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1.
FASEB J ; 35(12): e22000, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34731497

RESUMO

Extracellular vesicles (EVs) found in various biological fluids and particularly in reproductive fluids, have gained considerable attention for their possible role in cell- to- cell communication. Among, the different bioactive molecules cargos of EVs, MicroRNAs (miRNAs) are emerging as promising diagnostic biomarkers with high clinical potential. Aiming to understand the roles of EVs in bovine reproductive tract, we intended to characterize and profile the EVs of oviduct and uterine fluids (OF-EVs, UF-EVs) and their miRNA across the estrous cycle. Nanoparticle tracking analysis and transmission electron microscopy confirmed the existence of small EV population in OF and UF at all stages, (size between 30 and 200 nm; concentration: 3.4 × 1010  EVs/ml and 6.0 × 1010  EVs/ml for OF and UF, respectively, regardless of stage). The identification of EV markers (CD9, HSP70, and ALIX proteins) was confirmed by western blot. The miRNA analysis revealed the abundance of 310 and 351 miRNAs in OF-EVs and UF-EVs, respectively. Nine miRNAs were differentially abundant in OF-EVs between stages of the cycle, eight of them displayed a progressive increase from S1 to S4 (p < .05). In UF-EVs, a total of 14 miRNAs were differentially abundant between stages. Greater differences were observed between stage 1 (S1) and stage 3 (S3), with 11 miRNAs enriched in S3 compared to S1. Functional enrichment analysis revealed the involvement of these miRNAs in relevant pathways such as cell signaling, intercellular junctions, and reproductive functions that may be implicated in oviduct and uterus modulation across the cycle, but also in their preparation for embryo/conceptus presence and development.

2.
Biol Reprod ; 2021 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-34617564

RESUMO

During preimplantational embryo development, PI3K/AKT regulates cell proliferation and differentiation and nobiletin modulates this pathway to promote cell survival. Therefore, we aimed to establish whether, when the AKT cascade is inhibited using inhibitors III and IV, nobiletin supplementation to in vitro culture media during the minor (2 to 8-cell stage, MNEGA) or major (8 to 16-cell stage, MJEGA) phases of EGA is able to modulate the development and quality of bovine embryos. In vitro zygotes were cultured during MNEGA or MJEGA phase in SOF + 5% FCS or supplemented with: 15 µM AKT-InhIII; 10 µM AKT-InhIV; 10 µM nobiletin; nobiletin+AKT-InhIII; nobiletin+AKT-InhIV; 0.03% DMSO. Embryo development was lower in treatments with AKT inhibitors, while combination of nobiletin with AKT inhibitors was able to recover their adverse developmental effect and also increase blastocyst cell number. The mRNA abundance of GPX1, NFE2L2, and POU5F1 was partially increased in 8- and 16-cell embryos from nobiletin with AKT inhibitors. Besides, nobiletin increased the p-rpS6 level whether or not AKT inhibitors were present. In conclusion, nobiletin promotes bovine embryo development and quality and partially recovers the adverse developmental effect of AKT inhibitors which infers that nobiletin probably uses another signalling cascade that PI3K/AKT during early embryo development in bovine.

3.
Sex Dev ; : 1-11, 2021 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-34583366

RESUMO

During the process of sex determination, a germ-cell-containing undifferentiated gonad is converted into either a male or a female reproductive organ. Both the composition of sex chromosomes and the environment determine sex in vertebrates. It is assumed that transcription level regulation drives this cascade of mechanisms; however, transcription factors can alter gene expression beyond transcription initiation by controlling pre-mRNA splicing and thereby mRNA isoform production. Using the key time window in sex determination and gonad development in mice, it has been reported that new non-transcriptional events, such as alternative splicing, could play a key role in sex determination in mammals. We know the role of key regulatory factors, like WT1(+/-KTS) or FGFR2(b/c) in pre-mRNA splicing and sex determination, indicating that important steps in the vertebrate sex determination process probably operate at a post-transcriptional level. Here, we discuss the role of pre-mRNA splicing regulators in sex determination in vertebrates, focusing on the new RNA-seq data reported from mice fetal gonadal transcriptome.

4.
Transl Res ; 2021 Aug 27.
Artigo em Inglês | MEDLINE | ID: mdl-34461306

RESUMO

CD160 is a member of the immunoglobulin superfamily with a pattern of expression mainly restricted to cytotoxic cells. To assess the functional relevance of the HVEM/CD160 signaling pathway in allogeneic cytotoxic responses, exon 2 of the CD160 gene was targeted by CRISPR/Cas9 to generate CD160 deficient mice. Next, we evaluated the impact of CD160 deficiency in the course of an alloreactive response. To that aim, parental donor WT (wild-type) or CD160 KO (knock-out) T cells were adoptively transferred into non-irradiated semiallogeneic F1 recipients, in which donor alloreactive CD160 KO CD4 T cells and CD8 T cells clonally expanded less vigorously than in WT T cell counterparts. This differential proliferative response rate at the early phase of T cell expansion influenced the course of CD8 T cell differentiation and the composition of the effector T cell pool that led to a significant decreased of the memory precursor effector cells (MPECs) / short-lived effector cells (SLECs) ratio in CD160 KO CD8 T cells compared to WT CD8 T cells. Despite these differences in T cell proliferation and differentiation, allogeneic MHC class I mismatched (bm1) skin allograft survival in CD160 KO recipients was comparable to that of WT recipients. However, the administration of CTLA-4.Ig showed an enhanced survival trend of bm1 skin allografts in CD160 KO with respect to WT recipients. Finally, CD160 deficient NK cells were as proficient as CD160 WT NK cells in rejecting allogeneic cellular allografts or MHC class I deficient tumor cells. CD160 may represent a CD28 alternative costimulatory molecule for the modulation of allogeneic CD8 T cell responses either in combination with costimulation blockade or by direct targeting of alloreactive CD8 T cells that upregulate CD160 expression in response to alloantigen stimulation.

5.
Nutrients ; 13(7)2021 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-34209137

RESUMO

D-Pinitol (DPIN) is a natural occurring inositol capable of activating the insulin pathway in peripheral tissues, whereas this has not been thoroughly studied in the central nervous system. The present study assessed the potential regulatory effects of DPIN on the hypothalamic insulin signaling pathway. To this end we investigated the Phosphatidylinositol-3-kinase (PI3K)/Protein Kinase B (Akt) signaling cascade in a rat model following oral administration of DPIN. The PI3K/Akt-associated proteins were quantified by Western blot in terms of phosphorylation and total expression. Results indicate that the acute administration of DPIN induced time-dependent phosphorylation of PI3K/Akt and its related substrates within the hypothalamus, indicating an activation of the insulin signaling pathway. This profile is consistent with DPIN as an insulin sensitizer since we also found a decrease in the circulating concentration of this hormone. Overall, the present study shows the pharmacological action of DPIN in the hypothalamus through the PI3K/Akt pathway when giving in fasted animals. These findings suggest that DPIN might be a candidate to treat brain insulin-resistance associated disorders by activating insulin response beyond the insulin receptor.


Assuntos
Hipotálamo/metabolismo , Inositol/análogos & derivados , Fosfatidilinositol 3-Quinase/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Administração Oral , Animais , Glicemia/metabolismo , Ativação Enzimática/efeitos dos fármacos , Glucagon/sangue , Homeostase , Hipotálamo/efeitos dos fármacos , Inositol/administração & dosagem , Inositol/sangue , Inositol/química , Inositol/farmacologia , Insulina/sangue , Insulina/metabolismo , Resistência à Insulina , Fator de Crescimento Insulin-Like I/metabolismo , Masculino , Fosforilação/efeitos dos fármacos , Ratos Wistar , Transdução de Sinais/efeitos dos fármacos
6.
Animals (Basel) ; 11(6)2021 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-34205204

RESUMO

This report reviews current knowledge of sperm metabolomics analysis using proton nuclear magnetic resonance spectroscopy (1 H-NMR) with particular emphasis on human and farm animals. First, we present the benefits of NMR over other techniques to identify sperm metabolites and then describe the specific methodology required for NMR sperm analysis, stressing the importance of analyzing metabolites extracted from both the hydrophilic and lipophilic phases. This is followed by a description of advances produced to date in the use of NMR to diagnose infertility in humans and to identify metabolic differences among the sperm of mammalian herbivore, carnivore, and omnivore species. This last application of NMR mainly seeks to explore the possible use of lipids to fuel sperm physiology, contrary to previous theories that glycolysis and oxidative phosphorylation (OXPHOS) are the only sources of sperm energy. This review describes the use of NMR to identify sperm and seminal plasma metabolites as possible indicators of semen quality, and to examine the metabolites needed to maintain sperm motility, induce their capacitation, and consequently, to predict animal fertility.

7.
Int J Mol Sci ; 22(13)2021 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-34203454

RESUMO

Mutations in splicing factors are recurrent somatic alterations identified in myelodysplastic syndromes (MDS) and they frequently coincide with mutations in epigenetic factors. About 25% of patients present concurrent mutations in such pathways, suggesting a cooperative role in the pathogenesis of MDS. We focused on the splicing factor U2AF1 involved in the recognition of the 3' splice site during pre-mRNA splicing. Using a CRISPR/Cas9 system, we created heterozygous mice with a carboxy-terminal truncated U2af1 allele (U2af1mut/+), studied the U2af1mut/+ hematopoietic system, and did not observe any gross differences in both young (12-13 weeks) and old (23 months) U2af1mut/+ mice, except for a reduction in size of approximately 20%. However, hematopoietic stem/progenitor cells lacked reconstitution capacity in transplantation assays and displayed an aberrant RNA splicing by RNA sequencing. We also evaluated U2af1mut/+ in conjunction with Tet2-deficiency. Novel double mutant U2af1mut/+Tet2-/- mice showed increased monogranulocytic precursors. Hematopoietic stem/progenitor cells were also enhanced and presented functional and transcriptomic alterations. Nonetheless, U2af1mut/+Tet2-/- mice did not succumb to MDS disease over a 6-month observation period. Collectively, our data suggest that cooperation between mutant U2af1 and Tet2 loss is not sufficient for MDS initiation in mice.


Assuntos
Processamento Alternativo/fisiologia , Proteínas de Ligação a DNA/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Fator de Processamento U2AF/metabolismo , Processamento Alternativo/genética , Animais , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/fisiologia , Proteínas de Ligação a DNA/genética , Camundongos , Síndromes Mielodisplásicas/genética , Síndromes Mielodisplásicas/metabolismo , Proteínas Proto-Oncogênicas/genética , Fatores de Processamento de RNA/genética , Fatores de Processamento de RNA/metabolismo , Fator de Processamento U2AF/genética
8.
Curr Biol ; 31(16): 3648-3655.e9, 2021 Aug 23.
Artigo em Inglês | MEDLINE | ID: mdl-34171300

RESUMO

Mammalian spermatozoa are a notable example of metabolic compartmentalization.1 Energy in the form of ATP production, vital for motility, capacitation, and fertilization, is subcellularly separated in sperm cells. While glycolysis provides a local, rapid, and low-yielding input of ATP along the flagellum fibrous sheath, oxidative phosphorylation (OXPHOS), far more efficient over a longer time frame, is concentrated in the midpiece mitochondria.2 The relative weight of glycolysis and OXPHOS pathways in sperm function is variable among species and sensitive to oxygen and substrate availability.3-5 Besides partitioning energy production, sperm cell energetics display an additional singularity: the occurrence of sperm-specific gene duplicates and alternative spliced variants, with conserved function but structurally bound to the flagellar fibrous sheath.6,7 The wider selective forces driving the compartmentalization and adaptability of this energy system in mammalian species remain largely unknown, much like the impact of ecosystem resource availability (e.g., carbohydrates, fatty acids, and proteins) and dietary adaptations in reproductive physiology traits.8 Here, we investigated the Cetacea, an iconic group of fully aquatic and carnivorous marine mammals, evolutionarily related to extant terrestrial herbivores.9 In this lineage, episodes of profound trait remodeling have been accompanied by clear genomic signatures.10-14 We show that toothed whales exhibit impaired sperm glycolysis, due to gene and exon erosion, and demonstrate that dolphin spermatozoa motility depends on endogenous fatty acid ß-oxidation, but not carbohydrates. Such unique energetic rewiring substantiates the observation of large mitochondria in toothed whale spermatozoa and emphasizes the radical physiological reorganization imposed by the transition to a carbohydrate-depleted marine environment.

9.
Sci Rep ; 11(1): 11796, 2021 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-34083641

RESUMO

In vitro culture can alter the development and quality of bovine embryos. Therefore, we aimed to evaluate whether nobiletin supplementation during EGA improves embryonic development and blastocyst quality and if it affects PI3K/AKT signaling pathway. In vitro zygotes were cultured in SOF + 5% FCS (Control) or supplemented with 5, 10 or 25 µM nobiletin (Nob5, Nob10, Nob25) or with 0.03% dimethyl-sulfoxide (CDMSO) during minor (2 to 8-cell stage; MNEGA) or major (8 to 16-cell stage; MJEGA) EGA phase. Blastocyst yield on Day 8 was higher in Nob5 (42.7 ± 1.0%) and Nob10 (44.4 ± 1.3%) for MNEGA phase and in Nob10 (61.0 ± 0.8%) for MJEGA phase compared to other groups. Mitochondrial activity was higher and lipid content was reduced in blastocysts produced with nobiletin, irrespective of EGA phase. The mRNA abundance of CDK2, H3-3B, H3-3A, GPX1, NFE2L2 and PPARα transcripts was increased in 8-cells, 16-cells and blastocysts from nobiletin groups. Immunofluorescence analysis revealed immunoreactive proteins for p-AKT forms (Thr308 and Ser473) in bovine blastocysts produced with nobiletin. In conclusion, nobiletin supplementation during EGA has a positive effect on preimplantation bovine embryonic development in vitro and corroborates on the quality improvement of the produced blastocysts which could be modulated by the activation of AKT signaling pathway.


Assuntos
Técnicas de Cultura Embrionária , Desenvolvimento Embrionário/efeitos dos fármacos , Desenvolvimento Embrionário/genética , Flavonas/farmacologia , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Animais , Biomarcadores , Blastocisto/efeitos dos fármacos , Blastocisto/metabolismo , Bovinos , Feminino , Fertilização In Vitro , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Gravidez , Transdução de Sinais/efeitos dos fármacos
10.
Biol Reprod ; 105(2): 345-358, 2021 08 03.
Artigo em Inglês | MEDLINE | ID: mdl-33889937

RESUMO

We hypothesized that sexually dimorphic differences exist in the expression of miRNAs in amniotic fluid (AF) and maternal blood plasma (MP) in association with the process of sex determination and gonad differentiation in cattle. Amniotic fluid and MP were collected from six pregnant heifers (three carrying a single male and three a single female embryo) following slaughter on Day 39 postinsemination, coinciding with the peak of SRY expression. Samples (six AF and six MP) were profiled using an miRNA Serum/Plasma Focus PCR Panel. Differentially expressed (DE) miRNAs were identified in AF (n = 5) and associated MP (n = 56) of male vs. female embryos (P < 0.05). Functional analysis showed that inflammatory and immune response were among the 13 biological processes enriched by miRNAs DE in MP in the male group (FDR < 0.05), suggesting that these sex-dependent DE miRNAs may be implicated in modulating the receptivity of the dam to a male embryo. Further, we compared the downstream targets of the sex-dependent DE miRNAs detected in MP with genes previously identified as DE in male vs. female genital ridges. The analyses revealed potential targets that might be important during this developmental stage such as SHROOM2, DDX3Y, SOX9, SRY, PPP1CB, JARID2, USP9X, KDM6A, and EIF2S3. Results from this study highlight novel aspects of sex determination and embryo-maternal communication in cattle such as the potential role of miRNAs in gonad development as well as in the modulation of the receptivity of the dam to a male embryo.

11.
Animals (Basel) ; 11(3)2021 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-33804105

RESUMO

Natural service remains the main breeding method in extensively managed beef herds. Although the bull might be the most important factor in determining herd fertility, its importance has been largely overlooked, focusing instead on female fertility. Management of the bull is critical to maximize the opportunities for cow conception. Infectious bovine rhinotracheitis (IBR) and bovine viral diarrhea (BVD) are infections associated with reduced conception rates. This study aimed to determine the effect of both IBR and BVD infection, and bull: cow ratio on seminal parameters in the bull and herd fertility. The presence of antibodies to IBR and BVD, seminal parameters (volume, concentration, mass, and progressive motility), and herd fertility were analyzed in 158 extensively managed bulls. Sperm concentration and mass motility, as well as herd fertility, were significantly lower in BVD-positive bulls. No significant differences were found between IBR-positive and -negative bulls in any reproductive parameter. Sperm concentration was negatively affected by BVD infection in both Charolais and Limousin bulls, whereas mass motility and herd fertility were reduced in Limousin bulls only. No differences were observed in the cow: bull ratio between BVD+ and BVD- bulls. A significant negative correlation was detected between the number of cows per bull and herd fertility, which was negatively affected when herds had more than 40 cows per bull. In conclusion, BVD and bull overuse negatively affect the reproductive performance of the herd.

12.
Int J Mol Sci ; 22(7)2021 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-33915805

RESUMO

Low birth weight and rapid postnatal weight gain are independent predictors of obesity and diabetes in adult life, yet the molecular events involved in this process remain unknown. In inbred and outbred mice, this study examines natural intrauterine growth restriction (IUGR) in relation to body weight, telomere length (TL), glucose tolerance, and growth factor gene (Igf1, Igf2, Insr, Igf1r, and Igf2r) mRNA expression levels in the brain, liver, and muscle at 2- and 10 days of age and then at 3- and 9 months of age. At birth, ~15% of the animals showed IUGR, but by 3 and 9 months, half of these animals had regained the same weight as controls without IUGR (recuperated group). At 10 days, there was no difference in TL between animals undergoing IUGR and controls. However, by 3 and 9 months of age, the recuperated animals had shorter TL than the control and IUGR-non recuperated animals and also showed glucose intolerance. Further, compared to controls, Igf1 and Igf2 growth factor mRNA expression was lower in Day 2-IUGR mice, while Igf2r and Insr mRNA expression was higher in D10-IUGR animals. Moreover, at 3 months of age, only in the recuperated group were brain and liver Igf1, Igf2, Insr, and Igf2r expression levels higher than in the control and IUGR-non-recuperated groups. These data indicate that catch-up growth but not IUGR per se affects TL and glucose tolerance, and suggest a role in this latter process of insulin/insulin-like growth signaling pathway gene expression during early development.


Assuntos
Peso Corporal , Retardo do Crescimento Fetal/metabolismo , Intolerância à Glucose/etiologia , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Homeostase do Telômero , Animais , Encéfalo/metabolismo , Modelos Animais de Doenças , Feminino , Retardo do Crescimento Fetal/fisiopatologia , Regulação da Expressão Gênica no Desenvolvimento , Fígado/metabolismo , Masculino , Camundongos , Músculos/metabolismo
13.
Reproduction ; 161(3): 343-352, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33600356

RESUMO

The objective of this work was to elucidate whether a sperm selection method that combines rheotaxis and microfluidics can improve the selection of spermatozoa over density gradient and swim-up. For this purpose human sperm selected by rheotaxis were compared against density gradient, swim-up and a control group of non-selected spermatozoa in split frozen-thawed (FT) and fresh (F) semen samples. Sperm quality was assessed in terms of motility, morphology, DNA fragmentation index (DFI), viability, acrosome integrity and membrane fluidity. Using a mouse model, we compared fertilisation and embryo development rates after performing ICSI with spermatozoa, sorted using rheotaxis or swim-up. Selection by rheotaxis yielded a sperm population with reduced DFI than the control (P < 0.05), improved normal morphology (P < 0.001) and higher total motility (TM; P < 0.001) than the other techniques studied in F and FT samples. Swim-up increased TM compared to density gradient and control in FT or F samples (P < 0.001), and yielded lower DFI than the control with F samples (P < 0.05). In FT samples, selection by rheotaxis yielded sperm with higher viability than control, density gradient and swim-up (P < 0.01) while acrosomal integrity and membrane fluidity were maintained. When mouse spermatozoa were selected for ICSI using rheotaxis compared to swim-up, there was an increase in fertilisation (P < 0.01), implantation (P < 0.001) and foetal development rates (P < 0.05). These results suggest that, in the absence of non-destructive DNA testing, the positive rheotaxis can be used to select a population of low DNA fragmentation spermatozoa with high motility, morphology and viability, leading to improved embryo developmental rates.

14.
Clin Epigenetics ; 13(1): 27, 2021 02 03.
Artigo em Inglês | MEDLINE | ID: mdl-33536045

RESUMO

BACKGROUND: Prior work in mice has shown that some retrotransposed elements remain substantially methylated during DNA methylation reprogramming of germ cells. In the pig, however, information about this process is scarce. The present study was designed to examine the methylation profiles of porcine germ cells during the time course of epigenetic reprogramming. RESULTS: Sows were artificially inseminated, and their fetuses were collected 28, 32, 36, 39, and 42 days later. At each time point, genital ridges were dissected from the mesonephros and germ cells were isolated through magnetic-activated cell sorting using an anti-SSEA-1 antibody, and recovered germ cells were subjected to whole-genome bisulphite sequencing. Methylation levels were quantified using SeqMonk software by performing an unbiased analysis, and persistently methylated regions (PMRs) in each sex were determined to extract those regions showing 50% or more methylation. Most genomic elements underwent a dramatic loss of methylation from day 28 to day 36, when the lowest levels were shown. By day 42, there was evidence for the initiation of genomic re-methylation. We identified a total of 1456 and 1122 PMRs in male and female germ cells, respectively, and large numbers of transposable elements (SINEs, LINEs, and LTRs) were found to be located within these PMRs. Twenty-one percent of the introns located in these PMRs were found to be the first introns of a gene, suggesting their regulatory role in the expression of these genes. Interestingly, most of the identified PMRs were demethylated at the blastocyst stage. CONCLUSIONS: Our findings indicate that methylation reprogramming in pig germ cells follows the general dynamics shown in mice and human, unveiling genomic elements that behave differently between male and female germ cells.

15.
Methods Mol Biol ; 2273: 219-238, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33604857

RESUMO

Intercellular communication can be carried out by circulating systemic and/or locally released extracellular vesicles (EVs), produced by nearly every cell type and tissue, and are involved in physiological and pathological processes. In recent years, EVs have been identified in reproductive tissues, such as oviduct and uterus, and have been shown to be related to several events important for reproductive success. The understanding of their functions in reproduction has important implications for assisted reproductive technologies, for the treatment of infertility in humans and improvement of reproduction efficiency in animals. To study such EVs, it is necessary to isolate and concentrate them from fluid samples, which in the case of reproductive tissues, are usually of limited volume. Several methods for EV isolation are available such as chromatography, ultracentrifugation, polymer-based precipitation, and immunoaffinity.Outcomes can be variable in terms of the amount and quality of isolated EVs, due to the type of isolation method. The choice of method, or a different combination of methods, may depend on the type of sample and scientific question to be addressed in a given study. In this chapter, we describe a method for isolation of EVs from bovine oviductal and uterine fluids for use in functional studies. The method combines size exclusion chromatography and ultracentrifugation. We also describe the different protocols for characterization of isolated EVs (transmission electron microscopy, nanoparticle tracking analysis, and western blot), as well as the isolation of RNA content in EVs, and their miRNAs profiling for functional studies.


Assuntos
Bovinos/genética , Vesículas Extracelulares/genética , Tubas Uterinas/metabolismo , MicroRNAs/genética , Animais , Western Blotting/métodos , Cromatografia em Gel/métodos , Feminino , Perfilação da Expressão Gênica/métodos , MicroRNAs/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Transcriptoma , Ultracentrifugação/métodos , Útero/metabolismo
16.
Theriogenology ; 162: 15-21, 2021 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-33388725

RESUMO

Early embryonic development may be affected by adrenal hyperactivity in stressful situations which may lead to endocrine changes in the embryo environment. A sensitive period in porcine embryo development is the 4-cell stage when the embryo genome activation occurs. A mixed in vivo-in vitro system was implemented to test whether an altered milieu around this stage could affect embryo development and blastocyst quality in the porcine model. After in vitro maturation and fertilisation, presumptive zygotes were exposed for 24 h to plasma collected after ovulation from adrenocorticotropic hormone (ACTH)-treated, non-ACTH-treated sows; and, medium without plasma, supplemented with bovine serum albumin. Subsequently, embryo development and differences in gene expression were tested among treatments. Cleavage and blastocyst rates did not differ between treatments. Blastocyst quality by morphology assessment was similar when all the resulting blastocysts were included in the analysis. However, when only expanded blastocysts (and onwards) were included in the analysis, the blastocysts from the non-ACTH plasma group showed better quality score. Blastocyst quality by morphological assessment was not mirrored by the transcription levels of various important genes for embryo development whose gene expression profile did not significantly differ among groups. It is likely that the effect of the altered environment provided by plasma from ACTH-treated sows was too short to affect embryo development. Therefore, a brief exposure to an altered endocrine environment may not have harmful consequences for the embryo once fertilisation occurs.


Assuntos
Blastocisto , Desenvolvimento Embrionário , Animais , Técnicas de Cultura Embrionária/veterinária , Embrião de Mamíferos , Feminino , Fertilização In Vitro/veterinária , Expressão Gênica , Plasma , Gravidez , Suínos
17.
Equine Vet J ; 53(4): 787-795, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32881040

RESUMO

BACKGROUND: Equine embryos exhibit an unusual pattern of glucose tolerance in vitro and are currently cultured in hyperglycaemic conditions. OBJECTIVE: Our main objective was to analyse the effect of different glucose concentrations on in vitro-produced equine embryo development and quality. STUDY DESIGN: Experiments comparing in vitro and in vivo produced embryos. METHODS: Oocytes (n = 641) were collected from post-mortem ovaries, matured in vitro and fertilised by intracytoplasmic sperm injection (ICSI). Embryo culture was divided from Day 0 to Day 4 and from Day 4 to Day 9 in three groups: 5-10 (5 and 10 mmol/L glucose respectively; n = 87); 5-17 (5 and 17.5 mmol/L; n = 66); and 10-17 (10 and 17.5 mmol/L; n = 117). A control group of 20 in vivo produced blastocysts was included. Cleavage and blastocyst rates were evaluated and embryos were snap-frozen for analysis of the relative mRNA expression of genes related to mitochondrial function, DNA methylation, apoptosis, glucose transport and metabolism. RESULTS: No differences were observed in the cleavage or blastocyst rates among in vitro groups. Under high glucose conditions in vitro (10-17 group), BAX/BCL2 was higher, and PFKP, LDHA and COX2 were overexpressed compared to all other groups. The two groups with 5 mmol/L glucose concentration during the first culture stage (5-10 and 5-17) displayed similar patterns which differed to the 10-17 group. MAIN LIMITATIONS: Conclusions related to embryo quality are based on gene expression patterns. Transfer of in vitro-produced embryos would reveal whether the observed differences improve embryo developmental competence. CONCLUSIONS: Five mM glucose during the first days of culture seems to be preferable to avoid over-activation of embryonic glycolytic pathways. Further studies are necessary to determine whether this improves embryo developmental competence.


Assuntos
Blastocisto , Desenvolvimento Embrionário , Animais , Técnicas de Cultura Embrionária/veterinária , Glucose , Cavalos , Oócitos , Injeções de Esperma Intracitoplásmicas/veterinária
18.
Int J Mol Sci ; 21(17)2020 Aug 22.
Artigo em Inglês | MEDLINE | ID: mdl-32842637

RESUMO

Polycystic ovarian syndrome (PCOS) is the main cause of female infertility. It is a multifactorial disorder with varying clinical manifestations including metabolic/endocrine abnormalities, hyperandrogenism, and ovarian cysts, among other conditions. D-Chiro-inositol (DCI) is the main treatment available for PCOS in humans. To address some of the mechanisms of this complex disorder and its treatment, this study examines the effect of DCI on reproduction during the development of different PCOS-associated phenotypes in aged females and two mouse models of PCOS. Aged females (8 months old) were treated or not (control) with DCI for 2 months. PCOS models were generated by treatment with dihydrotestosterone (DHT) on Days 16, 17, and 18 of gestation, or by testosterone propionate (TP) treatment on the first day of life. At two months of age, PCOS mice were treated with DCI for 2 months and their reproductive parameters analyzed. No effects of DCI treatment were produced on body weight or ovary/body weight ratio. However, treatment reduced the number of follicles with an atretic cyst-like appearance and improved embryo development in the PCOS models, and also increased implantation rates in both aged and PCOS mice. DCI modified the expression of genes related to oocyte quality, oxidative stress, and luteal sufficiency in cumulus-oocyte complexes (COCs) obtained from the aged and PCOS models. Further, the phosphorylation of AKT, a main metabolic sensor activated by insulin in the liver, was enhanced only in the DHT group, which was the only PCOS model showing glucose intolerance and AKT dephosphorylation. The effect of DCI in the TP model seemed mediated by its influence on oxidative stress and follicle insufficiency. Our results indicate that DCI works in preclinical models of PCOS and offer insight into its mechanism of action when used to treat this infertility-associated syndrome.


Assuntos
Blastocisto/efeitos dos fármacos , Infertilidade Feminina/tratamento farmacológico , Inositol/farmacologia , Oócitos/efeitos dos fármacos , Síndrome do Ovário Policístico/tratamento farmacológico , Envelhecimento , Animais , Blastocisto/fisiologia , Células do Cúmulo/efeitos dos fármacos , Di-Hidrotestosterona/toxicidade , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Intolerância à Glucose/tratamento farmacológico , Infertilidade Feminina/etiologia , Infertilidade Feminina/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Camundongos Endogâmicos , Oócitos/fisiologia , Fosforilação/efeitos dos fármacos , Síndrome do Ovário Policístico/induzido quimicamente , Síndrome do Ovário Policístico/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Propionato de Testosterona/toxicidade
19.
Animals (Basel) ; 10(9)2020 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-32825582

RESUMO

Assisted reproductive technologies (ARTs) in the horse still yield suboptimal results in terms of pregnancy rates. One of the reasons for this is the lack of optimal conditions for the sperm capacitation in vitro. This study assesses the use of synthetic human tubal fluid (HTF) supplemented with D-penicillamine (HTF + PEN) for the in vitro capacitation of frozen/thawed stallion spermatozoa by examining capacitation-related events over 180 min of incubation. Besides these events, we explored the in vitro capacity of the spermatozoa to migrate by thermotaxis and give rise to a population of high-quality spermatozoa. We found that HTF induced higher levels of hyperactive-like motility and protein tyrosine phosphorylation (PTP) compared to the use of a medium commonly used in this species (Whitten's). Also, HTF + PEN was able to maintain this hyperactive-like motility, otherwise lost in the absence of PEN, for 180 min, and also allowed for sperm selection by thermotaxis in vitro. Remarkably, the selected fraction was enriched in spermatozoa showing PTP along the whole flagellum and lower levels of DNA fragmentation when compared to the unselected fraction (38% ± 11% vs 4.4% ± 1.1% and 4.2% ± 0.4% vs 11% ± 2% respectively, t-test p < 0.003, n = 6). This procedure of in vitro capacitation of frozen/thawed stallion spermatozoa in HTF + PEN followed by in vitro sperm selection by thermotaxis represents a promising sperm preparation strategy for in vitro fertilization and intracytoplasmic sperm injection in this species.

20.
Int J Mol Sci ; 21(15)2020 Jul 27.
Artigo em Inglês | MEDLINE | ID: mdl-32727154

RESUMO

Nobiletin is a polymethoxylated flavonoid isolated from citrus fruits with wide biological effects, including inhibition of reactive oxygen species (ROS) production and cell cycle regulation, important factors for oocyte in vitro maturation (IVM). Therefore, the objective of the present study was to evaluate the antioxidant activity of nobiletin during IVM on matured bovine oocyte quality (nuclear and cytoplasmic maturation; oocyte mitochondrial activity; intracellular ROS and glutathione (GSH) levels) and their developmental competence, steroidogenesis of granulosa cells after maturation, as well as quantitative changes of gene expression in matured oocytes, their cumulus cells, and resulting blastocysts. Bovine cumulus-oocyte complexes were in vitro matured in TCM-199 +10% fetal calf serum (FCS) and 10 ng/mL epidermal growth factor (EGF) (Control) supplemented with 10, 25, 50, or 100 µM of nobiletin (Nob10, Nob25, Nob50, and Nob100, respectively) or 0.1% dimethyl sulfoxide (CDMSO: vehicle for nobiletin dilution). A significantly higher percentage of matured oocytes in metaphase II was observed in Nob25 and Nob50 compared to other groups. Similarly, cleavage rate and cumulative blastocyst yield on Days 7 and 8 were significantly higher for Nob25 and Nob50 groups. Oocytes matured with 25 and 50 µM nobiletin showed a higher rate of migration of cortical granules and mitochondrial activity and a reduction in the ROS and GSH content in comparison with all other groups. This was linked to a modulation in the expression of genes related to metabolism (CYP51A1), communication (GJA1), apoptosis (BCL2), maturation (BMP15 and MAPK1), and oxidative stress (SOD2 and CLIC1). In conclusion, nobiletin offers a novel alternative for counteracting the effects of the increase in the production of ROS during IVM, improves oocyte nuclear and cytoplasmic maturation, and subsequent embryo development and quality in cattle.


Assuntos
Antioxidantes/farmacologia , Blastocisto/metabolismo , Desenvolvimento Embrionário/efeitos dos fármacos , Flavonas/farmacologia , Oócitos/metabolismo , Animais , Blastocisto/citologia , Bovinos , Células do Cúmulo/citologia , Células do Cúmulo/metabolismo , Feminino , Técnicas de Maturação in Vitro de Oócitos , Oócitos/citologia
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