Restoring Impaired Fertility Through Diet: Observations of Switching From High-Fat Diet During Puberty to Normal Diet in Adulthood Among Obese Male Mice

Abstract

Background: Obesity is associated with a decrease in testicular function, yet the effects and mechanisms relative to different stages of sexual development remain unclear. The aim of this study is to determine whether high-fat diet-induced obesity impairs male fertility during puberty and in adulthood, and to ascertain its underlying mechanisms. This study aims to further reveal whether restoring to a normal diet can improve impaired fertility.

Methods: Male mice were divided into 6 groups: the group N and H exposed to a normal diet or high-fat diet during puberty. The group NN or NH were further maintained a normal diet or exposed to high-fat diet in adulthood, the group HH or HN were further maintained high-fat diet or switched to normal diet in adulthood. Metabolic parameters, fertility parameters, testicular function parameters, TUNEL staining and testicular function-related proteins were evaluated, respectively.

Results: The fertility of the mice in the high-fat diet group was impaired, which validated by declines in pregnancy rates and litter weight loss. Further analysis demonstrated the increased level of oxidative stress, the increased number of spermatogenic cell apoptosis and decreased number of sperm and decreased acrosome integrity. The expression of steroidogenic acute regulatory (StAR) and spermatogenesis related proteins (WT-1) decreased. Fertility among the HN group recovered, accompanied by the recovery of metabolism, fertility and testicular function parameters, StAR and WT-1 expression.

Conclusions: The findings suggest that high-fat diet-induced obesity impairs male fertility during puberty and in adulthood. The loss of acrosome integrity, the increase of oxidative stress, the increase of cells apoptosis and the down-regulation of StAR and WT-1 may be the underlying mechanisms. Switching from high-fat diets during puberty to normal diets in adulthood can improve male fertility.

Keywords: fertility; high fat diet; obesity; puberty; restoring to low fat diet; testis.

Figure 1

General conditions induced by high-fat diet during sexual development (A) Comparison of time-dependent changes in body weight between normal diet (N) and high-fat diet (H) group of 8-week age mice (n = 8 for each group). (B) Representative gross inspection of normal diet of (N) and H group at the 8th week. (C). Comparison of time-dependent changes in body weight among NN group, HN group, NH group and HH group at 16th week (n = 8 for each group). (D) Representative gross inspection of NN group, HN group, NH group and HH group at the 16th week. (E) testicular weight and (F) testicular coefficient and (G) epididymal adipose in normal diet group (N) and high-fat diet group (H) of 8th week of feeding (n = 8 for each group). The comparison of (H) testicular weight and (I) testicular coefficient and (J) epididymal adipose among NN group, HN group, NH group and HH group at 16th week (n = 3 for each group). Results are presented as mean ± SEM. *p < 0.05, **p < 0.01 vs the N group. #p < 0.05, ##p < 0.01 vs the NN group.

Figure 2

Lipid profile induced by high-fat diet during sexual development the level of serum TC, TG, LDL-C, HDL-C and serum glucose between normal diet (N) and high-fat diet (H) group of 8-week old mice (n = 8 for each group). (A–E). The level of serum TC, TG, LDL-C, HDL-C and serum glucose of 16-week old mice among NN group, HN group, NH group and HH group (n = 8 for each group). (F–J) Results are presented as mean ± SEM. **p < 0.01 vs the N group. ##p < 0.01 vs the NN group.

Figure 3

Reproduction function parameters and acrosome reaction and integrity induced by high-fat diet during sexual development Fertility (A), pups per litter (B) and average litter weight (C) were observed, testosterone levels (G) (n = 8 for each group), sperm parameters (H–J) (n = 4 for each group) were assayed at the 8th week of feeding. Fertility (D), pups per litter (E) and average litter weight (F) were observed, testosterone levels (K) and sperm parameters (L–N) were assayed at the 16th week of feeding (n = 8 for each group). *p < 0.05, **p <0.01 vs the N group. #p < 0.05, ##p < 0.01 vs the NN.

Figure 4

Acrosome reaction and integrity induced by high-fat diet during sexual development Testicular tissue sperm PNA—FITC staining of 8-week and 16-week old mice (A, C). The percentage of lack of acrosome were calculated (B, D) (n = 3 for each group). Results are presented as mean ± SEM. **p < 0.01 vs the N group. ^##p < 0.01 vs the NN group.

Figure 5

Testicular lipid deposition and morphological induced by high-fat diet during sexual development (A) Oil red O stained testicular sections at the 8th -week age of mice, and lipid abundance in the testis was analysed (B) (n = 3 for each group). (C) Oil red O stained testicular sections at the 16-week age of mice, and lipid abundance in the testis was analysed among NN group, HN group, NH group, HH group (D) (n = 3 for each group). (E) Representative photomicrographs of H&E staining of testicular sections at 8th week were shown, H&E stained testicular sections at the 16th week of feeding were observed among NN group, HN group, NH group, HH group (F) (n = 3 for each group). All of the above micrographs were from at least three independent experiments, magnification were 200 times. Results are presented as mean ± SEM. **p < 0.01 vs the N group. ^##p < 0.01 vs the NN group.

Figure 6

Oxidative levels and germ cell apoptosis induced by high-fat diet during sexual development. Testicular MDA content of 8-week and 16-week old mice (A, B) Testicular tissue TUNEL staining of 8-week and 16-week old mice (C, D). The TUNEL+ cells/filed of spermatogenic cells were calculated (E, F) (n = 3 for each group). Arrows show TUNEL-positive spermatogonia, arrows head show TUNEL-positive primary spermatocyte. Results are presented as mean ± SEM. *p < 0.05, **p < 0.01 vs the N group. #p < 0.05, ##p < 0.01 vs the NN group.

Figure 7

The expression of synthetic testosterone and spermatogenic enzymes induced by a high-fat diet during sexual development. (A) The bands of Western blotting analysis of StAR, WT-1, DAZL in mice testes. Testis lysates (40 µg/well) were loaded onto gel and reacted with primary and later secondary antibodies; GAPDH was set as loading control. The panel (B–D) is a densitometric histogram of protein bands and results were expressed as ratio of corresponding protein to GAPDH (n = 3 for each group). Results are presented as mean ± SEM. #p < 0.05, ##p < 0.01 vs the NN group. All of the above panels are representative of 3 independent experiments.