Participation of the sperm proteasome during in vitro fertilisation and the acrosome reaction in cattle

Abstract

In this work, we have investigated the role of the bovine sperm proteasome during in vitro fertilisation (IVF) and the acrosome reaction (AR). Motile spermatozoa, obtained by a swim-up method in Sperm-Talp medium, were capacitated for 3.5 h and incubated in the presence or absence of the specific proteasome inhibitor epoxomicin for 30 and 60 min. Then, the spermatozoa were co-incubated with mature bovine cumulus oocytes and after 48 h the cleavage rate of inseminated oocytes was evaluated. In addition, we evaluated the participation of the sperm proteasome during the progesterone-induced AR. Capacitated spermatozoa were incubated for 30 min with or without epoxomicin, then progesterone was added and the ARs were evaluated using the dual fluorescent staining technique 'Hoechst and chlortetracycline'. The results indicate that the proteasome inhibitor decreased the cleavage rate of oocytes inseminated with treated spermatozoa. In addition, acrosomal exocytosis levels were statistically significantly higher in the samples treated with the AR inducer progesterone than in control samples in the absence of the inducer. However, the progesterone-induced AR was significantly reduced by previous treatment of the spermatozoa with epoxomicin (P < 0.001). These observations indicate that the bovine sperm proteasome participates in the IVF and AR processes.

Sperm proteasome and fertilization

“…Removal of ZP eliminated the effect of MG132 and lactacystin on porcine fertilization, an indication that these compounds specifically inhibit sperm-zona penetration step of fertilization (Sutovsky et al 2004). Similar effect on sperm-VC penetration was observed with a related compound, MG115 in ascidian and sea urchin studies (Sawada et al 1998, 2002b, Yokota & Sawada 2007a, with lactacystin in the pig (Sutovsky et al 2003, Sun et al 2004 and with epoxomicin in the bovine model (Sanchez et al 2011). Proteasome inhibition by the above compounds does not alter sperm-VC/ZP binding or sperm motility and specifically occurs at the time of sperm penetration through VC/ZP in all of the above species except the mouse, wherein fertilization was performed with zona free oocytes.…”

“…Authors of the first study attempting to isolate the human and mouse sperm proteasomes observed that 'in comparison with less mature germ cells, the (mature human spermatozoa) retain relatively high values of 26S proteasome activity, suggesting that they have further roles to play in normal sperm physiology' (Tipler et al 1997). Since then, the substrate-specific enzymatic activities of 26S proteasomes have been measured in spermatozoa of livestock species (Sutovsky et al 2004, Yi et al 2007b, 2009a, Rawe et al 2008, Sanchez et al 2011, humans (Morales et al 2003, Chakravarty et al 2008, Kong et al 2009), and rodents (Pasten et al 2005, Rivkin et al 2009). The proteasomedependent mechanism of sperm VC penetration is …”

Global proteomic profiling of the membrane compartment of bovine testis cell populations

“…Interestingly, these proteins were also identified in a recent study in our laboratory examining the plasma membrane fraction of bovine sperm, implying that they are indeed associated with the plasma membrane [57]. The CCT complex is present on the surface of capacitated spermatozoa playing a role in binding to the zona pellucida during fertilization [58], and the membrane proteasome of mammalian sperm is required for the acrosome reaction and fertilization [59].…”

Ram seminal plasma and its functional proteomic assessment

“…Large proline-rich protein BAG6 reorganisation and degradation of proteins occurring during spermatogenesis (Tipler et al 1997). However, high levels of proteasomal activity have since been found in mature spermatozoa and found to regulate later events such as capacitation, the acrosome reaction and zona pellucida penetration (Arcelay et al 2008, Kong et al 2009, Yi et al 2010, Sanchez et al 2011, Zimmerman et al 2011. Similar to the CTT complex, in spermatozoa, the 26 proteasome shows a unique sperm surface localisation (Redgrove et al 2011, Byrne et al 2012 rather than a traditional intracellular localisation.…”