Cyclic AMP and glycogen synthase kinase 3 form a regulatory loop in spermatozoa

Abstract

The multifaceted glycogen synthase kinase (GSK3) has an essential role in sperm and male fertility. Since cyclic AMP (cAMP) plays an important role in sperm function, we investigated whether GSK3 and cAMP pathways may be interrelated. We used GSK3 and soluble adenylyl cyclase (sAC) knockout mice and pharmacological modulators to examine this relationship. Intracellular cAMP levels were found to be significantly lower in sperm lacking GSK3α or GSK3β. A similar outcome was observed when sperm cells were treated with SB216763, a GSK3 inhibitor. This reduction of cAMP levels was not due to an effect on sperm adenylyl cyclase but was caused by elevated phosphodiesterase (PDE) activity. The PDE4 inhibitor RS25344 or the general PDE inhibitor IBMX could restore cAMP levels in sperm lacking GSK3α or β-isoform. PDE activity assay also showed that hyperactivated PDE4 contributes in lowering of cAMP levels in GSK3α null sperm suggesting that in wild-type sperm PDE4 activity is kept in check by GSK3. Conversely, PKA being triggered by cAMP, affected GSK3 activity through increasing its phosphorylation. Increased GSK3 phosphorylation also occurred by inhibition of sperm specific protein phosphatase type 1, PP1γ2. The relationship between cAMP, GSK3, and PP1γ2 activities was also confirmed in sperm from sAC null mice. Pull-down assay using recombinant PP1γ2 indicated that PKA, GSK3, and PP1γ2 could exist as a complex. Pharmacological inhibition of GSK3 in mature spermatozoa resulted in significantly reduced fertilization of eggs in vitro. Our results show that cAMP, PKA, and GSK3 are interrelated in regulation of sperm function.

Roles of glycogen synthase kinase 3 alpha and calcineurin in regulating the ability of sperm to fertilize eggs

“…19 Sperm were incubated with Fura 2-AM (final conc. 19 Sperm were incubated with Fura 2-AM (final conc.…”

“…19 GSK3 activity was measured by the amount of 32 PO 4 transferred from [ 32 P] γ-ATP to phospho-glycogen synthase peptide-2 (GSK3 substrate, Millipore). 19 GSK3 activity was measured by the amount of 32 PO 4 transferred from [ 32 P] γ-ATP to phospho-glycogen synthase peptide-2 (GSK3 substrate, Millipore).…”

“…19 Extracts were incubated overnight in a rotating shaker at 4°C with 4 µg each of either recombinant PPP3CC-His tag (US Biological, Cat# 374827).…”

“…[11][12][13] In the female reproductive tract, intra-sperm calcium levels are elevated subsequent to a change in membrane potential and intracellular pH. 19 The two isoforms of GSK3α and β are ubiquitous and redundant in most contexts. These models however fall short because they do not incorporate protein phosphatases, which are known to be initiators, modifiers, and terminators of signaling responses in cells.…”

“…16,17 The protein phosphatase isoform PP1γ2, present only in mammals, is regulated by its binding proteins-PPP1R2, PPP1R7, and PPP1R11. 19 Calcineurin (also known as PP2B or PPP3) is a serine/ threonine phosphatase regulated by calcium. GSK3, PKA, and PP1γ2 appear mechanistically interrelated in regulating sperm motility.…”