Fatty Acid Degradation Plays An Essential Role In The Proliferation Of Mouse Female Primordial Germ Cells

Primordial germ cells(PGCs)are the founders of germ cells in embryos and ultimately differentiate into sperms or eggs,which could produce offspring.PGCs are derived from proximal epiblast cells,reach to the genital ridge between E10.5 and E 11.5 and subsequently start to proliferate from E 11.5 to E 13.5.The proliferation of PGCs assures the number of germ cell after birth especially in female mammals because the oocytes in the ovary continue to reduce after proliferation.Abnormalities in the proliferation can cause depletion of germ cells leading to infertility in females.To better understand the proliferation of PGCs in female mouse,we constructed a proteome profile of female mouse gonads at E 11.5 and a total of 3662 proteins were quantified in preliminary work.Then we performed the KEGG pathway analysis of 3662 proteins in genital ridge.Based on the analysis,pathways involved in fatty acid degradation are enriched and the ratio of enrichment was 9.We successfully identified 27 of the total 48 proteins participated in fatty acid degradation.The results of real-time PCR showed that the expression of some of them reached the peak in E 12.5,and the expression between E 11.5 and 13.5 were obviously higher than in E 14.5,which suggests the important role of fatty acid degradation in the proliferation of PGCs.Carnitine palmitoyltransferase 1(cpt1)was identified in the proteome profile and was the rate-limiting enzyme and regulated the entry into the mitochondria of the fatty acid.Fatty acid degradation could be inhibited by repressing the activity of CPT1.To explore the effects of the fatty acid degradation on the development of PGCs,we inhibited the activity of CPT1 by adding different doses of etomoxir including 2μM,10μM and 50μM,a nonreversible inhibitor of CPT1.Then we found the number of PGCs decreased largely at E 14.5 though the supplement with Etomoxir.The decreasing number of PGCs may be caused by the the abnormal proliferation or apoptosis.The results of EDU incorporation assay showed that the ratio of EDU-positive PGC reduced and there was a does-dependent decrease in EDU incorporation with the addition of etomoxir.However,there were a few co-localized positive signals between cleaved caspase-3 and DDX4 in all four groups and no statistical significance among them.So we verified the mechanism underlying the deceasing PGCs was a result of reduced proliferation,but not apoptosis.AMPK is thought to serve as a power switch to protect against energy deprivation and is sensitive to variety of ATP;what’s more,the activation of AMPK could induce the P53 dependent G1 arrest.Both the expression levels of P-AMPK and P-P53 increased successively along with the increasing doses of etomoxir by western blot detection.To further confirm that P53-dependent G1 arrest was the reason of the decrease of PGCs,genital ridges were cultured by simultaneously adding 10μM etomoxir and 10μM Pifithrin-α,which was a inhibitor of P53 pathway.There were no statistical difference in the numbers of PGC between Pifithrin-α group and the normal group,but the combined use of 10μM etomoxir and Pifithrin-α rescued the number of PGCs.The number of PGCs reached near to the normal level.These data suggest that fatty acid degradation play a key role during the proliferation of PGC via P53 dependent G1 arrest.In summary,our study verified that the inhibition of fatty acid degradation could induce the P53 dependent G1 arrest and the reduced proliferation of PGCs,which caused the decrease of PGCs number ultimately.The exploration of the biological functions of CPT1 A could not only lay the foundation of an in-depth understanding of the important role of fatty acid degradation in the primordial germ cell proliferation,but also offered some new ideas to study the primordial germ cell development events and the regulatory mechanisms of ovarian reserve capacity.