The Full CDNA Cloning And Expression Regulation Of Rabbit Meiosis Activating Sterol (MAS) Synthetase CYP51 And Metabolic Enzyme Δ 14-SR,C4MO

Meiosis activating sterol (MAS), the intermediate of the postsqualene part of cholesterol biosynthesis, was proved to have the ability to trigger resumption of oocyte meiosis in vitro. Lanosterol 14a-demethylase (CYP51) converted lanosterol to follicular fluid meiosis activating sterol (FF-MAS), which accumulated in ovaries. FF-MAS was further metabolized by sterol Δ14-reductase (Δ14-SR) to testis meiosis activating sterol (T-MAS), which accumulated in testis. T-MAS was converted to cholesterol by several enzymatic steps, in which C-4 sterol methyl oxidase (C4MO) was the first enzyme. One of the reasons for accumulation of MAS in germ cells but not in somatic tissue was lacking of a coordinate transcriptional regulation of cholesterogenic genes. But the accumulation mechanism of MAS in ovaries had not been fully understood. In this paper, we wanted to explain the mechanism by studying the expression regulation of CYP51, Δ14-SR, C4MO in ovaries.We characterized the full-length cDNA of rabbit three enzymes mentioned above with RT-PCR and RACE tech. The results showed that rabbit CYP51 full-length cDNA was about 2.7kb coding a 503 amino acid protein with 57kDa molecular weight, PI 8.79. The protein had highly conserved six putative substrate recognition sites (SRSs) and an iron-binding cysterine. The length of rabbit A14-SR cDNA was 1370bp coding a 417 amino acid protein with 46kDa molecular weight, PI 8.87. The Δ14-SR protein belonged to sterol reductase super family. The rabbit C4MO is 1735bp long coding a 292 amino acid protein with 35kDa molecular weight, PI 6.79. It was characterized by the presence of three histidinerich motifs, such as HX₃H, HX₂HH and HX₂HH.We detected the expression modes of MAS synthetase CYP51, MAS metabolic enzymeΔ14-SR and C4M0, and sterol transcription regulator SREBP in rabbit developing gonads, growing follicles and gonadotropin treated ovaries by Northern Blot. The results showed that: in rabbit testis, expression of CYP51 mRNA increased, while C4MO mRNA decreased with the gonad development, which further proved the hypothesis that it was the inconsistence of transcriptional regulation between upstream and downstream MAS synthases to cause T-MAS accumulation in testis. In gonadotropins (FSH/LH) treated rabbit ovary, expression of CYP51 mRNA in antral follicle increased along with follicle diameter growth compared with untreated ovary. In follicles, expression of enzyme Δ14-SR mRNA decreased along with follicle growth. The decrease continuously even after gonadotropins treatment. Therefore, we deduced that after LH surge in vivo, CYP51 mRNA expression increased and Δ14-SR mRNA expression decreased in preovulatory follicle, which could be the reason of FF-MAS accumulation in preovulatory follicles. With or without gonadotropins treatment, SREBP1 expression level changed slightly in all growing follicles, which suggested that the regulation effect of gonadotropins on ovary CYP51, Δ14-SR and C4MO mRNA expression did not through SREBP1 signal pathway.To study whether testis leydig cells took part in MAS accumulation in testis, we used EDS to establish rabbit leydig cell destructive model. Results showed that after 20mg/kg EDS treatment, the serum testosterone level first decreased and then increased; 3 β -HSD activity was inhibited and the quantity decreased to the lowest level after 12h treatment and then gradually increased. By usingelectron microscope and DNA ladder, we found that rabbit leydig cells did not show any significant apoptosis, which illuminated that the EDS toxicity on rabbit leydig cells at the used dose (20mg/kg) was too slender to be used in producing rabbit leydig cell destructive model.To further study the mechanism of MAS accumulation in ovaries on protein level, we managed to prepare the CYP51 polyclonal antibodies with gene engineering tech. Firstly, we reconstructured the expression vector CYP51-pGEX4T-3, and got the fusing protein GST-CYP51 after inducing. After immunizing the KM mouse with purified induced GST-CYP51 protein we obtained the anti serum. With...