RESUMO
Several studies have recently investigated the role of leptin, the adipocyte-secreted hormone, in the growth and reproduction of rodents, humans, and domestic animals. The present study was designed to explore the expression of leptin and its receptor in pig spermatozoa. Successful Western blot evidenced a 16 kDa band for leptin and six isoforms, ranging from 120 to 40 kDa, for the leptin receptor. Both leptin and leptin receptor were interestingly located at sperm acrosomal level, suggesting their involvement in the oocyte fertilization events. In fact, both capacitation indexes and acrosin activity were enhanced by leptin, and these effects were reduced by the anti-leptin receptor antibody. Afterwards, we investigated the main transduction pathways regulated by the hormone. Our results showed that, in pig sperm, leptin can trigger the signal transducer and activator of transcription 3, a classical component of cytokine signal transduction pathways, whose expression has not been previously reported in male gamete; in addition it was found constitutively activated. Besides, leptin was able to induce the activation of phosphatidylinositol phosphate kinase 3 and MAP kinase pathways as well as of BCL2, a known antiapoptotic protein. These data address to a role of leptin and its receptor on pig sperm survival. The presence of leptin and its receptor in pig sperm suggests that they, through an autocrine short loop, may induce signal transduction and molecular changes associated with sperm capacitation and survival.
Assuntos
Acrossomo/metabolismo , Leptina/fisiologia , Isoformas de Proteínas/fisiologia , Receptores para Leptina/metabolismo , Transdução de Sinais/fisiologia , Capacitação Espermática/fisiologia , Sus scrofa/fisiologia , Animais , Western Blotting/métodos , Sobrevivência Celular , Leptina/análise , Leptina/genética , Masculino , Proteínas Quinases Ativadas por Mitógeno/análise , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosfatidilinositol 3-Quinases/análise , Fosfatidilinositol 3-Quinases/metabolismo , Isoformas de Proteínas/análise , Isoformas de Proteínas/genética , Proteínas Proto-Oncogênicas c-akt/análise , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores para Leptina/análise , Receptores para Leptina/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Fator de Transcrição STAT3/metabolismo , Proteína de Morte Celular Associada a bcl/análise , Proteína de Morte Celular Associada a bcl/metabolismoRESUMO
BACKGROUND: Results from mice lacking the androgen receptor (AR) showed that it is critical for the proper development and function of the testes. The aim of this study was to investigate whether a functional AR is present in human sperm. METHODS: The expression of AR and its effects on sperm were evaluated by RT-PCR, Western Blot, Immunocytochemistry, PI3Kinase and DNA laddering assays. RESULTS: We showed in human sperm that AR is located at the head region. Dihydrotestosterone (DHT), in a dose-dependent manner, leads to the rapid phosphorylation of the AR on tyrosine, serine and threonine residues and this effect was reduced by the AR antagonist hydroxyflutamide (OH-Flut). The effects of AR were evaluated on the phosphoinositide-3 kinase/protein kinase B (PI3K/AKT) pathway. Specifically, 0.1 and 1 nM DHT stimulated PI3K activity, whereas 10 nM DHT decreased PI3K activity and levels of p-AKT S473 and p-AKT T308, p-BCL2, and enhanced phosphatase and tensin homologue (PTEN) phosphorylation. In addition, 10 nM DHT was able to induce the cleavage of caspases 8, 9 and 3 and cause DNA laddering, and these effects were reversed either by casodex or OHFlut. By using wortmannin, a specific PI3K inhibitor, the cleavage of caspase 3 was reproduced, confirming that in sperm the PI3K/AKT pathway is involved in caspase activation. CONCLUSIONS: Human sperm express a functional AR that have the ability to modulate the PI3K/AKT pathway, on the basis of androgen concentration.