Papel da EPPIN (Epididymal protease inhibitor) sobre a maturação pós-testicular de espermatozoides de camundongos: consequências para a função espermática.
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Data
2021-09-09
Autores
Silva, Alan Andrew S. [UNESP]
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Universidade Estadual Paulista (Unesp)
Resumo
Cerca de 40% das 215 milhões de gestações que ocorrem anualmente no planeta não são planejadas pelos casais, das quais 20% acabam em abortamentos. Este cenário reforça a necessidade do desenvolvimento de novos métodos contraceptivos, em especial os masculinos, cujas opções atuais limitam-se ao preservativo e vasectomia. A EPPIN (Epididymal protease inhibitor) é uma proteína rica em resíduos de cisteína que contém em sua sequência primária os domínios inibidores de protease do tipo WFDC (N-terminal) e Kunitz (C-terminal). A EPPIN é encontrada na superfície do espermatozoide, onde exerce um papel fundamental essencial na função do gameta masculino. A relevância da EPPIN como alvo espermático para contracepção masculina foi demonstrada pelos efeitos inibitórios de anticorpos anti-EPPIN e de ligantes orgânicos de baixo peso molecular sobre a motilidade do espermatozoide humano e de primatas não-humanos. O desenvolvimento de fármacos contraceptivos com alto grau de eficácia e segurança baseados na EPPIN como alvo molecular requer um profundo entendimento de suas funções e mecanismos moleculares pelos quais esta proteína regula a fertilidade masculina. Visando fomentar avanços nesse sentido, investigamos o papel da EPPIN em eventos associados à motilidade e capacitação espermática, bem como impacto da maturação póstesticular sobrea distribuição celular da EPPIN em espermatozoides de camundongos. Para determinar o efeito da maturação e capacitação espermática sobre a localização da EPPIN no espermatozoide murino, realizamos ensaios de Western blot e imunofluorescência. Para investigar o papel da EPPIN no controle da motilidade espermática, avaliamos o efeito de anticorpos anti-EPPIN que reconhecem epítopos nos domínios Kunitz (anticorpo S21C e F21C) e WFDC (anticorpo Q20E) sobre parâmetros de motilidade e cinemática por análise computadorizada (CASA). Detectamos uma dinâmica na presença da EPPIN na fração solúvel em Triton X-100, na fração enriquecida de membrana, e em associação com estruturas do citoesqueleto de espermatozoides epididimários, sugerindo uma associação entre o grau de maturação espermática e a distribuição celular da EPPIN. Observamos que o anticorpo S21C, mas não o F21C, inibiu a motilidade espermática progressiva e hiperativada. Em suporte a estes achados, o anticorpo S21C alterou os parâmetros de cinemática que descrevem a progressividade (VSL, VAP e STR) e o vigor (VCL, ALH e LIN) do movimento espermático. Interessantemente, o fragmento S21C Fab reproduziu efeitos similares ao total, indicando que a ligação monovalente ao epítopo S21C é suficiente para afetar a motilidade e hiperativação espermática. Por sua vez, o anticorpo Q20E (inteiro e fragmento Fab) inibiu a motilidade progressiva e os parâmetros de cinemática (VAP, VCL, ALH) de forma menos intensa em comparação ao anticorpo S21C. Visando investigar o mecanismo de ação associado aos efeitos dos anticorpos anti-EPPIN sobre a motilidade progressiva e hiperativada, avaliamos a fosforilação da proteína quinase A (PKA) e a produção de adenosina 3’-5’- monofosfato cíclico (AMPc) associados à capacitação por Western blot e AlphaScreen. Nossos resultados demonstraram que o anticorpo S21C induziu aumento na PKA total/PKA fosforilada, sem alterar e os níveis intracelulares de AMPc. Em conclusão, o domínio Kunitz da EPPIN desempenha papel essencial na regulação da motilidade espermática e hiperativação, e seu domínio WFDC pode contribuir para com estes efeitos. A presença da EPPIN em estruturas associadas ao citoesqueleto celular reforça o seu envolvimento em eventos associados à motilidade espermática. Os achados deste estudo contribuem para o desenvolvimento da EPPIN como alvo farmacológico para contracepção masculina.
Around 40% of pregnancies globally are unintended; among them, 20% are terminated in abortion. This scenario reinforces the need for new contraception methods, especially male options, which are currently limited to condoms and vasectomy. EPPIN (Epididymal protease inhibitor) is a cysteine-rich protein containing both WFDC (N-terminal) and Kunitz (C-terminal) protease inhibitor consensus sequences. EPPIN is present on the surface of spermatozoa, where it plays a critical role in sperm function. The suitability for male contraceptive pharmacological interventions targeting EPPIN was demonstrated by the inhibitory effects of anti-EPPIN antibodies and small molecular ligands on sperm motility in human and non-human primate models. Developing effective and safe contraceptive drugs targeting EPPIN functions requires a deeper understanding of its physiological roles and mechanisms regulating male fertility. Here, we investigated the role of EPPIN in events associated with sperm motility and capacitation and the impact of pos-testicular maturation on the relative stability of EPPIN in mouse spermatozoa. We evaluated whether epididymal transit and capacitation modulate EPPIN distribution and localization in mouse spermatozoa by Western blot and immunofluorescence assays. We evaluated the effects of antibodies anti-EPPIN (IgG and Fab fragments) mapping sequences in EPPIN WFDC (Q20E antibody) and Kunitz (S21C and F21C antibody) domains on mouse sperm motility and kinematic parameters by computer-assisted sperm analysis (CASA). We detected the presence of EPPIN in different protein fractions of mouse spermatozoa: (i) soluble fraction, (ii) membrane-enriched fraction, and (iii) in association with sperm cytoskeleton and flagellar accessory structures. The presence of EPPIN associated with membrane and cytoskeleton structures reinforces its involvement in events related to mouse sperm motility. Moreover, epididymal sperm maturation, but not capacitation, was associated with changes in the presence of EPPIN in association with sperm cytoskeleton and flagellar accessory structures. We showed that the S21C antibody, but not the F21C, inhibited progressive and hyperactivated sperm motility. In addition, the S21C antibody affected the kinematics parameters that describe the progressivity (VSL, VAP, STR) and vigor (VCL, ALH, LIN) of the sperm movement. Interestingly, S21C Fab fragments reproduced these outcomes, indicating that monovalent binding to the S21C epitope is sufficient to affect sperm motility and hyperactivation. Conversely, the Q20E antibody (whole IgG and Fab fragment) induced a milder effect on inhibition of progressive motility and kinematic parameters (VAP, VCL, ALH). To provide insights into the underlying mechanisms by which antiEPPIN antibodies impair sperm motility parameters, we evaluated their effects on sperm capacitation-induced phosphorylation of protein kinase A (PKA) and the production of adenosine 3'-5'-cyclic monophosphate (cAMP) by Western blot and AlphaScreen, respectively. Our results suggest that the S21C antibody induced an increase in PKA/phospho-PKA ratio without changing the intracellular levels of cAMP. In conclusion, our results demonstrate the key roles of EPPIN Cterminal (Kunitz domain) sequences on the regulation of mouse sperm motility and hyperactivation and that its WFDC domain also contributes to these outcomes. Our findings contribute to the development of EPPIN as a sperm drug target for non-hormonal male contraception.
Around 40% of pregnancies globally are unintended; among them, 20% are terminated in abortion. This scenario reinforces the need for new contraception methods, especially male options, which are currently limited to condoms and vasectomy. EPPIN (Epididymal protease inhibitor) is a cysteine-rich protein containing both WFDC (N-terminal) and Kunitz (C-terminal) protease inhibitor consensus sequences. EPPIN is present on the surface of spermatozoa, where it plays a critical role in sperm function. The suitability for male contraceptive pharmacological interventions targeting EPPIN was demonstrated by the inhibitory effects of anti-EPPIN antibodies and small molecular ligands on sperm motility in human and non-human primate models. Developing effective and safe contraceptive drugs targeting EPPIN functions requires a deeper understanding of its physiological roles and mechanisms regulating male fertility. Here, we investigated the role of EPPIN in events associated with sperm motility and capacitation and the impact of pos-testicular maturation on the relative stability of EPPIN in mouse spermatozoa. We evaluated whether epididymal transit and capacitation modulate EPPIN distribution and localization in mouse spermatozoa by Western blot and immunofluorescence assays. We evaluated the effects of antibodies anti-EPPIN (IgG and Fab fragments) mapping sequences in EPPIN WFDC (Q20E antibody) and Kunitz (S21C and F21C antibody) domains on mouse sperm motility and kinematic parameters by computer-assisted sperm analysis (CASA). We detected the presence of EPPIN in different protein fractions of mouse spermatozoa: (i) soluble fraction, (ii) membrane-enriched fraction, and (iii) in association with sperm cytoskeleton and flagellar accessory structures. The presence of EPPIN associated with membrane and cytoskeleton structures reinforces its involvement in events related to mouse sperm motility. Moreover, epididymal sperm maturation, but not capacitation, was associated with changes in the presence of EPPIN in association with sperm cytoskeleton and flagellar accessory structures. We showed that the S21C antibody, but not the F21C, inhibited progressive and hyperactivated sperm motility. In addition, the S21C antibody affected the kinematics parameters that describe the progressivity (VSL, VAP, STR) and vigor (VCL, ALH, LIN) of the sperm movement. Interestingly, S21C Fab fragments reproduced these outcomes, indicating that monovalent binding to the S21C epitope is sufficient to affect sperm motility and hyperactivation. Conversely, the Q20E antibody (whole IgG and Fab fragment) induced a milder effect on inhibition of progressive motility and kinematic parameters (VAP, VCL, ALH). To provide insights into the underlying mechanisms by which antiEPPIN antibodies impair sperm motility parameters, we evaluated their effects on sperm capacitation-induced phosphorylation of protein kinase A (PKA) and the production of adenosine 3'-5'-cyclic monophosphate (cAMP) by Western blot and AlphaScreen, respectively. Our results suggest that the S21C antibody induced an increase in PKA/phospho-PKA ratio without changing the intracellular levels of cAMP. In conclusion, our results demonstrate the key roles of EPPIN Cterminal (Kunitz domain) sequences on the regulation of mouse sperm motility and hyperactivation and that its WFDC domain also contributes to these outcomes. Our findings contribute to the development of EPPIN as a sperm drug target for non-hormonal male contraception.
Descrição
Palavras-chave
Espermatozoides, EPPIN, Camundongo, Contracepção masculina, Motilidade espermática, Spermatozoa, Male contraception, Sperm motility