RESUMO
Progesterone is a physiological agonist for mammalian sperm, modulating its flagellar movement and facilitating the acrosome reaction. To study the initial action of progesterone, we developed a caged analog with a photosensitive group: nitrophenylethanediol, at position 20. Using this compound combined with stroboscopic illumination, we performed Ca(2)(+) imaging of human spermatozoa and analyzed the effects of progesterone on the intracellular Ca(2)(+) concentration ([Ca(2)(+)](i)) of beating flagella for the first time. We observed a transient [Ca(2)(+)](i) increase in the head and the flagellum upon photolysis of the caged progesterone and an increase in flagellar curvature. Detailed kinetic analysis revealed that progesterone elicits an increase in the [Ca(2)(+)](i) immediately in the flagellum (mid-piece and principal piece), thereafter in the head with a short time lag. This observation is different from the progesterone-induced Ca(2)(+) mobilization in mouse spermatozoa, where the Ca(2)(+) rise initiates at the base of the sperm head. Our finding is mostly consistent with the recent discovery that progesterone activates CatSper channels in human spermatozoa, but not in mouse spermatozoa.
Assuntos
Cálcio/análise , Progesterona/análogos & derivados , Progesterona/farmacologia , Cauda do Espermatozoide/efeitos dos fármacos , Espermatozoides/efeitos dos fármacos , Canais de Cálcio/efeitos dos fármacos , Corantes Fluorescentes , Humanos , Masculino , Nitrobenzenos/química , Fotólise , Progesterona/química , Espectrometria de Fluorescência , Cabeça do Espermatozoide/química , Cabeça do Espermatozoide/efeitos dos fármacos , Cauda do Espermatozoide/química , Cauda do Espermatozoide/fisiologia , Espermatozoides/química , Espermatozoides/fisiologiaRESUMO
Examination of the type and frequency of damage to the head of spermatozoa using electron microscopy can be used to evaluate the quality of differently treated sperm. This report describes a systematic approach based on 29 morphological categories of sperm heads assessed from discrete regions in raw, chilled and frozen-thawed spermatozoa. Injury occurred principally at the plasma membrane and could be present or absent in all regions. In the anterior segment, when the plasma membrane is present, it can be intact, dilated, very dilated, disrupted, or contain vesicles characteristic of acrosomal reaction-like capacitation changes. When the plasma membrane is absent, the acrosome may be intact, exhibit a complete loss of contents, or retain some contents of the apical ridge and present a very dilated outer acrosomal membrane. The plasma membrane in the equatorial segment and the boundary between regions can be intact, dilated, very dilated or disrupted. The post-acrosomal plasma membrane is classified as intact, dilated or very dilated, whereas the dense lamina is intact, dilated or fragmented. The morphology of the heads most frequently observed in chilled spermatozoa consists of anterior and equatorial segments with a dilated, or dilated and disrupted plasma membrane; a boundary between regions with an intact and dilated plasma membrane; and a post-acrosomal region with an intact plasma membrane and dense lamina, both dilated. In frozen-thawed spermatozoa, the morphology of the heads is more frequently characterised by no plasma membrane and an acrosome showing complete or some loss of contents in the apical ridge and very dilated outer acrosomal membrane, presenting mostly dilated and fragmented dense lamina in the post-acrosomal region. These findings are consistent with the conclusion that the freezing process produces an increase in the degree of damage to the cells when they are subjected to increasing degrees of cold shock. There are still difficulties in developing a good diluent and process for preserving the plasma membrane in ram spermatozoa. This systematisation, using different categories, allows characterisation of multiple transmission electron microscopy images. Thus, the different changes observed due to cryopreservation may be correlated.
Assuntos
Membrana Celular/ultraestrutura , Criopreservação , Cabeça do Espermatozoide/ultraestrutura , Animais , Membrana Celular/efeitos dos fármacos , Crioprotetores/farmacologia , Congelamento , Processamento de Imagem Assistida por Computador , Masculino , Microscopia Eletrônica de Transmissão , Carneiro Doméstico , Cabeça do Espermatozoide/efeitos dos fármacosRESUMO
Llama semen is characterized by great structural viscosity and minimal sperm progressive motility. These characteristics, inherent to South American Camelid ejaculates, have slowed down the development of assisted reproductive techniques in these species. The aim of the present research was to evaluate the effect of different combinations of dilutions and incubation time with H-TALP-BSA medium, with and without adding 0.1% collagenase, on the qualitative and quantitative semen characteristics, for its use in assisted fertility techniques. Ejaculates (n=8; r=3) were obtained using electroejaculation. Each ejaculate was evaluated and then split into four aliquots. Two of these were diluted 4:1 and 8:1 in 0.1% collagenase in H-TALP-BSA (treatments 1 and 3) and the other two 4:1 and 8:1 in H-TALP-BSA without collagenase (treatments 2 and 4). Treatments 1 and 2 were incubated 4 min at 37 degrees C while treatments 3 and 4 were incubated 8 min. All aliquots were centrifuged at 800 x g for 4 min immediately after incubation. Supernatants were pipetted to observe thread formation and pellets were re-diluted in H-TALP-BSA. Supernatants from samples treated with collagenase did not form a thread when pipetted, while the ones from samples that were not treated with the enzyme did. Only semen samples treated with collagenase showed progressive sperm motility, with averages over 40%. There were no significant differences (P>0.05) for the percentage of live spermatozoa and for the percentage of detached heads between raw and treated semen samples. Percentages of spermatozoa with functional membranes were significantly higher (P< or =0.05) in samples treated with collagenase than in raw semen and in samples incubated without collagenase. These results show that treating semen with 0.1% collagenase in H-TALP-BSA improves semen rheological properties while facilitates the separation of spermatozoa from seminal plasma in llama; it also promotes sperm progressive motility, while maintaining sperm membrane functionality and integrity. Consequently, this protocol could be used for in vitro llama embryo production with ejaculated spermatozoa.
Assuntos
Camelídeos Americanos/fisiologia , Colagenases/farmacologia , Sêmen/efeitos dos fármacos , Animais , Relógios Biológicos/efeitos dos fármacos , Ejaculação , Masculino , Reologia , Sêmen/citologia , Sêmen/fisiologia , Análise do Sêmen , Cabeça do Espermatozoide/efeitos dos fármacos , Cabeça do Espermatozoide/fisiologia , Fatores de TempoRESUMO
Atomic force microscopy (AFM) has been employed to examine morphological and topographical changes caused by human immunodeficiency virus (HIV) and the effects of highly active antiretroviral therapy (HAART) on spermatozoon of HIV infected patients. This powerful technique has allowed us to visualize morphological alterations present in the spermatozoa of patients either with or without treatment. In addition to this, even the minute details, such as viral particles, located on the membrane of the spermatozoa, and the merging of such particles on the surface of the spermatozoa were detected with precision. The most important aspect is that AFM, unlike electron microscopy, permits to image virions in their nearly natural environment. Excess of damage of spermatozoon is due to the chemicals involved in HAART rather than the damage made by virus.
Assuntos
Terapia Antirretroviral de Alta Atividade/efeitos adversos , Infecções por HIV/tratamento farmacológico , HIV/isolamento & purificação , Microscopia de Força Atômica/métodos , Cabeça do Espermatozoide/efeitos dos fármacos , Cauda do Espermatozoide/efeitos dos fármacos , HIV/ultraestrutura , Infecções por HIV/patologia , Humanos , Masculino , Cabeça do Espermatozoide/ultraestrutura , Cabeça do Espermatozoide/virologia , Motilidade dos Espermatozoides/efeitos dos fármacos , Cauda do Espermatozoide/ultraestrutura , Cauda do Espermatozoide/virologia , Vírion/isolamento & purificação , Vírion/ultraestruturaRESUMO
Los espermatozoides presentan estructuras glicoesfingolipídicas asociadas a la membrana que son propias de los antígenos ABH. Los glúcidos que conforman estas moléculas participan en la adhesión, reconocimiento e inhibición del contacto celular. Por esta razón los antígenos de grupo sanguíneo podrían tener relevancia en los mecanismos moleculares del proceso reproductivo. El objetivo de este trabajo fue evaluar si exite asociación entre los tests de integridad de la membrana espermática y la alteración en la expresión de los glicoesfingolípidos ABH. Se seleccionaron 50 pacientes infértiles cuyas muestras de semen fueron procesadas según normas OMS. La expresión ABH en los espermatozoides se estudió por técnicas inmunohematológicas y se clasificó según estuviera disminuida o ausente (grupo 1), o normal (grupo 2). La integridad de la membrana se evaluó con Test de Burgos y De Paola y Test Hiposmótico. Se efectuó la comparación de los dos grupos respecto de ambos test de viabilidad espermática. El análisis estadistico demostró que: 1) Existe diferencia significativa en la variable porcentaje de muertos (Burgos De Paola) para los dos grupos (p<0.001). 2) No existe diferencia significativa en la variable porcentaje de hinchados (Test Hiposmótico) para los dos grupos. Sobre la base de los resultados obtenidos podemos concluir que existe asociación entre expresión ABH disminuida e integridad de la menbrana del espermatozoide, principalmente a nivel de la cabeza. Lo que nos lleva a pensar que los glicoesfingolípidos ABH, se localizan preferentemente en esta zona del espermatozoide, involucrada fundamentalmente en la interacción con el ovocito. Nos proponemos ampliar la población en estudio e investigar el gen responsable de la estructura final de los antígenos del ABO tratando de profundizar en las alteraciones del plasmalema a nivel molecular
Assuntos
Humanos , Masculino , Glicoesfingolipídeos/deficiência , Espermatozoides/patologia , Antígenos , Interações Espermatozoide-Óvulo/imunologia , Cabeça do Espermatozoide/efeitos dos fármacosRESUMO
Los espermatozoides presentan estructuras glicoesfingolipídicas asociadas a la membrana que son propias de los antígenos ABH. Los glúcidos que conforman estas moléculas participan en la adhesión, reconocimiento e inhibición del contacto celular. Por esta razón los antígenos de grupo sanguíneo podrían tener relevancia en los mecanismos moleculares del proceso reproductivo. El objetivo de este trabajo fue evaluar si exite asociación entre los tests de integridad de la membrana espermática y la alteración en la expresión de los glicoesfingolípidos ABH. Se seleccionaron 50 pacientes infértiles cuyas muestras de semen fueron procesadas según normas OMS. La expresión ABH en los espermatozoides se estudió por técnicas inmunohematológicas y se clasificó según estuviera disminuida o ausente (grupo 1), o normal (grupo 2). La integridad de la membrana se evaluó con Test de Burgos y De Paola y Test Hiposmótico. Se efectuó la comparación de los dos grupos respecto de ambos test de viabilidad espermática. El análisis estadistico demostró que: 1) Existe diferencia significativa en la variable porcentaje de muertos (Burgos De Paola) para los dos grupos (p<0.001). 2) No existe diferencia significativa en la variable porcentaje de hinchados (Test Hiposmótico) para los dos grupos. Sobre la base de los resultados obtenidos podemos concluir que existe asociación entre expresión ABH disminuida e integridad de la menbrana del espermatozoide, principalmente a nivel de la cabeza. Lo que nos lleva a pensar que los glicoesfingolípidos ABH, se localizan preferentemente en esta zona del espermatozoide, involucrada fundamentalmente en la interacción con el ovocito. Nos proponemos ampliar la población en estudio e investigar el gen responsable de la estructura final de los antígenos del ABO tratando de profundizar en las alteraciones del plasmalema a nivel molecular (AU)
Assuntos
Humanos , Masculino , Espermatozoides/patologia , Glicoesfingolipídeos/deficiência , Antígenos/diagnóstico , Interações Espermatozoide-Óvulo/imunologia , Cabeça do Espermatozoide/efeitos dos fármacosRESUMO
Se realizó la evaluación genotóxica de un extracto fluido de Indigofera suffructicosa Mill (añil cimarrón), ante la posibilidad de que el extracto indujera daño en las células germinales, mediante el ensayo de anomalías en la cabeza de los espermatozoides. Se utilizaron animales de la línea OF-1, a los cuales se les administraron por vía intragástrica 4 dosis, la dosis máxima fue de 1,857 mg/kg de peso corporal, que representa 80 porciento de la LD50. No se contraron diferencias significativas en el porcentaje de anomalías morfológicas de los espermatozoides, lo cual implica que no hubo actividad genotóxica