RESUMO
BACKGROUND/AIMS: Among the assisted reproductive techniques, the in vitro maturation of oocytes (IVM) is less developed than other techniques, but its implementation would entail a qualitative advance. This technique consists in the extraction of immature oocytes from antral ovarian follicles with the patient under low hormone stimulation or without hormone to mature exogenously in culture media supplemented with different molecules to promote maturation. In this sense, we are interested in the role that cannabinoids could have as IVM promoters because cannabinoid's molecular pathway is similar to the one by which oocyte's meiosis resumption is activated. With the intention of advancing in the possible use of cannabinoids as supplements for the media for in vitro maturation of oocytes, we intend to deepen the study of the function of the phytocannabinoid Δ-9-tetrahydrocannabinol (THC) in the IVM process. METHODS: By immunocytochemistry, we detected the location pattern of cannabinoid receptor type 1 (CB1) and type 2 (CB2) during oocyte maturation in presence or absence of THC, as well as, the staining pattern of p-AKT and p-ERK. We used a genetic/ pharmacological approach generating knockout oocytes for CB1 and/or CB2 and they were incubated with THC during the oocyte maturation to visualize the physiological effects of THC, observing the rate of blastocyst achieved by oocyte. RESULTS: This study confirms that the incubation of oocytes with THC during IVM accelerated some events of that process like the phosphorylation pattern of ERK and AKT and was able to increase the blastocyst rate in response to IVF. Moreover, it seems that both CB1 and CB2 are necessary to maintain a healthy oocyte maturation. CONCLUSION: Our data suggest that THC may be useful IVM supplements in clinic as is more feasible and reliable than any synthetic cannabinoid.
Assuntos
Blastocisto/efeitos dos fármacos , Dronabinol/farmacologia , Oócitos/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Blastocisto/citologia , Blastocisto/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Fertilização in vitro , Técnicas de Maturação in Vitro de Oócitos , Meiose/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Oócitos/citologia , Oócitos/metabolismo , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor CB1 de Canabinoide/genética , Receptor CB1 de Canabinoide/metabolismo , Receptor CB2 de Canabinoide/genética , Receptor CB2 de Canabinoide/metabolismoRESUMO
Endocannabinoids have been recognized as mediators of practically all reproductive events in mammals. However, little is known about the role of this system in oocyte maturation. In a mouse model, we observed that activation of cannabinoid receptor 1 (CB1) during in vitro oocyte maturation modulated the phosphorylation status of Akt and ERK1/2 and enhanced the subsequent embryo production. In the absence of CB1, in vivo oocyte maturation was impaired and embryo development delayed. Cannabinoid receptor 2 (CB2) was unable to rescue these effects. Finally, we confirmed abnormal oocyte maturation rather than impaired embryonic transport through the oviduct in CB1 knockouts. Our data suggest that cannabinoid agonists may be useful in vitro maturation supplements. For in vitro fertilization patients intolerant to gonadotropins, this could be a promising and only option.-López-Cardona, A. P., Pérez-Cerezales, S., Fernández-González, R., Laguna-Barraza, R., Pericuesta, E., Agirregoitia, N., Gutiérrez-Adán, A., Agirregoitia, E. CB1 cannabinoid receptor drives oocyte maturation and embryo development via PI3K/Akt and MAPK pathways.
Assuntos
Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Oócitos/fisiologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor CB1 de Canabinoide/metabolismo , Animais , Desenvolvimento Embrionário , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Maturação in Vitro de Oócitos , Camundongos , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Fosfatidilinositol 3-Quinases/genética , Receptor CB2 de CanabinoideRESUMO
During the transit through the oviduct, the early embryo initiates an extensive DNA methylation reprogramming of its genome. Given that these epigenetic modifications are susceptible to environmental factors, components present in the oviductal milieu could affect the DNA methylation marks of the developing embryo. The aim of this study was to examine if culture of bovine embryos with oviductal fluid (OF) can induce DNA methylation changes at specific genomic regions in the resulting blastocysts. In vitro produced zygotes were cultured in medium with 3 mg/mL bovine serum albumin (BSA) or 1.25% OF added at the one- to 16-cell stage (OF1-16), one- to 8-cell stage (OF1-8) or 8- to 16-cell stage (OF8-16), and then were cultured until Day 8 in medium with 3 mg/mL BSA. Genomic regions in four developmentally important genes (MTERF2, ABCA7, OLFM1, GMDS) and within LINE-1 retrotransposons were selected for methylation analysis by bisulfite sequencing on Day 7-8 blastocysts. Blastocysts derived from OF1-16 group showed lower CpG methylation levels in MTERF2 and ABCA7 compared with the BSA group. However, CpG sites within MTERF2, ABCA7 and OLFM1 showed higher methylation levels in groups OF1-8 and OF8-16 than in OF1-16. For LINE-1 elements, higher CpG methylation levels were observed in blastocysts from the OF1-16 group than in the other experimental groups. In correlation with the methylation changes observed, mRNA expression level of MTERF2 was increased, while LINE-1 showed a decreased expression in blastocysts from OF1-16 group. Our results suggest that embryos show transient sensitivity to OF at early stages, which is reflected by specific methylation changes at the blastocyst stage.
Assuntos
Blastocisto/metabolismo , Líquidos Corporais/fisiologia , Bovinos/embriologia , Metilação de DNA , Técnicas de Cultura Embrionária/veterinária , Tubas Uterinas/fisiologia , Animais , Blastocisto/química , Clonagem Molecular , Meios de Cultura , Desenvolvimento Embrionário/fisiologia , Feminino , Fertilização in vitro/veterinária , Técnicas de Maturação in Vitro de Oócitos/veterinária , Elementos Nucleotídeos Longos e Dispersos/genética , Reação em Cadeia da Polimerase/veterinária , RNA Mensageiro/análiseRESUMO
The contribution of the contents of spermatozoa to the development of the embryo is currently being considered wider than was previously thought. Recent findings point to the participation of epigenetic marks present in the retained histones of mature spermatozoa on embryo and fetal development. Here we created a novel conditional transgenic mouse that expresses lysine (K) demethylase 1a (Kdm1a) during spermatogenesis when the testicles are subjected to heat stress. Using these animals under these conditions we were able to reduce the methylation level of histone 3 at lysines 4 and 9 (H3K4 and H3K9, respectively) in mature spermatozoa. The offspring of these transgenic mice were followed for correct development and growth after birth. We found that the offspring of males expressing Kdm1a suffered 20% of reabsorptions at Day 15 after implantation (vs 0.3% in the control). In addition, 35% of the offspring sired by these males showed some kind of abnormality (suckling defects, lack of movement coordination, dropping forelimbs, abnormal body curvature, absence of eyes, gigantisms and neuromuscular defects) and 25% died before postnatal Day 21. Some abnormalities were maintained to adulthood. These results show that alteration of epigenetic marks present in the retained histones of mature spermatozoa affect fetal development and have phenotypic consequences in the newborn.
Assuntos
Metilação de DNA/genética , Histonas/metabolismo , Espermatozoides/metabolismo , Animais , Histona Desmetilases/genética , Histona Desmetilases/metabolismo , Histonas/genética , Masculino , Camundongos , Camundongos Transgênicos , Fenótipo , Espermatogênese/genéticaRESUMO
Asynchronous embryo transfer (ET) is a common assisted reproduction technique used in several species, but its biological effects on postnatal and early development remain unknown. The aim of this study was to determine whether asynchronous ET produces long-term effects in mice. Postnatal development, animal weight, systolic blood pressure (SBP), relative organ weight (liver, spleen, kidneys, heart, lungs, brain, and testicles), and behavior (assessed in open-field and elevated plus maze tests) were assessed in CD1 mice produced by different ET procedures: 1) the transfer of Day 3.5 (D3.5) blastocysts to the uterus (BL-UT); 2) the transfer of D3.5 blastocysts to the oviduct (BL-OV); or 3) the transfer of D0.5 zygotes to the oviduct (Z-OV). In vivo conceived animals served as controls (CT). The transfer of blastocysts to the uterus or zygotes to the oviduct was defined as synchronous, and transfer of blastocysts to the oviduct was defined as asynchronous. Both synchronous and asynchronous ET resulted in increased weight at birth that normalized thereafter with the exception of asynchronous ET females. In this group, female BL-OV, a clear lower body weight was recorded along postnatal life when compared with controls (P < 0.05). No effects on animal weight were produced during postnatal development in the synchronous ET groups (BL-UT, Z-OV, and CT). Both synchronous and asynchronous ET had impacts on adult (Wk 30) organ weight. SBP was modified in animals derived from blastocyst but not zygote ET. Effects on behavior (anxiety in the plus maze) were only detected in the BL-UT group (P < 0.05). Our findings indicate that zygotes are less sensitive than blastocysts to ET and that both synchronous and asynchronous blastocyst ET may have long-term consequences on health, with possible impacts on weight, arterial pressure, relative organ weight, and behavior.
Assuntos
Comportamento Animal/fisiologia , Transferência Embrionária/métodos , Crescimento/fisiologia , Animais , Animais Recém-Nascidos , Ansiedade , Blastocisto/fisiologia , Pressão Sanguínea/fisiologia , Peso Corporal/fisiologia , Técnicas de Cultura Embrionária , Tubas Uterinas , Feminino , Nível de Saúde , Masculino , Camundongos , Atividade Motora/fisiologia , Tamanho do Órgão/fisiologia , Gravidez , Caracteres Sexuais , Zigoto/fisiologiaRESUMO
BACKGROUND: It is believed that the main factors of low prenatal growth in mammals are genetic and environmental. We used isogenic mice maintained in standard conditions to analyze how natural non-genetic microsomia (low birth weight) is produced in inbred mice and its long term effect on health. To better understand the molecular basis of non-genetic microsomia, we undertook transcriptome profiling of both male and female livers from small and normal size mice at birth. RESULTS: Naturally occurring neonatal microsomia was defined as a gender-specific weanling weight under the 10th percentile of the colony. Birth weight variation was similar in inbred and outbred lines. Mice were phenotyped by weight, size, blood pressure, organ size, their response to a glucose challenge, and survival rates. Regardless of diet, adult mice born with microsomia showed a significantly lower body weight and size, and differences in the weight of several organs of microsomic adult mice compared to normal birth weight adults were found. After a high-fat diet, microsomic mice were less prone to obesity, showing a better glucose tolerance and lower blood pressure. Through a transcriptome analysis, we detected a different pattern of mRNA transcription in the liver at birth comparing male vs female and microsomic vs normal mice, noting some modifications in epigenetic regulatory genes in females and modifications in some growth factor genes in males. Finally, using embryo transfer of embryos of different quality and age, we identified a putative preimplantation origin of this non-genetic microsomia. CONCLUSIONS: (1) neonatal microsomia is not always a risk factor for adult metabolic syndrome, (2) neonatal non-genetic microsomia displays changes in the expression of important epigenetic genes and changes in liver mRNA transcription profile at birth, exaggerating sexual dimorphism, and (3) random preimplantation phenotypic variability could partially explain body birth weight variation in isogenic lines.
Assuntos
Peso ao Nascer , Perfilação da Expressão Gênica , Fígado/metabolismo , Animais , Animais Recém-Nascidos , Estatura , Dieta Hiperlipídica , Feminino , Peso Fetal , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica , Humanos , Recém-Nascido de Baixo Peso/crescimento & desenvolvimento , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Tamanho do Órgão , Fenótipo , Caracteres SexuaisRESUMO
In the present study, we identify and describe an obese phenotype in mice as a long-term consequence of a suboptimal in vitro culture that resulted from the addition of fetal calf serum (FCS) into the culture medium. Mice produced with FCS displayed a high mortality rate (approximately 55% versus 15% in control mice within 20 mo) and increased sensitivity to the development of obesity in adulthood when fed either a standard or a high-fat diet. These mice developed hyperplastic obesity that was characterized by a significant expansion of the fat pads (approximately 25% and 32% higher body weight in male and female mice over controls, respectively) with unchanged adipocyte size. We observed a sexual dimorphism in the development of obesity in the mice produced with FCS. Whereas the female mice displayed hypertension, hyperleptinemia, and fatty liver, the male mice only displayed glucose intolerance. The mRNA expression of metabolically relevant genes in the adipose tissue was also affected. The males produced with FCS expressed higher mRNA levels of the genes that activate fatty acid oxidation (peroxisome proliferator-activated receptor alpha [Ppara, PPARalpha] and acyl-CoA oxidase 1 [Acox1, ACOX1]) and thermogenesis (uncoupling protein 1 [Ucp1, UCP1]), which may counteract the metabolic phenotype. Conversely, the females produced with FCS generally expressed lower levels of these metabolic genes. In the females, the obese phenotype was associated with inhibition of the lipogenic pathway (peroxisome proliferator-activated receptor gamma [Pparg, PPARgamma] and fatty acid synthase [Fasn, FAS]), indicating a saturation of the storage capacity of the adipose tissue. Overall, our data indicate that the exposure to suboptimal in vitro culture conditions can lead to the sexually dimorphic development of obesity in adulthood.
Assuntos
Meios de Cultura/química , Gorduras na Dieta , Técnicas de Cultura Embrionária/métodos , Fígado Gorduroso/metabolismo , Sangue Fetal , Obesidade , Tecido Adiposo Branco , Animais , Bovinos , Meios de Cultura/efeitos adversos , Feminino , Fígado/metabolismo , Masculino , Camundongos , Gravidez , Efeitos Tardios da Exposição Pré-NatalRESUMO
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/veterináriaRESUMO
The U2AF35-like ZRSR1 has been implicated in the recognition of 3' splice site during spliceosome assembly, but ZRSR1 knockout mice do not show abnormal phenotypes. To analyze ZRSR1 function and its precise role in RNA splicing, we generated ZRSR1 mutant mice containing truncating mutations within its RNA-recognition motif. Homozygous mutant mice exhibited severe defects in erythrocytes, muscle stretch, and spermatogenesis, along with germ cell sloughing and apoptosis, ultimately leading to azoospermia and male sterility. Testis RNA sequencing (RNA-seq) analyses revealed increased intron retention of both U2- and U12-type introns, including U12-type intron events in genes with key functions in spermatogenesis and spermatid development. Affected U2 introns were commonly found flanking U12 introns, suggesting functional cross-talk between the two spliceosomes. The splicing and tissue defects observed in mutant mice attributed to ZRSR1 loss of function suggest a physiological role for this factor in U12 intron splicing.
Assuntos
Azoospermia/genética , Eritropoese , Contração Muscular , Proteínas do Tecido Nervoso/genética , Proteínas Nucleares/genética , Splicing de RNA , Ribonucleoproteínas/genética , Espermatogênese , Animais , Azoospermia/patologia , Células Cultivadas , Masculino , Camundongos , Mutação , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/metabolismo , Ribonucleoproteínas/metabolismo , Fator de Processamento U2AFRESUMO
Cannabinoid receptors are able to couple to different families of G proteins when activated by an agonist drug. It has been suggested that different intracellular responses may be activated depending on the ligand. The goal of the present study was to characterize the pattern of G protein subunit stimulation triggered by three different cannabinoid ligands, Δ9-THC, WIN55212-2, and ACEA in mouse brain cortex. Stimulation of the [35S]GTPγS binding coupled to specific immunoprecipitation with antibodies against different subtypes of G proteins (Gαi1, Gαi2, Gαi3, Gαo, Gαz, Gαs, Gαq/11, and Gα12/13), in the presence of Δ9-THC, WIN55212-2 and ACEA (submaximal concentration 10 µM) was determined by scintillation proximity assay (SPA) technique in mouse cortex of wild type, CB1 knock-out, CB2 knock-out and CB1/CB2 double knock-out mice. Results show that, in mouse brain cortex, cannabinoid agonists are able to significantly stimulate not only the classical inhibitory Gαi/o subunits but also other G subunits like Gαz, Gαq/11, and Gα12/13. Moreover, the specific pattern of G protein subunit activation is different depending on the ligand. In conclusion, our results demonstrate that, in mice brain native tissue, different exogenous cannabinoid ligands are able to selectively activate different inhibitory and non-inhibitory Gα protein subtypes, through the activation of CB1 and/or CB2 receptors. Results of the present study may help to understand the specific molecular pathways involved in the pharmacological effects of cannabinoid-derived drugs.
RESUMO
Artificial reproductive techniques are currently responsible for 1.7-4% of the births in developed countries and intracytoplasmatic sperm injection (ICSI) is the most commonly used, accounting for 70-80% of the cycles performed. Despite being an invaluable tool for infertile couples, the technique bypasses several biological barriers that naturally select the gametes to achieve an optimal embryonic and fetal development. In this perspective, ICSI has been associated with an increased risk for diverse health problems, ranging from premature births and diverse metabolic disorders in the offspring to more severe complications such as abortions, congenital malformations, and imprinting disorders. In this review, we discuss the possible implications of the technique per se on these adverse outcomes and highlight the importance of several experiments using mammalian models to truthfully test these implications and to uncover the molecular base that origins these health problems. We also dissect the specific hazards associated to ICSI and describe some strategies that have been developed to mimic the gamete selection occurring in natural conception in order to improve the safety of the procedure.
RESUMO
OBJECTIVE: To study how long-term elevated non-esterified fatty acid (NEFA) concentrations, typical in metabolic disorders such as obesity or type 2 diabetes, affect murine follicular development, follicle quality, and subsequent oocyte developmental competence in vitro. DESIGN: Experimental study. SETTING: In vitro culture setting. ANIMAL(S): Female and male 13-day old, B6CBAF1 mice of proven fertility were sacrificed for harvesting ovaries and epididymal sperm, respectively. INTERVENTION(S): Early secondary murine follicles were cultured in vitro in the presence of NEFAs until the antral stage (12 days). Treatments consisted of one or a mixture of NEFAs (stearic acid [SA], palmitic acid [PA], oleic acid [OA]) in physiological (basal) or pathological (high SA, high OA, high NEFA) concentrations. MAIN OUTCOME MEASURE(S): Follicular development; follicle and oocyte diameters; secretion of progesterone, estradiol, and inhibin B; and luteinized granulosa cell gene expression patterns were investigated. Oocytes from NEFA-exposed follicles were fertilized in vitro, and presumptive zygotes were cultured until the blastocyst stage. RESULT(S): Exposure to high SA reduced follicle diameters and day-12 antrum formation. Elevated NEFA concentrations changed luteinized granulosa cell messenger-ribonucleic acid abundance of genes related to energy/fatty acid/steroid metabolism, apoptosis, and oxidative stress. High NEFA and high SA treatments increased progesterone synthesis, compared with high OA follicles. Oocyte developmental competence was substantially reduced in oocytes retrieved from high OA-, high SA-, and high NEFA-exposed follicles compared with basal-treated follicles. CONCLUSION(S): This study showed, for the first time, that lipolysis-linked, elevated NEFA concentrations can potentially impair fertility, by altering follicular physiology and reducing oocyte developmental competence.