RESUMO
In brief: Conditioned medium from Wharton's jelly mesenchymal stem cells improved tissue and preantral follicle outcomes, preventing adverse effects of oxidative stress, apoptosis, and epigenetic changes. Abstract: This study investigated the methylation patterns of H3K4me3 and H3K9me3, as well as the mRNA expression of genes encoding the epigenetic regulators KDM1AX1, KDM1AX2, and KDM3A in goat preantral follicles developed in vivo (Uncultured control) or after in vitro culture for 7 days in either the absence (α-MEM+) or presence of conditioned medium (α-MEM+ + CM) from Wharton's jelly mesenchymal stem cells (WJ-MSCs). In the invivo setting, all follicular categories exhibited similar H3K4me3 and H3K9me3 patterns, and transcripts of KDM1AX1, KDM1AX2, and KDM3A were detected in all samples. During in vitro culture, α-MEM+ + CM enhanced several important aspects. It increased the percentage of normal growing follicles, oocyte diameters across all categories, stromal cell density, and the H3K4me3 methylation pattern in preantral follicles. Simultaneously, it decreased the levels of reduced thiols and reactive oxygen species in the spent media, diminished the presence of lipofuscin aggresomes, lowered granulosa cell apoptotic rates, and reduced the H3K9me3 methylation pattern in preantral follicles. In conclusion, the findings from this study provide compelling evidence that supplementing the in vitro culture medium (α-MEM+) with CM from WJ-MSCs has a protective effect on goat preantral follicles. Notably, CM supplementation preserved follicular survival, as evidenced by enhanced follicular and oocyte growth and increased stromal cell density when compared to the standard culture conditions in the α-MEM+ medium. Furthermore, CM reduced oxidative stress and apoptosis and promoted alterations in H3K4me3 and H3K9me3 patterns.
Assuntos
Apoptose , Epigênese Genética , Cabras , Células-Tronco Mesenquimais , Folículo Ovariano , Estresse Oxidativo , Animais , Feminino , Cabras/fisiologia , Meios de Cultivo Condicionados/farmacologia , Folículo Ovariano/metabolismo , Folículo Ovariano/citologia , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/citologia , Metilação , Células Cultivadas , Histonas/metabolismoRESUMO
This work investigated the effect of zinc oxide nanoparticles functionalized with curcumin (ZnO(np) + CUR) supplementation during the in vitro embryo culture (IVC) on the bovine in vitro embryo production, and the cellular antioxidant response. The cumulus-oocyte complexes (COCs) were matured, fertilized and then the presumptive zygotes were cultured in the medium in the absence (0 µM-control) or presence of different concentrations of ZnO(np) + CUR (3, 6 or 12 µM). After IVC, the embryos were destined either to assay intracellular ROS levels and mitochondrial membrane potential. The results demonstrated that only the addition of 12 µM ZnO(np) + CUR during IVC decreased intracellular ROS production and the rate of blastocyst production when compared to the control (p < .05). In conclusion, ZnO(np) + CUR addition during the IVC impaired in concentration-dependent-manner bovine in vitro embryo production.
Assuntos
Curcumina , Óxido de Zinco , Animais , Bovinos , Técnicas de Maturação in Vitro de Oócitos/veterinária , Técnicas de Maturação in Vitro de Oócitos/métodos , Óxido de Zinco/farmacologia , Curcumina/farmacologia , Espécies Reativas de Oxigênio , Oócitos , Blastocisto , Suplementos Nutricionais , Técnicas de Cultura Embrionária/veterinária , Técnicas de Cultura Embrionária/métodos , Fertilização in vitro/veterinária , Desenvolvimento EmbrionárioRESUMO
The present study evaluated the effects of in vitro maturation (IVM) on the proteome of cumulus-oocyte complexes (COCs) from ewes. Extracted COC proteins were analyzed by LC-MS/MS. Differences in protein abundances (p < 0.05) and functional enrichments in immature versus in vitro-matured COCs were evaluated using bioinformatics tools. There were 2550 proteins identified in the COCs, with 89 and 87 proteins exclusive to immature and mature COCs, respectively. IVM caused downregulation of 84 and upregulation of 34 proteins. Major upregulated proteins in mature COCs were dopey_N domain-containing protein, structural maintenance of chromosomes protein, ubiquitin-like modifier-activating enzyme 2. Main downregulated proteins in mature COCs were immunoglobulin heavy constant mu, inter-alpha-trypsin inhibitor heavy chain 2, alpha-2-macroglobulin. Proteins exclusive to mature COCs and upregulated after IVM related to immune response, complement cascade, vesicle-mediated transport, cell cycle, and extracellular matrix organization. Proteins of immature COCs and downregulated after IVM were linked to metabolic processes, immune response, and complement cascade. KEGG pathways and miRNA-regulated genes attributed to downregulated and mature COC proteins related to complement and coagulation cascades, metabolism, humoral response, and B cell-mediated immunity. Thus, IVM influenced the ovine COC proteome. This knowledge supports the future development of efficient IVM protocols for Ovis aries.
Assuntos
Células do Cúmulo , MicroRNAs , Ovinos , Animais , Feminino , Células do Cúmulo/metabolismo , Proteoma/metabolismo , Carneiro Doméstico , Cromatografia Líquida , Espectrometria de Massas em Tandem , Oócitos/metabolismo , Ubiquitinas/metabolismo , Ubiquitinas/farmacologia , Imunoglobulinas/metabolismo , Macroglobulinas/metabolismo , Macroglobulinas/farmacologia , MicroRNAs/metabolismo , Técnicas de Maturação in Vitro de Oócitos/métodosRESUMO
The present study was conducted to characterize the major proteome of preimplantation (D6) ovine embryos produced in vitro. COCs were aspirated from antral follicles (2-6 mm), matured and fertilized in vitro and cultured until day six. Proteins were extracted separately from three pools of 45 embryos and separately run in SDS-PAGE. Proteins from each pool were individually subjected to in-gel digestion followed by LC-MS/MS. Three 'raw files' and protein lists were produced by Pattern Lab software, but only proteins present in all three lists were used for the bioinformatics analyses. There were 2,262 proteins identified in the 6-day-old ovine embryos, including albumin, zona pellucida glycoprotein 2, 3 and 4, peptidyl arginine deiminase 6, actin cytoplasmic 1, gamma-actin 1, pyruvate kinase, heat shock protein 90 and protein disulfide isomerase, among others. Major biological processes linked to the sheep embryo proteome were translation, protein transport and protein stabilization, and molecular functions, defined as ATP binding, oxygen carrier activity and oxygen binding. There were 42 enriched functional clusters according to the 2,147 genes (UniProt database). Ten selected clusters with potential association with embryo development included translation, structural constituent of ribosomes, ribosomes, nucleosomes, structural constituent of the cytoskeleton, microtubule-based process, translation initiation factor activity, regulation of translational initiation, cell body and nucleotide biosynthetic process. The most representative KEEG pathways were ribosome, oxidative phosphorylation, glutathione metabolism, gap junction, mineral absorption, DNA replication and cGMP-PKG signalling pathway. Analyses of functional clusters clearly showed differences associated with the proteome of preimplantation (D6) sheep embryos generated after in vitro fertilization in comparison with in vivo counterparts (Sanchez et al., 2021; https://doi.org/10.1111/rda.13897), confirming that the quality of in vitro derived blastocysts are unlike those produced in vivo. The present study portrays the first comprehensive overview of the proteome of preimplantational ovine embryos grown in vitro.
Assuntos
Proteoma , Proteômica , Animais , Blastocisto/fisiologia , Cromatografia Líquida/veterinária , Fertilização in vitro/veterinária , Oxigênio , Ovinos , Espectrometria de Massas em Tandem/veterináriaRESUMO
The cryopreservation of secondary follicles (SF) is a promising alternative to preserve the reproductive potential both in humans and animals in situations in which the transplantation of ovarian tissue is not possible. The objective of the present study was cryopreserved SF isolated sheep. Beyond follicular morphology, viability and development, we investigated proteins related to steroidogenic function and basement membrane remodeling [metalloproteinases 2 (MMP-2) and 9 (MMP-9)] in fresh SF (FSF) and vitrified SF (VSF) followed by in vitro culture for 6 (D6) or 12 days (D12). The percentage of intact follicles, follicular and oocyte diameter of the VSF were lower than FSF on both days of culture (P < 0.05). The VSF viability was statistically reduced from D6 (95.5%) to D12 (77.3%) but did not differ from the FSF on both days (D6:96.2% to D12:86.5%). Antrum formation in the VSF (D6: 59.13%; D12: 79.56%) was significantly lower than the FSF (D6: 79.61%; D12: 92.23%). However, an increase in this percentage was observed from D6 to D12 in both groups. Aromatase showed stronger labeling on FSF D6 and VSF D12 compared to other treatments (P < 0.05). MMP-2 showed a similar pattern of labeling in FSF D6 and VSF D12, similarly to that observed in FSF D12 and VSF D6. MMP-9 was similar in FSF and VSF cultivated for 6 and 12 days. In conclusion, VSF are able to grow and develop during 12 days of in vitro culture and showed evidence of preservation of steroidogenic function and remodeling of the basement membrane.
Assuntos
Metaloproteinase 9 da Matriz , Vitrificação , Animais , Aromatase/metabolismo , Criopreservação/veterinária , Feminino , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Folículo Ovariano/metabolismo , OvinosRESUMO
This study evaluated the effects of bioidentical nanostructured progesterone alone or in association with human chorionic gonadotropin (hCG) on the in vitro survival and development of preantral follicles (experiment 1) and oocyte maturation (experiment 2). Bioidentical hormones have a molecular structure identical with that of endogenous hormones; nanostructured substances refer to those reduced to a nanoscale. In experiment 1, fragments of goat ovarian tissue were cultured for 7 days in α-MEM+ alone or supplemented with nanoprogesterone (MEM+ + P4) or P4 and hCG (MEM+ + P4 + hCG). In experiment 2, two mediums of oocyte in vitro maturation (IVM) were compared. Medium 1 consisted of TCM 199+ + LH, and medium 2 consisted of TCM 199+ with nanoprogesterone and hCG. The MEM+ + P4 + hCG treatment showed the lowest percentage of follicular survival after 7 days of culture. MEM+ + P4 and MEM+ + P4 + hCG treatments showed higher percentage of follicular activation than MEM+. In experiment 2, there were no differences between mediums 1 and 2 for all endpoints evaluated. In conclusion, the addition of nanoprogesterone is advisable for in vitro culture of preantral follicles and oocyte maturation. However, the association of nanoprogesterone with hCG causes the cellular death of initial follicles but shows efficacy in IVM.
Assuntos
Técnicas de Maturação in Vitro de Oócitos/métodos , Nanoestruturas/normas , Folículo Ovariano/fisiologia , Progesterona/metabolismo , Técnicas de Cultura de Tecidos/métodos , Animais , Feminino , HumanosRESUMO
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.
Assuntos
Criopreservação/métodos , Crioprotetores/farmacologia , Ovário , Polímeros/farmacologia , Vitrificação/efeitos dos fármacos , Animais , Bovinos , FemininoRESUMO
The present study aimed to evaluate the effect of three culture systems on caprine primordial follicle activation in vitro: follicles cultured either in the isolated form within alginate (Isolated follicles + Alginate treatment), or enclosed in ovarian tissue (in situ), with or without alginate (Fragment + Alginate, and Fragment alone treatments, respectively). After culture, the Isolated follicles + Alginate treatment presented a percentage of morphologically normal follicles (MNF) similar to both the non-cultured control and the Fragment Alone treatments. Nevertheless, Fragment + Alginate treatment showed a significant reduction in the number of MNF when compared to the other treatments. Regarding follicle development, our results showed that regardless of the alginate, the presence of ovarian tissue limited primordial follicle activation during in vitro culture. Remarkably, the Isolated primordial follicle + Alginate treatment was the only one that significantly promoted follicle activation and increased both follicle and oocyte diameters during IVFC, pointing out a higher cell proliferation. In conclusion, the presence of ovarian tissue with or without alginate limited follicle development (activation) after culture. Nevertheless, when primordial follicles were isolated and encapsulated in alginate they presented suitable survival rates, higher rates of follicle activation and continued to grow throughout the culture period.
Assuntos
Cabras/fisiologia , Folículo Ovariano/fisiologia , Técnicas de Cultura de Tecidos/veterinária , Alginatos/farmacologia , Animais , Meios de Cultura , Feminino , Oócitos , Folículo Ovariano/efeitos dos fármacos , Técnicas de Cultura de Tecidos/métodosRESUMO
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/fisiologiaRESUMO
This study aimed to examine the in vitro culture of secondary preantral follicles, using reused ovaries, to compare both the 2D and 3D methods of in vitro culture of preantral follicles, and the system of medium replacement. Twenty-five pairs of ovaries from mixed-breed goats were used for the experiment. Follicular puncture of antral follicles was performed for in vitro production. After this procedure, the secondary preantral follicles were submitted to a microdissection procedure. The isolated preantral follicles were randomly divided into three treatments: (a) Two-dimensional culture with partial replacement of medium during culture (2D PR), (b) Three-dimensional culture with addition of medium during culture (3D AD) and (c) Three-dimensional culture with partial replacement of medium (3D PR). The culture period was 18 days. All treatments at the end of the in vitro culture period (18 days) presented a follicular survival rate which ranged from 59% to 70%, demonstrating that it was possible to perform an experiment with preantral follicles using ovaries that had previously been used in another reproductive biotechnique. The 3D AD treatment showed a survival percentage and follicular diameter higher than the 2D PR treatment, however, it did not differ from the 3D PR treatment. In conclusion, experiments employing the use of preantral follicles can be performed with success after the ovaries have been used for experiments with antral follicles. Moreover, the three-dimensional system with the addition of medium is recommended for in vitro culture of preantral follicles, since this system is more practical and financially feasible.
Assuntos
Folículo Ovariano/crescimento & desenvolvimento , Ovário/fisiologia , Técnicas de Cultura de Tecidos/veterinária , Animais , Meios de Cultura , Feminino , Hormônio Foliculoestimulante/farmacologia , Cabras , Oócitos/metabolismo , Reprodução , Técnicas de Cultura de Tecidos/métodosRESUMO
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/veterináriaRESUMO
The objective of this study was to determine whether preantral follicles cultured in vitro for 7 days within ovine ovarian cortical strips could be isolated at the secondary follicles (SF) and grown until antral stage during an additional 6 days period of in vitro culture in the presence of aqueous extract of Justicia insularis. Fresh ovarian fragments from 16 adult sheep were fixed for histological analysis (Control 1) or in vitro cultured individually in α-MEM+ supplemented with 0.3 mg/ml J. insularis (Step 1) for 7 days. Part of the fragments then were fixed for histological analysis (in vitro culture group). Remaining fragments were exposed stepwise to increasing trehalose concentrations before immediate isolation of SF and viability assessment (Control 2) or after 6 days of culture in α-MEM++ supplemented with 0.3 mg/ml J. insularis (Step 2). In Step 1, percentage of follicular activation was 80%. In Step 2, a significant increase (p < 0.05) in follicular diameter and antrum formation within 6 days in vitro culture of isolated follicles was achieved. The total antioxidant capacity from both steps significantly increase (p < 0.05) from day 2 to day 6. Confocal analysis of oocytes showed 57.14% oocytes with homogeneous distribution and 42.86% with peri-cortical distribution. In conclusion, SF can be successfully isolated from sheep ovarian cortex after 7 days of culture and are capable of surviving and forming an antral cavity if cultured in vitro for an additional 6 days in the presence of 0.3 mg/ml J. insularis.
Assuntos
Justicia/química , Folículo Ovariano/crescimento & desenvolvimento , Extratos Vegetais/farmacologia , Ovinos , Animais , Meios de Cultura/química , Feminino , Extratos Vegetais/química , Técnicas de Cultura de Tecidos , Trealose/química , Trealose/farmacologiaRESUMO
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 RotulagemRESUMO
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 , OvinosRESUMO
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 , OvinosRESUMO
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/genéticaRESUMO
This study aimed to investigate the effect of including mouse feed with different concentrations (5, 10, or 20%) of Pereskia aculeata Miller (PAM) leaves on the morphology and development of preantral ovarian follicles and ovarian stromal cell density. The oral toxicity was performed using repeated dose toxicity assays subdivided into experiments of 30 days and 90 days of treatment. After the experiments, the ovaries of each animal were collected and submitted to classical histology. At 30 and 90 days, there was an equivalent percentage of normal, primordial, and developing follicles (P > 0.05) between PAM treatments compared to the control. Regarding the different stages of follicular development, after 90 days, there was a higher percentage (P < 0.05) of developing follicles only in the control group compared to day 30. The PAM 5% treatment was the only one that affected the cell density in the stroma after 90 days of treatment. Thus, we observed that supplementing the diet with P. aculeata did not pose any risk concerning animal consumption; specifically, there were no toxic reproductive effects observed from adding Pereskia aculeata Miller to the mouse diet.
RESUMO
The present study investigated the effect of adding allopathic doxorubicin (DOX 0.3⯵g/mL), the vehicle of ultradiluted/dynamized doxorubicin (0.2â¯% ethanol), different dynamizations of ultradiluted/dynamized doxorubicin (DOX 6CH, DOX 12CH and DOX 30CH), both in the absence or presence of chemical stress induced by doxorubicin at 0.3⯵g/mL on follicular survival and activation, antioxidant capacity of the medium, Catalase activity (CAT), production of reactive protein thiol, maintenance of type I and III collagen fibers and accumulation of lipofuscin in porcine ovarian tissue cultured in vitro for 48â¯hours. To do this, part of the ovarian tissue fragments was fixed for the uncultured control and the rest were cultured in: MEM (cultured control), DOX 0.3⯵g/mL, Ethanol, DOX 6CH, DOX 12CH, DOX 30CH, DOX (0.3⯵g/mL) + DOX 6CH, DOX (0.3⯵g/mL) + DOX 12CH, DOX (0.3⯵g/mL) + DOX 30CH treatments. The results showed that, in general, ultradiluted/dynamized doxorubicin (DOX 6CH, DOX 12CH and DOX 30CH) mitigated the toxic effect of allopathic doxorubicin (0.3⯵g/mL) on the morphology of preantral follicles, the content of type I and III collagen fibers, and the production of lipofuscin in the tissue. However, only DOX (0.3⯵g/mL) + DOX 6CH attenuated the oxidative stress induced by DOX (0.3⯵g/mL), maintaining adequate CAT activity that was similar to the uncultured control. Additionally, when the three isolated ultradiluted/dynamized doxorubicin were considered, only DOX 12CH increased the reduced thiol levels compared to the uncultured control and MEM. In conclusion, supplementing the culture medium with ultradiluted/dynamized DOX (DOX 6CH, DOX 12CH and DOX 30CH) attenuated the toxicity induced by allopathic doxorubicin during the in vitro culture of pig preantral follicles enclosed in ovarian tissue.
RESUMO
Understanding of gene expression and metabolic, biological and physiological pathways in ovarian follicular development can have a significant impact on the dynamics of follicular atresia or survival. In fact, some oocyte loss occurs during the transition from secondary to early tertiary follicles. This study aimed to understand, by microarray analysis, the temporal changes in transcriptional profiles of secondary and early antral (tertiary) follicles in caprine ovaries. Ovarian follicles were microdissected and pooled to extract total RNA. The RNA was cross hybridized with the bovine array. Among 23,987 bovine genes, a total of 14,323 genes were hybridized with goat mRNAs while 9,664 genes were not. Of all the hybridized genes, 2,466 were stage-specific, up- and down-regulated in the transition from secondary to early tertiary follicles. Gene expression profiles showed that three major metabolic pathways (lipid metabolism, cell death, and hematological system) were significantly differentiated between the two follicle stages. In conclusion, this study has identified important genes and pathways which may potentially be involved in the transition from secondary to early tertiary follicles in goats.
RESUMO
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.