Human sperm degradation of zona pellucida proteins contributes to fertilization.
BACKGROUND
The mammalian oocyte extracellular matrix known as the zona pellucida (ZP) acts as a barrier to accomplish sperm fusion with the female gamete. Although penetration of the ZP is a limiting event to achieve fertilization, this is one of the least comprehended stages of gamete interaction. Even though previous studies suggest that proteases of sperm origin contribute to facilitate the passage of sperm through the ZP, in human this process is not yet fully understood. The aim of this study was to determine the ability of human sperm to degrade recombinant human ZP (rhZPs) proteins and to characterize the proteases involved in this process.


METHODS
Purified rhZP2, rhZP3 and rhZP4 proteins were incubated with capacitated sperm and the proteolytic activity was determined by Western blot analysis. To further characterize the proteases involved, parallel incubations were performed in the presence of the protease inhibitors o-phenanthroline, benzamidine and MG-132 meant to block the activity of metalloproteases, serine proteases and the proteasome, respectively. Additionally, protease inhibitors effect on sperm-ZP binding was evaluated by hemizona assay.


RESULTS
The results showed that rhZPs were hydrolyzed in the presence of capacitated sperm. O-phenanthroline inhibited the degradation of rhZP3, MG-132 inhibited the degradation of rhZP4 and benzamidine inhibited the degradation of the three proteins under investigation. Moreover, hemizona assays demonstrated that sperm proteasome inhibition impairs sperm interaction with human native ZP.


CONCLUSIONS
This study suggests that sperm proteasomes could participate in the degradation of ZP, particularly of the ZP4 protein. Besides, metalloproteases may be involved in specific degradation of ZP3 while serine proteases may contribute to unspecific degradation of the ZP. These findings suggest that localized degradation of ZP proteins by sperm is probably involved in ZP penetration and may be of help in understanding the mechanisms of fertilization in humans.
Departamento de Biología de la Reproducción, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, México, D.F. 14080, Mexico. analilia.saldivar@gmail.com.
Analilia
A
Saldívar-Hernández
Departamento de Biología de la Reproducción, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, México, D.F. 14080, Mexico. helen_1616@hotmail.com.
María E
ME
González-González
Departamento de Genética del Desarrollo y Fisiología Molecular, Instituto de Biotecnología, UNAM, Cuernavaca, 62210, Mexico. anatusie@gmail.com.
Ana
A
Sánchez-Tusié
Instituto Mexicano de Alta Tecnología Reproductiva (Inmater), Mexico, D.F. 11000, Mexico. imaldonado@ingenes.com.
Israel
I
Maldonado-Rosas
Instituto Mexicano de Alta Tecnología Reproductiva (Inmater), Mexico, D.F. 11000, Mexico. plopez@inmater.com.
Pablo
P
López
Departamento de Genética del Desarrollo y Fisiología Molecular, Instituto de Biotecnología, UNAM, Cuernavaca, 62210, Mexico. ctrevino@ibt.unam.mx.
Claudia L
CL
Treviño
Departamento de Biología de la Reproducción, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, México, D.F. 14080, Mexico. fernando.larreag@incmnsz.mx.
Fernando
F
Larrea
Departamento de Biología de la Reproducción, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, México, D.F. 14080, Mexico. mayel.chirinose@incmnsz.mx.
Mayel
M
Chirinos
D004527:Egg Proteins / Q000378:metabolism*; D005260:Female; D005306:Fertilization / Q000502:physiology*; D006801:Humans; D008297:Male; D008562:Membrane Glycoproteins / Q000378:metabolism*; D009865:Oocytes / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000378:metabolism*; D013075:Sperm Capacitation / Q000502:physiology*; D013094:Spermatozoa / Q000378:metabolism*; D015044:Zona Pellucida / Q000378:metabolism*; D000072216:Zona Pellucida Glycoproteins
D004527:Egg Proteins; D008562:Membrane Glycoproteins; D011956:Receptors, Cell Surface; C000606517:ZP3 protein, human; D000072216:Zona Pellucida Glycoproteins