Study On Ovarian Development And Related Gene Expression Regulation Of Bamboo Rat

Hoary bamboo rat(Rhizomys pruinosus)is a subterranean rodent which belongs to family of Rhizomyidae,Rodentia.Due to the long term underground life,subterranean rodents have evolved a series of adaptive characteristics different from the ground rodents,the typical feature is the fertility decline.Animal reproduction has great significance for maintaining population stability and species diversity.As a subterranean rodent,hoary bamboo rat has an average litter size of only 2-4,which is an ideal species for studying the regulation of reproduction in subterranean rodents.The ovary as a reproductive organ,the study of its developmental law can provide a theoretical basis for exploring the reproductive regulation of the hoary bamboo rat.The development of hoary bamboo rat ovary in several key periods were initially explored.The ovarian development-related genes and the genetic information of transcriptome were identified,which have reference value for the breeding research of hoary bamboo rat and subterranean rodents,and lays a theoretical foundation for exploring the relationship between ovarian development and the decline in litter size in underground rodents.A total of 16 healthy individuals in 2 weeks,1 month,3 months,sexual maturity and pregnancy were purchased at the Dapan farm in Chenggu County,Hanzhong,Shaanxi.They were divided into five groups(n=3 or 4):two-week-old group,one-month-old group,three-month-old group,sexual maturity group and reproductive period group.One side of the ovary was collected for making paraffin sections and HE staining,and the other side was collected for transcriptome sequencing.Functional annotation and differentially expressed genes screening were performed on the sequencing results,and the differentially expressed genes were enriched in the KEGG database to obtain the two pathways with the most significant enrichment:Ovarian steroidogenesis and Renin-angiotensin system.q-PCR vertification of the differentially expressed genes on these two pathways were performed.The purpose of this study is to initially reveal the ovarian development characteristics of hoary bamboo rat.The main results are as follows:1.The paraffin sections were performed to study the follicular structure of the ovaries of hoary bamboo rat in different periods.It was found that only the primordial follicles were found in the ovaries of the two-week old individuals.The primary and secondary follicles appeared in the ovaries of the first and third months individuals,only a little antral follicles andmature follicles were found in the ovaries of the sexually mature and reproductive stages.There were only 2-3 corpus luteum in the ovary of pregnant hoary bamboo rat.These results indicate that the ovaries of hoary bamboo rat develop late,the ovary enters the growing follicle stage at the age of one month;the number of ovulation and litters is limited,only 2-3,hoary bamboo rat has a low fertility.2.Analysis of ovarian transcriptome data from five age stages of hoary bamboo rat,3339 DEGs were screened in 2 weeks old vs 1 month old,of which 2240 were up-regulated and 1099 were down-regulated;1820 DEGs were screened in 1 month old vs three months old,of which 906 were up-regulated and 914 were down-regulated;1470 DEGs were screened in 3 months old vs sexual maturity,of which 800 were up-regulated,and 670 down-regulated;63 DEGs were screened in sexual maturity vs pregnancy,of which 55 up-regulated,and 8 down-regulated.A large number of identified genes related to ovarian development lays a theoretical foundation for studying the development and reproduction of ovary in hoary bamboo rat.After all DEGs were aligned in the KEGG database,the ovarian steroid synthesis pathway and the renin angiotensin system(RAS)were significantly enriched.The first pathway was significantly enriched in different DEGs groups(except sexual maturity vs pregnancy),and the second pathway was only enriched in sexual maturity vs pregnancy.The results indicated that the ovarian development of hoary bamboo rat was regulated by the hypothalamic-pituitary-gonadal axis and RAS.The ovary was significantly regulated by the ovarian steroidogenesis pathway during the whole development process,while the RAS may mainly regulate the ovary during pregnancy.3.The DEGs in ovarian steroidogenesis pathway and renin angiotensin system were verified by q-PCR.Compared with 2-weeks-old,Cypl7 were significantly up-regulated in one-month-old,three-month-old and sexual maturity.There was no difference in the expression of Cyp11a,17?-hsd and 3?-hsd between two weeks old and 1 month old.Compared with two-week-old,17?-hsd and 3?-hsd were significantly up-regulated at 3 months old and sexual maturity.Five DEGs(Ace,Agt,Agt?r2,Cpa3 and Cmal)in renin angiotensin system were up-regulated in varying degrees during pregnancy compared with sexual maturity.The expression trend of all genes was consistent with that of transcriptome results.These results show that the ovarian steroidogenesis pathway plays an important role in the regulation of ovarian development,and the RAS system regulates the ovary in the late ovulation.The ovarian steroidogenesis pathway and the renin angiotensin system act together on the ovary to regulate ovarian development and function.In conclusion,we found that hoary bamboo rat develop slowly,enter the follicular growth stage at the age of one month by morphological observation,transcriptome sequencing and q-PCR verification.Hoary bamboo rat may slow down the population growth by delaying the development of the ovary and the time of childbearing to maintain the stability of the population.Hoary bamboo rat may reduce the number of litters actively by reducing ovulation,which may make it adapted to the environment with low underground capacity.In addition,a large number of genes related to ovarian development identified in this study have laid a theoretical foundation for studying the relationship between ovarian development and reproduction regulation in subterranean rodents.