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1.
Nat Commun ; 12(1): 841, 2021 02 05.
Artigo em Inglês | MEDLINE | ID: mdl-33547291

RESUMO

A new life begins with the unification of the maternal and paternal chromosomes upon fertilization. The parental chromosomes first become enclosed in two separate pronuclei near the surface of the fertilized egg. The mechanisms that then move the pronuclei inwards for their unification are only poorly understood in mammals. Here, we report two mechanisms that act in concert to unite the parental genomes in fertilized mouse eggs. The male pronucleus assembles within the fertilization cone and is rapidly moved inwards by the flattening cone. Rab11a recruits the actin nucleation factors Spire and Formin-2 into the fertilization cone, where they locally nucleate actin and further accelerate the pronucleus inwards. In parallel, a dynamic network of microtubules assembles that slowly moves the male and female pronuclei towards the cell centre in a dynein-dependent manner. Both mechanisms are partially redundant and act in concert to unite the parental pronuclei in the zygote's centre.


Assuntos
Núcleo Celular/metabolismo , Fertilização/genética , Forminas/genética , Proteínas dos Microfilamentos/genética , Proteínas do Tecido Nervoso/genética , Zigoto/metabolismo , Proteínas rab de Ligação ao GTP/genética , Actinas/genética , Actinas/metabolismo , Animais , Núcleo Celular/ultraestrutura , Feminino , Forminas/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Proteínas dos Microfilamentos/metabolismo , Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , Movimento , Proteínas do Tecido Nervoso/metabolismo , Oócitos/metabolismo , Oócitos/ultraestrutura , Espermatozoides/metabolismo , Espermatozoides/ultraestrutura , Zigoto/ultraestrutura , Proteínas rab de Ligação ao GTP/metabolismo
2.
Am J Hum Genet ; 108(2): 309-323, 2021 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-33472045

RESUMO

Asthenoteratozoospermia characterized by multiple morphological abnormalities of the flagella (MMAF) has been identified as a sub-type of male infertility. Recent progress has identified several MMAF-associated genes with an autosomal recessive inheritance in human affected individuals, but the etiology in approximately 40% of affected individuals remains unknown. Here, we conducted whole-exome sequencing (WES) and identified hemizygous missense variants in the X-linked CFAP47 in three unrelated Chinese individuals with MMAF. These three CFAP47 variants were absent in human control population genome databases and were predicted to be deleterious by multiple bioinformatic tools. CFAP47 encodes a cilia- and flagella-associated protein that is highly expressed in testis. Immunoblotting and immunofluorescence assays revealed obviously reduced levels of CFAP47 in spermatozoa from all three men harboring deleterious missense variants of CFAP47. Furthermore, WES data from an additional cohort of severe asthenoteratozoospermic men originating from Australia permitted the identification of a hemizygous Xp21.1 deletion removing the entire CFAP47 gene. All men harboring hemizygous CFAP47 variants displayed typical MMAF phenotypes. We also generated a Cfap47-mutated mouse model, the adult males of which were sterile and presented with reduced sperm motility and abnormal flagellar morphology and movement. However, fertility could be rescued by the use of intra-cytoplasmic sperm injections (ICSIs). Altogether, our experimental observations in humans and mice demonstrate that hemizygous mutations in CFAP47 can induce X-linked MMAF and asthenoteratozoospermia, for which good ICSI prognosis is suggested. These findings will provide important guidance for genetic counseling and assisted reproduction treatments.


Assuntos
Astenozoospermia/genética , Infertilidade Masculina/genética , Animais , Astenozoospermia/patologia , Astenozoospermia/fisiopatologia , Estudos de Coortes , Feminino , Deleção de Genes , Genes Ligados ao Cromossomo X , Hemizigoto , Humanos , Infertilidade Masculina/metabolismo , Infertilidade Masculina/patologia , Infertilidade Masculina/fisiopatologia , Masculino , Camundongos Endogâmicos C57BL , Mutação , Mutação de Sentido Incorreto , Linhagem , Fenótipo , Injeções de Esperma Intracitoplásmicas , Motilidade Espermática , Cauda do Espermatozoide/ultraestrutura , Espermatozoides/patologia , Espermatozoides/fisiologia , Espermatozoides/ultraestrutura , Sequenciamento Completo do Exoma
3.
Methods Mol Biol ; 2233: 139-168, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33222133

RESUMO

Acrosome reaction is an exocytic process that enables a sperm to penetrate the zona pellucida and fertilize an egg. The process involves the fenestration and vesiculation of the sperm plasma membrane and outer acrosomal membrane, releasing the acrosomal content. Given the importance of the acrosome secretion in fertilization, many different methods have been developed to detect the acrosome reaction of sperm. In this chapter, we describe detailed practical procedures to assess the acrosomal status of human spermatozoa. To do this, we resorted to light optical and epifluorescence microscopy, flow cytometry, and transmission electron microscopy. We also itemize the protocol for real-time measurements of the acrosome reaction by confocal microscopy. Further, we discuss the level of complexity, costs, and the reasons why a researcher should choose each technique.This chapter is designed to provide the user with sufficient background to measure acrosomal exocytosis in human sperm.


Assuntos
Reação Acrossômica/genética , Membrana Celular/ultraestrutura , Exocitose/genética , Espermatozoides/ultraestrutura , Acrossomo/metabolismo , Membrana Celular/genética , Humanos , Masculino , Espermatozoides/patologia , Zona Pelúcida/metabolismo
4.
Proc Natl Acad Sci U S A ; 117(31): 18302-18309, 2020 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-32690677

RESUMO

The ability to evaluate sperm at the microscopic level, at high-throughput, would be useful for assisted reproductive technologies (ARTs), as it can allow specific selection of sperm cells for in vitro fertilization (IVF). The tradeoff between intrinsic imaging and external contrast agents is particularly acute in reproductive medicine. The use of fluorescence labels has enabled new cell-sorting strategies and given new insights into developmental biology. Nevertheless, using extrinsic contrast agents is often too invasive for routine clinical operation. Raising questions about cell viability, especially for single-cell selection, clinicians prefer intrinsic contrast in the form of phase-contrast, differential-interference contrast, or Hoffman modulation contrast. While such instruments are nondestructive, the resulting image suffers from a lack of specificity. In this work, we provide a template to circumvent the tradeoff between cell viability and specificity by combining high-sensitivity phase imaging with deep learning. In order to introduce specificity to label-free images, we trained a deep-convolutional neural network to perform semantic segmentation on quantitative phase maps. This approach, a form of phase imaging with computational specificity (PICS), allowed us to efficiently analyze thousands of sperm cells and identify correlations between dry-mass content and artificial-reproduction outcomes. Specifically, we found that the dry-mass content ratios between the head, midpiece, and tail of the cells can predict the percentages of success for zygote cleavage and embryo blastocyst formation.


Assuntos
Doenças dos Bovinos/diagnóstico , Processamento de Imagem Assistida por Computador/métodos , Infertilidade Masculina/veterinária , Redes Neurais de Computação , Espermatozoides/ultraestrutura , Animais , Bovinos , Feminino , Infertilidade Masculina/diagnóstico , Masculino , Folículo Ovariano , Óvulo/fisiologia , Análise do Sêmen
5.
PLoS One ; 15(5): e0232156, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32357155

RESUMO

PURPOSE: To examine the efficacy of motile sperm organelle morphology examination (MSOME) and intracytoplasmic morphologically-selected sperm injection (IMSI) for unexplained infertility. METHODS: This historical study, included 271 couples with primary, unexplained infertility/male subfertility, treated at an outpatient, IVF clinic, 2015-2018. These couples underwent MSOME after ≥3 failed intrauterine insemination (IUI) cycles and ≥1 failed IVF-ICSI cycle. They proceeded to intracytoplasmic morphologically-selected sperm injection (IMSI) within 6 months of MSOME. IMSI is conducted on the day of oocyte pick-up with a fresh semen sample. Pregnancy and delivery rates were analyzed. RESULTS: The cohort was divided based on percentage of normal cells at MSOME: Group A included 55 with no normal cells, Group B, 184 with 0.5%≤ normal cells ≤1.5% and Group C, 32 with ≥2% normal cells. Normal spermatozoa were found in 49 (89%) of Group A after extensive search. Group A had higher pregnancy rate (62.7%) compared to B (47.2%, P = 0.05) and C (28.1%, P = 0.002). Group B had higher pregnancy rate than C (p = 0.045). Delivery rate was higher in Group A (52.1%) compared to B (34.1%, p = 0.023) and C (21.9%, p = 0.007). Pregnancy and delivery rates were higher in A compared to B+C (p = 0.018, p = 0.01, respectively). CONCLUSIONS: MSOME may be useful for evaluating unexplained infertility. IMSI can be recommended for men with <2% normal spermatozoa at MSOME.


Assuntos
Infertilidade Masculina/terapia , Organelas , Injeções de Esperma Intracitoplásmicas , Motilidade Espermática , Espermatozoides/ultraestrutura , Adulto , Estudos de Coortes , Feminino , Humanos , Masculino , Gravidez , Taxa de Gravidez , Análise do Sêmen
6.
PLoS One ; 15(5): e0232592, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32365118

RESUMO

Chromosomal aberrations are relatively frequent pathologies in both humans and animals. Among them, translocations present a specific meiotic segregation pattern able to give a higher percentage of unbalanced gametes that can induce fertility problems. In this study, the meiotic segregation patterns of 1p, 1q and 18 Bubalus bubalis chromosomes were analyzed in both total sperm fraction and motile sperm fraction of a t(1p;18) carrier and a control bulls by triple-color FISH analysis with a pool of specific BAC probes. The frequencies of each total sperm fraction products in the carrier resulting from alternate, adjacent I, adjacent II and 3:1 segregation were 39%, 20%, 1% and 38%, respectively. On the other hand, the frequencies of each motile sperm fraction products in the carrier resulting from alternate, adjacent I, adjacent II and 3:1 segregation were 93%, 5%, 0% and 2%, respectively. The frequencies of normal sperms in the carrier were 27% and 69% in total sperm fraction and motile sperm fraction, respectively. The frequencies detected in motile sperm fraction were also validated by comparison with bull's progeny. To our knowledge, this is the first report on the meiotic segregation patterns in motile sperm fractions of B. bubalis bull carrying a chromosomal translocation. These data suggest that translocation has a very limited effect on aneuploidy in the gametes, and therefore, on the reproductive abilities of the bull.


Assuntos
Búfalos/genética , Meiose , Motilidade Espermática , Espermatozoides/fisiologia , Espermatozoides/ultraestrutura , Translocação Genética , Aneuploidia , Animais , Búfalos/fisiologia , Aberrações Cromossômicas , Segregação de Cromossomos , Cromossomos Artificiais Bacterianos , Criopreservação , Hibridização in Situ Fluorescente , Masculino , Reprodução
7.
Arthropod Struct Dev ; 55: 100919, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32114289

RESUMO

The sperm of three coccinellid species belonging to the subfamilies Chilocorinae, Coccinellinae and Epilachninae were studied under light and transmission electron microscopy. The basic sperm structure of these ladybirds is common to that of the other previously studied species, especially the acrosome in front of the basal body and not the nucleus, with this latter running parallel with the flagellar components. In the Chilocorinae Platynaspis luteorubra (Platynaspidini) the sperm are of the type 1, as in Scymnini and Coccinellini, since they exhibited a cylindrical basal body with 9 + 0 and then 9 + 2, microtubules continuing further in an initial flagellar portion with the only axoneme devoid of accessory structures. The sperm exhibits a thin nucleus and mitochondrial derivatives. Such uniformity of sperm ultrastructure could be indicative of the occurrence of a close relationship between Platynaspidini and Scymnini as also proposed in the previous studies. Conversely, they differ markedly from the sperm of type 3 observed in the Chilocorini Exochomus quadripustulatus. In the Coccinellini Propylea quatuordecimpunctata the sperm are also of the type 1, but they can be easily differentiated from those of the other Coccinellinae studied so far, because of their very short acrosome without the posterior extension, the relatively thicker mitochondrial derivatives and the cylindrical nucleus. Epilachna clandestina sperm resemble those of E. quadripustulatus but differ from them, because they exhibit an elliptical nucleus which is anteriorly very thin, and the asymmetrical mitochondrial derivatives in the anterior extremity, with the greater one starting at the same level of the basal body, rather than at the nucleus level, as it occurs in E. quadripustulatus. Because of the differences observed in the sperm ultrastructure we propose a new sperm type (Type 4) for E. clandestina. This study on ladybird spermatozoon ultrastructure clearly indicates that the current classifications of coccinellids do not reflect the natural history of this well-known insect family.


Assuntos
Besouros/ultraestrutura , Espermatozoides/ultraestrutura , Acrossomo/ultraestrutura , Animais , Axonema/ultraestrutura , Masculino , Microscopia Eletrônica de Transmissão , Filogenia , Especificidade da Espécie
8.
Micron ; 133: 102853, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32114398

RESUMO

Nemertea is a phylum of worms with a simple internal morphology; nemerteans' spermatozoon morphology can be used for their classification and phylogenetic analyses. The aim of the present study was to describe spermatozoa of the nemerteans Hubrechtella juliae and Sonnenemertes cantelli from the basal groups of the class Pilidiophora at the ultrastructure level. Both species have primitive ('compact-head' sensu Stricker and Folsom, 1998) spermatozoa with ovoid head and five mitochondria in the midpiece, but differ in the structure of acrosomal complex: in Hubrechtella juliae, the single lens-shaped acrosomal vesicle contains an area of moderate electron density not enclosed by a separate membrane; in Sonnenemertes cantelli, the acrosome shows a unique morphology and contains a few electron-dense vesicles with irregular shapes and positions and one more electron-lucent elongated vesicle. Such a pattern of the acrosomal complex organization is described for Nemertea for the first time. An assumption is made that the states "two or more mitochondria" and "posterior acrosomal ring component" may be synapomorphies of Hubrechtiiformes+Heteronemertea (class Pilidiophora), whereas "the posterior margin of the acrosomes forms an acrosomal ring component" is presumably an autapomorphy of the family Lineidae s.l. The results suggest that spermatozoa provide a useful source of characters for nemertean systematics.


Assuntos
Invertebrados/anatomia & histologia , Espermatozoides/ultraestrutura , Acrossomo/ultraestrutura , Animais , Invertebrados/classificação , Masculino , Microscopia Eletrônica de Varredura , Filogenia
9.
Biochem Biophys Res Commun ; 525(3): 706-713, 2020 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-32139124

RESUMO

GGNBP1 is known as gametogenetin protein 1 (GGN1)-interacting protein. It is specifically expressed in the mitochondria of the testis, while its functional role during spermatogenesis is still unknown. Here, we showed that the disruption of Ggnbp1 resulted in abnormal spermiogenesis in around 40% mice, while the others show no defects in the genital system. Moreover, upon treatment with low dose of bisphenol A (BPA), Ggnbp1 knockout mice were more sensitive to environmental pollutant than control mice. The treatment led to decrease in sperm motility and production of abnormal spermatozoa. These results suggest that GGNBP1 mainly ensures proper spermiogenesis in response to various stresses in male mice.


Assuntos
Proteínas de Transporte/metabolismo , Espermatogênese , Estresse Fisiológico , Animais , Sequência de Bases , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Espermatozoides/metabolismo , Espermatozoides/ultraestrutura
10.
Life Sci ; 242: 117250, 2020 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-31899225

RESUMO

BACKGROUND: Endocrine disruptor such as cadmium has been widely reported to cause testicular toxicity, which contributes to recent decline in male fertility worldwide. Glutamine, the most abundant amino acid in the body has been demonstrated to exert protective effects in cellular toxicity. However, its role in testicular toxicity is unknown. The present study is therefore aimed at investigating the effects of glutamine supplementation on cadmium-induced testicular toxicity, and the possible involvement of glucose-6-phosphate dehydrogenase (G6PD) activity. MATERIALS AND METHOD: Male Wistar rats weighing 160-190 g were allotted into 4 groups (n = 5/group): The groups received vehicle (distilled water; p.o.), glutamine (1gkg-1; p.o.), cadmium chloride (5mgkg-1p.o.) and Cadmium chloride plus glutamine respectively, daily for 30 days. Biochemical and histological analyses were performed with appropriate method. RESULTS: Administration of cadmium significantly decreased body weight, sperm count, motility and viability, as well as altered sperm morphology and progressivity. Cadmium also caused atrophy of the seminiferous tubule in addition to disrupted testicular architecture, lumen, Sertoli cells and spermatogonia. Similarly, serum and testicular aspartate transaminase, and malondialdehyde significantly increased, and G6PD, glutathione, nicotinamide adenine dinucleotide phosphate and nitric oxide significantly decreased with corresponding decrease in follicle stimulating hormone, luteinizing hormone and testosterone in cadmium-treated animals compared with control groups. However, supplementation with glutamine attenuated these alterations. CONCLUSION: The present study demonstrates that cadmium induces testicular dysfunction that is attributable to defective G6PD and accompanied by increased lipid peroxidation and impaired NO-dependent endothelial function. Interestingly, glutamine supplementation ameliorates cadmium-induced testicular dysfunction through enhancement of G6PD activity.


Assuntos
Cloreto de Cádmio/toxicidade , Glucosefosfato Desidrogenase/metabolismo , Glutamina/farmacologia , Testículo/efeitos dos fármacos , Animais , Glucosefosfato Desidrogenase/efeitos dos fármacos , Peroxidação de Lipídeos/efeitos dos fármacos , Masculino , Estresse Oxidativo/efeitos dos fármacos , Ratos , Ratos Wistar , Contagem de Espermatozoides , Motilidade Espermática/efeitos dos fármacos , Espermatozoides/efeitos dos fármacos , Espermatozoides/ultraestrutura , Testículo/enzimologia
11.
Development ; 147(2)2020 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-31969357

RESUMO

The development and maintenance of the correct morphology of sperm is important for their functions. Cellular morphogenesis of sperm occurs during the post-meiotic developmental stage; however, little is known about what coordinates this process. In the present study, we investigated the role of A-kinase anchoring protein 3 (AKAP3) during mouse spermiogenesis, using both mouse genetics and proteomics. It was found that AKAP3 is essential for the formation of the specific subcellular structure of the sperm flagellum, motility of sperm and male fertility. Additionally, lack of AKAP3 caused global changes of the sperm proteome and mislocalization of sperm proteins, including accumulation of RNA metabolism and translation factors and displacement of PKA subunits in mature sperm, which may underlie misregulated PKA activity and immotility in sperm. Interestingly, sperm lacking a complete fibrous sheath from both Akap3 and Akap4 null mice accumulated F-actin filaments and morphological defects during post-testicular maturation in the epididymis. These results suggest that the subcellular structures of sperm could be formed via independent pathways, and elucidate the roles of AKAP3 during the coordinated synthesis and organization of the sperm proteome and sperm morphology.


Assuntos
Proteínas de Ancoragem à Quinase A/metabolismo , Infertilidade Masculina/metabolismo , Espermatozoides/metabolismo , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Animais , Sequência de Bases , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Epididimo/metabolismo , Deleção de Genes , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação/genética , Proteoma/metabolismo , Transdução de Sinais , Espermatozoides/anormalidades , Espermatozoides/patologia , Espermatozoides/ultraestrutura , Frações Subcelulares/metabolismo
12.
Parasitol Res ; 119(3): 991-999, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31989239

RESUMO

The present work provides the first ultrastructural analysis of spermatozoa of two digeneans (Aphanurus stossichii (Monticelli, 1891) and Ectenurus lepidus Looss, 1907) belonging to the unexplored subfamilies of the Hemiuridae, namely, the Aphanurinae and the Dinurinae. In March 2019, these hemiurids were collected respectively from the digestive tract of the bogue Boops boops (Teleostei, Sparidae) and the Atlantic horse mackerel Trachurus trachurus (Teleostei, Carangidae) captured in the coastal zone of the Mediterranean Sea, off La Chebba (Tunisia). The ultrastructural study reveals that both spermatozoa exhibit the Bakhoum et al.'s type II of the digenean sperm cells characterized by the presence of two 9+'1' axonemes, an external ornamentation of the plasma membrane not associated with cortical microtubules and located in the anterior part of the spermatozoon, a single bundle of cortical microtubules, the maximum number of cortical microtubules located in a middle part of the sperm cell, and one mitochondrion. Moreover, they share several ultrastructural features with the studied spermatozoa of Hemiuridae such as the presence of two axonemes with the 9+'1' trepaxonematan pattern, a reduced number of parallel cortical microtubules organized into one field with their maximum number located in the median (A. stossichii) or posterior (E. lepidus) part of the spermatozoon, an external ornamentation of the plasma membrane in the anterior part of the spermatozoon, one mitochondrion, a nucleus, and a small amount of glycogen granules. However, the two studied hemiurids could be distinguished by the morphology of the anterior and posterior spermatozoon extremities and the presence of mitochondrial matrix granules in A. stossichii.


Assuntos
Espermatozoides/ultraestrutura , Trematódeos/citologia , Animais , Axonema/ultraestrutura , Membrana Celular/ultraestrutura , Núcleo Celular/ultraestrutura , Peixes/parasitologia , Trato Gastrointestinal/parasitologia , Masculino , Mar Mediterrâneo , Microtúbulos/ultraestrutura , Mitocôndrias/ultraestrutura , Trematódeos/ultraestrutura , Infecções por Trematódeos/parasitologia , Infecções por Trematódeos/veterinária , Tunísia
13.
Arthropod Struct Dev ; 54: 100912, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31991324

RESUMO

The sperm structure of several species belonging to different tribes of the large Carabidae family is described. Some species of Nebriinae, such as Nebria brevicollis and Notiophilus biguttatus, have free conventional insect sperm. Their sperm type can be regarded as the ancestral model for Carabidae. All the other species examined, either with isolated sperm such as Calomera nemoralis, Scarites sp., Duvalius andreinii and Anillus florentinus or with spermatozeugmata and sperm associated to spermatostyles such as Typhloreicheia usslaubi, Brachinus italicus, Carabus convexus, Calathus fuscipes, Calathus montivagus, and Paraphorus mendax, showed sperm with long nucleus and a parallel axoneme running the length of the tail starting from the apical bell-like acrosome. C. nemoralis, like Cicindela campestris previously studied, has a sperm structure similar to that of several other Carabidae, confirming their correct assignment to the family. C. convexus has the same sperm structure as previously studied C. preslii and C. interstitialis, indicating that the spermatozeugmata of the group consist only of an apical cap in which the anterior sperm regions are embedded. Unlike other Carabidae with spermatozeugmata, Carabini have the typical sperm organization with acrosome, nucleus and flagellum in a regular sequence. A. florentinus, (Trechinae) shows major differences, such as the absence of an acrosome and an extremely long nucleus that reaches the end of the tail in close association with the axoneme. T. usslaubi (Scaritinae) has slender spermatozeugmata with orderly quartets of sperm. The posterior region of the sperm tail is also unusual, showing a perfect circular section and a plasma membrane reinforced by a dense underlying layer. The present observations confirm that spermatozeugmata, can vary in shape and size among different species of the Carabidae. Such diversity may be the result of the male reproductive strategy, different in each species, that enhances the efficiency of sperm transfer to the female.


Assuntos
Besouros/ultraestrutura , Espermatozoides/ultraestrutura , Animais , Masculino , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência
14.
Arq. bras. med. vet. zootec. (Online) ; 72(1): 253-262, Jan.-Feb. 2020. tab, ilus
Artigo em Português | LILACS, VETINDEX | ID: biblio-1088914

RESUMO

Os objetivos do presente estudo foram analisar a ultraestrutura do espermatozoide do jundiá amazônico e avaliar a sua criopreservação com três agentes crioprotetores (metanol 10%, DMSO 10% e etilenoglicol 10%) e duas soluções ativadoras (NaCl 0,29% e NaHCO3 1%). Como diluente, foi utilizada uma solução de glicose a 5%, sendo o sêmen envasado em palhetas de 0,25mL e congelado em vapor de nitrogênio (botijão dry shipper). No sêmen fresco, o espermatozoide apresentou comprimento de 25,46±2,54µm, cabeça esférica (1,51±0,18µm), ausência de acrossoma, peça intermediária com formato cônico (0,93±0,17µm), ligeiramente assimétrica, com presença de vesículas, e flagelo único (21,48±2,45µm). O sêmen descongelado apresentou valores mais altos (P<0,05) para duração, vigor e taxa de motilidade espermática com os crioprotetores metanol 10% e DMSO 10%. A duração da motilidade espermática foi maior (P<0,05) com o ativador NaHCO3 1% (21-96 s). O sêmen de Leiarius marmoratus criopreservado com DMSO e metanol apresentou, respectivamente, 7,32±4,21% e 8,94±6,69% de taxa de motilidade. No entanto, os resultados não foram satisfatórios para estabelecer um protocolo para a espécie.(AU)


The aims of this study were to describe the spermatozoon ultrastructure and to evaluate the sperm cryopreservation of the amazon catfish with three cryoprotectant agents (10% methanol, 10% DMSO, and 10% ethylene glycol) and two activator agents (0.29% NaCl and 1% NaHCO3). Glucose 5% extender was used as a diluent solution and sperm loaded in 0.25 straws was frozen in nitrogen vapor (dry shipper). Fresh spermatozoon was 25.46±2.54µm long, the head was spherical (1.51±0.18µm) with no acrosome, the midpiece was cone shaped (0.93±0.17µm) with presence of vesicles, slightly asymmetric, and the flagellum was single (21.48±2.45µm). Post-thawed semen presented higher values (P< 0.05) for duration, vigor and sperm motility rate with cryoprotectants 10% methanol and 10% DMSO. The duration of sperm motility was longer (P< 0,05) when triggered in 1% NaHCO3 (96-21 s). Leiarius marmoratus semen cryopreserved with DMSO and methanol, presented respectively 7.32±4.21% and 8.94±6.69% of motility. However, the results were not satisfactory to establish a protocol for the specie.(AU)


Assuntos
Animais , Masculino , Preservação do Sêmen , Espermatozoides/ultraestrutura , Peixes-Gato , Crioprotetores
15.
Cryobiology ; 92: 53-61, 2020 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-31704199

RESUMO

Due to the global decrease in jaguar population, conservation strategies are essential and the development of effective semen cryopreservation protocols would contribute to the formation of germplasm banks. Therefore, the objectives were to (1) evaluate the use of TRIS and ACP-117c extenders for jaguar semen freezing, (2) describe the ultrastructural changes in sperm after cryopreservation, and (3) evaluate the binding capacity of the thawed sperm. Eight ejaculates from five mature individuals were collected by electroejaculation, extended in TRIS or a coconut based-extender (ACP-117c), and frozen in liquid nitrogen. Samples were evaluated for sperm motility, vigor, membrane functionality, mitochondrial activity, morphology (using light microscopy, scanning electron microscopy - SEM and transmission electron microscopy - TEM), sperm kinetic parameters (by computerized analysis - CASA), and sperm binding capability using an egg yolk perivitelline membrane assay. Samples preserved in TRIS presented better post-thaw motility (46.0 ±â€¯7.7%) and membrane functionality (60.5 ±â€¯4.2%) and higher mitochondrial activity (21.5 ±â€¯3.7%) than those preserved in ACP-117c (20.9 ±â€¯5.4% motile sperm; 47.1 ±â€¯2.5% functional membrane; 11.8 ±â€¯1.7% mitochondrial activity). Regarding ultrastructural evaluations, SEM showed that both extenders were able to preserve the superficial membrane of the sperm, but TEM revealed the occurrence of nuclear electron lucent points, especially in samples extended in ACP-117c. Additionally, TRIS also provided a higher number of sperm bound to the perivitelline membrane (29.5 ±â€¯3.3%) in comparison to samples diluted in ACP-117c (18.6 ±â€¯1.5%). Overall, we suggest the use of a TRIS-based extender for cryopreservation of jaguar semen.


Assuntos
Crioprotetores/farmacologia , Panthera/embriologia , Preservação do Sêmen/métodos , Espermatozoides/ultraestrutura , Trometamina/farmacologia , Animais , Cocos/química , Criopreservação/métodos , Crioprotetores/química , Gema de Ovo/química , Congelamento , Humanos , Masculino , Microscopia Eletrônica de Transmissão , Sêmen/fisiologia , Análise do Sêmen , Motilidade Espermática
16.
Parasitol Res ; 119(1): 137-144, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31760497

RESUMO

The ultrastructural characteristics of the mature spermatozoon of the aspidogastrean Rohdella amazonica (Aspidogastridae, Rohdellinae) were studied by means of transmission electron microscopy. The sperm cell shows two axonemes of the 9 + '1' trepaxonematan pattern of Platyhelminthes, parallel cortical microtubules, a well-developed lateral expansion, external ornamentation of the plasma membrane, one mitochondrion, an electron-dense ring, a nucleus and granules of glycogen. The present results were compared with those observed in the aspidogastreans studied to date and in other Platyhelminthes. The lateral expansion and the electron-dense ring are typical characters for aspidogastreans. Although a lateral expansion has been described in other Platyhelminthes such as monogeneans and digeneans, the Aspidogastrea shows a much larger lateral expansion with both peripheral and internal microtubules. The dense ring is observed as a cylinder in a longitudinal view and shows a more granular appearance in sperm cells from the seminal vesicle in comparison to a more electron-dense appearance in sperm cells from the seminal uterine receptacle.


Assuntos
Axonema/ultraestrutura , Espermatozoides/ultraestrutura , Tetraodontiformes/parasitologia , Trematódeos/ultraestrutura , Animais , Membrana Celular/ultraestrutura , Núcleo Celular/ultraestrutura , Masculino , Microscopia Eletrônica de Transmissão , Microtúbulos/ultraestrutura , Mitocôndrias/ultraestrutura
17.
Reprod Domest Anim ; 55(1): 29-37, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31626708

RESUMO

Freeze-drying (FD) has been exhaustively tried in several mammalian species as an alternative technique to sperm cryopreservation, but few studies have been done in rabbits (Oryctolagus cuniculus). The main objective of this study was to compare the protective effect of various antioxidants added to EDTA medium on structural and functional components of FD rabbit spermatozoa and on their status of global DNA methylation. FD media used were composed of basic FD medium (10 mM Tris-HCl buffer and 50 mM NaCl) supplemented with either 50 mM EDTA alone (EDTA) or added with 105 µM of rosmarinic acid (RA, EDTA-RA) or 10 µM of melatonin (MLT, EDTA-MLT). The effect of each medium on the preservation of FD spermatozoon structure was evaluated with light and scanning electron microscopy (SEM). Global DNA methylation was quantified in all FD sperm samples as well as in fresh spermatozoa. Morphologically, fracture points were evidenced in the neck, mid and principal piece of the spermatozoon tail. No differences in spermatozoon fracture points were evidenced among FD treatments: intact spermatozoa were the largest (p < .01) category, whereas the most frequent (p < .01) injury was the neck fracture, resulting in tailless heads. At SEM, the head of spermatozoa showed a well-conserved shape and intact membrane in all treatments. DNA methylation status was the same in all FD treatments. In conclusion, supplementation of EDTA, EDTA-RA and EDTA-MLT during FD preserved rabbit sperm morphological integrity and methylation status as well. Therefore, the difficulty of getting viable offspring using FD semen is likely unrelated to the impact of the lyophilization process on DNA methylation and morphology of lyophilized spermatozoa.


Assuntos
Antioxidantes/farmacologia , Quelantes/farmacologia , Liofilização/veterinária , Espermatozoides/efeitos dos fármacos , Animais , Cinamatos/farmacologia , Metilação de DNA/efeitos dos fármacos , Depsídeos/farmacologia , Ácido Edético/farmacologia , Liofilização/métodos , Masculino , Melatonina/farmacologia , Microscopia Eletrônica de Varredura/veterinária , Coelhos , Preservação do Sêmen/métodos , Preservação do Sêmen/veterinária , Espermatozoides/citologia , Espermatozoides/ultraestrutura
18.
EMBO J ; 39(4): e102723, 2020 02 17.
Artigo em Inglês | MEDLINE | ID: mdl-31880004

RESUMO

Cilia serve as cellular antennae that translate sensory information into physiological responses. In the sperm flagellum, a single chemoattractant molecule can trigger a Ca2+ rise that controls motility. The mechanisms underlying such ultra-sensitivity are ill-defined. Here, we determine by mass spectrometry the copy number of nineteen chemosensory signaling proteins in sperm flagella from the sea urchin Arbacia punctulata. Proteins are up to 1,000-fold more abundant than the free cellular messengers cAMP, cGMP, H+ , and Ca2+ . Opto-chemical techniques show that high protein concentrations kinetically compartmentalize the flagellum: Within milliseconds, cGMP is relayed from the receptor guanylate cyclase to a cGMP-gated channel that serves as a perfect chemo-electrical transducer. cGMP is rapidly hydrolyzed, possibly via "substrate channeling" from the channel to the phosphodiesterase PDE5. The channel/PDE5 tandem encodes cGMP turnover rates rather than concentrations. The rate-detection mechanism allows continuous stimulus sampling over a wide dynamic range. The textbook notion of signal amplification-few enzyme molecules process many messenger molecules-does not hold for sperm flagella. Instead, high protein concentrations ascertain messenger detection. Similar mechanisms may occur in other small compartments like primary cilia or dendritic spines.


Assuntos
Arbacia/fisiologia , Quimiotaxia , Proteômica , Transdução de Sinais , Animais , Arbacia/ultraestrutura , Cálcio/metabolismo , Cílios/fisiologia , Cílios/ultraestrutura , GMP Cíclico/metabolismo , Tomografia com Microscopia Eletrônica , Flagelos/fisiologia , Flagelos/ultraestrutura , Guanilato Ciclase/metabolismo , Masculino , Espectrometria de Massas , Espermatozoides/fisiologia , Espermatozoides/ultraestrutura
19.
Parasitol Res ; 119(1): 177-187, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31811425

RESUMO

The spermatozoon ultrastructure of the progenetic cestode Diplocotyle olrikii (Spathebothriidea) has been examined using transmission electron microscopy and cytochemical staining with periodic acid-thiosemicarbazide-silver proteinate (PA-TSC-SP) for glycogen. The spermatozoon is a filiform cell, tapered at both extremities. Its moderately electron-dense cytoplasm possesses two parallel axonemes of unequal lengths. New for the Cestoda is a finding of three types of the mature spermatozoa with respect to different axonemal structure. The first type has both axonemes with standard 9 + '1' trepaxonematan pattern. The second type is represented by a spermatozoon having one axoneme with 9 + '1' structure and the second one with 9 + 0 pattern. The third type includes the two axonemes with 9 + 0 pattern. Microtubule doublets of the 9 + 0 axonemes contain either inner dynein arms or no dynein arms. In addition to the two axonemes, all three types of the mature sperm cells contain parallel nucleus, parallel cortical microtubules, four electron-dense plaques/attachment zones, and glycogen. The anterior extremity of the gamete exhibits a centriole surrounded by a semiarc of up to five electron-dense tubular structures. The distal end of the first type spermatozoa exhibits two morphological variants, represented either by (i) nucleus or (ii) remnants of the disorganized axoneme. Distal extremity of the spermatozoa of the second and third types contains doublets and singlets of disorganized axoneme. The ultrastructural characters of the spermatozoon of progenetic D. olrikii support the basal position of the Spathebothriidea within the Eucestoda.


Assuntos
Axonema/ultraestrutura , Núcleo Celular/ultraestrutura , Centríolos/ultraestrutura , Cestoides/ultraestrutura , Espermatozoides/ultraestrutura , Animais , Citoplasma/ultraestrutura , Masculino , Microscopia Eletrônica de Transmissão , Espermatogênese/fisiologia
20.
Sci Rep ; 9(1): 18537, 2019 12 06.
Artigo em Inglês | MEDLINE | ID: mdl-31811199

RESUMO

Manipulating mosquito reproduction is a promising approach to reducing mosquito populations and the burden of diseases they carry. A thorough understanding of reproductive processes is necessary to develop such strategies, but little is known about how sperm are processed and prepared for fertilization within female mosquitoes. By employing cryo-electron microscopy for the first time to study sperm of the mosquito Aedes aegypti, we reveal that sperm shed their entire outer coat, the glycocalyx, within 24 hours of being stored in the female. Motility assays demonstrate that as their glycocalyx is shed in the female's sperm storage organs, sperm transition from a period of dormancy to rapid motility-a critical prerequisite for sperm to reach the egg. We also show that females gradually become fertile as sperm become motile, and that oviposition behavior increases sharply after females reach peak fertility. Together, these experiments demonstrate a striking coincidence of the timelines of several reproductive events in Ae. aegypti, suggesting a direct relationship between sperm modification and female reproductive capacity.


Assuntos
Aedes/fisiologia , Fertilidade/fisiologia , Mosquitos Vetores/fisiologia , Oviposição/fisiologia , Espermatozoides/ultraestrutura , Aedes/citologia , Animais , Microscopia Crioeletrônica , Feminino , Glicocálix/ultraestrutura , Masculino , Controle de Mosquitos/métodos , Mosquitos Vetores/citologia , Motilidade Espermática/fisiologia , Espermatozoides/fisiologia
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