The Sarcoplasmic/Endoplasmic reticulum Ca2+-ATPase (SERCA) is present in pig sperm and modulates their physiology over liquid preservation

Abstract

Liquid storage is the primary preservation method in the swine breeding industry because of its advantages over cryopreservation. Calcium (Ca²⁺), a key regulator of cell physiology, plays a crucial role during liquid preservation. Sarcoplasmic/Endoplasmic Reticulum Ca²⁺ ATPases (SERCA) belong to a family of P-type ATPases that regulate Ca²⁺ homeostasis within cells and have been previously described to play a function in the sperm of various mammalian species. Herein, we hypothesized that SERCA2 is present in pig sperm and is involved in the resilience of this cell to liquid preservation at 17 °C. For this purpose, sperm were incubated with different concentrations of thapsigargin (Thg; 0, 5, 25, and 50 µM) and stored at 17 °C for ten days. The presence and localization of SERCA2 were evaluated using immunoblotting and immunofluorescence, respectively. On days 0, 4, and 10, sperm motility was assessed using a computer-assisted sperm analysis (CASA) system, and sperm viability, membrane lipid disorder, acrosome integrity, mitochondrial membrane potential (MMP), and intracellular levels of Ca²⁺, superoxides and total reactive oxygen species (ROS) were evaluated by flow cytometry. We localized SERCA2 in the acrosome and midpiece of pig sperm. Furthermore, inhibition of SERCA with Thg resulted in reduced sperm viability and membrane stability, and increased MMP, and Ca²⁺ and ROS levels. In conclusion, the activity of SERCA prevents the accumulation of intracellular Ca²⁺ in sperm, which is detrimental to sperm quality and function during liquid storage at 17 °C. We thus suggest that the function of SERCA is crucial for the preservation of pig semen.

Keywords: Calcium; Liquid storage; Pig; SERCA; Semen; Thapsigargin.

Fig. 1

Presence and localization of SERCA2 in pig sperm. (a) Representative immunoblotting of SERCA2 in pig sperm after incubation with the anti-SERCA2 antibody. MW: Molecular Weight; 1–3 correspond to different samples. The black box indicates the SERCA2 band. (b) Representative immunofluorescence analysis of SERCA2 in pig sperm. Sperm nuclei are shown in blue (DAPI), whereas SERCA2 is shown in green. The white arrow shows the localization of SERCA2 in the midpiece, whereas arrowheads show its presence in the acrosome. Scale bars: 20 μm.

Fig. 2

Effect of inhibiting SERCA activity with Thg on the plasma membrane and acrosome integrity, and lipid membrane stability. (a) Viable sperm (SYBR-14⁺/PI⁻, %); (b) viable sperm with an intact acrosome (PNA⁻/PI⁻, %); and (c) viable sperm with high lipid membrane stability (M540⁻/Yo-Pro-1⁻, %) after 0, 4 and 10 days of liquid storage at 17 °C in the presence (5, 25 and 50 µM) or absence (0 µM; control) of Thg. Different letters (a-c) indicate significant differences between experimental groups at a given time point. Results are expressed as the mean ± SEM (n = 7).

Fig. 3

Effect of inhibiting SERCA activity with Thg on reactive oxygen species (ROS) levels. (a) E⁺ intensity in viable sperm (Yo-Pro-1⁻); and (b) DCF⁺ intensity in viable sperm (PI⁻) after 0, 4 and 10 days of liquid storage at 17 °C in the presence (5, 25 and 50 µM) or absence (0 µM; control) of Thg. Different letters (a-c) indicate significant differences between experimental groups at a given time point. Results are expressed as the mean ± SEM (n = 7).

Fig. 4

Effect of inhibiting SERCA activity with Thg on intracellular calcium (Ca²⁺) and mitochondrial membrane potential (MMP). (a) Fluo4 intensity in viable sperm (PI⁻); and b) ratio between JC-1_agg and JC-1_mon in viable sperm (LD⁻) after 0, 4 and 10 days of liquid storage at 17 °C in the presence (5, 25 and 50 µM) or absence (0 µM; control) of Thg. Different letters (a-c) indicate significant differences between experimental groups at a given time point. Results are expressed as the mean ± SEM (n = 7).

Fig. 5

Effect of inhibiting SERCA activity with Thg on sperm motility. (a) Percentages of total motile sperm; and (b) percentages of progressively motile sperm after 0, 4 and 10 days of liquid storage at 17 °C in the presence (5, 25 and 50 µM) or absence (0 µM; control) of Thg. No significant differences between groups were observed Results are expressed as the mean ± SEM (n = 7).