Research On Effects Of Gonadotropins On Oocyte Developmental Competence In Vitro And Cholesterol Metabolism In Rat Liver S9 Cultured System

Gonadotropins are commonly used in assisted reproductive technology (ART) for control ovarian stimulation (COS). So far, more than 1% of newborn babies in America are resulted from ART, which relies heavily on gonadotropins, i.e. Follicle-stimulating hormone (FSH) mainly.FSH plays a critical role in the development of antral follicles to dominant follicles containing mature oocytes and corresponding hormone environment, gonadotropins in this included Gonal-F, Follism, Bravelle, Luveris, Menopur, Repronex, Norvarel and so on.FSH is complex heterodimeric glycoproteins, which consist of two non-covalently linked, non-identical protein components designated as α and β subunits. The molecular weight of FSH is around 30 000 Da, of which sugar residues count one-third. FSH is necessary for ovarian follicular maturation and production of ovarian steroids by stimulating granulosa cells to increase expression of the cyctochrome P450 enzyme, aromatase.In oocyte development, morphologically, resumption of meiosis is marked by disappearance of oocyte nuclear membrane (germinal vesicle, GV) i.e., germinal vesicle breakdown (GVBD). FSH induces production of a meiosis activating substance from cumulus cells signaling from cumulus cells to oocytes. Various studies suggested that extremely high FSH levels in the follicular fluid may cause fertilization failure, abnormal embryo development and implantation failure. However, the dose-dependent effects of gonadotropins on oocyte maturation in vitro and in vivo remain uncertain.In vitro, it has been shown that FSH induces synthesis of a signal in the cumulus cells, which overcomes the meiosis arresting effect of hypoxanthine. Studies showed that meiosis activating sterols (MAS) induces oocyte maturation in vitro even in oocytes depleted of cumulus cells. MAS were identified as intermediates in cholesterol biosynthesis between lanosterol and cholesterol. The two best characterized members of the MAS family are FF-MAS purified from human follicular fluid and T-MAS purified from bull testicular tissue. Whether FSH actually uses MAS as a signal transduction molecule for inducing oocyte maturation or the mechanism by which MAS induces resumption of meiosis is currently unknown.To determine the effect of gonadotropins either individually or in combination for IVM in vitro on bovine oocyte maturation competence. To select the optimisation gonadotropins, which could be applied to therapeutic clinical stimulation and improve IVF success rates.To determine the effect of combined gonadotropins, which were divided into 6 groups for IVM in vitro, on bovine oocyte maturation competence, subsequent fertilisation and blastocyst development. To select the optimized combination of gonadotropins, which could be applied to therapeutic clinical and improve IVF success rates.To explore the effect of FSH on cholesterol metabolism in rat liver S9 cultured system. The research results may help to have better understanding the mechanism of gonadotropins.In total,3921 bovine cumulus-oocyte complexes (COCs) were purchased commercially (Minitube, Verona, WI, USA) and cultured in TCM-199 with 10% fetal bovine serum (FBS) together with gonadotropins(0,5,10,20,40 IU/mL) and Norvarel (0,5,10,20,40 United States Pharmacopoeia (USP)/mL) for maturation in vitro.3921 Oocytes were divided evenly into 10 groups based on the kind of gonadotropins or the combination of gonadotropins administered, i.e., the Bravelle only, the Luveris only, the Menopur only, the Repronex only, the Norvarel only, the Gonal-F only, the Follism only, the Bravelle+Menopur, the Bravelle+Repronex, and the Bravelle+Norvarel group. After 24 and 48h culture, cumulus expansion was assessed under dissecting microscopes. Oocyte maturation (GV, MⅠ, MⅡ) was determined by examining nuclear maturation and polar body formation under inverted microscopes.In total,1281 bovine COCs with three to four layers of cumulus cells were cultured in TCM-199 supplemented with 10% FBS for IVM. The culture in the absence of gonadotropins was set as the control group. In the experimental groups, bovine COCs were matured in the presence of 40 IU/mL of Follism+Repronex, Follism+Menopur, Bravelle+Repronex, Bravelle+Menopur; Gonal-F+Kepronex, and Gonal-F+Menopur respectively. After 24h maturation, oocytes were inseminated with bull spermatozoa. Cleavage rate of zygotes and embryo quality at the 2-,4- and 8-16-cell stage were determined after incubation for 24,48 and 72-96h, respectively. On Day 7/8 of culture, the number and quality of embryos developing to morula and blastocyst stage were assessed.After the preparation of rat liver S9 cultured system utilized 20 adult rats, three kinds of FSH (Gonal-F, Follism and Fostimon) were added with different concentrations (low, middle, high) and different incubation time to investigate the cholesterol concentration by LC-MSMS.Statistical comparisons between the experimental and control groups were conducted with Pearson Chi-squared or Fisher’s exact tests. Differences were considered significant at P≤0.05.In vitro MaturationAll of the gonadotropins, either individually or in combination enhanced oocyte maturation in vitro in a dose-dependently manner. Culture media supplemented with Bravelle, Luveris, Menopur and Repronex significantly improved COC expansion after culture for 24h in comparison with the control group. However, the culture medium supplemented with Norvarel menifested dose-dependent expansion of COCs from 10 to 40 IU/mL (P<0.05). For the oocytes exposed to the dosage of 20 or 40 IU/mL Bravelle (B), Luveris (L), Menopur (M), Repronex (R), Gonal-F (G), Follism (F) or 20 or 40 USP/mL, Norvarel (N), the respective maturation rates after culture for 24 and 48h were 13.1% and 45.0% for 20 IU/mL B,25.0% and 57.1% for 40 IU/mL B,333% and 37.5% for 20 IU/mL L,33.3% and 53.6% for 40 IU/mL L, 50.0% and 71.4% for 20 IU/mL M,56.3% and 76.2% for 40 IU/mL M,53.8% and 81.3% for 20 IU/mLR,55.0% and 81.8% for 40 IU/mL R,15.1% and 16.2% for 20 IU/mL G,38.9% and 39.5% for 40 IU/mL G,22.2% and 39.2% for 20 IU/mL F, 37.3% and 40.6% for 40 IU/mL F,37.5% and 44.4% for 20 USP/mL N, and 40.0% and 71.4% for 40 USP/mL N. In combination treatments, oocytes were cultured in the presence of either 10 or 40 IU/mL of drug combinations, M+B, R+B and B+N. Oocyte maturation rates after culture for 24 and 48h were 37.9% and 66.7% for M+B (10 IU/mL each),80.0% and 73.9% for M+B (40 IU/mL each),50.0% and 73.9% for R+B(10 IU/mL each),81.8% and 78.9% for R+B (40 IU/mL each),52.4% and 75.0% for B+N (10 IU/mL each), and 56.2% and 62.5% B+N (40 IU/mL each). The results suggested that high concentration of gonadotropins, either individually (such as N and F) or in combination (such as B+N), may result in maturation arrest at 48h and the optimum maturation time in vitro should be around 24h for any gonadotropins combination at the concentration of 40 IU/mL.2 IVF and Early Embryo DevelopmentThe maturaion rates after culture for 24h in different gonadotropins combinations (40 IU/mL) were 88.4%,93.4%,80.7%,80.9%,82.0%,81.7% and 14.19% for the R+F, M+F, R+B, M+B, R+G, M+G and CG groups, respectively (P<0.05 for all experimental groups vs CG). The fertilization rates (presumptive zygotes) were 67.8%,68.2%,71.5%,68.7%,72.1%,63.1% and 33.9% in the R+F, M+F, R+B, M+B, R+G, M+G and CG groups, respectively (P<0.05 for all experimental groups vs CG). The rate of 8-16-cell embryos in the R+F, M+F, R+B, M+B, R+G, M+G and CG groups was 33.3%,34.1%,40.0%,50.0%,41.5% and 31.3%, respectively (P<0.05 for all experimental groups vs CG); the rates of embryos at morula or blastocyst stages in the R+F, M+F, R+B, M+B, R+G, M+G and CG groups were 21.2%,20.5%,26.0%,30.0%,26.8% and 25.0%, respectively (P<0.05 for all experimental groups vs CG).3 The linear range of cholesterol was 62.5-1000 ng/ml. LC-MSMS method was obtained with high recovery rate, and good precision,97.5 to 102.4%, RSD< 3%.4 Follism and Gonal-F Enhanced Cholesterol Synthesis in Rat Liver S9 Cultured System. Fostimon Promoted Cholesterol Catabolism in Rat Liver S9 Cultured System.The rat liver S9 cultured system manifested enhanced cholesterol synthesis after exposed to Follism for 0-20min in a dose-depended pattern. However, the concentration of cholesterol declined with dosages going up from the 20min to the 40min. The concentration of cholesterol increased after 40 min incubation with low concentration Follism. On the contrary, the concentration of cholesterol increased when the rat liver S9 incubated with middle or high concentration Follism from the time 40 to 80min. After 80min, the cholesterol was reduced again.The concentration of cholesterol was gradually increased in the beginning of incubation with low concentration Gonal-F in rat liver S9 cultured system. It reached the peak after 40 min and then gradually decline close to the level of the control group. In the high and middle concentration groups, although the trend in the middle and high concentration groups was similar to the low concentration group, the changes were less dramatic than in the lower concentration group.The cholesterol level in the low concentration Fostimon group was reduced compared with the control group between the 0 to 100 min. It reached the lowest valley after 20 min of culture and then increased with a dose-depended manner, and finally obtained closely to the level of the control group.Gonadotropins dose-dependently enhanced COCs expansion and oocyte maturation in vitro. Alone, Menopur and Repronex were stronger in promoting oocyte maturation. The combination of Menopur+Follismall enhanced oocyte maturation effectively.Gonadotropins including FSH、LH and hCG either individually or in combination, dose-dependently enhanced oocyte nucleus maturation in vitro.The combination of gonadotropins had effects on oocyte maturation and early embryo development until 2 cell stage but the 8-16-cell and morula-blastocyst stages.LC-MSMS was a simple, rapid and highly sensitivity determination method for cholesterol test in the rat liver S 9 cultured system. r-hFSH and u-FSH had different effects on cholesterol synthesis metabolism, which may be related to facilitating oocyte maturation mechanisms.