Papel das proteínas do plasma seminal SVS2 e SVS3 sobre a motilidade de espermatozoides de camundongos: repercussões para a função espermática e fertilidade
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Data
2020-07-27
Autores
Andrade, Juliana de Jesus
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Universidade Estadual Paulista (Unesp)
Resumo
Proteínas do plasma seminal possuem o importante papel de regular a função espermática, sendo fundamentais para a fertilidade masculina. Em primatas e roedores, as proteínas mais abundantes do plasma seminal são as semenogelinas (SEMG1 e SEMG2) e as SVS’s (SVS2 e SVS3; seminal vesicle-secretory proteins), respectivamente, as quais possuem similaridades funcionais e estruturais. Essas proteínas fazem parte de uma família conhecida como REST (Rapidly-evolving seminal vesicle-transcribed), sendo abundantemente expressas pela glândula seminal de mamíferos. A SEMG1 interage com o espermatozoide ejaculado e inibe a sua motilidade, capacitação e reação acrossômica. Por sua vez, a SVS2 também inibe a capacitação e a reação acrossômica no espermatozoide murino, sendo estes efeitos inibitórios potencializados pela SVS3. Entretanto, pouco se sabe sobre os efeitos da SVS2 e SVS3 na motilidade espermática. Neste estudo, testamos a hipótese que a SVS2 inibe a motilidade espermática em camundongos, e que a SVS3 facilita esses efeitos inibitórios. Também caracterizamos o perfil de expressão e distribuição da SVS2 e SVS3 (RNAm e proteína) no sistema reprodutor de camundongos machos, bem como sua regulação por androgênios. Para isto, processamos órgãos reprodutivos para ensaios de RT-PCR, RT-qPCR, Western blot e imuno-histoquímica, e espermatozoides epididimários para ensaios de imunofluorescência após incubação com a SVS2 e SVS3A murinas recombinantes (SVS2m e SVS3m) ou fluído da glândula seminal. Além disso, para avaliar o impacto da SVS2m e SVS3Am sobre a motilidade espermática, incubamos espermatozoides da cauda do epidídimo com diferentes concentrações de SVS2m e SVS3m recombinantes, isoladas ou em associação, e avaliamos a motilidade pela plataforma CASA (computer-assisted sperm analysis). Nossos resultados demonstraram que SVS2 e SVS3 (RNAm e proteína) são diferencialmente expressos em tecidos reprodutivos de camundongos machos. Os transcritos Svs2 e Svs3 foram detectados na glândula seminal, mas também em outros tecidos do sistema reprodutor, como cauda do epidídimo, ducto deferente, próstata ventral, e no testículo (somente Svs3). Os resultados de Western blot revelaram a expressão da SVS2 pela presença de uma banda única, com massa molecular aparente de 45 kDa na glândula seminal (tecido e fluido). Na cauda do epidídimo, entretanto, observamos a presença de uma banda menor, de ~11 kDa. Para a SVS3, observamos a presença de uma banda de 35 kDa no fluído da glândula seminal. Já em amostras de testículo, cauda do epidídimo e glândula seminal, observamos a presença de múltiplas bandas com massas moleculares aparentes de 40-43, 28-29 e 14 kDa, respectivamente. Análises de RT-qPCR e imuno-histoquímica utilizando amostras da glândula seminal de animais em diferentes fases da maturação sexual ou submetidos à orquiectomia bilateral com ou sem reposição hormonal com testosterona (8 mg/kg, s.c.) demonstraram a correlação positiva entre a expressão da SVS2 e SVS3 com os níveis plasmáticos de androgênios. Tanto a SVS2m quanto a SVS3m interagiram com espermatozoides maduros, apresentando sítios de ligação específica na cabeça e flagelo. Observamos que a SVS2m inibiu os parâmetros de motilidade e cinemática espermática de forma dependente da concentração (IC50 = ~5 µM) e tempo de incubação, os quais afetaram a motilidade progressiva e hiperativada. Por outro lado, a SVS3m apresentou apenas efeitos modestos sobre a motilidade hiperativada. Em adição, a SVS3m não demonstrou efeito sinérgico sobre os efeitos inibitórios da SVS2m sobre a motilidade de espermatozoides de camundongos. Em conjunto, nossos dados demonstram que a SVS2 é um fator endógeno do plasma seminal de camundongos com atividade inibitória da motilidade espermática. Considerando que a SVS2 é a ortóloga murina à SEMG1 humana, propomos a existência de mecanismos conservados para o controle da motilidade espermática a partir de proteínas oriundas do plasma seminal em primatas e roedores. Nossas descobertas abrem novas fronteiras para o estudo do papel das proteínas da família REST na regulação das funções espermáticas, principalmente nos parâmetros de motilidade espermática, bem como sua exploração como alvos para contracepção masculina, utilizando modelos murinos.
Seminal plasma proteins play important roles on the regulation of sperm function and male fertility. In primates and rodents, the most abundant proteins in the seminal plasma are the semenogelins (SEMG1 and SEMG2) and seminal vesicle-secreted proteins (SVS2 and SVS3), respectively, which display functional and structural similarities. These proteins are members of the REST (Rapidly-evolving seminal vesicle-transcribed) family, which are abundantly expressed in the seminal vesicles. SEMG1 interacts with the ejaculated spermatozoa, thus inhibiting its motility, capacitation and acrosomal reaction. In turn, SVS2 inhibits capacitation and acrosome reaction in murine spermatozoa. SVS2 inhibitory effects on mouse sperm capacitation and acrosome reaction are potentiated by SVS3. However, little is known about the effects of SVS2 and SVS3 on sperm motility. In this study, we tested the hypothesis that SVS2 inhibits mouse sperm motility, and that SVS3 facilitates these inhibitory effects. We also characterized the expression and distribution profile of SVS2 and SVS3 (mRNA and protein) in the male mouse reproductive tract, as well as their regulation by androgens. We processed reproductive tissues for RT-PCR, qPCR, Western blot and immunohistochemistry assays, and epididymal spermatozoa incubated with recombinant murine SVS2 and SVS3A (SVS2m and SVS3m) or seminal vesicle fluid for immunofluorescence assays. To assess the impact of SVS2m and SVS3m on sperm motility, we incubated spermatozoa with different concentrations of recombinant SVS2m and SVS3m, isolated or in combination, and evaluated their motility parameters using the computer-assisted sperm analysis (CASA). Our results demonstrated that SVS2 and SVS3 (mRNA and protein) are differentially expressed in male mouse reproductive tissues. Svs2 and Svs3 transcripts were detected in the seminal vesicle, but also testis (only Svs3), cauda epididymis, vas deferens and ventral prostate. Western blot results revealed the expression of SVS2 by the presence of a single band, with apparent molecular mass of 45 kDa in the seminal vesicle (tissue and fluid). In cauda epididymis protein extracts, however, we observed the presence of a smaller band with apparent molecular mass of 11 kDa. For SVS3, we observed the presence of a 35-kDa-band in the seminal vesicle fluid. In testis, epididymis and seminal vesicle samples, however, we observed the presence of multiple bands with apparent molecular masses of 40-43, 28-29 and 14 kDa depending on the tissue analyzed. RT- qPCR and immunohistochemistry analyzes using seminal vesicle samples from animals at different stages of sexual maturation or subjected to bilateral orchiectomy with or without hormone replacement with testosterone (8 mg/kg, s.c.) demonstrated that both Svs2 and Svs3 are androgen-dependent genes. Both SVS2m and SVS3m bound to the head and flagellum of mature spermatozoa. We observed that SVS2m inhibited sperm motility and kinematics in a concentration- (IC50 = ~5 µM) and time-dependent manner, thus leading to an inhibition of progressive motility and hyperactivation. On the other hand, SVS3m displayed modest effects on hyperactivated, but not progressive, motility. Furthermore, SVS3m did not demonstrate a synergistic effect on the inhibitory effects of SVS2m on mouse sperm motility. Altogether, our data demonstrate that SVS2 is a seminal plasma-derived inhibitory factor of mouse sperm motility. Since SVS2 is the murine orthologue of human SEMG1, we propose the existence of conserved mechanisms for the control of sperm motility endogenous to the seminal plasma in primates and rodents. Our findings open new frontiers for the study on the roles of REST proteins on the regulation of sperm functions, which could be explored for the development of new targets for male contraception, using murine models.
Seminal plasma proteins play important roles on the regulation of sperm function and male fertility. In primates and rodents, the most abundant proteins in the seminal plasma are the semenogelins (SEMG1 and SEMG2) and seminal vesicle-secreted proteins (SVS2 and SVS3), respectively, which display functional and structural similarities. These proteins are members of the REST (Rapidly-evolving seminal vesicle-transcribed) family, which are abundantly expressed in the seminal vesicles. SEMG1 interacts with the ejaculated spermatozoa, thus inhibiting its motility, capacitation and acrosomal reaction. In turn, SVS2 inhibits capacitation and acrosome reaction in murine spermatozoa. SVS2 inhibitory effects on mouse sperm capacitation and acrosome reaction are potentiated by SVS3. However, little is known about the effects of SVS2 and SVS3 on sperm motility. In this study, we tested the hypothesis that SVS2 inhibits mouse sperm motility, and that SVS3 facilitates these inhibitory effects. We also characterized the expression and distribution profile of SVS2 and SVS3 (mRNA and protein) in the male mouse reproductive tract, as well as their regulation by androgens. We processed reproductive tissues for RT-PCR, qPCR, Western blot and immunohistochemistry assays, and epididymal spermatozoa incubated with recombinant murine SVS2 and SVS3A (SVS2m and SVS3m) or seminal vesicle fluid for immunofluorescence assays. To assess the impact of SVS2m and SVS3m on sperm motility, we incubated spermatozoa with different concentrations of recombinant SVS2m and SVS3m, isolated or in combination, and evaluated their motility parameters using the computer-assisted sperm analysis (CASA). Our results demonstrated that SVS2 and SVS3 (mRNA and protein) are differentially expressed in male mouse reproductive tissues. Svs2 and Svs3 transcripts were detected in the seminal vesicle, but also testis (only Svs3), cauda epididymis, vas deferens and ventral prostate. Western blot results revealed the expression of SVS2 by the presence of a single band, with apparent molecular mass of 45 kDa in the seminal vesicle (tissue and fluid). In cauda epididymis protein extracts, however, we observed the presence of a smaller band with apparent molecular mass of 11 kDa. For SVS3, we observed the presence of a 35-kDa-band in the seminal vesicle fluid. In testis, epididymis and seminal vesicle samples, however, we observed the presence of multiple bands with apparent molecular masses of 40-43, 28-29 and 14 kDa depending on the tissue analyzed. RT- qPCR and immunohistochemistry analyzes using seminal vesicle samples from animals at different stages of sexual maturation or subjected to bilateral orchiectomy with or without hormone replacement with testosterone (8 mg/kg, s.c.) demonstrated that both Svs2 and Svs3 are androgen-dependent genes. Both SVS2m and SVS3m bound to the head and flagellum of mature spermatozoa. We observed that SVS2m inhibited sperm motility and kinematics in a concentration- (IC50 = ~5 µM) and time-dependent manner, thus leading to an inhibition of progressive motility and hyperactivation. On the other hand, SVS3m displayed modest effects on hyperactivated, but not progressive, motility. Furthermore, SVS3m did not demonstrate a synergistic effect on the inhibitory effects of SVS2m on mouse sperm motility. Altogether, our data demonstrate that SVS2 is a seminal plasma-derived inhibitory factor of mouse sperm motility. Since SVS2 is the murine orthologue of human SEMG1, we propose the existence of conserved mechanisms for the control of sperm motility endogenous to the seminal plasma in primates and rodents. Our findings open new frontiers for the study on the roles of REST proteins on the regulation of sperm functions, which could be explored for the development of new targets for male contraception, using murine models.
Descrição
Palavras-chave
Espermatozoides, Motilidade espermática, Proteínas de plasma seminal, Camundongos