The effects of oxidative stress and intracellular calcium on mitochondrial permeability transition pore formation in equine spermatozoa

Abstract

The in vitro storage of stallion spermatozoa for use in artificial insemination leads to oxidative stress and imbalances in calcium homeostasis that trigger the formation of the mitochondrial permeability transition pore (mPTP), resulting in premature cell death. However, little is understood about the dynamics and the role of mPTP formation in mammalian spermatozoa. Here, we identify an important role for mPTP in stallion sperm Ca²⁺ homeostasis. We show that stallion spermatozoa do not exhibit "classical" features of mPTP; specifically, they are resistant to cyclosporin A-mediated inhibition of mPTP formation, and they do not require exogenous Ca²⁺ to form the mPTP. However, chelation of endogenous Ca²⁺ prevented mPTP formation, indicating a role for intracellular Ca²⁺ in this process. Furthermore, our findings suggest that this cell type can mobilize intracellular Ca²⁺ stores to form the mPTP in response to low Ca²⁺ environments and that under oxidative stress conditions, mPTP formation preceded a measurable increase in intracellular Ca²⁺, and vice versa. Contrary to previous work that identified mitochondrial membrane potential (MMP) as a proxy for mPTP formation, here we show that a loss of MMP can occur independently of mPTP formation, and thus MMP is not an appropriate proxy for the detection of mPTP formation. In conclusion, the mPTP plays a crucial role in maintaining Ca²⁺ and reactive oxygen species homeostasis in stallion spermatozoa, serving as an important regulatory mechanism for normal sperm function, thereby contraindicating the in vitro pharmacological inhibition of mPTP formation to enhance sperm longevity.

Keywords: JC‐1; horse; mitochondrial permeability transition pore; oxidative stress; spermatozoa.

FIGURE 1

Representative flow cytometric dot plots of triple‐stained (Far Red LIVE/DEAD, JC‐1, and calcein AM Violet [C‐AM‐V]) stallion spermatozoa. (A) Spermatozoa were gated from debris (using a forward scatter; FSC/side‐scatter; SSC dot plot) into a second plot to gate viable spermatozoa (Far Red LIVE/DEAD dim) into subsequent JC‐1 plots to determine mitochondrial membrane potential (MMP). (B–D) Representative flow cytometric dot plots showing the control treatment (B), CCCP negative control for JC‐1 gating (C), and 1 μM ionomycin treatment at 1 h (D). From the JC‐1 plots, high or low MMP spermatozoa were gated into C‐AM‐V/SSC plots to determine the mPTP formation status of these populations. Created using Biorender.com. CCCP, carbonyl cyanide m‐chlorophenylhydrazone; mPTP, mitochondrial permeability transition pore.

FIGURE 2

Mitochondrial permeability transition pore (mPTP) formation in stallion spermatozoa is not immediately accompanied by a loss of mitochondrial membrane potential (MMP). (A) Percentage of C‐AM‐V bright (no mPTP formation) and high MMP of stallion spermatozoa exposed to various ionomycin doses. (B) Percentage of C‐AM‐V bright (no mPTP formation) and high MMP of stallion spermatozoa exposed to 1 μM ionomycin for 0, 1, 2, and 3 h. N = 3 split ejaculates. Significant difference from the control (A) or t = 0 h (B) denoted by *p ≤ 0.05 or ***p ≤ 0.001. C‐AM‐V, calcein AM Violet.

FIGURE 3

Stallion spermatozoa do not exhibit classical mitochondrial permeability transition pore (mPTP) formation. The stallion sperm mPTP formed in response to ionomycin‐induced calcium (Ca²⁺) influx (A), but the mPTP could not be inhibited by pretreatment with cyclosporin A (B). The stallion sperm mPTP is not dependent on an exogenous source of Ca²⁺; complete mPTP formation occurs in a Ca²⁺‐free medium without any additional stimuli [(C) mean of the median calcein AM signal, (D) representative flow cytometric dot plots]. N = 9 split ejaculates. Significant difference from the control (gray bar) denoted by ***p ≤ 0.001.

FIGURE 4

The stallion sperm mitochondrial permeability transition pore (mPTP) is responsible for maintaining calcium (Ca²⁺) homeostasis‐assessed using calcein AM Green (C‐AM‐G) staining and flow cytometry. (A) Mean ± SEM of the median C‐AM‐G signals from spermatozoa which have been incubated in normal BWW (“Control” containing 1.7 mM Ca²⁺ for 1 h; gray bar), and treatments (black bars) which have been incubated in Ca²⁺‐free BWW for 30 min, after which Ca²⁺ was reintroduced in a dose‐dependent manner for an additional 30 min. (B) Representative histogram showing fluorescence signals from the treatments shown in (A). N = 3 split ejaculates. Significant difference from the control [gray bar; (A)] denoted by **p ≤ 0.01 and ***p ≤ 0.001.

FIGURE 5

Stallion sperm mitochondrial permeability transition pore (mPTP) formation in response to oxidative stress using exogenous [(A) H₂O₂] and endogenous [(B) stimulated by AA] sources of reactive oxygen species. The sperm mPTP formed in response to oxidative stress using physiological doses of AA, but the IC₅₀ was achieved only at supraphysiological doses of H₂O₂. N = 9 split ejaculates. Significant difference from the control [gray bar; (A)] denoted by *p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001. AA, arachidonic acid.

FIGURE 6

Characterizing the dynamic relationship between oxidative stress and mitochondrial permeability transition pore (mPTP) formation. A triple staining technique (LIVE/DEAD Violet to gate viable spermatozoa into a MitoSox Red [MSR] and C‐AM‐G analysis dot plot) revealed that the mPTP does not begin to form until the cell is in a state of oxidative stress (MSR bright). Each series in the graph (bottom) corresponds to a quadrant from the flow cytometry output (shown in the representative dot plots above each treatment group), denoted by the same color; orange = MSR bright/C‐AM‐G dim; blue = MSR bright/C‐AM‐G bright; gray = MSR dim/C‐AM‐G dim; and green = MSR dim/C‐AM‐G dim. N = 9 split ejaculates. Significant difference from the control (pairwise comparisons using Bonferroni corrections) denoted by *p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001. C‐AM‐G, calcein AM Green.

FIGURE 7

The stallion sperm mitochondrial permeability transition pore (mPTP) is more sensitive to increases in calcium [Ca²⁺; (A)] than increases in ROS (B), though mPTP formation via either mechanism is independent of the other. (A) The mPTP forms (loss of calcein AM Violet [C‐AM‐V] fluorescence) immediately in response to elevated Ca²⁺ (Fluo‐4), both of which become significantly different from the control at 40 nM of ionomycin with no increase in mitochondrial ROS. (B) Mitochondrial ROS increases significantly at 2.5 μM arachidonic acid (AA), which is followed by mPTP formation at doses at or above 10 μM AA, while intracellular Ca²⁺ does not increase until a dose of 20 μM AA. N = 3 split ejaculates. Significant difference from the control (Dunnett's test) denoted by “†” (p ≤ 0.05); “**” or “◊◊” (p ≤ 0.01); and “***”, “◊◊◊” or “†††” (p ≤ 0.001) for intracellular Ca²⁺ (Fluo‐4), mitochondrial ROS (MSR), and mPTP formation (C‐AM‐V fluorescence loss), respectively. ROS, reactive oxygen species.

FIGURE 8

BAPTA‐AM inhibits stallion sperm mitochondrial permeability transition pore (mPTP) formation in response to oxidative stress stimulated by arachidonic acid (AA). Formation of the sperm mPTP in response to 20 μM AA can be inhibited via pretreatment with BAPTA‐AM (5 μM), suggesting that reactive oxygen species‐induced mPTP formation is mediated by intracellular Ca²⁺. N = 10 split ejaculates. Significant difference from the control (gray bar) denoted by different letter superscripts (p ≤ 0.05). BAPTA‐AM, 1,2‐bis(2‐aminophenoxy)ethane‐N,N,N′,N′‐tetraacetic acid tetrakis(acetoxymethyl ester).