RESUMEN
Beauvericin (BEA) is a mycotoxin produced by Beauveria bassiana and Fusarium species recently reported as toxic on porcine oocyte maturation and embryo development. The aim of this study was to assess, in the juvenile sheep, whether its effects are due to alterations of oocyte and/or embryo bioenergetic/oxidative status. Cumulus-oocyte-complexes (COCs) were exposed to BEA during in vitro maturation (IVM), evaluated for cumulus cell (CC) apoptosis, oocyte maturation and bioenergetic/oxidative status or subjected to in vitro fertilization (IVF) and embryo culture (IVEC). Oocyte nuclear maturation and embryo development were assessed after Hoechst staining and CC apoptosis was analysed by terminal deoxynucleotidyl transferase-mediated dUTP nick-End labeling assay and chromatin morphology after Hoechst staining by epifluorescence microscopy. Oocyte and blastocyst bioenergetic/oxidative status were assessed by confocal microscopy after mitochondria and reactive oxygen species labelling with specific probes. BEA showed various toxic effects, that is, short-term effects on somatic and germinal compartment of the COC (CCs and the oocyte) and long-term carry-over effects on developing embryos. In detail, at 5 µM, it significantly reduced oocyte maturation and immature oocytes showed increased late-stage (Type C) CC apoptosis and DNA fragmentation while matured oocytes showed unaffected CC viability but abnormal mitochondrial distribution patterns. At lower tested concentrations (3-0.5 µM), BEA did not affect oocyte maturation, but matured oocytes showed reduced mitochondrial activity. At low concentrations, BEA impaired embryo developmental capacity and blastocyst quality after IVF and IVEC. In conclusion, in the juvenile sheep, COC exposure to BEA induces CC apoptosis and oocyte mitochondrial dysfunction with negative impact on embryo development.
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Depsipéptidos/toxicidad , Desarrollo Embrionario/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Micotoxinas/toxicidad , Oocitos/efectos de los fármacos , Animales , Apoptosis/efectos de los fármacos , Embrión de Mamíferos/efectos de los fármacos , Femenino , Estrés Oxidativo/efectos de los fármacos , Embarazo , OvinosRESUMEN
Sperm motility, a feature essential for in vivo fertilization, is influenced by intracellular pH (pHi) homeostasis. Several mechanisms are involved in pHi regulation, among which sodium-hydrogen exchangers (NHEs), a family of integral transmembrane proteins that catalyze the exchange of Na+ for H+ across lipid bilayers. A preliminary characterization of NHE activity and kinetic parameters, followed by analysis of the expression and localization of the protein in ram spermatozoa was performed. NHE activity showed an apparent Km for external Na+ of 17.61 mM. Immunoblotting revealed a molecular mass of 85 kDa. Immunolocalization pattern showed some species-specific aspects, such as positive labeling at the equatorial region of the sperm head. Cariporide, a selective NHE1 inhibitor, significantly reduced pHi recovery (85%). Similarly, exposure to cariporide significantly inhibited different motility parameters, including those related to sperm capacitation. In vitro fertilization (IVF) was not affected by cariporide, possibly due to the non-dramatic, although significant, drop in motility and velocity parameters or due to prolonged exposure during IVF, which may have caused progressive loss of its inhibitory effect. In conclusion, this is the first study documenting, in a large animal model (sheep) of well-known translational relevance, a direct functional role of NHE on sperm pHi and motility. The postulated specificity of cariporide toward isoform 1 of the Na+/H+ exchanger seems to suggest that NHE1 may contribute to the observed effects on sperm cell functionality.
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Guanidinas/farmacología , Intercambiador 1 de Sodio-Hidrógeno/metabolismo , Motilidad Espermática/efectos de los fármacos , Espermatozoides/efectos de los fármacos , Sulfonas/farmacología , Animales , Concentración de Iones de Hidrógeno , Masculino , Ovinos , Capacitación Espermática/efectos de los fármacos , Capacitación Espermática/fisiología , Motilidad Espermática/fisiología , Espermatozoides/metabolismoRESUMEN
Most wild equids and many domestic horse breeds are at risk of extinction, so there is an urgent need for genome resource banking. Embryos cryopreservation allows the preservation of genetics from male and female and is the fastest method to restore a breed. In the equine, embryo production in vitro would allow the production of several embryos per cycle. Intracytoplasmic sperm injection (ICSI) is used to generate horse embryos, but it requires expensive equipment and expertise in micromanipulation, and blastocyst development rates remain low. No conventional in vitro fertilization (IVF) technique for equine embryo production is available. The development of culture conditions able to mimic the maturation of the oocyte in preovulatory follicular fluid (pFF) and the post-maturation in oviductal fluid (OF) may improve embryo production in vitro. Our aim was to analyse the effect of in vitro maturation in pFF and incubation in OF on in vitro maturation of equine oocytes, fertilization using conventional IVF or ICSI, and embryo development after culture in synthetic oviductal fluid (SOF) or DMEM-F12. Oocytes collected from slaughtered mares or by ovum pick up were matured in vitro in pFF or semi-synthetic maturation medium (MM). The in vitro maturation, fertilization and development rates were not statistically different between pFF and MM. After in vitro maturation, oocytes were incubated with or without OF. Post-maturation in OF did not significantly improve the fertilization and development rates. Thus, in our study, exposure to physiological fluids for oocyte maturation and post-maturation does not improve in vitro embryo production in the horse.
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Líquidos Corporales/química , Medios de Cultivo/farmacología , Líquido Folicular/química , Técnicas de Maduración In Vitro de los Oocitos/métodos , Oocitos/efectos de los fármacos , Animales , Blastocisto/citología , Blastocisto/efectos de los fármacos , Blastocisto/fisiología , Medios de Cultivo/química , Técnicas de Cultivo de Embriones/métodos , Técnicas de Cultivo de Embriones/veterinaria , Desarrollo Embrionario/efectos de los fármacos , Femenino , Fertilización In Vitro/métodos , Fertilización In Vitro/veterinaria , Caballos , Técnicas de Maduración In Vitro de los Oocitos/veterinaria , Masculino , Oocitos/citología , Oocitos/fisiología , Oviductos , Inyecciones de Esperma Intracitoplasmáticas/métodos , Inyecciones de Esperma Intracitoplasmáticas/veterinariaRESUMEN
The high complexity of glycome, the repertoire of glycans expressed in a cell or in an organism, is difficult to analyze and the use of new technologies has accelerated the progress of glycomics analysis. In the last decade, the microarray approaches, and in particular glycan and lectin microarrays, have provided new insights into evaluation of cell glycosylation status. Here we present a cell microarray method based on cell printing on microarray slides for the analysis of the glycosylation pattern of the cell glycocalyx. In order to demonstrate the reliability of the developed method, the glycome profiles of equine native uncultured mural granulosa cells (uGCs) and in vitro cultured mural granulosa cells (cGCs) were determined and compared. The method consists in the isolation of GCs, cell printing into arrays on microarray slide, incubation with a panel of biotinylated lectins, reaction with fluorescent streptavidin and signal intensity detection by a microarray scanner. Cell microarray technology revealed that glycocalyx of both uGCs and cGCs contains N-glycans, sialic acid terminating glycans, N-acetylglucosamine and O-glycans. The comparison of uGCs and cGCs glycan signals indicated an increase in the expression of sialic acids, N-acetylglucosamine, and N-glycans in cGCs. Glycan profiles determined by cell microarray agreed with those revealed by lectin histochemistry. The described cell microarray method represents a simple and sensitive procedure to analyze cell surface glycome in mammalian cells.
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Glicocálix/metabolismo , Células de la Granulosa/metabolismo , Lectinas/química , Análisis de Matrices Tisulares/instrumentación , Análisis de Matrices Tisulares/métodos , Animales , Femenino , Células de la Granulosa/citología , CaballosRESUMEN
Glycoprotein oligosaccharides play major roles during reproduction, yet their function in gamete interactions is not fully elucidated. Identification and comparison of the glycan pattern in cumulus-oocyte complexes (COCs) from species with different efficiencies of in vitro spermatozoa penetration through the zona pellucida (ZP) could help clarify how oligosaccharides affect gamete interactions. We compared the expression and localization of 12 glycosidic residues in equine and porcine in vitro-matured (IVM) and preovulatory COCs by means of lectin histochemistry. The COCs glycan pattern differed between animals and COC source (IVM versus preovulatory). Among the 12 carbohydrate residues investigated, the IVM COCs from these two species shared: (a) sialo- and ßN-acetylgalactosamine (GalNAc)-terminating glycans in the ZP; (b) sialylated and fucosylated glycans in cumulus cells; and (c) GalNAc and N-acetylglucosamine (GlcNAc) glycans in the ooplasm. Differences in the preovulatory COCs of the two species included: (a) sialoglycans and GlcNAc terminating glycans in the equine ZP versus terminal GalNAc and internal GlcNAc in the porcine ZP; (b) terminal galactosides in equine cumulus cells versus terminal GlcNAc and fucose in porcine cohorts; and (c) fucose in the mare ooplasm versus lactosamine and internal GlcNAc in porcine oocyte cytoplasm. Furthermore, equine and porcine cumulus cells and oocytes contributed differently to the synthesis of ZP glycoproteins. These results could be attributed to the different in vitro fertilization efficiencies between these two divergent, large-animal models.
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Células del Cúmulo/metabolismo , Caballos/metabolismo , Oligosacáridos/metabolismo , Oocitos/metabolismo , Porcinos/metabolismo , Zona Pelúcida/metabolismo , Animales , Femenino , Histocitoquímica , Técnicas In Vitro , Lectinas , Especificidad de la Especie , Estadísticas no ParamétricasRESUMEN
Fetal adnexa are a non-controversial source of mesenchymal stem cells (MSCs) that have high plasticity, a high proliferation rate, and the ability to differentiate towards multiple lineages. MSC populations have been characterized for their stemness and differentiation capabilities; more recent work has focused on MSC selection and on establishing predictable elements to discriminate the cells with the most potential for regenerative medicine. In this study, we cytogenetically and molecularly characterized and followed the in vitro proliferation and differentiation potential of early-passage canine amniotic membrane MSCs (AM-MSCs) and umbilical cord matrix MSCs (UCM-MSCs) isolated from fetuses at early (35-40 days) and late (45-55 days) gestational ages. We found that cells from both fetal gestational ages showed similar features. In all examined cell lines, the morphology of proliferating cells typically appeared fibroblast-like. Population doublings, passaged up to 10 times, increased significantly with passage number. In both cell types, cell viability and chromosomal number and structure were not affected by gestational age at early passages. Passage-3 AM- and UCM-MSCs from both gestational phases also expressed embryonic (POU5F1) and mesenchymal (CD29, CD44) stemness markers, whereas hematopoietic and histocompatibility markers were never found in any sample. Passage-3 cell populations of each cell type were also multipotential as they could differentiate into neurocytes and osteocytes, based on cell morphology, specific stains, and molecular analysis. These results indicated that MSCs retrieved from the UCM and AM in the early and late fetal phases of gestation could be used for canine regenerative medicine.
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Amnios , Antígenos de Diferenciación/metabolismo , Diferenciación Celular/fisiología , Edad Gestacional , Células Madre Mesenquimatosas , Cordón Umbilical , Amnios/citología , Amnios/metabolismo , Animales , Células Cultivadas , Perros , Femenino , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Embarazo , Cordón Umbilical/citología , Cordón Umbilical/metabolismoRESUMEN
The gastrointestinal tract is the main target of exposure to mycotoxin fumonisin B1 (FB1), common natural contaminant in food. Previous studies reported that proliferating cells are more sensitive than confluent cells to the toxic effect of FB1. This study aims to investigate, by dose- and time-dependent experiments on human colon proliferating intestinal cell line (HT-29), the modifications induced by FB1 at concentrations ranging from 0.25 to 69 µM. The choice of highest FB1 concentration considered the low toxicity previously reported on intestinal cell lines, whereas the lowest one corresponded to the lower FBs levels permitted by European Commission Regulation. Different functional parameters were tested such as cell proliferation, oxidative status, immunomodulatory effect and changes in membrane microviscosity. In addition FB1-FITC localization in this cell line was assessed by using confocal laser scanning microscopy. Lipid peroxidation induction was the main and early (12 h) effect induced by FB1 at concentrations ranging from 0.5 to 69 µM, followed by inhibition of cell proliferation (up to 8.6 µM), the immunomodulatory effect (up to 17.2 µM), by assessing IL-8 secretion, and increase in membrane microviscosity (up to 34.5 µM). The toxic effects observed in different functional parameters were not dose-dependent and could be the consequence of the FB1 intracytoplasmatic localization as confirmed by confocal microscopy results. The different timescales and concentrations active of different functional parameters could suggest different cellular targets of FB1.
Asunto(s)
Carcinógenos Ambientales/toxicidad , Fumonisinas/toxicidad , Línea Celular , Proliferación Celular , Humanos , Intestinos , Peroxidación de LípidoRESUMEN
The purpose of this study was to improve the quality of frozen-thawed Piedmontese bull semen by incorporating MitoTEMPO (MT) in extended semen before cryopreservation. Semen was collected from 4 fertile bulls, using an artificial vagina, once weekly for 6 consecutive weeks. Semen samples were pooled, diluted with Bullxcell® extender, and supplemented with different concentrations of MT (0 as control, 5, 10, 20, 40, and 80 µM) before cooling, equilibration, and freezing procedures. The frozen-thawed semen was assessed for motility, vitality, acrosome intactness, plasma membrane integrity, DNA integrity, apoptosis, mitochondrial membrane potential, intracellular ROS level and in vitro fertilizing capability. The results showed that MT at concentrations of 10, 20, and 40 µM improved the total, progressive, and rapid motility directly after thawing while, at the highest tested concentration (80 µM), it decreased the progressive and rapid motility after 1, 2, and 3 h of incubation. The sperm kinetics including STR and LIN were noticeably increased at concentrations of 10, 20, and 40 µM directly after thawing (0 h), whereas the MT effect was variable on the other sperm kinetics during the different incubation periods. MitoTEMPO improved the sperm vitality at all tested concentrations, while the acrosomal and DNA integrity were improved at 20 µM and the mitochondrial membrane potentials was increased at 80 µM. The cleavage and blastocyst formation rates were significantly increased by using semen treated with 20 µM MT compared with controls. These findings suggest a potential use of MT mainly at a concentration of 20 µM as an additive in the cryopreservation media of bull semen to improve sperm quality.
RESUMEN
Reproductive biotechnologies can be used as a supporting tool, through gamete conservation and in vitro embryo production, in the preservation of invaluable and irreplaceable animal genetic resources. In the present study, immature mouflon cumulus-oocyte complexes (COCs) collected from ovariectomized female ovaries underwent short- or long-term conservation (24 h maintained in Earle's/Hank's (EH) medium or vitrification) under field conditions and afterwards transported to the laboratory where they were cultured for in vitro maturation (IVM) and assessed for oocyte meiotic competence and bioenergetic-oxidative status. Utilization of both storage techniques led to COC morphology preservation, as well as cumulus expansion and oocyte meiotic resumption after the IVM procedure. Quantitative bioenergetic-oxidative parameters were reduced in vitrified oocytes compared with EH ones. Immature COC storage needs to be optimized in both domesticated and non-domesticated sheep as a part of the strategy to avoid the loss of valuable genotypes of these animal species.
RESUMEN
BACKGROUND: Infertility affects ~10-15% of couples trying to have children, in which the rate of male fertility problems is approximately at 30-50%. Copy number variations (CNVs) are DNA sequences greater than or equal to 1 kb in length sharing a high level of similarity, and present at a variable number of copies in the genome; in our study, we used the canine species as an animal model to detect CNVs responsible for male infertility. We aim to identify CNVs associated with male infertility in the dog genome with a two-pronged approach: we performed a sperm analysis using the CASA system and a cytogenetic-targeted analysis on genes involved in male gonad development and spermatogenesis with fluorescence in situ hybridization (FISH), using dog-specific clones. This analysis was carried out to evaluate possible correlations between CNVs on targeted genes and spermatogenesis impairments or infertility factors. RESULTS: We identified two genomic regions hybridized by BACs CH82-321J09 and CH82-509B23 showing duplication patterns in all samples except for an azoospermic dog. These two regions harbor two important genes for spermatogenesis: DNM2 and TEKT1. The genomic region encompassed by the BAC clone CH82-324I01 showed a single-copy pattern in all samples except for one dog, assessed with low-quality sperm, displaying a marked duplication pattern. This genomic region harbors SOX8, a key gene for testis development. CONCLUSION: We present the first study involving functional and genetic analyses in male infertility. We set up an extremely reliable analysis on dog sperm cells with a highly consistent statistical significance, and we succeeded in conducting FISH experiments on sperm cells using BAC clones as probes. We found copy number differences in infertile compared with fertile dogs for genomic regions encompassing TEKT1, DNM2, and SOX8, suggesting those genes could have a role if deleted or duplicated with respect to the reference copy number in fertility biology. This method is of particular interest in the dog due to the recognized role of this species as an animal model for the study of human genetic diseases and could be useful for other species of economic interest and for endangered animal species.
Asunto(s)
Variaciones en el Número de Copia de ADN/genética , Procesamiento de Imagen Asistido por Computador , Infertilidad Masculina/genética , Espermatozoides/patología , Animales , Mapeo Cromosómico , Perros , Humanos , Hibridación Fluorescente in Situ , Infertilidad Masculina/patología , Masculino , Espermatogénesis/genéticaRESUMEN
Dairy cows diagnosed with metritis may experience a greater degree of oxidative stress (OS) and a deficit in the antioxidative capacity compared to healthy cows. We aimed to assess circulating OS markers and endometrial cell mitochondrial function, intracellular reactive oxygen species (ROS) production, and mean endometrial nuclear cell area in postpartum cows diagnosed with metritis or as healthy. From an initial pool of 121 Holstein cows, we retrospectively selected 34 cows and balanced for metritis (n = 17) or healthy (n = 17). Metritis was defined as an enlarged uterus with red-brown watery or thick off-white purulent discharge occurring within 21 days postpartum. Cows with no signs of clinical disease (including dystocia or retained placenta) were referred to as healthy. Blood samples for serum reactive oxygen metabolites (d-ROM), antioxidants (OXY), and oxidative status index (OSI) tests, evaluated via photometric determination of plasma thiols, were performed at 7, 14, 21, 28, and 35 days postpartum. Furthermore, from the initial pool, a random subset of 5 cows diagnosed with metritis and 6 diagnosed as healthy we collected (at the same time points as for the blood samples) endometrial cytology samples using the cytobrush technique. From the uterine samples, we evaluated the endometrial cell mitochondrial function, intracellular ROS levels, and the endometrial cell nuclear area using MitoTracker Orange, dichlorodihydrofluorescein diacetate, and Hoechst 33258, respectively. Mixed linear regression models, accounting for repeated measurements, were fitted to assess the effect of metritis versus healthy on circulating and endometrial cell OS parameters and endometrial cell size. The effect of days postpartum and its interaction with uterine health status were forced into each model. Serum concentrations of d-ROMs and OSI were greater in metritis at 7, 14, and 35 days postpartum than in healthy cows. Interestingly, the mean endometrial cell nuclear area was lower in metritis than healthy cows at 14 and 21 days postpartum. We found no differences between metritis and healthy for endometrial cell mitochondrial function and intracellular ROS production. In conclusion, cows diagnosed with metritis experienced greater systemic OS levels than healthy cows, but their OS was not higher in the uterine milieu.
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Enfermedades de los Bovinos , Endometritis , Embarazo , Femenino , Bovinos , Animales , Endometritis/veterinaria , Lactancia , Especies Reactivas de Oxígeno , Estudios Retrospectivos , Enfermedades de los Bovinos/diagnóstico , Periodo Posparto , Antioxidantes/metabolismo , Estrés OxidativoRESUMEN
Gentile di Puglia (GdP) is an autochthonous sheep breed of Southern Italy included among ovine breeds threatened by genetic erosion and extinction risk, which have been given attention by local and international institutions, thus emphasizing the need for germplasm conservation actions. In the present study, two assisted reproduction approaches, finalized for GdP conservation, were performed: (1) on-farm reproductive efficiency evaluation, expressed as pregnancy rate (PR), twin pregnancy rate (tPR), and body condition score (BCS), for three consecutive breeding cycles and (2) pre-pubertal lambs' immature cumulus-oocyte complex (COC) retrieval, vitrification, in vitro maturation (IVM), and assessment of meiotic stage and bioenergetic-oxidative status compared with those of other Italian and European commercial breeds. PR and tPR were progressively reduced over time. In all clinical examination times, BCS was significantly lower in nonpregnant ewes compared with pregnant ones. Fresh GdP pre-pubertal lamb COCs achieved meiotic maturation and showed healthy bioenergetic-oxidative status after IVM. Vitrification reduced the oocyte maturation rate in all groups. However, mature oocytes retained their cytoplasmic maturity, expressed as a mitochondria distribution pattern and activity, indicating promising developmental competence. In conclusion, clinical- and biotechnological-assisted reproduction approaches can support conservation strategies of GdP and other local sheep breeds in Southern Italy.
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Cadmium (Cd), a highly toxic pollutant, impairs oocyte fertilization, through oxidative damage on cumulus cells (CCs). This study analysed the transcriptomic profile of CCs of cumulus-oocyte complexes (COCs) from adult and prepubertal sheep, exposed to Cd nanomolar concentration during in vitro maturation. In both age-groups, CCs of matured oocytes underwent RNA-seq, data analysis and validation. Differentially expressed genes (DEGs) were identified in adult (n = 99 DEGs) and prepubertal (n = 18 DEGs) CCs upon Cd exposure. Transcriptomes of adult CCs clustered separately between Cd-exposed and control samples, whereas prepubertal ones did not as observed by Principal Component Analysis. The transcriptomic signature of Cd-induced CC toxicity was identified by gene annotation and literature search. Genes associated with previous studies on ovarian functions and/or Cd effects were confirmed and new genes were identified, thus implementing the knowledge on their involvement in such processes. Enrichment and validation analysis showed that, in adult CCs, Cd acted as endocrine disruptor on DEGs involved in hormone biosynthesis, cumulus expansion, regulation of cell signalling, growth and differentiation and oocyte maturation, whereas in prepubertal CCs, Cd affected DEGs involved in CC development and viability and CC-oocyte communications. In conclusion, these DEGs could be used as valuable non-invasive biomarkers for oocyte competence.
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This work aimed to determine the effect of cysteamine (25, 50, 100 and 200 µM) incorporated during dilution on frozen thawed buffalo semen quality. Semen was collected twice weekly for 7 consecutive weeks from three Egyptian buffalo bulls using an artificial vagina. Semen samples were pooled and extended with a Tris-based extender, cooled, equilibrated and finally frozen in liquid nitrogen. The diluted semen was evaluated for motility, viability, morphology, plasma membrane and DNA integrity, in addition to oxidative stress and in vitro fertilizing capability. The post thaw motility and velocity parameters noticeably increased with different concentrations of cysteamine (mainly 100 µM) during different incubation periods. The post thaw sperm viability and normality significantly (p < 0.05) improved with concentrations of 50 and 100 µM. Plasma membrane integrity substantially increased at all concentrations of cysteamine. Cysteamine reduced alanine aminotransferase (at all concentrations), aspartate aminotransferase (at 25-100 µM), and creatine kinase (at 100 and 200 µM). Cysteamine at a concentration of 100 µM noticeably enhanced the total antioxidant capacity and glutathione peroxidase and decreased nitric oxide production. Cysteamine, at concentrations of 100 and 200 µM, increased the DNA intensity in the comet head (%) and decreased the DNA % in the comet tail. The comet tail length and moment substantially decreased at concentrations of 50-200 µM. Cysteamine did not affect the in vitro fertilizing capability of sperm. In conclusion, cysteamine incorporation (mainly at a concentration of 100 µM) in buffalo semen extender showed varying protective effects on different sperm parameters against cryo-damage; however, it did not affect the in vitro fertilizing capacity of sperm.
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Bison , Preservación de Semen , Alanina Transaminasa , Animales , Antioxidantes/farmacología , Aspartato Aminotransferasas , Búfalos , Creatina Quinasa , Criopreservación/veterinaria , Crioprotectores/farmacología , Cisteamina/farmacología , Suplementos Dietéticos , Femenino , Glutatión Peroxidasa , Masculino , Óxido Nítrico , Nitrógeno/farmacología , Semen , Análisis de Semen/veterinaria , Preservación de Semen/veterinaria , Motilidad Espermática , EspermatozoidesRESUMEN
BACKGROUND: Heavy metal cadmium (Cd) is a widespread environmental contaminant with a potential toxicity that might negatively affect female reproduction and fertility. It has been reported that Cd exposure impaired the quality of oocytes and led to a defective maturation and fertilization, through oxidative stress induction. Resveratrol (Res) is a natural polyphenol with strong antioxidant properties that exhibited protective role in preventing oocyte redox homeostasis disruption and quality decline. Here, we explored whether the addition of Res to in vitro maturation (IVM) medium might act as a protection against Cd-induced toxicity on ovine oocyte maturation and fertilization. Firstly, we evaluated the effect of supplementing IVM medium with two different Res concentrations (1 and 2 µmol/L) on nuclear maturation and fertilization of oocytes matured under CdCl2 (2 µmol/L) exposure. Therefore, the concentration of 1 µmol/L Res was selected to analyse the effects of this compound on intracellular ROS levels, mitochondrial (mt) distribution and activity, chromatin configuration, cytoskeleton morphology, cortical granules (CGs) distribution and mRNA expression of genes associated with cellular response to oxidative stress (i.e. SIRT1, SOD 1, GPX1, GSR, CAT) in Cd-exposed in vitro matured oocytes. RESULTS: We found that 1 µmol/L Res restored the reduced oocyte meiotic competence induced by Cd exposure as well as, Res sustained oocyte ability to be normally fertilized and decreased polyspermic fertilization at both tested concentrations. Moreover, we demonstrated that 1 µmol/L Res mitigated Cd-induced alterations of oocyte cytoplasmic maturation by reducing reactive oxygen species (ROS) accumulation, preventing mt dysfunction, maintaining the correct meiotic spindle and cortical F-actin assembly and the normal cortical granule distribution as well as up-regulating SIRT1, SOD1 and GPX1 genes. CONCLUSIONS: Taken together, our findings highlighted the beneficial influence exerted by Res in preventing Cd-induced disturbance of nuclear and cytoplasmic maturation and subsequent fertilization in ovine oocytes. Res treatment may help to establish defence strategies counteracting Cd-induced toxicity on the female gamete.
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In conventional assisted reproductive technologies (ARTs), oocytes are in vitro cultured in static conditions. Instead, dynamic systems could better mimic the physiological in vivo environment. In this study, a millifluidic in vitro oocyte maturation (mIVM) system, in a transparent bioreactor integrated with 3D printed supports, was investigated and modeled thanks to computational fluid dynamic (CFD) and oxygen convection-reaction-diffusion (CRD) models. Cumulus-oocyte complexes (COCs) from slaughtered lambs were cultured for 24 h under static (controls) or dynamic IVM in absence (native) or presence of 3D-printed devices with different shapes and assembly modes, with/without alginate filling. Nuclear chromatin configuration, mitochondria distribution patterns, and activity of in vitro matured oocytes were assessed. The native dynamic mIVM significantly reduced the maturation rate compared to the static group (p < 0.001) and metaphase II (MII) oocytes showed impaired mitochondria distribution (p < 0.05) and activity (p < 0.001). When COCs were included in a combination of concave+ring support, particularly with alginate filling, oocyte maturation and mitochondria pattern were preserved, and bioenergetic/oxidative status was improved (p < 0.05) compared to controls. Results were supported by computational models demonstrating that, in mIVM in biocompatible inserts, COCs were protected from shear stresses while ensuring physiological oxygen diffusion replicating the one occurring in vivo from capillaries.
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Técnicas de Maduración In Vitro de los Oocitos , Ovario , Femenino , Ovinos , Animales , Técnicas de Maduración In Vitro de los Oocitos/métodos , Oocitos/fisiología , Oxígeno , Alginatos/farmacologíaRESUMEN
Diagnostic imaging has significantly grown over the last thirty years as indispensable support for diagnostic, prognostic, therapeutic and monitoring procedures of human diseases. This study explored the effects of low-dose X-ray medical diagnostics exposure on female fertility. To aim this, cumulus-oocyte complexes (COCs) recovered from the ovaries of juvenile sheep and human ovaries were used as complementary models for in vitro studies. In the sheep model, the effects of low-dose X-rays on oocyte viability and developmental competence were evaluated. In human ovaries originated from two age group (21-25 and 33-36 years old) subjects with gender dysphoria, X-rays effects on tissue morphology, follicular density and expression of apoptosis-related (NOXA, PUMA, Bcl2, Bak, γH2AX) and cell cycle-related genes (p21 and ki67) were investigated. It was noted that in sheep, the minimum dose of 10 mGy did not influence most of examined parameters at oocyte and embryo levels, whereas 50 and 100 mGy X-ray exposure reduced oocyte bioenergetic/oxidative activity but without any visible effects on oocyte and embryo development. In addition, blastocyst bioenergetic/oxidative status was reduced with all used doses. Overall data on human ovaries showed that low-dose X-rays, similarly as in sheep, did not alter any of examined parameters. However, in women belonging to the 33-36 year group, significantly reduced follicular density was observed after exposure to 50 and 100 mGy, and increased NOXA and Bax expression after exposure at 50 mGy. In conclusion, used low-doses of X-ray exposure, which resemble doses used in medical diagnostics, produce weak damaging effects on female fertility with increased susceptibility in advanced age.
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Embrión de Mamíferos/metabolismo , Desarrollo Embrionario/efectos de la radiación , Metabolismo Energético/efectos de la radiación , Oocitos/metabolismo , Ovario/metabolismo , Rayos X , Adulto , Animales , Femenino , Humanos , Técnicas de Maduración In Vitro de los Oocitos , Ovario/diagnóstico por imagen , Oxidación-Reducción/efectos de la radiación , Radiografía , OvinosRESUMEN
The genotoxic and nephrotoxic mycotoxin Ochratoxin A (OTA) has also been reported to have adverse effects on oocyte maturation and embryo development. Previous studies on the effects of OTA on female fertility have used micromolar concentrations, but no information is available to date on effects in a more relevant nanomolar range. This study used a juvenile sheep model to evaluate the effects of oocyte exposure to low levels of OTA on maturation, fertilization, and embryo development. Further, it was investigated whether different mechanisms of action of OTA could be responsible for varying toxic effects at different levels of exposure. Cumulus-oocyte-complexes (COCs) were exposed to 10 µmol/L-0.1 nmol/L OTA during in vitro maturation and evaluated for cumulus viability, oocyte maturation, and bioenergetic/oxidative status. COCs were subjected to in vitro fertilization, embryo culture, and embryo quality assessment via morphology, viability, bioenergetic/oxidative status, and time-lapse monitoring. At micromolar concentrations, OTA induced cytotoxic effects, by reducing cumulus expansion and oocyte maturation. OTA altered temporospatial dynamics of zygote pronuclear formation and embryo morphokinetics. Blastocysts, even morphologically normal, were found to undergo collapse events, which were probably related to boosted blastocyst mitochondrial activity. At nanomolar concentrations, OTA did not affect COC morpho-functional parameters, but impaired oocyte ability to prevent polyspermy and increased blastocyst apoptosis. In conclusion, in the female germ cell, cytotoxic nonspecific effects characterize OTA-induced toxicity at high exposure levels, whereas fine tuning-mode effects, not associated with altered cell viability and integrity, characterize OTA toxic action at low levels.
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Células del Cúmulo/efectos de los fármacos , Desarrollo Embrionario/efectos de los fármacos , Ocratoxinas/farmacología , Oocitos/efectos de los fármacos , Oocitos/crecimiento & desarrollo , Factores de Edad , Animales , Apoptosis/efectos de los fármacos , Femenino , Modelos Animales , OvinosRESUMEN
BACKGROUND: Equine amniotic mesenchymal stromal cells (AMSCs) and their conditioned medium (CM) were evaluated for their ability to inhibit in vitro proliferation of peripheral blood mononuclear cells (PBMCs) with and without priming. Additionally, AMSC immunogenicity was assessed by expression of MHCI and MHCII and their ability to counteract the in vitro inflammatory process. METHODS: Horse PBMC proliferation was induced with phytohemagglutinin. AMSC priming was performed with 10 ng/ml of TNF-α, 100 ng/ml of IFN-γ, and a combination of 5 ng/ml of TNF-α and 50 ng/ml of IFN-γ. The CM generated from naïve unprimed and primed AMSCs was also tested to evaluate its effects on equine endometrial cells in an in vitro inflammatory model induced by LPS. Immunogenicity marker expression (MHCI and II) was evaluated by qRT-PCR and by flow cytometry. RESULTS: Priming does not increase MHCI and II expression. Furthermore, the inhibition of PBMC proliferation was comparable between naïve and conditioned cells, with the exception of AMSCs primed with both TNF-α and IFN-γ that had a reduced capacity to inhibit T cell proliferation. However, AMSC viability was lower after priming than under other experimental conditions. CM from naïve and primed AMSCs strongly inhibited PBMC proliferation and counteracted the inflammatory process, rescuing about 65% of endometrial cells treated by LPS. CONCLUSION: AMSCs and their CM have a strong capacity to inhibit PBMC proliferation, and priming is not necessary to improve their immunosuppressive activity or reactivity in an inflammatory in vitro model.
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Células Madre Mesenquimatosas , Amnios , Animales , Células Cultivadas , Medios de Cultivo Condicionados , Citocinas/genética , Caballos , Leucocitos MononuclearesRESUMEN
Three-dimensional in vitro maturation (3D IVM) is a promising approach to improve IVM efficiency as it could prevent cumulus-oocyte complex (COC) flattening and preserve its structural and functional integrity. Methods reported to date have low reproducibility and validation studies are limited. In this study, a bioprinting based production process for generating microbeads containing a COC (COC-microbeads) was optimized and its validity tested in a large animal model (sheep). Alginate microbeads were produced and characterized for size, shape and stability under culture conditions. COC encapsulation had high efficiency and reproducibility and cumulus integrity was preserved. COC-microbeads underwent IVM, with COCs cultured in standard 2D IVM as controls. After IVM, oocytes were analyzed for nuclear chromatin configuration, bioenergetic/oxidative status and transcriptional activity of genes biomarker of mitochondrial activity (TFAM, ATP6, ATP8) and oocyte developmental competence (KHDC3, NLRP5, OOEP and TLE6). The 3D system supported oocyte nuclear maturation more efficiently than the 2D control (P<0.05). Ooplasmic mitochondrial activity and reactive oxygen species (ROS) generation ability were increased (P<0.05). Up-regulation of TFAM, ATP6 and ATP8 and down-regulation of KHDC3, NLRP5 expression were observed in 3D IVM. In conclusion, the new bioprinting method for producing COC-microbeads has high reproducibility and efficiency. Moreover, 3D IVM improves oocyte nuclear maturation and relevant parameters of oocyte cytoplasmic maturation and could be used for clinical and toxicological applications.