| Rabbit,as an important food-saving herbivore,has gradually become a pillar industry of increasing farmers’ income and getting rich in poor areas in China due to its advantages of low investment,quick effect and high income.Rabbit fecundity is a key factor affecting the production efficiency and economic benefits of rabbits,and the level of fecundity depends on the development and maturation of ovarian follicles and ovulation process.In this study,60-day-old Tianfu Black Rabbits were used to induce follicular development,maturation and ovulation by combined injection of PMSG and h CG in vivo.At five time points,namely,24 and 72 hours before PMSG treatment(C),24 and 72 hours after PMSG treatment(P24 and 722)and 12 and 48 hours after h CG treatment(H12 and H48).Six rabbits were randomly selected and slaughtered after weighing,and plasma and bilateral ovaries were collected for hormone determination.HE staining and transcriptome sequencing were used to reveal the changes of expression profiles of RNA and micro RNAs during follicular maturation and ovulation in rabbits,and to construct a core regulatory network of micro RNAs.The main test results are as follows:(1)The results of reproductive organ index showed that PMSG or h CG injection had no significant effect on rabbit body weight and oviduct weight;ovarian weight and organ index increased about 3-5 times after 72 hours of PMSG treatment(p<0.05),and reached the peak after 12 hours of h CG treatment(p<0.05);uterine weight and organ index also showed a gradual upward trend,and reached the maximum after 48 hours of h CG treatment(p<0.05).)These results indicate that PMSG and h CG stimulation can significantly increase the reproductive organ index of rabbits.(2)HE staining showed that the ovaries of rabbits in the control group were mainly composed of primary follicles and secondary follicles.After 24 hours of PMSG treatment,the number of tertiary follicles increased significantly.When PMSG treatment increased to 72 hours,mature follicles appeared on the ovaries.The formation of ovulation and corpus luteum was further promoted by h CG treatment,and the number of corpus luteum reached its maximum after 48 hours of h CG treatment.These results indicated that the combination of PMSG and h CG successfully induced follicular maturation and ovulation in rabbits.(3)The results of serum reproductive hormone determination showed that: Under the stimulation of PMSG or h CG,the melatonin level continued to increase compared with the control group,and reached the highest level at 12 h of h CG treatment(P <0.05).The levels of prostaglandin and progesterone continued to increase compared with the control group,and reached the highest level at 48 h after h CG treatment(P <0.05).Estradiol level decreased gradually compared with the control group,but increased sharply at 12 h of h CG treatment,reaching the maximum at 48 h of h CG treatment(P < 0.05).These results indicate that the dynamic changes of serum levels of melatonin,prostaglandin,progesterone and estradiol are closely related to follicular maturation and ovulation.Melatonin and prostaglandin are more closely related to follicular maturation,while estradiol and progesterone are directly related to ovulation.(4)The results of RNA-seq sequencing showed that after the quality control and filtering of the original data,an average of 47.37 million clean reads were obtained per library,of which 75.24%-78.30% could be compared with the reference genome of rabbits.A total of 20,719 expressed genes were detected,of which 18,597,18,424 and 18,515 genes were detected in C,P72 and H48 groups respectively.1,122 differentially expressed genes were screened out from the three groups.Further analysis of time series expression patterns revealed that 250 and 103 genes were significantly up-regulated and down-regulated with treatment time.KEGG enrichment analysis showed that differentially expressed genes were mainly concentrated in PI3K-Akt,macula adhesion,ovarian steroid production and other signaling pathways.In addition,compared with C,279 and 103 genes were up-regulated and down-regulated in P72;compared with P72,11 and 5 genes were up-regulated and down-regulated in H48,and the expression levels of PPP1R3 C,MMP13 and SLC2A12 were significantly different between any two groups;KEGG analysis showed that these genes were significantly enriched in PI3K-Akt and protein elimination.Chemical and absorption pathways.In addition,the expression patterns of nine genes verified by q RT-PCR were consistent with the sequencing results.(5)The results of micro RNA-seq sequencing showed that after the quality control and filtering of the original data,the average number of clean reads per library was 13.75 million,and about 89.54%-91.11% could be compared with the reference genome of rabbits.591,592 and 595 known micro RNAs and 325,326 and 334 new micro RNAs were detected in the three groups.Twelve differentially expressed micro RNAs were screened.KEGG enrichment analysis revealed that the predicted target genes were mainly concentrated in PI3K-Akt signal and HIF-1 signal pathways.There were 56 differentially expressed micro RNAs between C and P72,36up-regulated micro NAs and 20 down-regulated micro NAs,respectively.P72 and H48 had 49 differentially expressed micro NAs,with 33 up-regulated micro NAs and 16down-regulated micro NAs,respectively.The expression levels of micro NA205-1 and micro NA34 c were significantly different between any two groups.The expression patterns of six micro RNAs verified by q RT-PCR were consistent with the sequencing results.(6)Joint analysis showed that single micro RNAs could regulate the development and maturation of rabbit follicles and ovulation process by regulating the expression of multiple target genes.At the same time,multiple micro RNAs could also regulate the expression of the same target gene.The core regulatory networks of micro RNAs were constructed for the top 10 micro RNAs which were most significantly up-regulated or down-regulated after PMSG or h CG treatment.For example,for ovulation,the expression of micro RNA-107-1 was down-regulated under the stimulation of h CG,while its target genes MMP13 and PPP1R3 C were up-regulated.It is suggested that micro RNA-107-1 can up-regulate the expression of PPP1R3 C,then activate the c AMP-PKA signaling pathway and participate in the regulation of follicular development. |
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