| The oocytes harbored in the primordial follicles are dormant in the ovaries.The pool of primordial follicle is crucial for fertility because it provides all of the oocytes for postpubertal ovulations and is the approximate determinant of reproductive lifespan.The newborn porcine ovaries contain about one million oocytes,however,only one hundred of those oocytes mature and develop into an embryo.The insufficient activation or over-activation of the primordial follicle caused by mismatched nutrition or management might be one of the main reasons leading to the shortening of reproductive carrier in sows.The activation of dormant primordial follicles requires an ovarian mTOR signaling,which is closely related to nutritional metabolic signals.However,very few researches reported the nutritrient-dependent regulation of primordial follicle activation via mTOR signaling.Recent evidences in invertebrates revealed that dietary protein,but not carbohydrates or lipids,was the major determint of reproductive efforts and lifespan,yet whether dietary protein could influence the activation of ovarian primordial follicles and the subsequent reproductive lifespan remains largely unknown.The circulating FGF21,which was mainly(80%)derived from liver expressions and secretions,was essential for the control of whole body energy metabolism by dietary protein restriction,however,whether liver FGF21 was also required for the control of ovarian primordial follicle activation by dietary protein is uncertain.Therefore,in this study,the effects of dietary protein restriction and excess on the activation of ovarian primordial follicle will be investigated using porcine and mouse as experimental models.Further,the underlying mechanisms of ovarian primordial follicle activation will be revealed based on in vitro ovary culture,liver-specific knockout of FGF21(FGF21LKO)model and exogenous nutritional inventions.According to the scientific issues targeted,five experiments were included as follows:Experiment 1,the effects of dietary protein restriction on the ovarian primordial follicle activation and the oocyte quality in gilts.A total of forty LY replacement gilts at the age of d 90 and body weight of 33 kg were fed an NRC-suggested normal protein to contain 100%SIDLys(NP,n =20)or a protein-restricted diet to contain 50%SIDLys(LP,n =20).Results showed that:(1)Compared with the NP gilts,the LP gilts had significantly elevated circulating FGF21 concentrations.(2)The LP gilts attained the first observed puberty with the age 9.9 days later(P = 0.14),the bodyweight 8.71 kg lower(P<0.05)and backfat thickness 1.78 mm lower(P<0.05)compared with the NP gilts.(3)At 30 days post dietary treatment,the paired ovaries were collected by ovariectomy and ovarian histology revealed that the percentage of ovarian primordial follicle was increased(68.74 vs 80.39%,P=0.012),the percentage of primary follicle(20.87 vs 15.47%,P = 0.062)and secondary follicle(9.46 vs 3.31%,P = 0.040),was decreased by the LP diet compared with the NP diet.Yet the number of antral follicle with the diameter between 1?3 mm on the survace of ovaries was not affected by dietary treatment.When the dietary treatment preceeded to the 3rd estrus,the percentage of ovarian primordial,primary and secondary follicle were not affected by dietary treatment.The number of antral follicle with the diameter between 1?3 mm,and the number of antral follicle with the diameter ? 3 mm on the survace of ovaries was not affected by dietary treatment.(4)At the the 3rd estrus,the cumulus cell-oocyte complexes(COCs)retrieved from antral follicle with diameter>3 mm were used for in vitro culture.The oocyte quality,represented by the expansion rate of COCs and the rate of oocytes with first polar body,was not affected by dietary treatment.(5)Western blot analysis of the proteins that were related to ovarian primordial follicle activation revealed that the mTORCl signaling(featured by p-mTOR/mTOR,p-S6/S6)and ERK1/2 signaling were down-regulated,and AMPK signaling was up-regulated,by the LP diet compared with the NP diet.The results of the experiment 1 demonstrated that:(1)Dietary protein restriction decreased the ovarian primordial follicle activation without affecting the oocyte quality in gilts;(2)The elevated circulating FGF21 concentrations,as well as the down-regulated ovarian mTORCl pathway,might be involved in the control of primordial follicle activation by dietary protein in gilts.Experiment 2,the effects of dietary protein restriction or excess on the ovarian primordial follicle activation and the subsequent lifetime reproductive performance in mice.4-week-old C57BL/6 female mice were fed a protein restricted diet(LP,8%CP),a normal protein diet(NP,20%CP)or a protein excess diet(HP,50%CP)for 4 weeks,12 weeks,24 weeks and 48 weeks,at which time the ovarian follicles were detected,and mice were mated with male mice to detect their subsequent reproductive performance.(1)Compared with the NP mice,the LP mice had greater liver Fgf21 gene expressions and circulating FGF21 concentrations,and the HP mice had lower liver Fgf21 gene expressions and circulating FGF21 concentrations.The LP mice also had greater adipose adiponectin gene expressions and circulating adiponectin concentrations,and the HP mice had lower adipose adiponectin gene expressions and circulating adiponectin concentrations.(2)Ovarian HE histology revealed that,the ovarian primordial follicle activation was decreased by the LP diet,evidenced by greater number and percentage of primordial follicles,lower number and percentage of primary follicles,and lower ratio of primary to primordial follicles;On the other side,the ovarian primordial follicle activation was increased by the HP diet,evidenced by lower number and percentage of primordial follicles,greater number and percentage of primary follicles,and greater ratio of primary to primordial follicles.Addtionally,the number and percentage of atresia follicles was decreased by the LP diet but was increased by the HP diet.(3)Gene expressions of Pten,Lhx8,Foxo3a in oocytes,that were observed to maintain the dormancy of primordial follicle,were greater in the LP mice,but was lower in the HP diet.(4)Western blot analysis of the proteins that were related to ovarian primordial follicle activation revealed that the phosphorylation of ovarian mTOR and its downstream target S6 protein were decreased in mice fed the LP diet,and the phosphorylation of ovarian ERK1/2,that is required for granulosa cell proliferation and oocyte growth,were decreased in mice fed the LP diet,on the other side,the phosphorylation of ovarian mTOR,S6 and EKR1/2,were elevated in mice fed the HP diet.(5)Localization of p-S6 protein expressions was analyzed by IHC analysis,and found that the positive signals of p-S6 was mainly localized in oocytes.(6)After dietary treatment for 4,12,24 and 48 weeks,the mice(12-20 mice per treatment group)were tested for their ovulations.The number of mature COCs ovulated from mice on diets for 4,12 and 24 weeks were not affected.However,the number of mature COCs ovulated from mice fed the LP diet was greater than that fed the HP diet after dietary treatment for 48 weeks.(7)The mice(n = 20 per group)on diets for 12 weeks were mated with the male mice(aged 4?8 month,1:1)for 32 weeks.The cumulative parities(LP:5.74?NP:6.32?HP:5.55)farrowed in different groups were not affected during the 32-weeks of mating.The cumulative pups of LP mice after mating for 16,20 and 24 weeks were significantly lower than that in NP group,but the cumulative pups after mating for 28 and 32 weeks were similar among different groups.The mice(n = 20 per group)on diets for 24 weeks were mated with the male mice(aged 4?8 month,1:1)for 28 weeks.The cumulative parities from LP group was greater(3.78 vs 2.64,P = 0.081)that that in HP group during the 28-weeks of mating.The cumulative pups of HP mice after mating for 28 weeks were lower than that in NP or LP groups(P = 0.067).The mice(n = 20 per group)on diets for 48 weeks were mated with the male mice(aged 4?8 month,1:1)for 8 weeks.The farrowing percentage of mice in the first parity(four weeks after mating)was greater in LP mice than that in HP mice(35%vs 5%,P<0.05).The cumulative pups of HP mice after mating for 8 weeks were lower than that in NP or LP groups(LP:25,NP:20,HP:2).Results of experiment 2 demonstrated that:(1)Protein restriction decreased the ovarian mTORCl signaling pathway and primordial follicle activation,and protein excess increased the mTORCl signaling pathway and primordial follicle activation;(2)Short-term(12 week)overconsumption of protein did not affect the infertility,but a long-term intake of protein excess diet impaired the subsequent fertility.Experiment 3,regulation of ovarian primordial follicle activation by dietary protein derived metabolic signals using in vitro ovary organ culture.In this part of study,in vitro culture of newborn mouse ovaries was used to test which kind of metabolic signals by dietary protein,including amino acids,FGF21 and adiponectin,was responsible for the control of ovarian primordial follicle activation by dietary protein.(1)Four culture media with different levels of amino acids were formulated.Including amino acid-free media(0-AA),and another three levels of amino acids(LP-AA:3.93 mM,LP-AA:5.14 mM,LP-AA:7.74 mM)based on the profile of circulating amino acid composition derived from mice fed the LP,NP and HP diets in experiment 2.Results:the ovarian primordial follicle activation,as well as the ovarian phosphorylation level of mTOR and its downstream target S6,were significantly inhibited by 0-AA media compared with other three groups.However,the ovarian primordial follicle activation,as well as the ovarian phosphorylation level of mTOR and its downstream target S6,were not different among the LP-AA,LP-AA and LP-AA groups.(2)Graded level of FGF21(0,50,200,1000 ng/ml)were supplemented to treat the in vitro cultured mouse ovaries and found that FGF21 treatment could not affect the ovarian primordial follicle activation as well as the protein expressions of mTORCl and AMPK signaling.(3)Graded level of adiponectin(0,5,15,30 ?g/ml)were supplemented to treat the in vitro cultured mouse ovaries and found that adiponectin treatment at the level of 15 and 30?g/ml significantly inhibited the activation of primordial follicles.Treatment of adiponectin elevated the ovarian phosphorylation level of AMPK,and down-regulated the ovarian phosphorylation levels of mTOR and its downstream target S6 protein.Further,treatment of compound C,an inhibitor of AMPK signaling,was used to test whether AMPK could mediate the effects of adiponectin on the primordial follicle activation.Results revealed that compound C treatment significantly abragated the adiponectin induced AMPK phosphorylation,but compound C treatment did not abolish the inhibitory effects of adiponectin on the phosphorylation of S6 protein.Meanwhile,compound C treatment did not affect the inhibitory effects of adiponectin on the primordial follicle activation.These results demonstrated that:(1)Amino acids is the essential nutrients required for the activation of primordial follicle,however,when the amino acid reached the minimal threshold for the activation of primordial follicle,further elevation of amino acids was not able to influence the activation of primordial follicle;(2)FGF21 exerted no direct effects on the activation of primordial follicles;(3)Adiponectin could inhibit the activation of primordial follicles independent of AMPK signaling.Experiment 4,role of liver FGF21 in the control of ovarian primordial follicle activation by dietary protein.Because the adipose tissue is the direct target of liver FGF21,and the control of energy metabolism by FGF21 requires adiponection expression and secretion.In the present study,we hypothesized that liver FGF21 could mediate the effects of dietary protein on the secretion of adiponection and the control of ovarian primordial follicle activation.According to a 2 × 3 experimental design,WT and FGF21LKO mice were fed the LP,NP and HP diets for 12 weeks.Results:(1)The WT mice fed the LP diet had greater mRNA expression of Adiponectin in adipose tissues and elevated circulating adiponectin concentration.In the FGF21LKO mice,the dietary protein-dependent regulation of Adiponectin mRNA expression and circulating adiponectin concentration was abrogated.(2)The WT mice fed the LP diet had greater number and percentage of ovarian primordial follciles,and lower percentage of primary follicle.The WT mice fed the HP diet had greater number and percentage of ovarian primary follciles,and lower number and percentage of primordial follicles.In the FGF21LKO mice,no difference of the number and percentage of primary and primordial follicles were observed among different dietary groups.(3)The phosphorylation levels of ovarian mTOR were significantly affected by diets,liver FGF21,and the interaction between diets and liver FGF21 for WT mice.In the FGF21LKO mice,the phosphorylation levels of ovarian mTOR were further elevated in mice fed the NP and HP diets.The phosphorylation levels of ovarian S6 in WT mice fed the LP diet were significantly lower than that fed the NP and HP diets.In the FGF21LKO mice,the phosphorylation levels of ovarian S6 were not affected by diets.These results demonstrated that:(1)Liver FGF21 was involved in the control of ovarian x primordial follicle by dietary protein;(2)The elevation of adiponection secretion stimulated by FGF21,was probably the reason for the inhibitory effects on primordial follicle activation by dietary protein restriction.Experiment 5,Protection of adiponectin receptor agonist AdipoRon on the primordial follicle activation in mice fed a protein-excess diet.To further investigate the role of adiponectin in the dietary protein-dependent regulation of ovarian primordial follicle activation,this study was designed to test whether AdipoRon,an adiponectin receptor agonist,was able to attenuate the HP-induced over-activation of ovarian primordial follicle.Two parts of researches were included in this experiment:(1)Firstly,the comparision of AdipoRon and adiponectin treatment on the ovarian primordial follicle activation was investigated by in vitro cultured ovaries and found that the adiponectin(15 and 30 ?g/ml)treatment decreased the activation of primordial follicles,as well as up-regulated phosphorylation levels of ovarian AMPK and down-regulated phosphorylation levels of ovarian S6.Meanwhile,AdipoRon treatment(25 and 50 mM)had comparable effects on ovarian primordial follicle activation as well as the phosphorylation levels of ovarian mTOR and its downstream target S6 with adiponectin treatment.(2)Further,according to a 2 × 3 experimental design,AdipoRon or DMSO(as control)was administered orally to mice fed the LP,NP and HP diets at a dosage of 50 mg/kg per day for 28 consecutive days.Results:the number and percentage of ovarian primordial follicles were greater,and percentage of primary follicle were lower,in LP mice than that in NP and HP mice.Additionally,the phosphorylation levels of mTOR and S6 were down regulated,the phosphorylation level of AMPK was up-regulated in LP mice.In the AdipoRon treatment mice,the number and percentage of ovarian primordial follicles in mice fed the HP diet was reversed to the level of that in mice fed the LP diet,and the phosphorylation levels of mTOR,S6 and AMPK were not different among the three dietary groups.Results from experimental 3 revealed that AdipoRon had comparable effects on the control of ovarian primordial follicle activation,and AdipoRon was able to protect the over-activation of ovarian primordial follicle induced by the excess intake of protein.Collectively,the results of experimental 1 to 5 demonstrated that:1)The ovarian primordial follicle activation was attenuated by dietary protein restriction but was accelerated by dietary protein excess.2)The ovarian mTORCl signaling was crucial for ovarian primordial follicle activation by dietary protein.3)Liver FGF21 secretion is the key endocrine signal of dietary protein,and the regulations of FGF21 on ovarian mTORCl signaling and primordial follicle activation requires adipose adiponectin. |
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