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Effect of ovarian angiogenic factor on estrogen production by granulosa cells
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ANDROGEN AND 19-NORSTEROID PROFILES IN HUMAN PREOWLATORY FOLLICLES FROM STIMULATEDCYCLES : ANALYSIS BY ISOTOPE DILUTION-MASS SPECTROMETRY. Dehennin, L., Jondet M., Scholler R,, Fondation da Recherche en Hormonologie, 94260 Fresnes, FRANCE. Fofficular fluid CFF) was aspirated from preovulatory follicles of women under ovarian stimulation for in vitro fertilization and analyzed by a reference technique based on gas chromatography-massspectrometry associated with stable isotope dilution. l9-Nortestosterone (NT) and 19-norandrostenedione(NA) were identified and quantified for the first time in human FF. There was a strong positive correlation between NT and estradiol (E ) and between NA and estrone concentrations, thus Indicating a common cellular origin and a &ssible regulatory action of 19-norsteroids on ovarian aromatase, rather than being active intermediates of the multistep enzymatic conversion of androgen to estrogen. Testosterone concentrations were significantly lower than those obtained by radioimmunoassay ; cross-reactionwith substantially higher levels of NT seems to be at the origin of this discrepancy. Androstenedfone (A) concentrations were similar to those reported in the literature, therefore was it confirmed that a concentration ratio E2/A > 20 is favorable for oocyte cleavage. Other newly estimated androgens are : testosterone sulphate, androstenediol mono- and disulphate, DHT sulphate, epitestosterone. 19-hydroxy-androstenedione,5aandrostanedione, 5oandrostanediols and androsterone, DHEA sulphate was by far the most abundant androgen in this type of follicles in which sulphokinase enzyme activity catalyzes the conversion of testosterone and DHT to their sulpho-conjugates.Thus the scheme of metabolic pathways for follicular steroid biosynthesis may be extended somewhat more. -_ -164 EFFECT OF OVARIAN ANCIOGENIC FACTOR ON ESTROGEN PRODUCTION BY GRANULOSA CELLS; MAKRIS, A.; R.; Ryan, K.J. Obstetrics and Gynecology, Harvard Medical Sohool; 45 Shattuck Street;/ Boston, Massachusetts 02115; U.S.A. The effect of highly purified porcine ovarian angiogenic factor (OAF) on granulosa cell (CC) steroidogenesis was investigated. Estrogen (E2) synthesis was measured in cultures of CC derived from DES-treated hypophysectomized rat ovaries, and from pig and human ovaries. Choriocarcinoma cells, which have high endogenous levels of aromatase activity, were used for. cornperativepurposes. GC were plated in serum-free tissue culture medium (supplemented with insulin, transferrin, fibroneotin and gluoocorticoid), and _Ez production was assessed every 2 from days over a b-day period. FSH (50 nglml) and CAMP (lo-' M) stimulated E2 production exogenous substrate (A&-androstenedfonelin rat GC. Ez was not detected in the control cells. OAF, in doses ranging from 0.1 to 100 u/ml inhibited Ez production from 50 to 801. There was no significant effect by OAF on FSH or CAMP-stimulated progesterone synthesis. CC from large porcine i>5 sxn) follicles or from humans undergoing IVF procedure had significant levels of aromataseactivity. LH or FSH had s slight stimulating effect on these cells and OAF t gonadotropin either had no effect or enhanced E2 production. Aromatase activity in choriocarcinoma cells was not affected by OAF. It is concluded that OAF may have a modulatingeffect on the inductionof aromatasei in immature cells that is inde endentof the FSH-adenyl cyclase mechanism. activi+&s research was supported by NIR Grant HD07923.
BIOSYNTHESIS OF 4- ESTREN-3,17-DIONE (19-NO~ROST~EDIONE) BY THE OVARY OF P~PUBERT~ GILTS TREATED WITH PMSG AND HCG. Khalil, W; Clausen, D; and Walton, J., MRC Group in Reproductive Biology, Department of Obstetrics and Gynecology, University of Western Ontario, London, Ontario N6A 5A5, CANADA Department of Animal and Poultry Science, University of Guelph, Guelph, Ontario CANADA. 19-Norandrostenedione (19-nor A) has recent& been identified in pig ovarian follicular fluid (Khalil and Walton, J. Endocrinology, 1985). We Investigated its formation from 13H)pregnenolone, using either a crude homogenate, 3000 g and 10,000 g supernatents from ovaries o: prepubertal gilts treated thus; 1: no gonadotropin; 2: PMSG (1000 I.U.) for 72h; 3: PMSG (1000 I.U.) for 96h, HCG, (500 I.U.) given 72h after PMSG. Incubations were carried out for 60 min. in 3.Oml of Tris-HCL buffer (pH 7.41, and products were analyzed by HPLC (CIBuBondapak 300 x 3.9 mm column, 70% MeOH/H20), and TLC (CHzClz/Et20:5:2 V/V). Progesterone (P) (62X), androstenedione (A) and 19-nor A (10%) and 19-hydroxyandrostenedione(19-OH A, 4.5%) were the major products produced by ovarian homogenates suggesting high endogenous 30-HSD and A4-isomerase activity prior to gonadotropin treatment. Homogenate, 3000 g and 10,000 g supernatents from treatments 2 and to a lesser extent 3 converted [3H)-pregnenolonemainly to androgens, tentatively identified as 19-OH A (34%, HPLC relative retention time (RRT) 0.73, A-l) (acetate RRT-0.88), A/19-nor A (36X, RRT 1.15) P, (6% RRT 1.76) and minor metabolites with RRT 1.5 and 2.2. Four recrystalllzationsof the 19-nor A "peak" from HPLC purified further by TLC gave specific activities of 245, 279, 215 and 117 CPM mg- These data suggest that 19-nor A may be formed from pregnenolone and this conversion may be enchanced by prior exposure of the ovary to PMSG. Supported by MRC of Canada. SE 25
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ANDROGEN AND 19-NORSTEROID PROFILES IN HUMAN PREOWLATORY FOLLICLES FROM STIMULATEDCYCLES : ANALYSIS BY ISOTOPE DILUTION-MASS SPECTROMETRY. Dehennin, L., Jondet M., Scholler R,, Fondation da Recherche en Hormonologie, 94260 Fresnes, FRANCE. Fofficular fluid CFF) was aspirated from preovulatory follicles of women under ovarian stimulation for in vitro fertilization and analyzed by a reference technique based on gas chromatography-massspectrometry associated with stable isotope dilution. l9-Nortestosterone (NT) and 19-norandrostenedione(NA) were identified and quantified for the first time in human FF. There was a strong positive correlation between NT and estradiol (E ) and between NA and estrone concentrations, thus Indicating a common cellular origin and a &ssible regulatory action of 19-norsteroids on ovarian aromatase, rather than being active intermediates of the multistep enzymatic conversion of androgen to estrogen. Testosterone concentrations were significantly lower than those obtained by radioimmunoassay ; cross-reactionwith substantially higher levels of NT seems to be at the origin of this discrepancy. Androstenedfone (A) concentrations were similar to those reported in the literature, therefore was it confirmed that a concentration ratio E2/A > 20 is favorable for oocyte cleavage. Other newly estimated androgens are : testosterone sulphate, androstenediol mono- and disulphate, DHT sulphate, epitestosterone. 19-hydroxy-androstenedione,5aandrostanedione, 5oandrostanediols and androsterone, DHEA sulphate was by far the most abundant androgen in this type of follicles in which sulphokinase enzyme activity catalyzes the conversion of testosterone and DHT to their sulpho-conjugates.Thus the scheme of metabolic pathways for follicular steroid biosynthesis may be extended somewhat more. -_ -164 EFFECT OF OVARIAN ANCIOGENIC FACTOR ON ESTROGEN PRODUCTION BY GRANULOSA CELLS; MAKRIS, A.; R.; Ryan, K.J. Obstetrics and Gynecology, Harvard Medical Sohool; 45 Shattuck Street;/ Boston, Massachusetts 02115; U.S.A. The effect of highly purified porcine ovarian angiogenic factor (OAF) on granulosa cell (CC) steroidogenesis was investigated. Estrogen (E2) synthesis was measured in cultures of CC derived from DES-treated hypophysectomized rat ovaries, and from pig and human ovaries. Choriocarcinoma cells, which have high endogenous levels of aromatase activity, were used for. cornperativepurposes. GC were plated in serum-free tissue culture medium (supplemented with insulin, transferrin, fibroneotin and gluoocorticoid), and _Ez production was assessed every 2 from days over a b-day period. FSH (50 nglml) and CAMP (lo-' M) stimulated E2 production exogenous substrate (A&-androstenedfonelin rat GC. Ez was not detected in the control cells. OAF, in doses ranging from 0.1 to 100 u/ml inhibited Ez production from 50 to 801. There was no significant effect by OAF on FSH or CAMP-stimulated progesterone synthesis. CC from large porcine i>5 sxn) follicles or from humans undergoing IVF procedure had significant levels of aromataseactivity. LH or FSH had s slight stimulating effect on these cells and OAF t gonadotropin either had no effect or enhanced E2 production. Aromatase activity in choriocarcinoma cells was not affected by OAF. It is concluded that OAF may have a modulatingeffect on the inductionof aromatasei in immature cells that is inde endentof the FSH-adenyl cyclase mechanism. activi+&s research was supported by NIR Grant HD07923.
BIOSYNTHESIS OF 4- ESTREN-3,17-DIONE (19-NO~ROST~EDIONE) BY THE OVARY OF P~PUBERT~ GILTS TREATED WITH PMSG AND HCG. Khalil, W; Clausen, D; and Walton, J., MRC Group in Reproductive Biology, Department of Obstetrics and Gynecology, University of Western Ontario, London, Ontario N6A 5A5, CANADA Department of Animal and Poultry Science, University of Guelph, Guelph, Ontario CANADA. 19-Norandrostenedione (19-nor A) has recent& been identified in pig ovarian follicular fluid (Khalil and Walton, J. Endocrinology, 1985). We Investigated its formation from 13H)pregnenolone, using either a crude homogenate, 3000 g and 10,000 g supernatents from ovaries o: prepubertal gilts treated thus; 1: no gonadotropin; 2: PMSG (1000 I.U.) for 72h; 3: PMSG (1000 I.U.) for 96h, HCG, (500 I.U.) given 72h after PMSG. Incubations were carried out for 60 min. in 3.Oml of Tris-HCL buffer (pH 7.41, and products were analyzed by HPLC (CIBuBondapak 300 x 3.9 mm column, 70% MeOH/H20), and TLC (CHzClz/Et20:5:2 V/V). Progesterone (P) (62X), androstenedione (A) and 19-nor A (10%) and 19-hydroxyandrostenedione(19-OH A, 4.5%) were the major products produced by ovarian homogenates suggesting high endogenous 30-HSD and A4-isomerase activity prior to gonadotropin treatment. Homogenate, 3000 g and 10,000 g supernatents from treatments 2 and to a lesser extent 3 converted [3H)-pregnenolonemainly to androgens, tentatively identified as 19-OH A (34%, HPLC relative retention time (RRT) 0.73, A-l) (acetate RRT-0.88), A/19-nor A (36X, RRT 1.15) P, (6% RRT 1.76) and minor metabolites with RRT 1.5 and 2.2. Four recrystalllzationsof the 19-nor A "peak" from HPLC purified further by TLC gave specific activities of 245, 279, 215 and 117 CPM mg- These data suggest that 19-nor A may be formed from pregnenolone and this conversion may be enchanced by prior exposure of the ovary to PMSG. Supported by MRC of Canada. SE 25
SuppI.--E
59s