RESUMO
EPPIN (epididymal protease inhibitor) is a mammalian conserved sperm-binding protein displaying an N-terminal WFDC (whey-acidic protein four-disulfide core) and a C-terminal Kunitz protease inhibitor domains. EPPIN plays a key role in regulating sperm motility after ejaculation via interaction with the seminal plasma protein SEMG1 (semenogelin-1). EPPIN ligands targeting the SEMG1 binding site in the Kunitz domain are under development as male contraceptive drugs. Nevertheless, the relative contributions of EPPIN WFDC and Kunitz domains to sperm function remain obscure. Here, we evaluated the effects of antibodies targeting specific epitopes in EPPIN's WFDC (Q20E antibody, Gln20-Glu39 epitope) and Kunitz (S21C and F21C antibodies, Ser103-Cys123 and Phe90-C110 epitopes, respectively) domains on mouse sperm motility and fertilizing ability. Computer-assisted sperm analysis showed that sperm co-incubation with S21C antibody (but not F21C antibody) lowered progressive and hyperactivated motilities and impaired kinematic parameters describing progressive (straight-line velocity; VSL, average path velocity; VAP and straightness; STR) and vigorous sperm movements (curvilinear velocity; VCL, amplitude of lateral head movement; ALH, and linearity; LIN) compared with control. Conversely, Q20E antibody-induced milder inhibition of progressive motility and kinematic parameters (VAP, VCL and ALH). Sperm co-incubation with S21C or Q20E antibodies affected in vitro fertilization as revealed by reduced cleavage rates, albeit without changes in capacitation-induced tyrosine phosphorylation. In conclusion, we show that targeting specific epitopes in EPPIN Kunitz and WFDC domains inhibits sperm motility and capacitation-associated events, which decrease their fertilizing ability; nevertheless, similar observations in vivo remain to be demonstrated. Simultaneously targeting residues in S21C and Q20E epitopes is a promising approach for the rational design of EPPIN-based ligands with spermostatic activity.
Assuntos
Anticorpos/farmacologia , Anticoncepcionais Masculinos/farmacologia , Desenho de Fármacos , Proteínas Secretadas Inibidoras de Proteinases/antagonistas & inibidores , Capacitação Espermática/efeitos dos fármacos , Motilidade dos Espermatozoides/efeitos dos fármacos , Espermatozoides/efeitos dos fármacos , Animais , Sítios de Ligação , Fenômenos Biomecânicos , Epitopos , Feminino , Ligantes , Masculino , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Fosforilação , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteínas Secretadas Inibidoras de Proteinases/química , Proteínas Secretadas Inibidoras de Proteinases/metabolismo , Espermatozoides/metabolismo , TirosinaRESUMO
Bacterial infections are the most prevalent etiological factors of epididymitis, a commonly diagnosed inflammatory disease in the investigation of male infertility factors. The influence of early pathogenic mechanisms at play during bacterial epididymitis on reproductive outcomes is little understood. We report here that experimental epididymitis induced in rats by Gram-negative (LPS) and Gram-positive (LTA) bacterial products resulted in differential patterns of acute inflammation in the cauda epididymis. LPS elicited a strong inflammatory reaction, as reflected by upregulation of levels of mRNA for seven inflammatory mediators (Il1b, Tnf, Il6, Ifng, Il10, Nos2 and Nfkbia), and tissue concentration of six cytokines/chemokines (IL1A, IL1B, IL6, IL10, CXCL2 and CCL2) within the first 24 h post-treatment. Conversely, LTA induced downregulation of one (Nfkbia) and upregulation of six (Il1b, Il6, Nos2, Il4 Il10 and Ptgs1) inflammatory gene transcripts, whereas increased the tissue concentration of three cytokines/chemokines (IL10, CXCL2 and CCL2). The stronger acute inflammatory response induced by LPS correlated with a reduction of epididymal sperm count and transit time that occurred at 1, 7, and 15 days post-treatment. Our study provides evidence that early epididymal inflammatory signaling events to bacterial activators of innate immunity may contribute to the detrimental effects of epididymitis upon male fertility.