ABSTRACT REGULATION OF THE ACROSOME REACTION BY THE TRANSMEMBRANE ADENYLYL CYCLASE SEPTEMBER 2012 DOUGLAS HADDAD, B.S., UNIVERSITY OF MASSACHUSETTS AMHERST M.Sc., UNIVERSITY OF MASSACHUSETTS AMHERST Directed by: Pablo Visconti Capacitation prepares mammalian sperm to undergo a process known as the acrosome reaction, which enables them to penetrate the zona pellucida. The standard method of measuring the acrosome reaction has been over the years the staining of the acrosome and visual counting using light or fluorescence microscopy. In this study we explored the intracellular signaling that results in the acrosome reaction using a novel method. This method employs the use of transgenic mice that contain green fluorescent protein, GFP, in the sperm acrosome. The quantity of sperm either containing or lacking GFP is precisely and rapidly quantified with flowcytometry. Currently little is known about the signaling processes that lead to the acrosome reaction. It has been proposed that this reaction is regulated by the cAMP activated guanine nucleotide exchange factor, Epac. In human sperm, stimulation of this pathway leads to an increase in acrosomal exocytosis. Furthermore, previous studies from our laboratory indicated that Gαs is present in the mouse sperm anterior head. These results suggest that in mouse sperm, the cAMP pathway leading to the acrosome reaction could be stimulated by Gαs associated with a transmembrane adenylyl cyclase. In this study we first validated the ability of known reaction inducers to increase the rate of the acrosome exocytosis in mouse sperm. Progesterone, solubilized zona pellucida and calcium ionophore A23187 all showed to be very effective at inducing the acrosome reaction in mouse sperm. We then investigated the role of the cAMP pathway using a battery of cAMP agonists and antagonists. We observed that stimulation of the cAMP dependent pathway through the transmembrane adenylyl cyclases, using forskolin, inhibit the increase in acrosome reaction induced by soluble zona pellucida, progesterone and the calcium ionophore A23187. This inhibitory effect was observed only when forskolin was used before the start of capacitation. Consistent with this observation, addition of cAMP analogues including an Epac specific cAMP analogue 8-pCPT-2’-O-Me-cAMP can also inhibit the increase in acrosome reaction by progesterone. This inhibition was seen only when the pathway was stimulated from the beginning of capacitation. Altogether, these data suggest that transmembrane cyclases are involved in the regulation of mouse sperm acrosome reaction.

Eine wichtige Eigenschaft der Plasmamembran eukaryotischer Zellen ist die stabile transversale Asymmetrie der Phospholipide. Sie wird energieabhängig durch die Aktivität einer Aminophospholipidtranslokase aufrechterhalten und gilt als wichtige Voraussetzung für die Homöostasis der Zellen. Die Plasmamembran einiger Säugerzellen weist zudem laterale Lipiddomänen auf, denen eine wesentliche Bedeutung bei der Signaltransduktion zugeschrieben wird. Während der Genese durchlaufen die Membranen der Säugerspermien intensive Veränderungen. Um die Bedeutung der Phospholipidasymmetrie für die Funktion der Spermien zu untersuchen, wurde die Lokalisation und Dynamik von Phosphatidylserin in der Zell- und Akrosommembran von Eberspermien im Verlauf von Kapazitation und Akrosomreaktion betrachtet. Unter Ausnutzung der selektiven, kalziumabhängigen Bindung von AnnexinV an endogenes Phosphatidylserin konnte dessen Lokalisation an morphologisch differenzierten Zellen verfolgt werden. Eine Markierung der Zellen mit NBD-markierten Phospholipidanaloga lieferte zudem Informationen zur Dynamik der Phospholipide in der Plasmamembran. Die Differenzierung der Zellen erfolgte entweder am Durchflusszytometer oder fluoreszenz- bzw. elektronenmikroskopisch. Die Ergebnisse der vorliegenden Arbeit weisen sowohl auf eine transversale als auch laterale Ungleichverteilung der Lipide in der Zell- und Akrosommembran während der Genese der Spermien hin. Neben der stabilen transversalen Phospholipidasymmetrie der Plasmamembran konnte erstmals eine zytoplasmatische Lokalisation von Phosphatidylserin auf der äußeren Akrosommembran nachgewiesen werden. Somit akkumulieren die beiden einander zugewandten zytoplasmatischen Monolayer von Plasmamembran und äußerer Akrosommembran Phosphatidylserin. Kapazitationsbedingt kommt es zu einer engen Wechselwirkung zwischen Plasmamembran und äußerer Akrosommembran. Die Ausbildung lateraler Membrandomänen, in denen Phosphatidylserin zytoplasmatisch akkumuliert, wird als Voraussetzung für diese enge Assoziation diskutiert. Weitere Hinweise auf eine funktionelle Bedeutung lateraler Membrandomänen lieferten die Arbeiten zur Isolation Triton-unlöslicher Lipiddomänen aus der Plasmamembran von Forellenspermien. / One of the essential qualities of cell membranes in Eucaryotae is a stable transverse phospholipid asymmetry. It is regulated and maintained by ATP-dependent action of an aminophospholipid translocase and is a major prerequisite for cell homeostasis. The plasma membranes of several mammalian cells show moreover lateral lipid domains, which are imputed to play a significant role in signal transduction. The membranes of mammalian spermatozoa undergo significant changes during genesis. The localisation and dynamics of phosphatidylserine in the cell as well as acrosome membranes of boar sperm cells was studied during capacitation and acrosome reaction to assess the relevance of lipid asymmetry for sperm function. The localisation of endogenous phosphatidylserine in morphologically differentiated cells was followed using the selective calcium depending binding of annexinV. Information on the transverse dynamics of phospholipids in the plasma membrane was obtained by labelling the cells with a NBD-phospholipid analogues. The morphological status of the cells was assessed by flow cytometry, fluorescence and electron microscopy. The results of this study indicate both a transversal and lateral inhomogenous distribution of lipids in the cell membrane as well as in the outer acrosome membrane during sperm genesis. The plasma membrane of boar sperm shows a stable transversal lipid asymmetry characterised by an accumulation of phosphatidylserine in the cytoplasmic monolayer. Moreover a cytoplasmic localisation of phosphatidylserine on the outer acrosome membrane could be detected for the first time. Therefore the two facing cytoplasmic leaflets of the outer acrosome and cell membrane contain phosphatidylserine. Applying microscopy substantiated the hypothesis that there are close interactions between the cell membrane and the outer layer of the acrosome membrane because of capacitation. The cytoplasmic accumulation of phosphatidylserine in lateral lipid domains is probably essential for the strong association of plasma and outer acrosome membrane finally leading to local fusions of both membranes. An indication for the functional meaning of lateral membrane domains in sperm cells was futher deduced from the isolation of Triton-insoluble lipid domains from membranes of trout sperm cells.

Dynamics of acrosome reaction during intra-specific sperm competition in rodents Sperm acrosome integrity is disturbed in promiscuous species field mice (Apodemus) and more than half of the spermatozoa undergoing spontaneous acrosome reaction (AR) before binding to the zona pellucida. In Muridae it is documented a generally high rate of spontaneous AR, and the percentage increases in promiscuous species up to 60 % in 60 min capacitation in vitro. The acrosome integrity positively corellates with presence of CD46 protein which absence in wood mouse is fenotypicaly same as in CD46 knock-out mouse leading to accelerated spontaneous AR. It is necessary to clarify whether for mouse sperm it is essential the primary binding of intact sperm to zona pellucida of the egg or whether it is preferred secondary sperm binding after spontaneous AR. In this context, the question is whether there is a relocalization of the key fusion protein IZUMO in sperm during spontaneous AR. IZUMO relocalization was monitored by immunofluorescence at specific times of capacitation in vitro during spontaneous and induced AR. IZUMO relocalization as closely connected to actin cytoskeleton, and β1 integrins. Dynamics and localization of β1 integrin during spontaneous and induced AR was also detected by immunofluorescence. Our results...

O objetivo deste estudo foi avaliar os efeitos de três diluidores diferentes na criopreservação do sêmen bovino sobre a motilidade, hiperativação espermática, reação acrossomal, capacitação e peroxidação lipídica das membranas espermáticas. Foram realizadas seis colheitas de sêmen em intervalos quinzenais de dez touros zebuínos da raça Nelore. O sêmen in natura foi avaliado, dividido em três tratamentos: uma fração foi diluída em meio a base de gema de ovo (Botu-Bov® - diluidor 1), a segunda fração diluída em meio a base de lecitina de soja (Botu-Bov® - diluidor 2) e a terceira fração em meio AndroMed® (diluidor 3) também a base de lecitina de soja. Logo após as amostras foram submetidas à congelação. As avaliações do sêmen após descongelação consistiram na análise computadorizada das características de motilidade e nas análises pela citometria de fluxo quanto à reação acrossomal (PI/FITC-PSA/H33342); peroxidação lipídica (PI/C11-BODIPY581/591/H33342) e capacitação espermática através da estabilidade da membrana plasmática (Merocianina 540/Yo-Pro1/H33342). As variáveis foram submetidas à análise de variância e ao teste LSD para comparação das médias, ao nível de 5% de significância. O diluidor a base de gema de ovo preservou melhor a motilidade espermática, a população de células com integridade de membrana plasmática e acrossomo não reagido no sêmen bovino pós-descongelação que os diluidores a base de lecitina de soja. Os espermatozóides criopreservados com o diluidor a base de gema de ovo apresentou menor sub população de células hiperativas e com membrana plasmática desestabilizada quando comparados com os diluidores a base de lecitina de soja e o diluidor a base de gema de ovo possibilitou uma diminuição da peroxidação lipídica das membranas espermáticas / The objective of this experiment was evaluate the effects of three diferent extenders in bovine semen cryopreservation about motility, sperm hiperactivation, acrosome reaction, capacitation, and sperm membrane lipid peroxidation. It was made six collection of semen samples each 15 days of 10 Nelore bulls. Semen samples in natura was verify and divided in three treatments: The first one was extended in egg yolk base (Botu-Bov® Extender 1), second one was extended in soy lecithin base (Botu-Bov® Extender 2) and the third was extended in AndroMed® soy lecithin base too. After that, semen samples were submitted to freeze process. Semen evaluations after thawing were made with computer assisted sperm analysis (CASA) and flow citometry for acrosome reaction (PI/FITC-PSA/H33342), lipid peroxidation (PI/C11-BODIPY581/591/H33342) and sperm capacitation by plasma membrane stability (Merocianina 540/Yo-Pro1/H33342). Data obtained from experimental proceeding were submitted to variance analysis and LSD test to compare means with 5% of significance. Egg yolk base extender preserved better sperm motility, cell population with plasma membrane integrity and non-reacted acrosome in bovine semen after thawing than soy lecithin extenders. Cryopreserved sperm with egg yolk base extender displayed less subpopulation of hiperactivated cells, less destabilized plasma membrane and permitted a decrease of lipid peroxidation of membranes when it was compared with soy lecithin extender

Submitted by LUÍS LENIN VICENTE PEREIRA null (luislenin@gmail.com) on 2017-03-02T15:07:34Z No. of bitstreams: 1 Tese Luis Lenin Vicente Pereira.pdf: 2894913 bytes, checksum: 01d77fed0e0eb6b5cfb7979e52829667 (MD5) / Approved for entry into archive by LUIZA DE MENEZES ROMANETTO (luizamenezes@reitoria.unesp.br) on 2017-03-08T13:17:27Z (GMT) No. of bitstreams: 1 pereira_llv_dr_sjrp.pdf: 2894913 bytes, checksum: 01d77fed0e0eb6b5cfb7979e52829667 (MD5) / Made available in DSpace on 2017-03-08T13:17:27Z (GMT). No. of bitstreams: 1 pereira_llv_dr_sjrp.pdf: 2894913 bytes, checksum: 01d77fed0e0eb6b5cfb7979e52829667 (MD5) Previous issue date: 2017-02-17 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A subordem Heteroptera possui sete infraordens com aproximadamente 80 famílias. A maioria ocorre em todos os continentes (exceto Antártica) e algumas ilhas. Além dos Heteroptera terrestres, há os aquáticos e semi-aquáticos que são amplamente distribuídos, e surpreendem por sua capacidade de habitar uma extraordinária variedade de ecossistemas, sendo encontrados em habitats de água doce e marinho, e variada faixa de altitude entre 0 e 4.700 m. Estudos sobre aspectos ultraestruturais da espermatogênese e, especificamente, a estrutura do espermatozoide em Heteroptera ainda são escassos, por este motivo o objetivo do presente estudo foi o de analisá-los, por meio de cortes semifinos corados com azul de toluidina ou impregnados por íons prata, e cortes ultra-finos analisados em microscopia eletrônica de transmissão, utilizando testículos de machos adultos das famílias Belostomatidae, Gelastocoridae, Gerridae, Mesoveliidae, Notonectidae e Veliidae. Após a análise ultraestrutural da espermatogênese foi possível determinar que o padrão flagelar do axonema é de 9+9+2 para todas as espécies analisadas sendo, portanto, o padrão para essa subordem, as mitocôndrias durante a espermatogênese assumem diferentes morfologias, sendo que inicialmente as mitocôndrias se unem formando o complexo mitocondrial e, posteriormente, se divide em dois derivados mitocondriais que estão posicionados bilateralmente em relação ao axonema. Os derivados mitocondriais apresentaram tamanhos diferentes para as espécies B. amnigenus (Notonectidae) e R. c. crassifemur (Gerridae) e para as demais espécies o tamanho foi semelhante. As células germinativas possuem em seu citoplasma o acúmulo de um material denominado corpo cromatóide estando localizado próximo ao núcleo. Com relação ao comportamento nucleolar da espécie Martarega brasiliensis foi observado de um a quatro corpúsculos nucleolares em células de Prófase I comprovando uma grande atividade sintética das células nessa fase da divisão celular. Células em Metáfase I apresentaram regiões organizadoras nucleolares na região telomérica de um dos autossomos. Ainda, nessa espécie, foi possível observar, em Anáfase I, vários corpúsculos nucleolares persistindo até a fase de Telófase I. Todas as ultraestruturas descritas nas espécies analisadas foram semelhantes às descritas na literatura para Heteroptera, corroborando as características sinapomórficas dessa subordem sendo elas: a) a presença de duas pontes que ligam o material intertubular do axonema flagelar às cisternas achatadas que aderem aos lados internos dos derivados mitocondriais; b) padrão flagelar do axonema de 9+9+2 e c) ausência de corpos acessórios. / The suborder Heteroptera has seven infraorders with approximately 80 families. Most occur in all continents, except Antarctica and some islands. In addition to terrestrial Heteroptera, there are also widely distributed aquatic and semi-aquatic species. This suborder have adapted to live in an extraordinary variety of ecosystems as freshwater and marine habitats and at altitudes ranging from 0 m to 4,700 m. The research concerning the ultrastructural aspects of spermatogenesis is a large and growing field of study, however, in the case of Heteroptera, research is still scarce. For this reason, the aim of this study was to analyze the ultrastructures and spermatogenesis through semi-thin sections stained with toluidine blue or silver ions (Ag-NOR) and ultrathin sections examined in transmission electron microscopy, using testes of adult males ofthe following families: Belostomatidae, Gelastocoridae, Gerridae, Mesoveliidae, Notonectidae and Veliidae. After ultrastructural analysis of spermatogenesis, it was possible to determine that the flagellar pattern of the axoneme is 9+9+2 for all species, being therefore, the pattern for this suborder. As spermatogenesis progresses, the mitochondria begins to cluster and concentrate on only one side of the cell. Then, the mitochondria combine to form a single mitochondrial complex, which subsequently divides into two mitochondrial derivatives. They are positioned on opposite sides of the axoneme. The mitochondrial derivatives presented different sizes for the species B. amnigenus (Notonectidae) and R. c. crassifemur (Gerridae) and for the other species the size was similar. The germ cells have in their cytoplasm the accumulation of a material denominated the chromatoid body, being located near the nucleus. Regarding the nucleolar behavior, M. brasiliensis showed nucleus in prophase I composed by the nucleolus and nucleolar corpuscles that varied from one to four, emphasizing that this insect has great synthetic activity during meiosis. The analysis of cells in metaphase I, showed that M. brasiliensis presents nucleolar organizing region (NOR) in at least one autosome. Furthermore, was not observed the phenomenon of nucleolar persistence. All the ultrastructures described in the analyzed species were similar to those described in the literature for Heteroptera, corroborating the synapomorphic characteristics of this suborder, being them: a) two opposite bridges in the axoneme connect the flattened cisterns adherent to the internal side of each mitochondrial derivative to the intertubular material; b) flagellar pattern of the axoneme of 9+9+2; c) accessory bodies are absent all along the flagellum.

Submitted by Erika Demachki (erikademachki@gmail.com) on 2015-02-13T16:00:10Z No. of bitstreams: 2 Dissertação - Rogério Orfaly Addad da Silva - 2006.pdf: 843452 bytes, checksum: 27a7d9be950071d5241ff583004bbe06 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Erika Demachki (erikademachki@gmail.com) on 2015-02-13T16:00:31Z (GMT) No. of bitstreams: 2 Dissertação - Rogério Orfaly Addad da Silva - 2006.pdf: 843452 bytes, checksum: 27a7d9be950071d5241ff583004bbe06 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2015-02-13T16:00:31Z (GMT). No. of bitstreams: 2 Dissertação - Rogério Orfaly Addad da Silva - 2006.pdf: 843452 bytes, checksum: 27a7d9be950071d5241ff583004bbe06 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2006-12-08 / Thirty six Nelore bulls with age between 30 and 120 months were used to evaluation of the seminal quality and the reproductive aptitude. The animals had been kept in extensive conditions, with feeding the grass, receiving mineral supplementation and serving a herd with 3,782 females of some categories (heifers and cows). Data enclosed measured of scrotal circumference and evaluation of the semen collected by eletroejaculation and evaluated the physical characteristics (volume, mass activity, motility, strong), morphologic (major, minor and total defects) and spermatic structure integrity (integrity of plasmatic membrane, acrosome and chromatin) in this period had been carried through that preceded three consecutive mating season (2003, 2004 and 2005). The data had been submitted analyze of variance, the averages compared for test SNK and the coefficient of correlation of Pearson was calculated using the SAS. The bulls presented scrotal circumference of 33.8 ± 2.8cm, 15.6 ± 8.5% of total spermatic defects, 40.1 ± 23.1% of gametes with normal membrane and acrosome and 8.8 ± 3.8% of fragmented chromatin. The relations between scrotal circumference, physical and morphologic characteristics, spermatic integrity and the reproductive performance of the herd had been low and not significant (P< 0.05). In sexually mature bulls the integrity of plasmatic membrane, acrosome and nuclear chromatin had not been the most frequent trouble of the spermatic morphology. / Foram utilizados 36 touros da raça Nelore com idade entre 30 e 120 meses, para avaliação da qualidade seminal e da aptidão reprodutiva. Os animais foram mantidos em condições extensivas, com alimentação a pasto, recebendo suplementação mineral e servindo um rebanho com 3782 fêmeas de várias categorias (novilhas, vacas primíparas e pluríparas). Foram realizadas colheitas de dados que abrangiam medidas de circunferência escrotal e avaliação do sêmen colhido por eletroejaculação e avaliadas as características físicas (volume, turbilhonamento, motilidade, vigor), morfológicas (defeitos maiores, menores e totais) e de integridade espermática (integridade de membrana plasmática, de acrossoma e de cromatina) no período que antecedeu três estações de monta consecutivas (2003, 2004 e 2005). Os dados foram submetidos a analise de variância, as médias comparadas pelo teste SNK e o coeficiente de correlação de Pearson foi calculado utilizando o SAS. Os touros apresentaram circunferência escrotal de 33,8 ± 2,8cm, 15,6 ± 8,5% de defeitos espermáticos totais, 40,1 ± 23,1% de gametas com membrana e acrossoma íntegros e 8,8 ± 3,8% de cromatina fragmentada. As relações entre circunferência escrotal, as características físicas, morfológicas e de integridade espermática e o desempenho reprodutivo do rebanho foram baixas e não significativas (P>0,05). Em touros sexualmente maduros a integridade de membrana plasmática, acrossoma e fragmentação de cromatina nuclear não foram os distúrbios mais freqüentes da morfologia espermática.

Orientador: Fabiana Ferreira Souza / Resumo: As proteínas dos fluido e células do sitema reprodutor são constantes objetos de estudo, a fim de elucidar eventos fisiológicos e buscar biomarcadores das funções reprodutivas, facilitando a escolha de reprodutores. Em vista disso, este estudo objetivou caracterizar as proteínas dos espermatozoides do ejaculado e da cauda do epidídimo, do plasma seminal e do fluido epididimário de touros. Foram utilizados 10 touros adultos da raça Brangus. O sêmen foi colhido por eletroejaculação e, posteriormente os machos foram orquiectomizados para a colheita dos espermatozoides e fluido do epidídimo. As células do ejaculado e da cauda do epidídimo foram analisadas subjetivamente após a colheita, e o plasma seminal e fluido do epidídimo foram separados por centrifugação. Então, as amostras foram preparadas para a espectrometria de massas (ESI-QTof MS/MS), com um pool de cada grupo. A concentração de proteínas totais não diferiu entre os grupos. Foram encontradas 67 e 66 proteínas nos espermatozoides do ejaculado e da cauda do epidídimo, e 20 e 16 no plasma seminal e líquido epididimário, respectivamente. Além disso, 52 proteínas foram comuns entre os espermatozoides obtidos do ejaculado e epidídimo, e 9 entre o plasma seminal e fluido epididimário. Atividade catalítica foi a principal função molecular nas células espermáticas; já no plasma seminal foi de ligação e no fluido epididimário, atividade catalítica. As proteínas que se destacaram foram: 14-3-3 protein zeta/delta, A-kinase anchor ... (Resumo completo, clicar acesso eletrônico abaixo) / Mestre

Submitted by (edna.saturno@ufrpe.br) on 2016-11-03T14:12:07Z No. of bitstreams: 1 Sildivane Valcacia Silva.pdf: 2091723 bytes, checksum: a3f1920b1bf7539ac7a5652276f36b57 (MD5) / Made available in DSpace on 2016-11-03T14:12:07Z (GMT). No. of bitstreams: 1 Sildivane Valcacia Silva.pdf: 2091723 bytes, checksum: a3f1920b1bf7539ac7a5652276f36b57 (MD5) Previous issue date: 2010-04-30 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The objective of this work was to evaluate the effect of addition of antioxidants superoxide dismutase (SOD), glutathione (GSH), catalase (CAT), vitamin E (Trolox), and the association between CAT and SOD to Tris egg-yolk ram semen freezing extender. We used five Santa Inês breed rams, with history of fertility, and the ejaculates obtained by artificial vagina. The pool of semen samples were diluted in Tris egg-yolk plus glycerol 5% supplemented with antioxidants, according to the experiments and experimental groups: Exp 1 (G1= control group, G2= 25 U/mL SOD; G3= 50 U/mL SOD; G4= 100 U/mL SOD; G5= 2 mM GSH; G6= 5 mM GSH and G7= 7 mM GSH) and Exp 2 (G1= control group, G2= 30 μM Trolox, G3= 60 μM Trolox, G4= 120 μM Trolox, G5= 25 U/mL CAT, G6= 50 U/mL CAT; G7= 100 U/mL CAT, G8= 100 U/mL SOD + 25 U/mL CAT), at concentration of 240 x 106 spermatozoa/mL. Semen was stored in straws (0.25 mL), frozen using an automated system and stored in liquid nitrogen (- 196 °C). After thawing (37 ºC/30 seconds), samples were analyzed for plasma membrane integrity (iMP), acrosome (IAC) and mitochondrial membrane potential (MMP) with fluorescent probes, kinematics sperm by CASA, and ultrastructure spermatozoa by transmission electron microscopy (TEM). In Exp 1, evaluation was performed in vivo, with the semen used in embryo transfer program. In the Exp 1, significant differences (P<0.05) were observed among groups for total motility (MT), straightness (STR) and oscillation index (WOB) and the GSH 7 mM was lower in MT and higher in STR, and GSH 5 e 7 mM groups were greater in WOB when compared to control, SOD 25 and 100 U/mL. Ultrastructure study showed acrosome better (P<0.05) preserved after freezing in SOD (50 and 100 U/mL) and GSH (5 and 7 mM), whereas mitochondria from control group together with 7 mM GSH suffered further damage. The plasma membrane remained preserved after freezing, regardless of group. For in vivo fertilization, SOD group gave better results than GSH (P>0.05). For Exp 2, CAT 100 U/mL showed a lower percentage (P<0.05) of spermatozoa with intact acrosome. Significant differences (P<0.05) were observed among groups on the kinematics sperm (progressive motility, linearity, straightness, oscillation index, straight line velocity and velocity average path), and were higher for Trolox (30 and 60 μM) and lower for CAT (50 and 100 U/mL). On ultrastructural evaluation, CAT (50 and 100 U/mL) had lower acrosome preservation (P<0.05) and CAT 25 U/mL was higher (P<0.05) than CAT 100 U/ml for the preservation of plasma membrane. Thus, we conclude that the addition of GSH at a concentration of 7mm and 50 CAT and 100 U/mL do not preserve the structural integrity of spermatozoa after cryopreservation; the addition of SOD 100 U/mL, Trolox 60 and 120 mM, and association of CAT 25 U/mL + SOD 100 U/mL provide better preservation of sperm membranes of ram after freezing. / Objetivou-se com este trabalho avaliar o efeito da adição dos antioxidantes superóxido dismutase (SOD), glutationa reduzida (GSH), catalase (CAT), vitamina E (Trolox), e a associação entre CAT e SOD ao diluente Tris-gema de congelação do sêmen ovino. Foram utilizados cinco reprodutores ovinos da raça Santa Inês, com histórico de fertilidade, sendo os ejaculados obtidos pelo método de vagina artificial. Os ejaculados foram avaliados e submetidos à formação do pool, diluído em Tris-gema e glicerol 5%, acrescido de antioxidantes de acordo com o experimento e o grupo experimental: Exp. 1 (G1= Controle; G2= 25 U/mL de SOD; G3= 50 U/mL de SOD; G4= 100 U/mL de SOD; G5= 2 mM de GSH; G6= 5 mM de GSH; G7= 7 mM de GSH) e Exp. 2 (G1= Controle; G2= 30 μM de Trolox; G3= 60 μM de Trolox; G4= 120 μM de Trolox; G5= 25 U/mL de CAT; G6= 50 U/mL de CAT; G7= 100 U/mL de CAT; G8= 25 U/mL de CAT + 100 U/mL de SOD), na concentração de 240 x 106 espermatozoides/mL. O sêmen foi acondicionado em palhetas (0,25 mL), congelado utilizando sistema automatizado e armazenado em nitrogênio líquido (- 196 ºC). Após descongelação (37 oC/30 segundos), as amostras foram submetidas à análise de integridade da membrana plasmática (iMP), acrossoma (iAc) e potencial de membrana mitocondrial (PMM) com sondas fluorescentes; cinética espermática, pelo CASA; e ultraestrutura dos espermatozoides, por microscopia eletrônica de transmissão. No Exp. 1, foi realizada avaliação in vivo, com o sêmen utilizado em programa de transferência de embriões. Diferenças significativas (P<0,05) foram observadas entre grupos para motilidade total (MT), retilinearidade (STR) e índice de oscilação (WOB), sendo o GSH 7mM inferior (P<0,05) na MT e superior na STR, e os grupos GSH 5 e 7 mM commaior (P<0,05) WOB em comparação ao controle, SOD 25 e 100 U/mL. Na análise ultraestrutural, evidenciou-se que o acrossoma foi melhor preservado (P<0,05), pós-congelação, nos grupos SOD 50 e 100 U/mL e GSH 2 e 5 mM, enquanto que o acrossoma e as mitocôndrias do grupo Controle, juntamente com o GSH 7 mM, sofreram maiores danos (P<0,05). Para a fertilização in vivo, o grupo SOD conferiu resultados numericamente superiores aos grupos controle e GSH. Para o Exp. 2, o CAT 100 U/mL apresentou menor porcentual (P<0,05) de espermatozoides com acrossoma íntegros. Diferenças significativas (P<0,05) foram observadas entre grupos na cinética espermática (motilidade progressiva, linearidade, retilinearidade, índice de oscilação, velocidade em linha reta e velocidade média do percurso), sendo superiores para os grupos Trolox 30 e 60 μM e inferiores para os grupos CAT 50 e 100 U/mL. Na avaliação ultraestrutural, os grupos CAT 50 e 100 U/mL apresentaram menor preservação (P<0,05) do acrossoma, enquanto o CAT 25 U/mL foi superior (P<0,05) ao CAT 100 U/mL para a preservação da membrana plasmática e o SOD 25 U/mL foi inferior (P<0,05) aos demais grupos na preservação da mitocôndria. Assim, conclui-se que a adição de GSH na concentração de 7mM e CAT 50 e 100 U/mL não preservam a integridade estrutural de espermatozoides ovinos pós-criopreservação; a adição de SOD 100 U/mL, Trolox 60 e 120 μM, e a associação de CAT 25 U/mL + SOD 100 U/mL proporcionam maior preservação das membranas de espermatozóides congelados de ovinos.

O objetivo deste estudo foi avaliar os efeitos de três diluidores diferentes na criopreservação do sêmen bovino sobre a motilidade, hiperativação espermática, reação acrossomal, capacitação e peroxidação lipídica das membranas espermáticas. Foram realizadas seis colheitas de sêmen em intervalos quinzenais de dez touros zebuínos da raça Nelore. O sêmen in natura foi avaliado, dividido em três tratamentos: uma fração foi diluída em meio a base de gema de ovo (Botu-Bov® - diluidor 1), a segunda fração diluída em meio a base de lecitina de soja (Botu-Bov® - diluidor 2) e a terceira fração em meio AndroMed® (diluidor 3) também a base de lecitina de soja. Logo após as amostras foram submetidas à congelação. As avaliações do sêmen após descongelação consistiram na análise computadorizada das características de motilidade e nas análises pela citometria de fluxo quanto à reação acrossomal (PI/FITC-PSA/H33342); peroxidação lipídica (PI/C11-BODIPY581/591/H33342) e capacitação espermática através da estabilidade da membrana plasmática (Merocianina 540/Yo-Pro1/H33342). As variáveis foram submetidas à análise de variância e ao teste LSD para comparação das médias, ao nível de 5% de significância. O diluidor a base de gema de ovo preservou melhor a motilidade espermática, a população de células com integridade de membrana plasmática e acrossomo não reagido no sêmen bovino pós-descongelação que os diluidores a base de lecitina de soja. Os espermatozóides criopreservados com o diluidor a base de gema de ovo apresentou menor sub população de células hiperativas e com membrana plasmática desestabilizada quando comparados com os diluidores a base de lecitina de soja e o diluidor a base de gema de ovo possibilitou uma diminuição da peroxidação lipídica das membranas espermáticas / The objective of this experiment was evaluate the effects of three diferent extenders in bovine semen cryopreservation about motility, sperm hiperactivation, acrosome reaction, capacitation, and sperm membrane lipid peroxidation. It was made six collection of semen samples each 15 days of 10 Nelore bulls. Semen samples in natura was verify and divided in three treatments: The first one was extended in egg yolk base (Botu-Bov® Extender 1), second one was extended in soy lecithin base (Botu-Bov® Extender 2) and the third was extended in AndroMed® soy lecithin base too. After that, semen samples were submitted to freeze process. Semen evaluations after thawing were made with computer assisted sperm analysis (CASA) and flow citometry for acrosome reaction (PI/FITC-PSA/H33342), lipid peroxidation (PI/C11-BODIPY581/591/H33342) and sperm capacitation by plasma membrane stability (Merocianina 540/Yo-Pro1/H33342). Data obtained from experimental proceeding were submitted to variance analysis and LSD test to compare means with 5% of significance. Egg yolk base extender preserved better sperm motility, cell population with plasma membrane integrity and non-reacted acrosome in bovine semen after thawing than soy lecithin extenders. Cryopreserved sperm with egg yolk base extender displayed less subpopulation of hiperactivated cells, less destabilized plasma membrane and permitted a decrease of lipid peroxidation of membranes when it was compared with soy lecithin extender

A sperm specific and species conserved monoclonal antibody (HS-63) was shown to inhibit in vitro fertilization of mouse oocytes and human sperm penetration to zona-free hamster ova. The sperm antigen (SA-63) which reacts with HS-63 was found to be localized on the sperm acrosome. Following sperm capacitation, this antigen becomes exposed and is shed after the acrosome reaction. SA-63 may be involved in the sperm acrosome reaction during the initial fertilization process. Sperm antigen (SA-63) from mouse (MSA-63) was purified from mouse testes with soluble and detergent extraction procedures followed by immunoaffinity chromatography. The purified MSA-63 antigen was shown to be a group of proteins with a size ranging from 25 Kd to 50 Kd and pIs of about 4.2 when analyzed by two dimensional gel electrophoresis. MSA-63 antigen may be associated with actins in its native form. A proteolytic activity was found in the solution of purified MSA-63 preparation. Purified MSA-63 was used for immunization of mice and rabbits. Following successive immunizations, antisera of high titres were raised and reacted specifically with sperm acre-some. The isoimmune sera from immunized mice exhibited significant inhibition on in vitro fertilization of mouse oocytes. Complementary deoxyribonucleic acid (cDNA) fragments encoding the MSA-63 were cloned from a mouse testis cDNA library by using an immunoscreening method with rabbit antisera against MSA-63 as the detecting probe. When a specific cDNA probe was used for Northern blot analysis, an mRNA of 1.5 Kb in size was detected only in the adult mouse testis, but not in any other somatic tissues. By Southern blot analysis, it was also demonstrated that the gene encoding for SA-63 protein is conserved among different mammalian species. The location of SA-63 antigen gene was determined to be on human chromosome 11 when analyzed with a blot of a human-hamster somatic cell hybrid panel. By DNA sequence analysis, a protein of 28 Kd in size was deduced from the MSA-63 cDNA. The amino acid sequences of trypsin-digested peptide fragments of MSA-63 were used to verify that deduced amino acid sequence from the cDNA. The recombinant fusion proteins containing MSA-63 protein fragment were produced in E. coli and used to immunize female mice. Similar to the original HS-63 monoclonal antibody, the antisera thus produced reacted only with the sperm acrosome and revealed significant inhibition of the in vitro fertilization of mouse oocytes. In the developing mouse testis, the expression of MSA-63 gene was found to be post-meiotic. Protein and mRNA of MSA-63 were not produced until day 20 after birth. / Medicine, Faculty of / Obstetrics and Gynaecology, Department of / Graduate