| Ketosis is a nutritional metabolic disease with high prevalence in periparturient dairy cows,which affects the reproductive performance in the long term.High concentrations of ketone bodies（KBs）,represented byβ-hydroxybutyric acid（BHBA）in the blood of ketotic cows,may enter the follicular fluid（FF）and affect follicular granulosa cells（GCs）and follicular development,resulting in low fertility performance such as reduced estrous activity and prolonged first postpartum estrus in cows.However,the effects of high concentrations of BHBA in follicular fluid on GCs and their regulatory mechanisms remain unclear.Therefore,we firstly identified the differential expression genes（DEGs）and their functions of GCs under different concentrations of BHBA stress via RNA-seq technique and bioinformatics analysis.Then,we further used molecular biology techniques to reveal the mechanism of the effect of high concentration of BHBA on GCs in cow follicles.Finally,we explored the role of exosomes（Exos）in restoring the proliferation rate of granulosa cells under high BHBA stress,and clarified the possible regulatory mechanisms of Exos micro RNAs（mi RNAs）derived from FF on the proliferation of GCs.The main research contents are as follows:1.Preliminary exploration of the effect and possible mechanism of BHBA on the proliferation of follicular GCs in dairy cows.In this study,we initially evaluated the effects of BHBA stress at 0 m M,1.2 m M and 2.4 m M on the proliferation and cell cycle in the follicular GCs of dairy cows,resulting in 1.2 m M and 2.4 m M BHBA stress leading to GCs cell cycle arrest in G1 phase and abnormal GCs proliferation.RNA-seq results showed that DEGs were clustered into eight expression profiles with different trends.Among them,DEGs in profiles 1 and 6 responded to 1.2 m M BHBA already;DEGs in profiles 3 and 4responded only when the BHBA concentration reached 2.4 m M and DEGs in profiles 0 and 7 showed the same expression trend in response to increasing BHBA concentrations.The results of GO and KEGG functional enrichment analysis suggested that 1.2 m M BHBA may inhibit the cell cycle,while2.4 m M BHBA was more likely to induce apoptosis in GCs;in addition,estrogen synthesis may be blocked with the continuous increase of BHBA concentration.2.The effect and related mechanism of BHBA on the mitochondrial function of follicular GCs in dairy cows.We further found that 2.4 m M BHBA stress led to a significant increase in apoptosis rate and mitochondrial reactive oxygen species levels in GCs.Meanwhile,we found that 2.4 m M BHBA stress led to mitochondrial dysfunction,including increased mitochondrial abnormalities in GCs,opening of the mitochondrial permeability transition pore（MPTP）,decreased mitochondrial membrane potential（MMP）and Ca2+release.In contrast,overexpression of the mitochondrial deacetylase Sirtuin 3（Sirt3）enhanced autophagy of follicular GCs via the AMPK-m TOR-Beclin-1 pathway,thereby alleviating BHBA-induced mitochondrial damage.3.Mechanism of follicular fluid-derived Exos mi RNA regulating proliferation of follicular GCs in dairy cows.Co-incubation of follicular fluid Exos with GCs from dairy cows showed to significantly increase the proliferation rate of GCs.A total of 95 differentially expressed mi RNAs（76 up-regulated and 19 down-regulated）were identified in follicular fluid Exos of ketosis and non-ketosis cows by mi RNA-seq technique.Further studies revealed that bta-mi R184 may play an important regulatory role in the proliferation of follicular GCs.And GO and KEGG functional enrichment analysis of the target genes of bta-mi R184 showed that the possible involvement in the regulation of metabolism,redox homeostasis and Notch signaling pathway,etc.In this study,firstly,we isolated Exos from bovine FF and found that Exos could significantly increase the proliferation rate of GCs by co-incubation with GCs,with the best effect of Exos from ketosis cows.We further screened the FF-derived Exos-mi RNAs from ketosis and non-ketosis cows by mi RNA-seq technique.A total of 95 differentially expressed mi RNAs（76 up-regulated and 19 down-regulated）were identified.One of the down-regulated bta-mi R184 may play an important regulatory role in the proliferation of follicular GCs.The GO and KEGG functional enrichment analysis of bta-mi R184-regulated target genes,revealed a possible involvement in metabolic regulation,regulation of redox homeostasis,Notch pathway,etc.The present study elucidated the molecular mechanism of GCs damage induced by high concentration of BHBA in FF of ketotic cows,which provides a theoretical basis for improving the reproduction rate of ketotic cows.Meanwhile,we also provide a reference for research on human diseases（such as premature ovarian failure（POF）and polycystic ovary syndrome（PCOS）,etc..）,which are related to follicular development disorders. |
