Studies On Mechanisms Of Fish Spermatogenesis Regulated By Progestin (DHP)

In vertebrates,spermatogenesis is a highly coordinated and conservative process,in which diploid spermatogonia proliferation and differentiation into mature sperm through the three main steps:mitosis or different types of spermatogonia;meiosis of primary or secondary spermatocytes;the haploid spermatozoa differentiates into a motile sperm with flagellum stage.As we all know,progestin?DHP?binds to nuclear progesterone receptor?pgr?to control many physiological processes such as ovulation,pregnancy and childbirth in mammals.In teleost fish,various research results confirmed that progesterone including 17?,20?-dihydroxy-4-pregnen-3-one?17?,20?-DP,DHP?and 17?,20?,21-trihydroxy-4-pregnen-3-one?17?,20?,21-P?might be involved in oocyte maturation,ovulation,and sperm production,sperm motility,spermiation and the proliferation of spermatogonia.It is reported that DHP is a fish specific progestin and promotes sperm maturation and sperm emission through activating nuclear receptor.This indicates the importance of DHP in spermatogenesis.However,there is no direct evidence to demonstrate the function of DHP in the process of fish spermatogenesis.Therefore,the molecular mechanism between progestin and male fertility needs further studied.To elucidate the role of DHP in teleost fish,we used the CRISPR/Cas9technology to knock out the pgr gene and obtained the homozygous mutation XY Nile tilapia?Oreochromis niloticus?.And studied the histological of testis,sperm characteristics,levels of steroid hormones and changes of gene expression in male pgr mutant fish.The main results are as follows:?1?Histologically,various stages spermatogenic cells were observed in the control testis,while only spermatogonia were observed in the pgr homozygous knockout testis at 75 days after incubation,a critical period of spermatogenesis in control group.At the same time,hyper-proliferation of Leydig cells was also observed in the pgr deficient testis.However,histological observation indicated that spermatocyte and spermtid appeared in 4 months pgr homozygous mutuant testis,indicating that spermatogenesis has been restored partily.This suggests that DHP through the nuclear receptor pgr,plays an important role in early spermatogenesis stage,and the deletion of pgr gene leads to the delay of the process.?2?Pgr gene mutation resulted in the development of a smaller,thinner testis and a lower GSI compared with normal testis at 300 dah.Histological examination and quantitative analysis of spermatogenic cells indicated that pgr gene knockout led to irregular arrangement of spermatogenic cysts,and the significantly decreased of spermatogonia,spermatocytes,spermatid and spermatozoa.Immunohistochemistry?IHC?showed that the spermatocyte marker gene?PCNA and Ph3?was significantly lower in the pgr deficient testis than that of the control group.Real-time PCR showed that the expression of oct4?spermatogonia marker gene?,sycp3?meiotic marker gene?,prm?spermatid markergene?and some cell cycle markersgene cdk1,cyclinB1,cdc25and cyclinB2 significantly decreased.And then,increased TUNEL staining and enhanced apoptosis maker gene?bax?expressions were also observed in pgr knockout fish.This suggests that the absence of pgr affects the process of spermatogenesis and sperm maturation in the male.?3?In pgr knockout XY fish,sperm characters such as sperm count,sperm motility and straight line velocity?VSL?significantly decreased when compared with control fish.In addition,significantly increase in the mortality of offspring when crossing pgr^-/-XY fish with wild type XX fish.It demonstrates that pgr knockout affects male fertility.?4?We further studied the molecular mechanism of DHP/pgr signal in the process of spermatogenesis and sperm maturation.Real-time PCR analysis demonstrated that mutation of pgr gene resulted in a significant up-regulation of steroidogenesis-related genes of cyp17a,cyp11b2,StAR,scc,20?-HSD,and sf1,Furthermore,both Immunohistochemistry and Western blotting experiments revealed the remarkable increase of Cyp17a1,Cyp17a2 and Cyp11b2 expressions in the pgr^-/-testis.This suggests that pgr knockout would cause disordered steroidogenesis in Leydig cell of male.However,the expression of fshb in pituitary and fshr in testis decreased significantly,which indicates that the absence of pgr in the pituitary and gonad would affects the level of gonadotropin,and it further affects the process of spermatogenesis and sperm maturation.In summary,we established pgr mutant lines in XY Nile tilapia by CRISPR/Cas9.And clarify the molecular mechanism of progestin in the early initiation of spermatogenesis,sperm maturation and male feritility by binding to the receptor pgr gene.Our data suggested that DHP-activated physiology via pgr is crucial for the fertility in the XY tilapia.