Protein Kinase A (PRKA) Activity Is Regulated by the Proteasome at the Onset of Human Sperm Capacitation.

The proteasome increases its activity at the onset of sperm capacitation due to the action of the SACY/PRKACA pathway; this increase is required for capacitation to progress. PRKA activity also increases and remains high during capacitation. However, intracellular levels of cAMP decrease in this process. Our goal was to evaluate the role of the proteasome in regulating PRKA activity once capacitation has started. Viable human sperm were incubated in the presence and absence of epoxomicin or with 0.1% DMSO. The activity of PRKA; the phosphorylation pattern of PRKA substrates (pPRKAs); and the expression of PRKAR1, PRKAR2, and AKAP3 were evaluated by Western blot. The localization of pPRKAs, PRKAR1, PRKAR2, and AKAP3 was evaluated by immunofluorescence. Treatment with epoxomicin changed the localization and phosphorylation pattern and decreased the percentage of pPRKAs-positive sperm. PRKA activity significantly increased at 1 min of capacitation and remained high throughout the incubation. However, epoxomicin treatment significantly decreased PRKA activity after 30 min. In addition, PRKAR1 and AKAP3 were degraded by the proteasome but with a different temporal kinetic. Our results suggest that PRKAR1 is the target of PRKA regulation by the proteasome.

Periovulatory oviductal fluid decreases sperm protein kinase A activity, tyrosine phosphorylation, and in vitro fertilization in pig.

BACKGROUND: Molecules from the female reproductive tract modulate capacitation and function of sperm cells in vivo. These molecules vary in a quantitative and qualitative manner throughout the estrous cycle. OBJECTIVES: This work evaluates the effect of using various female reproductive fluids on capacitation and fertilization of pig spermatozoa in vitro. MATERIAL AND METHODS: The effects of culturing spermatozoa in different fluids on the levels of sperm protein kinase A (pPKA), tyrosine phosphorylation, acrosome reaction, and in vitro fertilization (IVF) were evaluated. The fluids tested were as follows: oviductal fluid (OF) from five phases of the estrous cycle, namely early and late follicular (OF-EF, OF-LF), early and late luteal (OF-EL, OF-LL) and periovulatory (pOF), follicular fluid from medium-sized follicles, and secretions of cumulus-oocyte complexes (conditioned medium). RESULTS: The pPKAs and tyrosine phosphorylation were decreased by OF-EF, OF-LF, OF-EL, and pOF but not by follicular fluid and conditioned medium. OF-EF, OF-LF, and pOF also decreased the sperm acrosome reaction. Moreover, the effect of pOF on pPKAs and tyrosine phosphorylation was reversible. In in vitro fertilization, OF-EF, OF-LF, OF-EL, and pOF reduced the percentage of penetrated oocytes, the mean number of spermatozoa per penetrated oocyte, and increased monospermy. CONCLUSION: OF from follicular, early luteal, and periovulatory phases of the estrous cycle modulates the sperm protein phosphorylation as well as the acrosome reaction involved in capacitation and increases monospermic fertilization in in vitro fertilization. Our findings suggest that fluids from the female reproductive tract could be used as additives in porcine IVF systems to modulate sperm-oocyte interaction.

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.

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 < 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 < 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.