RESUMO
High-fat diet is associated with hypercholesterolemia and seminal alterations in White New Zealand rabbits. We have previously reported disorders in the development of the manchette-acrosome complex during spermiogenesis and decreased testicular efficiency in hypercholesterolemic rabbits. On the other hand, olive oil incorporated into the diet improves cholesterolemia and semen parameters affected in hypercholesterolemic rabbits. In this paper, we report the recovery-with the addition of olive oil to diet-from the sub-cellular mechanisms involved in the shaping of the sperm cell and testicular efficiency altered in hypercholesterolemic rabbits. Using morphological (structural, ultra-structural and immuno-fluorescence techniques) and cell biology techniques, a reorganization of the manchette and related structures was observed when olive oil was added to the high-fat diet. Specifically, actin filaments, microtubules and lipid rafts-abnormally distributed in hypercholesterolemic rabbits-were recovered with dietary olive oil supplementation. The causes of the decline in sperm count were studied in the previous report and here in more detail. These were attributed to the decrease in the efficiency index and also to the increase in the apoptotic percentage in testis from animals under the high-fat diet. Surprisingly, the addition of olive oil to the diet avoided the sub-cellular, efficiency and apoptosis changes observed in hypercholesterolemic rabbits. This paper reports the positive effects of the olive oil addition to the diet in the recovery of testicular efficiency and normal sperm shaping, mechanisms altered by hypercholesterolemia.
Assuntos
Acrossomo/efeitos dos fármacos , Dieta Hiperlipídica/efeitos adversos , Hipercolesterolemia/tratamento farmacológico , Azeite de Oliva/administração & dosagem , Doenças Testiculares/prevenção & controle , Acrossomo/patologia , Animais , Modelos Animais de Doenças , Hipercolesterolemia/induzido quimicamente , Hipercolesterolemia/complicações , Infertilidade Masculina/etiologia , Infertilidade Masculina/prevenção & controle , Masculino , Microdomínios da Membrana/efeitos dos fármacos , Azeite de Oliva/farmacologia , Coelhos , Contagem de Espermatozoides , Espermatogênese/efeitos dos fármacos , Doenças Testiculares/etiologia , TestículoRESUMO
Fat increment (0.05% cholesterol, chol) in standard diet promoted a significant increase in serum and sperm membrane chol, which ultimately altered membrane-coupled sperm specific functions: osmotic resistance, acrosomal reaction, and sperm capacitation in White New Zealand rabbits. These changes were also associated with a reduction in motility percentage and appearance of abnormal sperm morphology. The present study was carried out to evaluate the effect of dietary olive oil (OO, 7% v/w) administration to several male hypercholesterolemic rabbits (hypercholesterolemic rabbits, HCR) with altered fertility parameters. These HCR males were achieved by feeding normal rabbits with a high-fat diet (0.05% chol). HCR were associated with a modest non-significant increase in body weight (standard diet, 4.08±0.17 Kg, versus high-fat diet, 4.37±0.24 Kg). Hypercholesterolemic rabbits presented a marked decrease in semen volume, sperm cell count, and percentage of sperm motility, associated with a significant increase in sperm cell abnormalities. Moreover, sperm capacitation measured by the characteristic phosphorylated protein pattern in and induced acrosomal reaction were also altered suggesting sperm dysfunction. However, the administration of OO (for 16 weeks) to rabbits that were fed with 50% of the high-fat diet normalized serum chol. Curiously, OO supply succeeded to attenuate the seminal and sperm alterations observed in HCR group. Administration of OO alone did not cause any significant changes in above mentioned parameters. These data suggest that OO administration to HCR male rabbits recovers the loss of semen quality and sperm functionality.
Assuntos
Hipercolesterolemia/prevenção & controle , Óleos de Plantas/farmacologia , Análise do Sêmen , Espermatozoides/efeitos dos fármacos , Reação Acrossômica/efeitos dos fármacos , Animais , Colesterol na Dieta/administração & dosagem , Colesterol na Dieta/efeitos adversos , Dieta Hiperlipídica/efeitos adversos , Gorduras Insaturadas na Dieta/administração & dosagem , Gorduras Insaturadas na Dieta/farmacologia , Eletroforese em Gel de Poliacrilamida , Hipercolesterolemia/etiologia , Hipercolesterolemia/fisiopatologia , Masculino , Azeite de Oliva , Fosforilação/efeitos dos fármacos , Fosfotirosina/metabolismo , Óleos de Plantas/administração & dosagem , Coelhos , Capacitação Espermática/efeitos dos fármacos , Contagem de Espermatozoides , Motilidade dos Espermatozoides/efeitos dos fármacos , Espermatozoides/fisiologiaRESUMO
Mammalian sperm proteins undergo thiol group (SH) oxidation to form disulfides bonds (SS) as they travel through the epididymis during cell maturation. Disulfide bonds are involved in chromatin condensation and tail organelle stabilization. In this work, we used a fluorescent thiol-selective labeling agent, monobromobimane (mBBr), to study the protein thiol status of rat sperm during maturation. Fluorescence signal decrease along the epididymal trip, more evidently in the head, but also in the tail, indicates that both sub cellular regions participate in the thiol changes. The sources of the fluorescence signal are sulfhydryls sperm proteins labeled by mBBr (mBBr-spp). Initial attempts to identify the mBBr-spp labeled were detected in the initial-caput, but not in the distal cauda-segment of the epididymis in sodium dodecyl sulfate (SDS)-PAGE analysis. This phenomenon could be due to protein resistance to solubilization. For this reason, disulfide bond reduction was accomplished by sodium dodecyl sulfate plus dithiothreitol treatment to recover the mBBr signal in SDS-PAGE. Under this protocol, a major 27 kDa protein band displays a strong signal. Protein identification by mass spectrometry and sequence database searching correlated this protein with the outer dense fiber 1 (ODF1). The mBBr specifically bound to N-terminal domain cysteine of ODF1. The mBBr reduces rat sperm motility, quantitatively and qualitatively, and the effects are dose dependent, without significantly increasing the percentage of dead sperm. Thus, we found that ODF1 is highly responsible for mBBr fluorescence detection in the sperm tail, and the motility inhibition by the fluorescence marker indicates that ODF1 N-terminal domain are related to sperm motility. © 2011 Wiley-Liss, Inc.
Assuntos
Proteínas de Choque Térmico/metabolismo , Motilidade dos Espermatozoides/fisiologia , Cauda do Espermatozoide/metabolismo , Animais , Compostos Bicíclicos com Pontes/farmacologia , Relação Dose-Resposta a Droga , Corantes Fluorescentes/farmacologia , Masculino , Oxirredução/efeitos dos fármacos , Estrutura Terciária de Proteína , Ratos , Ratos Wistar , Motilidade dos Espermatozoides/efeitos dos fármacosRESUMO
Hypercholesterolemia represents a high risk factor for frequent diseases and it has also been associated with poor semen quality that may lead to male infertility. The aim of this study was to analyze semen and sperm function in diet-induced hypercholesterolemic rabbits. Twelve adult White New Zealand male rabbits were fed ad libitum a control diet or a diet supplemented with 0.05% cholesterol. Rabbits under cholesterol-enriched diet significantly increased total cholesterol level in the serum. Semen examination revealed a significant reduction in semen volume and sperm motility in hypercholesterolemic rabbits (HCR). Sperm cell morphology was seriously affected, displaying primarily a "folded head"-head fold along the major axe-, and the presence of cytoplasmic droplet on sperm flagellum. Cholesterol was particularly increased in acrosomal region when detected by filipin probe. The rise in cholesterol concentration in sperm cells was determined quantitatively by Gas chromatographic-mass spectrometric analyses. We also found a reduction of protein tyrosine phosphorylation in sperm incubated under capacitating conditions from HCR. Interestingly, the addition of Protein Kinase A pathway activators -dibutyryl-cyclic AMP and iso-butylmethylxanthine- to the medium restored sperm capacitation. Finally, it was also reported a significant decrease in the percentage of reacted sperm in the presence of progesterone. In conclusion, our data showed that diet-induced hypercholesterolemia adversely affects semen quality and sperm motility, capacitation and acrosomal reaction in rabbits; probably due to an increase in cellular cholesterol content that alters membrane related events.
Assuntos
Hipercolesterolemia/fisiopatologia , Infertilidade Masculina/fisiopatologia , Espermatozoides/fisiologia , Reação Acrossômica , Animais , Cromatografia Gasosa-Espectrometria de Massas , Masculino , Fosforilação , Coelhos , Capacitação Espermática , Motilidade dos Espermatozoides , Espermatozoides/metabolismo , Tirosina/metabolismoRESUMO
It has been shown that seasonal changes, especially in arid areas have a large influence on gonadal changes of the species that inhabit these areas. We studied a wild hystricomorph Microcavia australis in its natural habitat in the arid Andes Mountains. Sampling of adult males was carried out every 2 months. After autopsy, testes and epididymides were weighed and processed to obtain histological samples. Testes were analyzed with a microscope to measure seminiferous tubule area and diameter for each sampled month. Epididymides were used to determine spermatozoon storage in the cauda region. Results illustrate morphological changes in the testis and epididymis along the year. A high output of sperm cells was detected from middle winter to middle summer and a complete shutdown of spermatogenesis at the end of summer. The initiation of testicular activity coincided with month with the shortest day length, in dry season and very low temperature. On the other hand, gonadal regression started in the middle of summer with long day length, in the wet season, and high temperatures. Rainfall, temperature, and day length seem to be important for the testis cycle. We suggest that photoperiod could be a good predictor for an oncoming period suitable for breeding, and males may probably use it as a signal to regulate gonadal activity.
Assuntos
Epididimo/anatomia & histologia , Roedores/anatomia & histologia , Espermatogênese/fisiologia , Testículo/anatomia & histologia , Animais , Argentina , Clima Desértico , Epididimo/fisiologia , Histocitoquímica , Masculino , Tamanho do Órgão/fisiologia , Roedores/fisiologia , Estações do Ano , Estatísticas não Paramétricas , Testículo/fisiologiaRESUMO
In many mammalian species, sperm associate as a consequence of the epididymal transit. From the classic Rouleaux in guinea pig to the most recent work in mouse and echidna, authors have focused mainly on a detailed morphological description of this phenomenon. Some of these articles have also begun to describe the nature of the material present between sperm heads. Here, we try to better understand the factor/s involved in rat sperm association (Rosette). Based on previous work describing the appearance of Rosettes in the distal segments of the rat epididymis, we consider that sperm during their transit must be in contact with factor/s present in the caudal lumen in order to associate with each other. By an in vitro sperm re-associating assay, we try to determine the in vivo phenomenon observed in the lumen. The assay consists of co-incubating non-associated sperm with several protein fractions obtained from epididymal caudal fluid. After establishing the most active fraction, the proteins were characterized by MALDI-TOF mass spectrometry. Among the proteins we found two members of the serine protease inhibitors family; an alpha-1 antitrypsin and a new protein with an alpha-1 antitrypsin like domain which includes a sequence compatible with the serpins' reactive center loop. These serpins may play a role in the assembly/disassembly process of Rosettes by modulating lumenal protease activity. Finally, a biochemical-morphological model which explains the sperm-proteases interaction was proposed.