Abstract
The physiological processes of organisms in this rotating planet can adjust according to the time of day via built-in circadian clocks. However, more people are having different shift works, which can increase the risk of pathological conditions including altered reproductive function. Thus, circadian rhythm disturbance has become prevalent in the modern society. Specifically, epidemiological evidence has shown that shift-working women are at high risk of spontaneous abortions, irregular menstrual cycles, and low-birth-weight babies. The current study aimed to investigate the effects of circadian rhythm disturbances on the reproductive function of mice caused by dietary time shift, which is common among night-shift workers. According to the schedule of restricted feeding, the mice were classified into the free feeding, daytime feeding, and night feeding groups. The fertility indices of each group were then evaluated. Activity monitoring was performed to determine whether pregnancy delay might be attributed to mealtime shift. Moreover, the estrous cycle of female mice and the reproductive phenotype of male mice were investigated. Results showed that a 12-h mealtime shift significantly delayed successful conception, which could be attributed to a disrupted estrous cycle, in adult female mice.
Introduction
Organisms in this rotating planet have successfully evolved. That is, they possess endogenous mechanisms in response to daily environmental changes. Hence, they can modify almost every aspect of their behavior and physiology according to the time of day (Takahashi 2017, Pilorz et al. 2018). The circadian clock at the molecular level allows rhythmic expression of specific target genes within approximately 24 h. At the system level, circadian signals generated as neural or hormonal outputs from the master clock are transmitted to the peripheral clocks located in almost every tissue within the body, thereby achieving temporal homeostasis in life. In this way, the hierarchical system of master and peripheral clocks can synchronize almost all physiological and behavioral processes within approximately a day (Partch et al. 2014).
Notably, reproductive function is one of the key physiological processes essential for species survival. Several data have shown that circadian clocks are significantly involved in these processes (Boden et al. 2013, Sen & Sellix 2016, Simonneaux et al. 2017, Evans & Anderson 2018, Pilorz et al. 2018, Swamy et al. 2018). For example, destruction of the suprachiasmatic nucleus, which is the master circadian pacemaker in mammals, or inhibition of the communication between the suprachiasmatic nucleus and gonadotropin-releasing hormone neurons can lead to estrous acyclicity and infertility (Brown-Grant & Raisman 1977, Wiegand et al. 1980). Mutations, global knockouts, and polymorphisms in the core clock genes can cause reproductive issues including infertility, estrous cyclicity disruption, and miscarriages (Boden et al.2010, Liu et al. 2014, Hodžić et al. 2018). Circadian peripheral clocks tick in almost every tissue across the hypothalamic-pituitary-gonadal axis, thereby indicating that peripheral clocks have tissue-specific roles in regulating reproduction. Moreover, epidemiological data have revealed that shift-working women are at high risk of irregular menstruation, reduced fertility, and pregnancy issues including spontaneous abortions, premature birth, and low-birth-weight babies (Nurminen et al. 1998, Wang et al. 2016). Thus, the circadian systems might be closely associated with the different aspects of reproductive functions.
In the modern society, shift work or forced desynchronization between environmental light/dark (LD) cycle and working hours has become increasingly prevalent. Since shift workers frequently need to adjust their sleep/wake cycle and food intake according to the imposed working schedules (Balieiro et al. 2014, Bekkers et al. 2015, De Freitas Eda et al. 2015), the physiological rhythms of hormonal secretion and body temperature can be altered. The resulting circadian disturbance may lead to some pathological consequences. Indeed, shift work can cause shift work disorders encompassing metabolic disturbances, gastrointestinal symptoms, sleep disorders, and mental disorders (Boivin & Boudreau 2014, Deng et al. 2018, Zimmet et al. 2019).
To mimic the inevitable shift work in the modern society, we conducted experiments with a shift work model of mice by changing food intake time. Nocturnal mice consume most of their food during the early scotophase (Sanchez-Alavez et al. 2007, Yoon et al. 2012). Our recent study showed that a 6-h advance or delay in usual mealtime can disrupt circadian rhythmicity with metabolic and behavioral consequences in young adult male mice (Yoon et al. 2012). The feeding/fasting cycle plays a critical role in metabolism. Thus, time-restrictive feeding can effectively mimic not only shift-working condition but also metabolic effect in shift workers (Guerrero-Vargas et al. 2018).
The current study aimed to assess the effect of mealtime shift reproductive success and its associated mechanism.
Materials and methods
Ethics statement
All animal experiments were approved by the Institutional Animal Care and Use Committee of Kyung Hee University (permit number: KHUASP(SE)-13-022). Moreover, they were performed in accordance with the guidelines of the ethical committee and were designed to reduce the number of animals used and to minimize suffering.
Animal care and handling
C57BL/6J mice were purchased from DBL (Seoul, Korea) and acclimatized to an ambient temperature of 23 ± 1°C with a 12 h light:12 h darkness cycle. To perform the experiment, animals were housed individually in light-proof clean animal rack cabinets (Shin Biotech, Seoul, Korea) with constant air ventilation. Light intensity was maintained at 350–450 lux at the bottom of the cage during the light phase. Mice were continuously fed with standard chow (Purina chow food: 20% crude protein, 4.5% crude fat, 6% crude fiber, 7% crude ash, 0.5% calcium, and 1% phosphorus) and water ad libitum until time-restrictive feeding ensued.
Effect of mealtime shift on fertility
Figure 1A shows the experimental timeline. Eight-week-old male and female mice were entrained to a 12 h light:12 h darkness cycle for 1 week with food and water ad libitum. Sixteen female and eight male mice were continuously fed ad libitum (free feeding (FF) schedule). The remaining mice were randomly divided into two groups that received a time-restricted feeding schedule in which they could eat for 5 h per day. Mice in the daytime feeding (DF) group ate during the light early phase (Zeitgeber time, ZT1– ZT6). Eight female and four male mice were included in this group. The number of mice in the nighttime feeding (NF) group was similar to that in the DF group (Fig. 1B). After a week of adaptation, mating cages were set up to ensure that one male and two female mice were housed in one cage according to the intended feeding schedule. The presence of vaginal plug every morning at ZT1 in female mice was evaluated. Plug-positive female mice were transferred to new individual cages according to the feeding schedule until delivery. In addition, the gestational period of each female mouse was recorded from plug check to delivery. After giving birth, the pup size was recorded. Moreover, the sex was identified to determine the male-to-female (M/F) ratio.
Experimental scheme. (A) Experimental timeline. Eight-week-old male and female mice were entrained to a 12 h light:12 h darkness cycle for a week with food and water available ad libitum. On the following week, they were randomly divided into the free feeding (FF), daytime feeding (DF), and nighttime feeding (NF) groups. All restrictive feeding was conducted for 4 weeks or longer. Via an experiment about the estrus cycle of female mice, vaginal cytology was performed on ZT5 every morning for 15 days under the same environment as the adaptation period in female mice after 1 week of adaptation to validate the estrus cycle. In total, 34 mice with a stable estrus cycle were selected and randomly divided into three groups, and the estrous cycle was checked while restrictive feeding was performed. (B) Food was provided all day for FF schedule, 5 h a day from ZT1 to ZT6 for DF, and 5 h a day from ZT13 to ZT18 for NF. Water was available all the time for all feeding schedules.
Citation: Reproduction 163, 5; 10.1530/REP-21-0336
Experimental scheme. (A) Experimental timeline. Eight-week-old male and female mice were entrained to a 12 h light:12 h darkness cycle for a week with food and water available ad libitum. On the following week, they were randomly divided into the free feeding (FF), daytime feeding (DF), and nighttime feeding (NF) groups. All restrictive feeding was conducted for 4 weeks or longer. Via an experiment about the estrus cycle of female mice, vaginal cytology was performed on ZT5 every morning for 15 days under the same environment as the adaptation period in female mice after 1 week of adaptation to validate the estrus cycle. In total, 34 mice with a stable estrus cycle were selected and randomly divided into three groups, and the estrous cycle was checked while restrictive feeding was performed. (B) Food was provided all day for FF schedule, 5 h a day from ZT1 to ZT6 for DF, and 5 h a day from ZT13 to ZT18 for NF. Water was available all the time for all feeding schedules.
Citation: Reproduction 163, 5; 10.1530/REP-21-0336
Experimental scheme. (A) Experimental timeline. Eight-week-old male and female mice were entrained to a 12 h light:12 h darkness cycle for a week with food and water available ad libitum. On the following week, they were randomly divided into the free feeding (FF), daytime feeding (DF), and nighttime feeding (NF) groups. All restrictive feeding was conducted for 4 weeks or longer. Via an experiment about the estrus cycle of female mice, vaginal cytology was performed on ZT5 every morning for 15 days under the same environment as the adaptation period in female mice after 1 week of adaptation to validate the estrus cycle. In total, 34 mice with a stable estrus cycle were selected and randomly divided into three groups, and the estrous cycle was checked while restrictive feeding was performed. (B) Food was provided all day for FF schedule, 5 h a day from ZT1 to ZT6 for DF, and 5 h a day from ZT13 to ZT18 for NF. Water was available all the time for all feeding schedules.
Citation: Reproduction 163, 5; 10.1530/REP-21-0336
Activity monitoring
Six-week-old C57BL/6J male and female mice were entrained to a 12 h light:12 h darkness cycle for 1 week, and they had free access to food and water ad libitum. To monitor the activity of 7-week-old mice, the G2 E-mitter probe (Mini Mitter, Bend, OR, USA) was surgically implanted into the dorsal neck under the skin (Son et al. 2008, Park et al. 2012, Yoon et al. 2012). After 2 weeks of recovery from the 12 h light:12 h darkness, the mice were randomly divided into three groups (Fig. 1B) and activity recording was initiated. Body temperature (BT), home cage activity (HCA), and wheel running activity (WRA) were monitored simultaneously using the activity monitoring system (Mini Mitter). The BT and HCA obtained using the E-mitter probe were recorded with a receiver placed under the cage. WRA was assessed with a magnetic bar and switch placed on the side of the running wheel (diameter: 12 cm and width: 5.4 cm). These data were continuously recorded at 6-min intervals using VitalView® Data Acquisition System. Individual actograms were obtained using ActiView®. To generate daily patterns, the resulting 28-day profiles were pooled according to the ZT indicated after the average of the monitoring results retrieved from the Excel file during the whole timeout period was obtained (in cage of BT) or summed up (in cage of WRA, HCA) into 1-h bins. The activity parameters of the mice in all groups were evaluated for 4 weeks. The animals used in the experiment were classified into the female FF group (n = 4), male FF group (n = 4), female DF group (n = 5), male DF group (n = 5), female NF group (n = 5), and male NF group (n = 4).
Effect of DF on male reproductive function
Adult male mice (n = 35) were entrained to a 12 h light:12 h darkness cycle. They were provided with free access to food and water ad libitum. After 1 week of entrainment, the mice were randomly divided into two groups and subjected to either FF or DF for 4 weeks (Fig. 1B). Then, they were sacrificed via restrictive feeding at 0, 1, 2, and 4 weeks (n = 5 for each time point). The testicular and seminal vesicle weights were determined. Moreover, the sperm contents in the cauda epididymis and vas deferens were determined (as shown below).
Sperm counting
Sperm counting was performed according to the World Health Organization laboratory manual (Damiola et al. 2000). Briefly, the cauda epididymis and vas deferens were squeezed out and the resulting fluid was incubated for 10 min in a plate containing PBS. After centrifugation, sperms were resuspended in PBS and counted using a hemocytometer under a light microscope.
Effect of DF or NF on estrous cycle
Figure 1A depicts the experimental timeline. Eight-week-old C57BL/6J female mice were entrained to a 12 h light:12 h darkness cycle with food and water available ad libitum. After 1 week of entrainment, the estrous cycle was determined every morning at ZT5 for 15 days. Using a 20-μL pipette, 10 μL of 0.9% saline solution was introduced into the vagina for flushing several times. The saline solution containing cellular contents was retrieved into a slide glass. The slide was then stained with 0.02% toluidine blue (Sigma) and air-dried. Upon microscopic observation, the estrous cycle was identified according to the presence of leukocytes, cornified cells, and nucleated epithelial cells (Goldman et al. 2007, Cora et al. 2015). Proestrus (P) was characterized by the presence of both leukocytes and nucleated epithelial cells. Estrus (E) was defined as the presence of several large cornified cells without nuclei. Metestrus (M) was characterized by approximately equal numbers of leukocytes and epithelial cells. Diestrus (D) was considered when almost exclusive leukocytes were found. The appearance of estrus within 3–4 days and the appearance of diestrus within 4–5 days were defined as a normal estrous cycle (Goldman et al. 2007). Evaluation was performed to check for 15-day estrous cycle, and the most normal female mice (n = 34) were divided into three groups. The DF group (n = 15) was subjected to restrictive feeding for 40 days and the NF group (n = 9) to restrictive feeding for 35 days. The estrous cycle was observed in ZT5 every day. After 40 or 35 days of restrictive feeding, mice were provided with free access to food ad libitum and the estrous cycle was determined every morning for 20 or 15 more days. During the same period, the experiment was continued for 75 or 65 days in the FF (n = 10) group. The estrus cycle of mice with restrictive feeding was evaluated. The total number of days in each stage was summed up and then divided by 40 or 35 days to quantify data as percentage and graph. Finally, the number of mice with normal cycle changes over the last 5 or 10 days was expressed as percentage. ANOVA and the Duncan’s post hoc test were used for statistical analysis.
Statistical analysis
All data were expressed as mean ± s.e.m. For statistical comparisons, one-way ANOVA, followed by the Duncan’s post hoc test, was conducted. A Pvalue of < 0.05 was considered statistically significant. GraphPad Prism 5 and the t-test were used for all statistical analyses.
Results
DF significantly delays conception in young adult mice
We initially determined the effects of 12-h mealtime shifts on fertility in mice (as shown in Fig. 1A for the experimental scheme). Three different feeding regimens were adopted. That is, foods were provided all the time in the control group (FF), for 5 h during the early light phase (DF), or for 5 h during the early scotophase (NF) (Fig. 1B). DF was defined as restricted food availability that is 12-h out of phase from their major mealtime. NF was defined as restrictive feeding in which food availability is limited to their major mealtime. After full acclimatization to each feeding regimen for a week, mating cages were arranged to ensure that one male and two female mice were housed in each cage. Concurrently, both male and female participants were subjected to restrictive feeding. The presence of vaginal plug and other fertility parameters was then determined every morning (as shown in the ‘Experimental outline’ in ‘Materials and methods’ section). When mice were fed ad libitum, the duration from the mating cage set up to the presence of a vaginal plug was 5.4 ± 1.6 days. However, it took approximately 10–15 days if mice were fed at a limited time: 16.9 ± 5.3 days in the NF group (P < 0.05 vs FF) and 30.8 ± 3.9 days in the DF group (P < 0.001 vs FF) (Table 1), thereby indicating that the duration of feeding restriction itself negatively affected successful conception in mice. However, DF had a significantly stronger influence than NF on the duration (days) of vaginal plug development. Thus, the timing of feeding could be a more important factor than the feeding duration. The duration from pup delivery did not significantly differ among three groups: 20.5 ± 1.7, 20.8 ± 2.4, and 19.0 ± 1.2 days in the NF, DF, and FF groups, respectively (Table 1). Moreover, there was no significant difference in terms of litter size or M/F ratio of the offspring among the three groups. Thus, time-restrictive feeding, particularly DF rather than NF, can significantly delay conception without affecting the litter size or M/F ratio of the offspring.
Effects of time-restrictive feeding on fertility indices. Data were expressed as mean ± s.e.m. Statistical comparisons were performed using ANOVA, followed by the Turkey’s post hoc test.
| Feeding schedule (no. of female mice) | Days to vaginal plug1 | Days to delivery2 | Average litter size (cm) | Sex ratio (M/F) |
|---|---|---|---|---|
| FF (n = 16) | 5.4 ± 1.6a | 19.0 ± 1.2 | 5.9 ± 0.3 | 1.2 |
| DF (n = 8) | 30.8 ± 3.9b, ### | 20.8 ± 2.4 | 5.8 ± 0.8 | 1.5 |
| NF (n = 8) | 16.9 ± 5.3c, # | 20.5 ± 1.7 | 5.2 ± 1.0 | 1.5 |
1Duration (days) from mating cage setup to the presence of vaginal plug. 2Duration (days) from the presence of vaginal plug to parturition. Each alphabet indicates significant differences between the FF, DF, and NF groups. #P < 0.05 vs the FF group, ###P < 0.001 vs the FF group.
Movement time shift due to DF does not cause fertility delay
Based on the experimental results of delayed conception between DF and NF groups and the FF group (Table 1), we then assessed how restrictive feeding affected mice in a 24-h cycle. Based on the feeding data of female and male mice (Supplementary Fig. 1A and B, see section on supplementary materials given at the end of this article), the DF and NF groups had a lower food intake than the FF group until about 7–8 days after the start of restrictive feeding. However, at 1 week from the last time point, there was no significant difference between food intake among the three groups. Although the body weight did not significantly differ between the FF and DF/NF groups for 1 week until the 2nd week, it did not remarkably differ thereafter among the three groups (Supplementary Fig. 1C and D). Based on this finding, an experiment was conducted using an activity monitoring system to assess for pregnancy delay due to changes in activity/rest patterns caused by restrictive feeding in each group. The E-mitter was surgically implanted into 7-week-old C57BL/6J female and male mice. After 2 weeks of recovery and entrainment period, WRA and HCA were constantly recorded for 4 weeks (as shown in the ‘Experimental outline’ in ‘Materials and methods’ section). We used the activity monitoring system to check the activity of mice to confirm if mating was delayed by time-restrictive feeding. The WRA could measure exercise rhythms. However, it did not significantly differ in female or male mice among the three groups (Fig. 2A and C). HCA showing movement in the cage increased significantly in female mice in the NF group at nighttime, thereby increasing their all-day activities (Fig. 2B). By contrast, the HCA of male mice did not significantly differ among the three groups.
All-day activity and nighttime activity of young adult mice under time-restrictive feeding. Mice were entrained to a 12 h light:12 h darkness photoperiodic cycle for a week with food and water available ad libitum. E-mitter probes were surgically implanted into young adult female and male mice. After 2 weeks of recovery, female mice were divided into the free feeding (FF, n = 4), daytime feeding (DF, n = 5), and nighttime feeding (NF, n = 5) groups. Wheel running activity (WRA) and home cage activity (HCA) were recorded at 6-min intervals for 4 weeks. Moreover, the WRA and HCA of male mice in the FF (n = 4), DF (n = 5), and NF (n = 4) groups were assessed using the same method (C and D). All data were expressed as mean ± s.e.m. *P < 0.05 vs the FF group, ***P < 0.001 vs the FF group, ### P < 0.001 vs the DF group.
Citation: Reproduction 163, 5; 10.1530/REP-21-0336
All-day activity and nighttime activity of young adult mice under time-restrictive feeding. Mice were entrained to a 12 h light:12 h darkness photoperiodic cycle for a week with food and water available ad libitum. E-mitter probes were surgically implanted into young adult female and male mice. After 2 weeks of recovery, female mice were divided into the free feeding (FF, n = 4), daytime feeding (DF, n = 5), and nighttime feeding (NF, n = 5) groups. Wheel running activity (WRA) and home cage activity (HCA) were recorded at 6-min intervals for 4 weeks. Moreover, the WRA and HCA of male mice in the FF (n = 4), DF (n = 5), and NF (n = 4) groups were assessed using the same method (C and D). All data were expressed as mean ± s.e.m. *P < 0.05 vs the FF group, ***P < 0.001 vs the FF group, ### P < 0.001 vs the DF group.
Citation: Reproduction 163, 5; 10.1530/REP-21-0336
All-day activity and nighttime activity of young adult mice under time-restrictive feeding. Mice were entrained to a 12 h light:12 h darkness photoperiodic cycle for a week with food and water available ad libitum. E-mitter probes were surgically implanted into young adult female and male mice. After 2 weeks of recovery, female mice were divided into the free feeding (FF, n = 4), daytime feeding (DF, n = 5), and nighttime feeding (NF, n = 5) groups. Wheel running activity (WRA) and home cage activity (HCA) were recorded at 6-min intervals for 4 weeks. Moreover, the WRA and HCA of male mice in the FF (n = 4), DF (n = 5), and NF (n = 4) groups were assessed using the same method (C and D). All data were expressed as mean ± s.e.m. *P < 0.05 vs the FF group, ***P < 0.001 vs the FF group, ### P < 0.001 vs the DF group.
Citation: Reproduction 163, 5; 10.1530/REP-21-0336
DF affects the reproductive phenotype in male mice
Whether DF has significant effects on male reproductive function was evaluated. To address issue, young adult male mice entrained to an LD cycle were subjected to either FF or DF for 4 weeks. Time-course changes in testicular and seminal vesicular weight and sperm contents in the cauda epididymis and vas deferens were then analyzed (Fig. 3). During 4 weeks of experiment, we found no significant difference in terms of sperm content (Fig. 3C) between the FF and DF groups. The testicular weight was likely to decrease in the DF group (Fig. 3A). However, the result did not significantly differ even if the left and right testes were analyzed individually (Supplementary Fig. 4). Only the weight of the seminal vesicles significantly differed after 2 weeks of DF and thereafter. As shown in Fig. 3B, the weight of the seminal vesicles decreased by over 30% at 2 and 4 weeks in the DF group. Thus, DF did not cause an immediate decrease in male reproductive function.
Effects of daytime feeding on the reproductive indices of male mice. Male mice were entrained to a 12 h light:12 h darkness cycle and then randomly divided into the free feeding (FF) and daytime feeding (DF) groups for restrictive feeding for 4 weeks. Mice were sacrificed at 0, 1, 2, and 4 weeks after restrictive feeding (n = 5 for each time point). The testicular (A) and seminal vesicle (B) weight were determined. Sperm contents were assessed from the cauda epididymis and vas deferens (C). All data were expressed as mean ± s.e.m. *P < 0.05 vs the FF group.
Citation: Reproduction 163, 5; 10.1530/REP-21-0336
Effects of daytime feeding on the reproductive indices of male mice. Male mice were entrained to a 12 h light:12 h darkness cycle and then randomly divided into the free feeding (FF) and daytime feeding (DF) groups for restrictive feeding for 4 weeks. Mice were sacrificed at 0, 1, 2, and 4 weeks after restrictive feeding (n = 5 for each time point). The testicular (A) and seminal vesicle (B) weight were determined. Sperm contents were assessed from the cauda epididymis and vas deferens (C). All data were expressed as mean ± s.e.m. *P < 0.05 vs the FF group.
Citation: Reproduction 163, 5; 10.1530/REP-21-0336
Effects of daytime feeding on the reproductive indices of male mice. Male mice were entrained to a 12 h light:12 h darkness cycle and then randomly divided into the free feeding (FF) and daytime feeding (DF) groups for restrictive feeding for 4 weeks. Mice were sacrificed at 0, 1, 2, and 4 weeks after restrictive feeding (n = 5 for each time point). The testicular (A) and seminal vesicle (B) weight were determined. Sperm contents were assessed from the cauda epididymis and vas deferens (C). All data were expressed as mean ± s.e.m. *P < 0.05 vs the FF group.
Citation: Reproduction 163, 5; 10.1530/REP-21-0336
DF disturbs the estrous cyclicity of young adult female mice in a reversible manner
To further understand the significant delay caused by time-restrictive feeding in conception, we examined the effects of both DF and NF on the reproductive cycle of female mice. Meanwhile, the estrus cycle was determined by performing vaginal cytology (as shown in the ‘Experimental outline’ in ‘Materials and methods’ section’). Our main question was whether DF or NF could disrupt the normal estrous cycle of female mice. If so, we examined if such a disturbance was reversible. To answer these questions, we selected 34 mice with regular cycle that previously underwent the 15-day cycle test. Regular cycle was defined as the appearance of estrous within 3–4 days and diestrus within 4–5 days. In total, 15 mice received DF for 40 days, 9 mice received NF for 35 days, and the remaining 10 control mice received FF during the same period. The diet of mice receiving DF or NF was again shifted to FF, and the reversibility of the estrous cycle was assessed for 20 or 15 more days. As shown in Fig. 4A and B, control female mice receiving FF showed a robust and consistent cyclicity during the whole examination period (75 or 65 days) (Supplementary Fig. 5 for individual data). However, in mice subjected to DF, immediate disturbance of the estrous cycle was evident in all DF animals (Fig. 4C and Supplementary Fig. 6). In several cases, continued diestrus was evident for at least 10 days upon DF. Occasional completion of the estrous cycle was irregular thereafter. While DF was shutting out the estrous cycle almost completely, NF had a milder effect (Fig. 4D and Supplementary Fig. 7 for individual data). Some mice maintained their estrous cyclicity during the NF period or regained the rhythm within almost 10 days upon NF. Only two animals in the NF group exhibited a disrupted estrous cycle throughout the restrictive feeding period. This explains at least in part, why the s.e.m. of days to vaginal plug was higher in the NF group than in the FF and DF groups (Table 1). As summarized in Fig. 4E, the high incidence of diestrus and low frequency of estrus were prominent during the DF period, and these findings were not observed if mice receiving DF were again subjected to FF. A similar tendency was observed in the NF group. However, it was not as severe as that in the DF group (Fig. 4F). In particular, the frequency of estrus was slightly reduced. To visualize time course changes caused by time-restrictive feeding and subsequent Re-FF, the percentage of cycling mice was quantified by a criterion showing normally cyclicity if the mice exhibited normal cyclic changes for the last 5 or 10 days (Fig. 4G and H). The quantification revealed a rapid decline in the percentage of cycling mice caused by DF within 10 days and a gradual increase in the percentage of cycling mice upon returning to FF (Fig. 4G). However, the percentage of cycling mice transiently increased after 25–35 days in the DF group and it then declined thereafter. Interestingly, the transient increase in the percentage of cycling mice was in good agreement with the observed delay in conception caused by DF (Table 1). In the NF group, most mice had restored estrous rhythm before the termination of NF, and all had complete estrous cycle within 15 days after returning to FF (Fig. 4H). Therefore, DF could strongly disrupt the female reproductive cycle in a reversible manner, which is a remarkable cause of fertilization delay caused by DF.
Effects of daytime or nighttime feeding on the estrous cycle of female mice. (A, B, C, and D) After 40 days of daytime feeding (n = 15) and 35 days of nighttime feeding (n = 9), mice were fed ad libitum and their estrus cycles were assessed for 20 or 15 more days. The estrous cycles of the free feeding (FF) group (n = 10) was assessed for 55 days. E, estrus; M, metestrus; D, diestrus; P, proestrus. (E and F) Frequency of vaginal cytology in the FF, DF/NF, and Re-FF groups (when the diet of DF/NF mice shifted to FF). The letters above each bar represent statistically different groups according to ANOVA and the Duncan's post hoc test. (G and H) Percentage of cycling mice during the whole experiment period. Female mice had a normal cycle if they presented with normal cyclic changes over the last 5 days (open circle) or 10 days (closed circle). *P < 0.5, **P < 0.01, ***P < 0.001
Citation: Reproduction 163, 5; 10.1530/REP-21-0336
Effects of daytime or nighttime feeding on the estrous cycle of female mice. (A, B, C, and D) After 40 days of daytime feeding (n = 15) and 35 days of nighttime feeding (n = 9), mice were fed ad libitum and their estrus cycles were assessed for 20 or 15 more days. The estrous cycles of the free feeding (FF) group (n = 10) was assessed for 55 days. E, estrus; M, metestrus; D, diestrus; P, proestrus. (E and F) Frequency of vaginal cytology in the FF, DF/NF, and Re-FF groups (when the diet of DF/NF mice shifted to FF). The letters above each bar represent statistically different groups according to ANOVA and the Duncan's post hoc test. (G and H) Percentage of cycling mice during the whole experiment period. Female mice had a normal cycle if they presented with normal cyclic changes over the last 5 days (open circle) or 10 days (closed circle). *P < 0.5, **P < 0.01, ***P < 0.001
Citation: Reproduction 163, 5; 10.1530/REP-21-0336
Effects of daytime or nighttime feeding on the estrous cycle of female mice. (A, B, C, and D) After 40 days of daytime feeding (n = 15) and 35 days of nighttime feeding (n = 9), mice were fed ad libitum and their estrus cycles were assessed for 20 or 15 more days. The estrous cycles of the free feeding (FF) group (n = 10) was assessed for 55 days. E, estrus; M, metestrus; D, diestrus; P, proestrus. (E and F) Frequency of vaginal cytology in the FF, DF/NF, and Re-FF groups (when the diet of DF/NF mice shifted to FF). The letters above each bar represent statistically different groups according to ANOVA and the Duncan's post hoc test. (G and H) Percentage of cycling mice during the whole experiment period. Female mice had a normal cycle if they presented with normal cyclic changes over the last 5 days (open circle) or 10 days (closed circle). *P < 0.5, **P < 0.01, ***P < 0.001
Citation: Reproduction 163, 5; 10.1530/REP-21-0336
Discussion
We investigated changes in fertility caused by alteration in mealtime and their causes in young adult mice. Interestingly, DF, which reflects disrupted eating patterns during shift work, had a profound effect on the reproductive performance of adult mice.
Several studies have shown that long-term hunger and food intake only during active periods may be effective in addressing metabolic syndromes such as obesity and diabetes by modulating the circadian rhythm of organisms in case of intermittent fasting (Patterson et al. 2015, Chaix et al. 2019). Humans have evolved. That is, they feed in the active phase and fast in the rest phase. However, mealtime shift disrupts the circadian rhythm in the living body by ingesting food in the rest phase rather than the active phase. These restrictive feeding models are devised based on the fact that food intake occurs during the rest phase of the circadian rhythm that is experienced by shift workers (Lennernäs et al. 1995). Breaking the actual 24-h rhythm can increase the risk of developing various metabolic and chronic disorders (Hatori & Panda 2015, Deng et al. 2018, Zimmet et al. 2019). Shift work can affect the 24-h rhythm. Shift workers experience mealtime shift, and they are also affected by different unusual environmental factors such as degree of light exposure and sleep rhythm. Although mealtime shift alone is not a perfect model for shift work, it can strongly change the circadian rhythm (Yoon et al. 2012). Such alteration in the circadian rhythm is associated with illnesses experienced by shift workers. Currently, active research about shift work and reproductive function is conducted using not only mealtime shift but also LD cycle. Various models of shift work can identify reproductive function problems experienced by shift workers. The current study aimed to determine the cause of pregnancy delay in mice that underwent mealtime shift, which might shed light on the reproductive function issue of shift workers.
Reproduction is an energy-consuming process. Undernourishment can adversely affect female reproductive function (Martin et al. 2008). Decreased food consumption and increased energy output (i.e., higher physical activity) can result in infertility. However, the initial reduction in daily food consumption caused by DF or NF disappeared after 1 week. According to the difference in the amount of food intake based on the data of female and male mice measured under the same conditions, female mice had lesser individual variations than male mice. In addition, they had faster restrictive feeding adaptation (Supplementary Fig. 1A and B). There was no difference in terms of weight among the FF, DF, and NF groups from the second week of restrictive feeding and between male and female mice (Supplementary Fig. 1C and D). However, the current study assessed food consumption and for activity monitoring. Data could be more accurate if group sizes were increased and if mice did not undergo surgeries. Due to restricted feeding, there were changes in food intake and body weight, which are both metabolic factors essential for the survival of mice. Hence, these changes were temporary phenomena during the adaptation period, and these did not significantly affect pregnancy delay in mice after the adaptation period. The DF group had a conception delay of about 25 days (Table 1). Such difference was evident after a simple meal-time change. Nevertheless, this was not consistent based on the graphs of food intake, weight gain, and other physiological effects. In terms of the number of pups born and their M/F ratio, there was no significant difference between the DF and FF groups. Thus, restrictive feeding had no significant effect on pregnancy maintenance. However, results should be validated via concrete experiments to determine how restrictive feeding during pregnancy could affect offspring.
To identify the cause of delayed conception attributed to restrictive feeding, we hypothesized that restrictive feeding shifted the activity time of mice and caused delayed conception. Thus, an activity monitoring experiment was conducted (representative data: Supplementary Figs. 2 and 3). Mice are nocturnal animals. That is, the active phase of mice is during nighttime, which is in contrast to that of humans. Mating occurs primarily at night (Landry et al. 2012). By focusing on these characteristics, experiments were performed based on the assumption that changes in food intake from nighttime to daytime could result in insufficient rest during the rest phase, thereby delaying mating behavior and pregnancy. However, the HCA of the DF group was more likely to be similar to that of FF group. In the NF group, interesting outcomes such as an increased absolute amount of overall activity were observed (Fig. 2B). In this experiment, compared with FF, DF delayed pregnancy. It was impossible to identify the cause of such delay based on the results of activity monitoring. Therefore, future studies must be conducted to assess the causes of the significant increase in the activity of female mice that received NF. Male mice that received DF were more likely to have a lower overall activity and HCA than female mice that received FF. However, the results did not significantly differ. The NF group had lower activities in the night phase than the FF group (Fig. 2D). Nevertheless, further research about the effects of NF in female and male mice must be performed.
A recent study (Demirkol et al. 2021) has shown that shift work can adversely affect the circadian rhythm, which can reduce semen quality. This result is consistent with that of the current study showing that the reproduction phenotype was reduced in male mice that received DF than in those that received FF. However, since sperm mature after 3 weeks, a more detailed evaluation of the reproductive function of male mice, including the assessment of semen quality, is required. Male mice should be viewed in terms of mating behavior, in addition to physiological indicators associated with reproduction. According toSwamy et al. (2018), successful mistimed feeding interferes with mating behavior. This study reported a significant reduction in mating behavior in the group that ate food during daytime, which is consistent with our experimental results (Table 1). However, the estrous cycle pattern of female mice is controversial. We assumed that the difference was attributed to the characteristics of estrous cycle in female mice, which is often irregular. It varies based on several environmental factors (Goldman et al. 2007). To control these variables, our group assessed the estrus cycle for 15 days prior to the onset of restrictive feeding in 55 female mice, of which 34 had normal functioning. The estrous cycle of these mice was assessed during restrictive feeding (Fig. 4A, B, C, D and Supplementary Figs 5, 6, 7). After restrictive feeding was finished, the diet was shifted again to FF. After evaluating the estrous cycle, several regular cycles were selected (Fig. 4E, F, G and H). In a controlled experimental environment, the estrous cycle disrupted by restrictive feeding was found to cause delayed pregnancy. During the experiment, the estrous cycle of the FF group was normal (Fig. 4A, B and Supplementary Fig. 5). A disrupted estrous cycle in female mice is among the several causes of delated pregnancy. Hence, further studies should be conducted to validate the causes of delayed pregnancy from various standpoints via different experiments, such as uterine weight, ovarian weight, and ovarian function evaluations.
In conclusion, 12-h mealtime shift can adversely affect fertility in young adult mice. Such mealtime shift decreased seminal vesicle weights and disrupted estrous cycles. Hence, infertility and decreased menstrual function might be associated with restrictive feeding in people with shift work. However, more extensive studies should be conducted to validate these findings.
Supplementary materials
This is linked to the online version of the paper at https://doi.org/10.1530/REP-21-0336.
Declaration 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 work was supported by the Korea Research Foundation (KRF) grants funded by Korea government (MEST) (Nos. 2008-0062417, 2009-0088886,-2-C00625). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Author contribution statement
Conceived and designed the experiments: J H P, M H K, D H H, S C. Performed the experiments: J H P, M H K, D H H, J Y N, E S J, J H P, S C. Analyzed the data: J H P, M H K, D H H, S H L, C J K, S C. Contributed reagents/materials/analysis tools: J H P, S H L, C J K, S C. Wrote the paper: J H P, M H K, S H L, C J K, S C.
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