The achievement of boar spermin vitrocapacitation is related to an increase of disrupted disulphide bonds and intracellular reactive oxygen species levels

Abstract

The aim of this work was to determine the relationship of intracellular reactive oxygen species (ROS) and the disulphide bonds established between sperm proteins with the achievement of capacitation in boar spermatozoa. With this purpose, spermatozoa were incubated in a specifically designed in vitro capacitation medium (CM) in the presence or absence of reduced glutathione (GSH). Incubation of boar spermatozoa in CM for 4 h significantly (p < 0.05) increased free cysteine residues, which is a marker of disrupted disulphide bonds, and also intracellular ROS levels. The addition of GSH to the medium prevented most capacitation-like changes in sperm motility, membrane lipid disorder, mitochondrial membrane potential, intracellular calcium levels and localization of tyrosine-phosphorylated proteins (pTyr), but not in tyrosine phosphorylation of P32. These effects were accompanied by the inhibition of the ability of sperm cells to trigger the acrosome exocytosis in response to progesterone. When GSH was added together with progesterone after 4 h of incubation, acrosome exocytosis was not altered, but the subsequent decrease in intracellular calcium observed in controls cells was inhibited. Furthermore, co-incubation of oocytes with spermatozoa previously incubated in CM in the presence of GSH for 4 h significantly (p < 0.05) increased the number of spermatozoa attached to the oocyte surface but decreased normal fertilization rates. Our results suggest that boar sperm capacitation is related to an increase in disrupted disulphide bonds and intracellular ROS levels and that both events are related to the regulation of hyperactivated motility, intracellular calcium dynamics, sperm binding ability to the oocyte and achievement of proper nuclear decondensation upon oocyte penetration.

Unravelling how in vitro capacitation alters ram sperm chromatin before and after cryopreservation

“…However, only a few studies have investigated the effect of sperm capacitation on the chromatin structure. These studies found that spermatozoa showed a less compacted chromatin, sperm DNA integrity remained unchanged and the disulphide bonds between protamines decreased during in vitro capacitation 21‐24,46‐48 . Nevertheless, to the best of our knowledge, no previous studies have investigated sperm DNA methylation during this event.…”

“…These studies found that spermatozoa showed a less compacted chromatin, sperm DNA integrity remained unchanged and the disulphide bonds between protamines decreased during in vitro capacitation. [21][22][23][24][46][47][48] Sperm chromatin condensation can be evaluated rapidly with aniline blue (AB). This cytochemical dye selectively stains persisting lysine-rich histones 49,50 and has been widely used to identify an excessive histone retention in the chromatin of mature spermatozoa, which has been related to infertility, poor fertilization rates and morphological defects in the sperm head.…”

“…Some authors have investigated whether the acquisition of sperm fertilizing ability through the physiological process known as capacitation 19,20 might prepare the sperm chromatin for optimal remodelling after oocyte penetration. In boar spermatozoa, a reduction in disulphide bonds between protamines was observed under capacitating conditions, 21 while in bull and human spermatozoa, the degree of chromatin condensation decreased during in vitro capacitation without affecting DNA integrity 22‐24 . Such changes in nuclear compaction could facilitate the events surrounding and following fertilization, as has been suggested by De Lamirande et al 22 In line with these findings, Pérez‐Cerezales et al 25 demonstrated that a specific subpopulation of capacitated spermatozoa was selected by in vitro thermotaxis in humans and mice.…”

Parkinson Disease Protein 7 (PARK7) Is Related to the Ability of Mammalian Sperm to Undergo In Vitro Capacitation

“…Despite being mature and motile once ejaculated, mammalian sperm must reside in the female reproductive tract to acquire their fertilizing ability, in a process known as capacitation [ 1 , 2 , 3 ]. During capacitation, sperm undergo an assortment of physiological and biochemical changes, including alterations in plasma membrane permeability and fluidity [ 4 , 5 ], acrosome integrity [ 6 ], intracellular pH and calcium levels [ 7 , 8 ], mitochondrial activity [ 9 ], and the generation of reactive oxygen species (ROS) [ 10 ]. Oxidative stress, caused by excessive ROS formation, has detrimental consequences for sperm cells and fertility, including a decrease in sperm motility and their ability to interact with the oocyte, an increase in plasma membrane lipid peroxidation, protein damage and DNA fragmentation, and even the activation of apoptotic-like pathways [ 11 ].…”

“…Oxidative stress, caused by excessive ROS formation, has detrimental consequences for sperm cells and fertility, including a decrease in sperm motility and their ability to interact with the oocyte, an increase in plasma membrane lipid peroxidation, protein damage and DNA fragmentation, and even the activation of apoptotic-like pathways [ 11 ]. Notwithstanding, moderate levels of ROS are needed to perform some physiological processes, such as sperm capacitation [ 10 , 12 ]. Indeed, ROS generation during early capacitation events induces a rise of cAMP levels and the activation of protein kinases, which, in turn, trigger the phosphorylation of tyrosine residues of certain proteins, stimulating specific downstream pathways [ 13 , 14 ].…”