Transcriptome Analysis Of Oocyte Maturation In Pig Antrum Follicles

Ovary is an important reproductive organ in mammals,and the number of ovulation determines the number of offspring,which effects the reproductive performance.Follicle is a basic function unit of ovary,one of which main functions is to produce secondary oocytes.During the maturation of follicles,the diameter,chromatin configuration and maturation state of primary oocytes change significantly.The primary oocyte stagnates in the prophase of the first meiotic division in the preantral follicle.As the diameter of the antrum follicles increases from 0.5 mm to 8 ～ 10 mm,the diameter of the primary oocytes increases from 100 mm to 120 mm,and the chromatin of primary oocytes produces great changes to complete the first meiosis.During chromatin agglutination,the expression of histone H3K4me3 gradually decreases,affecting the transcriptional silencing of genes.H3K27me3,on the other hand,is involved in the regulation of chromatin aggregation in a coordinated manner.However,it is unclear that changes in primary oocyte diameter and meiosis and the mechanism between antrum follicles and oocyte maturation in pig.Hence,this study investigated the mechanism of maturation of primary oocyte in pig by measuring the diameter and karyotype of primary oocyte and screening the candidate genes in porcine antrum follicles of different sizes.According to the follicular diameter in pig,this study collected and classified the antrum follicles into five stages: 0.5 ～ 1.8,1.8 ～ 3,3 ～ 5,5 ～ 7 and ≥ 7 mm.For each stage,the diameter of primary oocytes was measured.The chromatin pattern of primary oocytes was then detected and confirmed using immunofluorescence.Finally,the candidate genes affecting the primary oocytes maturation of pig were screened using sc RNAseq.The results of this study are as follows:(1)The primary oocyte diameter increased gradually with the follicle increased in the first three stages(i.e.0.5 ～ 1.8 mm,1.8 ～ 3 mm,and 3 ～ 5 mm).The opposite result was observed while the follicle diameter is greater than 5 mm.(2)Using immunofluorescence,the results showed that histone H3K4me3 and H3K27me3 were expressed across five stages in the oocytes.The fluorescence intensity of H3K4me3 decreased gradually and the opposite result was observed for H3K27me3,which indicated that H3K4me3 and H3K27me3 coordinated involvement agglutination of chromatin.α-Tubulin has not significant change across different stages,which indicated that oocytes were at the GV stage.In 0.5-1.8 mm follicle,the chromatin was filamentous and had obvious nucleoli and nuclear membranes,which indicated that oocytes were at the GV0 stage.In 1.8-3 mm follicle,the chromatin morphologic of ringlike or horseshoe shape indicated that oocytes were at the GV1 stage.In 3-5 mm follicle,there were heterochromatin clumps in the oocytes indicating that oocytes were at the GV2 stage.In the 5-7 mm follicle,the cell chromatin block around the nucleolus indicated that oocytes were at the GV3 stage.While the follicle diameter ≥ 7 mm,the nucleoli and the cell nuclear membrane disappeared,and the cell was at the GV4 stage.(3)Comparing the oocytes in 0.5 ～ 1.8 and 1.8 ～ 3 mm stages follicle,the DEGs number decreased from 1430 to 136 after the correction of P value,of which one gene was up-regulated.These genes promoting primary oocyte maturation including NADPH,DHRS3 and CCL21 were screened out by q RT-PCR,and were significantly enriched in arachidonic acid metabolic pathway and cell adhesion molecular pathway.(4)Comparing the oocytes in 1.8-3 and 3 ～ 5 mm stages follicle,the DEGs number decreased from 616 to 12 after the correction of P value,of which four genes were downregulated.These genes SERPINI1 and PSMD4 were screened out,relating to the process of primary oocyte meiosis,and were significantly enriched in ubiquitin-proteasome pathway.(5)Comparing the oocytes in 3 ～ 5 and 5 ～ 7 mm stages follicle,the DEGs number decreased from 736 to 10 after the correction of P value,among them three genes were downregulated.The gene TSPO was screened out,relating to the maturation of primary oocyte,and was significantly enriched cholesterol metabolism pathway.(6)Comparing the oocytes in 5 ～ 7 and ≥ 7 mm stages follicle,the DEGs number decreased from 700 to 271 after the correction of P value,among them one gene was downregulated.These genes TSPO and ST3GAL1 were screened out,affecting the ovulation process,and screened out significantly enriched in Metabolic pathways involved in biofilm synthesis.Overall,the findings of this study showed that the diameter of primary oocytes in porcine antrum follicle increases first and then decreases.The chromatin configuration of primary oocyte gradually changed from GV0 to GV4 to complete the first meiotic.The DEGs of oocytes across all stages decreased from 3482 to 452 after the correction of P value.Furthermore,these genes NADPH、DHRS3、CCL21、SERPINI1 and PSMD4 affecting the maturation of primary oocytes were screened out.This study further explored the mechanism of maturation of porcine oocyte,and also provides a theoretical grounding of biology for future breeding improvement of reproductive traits in pig.