Micromorphological and ultrastructural description of spermatozoa from squirrel monkeys (Saimiri collinsi Osgood, 1916).

Saimiri collinsi is used as an animal model in biotechnology research for conservation of species from the genus Saimiri. However, the development of biotechnologies depends on a proper knowledge of the sperm morphology to understand the basic aspects of sperm physiology, as potential male fertility depends on different cellular sperm structures. With this purpose, this study characterized the micromorphological and ultrastructural characteristics of squirrel monkeys (Saimiri collinsi) sperm using scanning electron microscopy (SEM) and transmission electron microscopy (TEM). SEM electromyography revealed that a normal Saimiri collinsi sperm measures 71.7 ± 0.7 µm with lateral tail insertion, a paddle-shaped flattened head and an acrosome occupying most of the head. TEM also showed that the middle piece is characterized by a central 9 + 2 microtubule axoneme surrounded by nine dense fibres, and that the mitochondria were juxtaposed, forming the mitochondrial sheath. Here we provide the first micromorphological and ultrastructure description of S. collinsi sperm.

Sperm swimming behaviors are correlated with sperm haploid genetic variability in the Mexican tetra, Astyanax mexicanus.

The diploid genotypes of males are widely thought to determine sperm phenotypes, yet recent work shows that the haploid genetics of the individual sperm cell also contributes significantly. We tested seven sperm phenotypes, flagellar length and six behaviors, looking for correlations between genetic and phenotypic variability. While flagellar length appears to be controlled by the diploid genotype of the source, variation in three of the behavioral phenotypes, linearity, wobble, and progression are significantly correlated with the heterozygosity of the male producer. Because males that are more genetically variable produce a sperm set that is more diverse in its haploid genotypes, we suggest that the correlations may reflect significant haploid genetic control of sperm swimming behaviors.

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)

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)

Are there intracellular Ca2+ oscillations correlated with flagellar beating in human sperm? A three vs. two-dimensional analysis.

STUDY QUESTION: Are there intracellular Ca2+ ([Ca2+]i) oscillations correlated with flagellar beating in human sperm? SUMMARY ANSWER: The results reveal statistically significant [Ca2+]i oscillations that are correlated with the human sperm flagellar beating frequency, when measured in three-dimensions (3D). WHAT IS KNOWN ALREADY: Fast [Ca2+]i oscillations that are correlated to the beating flagellar frequency of cells swimming in a restricted volume have been detected in hamster sperm. To date, such findings have not been confirmed in any other mammalian sperm species. An important question that has remained regarding these observations is whether the fast [Ca2+]i oscillations are real or might they be due to remaining defocusing effects of the Z component arising from the 3D beating of the flagella. STUDY DESIGN, SIZE, DURATION: Healthy donors whose semen samples fulfill the WHO criteria between the age of 18-28 were selected. Cells from at least six different donors were utilized for analysis. Approximately the same number of experimental and control cells were analyzed. PARTICIPANTS/MATERIALS, SETTING, METHODS: Motile cells were obtained by the swim-up technique and were loaded with Fluo-4 (Ca2+ sensitive dye) or with Calcein (Ca2+ insensitive dye). Ni2+ was used as a non-specific plasma membrane Ca2+ channel blocker. Fluorescence data and flagella position were acquired in 3D. Each cell was recorded for up to 5.6 s within a depth of 16 microns with a high speed camera (coupled to an image intensifier) acquiring at a rate of 3000 frames per second, while an oscillating objective vibrated at 90 Hz via a piezoelectric device. From these samples, eight experimental and nine control sperm cells were analyzed in both 2D and 3D. MAIN RESULTS AND THE ROLE OF CHANCE: We have implemented a new system that allows [Ca2+]i measurements of the human sperm flagellum beating in 3D. These measurements reveal statistically significant [Ca2+]i oscillations that correlate with the flagellar beating frequency. These oscillations may arise from intracellular sources and/or Ca2+ transporters, as they were insensitive to external Ni2+, a non-specific plasma membrane Ca2+ channel blocker. LARGE SCALE DATA: N/A. LIMITATIONS REASONS FOR CAUTION: Analysis in 3D needs a very fast image acquisition rate to correctly sample a volume containing swimming sperm. This condition requires a very short exposure time per image making it necessary to use an image intensifier which also increases noise. The lengthy analysis time required to obtain reliable results limited the number of cells that could be analyzed. WIDER IMPLICATIONS OF THE FINDINGS: The possibility of recording flagellar [Ca2+]i oscillations described here may open a new avenue to better understand ciliary and flagellar beating that are fundamental for mucociliary clearance, oocyte transport, fertilization, cerebrospinal fluid pressure regulation and developmental left-right symmetry breaking in the embryonic node. STUDY FUNDING AND COMPETING INTEREST(S): This work was supported by Consejo Nacional de Ciencia y Tecnología (CONACyT) (grants 253952 to G.C.; 156667 to F.M.M. and Fronteras 71 39908-Q to A.D. and Post-doctoral scholarships 366844 to P.H.-H. and 291028 to F.M.) and the Dirección General de Asuntos del Personal Académico of the Universidad Nacional Autónoma de México (DGAPA-UNAM) (grants CJIC/CTIC/4898/2016 to F.M. and IN205516 to A.D.). There are no conflicts of interest to declare.

Morphometry, Morphology and Ultrastructure of Ring-tailed Coati Sperm (Nasua nasua Linnaeus, 1766).

The ring-tailed coati (Nasua nasua) is a procyonid whose population is in sharp decline. Therefore, studies are needed to better understand the reproduction of this animal. For this reason, this study aimed to evaluate the morphology, morphometry and sperm ultrastructure of ring-tailed coati sperm. Four captive adult males were used for this study. Slides stained with Bengal Rose were used for the morphometric and morphologic analyses. The length and width of the head were measured, as well as the length of the midpiece and tail and the total length of the sperm. Scanning electron microscopy and transmission electron microscopy were used for the ultrastructural analyses. The most obvious morphological abnormalities observed were coiled tails (6.1 ± 8.7%) and the lack of acrosomes (5.4 ± 4.4%). Regarding the morphometry, the measurements of the head (length × width), midpiece (length) and tail (length) were (mean ± SD) 6.2 ± 0.4 × 8.1 ± 0.6 µm, 14.1 ± 0.5 and 63.9 ± 4.1 µm, respectively, and the total length of the sperm was 86.1 ± 4.3 µm. Through electron microscopy, the presence of electron-lucent points in the nucleus and the presence of approximately 55 mitochondrial spirals in the midpiece were identified. The data obtained in this study provide detailed information on the sperm characteristics of coatis and may inform future research on germplasm conservation, both for this species and other threatened procyonids.

Ultrastructural features of spermatogenesis in Melanorivulus punctatus (Cyprinodontiformes: Rivulidae).

Fish belonging to the family Rivulidae possess one of the most complex reproductive systems. Rivulus, a genus of freshwater fish in the Rivulidae family, was recently reclassified into five genera, including Melanorivulus. Its type species, M. punctatus, is widely distributed and probably represents a species complex. The ultrastructure of sperm has been broadly used in systematics, and we hereby describe the ultrastructural features of spermatogenesis in M. punctatus. Ten M. punctatus males were collected from the reservoir of Parque Estadual da Quineira, municipality of Chapada dos Guimarães, Mato Grosso, Brazil, and prepared for analysis by light microscopy and transmission electron microscopy. M. punctatus undergoes cystic spermatogenesis. Its cysts consist of groups of germ cells that are in synchronous development and are surrounded by cytoplasmic projections of Sertoli cells. With the breakdown of the cysts, the spermatozoa are released and their maturation is completed in the duct, where part of the cytoplasmic material is discarded through the vesicles. The mature spermatozoon is characterized by a spherical head with homogeneously condensed chromatin, a symmetric midpiece consisting of a pair of perpendicular centrioles, a ring of mitochondria, several vesicles, and one flagellum medial to the nucleus. Early stages of spermatogenesis show no peculiarities; however, in spermiogenesis, we observed that the spermatids remain interconnected by cytoplasmic bridges and have pockets of residual cytoplasm. The sperm is of the aquasperm type and is similar to that observed in the members of the family Rivulidae. The spermatozoa have a single flagellum that consists of a classic axoneme (9+2), as found in most groups of fish, despite the lateral extensions.

Analysis of spermiogenesis like a tool in the study of the triatomines of the Brasiliensis subcomplex.

The specific identification and systematic of triatomines have been based fundamentally on morphological observations. These organisms are classified into complexes and specific subcomplexes, principally for morphological parameters and geographical disposition. The use of cytogenetic analyzes has been represented as a tool in systematic and taxonomy of triatomines. Thus, the present work, through the analysis of spermiogenesis, aims to characterize this stage of spermatogenesis in triatomines little studied, and especially to compare it among the species Triatoma lenti and T. sherlocki, to assist in the diagnosis of differentiation of these insects. The presence of the heteropyknotic corpuscle is shown as a diagnostic tool to differentiate T. sherlocki and T. lenti, since it is absent in T. lenti. The analysis of the spermiogenesis in T. sherlocki also allowed us to address morphological differences between elongating cells, which were relatively smaller and more filamentous when compared to T lenti. Furthermore, the flagellum was observed in all stages of cell differentiation and elongation. This structure, which helps in the locomotion of the sperm, is hardly observed in cytogenetic analysis, especially throughout spermiogenesis. Thus, although other comparative approaches should be taken, this paper allowed emphasizing the analysis of spermiogenesis as an important cytotaxonomic tool that assists in the differentiation of morphologically related species, such as T. lenti and T. sherlocki.

Morphology of spermatozoa of Dissomphalus connubialis (Ashmead, 1893) (Hymenoptera: Bethylidae).

In this study, the morphology of spermatozoa of Bethylidae Dissomphalus connubialis (Pristocerinae) was analyzed using light and transmission electron microscopy. Spermatozoa of this species are thin, measure approximately 130 µm in length and comprise a head region and flagellum region. The head is formed by the acrosome and nucleus. The acrosome consists of the acrosome vesicle and the perforatorium, the posterior portion of which is inserted into a cavity at the anterior extremity of the nucleus. The nucleus is compact, electron-dense and measures 15 µm in length. The flagellum possesses two mitochondrial derivatives, two accessory bodies and one axoneme with a 9+9+2 microtubular pattern. The nucleus is connected to the flagellum by the centriole adjunct. Mitochondrial derivatives are compact, apparently without paracrystalline material and with rare mitochondrial cristae. They are asymmetric in length, such that the larger mitochondrial derivative begins parallel to the posterior region of the nucleus and the smaller mitochondrial derivative begins just below the centriole adjunct. The basic structure of spermatozoa of D. connubialis is similar to that of other Aculeata studied. However, this species shows characteristics not seen in other Hymenoptera, such as the wide electron-lucid region that separates the acrosomal vesicle from the perforatorium and the depth of the cavity in the anterior extremity of the nucleus, into which the base of the perforatorium is inserted. There are also characteristics that distinguish this species from Bethylidae Prorops nasuta, including the fact that one of the mitochondrial derivatives lies to parallel to the nucleus over a long distance, the small quantity of cristae, the absence of paracrystalline material in these organelles, and the fact that the accessory microtubules are the first to terminate in the final portion of the flagellum.

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.(AU)

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.

Value of quantitative ultramorphological sperm analysis in infertile men.

A specific cause of infertility cannot be identified in at least 25% of men referred to a specialized clinic. Diagnosis of infertile men is based mainly on standard semen analysis and the observation of sperm under light microscope. The aim of our study was to find the subcellular sperm characteristics that could explain infertility in a group of teratozoospermic infertile men. Morphological characteristics of sperm from non-teratozoospermic (control donors) and teratozoospermic infertile men were analyzed by transmission electron microscopy (TEM) and quantified. Our analysis showed that sperm cells from control donors presented a higher number of normal heads and tails than infertile men. Regarding subcellular characteristics of nucleus and tails, only the percentage of vacuolated nucleus, the absence of at least one pair of microtubules of the axoneme and the total distortion of the tail were statistically higher in infertile men than in control donors. There were no differences in the number of normal acrosomes between the groups. Although the ultrastructural sperm defects overlapped between control donors and infertile men, TEM permits the identification and differentiation of a larger amount of defects than light microscopy. Vacuolated nucleus and gross alterations of the tail are the major sperm defects that seem to have prognostic value in teratozoospermic men.

Characterization of Mongolian gerbil chromatoid bodies and their correlation with nucleolar cycle during spermatogenesis.

The aims of the present study were to monitor the nucleolar cycle in Mongolian gerbil spermiogenesis, to verify the relationship between the nucleolar component and chromatoid body (CB) formation and to investigate the function of this cytoplasmic supramolecular structure in spermatogenic cells. Histological sections of adult seminiferous tubules were analysed cytochemically by light microscopy and ultrastructurally by transmission electron microscopy. The results reveal that in early spermatids, the CB was visualized in association with Golgi vesicles indicating that this structure may participate in the acrosome formation process as had been reported in other rodents. In late spermatids, the CB was observed near the axoneme region suggesting that this structure may support spermatozoon tail formation as happens in other species. Chromatoid body was joined with lipid droplets in this same cell type. This observation should be investigated to verify whether CB may be related to steroidal hormone metabolism. In conclusion, our data showed that there is disintegration of primary spermatocyte nucleoli at the beginning of prophase I and a fraction of this nucleolar material migrates to the cytoplasm, where a specific structure is formed, known as the 'chromatoid body', which apparently participates in some parts of the gerbil spermiogenesis process.

Sperm morphology of beef bulls evaluated by two different methods.

The objectives of this study were to compare two different methods of evaluating bull sperm morphology, bright-field (BF) microscopy of eosin-nigrosin (EN) stained dry-mount semen smears and differential interference phase contrast (DIC) microscopy of wet-mount semen 'fixed' in isotonic formal saline, both at 1000x. Ejaculates (n=72) were evaluated, representing both pre- and post-breeding season ejaculates collected from 40 2-yr-old beef bulls via electro-ejaculation. For both methods, 200 sperm were counted in random fields with defects categorized as major (MAD) and minor (MID). Sperm abnormalities were also placed into two other categories: those considered to be most influenced by process (wet or dry, METHDEF) and those with depictions that could be influenced by optics (BF or DIC, OPTIDEF). Differences (P<0.05) occurred between DIC and BF methods respectively: MAD 23.3/16.1, MID 7.6/13.4, acrosome 3.8/1.1, midpiece 9.2/11.7, tail 2.0/4.7, droplets 8.3/4.2, METHDEF 14.2/21.4 and OPTIDEF 13.0/5.5, but not (P>0.05) in percent normal sperm 69.1/70.4 or sperm head defects 7.5/8.3. Acrosome, tail and droplet defects were observed in 98.2/80.5, 86.1/100 and 98.2/94.4 percent of bulls for DIC and BF, respectively (P<0.05). As percent normal sperm did not differ between methods, bright-field microscopy assessment of EN preparations was considered to be a satisfactory method to categorize breeding soundness of bulls. However, DIC was more effective in visualizing major defects, while BF (which included stained smear preparation) was considered to cause more minor defects. Thus DIC was considered to be the preferred method of semen assessment for accurate assessment of sperm morphology in bulls.

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.

Ultrastructure of sperm 'tail stump' defect in wild boar.

Sperm 'tail stump' defect was found in ejaculates of a wild boar maintained in captivity. It was in good physical condition, the testes and genital tract were found to be of normal size and consistency. There was no evidence of macroscopic abnormalities at the clinical analysis and at necropsy. The volume and concentration of the semen samples obtained by electroejaculation were lower than normal. The slides examined contained a high level of abnormal spermatozoa (52.7%). The most frequent morphological finding was a droplet-like form attached to the base of the head or a very short stump. The non-stumped spermatozoa had no normal tail but a shortened one. Analysing the histological structure with light microscopy, no ring of spermatozoa was observed lining the lumen of the seminiferous tubules and the characteristically cellular structure was not conserved. The ultrastructural examination evidenced a disorganisation of the normal tubular structure of the flagellum, with lost of regular pattern of the axial bundle of fibrils and the mitochondrial helix. The origin of this abnormality is unknown.

Exploring the cytoskeleton during intracytoplasmic sperm injection in humans.

Understanding the cellular events during fertilization in mammals is a major challenge that can contribute to the improvement of future infertility treatments in humans and reproductive performance in farm animals. Of special interest is the role of the oocyte and sperm cytoskeleton during the initial interaction between gametes. The aim of this chapter is to describe methods for studying cytoskeletal features during in vitro fertilization after intracytoplasmic sperm injection (ICSI) in humans. The following protocols will provide a detailed description of how to perform immunodetection and imaging of human eggs, zygotes, and sperm by fluorescence (confocal and epifluorescence) and electron microscopy.

The role of sperm proteasomes during sperm aster formation and early zygote development: implications for fertilization failure in humans.

BACKGROUND Sperm aster organization during bovine and human fertilization requires a paternally-derived centriole that must first disengage from the sperm tail connecting-piece. We investigated the participation of the 26S proteasome in this process. METHODS Proteasome localization and enzymatic activity were studied in normal and pathological human spermatozoa by immunocytochemistry and enzyme-substrate assays. The role of proteasomes during bovine zygote development was investigated using a pharmacological proteasome-inhibitor, MG132, and with anti-proteasome antibodies delivered by Streptolysin O-permeabilization or with the Chariot reagent. Human zygotes discarded after ICSI failures (n = 28) were also examined. RESULTS Proteasomes were localized in the sperm acrosome and connecting-piece, as well as in the pronuclei of bovine and human zygotes. Proteasomal enzymatic activities were decreased in defective human spermatozoa. Disrupted sperm aster formation and pronuclear development were found after pharmacological and immunological block of proteasomes in human/bovine spermatozoa and oocytes, as well as in 28 discarded human post-ICSI fertilization failures. CONCLUSIONS Specific proteasome inhibition disrupts sperm aster formation and pronuclear development/apposition in bovine and human zygotes. Human spermatozoa with defective centriolar/pericentriolar structures have decreased proteasomal enzymatic activity. Release of a functional sperm centriole that acts as a zygote microtubule-organizing center probably relies on selective proteasomal proteolysis. These findings suggest an important role of sperm proteasomes in zygotic development.

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.(AU)

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.