First published October 12, 2020; doi:10.1152/ajpendo.00235.2020.— Childhood obesity is a serious concern associated with ill health later in life. Emerging data suggest that obesity has long-term adverse effects upon male sexual and reproductive health, but few studies have addressed this issue. We hypothesized that exposure to high-fat diet during early life alters testicular lipid content and metabolism, leading to permanent damage to sperm parameters. After weaning (day 21 af- ter birth), 36 male mice were randomly divided into three groups and fed with a different diet regimen for 200 days: a standard chow diet (CTRL), a high-fat diet (HFD) (carbohydrate: 35.7%, protein: 20.5%, and fat: 36.0%), and a high-fat diet for 60 days, then replaced by standard chow (HFDt). Biometric and metabolic data were monitored. Animals were then euthanized, and tissues were collected. Epididymal sperm parameters and endocrine parameters were evaluated. Testicular metabolites were extracted and characterized by 1H-NMR and GC- MS. Testicular mitochondrial and antioxidant activity were evaluated. Our results show that mice fed with a high-fat diet, even if only until early adulthood, had lower sperm viability and motility, and higher incidence of head and tail defects. Although diet reversion with weight loss during adulthood prevents the progression of metabolic syndrome, testicular content in fatty acids is irreversibly affected. Excessive fat intake promoted an overaccumulation of proinflammatory n-6 polyun- saturated fatty acids in the testis, which is strongly correlated with negative effects upon sperm quality. Therefore, the adoption of high- fat diets during early life correlates with irreversible changes in testic- ular lipid content and metabolism, which are related to permanent damage to sperm quality later in life.
Nonetheless, more studies will be needed to confirm this hypothesis, particularly by using a model in which HFD is replaced by a high-fat, low-PUFA diet. Moreover, we have not considered the potential contamination of testicular lipid profile by serum. Yet, testes were decapsulated before homogenization to hinder this issue. Besides, the interference of serum lipid pro- file is unlikely considering blood to testis volume (27) and a pre- vious study comparing testicular and serum lipid profiles (44).
HFD is the culprit for testicular lipid dysmetabolism. The proportion of SFAs is the lowest in testes of mice from the HFD
group, and the highest in testis from mice of the CTRL group. Contrastingly, MUFA and PUFA fractions were more abundant in HFD than in the CTRL group. As lard, one of the main com- ponents of the diet formulations, is rich in oleic acid (18:1n9), it is not surprising that HFD and HFDt testes are enriched in this fatty acid. Yet, the relative content of MUFAs compared with PUFAs, in both groups, suggests that oleic acid and other MUFAs are unsaturated into PUFAs. Besides, the detected n-3 and n-6 long-chain PUFAs can only be synthesized after their precursors, the linoleic and linolenic acid are obtained by diet, as they cannot be synthesized de novo in mammals (39). According to Koeberle et al. (28), mammals store large amounts of PUFAs in testes during puberty, by the action of the lyso- phosphatidic acid acyltransferase 3, resulting in a unique FA profile. For instance, docosapentaenoic acid (C22:5n-6, n-6 DPA) is more abundant in the testis than in other mammalian tissues (7, 74). Although several studies describe n-6 DPA- enrichment in various tissues due to HFD (16, 43, 50), testicu- lar content of DPA was unchanged in our model, but we observed testicular enrichment in other n-6 FAs (Supplemental Table S5).
Regarding PUFAs, the n-3/n-6 ratio in testis supports an amelioration in testicular metabolism after ceasing HFD feeding at early adulthood. The D6 desaturase (D6D) has been associ- ated with the deleterious effects of HFD, particularly regarding glucose resistance (23). The dietary n-3/n-6 ratio intake, for humans, is ideally close to 1, and lower ratios (n-6 enrichment) are associated with health deterioration, including increased car- diovascular risk (59). In human testis, a lower n-3/n-6 ratio has been reported in oligo and asthenozoospermia (73). Herein, we found a much lower n-3/n-6 ratio (0.22) in testicular FA content, in the CTRL group. Yet, this ratio was significantly lower (0.18) in HFD-fed mice during their lifetime. Considering the critical role of docosahexaenoic acid (C22:6n-3, DHA) in spermatogen- esis, the detrimental content of n-3 in testis may be associated with the observed phenotype, as it cannot balance the proinflam- matory environment induced by HFD. Effectively, DHA sup- plementation was shown to improve testicular n-3/n-6 ratio, concomitantly improving antioxidant balance (63). However, in our study, sperm parameters did not correlate to the testicular n- 3/n-6 ratio.
Although the HFD formulation had similar relative amounts of SFAs, MUFAs, and PUFAs comparing to the standard chow, the C18:3n-3/C18:2n-6 molar ratio is an order of magnitude higher in HFD (according to the manufacturer information). Interestingly, testicular cells responded to this difference by accumulating even more n-6 PUFAs, without changing their n-3 PUFA content (Fig. 4/Supplemental Table S5). Moreover, n-6 PUFA changes are distinguished not only by an increase in the relative abundance of dietary available linoleic acid (C18:2n-6) but also by an increase of dihomo-c-linolenic acid (C20:3n-6), and arachidonic acid (C20:4n-6; AA), which are obtained from linoleic acid by an enzyme-catalyzed desaturation-elongation process. This may reflect an overall inhibition of the D4Ds and D5D induced by HFD, notably by the relative enrichment in the dietary n-3. D6D is considered the rate-limiting enzyme in the desaturation-elongation processes in mammalian cells, but according to the relative abundance of n-6 PUFAs to n-3 PUFAs and the C20:4n-6/C18:2n-6 ratio, HFD does not change its activity nor is it overwhelmed by the extra dietary n-3. Therefore, testicular metabolic pathways involving n-6 PUFA
Ultimately, it leads to increased release of PUFAs, particularly AA, a central precursor of pro-inflammatory pathways (leukotrienes, throm- boxanes and prostaglandins). Overall, the present data indicate that HFD shifts the cellular redox environment toward a more prooxidant and pro-inflammatory state, enhancing testes suscep- tibility for oxidative stress and inflammatory processes develop- ment. Thus, the absence of changes in lipid peroxidation (TBARS) and in the activity of mitochondrial Electron-Transfer Chain activity complexes suggest that HFD by itself seems not to be enough stimulus to trigger oxidative stress. It was reported that HFD causes an increase in antioxidant metabolites in testis, notably GSH and betaine
