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Anim Reprod ; 17(2): e20190100, 2020 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-32714448


This study investigated the effect of Folliculinum 6 cH on the oocyte meiosis resumption and viability rates, progesterone production and mitochondrial activity after in vitro maturation of cumulus-oocyte complexes (COCs) in sheep. Sheep ovaries were collected at a local slaughterhouse and COCs were recovered by slicing technique. The selected COCs were maturated in TCM199 (Control treatment), or control medium supplemented with 0.05% ethanol (v/v) (the vehicle of the homeopathic preparation - Ethanol treatment) or with Folliculinum 6 cH. After 24 h of in vitro maturation (IVM), oocytes were mechanically denuded and incubated with Hoechst 33342 and MitoTracker (0.5 µM) Orange CMTMRos for analysis of viability and chromatin configuration, and mitochondrial activity, respectively. The results showed that Folliculinum 6 cH addition increased oocyte degeneration and reduced meiotic resumption compared to the control (P < 0.05). Interestingly, the percentages meiotic resumption and oocyte maturation were lower in the Folliculinum 6 cH treatment compared to its vehicle (Ethanol treatment) (P < 0.05). On the other hand, when the treatments were compared, higher mitochondrial activity was observed in the Ethanol treatment (P < 0.05). In conclusion, contrary to its vehicle, the addition of Folliculinum 6 cH to the IVM medium promoted oocyte degeneration and affected negatively the mitochondrial distribution, impairing meiosis resumption.

Forsch Komplementmed ; 23(5): 307-313, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27811472


BACKGROUND: Auxemma oncocalyx and its main component oncocalyxone A (onco A) have a high level of antioxidant and antitumor activity, but there are no studies on the action of both of these drugs regarding folliculogenesis. MATERIAL AND METHODS: Caprine ovarian tissue fragments were fixed (non-cultured control) or cultured for 1 or 7 days in α-MEM+ alone (cultured control) or supplemented with dimethyl sulfoxide (DMSO; 20% v/v), bone morphogenetic protein 15 (BMP-15; 100 ng/ml), doxorubicin (DXR; 0.3 g/ml), or different concentrations of A. oncocalyx (1.2, 12, or 34 g/ml) or onco A (1, 10, or 30 g/ml). We analyzed for follicular morphology and growth, apoptosis (terminal deoxynucleotidyl transferase dUTP-biotin nick end labeling (TUNEL) assay), and cell proliferation (silver staining of argyrophilic nucleolus organizer regions (AgNOR) and test for proliferating cell nuclear antigen (PCNA)). RESULTS: A. oncocalyx and onco A (in a concentration-dependent manner) and DXR decreased (P < 0.05) the number of morphologically normal follicles, with no effect (P > 0.05) on follicular growth. A. oncocalyx reduced (P < 0.05) the percentage of normal follicles compared to onco A, whereas DXR, A. oncocalyx 1.2 g/ml, and onco A 1 g/ml increased (P < 0.05) the percentage of TUNEL-positive follicles. DXR decreased (P < 0.05) the number of nucleolus organizer regions. CONCLUSION: A. oncocalyx and onco A affected the in vitro caprine folliculogenesis in a concentration-dependent manner. Onco A (1 g/ml) has a less harmful effect than DXR on goat preantral follicle survival.

Antraquinonas/farmacologia , Folículo Ovariano/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Proliferação de Células , Relação Dose-Resposta a Droga , Feminino , Cabras , Marcação In Situ das Extremidades Cortadas , Técnicas In Vitro , Folículo Ovariano/fisiologia , Antígeno Nuclear de Célula em Proliferação/análise
Theriogenology ; 85(7): 1203-10, 2016 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-26852069


Cryopreservation of preantral follicles is a promising technique to preserve female fertility. The aim of this study was to evaluate the effect of vitrification on the development of secondary follicles included in ovarian tissue or isolated after microdissection. An important end point included is the capacity of grown oocytes to resume meiosis. Sheep ovarian cortexes were cut into fragments and split into three different groups: (1) fresh (control): secondary follicles isolated without any previous vitrification; (2) follicle-vitrification (follicle-vit): secondary follicles vitrified in isolated form; and (3) tissue-vitrification (tissue-vit): secondary follicles vitrified within fragments of ovarian tissue (in situ former) and subsequently subjected to isolation. From the three groups, isolated secondary follicles were submitted to IVC for 18 days. After IVC, cumulus-oocyte complexes (COCs) were harvested from follicles. As an additional control group, in vivo grown, in vivo-grown COCs were collected from antral ovarian follicles. All, recovered COCs were matured and the chromatin configuration was evaluated. Data were analyzed by ANOVA, and the means were compared by Student-Newman-Keuls test, and by chi-square. Differences were considered to be significant when P < 0.05. Isolated preantral follicles from all treatments had normal morphology, antrum formation, and low follicle degeneration after IVC. The growth rate between control and follicle-vit did not differ (P > 0.05), and was higher (P < 0.05) than for tissue-vit. The percentage of follicles that decreased diameter during IVC was significantly higher in tissue-vit than the in follicle-vit. Recovery rate of oocytes from normal follicles was higher in follicle-vit than in tissue-vit. Furthermore, oocyte viability was lower in tissue-vit than other treatments, and follicle-vit did not differ from control and in vivo grown. The percentage of oocytes meiosis resuming was not different between treatments except for in vivo grown. After vitrification, only follicle-vit showed metaphase I oocyte. We conclude that secondary follicles vitrified after isolation displayed a better follicular growth rate, oocyte viability, percentage of oocytes reaching the metaphase I stage, and fewer follicles with decreased diameter after IVC.

Criopreservação , Folículo Ovariano/fisiologia , Ovinos/fisiologia , Preservação de Tecido/veterinária , Vitrificação , Animais , Feminino , Meiose , Preservação de Tecido/métodos
Theriogenology ; 85(8): 1457-67, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-26876055


Cryoinjuries caused by vitrification of tissues and organs lead to the loss of membrane proteins that mediate intercellular communications, such as connexins 37 (Cx37) and 43 (Cx43). Thus, the present study aimed to evaluate ovine Cx37 and Cx43 gene and protein expressions and developmental competence by in vitro-cultured secondary follicles retrieved from vitrified ovarian tissue. Ovarian fragments for the same ovary pair were distributed into six treatments: (1) fresh ovarian tissue (FOT); (2) vitrified ovarian tissue (VOT); (3) isolated follicles from fresh ovarian tissue (FIF); (4) isolated follicles from vitrified ovarian tissue; (5) isolated follicles from fresh ovarian tissue followed by in vitro culture (CFIF); (6) isolated follicles from vitrified ovarian tissue followed by in vitro culture (CVIF). In all treatments, Cx37 and Cx43 gene and protein expression patterns were evaluated by reverse transcription polymerase chain reaction and immunocytochemistry. In addition, secondary follicles were analyzed according to follicular integrity and growth, apoptosis, and cell proliferation. In vitro-cultured secondary follicles (CFIF and CVIF) were evaluated based on morphology (extruded follicles), antrum formation, and viability. The percentage of intact follicles was higher, whereas antrum formation, oocyte extrusion rate, and follicle viability were lower in CVIF than in CFIF treatment (P < 0.05). Terminal deoxynucleotidyl transferase-mediated biotinylated deoxyuridine triphosphates nick end-labeling assay demonstrated that apoptosis was absent in FIF, whereas follicles from all other treatments showed positive labeling. Cell proliferation index was higher in isolated follicles from vitrified ovarian tissue and CVIF treatments than in follicles from FIF. Expression of Cx43 messenger RNA was lower in CVIF treatment when compared with follicles from all other treatments (P < 0.05). Follicle Cx37 messenger RNA levels did not show alterations in any treatment (P > 0.05). Cx37 and Cx43 immunolabeling was localized mainly on granulosa cells and oocytes, respectively. In conclusion, isolation of ovine secondary follicles could be done successfully after vitrification of ovarian tissue, and the basement membrane integrity remained intact after in vitro culture. Although the gene and protein expression of Cx37 did not change after vitrification of ovarian tissue, Cx43 turned out to be altered in secondary follicles after vitrification and in vitro culture.

Conexina 43/metabolismo , Conexinas/metabolismo , Folículo Ovariano/crescimento & desenvolvimento , Ovinos , Animais , Apoptose , Técnicas de Cultura de Células/veterinária , Proliferação de Células , Conexina 43/genética , Conexinas/genética , Criopreservação/veterinária , Feminino , Imunofluorescência , Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Folículo Ovariano/citologia , Folículo Ovariano/metabolismo , RNA Mensageiro/metabolismo , Vitrificação
Theriogenology ; 85(6): 1019-29, 2016 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-26711701


The aim of this study was to determine the effectiveness of minimum essential medium alpha modification (α-MEM), tissue culture medium 199 (TCM-199), and McCoy's medium (McCoy's) on IVC of preantral follicles included in the bovine ovarian cortex (in situ). Bovine ovarian fragments were cultured in α-MEM, TCM-199, or McCoy supplemented ((+)) with glutamine, insulin, transferrin, selenium, ascorbic acid, BSA, penicillin, streptomycin, and HEPES buffer in 24-well plates, at 37 °C and 5% CO2 for 1 or 7 days. The morphology of follicles, normal, primordial and development (primary and secondary), as well as viability and morphometric variables of follicles and oocytes were assessed. The morphology and morphometry of preantral follicles were analyzed by ANOVA followed by the Tukey and Dunnett tests, and viability variables were determined by the chi-square test. The results showed that TCM-199(+) reduced significantly (P < 0.05) the percentage of morphologically normal and viable follicles after 7 days of culture compared to the control. Similar results were observed in McCoy(+), in which the percentage of viable follicles after 7 days of culture was significantly lower (P < 0.05) than in the control. However, it was similar (P > 0.05) between α-MEM(+) and TCM-199(+). Moreover, follicular diameters in McCoy(+) and TCM-199(+) were significantly smaller (P < 0.05) than in control and α-MEM(+) after 7 days of culture. In addition, the ultrastructure of preantral follicles was similar between the control and α-MEM(+) after 7 days of culture. In conclusion, α-MEM(+) showed to be the most effective medium to preserve morphology, morphometry and ultrastructure of bovine preantral follicles, ensuring their viability and growth after in situ culture.

Técnicas de Cultura de Células/veterinária , Meios de Cultura/química , Folículo Ovariano/fisiologia , Animais , Bovinos , Feminino , Folículo Ovariano/crescimento & desenvolvimento , Folículo Ovariano/ultraestrutura
Cell Tissue Res ; 362(1): 241-51, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25948481


The risk of reintroducing malignant cells after ovarian graft into patients following post-cancer treatment is an obstacle for clinical applications (autotransplantation). In this context, in vitro follicle culture would be an alternative to transplantation in order to minimize such risks. Therefore, the aim of this study was to compare the development of secondary follicles after vitrification in isolated form (without stroma) with vitrification in in situ form (within fragments of ovarian tissue). Follicles were first isolated from ovarian fragments from mixed-breed ewes and then vitrified; these comprised the Follicle-Vitrification group (Follicle-Vit), or fragments of ovarian tissue were first vitrified, followed by isolation of the follicles, resulting in the Tissue-Vitrification group (Tissue-Vit). Control and vitrified groups were submitted to in vitro culture (6 days) and follicular morphology, viability, antrum formation, follicle and oocyte diameter, growth rate, ultrastructural characteristics and cell proliferation were evaluated. The percentages of morphologically normal follicles and antrum formation were similar among groups. Follicular viability and oocyte diameter were similar between Follicle-Vit and Tissue-Vit. The follicular diameter and growth rate of Follicle-Vit were similar to the Control, while those of Tissue-Vit were significantly lower compared to the Control. Both vitrified groups had an augmented rate of granulosa cellular proliferation compared to Control. Secondary follicles can be successfully vitrified before or after isolation from the ovarian tissue without impairing their ability to survive and grow during in vitro culture.

Folículo Ovariano/crescimento & desenvolvimento , Vitrificação , Animais , Feminino , Técnicas In Vitro , Ovinos
Homeopathy ; 102(1): 41-8, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23290878


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.

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
Growth Horm IGF Res ; 23(1-2): 37-44, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23333247


OBJECTIVE: Evaluate the possible role of IGF-II alone or in association with FSH on in vitro development of isolated caprine preantral follicles. METHODS: Preantral follicles (≥150 µm) were isolated from goat ovaries and cultured for 18 days in basic αMEM medium (control) or supplemented with IGF-II alone at 20 or 50 ng/ml, named IGF20 and IGF50, respectively, or in combination with recombinant FSH (FSH, IGF20F or IGF50F). During in vitro culture, the follicles were analyzed by using morphology criteria, antrum formation and growth rate as parameters. After 18 days of follicular culture, oocytes equal to or larger than 110 µm were used for in vitro maturation (IVM). Oocyte viability and meiosis resumption were assessed by fluorescence microscopy after labeling with calcein-AM, ethidium homodimer and Hoechst 33342. RESULTS: The IGF20 treatment was the only treatment capable of maintaining the percentage of morphologically normal follicles from D0 until D6 and from D12 to D18 (p>0.05), while in all other treatments the percentage of morphologically normal follicles decreased progressively during 18 days of in vitro culture (p<0.05). At D18, all treatments with IGF-II or FSH resulted in a significantly higher percentage of normal follicles when compared to αMEM alone. The IGF50F treatment provided a significantly higher early antrum formation rate when compared to αMEM and FSH alone. The addition of IGF-II alone (20 or 50 ng/ml) or in combination with FSH prevented oocyte degeneration after IVM. Moreover, the FSH treatment demonstrated a lower percentage of oocyte degeneration when compared to control (4.35% vs. 26.3%, respectively; p<0.05). Regarding meiosis resumption, the IGF20F treatment was the only treatment that significantly differed from αMEM alone. All treatments except the control (αMEM alone) presented oocytes at metaphase II. CONCLUSION: IGF-II associated with FSH stimulated in vitro follicular development, oocyte viability and meiotic resumption of caprine oocytes after IVM.

Hormônio Foliculoestimulante/farmacologia , Cabras , Técnicas de Maturação in Vitro de Oócitos/métodos , Fator de Crescimento Insulin-Like II/farmacologia , Oócitos/efeitos dos fármacos , Folículo Ovariano/efeitos dos fármacos , Animais , Tamanho Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Meios de Cultura/farmacologia , Feminino , Hormônio Foliculoestimulante/administração & dosagem , Cabras/fisiologia , Técnicas de Maturação in Vitro de Oócitos/veterinária , Fator de Crescimento Insulin-Like II/administração & dosagem , Oócitos/citologia , Oócitos/fisiologia , Oogênese/efeitos dos fármacos , Oogênese/fisiologia , Folículo Ovariano/citologia , Folículo Ovariano/fisiologia
Ciênc. rural ; 43(1): 107-113, jan. 2013. ilus, tab
Artigo em Inglês | LILACS-Express | LILACS | ID: lil-659669


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.

Zygote ; 21(4): 394-405, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23174125


Tissue transplantation and in vitro ovarian follicle culture have been investigated as alternative techniques to restore fertility in young women who are facing fertility-threatening diseases or treatments following ovarian tissue cryopreservation. Although transplants of fresh or frozen ovarian tissue have successfully yielded healthy live births in different species including humans, the risks of reintroducing cancer cells back into the patient, post treatment, have limited its clinical purpose. The in vitro ovarian follicle culture minimizes these risks and provides a way to harvest more mature oocytes, however its clinical translation has yet to be determined. Not only is it possible for tissue cryopreservation to safeguard fertility in cancer patients, this technique also allows the maintenance of germplasm banks for animals of high commercial value or for those animals that are at risk of extinction. Given the importance of managing female genetic material, this paper reviews the progress of the methods used to preserve and restore female fertility in different species to demonstrate the results obtained in the past 50 years of research, the current achievements and the future directions on this field.

Pesquisa Biomédica , Criopreservação , Fertilidade/fisiologia , Oócitos/fisiologia , Ovário/fisiologia , Animais , Feminino , Humanos , Oócitos/citologia , Ovário/citologia
Zygote ; 21(3): 295-304, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21993013


Ovarian folliculogenesis in mammals is a complex process. Several compounds have been tested during in vitro culture of follicular cells for a better understanding of the mechanisms and factors related to ovarian folliculogenesis in mammals. From these compounds, vascular endothelial growth factor (VEGF) can be highlighted, as it is strongly associated with angiogenesis and, in recent years, its presence in ovarian cells has been investigated extensively. Previous studies have shown that the presence of VEGF protein, as well as mRNA expression of its receptor 2 (VEGFR-2) increases during follicular development. Therefore, it is likely that the interaction between VEGF and VEGFR-2 is crucial to promote follicular development. However, few studies on the influence of this factor on follicular development have been reported. This review addresses aspects related to the structural characterization and mechanism of action of VEGF and its receptors, and their biological importance in the ovary of mammals.

Oócitos/fisiologia , Ovário/fisiologia , Fator A de Crescimento do Endotélio Vascular/fisiologia , Animais , Sobrevivência Celular , Feminino , Humanos , Mamíferos , Ovário/irrigação sanguínea , Ovário/citologia , Estrutura Terciária de Proteína , Receptores de Fatores de Crescimento do Endotélio Vascular/química , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismo , Fator A de Crescimento do Endotélio Vascular/química
Zygote ; 19(3): 215-27, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-20979684


The aim of this study was to evaluate the influence of the number of follicles per drop (one or three) and antral follicles on in vitro development of isolated goat preantral follicles. Preantral follicles were isolated through microdissection and distributed individually (control) or in groups of three follicles (treatment) in microdroplets of α-MEM with or without 1000 ng/ml follicle stimulating hormone (FSH) for Experiments 1 and 2, respectively. Experiment 3 was divided into four treatments according to the presence of one or three preantral follicles, associated or not with antral follicles. After culture, oocytes were retrieved from morphologically normal follicles and submitted to in vitro maturation (IVM) and live/dead fluorescent labelling. Results of Experiment 1 (basic medium without FSH) showed that culture of preantral follicles in groups enhances viability, growth and antrum formation after 12 days. However, in the presence of FSH (Experiment 2), only the recovery rate of fully grown oocytes for IVM was significantly affected by grouping of follicles. In Experiment 3, in general, co-culture of preantral follicles with an early antral follicle had a detrimental effect on viability, antrum formation and production of oocytes for IVM. In conclusion, the performance of in vitro culture of goat preantral follicles is affected by the number of follicles per drop, the presence of an antral follicle and FSH.

Hormônio Foliculoestimulante/farmacologia , Folículo Ovariano/citologia , Folículo Ovariano/fisiologia , Animais , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Meios de Cultura , Feminino , Cabras , Hormônios/farmacologia , Técnicas In Vitro , Oócitos/citologia , Oócitos/efeitos dos fármacos , Folículo Ovariano/efeitos dos fármacos
Pesqui. vet. bras ; 30(9): 770-781, set. 2010. ilus, graf, tab
Artigo em Inglês | LILACS | ID: lil-562961


This study investigated the effects of bone morphogenetic protein 6 (BMP-6) on in vitro primordial follicle development in goats. Samples of goat ovarian cortex were cultured in vitro for 1 or 7 days in Minimum Essential Medium (control medium) supplemented with different concentrations of BMP-6. Follicle survival, activation and growth were evaluated through histology and transmission electron microscopy (TEM). After 7 days of culture, histological analysis demonstrated that BMP-6 enhanced the percentages of atretic primordial follicles when compared to fresh control (day 0). Nevertheless, BMP-6 increased follicular and oocyte diameter during both culture periods. As the culture period progressed from day 1 to day 7, a significant increase in follicle diameter was observed with 1 or 50ng/ml BMP-6. However, on the contrary to that observed with the control medium TEM revealed that follicles cultured for up to 7 days with 1 or 50ng/ml BMP-6 had evident signs of atresia. In conclusion, this study demonstrated that BMP-6 negatively affects the survival and ultrastructure of goat primordial follicles.

O presente estudo investigou os efeitos da proteína morfogenética óssea-6 (BMP-6) no desenvolvimento in vitro de folículos primordiais caprinos. Amostras de córtex ovariano de cabras foram cultivados por 1 ou 7 dias em Meio Essencial Mínimo (meio controle) suplementado com diferentes concentrações de BMP-6. As taxas de sobrevivência, ativação e crescimento foram avaliadas por histologia clássica e microscopia eletrônica de transmissão (MET). Após 7 dias de cultivo, a análise histológica demonstrou que a BMP-6 aumentou o percentual de folículos primordiais degenerados no dia 7 quando comparados ao controle fresco (D0). Além disso, houve um aumento significativo do diâmetro folicular e oocitário em ambos os períodos de cultivo em todos os tratamentos na presença de BMP-6. Com a progressão do cultivo do dia 1 para o dia 7, nos tratamentos com 1 ou 50ng/ml de BMP-6, foi observado um aumento significativo no diâmetro folicular. Entretanto, contrário ao observado no meio controle, a MET revelou que os folículos cultivados nesses tratamentos apresentavam sinais evidentes de atresia. Em conclusão, esse estudo demonstrou que a BMP-6 afeta negativamente a sobrevivência e a ultra-estrutura de folículos primordiais caprinos.

Animais , Folículo Ovariano/transplante , Folículo Ovariano/ultraestrutura , Proteínas Morfogenéticas Ósseas/efeitos adversos
Theriogenology ; 74(5): 749-55, 2010 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-20537700


The objective was to evaluate the effects of various concentrations of exogenous FSH during in vitro culture of isolated canine preantral follicles. Preantral secondary follicles (>200 microm) were isolated by microdissection and cultured for 18 d in supplemented alpha-Minimum Essential Medium (alpha-MEM). There were three treatment groups: 1) absence of FSH (control medium); 2) FSH100 (fixed concentration of 100 ng/mL throughout the entire culture period); and 3) sequential FSH (FSHSeq - 100, 500, and 1,000 ng/mL were added sequentially). Following culture, all follicles from all treatments were still viable (marked green by calcein-AM). The initial (D0) average follicle diameter for the control, FSH100, and FSHSeq was (mean +/- SEM) 298.96 +/- 7.02, 286.00 +/- 5.87, and 275.39 +/- 174 6.55 microm, respectively (P > 0.05). Mean diameter of follicles treated with FSHSeq on Day 18 (D18-439.80 +/- 14.08 microm) was greater than those of the other treatments (P < 0.05). Daily follicular growth rate (microm/d) of follicles in the FSHSeq treatment (6.47 +/- 0.55) was significantly faster than for both the control (3.67 +/- 0.32) and FSH100 (4.47 +/- 0.38) treatments. Furthermore, FSH100 and FSHSeq treatments had a significantly higher rate of antrum formation than the control group on D12 of culture, whereas after D12, FSH100 had a significantly higher rate of extrusion compared to the control (P < 0.05). In conclusion, the sequential addition of FSH to the culture medium maintained the survival of isolated canine preantral follicles and promoted an increased rate of follicular growth and antrum formation.

Cães , Hormônio Foliculoestimulante/farmacologia , Folículo Ovariano/efeitos dos fármacos , Técnicas de Cultura de Tecidos/veterinária , Animais , Meios de Cultura , Feminino , Folículo Ovariano/anatomia & histologia , Folículo Ovariano/crescimento & desenvolvimento
Braz. j. vet. res. anim. sci ; 46(5): 378-386, 2009. ilus, tab, graf
Artigo em Inglês | LILACS | ID: lil-538430


The aim of this study was to evaluate the effects of pituitary (pFSH) or recombinant (rFSH) FSH on the survival and growth of caprine preantral follicles. Caprine ovarian tissues were in vitro cultured for one or seven days in Minimum Essential Medium (MEM) alone or containing 10, 50, 100 and 1000 ng/ml of pFSH or rFSH. Control tissues (non-cultured) and those cultured were processed for histological and ultrastructural studies. In addition, follicular and oocyte diameter were analysed. After seven days of culture, only 50ng/ml of rFSH maintained the percentage of normal follicles similar to control. Moreover, 10 ng/ml of pFSH and all the concentrations of rFSH promoted primordial follicles activation. In addition, the presence of 50 ng/ml of rFSH promoted the highest follicular diameter at day seven of culture. In conclusion, 50 ng/ml of rFSH maintained the ultrastructural integrity of caprine preantral follicles, promoted primordial follicles activation and further growth of cultured follicles.

O objetivo deste estudo foi avaliar os efeitos do FSH pituitário (pFSH)ou recombinante (rFSH) sobre a sobrevivência e o crescimento de folículos pré-antrais caprinos. O tecido ovariano foi cultivado in vitro por um ou sete dias em Meio Essencial Mínimo (MEM) sozinho, ou contendo 10, 50, 100 e 1000 ng/ml de pFSH ou rFSH. O grupo controle (não cultivado) e aqueles cultivados foram processados para análises histológica e ultra-estrutural. Além disso, os diâmetros folicular e oocitário foram avaliados. Após sete dias de cultivo, apenas 50 ng/ml de rFSH manteve o percentual de folículos normais semelhante ao controle. Além disso, 10 ng/ml de pFSH e todas as concentrações de rFSH promoveram ativação de folículos primordiais. A presença de50 ng/ml de rFSH promoveu o maior diâmetro folicular após sete dias de cultivo. Em conclusão, 50 ng/ml de rFSH manteve a integridadede folículos pré-antrais caprinos e promoveu a ativação e o crescimento dos folículos cultivados.

Animais , Hormônio Foliculoestimulante , Folículo Ovariano/crescimento & desenvolvimento , Folículo Ovariano/ultraestrutura , Cabras , Meios de Cultura , Técnicas de Cultura de Órgãos