Studies On Mechanisms Of Progestin In Oogenesis And Ovulation In Tilapia

In teleost fish,the process of oogenesis was divided into primordial germ cell formation stage,oocyte formation stage,oocyte growth stage,maturity period and ovulation period.As we all known,17α,20β-dihydroxy-4-pregnen-3-one（DHP）banded with nuclear progesterone receptor（pgr）to play important role in many physiological processes.Previous studies have shown that DHP synthase gene and pgr began to express at 5 days after hatching（dah）,suggesting that DHP-PGR signaling pathway may play an important role in ovarian development,but it remained to be further exploration.Therefore,in order to further clarify the role of DHP-PGR signaling pathway in the early gonadal development and ovulation of female fish,we successfully established pgr homozygous mutation line,and further explored the effects of pgr mutation on ovarian development of early stage,hormone synthesis in the reproductive cycle and ovulation by the methods of histology,cytology and molecular biology.Eventually,it will enrich our knowledge of the relationship between progesterone and ovarian development,female fertility in Nile tilapia and even bony fish.The main results of this paper are as follows:1.The cellular localization of pgr gene in ovary of young and adult fishIn situ hybridization data showed that pgr was located in germ cells of the ovary at60 days after hatching.In the adult,pgr is detected in the oocytes of pre-vitellogenic,as well as in the follicular granulosa cells.2.The effects of pgr gene mutation in the early ovary developmentHistological examination was carried out on the ovaries of 30 dah,60 dah and 90dah,and the results showed that at 30 dah,fewer germ cells were found in the ovaries of pgr homozygous knockout compared with the control,and masculinization was not found.At 60 dah,most of the oocytes were in the oogonia stage and stage I oocytes,while there were a large number of stage II oocytes in the control group.The results of longitudinal section of gonad showed that the both sides ovary of pgr^-/-XX was filled with a large number of somatic cells and highly lacked of germ cells.The Real-time PCR also illustrated the expression of vasa and 42sp50 significantly decreased compared with pgr^+/+XX.The expressions of steroid synthesis relative genes（St AR1,St AR2 and scc）in the gonads of pgr^-/-XX were significantly up-regulated.Correspondingly,the content of testosterone（T）in serum was significantly higher than that in the control group.But the expression level of estrogen synthesis-related genes（cyp19a1a,foxl2）have no significant difference.At 90 dah,from the results of tissue section and Real-time,the development of germ cells has returned to normal.Among the genes related to steroid synthesis,except for St AR1,the expression level of other genes（St AR2,scc,cyp17a1）were also not significantly different from the control.Therefore,homozygous mutations in pgr could cause steroid synthesis and germ cell developmental disorders in young female fish.3.The effects of pgr gene mutation on the maturation and ovulation of folliclesAt 360 dah,the pgr^+/+XX had been able to spawn,while the pgr^-/-XX did not.By observing the ovarys of pgr^+/+and pgr^-/-XX,a large number of abnormal white oocytes were found in pgr^-/-ovary,meanwhile,the gonadosomatic index（GSI）was significantly higher than that of the control female.At 540 dah,due to pgr^-/-XX could not spawn,so,the oocytes were accumulated and necrosed,and the green liquid filled the entire ovary of pgr^-/-XX.When the oocytes were treated with human chorionic gonadotropin（h CG）in vitro for 0 h,obvious germinal vesicles could be seen in both the control group and knockout group after transparent treatment.After 24 hours of treatment,the nucleus disappeared in both,indicating that the oocytes had matured.However,it was worth noting that the oocyte ovulation process of the control group had also been completed,while the oocytes of the knockout group showed ovulation failure.The above results suggested that pgr knockout would not block oocytes maturation,but would block oocytes ovulation.4.The mechanism of pgr gene mutation affecting reproductive cycle and ovulationAs we all known,the average spawning cycle of Nile tilapia is 14 days,and DHP played a vital role in maturation and ovulation of oocytes.Therefore,Serum of pgr^+/+XX and pgr^-/-XX was collected on the 3rd,8th,12th and 14th day to detected DHP and 17β-estradiol（E2）level in a spawning cycle.The results showed that the DHP content of pgr^-/-XX was significantly higher than pgr^+/+XX.It was worth mentioning that between 8 and14 days after spawning,the serum DHP content of pgr^+/+XX increased sharply with a peak,but there was no such peak in the knockout group,showing the lack of DHP fluctuation.In addition,E2 results showed that,like DHP,the pgr gene knockout also resulted in the failure of the E2 fluctuation.Because the production of DHP was tightly regulated by the gonadotropin-releasing hormone,the expression of luteinizing hormone（Lh）in pituitary was detected by fluorescent immunohistochemical and Real-time PCR,the results showed that the expression level was significantly increased in pgr^-/-XX.It may be one of the reasons for the high level of DHP in the serum of pgr^-/-XX.In addition,the data of TUNEL staining showed that obvious positive signals were found in the follicular cells of pgr^+/+XX,while no signals were detected in pgr^-/-XX,meanwhile,the metalloproteinases genes（mmp2 and mmp9）were significantly lower than control females,suggesting that the follicular layer could not be rupture ang apoptosis after pgr knockout.The above results indicated that knockout of the pgr gene led to hormone synthesis disorder and the lack of a signal for fluctuation,which further affected the rupture and apoptosis of follicular cells,and ultimately resulted in failure of ovulation.In summary,in this study,it was found that pgr mutation led to disorder of steroid synthesis,resulted in a delaying of oogenesis in early ovarian development.In adult,the knockout of pgr led to the deficiency of the normal fluctuation signal of DHP and E2,which further affected the rupture and apoptosis of follicular cells,and ultimately resulted in ovulation failure.This study first explored the important role of DHP-PGR signaling pathway in the early stages of fish ovarian development through a knockout model.At the same time,by exploring the synthesis of steroid hormones in the reproductive cycle,the possible mechanism of the DHP-PGR signaling pathway affecting the ovulation process was revealed.