Polyamines protect porcine sperm from lipopolysaccharide-induced mitochondrial dysfunction and apoptosis via casein kinase 2 activation

Abstract

Bacterial contamination is an inevitable issue during the processing of semen preservation in pigs. As a prototypical endotoxin from Gram-negative bacteria in semen, lipopolysaccharide (LPS) undermines sperm function during liquid preservation. Spermine and spermidine could protect cells against LPS-induced injury, and the content of spermine and spermidine in seminal plasma is positively correlated with sperm quality. Thus, the present study aimed to clarify whether addition of spermine or spermidine is beneficial to porcine semen preservation and able to prevent LPS-induced sperm damage. The supplementation of spermine and spermidine in the diluent resulted in higher sperm motility, viability, acrosome integrity, and mitochondrial membrane potential (ΔΨ_m) after preservation in vitro at 17 °C for 7 d (P < 0.05). LPS-induced sperm quality deterioration, ΔΨ_m decline, cellular adenosine-triphosphate depletion, mitochondrial ultrastructure abnormality, mitochondrial permeability transition pore opening, phosphatidylserine (PS) translocation, and caspase-3 activation (P < 0.05). Interestingly, spermine and spermidine alleviated the LPS-induced changes of the aforementioned parameters and mitigated the decrease in the microtubule-associated protein light chain 3-II (LC3-II) to LC3-I ratio. Meanwhile, the α and β subunits of casein kinase 2 (CK2) were detected at the connecting piece and the tail. Significantly, addition of 4,5,6,7-tetrabromobenzotriazole, a specific CK2 inhibitor, counteracted the beneficial effects of spermine and spermidine on sperm quality, mitochondrial activity, and apoptosis. Together, these results suggest that spermine and spermidine improve sperm quality and the efficiency of liquid preservation of porcine semen. Furthermore, spermine and spermidine alleviate LPS-induced sperm mitochondrial dysfunction and apoptosis in a CK2-dependent manner.

Lipopolysaccharide released from the Gram-negative bacteria undermines sperm functionality and efficacy of semen preservation in vitro. Spermine and spermidine alleviate LPS-induced boar sperm mitochondrial dysfunction and apoptosis via casein kinase 2 activation, thereby maintaining sperm motility and viability.

Introduction

In view of the sperm biological characteristics and limited efficiency of cryopreservation, liquid semen preserved at 17 °C in vitro is used mainly for artificial insemination (AI) in the pig industry (Waberski et al., 2019). Bacterial contamination is unavoidable during the processing of semen storage. Gram-negative bacteria, the predominant pathogenic microorganism in semen, are detrimental to sperm quality and decrease the litter size obtained from sows inseminated with contaminated semen (Contreras et al., 2022).

In the contaminated semen, lipopolysaccharide (LPS) released from the outer membrane of Gram-negative bacteria undermines sperm functionality, consequently inducing sperm apoptosis (Eley et al., 2005; He et al., 2017) and immune response disorders in the female genital tract, eventually leading to subfertility or infertility (Makvandi et al., 2019). It has been demonstrated that LPS was present in porcine semen (0.62 µg/mL) and that it induced mitochondrial dysfunction and ultrastructure changes, resulting in decreased mitochondrial integrity and motility in porcine sperm (He et al., 2017). Although specific chemicals or antibiotics in semen extenders may inhibit bacterial proliferation, they do not eliminate LPS from the bacteria (Okazaki et al., 2010). As a result, supplementation with neutralization molecules that work against LPS activity, such as β-defensin 114, polymyxin B, polyhistidine, or alpha lipoic acid, is a potential protective strategy to alleviate LPS-induced sperm motility decline and apoptosis (Okazaki et al., 2010; Yu et al., 2013; Makvandi et al., 2019; Song et al., 2019).

Spermine and spermidine, as natural polyamine members, are derived from the prostate and abundant in seminal plasma (Lefevre et al., 2011). The concentration of polyamines in seminal plasma was positively correlated with sperm quality in humans (Vanella et al., 1978), dogs (Setyawan et al., 2016), and bulls (Saraf et al., 2020). Previous evidence has suggested that the anti-inflammatory and anti-apoptotic effects can be attributed to spermine and spermidine in cardiac cells, stem cells, intestinal epithelial cells, chondrocytes, and macrophages (Flamigni et al., 2007; Niechcial et al., 2023). The anti-apoptotic function of spermine has also been shown in canine and rat sperm (Setyawan et al., 2016; Shahin et al., 2019). In microglia, intestinal cells, and the mouse hippocampus, spermine and spermidine have been shown to reverse the inflammatory or pathological effects induced by LPS (Choi and Park, 2012; Fruhauf et al., 2015; Truzzi et al., 2023). On the basis of these findings, we hypothesize that spermine or spermidine may alleviate the negative effects of LPS on boar sperm. Thus, the present study aimed to elucidate whether supplementation of spermine and spermidine is beneficial to sperm survival and able to prevent LPS-induced sperm damage and, if so, to elucidate the related underlying mechanism.

Materials and Methods

All experimental procedures connected with the care and use of animals, as described herein, were approved by the Animal Care and Use Committee of Northwest A&F University (ethical approval code: H17-89), in accordance with the Regulations of the Executive Committee for Laboratory Animal Management and Ethical Review of Northwest A&F University.

Experimental design

Experiment 1 was devised to identify whether spermine or spermidine positively affected porcine sperm quality during liquid preservation at 17 °C. Sperm were diluted in Modena with different concentrations (0, 0.25, 0.5, 1, and 2 mM) of spermine or spermidine when preserved at 17 °C for 7 d. Sperm motility, viability, acrosome integrity, and mitochondrial membrane potential (ΔΨ_m) were assessed at 0 d (the immediately extended raw semen samples) and after 7 d of preservation.

Experiment 2 aims to determine whether spermine or spermidine can alleviate LPS-induced dysfunction in boar sperm. Sperm were purified and incubated in Modena with different concentrations (0, 0.25, 0.5, and 1 mM) of spermine or spermidine in the presence or absence of LPS (1 µg/mL) at 37 °C for 6 h. We evaluated sperm motility, viability, acrosome integrity, ΔΨ_m, adenosine-triphosphate (ATP) content, mitochondrial permeability transition pore (MPTP), mitochondrial ultrastructure, phosphatidylserine (PS) translocation, caspase-3 and microtubule-associated protein light chain-3 (LC3) levels after 6 h of incubation at 37 °C.

With experiment 3, we aimed to find out whether casein kinase 2 (CK2) mediated the protective role of spermine or spermidine on porcine sperm. The expression and localization of CK2 (including α and β subunits) in porcine sperm were determined by western blotting and immunofluorescence, respectively. Sperm were also incubated at 37 °C for 6 h with polyamines (0.5 mM spermine or spermidine) and LPS in the presence or absence of 60 µM 4,5,6,7-tetrabromobenzotriazole (TBB, a CK2-specific inhibitor). Sperm motility, viability, acrosome integrity, ΔΨ_m, MPTP, CK2α, CK2β, Bax, caspase-3, and LC3 levels were detected after the incubation.

In the present study, the sperm storage trial at room temperature (17 °C) was performed to assess the relevance of spermine- or spermidine-mediated changes in an applied sperm storage setting. With the exception of the sperm storage trial conducted at room temperature (17 °C), all experiments were performed by incubating sperm treatments at 37 °C. This temperature was chosen to facilitate the observation of biologically relevant changes in sperm physiology.

Reagents and media

All reagents were purchased from Sigma-Aldrich (China) unless otherwise stated. TBB was purchased from Santa Cruz. Modena solution was used as the basic diluent.

Semen collection and processing

Seven mature and fertile boars (2 Large White, 2 Landrace, and 3 Duroc), aged 17 to 30 mo, were used in the present study. These boars were provided individual houses with natural daylight, and the same commercial porcine ration and water were provided throughout the experimental duration. The temperature in the pig houses was controlled at 20 to 24 °C. The sperm-rich fraction was collected with the gloved hand technique twice a week, with fresh semen placed in a 37 °C bath and delivered to the laboratory within 15 min for the evaluation of sperm motility and concentration. The ejaculates were processed individually. Only fresh semen samples with over 80% total motility (TM) and 2 × 10⁸ sperm/mL were used for the present study. Sperm were diluted to a final concentration of 50 × 10⁶ sperm/mL with basic diluent in the subsequent procedures. Each experiment was conducted on at least 3 ejaculates, with the biological replicates presented as n = 3 or n = 5 in the legends of the figures or tables. For evaluation of sperm parameters, each sample of the treatments was analyzed in 3 technical replicates, and the values obtained from each replicate were averaged to determine the final measurement for each sample.

Sperm motility, viability, and acrosome integrity

Sperm motility was determined by computer-assisted sperm analysis (CASA; HVIEW, China). A Sperm Viability Kit (Invitrogen, USA) was used to evaluate sperm viability. Fluorescein isothiocyanate peanut agglutinin (FITC-PNA) and prodium iodide (PI) probes were used to evaluate sperm acrosome integrity. Detailed descriptions of the evaluation of sperm motility, viability, and acrosome integrity are given in Supplementary material, Materials and Methods section.

Sperm ΔΨ_m and ATP content

ΔΨ_m and ATP assay kits (Beyotime, China) were used to monitor ΔΨ_m and ATP content of sperm, respectively. Detailed descriptions of the evaluation of sperm ΔΨ_m and ATP content are given in Supplementary material, Materials and Methods section.

Transmission electron microscopy and scanning electron microscopy observation for sperm mitochondrial ultrastructure

Sperm mitochondrial ultrastructure was observed by a transmission electron microscope (Tecnai G2 Spirit, FEI, USA) as described in He et al. (2017). Briefly, the samples were fixed with 2% glutaraldehyde and then fixed with 1% osmium tetroxide, and processed by the electron microscopy service at the Life Science Research Center of Northwest A&F University, following a conventional protocol. The sperm mitochondrial ultrastructure was examined under a transmission electron microscope at magnifications ranging from 10,000 to 30,000×. Detailed descriptions of the evaluation of sperm mitochondrial ultrastructure are given in Supplementary material, Materials and Methods section.

Sperm mitochondrial morphology was determined by a scanning electron microscope (Nova NanoSEM450, FEI, USA), as described in Sun et al. (2020). Sperm were examined under a scanning electron microscope at 2,000× and 20,000× magnification. Detailed descriptions of the evaluation of sperm mitochondrial morphology are given in Supplementary material, Materials and Methods section.

Analysis of the MPTP by flow cytometry

The MPTP was evaluated with the MPTP Assay Kit (Beyotime, China), as reported by Fang et al. (2019). Briefly, sperm aliquots (2 × 10⁶ sperm) were incubated with 1 mL 1 × calcein-acetoxymethyl ester (calcein-AM) stain solution and 1 × CoCl₂ solution at 37 °C for 30 min in darkness, followed by the replacement of the staining solution with prewarmed Modena for another 30 min incubation at 37 °C in the dark. To exclude dead sperm from the analysis, PI was added at a final concentration of 1 mM. For each sperm, calcein (green fluorescence, FL1) and PI (red fluorescence, FL3) were monitored by flow cytometry (BD, FACS Aria III, USA). Detailed descriptions of the analysis of the MPTP by flow cytometry are given in Supplementary material, Materials and Methods section.

Analysis of sperm PS translocation by flow cytometry

PS translocation was examined by an Annexin V-FITC Apoptosis Kit (YEASEN, China), following the manufacturer’s instructions. Briefly, we re-suspended 5 × 10⁶ sperm in binding buffer and mixed them with Annexin V-FITC and PI, then incubated the mixture at room temperature for 15 min in darkness. For each sperm, FITC fluorescence (FL1) and PI fluorescence (FL3) were monitored by flow cytometry (BD, FACS Aria III, USA). Detailed descriptions of the analysis of sperm PS translocation by flow cytometry are given in Supplementary material, Materials and Methods section.

Western blotting

Briefly, samples (5 × 10⁷ sperm in 1 mL) under different treatments were washed with PBS, and resuspended in 200 µL radio immunoprecipitation assay (RIPA) lysis buffer with 1% phenylmethyl sulfonyl fluoride, 1% phosphatase inhibitor, and 1% protease inhibitor cocktail. During lysis at 4 °C for 30 min, the samples also underwent ultrasonic treatment. Subsequently, the samples were centrifuged at 12,000 × g for 10 min at 4 °C. The supernatant was mixed with sodium dodecyl sulfate (SDS) loading buffer and boiled. sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting were performed on lysates containing equivalent amounts of protein (30 µg) using antibodies against caspase-3 (Cell Signaling Technology, 9662S, diluted 1:1,000), Bax (Cell Signaling Technology, 2772S, diluted 1:1,000), LC3 (Sigma-Aldrich, L7543, diluted 1:1,000), CK2α (Santa Cruz, sc-373894, diluted 1:1,000), and CK2β (Santa Cruz, sc-12739, diluted 1:1,000). Polyvinylidene fluoride (PVDF) membranes were stripped and re-incubated with anti-alpha-tubulin antibody (Proteintech, 11224-1-AP, diluted 1:5,000) as loading control. Detailed descriptions of western blotting procedure for sperm samples are provided in the Supplementary material, Materials and Methods section.

Immunofluorescence

Briefly, aliquots of 200 µL containing 1 × 10⁷ sperm were fixed with 4% paraformaldehyde and permeabilized with 0.25% Triton X-100, followed by blocking with 10% bovine serum albumin (BSA). They were then incubated overnight at 4 °C with primary antibodies for CK2α (Santa Cruz, sc-373894, diluted 1:50) or CK2β (Santa Cruz, sc-12739, 1:50). A negative control was set up without the primary antibody. After washing, the samples were incubated with a secondary antibody (Alexa Fluor 488 rabbit anti-mouse IgG, Solarbio, diluted 1:100) and counterstained with 4’,6-diamidino-2-phenylindole (DAPI) (diluted 1:1000). Observations and photography were conducted using an epifluorescent microscope (Nikon 80i, 400× magnification), with at least 100 sperm observed from 5 random fields per sample. Detailed descriptions of immunofluorescence of sperm samples are given in Supplementary material, Materials and Methods section.

Statistical analysis

All values were demonstrated as mean ± standard error of the mean (SEM). Before statistical analysis, we examined all data for normality and variance homogeneity, and performed arc-sin square root transformation if necessary. We analyzed data using a one-way analysis of variance and performed multiple comparison analyses by the Duncan test. All analyses were performed with SPSS v26.0 for Windows (SPSS Inc., USA). For all analyses, P < 0.05 was considered significant.

Results

Spermine and spermidine improve sperm quality during liquid preservation in vitro

The administration of spermine or spermidine did not alter the motility parameters, viability, acrosome integrity, or ΔΨ_m of boar sperm at 0 d (P > 0.05; Tables 1 and 2 and Figure 1a–c). After preservation at 17 °C for 7 d, higher progressive motility (PM), straight-line velocity (VSL), curve-line velocity (VCL), and average path velocity (VAP) were observed in samples treated with 0.5 mM spermine or spermidine treatment (P < 0.05, Tables 1 and 2). There were no differences in TM between the control and polyamine (spermine or spermidine) treatments during the liquid preservation (P > 0.05, Tables 1 and 2). Also, supplementation of 0.5 mM spermine or spermidine to the basic diluent improved sperm viability, acrosome integrity, and ΔΨ_m (Figure 1a–c, P < 0.05).

Table 1.

Effect of spermine supplementation on sperm motility after liquid preservation at 17 °C for 7 d

Motility parameters	Preservation for 0 d
	Control	SM-0.25 mM	SM-0.5 mM	SM-1 mM	SM-2 mM
TM, %	92.7 ± 0.8	93.6 ± 0.4	91.3 ± 1.2	90.7 ± 0.7	92.8 ± 0.9
PM, %	81.4 ± 1.8	81.6 ± 0.9	79.6 ± 1.3	79.9 ± 1.8	81.5 ± 1.9
VSL, µm/s	74.5 ± 3.3	72.2 ± 4.4	74.0 ± 3.3	78.5 ± 3.0	73.5 ± 4.3
VCL, µm/s	147.9 ± 3.6	144.3 ± 5.1	148.2 ± 5.0	155.3 ± 6.7	137.7 ± 6.1
VAP, µm/s	85.1 ± 5.7	86.2 ± 4.8	93.2 ± 8.0	83.6 ± 7.3	78.4 ± 4.9

	Preservation for 7 d
TM, %	83.3 ± 1.5	83.9 ± 1.8	85.3 ± 1.0	82.7 ± 0.5	76.0 ± 5.6
PM, %	50.6 ± 0.9bc	59.1 ± 3.1ab	63.2 ± 2.1a	59.2 ± 3.5ab	42.3 ± 6.1c
VSL, µm/s	31.4 ± 2.6bc	43.0 ± 4.2a	45.4 ± 1.5a	38.7 ± 4.6ab	25.7 ± 4.7c
VCL, µm/s	80.8 ± 6.8bc	105.1 ± 7.8a	108.5 ± 3.6a	100.6 ± 8.4ab	75.5 ± 8.9c
VAP, µm/s	29.7 ± 1.6b	40.1 ± 2.7a	43.8 ± 0.9a	41.0 ± 2.8a	29.2 ± 3.4b

Sperm motility parameters (TM, PM, VSL, VCL, and VAP) were analyzed by the CASA (n = 5). Values are presented as mean ± SEM. Values within different superscript letters indicate a significant difference (P < 0.05) between treatments. SM, spermine; TM, total motility; VSL, straight-line velocity; VCL, curve-line velocity; VAP, average path velocity; PM, progressive motility.

Table 2.

Effect of spermidine supplementation on sperm motility after liquid preservation at 17 °C for 7 d

Motility parameters	Preservation for 0 d
	Control	SM-0.25 mM	SM-0.5 mM	SM-1 mM	SM-2 mM
TM, %	92.7 ± 0.8	92.8 ± 1.1	93.9 ± 1.5	91.4 ± 1.1	91.6 ± 2.0
PM, %	81.4 ± 1.8	79.9 ± 1.3	80.5 ± 1.9	78.2 ± 1.8	79.9 ± 1.7
VSL, µm/s	74.5 ± 3.3	75.0 ± 3.7	78.0 ± 1.1	69.5 ± 3.0	81.4 ± 4.2
VCL, µm/s	147.9 ± 3.6	144.6 ± 6.9	152.7 ± 3.1	138.5 ± 1.5	153.2 ± 3.8
VAP, µm/s	85.1 ± 5.7	93.5 ± 7.8	84.2 ± 5.7	90.8 ± 5.2	87.0 ± 5.8

	Preservation for 7 d
TM, %	83.3 ± 1.5	85.2 ± 0.7	83.6 ± 1.7	86.8 ± 1.0	84.9 ± 0.8
PM, %	50.6 ± 0.9b	59.4 ± 2.5a	64.3 ± 1.8a	61.6 ± 3.3a	60.3 ± 3.9a
VSL, µm/s	31.4 ± 2.6b	39.5 ± 2.9ab	46.1 ± 5.2a	44.3 ± 5.8ab	40.6 ± 3.7ab
VCL, µm/s	80.8 ± 6.8b	96.3 ± 5.1ab	107.2 ± 8.0a	106.3 ± 9.7a	102.0 ± 9.0ab
VAP, µm/s	29.7 ± 1.6b	37.4 ± 2.4ab	43.0 ± 3.0a	41.8 ± 4.4a	44.0 ± 4.2a

Sperm motility parameters (TM, PM, VSL, VCL, and VAP) were analyzed by the CASA (n = 5). Values are presented as mean ± SEM. Values within different superscript letters indicate a significant difference (P < 0.05) between treatments. SD, spermidine; TM, total motility; VSL, straight-line velocity; VCL, curve-line velocity; VAP, average path velocity; PM, progressive motility.

Figure 1.

Effects of spermine or spermidine on sperm quality after liquid preservation at 17 °C for 0 d and 7 d. Sperm were treated with different concentration polyamines in the basic extender. Sperm viability (a), acrosome integrity (b), and ΔΨ_m (c) were monitored by SYBR-14/PI, FITC-PNA, and ΔΨ_m assay kits after preservation at 17 °C for 0 d and 7 d, respectively (n = 3). Graph bars represent mean ± SEM. Asterisks indicate significant differences with the control (SM-0 mM or SD-0 mM). *P < 0.05. SM, spermine; SD, spermidine.

Spermine and spermidine alleviate LPS-induced decline of sperm quality

In comparison with the control, significant decreases in sperm TM and PM were observed in the LPS treatment after incubating at 37 °C for 6 h (P < 0.05, Tables 3 and 4). The supplementation of spermine (0.5 mM) or spermidine (0.5 mM) attenuated the decline of TM and PM induced by LPS (P < 0.05; Tables 3 and 4), and spermidine (0.5 mM) attenuated the decline of VCL induced by LPS (P < 0.05; Table 4). LPS treatment also led to decreases in viability and acrosome integrity after the incubation (P < 0.05; Figure 2a and b). Supplementation of spermine (0.5 mM) or spermidine (0.25, 0.5, 1 mM) respectively yielded higher viability and acrosome integrity (P < 0.05; Figure 2a and b).

Table 3.

Effects of LPS treatment alone or with spermine on sperm motility

Motility parameters		LPS treatment
	Control	SM-0	SM-0.25 mM	SM-0.5 mM	SM-1 mM
TM, %	75.0 ± 2.2a	57.7 ± 2.1b	76.9 ± 2.9a	79.4 ± 2.4a	75.5 ± 3.2a
PM, %	39.1 ± 0.8a	21.7 ± 3.4b	44.0 ± 7.0a	50.4 ± 1.2a	39.5 ± 6.0a
VSL, µm/s	17.6 ± 1.6ab	12.7 ± 1.5b	18.2 ± 2.2ab	21.4 ± 1.7a	17.1 ± 2.1ab
VCL, µm/s	54.2 ± 3.9ab	41.4 ± 2.0b	59.8 ± 6.3a	65.2 ± 4.8a	61.7 ± 6.9a
VAP, µm/s	23.7 ± 3.1ab	15.0 ± 3.7b	28.4 ± 4.1a	29.7 ± 3.5a	28.3 ± 2.7a

Sperm were pre-incubated with different concentrations of spermine in a basic diluent for 30 min and incubated with 1 µg/mL LPS for 6 h at 37 °C. Sperm motility parameters (TM, PM, VSL, VCL, and VAP) were analyzed by the CASA (n = 5). Values are presented as mean ± SEM. Values within different superscript letters indicate a significant difference (P < 0.05) between treatments. LPS, lipopolysaccharide, SM, spermine; TM, total motility; VSL, straight-line velocity; VCL, curve-line velocity; VAP, average path velocity; PM, progressive motility.

Table 4.

Effects of LPS treatment alone or with spermidine on sperm motility

Motility parameters		LPS treatment
	Control	SD-0	SD-0.25 mM	SD-0.5 mM	SD-1 mM
TM, %	75.0 ± 2.2a	57.7 ± 2.1b	65.1 ± 4.1b	79.0 ± 1.2a	61.6 ± 2.2b
PM, %	39.1 ± 0.8ab	21.7 ± 3.4c	30.9 ± 6.3bc	49.1 ± 7.6a	27.7 ± 2.2bc
VSL, µm/s	17.6 ± 1.6	12.7 ± 1.5	15.7 ± 4.8	20.9 ± 4.2	15.0 ± 4.4
VCL, µm/s	54.2 ± 3.9ab	41.4 ± 2.0c	44.7 ± 5.5bc	61.6 ± 2.7a	45.4 ± 2.5bc
VAP, µm/s	23.7 ± 3.1ab	15.0 ± 3.7b	15.3 ± 0.9b	27.8 ± 3.4a	15.1 ± 1.5b

Sperm were pre-incubated with different concentrations of spermidine in basic diluent for 30 min and incubated with 1 µg/mL LPS for 6 h at 37 °C. Sperm motility parameters (TM, PM, VSL, VCL, and VAP) were analyzed by the CASA (n = 5). Values are presented as mean ± SEM. Values within different superscript letters indicate a significant difference (P < 0.05) between treatments. LPS, lipopolysaccharide; SD, spermidine; TM, total motility; VSL, straight-line velocity; VCL, curve-line velocity; VAP, average path velocity; PM, progressive motility.

Figure 2.

Effects of LPS treatment alone or with polyamines on sperm viability and acrosome integrity. Sperm were pre-incubated with different concentration polyamines in the basic diluent for 30 min and incubated with vehicle or 1 µg/mL LPS for 6 h at 37 °C. Sperm viability (a) and acrosome integrity (b) were monitored by SYBR-14/PI and PNA-FITC kits, respectively (n = 3). Graph bars represent mean ± SEM. Asterisks indicate significant differences between treatments. *P < 0.05. LPS, lipopolysaccharide; SM, spermine; SD, spermidine.

Spermine and spermidine alleviate LPS-induced sperm ATP depletion and ΔΨ_m decline

LPS treatment provoked significant decreases in ΔΨ_m and ATP content after incubation at 37 °C for 6 h (P < 0.05; Figure 3). Higher values of ΔΨ_m and ATP content were observed in the groups supplemented with spermine (0.5 mM) or spermidine (0.5 mM) compared to the LPS treatment group (P < 0.05; Figure 3).

Figure 3.

Effects of LPS treatment alone or with polyamines on sperm ΔΨ_m and ATP content. Sperm were pretreated with different concentration polyamines in the basic diluent for 30 min and incubated with vehicle or 1 µg/mL LPS for 6 h at 37 °C. Sperm ΔΨ_m (a) and ATP content (b) were determined by ΔΨ_m and ATP assay kits, respectively (n = 3). Graph bars represent mean ± SEM. Asterisks indicate significant differences between treatments. *P < 0.05, **P < 0.01. LPS, lipopolysaccharide; SM, spermine; SD, spermidine.

Spermine and spermidine alleviate LPS-induced mitochondrial ultrastructure changes and MPTP opening

In the control, mitochondria with a well-defined and intact outer membrane, inner membrane, and distinct intermembrane space were tightly wrapped into the midpiece (Figure 4a and b). However, noticeable ultrastructural changes, such as swelling, vacuole structures, and detached outer membrane, were noted in LPS-treated sperm (Figure 4a and b). Importantly, mitochondrial damage was less severe when spermine or spermidine was added (Figure 4a and b). In order to monitor mitochondrial function, an MPTP opening assay was performed with an established calcein cobalt loading procedure by incubating sperm with calcein-AM (Supplementary Figure S1). LPS treatment provoked a significant increase in the MPTP opening rate (P < 0.05, Figure 4c and d). However, supplementation of spermine or spermidine attenuated the increase in MPTP opening rate induced by LPS (P < 0.05, Figure 4c and d).

Figure 4.

Effects of LPS treatment alone or with polyamines on sperm mitochondrial ultrastructure and MPTP. Sperm were pretreated with 0.5 mM spermine or spermidine in the basic diluent for 30 min and incubated with vehicle or 1 µg/mL LPS for 6 h at 37 °C. The mitochondrial ultrastructure in the sperm midpiece was observed by transmission electron microscopy (a) and scanning electron microscopy (b). Swelling (sw), vacuole (va) structures, and membrane dissociation (md) were evident in sperm mitochondria. Bars = 500 nm (a), Bars = 1 µm (b). Sperm MPTP was determined using an MPTP assay kit. (c) Representative overlay histograms of changes in calcein fluorescence measured by flow cytometry. (d) Calcein fluorescence levels are shown as the geometric mean of fluorescence intensity measured by flow cytometry. Graph bars represent mean ± SEM, n = 3. Asterisks indicate significant differences between treatments. *P < 0.05. LPS, lipopolysaccharide; SM, spermine; SD, spermidine.

Spermine and spermidine alleviate LPS-induced apoptotic activation and autophagy blocking

The rate of sperm with PS translocation and the level of cleaved caspase-3 in the LPS treatment were significantly higher than those in the control (P < 0.05; Figure 5a–d). However, spermine and spermidine supplementation significantly alleviated LPS-induced PS translocation and caspase-3 activation (P < 0.05; Figure 5c and d). LPS treatment provoked a significant decrease in the ratio of LC3II/LC3-I (P < 0.05; Figure 5c and e). The addition of spermine and spermidine led to a higher ratio than that in the LPS treatment (P < 0.05; Figure 5c and e), and no difference was detected between the added-polyamine (spermine or spermidine) and the control (Figure 5c and e).

Figure 5.

Effects of LPS treatment alone or with polyamines on sperm apoptosis and autophagy. Sperm were pretreated with 0.5 mM spermine or spermidine in the basic diluent for 30 min and incubated with vehicle or 1 µg/mL LPS for 6 h at 37 °C. Sperm PS translocation was determined using the Annexin-V/PI assay kit (a and b); expression of cleaved caspase-3 was determined with western blotting with anti-cleaved caspase-3 antibody (c and d); and LC3-I/II was determined with anti-LC3 antibody (c and e). Annexin-V/PI staining of sperm was evaluated by flow cytometry: viable sperm (Annexin-V⁻, PI⁻), early apoptotic sperm (Annexin-V⁺, PI⁻), necrotic sperm (Annexin-V⁻, PI⁺), and late apoptotic sperm (Annexin-V⁺, PI⁺) (a). Apoptosis rates were measured by flow cytometry (n = 3) (b). Western blotting images showing expression of apoptosis protein cleaved caspase-3 and autophagy protein LC3-I/II (c). Quantitative expression of cleaved caspase-3 over α-tubulin generated from western blotting. The loading control blotted with an anti-α tubulin antibody was performed for each experiment in the same PVDF membrane (n = 3) (d). Quantitative expression of LC3-II over LC3-I generated from western blotting (e). Graph bars represent mean ± SEM, n = 3. Asterisks indicate significant differences between treatments. *P < 0.05. LPS, lipopolysaccharide; SM, spermine; SD, spermidine.

Expression and localization of CK2α and CK2β in porcine sperm

The images of immunofluorescence staining for CK2α and CK2β are shown in Figure 6a. CK2α was mainly distributed at the midpiece, connecting, principal, and end pieces (Figure 6a), and CK2β mainly localized at the connecting piece and midpiece of the tail. CK2β staining was also detected at the principal and end pieces (Figure 6a). Western blotting analyses confirmed that CK2α and CK2β were expressed in porcine sperm (Figure 6b). However, administration of spermine, spermidine, LPS, TBB, or their combination did not alter the CK2α or CK2β levels in boar sperm (P > 0.05; Figure 6c–e).

Figure 6.

Subcellular localization and expression of CK2α and CK2β protein in porcine sperm. Sperm samples were stained with anti-CK2α or anti-CK2β antibodies, and sperm nuclei were stained with PI. CK2α and CK2β proteins in sperm were determined with secondary antibody FITC conjugated goat anti-mouse immunoglobulin G. Negative control: no addition of primary antibodies. Images were visualized using an epifluorescence microscope. Images on the right were obtained from a single representative sperm. Bars = 40 µm (a). Western blotting using anti-CK2α or anti-CK2β antibody. Samples were from fresh semen of 7 boars (n = 7) (b). Sperm were pretreated with 0, 0.5 mM spermine, or 0.5 mM spermidine in the basic diluent for 30 min and incubated for 6 h at 37 °C with or without 60 µM TBB in the presence of 1 µg/mL LPS (n = 3) (c–e). Western blotting using anti-CK2α antibody or anti-CK2β antibody (c). Densitometric quantification of CK2α or CK2β bands obtained in (c) (d and e). For each experiment performed on the same PVDF membrane, the loading control was blotted with an anti-α tubulin antibody.

Spermine and spermidine alleviate LPS-induced sperm quality decline, mitochondrial dysfunction, and apoptosis via CK2 activation

LPS induced decline in TM, PM, VSL, VCL, and VAP after incubation at 37 °C for 6 h. Supplementation of spermine or spermidine attenuated the LPS-induced decrease in motility parameters (P < 0.05; Table 5). However, the protective effects of spermine and spermidine were entirely abrogated by addition of the CK2 inhibitor TBB (Table 5). Due to addition of TBB, no difference was detected between the TBB, TBB + LPS, TBB + LPS + spermine, and TBB + LPS + spermidine groups (P > 0.05; Figure 7a). Additionally, no differences were found between LPS + polyamines and TBB + LPS + polyamines in sperm viability (P > 0.05; Figure 7a). The protective effect of spermine and spermidine on sperm viability was also abrogated by the TBB (Figure 7a). TBB addition led to a decrease of acrosome integrity in comparison to the control (P < 0.05; Figure 7b), but spermine and spermidine also mitigated the decrease of acrosome integrity provoked by LPS in the presence of TBB (P < 0.05; Figure 7b). Moreover, TBB suppressed the protective ability of spermine and spermidine on ΔΨ_m and MPTP (Figure 7c and d). LPS treatment led to a significant increase in the levels of Bax and cleaved caspase-3 protein after the incubation (P < 0.05; Figure 8a–c). Supplementation of spermine and spermidine alleviated the increase of Bax and cleaved caspase-3 levels provoked by LPS (P < 0.05; Figure 8a–c). Compared to LPS + polyamines group, significant increases in the Bax and cleaved caspase-3 levels were observed in TBB + LPS + polyamines group (P < 0.05; Figure 8a–c). Also, the inhibitive effects of spermine and spermidine on Bax and cleaved caspase-3 levels were abrogated by the addition of the inhibitor TBB (Figure 8a–c). However, a higher ratio of LC3-II/LC3-I was observed in the added-spermine treatment but not the added-spermidine in the presence of TBB and LPS (P < 0.05; Figure 8a and d).

Table 5.

Effects of polyamines, LPS, and TBB on sperm motility parameters after incubation at 37 °C for 6 h

Motility parameters	Control	LPS	SM + LPS	SD + LPS	TBB	TBB + LPS	TBB + SM + LPS	TBB + SD + LPS
TM, %	81.2 ± 4.1a	65.7 ± 5.2b	82.3 ± 2.4a	84.2 ± 3.3a	63.8 ± 5.4b	69.2 ± 2.3ab	64.7 ± 6.0b	61.2 ± 9.4b
PM, %	45.9 ± 4.8a	28.5 ± 5.3b	52.1 ± 3.0a	53.8 ± 5.4a	25.3 ± 7.2b	28.2 ± 1.8b	26.1 ± 5.8b	26.4 ± 6.8b
VSL, µm/s	17.6 ± 0.9a	12.6 ± 1.0b	20.0 ± 1.5a	20.9 ± 2.4a	9.2 ± 2.8bc	8.8 ± 0.9c	9.6 ± 0.6bc	9.2 ± 2.2bc
VCL, µm/s	54.0 ± 2.2a	43.8 ± 2.1b	62.7 ± 3.3a	62.4 ± 1.7a	37.1 ± 6.4b	38.0 ± 4.2b	38.0 ± 1.5b	36.6 ± 6.1b
VAP, µm/s	24.8 ± 1.8a	17.6 ± 2.6b	28.6 ± 2.5a	28.9 ± 2.1a	13.7 ± 3.0b	14.7 ± 0.9b	14.5 ± 2.0b	14.5 ± 3.7b

Sperm were pretreated with 0, 0.5 mM spermine, or 0.5 mM spermidine in basic diluent for 30 min and incubated for 6 h at 37 °C with or without 60 µM TBB in the presence of 1 µg/mL LPS. Sperm motility parameters (TM, PM, VSL, VCL, and VAP) were analyzed by the CASA (n = 5). Values are presented as mean ± SEM. Values within different superscript letters indicate a significant difference (P < 0.05) between treatments. LPS, lipopolysaccharide; SM, spermine; SD, spermidine; TBB: 4,5,6,7-tetrabromobenzotriazole; TM, total motility; VSL, straight-line velocity; VCL, curve-line velocity; VAP, average path velocity; PM, progressive motility.

Figure 7.

Effects of polyamines, LPS, and TBB on sperm quality and mitochondrial function. Sperm were pretreated with 0, 0.5 mM spermine, or 0.5 mM spermidine in the basic diluent for 30 min and incubated for 6 h at 37 °C with or without 60 µM TBB in the presence of 1 µg/mL LPS. Sperm viability, acrosome integrity, ΔΨ_m, and MPTP were monitored by SYBR-14/PI (a), PNA-FITC (b), ΔΨ_m (c), and MPTP (d) assay kits (n = 3). Graph bars represent mean ± SEM. Asterisks indicate significant differences between treatments. Values within different superscript letters indicate a significant difference (P < 0.05) between treatments. LPS, lipopolysaccharide; SM, spermine; SD, spermidine; TBB, 4,5,6,7-tetrabromobenzotriazole.

Figure 8.

Effects of polyamines, LPS, and TBB on sperm apoptosis and autophagy. Sperm were pretreated with 0, 0.5 mM spermine, or 0.5 mM spermidine in the basic diluent for 30 min and incubated for 6 h at 37 °C with or without 60 µM TBB in the presence of 1 µg/mL LPS (n = 3). Western blotting using anti-caspase-3 antibody, anti-Bax antibody, and anti-LC3 antibody (a). Densitometric quantification of cleaved caspase-3, Bax, and LC3-I/II bands obtained in (a) (b–d). The value obtained for cleaved caspase-3 (b) and Bax (c) bands was normalized to that of α-tubulin. For each experiment performed on the same PVDF membrane, the loading control was blotted with an anti-α tubulin antibody. Quantitative expression of LC3-II over LC3-I (d) generated from western blotting. Graph bars represent mean ± SEM. Asterisks indicate significant differences between treatments. Values within different superscript letters indicate a significant difference (P < 0.05) between treatments. LPS, lipopolysaccharide; SM, spermine; SD, spermidine; TBB, 4,5,6,7-tetrabromobenzotriazole.

Discussion

LPS released from Gram-negative bacteria during bacteriolysis is a challenge for liquid preservation and AI of porcine semen (He et al., 2017; Contreras et al., 2022). In the present study, we have demonstrated that supplementation of spermine and spermidine is beneficial to maintain sperm functionality during preservation in vitro at 17 °C. Spermine and spermidine prevent mitochondrial dysfunction and apoptosis induced by LPS in a CK2-dependent manner, which conduces to the maintenance of sperm structural and functional integrity from LPS-induced injury.

The level of LPS is likely to increase in the presence of either penicillin G or amikamycin that induces bacteriolysis during semen storage (Okazaki et al. 2010). LPS reduces sperm motility and fertility in humans (Urata et al., 2001; Fujita et al., 2011), pigs (He et al., 2017), and mice (Makvandi et al., 2019). LPS was detected in boar semen samples at an average level of 0.62 ± 0.14 µg/mL (He et al., 2016). A treatment with LPS at a concentration of 1 µg/mL at 37 °C for 6 h was used to investigate the mechanism of sperm deterioration caused by LPS (Okazaki et al., 2010; He et al., 2016). In the present study, LPS led to a decline in motility, viability, and acrosome integrity, consistent with the previous reports (Okazaki et al., 2010; He et al., 2017). Polyamines are considered to be a class of immunomodulatory factors (Chamoto et al., 2024). Spermine inhibits LPS-induced pro-inflammatory cytokine synthesis in mouse peritoneal macrophages, human mononuclear cells, and dendritic cells in vitro (Zhang et al., 1997; Hasko et al., 2000; Wawrzyniak et al., 2021). Similarly, spermidine reduces LPS-induced colonic inflammation in colitis mice (Ma et al., 2021). Furthermore, spermidine also inhibits LPS-induced inflammation in murine BV2 microglia (Choi and Park, 2012). In this study, we found that spermine and spermidine protected sperm from LPS-induced damage and maintained motility, viability, and acrosome integrity, suggesting that spermine and spermidine could cushion the detrimental effects of LPS on sperm quality in vitro.

In the present study, we found that LPS induced ATP depletion, ΔΨ_m decline, and ultrastructural changes in sperm mitochondria that are consistent with a previous report by He et al. (2017). We also observed that LPS induced MPTP opening that agrees with the previous studies on THP-1 macrophages and human endothelial cells (Fang et al., 2019; Wu et al., 2020; Lee et al., 2024). Therefore, it appears that mitochondria could be the key target of inflammatory-like injury stimulated by LPS and that maintenance of mitochondrial function and structural integrity can moderately alleviate inflammatory-like injury (Wang et al., 2019; Meng et al., 2023). The present study indicates that spermine and spermidine alleviate LPS-induced mitochondrial dysfunction and abnormal ultrastructure in porcine sperm. Spermine and spermidine play essential roles in the maintenance of mitochondrial function (Xue et al., 2018; Zhang et al., 2023). Spermidine improves mitochondrial function of oocytes and cardiac muscles in aged mice (Eisenberg et al. 2016; Zhang et al., 2023). As a physiological depressor of the MPTP in isolated mitochondria (Chen et al., 2019), spermine can prevent mitochondrial swelling, ΔΨ_m collapse, and MPTP opening in rat liver mitochondria (Sava et al., 2006; Xue et al., 2018). Evidence in other cells and our results demonstrate that spermine and spermidine protect the mitochondria from multiple physiological stressors.

Mitochondrial dysfunction is strongly correlated with induction of sperm apoptosis (Liu et al., 2019). It is worth noting that sperm are provided with a shortened apoptotic cascade, which does not appear in other cell types (Amaral et al., 2014). In this study, LPS treatment led to increased PS translocation and cleaved caspase-3 in porcine sperm, in agreement with previous studies by He et al. (2017) and Okazaki et al. (2010). Furthermore, we found that spermine and spermidine alleviate LPS-induced porcine sperm apoptosis. The depletion of polyamines or the impairment of polyamine transport mechanisms contributes to the activation of apoptosis in various cell lines (Seiler and Raul, 2005; Liu et al., 2024). It is reported that spermine maintains canine sperm function and prevents apoptosis during cryopreservation (Setyawan et al., 2016). Together, spermine and spermidine probably act as anti-inflammatory and anti-apoptotic factors for mammalian sperm in vitro.

Autophagy plays a key role in spermatogenesis and spermiogenesis (Wang et al., 2022). Blocking autophagy impairs sperm quality and promotes cell death (Uribe et al., 2022). Likewise, we found that LPS treatment inhibited autophagy and caused deterioration in sperm quality and upregulation of cell death markers. Spermine and spermidine possess autophagy-induction properties and play protective roles in human cells (Hofer et al. 2024; Luo et al., 2024). Here, we found that spermine and spermidine could activate autophagy in the presence of LPS in porcine sperm, accompanied by inhibition of apoptosis and improvement of sperm quality. It is evident that autophagy activated by polyamines also contributes to alleviating LPS-induced inflammatory-like injuries in sperm.

CK2 is a ubiquitously expressed kinase that is necessary for the survival of cells (Mannowetz et al., 2010). Sub-millimolar concentrations of polyamines, particularly spermine, contribute to CK2 activation (Filhol et al., 1991; Kreutzer et al., 2011). CK2 is expressed in mouse, rat, bovine, and ram sperm (Chaudhry et al., 1991; Alvarado-Diaz et al., 2009; Mannowetz et al., 2010; He et al., 2016), and CK2 activity in bovine sperm is also stimulated by spermine and spermidine (Chaudhry et al., 1991). The localization of CK2α in the mid-piece and principal piece of porcine sperm in the present study was similar to that observed in ram sperm (He et al., 2016), but slightly different from that observed in acrosome and mid-piece of mouse sperm (Mannowetz et al., 2010). The localization of CK2β in the flagellum was identical to that observed in mice and rats (Alvarado-Diaz et al., 2009; Mannowetz et al., 2010). The primary spermatogenic defect in Csnk2a2^−/− testes was a specific abnormality in the anterior head shaping of elongating spermatids, which contributed to globozoospermia in mice and demonstrated a unique role for a CK2 isoform in spermatogenesis (Xu et al., 1999). He et al. (2016) reported that the level of CK2α expression in fresh ram sperm was significantly higher than that in frozen-thawed sperm. In the present study, there was no difference in CK2 levels between spermine, spermidine, LPS, TBB or their combination treatments. TBB, as the most specific CK2 inhibitor known to date, has been widely applied in research related to CK2 function (Sarno et al., 2002; Tapia et al., 2006; Zhang et al., 2015, 2024). Zhang et al. (2024) found that TBB inhibited the CK2 activity but did not affect CK2 level in mouse embryonic fibroblasts, which corresponds to our results. Indeed, TBB inhibits the enzymatic activity of CK2 by competitive binding to its ATP/GTP binding site, but it does not affect the expression levels of CK2 protein (Sarno et al., 2001, 2005). Therefore, the quantity of CK2 detected in western blotting should remain unchanged, given that western blotting measures protein quantity, rather than activity.

CK2 is a vital protein kinase that determines cell survival or death (St-Denis and Litchfield, 2009; Manni et al., 2012; Silva-Pavez and Tapia, 2020). Phosphorylation of many protein substrates CK2 is stimulated several-fold in the presence of polyamines (Leroy et al., 1995). We observed that spermine and spermidine fail to protect sperm viability and mitochondrial functionality from LPS-induced injury when CK2 is inhibited by TBB, suggesting that the protective effect of spermine and spermidine on sperm viability and mitochondrial functionality is dependent on CK2 activity. We observed that spermine and spermidine fail to protect sperm survival and mitochondrial integrity from inflammatory-like injury upon inhibition of CK2 by TBB. This suggests that the protective effect of spermine and spermidine on sperm viability and mitochondrial functionality under chronic stress is dependent on CK2 activity. In addition, we found that spermine (but not spermidine) could induce autophagy in the presence of TBB, suggesting that CK2 is not involved in the regulation of autophagy induced by spermine in porcine sperm. Previous reports showed that LPS impacted mitochondrial functionality through TLR4 signaling in human and porcine sperm (Barbonetti et al., 2014; He et al., 2017). In the presence of LPS, suppression of CK2 activity results in impairment of the macrophage effector function, characterized by a decrease in the production of pro-inflammatory cytokines and induction of apoptosis (Glushkova et al., 2018). As CK2 and PP4 regulate TLR4 signaling by reversible phosphorylation in mouse primary macrophages (Yang et al., 2021), CK2 may modulate the activation of the TLR4 signaling pathway in sperm during the response to LPS.

Conclusion

Supplementation of spermine and spermidine is beneficial for maintaining sperm functionality during preservation in vitro at 17 °C. Spermine and spermidine alleviate LPS-induced ΔΨ_m decline, ATP depletion, MPTP opening, and apoptosis in a CK2-dependent manner, thereby maintaining sperm motility and viability in vitro. Supplementation of spermine and spermidine provides novel insights into the protection of porcine sperm against LPS-induced injury and improvement of the efficiency of semen preservation in pigs and potentially in other domestic animals.

Glossary

Abbreviations

AI

artificial insemination

ANOVA

analysis of variance

ATP

adenosine-triphosphate

BSA

bovine serum albumin

calcein-AM

calcein-acetoxymethyl ester

CASA

computer-assisted sperm analysis

CK2

casein kinase 2

DAPI

4’,6-diamidino-2-phenylindole

FITC-PNA

fluorescein isothiocyanate peanut agglutinin

LC3

light chain 3

LPS

lipopolysaccharide

MPTP

mitochondrial permeability transition pore

PI

prodium iodide

PM

progressive motility

PBS

phosphate buffer saline

PS

phosphatidylserine

PVDF

polyvinylidene fluoride

RIPA

radio immunoprecipitation assay

SD

spermidine

SEM

standard error of the mean

SM

spermine

SDS-PAGE

sodium dodecyl sulfate polyacrylamide gel electrophoresis

SDS

sodium dodecyl sulfate

TBB

4,5,6,7-tetrabromobenzotriazole

TM

total motility

VAP

average path velocity

VSL

straight-line velocity

VCL

curve-line velocity

ΔΨ_m

mitochondrial membrane potential

Author contributions

Rongnan Li (Conceptualization, Data curation, Formal analysis, Investigation, Writing—original draft), Xiaodong Wu (Investigation), Jia Cheng (Investigation), Zhendong Zhu (Investigation), Ming Guo (Investigation), Guochao Hou (Investigation), Tianjiao Li (Investigation), Yi Zheng (Investigation), Haidong Ma (Investigation), Hongzhao Lu (Investigation), Wenxian Zeng (Conceptualization, Writing—original draft, Writing—review & editing), Tao Zhang (Writing—review & editing), and Xiaoxu Chen (Writing—review & editing)

Conflict of interest statement

The authors declare no real or perceived conflicts of interest.