Microscopic sperm head damage and abnormalities as heat stress indicators in Australian Merino rams (Ovis aries) in Northern Patagonia, Argentina / Danos e anormalidades microscópicas das cabeças espermáticas como indicadores de estresse por calor em carneiros Merino Australiano (Ovis aries) da Patagônia Norte, Argentina

In Northern Patagonia, the mating season starts on March 15th, when rams are submitted to summer temperatures. Exposure of rams to heat stress increases the prevalence of microscopic damage to spermatozoa, morphological abnormalities, and reductions in fertility. This study assesses the adaptive capabilities of six unshorn and six shorn Australian Merino rams, half of which were treated in a heat chamber for eight hours for five days, gradually reaching a temperature of up to 40 °C. Microscopic damage, abnormalities and ultramicroscopic alterations of the plasma membrane and the acrosome of sperm head were analysed. There were significant differences in the percentage of tailless spermatozoa and proximal cytoplasmic droplets between post-treatment periods. Temperature primarily affected the shorn rams and the sperm heads during spermiogenesis. Submicroscopic alterations were observed when the plasma membrane was present in the anterior segment. These alterations can be intact, waved, or dilated. When the plasma membrane was absent, the acrosome might be intact, dilated, and waved. In addition, the outer acrosomal membrane may completely lose its contents or have a nude nucleus. The plasma membrane assumes a waved shape as a result of the effect of temperature on the epididymis. According to this study, the tailless head, proximal cytoplasmic droplets, and the ultramicroscopic categories studied were robust indicators of semen heat stress. After ten weeks, the sperm head recovered its normal shape. Unshorn rams are better adapted to summer heat stress than shorn ones. Microscopy and transmission electron microscopy alterations have been shown to be excellent indicators of thermal stress in Australian Merino rams and may be useful tools to help sheep farmers choose when to begin the mating season, which will vary depending on the environmental conditions of the summer.(AU)

Na Patagônia Norte, os ovinos têm sua estação de acasalamento iniciada em 15 de março, portanto, ficam sujeitos às temperaturas do verão. A exposição de carneiros a estresse térmico aumenta a prevalência de danos microscópicos e anomalias morfológicas nos espermatozoides, que implica uma redução na fertilidade. Este trabalho avaliou a capacidade adaptativa de carneiros Merino Australiano com lã (N = 6) e tosquiados (N = 6): metade ficou ao ar livre e outra metade foi mantida em uma câmara climática por oito horas, durante cinco dias, chegando gradualmente a uma temperatura máxima de 40 °C. Foram analisados danos microscópicos, anormalidades e alterações ultramicroscópicas da membrana plasmática e do acrossoma da cabeça dos espermatozoides. Os resultados microscópicos confirmaram a existência de diferença significativa na porcentagem de espermatozoides sem cauda e com gota citoplasmática proximal, entre os ejaculados pós-tratamento. A temperatura afetou os carneiros tosquiados, principalmente a cabeça de seus espermatozoides, durante a espermatogênese. Alterações submicroscópicas foram observados na membrana plasmática quando ela estava presente no segmento anterior: quando não intacta, ficava ondulada ou dilatada. Quando a membrana plasmática estava ausente, o acrossoma podia se apresentar ondulado ou dilatado. Além disso, sob efeito do calor, a membrana acrossomal externa pode perder completamente seu conteúdo ou apresentar núcleo desnudo. A membrana plasmática assume uma forma ondulada pelo efeito da temperatura no epidídimo. Depois de dez semanas, a cabeça dos espermatozoides recuperou sua forma normal. Como demonstrado neste estudo, a cabeça sem cauda, as gotas citoplasmáticas proximais e as categorias ultramicroscópicas estudadas são indicadores do efeito do estresse térmico no sêmen, e os carneiros com maior cobertura de lã se adaptam melhor ao estresse por calor. Alterações de microscopia e de microscopia eletrônica de transmissão têm se mostrado excelentes indicadores de estresse por calor em carneiros Merino Australiano e podem ser ferramentas úteis para ajudar criadores de ovelhas a escolher quando começar a época de acasalamento, o que irá variar de acordo com as condições ambientais do verão.(AU)

Structural modification of the black bengal buck (Capra hircus) acrosome during post-testicular maturation of spermatozoa / Modificación estructural del acrosoma de la cabra black bengal (Capra hircus) durante la maduración de los espermatozoides

The present study was conducted to measure various biometric parameters of intact/normal acrosomes (AC) collected respectively from caput, corpus and cauda epididymis and vas deferens of Black Bengal buck. Giemsa stained acrosomes were measured after camera lucida drawings. Observations revealed dimensional characters of the acrosomal cap diminished gradually and significantly (p<0.01, p<0.05) during spermatozoa maturation phases in the different regions of the excurrent duct. Shape and size of the AC were also found to be influenced significantly (p<0.01, p<0.05) by the age and body weight of the animals. The structural modification along with decrease in the morphology of the AC reflected one of the maturational indexes of the male gametes in Black Bengal buck.

El presente estudio se realizó para medir diversos parámetros biométricos del acrosoma (AC) intacto/normal recogido desde la cabeza, cuerpo y cola del epidídimo y vas deferens de la Cabra Black Bengal. Los AC teñidos con Giemsa fueron medidos después de la captura con cámara lúcida. Las observaciones revelaron caracteres dimensionales del capuchón acrosomal que disminuyeron gradualmente y de manera significativa (p <0,01, p <0,05) durante fases de maduración espermatática en las diferentes regiones del conducto. La forma y tamaño del AC también fueron influenciados de manera significativa (p <0,01, p <0,05) por la edad y el peso corporal de los animales. La modificación estructural junto con los cambios morfológicos del AC refleja uno de los índices de maduración de los gametos masculinos de la Cabra Black Bengal.

Sperm ultrastructure of Mytella (Bivalvia) populations from distinct habitats along the northern coast of São Paulo State, Brazil

Ultrastructural analyses of bivalve spermatozoa are relevant in studies that aim to identify taxonomic traits for the purposes of discriminating species and conducting phylogenetic studies. In the present work, spermatozoa of mussel specimens of the genus Mytella, collected from two populations living in distinct habitats, were examined by electron microscopy. The objective was to identify sperm ultrastructural taxonomic traits that could be used to differentiate Mytella species. The specimens were from populations that live in intertidal zones on the southeast coast of Brazil, either buried in muddy-sand sediment or anchored to rocky substrates. The acrosomal vesicle was conical and long, the axial rod extended from the nucleus to the acrosome, the nucleus was an oblate spheroid with a condensed chromatin, the intermediate portion contained mitochondria encircling a pair of centrioles, and there was a single flagellum. The sperm was of a primitive type. The spermatozoon ultrastructure did not distinguish the specimens buried in muddy-sand sediment from those anchored to rocky substrates. The data suggest that the specimens analyzed, despite living in distinct habitats, belong to the same species, which conchological analyses identified as M. charruana. The presence of an axial rod in their sperm cells supports the inclusion of M. charruana in the subfamily Mytilinae.

An ultrastructural study of spermiogenesis in two species of Sitophilus (Coleoptera: Curculionidae)

The spermiogenesis of Sitophilus zeamais and Sitophilus oryzae, the maize and the rice weevil, respectively, was studied by light microscopy and scanning and transmission electron microscopy. Sitophilus spp. is the most widespread and destructive primary pest of stored cereals in the world. The spermiogenesis occurs within cysts. There are approximately 256 germ line cells per cyst. Inside each cysts, all the spermatids are in the same stage of maturation. The ultrastructure of the spermatozoa of S. zeamais and S. oryzae is similar to that described for other beetles. The head is formed by a three-layered acrosome with the perforatorium, the acrosomal vesicle, the extra-acrosomal layer and the nucleus. The flagellum has the typical axoneme formed by a 9+9+2 microtubules arrangement, two mitochondrial derivatives and two accessory bodies. The typical pattern for Curculionidae spermatozoa described here may provide useful information for future phylogenetic analysis of the superfamily Curculionoidea.

The sperm of Hylodinae species (Anura, Leptodactylidae): ultrastructural characteristics and their relevance to interspecific taxonomic relationships.

Hylodinae leptodactylids (sensu Lynch 1971) form a group of diurnal frogs, which is hypothesized on the basis of morphological traits to be the closest relatives of the dendrobatid frogs. Our study describes ultrastructural characteristics of sperm from three hylodine species (Hylodes phyllodes, Crossodactylus sp. n. and Megaelosia massarti) to reassess the intergeneric relationships within the Hylodinae, as well as the supposed relationship between the Hylodinae and Dendrobatidae. The ultrastructure of the sperm is very similar among the three species and is indicative of its conserved nature within the Hylodinae. The structure of the acrosomal complex was very similar to that of other leptodactylid species, to most of the remaining species included in the Bufonoidea lineage, and also to that observed in the dendrobatid species examined so far. Since such a structure has been considered a plesiomorphic trait, it contributes little to our understanding of the relationships between the Hylodinae and Dendrobatidae. The flagellar apparatus of Crossodactylus sp. n. is very similar to that of most leptodactylids. The sperm of Megaelosia massarti and Hylodes phyllodes display a distinctive condition in their axial and juxtaxonemal fibers. This distinctive flagellar condition expands the already known variability in sperm structure within the Leptodactylidae.

Protein detection in spermatids and spermatozoa of the butterfly Euptoieta hegesia (Lepidoptera)

This study was undertaken to detect protein components in both sperm types of the butterfly Euptoieta hegesia. These spermatozoa possess complex extracellular structures for which the composition and functional significance are still unclear. In the apyrene sperm head, the proteic cap presented an external ring and an internal dense content; basic proteins were detected only in external portions. In the tail, the paracrystalline core of mitochondrial derivatives and the axoneme are rich in proteins. The extratesticular spermatozoa are covered by a proteic coat, which presented two distinct layers. In eupyrene spermatozoa, acrosome and nucleus were negatively stained, probably because of their high compaction. In the tail, there is no paracrystalline core and the axoneme presented a very specific reaction for basic proteins. The lacinate and reticular appendages are composed of cylindrical sub-units and presented a light reaction to E-PTA and a strong reaction to tannic acid. A complex proteic coat also covers the extratesticular spermatozoa. We found similarities between both extratesticular coats, indicating a possible common origin. Both spermatozoon types are rich in proteins, especially the eupyrene appendages and the extratesticular coats. We believe that both coats are related to the sperm maturation and capacitation processes.

Sperm ultrastructure and spermatogenesis in the lizard, Tropidurus itambere

Spermatogenesis, with emphasis on spermiogenesis, is described for the lizard, Tropidurus itambere, using light microscopy, phase contrast and epifluorescence, as well as scanning and transmission electron microscopy. Cellular differentiation involves events of chromatin condensation, nuclear elongation and the formation of structural complexes, such as the acrosomal and axonemal ones. Other new characteristics, exclusive for this species, include various aspects of the subacrosomal granule, the insertion of the pro-acrosomal vesicle and the development of these structures to participate in the acrosomal complex. Radial projections occurjust above the nuclear shoulders, which have been recognized already from the beginning of cellular elongation. The development of the midpiece, the dense bodies, formation of the flagellum and elimination of residual cytoplasm result in the final characterization of the mature spermatozoon. Comparisons between Tropiduridae and other lizard families are made.

Morphological study of the spermatogenesis in the teleost Piaractus mesopotamicus

The spermatogenesis of Piaractus mesopotamicus was investigated under light and transmission electron microscopy. The specimens were captured from their natural environment (Rio Miranda and Rio Aquidauana, Pantanal Matogrossense, Brazil) during April and September. The results were compared with the spermatogenic data of specimens under captivity condition. In both conditions, P. mesopotamicus presented the typical spermatogenesis pattern of the teleost fishes, showing no significative differences. The spermatozoon was classified as type I, which has a globular head without acrosome, a short middle piece and a long tail constituted only by the flagellum. This type of spermatozoon is considered the basic type in fishes.

Efecto protector de la albúmina sobre las membranas del espermatozoide humano en la capacitación espermática in vitro / Albumin protective effect on human spermatozoa membranes during in vitro sperm capacitation

Se estudió el efecto de la preparación in vitro de semen sobre la estructura y sobrevida del espermatozoide humano, con el objeto de valuar si la albúmina protege las membranas durante la centrifugación. Se analizaron de manera cegada y aleatoria 12 muestras seminales de hombres fértiles a las cuales se les trató por tres métodos diferentes (swim-up, gradientes de albúmina, gradientes de percoll), para separar la fracción móvil de espermatozoides. Las membranas plasmática y acrosomal externa y la sustancia acrosomal de los espermatozoides fueron observadas al microscopio electrónico de transmisión en 50 espermatozoides por muestra y la viabilidad y movilidad progresiva se analizó después de incubación durante 24 horas a 37ºC. Se presenta evidencia que sugiere que la albúmina estabiliza la membrana acrosomal externa del espermatozoide humano e incrementa la viabilidad de esta célula

Immunodetection of acrosin during the acrosome reaction of hamster, guinea-pig and human spermatozoa

Mammalian sperm acrosomes contain a trypsin-like protease called acrosin which causes limited and specific hydrolysis of the extracellular matrix of the mammalian egg, the zona pellucida. Acrosin was localized on hamster, guinea-pig and human sperm using monoclonal and polyclonal antibodies to human acrosin labelled with colloidal gold. This was visualized directly with transmission electron microscopy, and with light and scanning microscopy after silver enhancement of the colloidal gold probe. Four distinct labelling patterns were found during capacitation and the acrosome reaction in hamster and guinea-pig spermatozoa, and three patterns were found in human spermatozoa. In the hamster, acrosin was not detected on the inner acrosomal surface after the completion of the acrosome reaction, thus correlating with the observation that hamster spermatozoa lose the ability to penetrate the zona after the acrosome reaction. With guinea-pig and human spermatozoa, acrosin was still detected after the completion of the acrosome reaction, thus correlating with the observation that acrosome reacted guinea-pig spermatozoa bind to and penetrate the zona pellucida

Solubilizacion de la membrana del espermatozoide humano con n-dodecil sarcosinato de sodio: I.accion del detergente / Solubilization of the membrane human spermatozoa: I. action of the detergent

Espermatozoides humanos fueron lavados con PBS y tratados com n-dodecil sarcosinato de sodio (Sarkosyl). El insoluble remanente estaba compuesto por cabezas, cuyas colas fueron cortadas a la altura del cuello o del segmento intermedio y cuyas membranas no sufrieron mayores danos, estando el acrosoma intacto y por pequenos corpusculos que podrian ser acrosomas aislados. Los resultados indican que la accion del detergente es de dos tipos claramente diferenciados: a)diseccion del espermatozoide con separacion de la cola y b)disolucion del mismo dejando el acrosoma intacto. El residuo conserva actividad inmunologica e inmunobiologica

Plasma membrane changes during epididymal transit in the guinea pig spermatozoa

El presente trabajo explora en un estudio comparado los cambios que ocurrem en la membrana plasmática (MP) de espermatozoides extraídos del caput (inmaduros) comparados con los extraídos de la región caudal del epidídimo (maduros) mediante técnicas convencionales de réplicas por criofractura, TEM, SEM y morfometría. Al final del tránsito epididimario la MP del segmento principal del acrosoma adquiere una trama hexagonal. El segmento ecuatorial y su borde serrado no muestran variaciones significativas. Otro importante cambio que ocurre también al final del tránsito epididimario es el aumento de la concentración de partículas intramembranosas en la región postacrosomal. El cuello del espermatozoide maduro se adelgaza probablemente debido a la migración de la gota citoplasmática. Esta gota presenta en la fase P de la mmembrana plasmática, hileras de partículas pequeñas que en ocasiones foram conglomerados densos. La MP de la pieza media también contiene particulas intramembranosas en al fase P que aparentemente están mejor el annulus. Se presume que estas dos zonas tienen de común la fusión de la mmembrana plasmática con las estructuras subyacentes, envoltura nuclear y anillo de Jensen respectivamente. La MP de la pieza principal contiene conglomerados de grandes partículas de forma rectangular y de función desconocida que junto al "zipper" no cambian durante el transito epididimario