0.9 mL, suspended in (v /v) in MEM+with EG, DMSO, GLI or PROH at 1.5 or 3.0 M to the toxicity test and cryopreservation. After the end of each treatment, the follicular viability was analysed and the PF were classified as viable if not dyed or not viable if dyed with trypan blue. The analysis of the results showed that after the toxicity test and cryopreservation, using all cryoprotectants and at both concentrations, the percentage of viable PF was significandy reduced when compared to the control. At the toxicity test, when the cryoprotectants were compared at the same concentrations, the lowest percentage of viable preantral follicles was obtained when 3.0 M PRO H (38,9%) was used, being, more toxic when compared to the others cryoprotectants. After cryopreservation, significantly higher percentual of viable PF was observed when the EG and DMSO were used. In conclusion, sheep PF can be cryopreserved successfully using DMSO and EG at 1.5 and 3.0 M.


Assuntos
Criopreservação/veterinária , Folículo Ovariano/anatomia & histologia , Ovário/anatomia & histologia , Ovário/metabolismo , Óvulo/crescimento & desenvolvimento , Ovinos , Testes de Toxicidade/veterinária
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