The tyrosine kinase FER is responsible for the capacitation-associated increase in tyrosine phosphorylation in murine sperm

Abstract

Sperm capacitation is required for fertilization. At the molecular level, this process is associated with fast activation of protein kinase A. Downstream of this event, capacitating conditions lead to an increase in tyrosine phosphorylation. The identity of the tyrosine kinase(s) mediating this process has not been conclusively demonstrated. Recent experiments using stallion and human sperm have suggested a role for PYK2 based on the use of small molecule inhibitors directed against this kinase. However, crucially, loss-of-function experiments have not been reported. Here, we used both pharmacological inhibitors and genetically modified mice models to investigate the identity of the tyrosine kinase(s) mediating the increase in tyrosine phosphorylation in mouse sperm. Similar to stallion and human, PF431396 blocks the capacitation-associated increase in tyrosine phosphorylation. Yet, sperm from Pyk2 −/− mice displayed a normal increase in tyrosine phosphorylation, implying that PYK2 is not responsible for this phosphorylation process. Here, we show that PF431396 can also inhibit FER, a tyrosine kinase known to be present in sperm. Sperm from mice targeted with a kinase-inactivating mutation in Fer failed to undergo capacitation-associated increases in tyrosine phosphorylation. Although these mice are fertile, their sperm displayed a reduced ability to fertilize metaphase II-arrested eggs in vitro.

Disruption of protein kinase A localization induces acrosomal exocytosis in capacitated mouse sperm

“…When necessary, PVDF membranes were stripped at 60°C for 15 min in 2% SDS, 0.74% ␤-mercaptoethanol, and 62.5 mM Tris (pH 6.5) and washed six times for 5 min each time in T-TBS. In all experiments, molecular masses were expressed in kilodaltons (kDa) (38).…”

Signaling Enzymes Required for Sperm Maturation and Fertilization in Mammals

“…Capacitation is operationaly defined as functional modifications that enable sperm to fertilize eggs. Sperm undergo various changes during capacitation: (a) removal of membrane cholesterol followed leading to a decrease in cholesterol to phospholipid ratio (b) membrane hyperpolarization, rise in intra-sperm pH and cAMP levels, (c) increase in calcium uptake, and (d) an increase in protein tyrosine phosphorylation possibily due to increased protein kinase A (PKA) activity (Arcelay et al, 2008;Bailey, 2010;Alvau et al, 2016;Jin and Yang, 2017;Molina et al, 2018). It is generally accepted that these changes during capacitation lead to: (a) the ability of the sperm to bind the oocyte's extracellular matrix, the zona pellucida (ZP) (Si and Olds-Clarke, 1999;Topper et al, 1999) and subsequently undergo the acrosome reaction, (b) hyperactivation, a whiplash flagellar motion required to penetrate the egg (Ho and Suarez, 2001), and (c) the capacity to fuse with the oocyte (Evans and Florman, 2002).…”

The actin cytoskeleton of the mouse sperm flagellum is organized in a helical structure

“…This localization is in line with previous reports of F-actin in sperm (Bouchard et al, 2000; Flaherty et al, 1986; Romarowski et al, 2015); however, the narrow shape of the sperm flagellum together with the limitations in resolution of widefield microscopy, did not allow to observe details of the actin structure. To investigate the organization of F-actin in the flagellum in detail, we imaged phalloidin-stained sperm using STORM (Bates et al, 2008), a superresolution technique that was previously employed successfully to evaluate the localization of different proteins in mouse sperm (Alvau et al, 2016; Chung et al, 2014). Consistent with results obtained using widefield immunofluorescence imaging (Fig 1 A and B), STORM showed high density of F-actin in the tail.…”