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1.
Anim Reprod ; 17(2): e20190100, 2020 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-32714448

RESUMO

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.

2.
Cryobiology ; 94: 66-72, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32339491

RESUMO

Type and concentration of cryoprotective agents (CPAs) are important factors which influence the likelihood of a successful ovarian tissue vitrification outcome. In an attempt to address this factor, the present study was conducted to evaluate the impacts of different synthetic polymers (Supercool X-1000, Supercool Z-1000 and PVP K-12) on vitrification of bovine ovarian tissue. From each ovarian pair, fragments were recovered and immediately fixed for analysis (fresh control) or submitted to vitrification, either or not followed by in vitro culture for one or five days. Vitrification was performed using the ovarian tissue cryosystem (OTC) system. The ovarian tissues were intended for histological and viability analysis [Reactive oxygen species (ROS) production and degenerate cells assay (Ethidium homodimer-1)], as well as immunolocalization of AQP3 and AQP9 were measured. The results showed that during almost all the periods after warming, in treatment groups which contain polymer (X-1000, Z-1000 and PVP), the percentage of morphologically normal follicles was the highest in the X-1000 samples. Furthermore, post-thawed X-1000 group revealed stronger labeling for AQP9 in primordial and transitional follicles, when compared with others. However, morphology after cryopreservation did not correlate with follicle viability and function where the levels of degeneration and tissue damage of PVP K-12 group were lower in comparison with X-1000 group and only in PVP K-12 group, ROS level was similar to that of the fresh control group. We believe that in addition to permeating CPAs, the addition of one (Supercool X-1000) or maybe a combination (Supercool X-1000 and PVP K-12) of non-permeating polymers could be useful to improve the outcome for vitrified bovine ovarian tissue.

3.
Res Vet Sci ; 128: 261-268, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31837514

RESUMO

The culture of preantral follicles as an in vitro model to evaluate the toxicity of new anticancer drug has being established. Therefore, the aim of this study was to evaluate the effect of quinoxaline derivative the 2 2- (XYZC 6H 3 -CH=N-NH)-quinoxaline, 1 (QX) on caprine preantral follicles. We evaluate the follicular morphology and activation, proliferation and apoptosis of granulosa cells and finally the protein (ABCB1) and genes expression (cyclin/Cdks), respectively involved in multidrug resistance and cell cycle progression. Ovarian fragments containing primordial and developing follicles were exposed (in vitro culture) to different concentrations of QX (QX1.5, QX3.0 or QX6.0 µM/mL) during 6 days. To evaluate the effect of QX, the ovarian tissue was exposed to Paclitaxel 0.1 µg/mL (PTX - negative control) or in culture media without QX (MEM). At the end of exposure time, we realized that the QX (all concentrations) increased (P < .05) the normal morphology of preantral follicles compared to control (not treated ovarian tissue) or MEM. However, QX6.0 showed a enhanced (P < .05) on follicular activation (burnout) and apoptosis than QX1.5 and QX3.0. Expression of ABCB1 was similar between QX1.5 and QX6.0 and both were lower than control, MEM and PTX. Interestingly, the apoptosis rate in QX3.0 was similar to control and MEM and lower then QX1.5; QX6.0 and PTX. We conclude that quinoxaline may be a promising chemotherapeutic agent, however, other concentrations within a defined range (2-5.5 µM) could be widely investigated.


Assuntos
Células da Granulosa/efeitos dos fármacos , Folículo Ovariano/efeitos dos fármacos , Quinoxalinas/farmacologia , Animais , Antineoplásicos/farmacologia , Antineoplásicos/toxicidade , Apoptose/efeitos dos fármacos , Feminino , Expressão Gênica/efeitos dos fármacos , Cabras , Células da Granulosa/metabolismo , Células da Granulosa/patologia , Técnicas In Vitro , Folículo Ovariano/citologia , Quinoxalinas/toxicidade
4.
Toxins (Basel) ; 11(11)2019 11 09.
Artigo em Inglês | MEDLINE | ID: mdl-31717534

RESUMO

The impact of zearalenone (ZEN) on female reproduction remains an issue, since its effects may differ among exposed cell types. Besides the use of decontaminants in animal diet, other approaches should be considered to minimise ZEN effects after exposure. Since the first organ in contact with ZEN is the gastrointestinal tract, we hypothesise that products of microbiota metabolism may play a role in ZEN detoxification. We aimed to evaluate the effect of 1 µmol/L ZEN and 1 µmol/L equol (a microbial metabolite), alone or in combination, on the survival and morphology of in vitro cultured ovarian preantral follicles. Ovaries from 12 sheep were collected at a local abattoir and fragmented, and the ovarian pieces were submitted to in vitro culture for three days in the presence or absence of the test compounds. The follicular morphology was impaired by ZEN, but equol could alleviate the observed degeneration rates. While ZEN decreased cell proliferation in primary and secondary follicles, as well as induced DNA double-strand breaks in primordial follicles, all these observations disappeared when equol was added to a culture medium containing ZEN. In the present culture conditions, equol was able to counteract the negative effects of ZEN on ovarian preantral follicles.


Assuntos
Equol/farmacologia , Microbioma Gastrointestinal , Folículo Ovariano/efeitos dos fármacos , Zearalenona/toxicidade , Animais , Técnicas de Cultura de Células , Células Cultivadas , Meios de Cultura/química , Feminino , Folículo Ovariano/metabolismo , Folículo Ovariano/patologia , Ovinos
5.
Zygote ; 27(2): 55-63, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30871647

RESUMO

SummaryStudies have shown that daily exposure to different products, whether chemical or natural, can cause irreversible damage to women's reproductive health. Therefore it is necessary to use tests that evaluate the safety and efficacy of these products. Most reproductive toxicology tests are performed in vivo. However, in recent years, various cell culture methods, including embryonic stem cells and tissues have been developed with the aim of reducing the use of animals in toxicological tests. This is a major advance in the area of toxicology, as these systems have the potential to become a widely used tool compared with in vivo tests routinely used in reproductive biology and toxicology. The present review describes and highlights data on in vitro culture processes used to evaluate reproductive toxicity as an alternative to traditional methods using in vivo tests.


Assuntos
Técnicas de Cultura de Órgãos/métodos , Folículo Ovariano/citologia , Ovário/efeitos dos fármacos , Reprodução/efeitos dos fármacos , Testes de Toxicidade/métodos , Animais , Linhagem Celular , Feminino , Humanos , Oócitos/citologia , Oócitos/efeitos dos fármacos , Folículo Ovariano/efeitos dos fármacos , Ovário/fisiologia
6.
Anim Reprod ; 16(4): 819-828, 2019 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-32368259

RESUMO

The objective of this study was to test the efficiency of powdered coconut water (ACP-406®) base-medium without or with the addition of supplements on in vitro culture of isolated goat secondary follicles. Follicles were cultured for 18 days in α-MEM or in ACP-406®, both without supplements (referred to as α-MEM and ACP, respectively), or both supplemented with BSA, insulin, transferrin, selenium, glutamine, hypoxanthine, and ascorbic acid (referred to as α-MEM+ and ACP+). Follicular morphology, antrum formation, follicular and oocyte diameter, levels of glutathione (GSH), and chromatin configuration after in vitro maturation were evaluated. At the end of culture, ACP-406® base-medium (without or with supplements) showed a higher (P < 0.05) percentage of normal follicles than α-MEM (without or with supplements). Antrum formation was similar among α-MEM+, ACP and ACP+, and significantly higher than α-MEM without supplements. The follicular diameter was greater in ACP+ than α-MEM, and similar to other treatments. Moreover, fully and daily grown rates were higher (P < 0.05) in ACP-406® base-medium (without or with supplements) than α-MEM (without or with supplements). Levels of GSH were similar between ACP+ and α-MEM+ treatments. Both ACP+ and α-MEM+ allowed meiotic resumption without a significant difference between the two groups. In conclusion, supplemented ACP-406® base-medium maintained follicular survival and promoted the development as well as meiotic resumption of isolated goat secondary follicles cultured in vitro for 18 days.

7.
Anim Reprod ; 16(4): 838-845, 2019 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-32368261

RESUMO

This study evaluated a powdered coconut water solution (ACP 406®) as a base culture medium on the in vitro survival and development of in situ goat preantral follicles. The ovarian fragments were either immediately fixed in Carnoy solution (non-cultured control) or individually cultured for 2 or 6 days. The following culture media (all containing 100 µg/mL penicillin and 100 µg/mL streptomycin) were evaluated: α-MEM (α-MEM alone, without additional supplementation); α-MEM+ (supplemented α-MEM); ACP (ACP®406 alone); or ACP+ (supplemented ACP®406). Additional supplementation includes: 1.25 mg/mL bovine serum albumin, 10 µg/mL insulin, 5.5 µg/mL transferrin, 5 ng/mL selenium, 2 mM glutamine, and 2 mM hypoxanthine. The endpoints (i) follicular morphology; (ii) development; (iii) estradiol production; and (iv) reactive oxygen species (ROS) were recorded. Data were analyzed using chi-square, Turkey, t-test or One-Way ANOVA. Differences were considered significant when P < 0.05. At day 2 of culture, a greater (P < 0.05) percentage of morphologically normal follicles was observed between ACP+ and ACP treatments. Moreover, at day 2 of culture, no hormonal difference (P < 0.05) was observed between ACP+ and both α-MEM treatments. At day 6 of culture when ACP and α-MEM treatments were compared the percentage of healthy follicles were similar (P > 0.05) among treatments. Overall, all treatments had lower primordial follicles (P < 0.05) accompany by greater developing follicles (P < 0.05) percentages than non-cultured control treatment, indicating primordial follicle activation. However, at day 6 of culture, the percentage of primordial follicle development were similar (P > 0.05) among the treatments. Likewise, no differences (P > 0.05) were observed for ROS production and follicular and oocyte diameters among treatments. Therefore, ACP+ has the equivalent efficiency to MEM+ in maintaining the survival and development of goat preantral follicles, representing an alternative plant-based low-cost culture medium for in vitro culture.

8.
Reprod Toxicol ; 84: 18-25, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30579997

RESUMO

The Withanolide D is a chemotherapeutic potential against the human tumor cell. However, there is no report on the effect of this compound on ovarian function, especially on preantral folliculogenesis. The aim of this study was to evaluate the toxicity of a new candidate to anticancer drug, Withanolide D (WD) on morphologic integrity, development (activation and granulosa cell proliferation) and gene expression of ABCB1 protein of caprine preantral follicles. Ovarian fragments were cultured in vitro for 2 or 6 days in α-MEM or α-MEM added with paclitaxel (PTX -0.1 µg/mL; negative control) and different concentrations of WD (WD1.5, WD3.0 or WD6.0). The higher dose of WD showed a toxic effect similar to PTX and higher (P < 0.05) than other treatments after 2 and 6 days. In addition, WD6.0 reduced the cell proliferating compared to PTX or mild dose. The expression of ABCB1 remained unchanged in the presence of the chemotherapeutic agents (PTX and WD) throughout the culture period. In conclusion, WD exerted a toxic effect observed by decreasing follicular survival and cell proliferation, on the preantral caprine follicles similar to PTX, whose negative effect on folliculogenesis is already widely known.


Assuntos
Antineoplásicos/toxicidade , Folículo Ovariano/efeitos dos fármacos , Vitanolídeos/toxicidade , Animais , Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Feminino , Cabras
9.
Reprod Domest Anim ; 54(2): 216-224, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30203872

RESUMO

The aim of this study was to evaluate the caprine preantral follicles enclosed on vitrified/warmed ovarian cortex grafted to nude BALB/mice during 1 month. The ovarian cortex from goats was fragmented (3 × 3 × 0.5 mm) and divided into four groups: fresh control, vitrified control, fresh transplant and vitrified transplant. Follicular morphology, development and density, fibrosis as well as apoptosis, and tissue revascularization were evaluated. It was also observed a significant decrease in morphologically normal preantral (primordial, transition, primary and secondary) follicles in both vitrified control and vitrified transplant treatments when compared with both fresh control and fresh transplant. However, fresh control and fresh transplant exhibited a similar percentage of developing follicles. Additionally, Vitrified control showed a significant increase in developing follicles in comparison with both fresh control and fresh transplant. Follicular density significantly decreased in all treatments in comparison with fresh control. We observed high fibrosis in both fresh transplant and vitrified transplant. The mRNA expression of caspase 3 was lower in both fresh transplant and vitrified transplant in comparison with vitrified control. In conclusion, xenotransplantation is an excellent strategy to maintain normal preantral follicle morphology after vitrification/warming of goat ovarian tissue. Yet, in order to ensure the survival and development of these follicles, it is essential to improve the revascularization of the graft.


Assuntos
Cabras/fisiologia , Folículo Ovariano/crescimento & desenvolvimento , Folículo Ovariano/transplante , Transplante Heterólogo/veterinária , Vitrificação , Animais , Apoptose , Criopreservação/veterinária , Feminino , Expressão Gênica , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , RNA Mensageiro/análise , Técnicas de Cultura de Tecidos/veterinária
10.
Zygote ; 26(5): 350-358, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30289102

RESUMO

SummaryThe objectives were to develop an effective protocol for transfection of ovine secondary follicles and to assess the effect of attenuating aquaporin 3 (AQP3) using a small interfering RNA (siRNA-AQP3) on antrum formation and follicular growth in vitro. Various combinations of Lipofectamine® volumes (0.5, 0.75 or 1.0 µl), fluorescent oligonucleotide (BLOCK-iT ™) concentrations (3.18, 27.12 or 36.16 nM) and exposure times (12, 14, 16, 18 or 20 h) were tested. The BLOCK-iT™ was replaced by siRNA-AQP3 in the transfection complex. Ovine secondary follicles were isolated and cultured in vitro for 6 days using standard protocols. Follicles were transfected on day 0 or 3 or on both days (0 and 3) and then cultured for an additional 3 or 6 days. As revealed by the fluorescence signal, the Lipofectamine®/BLOCK-iT™ complex (0.75 µl + 27.12 nM by 12 h of incubation) crossed the basement membrane and granulosa cell and reached the oocytes. In general, the rate of intact follicles was higher and the rate of antrum formation was lower in transfected follicles compared with control follicles. In conclusion, ovine secondary follicles can be successfully transfected during in vitro culture, and siRNA-mediated attenuation of AQP3 gene reduced antrum formation of secondary follicles.


Assuntos
Aquaporina 3/genética , Folículo Ovariano/fisiologia , Transfecção/métodos , Animais , Aquaporina 3/metabolismo , Técnicas de Cultura de Células , Feminino , Técnicas de Silenciamento de Genes , Lipídeos , Folículo Ovariano/crescimento & desenvolvimento , Interferência de RNA , Ovinos
11.
Theriogenology ; 116: 83-88, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-29783047

RESUMO

This study aimed to evaluate the follicular morphology and development (follicular activation, cell proliferation, and hormone production), as well as the distribution pattern of Connexins 37 and 43 and SDF-1α after vitrification and in vitro culture of goat ovarian tissue. The study involved four experimental groups: fresh control, vitrified control, fresh culture and vitrified culture. The ovarian fragments were vitrified by a solid surface technique using the Ovarian Tissue Cryosystem and subsequently in vitro cultured for 7 days. The percentage of normal preantral follicles was similar between vitrified control and vitrified culture. However, both vitrified control and vitrified culture treatments showed a significant reduction of morphologically normal follicles in comparison to fresh control. A higher percentage of developing follicles (transition, primary and secondary) was observed in both fresh culture and vitrified culture treatments. Progesterone and estradiol production decreased (P < 0.05) during in vitro culture. SDF-1α and Cx37 proteins were detected in oocytes and granulosa cells from all the treatments. However, in vitrified cultured tissue, only granulosa cells were labeled with Cx37. Connexin 43 was detected in the granulosa, theca cells and zona pellucida in all the treatments. In conclusion, in vitro culture of vitrified goat ovarian cortex was able to promote follicle survival and did not alter the expression of SDF-1α and 43. However, the expression of Cx 37 was modified after in vitro culture of vitrified tissue.


Assuntos
Quimiocina CXCL12/metabolismo , Conexina 43/metabolismo , Conexinas/metabolismo , Cabras/fisiologia , Ovário/fisiologia , Animais , Proliferação de Células , Criopreservação/veterinária , Estradiol/metabolismo , Feminino , Ovário/citologia , Ovário/metabolismo , Progesterona/metabolismo , Técnicas de Cultura de Tecidos/veterinária , Vitrificação
12.
Cell Tissue Res ; 372(3): 611-620, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29488001

RESUMO

The multidrug resistance proteins ABCB1, ABCC2 and ABCG2 are an energy-dependent efflux pump that functions in systemic detoxification processes. Physiologically expressed in a variety of tissues, most abundantly in the liver and intestinal epithelia, placenta, blood-brain barrier and various stem cells, until now, these pumps were not identified in goat ovarian tissue. Therefore, the aim of this study is to analyze ABCB1, ABCC2, and ABCG2 mRNA and protein expression in goat preantral follicles. Fragments (3 × 3 × 1 mm) from five pairs of ovary (n = 10) obtained from five goat were collected and immediately submitted to qPCR, Western blot, and immunofluorescence assay for mRNA detection and identification and localization of the ABC transporters, respectively. mRNA for ABCB1, ABCC2, and ABCG2 and the presence of their proteins were observed on ovarian tissue samples. Positive marks were observed for the three transport proteins in all follicular categories studied. However, the marks were primarily localized in the oocyte of primordial, transition and primary follicle categories. In conclusion, goat ovarian tissue expresses mRNA for the ABCB1, ABCC2 and ABCG2 transporters and the expression of these proteins in the preantral follicles is a follicle-dependent stage.


Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Regulação da Expressão Gênica , Cabras/genética , Folículo Ovariano/metabolismo , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Feminino , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Coloração e Rotulagem
13.
Biopreserv Biobank ; 15(4): 321-331, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28394173

RESUMO

The vitrification of preantral follicles followed by in vitro growth (IVG) could be valuable to produce fertilizable oocytes. However, the meiotic resumption rates of oocytes cultured from vitrified secondary follicles (SF) have been reported as suboptimal. This study aimed to verify two base media (alpha modification of minimum essential medium, α-MEM, and tissue culture medium 199, TCM199) on vitrified SF regarding different requirements during IVG. Sheep ovarian fragments were divided in six groups: (1) Fresh groups (Control α-MEM and TCM199): SF without vitrification; (2) Follicle-Vitrified (Follicle-Vit α-MEM and TCM199): SF vitrified after isolation; and (3) Tissue-Vitrified (Tissue-Vit α-MEM and TCM199): SF vitrified enclosed in ovarian fragments and, subsequently, isolated. The isolated SF were submitted to IVG for 18 days. Thereafter, the recovered cumulus-oocyte complexes (COCs) underwent in vitro maturation (IVM) and evaluation of chromatin configuration. Follicular granulosa cells were analyzed for their gene expression of Bax, Bcl2, and Connexins (CX) 37 and 43. COCs from in vivo antral follicles were used as in vivo control. Data were analyzed by analysis of variance, Tukey, and chi-square tests. Differences were considered significant if p-value is <0.05. Follicle-Vit groups had higher (p < 0.05) percentage of antrum formation compared with Tissue-Vit groups. Vitrification did not affect (p > 0.05) oocyte diameter postmaturation. Oocytes from Follicle-Vit in α-MEM reached metaphase II stage after IVM. Gene expression for CX37, CX43, and Bax was lower in Tissue-Vit groups. For Bcl2, the gene expression was the opposite. In conclusion, during IVG for 18 days, maximal oocyte meiotic resumption was not negatively impacted by vitrification and was greatest for isolated SF using α-MEM as a medium.


Assuntos
Oócitos/citologia , Folículo Ovariano/citologia , Folículo Ovariano/metabolismo , Vitrificação , Animais , Células Cultivadas , Feminino , Perfilação da Expressão Gênica , Metáfase , Compostos Orgânicos/metabolismo , Ovinos , Fatores de Tempo
14.
Theriogenology ; 86(6): 1530-1540, 2016 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-27371972

RESUMO

This study investigated the effect of androstenedione (A4) alone or in association with different concentrations of bovine recombinant FSH on the IVC of isolated goat preantral follicles. Follicles were mechanically isolated from ovarian tissue and cultured for 18 days in α-minimum essential medium supplemented or not with A4 (10 ng/mL) alone or in association with fixed (A4 + FixFSH: 100 ng/mL) or sequential (A4 + SeqFSH: Day 0, 100 ng/mL; Day 6, 500 ng/mL; Day 12, 1000 ng/mL) concentrations of FSH. After 18 days, the oocytes were recovered for IVM and fluorescence analysis. At Day 18 of culture, only A4 + SeqFSH treatment showed a lower (P < 0.05) rate of intact follicles, survival probability, and meiotic resumption, as well as higher (P < 0.05) percentage of degeneration and/or extrusion after antrum formation. Taken together, these results reported a positive correlation between fast-growing follicles and follicles that degenerated and/or extruded after antrum formation. When compared with control, the addition of A4 alone or in association of FSH did not increase (P > 0.05) the estradiol production or androstenedione levels on Day 6. However, on Day 18, the androstenedione levels were significantly lower in A4 + SeqFSH treatment when compared with A4 alone or to A4 + FixFSH treatments, whereas the estradiol production did not differ (P > 0.05). In summary, this study found that accelerated follicle growth negatively impacted the morphology of caprine preantral follicle cultured in vitro. In addition, the association of androstenedione with increasing concentration of FSH was detrimental to follicular survival and oocyte meiotic resumption.


Assuntos
Androstenodiona/farmacologia , Cabras , Meiose/fisiologia , Oócitos/citologia , Folículo Ovariano/crescimento & desenvolvimento , Androstenodiona/biossíntese , Animais , Bovinos , Meios de Cultura , Estradiol/biossíntese , Feminino , Hormônio Foliculoestimulante/administração & dosagem , Hormônio Foliculoestimulante/análise , Técnicas de Maturação in Vitro de Oócitos/veterinária , Meiose/efeitos dos fármacos , Folículo Ovariano/efeitos dos fármacos , Folículo Ovariano/metabolismo , Proteínas Recombinantes , Técnicas de Cultura de Tecidos/veterinária
15.
Theriogenology ; 86(5): 1165-74, 2016 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-27160447

RESUMO

Cryopreservation of ovarian cortex is potentially an important tool for the conservation of endangered species. It will allow preserving the large pool of primordial and primary follicles to retrieve fertilizable oocytes in the future. The aim of this study was to evaluate the effects of slow freezing on the morphology and viability of canine follicles after thawing using DMSO or 1,3-propanediol (PROH) as cryoprotectants. Slices of canine ovarian tissue were equilibrated for 20 minutes at 20 °C in minimum essential medium containing either cryoprotectants at 1.5 M, and then frozen by a standardized protocol. Morphology of follicles after thawing was analyzed by means of histology and transmission electron microscopy, and viability was assessed using Trypan blue and fluorescent probes. The exposure of dog ovarian tissue to both cryoprotectants before freezing had no effect on follicular morphology and viability. Also after freezing, follicles remained histologically normal, but transmission electron microscopy revealed damage of ultrastructure in follicles, which were exposed to PROH. Postthaw viability was significantly reduced with 65.7% of the follicles remaining alive in DMSO and 48.7% in PROH. In conclusion, this study demonstrated the survival of canine oocytes within ovarian cortex cryopreserved by slow freezing using 1.5-M DMSO.


Assuntos
Criopreservação/veterinária , Crioprotetores/farmacologia , Dimetil Sulfóxido/farmacologia , Cães/fisiologia , Ovário/fisiologia , Propilenoglicóis/farmacologia , Animais , Sobrevivência Celular , Feminino , Coloração e Rotulagem
16.
Theriogenology ; 85(7): 1203-10, 2016 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-26852069

RESUMO

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.


Assuntos
Criopreservação , Folículo Ovariano/fisiologia , Ovinos/fisiologia , Preservação de Tecido/veterinária , Vitrificação , Animais , Feminino , Meiose , Preservação de Tecido/métodos
17.
Theriogenology ; 85(8): 1457-67, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-26876055

RESUMO

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.


Assuntos
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
18.
Cell Tissue Res ; 362(1): 241-51, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25948481

RESUMO

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.


Assuntos
Folículo Ovariano/crescimento & desenvolvimento , Vitrificação , Animais , Feminino , Técnicas In Vitro , Ovinos
19.
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
20.
Biopreserv Biobank ; 12(5): 317-24, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25340940

RESUMO

The aim of this study was to evaluate the efficiency of different media in the in vitro culture of bovine preantral follicles that were used either fresh or following slow freezing treatment. Frozen and fresh noncultured or cultured ovarian fragments were processed for histological, viability, and cell proliferation analyses. For cryopreservation, a solution containing 1.5 M ethylene glycol was frozen in a programmable biological freezer. After thawing, a portion of the samples was destined for frozen controls. The remainder were cultured in vitro for 5 days in three media: α-MEM, McCoy, or M199. Samples from these culture media were collected on days 1 and 5 for quantification of reactive oxygen species (ROS) and for hormonal assays. In fresh-cultured tissues, the percentage of morphologically normal follicles was significantly higher when cultured in M199 compared to that in the other media. In frozen-cultured tissues, McCoy medium was significantly superior to the other media, and was the only treatment that helped in maintaining the viability similar to fresh and frozen controls. Upon quantification of the nucleolus organizer region, we observed greater proliferation of granulosa cells in the frozen-cultured tissues with McCoy medium, and lesser proliferation in fresh-cultured tissues only with α-MEM. In frozen-cultured tissues, ROS levels were highest at day 1 and progressively reduced during culture, independent of the media used. In conclusion, under the conditions used in this study, the M199 and McCoy media are recommended for the culture of follicles derived from fresh and frozen ovarian tissues, respectively.


Assuntos
Criopreservação/métodos , Meios de Cultura/química , Folículo Ovariano/citologia , Técnicas de Cultura de Tecidos/métodos , Animais , Bovinos , Proliferação de Células , Sobrevivência Celular , Feminino , Hormônios/metabolismo , Humanos , Modelos Biológicos , Espécies Reativas de Oxigênio/metabolismo
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