The calcium‐sensing receptor regulates protein tyrosine phosphorylation through PDK1 in boar spermatozoa

Abstract

Regulation of protein tyrosine phosphorylation is required for sperm capacitation and oocyte fertilization. The objective of the present work was to study the role of the calcium‐sensing receptor (CaSR) on protein tyrosine phosphorylation in boar spermatozoa under capacitating conditions. To do this, boar spermatozoa were incubated in Tyrode's complete medium for 4 hr and the specific inhibitor of the CaSR, NPS2143, was used. Also, to study the possible mechanism(s) by which this receptor exerts its function, spermatozoa were incubated in the presence of specific inhibitors of the 3‐phosphoinositide dependent protein kinase 1 (PDK1) and protein kinase A (PKA). Treatment with NPS2143, GSK2334470, an inhibitor of PDK1 and H‐89, an inhibitor of PKA separately induced an increase in tyrosine phosphorylation of 18 and 32 kDa proteins, a decrease in the serine/threonine phosphorylation of the PKA substrates together with a drop in sperm motility and viability. The present work proposes a new signalling pathway of the CaSR, mediated by PDK1 and PKA in boar spermatozoa under capacitating conditions. Our results show that the inhibition of the CaSR induces the inhibition of PDK1 that blocks PKA activity resulting in a rise in tyrosine phosphorylation of p18 and p32 proteins. This novel signalling pathway has not been described before and could be crucial to understand boar sperm capacitation within the female reproductive tract.

Fluorescent analysis of boar sperm capacitation process in vitro

“…According to our results, the first observable change is the redistribution of calcium ions (CTC FM [30];), accompanied by highest accessibility of acrosin epitopes (ACR.2 FM, FC), which resulted from enzymatic and proteomic changes in acrosomal matrix. At later capacitation stages (180 min), the phosphorylation of sperm proteins [31] and actin polymerization [6, 7] are also well detectable by presented methods. At this point it is important to mention, that collecting samples at only five different times during capacitation is not sufficient for detailed characterization of the molecular changes, on which physiological process of capacitation is based and sperm life imaging is more appropriate method to study this in detail.…”

Calcium transport in male reproduction is possibly influenced by vitamin D and CaSR

“…Previous studies have shown CaSR expression in the testis and spermatozoa from different species (Mendoza et al 2012, Kong et al 2015, Sun et al 2015, Macías-García et al 2016, Wang et al 2017, Liu et al 2018, Macías-García et al 2019) and a proteomic study showed that the CaSR was the most differentially expressed protein when comparing good vs bad quality bull spermatozoa (Singh et al 2018). Moreover, treatment with different CaSR agonists increased spermatozoa motility in rodents (Mendoza et al 2012), which implies that the low Casr mRNA in the testis of Vdr −/− mice could be related to the low motility in Vdr −/− and Cyp27b1 −/− mice (Kinuta et al 2000).…”

Ritonavir Has Reproductive Toxicity Depending on Disrupting PI3K/PDK1/AKT Signaling Pathway

“…In another previous research, AKT inhibition with AKTi-2 in mouse spermatozoa during capacitation significantly reduced total and progressive motility, tyrosine phosphorylation, and acrosome reaction, whereas there was no significant change in cell viability [ 39 ]. In regards to the previous study on PDK1 in boar spermatozoa, inhibition of PDK1 phosphorylation at the site of Ser 241 increased tyrosine phosphorylation [ 40 ]. Similarly, soon after inducing full hyperactivation of boar spermatozoa, PDK1 abruptly became inactivated by dephosphorylation in the Ser 241 region, and protein tyrosine phosphorylation was increased [ 41 ].…”