Basic Study On The Molecular Basis Of Corpus Luteum Of Cynomolgus Monkey Based On Single Cell Sequencing

Corpus luteum(CL)is a temporary endocrine organ in the ovary of mammals.It is a heterogeneous gland composed of large and small luteal cells,endothelial cells,fibroblasts,smooth muscle cells and immune cells.Its main function is to secrete progesterone(P4).The regular formation and deterioration of luteal structure and function is essential for the preparation,realization,maintenance of pregnancy and maintenance of the normal reproductive cycle.The mechanism of luteinizing and its regression in primates is not clear,and is different from other mammals.The reproductive cycle of cynomolgus monkeys is similar to that of humans.The study on the formation and degradation mechanism of corpus luteum in cynomolgus monkeys is not only conducive to improving the efficiency of the assisted reproductive technology of cynomolgus monkeys,thus to improve the efficiency of the establishment of model animals with cynomolgus monkeys as the experimental subjects,but also provides a theoretical basis for the study of the mechanism of the formation and degradation of corpus luteum in humans.Experiment 1: To determine the exact time of luteum formation and the stage of its development.The ovarian reproductive status of cynomolgus monkey was monitored by B ultrasound and endoscopy to determine the relationship between the development of ovarian follicles and corpus luteum.The results showed that in the follicular stage of cynomolgus monkeys,there was only a small amount of linear medullary blood flow in the ovary,and sometimes no visible blood flow in ultrasonic examination.A small amount of blood flow was detected around the dominant follicle before ovulation.At the early stage of corpus luteum formation,abundant blood flow was detected around the corpus luteum.Blood vessels were immersed into the corpus luteum with the development of the corpus luteum,and blood flow increases.The blood flow in corpus luteum decreased with the degeneration of corpus luteum.Thus,the monitoring of blood flow in the ovary by ultrasonic examination accurately estimate the reproductive status of the ovary,distinguish the ovarian follicular stage from the ovarian luteal stage,to predict the development stage of the corpus luteum.However,B ultrasound images of luteal corpus were not clear about its physiological state.Experiment 2: Based on the methods above,the cynomolgus monkey ovaries at the 4th and 9th day of luteal phase were screened and isolated,and a preliminary digestion method was established for the preparation of luteal cell suspension conforming to the single-cell sequencing standards.The sequencing results of the obtained luteal cells were integrated,and 10 kinds of cell populations including luteal cells,endothelial cells,macrophages,natural killer cells,and B cells were obtained through t SNE visualized of 10 X genomics and Smart-seq2(SS2)sc RNA-seq analysis.By comparing the differentially expressed genes and their functions between luteal cells and other cells,it was found that Maestro(MRO),which is related to gonad development,may be used as a new marker gene for luteal cells,and the expression of MRO on small luteal cells decreases with luteal degeneration.According to the traditional theory,luteal cells were divided into two cell subgroups.It was found that the marker gene of granulosa cells,CYP19 A,was expressed only in one luteal cell subgroup.It was speculated that the CYP19 A positive luteal cells and negative luteal cells could be defined as large luteal cells and small luteal cells.In the luteal cells with positive expression of CYP19 A,MSMO1 and SCARB1 are largely and more specifically expressed,which may be used as marker genes to distinguish the large and small luteal cells.The main function of luteal cell subpopulation with high expression of CYP19 A may be related to steroid production,metabolism and secretion of luteum,and its gene expression changes little with the growth and development of luteum.The main function of luteal cell subsets with low expression of CYP19 A may be related to the occurrence of immune response and luteal regression,and its gene expression will change with the growth and development of luteal cells.Classical theory holds that the maintenance and deterioration of luteal function are related to the major hormones PGE and PGF.In this paper,we found that luteal cells and macrophages in the corpus luteum synthesized PGE,and macrophages also synthesized PGF.The induction enzyme PTGS2 for the synthesis of PG hormone precursor is mainly produced in large quantities in macrophages,suggesting that the regulation of PG hormone synthesis under inflammatory reaction of corpus luteum may be completed by macrophages.Single cell sequencing technology is an important tool to study the molecular basis of various cells in corpus luteum tissue and plays an important role in clarifying the mechanism of corpus luteum formation and regression in primates.