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The effects of low-density lifoprotein on steroidogenesis by human granulosa and theca cells
~tilh?IK ANALYSIS OF mOIDS OF m ORIANDPLASMAOF m AND FULLTERM INFANTSBY GC ANDGC-MS: AN INDEX OF ADRENAL MORPHOGENSIS;Pike, A; Hambidge,S;Gotlin, R; Fennessey, P. Departament of Pediatrics, University of Colorado Health Sciences Center; Denver, CO 80262; USA
Clinical signs suggestive of adrenal glucocorticoid and mineralocorticoid insufficiency are However, determination of adrenal function employing la commonfinding in the premature infant. ia variety of biochemical methods have reported normal to high blood and urine concentrations of these steroids. Our preliminary studies were designed to maesure urinary steroids of adrenal #origin and use them as markers of adrenal morphogensis. This was accomplished by collecting 24 hour urine specimens at weekly intervals from premature and term infants. Aliquots were
enzymaticaly hydrolysed and derivatized to allow analysis by gas chromatography - mass spectrometry (GC-MS). Similarly, blood plasma collected during the same time period was analyzed for cortisol using GC-MS(operated in the selected ion mode).
The results from the urinary steroid profiles suggest that the adrenal gland of the premature infant does not undergo the postnatal maturation that has been reported in the term baby. Rather, the gland of the premature infant maintains its architecture and appears to undergo maturation as a function of time and in a manner similar to term infant. Plasma cortisol data suggests that although these infants do not suffer from cortisol insufficiency, they do axhibit large fluctuations in plasma cortisol concentrations. These fluctuations may indicate why some premature infants exhibit a delayed recovery during periods of acute stress.
16
EFmmlffJGTERM mWITTiA==AWIIXX;UE
cNSITN0ID pIIDBxlcTIcNoP!L?ISHuMAN TESTIS TISSUE; Huhtzniemi, I.‘; Nikula, ?I.‘) Rar&kko, S.‘; Dept. of Physiolcgyl , University of Turku, SF-20520 Turku; Md 2nd Dept. of Surgery, Univ. of Helsinki, SF-00290 HelsMci; FIINLAND. Eight patients with advanced prostate cancerwaretreatadwithap0tentGnPuiagonistanalthe paague Oouserelin, Bu, Boechst) , 600 ug x 3/day intranasal ly . After 6 mo of treatment, tients were orciectanized, and steroi&qenesis of the m testis tissue was investigati ti vitro. Eight other prostate c~cer patients with nlatching ages and extent of disease were casr trated as the first therapy, and their testis tissue gervea as controls (Cl. Bu-trea~t had no significant effeot on testis tights: C, 13.3 + 1.7 g; Eu, 8.5 + 1.7 cI The intratesticular + 0.2 nrrol/g wet
lwel
of testosterone
decreaeed
by 95%. during the a&n.ist
trsatment:-C,
1.5 )/
tissue;Bu, 0.070 2 0.019 ml/g. % examine the site of blockade in steroidogenesis, slices of testis tissue f?X4nboth groups m inoubated in medium 199 in the presence of Wily stimulating concentration of hCG (100 ng/til , and seven steroids accmulated in the medium during a 3-h incubaticm were analyz& by RI&o (after chrmmatography on Lipidex 5000) The steroid ccncentraticm in the saqles were as follows (n110l/teetis) : prqnenob, C = 6.3 + 1.2, ml = 3.5 + 1.0; progesterone, C = 0.9 + 0.36, Bu = 0.34 + 0.13; 17-hy&oxyp~sterone, ? = 6.2 + 1.1, Bii = 0.30 + 0.024, CHEA,C = 377 + 0.42, Bu = 2.8 + 2.0; an&ostenedione, C = 1.3 + 0.77, Bu = 0.12 _t 07085; testosterone, C e-19 + 4.5, Bu = o‘r54 + 0.15; 5a-dtiydrotestoster&e, C = 0.90 2 0.18, BU = 0.12 + 0.033. Bu-trea-%nt decreased &a levels of all other steroids significantly (p < 0 .Ol 1 except the tw 38-hydrmxy-5-ene steroids prqnenolone and lXIE% and progesn. It is concluded that the long tezm treatment with GnINiagonists dramatically reduces the andrcqn product&~ of hm testis tissue, and the blockade is imst clear2 Jz~ZZredueec?actFvltvof~ 77 E ‘&‘NCTS OF LOW-DENSITY LIPOPROTEINON STENOIDOGENESIS BY HUMAN GRANULOSA AHD THECACELLSJ Stein, A. L. ; Tonetta, S.A.; and diEerega, C.S.; Livingston Reproductive Laboratory, of Obstetrics/Gynecology, USC Medical Center, 1321 N. Mission Rd., Los Angeles, CA.,
Department
90033, USA.
In steroidogenic cells, cholesterol from low-density lipoproteins (LDL) and/or high-density +DL is metabolized by human lipoproteins is ueed as a subetrate for steroid production. granuloea cells (CC) in vitro. Therefore, radiolabelled LDL ( Ii-LDL) was added to primary cultures of human CC and theta cells (TC) to examine steroidogenesis. Ovariea were obtained at the time of surgery from otherwise healthy, pre-menopausal women undergoing TC and CC were harvested from oopherectomy for benign gynecologic diseases. follicles, dispersed and plated a8 monolayer culture8 in serum-free media supplemented with stimulating hormone growth factors. Human chorionic gonadotropin (HCG, 5 IV/ml), human ollicle (hFSH, 100 mIU/ml) or media were added to cultures. At 24 hours, 3fH-LDL and LDL were added to each culture. Twenty-four hours later, media were collected and the cella were resuspended in Progesterone (P), 17-hydroxyprogesterone (170H-P) , media then disrupted by eonication. eetradiol (E), androstenedione (A), and testosterone(T) in cells and media were extracted and identified by thin layer chromatogrgphy. GC produced all steroids from H-LDL, but particularly P, 170H-P nd E. The hFSH increased 9H-LDL to manufacture all E production while HCC etimulated production of P. TC also utilized steroids but the most prominent steroids detected were P, 170H-P, T and A. HCGstimulated A and T production whereas hPSH had little or no effect. In summary, human granuloea and theoa cells utilize cholesterol from LDL for steroid production. In addition, gonadotropine can enhance this use of cholesterol by ovarian cells.
28s
Clinical signs suggestive of adrenal glucocorticoid and mineralocorticoid insufficiency are However, determination of adrenal function employing la commonfinding in the premature infant. ia variety of biochemical methods have reported normal to high blood and urine concentrations of these steroids. Our preliminary studies were designed to maesure urinary steroids of adrenal #origin and use them as markers of adrenal morphogensis. This was accomplished by collecting 24 hour urine specimens at weekly intervals from premature and term infants. Aliquots were
enzymaticaly hydrolysed and derivatized to allow analysis by gas chromatography - mass spectrometry (GC-MS). Similarly, blood plasma collected during the same time period was analyzed for cortisol using GC-MS(operated in the selected ion mode).
The results from the urinary steroid profiles suggest that the adrenal gland of the premature infant does not undergo the postnatal maturation that has been reported in the term baby. Rather, the gland of the premature infant maintains its architecture and appears to undergo maturation as a function of time and in a manner similar to term infant. Plasma cortisol data suggests that although these infants do not suffer from cortisol insufficiency, they do axhibit large fluctuations in plasma cortisol concentrations. These fluctuations may indicate why some premature infants exhibit a delayed recovery during periods of acute stress.
16
EFmmlffJGTERM mWITTiA==AWIIXX;UE
cNSITN0ID pIIDBxlcTIcNoP!L?ISHuMAN TESTIS TISSUE; Huhtzniemi, I.‘; Nikula, ?I.‘) Rar&kko, S.‘; Dept. of Physiolcgyl , University of Turku, SF-20520 Turku; Md 2nd Dept. of Surgery, Univ. of Helsinki, SF-00290 HelsMci; FIINLAND. Eight patients with advanced prostate cancerwaretreatadwithap0tentGnPuiagonistanalthe paague Oouserelin, Bu, Boechst) , 600 ug x 3/day intranasal ly . After 6 mo of treatment, tients were orciectanized, and steroi&qenesis of the m testis tissue was investigati ti vitro. Eight other prostate c~cer patients with nlatching ages and extent of disease were casr trated as the first therapy, and their testis tissue gervea as controls (Cl. Bu-trea~t had no significant effeot on testis tights: C, 13.3 + 1.7 g; Eu, 8.5 + 1.7 cI The intratesticular + 0.2 nrrol/g wet
lwel
of testosterone
decreaeed
by 95%. during the a&n.ist
trsatment:-C,
1.5 )/
tissue;Bu, 0.070 2 0.019 ml/g. % examine the site of blockade in steroidogenesis, slices of testis tissue f?X4nboth groups m inoubated in medium 199 in the presence of Wily stimulating concentration of hCG (100 ng/til , and seven steroids accmulated in the medium during a 3-h incubaticm were analyz& by RI&o (after chrmmatography on Lipidex 5000) The steroid ccncentraticm in the saqles were as follows (n110l/teetis) : prqnenob, C = 6.3 + 1.2, ml = 3.5 + 1.0; progesterone, C = 0.9 + 0.36, Bu = 0.34 + 0.13; 17-hy&oxyp~sterone, ? = 6.2 + 1.1, Bii = 0.30 + 0.024, CHEA,C = 377 + 0.42, Bu = 2.8 + 2.0; an&ostenedione, C = 1.3 + 0.77, Bu = 0.12 _t 07085; testosterone, C e-19 + 4.5, Bu = o‘r54 + 0.15; 5a-dtiydrotestoster&e, C = 0.90 2 0.18, BU = 0.12 + 0.033. Bu-trea-%nt decreased &a levels of all other steroids significantly (p < 0 .Ol 1 except the tw 38-hydrmxy-5-ene steroids prqnenolone and lXIE% and progesn. It is concluded that the long tezm treatment with GnINiagonists dramatically reduces the andrcqn product&~ of hm testis tissue, and the blockade is imst clear2 Jz~ZZredueec?actFvltvof~ 77 E ‘&‘NCTS OF LOW-DENSITY LIPOPROTEINON STENOIDOGENESIS BY HUMAN GRANULOSA AHD THECACELLSJ Stein, A. L. ; Tonetta, S.A.; and diEerega, C.S.; Livingston Reproductive Laboratory, of Obstetrics/Gynecology, USC Medical Center, 1321 N. Mission Rd., Los Angeles, CA.,
Department
90033, USA.
In steroidogenic cells, cholesterol from low-density lipoproteins (LDL) and/or high-density +DL is metabolized by human lipoproteins is ueed as a subetrate for steroid production. granuloea cells (CC) in vitro. Therefore, radiolabelled LDL ( Ii-LDL) was added to primary cultures of human CC and theta cells (TC) to examine steroidogenesis. Ovariea were obtained at the time of surgery from otherwise healthy, pre-menopausal women undergoing TC and CC were harvested from oopherectomy for benign gynecologic diseases. follicles, dispersed and plated a8 monolayer culture8 in serum-free media supplemented with stimulating hormone growth factors. Human chorionic gonadotropin (HCG, 5 IV/ml), human ollicle (hFSH, 100 mIU/ml) or media were added to cultures. At 24 hours, 3fH-LDL and LDL were added to each culture. Twenty-four hours later, media were collected and the cella were resuspended in Progesterone (P), 17-hydroxyprogesterone (170H-P) , media then disrupted by eonication. eetradiol (E), androstenedione (A), and testosterone(T) in cells and media were extracted and identified by thin layer chromatogrgphy. GC produced all steroids from H-LDL, but particularly P, 170H-P nd E. The hFSH increased 9H-LDL to manufacture all E production while HCC etimulated production of P. TC also utilized steroids but the most prominent steroids detected were P, 170H-P, T and A. HCGstimulated A and T production whereas hPSH had little or no effect. In summary, human granuloea and theoa cells utilize cholesterol from LDL for steroid production. In addition, gonadotropine can enhance this use of cholesterol by ovarian cells.
28s