Abstract
Bisphenol A (BPA) is an endocrine-disrupting compound, used as the key monomer of polycarbonate plastics and epoxy resins. BPA has been detected in both humans and farm animals and has been correlated with decreased sperm counts and motility. BPS and BPF are structural analogs of BPA and are increasingly being used in manufacturing as BPA substitutes. In this study, we aim to assess the direct outcomes of BPA, bisphenol S (BPS), and bisphenol F (BPF) exposure on bovine sperm parameters in vitro to elucidate how they affect sperm quality and fertilization potential, and to assess whether BPS and/or BPF are less harmful than BPA. Sperm from three or more bulls was obtained from either fresh samples or cryopreserved straws and exposed to 0.05 mg/mL of BPA, BPS, and BPF in vitro. After 4 h incubation, motility, capacitation, apoptosis/necrosis, and mitochondrial membrane potential levels were measured by computer-assisted sperm analysis or computational flow cytometry. Results showed that BPA exposure significantly reduced both fresh and cryopreserved sperm motility, capacitation, viability and mitochondrial membrane potential levels. Furthermore, BPF significantly decreased motility, capacitation and mitochondrial membrane potential in cryopreserved sperm only. BPS did not have any significant effects on any of the parameters measured. Our results suggest that BPA is the most harmful to sperm, while BPF is toxic under certain conditions, and BPS seems to be the least detrimental. Overall, this study provides an understanding of how the ubiquitous environmental chemicals, bisphenols, may impact male fertility even after ejaculation.
Lay summary
Bisphenol A (BPA) is a widespread man-made compound found in plastic products and the environment, and has been detected in human and animal biological fluids. BPA has been previously investigated for its negative effects on health, including its detrimental impacts on reproductive cells. Concerns over the use of BPA has led to its ban in some countries and has introduced the use of ‘BPA-free’ products, which typically contain substitutes such as bisphenols S and F (BPS and BPF). This current study investigates the effects of bull sperm exposure to BPA, BPS, and BPF to evaluate their impacts on sperm quality. After exposure, both BPA and BPF lowered sperm mobility, organelle function (by reducing mitochondrial activity), and overall survival; while BPS had no effects. Overall, this study shows that BPA and its common replacement, BPF, can both act on sperm directly, reducing their overall quality and function.
Introduction
Bisphenol A (BPA) is a widespread industrial chemical, found in a variety of household products, which has been detected in both human and bovine biological fluids (Mercogliano & Santonicola 2018, Basak et al. 2020, Cimmino et al. 2020). BPA is classified as an endocrine-disrupting compound (EDC), a term referring to exogenous agents that can interact and consequently interfere with endogenous hormonal function, having been correlated with numerous negative health outcomes including reproductive developmental defects in males (e.g. cryptorchidism and hypospadias) (Manfo et al. 2014). In addition to its endocrine disrupting properties, BPA has been investigated for its direct effects on cells and has been shown to disrupt vital cellular functions leading to the induction of apoptosis and necrosis (Huang et al. 2020, Harnett et al. 2021). Concerns regarding BPA’s negative effects have led to ‘BPA-free’ products with the use of analogs, such as bisphenols S and F (BPS and BPF) as the main substitutes, with detectable concentrations of both compounds also reported across countries (Rochester & Bolden 2015). The current trend toward the use of BPS and BPF raises concerns due to the lack of sufficient research on their reproductive effects (Catenza et al. 2021).
While bisphenol production continues to increase, fertility rates among men are reportedly declining, with male factor contributing to 30-40% of infertility cases (Practice Committee of the American Society for Reproductive Medicine 2015, Ravitsky & Kimmins 2019). Male fertility is not only relevant in humans, but has significant implications in cattle production, where one bull can be employed to inseminate thousands of cows with the advancement of Assisted Reproductive Technologies (ARTs). Thus, subfertility in males has major repercussions for humans and in agriculture. Direct exposure of spermatozoa (sperm) to BPA can occur in semen, through leeching from plastic labproducts (in the case of ART use), or in the female reproductive tract (Vitku et al. 2016).
Sperm require both motility and capacitation abilities in order to fertilize an egg. In 2015, Lukacova et al. reported decreased bovine sperm motility after in vitro exposure to BPA for 4h in a dose dependent manner (Lukacova et al. 2015). A recent study also reported that BPA can alter capacitation-related protein modifications in both boar and mouse (Hu et al. 2022). To our knowledge, no studies have investigated the effects of BPS and/or BPF on sperm capacitation. Previous studies have also described the relationship between BPA exposure and increased ROS generation in sperm (Yin et al. 2017). In 2022, Nguyen et al. also showed that spermatozoa treated with BPA had significantly higher levels of oxidative stress than BPS- or BPF-treated groups at the same dose used in this study, 50 ug/mL, which represents the Lowest Adverse Effect Level (LOAEL) for BPA (Nguyen et al. 2022).
In order for sperm to maintain motility, and produce balanced levels of ROS required for capacitation, sperm must have functional mitochondria. The energy status of the mitochondria is depicted by the inner mitochondrial membrane potential (MMP), which can be analyzed in vitro and can be used as a measure of sperm viability, as mitochondria are key activators of apoptosis (Barbagallo et al. 2020, Durairajanayagam et al. 2021).
Although ejaculated sperm does not follow the exact apoptotic cascade as somatic cells, there is evidence of apoptotic hallmarks in sperm, including a rapid loss of motility and externalization of phosphatidyl serine (Mahfouz et al. 2010). BPA has been shown to induce the intrinsic apoptotic pathway in spermatogonia, spermatocytes and other testicular cells (Gong et al. 2017, Wang et al. 2017, Zhang et al. 2022). However, BPA’s role in mature spermatozoal apoptotic events is less known, and even more so for BPS and BPF. The incidence of both apoptosis and necrosis has previously been shown in fresh and cryopreserved-thawed bovine sperm samples (Anzar et al. 2002). It is possible that spermatozoa are vulnerable to the pro-apoptotic effects of BPA observed in other cells types.
Cryopreservation is routinely employed in both human and bovine ART practices for various reasons (i.e. fertility preservation), despite the damaging effects it can have on sperm, including losses in motility and plasma membrane functionality. The underlying mechanisms behind these effects are not well understood, and whether bisphenols can exacerbate them is of interest for the field of ARTs (Hezavehei et al. 2018). Furthermore, cryopreserved sperm is often used in research to study the effects of various chemicals on sperm function due to the availability and ease of obtaining and storing a high number samples in liquid nitrogen tanks. However, this may affect experimental results considering the abundant research outlining changes to sperm membranes, viability, motility and capacitation levels after thawing (Ortega-Ferrusola et al. 2017).
In this study, both fresh and cryopreserved then thawed (cryo-thawed) bovine sperm were used to evaluate the effects of BPA and its analogs, BPS and BPF, on sperm functions through assessing motility rates, capacitation status, apoptosis/necrosis levels, and mitochondrial membrane potential. It was hypothesized that all three bisphenols would adversely affect sperm motility, viability, and mitochondrial function while inducing capacitation. Overall, the results from this study can provide a clinical understanding of how endocrine disrupting chemicals can directly affect male gametes and ultimately fertilization potential and pregnancy outcome. This research also provides insights into the suitability of replacing BPA with BPS or BPF while observing whether fresh and cryo-thawed sperm are equally vulnerable to in vitro treatments.
Materials and methods
Ethics approval
This research was carried out in accordance with the recommendations of the Animal Care Committee at the University of Guelph and adheres to the principles espoused by the Canadian Council on Animal Care (CCAC). This article does not contain any studies involving live animals; thus, no further ethics approvals were required.
Reagents
All chemicals and media were purchased from Sigma-Aldrich, unless otherwise specified.
Sperm preparation and in vitro treatment
Fresh or cryopreserved semen samples containing approximately 50 million sperm from three-four bulls with known fertility (Semex®; Guelph, ON, Canada) were collected fresh or thawed in a 37℃ water bath for 30 s. Total sample size (n) represents the total number of individual ejaculates analyzed. To isolate motile sperm from egg-yolk extender debris and/or dead spermatozoa, semen samples were washed using a discontinuous Percoll density gradient followed by a HEPES–sperm-Tyrode's albumin lactate pyruvate (HEPES–ST) wash containing 0.75% bovine serum albumin, NaCl, KCl, Na2HPO4·12H2O, CaCl·2H2O, and MgCl·6H2O (Truong et al. 2023). Sperm pellets were resuspended in 50 μL of HEPES-ST and equally divided among five treatment groups: control (1 mL HEPES-ST), vehicle (0.1% ethanol in 1 mL HEPES-ST), BPA (0.05 mg/mL final concentration of BPA (LOAEL dose) in 1 mL HEPES-ST), BPS (0.05 mg/mL final concentration of BPS in 1 mL HEPES-ST), and BPF (0.05 mg/mL final concentration of BPF in 1 mL HEPES-ST).
After 4 h incubation at 38.5℃ and 5% CO2, treated sperm were centrifuged for 7 min at 600 g and supernatant was removed (Truong et al. 2023). Pellets were resuspended in approximately 200 µL of prewarmed HEPES-ST to use in subsequent experiments, with each group containing approximately 1–2 million sperm cells.
Motility and morphology assessments
Motility analysis was carried out using a computer-assisted sperm analysis (CASA) system (Sperm Class Analyzer (SCA®) Evolution, Microptic, Barcelona, Spain) at Semex®, Canada. Two microliters taken from the 200 µL of washed well-mixed sperm were loaded onto a warmed chamber slide. A minimum of eight fields were analyzed with at least 200 sperm assessed per treatment group per replicate. Reports on total motility, progressive motility, and immotility rates were generated by the SCA software, based on cut-off values previously established at Semex®.
For the evaluation of sperm morphology, 10 µL were taken from the washed resuspension and smeared onto a prewarmed slide to cover the entire surface. Once air-dried, slides were fixed in 3:1 methanol–acetic acid and stained using a Giemsa solution (Sigma-Aldrich) as described by Nguyen et al. (2022). Morphology was manually determined by assessing at least 100 spermatozoa per treatment group where defects were categorized based on head, midpiece, and tail abnormalities. Head defects include pyriform heads, tapered heads, or detached heads, while midpiece anomalies include bent necks and proximal or distal cytoplasmic droplets. Lastly, tail defects included bent, coiled, or shortened tails (WHO Laboratory Manual for the Examination and Processing of Human Semen 2021).
Capacitation
To assess capacitation, merocyanine 540 (M540, stock solution of 25 µM in DMSO) (Sigma Aldrich; 323756) and Yo-Pro-1 (YP1, stock solution of 1 mM in DMSO) (Sigma-Aldrich; SML1792) were added to the sperm suspensions to detect capacitation status based on plasma membrane changes (Steckler et al. 2015). M540 intercalates into cell membranes with increasing membrane disorder, while YP1, a DNA-binding dye, enters apoptotic and dead cells with permeable membranes. Events that are negative for both stains represent the live and noncapacitating sperm, while M540-positive/YP1-negative events represent live capacitating sperm, and all YP1 positive events indicate apoptotic/dead sperm (Steckler et al. 2015). An additional group containing 10 µL of 2 U/mL heparin, a glycosaminoglycan known to induce capacitation in bovine sperm, was used as a positive control group (Rodríguez-Villamil et al. 2020). Samples were stained in a dark room with final concentrations of 50 nM M540 and 1.5 µM YP1 for 15 min at 38℃ and read on a BD Accuri C6 flow cytometer (BD Biosciences). Three additional groups consisting of no stain, only M540 and only YP1 were included as controls for flow cytometry. After gating, a minimum of 25,000 events were included for each treatment group in each replicate (n = 4 fresh; n = 5 cryo-thawed samples) and analyzed using FlowJoTM v10 (BD Biosciences).
Apoptosis
Staining to detect apoptosis and necrosis was performed using the annexin V–fluorescein isothiocyanate (FITC) apoptosis staining/detection kit according to the manufacturer’s instructions (Abcam, Cambridge, MA, USA; ab14085) and as previously described with minor modifications (Kourmaeva et al. 2022). Briefly, 5 µL of propidium iodide (PI) and 5 µL of annexin V-FITC (FITC) were added to each sample which was resuspended in 200µL of binding buffer. An additional group containing 0.7 µM of dithiothreitol (DTT) added 45 min before the end of the treatment period was used as a positive control for apoptosis. Samples were placed on a warming plate in darkness for 15 min and read by a BD Accuri C6 flow cytometer (BD Biosciences). FITC labels phosphatidylserine residues accumulated on the outer leaflet of the lipid bilayer under apoptotic conditions, while PI, a DNA dye, enters the cell upon membrane compromise during necrosis (Spinaci et al. 2005). FITC-negative/PI-negative staining represents the live sperm population, FITC-positive/PI-negative staining indicates early apoptosis, FITC-positive/PI-positive staining indicates late apoptosis, and FITC-negative/PI-positive staining indicates necrosis. Three additional groups consisting of no stain, only annexin and only PI were included as controls for flow cytometry. After gating, a minimum of 25,000 events were included for each treatment group in each replicate (n = 6 fresh; n = 5 cryo-thawed samples) and analyzed using FlowJoTM v10 (BD Biosciences).
Mitochondrial membrane potential
Mitochondrial membrane potential (MMP) was detected by staining with the lipophilic cationic dye 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolyl-carbocyanine iodide (JC-1). In healthy and motile sperm, JC-1 enters mitochondria and can be found in its aggregated form emitting orange fluorescence. In cells with damaged MMP, JC-1 remains in its monomeric form, emitting green fluorescence (Alamo et al. 2020). Therefore, sperm emitting orange staining represents the proportion of sperm considered to have high MMP (Jiang et al. 2017). A final concentration of 1.7 µM JC-1 was added to 200 µL of samples containing 1–2 million sperm for 15 min at 38℃ and read on a C6 BD Accuri flow cytometer (BD Biosciences). A potent mitochondrial oxidative phosphorylation uncoupler, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) was used as a positive control at a final concentration of 0.132 mM. After gating, a minimum of 25,000 events were included for each treatment group in each replicate (n = 6 fresh; n = 5 cryo-thawed samples) and analyzed using FlowJoTM v10 (BD Biosciences).
Statistical analysis
Statistical analysis was performed on GraphPad Prism 6 using data from at least three biological replicates. All data sets were subjected to the Shapiro–Wilk test for normality. Normally distributed data sets were analyzed using one-way analysis of variance (ANOVA) and nonparametric distributed data were analyzed using the Kruskal–Wallis test. Significant data sets (P < 0.05) were then subjected to Tukey’s post hoc (parametric) or Dunn’s multiple comparison test (nonparametric) to determine differences between each group (P < 0.05). Comparisons described between treatments and ‘control groups’ represent statistical differences against the vehicle. When there was no difference between treatment and vehicle, significant differences and P-value are against control. No differences were observed between control and vehicle in any of our experiments. Statistical differences among the bisphenol treatments are also described.
Results
Motility assessment
Fresh bovine sperm exposed to BPA, BPS, and BPF were assessed for progressive motility, nonprogressive motility and immotility rates after 4 h incubation. Our results indicated that only BPA exposure significantly decreased the proportion of both progressive (P < 0.001) and nonprogressive (P < 0.05) sperm motility rates when compared to controls (Fig. 1A and B). Correspondingly, the immotility rate for BPA-treated sperm was significantly increased compared to controls (P < 0.0001) (Fig. 1C). Progressive motility and immotility rates were also significantly different between the bisphenol groups, where BPA-treated sperm showed a significantly larger loss of motility than either BPS or BPF (P < 0.05).
Motility of fresh bovine sperm after 4 h treatment with BPA, BPS, and BPF (0.05 mg/mL) analyzed through CASA. (A) Represents the proportion (%) of sperm with progressive motility. (B) Represents the proportion (%) of sperm with nonprogressive motility. (C) Represents the proportion (%) of sperm that are immotile. Bars represent the mean ± s.e.m. (n = 6; >200 sperm assessed per replicate). Different letters indicate significant differences at P < 0.05.
Citation: Reproduction and Fertility 4, 4; 10.1530/RAF-23-0018
Cryo-thawed sperm groups were also assessed for the same motility parameters after 4 h of exposure, showing that two of the bisphenols had significant effects compared to controls. BPA (P < 0.001) and BPF (P < 0.05) both reduced the proportion of progressive motility and nonprogressive motility (P < 0.05) compared to the control groups. Correspondingly, both BPA and BPF had significantly higher proportions of immotile sperm compared to controls (P < 0.001) (Fig. 2A, B, and C). In comparison to BPS, both BPA and BPF had significantly higher (P < 0.05) levels of immotility (Fig. 2C). Overall for both fresh and cryo-thawed samples, BPS did alter any motility parameters compared to controls.
Cryo-thawed bovine sperm motility after 4 h treatment with BPA, BPS, and BPF (0.05 mg/mL) analyzed through CASA. (A) Represents the proportion (%) of sperm with progressive motility. (B) Represents the proportion (%) of sperm with nonprogressive motility. (C) Represents the proportion (%) of sperm that are immotile. Bars represent the mean ± s.e.m. (n = 6; >200 sperm assessed per replicate). Different letters indicate significant differences at P < 0.05.
Citation: Reproduction and Fertility 4, 4; 10.1530/RAF-23-0018
Sperm morphology, another traditional indicator of sperm quality, was also assessed in this study where no significant differences were observed between any groups (Supplementary Fig. 1, see section on supplementary materials given at the end of this article).
Capacitation
Fresh and thawed bovine sperm were stained with M540 and Yo-Pro1 to detect live sperm with membrane disorder, characteristic of capacitation. Flow cytometry density plots showed the distribution of the sperm populations after incubation with stains (Fig. 3A). The bottom left quadrant represents nonstained (noncapacitated and live) sperm populations, while the top left quadrant represents M540 (+) staining, indicating live, capacitated sperm. Bottom right and top right quadrants indicate all YP1 (+) apoptotic or dead sperm populations (Fig. 3A).
Bovine sperm capacitation levels after treatment with BPA, BPS, and BPF for 4 h. (A) Representative flow cytometry plots taken from a fresh sperm replicate for all treatment groups stained with M540/Yo-Pro1. Bottom left quadrants represent live and uncapacitated sperm populations. Top left quadrants represent M540 (+) live, capacitated sperm. Bottom right and top right quadrants indicate all YP1 (+) apoptotic/dead sperm populations. (B) Represents the live capacitated sperm populations for treatment and control groups when fresh sperm was used (n = 4; >25,000 events counted). (C) Represents the live capacitated sperm populations for treatment and control groups when cryo-thawed sperm was used (n = 5; >25,000 events counted). Bars represent the mean ± s.e.m.Different letters indicate significant differences at P < 0.05.
Citation: Reproduction and Fertility 4, 4; 10.1530/RAF-23-0018
Fresh sperm groups were compared for changes in the levels of capacitation after exposure to bisphenols A, S, and F. Differences were observed in the live-capacitated population after exposure to BPA and BPF where both bisphenols produced a marked decrease in capacitation levels compared to the control groups (P < 0.01) (Fig. 3B). Heparin was used as a positive control and also showed an increase in capacitation compared to controls (P < 0.01) (Fig. 3B). Between the bisphenols, BPA and BPF-treated groups both has significantly reduced populations of live capacitated sperm compared to BPS (P < 0.05; Fig. 3B).
Cryo-thawed sperm groups yielded similar results after the same treatments. We observed a marked decrease in live capacitated sperm after treatment with BPA and BPF (P < 0.05), while an increase in live capacitated sperm was observed after heparin treatment (P < 0.01) compared to controls (Fig. 3C). BPS-treated sperm did not show any statistical difference in live capacitated sperm populations compared to controls or BPA/BPF (Fig. 3C).
Apoptosis and necrosis
Fresh and thawed bovine sperm groups were assessed for apoptosis and necrosis rates after exposure to bisphenols using the annexin V/PI assay. Four populations of sperm were identified: annexin and PI (−) healthy/viable sperm, annexin (+) early apoptotic sperm, annexin and PI (+) late apoptotic/early necrotic sperm, and PI (+) necrotic sperm as seen in Fig. 4.
Representative flow cytometry plots of fresh treatment groups stained with annexin/PI. Bottom left quadrants represent viable sperm populations. Bottom right quadrants represent annexin (+) early apoptotic sperm. Top right quadrants represent late apoptotic/early necrotic sperm, and top left quadrants represent PI (+) necrotic sperm.
Citation: Reproduction and Fertility 4, 4; 10.1530/RAF-23-0018
In the fresh sperm groups, a significant decrease in viable sperm was observed after BPA treatment (P < 0.05), with a higher proportion of BPA-treated sperm identified in the late apoptotic (P < 0.001) and necrotic (P < 0.01) populations compared to controls (Fig. 5A, C, and D). BPA treatment groups also had significantly higher late apoptotic and necrotic sperm compared to BPS (P < 0.05) but not BPF (Fig. 5C and D). Similar trends were observed for DTT treatment, where there was a marked decrease (P < 0.05) in viable cells and increase in necrotic cells (P < 0.05) compared to controls (Fig. 5A and D). No significant differences were observed between any groups in the early apoptotic sperm populations (Fig. 5B).
Fresh sperm viability as determined through annexin V/PI staining after bisphenol treatment. Average percentages of (A) viable sperm, (B) early apoptotic sperm, (C) late apoptotic/early necrotic sperm, and (D) necrotic sperm. Bars represent the mean ± s.e.m.(n = 6; >25,000 events counted). Different letters indicate significant differences at P < 0.05.
Citation: Reproduction and Fertility 4, 4; 10.1530/RAF-23-0018
Similarly in the cryo-thawed sperm groups, both BPA (P < 0.05) and DTT (P < 0.01) decreased the proportion of viable sperm compared to controls and increased the proportion of necrotic sperm (P < 0.05) (Fig. 6A and D). There were no significant differences observed between BPS or BPF when compared to all other groups. Overall, no significant differences were observed for the early and late apoptotic populations (Fig. 6B and C).
Cryo-thawed sperm viability as determined through annexin V/PI staining after bisphenol treatment. Average percentages of (A) viable sperm, (B) early apoptotic sperm, (C) late apoptotic/early necrotic sperm, and (D) necrotic sperm. Bars represent the mean ± s.e.m.(n = 5; >25,000 events counted). Different letters indicate significant differences at P < 0.05.
Citation: Reproduction and Fertility 4, 4; 10.1530/RAF-23-0018
Mitochondrial membrane potential
The effects of BPA, BPS, and BPF on sperm MMP were assessed via JC-1 staining. Representative fluorescence plots exported after flow-cytometric analysis indicated populations of high MMP among the treatment groups (Fig. 7A, top quadrants). Overall, we saw a significant decrease in MMP after BPA exposure compared to controls (P < 0.01) and compared to BPS (P < 0.05). BPF-treated sperm was did not show a significant changes in MMP compared any of the groups. Exposure to the positive control, FCCP (P < 0.05), also decreased MMP in fresh bovine sperm compared to control groups (Fig. 7B).
JC-1 staining to determine mitochondrial membrane potential (MMP) in bovine sperm following treatment with BPA, BPS, and BPF. (A) Representative flow cytometry plots of fresh treatment groups stained with JC-1. Populations in the top portion of the plots indicate orange staining (high MMP).The effects of bisphenols on MMP in (B) fresh bovine sperm samples (n = 6; >25,000 events counted), and (C) cryopreserved/thawed bovine sperm samples (n = 5; >25,000 events counted). Bars represent the mean ± s.e.m.Different letters indicate significant differences at P < 0.05.
Citation: Reproduction and Fertility 4, 4; 10.1530/RAF-23-0018
In cryo-thawed sperm, we observed similar decreases to MMP compared to controls, marked by BPA (P < 0.01), BPF (P < 0.05), and FCCP (P < 0.05) exposure. BPS-treated sperm did not show any significant changes compared to controls or other bisphenols (Fig. 7C).
Discussion
Studies assessing the effects of bisphenols on ejaculated sperm function have almost exclusively focused on BPA. This study suggests that BPF exposure also negatively affects sperm function in similar manners to BPA, while BPS appears to have no effects on our tested parameters. On average, BPS was found to be significantly less harmful to our tested parameters than BPA, and most often resembled the results of the control groups. BPF, on the other hand, fell somewhere in the middle, where its effects were at times significantly different from controls, BPA and/or BPF depending on the experiment. BPF is more structurally similar to BPA than BPS, which may contribute to its ability to enter cells. Russo and colleagues in 2018 calculated the lipophilicity and phospholipophilicity of BPA and its alternatives and found that BPA had the highest affinity for membrane phospholipids followed by BPF then BPS (Russo et al. 2018). Thus, while BPS may not specifically be less cytotoxic to sperm, it appears to be less harmful in our in vitro model perhaps due to its inability to pass through the sperm membrane as readily as BPA.
Our first set of experiments demonstrated that BPA exposure significantly reduced motility in fresh bovine sperm, and both BPA and BPF exposure decreased motility in previously cryo-thawed bovine sperm samples. In line with our results, BPA has previously been shown to reduce motility parameters in birds, rats, bovine, and human at doses similar to the current study (Lukacova et al. 2015, Barbonetti et al. 2016, Alamo et al. 2020). The dose used in this study is the established lowest adverse effect level (LOAEL) for BPA (50 mg/mL in vivo), which is translated in vitro to be 0.05 mg/mL (Vom Saal & Vandernberg 2021). Presently, no established LOAEL dose exists for BPF, while animal studies have attempted to identify a LOAEL for BPS, but large discrepancies exist (0.001–0.3 mg/mL) (Beausoleil et al. 2022). Additionally, pilot studies conducted in our lab further confirmed that the lowest concentration where an effect was seen on sperm motility was 0.05 mg/mL (Davis OS, unpublished observations; Nguyen et al. 2022). Therefore, the known LOAEL dose for BPA was applied for all three bisphenols, which are used in similar concentrations in manufacturing (Peretz et al. 2011). Using this dose, our lab has previously reported that the progressive motility of cryopreserved bovine sperm was decreased after exposure to all three bisphenols, including BPS (Nguyen et al. 2022). Contrary to these results and our hypothesis, no difference in motility was observed for BPS-treated sperm in this current research, although a small trend in reduced progressive motility can be seen within the cryo-thawed groups. Nevertheless, the differences in these results are likely due to the increased sample size used in this current study and the use of CASA software that provided more precise and less subjective measurements of sperm movement.
Motility and capacitation in sperm are both highly dependent on metabolic and regulatory pathways, including the Ca2+ and cAMP/PKA pathways (Lefièvre et al. 2002, Pereira et al. 2017). We speculate that the observed decreases in both progressive and nonprogressive motility following BPA and BPF exposure may be due to a disruption in these pathways. This relationship is corroborated by reports of a dose-dependent inhibition of Ca2+ signaling when BPA-treated rat sperm were exposed to progesterone (Rehfeld et al. 2020). Perhaps BPA and BPF may be disrupting these vital signaling pathways, interfering with the sperm motility and capacitation abilities, therefore contributing to some of the effects observed in this study. Moreover, reductions in ATP, the energy source for flagellar movement in sperm, can also lead to a decrease in motility (Meyers et al. 2019). In fish and mice, a reduction in sperm ATP content was observed after BPA exposure, which could indicate disrupted mitochondrial function, which could further lead to disruptions in motility and capacitation (Chen et al. 2015, Rahman et al. 2017).
To our knowledge, this is the first study to assess capacitation levels in bovine sperm after bisphenol exposure and the first to assess capacitation levels in any species upon BPS or BPF treatment. Initially, we hypothesized that bisphenols may promote capacitation, as BPA is known to exert weak estrogenic effects and estradiol is a known inducer of capacitation (Ded et al. 2010). However, our study, and other studies in rodent models, have shown decreases in sperm capacitation after BPA exposure. For instance, Li and colleagues in 2021 saw a remarkable decrease in sperm hyperactivation and capacitation following BPA exposure (Li et al. 2021). This correlation, however, is not consistent, with another study reporting premature capacitation of spermatozoa in mice after in vivo BPA administration (Park et al. 2020). Both of these findings might be explained by the data of Hu and colleagues in 2022, who published results similar to the aforementioned studies, where they observed a marked acceleration in capacitation of boar sperm when BPA was administered at lower doses, but a decrease in tyrosine phosphorylation at higher doses (similar to our current study) (Hu et al. 2022). Overall, it is evident that BPA can affect capacitation at different levels, and here we show that BPF can also cause similar changes.
The annexin V/PI assay revealed detrimental effects to sperm viability after BPA exposure only, while the mitochondrial membrane potential assay showed significant decreases after BPA exposure in fresh groups, and both BPA and BPF exposure in cryo-thawed groups. It has been well-documented that BPA induces apoptosis through the intrinsic apoptotic pathway in spermatogenic cells including spermatogonia and spermatocytes, as well as in the supporting Sertoli cells (Wang et al. 2014, Meng et al. 2018, Harnett et al. 2021). Although the differentiated spermatozoon cannot undergo apoptosis in the same mode, a truncated apoptotic pathway has been identified which includes the externalization of phosphatidylserine (PS) to the sperm outer membrane (Lachaud et al. 2004). In this case, the loss of viability we observed after BPA exposure indicated an increase in late apoptosis via externalization of PS (high annexin V binding) and entry of propidium iodide (PI) into the cell in the fresh sperm. In the case of BPF treatment, we did not notice a total loss in viability; however, the observed reduction in mitochondrial membrane potential could possibly lead to increased ATP depletion and thus the observed reduction in motility. Perhaps given a higher dose of BPF or a longer time to exert its effects, we would also observe a loss in viability in a similar manner to BPA.
A final noteworthy observation of this study was that the same exposure conditions applied to both fresh and cryo-thawed sperm could produce differing results. Cryo-thawed sperm appeared overall more vulnerable to damage caused by BPF than fresh sperm. BPF had marked effects on motility and mitochondrial function in the cryo-thawed groups only. This may be due to the cryopreservation/thawing process, which can disrupt protein–lipid interactions or translocate vital proteins, such as ion channels, and ultimately lead to sperm membrane integrity damage (Yeste 2016). These modifications can also lead to capacitation-like changes in sperm that have been previously outlined in the literature (Ortega-Ferrusola et al. 2017). Thus, we speculate that the compromised membranes in the cryo-thawed groups allow greater entry of BPF molecules into the sperm. Overall, since cryopreservation of sperm involves ex vivo manipulation of these cells typically involving plastic products, the possibility of higher exposure to bisphenols should also be considered.
Conclusions
In conclusion, this study summarizes some of the outcomes of direct BPA, BPS, and BPF exposure on bovine sperm. Our results indicated a damaging effect on vital sperm functions after BPA and BPF exposure only, which was exacerbated when the exposed samples were previously cryopreserved. Therefore, this study contributes to our knowledge of how the ubiquitous chemicals, bisphenols, can negatively alter sperm capabilities and further indicates that BPA and BPF appear to be more harmful to sperm than BPS in our experimental model. Ultimately, additional research on the impacts of EDCs can provide further insights into their potential involvement in male infertility.
Supplementary materials
This is linked to the online version of the paper at https://doi.org/10.1530/RAF-23-0108.
Declarations of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
Funding
This research was funded by the Natural Sciences and Engineering Research Council of Canada (NSERC) grant (#401511) to LAF and the General Purpose Account (# 072049) to LAF through the Department of Biomedical Sciences, University of Guelph, and the Ontario Veterinary College (OVC) scholarship to OSD from the University of Guelph.
Author contribution statement
Conceptualization: OSD and LAF; methodology: OSD, VBT, KDH; formal analysis, investigation, writing—original draft preparation: OSD; writing—review and editing: OSD, VBT, KDH, LAF; supervision, project administration, funding acquisition: LAF All authors have read and agreed to the published version of the manuscript.
Acknowledgements
The authors wish to thank Monica Antenos, Elizabeth J. St John, Ed Reyes, and Allison MacKay for their technical assistance, along with Reem Sabry, the Koch Lab, and all past and present members of the Reproductive Health and Biotechnology Laboratory at the University of Guelph. The authors also wish to thank the Collection, Laboratory, and Research Teams at Semex®, Canada, for their support.
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