RESUMO
PURPOSE: To assess the impact of maternal age on the association between maternal basal FSH and aneuploidy. METHODS: A retrospective study including data from 1749 blastocysts diagnosed as euploid or aneuploid by PGT-A (preimplantation genetic testing for aneuploidy). Aneuploidy incidence was compared between embryos from mothers with high vs. low basal FSH levels (above and below the group median, respectively) in total, pre-AMA (advanced maternal age; < 35 years, 198 embryos) and AMA (≥ 35 years, 1551 embryos) patient groups, separately. To control for the interference of potentially confounding variables, the association between aneuploidy and high basal FSH levels was assessed by multivariate logistic analysis in overall, pre-AMA and AMA patient groups. RESULTS: Overall, aneuploidy rate was 9% higher (p = 0.02) in embryos from patients with high basal FSH (63.7%) compared to those with low basal FSH (58.4%). In the pre-AMA subgroup, aneuploidy incidence was 35% higher (p = 0.04) in embryos from patients with high basal FSH (53.5%) compared to those with low basal FSH (39.4%). Differently, aneuploidy occurrence did not vary between embryos from AMA patients with low (61.0%) and high (64.8%) basal FSH (p = 0.12). The multivariate analysis revealed that, in pre-AMA embryos, the association between aneuploidy occurrence and high basal FSH is independent of potential confounding variables (p = 0.04). CONCLUSION: Maternal basal FSH values are associated with embryo aneuploidy in pre-AMA but not in AMA patients. The present findings suggest that basal FSH is a useful parameter to assess aneuploidy risk in pre-AMA patients and reinforce the hypothesis that excessive FSH signalling can predispose to oocyte meiotic errors.
Assuntos
Aneuploidia , Hormônio Foliculoestimulante , Idade Materna , Humanos , Feminino , Adulto , Hormônio Foliculoestimulante/sangue , Gravidez , Diagnóstico Pré-Implantação , Estudos Retrospectivos , Incidência , Blastocisto/metabolismo , Fertilização in vitro , Transferência Embrionária , Testes Genéticos , Taxa de GravidezRESUMO
Oocyte in vitro maturation (IVM) is still a major challenge in human and animal assisted reproduction. Gradual instead of abrupt activation of the ovulatory cascade during IVM has been proposed to enhance nuclear-cytoplasmic synchrony and cumulus-oocyte communication, thus favoring oocyte developmental competence. Herein, we assessed the effects of neuregulin 1 (NRG1), an EGF-like factor that modulates EGFR signaling, on oocyte nuclear maturation dynamics, cumulus expansion and expression of mRNAs regulating these processes during IVM, as well as on post-IVF embryo development following AREG-stimulated IVM in cattle. In experiment 1, cumulus-oocyte complexes (COCs) were subjected to IVM with graded doses of NRG1 (1, 10 or 100 ng/mL) for 6, 9, 12, 20, and 24 h, after which oocyte nuclear status and cumulus mRNA expression were assessed. At 6 h of IVM, NRG1 at 1 ng/mL significantly decreased the percentage of GVBD (germinal vesicle breakdown) oocytes without altering later meiotic dynamics or the percentage of oocytes achieving meiosis II. In experiment 2, adding NRG1 (1 ng/mL) to the IVM medium did not affect cumulus expansion but increased the percentage of expanded and hatched blastocysts, and blastocyst total cell number following IVF/IVC. NRG1 decreased EGFR mRNA abundance while increasing NPR2 and PTX3 mRNA levels at 9 h, and TNFAIP6 mRNA abundance at 20 h of IVM. This is the first study that reports the modulatory effect of NGR1 during oocyte maturation in a mono-ovulatory species and demonstrates that this action may be applied during IVM to improve post-IVF embryo development.
Assuntos
Neuregulina-1 , Oócitos , Humanos , Animais , Bovinos , Neuregulina-1/farmacologia , RNA Mensageiro , Desenvolvimento Embrionário , Receptores ErbB , Fertilização in vitro/veterináriaRESUMO
In vitro embryo production (IVP) efficiency is reduced when compared to in vivo. The basic knowledge of bovine in vitro oocyte maturation (IVM) mechanisms provides support to improve in vitro embryo production yields. The present study assessed the effects of bone morphogenetic protein 15 (BMP15), fibroblast growth factor 16 (FGF16) and their combined action on cumulus cells (CC) expansion, oocyte and CC DNA fragmentation, oocyte nuclear maturation, energetic metabolism and progesterone production in bovine IVM. Cumulus-oocyte complexes (COC) were matured in control or supplemented media containing BMP15 (100 ng/ml), FGF16 (10 ng/ml) or BMP15 combined with FGF16; and assessed at 0 and 22 hr of IVM. BMP15 alone or its association with FGF16 enhanced cumulus expansion. BMP15 decreased DNA fragmentation in both CC and oocytes, and improved oocyte nuclear maturation rate. In addition, BMP15 increased CC progesterone production, an effect not previously reported. The present study reinforces previous data pointing to a beneficial influence of BMP15 during IVM, while providing novel evidence that the underlying mechanisms involve increased progesterone production.
Assuntos
Proteína Morfogenética Óssea 15/farmacologia , Fatores de Crescimento de Fibroblastos/farmacologia , Técnicas de Maturação in Vitro de Oócitos/veterinária , Animais , Bovinos , Células do Cúmulo/efeitos dos fármacos , Fragmentação do DNA , Feminino , Técnicas de Maturação in Vitro de Oócitos/métodos , Oócitos/efeitos dos fármacos , Progesterona/metabolismoRESUMO
In vitro maturation (IVM) has been applied in numerous different contexts and strategies in humans and animals, but in both cases it represents a challenge still far from being overcome. Despite the large dataset produced over the last two decades on the mechanisms that govern antral follicular development and oocyte metabolism and differentiation, IVM outcomes are still unsatisfactory. This review specifically focuses on data concerning the potential consequences of using supraphysiological levels of FSH during IVM, as well as on the regulation of oocyte chromatin dynamics and its utility as a potential marker of oocyte developmental competence. Taken together, the data revisited herein indicate that a significant improvement in IVM efficacy may be provided by the integration of pre-OPU patient-specific protocols preparing the oocyte population for IVM and more physiological culture systems mimicking more precisely the follicular environment that would be experienced by the recovered oocytes until completion of metaphase II.
Assuntos
Técnicas de Maturação in Vitro de Oócitos , Meiose , Animais , Bovinos , Feminino , Fertilização in vitro , Humanos , Técnicas de Maturação in Vitro de Oócitos/veterinária , Oócitos/metabolismo , OogêneseRESUMO
PURPOSE: We first assessed regulation of FGF2 expression in cumulus cells by FSH and oocyte-secreted factors during in vitro maturation (IVM). Then, we tested the hypothesis that FGF2 regulates meiotic progression, cumulus expansion, and apoptosis in cumulus-oocyte complexes (COC) undergoing IVM. METHODS: In vitro maturation of bovine COC was utilized as a model to assess regulation of FGF2 expression by FSH and oocyte-secreted factors (via microsurgical removal of the oocyte), as well as effects of graded doses of FGF2 on meiotic progression, degree of cumulus expansion, dissociation of cumulus cells, and cumulus cells apoptosis. Expression of genes regulating functional endpoints altered by FGF2 treatment was assessed in cumulus cells by real-time PCR. Cultures were replicated 4-5 times and effects of treatments were tested by ANOVA. RESULTS: FGF2 mRNA expression was increased by FSH and oocyte-secreted factors during IVM. Addition of FGF2 to the IVM medium advanced meiosis resumption, decreased the ease with which cumulus cells were dissociated, and inhibited cumulus cells apoptosis. Decreased cumulus dissociation was accompanied by decreased expression of TNFAIP6. CONCLUSIONS: This is the first study showing that FGF2 expression is regulated by the oocyte in cumulus cells. Moreover, we report novel effects of FGF2 on cumulus cell survival and extracellular matrix (ECM) quality during IVM that may favor acquisition of developmental competence and suggest physiological roles during the final steps of COC differentiation.
Assuntos
Blastocisto/citologia , Diferenciação Celular , Células do Cúmulo/citologia , Fator 2 de Crescimento de Fibroblastos/farmacologia , Técnicas de Maturação in Vitro de Oócitos/métodos , Oócitos/citologia , Animais , Apoptose , Blastocisto/efeitos dos fármacos , Blastocisto/metabolismo , Bovinos , Células do Cúmulo/efeitos dos fármacos , Células do Cúmulo/metabolismo , Técnicas de Cultura Embrionária , Desenvolvimento Embrionário , Feminino , Meiose , Oócitos/efeitos dos fármacos , Oócitos/metabolismoRESUMO
A large amount of data on the mechanisms regulating cumulus-oocyte maturation in mammals has been generated in the last 20 years. It has been made clear that oocyte-secreted factors play a central role in the control of cumulus differentiation and oocyte developmental competence. However, more recent data indicate that cumulus-derived factors are also involved. In this mini-review, we have compiled and discussed data produced in our laboratory about the involvement of oocyte and cumulus-derived peptides, including fibroblast growth factors, bone morphogenetic protein 15, Kit ligand and natriuretic peptide C, in the regulation of cumulus metabolism and oocyte nuclear maturation. In addition, we discuss the interaction of follicular steroids with natriuretic peptide C in the control of meiosis progression.(AU)
Assuntos
Animais , Feminino , Gravidez , Bovinos , Bovinos/embriologia , Técnicas de Maturação in Vitro de Oócitos/métodos , Técnicas de Maturação in Vitro de Oócitos/veterinária , Fase Folicular , Peptídeos , EsteroidesRESUMO
A large amount of data on the mechanisms regulating cumulus-oocyte maturation in mammals has been generated in the last 20 years. It has been made clear that oocyte-secreted factors play a central role in the control of cumulus differentiation and oocyte developmental competence. However, more recent data indicate that cumulus-derived factors are also involved. In this mini-review, we have compiled and discussed data produced in our laboratory about the involvement of oocyte and cumulus-derived peptides, including fibroblast growth factors, bone morphogenetic protein 15, Kit ligand and natriuretic peptide C, in the regulation of cumulus metabolism and oocyte nuclear maturation. In addition, we discuss the interaction of follicular steroids with natriuretic peptide C in the control of meiosis progression.
Assuntos
Feminino , Animais , Gravidez , Bovinos , Bovinos/embriologia , Técnicas de Maturação in Vitro de Oócitos/métodos , Técnicas de Maturação in Vitro de Oócitos/veterinária , Esteroides , Fase Folicular , PeptídeosRESUMO
In vivo, oocyte maturation is triggered by the ovulatory LH surge, whereas in vitro it is precociously induced when the cumulus-oocyte complex is removed from the follicle. Natriuretic peptide C (NPPC) delays germinal vesicle breakdown (GVBD) while increasing oocyte-cumulus communication during in vitro maturation (IVM) in cattle. In the present study we first tested the hypothesis that steroids secreted by the follicle (17ß-oestradiol, progesterone and androstenedione) interact with NPPC to delay GVBD and to maintain oocyte-cumulus communication as assessed by transfer of a dye (Lucifer Yellow) from the oocyte to cumulus cells. Then, we assessed the effects of steroid hormones and NPPC, alone and in combination in a pre-IVM culture, on embryo production. The combination of NPPC with steroids delayed GVDB, increased natriuretic peptide receptor 2 (NPR2) mRNA abundance in cumulus cells during culture, and maintained oocyte-cumulus communication at levels not different from non-cultured controls. The addition of steroids and/or NPPC to a pre-IVM culture did not alter blastocyst rates after IVF, but supplementation with steroids increased blastocyst total cell number. The present study provides evidence, for the first time in cattle, that steroids interact with NPPC to regulate oocyte nuclear maturation and oocyte-cumulus communication, and improve oocyte developmental competence.
Assuntos
Androstenodiona/farmacologia , Células do Cúmulo/metabolismo , Estradiol/farmacologia , Técnicas de Maturação in Vitro de Oócitos/veterinária , Peptídeo Natriurético Tipo C/farmacologia , Oócitos/metabolismo , Progesterona/farmacologia , Animais , Bovinos , Células do Cúmulo/efeitos dos fármacos , Feminino , Oócitos/efeitos dos fármacos , Oogênese/efeitos dos fármacos , Folículo Ovariano/metabolismoRESUMO
In vitro maturation (IVM) of oocytes in cattle is inefficient, and there is great interest in the development of approaches to improve maturation and fertilization rates. Intraovarian signalling molecules are being explored as potential additives to IVM media. One such factor is kit ligand (KITL), which stimulates the growth of oocytes. We determined if KITL enhances oocyte maturation in cattle. The two main isoforms of KITL (KITL1 and KITL2) were expressed in bovine cumulus-oocyte complexes (COC), and levels of mRNA increased during FSH-stimulated IVM. The addition of KITL to the culture medium increased the percentage of oocytes that reached meiosis II but did not affect cumulus expansion after 22 h of IVM. Addition of KITL reduced the levels of mRNA encoding natriuretic peptide precursor C (NPPC), a protein that holds oocytes in meiotic arrest, and increased the levels of mRNA encoding YBX2, an oocyte-specific factor involved in meiosis. Removal of the oocyte from the COC resulted in increased KITL mRNA levels and decreased NPPC mRNA levels in cumulus cells, and addition of denuded oocytes reversed these effects. Taken together, our results suggest that KITL enhances bovine oocyte nuclear maturation through a mechanism that involves NPPC, and that the oocyte regulates cumulus expression of KITL mRNA.
Assuntos
Células do Cúmulo/citologia , Técnicas de Maturação in Vitro de Oócitos/veterinária , Peptídeo Natriurético Tipo C/metabolismo , Oócitos/citologia , Oogênese/fisiologia , Fator de Células-Tronco/metabolismo , Animais , Bovinos , Células do Cúmulo/metabolismo , Feminino , Fertilização in vitro , Meiose/fisiologia , Oócitos/metabolismoRESUMO
Bone morphogenetic protein 15 (BMP15) and members of the fibroblast growth factor (FGF) family are expressed by the oocyte and are involved in the control of cumulus cell function. We tested the hypothesis that FGF17, alone or combined with BMP15 in the maturation medium, enhances cumulus expansion, meiosis progression, embryonic development, and expression of mRNA encoding key genes regulating expansion (prostaglandin-endoperoxide synthase 2 [PTGS2], hyaluronan synthase 2 [HAS2], tumor necrosis factor-stimulated gene 6 [TNFAIP6], and pentraxin 3 [PTX3]) and markers of oocyte developmental competence (phosphofructokinase [PFKP], gremlin [GREM1], versican [VCAN], and the genomic progesterone receptor [nPR]) in cumulus cells. Fibroblast growth factor 17 and BMP15 increased the percentage of fully expanded cumulus-oocyte complexes (COCs), but there was no additive effect when both were combined. Neither FGF17 nor BMP15 altered the percentage of oocytes reaching meiosis II at the end of COC culture or cleavage and blastocyst rates after IVF. However, embryo quality, as assessed by the number of cells in the inner cell mass, was improved by the combination of FGF17 with BMP15. Fibroblast growth factor 17 alone did not alter gene expression in cumulus cells at the end of IVM, whereas BMP15 increased PTGS2 and PTX3 mRNA levels. The combination of FGF17 and BMP15 increased nPR mRNA abundance in cumulus cells but did not change the expression of other markers of developmental competence. This study provides novel evidence that FGF17 enhances cumulus expansion in bovine COCs submitted to IVM and that the supplementation of the IVM medium with FGF17 and BMP15 may improve embryo quality.
Assuntos
Proteína Morfogenética Óssea 15/farmacologia , Bovinos/embriologia , Técnicas de Cultura Embrionária/veterinária , Embrião de Mamíferos/efeitos dos fármacos , Fatores de Crescimento de Fibroblastos/farmacologia , Animais , Embrião de Mamíferos/metabolismo , Embrião de Mamíferos/fisiologia , Desenvolvimento Embrionário/efeitos dos fármacos , Feminino , Perfilação da Expressão Gênica , MasculinoRESUMO
In a 2×2 factorial experimental design, embryo development, cryotolerance and global gene expression of Nellore (Bos taurus indicus) and Simmental (Bos taurus taurus) blastocysts produced in vitro (IVP) and in vivo (multiple ovulation derived embryo, MODE) were assessed. Blastocyst production was higher in Nellore than in Simmental (47.7±2.0% vs 27.0±2.0%) cows. The total numbers of ova or embryos recovered (5.5±0.9 vs 3.7±0.8) and transferable embryos (3.8±1.0 vs 2.3±0.8) per cow were not different between breeds. Simmental and MODE (34.6% and 38.5%, n=75 and 70) blastocysts had higher survival rates after cryopreservation compared with Nellore and IVP (20.2% and 18.1%, n=89 and 94) embryos, respectively. Differences between transcriptomes were addressed by principal-component analysis, which indicated that gene expression was affected by subspecies (158 genes), origin (532 genes) and interaction between both subspecies and origin (53 genes). Several functional processes and pathways relevant to lipid metabolism and embryo viability involving differentially expressed genes were identified. The lipid metabolism-related genes were upregulated in Simmental (AUH and ELOVL6) and IVP (ACSL3 and ACSL6) blastocysts. The expression profiles of genes related to mitochondrial metabolism (ATP5B), oxidative stress (GPX4), apoptosis (DAD1, DAP, PRDX2), heat shock (HSPA5), pregnancy (IFNT2, PAG2) and cell differentiation (KRT18) varied between experimental groups.
Assuntos
Blastocisto/fisiologia , Criopreservação/veterinária , Fertilização in vitro/veterinária , Fertilização , Perfilação da Expressão Gênica/veterinária , Regulação da Expressão Gênica no Desenvolvimento , Inseminação Artificial/veterinária , Animais , Blastocisto/metabolismo , Bovinos , Sobrevivência Celular , Técnicas de Cultura Embrionária/veterinária , Transferência Embrionária/veterinária , Feminino , Perfilação da Expressão Gênica/métodos , Redes Reguladoras de Genes , Técnicas de Maturação in Vitro de Oócitos/veterinária , Masculino , Análise de Sequência com Séries de Oligonucleotídeos/veterinária , Gravidez , Análise de Componente Principal , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Especificidade da EspécieRESUMO
Oocyte-secreted factors (OSFs) regulate differentiation of cumulus cells and are of pivotal relevance for fertility. Bone morphogenetic protein 15 (BMP15) and fibroblast growth factor 10 (FGF10) are OSFs and enhance oocyte competence by unknown mechanisms. We tested the hypothesis that BMP15 and FGF10, alone or combined in the maturation medium, enhance cumulus expansion and expression of genes in the preovulatory cascade and regulate glucose metabolism favouring hyaluronic acid production in bovine cumulus-oocyte complexes (COCs). BMP15 or FGF10 increased the percentage of fully expanded COCs, but the combination did not further stimulate it. BMP15 increased cumulus cell levels of mRNA encoding a disintegrin and metalloprotease 10 (ADAM10), ADAM17, amphiregulin (AREG), and epiregulin (EREG) at 12 h of culture and of prostaglandin (PG)-endoperoxide synthase 2 (PTGS2), pentraxin 3 (PTX3) and tumor necrosis factor alpha-induced protein 6 (TNFAIP6 (TSG6)) at 22âh of culture. FGF10 did not alter the expression of epidermal growth factor-like factors but enhanced the mRNA expression of PTGS2 at 4âh, PTX3 at 12âh, and TNFAIP6 at 22âh. FGF10 and BMP15 stimulated glucose consumption by cumulus cells but did not affect lactate production or levels of mRNA encoding glycolytic enzymes phosphofructokinase and lactate dehydrogenase A. Each growth factor increased mRNA encoding glucosamine:fructose-6-PO4 transaminases, key enzymes in the hexosamine pathway leading to hyaluronic acid production, and BMP15 also stimulated hyaluronan synthase 2 (HAS2) mRNA expression. This study provides evidence that BMP15 and FGF10 stimulate expansion of in vitro-matured bovine COCs by driving glucose metabolism toward hyaluronic acid production and controlling the expression of genes in the ovulatory cascade, the first acting upon ADAM10, ADAM17, AREG, and EREG and the second on downstream genes, particularly PTGS2.
Assuntos
Proteína Morfogenética Óssea 15/fisiologia , Células do Cúmulo/fisiologia , Fator 10 de Crescimento de Fibroblastos/fisiologia , Oócitos/fisiologia , Ovulação , Animais , Bovinos , Feminino , Expressão Gênica , Regulação da Expressão Gênica , Glucose/metabolismoRESUMO
Intrauterine dietary restriction may cause changes in the functioning of offspring organs and systems later in life, an effect known as fetal programming. The present study evaluated mRNA abundance and immunolocalization of nutrient transporters as well as enterocytes proliferation in the proximal, median and distal segments of small intestine of rats born to protein-restricted dams. Pregnant rats were fed hypoproteic (6% protein) or control (17% protein) diets, and offspring rats were evaluated at 3 and 16 weeks of age. The presence of SGLT1 (sodium-glucose co-transporter 1), GLUT2 (glucose transporter 2), PEPT1 (peptide transporter 1) and the intestinal proliferation were evaluated by immunohistochemical techniques and the abundance of specific mRNA for SGLT1, GLUT2 and PEPT1 was assessed by the real-time PCR technique. Rats born to protein-restricted dams showed higher cell proliferation in all intestinal segments and higher gene expression of SGLT1 and PEPT1 in the duodenum. Moreover, in adult animals born to protein-restricted dams the immunoreactivity of SGLT1, GLUT2 and PEPT1 in the duodenum was more intense than in control rats. Taken together, the results indicate that changes in the small intestine observed in adulthood can be programmed during the gestation. In addition, they show that this response is caused by both up-regulation in transporter gene expression, a specific adaptation mechanism, and intestinal proliferation, an unspecific adaptation mechanism.
Assuntos
Fenômenos Fisiológicos da Nutrição Animal , Dieta com Restrição de Proteínas , Intestino Delgado/metabolismo , Desnutrição/metabolismo , Fenômenos Fisiológicos da Nutrição Materna , Proteínas de Membrana Transportadoras/metabolismo , Adaptação Fisiológica , Adiposidade , Animais , Peso Corporal , Proliferação de Células , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica , Transportador de Glucose Tipo 2/metabolismo , Imuno-Histoquímica , Desnutrição/etiologia , Desnutrição/genética , Desnutrição/fisiopatologia , Proteínas de Membrana Transportadoras/genética , Transportador 1 de Peptídeos , Gravidez , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transportador 1 de Glucose-Sódio/metabolismo , Simportadores/metabolismoRESUMO
FSH induces expansion of bovine cumulus-oocyte complexes (COCs) in cattle, which can be enhanced by oocyte-secreted factors (OSFs). In this study it was hypothesised that FSH stimulates COC expansion in part from direct stimulation of the epidermal growth factor (EGF)-like ligands amphiregulin (AREG), epiregulin (EREG) and betacellulin (BTC), but also in part through regulation of OSFs or their receptors in cumulus cells. Bovine COCs were cultured in defined medium with graded doses of FSH. In the absence of FSH, COCs did not expand. FSH caused cumulus expansion, and increased the abundance of AREG and EREG mRNA in a time- and dose-dependent manner, but decreased BTC mRNA levels. FSH had modest stimulatory effects on the levels of mRNA encoding the bone morphogenetic protein 15 (BMP15) receptor, BMPR1B, in cumulus cells, but did not alter mRNA expression of the growth and differentiation factor 9 (GDF9) receptor, TGFBR1. More interestingly, FSH dramatically stimulated levels of mRNA encoding two receptors for fibroblast growth factors (FGF), FGFR2C and FGFR3C, in cumulus cells. FSH also stimulated mRNA expression of FGFR1B, but not of FGFR2B in cumulus cells. Based on dose-response studies, FGFR3C was the receptor most sensitive to the influence of FSH. This study demonstrates that FSH stimulates the expression of EGF-like factors in bovine cumulus cells, and provides evidence that FSH differently regulates the expression of distinct receptors for OSFs in cumulus cells.
Assuntos
Bovinos/fisiologia , Células do Cúmulo/efeitos dos fármacos , Fator de Crescimento Epidérmico/metabolismo , Fármacos para a Fertilidade Feminina/farmacologia , Hormônio Foliculoestimulante/farmacologia , Técnicas de Maturação in Vitro de Oócitos/veterinária , Oócitos/efeitos dos fármacos , Matadouros , Anfirregulina , Animais , Betacelulina , Receptores de Proteínas Morfogenéticas Ósseas Tipo I/biossíntese , Receptores de Proteínas Morfogenéticas Ósseas Tipo I/genética , Receptores de Proteínas Morfogenéticas Ósseas Tipo I/metabolismo , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Técnicas de Cocultura/veterinária , Células do Cúmulo/citologia , Células do Cúmulo/metabolismo , Fator de Crescimento Epidérmico/biossíntese , Fator de Crescimento Epidérmico/genética , Epirregulina , Feminino , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Glicoproteínas/biossíntese , Glicoproteínas/genética , Glicoproteínas/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/biossíntese , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Ligantes , Oócitos/citologia , Oócitos/metabolismo , Isoformas de Proteínas/biossíntese , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/biossíntese , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/genética , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/metabolismo , Sus scrofaRESUMO
Lipid droplets, subspecies (Bos taurus indicus vs. Bos taurus taurus), and in vitro culture are known to influence cryopreservation of bovine embryos. Limited information is available regarding differences in membrane lipids in embryo, such as phosphatidylcholines (PC) and sphingomyelins (SM). The objective of the present study was to compare the profiles of several PC and SM species and relate this information to cytoplasmic lipid levels present in Nellore (B. taurus indicus) and Simmental (B. taurus taurus) blastocysts produced in vitro (IVP) or in vivo (ET). Simmental and IVP embryos had more cytoplasmic lipid content than Nellore and ET embryos (n = 30). Blastocysts were submitted to matrix-assisted laser desorption/ionization mass spectrometry. Differences in the PC profile were addressed by principal component analysis. The lipid species with PC (32:1) and PC (34:1) had higher ion abundances in Nellore embryos, whereas PC (34:2) was higher in Simmental embryos. IVP embryos had less abundant ions of PC (32:1), PC (34:2), and PC (36:5) compared to ET embryos. Moreover, ion abundance of PC (32:0) was higher in both Nellore and Simmental IVP embryos compared to ET embryos. Therefore, mass spectrometry profiles of PC and SM species significantly differ with regard to unsaturation level and carbon chain composition in bovine blastocysts due to subspecies and in vitro culture conditions. Because PC abundances of Nellore and Simmental embryos were distinct (34:1 vs. 34:2), as were those of IVP and ET embryos (32:0 vs. 36:5), they are potential markers of postcryopreservation embryonic survival.
Assuntos
Blastocisto/metabolismo , Bovinos/fisiologia , Membrana Celular/metabolismo , Grânulos Citoplasmáticos/metabolismo , Fertilização in vitro/veterinária , Fosfatidilcolinas/metabolismo , Esfingomielinas/metabolismo , Animais , Biomarcadores/química , Biomarcadores/metabolismo , Blastocisto/citologia , Brasil , Criopreservação/veterinária , Ectogênese , Feminino , Fertilização , Fertilização in vitro/efeitos adversos , Técnicas de Maturação in Vitro de Oócitos/veterinária , Fosfatidilcolinas/química , Gravidez , Análise de Componente Principal , Especificidade da Espécie , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/veterinária , Esfingomielinas/química , Espectrometria de Massas em Tandem/veterináriaRESUMO
Several growth factors have been identified as local regulators of follicle development and ovulation. Fibroblast growth factor (FGF) family members are potent mitogens and are involved in cell differentiation, cell migration and angiogenesis in many tissues and organs. In addition to FGF-2, which is the most-studied FGF, other important members are FGF-1, -5, -7, -8, -9 and -10. A number of studies have indicated that FGFs play important roles in regulating the initiation of primordial follicle growth, oocyte and follicle survival, granulosa and theca cell proliferation and differentiation, corpus luteum formation, steroidogenesis and angiogenesis. The purpose of this review is to highlight the importance of the FGFs on mammalian female reproduction, providing a better understanding of the roles of this family in ovarian physiology and female fertility.
Assuntos
Fatores de Crescimento de Fibroblastos/metabolismo , Folículo Ovariano/metabolismo , Ovulação , Animais , Sobrevivência Celular , Feminino , Fertilidade , Humanos , Ligantes , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Transdução de SinaisRESUMO
The first cleavage divisions and preimplantation embryonic development are supported by mRNA and proteins synthesized and stored during oogenesis. Thus, mRNA molecules of maternal origin decrease and embryonic development becomes gradually dependent on expression of genetic information derived from the embryonic genome. However, it is still unclear what the role of the sperm cell is during this phase and whether the absence of the sperm cell during the artificial oocyte activation affects subsequent embryonic development. The objective of this study was to determine, in bovine embryos, changes in cell cycle-associated transcript levels (cyclin A, cyclin B, cyclin E, CDC2, CDK2, and CDK4) after oocyte activation in the presence or absence of the sperm cell. To evaluate that, in vitro-produced (IVP) and parthenogenetically activated (PA) embryos (2-4 cells (2-4C), 8-16 cells (8-16C) and blastocysts) were evaluated by real-time PCR. There was no difference in cleavage and blastocyst rates between IVP and PA groups. Transcript level was higher in oocytes than in IVP and PA embryos. Cleaved PA embryos showed higher expression of cyclin A, cyclin B, cyclin E, and CDK2 and lower expression of CDC2 when compared with that from the IVP group. At the time of activation, all transcripts were expressed less in PA than in IVP embryos, whereas at the blastocyst stage, almost all genes were expressed at a higher level in the PA group. These results suggest that in both groups there is an initial consumption of these transcripts in the early stages of embryonic development. Furthermore, 8-16C embryos seem to synthesize more cell cycle-related genes than 2-4C embryos. However, in PA embryos, activation of the cell cycle genes seems to occur after the 8- to 16-cell stage, suggesting a failure in the activation process.
Assuntos
Blastocisto , Genes cdc , Oócitos/metabolismo , Animais , Sequência de Bases , Bovinos , Primers do DNA , Desenvolvimento Embrionário , Perfilação da Expressão Gênica , Partenogênese , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Fibroblast growth factors (FGFs) are involved in paracrine control of follicle development. It was previously demonstrated that FGF10 decreases estradiol (E(2)) secretion in granulosa cell culture and that theca cell FGF10 mRNA expression is decreased in healthy follicles from abattoir ovaries. The main objectives of this study were to evaluate FGF10 and FGFR2b mRNA expression during follicular development in vivo, to evaluate the effect of FGF10 on follicle growth using Bos taurus taurus cows as a model, and to gain more insight into the mechanisms through which FGF10 inhibits steroidogenesis. Messenger RNA encoding both FGF10 and FGFR2b (main FGF10 receptor) was significantly more expressed in subordinate follicles (SFs) than in dominant follicles (DFs). The intrafollicular injection of FGF10 into the largest growing follicle at 7-8âmm in diameter interrupted the DF growth in a dose-dependent manner (11±0.4, 8.3±1 and 5.9±0.3âmm for 0, 0.1, and 1âµg/ml FGF10, respectively, at 72âh after treatment; P<0.05). In a third experiment, follicles were obtained 24âh after FGF10 (1âµg/ml) or PBS treatment through ovariectomy. In theca cells, FGF10 treatment did not affect mRNA encoding steroidogenic enzymes, LHCGR and IGFBPs, but significantly upregulated FGF10 mRNA expression. The expression of CYP19A1 mRNA in granulosa cells was downregulated by FGF10 treatment, which was accompanied by a 50-fold decrease in E(2) production, and decreased cyclin D2 mRNA. These results have shown that FGF10 and its receptor FGFR2b are more expressed in SFs and provide solid in vivo evidence that FGF10 acts as an important regulator of follicular growth in cattle.
Assuntos
Bovinos , Estradiol/metabolismo , Fator 10 de Crescimento de Fibroblastos/farmacologia , Folículo Ovariano/efeitos dos fármacos , Animais , Bovinos/genética , Bovinos/metabolismo , Bovinos/fisiologia , Relação Dose-Resposta a Droga , Regulação para Baixo/efeitos dos fármacos , Feminino , Fator 10 de Crescimento de Fibroblastos/administração & dosagem , Fator 10 de Crescimento de Fibroblastos/genética , Fator 10 de Crescimento de Fibroblastos/metabolismo , Expressão Gênica/efeitos dos fármacos , Células da Granulosa/efeitos dos fármacos , Células da Granulosa/metabolismo , Células da Granulosa/fisiologia , Microinjeções , Oogênese/efeitos dos fármacos , Oogênese/genética , Oogênese/fisiologia , Folículo Ovariano/crescimento & desenvolvimento , Folículo Ovariano/metabolismo , Folículo Ovariano/fisiologia , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/genética , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/metabolismo , Células Tecais/efeitos dos fármacos , Células Tecais/metabolismo , Células Tecais/fisiologiaRESUMO
Oocyte meiotic resumption is triggered by the ovulatory gonadotropin surge; in cattle, angiotensin II (AngII) and prostaglandins (PG) are key mediators of this gonadotropin-induced event. Here, we tested the hypothesis that progesterone (P(4)) is also involved in oocyte meiotic resumption induced by the gonadotropin surge. In Experiment I, P(4) induced nuclear maturation in a dose-dependent manner using a coculture of follicular hemisections and cumulus-oocyte complexes. In the second experiment, using an in vivo model, an injection of mifepristone (MIFE; P(4) receptor antagonist) at the antrum of preovulatory follicles prevented GnRH-induced oocyte meiotic resumption in vivo. In Experiment III (coculture system similar to that of Experiment I), MIFE prevented stimulatory effects of AngII on resumption of meiosis, but saralasin (AngII receptor antagonist) did not inhibit P(4) actions. In Experiments IV and V, fibroblast growth Factor 10 (FGF10; known to suppress steroidogenesis in granulosa cells), blocked AngII-but not P(4)-induced oocyte meiotic resumption. Therefore, we inferred that AngII is upstream to P(4) in a cascade to induce meiotic resumption. Previously, we had reported that AngII acted throughout the PGs pathway to modulate nuclear progression. In Experiment V, indomethacin inhibited resumption of meiosis induced by P(4), providing further support to the AngII-P(4) sequential effect on meiotic resumption. In conclusion, we inferred that AngII, P(4) and PGs are sequential steps in the same pathway that culminates with bovine oocyte maturation.
Assuntos
Angiotensina II/metabolismo , Bovinos/sangue , Técnicas de Maturação in Vitro de Oócitos/veterinária , Oócitos/fisiologia , Progesterona/metabolismo , Prostaglandinas/metabolismo , Animais , Fator 10 de Crescimento de Fibroblastos/farmacologia , Indometacina/farmacologia , Luteolíticos/farmacologia , Meiose/fisiologia , Mifepristona/farmacologia , Receptores de Progesterona/antagonistas & inibidoresRESUMO
The objective was to determine the relationship among the diameter of ovarian follicles, ovulation rate, and gene expression of the LH receptor (LHR) in Nelore cattle. In Experiment 1, ovulation was synchronized in 53 Nelore cows. Three days after ovulation, ovaries were assessed with ultrasonography, all cows were given 6.25 mg LH im, and they were allocated into three groups, according to diameter of their largest ovarian follicle: G1 (7.0-8.0 mm); G2 (8.1-9.0 mm); and G3 (9.1-10.0 mm). For these three groups, ovulation rates were 9, 36, and 90%, respectively, (P<0.03; each rate differed significantly from the other two). In Experiment 2, granulosa and theca cells were subjected to total RNA extraction, and gene expression of the LHR was determined by RT-PCR. Follicles were allocated in three groups based on their diameter (similar to the Experiment 1), which were denoted Groups A, B, and C. Expression of the LHR gene in granulosa cells was lower in Group A than Group C (P<0.05). However, there were no significant differences among groups in expression of the LHR gene in theca cells. We concluded that ovulatory capacity in Nelore cattle was related to increased follicular diameter and expression of the LHR gene in granulosa cells.