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Potential roles of EPPIN (Epididymal protease inhibitor) on post-testicular maturation of mouse spermatozoa: consequences for sperm function

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Alan Andrew S. Silva
Total Authors: 1
Document type: Master's Dissertation
Press: Botucatu. 2021-10-18.
Institution: Universidade Estadual Paulista (UNESP). Instituto de Biociências. Botucatu
Defense date:
Advisor: Erick Jose Ramo Silva

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. (AU)

FAPESP's process: 19/13661-1 - Potential roles of EPPIN (Epididymal Protease Inhibitor) on post-testicular maturation of mouse spermatozoa: consequences for sperm function
Grantee:Alan Andrew dos Santos Silva
Support type: Scholarships in Brazil - Master