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1.
Sci Rep ; 11(1): 22563, 2021 11 19.
Artigo em Inglês | MEDLINE | ID: mdl-34799600

RESUMO

The adaptive ability of sperm in the female reproductive tract micromilieu signifies the successful fertilization process. The study aimed to analyze the preparedness of sperm to the prevailing osmotic and pH stressors in the female reproductive tract. Fresh bovine sperm were incubated in 290 (isosmotic-control), 355 (hyperosmotic-uterus and oviduct), and 420 (hyperosmotic-control) mOsm/kg and each with pH of 6.8 (uterus) and 7.4 (oviduct). During incubation, the changes in sperm functional attributes were studied. Sperm kinematics and head area decreased significantly (p < 0.05) immediately upon exposure to hyperosmotic stress at both pH. Proportion of sperm capacitated (%) in 355 mOsm/kg at 1 and 2 h of incubation were significantly (p < 0.05) higher than those in 290 mOsm media. The magnitude and duration of recovery of sperm progressive motility in 355 mOsm with pH 7.4 was correlated with the ejaculate rejection rate (R2 = 0.7). Using this information, the bulls were divided into good (n = 5) and poor (n = 5) osmo-adapters. The osmo-responsive genes such as NFAT5, HSP90AB1, SLC9C1, ADAM1B and GAPDH were upregulated (p < 0.05) in the sperm of good osmo-adapters. The study suggests that sperm are prepared for the osmotic and pH challenges in the female reproductive tract and the osmoadaptive ability is associated with ejaculate quality in bulls.


Assuntos
Osmorregulação , Capacitação Espermática , Motilidade Espermática , Espermatozoides/metabolismo , Animais , Fenômenos Biomecânicos , Bovinos , Sobrevivência Celular , Ejaculação , Fertilinas/genética , Fertilinas/metabolismo , Gliceraldeído-3-Fosfato Desidrogenases/genética , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Proteínas de Choque Térmico HSP90/genética , Proteínas de Choque Térmico HSP90/metabolismo , Concentração de Íons de Hidrogênio , Masculino , Fatores de Transcrição NFATC/genética , Fatores de Transcrição NFATC/metabolismo , Pressão Osmótica , Trocadores de Sódio-Hidrogênio/genética , Trocadores de Sódio-Hidrogênio/metabolismo
2.
Biopreserv Biobank ; 19(6): 470-482, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-33956503

RESUMO

According to various reports, current methods of sperm freezing destroy the integrity of the sperm plasma membrane and acrosome. This study aimed to determine the changes in the existence and location of three proteins, namely fertilin ß, IZUMO1, and P34H, in ram spermatozoa. By using frozen-thawed spermatozoa, ejaculated fresh spermatozoa, and testicular and epididymal spermatozoa (obtained from caput, corpus, and caudal regions), the localizations of the mentioned proteins were performed using signal labeling with indirect immunofluorescence, and the quantification of these proteins was compared using Western blot analyses. Moreover, protein localization and signal labeling in fresh and frozen-thawed spermatozoa subjected to in vitro capacitation and acrosome reaction were compared. Using chlortetracycline (CTC) staining, as expected, it was detected that after incubating for 4 hours under capacitating conditions related to the control sample (0 hour), capacitated and acrosome-reacted sperm were increased (p < 0.001). Frozen-thawed samples had a lower density and expression than the ejaculate samples. Expression was not obtained, except for IZUMO1, from samples that underwent in vitro capacitation/acrosome reactions. Expression of IZUMO1 was seen as an increasing band formation from the equatorial region through the acrosome, after in vitro capacitation. However, after the acrosome reaction, the band formation was only on the equatorial region. Region-specific differences of proteins at the kDa level were obtained using Western blot analysis and possible isoforms specific to ram spermatozoa or proteins with similar epitopes were expressed. Considering the changes in surface proteins in frozen-thawed sperm, it is suggested that fertilin ß and P34H can be used as fertility or freezability markers.


Assuntos
Fertilinas , Proteínas de Membrana , Capacitação Espermática , Espermatozoides , Desidrogenase do Álcool de Açúcar , Acrossomo , Reação Acrossômica , Animais , Imunoglobulinas , Masculino , Ovinos
3.
Reprod Biol ; 20(4): 589-594, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32972883

RESUMO

The a disintegrin and metalloprotease (ADAM) family proteins comprise a group of membrane-anchored proteins. ADAM32 is expressed specifically in testis and is closely related phylogenetically to ADAM2 and ADAM3, which are known to be critical for fertilization in mice. To assess the biological role of ADAM32, we analyzed Adam32-mutant mice. We found that male mice lacking ADAM32 have normal fertility, testicular integrity, and sperm characteristics. ADAM32 was found to exist at lower levels than ADAM2 and ADAM3 in wild-type testis and sperm, respectively. The present study demonstrates that ADAM32 is dispensable for fertility and appears to be functionally unrelated to ADAM2 and ADAM3 in mice.


Assuntos
Proteínas ADAM/deficiência , Proteínas ADAM/fisiologia , Fertilidade/fisiologia , Expressão Gênica/fisiologia , Testículo/metabolismo , Proteínas ADAM/análise , Proteínas ADAM/genética , Animais , Cruzamento , Epididimo/anatomia & histologia , Feminino , Fertilinas/análise , Masculino , Glicoproteínas de Membrana/análise , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Motilidade Espermática/fisiologia , Espermatozoides/química , Espermatozoides/fisiologia , Testículo/anatomia & histologia , Testículo/química
4.
Hum Fertil (Camb) ; 23(2): 123-133, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30463455

RESUMO

Selection of the best sperm, with the least defects, is a critical factor in the success of ART especially in male factor infertility. This study assessed the potential Heat shock protein (HSPA2) and metallopeptidase domain2 (ADAM2) biomarkers for sperm selection. Sperm were obtained from 72 asthenoteratozoospermic and 42 normospermic ejaculates. The semen characteristic, DNA fragmentation (DFI), chromatin maturation index (CMI), ADAM2 and HSPA2 levels on sperm, and their correlation with embryo quality were assessed in both groups. Results showed the significant reduction in HSPA2 and ADAM2 in asthenoteratozoospermic compared to normazoospermic ejaculates regarding the cut-off value of 14 and 13% for these two biomarkers. The specificity of HSPA2 and ADAM2 separately, and the combination of these two biomarkers, were 95.2, 90.5 and 93.5%, respectively, for sperm from normozoospermic ejaculates. However, they were 48.6, 50.0 and 54.5% for asthenoteratozoospermic ones. A significant correlation was observed with HSPA2, ADAM2 and a combination of these two biomarkers with CMI, DFI and embryo quality. Although a combination of these two biomarkers have the potential to be a good choice for selecting sperm with the lowest level of chromatin damage, it seems that selection according to HSPA2 has priority over ADAM2 or a combination of the two.


Assuntos
Fertilinas/genética , Proteínas de Choque Térmico HSP70/genética , Espermatozoides/fisiologia , Estudos de Casos e Controles , Fragmentação do DNA , Marcadores Genéticos , Humanos , Infertilidade Masculina/genética , Masculino , Técnicas de Reprodução Assistida , Análise do Sêmen
5.
Front Biosci (Landmark Ed) ; 24: 735-749, 2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30844709

RESUMO

Mammalian fertilization that culminates by fusion of the male and female gametes is intricately regulated within the female reproductive tract. To become competent to fertilize an egg, the mammalian spermatozoa that enter the female reproductive tract must undergo a series of physiological changes, including hyperactivation, and capacitation. For reaching full competency, the acrosome, a specialized membrane-bound organelle that covers the anterior part of the sperm head, must undergo an acrosome reaction. For becoming competent to bind an ovum, and to penetrate the zona pellucida and cumulus, many sperm proteins are released in the course of the acrosome reaction. Ultimately, the acrosome binds to the oolemma and fusion of sperm and egg occurs. In this review, we outline current understanding of the roles and effects of some essential sperm proteins and their functions during fertilization in the female reproductive tract.


Assuntos
Fertilização/fisiologia , Genitália Feminina/fisiologia , Espermatozoides/fisiologia , Reação Acrossômica , Animais , Antígenos/metabolismo , Moléculas de Adesão Celular/metabolismo , Feminino , Fertilinas/metabolismo , Humanos , Hialuronoglucosaminidase/metabolismo , Imunoglobulinas/metabolismo , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Receptores de Superfície Celular/metabolismo , Zona Pelúcida/metabolismo
6.
Biomed Pharmacother ; 109: 217-225, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30396079

RESUMO

Volatile anesthetics, including isoflurane, have been reported to have negative effects on cognitive dysfunction characterized by cognitive deficits following anesthesia. The aim of the current study was to investigate the effects involved with disintegrin and metallopeptidase domain 2 (ADAM2) silencing on isoflurane-induced cognitive dysfunction via the P13 K/Akt signaling pathway in immature rats. One week old healthy Sprague-Dawley (SD) rats were recruited and administered isoflurane anesthesia. The rats were then subjected to shADAM2 or wortmannin (PI3K/Akt signaling pathway inhibitor) to identify the effects of ADAM2 and the PI3K/Akt signaling pathway on the cognitive function of rats. Morris water maze and passive-avoidance tests were performed to examine the cognitive function of the rats. TUNEL staining was conducted to detect neuronal apoptosis in the hippocampal CA1 region. The obtained experimental results demonstrated that isoflurane anesthesia led to increased escape latency, reaction time, number of errors and TUNEL-positive neurons, along with a decreased latency time. In response to treatment with shADAM2, escape latency, reaction time, number of errors and TUNEL-positive cells were all noted to have decreased, in addition to elevated latency time, while contrasting trends were observed in regard to treatment with wortmannin. Taken together, the key findings of the present study revealed that shADAM2 activated the PI3K/Akt signaling pathway, resulting in elevated expressions of PI3K and Akt. Our study ultimately identified that ADAM2 silencing alleviates isoflurane-induced cognitive dysfunction by activating the P13 K/Akt signaling pathway in immature rats.


Assuntos
Disfunção Cognitiva/metabolismo , Fertilinas/antagonistas & inibidores , Fertilinas/metabolismo , Isoflurano/toxicidade , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fatores Etários , Anestésicos Inalatórios/toxicidade , Animais , Disfunção Cognitiva/induzido quimicamente , Feminino , Inativação Gênica , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Masculino , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia
7.
Brasília; CONITEC; dez. 2018. graf, ilus, tab.
Não convencional em Português | BRISA/RedTESA, BRISA/RedTESA | ID: biblio-997651

RESUMO

INTRODUÇÃO: A púrpura trombocitopênica idiopática crônica (PTI), atualmente denominada trombocitopenia imune primária (TIP) é uma doença autoimune adquirida, de causa desconhecida, caracterizada predominantemente pela presença isolada de trombocitopenia (baixas contagens de plaquetas). Fisiologicamente, observa-se a destruição aumentada das plaquetas causada por autoanticorpos contra os antígenos das plaquetas e por linfócitos T citotóxicos, associada a uma produção prejudicada de plaquetas pela medula óssea. Não existe exame laboratorial específico para o diagnóstico de PTI, sendo feito diagnóstico por exclusão: presença de trombocitopenia isolada, sem alterações nas outras séries do hemograma e no esfregaço de sangue periférico; e a exclusão de outras causas de trombocitopenia. O tratamento é recomendado apenas para pacientes com trombocitopenia grave (plaquetas < 20 x 109 /L) ou àqueles com sangramentos associados à trombocitopenia (plaquetas < 50 x 109 /L) e tem como objetivo controlar precocemente os sintomas, diminuir o risco de sangramento e causar menos impacto na qualidade de vida. Os agonistas do receptor de trombopoietina (TPO) aumentam a produção de plaquetas estimulando o receptor de TPO em pessoas com ITP crônica. Segundo o Protocolo Clínico e Diretrizes Terapêuticas - púrpura trombocitopênica idiopática (PCDT - PTI) do Ministério da Saúde, de 2013, as práticas de tratamento atuais para pacientes adultos com PTI crônica devem ter a seguinte abordagem: corticosteroides, imunoglobulina humana intravenosa, esplenectomia, azatioprina, ciclofosfamida, danazol e vincristina. TECNOLOGIA: Romiplostim (Nplate®). PERGUNTA: O romiplostim é seguro, eficaz e custo-efetivo em pacientes com PTI crônica, esplenectomizados, refratários e em alto risco de sangramento, quando comparado ao tratamento atual disponível no SUS? EVIDÊNCIAS CIENTÍFICAS: Baseada em dois ensaios clínicos randomizados e uma revisão sistemática da Cochrane (2011) com 6 ensaios clínicos randomizados (ECR) que avaliaram a eficácia e segurança dos agonistas do receptor de TPO em pacientes com PTI crônica. O ECR realizado por Bussel et. al. (2006) demostrou que o romiplostim causa um aumento significativo na contagem de plaquetas quando comparado ao placebo (RR= 3,00; IC95% 0,54 - 16,77), achado que foi corroborado pelo estudo de Kuter et. al. (2008) que encontrou taxas de duração da resposta plaquetária significativamente maiores (RR= 16,88; IC95% 1,06 - 268,42) e melhora significativa na taxa de resposta plaquetária global (RR= 34,28; IC95% 2,20 ­ 533,41), quando comparado o romiplostim com o placebo. A revisão sistemática confirmou esse achado, visto que, na análise combinada o uso de agonistas do receptor de TPO (romiplostim e eltrombopague) apresentou melhora significativa nas taxas de resposta plaquetária global comparado ao placebo (RR= 4,06; IC95% 2,93 - 5,63; valor de p< 0,00001) e ao tratamento padrão (RR= 1,81; IC95% 1,37 - 2,37; valor de p< 0,0001) , e maiores taxas de duração da resposta no número de plaquetas, com significância estatística, comparado ao placebo (RR= 14,16; IC95% 2,91 - 69,01; valor de p=0,001). Apesar desse aumento significativo na contagem de plaquetas, a RS demonstrou que não houve diferença significativa para incidência de eventos hemorrágicos importantes (classificados como graves, com risco de morte ou fatais) na PTI crônica quando comparado com placebo (RR= 0,48; IC95% 0,19 - 1,16; valor de p=0,10) ou tratamento padrão (RR= 0,49; IC95% 0,15 - 1,63; valor de p=0,24), resultado também encontrado no estudo de Kuter et. al. (2008) comparado com o placebo (RR= 0,50; IC95% 0,14 - 1,80). Nenhum dos estudos incluiu sobrevida global como desfecho, portanto, não foi possível avaliar se o romiplostim ajuda a prolongar a vida dos pacientes. Embora faltem estudos de longo prazo, a RS demonstrou que os agonistas do receptor de TPO são bem tolerados, apresentando o total de efeitos adversos semelhantes ao do grupo placebo (RR= 1,04; IC95% 0,95 - 1,15; valor de p=0,35). No que se refere ao total de eventos adversos graves, os 3 estudos não encontraram diferença significativa entre o romiplostim comparado ao placebo (RR= 0,12, IC 95% 0,01 - 1,00) (Bussel, 2006), (RR= 0,94, IC 95% 0,48 - 1,85) (Kuter, 2008) e (RR= 0,92; IC95% 0,61 - 1,38; valor de p= 0,68) (Zeng, 2011), entretanto a RS demonstrou uma redução significativa entre o romiplostim e o tratamento padrão (RR= 0,61; IC95% 0,40 - 0,92; valor de p=0,02). Não existe evidência suficiente para afirmar a eficácia do romiplostim na PTI crônica, sendo necessários mais estudos para explorar o papel desse medicamento no tratamento da PTI crônica de forma mais completa. AVALIAÇÃO ECONÔMICA: O demandante realizou avaliação de custo-utilidade, com modelo de Markov, com ciclo de 4 semanas, em um horizonte temporal de até o fim da vida, na perspectiva do SUS. Foram definidos os seguintes parâmetros: população de pacientes esplenectomizados, com PTI crônica, recebendo um tratamento a partir de segunda linha, com contagem de plaqueta (CP) < 20x109/L; possibilidade de 3 estados de saúde abrangentes (Plaquetas ≥ 50×109/L; Plaquetas < 50×109/L; Morto); comparação entre romiplostim e a terapia de regaste usada nos pacientes em observação e sem tratamento (imunoglobulina intravenosa - IVig); resposta ao tratamento (CP > 50x109/L); eficácia de cada tratamento (probabilidade de resposta inicial ao tratamento; tempo médio à resposta (CP > 50x109/L); tempo até a falha do tratamento (CP < 50x109/L durante 4 semanas consecutivas)); e desfechos primários (custos incrementais incorridos e os anos de vida ajustados por qualidade (QALYs) ganhos a partir da introdução do romiplostim). Os custos foram avaliados em Reais (R$) com base nos anos 2016 e 2017, extraídos do banco de dados SIGTAP do Ministério da Saúde (MS), lista de preço de medicamentos publicada pela Câmara de Regulação do Mercado de Medicamentos e parecer de especialistas locais. O custo do tratamento para cada intervenção incluiu: custos de compra dos medicamentos, administração de testes laboratoriais e monitoramento. Taxa de desconto de 5% para efetividade e custos. A razão de custo-efetividade incremental (RCEI) foi de R$ - 121.213,45 /QALY, mostrando que o tratamento da PTI com o romiplostim quando comparado com o grupo de pacientes em observação e sem tratamento é uma estratégia custo-efetiva (dominante). Foram realizadas análises de sensibilidade determinista e probabilística cujos resultados demonstraram que a RCEI foi robusta, visto que a estratégia de tratamento com o romiplostim continuou dominante, mesmo com a variação dos valores dos parâmetros utilizados no modelo. AVALIAÇÃO DE IMPACTO ORÇAMENTÁRIO: Foi estimada considerando duas possibilidades de prevalência de PTI, uma de "baixa prevalência" e outra de "alta prevalência", e dois panoramas de difusão da tecnologia, um conservador e outro otimista, totalizando a possibilidade de 4 diferentes cenários. Como resultado, o demandante encontrou que, durante 5 anos, em todos os quatro cenários, a incorporação do romiplostim resultaria em economias de R$ 49.386.749 a R$ 196.521.197 no cenário de "alta prevalência" e de R$ 49.725.986 a R$ 79.164.764 no cenário de "baixa prevalência". Na análise de sensibilidade unilateral, a incorporação do romiplostim permaneceu resultando em economias, mesmo com a variação dos valores dos dois parâmetros direcionadores do modelo. MONITORAMENTO DO HORIZONTE TECNOLÓGICO: Há tecnologias em fase de desenvolvimento clínico para tratamento de púrpura trombocitopênica idiopática. Porém, essas novas tecnologias ainda não tiveram seu registro aprovado pela Anvisa para a indicação clínica pesquisada. RECOMENDAÇÃO PRELIMINAR DA CONITEC: Os membros do Plenário presentes em sua 65ª reunião ordinária, no dia 04 de abril de 2018, indicaram que o tema seja submetido à Consulta Pública com recomendação preliminar a não incorporação no SUS do romiplostim para PTI crônica e refratária em alto risco de sangramento. Considerou-se que apesar do aumento significativo na contagem de plaquetas, a evidência atualmente disponível não foi suficiente para afirmar que o romiplostim reduz significativamente a incidência de eventos hemorrágicos importantes, comparado ao placebo e ao tratamento padrão e que os estudos econômicos apresentaram fragilidades, sobretudo, devido ao comparador utilizado. CONSULTA PÚBLICA: Foram recebidas 7 contribuições técnico-científicas e 14 contribuições de experiência ou opinião, sendo que todas discordaram totalmente da recomendação preliminar da CONITEC. De maneira geral, as contribuições apresentarem oito artigos e alguns comentários acerca: do PCDT vigente, do desfecho avaliado e da população alvo, entretanto não acrescentaram e nem mudaram os aspectos anteriormente discutidos e apresentados no relatório inicial da CONITEC. Assim, a CONITEC entendeu que não houve argumentação suficiente para alterar sua recomendação preliminar. RECOMENDAÇÃO FINAL: Os membros da CONITEC em 08/11/2018 deliberaram por unanimidade recomendar a não incorporação do romiplostim para tratamento de púrpura trombocitopênica idiopática, ao SUS. Foi assinado o Registro de Deliberação nº 393/2018. DECISÃO: Não incorporar o romiplostim para púrpura trombocitopênica idiopática (PTI) crônica e refratária em alto risco de sangramento, no âmbito do Sistema Único de Saúde - SUS. Dada pela Portaria nº 69, publicada no DOU nº 238, seção 1, página 71, em 11 de dezembro de 2018.


Assuntos
Humanos , Peptídeos/administração & dosagem , Púrpura Trombocitopênica Idiopática/complicações , Púrpura Trombocitopênica Idiopática/tratamento farmacológico , Fertilinas/uso terapêutico , Avaliação da Tecnologia Biomédica , Avaliação em Saúde/economia , Sistema Único de Saúde , Brasil , Análise Custo-Benefício/economia
8.
Int J Mol Med ; 40(4): 1105-1113, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28765881

RESUMO

The degradation of cruciate ligaments is frequently observed in degenerative joint diseases, such as osteo-arthritis (OA). The present study aimed to identify the differentially expressed microRNAs (miRNAs or miRs) in knee anterior cruciate ligament (ACL) tissues derived from patients with OA and in health subjects (non-OA). By using Affymetrix miRNA 4.0 microarrays, a total of 22 miRNAs (including let-7f-5p, miR-26b-5p and miR-146a-5p) were found to be upregulated, while 17 (including miR-18a-3p, miR-138-5p and miR-485-3p) were downregulated in the osteoarthritic ACL tissues (fold change ≥2, P-value <0.05). The expression levels of 12 miRNAs were validated by quantitative PCR, and the corresponding results revealed an excellent correlation with the microarray data (R2=0.889). Genes (such as a disintegrin and metalloproteinase domain with thrombospondin type-1 motifs, bone morphogenetic protein-2, runt related transcription factor-2, collagen-1A1 and 2, interleukin-6 and transforming growth factor-ß) involved in cartilage development and remodeling, collagen biosynthesis and degradation, inflammatory response and extracellular matrix homeostasis were predicted as potential targets of the dysregulated miRNAs. Moreover, a large set of putative genes were enriched in OA pathogenesis­associated pathways (such as mitogen-activated protein kinase and vascular endothelial growth factor signaling pathway). Collectively, the data from our study provides novel insight into the ligament injury-related miRNA dysregulation in patients with OA.


Assuntos
Ligamento Cruzado Anterior/metabolismo , Regulação da Expressão Gênica , Articulação do Joelho/metabolismo , MicroRNAs/genética , Osteoartrite/genética , Idoso , Ligamento Cruzado Anterior/patologia , Ligamento Cruzado Anterior/cirurgia , Proteína Morfogenética Óssea 2/genética , Proteína Morfogenética Óssea 2/metabolismo , Estudos de Casos e Controles , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Feminino , Fertilinas/genética , Fertilinas/metabolismo , Perfilação da Expressão Gênica , Ontologia Genética , Humanos , Interleucina-6/genética , Interleucina-6/metabolismo , Articulação do Joelho/patologia , Articulação do Joelho/cirurgia , Masculino , MicroRNAs/classificação , MicroRNAs/metabolismo , Pessoa de Meia-Idade , Anotação de Sequência Molecular , Análise de Sequência com Séries de Oligonucleotídeos , Osteoartrite/metabolismo , Osteoartrite/patologia , Osteoartrite/cirurgia , Reação em Cadeia da Polimerase em Tempo Real , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismo
9.
Acta Biochim Biophys Sin (Shanghai) ; 48(11): 1058-1065, 2016 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-27649891

RESUMO

PRSS37, a putative trypsin-like serine protease, is highly conserved during mammalian evolution as revealed by multiple sequence alignment. Mice deficient for Prss37 gene exhibit male infertility, but their mating behavior, spermatogenesis, sperm morphology, and motility remain unaffected, similar to a situation called unexplained male infertility (UMI) in men (human being). Here, we demonstrated that PRSS37 is restrictively expressed in human testis, where it is mainly located in the elongating and elongated spermatids during spermiogenesis as shown by immunohistochemical analysis of normal human testicular sections. In mature sperm, PRSS37 appears in the acrosome region and diminishes during acrosome reaction. Further examination reveals that PRSS37 contents in sperm from patients with UMI are dramatically lower than those in sperm from men with proven fertility or from sperm donors. Sperm with low PRSS37 contents exhibit abnormal activation of the proacrosin/acrosin system and premature proteolysis of ADAM2, which may impair the functional competence of human sperm in vivo However, the in vitro fertilization outcomes of sperm with low PRSS37 contents are not affected. Together, these data implicate an important role of PRSS37 for male fertility. PRSS37 can be used as a potential molecular biomarker for evaluating sperm fertilization capability in vivo but not in vitro.


Assuntos
Infertilidade Masculina/metabolismo , Serina Proteases/metabolismo , Espermatozoides/metabolismo , Acrossomo/metabolismo , Reação Acrossômica , Estudos de Casos e Controles , Fertilinas/metabolismo , Fertilização In Vitro , Perfilação da Expressão Gênica , Humanos , Masculino , Proteólise , Serina Proteases/genética
10.
PLoS One ; 11(6): e0158321, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27341348

RESUMO

The members of the ADAM (a disintegrin and metalloprotease) family are membrane-anchored multi-domain proteins that play prominent roles in male reproduction. ADAM2, which was one of the first identified ADAMs, is the best studied ADAM in reproduction. In the male germ cells of mice, ADAM2 and other ADAMs form complexes that contribute to sperm-sperm adhesion, sperm-egg interactions, and the migration of sperm in the female reproductive tract. Here, we generated specific antibodies against mouse and human ADAM2, and investigated various features of ADAM2 in mice, monkeys and humans. We found that the cytoplasmic domain of ADAM2 might enable the differential association of this protein with other ADAMs in mice. Western blot analysis with the anti-human ADAM2 antibodies showed that ADAM2 is present in the testis and sperm of monkeys. Monkey ADAM2 was found to associate with chaperone proteins in testis. In humans, we identified ADAM2 as a 100-kDa protein in the testis, but failed to detect it in sperm. This is surprising given the results in mice and monkeys, but it is consistent with the failure of ADAM2 identification in the previous proteomic analyses of human sperm. These findings suggest that the reproductive functions of ADAM2 differ between humans and mice. Our protein analysis showed the presence of potential ADAM2 complexes involving yet-unknown proteins in human testis. Taken together, our results provide new information regarding the characteristics of ADAM2 in mammalian species, including humans.


Assuntos
Fertilinas/metabolismo , Mamíferos/metabolismo , Espermatozoides/metabolismo , Sequência de Aminoácidos , Animais , Cromatografia Líquida , Fertilinas/química , Fertilinas/genética , Humanos , Macaca fascicularis , Masculino , Camundongos , Domínios e Motivos de Interação entre Proteínas , Espectrometria de Massas em Tandem , Testículo/metabolismo
11.
Reproduction ; 151(5): 491-500, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-26860122

RESUMO

Head-to-head agglutination of ram spermatozoa is induced by dilution in the Tyrode's capacitation medium with albumin, lactate and pyruvate (TALP) and ameliorated by the addition of the thiol d-penicillamine (PEN). To better understand the association and disassociation of ram spermatozoa, we investigated the mechanism of action of PEN in perturbing sperm agglutination. PEN acts as a chelator of heavy metals, an antioxidant and a reducing agent. Chelation is not the main mechanism of action, as the broad-spectrum chelator ethylenediaminetetraacetic acid and the copper-specific chelator bathocuproinedisulfonic acid were inferior anti-agglutination agents compared with PEN. Oxidative stress is also an unlikely mechanism of sperm association, as PEN was significantly more effective in ameliorating agglutination than the antioxidants superoxide dismutase, ascorbic acid, α-tocopherol and catalase. Only the reducing agents cysteine and DL-dithiothreitol displayed similar levels of non-agglutinated spermatozoa at 0 h compared with PEN but were less effective after 3 h of incubation (37 °C). The addition of 10 µM Cu(2+) to 250 µM PEN + TALP caused a rapid reversion of the motile sperm population from a non-agglutinated state to an agglutinated state. Other heavy metals (cobalt, iron, manganese and zinc) did not provoke such a strong response. Together, these results indicate that PEN prevents sperm association by the reduction of disulphide bonds on a sperm membrane protein that binds copper. ADAM proteins are possible candidates, as targeted inhibition of the metalloproteinase domain significantly increased the percentage of motile, non-agglutinated spermatozoa (52.0% ± 7.8) compared with TALP alone (10.6% ± 6.1).


Assuntos
Proteínas de Transporte/metabolismo , Quelantes/farmacologia , Cobre/farmacologia , Dissulfetos/química , Penicilamina/farmacologia , Aglutinação Espermática/efeitos dos fármacos , Animais , Dissulfetos/metabolismo , Fertilinas/metabolismo , Masculino , Ovinos
12.
Urology ; 86(6): 1241.e1-9, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26408501

RESUMO

OBJECTIVE: To determine whether insulin-like growth factor (IGF-1) deficiency can cause testicular damage and to examine changes of the testicular morphology and testicular function-related gene expression caused by IGF-1 deficiency. Therefore, this study aims to determine the benefits of low doses of IGF-1 and to explore the mechanisms underlying the IGF-1 replacement therapy. MATERIALS AND METHODS: A murine model of IGF-1 deficiency was used to avoid any factor that could contribute to testicular damage. Testicular weight, score of histopathological damage, and gene expressions were studied in 3 experimental groups of mice: controls (wild-type Igf1(+/+)), heterozygous Igf1(+/-) with partial IGF-1 deficiency, and heterozygous Igf1(+/-) treated with IGF-1. RESULTS: Results show that the partial IGF-1 deficiency induced testicular damage and altered expression of genes involved in IGF-1 and growth hormone signaling and regulation, testicular hormonal function, extracellular matrix establishment and its regulation, angiogenesis, fibrogenesis, inflammation, and cytoprotection. In addition, proteins involved in tight junction expression were found to be reduced. However, low doses of IGF-1 restored the testicular damage and most of these parameters. CONCLUSION: IGF-1 deficiency caused the damage of the blood-testis barrier and testicular structure and induced the abnormal testicular function-related gene expressions. However, low doses of IGF-1 constitute an effective replacement therapy that restores the described testicular damage. Data herein show that (1) cytoprotective activities of IGF-1 seem to be mediated by heat shock proteins and that (2) connective tissue growth factor could play a relevant role together with IGF-1 in the extracellular matrix establishment.


Assuntos
Barreira Hematotesticular/química , Proteínas da Matriz Extracelular/genética , Expressão Gênica/efeitos dos fármacos , Fator de Crescimento Insulin-Like I/deficiência , Fator de Crescimento Insulin-Like I/farmacologia , Proteoglicanas/genética , Testículo/patologia , Testículo/fisiopatologia , Proteínas ADAM/genética , Animais , Antígenos CD18/genética , Caderinas/análise , Fator de Crescimento do Tecido Conjuntivo/genética , Citocromo P-450 CYP3A/genética , Modelos Animais de Doenças , Fertilinas , Expressão Gênica/genética , Genótipo , Inibinas/genética , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina/genética , Fator de Crescimento Insulin-Like I/genética , Masculino , Glicoproteínas de Membrana/genética , Metaloproteases/genética , Camundongos , Tamanho do Órgão , Receptor IGF Tipo 1/genética , Receptores do FSH/análise , Receptores da Somatotropina/análise , Receptores da Somatotropina/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Testículo/química , Junções Íntimas/química , Inibidor Tecidual de Metaloproteinase-1/genética , Fator de Crescimento Transformador alfa/genética , Fator de Crescimento Transformador beta/genética , Fator A de Crescimento do Endotélio Vascular/genética , Proteína da Zônula de Oclusão-1/análise , beta Catenina/análise
13.
PLoS One ; 10(8): e0134967, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26252478

RESUMO

Immunotherapy is emerging as a supplement to conventional cancer treatment, and identifying antigen targets for specific types of cancer is critical to optimizing therapeutic efficacy. Cancer/testis antigens are highly promising targets for immunotherapy due to their cancer-specific expression and antigenic properties, but the expression patterns of most of the more than 200 identified cancer/testis antigens in various cancers remain largely uncharacterized. In this study, we investigated the expression of the cancer/testis antigens ADAM2, CALR3 and SAGE1 in lung and breast cancer, the two most frequent human cancers, with the purpose of providing novel therapeutic targets for these diseases. We used a set of previously uncharacterized antibodies against the cancer/testis antigens ADAM2, CALR3 and SAGE1 to investigate their expression in a large panel of normal tissues as well as breast and lung cancers. Staining for the well-characterized MAGE-A proteins was included for comparison. Immunohistochemical staining confirmed previous mRNA analysis demonstrating that ADAM2, CALR3 and SAGE1 proteins are confined to testis in normal individuals. Negative tissues included plancenta, which express many other CT antigens, such as MAGE-A proteins. Surprisingly, we detected no ADAM2, CALR3 and SAGE1 in the 67 lung cancers (mainly non-small lung cancer) and 189 breast cancers, while MAGE-A proteins were present in 15% and 7-16% of these tumor types, respectively. Treatment with DNA methyltransferase inhibitors has been proposed as an attractive strategy to increase the expression of cancer/testis antigens in tumors before immunotargeting; however, neither ADAM2, CALR3 nor SAGE1 could be significantly induced in lung and breast cancer cell lines using this strategy. Our results suggest that ADAM2, CALR3 and SAGE1 cancer/testis antigens are not promising targets for immunotherapy of breast and lung cancer.


Assuntos
Proteínas ADAM/metabolismo , Antígenos de Neoplasias/metabolismo , Neoplasias da Mama/metabolismo , Calreticulina/metabolismo , Neoplasias Pulmonares/metabolismo , Glicoproteínas de Membrana/metabolismo , Azacitidina/farmacologia , Azacitidina/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Estudos de Coortes , Feminino , Fertilinas , Humanos , Imuno-Histoquímica , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Antígenos Específicos de Melanoma/metabolismo
14.
J Reprod Immunol ; 112: 38-45, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26226211

RESUMO

The a2 isoform of vacuolar-ATPase (ATP6V0A2, referred to as a2V) is required for normal spermatogenesis and maturation of sperm. Treatment of male mice with anti-a2V disturbs the testicular cytokine/chemokine balance and leads to severe deficiencies of spermatogenesis. The aim of the present study was to investigate the role of a2V in male fertility and in the regulation of apoptotic pathways required for normal spermatogenesis in mice. To study the role of a2V single dose of anti-a2V monoclonal antibody or mouse IgG isotype (3µg/animal) was injected i.p. into males on alternate days for 10 days. The expression of sperm maturation-related molecules and pro-apoptotic molecules was measured by real-time PCR or immunohistochemistry in control and anti-a2V-treated testes. The caspase levels and their activity were measured by western blot and fluorometry. We found that the expression of the sperm maturation-related molecules SPAM1, ADAM1, and ADAM2 was significantly decreased in testes from anti-a2V-treated males. The expression of pro-apoptotic molecules (Bax, p53, and p21) and molecules involved in the intrinsic pathway of apoptosis (caspase-9, caspase-3, and PARP), which are crucial for normal spermatogenesis was significantly reduced in testes from anti-a2V-treated males compared with the control. The total ATP level was significantly lower in anti-a2V-treated testes. The data provide novel evidence showing that a2V can regulate the apoptotic pathways, an essential testicular feature, and is necessary for efficient spermatogenesis.


Assuntos
Apoptose/imunologia , Fertilidade/imunologia , ATPases Translocadoras de Prótons/imunologia , Espermatogênese/imunologia , Espermatozoides/imunologia , Proteínas ADAM/imunologia , Animais , Anticorpos Monoclonais Murinos/imunologia , Anticorpos Monoclonais Murinos/farmacologia , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/imunologia , Caspases/imunologia , Moléculas de Adesão Celular/imunologia , Fertilinas , Fertilidade/efeitos dos fármacos , Hialuronoglucosaminidase/imunologia , Masculino , Glicoproteínas de Membrana/imunologia , Camundongos , Camundongos Endogâmicos BALB C , ATPases Translocadoras de Prótons/antagonistas & inibidores , Espermatogênese/efeitos dos fármacos
15.
Reprod Fertil Dev ; 25(5): 807-17, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-22951102

RESUMO

Although it is well known that mRNA is present in mammalian spermatozoa, the relevance of mRNA to capacitation and early embryo development in the pig remains unclear. In the present study, we investigated differences in the abundance of selected mRNAs coding for MYC, CYP19, ADAM2, PRM1 and PRM2 in purified porcine spermatozoa depending on embryo cleavage rate and capacitation (n=20 semen samples). Semen samples were used in IVF procedures, with subsequent embryo development classified into one of two groups based on cleavage rate (i.e. high (>75%) and low (<75%) cleavage groups) and mRNA abundance in purified spermatozoa compared between these two groups. In addition, mRNA abundance was compared between capacitated and non-capacitated spermatozoa. Comparison of mRNA levels between porcine spermatozoa revealed that the abundance of MYC, CYP19, ADAM2, PRM1 and PRM2 mRNA was significantly greater in the high cleavage group (n=10 high cleavage group semen samples) than in the low cleavage group (n=10; P<0.05). Significant downregulation of MYC mRNA was observed in capacitated spermatozoa (n=12; P<0.05). The results of the present study suggest that the amount of specific mRNAs could be used for estimating the quality of spermatozoa in the pig.


Assuntos
Regulação da Expressão Gênica/fisiologia , RNA Mensageiro/metabolismo , Capacitação Espermática/fisiologia , Espermatozoides/citologia , Suínos/embriologia , Suínos/genética , Proteínas ADAM/genética , Animais , Aromatase/genética , Primers do DNA/genética , Fertilinas , Regulação da Expressão Gênica/genética , Genes myc/genética , Modelos Lineares , Masculino , Glicoproteínas de Membrana/genética , Protaminas/genética , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária , Espermatozoides/metabolismo
16.
Mol Biol Rep ; 40(2): 787-96, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23065232

RESUMO

ADAM2, a member of the 'a disintegrin and metalloprotease' (ADAM) family, is a key protein in mammalian fertilization that is specifically expressed in testicular germ cells. Here, we investigated the transcriptional regulation of the mouse Adam2 gene. An in silico analysis identified two conserved non-coding sequences located upstream of the mouse and human ADAM2 genes. The upstream region of the mouse Adam2 gene was found to lack typical TATA and CAAT boxes, and to have a high GC content. Our in vitro transient transfection-reporter analysis identified a promoter in this region of the mouse Adam2 gene, along with regulatory regions that inhibit the activity of this promoter in somatic cells. Site-directed mutagenesis revealed that the caudal-type homeobox 1 and CCTC-binding factor motifs are responsible for the inhibitory activities of the repressor regions. Finally, electrophoretic mobility shift assays showed putative transcription factor-promoter DNA complexes, and DNA-affinity chromatography and proteomic analyses identified myelin gene regulatory factor as a binding partner of the Adam2 promoter. This provides the first identification and characterization of promoter and repressor regions that regulate the transcription of the mouse Adam2 gene, and offers insights into the regulation of this germ-cell-specific gene.


Assuntos
Proteínas ADAM/genética , Regulação Enzimológica da Expressão Gênica , Glicoproteínas de Membrana/genética , Regiões Promotoras Genéticas , Proteínas ADAM/metabolismo , Animais , Sequência de Bases , Sítios de Ligação , Ensaio de Desvio de Mobilidade Eletroforética , Fertilinas , Genes Reporter , Células HEK293 , Humanos , Luciferases de Renilla/biossíntese , Luciferases de Renilla/genética , Masculino , Glicoproteínas de Membrana/metabolismo , Camundongos , Anotação de Sequência Molecular , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Células NIH 3T3 , Ligação Proteica , Análise de Sequência de DNA , Testículo/citologia , Fatores de Transcrição/metabolismo
17.
Bull Exp Biol Med ; 153(4): 513-5, 2012 Aug.
Artigo em Inglês, Russo | MEDLINE | ID: mdl-22977858

RESUMO

We studied the relationship between the levels of protamines 1 and 2 (PRM1 and PRM2) and fertilin-ß (ADAM-2) mRNA expression and outcomes of infertility treatment using assisted reproductive technologies was studied. Analysis of the relationships between the outcomes of in vitro fertilization and embryo transfer and profiles of the expression of seminal genes PRM1, PRM2, ADAM-2 mRNA, evaluated by reverse transcription quantitative PCR was carried out in 79 couples. Significant differences in the expression of seminal PRM1, PRM2, ADAM-2 mRNA were detected in couples with different outcomes of in vitro fertilization and embryo transfer. The levels of seminal gene expression are potential predictors of the efficiency of in vitro fertilization and embryo transfer.


Assuntos
Proteínas ADAM/metabolismo , Biomarcadores/metabolismo , Transferência Embrionária , Fertilização In Vitro , Infertilidade/terapia , Glicoproteínas de Membrana/metabolismo , Protaminas/metabolismo , Proteínas ADAM/genética , Fertilinas , Humanos , Glicoproteínas de Membrana/genética , Protaminas/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Estatísticas não Paramétricas , Resultado do Tratamento
18.
Cancer Lett ; 326(1): 79-87, 2012 Dec 29.
Artigo em Inglês | MEDLINE | ID: mdl-22841667

RESUMO

It has been proposed that gliomas contain a subpopulation of 'Brain Tumor Stem Cells' (BTSCs), which demonstrate resistance to conventional therapies. A potential component of the environment governing the behavior of these BTSCs is a class of transmembrane proteins with structural and signaling functions, the A-Disintegrin And Metalloproteases (ADAMs). In this study we confirm overexpression of ADAM10 and 17 in human glioma tissue compared to human controls, and especially in tumor sphere cultures thought to enrich for BTSCs. Inhibition of ADAM10/17 function impairs the growth of tumor spheres with evidence of depletion of the sphere forming cell population. This results from a combination of reduced proliferation, cell death and a switch of sphere-forming cells away from symmetric self-renewal division towards neuronal differentiation. A developing appreciation of the role of ADAMs in BTSC promises insights into pathophysiology and potential therapeutic avenues in this intractable group of tumors.


Assuntos
Proteínas ADAM/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Neoplasias Encefálicas/metabolismo , Glioma/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/metabolismo , Células-Tronco Neoplásicas/metabolismo , Esferoides Celulares , Células Tumorais Cultivadas , Proteínas ADAM/antagonistas & inibidores , Proteína ADAM10 , Diferenciação Celular , Proliferação de Células , Sobrevivência Celular , Fertilinas , Humanos
19.
Reprod Biol Endocrinol ; 9: 96, 2011 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-21718510

RESUMO

Fertilin alpha (ADAM-1) and beta (ADAM-2) are integral membrane proteins of the ADAM family that form a fertilin complex involved in key steps of the sperm-oocyte membrane interaction. In the present work, we analyzed the presence of ADAM-1 and ADAM-2 mRNAs, the spermatozoa proteins' processing and their sub-cellular localization in epididymal samples from adult boars. ADAM-1 and ADAM-2 mRNAs were highly produced in the testis, but also in the vas efferens and the epididymis. On immunoblots of sperm extracts, ADAM-1 subunit appeared as a main reactive band of ~50-55 kDa corresponding to occurrence of different isoforms throughout the epididymal duct, especially in the corpus region where isoforms ranged from acidic to basic pI. In contrast, ADAM-2 was detected as several bands of ~90 kDa, ~75 kDa, ~50-55 kDa and ~40 kDa. The intensity of high molecular mass bands decreased progressively in the distal corpus where lower bands were also transiently observed, and only the ~40 kDa was observed in the cauda. The presence of bands of different molecular weights likely results from a proteolytic processing occurring mainly in the testis for ADAM-1, and also throughout the caput epididymis for ADAM-2. Immunolocalization showed that fertilin migrates from the acrosomal region to the acrosomal ridge during the sperm transit from the distal corpus to the proximal cauda. This migration is accompanied by an important change in the extractability of a part of ADAM-1 from the sperm membrane. This suggests that the fertilin surface migration may be triggered by the biochemical changes induced by the epididymal post-translational processing of both ADAM1 and ADAM-2. Different patterns of fertilin immunolocalization then define several populations of spermatozoa in the cauda epididymis. Characterization of such fertilin complex maturation patterns is an important step to develop fertility markers based on epididymal maturation of surface membrane proteins in domestic mammals.


Assuntos
Proteínas ADAM/metabolismo , Epididimo/crescimento & desenvolvimento , Glicoproteínas de Membrana/metabolismo , Espermatozoides/metabolismo , Sequência de Aminoácidos , Animais , Fertilinas , Masculino , Dados de Sequência Molecular , Maturação do Esperma , Suínos , Testículo/metabolismo
20.
J Cell Physiol ; 226(11): 2817-26, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21302280

RESUMO

Proprotein convertase subtilisin/kexin 4 (PCSK4) is implicated for sperm fertilizing ability, based on studies using Pcsk4-null mice. Herein we demonstrated proprotein convertase (PC) activity in intact sperm and acrosomal vesicles. To determine whether this activity was important for sperm fertilizing ability, a peptide inhibitor was designed based on PCSK4 prodomain sequence (proPC4(75-90)), which contains its primary autocatalytic cleavage site. ProPC4(75-90) inhibited recombinant PCSK4's activity with a K(i) value of 5.4 µM, and at 500 µM, it inhibited sperm PC activity almost completely. Treatment of sperm with proPC4(75-90) inhibited their egg fertilizing ability in a dose dependent manner. Correlation between sperm PC activity and fertilizing ability showed a high co-efficient value (>0.9), indicating the importance of sperm PC activity in fertilization. In particular, sperm PC activity was important for capacitation and zona pellucida (ZP)-induced acrosome reaction, since proPC4(75-90) -treated sperm showed markedly decreased rates in these two events. These results were opposite to those observed in Pcsk4-null sperm, which contained higher PC activity than wild type sperm, possibly due to overcompensation by PCSK7, the other PCSK enzyme found in sperm. ADAM2 (45 kDa), a sperm plasma membrane protein, involved in sperm-egg plasma membrane interaction, was also processed into a smaller form (27 kDa) during capacitation at a much reduced level in proPC4(75-90) -treated sperm. This result suggested that ADAM2 may be a natural substrate of sperm PCSK4 and its cleavage by the enzyme during acrosome reaction may be relevant to the fertilization process.


Assuntos
Proteínas ADAM/metabolismo , Fertilização , Glicoproteínas de Membrana/metabolismo , Serina Endopeptidases/metabolismo , Espermatozoides/enzimologia , Acrossomo/efeitos dos fármacos , Acrossomo/enzimologia , Reação Acrossômica/efeitos dos fármacos , Clorometilcetonas de Aminoácidos/farmacologia , Animais , Inibidores Enzimáticos/farmacologia , Feminino , Fertilinas , Masculino , Camundongos , Camundongos Knockout , Pró-Proteína Convertases , Capacitação Espermática/efeitos dos fármacos , Espermatozoides/efeitos dos fármacos , Especificidade por Substrato , Subtilisinas , Zona Pelúcida/efeitos dos fármacos , Zona Pelúcida/enzimologia
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