Lifestyle, environment, and genetic factors all contribute to sub-fecundity (1). Increasing evidence suggests that environmental chemical contaminants (e.g., heavy metals and anthropogenic chemicals) disrupt testicular physiology. Among different environmental chemical pollutants, per- and poly-fluoroalkyl substances (PFASs) are one of the prioritized family, known to perturb metabolic and reproductive health (2, 3). PFASs are coined as “forever chemicals” that contaminate public water systems, and linger in the environment, wildlife, and humans (4). The legacy PFAS, including PFOA, PFOS, PFNA, PFHxS share in common the lipophobic C-F chain and hydrophilic functional groups (5). PFASs are used in industrial and consumer products and have unique physicochemical properties, including heat and oil resistance, water repellence, and surfactant properties. They exhibited proteinophilic attraction toward albumin and various fatty acid binding proteins, resulting in their long biological half-lives and bioaccumulation in humans (6, 7). In recent decades, the direct effects of PFOS on testicular physiology have been studied in animal and cell culture models, revealing that it disrupted hormonal signaling in the testicles and perturbed the dynamics of tight junctional protein during spermatogenesis, which significantly affected males’ fertility and health (8–10). Even so, little is known about how the systemic impact of PFASs affects testicular function.

Over the past few years, patients seeking reproductive health care increasingly suffer from metabolic disorders, including obesity and insulin resistance (11–13). Since nutritional and hormonal factors influence energy metabolism and reproductive activity, to improve fertility rates, it is crucial to understand the underlying correlation of metabolic syndrome (14). The liver is a primary metabolic tissue that maintains energy and nutrient balance. Further, the liver metabolizes hormones and chemical contaminants, which may be degraded for excretion or bio-activated for more significant toxicity (15). Changes in metabolism affect hormone signals and nutrient flow, which can directly or indirectly affect gonadal function (16). One of the most notable effects of PFOS is the disruption of hepatic liver energy homeostasis, particularly fatty acid metabolism and the signaling of nuclear receptors (17–19). It is unclear how the disruption is related to the perturbing effect of testicular function. It was hypothesized that the disruption in fatty acid metabolism caused by PFASs could affect systemic energy metabolism and fatty acid metabolites in the testes. An integrated approach involving mass-spectrometry imaging, gene expression analysis, and biochemical testing were used in this study to investigate how PFAS affects the mammalian liver and testes. An analysis of the association between PFAS-induced metabolic perturbations and testicular dysfunction was conducted.

The exposure regime revealed a significant increase of liver weights and relative liver weights at the high-dose (3μg/g) of PFOS exposure while there was no noticeable effect on the changes of body weights among the control and PFOS treatment groups (Figure 1A). Additionally, hepatic triglyceride levels were found to be significantly increased at the high-dose exposure (Figure 1B). To underpin the underlying process of PFOS-elicited perturbation to hepatic lipid metabolism, western blot analysis of key metabolic enzymes for lipogenesis were conducted. The data revealed significant upregulation in the expression levels of ATP citrate lyase (ACLY), acetyl CoA carboxylase (ACC), phosphorylated ACC, and fatty acid synthase (FASN) at the high-dose PFOS-exposed groups (Figure 1C). The expression of ACLY and the ratio of pACC to ACC (Supplementary Figure 1A) did not change significantly.

As the liver is the primary metabolic tissue, PFOS-induced disruptions in energy metabolism are likely to have a systemic effect. Fasting insulin and glucose levels were measured on day 21 of post-PFOS exposure. A significant increase in serum insulin levels was observed in the high-dose PFOS group, but no significant changes were observed in fasting blood glucose levels (Figure 2A). In western blot analysis, high-dose PFOS treatment significantly reduced hepatic expression levels of insulin receptor (IR) but not the insulin-like growth factor-1 receptor (IGF-1R) (Figure 2B). Considering the liver’s role in regulating systemic energy homeostasis, the expression levels of hepatokines, the major liver-to-tissue messengers that responds to perturbed energy metabolism, were investigated. Upon PFOS exposure, there was a dose-dependent reduction in the expression levels of the hepatokines, fibroblast growth factor-21 (Fgf-21), leukocyte cell derived chemotaxin-2 (Lect-2), retinol binding protein-4 (Rbp-4), but a significant induction of angiopoietin-like 4 (Angptl4) (Figure 2C). The expression levels of the other measured hepatokines, Angptl-3, Angptl-6, Selenop, and Smoc-1 showed no noticeable differences (Supplementary Figure 1B).

Due to PFOS’ effects on hepatic metabolism and serum insulin levels, it likely perturbed testicular functions. Western blot analysis showed that there were no significant changes in the expression levels of IR and phosphorylated IR in testes (Supplementary Figure 1C). However, PFOS-exposed groups showed a significant dose-dependent decrease of testicular ATP, associated with a significant increase in the expression of peroxisome proliferator-activated receptor γ coactivator 1α, Pgc1α (Figure 3A), a transcription coactivator in the regulation of cellular energy metabolism. Additionally, there was a significant reduction in testosterone levels in the high-dose PFOS exposed groups (Figure 3B, left panel). Yet, no significant change in testicular weight and epididymal sperm counts were noted (Figure 3B, right panel). Nonetheless, in testing the expression levels of endocrine and paracrine factors, there were no significant changes in the testicular gene expression of follicle-stimulating hormone receptor (Fshr) and luteinizing hormone receptor (Lhr) (Supplementary Figure 2A), growth hormone receptor (Ghr), insulin-like growth factor (Igf) and its receptor, Igfr (Supplementary Figure 2B), hepatocyte growth factor (Hgf) and its receptor, Hgf-r (Supplementary Figure 2C), and the steroidogenic enzymes (Star, Cyp11a1, Cyp17a1, Hsd-3β, & Hsd-17β) (Supplementary Figure 3) as compared with the control. Interesting, a significant reduction in the expression levels of Srd5α2 was noted in the high-dose group vs low-dose group.

PFOS is known to perturb fatty acid metabolism via PPARs and fatty acid-mimicry pathways. To unravel the possible impact of PFOS on fatty acid metabolites in testes, we examined PFOS uptake and its perturbation to lipid profiles using MS-imaging. Figure 4A showed a significant increase of PFOS levels in the testes of low- and high-dose exposed mice. MS-imaging data identified significant increases in the levels of the poly-unsaturated fatty acids, eicosa-5, 8, 11-trienoic acid (ETA), eicosa-5, 11, 14-trienoic acid (arachidonic acid, AA), dihomo-α-linolenic acid (DALA), and dihomo-γ-linolenic acid (DGLA) (Figure 4B right panel, Supplementary Table 2), oxidized ceramide (CER) (Figure 4C; Supplementary Table 3), diacylglycerol (DAG) (Figure 4D; Supplementary Table 4), and the phospholipids (phosphatidylcholine & phosphatidylethanolamine) (Figure 4E; Supplementary Table 5). The ion signal of phosphatidylinositol, which is a common and stable component of cell membranes (Figure 4B, left panel), was used for data normalization.

With hindsight, the effects of PFOS on both PPAR-dependent and PPAR-independent fatty acid mimicry pathways disrupt multiple metabolic pathways in multiple tissues, resulting in dyslipidemia, insulin resistance, and inflammation (21–24). This study showed that PFOS exposure at the high-dose (3μg/g b.w.) induced hepatic lipid accumulation which exhibited the early sign of non-alcoholic fatty liver disease (NAFLD). The observation is consistent with the previous studies, showing PFOS perturbed lipid metabolism and induced hepatotoxicity (22, 25). The hepatic liver accumulation was associated with the significant increase in the expression levels of ACLY and FASN. A combination of elevated expression of these enzyme activities promoted fatty acid synthesis from cytosolic acetyl-CoA. Despite this, the expression level of phosphorylated ACLY (pACLY) with reported 6-fold higher activity of ACLY (26) remained unchanged. The expression levels of both total ACC (active form) and pACC (inactive form) increased significantly with high-dose PFOS treatment. There was no significant change in the ratio of pACC to ACC, presumably no significant alteration in ACC activity. Nonetheless, it was unanticipated to see a significant increase in pACC with a functional outcome of hepatic lipogenesis in the group receiving high doses of PFOS. In retrospect, AMP-activated protein kinase (AMPK) is the major kinase known to phosphorylate ACC to promote fatty acid oxidation (27). In a recent study, PFOS-induced deranged hepatic metabolism stimulated both lipogenesis and lipid catabolism, as well as an activation of AMPK pathway (28). Therefore, in this study the observation of an increased phosphorylation of ACC might be associated with the dysregulation of hepatic metabolism induced by PFOS. The dysregulation was further demonstrated with a significant reduction in hepatic IR expression and a significant increase in blood insulin levels. This shows that PFOS perturbed insulin signaling in the liver and systemically.

Further illustrating the liver’s effect on bodily function, the secreted hepatic metabolic factors (hepatokines) were measured. In the PFOS-exposed groups, the expression levels of the four hepatokines (Fgf-21, Lect-2, Rbp-4, and Angptl4) were significantly altered. The four hepatokines were found to be associated with the progression of NAFLD (29–33). Apart from being a metabolic regulator, Fgf-21 was also found to play roles in promoting spermatogenesis, protecting germ cells from diabetes-induced apoptosis (34, 35), and increasing sperm motility (36). Lect-2 was linked to tissue inflammatory responses (37), coherent with the early sign of PFOS-elicited liver steatosis (22). Another perturbed hepatokine, Rbp-4 regulated the physiological functions of testosterone receptors in the testicles, including the production of testosterone and spermatogenesis (38). In obese adolescents, Rbp-4 expression was associated with gonadal functions (39). The decreased expression levels of Fgf-21 and Rbp4 imposed negative influence on testicular functions. In fact, fatty liver disease has also been linked to impaired testicular function (40, 41). There were reports indicating an association between NAFLD and low blood levels of testosterone, which caused accumulation of visceral adipose tissues, elevated free fatty acid levels, inflammation, and increased insulin resistance (42, 43). The observations suggest that systemic metabolism and gonadal function are mutually interdependent.

In testes, metabolic perturbation was evidenced with significant reduction in ATP levels and the increased expression of Pgc-1α, which was reported to protect against oxidative stress and energy metabolism dysfunction in the testes (44). Previous studies had shown that men with fatty liver disease exhibited lower levels of testosterone and sperm count (41, 45, 46). In this study, our data only showed the reduction of blood testosterone levels at the high-dose group. In our previous studies, a higher dose of PFOS exposure (5 μg/g b.w. for 21 days) in mice, caused significant reduction in testosterone levels, sperm count, and sperm swimming activities (8, 9, 47). To decipher the underlying process of the reduced testosterone levels in the high-dose group, the analysis of the steroidogenic enzymes did not reveal noticeable changes, except a significant reduction of Srd5α2 (steroid 5α-reductase 2, a membrane enzyme catalyzes testosterone to dihydrotestosterone). From a pharmacological perspective, an inhibition of Srd5α2 activity might be linked to an increase in serum testosterone levels (48). This may explain an increase in serum testosterone in the high-dose group of our study. In order to further investigate the impact of PFOS on testicular physiology, we focused on the notorious action of PFOS in perturbing fatty acid homeostasis, which may negatively impact testicular functions (49, 50).

The PFOS exposure increased the testicular levels of the poly-unsaturated fatty acids (ETA and AA), the important metabolites in regulating gonadal functions (51, 52). This observation is related to our previous study showing an increase of hydroxyeicosatetraenoic acids in neonatal testes upon PFOS exposure (49). Additionally, PFOS-induced an increase of the poly-unsaturated fatty acids (DGLA and DGLA), which were reported to be directly associated with the sign of tissue inflammation (53, 54). Moreover, our data showed a significant increase in oxidized ceramides and DAG, the important messengers for spermatogenesis, and apoptosis (55, 56). Furthermore, significant increases in total phospholipids, especially phosphatidylcholine, and phosphatidylethanolamine, were associated with the post-testicular causes of infertility (57). Overall, the data suggest that PFOS disrupted fatty acid metabolites’ homeostasis, altering the fatty acid signaling pathway in testes.

This study aimed to better understand the pleiotropic effects of PFOS on tissue functions. As shown by our data, PFOS affects hepatic lipid metabolism, hepatokine expression, blood insulin, testosterone levels, and testicular fatty acid metabolism. This study provided a mechanistic link between disrupted lipid metabolism and perturbed testicular physiology. These findings may help identify PFOS disregard targets and develop targeted interventions to restore and protect testicular function in mammals exposed to these pollutants. As a result of this study, we have gained a better understanding of the pathology, the molecular mechanisms, and the biochemical changes resulting from PFAS-induced testicular toxicity.

The original contributions presented in the study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding authors.

The procedure for animal handling was followed according to guidelines and regulations approved by the animal ethics committee (REC/20-21/0234) of Hong Kong Baptist University. The study was conducted in accordance with the local legislation and institutional requirements.

WL: Data curation, Formal Analysis, Investigation, Writing – review & editing. TL: Formal Analysis, Investigation, Methodology, Software, Writing – review & editing. HT: Investigation, Methodology, Writing – review & editing. TH: Data curation, Investigation, Writing – review & editing. HW: Conceptualization, Investigation, Methodology, Project administration, Supervision, Writing – review & editing. CW: Conceptualization, Funding acquisition, Supervision, Writing – original draft, Writing – review & editing.