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Specific androgen binding in human fetal epiphyseal chondrocytes in culture
-244 BIOLOGICAL EFFECTS OF ANDROGENS ON HUMAN FETAL EPIPAYSEAL CHONDROCYTES IN CVL Carrascosa A. , Audi L. , Ballabriga A. . TVRE . Hospital Infentil de la Vail 1 d -Hebron , Autonomous University of Barcelona ; 08035 , Barcelona ; SPAIN . Recently we have cultured human fetal epiphyseal chondrocytes (Ped.Res. 19: ?20,1985) and shown that 5-alpha-reductase activity is present in both human fetal epiphyseal cartilage and human fetal epiphyseal chondrocytes in culture (J. Clin.Endocrinol.Metab.S8:8l9,l984~ . The biological activity of androgens T and DBT on human fetal epiphyseal chon drocytes in culture has been investigated by studying their capacity to stimulate chondrocyte proliferation in a chemically defined serum-free medium . Chondrocytes (70,000 cells/well) were incubated with HAM F-12 serum and antibiotic free medium for 48h . The medium was then _qspifffed and replaced by MCDB 104 se-1 Ml . Cells were then incubated rum and antibiotic free with T or DHT (10 3-10 for 48h , the last 24h in the presence of H-thymidine (SuCi/ml). In each experiment five different wells were used for ch molarity and five as controls , -9 -?0 10 In male fetuses,l6-40 weeks-old,DHT M and T 10 M significantly (pcO.01) stimulated the proliferation of epiphyseal chondrocytes in culture (193238 % and 169I18 % MTSD , respectively; nm4 ) . In female fetuses only DET 10 M significantly (p
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._ 244 bis SPECIFIC ANDROGEN BINDING IN HUMAN FETAL EPIPHYSEAL CRONDROCYTES IN CULTURE . Hospital Infantil de la Vall d.Hebron A. , Ballabriga A. Audi L. , Carrascosa - SPAIN . Autonomous University of Barcelona r 08035 - Barcelona
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We have cultured human fetal epiphyseal chondrocytes C Ped. Res. 19:720,1985) and shown that 5-alpha-reductase activity is present in epiphyseal cartilage and In addition their chondrocytes in culture (J.Clin.Endocrinol.Metab.58:819,1984~. DHT significantly stimulates chondrocyte proliferation . In order to study the mechanism of androgen action on human fetal epiphyseal chondrocytes in culture, the presence of specific DRT binding hav3 been investigated . Chondrocytes were incubated with DHT- H i 0.1-5x10 M 1 with and without 200fold unlabelled DHT for 30*at 379C . Incubation was stopped in ice . Cells were sonicated in TrisO.OZM-HCl pB=7.5,0.5M KCl,t.SmM EDTA,ZmM Mercaptoethanol . Unbound DHT was separated with DCC buffer . Maximal binding capacity and Kd were from calculated according to a Scatchard plot . In human epiphyseal chondrocytes fetuses ( 20-40 weeks-old; 5 #and 2 Q 1 Bmax is 4.7 ; 1.2 CMTSD) fmol/mg prowas found for sex nor for gestational tein and Kd 0.45 ; 0.22 nM . No difference age . DHT receptors seem therefore to be present in human fetal epiphyseal chondroactivity of androgens cytes in culture _ These results suggest that biological chondrocytes in culture may be mediated through specion human fetal epiphyseaf fic receptors .
-245 OVARIC AND ADRENAL INFLUENCES ON UTERINE ESTROGEN RECEPMR IN PRHPUBERTAL PATS. M.I.Gonz~lez-Quijano,E.Castellano and B.N.D&z-Chico. Adapts.Physiology and Pharmacology. Cole gio Vniversitario de Las Palmas. P.O.Box 550. 35016 Las Palmas de Gran Canaria. SPAIN. Ovaric and adrenal influences on uterine estrogenic receptor content (PE) both cytosolic (RcE) and nuclear (RnE) as well as on the changes on this content responding to exogen estradio (E21, in 30-day old rats, have been studied in this work. The animals were ovariectomized (OVX) and adrenalectomised (ADX) or only OVX. Some of them were also implanted with E2. They were decapitated 3 or 7 days later. RE were measured by means of the 3H-E2 exchange method. OVX leads to a decrease in both uterine RcE (674 to 166 fmol/mg p, 3 days later and 293 fmol/mg p. 7 days later) and RnE (397 to 32 fmol/mg DNA on the 3rd day and 208 fmollmg DNA on the i'th),showing that uterine Rl?are strongly dependent on the ovaries at this stage. WX plus ADX leads to asma ller decrease in uterine RcE 3 days later (268 fmol/mg p) and specially in RnE (258 vs 32 fmol/ /mg DNA). On the 7th day there is no significant difference. A different responsiveness toE2wa determined by the presents or absence of adrenals in OVX rats, referring to uterine RE. E2 implant causes a still bigger decrease on the uterine RcE in only OVX rats (35 fmol/mg p), while in OVX and &RX ones there is no significant difference with the oil implanted. E2 causes a rise in uterine RnE in OVX and ADX rats (1827 and 826 fmol/mg DNA 3 and 7 days later respectively), while in only OVX ones it causes a big decrease (34 fmol/mg DNA) 3 days later. On the 7th day the value is not so small (303 fmol/mg DNA) but it is still lower than in the first group. These results suggest that some adrenal factor, possibly the progesterone, may participate in uterine PE regulation, causing a decrease in their concentration, specially on the nucleus.
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._ 244 bis SPECIFIC ANDROGEN BINDING IN HUMAN FETAL EPIPHYSEAL CRONDROCYTES IN CULTURE . Hospital Infantil de la Vall d.Hebron A. , Ballabriga A. Audi L. , Carrascosa - SPAIN . Autonomous University of Barcelona r 08035 - Barcelona
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We have cultured human fetal epiphyseal chondrocytes C Ped. Res. 19:720,1985) and shown that 5-alpha-reductase activity is present in epiphyseal cartilage and In addition their chondrocytes in culture (J.Clin.Endocrinol.Metab.58:819,1984~. DHT significantly stimulates chondrocyte proliferation . In order to study the mechanism of androgen action on human fetal epiphyseal chondrocytes in culture, the presence of specific DRT binding hav3 been investigated . Chondrocytes were incubated with DHT- H i 0.1-5x10 M 1 with and without 200fold unlabelled DHT for 30*at 379C . Incubation was stopped in ice . Cells were sonicated in TrisO.OZM-HCl pB=7.5,0.5M KCl,t.SmM EDTA,ZmM Mercaptoethanol . Unbound DHT was separated with DCC buffer . Maximal binding capacity and Kd were from calculated according to a Scatchard plot . In human epiphyseal chondrocytes fetuses ( 20-40 weeks-old; 5 #and 2 Q 1 Bmax is 4.7 ; 1.2 CMTSD) fmol/mg prowas found for sex nor for gestational tein and Kd 0.45 ; 0.22 nM . No difference age . DHT receptors seem therefore to be present in human fetal epiphyseal chondroactivity of androgens cytes in culture _ These results suggest that biological chondrocytes in culture may be mediated through specion human fetal epiphyseaf fic receptors .
-245 OVARIC AND ADRENAL INFLUENCES ON UTERINE ESTROGEN RECEPMR IN PRHPUBERTAL PATS. M.I.Gonz~lez-Quijano,E.Castellano and B.N.D&z-Chico. Adapts.Physiology and Pharmacology. Cole gio Vniversitario de Las Palmas. P.O.Box 550. 35016 Las Palmas de Gran Canaria. SPAIN. Ovaric and adrenal influences on uterine estrogenic receptor content (PE) both cytosolic (RcE) and nuclear (RnE) as well as on the changes on this content responding to exogen estradio (E21, in 30-day old rats, have been studied in this work. The animals were ovariectomized (OVX) and adrenalectomised (ADX) or only OVX. Some of them were also implanted with E2. They were decapitated 3 or 7 days later. RE were measured by means of the 3H-E2 exchange method. OVX leads to a decrease in both uterine RcE (674 to 166 fmol/mg p, 3 days later and 293 fmol/mg p. 7 days later) and RnE (397 to 32 fmol/mg DNA on the 3rd day and 208 fmollmg DNA on the i'th),showing that uterine Rl?are strongly dependent on the ovaries at this stage. WX plus ADX leads to asma ller decrease in uterine RcE 3 days later (268 fmol/mg p) and specially in RnE (258 vs 32 fmol/ /mg DNA). On the 7th day there is no significant difference. A different responsiveness toE2wa determined by the presents or absence of adrenals in OVX rats, referring to uterine RE. E2 implant causes a still bigger decrease on the uterine RcE in only OVX rats (35 fmol/mg p), while in OVX and &RX ones there is no significant difference with the oil implanted. E2 causes a rise in uterine RnE in OVX and ADX rats (1827 and 826 fmol/mg DNA 3 and 7 days later respectively), while in only OVX ones it causes a big decrease (34 fmol/mg DNA) 3 days later. On the 7th day the value is not so small (303 fmol/mg DNA) but it is still lower than in the first group. These results suggest that some adrenal factor, possibly the progesterone, may participate in uterine PE regulation, causing a decrease in their concentration, specially on the nucleus.
93s