Role Of FGF21 In The Regulation Of Female Reproductive Function Affected By Meal Frequency And Its Mechanism

The low reproductive efficiency of sows restricts the efficient production of pigs in China.The obstruction of follicle development and poor quality of oocytes in gilts are the main factors leading to the elimination of sows,which brings huge economic losses to pig production.Therefore,improving sow follicle development and promoting sow’s reproductive potential has important theoretical and practical significance.The feeding system for gilts is based on growing pigs,and follows the principles of “less feeding and frequent feeding” and “ad libitum”.The purpose is to improve the growth performance of pigs.However,this feeding strategy may not conducive to display the reproductivepoten tial of gilts.Recent studies have found that less meal frequency is beneficial to increasing the growth performance of growing pigs and improving the metabolism health.Metabolic health is closely linked to reproductive performance,but at present,whether meal frequency affects the follicular development and reproductive performance of gilts remains to be confirmed.Female reproductive activity is regulated by the secretion of Gn RH.However,it is still unclear whether meal frequency can affect the Gn RH secretion in gilts.The liver is the main organs that sense external nutrition signals and liver FGF21 is induces by fasting.Whether the liver FGF21 participate in meal frequency regulation of Gn RH secretion has not been reported.Therefore,this research focuses on the scientific question of "how does meal frequency regulate the metabolism and follicular development in gilts and its influence and mechanism on female reproductive performance".Experiment 1,Effects of meal frequency on metabolism,and ovarian follicular development in giltsA total of 36 gilts（Landrace × Yorkshire）at age of 150（±3）d and body weight at 77.6（±3.8）kg were randomly allocated into one of three meal frequencies to feed one meal（T1）;two meals（T2）,or six meals per day（T6）.Results showed that:（1）The average daily gain of gilts in the T1 group was greater than T6 gilts during the experiment（T1 vs T6 = 701.5 g vs 653.4 g,P < 0.05）,the body weight at the final of experiment was greater in T1 gilts compared with that in T6 gilts（+4.9kg,P < 0.05）,but no difference in backfat thickness at the final of experiment between different meal frequency（P > 0.05）.（2）Digestibility of NDF in T1 pigs were greater compared with the T6 pigs（P < 0.05）.Time-course changes of SCFAs concentrations in feces at different time points（P < 0.05）,and the total SCFAs contents in feces were greater in T1 gilts compared with the other two groups（P < 0.05）.（3）Meal frequency has no effect on nitrogen digestibility and fecal nitrogen excretion（P > 0.05）,but the urine excretion in the T6 gilts was greater than T1 gilts（P < 0.05）.The T1 gilts had increased amount of retained nitrogen and the biological value of nitrogen（P < 0.05）.（4）Meal frequency changed the postprandial serum insulin concentrations,T1 gilts had a greater serum insulin concentration compared with the pigs in T6 group（P < 0.05）,the gilts in T6 group had a greater serum glucose level than in T1 gilts（P < 0.05）.In T6 gilts,serum α-amino nitrogen levels were independent on meal ingestion,and were higher than T1 gilts（P < 0.05）.Urea levels were lower in T1 gilts than in T6 gilts（P < 0.05）.（5）The serum FGF21 levels of gilts in the T1 group was greater than T6 gilts during the experiment（P < 0.05）,but meal frequency had no effect on serum IGF-1 and leptin levels（P > 0.05）.（6）The difference of puberty between different meal frequency was not significant（the age,body weight and back-fat thickness at puberty onset）（P > 0.05）.The gilts in T1 group exhibited a substantially greater LH levels but not FSH levels,compared to gilts in T2 and T6 groups（P < 0.05）in 18 d of 3rd estrus cycle.（7）The number of growing follicles,and corpora lutea were greater in pigs fed one meal per day compared with gilts fed six meals per day（P < 0.05）.（8）The serum estrogen levels were greater in gilts fed one meal per day compared with gilts fed six meals per day（P < 0.05）.The m RNA levels of ovarian gonadotropin receptors FSHR and LHCGR,were greater in T1 and T2 gilts compared with T6 gilts（P < 0.05）.The gene expression levels related to ovarian follicular development,BMP15 and GDF9 were elevated by less meal frequency in gilts（P < 0.05）.The results of the experiment 1 demonstrated that: less meal frequency increased nutrient utilization,promoted the secretion of luteinizing hormones and serum FGF21,and improved ovarian follicular development in gilts,but no difference in the puberty of gilts.Experiment 2,Effects of meal frequency（intermittent feeding）on reproductive performance in femaleExperiment 1 shows that less frequency promoted follicular development in gilts,but the mechanism is still unclear.Therefore,in this part,C57BL/6J female mice were used to investigate the effect of meal frequency on female reproductive performance and its possible mechanism.Due to the different feeding habits of different animals,the different feeding frequencies in this experiment were achieved by intermittent feeding（the mice were free to access food during the 10 h feeding period every day,and the remaining 14 h were fasted）.The experiment was a 2×2 experimental design with fed with normal diet or high-fat diet,and ad libitum or intermittent feeding（IF）.Results showed that:（1）Compared to mice fed on the ND-ad libitum（NA）,mice fed on the HFD-ad libitum（HA）increased the cumulative energy intake（P < 0.05）,Compared to ad libitum,IF had no difference in the cumulative energy intake（P > 0.05）.（2）Compared to HA mice,HFD-IF（HT）mice had reduced body weight gain（P < 0.05）.Circulating glucose clearance were improved in HT mice compared to HA mice（P < 0.05）,but no difference in the normal chow diet with time-restricted access to food（NT）mice compared to NA mice（P > 0.05）.（3）HA mice exhibited irregular estrus cycles compared to HT mice,with a significant increase of the time spent in diestrus（P < 0.05）.In addition,compared to HA mice,HT mice exhibited a shorter cycle length（P < 0.05）.（4）Compared to the NA or HA mice,IF increased the number of primordial follicle,antral follicle,total follicle,and corpora lutea in NT and HT mice（P < 0.05）.（5）IF improved the female fertility in mice fed both normal diets and HFD（P < 0.05）.Decrease in the cumulative number of pups subjected to the high-fat diet was rescued by the IF regimen（P < 0.05）.（6）Serum FGF21 levels（P < 0.05）,but not the levels of IGF-1,leptin,and adiponectin（P > 0.05）,underwent cyclic changes during the estrus cycle,with the high levels at proestrus.Less meal frequency（IF）preserved the normal cyclic FGF21 rise of mice fed the HFD.（7）IF treatments stimulation enhanced the Gn RH secretion（P < 0.05）,independent of Kisspeptin expression levels（P > 0.05）.The results of the experiment 2 demonstrated that: less meal frequency promoted follicular development and improved female fertility through increased Gn RH secretion,and FGF21 may involved in the beneficial effect of less meal frequency on female reproductive performance.Experiment 3,The role of liver FGF21 in the beneficial effect of less meal frequency on fertility and its mechanismThe wild type（WT）and liver FGF21 knockout mice（FGF21LKO）were used to study the role of liver FGF21 in the beneficial effect of less meal frequency on fertility.The experiment was a 2×2 experimental design with fed with high-fat diet in wild type or liver FGF21 knockout mice,and ad libitum or intermittent feeding（IF）.Meantime,Gn RH neurons and primary granule cells were used as models to investigate the effects of FGF21 on Gn RH secretion in Gn RH neurons and estrogen synthesis in primary granule cells.Results showed that:（1）Compared to WT mice,FGF21 LKO had no difference in the cumulative energy intake（P > 0.05）.Compared to ad libitum mice,IF had no difference in the cumulative energy intake（P > 0.05）.（2）In WT mice,glucose clearance were improved in time-restricted access to food mice（HT）compared to ad libitum mice（HA）（P < 0.05）,but time-restricted access to food had no effect in FGF21 LKO mice（P > 0.05）.（3）In WT mice,estrus cycle,ovarian development were improved in HT mice compared to HA mice（P < 0.05）,but time-restricted access to food had no effect in FGF21 LKO mice（P > 0.05）.（4）In WT mice,IF improved the female litter sizes（P < 0.05）,but time-restricted access to food had no effect in FGF21 LKO mice（P > 0.05）.（5）FGF21 directly induced Gn RH secretion through ERK_1/2 signaling（P < 0.05）.（6）FGF21 has no effect on synthesis of estrogen in primary granulosa cells（P > 0.05）.The results of the experiment 3 demonstrated that: Less meal frequency improved the reproductive function of female mice via FGF21 modulated Gn RH secretion,and FGF21 directly modulated Gn RH secretion via ERK_1/2 pathway in vitro.Experiment 4,Effects of liver FGF21 in the cross talk of ovary,liver,and adipose during estrous cycleIn this experiment,C57BL/6J female mice were used to study the expression levels of FGF21 in different tissues during estrous cycle;Bilateral ovariectomized mice and primary hepatocytes were used to study the effect of estrogen on liver FGF21 expression.WT and FGF21 LKO mice were used to study the role of liver FGF21 in the beneficial effect of estrogen on adipose metabolism.Results showed that:（1）Liver and serum FGF21 expression underwent cyclic changes with the E2 during estrus cycle,with the highest levels at proestrus and the lowest levels at diestrus（P < 0.05）.（2）E2 robustly increased liver FGF21 expression through an ERα/mTOR-dependent manner（P < 0.05）.（3）Hepatic FGF21 deficiency abolished E2-induced subcutaneous adipose tissue browning（P < 0.05）.The results of the experiment 4 demonstrated that: E2 robustly increased liver FGF21 expression through an ERα/mTOR-dependent manner.Liver FGF21 expression underwent cyclic changes and has a beneficial effect in adipose metabolism,which promotes the synchronized metabolism of body tissues during testrus cycle.In summary:（1）Less meal frequency increased the body weight gain in gilts.The less meal frequency gilts have a faster glucose clearance and lower serum urea andα-amino nitrogen level, which suggested less meal frequency increased nutrient utilization.The less meal frequency gilts had decreased the urine excretion,increased the biological value of nitrogen,which suggested less meal frequency increased the lean deposition.（2）Less meal frequency improves the reproductive function of female mice via liver FGF21 modulated GnRH secretion through ERK_1/2 pathway.（3）Liver FGF21 promotes the synchronized metabolism of body tissues during estrus cycle.Liver FGF21 expression underwent cyclic changes and has a beneficial effect in adipose metabolism during estrus cycle.