RESUMO
Heparin is the most commonly used in vitro capacitation inducer in the bovine. However, hyaluronic acid (HA) has been recently used for capacitation induction as well as for other reproductive biotechnologies, such as sperm selection and in vitro fertilization (IVF). Our aim was to induce sperm capacitation with heparin or HA in order to study mAC and TK intracellular signals and their relation with cleavage and blastocyst rates after IVF as well as with the oxidative status of the potential bovine embryos. 2,5-dideoxyadenosine and genistein were used as mAC and TK inhibitors, respectively. Sperm capacitation was analyzed using CTC technique, sperm plasma membrane and acrosome integrity were determined using trypan blue stain and differential interference contrast, and mitochondrial activity was evaluated using fluorochrome JC-1. Cleavage rate was analyzed 48h and blastocyst production 7-8 days after IVF, while cytosolic oxidative activity was determined using RedoxSensor Red CC-1 fluorochrome 7h after IVF. When mAC and TK inhibitors were added to sperm samples, only capacitation decreased significantly both in HA and heparin treated samples (P < 0.05), but plasma membrane and acrosome integrity percentages were not affected in any of these groups (P > 0.05). Sperm mitochondrial membrane potential only decreased in heparin treated samples in the presence of both inhibitors (P < 0.05). Oocytes activated with HA sperm treated samples with the addition of 2,5-dideoxyadenosine and genistein presented a lower cytosolic oxidative status than those activated with sperm treated with HA alone (P < 0.05). On the other hand, oocytes activated with heparin treated sperm samples presented a lower cytosolic oxidative status only in the presence of 2,5-dideoxyadenosine (P < 0.05). Therefore, mAC and TK present a differential participation in heparin and HA sperm induced capacitation and mitochondrial function as well as in IVF.
Assuntos
Adenilil Ciclases/metabolismo , Fertilização in vitro/veterinária , Ácido Hialurônico/farmacologia , Proteínas Tirosina Quinases/metabolismo , Capacitação Espermática/efeitos dos fármacos , Animais , Bovinos , Membrana Celular/efeitos dos fármacos , Membrana Celular/enzimologia , Criopreservação/veterinária , Didesoxiadenosina/administração & dosagem , Didesoxiadenosina/farmacologia , Quimioterapia Combinada , Genisteína/administração & dosagem , Genisteína/farmacologia , Heparina/administração & dosagem , Heparina/farmacologia , MasculinoRESUMO
The objectives of this study were firstly to determine whether the stimulatory function of equine growth hormone (eGH) on equine oocyte maturation in vitro is mediated via cyclic adenosine monophosphate (cAMP); and secondly if the addition of eGH in vitro influences oocyte nuclear maturation and if this effect is removed when GH inhibitors are added to the culture. Cumulus-oocyte complexes (COCs) were recovered from follicles <25 mm in diameter and randomly allocated as follows: (i) control (no additives); and (ii) 400 ng/ml of eGH. A specific inhibitor against cyclic AMP-dependent protein kinase (H-89; 10-9, 10-11 or 10-15 M concentration) and a specific adenylate cyclase inhibitor, 2',3'-dideoxyadenosine (DDA; 10-8, 10-10 or 10-14 M concentration) were used to observe whether they could block the eGH effect. After 30 h of in vitro maturation at 38.5°C with 5% CO2 in air, oocytes were stained with 10 µg/ml of Hoechst to evaluate nuclear status. More mature oocytes (P < 0.05) were detected when COCs were incubated with eGH (29 of 84; 34.5%) than in the control group (18 of 82; 21.9%). The H-89 inhibitor used at a concentration of 10-9 M (4 of 29; 13.8%) decreased (P < 0.05) the number of oocytes reaching nuclear maturation when compared with eGH (11 of 29; 38%). The DDA inhibitor at a concentration of 10-8 M (2 of 27; 7.4%) also reduced (P < 0.05) the number of oocytes reaching maturity when compared with the eGH group (9 of 30; 30%). Results from the present study show that H-89 and DDA can be used in vitro to block the eGH effect on equine oocyte maturation.
Assuntos
Inibidores de Adenilil Ciclases/farmacologia , Didesoxiadenosina/farmacologia , Hormônio do Crescimento/farmacologia , Técnicas de Maturação in Vitro de Oócitos/métodos , Isoquinolinas/farmacologia , Oócitos/efeitos dos fármacos , Sulfonamidas/farmacologia , Animais , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Feminino , Cavalos , Oócitos/fisiologia , Inibidores de Proteínas Quinases/farmacologiaRESUMO
The aim of this work was to study the participation of membrane adenylyl cyclase in heparin-induced capacitation in cryopreserved bovine spermatozoa. Sperm suspensions were incubated in Tyrode's albumin lactate pyruvate medium in the presence of heparin (10 IU ml(-1) ) or forskolin (1-75 µm), a well-known membrane adenylyl cyclase activator. The participation of membrane adenylyl cyclase was confirmed using a specific inhibitor, 2',5'-dideoxyadenosine (6-25 µm). Spermatozoa capacitated with forskolin (25 µm) were incubated with bovine follicular fluid to evaluate their ability to undergo acrosome reaction. Capacitation percentages were determined by the fluorescence technique with chlortetracycline, and true acrosome reaction was determined by trypan blue and differential interferential contrast. The forskolin concentrations employed had no effect on progressive motility or sperm viability. Capacitation values induced by 25-µm forskolin treatment (27.80 ± 2.59%) were significantly higher respect to the control (4.80 ± 1.30%). The inhibitor 2',5'-dideoxyadenosine prevented forskolin-induced capacitation and significantly diminished capacitation induced by heparin. Follicular fluid induced physiological acrosome reaction in spermatozoa previously capacitated with 25-µm forskolin (P < 0.05). Forskolin acts as a capacitation inducer and involves the participation of membrane adenylyl cyclase as part of the intracellular mechanisms that lead to capacitation in cryopreserved bovine spermatozoa.
Assuntos
Reação Acrossômica/efeitos dos fármacos , Adenilil Ciclases/fisiologia , Criopreservação , Fibrinolíticos/farmacologia , Heparina/farmacologia , Preservação do Sêmen , Capacitação Espermática/efeitos dos fármacos , Espermatozoides/efeitos dos fármacos , Reação Acrossômica/fisiologia , Inibidores de Adenilil Ciclases , Animais , Antimetabólitos/farmacologia , Bovinos , Sobrevivência Celular , Colforsina/farmacologia , Didesoxiadenosina/farmacologia , Masculino , Capacitação Espermática/fisiologia , Motilidade dos Espermatozoides/efeitos dos fármacos , Motilidade dos Espermatozoides/fisiologia , Espermatozoides/fisiologiaRESUMO
The effects of glucagon-like peptide 2 (GLP-2) on expression and activity of jejunal multidrug resistance-associated protein 2 (Mrp2; Abcc2) and glutathione transferase (GST) were evaluated. After GLP-2 treatment (12 µg/100 g b.wt. s.c., every 12 h, for 5 consecutive days), Mrp2 and the α class of GST proteins and their corresponding mRNAs were increased, suggesting a transcriptional regulation. Mrp2 was localized at the apical membrane of the enterocyte in control and GLP-2 groups, as detected by confocal immunofluorescence microscopy. As a functional assay, everted intestinal sacs were incubated in the presence of 1-chloro-2,4-dinitrobenzene in the mucosal compartment, and the glutathione-conjugated derivative, dinitrophenyl-S-glutathione (DNP-SG; model Mrp2 substrate), was detected in the same compartment by high-performance liquid chromatography. A significant increase in apical secretion of DNP-SG was detected in the GLP-2 group, consistent with simultaneous up-regulation of Mrp2 and GST. GLP-2 also promoted an increase in cAMP levels as detected in homogenates of intestinal mucosa. Treatment of rats with 2',3'-dideoxyadenosine (DDA), a specific inhibitor of adenylyl cyclase, abolished the increase in cAMP levels and Mrp2 protein promoted by GLP-2, suggesting cAMP as a mediator of Mrp2 modulation. Increased expression of Mrp2 and cAMP levels in response to GLP-2 occurred not only at the tip but also at the middle region of the villus, where constitutive expression of Mrp2 is normally low. In conclusion, our study suggests a role for GLP-2 in the prevention of cell toxicity of the intestinal mucosa by increasing Mrp2 chemical barrier function.
Assuntos
Transportadores de Cassetes de Ligação de ATP/biossíntese , Peptídeo 2 Semelhante ao Glucagon/farmacologia , Mucosa Intestinal/efeitos dos fármacos , Jejuno/efeitos dos fármacos , Inibidores de Adenilil Ciclases , Animais , Western Blotting , Cromatografia Líquida de Alta Pressão , AMP Cíclico/metabolismo , Didesoxiadenosina/farmacologia , Enterócitos/efeitos dos fármacos , Enterócitos/enzimologia , Enterócitos/metabolismo , Enterócitos/patologia , Feminino , Imunofluorescência , Peptídeo 2 Semelhante ao Glucagon/fisiologia , Glutationa Transferase/biossíntese , Mucosa Intestinal/enzimologia , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Jejuno/enzimologia , Jejuno/metabolismo , Jejuno/patologia , Lactação/metabolismo , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Vascular smooth muscle cell proliferation and migration play an important role in the pathophysiology of several vascular diseases, including atherosclerosis. Prostaglandins that have been implicated in this process are synthesized by two isoforms of cyclooxygenase (COX), with the expression of the regulated COX-2 isoform increased in atherosclerotic plaques. Bradykinin (BK), a vasoactive peptide increased in inflammation, induces the formation of prostaglandins through specific receptor activation. We hypothesized that BK plays an important role in the regulation of COX-2, contributing to the increase in production of prostaglandins in vascular smooth muscle cells. Herein we examined the signaling pathways that participate in the BK regulation of COX-2 protein levels in primary cultured aortic vascular smooth muscle cells. We observed an increase in COX-2 protein levels induced by BK that was maximal at 24 h. This increase was blocked by a B2 kinin receptor antagonist but not a B1 receptor antagonist, suggesting that the B2 receptor is involved in this pathway. In addition, we conclude that the activation of mitogen-activated protein kinases p42/p44, protein kinase C, and nitric oxide synthase is necessary for the increase in COX-2 levels induced by BK because either of the specific inhibitors for these enzymes blocked the effect of BK. Using a similar approach, we further demonstrated that reactive oxygen species and cAMP were not mediators on this pathway. These results suggest that BK activates several intracellular pathways that act in combination to increase COX-2 protein levels. This study suggests a role for BK on the evolution of the atheromatous plaque by virtue of controlling the levels of COX-2.