Lipid remodeling in swine spermatozoa: Innovative model for the analysis of oxysterols during capacitation

Highlights

• •Oviductal epithelial cells and progesterone regulate sperm capacitation.
• •7KC potentially increases under capacitation in sperm membrane.
• •7α- and 7β-OHC show no change in concentration.
• •7KC could serve as an oxidative stress biomarker in capacitation.

Abstract

Capacitation is a vital process that prepares spermatozoa for successful fertilization by inducing significant biochemical and biophysical changes in the plasma membrane. Among these, the role of oxysterols — oxygenated derivatives of cholesterol — has garnered increasing attention due to their influence on membrane dynamics, signaling pathways, and capacitation-related modifications. This study aimed to evaluate the temporal and treatment-dependent changes in oxysterol concentrations during in vitro capacitation of swine spermatozoa. Using ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS), three key oxysterols were quantified: 7β-hydroxycholesterol (7β-OHC), 7α-hydroxycholesterol (7α-OHC), and 7-ketocholesterol (7-KC). Spermatozoa were subjected to three experimental conditions: a control group (Ctrl), exposure to swine oviductal epithelial cells (SOEC), and SOEC pre-treated with progesterone (SOEC P4), at three time points (t0, t2h, t4h). Findings reveal baseline oxysterol concentrations in non-capacitated spermatozoa and demonstrate condition- and time-dependent dynamics during capacitation. Notably, Principal Component Analysis (PCA) highlighted 7-KC as the oxysterol showing the largest variation across conditions and time points, particularly under the SOEC P4 condition. 7β- and 7α-OHC showed stability as possibly involved in distinct contributions to capacitation processes. These results suggest a potential role of oxysterols in sperm membrane dynamics, especially for 7-KC, and offer insights into their modulation by physiological and biochemical cues during capacitation. This study provides a foundation for understanding the implications of oxysterol dynamics in fertility and reproductive biology.

1. Introduction

Capacitation is a critical process that transforms quiescent spermatozoa into cells capable of fertilizing the oocyte through a series of finely tuned biochemical and biophysical modifications (Visconti et al., 1995). Central to this transformation is a comprehensive remodeling of the sperm plasma membrane, in which alterations in lipid and protein composition lead to increased fluidity and permeability. These changes are essential for the acrosome reaction and the subsequent penetration of the zona pellucida (Zaneveld et al., 1991).

A defining feature of sperm capacitation is lipid remodeling, during which cholesterol is actively removed from the membrane via interactions with a cholesterol acceptor such as albumin or high density lipoprotein. This cholesterol efflux not only enhances membrane fluidity but also facilitates the reorganization of membrane microdomains, thereby optimizing the conditions necessary for successful sperm–oocyte interaction (Kawano et al., 2011). Collectively, these orchestrated modifications prime the spermatozoa for fertilization, highlighting the remarkable precision of the capacitation process.

Recent research has highlighted the role of oxysterols (Aitken, 2017; Brouwers et al., 2011; Gadella and Harrison, 2002; Zerbinati et al., 2017), which were once regarded merely as byproducts of cholesterol metabolism but are now acquiring more interest as possible crucial regulators of membrane dynamics and cellular processes. This concept is reinforced by several studies. For instance, Zerbinati et al. (Zerbinati et al., 2017) carried out analysis in human spermatozoa, where incubation with 25-hydroxycholesterol induced calcium and cholesterol transients associated with the acrosome reaction. In addition, this study detected in the connecting piece and post acrosomal area of the spermatozoa, the enzyme cholesterol 25-hydroxylase, which is primarily responsible for the synthesis of this oxysterol. Complementing these findings, Gadella and Harrison (Gadella and Harrison, 2002) observed that sperm capacitation induces significant reorganization of membrane components, which may be modulated by oxysterol dynamics. Furthermore, Aitken (Aitken, 2017) provided evidence that oxidative stress, a key element in capacitation, contributes to oxysterol formation, thereby linking reactive oxygen species to the regulation of membrane integrity and signaling during sperm hyperactivation. Brouwers et al. (Brouwers et al., 2011) also detected oxysterols in sperm cells, when oxysterols concentrations varied after capacitation, suggesting that they could contribute to the structural and functional changes during sperm capacitation, required for successful fertilization.

Although studies have demonstrated that oxysterols are crucial regulators in modulating membrane fluidity, signaling cascades, and other physiological functions in somatic cells (Brown and Jessup, 2009; Olkkonen et al., 2012), significant gaps remain regarding the specific roles and temporal dynamics in spermatozoa during sperm capacitation, particularly in the swine model. Moreover, research investigating their role during capacitation remains largely limited to in vitro conditions, in which physiological factors are often not considered, and therefore, the physiological environment is not accurately mimicked. In vivo, multiple factors, including the presence of oviductal epithelial cells and hormonal regulators such as progesterone (P4), are likely to influence oxysterol concentrations and their functional impact on spermatozoa. Indeed, oviductal epithelial cells play a crucial role in sperm survival and function by secreting factors that modulate the sperm membrane, regulate oxidative stress, and provide an optimal microenvironment for capacitation by also influencing the dynamics of lipid remodeling (Fazeli et al., 1999). Furthermore, these cells contribute to the formation of a sperm reservoir, a specialized region within the oviduct that is critical for prolonging sperm viability and ensuring the gradual release of capacitated sperm at the optimal time for fertilization (Coy et al., 2012). The reservoir not only sustains sperm presence but also facilitates continuous interaction between spermatozoa and epithelial secretions, which modulates capacitation and enhances fertilizing potential (Leemans et al., 2016). Similarly, P4 is a well-established modulator of sperm function, known for its ability to enhance hyperactivation, induce acrosome reaction by triggering intracellular calcium signaling (Sumigama et al., 2015), possibly modulating enzymatic activity related to cholesterol oxidation.

Despite growing evidence that oxysterols participate in sperm membrane remodeling during capacitation, their temporal behavior in swine spermatozoa under physiologically relevant in vitro conditions remains poorly characterized. Because sperm capacitation in vivo occurs within the oviductal microenvironment, we used swine oviductal epithelial cells (SOEC) and P4-pretreated SOEC to reproduce biologically relevant modulatory conditions. We hypothesized that exposure to SOEC, with or without P4 pretreatment, would differentially influence oxysterol profiles during sperm capacitation.

Therefore, the aim of this study is to quantify 7β-hydroxycholesterol, 7α-hydroxycholesterol, and 7-ketocholesterol in swine spermatozoa during in vitro sperm capacitation under control, SOEC, and SOEC P4 conditions at defined time points by UHPLC-MS/MS.

2. Materials and methods

2.1. Chemicals

All chemicals, unless specified otherwise, were obtained from Sigma Aldrich (St. Louis, MO, USA).

The oxysterols standards used in this study were 22-hydroxycholesterol (22-OHC) (CAS 17954-98-2), 25-hydroxycholesterol (25-OHC) (CAS 2140-46-7), 24-hydroxycholesterol (24-OHC) (CAS 474-73-7), 27-hydroxycholesterol (27-OHC) (CAS 20380-11-4), 7α-hydroxycholesterol (7α-OHC) (CAS 566-26-7), 7β-hydroxycholesterol (7β-OHC) (CAS 566-27-8), 7β-hydroxycholesterol-deuterated-7 (7β-OHC-d7) (CAS 349553-97-5), 27-hydroxycholesterol-d6 (27-OHC-d6) (CAS 1246302-95-3) and 7-ketocholesterol (7-KC) (CAS 566-28-9). 6α-hydroxycholesterol (6α-OHC) (CAS 41083-73-2) and 19-hydroxycholesterol (19-OHC) (CAS 561-63-7) were purchased from Vinci-Biochem Srl (Florence, Italy). The working standard mixtures were prepared by appropriate dilutions in MeOH (10, 1, 0.1 µg mL⁻¹) with storage at −20°C. Strata-XL 100 μm, 30mg/mL Polymeric Reverse-phase cartridges were purchased from Phenomenex (Torrance, California, USA). Formic acid (HCOOH) (LC-MS grade) was obtained from Sigma Aldrich (Darmstadt, Germany). Methanol (MeOH), acetonitrile (ACN), and water (H₂O) were purchased as UHPLC-MS grade solvents from VWR (Radnor, Pennsylvania, USA).

2.2. Experimental design

The experimental design was structured to investigate the temporal and treatment-dependent changes in oxysterol concentrations during in vitro capacitation of swine spermatozoa, and it is fully described in Fig. 1. SOEC were cultured under two conditions: untreated SOEC and progesterone-pretreated SOEC (SOEC P4) (Fig. 1.A). In the SOEC P4 group, epithelial cells were exposed to 100 ng/mL progesterone, a concentration previously shown to preserve SOEC viability and phenotype while improving sperm fertilizing ability in an IVF swine model (Cimini et al., 2022).

Fig. 1.

Experimental design for the evaluation of oxysterol concentrations during in vitro capacitation of swine spermatozoa. (A) Collection of oviductal epithelial cells and cultures under two conditions: only Swine Oviductal Epithelial Cells (SOEC), SOEC with P4 supplementation 100 ng/mL (SOEC P4). (B) Swine spermatozoa preparation by centrifugation and capacitation induced in SOEC, SOEC P4 and Ctrl (only capacitation media) groups. (C) Collection of spermatozoa at three time points: baseline (t0); 2 hours post-capacitation induction (t2h); and 4 hours post-capacitation induction (t4h). Oxysterols extraction from sperm membranes and quantification using UHPLC-MS/MS.

Spermatozoa were washed (Fig. 1.B) and capacitated under three experimental conditions (Fig. 1.A):

• (i)Ctrl, consisting of capacitation medium alone.
• (ii)SOEC, consisting of spermatozoa incubated with untreated SOEC monolayers after replacement of the culture medium with capacitation medium.
• (iii)SOEC P4, consisting of spermatozoa incubated with progesterone-pretreated SOEC monolayers after replacement of the culture medium with capacitation medium.

Samples were collected at baseline (t0, Ctrl only), after 2 h (t2h), and after 4 h (t4h) by gentle pipetting as described in Section 4.5 to capture early and later temporal changes during sperm capacitation and oxysterols were extracted from sperm membranes and quantified using UHPLC-MS/MS, ensuring high sensitivity and specificity for each compound (Fig. 1.C). The just described methodology for cell culture and sperm incubation allows that the final product of the extraction is representative of the only sperm pellet, not containing any lipid profiles of the investigated from SOECs (Banliat et al., 2019; Banliat et al., 2020; Ormsby et al., 2022).

2.3. Swine oviductal epithelial cells collection and incubation

Swine oviducts at the peri-ovulatory stage were collected from a local slaughterhouse and carefully dissected. This stage was selected because it is the most physiologically relevant for sperm storage, sperm–oviduct interaction, and sperm capacitation-related signalling within the female reproductive tract (Rodriguez-Martinez et al., 2001).

Oviductal epithelial cells were isolated by scraping the entire oviduct (including both ampulla and isthmus segments) in accordance with established protocols (Lamy et al., 2017; Ramal-Sanchez et al., 2020). The collected cells were pooled together and washed three times in a medium composed of TCM 199 (Sigma-Aldrich / Merck ©, catalogue no. 4530) with 10% fetal bovine serum (FBS), 2% penicillin/streptomycin, and 1% amphotericin B. Subsequently, SOEC were cultured in TCM 199 medium supplemented with 10% FBS and 2% penicillin/streptomycin (Ferraz et al., 2017) in a humidified 5% CO2 atmosphere at 38.5°C (Hera Cell Thermo Fisher Scientific, Monza, MB, Italy). The culture medium was refreshed every 48 hours. During the culture period to reach the confluence, the phenotype of SOEC is maintained as validated by Cimini et al. (2022) for SOEC treated with P4 (Cimini et al., 2022) and was further assessed by immunofluorescence. As shown in Fig. 2, immunofluorescence indicated that exposure to P4 did not change the cytoskeletal arrangement of SOEC: microtubules (Fig. 2.A) and actin filaments (Fig. 2.B) morphology remained essentially intact and indistinguishable from untreated cells.

Fig. 2.

SOEC cytoskeleton morphology after reaching confluence. (A) Microtubules organization of SOEC. Immunofluorescence analysis of α-tubulin. Monoclonal anti-mouse-α-tubulin antibody and secondary antibody anti-mouse cy3 TRICT-conjugated (red) were used to detect α-tubulin; DAPI was used to stain the nuclei (blue). (B) Actin filaments of SOEC. DAPI (blue, nuclei), TRITC-conjugated phalloidin (red, F-actin).

2.4. Preparation of sperm samples and capacitation

Sperm samples were sourced from Geneetic Srl (Reggio Emilia, Italy) as commercial artificial insemination extended doses produced by approved boar semen collection and distribution centers. A total of ten commercial semen doses obtained from two breeding boars were included in the study. Each biological replicate consisted of two mixed semen doses, one from each boar. The boars were routinely used for commercial artificial insemination programs and semen doses were supplied only after standard quality-control assessment by the provider. Doses were prepared according to a standardized protocol (Maccarrone et al., 2005). Sperm capacitation was induced by incubating spermatozoa in a capacitating medium, composed of TCM 199, supplemented with 13.9 mM glucose, 1.25 mM sodium pyruvate, 2.25 mM calcium lactate, and 1 mM caffeine (Bernabò et al., 2019). The spermatozoa were suspended at a final concentration of 1 × 10⁸ spermatozoa/mL in a final volume of 2 ml per plate (standard 35 mm cell-culture dish, Corning, ref 430165), resulting in a final number of spermatozoa of 2 × 10⁸ spermatozoa per plate and incubated at 38.5°C in a humidified atmosphere with 5% CO₂ for all the different experimental time points and conditions just substituting the culture medium with sperm capacitation medium. Sperm motility was visually estimated before the capacitation by light microscopy (20x magnification, 38.5°C) before each experiment and only samples with sperm motility > 90% were considered for further analyses. The use of this validated protocol induced successfully capacitation on porcine spermatozoa as reported by Cimini et al., 2023 (Cimini et al., 2023).

2.5. Assessment of sperm–SOEC interaction

Sperm–SOEC interaction was assessed as a technical confirmation of sperm presence on the SOEC monolayer under the co-incubation conditions used in the experimental workflow. After incubation of spermatozoa with SOEC under the indicated experimental conditions, samples were stained with Hoechst 33342 to visualize nuclei, DiIC12 to label membranes, and CFDA to identify viable spermatozoa.

Image analysis was performed using ImageJ/Fiji. RGB channels were split, and regions of interest (ROIs) were manually selected to compare SOEC areas exposed to spermatozoa with SOEC-only control areas. Raw integrated density values were extracted from the fluorescence signal and used as a semi-quantitative readout of sperm presence and interaction with the epithelial monolayer.

2.6. Sample extraction

At the end of each incubation period, spermatozoa were gently recovered from the culture dish by collecting the sperm-containing medium and washing the dish with Dulbecco’s phosphate-buffered saline (DPBS), without scraping or mechanically detaching the SOEC monolayer. The recovered suspension was centrifuged at 12500 rpm for 2 minutes to remove capacitation media, then resuspended in 600uL PBS (4°C) and as final step centrifuged again at 13400 rpm at 4°C. The resulting sperm-enriched pellet was used for subsequent oxysterol extraction. This procedure follows established boar sperm preparation and washing workflows, in which sperm samples are washed in DPBS before biochemical or lipid-related analyses (Bernabò et al., 2019; Maccarrone et al., 2005).

For SOEC co-incubation conditions, this recovery procedure was designed to minimize the carry-over of epithelial material by avoiding disruption of the SOEC monolayer.

Sperm samples were then mixed with 500 µL of MeOH, to which an internal standard (IS) solution containing 7β-OHC-d7 and 27-OHC-d6 was added to achieve a final concentration of 10 ng/mL. The mixture was vortexed and homogenized using a Precellys Evolution homogenizer (Bertin Technologies SAS, Montigny-le-Bretonneux, France) under the following conditions: 3 cycles of 10 s at 5200 rpm, with a 20 s pause between cycles. The homogenized samples were then centrifuged at 12000 rpm for 10 minutes at 4°C. The resulting supernatant was collected, combined with 500 μL of H2O, vortexed and subjected to solid-phase extraction (SPE) for clean-up.

2.7. Solid phase extraction clean-up

To address the complexity of the matrix, a SPE procedure was conducted using Strata XL cartridges, which were first conditioned with 1 mL of MeOH and subsequently equilibrated with 1 mL of a 50:50 (v:v) MeOH:H2O mixture. Then each previously diluted extract was loaded onto the cartridge. For the washing step, 1 mL of 50:50 (v:v) MeOH:H2O solution was applied. Analytes were eluted using 1 mL of an ACN:MeOH 80:20 (v:v) solution containing 0.1% HCOOH. The eluted solution was dried using a SpeedVac vacuum concentrator (ThermoFisher, Waltham, Massachusetts, USA) and reconstituted in 50 μL of the elution phase. Finally, the prepared samples were submitted for UHPLC-MS/MS analysis.

2.8. UHPLC-MS/MS analysis

The analysis of oxysterols was conducted according to Fanti et al. (Fanti et al., 2020) with some modifications, using an ACQUITY UPLC H-Class System (Waters, Milford, Connecticut, United States) coupled to a QTrap 4500 mass spectrometer (Sciex, Toronto, ON, Canada) equipped with an Atmospheric Pressure Chemical Ionization (APCI) source operating in positive mode (APCI+). The ion spray voltage was set at 5500 V, with curtain gas at 50 psi, ion source gas 1 at 60 psi and source temperature at 500°C. Instrumental parameters for the selected analytes, including the declustering potential (DP), focusing potential (FP), entrance potential (EP), collision energy (CE) and cell exit potential (CXP) were tuned by infusion of a standard methanolic solution (100 ng/mL) at a flow rate of 10 µL/min. Ion current acquisition and quantification were performed in Multiple Reaction Monitoring (MRM) mode, with the first quadrupole (Q1) set to unit resolution and the third quadrupole (Q3) set to high resolution. Detailed LC-MS/MS parameters for MRM acquisition are reported in Table 1. The Total Ion Current of MRM acquisition is reported as Fig. 3. Chromatographic separation was achieved using a Kinetex C18 column from Phenomenex (Torrance, CA, USA) packed with Core Shell particles (1.7 µm, 2.1 × 100 mm), with the column oven maintained at 40°C. The flow rate was set at 0.6 mL/min and the mobile phases consisted of H2O (A) and ACN:MeOH 80:20 (v:v) (B), both containing 0.1 % HCOOH. The gradient elution was set as follows: start with 55 % of B phase for 0.10 min, then a linear increase to 60 % B in 1.90 min, then a further increase to 100 % B in 4.25 min and kept for 1.75 min. The gradient was then returned to the initial conditions at 55 % B in 0.25 min, with a final equilibration step of 0.50 minutes. The total run time was 8.75 minutes. The injection volume was set to 5 µL. The quantification of the analytes was normalized using deuterated ISs (7β-OHC-d7 and 27-OHC-d6). Data acquisition and processing were performed using Analyst 1.7.2, while analyte quantification was carried out using MultiQuant 3.0.3 software (both from Sciex, Toronto, ON, Canada). All samples were processed using the same extraction, SPE clean-up, internal standard normalization, and UHPLC-MS/MS workflow to minimize extraction-related and analytical variability across experimental conditions.

Table 1.

MS/MS parameters of the selected analytes for the MRM acquisition.

Oxysterols	Q1 (Da)	DP (Volts)	EP (Volts)	Q3 (Da)	CE (Volts)	CXP (Volts)
OX-H2O	384.9	40	7	109.0	40	8
				163.1	33	7
OX-2H2O	367.1	44	6	159.1	32	6
				133.2	33	5
7-KC	401.1	33	10	135.0	43	7
				175.1	36	6
27-OHC-d6	391.1	20	10	147.0	39	6
7β-OHC-d7	374.6	12	5	159.1	34	5

Since oxysterols tend to lose one or two water molecules during ionization, m/z value of precursor compounds in first quadrupole (Q₁) is reported as [M-H₂O+H]⁺ and [M-2H₂O+H]⁺, for exception of 7-KC for which the best-performing molecular ion was [M+H]+; also, m/z values of the ion fragments in the third quadrupole (Q₃) are reported.

Fig. 3.

Chromatographic profiles of the selected oxysterols analyzed by UHPLC-MS/MS. (A) Total ion chromatogram (TIC) of the MRM transition 367.1 → 159.1, common to all analytes except 7-ketocholesterol (7-KC). (B) Chromatographic peak of 7-KC detected using its MRM transition 401.1 → 175.1.

2.9. Statistical analysis and data visualization

Statistical analysis was conducted using Python 3.11.4 within a Jupyter Notebook environment (Kluyver et al., 2016). The analysis employed several libraries, including Pandas (McKinney, 2010) for data manipulation, SciPy (Virtanen et al., 2020) and NumPy (Harris et al., 2020) for statistical tests and numerical operations, and Matplotlib (Hunter, 2007) and Seaborn (Waskom, 2021) for data visualization. To assess the normality of the data, the Shapiro-Wilk test was applied to measurements from 7β-OHC, 7α-OHC, and 7-KC across five independent experiments. As the data were not normally distributed, Wilcoxon’s test was chosen as a non-parametric approach to compare paired observations within groups: Wilcoxon’s test was used to evaluate all possible combinations of time points and sperm capacitation treatments for each oxysterol, offering insight into significant differences across conditions.

3. Results

This study investigated how different experimental conditions influence the concentration of three oxysterols —7α-OHC, 7β-OHC, and 7-KC — within the spermatozoa membrane during in vitro capacitation. The research focused on tracking temporal changes and examining how the presence of SOEC and P4 modulate membrane lipid composition in vitro, providing insights into treatment-specific alterations in oxysterol levels. Data from five biological replicates were analysed. All data collected are summarized in Table 2.

Table 2.

Median, first quartile (Q1), and third quartile (Q3) of oxysterol concentrations (ng/10⁸ cells) in swine spermatozoa across experimental conditions and time points.

		7b-hydroxycholesterol			7a-hydroxycholesterol			7-Keto-hydroxycholesterol
Time point	Sample Name	Median	Q1	Q3	Median	Q1	Q3	Median	Q1	Q3
t0	Ctrl	1.596	1.368	2.129	0.8235	0.66625	1.03625	5.4925	5.1225	6.005
t2	Ctrl	1.3055	1.237	1.4425	0.777	0.62775	1.0355	4.355	3.7755	6.425
	SOEC	1.565	1.2295	1.7025	0.752	0.55275	1.1025	7.395	4.883	7.7575
	SOEC P4	1.92025	1.018	2.82	1.1025	0.6215	1.893	6.78	3.905	10.4
t4	Ctrl	1.516	0.7575	1.6935	0.788	0.5855	0.7895	5.2625	2.75	6.425
	SOEC	1.5915	1.036	1.7985	0.7485	0.64525	0.7875	6.585	3.6975	8.9525
	SOEC P4	1.7475	1.4885	4.3925	0.865	0.6875	1.1995	8.01	3.928	16.9725

3.1. Assessment of sperm–SOEC interaction

Quantitative analysis of sperm–SOEC interaction (Fig. 4) showed a significantly higher number of adhered spermatozoa and increased fluorescence intensity in sperm–cell contact regions compared with non-contact areas (p < 0.01), confirming effective sperm binding to the epithelial monolayer under the adopted co-incubation conditions.

Fig. 4.

Spermatozoa interaction with SOEC after incubation. (A-C) Confocal images showing the interaction of live spermatozoa with SOEC. Blue stain: Hoechst 33342 (nuclei); Red stain: DilC12 (membranes); Green stain CFDA (living spermatozoa). (D-F) Images were processed in ImageJ by splitting the RGB channels (Image > Color > Split Channels) and to quantify sperm binding and fluorescence intensity. (G) Boxplot of Raw Integrated Density values for groups of regions of interest (ROIs) considered as epithelial cells area: The y axis represents fluorescence intensity (arbitrary units, AU). SOEC incubated with spermatozoa exhibit a markedly higher median (∼1.5 × 10⁶ AU) versus SOEC only (∼1.6 × 10⁴ AU) (p < 0.01) indicating an effective interaction between the in vitro monolayer and spermatozoa.

3.2. Baseline levels of oxysterols at t0

At t0, oxysterol concentrations were assessed exclusively in the Ctrl group, establishing a baseline reference for subsequent analyses. The data indicate that 7β-OHC is present at moderate levels (median = 1.596 ng/10⁸ cells), whereas 7α-OHC is detected at relatively lower levels (median = 0.8235 ng/10⁸ cells). In contrast, 7-KC exhibits a markedly higher presence (median = 5.4925 ng/10⁸ cells), revealing a distinct baseline profile of oxysterols.

3.3. Dynamics of oxysterols at t2h

After two hours of sperm capacitation, oxysterol concentrations were measured under all experimental conditions to assess their dynamic behavior. For 7β-OHC, Ctrl exhibited a stable profile (median = 1.3055 ng/10⁸ cells), with median concentrations remaining steady in the SOEC (median = 1.565 ng/10⁸ cells) and SOEC P4 (median = 1.92025 ng/10⁸ cells) conditions too. 7α-OHC levels were generally lower across all groups (Ctrl median = 0.777 ng/10⁸ cells; SOEC median = 0.752 ng/10⁸ cells; SOEC P4 median = 1.1025 ng/10⁸ cells). Among the three oxysterols, 7-KC showed the largest numerical variation at t2h, with higher median values in the SOEC condition than in Ctrl (SOEC median = 7.395 ng/10⁸ cells; Ctrl median = 4.355 ng/10⁸ cells; approximately 1.70-fold higher). In the SOEC P4 condition, the median 7-KC concentration was 6.780 ng/10⁸ cells. However, these differences did not reach statistical significance (p = 0.0625).

3.4. Dynamics of oxysterols at t4h

At t4h, 7β-OHC and 7α-OHC remained broadly stable across conditions, with only modest numerical variation. By contrast, 7-KC again displayed the highest median value in the SOEC P4 group, although no statistically significant pairwise differences were detected (p = 0.0625).

3.5. Statistical analysis results

The statistical evaluation of oxysterol concentrations revealed that the data was not normally distributed across all groups and conditions. This conclusion was drawn after performing the Shapiro-Wilk test for normality (Mohd Razali and Yap, 2011) on each oxysterol dataset (7β-OHC: p-value = 4.9592 × 10⁻⁶; 7α-OHC: p-value = 6.3758 × 10⁻⁶; 7-KC: p-value = 0.0001; all datasets are reported in Supplemental data 1 and complete statistical analysis is available in Supplemental data 2. This finding underscores the necessity of employing non-parametric statistical methods to analyze the oxysterol data. Wilcoxon’s signed-rank test was applied to further explore pairwise differences across time points and conditions for each oxysterol. This non-parametric test is particularly suited for paired or matched data when the normality assumption is violated (McCrum-Gardner, 2008). It was used to compare all possible combinations of time and conditions for the three oxysterols investigated. No significant differences were observed (all p-values > 0.05 and reported in Supplemental data 2). Therefore, the observed differences should be interpreted as descriptive trends rather than statistically supported treatment effects. Data distributions are reported in Fig. 5.

Fig. 5.

Boxplot of oxysterol concentrations in swine spermatozoa membranes by time point and condition.

Principal Component Analysis (PCA) was also conducted to explore the relationships among all the variables, to reduce the dimensionality of the dataset while retaining most of the variance. This approach allows for the visualization of patterns within the dataset, facilitating the identification of key trends and potential outliers. As shown in Fig. 6, the PCA biplot shows the projection of the data on the first two principal components (PC1 and PC2), which together explain 91.19% of the total variance (68.03% by PC1 and 23.16% by PC2). This indicates that these two components effectively summarize the variability in the dataset.

Fig. 6.

PCA biplot of oxysterol profiles in swine spermatozoa membrane across experimental conditions and time points. Each point represents a biological replicate projected onto the first two principal components. Red vectors represent the loadings of the original oxysterol variables and indicate both their direction and relative contribution to the variance observed in the dataset.

4. Discussion

In this study, oxysterol concentrations in swine spermatozoa were quantified during in vitro capacitation under control, SOEC, and SOEC P4 conditions. The descriptive and multivariate analyses indicate that 7-KC may be the oxysterol most responsive to the experimental conditions tested, whereas 7β-OHC and 7α-OHC remained comparatively stable. In addition, the SOEC-based model provided a biologically relevant context to explore oxysterol behavior under conditions that more closely resemble the female reproductive environment than sperm capacitation medium alone.

The inclusion of SOEC co-incubation and P4-pretreated SOEC allowed oxysterol profiles to be evaluated not only in capacitating medium alone, but also under conditions incorporating oviductal epithelial contact and hormonal modulation of the epithelial environment. This design therefore provides a more physiologically inspired in vitro context for exploratory oxysterol profiling.

The inclusion of multiple time points was intended to capture the dynamic nature of sperm capacitation, which is a progressive process involving membrane remodeling, and cholesterol efflux. In this context, assessing oxysterol levels at baseline, 2 hours, and 4 hours allows to distinguish relatively stable components from those showing greater temporal variability, as these two timepoints are relevant in sperm capacitation itself (Martín-Hidalgo et al., 2022; Romar et al., 2001). At the same time, P4 is used as a known modulator of sperm function, which enhance the capacitation process by activating signaling pathways resulting in Ca²⁺ influx through specific channels, while SOEC mimic the physiological environment of the female reproductive tract (Suarez, 2016). These experimental conditions enable a comprehensive investigation of how the said factors influence membrane lipid composition, offering insights into both fundamental biology and potential applications in fertility enhancement (Aitken, 2020).

Indeed, SOEC provide essential factors that support sperm longevity, such as oviductal extracellular vesicles and soluble paracrine factors (Alcântara-Neto et al., 2020; Saint-Dizier et al., 2025), while P4 acts as a potent activator of sperm calcium channels, further enhancing the capacitation process (Lishko et al., 2012). The interplay between spermatozoa and somatic cells of the female reproductive tract fine-tunes the balance between sperm capacitation and apoptosis, ensuring that only functionally competent sperm reach the oocyte. This dynamic interaction underscores the pivotal role of environmental modulators such as SOEC and P4 in shaping the biochemical and biophysical properties of spermatozoa. The oviduct is not merely a passive conduit but an active regulator of sperm function, providing essential biochemical cues that influence sperm survival, capacitation, and fertilization potential (Coy et al., 2012). In addition to supporting sperm longevity, SOEC facilitate selective sperm storage in the so-called sperm reservoirs, allowing for prolonged sperm viability and the gradual release of spermatozoa at the optimal time for fertilization (Killian, 2004). Although two-dimensional SOEC cultures exhibit limited ciliation and polarization compared with the in vivo epithelium, they nevertheless retain critical secretory and paracrine activities that substantially affect sperm lipid remodeling. This proof-of-concept demonstrates that even partially differentiated oviductal cells can modulate sperm oxysterol profiles.

Oxysterol concentrations in sperm plasma membrane exhibit temporal trend during sperm capacitation, with 7β-OHC and 7α-OHC remaining stable, while 7-KC slightly incrementing, especially in SOEC P4 at t4h. The temporal stability for 7β-OHC and 7α-OHC mirrors the findings in bovine spermatozoa, where minimal variation of these isomers was reported during sperm capacitation, suggesting a conserved role in maintaining membrane stability across species (Brouwers et al., 2011), whereas 7-KC may promote destabilization for the acrosome reaction. These observations are consistent with previous porcine data reporting an association between 7-KC formation during sperm capacitation and fertilization-related outcomes (Boerke et al., 2013). However, the present study did not assess zona binding, IVF outcomes, or sperm fertilizing capacity; therefore, our data should not be interpreted as evidence of a causal relationship between 7-KC accumulation and sperm fertilizing potential. The interplay between these oxysterols underscores the intricate balance required for sperm capacitation, where membrane integrity must be maintained until the precise moment when destabilization is necessary for fertilization. Because no statistically significant differences were detected for 7α-OHC and 7β-OHC, the apparent numerical stability of these oxysterols should be interpreted descriptively and does not allow conclusions regarding differential regulatory mechanisms during sperm capacitation. Differences in stereospecific formation pathways and oxidative susceptibility may contribute to oxysterol profiles in spermatozoa, but the present study did not directly investigate the enzymatic or oxidative mechanisms responsible for these patterns. The predominance of one isomer over the other may reflect selective enzymatic activity or an adaptive response to oxidative stress, both of which are critical during sperm capacitation-associated membrane remodeling (Cheng et al., 2023). This highlights the possibility that oxysterols function as modulators of sperm membrane reorganization, fine-tuning the balance between stability and the controlled destabilization necessary for successful fertilization.

PCA showed that 7-KC contributed substantially to the variance observed in the dataset, particularly in relation to conditions involving SOEC P4. This result should be interpreted as an exploratory multivariate observation rather than as evidence that 7-KC directly modulates sperm plasma membrane composition or oxidative balance. Indeed, the present study quantified selected oxysterols but did not directly assess sperm plasma membrane composition, redox status, or the enzymatic pathways involved in 7-KC formation.Furthermore, the distinct variation of t4h Ctrl suggests unique membrane dynamics in non-capacitated spermatozoa, potentially linked to reduced oxidative stress or altered lipid metabolism (Rejraji et al., 2006). As 7β- and 7α-OHC contributed less prominently to variability, their roles in maintaining membrane fluidity and regulating oxidative stress are still not well understood.

The results reported in this manuscript suggest that oxysterols are part of the complex mechanism of membrane dynamics. The higher numerical 7-KC values observed in the SOEC P4 condition at t4h may reflect condition-associated differences in the sperm-enriched fraction analyzed in this study. However, functional consequences on membrane fusogenicity, lipid raft organization, receptor clustering, zona pellucida interaction, or oocyte fusion were not directly tested. Moreover, because a P4-only group was not included, the present design cannot determine whether the observed pattern in the SOEC P4 condition was related to direct effects of P4 on spermatozoa or to indirect effects mediated by P4-pretreated SOEC.

The formation of 7-KC directly ties its variability to oxidative stress experienced during sperm capacitation. The descriptive variation observed for 7-KC suggests that this oxysterol may be a candidate for future studies evaluating lipid-related indicators of sperm functional status. High levels of oxidative stress, while potentially damaging, are also essential for triggering membrane lipid remodeling necessary for functional activation (Bailey, 2010). Future studies combining oxysterol profiling with direct measurements of redox status, sperm plasma membrane remodeling, and fertilization outcomes will be required to test whether 7-KC or related oxysterols reflect the balance between physiological redox signaling and oxidative damage during sperm capacitation.

Although exploratory, these results support the value of investigating oxysterol profiles in spermatozoa under in vitro capacitation-related conditions. From an applied perspective, the SOEC-based model may represent a useful platform for studying how oviductal epithelial conditions are associated with sperm lipid-related profiles in vitro.

This study has some limitations that should be acknowledged. First, the sample size was limited to five biological replicates, reducing statistical power for pairwise comparisons. Second, the study was conducted in vitro and therefore cannot fully reproduce the complexity of the in vivo oviductal environment. Third, sperm capacitation status was inferred from the use of a previously established porcine sperm in vitro capacitation protocol, but it was not directly assessed in the present samples by chlortetracycline staining, protein tyrosine phosphorylation analysis, or other functional assays. Therefore, the experimental conditions should be interpreted as in vitro capacitation-related conditions rather than as direct evidence of a fully capacitated state. Fourth, the absence of a P4-only group prevents discrimination between direct effects of progesterone on spermatozoa and indirect effects mediated by progesterone-pretreated SOEC; consequently, the SOEC P4 condition should be interpreted as a composite epithelial/hormonal condition. Finally, the analysis was restricted to three oxysterols, and broader lipidomic investigations will be needed to better define the role of membrane oxidation products during sperm capacitation.

In conclusion, this study provides a targeted UHPLC-MS/MS assessment of 7β-OHC, 7α-OHC, and 7-KC in swine spermatozoa during in vitro capacitation under progressively more physiological experimental conditions. Within the limits of the study, 7-KC emerged as the oxysterol showing the greatest variability across time and treatment conditions, whereas 7β-OHC and 7α-OHC remained relatively stable. These findings support further investigation of oxysterol profiling in spermatozoa under in vitro capacitation-related conditions. However, the present data do not establish a causal role for 7-KC or other oxysterols in sperm plasma membrane remodeling, redox regulation, or fertilizing competence.

Ethics

This study did not involve human participants or animals; therefore, ethics committee approval was not required.

Funding sources

This work was supported by the European Union - Next Generation EU. Project Code: 337 ECS00000041; Project CUP: C43C22000380007; Project Title: Innovation, digitalization, and sustainability for the diffused economy in Central Italy - VITALITY.

CRediT authorship contribution statement

Di Carlo Carlo: Writing – original draft, Validation, Software, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. Eugelio Fabiola: Writing – original draft, Methodology, Investigation, Conceptualization. Belda-Perez Ramses: Writing – original draft, Methodology, Investigation. Cimini Costanza: Methodology, Conceptualization. Fanti Federico: Writing – original draft, Validation, Methodology, Investigation, Formal analysis. Valbonetti Luca: Visualization. Bernabò Nicola: Writing – review & editing, Validation, Supervision, Resources, Project administration, Conceptualization. Compagnone Dario: Supervision. Barboni Barbara: Supervision, Resources, Project administration.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability

• All data supporting the findings of this study are included in the manuscript and its supplementary material. Supplemental Data 1 and Supplemental Data 2 are freely accessible as part of this submission. No additional datasets or software were generated or analyzed in this study.