RESUMO
BACKGROUND: Cyclic adenosine monophosphate (cAMP) plays a crucial role as a signaling molecule for sperm functions such as capacitation, motility and acrosome reaction. It is well known that cAMP degradation by phosphodiesterase (PDE) enzyme has a major impact on sperm functions. The present study was undertaken to characterize cAMP-PDE activity in human semen. METHODS: cAMP-PDE activity was measured in human sperm and seminal plasma using family specific PDE inhibitors. Three sperm fractionation methods were applied to assess cAMP-PDE activity in spermatozoa. Western blots were used to validate the presence of specific family in sperm and seminal plasma. RESULTS: Using three sperm fractionation methods, we demonstrated that in human sperm, the major cAMP-PDE activity is papaverine-sensitive and thus ascribed to PDE10. In seminal plasma, total cAMP-PDE activity was 1.14±0.39fmol of cAMP hydrolyzed per minute per µg of protein. Using specific inhibitors, we showed that the major cAMP-PDE activity found in human seminal plasma is ascribed to PDE4 and PDE11. Western blot analysis, immunoprecipitation with a specific monoclonal antibody, and mass spectrometry confirmed the presence of PDE10 in human spermatozoa. CONCLUSION: This study provides the first demonstration of the presence of functional PDE10 in human spermatozoa and functional PDE4 and PDE11 in human seminal plasma. GENERAL SIGNIFICANCE: Since the contribution of cyclic nucleotides in several sperm functions is well known, the finding that PDE10 is an active enzyme in human spermatozoa is novel and may lead to new insight into fertility.
Assuntos
Líquidos Corporais/metabolismo , AMP Cíclico/metabolismo , Diester Fosfórico Hidrolases/metabolismo , Sêmen/metabolismo , Espermatozoides/metabolismo , Reação Acrossômica/efeitos dos fármacos , Reação Acrossômica/fisiologia , Sequência de Aminoácidos , Líquidos Corporais/efeitos dos fármacos , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/metabolismo , Humanos , Masculino , Inibidores de Fosfodiesterase/farmacologia , Sêmen/efeitos dos fármacos , Alinhamento de Sequência , Capacitação Espermática/efeitos dos fármacos , Capacitação Espermática/fisiologia , Motilidade dos Espermatozoides/efeitos dos fármacos , Motilidade dos Espermatozoides/fisiologia , Espermatozoides/efeitos dos fármacosRESUMO
Lipids rafts are specialised membrane microdomains involved in cell signalling that can be isolated as detergent-resistant membranes (DRMs). The second messenger cyclic AMP (cAMP) has a central role in cell signalling in the ovary and its degradation is carried out by the phosphodiesterase (PDE) enzyme family. We hypothesised that PDEs could be functionally present in the lipid rafts of porcine mural granulosa cell membranes. PDE6C, PDE8A and PDE11A were detected by dot blot in the DRMs and the Triton-soluble fraction of the mural granulosa cells membrane and the cytosol. As shown by immunocytochemistry, PDEs showed clear immunostaining in mural granulosa cell membranes and the cytosol. Interestingly, cAMP-PDE activity was 18 times higher in the DRMs than in the Triton-soluble fraction of cell membranes and was 7.7 times higher in the cytosol than in the DRMs. cAMP-PDE activity in mural granulosa cells was mainly contributed by the PDE8 and PDE11 families. This study shows that PDEs from the PDE8 and PDE11 families are present in mural granulosa cells and that the cAMP-PDE activity is mainly contributed by the cytosol. In the cell membrane, the cAMP-PDE activity is mainly contributed by the DRMs. In addition, receptors for prostaglandin E2 and LH, two G-protein-coupled receptors, are present in lipid rafts and absent from the non-raft fraction of the granulosa cell membrane. These results suggest that in these cells, the lipid rafts exist as a cell-signalling platform and PDEs are one of the key enzyme families present in the raft.
Assuntos
Membrana Celular/enzimologia , Células da Granulosa/enzimologia , Diester Fosfórico Hidrolases/metabolismo , Animais , Colesterol/metabolismo , AMP Cíclico/metabolismo , Feminino , SuínosRESUMO
C-type natriuretic peptide (CNP) and its cognate receptor, natriuretic peptide receptor (NPR) B, have been shown to promote cGMP production in granulosa/cumulus cells. Once transferred to the oocyte through the gap junctions, the cGMP inhibits oocyte meiotic resumption. CNP has been shown to bind another natriuretic receptor, NPR-C. NPR-C is known to interact with and degrade bound CNP, and has been reported to possess signaling functions. Therefore, NPR-C could participate in the control of oocyte maturation during swine in vitro maturation (IVM). Here, we examine the effect of CNP signaling on meiotic resumption, the amount of cGMP and gap junctional communication (GJC) regulation during swine IVM. The results show an inhibitory effect of CNP in inhibiting oocyte meiotic resumption in follicle-stimulating hormone (FSH)-stimulated IVM. We also found that an NPR-C-specific agonist (cANP([4-23])) is likely to play a role in maintaining meiotic arrest during porcine IVM when in the presence of a suboptimal dose of CNP. Moreover, we show that, even if CNP can increase intracellular concentration of cGMP in cumulus-oocyte complexes, cANP((4-23)) had no impact on cGMP concentration, suggesting a potential cGMP-independent signaling pathway related to NPR-C activation. These data support a potential involvement of cANP((4-23)) through NPR-C in inhibiting oocyte meiotic resumption while in the presence of a suboptimal dose of CNP. The regulation of GJC was not altered by CNP, cANP((4-23)), or the combination of CNP and cANP((4-23)), supporting their potential contribution in sending signals to the oocytes. These findings offer promising insights in to new elements of the signaling pathways that may be involved in inhibiting resumption of meiosis during FSH-stimulated swine IVM.
Assuntos
Meiose/fisiologia , Peptídeo Natriurético Tipo C/farmacologia , Oócitos/metabolismo , Oogênese/fisiologia , Transdução de Sinais/fisiologia , Animais , Células do Cúmulo/efeitos dos fármacos , Células do Cúmulo/metabolismo , GMP Cíclico/metabolismo , Feminino , Meiose/efeitos dos fármacos , Oócitos/efeitos dos fármacos , Oogênese/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , SuínosRESUMO
The serine/threonine kinase 5' adenosine monophosphate-activated protein kinase (AMPK), a heterotrimeric protein known as a metabolic switch, is involved in oocyte nuclear maturation in mice, cattle, and swine. The present study analyzed AMPK activation in cumulus cell expansion during in vitro maturation (IVM) of porcine cumulus-oocyte complexes (COC). 5-Aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR) is a well-known activator of AMPK. It inhibited oocyte meiotic resumption in COC. Moreover, cumulus cell expansion did not occur in the presence of AICAR, demonstrating its marked impact on cumulus cells. Activation of AMPK was supported by AICAR-mediated phosphorylation of alpha AMPK subunits. Furthermore, the presence of AICAR increased glucose uptake, a classical response to activation of this metabolic switch in response to depleted cellular energy levels. Neither nuclear maturation nor cumulus expansion was reversed by glucosamine, an alternative substrate in hyaluronic acid synthesis, through the hexosamine biosynthetic pathway, which ruled out possible depletion of substrates. Both increased gap junction communication and phosphodiesterase activity in COC are dependent on protein synthesis during the initial hours of IVM; however, both were inhibited in the presence of AICAR, which supports the finding that activation of AMPK by AICAR mediated inhibition of protein synthesis. Moreover, this protein synthesis inhibition was equivalent to that of the well-known protein synthesis inhibitor cycloheximide, as observed on cumulus expansion and protein concentration. Finally, the phosphorylation level of selected kinases was investigated. The pattern of raptor phosphorylation is supportive of activation of AMPK-mediated inhibition of protein synthesis. In conclusion, AICAR-mediated AMPK activation in porcine COC inhibited cumulus cell expansion and protein synthesis. These results bring new considerations to the importance of this kinase in ovarian physiology and to the development of new oocyte culture medium.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Células do Cúmulo/citologia , Células do Cúmulo/efeitos dos fármacos , Ativação Enzimática/fisiologia , Técnicas de Maturação in Vitro de Oócitos/veterinária , Suínos/fisiologia , Proteínas Quinases Ativadas por AMP/genética , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/farmacologia , Animais , Células do Cúmulo/fisiologia , Feminino , Regulação Enzimológica da Expressão Gênica/fisiologia , Glucose/metabolismo , Ribonucleotídeos/farmacologiaRESUMO
The phosphodiesterase (PDE) family is a group of enzymes that catalyzes the transformation of cyclic nucleotides into 5' nucleotides. Based on rodents, the current mammalian model of PDE distribution in the ovarian follicle predicts Pde3a in the oocyte and Pde4d in the somatic cells. Using bovine as an experimental model, the present results showed that PDE3 was the predominant PDE activity in oocytes. However, cumulus cell cAMP-PDE activity was predominantly resistant to inhibition by 3-isobutyl-methylxantine, indicating PDE8 activity (60% of total PDE activity) and a minor role for PDE4 (<5%). A total of 20% of total oocyte PDE activity was also attributed to PDE8. The PDE activity measurements in mural granulosa cells from 2 to 6 mm in diameter suggest the presence of PDE4 and PDE8. In granulosa cells from follicles >10 mm, total PDE and PDE8 activities along with PDE8A protein level were increased compared with smaller follicles. The RT-PCR experiments showed that cumulus cells expressed PDE8A, PDE8B, and PDE10A. Western blot experiments showed PDE8A, PDE8B, and PDE4D proteins in mural granulosa cells and cumulus-oocyte complexes. PDE8 inhibition using dipyridamole in a dose-dependent manner increased cAMP levels in the cumulus-oocyte complexes and delayed oocyte nuclear maturation. These results are the first to demonstrate the functional presence of PDE8 in the mammalian ovarian follicle. This challenges the recently described cell-specific expression of cAMP-PDEs in the ovarian follicle and the notion that PDE4 is the predominant granulosa/cumulus cell PDE. These findings have implications for our understanding of hormonal regulation of folliculogenesis and the potential application of PDE inhibitors as novel contraceptives.
Assuntos
Células da Granulosa/enzimologia , Oócitos/enzimologia , Oogênese/efeitos dos fármacos , Folículo Ovariano/enzimologia , Inibidores de Fosfodiesterase/farmacologia , Diester Fosfórico Hidrolases/metabolismo , Adenilil Ciclases/metabolismo , Análise de Variância , Animais , Bovinos , Núcleo Celular/fisiologia , Anticoncepcionais Femininos , AMP Cíclico/metabolismo , Técnicas de Cultura Embrionária , Feminino , Fertilização in vitro , Células da Granulosa/efeitos dos fármacos , Isoenzimas/genética , Isoenzimas/metabolismo , Masculino , Oócitos/efeitos dos fármacos , Oócitos/fisiologia , Diester Fosfórico Hidrolases/genética , Transporte Proteico , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The means by which cumulus cells react to gonadotropin stimulation and regulate the subsequent production and degradation of cAMP are largely unknown. In this article, we report that cyclic nucleotide phosphodiesterase (PDE) type 3A (Pde3a) is transcriptionally regulated in porcine cumulus cells by a cAMP-dependent pathway during in vitro maturation (IVM). cAMP-PDE activity was increased in the cumulus-oocyte complex (COC) after 10 h of IVM, and 78% of this increase was sensitive to a Pde3-specific inhibitor, cilostamide. Although no variation was observed in the oocyte, cilostamide-sensitive cAMP-PDE activity increased in the cumulus cells after IVM. This was supported by Western blotting, which showed that the intensity of a 135-kDa anti-Pde3a immunoreactive band was increased in COC after IVM. The Pde3a mRNA level was up-regulated 28-fold in the COC after 4 h of IVM and remained high up to 12 h. The mRNA up-regulation and increased activity were inhibited by an RNA synthesis inhibitor, alpha-amanitin. The cilostamide-sensitive increase in PDE activity was inhibited by a protein synthesis inhibitor, cycloheximide. Pregnant mare serum gonadotropin (PMSG) caused dose-dependent activation of Pde3. The PMSG-dependent increase in Pde3 activity and Pde3a mRNA were mimicked by the adenylyl cyclase activator forskolin or prostaglandin E2. PMSG-dependent Pde3 activation was inhibited by the protein kinase A-specific inhibitor H89. Collectively, our results show for the first time that degradation of the intracellular cyclic nucleotide by Pde3a is transcriptionally up-regulated via a cAMP-dependent pathway in cumulus cells, suggesting that it has a functional role during the ovulatory gonadotropin surge.
Assuntos
3',5'-AMP Cíclico Fosfodiesterases/genética , AMP Cíclico/farmacologia , Células da Granulosa/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos , 3',5'-AMP Cíclico Fosfodiesterases/metabolismo , Animais , Células Cultivadas , Nucleotídeo Cíclico Fosfodiesterase do Tipo 3 , Ativação Enzimática/efeitos dos fármacos , Feminino , Gonadotropinas/farmacologia , Células da Granulosa/enzimologia , Oócitos/citologia , Oócitos/enzimologia , SuínosRESUMO
Members of the M13 family of zinc metalloendopeptidases have been shown to play critical roles in the metabolism of various neuropeptides and peptide hormones, and they have been identified as important therapeutic targets. Recently, a mouse NL1 protein, a novel member of the family, was identified and shown to be expressed mainly in the testis as a secreted protein. To define its physiological role(s), we used a gene targeting strategy to disrupt the endogenous murine Nl1 gene by homologous recombination and generate Nl1 mutant mice. The Nl1(-/-) mice were viable and developed normally, suggesting that zygotic expression of Nl1 is not required for development. However, Nl1(-/-) males produced smaller litters than their wild-type siblings, indicating specific male fertility problems. Reduced fertility may be explained by two impaired processes, decreased egg fertilization and perturbed early development of fertilized eggs. These two phenotypes did not result from gross anatomical modifications of the testis or from impaired spermatogenesis. Basic sperm parameters were also normal. Thus, our findings suggest that one of the roles of NL1 in mice is related to sperm function and that NL1 modulates the processes of fertilization and early embryonic development in vivo.
Assuntos
Infertilidade Masculina/enzimologia , Metaloendopeptidases/deficiência , Animais , Sequência de Bases , DNA Complementar/genética , Desenvolvimento Embrionário e Fetal/genética , Desenvolvimento Embrionário e Fetal/fisiologia , Feminino , Fertilização/genética , Fertilização/fisiologia , Marcação de Genes , Infertilidade Masculina/genética , Infertilidade Masculina/patologia , Masculino , Metaloendopeptidases/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Knockout , Fenótipo , Gravidez , Testículo/patologiaRESUMO
BACKGROUND: The ovulatory surge of gonadotropins triggers oocyte maturation and rupture of the ovarian follicle. The resumption of nuclear maturation in the oocyte from the prophase stage is characterized by germinal vesicle breakdown (GVBD). It has previously been shown that specific inhibition of cAMP degradation by PDE3 prevents the resumption of oocyte meiosis. However, no report has characterized the activity of PDE3 in the porcine oocyte, or the implication of the cAMP-PDE3 pathway in the entire nuclear maturation process. In this study, PDE3 activity in the oocyte was assessed during in vitro maturation (IVM) and the possible roles of the cAMP-PDE3 pathway in the resumption and progression of meiosis were investigated in terms of different models of oocyte maturation. RESULTS: Cyclic AMP-degrading PDE activity was detected in the cumulus-oocyte complex (COC) and was partially inhibited by a specific PDE3 inhibitor, cilostamide. When measured only in the denuded oocyte, PDE activity was almost completely inhibited by cilostamide, suggesting that cAMP-PDE3 activity is the major cAMP-PDE in porcine oocytes. PDE3A mRNA was detected by RT-PCR. PDE3 activity did not vary significantly during the early hours of IVM, but a maximum was observed at 13 hours. In cumulus-oocyte complexes, meiosis resumed after 20.81 hours of culture. PDE3 inhibition no longer maintained meiotic arrest if sustained beyond 17.65 hours of IVM, 3 hours prior to resumption of meiosis. Thereafter, PDE3 inhibition progressively lost its efficacy in GVBD. When the protein phosphatase 1 and 2A inhibitor okadaic acid was continuously or transiently (3 hours) present during IVM, meiosis resumed prematurely; PDE3 inhibition was unable to prevent GVBD. However, PDE3 inhibition in COC treated with OA for 3 hours significantly delayed meiosis at the intermediate stage. CONCLUSION: The present investigation has demonstrated that PDE3A is the major cAMP-degrading PDE in the oocyte. It regulates the resumption of meiosis until 3 hours prior to GVBD and transiently affects meiotic progression.
Assuntos
3',5'-AMP Cíclico Fosfodiesterases/metabolismo , Oócitos/enzimologia , 1-Metil-3-Isobutilxantina/farmacologia , 3',5'-AMP Cíclico Fosfodiesterases/antagonistas & inibidores , 3',5'-AMP Cíclico Fosfodiesterases/genética , Animais , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , AMP Cíclico/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 3 , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , Meiose/efeitos dos fármacos , Ácido Okadáico/farmacologia , Oócitos/crescimento & desenvolvimento , Oócitos/metabolismo , Inibidores de Fosfodiesterase/farmacologia , Quinolonas/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Suínos , Fatores de TempoRESUMO
Androhep Plus, a long-term extender (up to 7 days) and Beltsville Thawing Solution (BTS), a short-term extender (up to 3 days), are commonly used for liquid storage of porcine semen. To test the hypothesis that modifications in sperm viability, motility, chlortetracycline (CTC) fluorescence patterns, and protein tyrosine phosphorylation occur during semen storage in extenders, we compared these end points at different periods of storage in either Androhep Plus or BTS. Sperm from five boars were assessed daily over 12 days of storage (n = 5 ejaculates from different boars). Viability was not different (P < 0.05 between extenders, except on Day 2, when Androhep Plus maintained better viability. Differences in the percentage of motile (total) sperm due to extender were evident on Days 2, 4, 5, and 6, when Androhep Plus was superior to BTS (P < 0.05). The percentages of progressively motile sperm also differed, with Androhep Plus supporting higher rates on Days 2, 4, 5, 7, 8, 9, 10, and 11 (P < 0.05). The CTC fluorescence pattern distribution differed due to extender as early as Day 2; storage in Androhep Plus induced higher levels of pattern B sperm (P < 0.05) than storage in BTS. A tyrosine-phosphorylated protein of Mr 21,000 appeared after 10 days in sperm incubated in BTS, and was identified as a phospholipid hydroperoxide glutathione peroxidase. Therefore, modifications in viability, motility, CTC fluorescence patterns, and sperm protein tyrosine phosphorylation were apparent during sperm storage in extenders; these may affect the fertilizing capacity of the semen.
Assuntos
Preservação do Sêmen/veterinária , Capacitação Espermática , Motilidade dos Espermatozoides , Espermatozoides/fisiologia , Suínos , Tirosina/metabolismo , Animais , Sobrevivência Celular , Clortetraciclina , Eletroforese em Gel Bidimensional , Corantes Fluorescentes , Masculino , Fosfoproteínas/análise , Fosforilação , Preservação do Sêmen/métodos , Soluções , Espermatozoides/química , Fatores de TempoRESUMO
Organochlorine chemicals are present in the environment worldwide; however, populations living in the Far North are particularly at risk because their traditional diets are mainly composed of contaminated animals (fish, seals, whales, and polar bears). It has been suggested that male fertility is globally declining, possibly because of chronic, low-level exposure to environmental contaminants. This study was designed to assess the effects on fresh sperm fertility parameters using the porcine model of 1) an environmentally relevant mixture of 15 organochlorines and 2) the metabolized extract of this mixture. In the first experiment, the organochlorine mixture (at relative concentrations of 10.5, 14.7, and 21 microg/mL polychlorinated biphenyls [PCBs]) reduced sperm total motility, progressive motility, and viability, and increased capacitation, spontaneous acrosome reaction rates, and cytosolic calcium levels, suggesting that the mixture alters the sperm membrane and is detrimental to sperm function. In the second experiment, the metabolized extract of this organochlorine mixture (at relative concentrations of 0.9, 1.8, 2.7, 3.6, and 4.5 microg/L OH-PCBs) tended to decrease only sperm total motility. In an in vitro porcine model, the mixture of organochlorines, as found in the Arctic food chain, was rapidly detrimental to sperm function at concentrations above environmental levels. In contrast, short and physiologically relevant exposure to the metabolized extract of this mixture induced only limited adverse effects on sperm motility.
Assuntos
Hidrocarbonetos Clorados/toxicidade , Espermatozoides/efeitos dos fármacos , Animais , Sobrevivência Celular/efeitos dos fármacos , Poluentes Ambientais/toxicidade , Masculino , Motilidade dos Espermatozoides/efeitos dos fármacos , SuínosRESUMO
Gap-junctional communication (GJC) plays a central role in oocyte growth. However, little is known about the regulation of connexin 43 (Cx43)-based gap-junction channels in cumulus-oocyte complexes (COCs) during in vitro maturation. We show that rupture of COCs from mural granulosa cells up-regulates Cx43-mediated GJC and that gonadotropins signal GJC breakdown by recruiting Cx43 to lipid rafts when oocyte meiosis resumes. Oocyte calcein uptake through gap junctions increases during early in vitro oocyte maturation and remains high until 18 h, when it falls simultaneously with the oocyte germinal vesicle breakdown. Immunodetection of Cx43 and fluorescence recovery after photobleaching assays revealed that the increase of GJC is independent of gonadotropins but requires RNA transcription, RNA polyadenylation, and translation. GJC rupture, in contrast, is achieved by a gonadotropin-dependent mechanism involving recruitment of Cx43 to clustered lipid rafts. These results show that GJC up-regulation in COCs in in vitro culture is independent of gonadotropins and transcriptionally regulated. However, GJC breakdown is gonadotropin dependent and mediated by the clustering of Cx43 in lipid raft microdomains. In conclusion, this study supports a functional role of lipid raft clustering of Cx43 in GJC breakdown in the COCs during in vitro maturation.
Assuntos
Junções Comunicantes/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Gonadotropinas/metabolismo , Células da Granulosa/metabolismo , Microdomínios da Membrana/metabolismo , Oócitos/metabolismo , Suínos/metabolismo , Animais , Western Blotting , Células Cultivadas , Conexina 43/metabolismo , Feminino , Lamina Tipo A/metabolismo , Proteínas de Membrana/metabolismo , Microscopia Confocal , Tubulina (Proteína)/metabolismoRESUMO
The purpose of this study was to examine the effects of an activator of AMPK (5-aminoimidazole-4-carboxamide 1-beta-D-ribofuranoside (AICAR)) on bovine oocyte nuclear maturation in vitro. After 7 hr of culture, AICAR (1 mM) significantly increased the percentages of cumulus-enclosed oocytes (CEO) and denuded oocytes (DO) remaining at the germinal vesicle stage. After 22 hr of culture, AICAR significantly reduced the percentage of CEO reaching metaphase II (MII). AICAR at 1.0 mM also increased the inhibitory effect of the adenylate cyclase activator forskolin in CEO; however, at 0.05 mM, AICAR increased the percentage of oocytes at MII after 22 hr of culture compared to forskolin alone. The adenosine kinase inhibitor 5'-aminodeoxyadenosine reversed the effect of AICAR in CEO and DO showing that phosphorylation of AICAR by adenosine kinase is required for its inhibitory activity. GMP, but not AMP, inhibited meiosis in CEO and DO; however, inhibition of guanyl and adenyl nucleotides synthesis did not reverse the effect of AICAR suggesting that the inhibitory effect of AICAR is not due to increased synthesis of these nucleotides. Metformin, another activator of AMPK, also inhibited GVBD in CEO and DO. The alpha-1 isoform of the catalytic subunit of AMPK was detected in oocytes and cumulus cells, and reverse transcription-polymerase chain reaction experiments showed the presence of transcripts for alpha-1, alpha-2, beta-1, and gamma-3 isoforms of the regulatory subunits in cumulus cells and oocytes. These data show that the AMPK activator AICAR is inhibitory to nuclear maturation in bovine oocytes due to activation of AMPK.
Assuntos
Aminoimidazol Carboxamida/análogos & derivados , Núcleo Celular/fisiologia , Complexos Multienzimáticos/metabolismo , Oócitos/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Subunidades Proteicas/metabolismo , Ribonucleotídeos/metabolismo , Proteínas Quinases Ativadas por AMP , Alanina/análogos & derivados , Alanina/metabolismo , Aminoimidazol Carboxamida/metabolismo , Animais , Antibióticos Antineoplásicos/metabolismo , Bovinos , Colforsina/metabolismo , AMP Cíclico/metabolismo , Ativação Enzimática , Feminino , Hipoglicemiantes/metabolismo , Meiose , Metformina/metabolismo , Ácido Micofenólico/metabolismo , Oócitos/citologiaRESUMO
Adenosine monophosphate-activated kinase (PRKA) is a serine/threonine kinase that functions as a metabolic switch in a number of physiological functions. The present study was undertaken to assess the role of this kinase in nuclear maturation of porcine oocytes. RT-PCR and immunoblotting revealed the expression of the PRKAA1 subunit in granulosa cells, cumulus-oocyte complexes (COC), and denuded oocytes (DO). Porcine COC and DO contained transcripts that corresponded to the expected sizes of the designed primers for PRKAB1 and PRKAG1. The PRKAA2 subunit was detected in granulosa cells and COC, whereas the PRKAG3 subunit was not detected in granulosa cells, COC or DO, whereas it was detected in the heart. The PRKAA1 protein was detected in granulosa cells, COC, DO, and zona pellucida (ZP). In the presence of the pharmacological activator of PRKA 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranosyl 5'-monophosphate (ZMP), COC were transiently maintained in meiotic arrest in a fully reversible manner. This inhibitory effect was not observed in DO. Other known PRKA activators, 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR) and metformin, also blocked meiotic resumption in COC. In contrast to mouse oocytes, in which PRKA activators reverse the inhibitory effect of PDE3 inhibitors, this combination still blocked meiotic resumption in porcine COC. These results demonstrate that the meiotic resumption of porcine COC is transiently blocked by PRKA activators in a dose-dependent manner, and that this effect is dependent on PRKA activity in cumulus cells. The present study describes a new role for PRKA in regulating meiotic resumption in COC and strongly suggests that cumulus cells play an essential role in the control of porcine oocyte maturation through the PRKA metabolic switch.
Assuntos
Aminoimidazol Carboxamida/análogos & derivados , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Meiose/efeitos dos fármacos , Oócitos/efeitos dos fármacos , Inibidores de Fosfodiesterase/farmacologia , Ribonucleotídeos/farmacologia , Aminoimidazol Carboxamida/farmacologia , Animais , Feminino , Isoenzimas/metabolismo , Oócitos/fisiologia , Técnicas de Cultura de Órgãos , Folículo Ovariano/metabolismo , Sus scrofaRESUMO
We hypothesized that in utero and lactational exposure of male rats to a mixture of more than 15 organochlorines, resembling that found in blubber from northern Quebec seals, alters reproductive development and function. Female rats were gavaged with either corn oil (controls) or the organochlorine mixture in increasing doses (low, medium, and high) for 5 wk before mating and through gestation. Developmental effects were monitored in the male offspring from Postnatal Day (PND) 2 until PND 90. The high-dose mixture reduced the number of pups per litter, percentage of live offspring, and pup weights (P < 0.05). Because only three rats from the high-dose treatment survived, data from this group beyond PND 2 were not included in the statistical analyses. As assessed by the time of preputial separation, puberty was delayed in the pups from treated dams (P < 0.05). Testes weights in the medium-dose group were greater than those in controls on PND 21 (P < 0.05). Ventral prostate weights were lower for the medium-dose group on PND 60 (P < 0.05). On PND 90, weights of the epididymis, ventral prostate, and seminal vesicle of the medium-dose rats were reduced compared to those of controls (P < 0.05). On PND 90, sperm motility parameters assessed by computer-assisted sperm analysis were altered in the low- and medium-dose groups (P < 0.05). Testicular and epididymal morphology was severely affected in rats exposed to the high dose of the mixture. Serum testosterone, LH, FSH, prolactin, and total thyroxine levels did not differ because of organochlorine treatment. Therefore, in utero and lactational exposure to an environmentally relevant organochlorine mixture adversely affects the reproductive system of male rats, perhaps via antiandrogenic effects during testis development, suggesting a possible reproductive health hazard for humans and other species.
Assuntos
Genitália Masculina/efeitos dos fármacos , Hidrocarbonetos Clorados/toxicidade , Exposição Materna , Efeitos Tardios da Exposição Pré-Natal , Espermatozoides/efeitos dos fármacos , Animais , Relação Dose-Resposta a Droga , Feminino , Genitália Masculina/crescimento & desenvolvimento , Hidrocarbonetos Clorados/sangue , Lactação , Fígado/efeitos dos fármacos , Masculino , Tamanho do Órgão , Gravidez , Ratos , Ratos Sprague-Dawley , Baço/efeitos dos fármacosRESUMO
We evaluated the effects of an environmentally relevant mixture of more than 15 organochlorines on the development of pig oocytes and sperm during in vitro fertilization (IVF). Oocytes were cocultured with sperm in IVF medium containing increasing concentrations of an organochlorine mixture, similar to that found in women of highly exposed populations. Exposure to the organochlorine mixture diminished oocyte penetration rates and polyspermy in a linear manner. The mixture did not affect rates of cleavage nor development to multicell embryos. However, rates of development to the blastocyst stage were lower at the highest concentration at which oocyte penetration was observed. The same experiment was performed using oocytes that were preexposed during in vitro maturation. This greater exposure to the mixture also reduced penetration in a dose-response manner and affected polyspermy. Frozen-thawed pig sperm were also cultured in IVF medium containing the same organochlorine concentrations. Sperm motility parameters were immediately reduced in a dose-dependent manner by the organochlorines, followed by diminished viability 2 h later. From these results, it appears that reduced sperm quality would account for decreases in fertilization, polyspermy, and blastocyst formation. These results suggest that exposing porcine oocytes and sperm to an environmentally pertinent organochlorine mixture in vitro disrupts the oocyte block to polyspermy, sperm fertility, and further embryonic development, and supports recent concerns that such pollutants harm reproductive health in humans and other species.