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. 2022 Apr 29;12(1):7064.
doi: 10.1038/s41598-022-11251-3.

Effects of time-restricted feeding and type of food on fertility competence in female mice

Nafuko Konishi  1   2 , Hiroshi Matsumoto  2   3 , Shu Hashimoto  4 , Udayanga Sanath Kankanam Gamage  5 , Daisuke Tachibana  1 , Aisaku Fukuda  3 , Yoshiharu Morimoto  5 , Masayasu Koyama  1
Affiliations

Effects of time-restricted feeding and type of food on fertility competence in female mice

Nafuko Konishi et al. Sci Rep. .

Abstract

We assessed the effects of feeding regimen (ad libitum vs. time-restricted food access) and type of food (normal chow (NC: 12% fat) vs. moderately high calorie diet (mHCD: 31% fat)) on fertility competence of female mice. Mice fed mHCD had higher number of oocytes than mice fed NC. On the other hand, when mice were fed NC under time-restricted access to food (NT), the developmental rate to the blastocyst per number of normally fertilized ova was significantly decreased compared to others. The reactive oxygen species (ROS) level in oocytes increased in time-restricted food access and NC group. Transcriptome analysis of whole ovarian tissues from these mice showed a change in the cholesterol metabolism among the four groups. Time-restricted food access decreased serum LDL cholesterol level in both NC and mHCD groups. Moreover, the number of atretic follicles increased in NT mice compared to ad libitum food access mice. The present study shows that mHCD feeding increases the number of ovulated oocytes and that time-restricted feeding of NC impairs the developmental competence of oocytes after fertilization, probably due to the changes in serum cholesterol levels and an increase in the ROS content in oocytes.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Feeding conditions and effects of TRF or mHCD regimen on energy intake, serum insulin levels, and body weight gain. (a) 6-week-old female C57BL/6J mice were housed in groups under a 12 h light:12 h dark schedule for 11 weeks to adapt to the housing condition. They were fed normal chow (NC: 339.4 kcal/100 g) or moderately high-calorie diet (mHCD: 409 kcal/100 g) either with unrestricted (ad libitum: ad lib) or time restricted access to food (TRF). Under TRF, mice were allowed access to food between Zeitgeber time 16 (ZT16, 4 h after lights off) and ZT0 (the time the lights are switched on). Food access was regulated by feeding mice at ZT16 and removing the remaining food at ZT0 daily. Daily energy intake was measured by monitoring the weight of the remaining food at ZT0. Weekly body weight gain was measured at ZT0 once a week. Feeding a normal chow without feeding time limits is a common way of rearing mice. (b) The x-axis shows the median value of the measurement time and the y-axis shows the amount of calorie intake per 2 h (kcal). Changes in energy intake of 5 mice/cage were measured every 2 h for 24 h. This measurement was repeated four times. Mice fed NC under an ad lib regimen (NA) displayed diurnal rhythms and nocturnal increase in their energy intake at ZT14. On the other hand, mice fed mHCD under an ad lib regimen (mHCDA) showed dampened diurnal rhythms in energy intake. Because of feeding initiation at ZT16, mice fed NC or mHCD under a TRF regimen (NT and mHCDT) exhibited at least 2 h delay in increase in energy intake compared to their ad lib-fed counterparts. (c) The x-axis shows ZT and the y-axis shows the serum insulin levels (ng/mL). NA and mHCDA displayed a clear nocturnal increase in insulin levels. In contrast, due to the initiation of feeding at ZT16, NT and mHCDT exhibited at least 2 h delay in insulin level increase compared to their ad lib-fed counterparts and displayed a rapid increase in insulin levels at ZT18.5. In addition, the insulin levels in TRF regimen sharply rose due to an abrupt increase in energy intake. Data were obtained from 3 replicates at each time for each group. (d) The x-axis shows breeding period, and the y-axis shows the body weight. The body weights of mice in NT (n = 35), mHCDA (n = 35), and mHCDT (n = 34) groups increased compared to those in NA group (n = 36, P < 0.01, Supplementary Table 1).
Figure 2
Figure 2
Effects of TRF or mHCD regimen on the number of follicles of female mice (a). The data were analyzed by two-way ANOVA following the confirmation of normal distribution. (b) The model included the main effects of feeding regimen (ad lib vs. TRF) and type of food (NC vs. mHCD) and their interaction. The number of atretic follicles increased due to TRF (P < 0.01) and decreased due to mHCD (P < 0.05).Meanwhile, the number of primary and secondary follicles increased due to TRF (P < 0.01). The number of antral follicles increased in mHCDT compared with NT by Steel–Dwass test (P < 0.05, a). NA mice fed NC under ad lib regimen, NT mice fed NC under time-restricted food access, mHCDA mice fed mHCD under ad lib regimen, mHCDT mice fed mHCD under time-restricted food access. Data were obtained from ovarian sections of 15 mice in each group. Data were shown mean ± SD. *P < 0.05.
Figure 3
Figure 3
Moderately high calorie diet increased the number of ovulated oocytes, while time-restricted feeding constrained embryonic development and increased reactive oxygen species (ROS) in oocytes and mitochondrial function in oocytes in normal chow. (a) The number of ovulated oocytes per mouse increased due to mHCD (P < 0.01) by a two-way ANOVA as shown in (b). ad lib ad libitum feeding, TRF time-restricted feeding, NC normal chow, mHCD moderately high calorie diet. NA mice fed NC under ad lib regimen, NT mice fed NC under time-restricted food access, mHCDA mice fed mHCD under ad lib regimen, mHCDT mice fed mHCD under time-restricted food access. Data were obtained from 22 (NA), 21 (NT), 19 (mHCDA), and 20 (mHCDT) mice. The rates of blastocyst formation (c) and morphologically-good blastocysts (d) per fertilized ova in NT were significantly lower than others (P < 0.001) by chi-square tests with Bonferroni corrections of the P values. The value in parentheses was number of fertilized ova examined. (e) Representative images of blastocysts. Expanded, hatching, and hatched stage blastocysts were defined as morphologically good blastocysts. (f) ROS levels were significantly increased due to TRF or NC diet (g) (P < 0.01) by two-way ANOVA. (h) The ratio of red to green fluorescence (mitochondrial activity) in oocytes obtained from NT group was significantly higher than those of NA and mHCDA groups (P < 0.05) by Steel–Dwass test. Each value was divided by the average value of NA oocytes in the same experiment (f,h). The data were obtained from 3 independent experiments. The number of oocytes examined were 36 (NA), 27 (NT), 30 (mHCDA), and 35 (mHCDT), respectively in (f) and 19 (NA), 17 (NT), and 40 (mHCDA), respectively in (h). Data were shown mean ± SD. *P < 0.05 and **P < 0.01.
Figure 4
Figure 4
Time-restricted feeding delayed circadian rhythm and increased the amplitude of circadian rhythm. Changes in expression level of Period 2 (Per2) over time in the liver (a), fat (b), and ovaries (c), and Reverb-alfa gene over time in the liver (d), fat (e), and ovaries (f) were seen. The x-axis shows Zeitgeber time while the y-axis shows the ratio of the expression level of each gene per actin. The circadian rhythm (Per2 and Reverb-alfa) in time-restricted feeding groups was delayed compared to their ad lib counterparts in the liver, fat and ovaries. The amplitude of circadian genes was increased by time-restricted feeding in the liver and fat. Data were obtained from 3 replicates at each time for each group. NA mice fed NC under ad lib regimen, NT mice fed NC under time-restricted food access, mHCDA mice fed mHCD under ad lib regimen, mHCDT mice fed mHCD under time-restricted food access.
Figure 5
Figure 5
Feeding regimen changed gene expression pattern in ovarian tissues. (a) Heatmap for DEG list. (b) Gene Ontology (GO) terms related to biological process. (c) GO Terms related to cellular component. (d) GO Terms related to molecular function. DEG list was further analyzed with GO (http://geneontology.org/) for gene set enrichment analysis per biological process, cellular component and molecular function. (b–d) The significant gene set by each category.
Figure 6
Figure 6
Feeding regimen changed serum lipid levels. The total (a), high-density lipoprotein (HDL, b), and low-density lipoprotein (LDL, c) cholesterol in the serum was measured. Analysis by Tukey Kramer test following ANOVA showed higher levels of total and HDL cholesterol in mice fed mHCD than mice fed NC (a,b). The analysis by two-way ANOVA revealed that time restricted regimen decreased the LDL levels (P < 0.01, c). Moreover, mHCD increased LDL levels (P < 0.05, c). Data were obtained from 8 (NA), 9 (NT), 10 (mHCDA), and 9 (mHCDT) mice. The data of cholesterols were analyzed by two-way ANOVA. The model included the main effects of feeding regimen (ad lib vs. TRF) and type of food (NC vs. mHCD) and their interaction. When the interaction was significant, the data were analyzed by Tukey Kramer test following ANOVA (a,b). **P < 0.01.
Figure 7
Figure 7
Feeding regimen affected fat degradation and synthesis in liver. (a) Changes of carnitine palmitoyltransferase 1 (CPT1) gene expression in liver over time. CPT1 is the rate-limiting enzyme in fatty acid metabolism. A peak in CPT1 gene expression was observed before the increase in energy intake. The peak of CPT1 gene expression in NT mice was remarkably high among the 4 groups. (b) Changes in fatty acid synthase (FAS) gene expression in liver over time. FAS catalyzes fatty acid synthesis. A peak in FAS gene expression was observed after an increase in energy intake. Data were obtained from 3 replicates at each time for each group. (c) Representative images of HE sections of livers were shown from 4 groups. There were no signs of fatty liver in any of the groups.
Figure 8
Figure 8
Time-restricted feeding and NC increased the ratio of Bax to Bcl2 in ovaries by a two-way ANOVA (P < 0.05, a). (b) The model included the main effects of feeding regimen (ad lib vs. TRF) and type of food (NC vs. mHCD) and their interaction. Data were obtained from 18 mice in each experimental group.
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