Investigação do papel da EPPIN no controle da função espermática: impacto na capacitação e no potencial fértil de espermatozoides de camundongos.
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Data
2021-08-30
Autores
Raimundo, Tamiris Rocha Fanti
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Universidade Estadual Paulista (Unesp)
Resumo
Proteínas do plasma seminal se ligam aos espermatozoides e controlam eventos associados à motilidade e capacitação espermática. Dentre essas proteínas, destacamos a semenogelina-1 (SEMG1), que é a proteína mais abundante do plasma seminal humano e inibe a motilidade progressiva e a capacitação do espermatozoide. A SEMG1 regula a função do espermatozoide ligando-se à proteína EPPIN (do inglês, Epididymal protease inhibitor protein), presente na superfície espermática. A demonstração de que ligantes da EPPIN, como anticorpos e moléculas orgânicas de baixo peso molecular mimetizaram os efeitos da SEMG1 na inibição da motilidade espermática tornou a EPPIN um alvo para a contracepção masculina. Visando avançar no entendimento dos aspectos funcionais e moleculares associados ao controle da função espermática pela EPPIN, nosso laboratório vem utilizando o camundongo como modelo experimental. Demonstramos anteriormente que a SVS2 (do inglês, seminal vesicle secretory protein 2), ortóloga à SEMG1, é uma proteína de interação da EPPIN em espermatozoides de camundongos, sugerindo que os papéis da EPPIN em eventos relacionados à função espermática são conservados entre camundongos e humanos. Essa hipótese é sustentada por observações de que a SEMG1 humana e a SVS2 murina inibem eventos associados à capacitação espermática, como a reação acrossômica e a hiperativação, via mecanismos ainda pouco conhecidos. Considerando: (i) o efeito inibitório de anticorpos anti-EPPIN miméticos à SEMG1 sobre a motilidade espermática; e (ii) o efeito inibitório da SEMG1 e da SVS2 sobre a capacitação espermática, neste trabalho testamos a hipótese de que anticorpos anti-EPPIN modulam efeitos moleculares e funcionais associados à capacitação espermática. Para isto, induzimos a capacitação de espermatozoides de camundongos na presença e ausência de anticorpos anti-EPPIN (10-300 μg/ml) que reconhecem epítopos específicos na região N-terminal (anticorpos Q20E e F15C) e C-terminal (anticorpo S21C) ou da SVS2 murina recombinante (1-25 μM) e avaliamos os seguintes parâmetros: (i) fosforilação de resíduos de tirosina, serina/treonina e de substratos da proteína quinase A (PKA); (ii) taxa de reação acrossômica espontânea e induzida por ionóforo de cálcio (A23187); e (iii) taxa de fertilização in vitro (FIV). Observamos que a incubação com a SVS2r diminuiu a fosforilação de proteínas induzida pela capacitação. Por outro lado, os anticorpos Q20E, F15C e S21C não afetaram o perfil fosfoproteico dos espermatozoides. Com relação à atividade da PKA, que foi avaliada de forma indireta pela abundância dos seus substratos fosforilados, identificamos que o anticorpo S21C, mas não o Q20E, aumentou a atividade da PKA em espermatozoides capacitados. Além disso, os anticorpos S21C e Q20E não modificaram as taxas de reação acrossômica espontânea e induzida (A23187); porém, o anticorpo F15C induziu aumento na taxa de reação acrossômica induzida por A23187. Por fim, observamos que o anticorpo S21C promoveu diminuição das taxas de FIV. Em conjunto, nossas descobertas expandem os caminhos pelas quais a EPPIN deve ser explorada com relação aos seus papéis em eventos moleculares e funcionais do espermatozoide e para o desenvolvimento de um contraceptivo masculino não hormonal.
Seminal plasma proteins bind to spermatozoa and control associated events with sperm motility and capacitation. Among these seminal proteins, we highlight semenogelin-1 (SEMG1), the most abundant protein in the human seminal plasma, inhibits sperm progressive motility and capacitation. SEMG1 regulates sperm function by binding to EPPIN, a protein present on the sperm surface. The demonstration that EPPIN ligands such as antibodies and organic molecules mimicked the effects of SEMG1 in inhibiting sperm motility has lifted EPPIN to the top of the list of male contraceptive drug targets. Aiming to advance the understanding of functional and molecular aspects associated with the control of spermatic function by EPPIN, we have been using the mouse as an experimental model. We have previously demonstrated that SVS2 (seminal vesicle secretory protein 2), the mouse ortholog to SEMG1, is an interacting protein of EPPIN in mouse spermatozoa, suggesting that EPPIN roles in events related to sperm function are conserved between mice and humans. This hypothesis is supported by observations that human SEMG1 and mouse SVS2 inhibit events associated with sperm capacitation, such as acrosome reaction and hyperactivation, via mechanisms that are still poorly understood. Considering: (i) the inhibitory effects of SEMG1 mimetic anti-EPPIN antibodies on sperm motility; and (ii) the inhibitory effect of SEMG1 and SVS2 on sperm capacitation, we tested the hypothesis that anti-EPPIN antibodies modulate molecular and functional effects associated with sperm capacitation. For that, we induced mouse sperm capacitation in the absence or presence of anti-EPPIN (10-300 μg/ml) that recognize specific epitopes in the N-terminal (Q20E and F15C) and C-terminal (S21C) or recombinant murine SVS2 region (1-25 μM) and evaluated the following parameters: (i) phosphorylation of tyrosine, serine/threonine residues, and PKA substrates; (ii) acrosome reaction rates of spontaneous samples and calcium ionophore-induced reaction (A23187); and (iii) in vitro fertilization rates. We observed that the incubation with SVS2r decreased capacitation-induced protein phosphorylation. On the other hand, Q20E, F15C, and S21C did not affect the phosphoprotein profile of spermatozoa. Regarding PKA activity, which was indirectly assessed by the source of its phosphorylated substrates, we found that the S21C antibody, but not Q20E, increased PKA activity in capacitated spermatozoa. Furthermore, S21C and Q20E did not change spontaneous and A23187-induced acrosome reaction rates; however, the F15C antibody increased A23187-induced acrosome reaction. Finally, we observed that spermatozoa incubated with S21C antibody during capacitation showed decreased in vitro fertilization rates. Together, our findings expand the understanding of how EPPIN modulates molecular events associated with sperm function, contributing to its development as a non-hormonal male contraceptive.
Seminal plasma proteins bind to spermatozoa and control associated events with sperm motility and capacitation. Among these seminal proteins, we highlight semenogelin-1 (SEMG1), the most abundant protein in the human seminal plasma, inhibits sperm progressive motility and capacitation. SEMG1 regulates sperm function by binding to EPPIN, a protein present on the sperm surface. The demonstration that EPPIN ligands such as antibodies and organic molecules mimicked the effects of SEMG1 in inhibiting sperm motility has lifted EPPIN to the top of the list of male contraceptive drug targets. Aiming to advance the understanding of functional and molecular aspects associated with the control of spermatic function by EPPIN, we have been using the mouse as an experimental model. We have previously demonstrated that SVS2 (seminal vesicle secretory protein 2), the mouse ortholog to SEMG1, is an interacting protein of EPPIN in mouse spermatozoa, suggesting that EPPIN roles in events related to sperm function are conserved between mice and humans. This hypothesis is supported by observations that human SEMG1 and mouse SVS2 inhibit events associated with sperm capacitation, such as acrosome reaction and hyperactivation, via mechanisms that are still poorly understood. Considering: (i) the inhibitory effects of SEMG1 mimetic anti-EPPIN antibodies on sperm motility; and (ii) the inhibitory effect of SEMG1 and SVS2 on sperm capacitation, we tested the hypothesis that anti-EPPIN antibodies modulate molecular and functional effects associated with sperm capacitation. For that, we induced mouse sperm capacitation in the absence or presence of anti-EPPIN (10-300 μg/ml) that recognize specific epitopes in the N-terminal (Q20E and F15C) and C-terminal (S21C) or recombinant murine SVS2 region (1-25 μM) and evaluated the following parameters: (i) phosphorylation of tyrosine, serine/threonine residues, and PKA substrates; (ii) acrosome reaction rates of spontaneous samples and calcium ionophore-induced reaction (A23187); and (iii) in vitro fertilization rates. We observed that the incubation with SVS2r decreased capacitation-induced protein phosphorylation. On the other hand, Q20E, F15C, and S21C did not affect the phosphoprotein profile of spermatozoa. Regarding PKA activity, which was indirectly assessed by the source of its phosphorylated substrates, we found that the S21C antibody, but not Q20E, increased PKA activity in capacitated spermatozoa. Furthermore, S21C and Q20E did not change spontaneous and A23187-induced acrosome reaction rates; however, the F15C antibody increased A23187-induced acrosome reaction. Finally, we observed that spermatozoa incubated with S21C antibody during capacitation showed decreased in vitro fertilization rates. Together, our findings expand the understanding of how EPPIN modulates molecular events associated with sperm function, contributing to its development as a non-hormonal male contraceptive.
Descrição
Palavras-chave
Espermatozoide, Capacitação, Contracepção masculina, EPPIN, Spermatozoa, Capacitation, Male contraception