Intracellular calcium movements of boar spermatozoa during ‘in vitro’ capacitation and subsequent acrosome exocytosis follow a multiple-storage place, extracellular calcium-dependent model

Abstract

SUMMARYThis work analysed intracellular calcium stores of boar spermatozoa subjected to 'in vitro' capacitation (IVC) and subsequent progesterone-induced acrosome exocytosis (IVAE). Intracellular calcium was analysed through two calcium markers with different physico-chemical properties, Fluo-3 and Rhod-5N. Indicative parameters of IVC and IVAE were also evaluated. Fluo-3 was located at both the midpiece and the whole head. Rhod-5N was present at the sperm head. This distribution did not change in any of the assayed conditions. Induction of IVC was concomitant with an increase in both head and midpiece Ca 2+ signals. Additionally, while IVC induction was concurrent with a significant (p < 0.05) increase in sperm membrane permeability, no significant changes were observed in O 2 consumption and ATP levels. Incubation of boar spermatozoa in the absence of calcium showed a loss of both Ca 2+ labellings concomitantly with the sperm's inability to achieve IVC. The absence of extracellular calcium also induced a severe decrease in the percentage of spermatozoa exhibiting high mitochondrial membrane potential (hMMP). The IVAE was accompanied by a fast increase in both Ca 2+ signalling in control spermatozoa. These peaks were either not detected or much lessened in the absence of calcium. Remarkably, Fluo-3 marking at the midpiece increased after progesterone addition to sperm cells incubated in a medium without Ca 2+. The simultaneous addition of progesterone with the calcium chelant EGTA inhibited IVAE, and this was accompanied by a significant (p < 0.05) decrease in the intensity of progesterone Ca 2+ -induced peak, O 2 consumption and ATP levels. Our results suggest that boar spermatozoa present different calcium deposits with a dynamic equilibrium among them and with the extracellular environment. Additionally, the modulation role of the intracellular calcium in spermatozoa function seems to rely on its precise localization in boar spermatozoa.

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

“…In the case of CM, but not in that of NCM, ALH decreased. These changes in sperm kinematic parameters are related to the achievement of capacitation status and are similar to those published previously in the same conditions (Garc ıa-Herreros et al, 2005; Yeste et al, 2013Yeste et al, , 2015. The addition of progesterone after 4 h of incubation in CM induced a rapid increase in VCL, VAP and ALH that was concomitant with a decrease in LIN and STR (Tables 2-4).…”

“…In fact, a sudden peak of O 2 consumption and ATP synthesis has been described immediately after the induction of acrosome exocytosis with progesterone (Rami o-Lluch et al, 2013). This peak seems to be related to a coupled status of boar sperm mitochondria upon acrosome reaction, whereas mitochondria are uncoupled before and after acrosome reaction (Rami o-Lluch et al, 2013;Yeste et al, 2015).…”

“…Capacitation-like membrane lipid changes of boar sperm membrane was evaluated by merocyanine-540 (M540) and YO-PRO-1 co-staining, as described by Rathi et al (2001) and Yeste et al (2015). A total of four sperm populations were distinguished: (i) viable spermatozoa with low membrane lipid disorder (M540 À /YO-PRO-1 À ); (ii) viable spermatozoa with high, capacitation-like membrane lipid changes (M540 + /YO-PRO-1 À ); (iii) non-viable spermatozoa with low membrane lipid disorder (M540 À /YO-PRO-1 + ) and (iv) non-viable spermatozoa with high membrane lipid changes (M540 + /YO-PRO-1 + ).…”

“…Evaluation of intracellular calcium levels Intracellular calcium levels were determined through two separate, specific stains: Rhod-5N-AM (Rhod5) and Fluo-3-AM (Flu-o3), following the protocols described previously (Harrison et al, 1993;Kadirvel et al, 2009;Yeste et al, 2015). Rhod5 mainly stains the calcium of sperm head, whereas Fluo3 mainly stains that of the mitochondrial piece .…”

Preservation of boar semen: An update

“…The addition of seminal plasma at 10% (v/v) to the thawing solution increased the pregnancy rate in AI with frozen sperm lacking SP, significantly improved the acrosomal integrity, and suppressed the expression of a phosphoprotein used as a sperm capacitation marker. The latter was associated with an increase in intracellular flow of Ca 2+ (Okazaki, Abe, Yoshida, & Shimada, ; Yeste et al, ). The addition of EDTA and EGTA, Ca 2+ ‐chelating agents, to the thawing solution improves the fertilization capacity in frozen and thawed boar sperm (Okazaki, Yoshida, Teshima, & Shimada, ).…”