The activation of the chymotrypsin-like activity of the proteasome is regulated by soluble adenyl cyclase/cAMP/protein kinase A pathway and required for human sperm capacitation
Authors: Milene Kong
Patricio Morales
Lidia M Zuñiga
Erma Z Drobnis
Lina Barón
Peter Sutovsky
Emilce Silvina Díaz
Héctor Zapata-Carmona
Journal: Molecular Human Reproduction (2019)
Discipline: MOLECULAR BIOLOGY & GENETICS
Keywords: SACY/cAMP/PKA; capacitation; human; proteasome; protein kinase A
Affiliations: Laboratorio de Biología de la Reproducción, Departamento Biomédico, Facultad de Ciencias de la Salud, Universidad de Antofagasta, Antofagasta, Chile, Department of Obstetrics, Gynecology and Women’s Health, University of Missouri, Columbia, MO, USA, Division of Animal Sciences, University of Missouri, Columbia, MO, USA, Instituto Antofagasta, Universidad de Antofagasta, Antofagasta, Chile

Abstract:  One of the first events of mammalian sperm capacitation is the activation of the soluble adenyl cyclase/cAMP/protein kinase A (SACY/cAMP/PKA) pathway. Here, we evaluated whether the increase in PKA activity at the onset of human sperm capacitation is responsible for the activation of the sperm proteasome and whether this activation is required for capacitation progress. Viable human sperm were incubated with inhibitors of the SACY/cAMP/PKA pathway. The chymotrypsin-like activity of the sperm proteasome was evaluated using a fluorogenic substrate. Sperm capacitation status was evaluated using the chlortetracycline assay and tyrosine phosphorylation. To determine whether proteasomal subunits were phosphorylated by PKA, the proteasome was immunoprecipitated and tested on a western blot using an antibody against phosphorylated PKA substrates. Immunofluorescence microscopy analysis and co-immunoprecipitation (IPP) were used to investigate an association between the catalytic subunit alpha of PKA (PKA-Cα) and the proteasome. The chymotrypsin-like activity of the sperm proteasome significantly increased after 5 min of capacitation (P &lt; 0.001) and remained high for the remaining incubation time. Treatment with H89, KT5720 or KH7 significantly decreased the chymotrypsin-like activity of the proteasome (P &lt; 0.001). IPP experiments indicated that PKA inhibition significantly modified phosphorylation of proteasome subunits. In addition, PKA-Cα colocalized with the proteasome in the equatorial segment and in the connecting piece, and co-immunoprecipitated with the proteasome. This is the first demonstration of sperm proteasome activity being directly regulated by SACY/PKA-Cα. This novel discovery extends our current knowledge of sperm physiology and may be used to manage sperm capacitation during assisted reproductive technology procedures. 

Retracted: No

PubPeer comments:
 Figure 1. * Bands in the PSMA7 and b-tubulin panels appear to look more similar to each other than expected. Bands that look similar are marked with boxes of the same color. ![file] , 
 I am a coauthor on this study, but not the one who ran these blots. I lined up some of the questioned bands, just for the sake of imaging exercise. They do not line up, and even if the dimension were altered (stretched), the pixel patterns differ. Nothing wrong with critical reading :-), stay sharp.![file] , 
 #2 Thank you for responding. Respectfully, this analysis is not convincing. JPG compression can cause differences of the type you are describing. However, the original data should answer these serious concerns. As this is a recent paper, you should have original uncropped scans. You may post those here or use a link to an external site (such as Imgur.com) for larger files. ,