STEROID METABOLISM IN GENETIC MUTANT MICE

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1 " /.. STEROID METABOLISM IN GENETIC MUTANT MICE

2 _--'"J ::_. ~...:--- I -... ~ METABOLISr.1 OF TESTOS!l'ERONE, J:'R0GESTERONE AND ANDROSTENEDIONE IN THE LIVER,. TESTES, HYPO THALAMUS AND CORTEX OF NORMAL AND TESTICULAR FEMINIZED MICE By.I!J JAMES DANIEL DALEY,. B.Sc A Thesis Submitted to the School o~ Graduate ~tudies In Partial Pul~ilment o~ the Requirements ~or the Degree Master o~ Science Mc~aster University September, 1976 \. JAMES DAllIa DALEY 1977

3 > 1... MASTER OF SCIENCE ) Medical Sciences (Growth and Deve1:opment) McMASTER UNIVERSJ;TY Hamilton, Ontario. September, c TITLE: Metabolism of Testosterone, Progesterone and Androstenedione in the Liver, Testes, Hypothalamus and Cortex of Normal and Testicular Feminized (Tfm) Mice AUTHOR: James Daniel Daley, B.Sc. SUPERVISOR: Dr. E. V. YoungLai.NUMBER OF PAGES: xvii, 182 " 11 '.

4 ABSTRACT Thein vitro metabolism of testosterone, progesterone and androstenedione was studied in liver, testes, hypothalamus and cortex incubations of normal BALB/c d, BALB/C9 and a number of sex-mutant mice carrying the testicular feminization (m) and sex-reversal (~) genes. The metabolism of testosterone was studied in liver'homogenates of BALB/co",T:fln (o+)f and T:fln (ohv)(. Androstenedione was identified as the major metabolite of testosterone, in all incubations, indicating the presence of l7a-hydroxysteroid dehydrogenase activity. C~parison of the. percentage to androstenedi~~e suggested conversions of testosterone ~hat of the three genotypes studie~, the activity of l7~-hydroxysteroiddehydrogenase was lowest in the!!,!!! (ohv) liver. Similarly, the metabolism of testosterone and progesterone was studied by double-labelled tracer experiments using testes minces from BALB/c~, m (0+) 't and T:fln (ohv)'f mice. The conversion of Jlr?gesterone to testosterone was greatel>t in BALB/c d'. in~bations and least in the T:fln (0+) incubations. These data. (. I' iii

5 suggest that the relative ac~ivities of the l7~.. ketoreductase for the conversion of androstenedione to testosterone are in the order I BALB/c ci' > m (ohv) rt > m (0+). Progesterone metabolism was studied in testis incubations of normal male (]ALB/c 0'), testieularfeminized, sex-reversed (m+(ohv) Blo/+++, and sex-reversed (+Ta++/++l:. sxr/+ d') mice. sxr/+ d') Comparison of the amounts of androstenedione and testosterorle formed from P suggests that differences are present in the relative activity of the testis 3-ketoreductase. The. relative activity is in the orde~ BALB/c 0' > +Ta++/+++, sxra' > Tfm+(ohV) Blo/+++,.2.2Q;: d' The metabolism of androstenedione was also studied in testes.incubation of m (0+)t' and Tfm (ohv) 't mice. The formation of testosterone was similar in all incubations. A number of unidentified metabolites'of androstenedione were detected in the Tfm (ohv) incubations which were not present in the Tfm (0+) incubations. Finally, the aromatization of testosterone was studied in hypothalamus and cortex incubations of norma! iv..

6 ' BALBIc t:1', BALBIc ~, and testicular feminized Tfm (0+)t. and Tfm (o~v~mice.. A new, rapid method was developed for studying aromatization-base~toluene-sodium hyd~xide partitioning and a nove~n methylation procedure known as extractive alkylation. method the formation of estrone-,and estradiol was Using this detected. The conversion of testosterone to estradiol - by hypothalamus minces was abou"t'1.5 times greater in normal BALBIc ci" than BALBIc ~ a~id about the s~e as ij? -- --, ( +) ""- ( hv) tal". Tfm 0 and TJ.w 0 - To ': aromat~zat~on by cortex minces was about 30-50% that of the hypothalamus. major metabolite of testosterone.in tl;0rm2.l BALBlc cj'.. -~ The and / ""BALB/c ~ incubations was estrone while in the Tfm (0+) and Tfm (ohv) incubations it was estradiol The studies. confirm the work of others which suggests that the-metabolism of steroids is altered in the livej" and testis of the Tfm (0~)/Y1 mouse. The present work extends these observations' to include a number of other mice carrying the -- Tfm and,-- sxr genes. US~ the extractive alkylation technique aromatization was detected in Tfm hypq..thalamus and although the overall,/ quantitative yields of estrog~ from testosterone were similar to normals, qualitative differences were evident v "I! II

7 which may be signific~t to the explanation of.. altered hypothalamic-pituitary gonadotrophin relationships in 1f!!! (0+)' and 1f!!! (ohv) mice. vi./

8 / TABLE OF CONTENTS.. Page Acknowledgements viii Abbreviations and Triv.ia1 Names xiv List of Tables ix List of Figures xi In~oduction General Methods 26 Experimental and Results Discus ion Appendix 157 Bibliography 161 b-o /" '- '~ v.ii

9 , ACKNOWLEDGEMENTS.' The 'author. expresses his sincere appreciation and gratitude to Dr. Edward YoungLai tor his guidance, encouragement and patience in the su~rvision of this.' investigation. The author would also like to thank Dr.Jack Rosenfeld and Dr.Vince Taguchi for their suggestions and advice in. the develo:plllent.of the extractive alkylation techniques for the synthesis of estrogen methyl ethers, and to Dr.K.I.Williams for his generous gift of authentic catechol estrone and derivatives. This research was financially supported by the Medical Research Council of Canada. Qrtho Pharmaceuticals /. and a McMaster University Graduate Studies Scholarship. The thesis was typed by Mrs.Barbara Billington to whom gratitude is here expressed. ~ i l \ ;, i,,, j!, ~ 'I i J! viii \ t I ; 1.

10 LIST OF TABLES Table 1 4 Title Hormonal responses in testicular feminization. "Steroid receptors" in testicular feminization. ' Steroid metabolism by tissues in testicular feminization.. Composition of chromatography systems. Page Recrystallization of ~-5a-andr~stanediol from liver. incubations with 7CI--'H-. 54 testosterone. 6 Recrystallization of 3H-testosterone from liver incubations with 7CI-3Htestosterone Recrystal:izationof ~-androst~nedione from liver incubations with 7CI-JHtestosterone. 7CI-~-Testosteronemetabolism in liver homogenates. Recovery of ~- and l4c_ in testes incubations with 7CI-3H-testosterone and 4- l4 c-progesterone. Recrystallization of ~- and l4c _ testosterone from testes incubations with 7CI~3H-testosteroneand 4-L4cprogesterone. 7CI-~-Testosterone and 4-l4c-p~gesterone metabolism in testes incubations ix \

11 / ''-. Table Title Page 12, 4_ 14 c-progesterone, ;~tabolism in testes incubations. 1) 7~_14C-Androstenedionemetabolism in testes incubations Gas chromatography or estrogen methyl ethers ' 18 o Thin-layer and paper chromatography or estrogen extractive alkylations. 107 Gas chromatography-mass spectroscopy' or estrogen methyl ethers.l. estrone and estrone-)-methylether. 108 Gas chromatography~mass spectroscopy or estrogen methyl-ethers. 2. estradiol-17~ and estradiol-17~-)-methylether. 109 Thin-layer chromatography' of estriol extractive alky~tion. 110 "- '-,'-.. ;/ I i \ 19, Gas chromatography of catechol es ';rogen and methylated derivatives. ' Recrystallization of 14C-estrone and 14C_ estradiol-17~ derivatives from thin-layer chromatography of extractive alkylation of phenolic fractions from normal mouse hyppthalamus and cortex incubations with 4:'14C-testosterone Aromatization or 4-14C-~estosteroneby hypothalamus and cortex incubations of day old BALB/c mice. ', 22 Aromatization of ~_14C-testosterone by hypothalamus and cortex incubations of )0 day old Tfm (0+) and ~ (ohv) mice. 126 x

12 ;, LIST OF FIGURES Figure Title Page 1 2 Genetics o:f T:fln in the mouse. 6 Metabolic pathways :for the production of' progestogens. androgens 14 and estrogens. 4 Paper chromatography o:f 5ct-andrQstanediol :fi"9m liver incubations with 7ct-JHtestosterone. 47 Paper chromatography o:f testosterone :from liver incubations with 7ct-3Htestosterone. ' 5 Paper ch..-omatography o:f androstenedione :from liver incubations with 7ct-3Htestosterone Change in %-an~stenedionephtestosterone ratio with time in liver incubations with 7ct-3H-testosterone " 7 Paper chromatography o:f neutral steroids extract':from testes incubat~ons with 7ct-3H-testosterone and 4-1~-progesterone., 62 ( ;, ", 8 Paper chromatography o:f phenolic steroids extract :from testes incubat~ons with 7ct~-testosteroneand 4-1~-progesterone "., 9 Thin-layer chromatography o:f organic extract :from testes incubations with 4J4C-progesterone. ' 10 Paper chromatography o:f neutral steroids extract :from testes incubations with-7ct-3h-androstenedione. 76 :i.,.;

13 TiUe Page' Pa~r chromat6"graphy of phenolic steroids extract frow testes ' incubations with 7a-JH-androstenedione. 79 Gas chromatography of estrone and estradiol-17~ extractive alkylation. ' 89 Column chromatography of ~-estrone and JH-estradiol-17~ extractive 91 alkylation.,. ' Mass s~ctrum of estriol extractive alkylation: non deuterated derivative Mass spectrum of estriol extractive alkylation: deuterated derivative. Gas 'chromatography of catechol estrogen standards. Gas chromatography of catechol estrogen extractive alkylation. Mass spectrum 9f 2-hydroxyestrone extractive alqlation. ' Absorbance spectrum of 2.3 dimethoxyestrone in me;thanol., d I \ Absorbance of 2.3-dllnethoxyestrone in at 2]4 mu. methan0r Gas chromatography of extractive alkylation of BALB/c hypothalf!ilius and cortex incubations with-1'+ctestosterone. Thin-layer chromatography of' extractive alkylation of BALB/c hypothalamus and cortex incubations with 4_l4c-testosterone.... Thin-layer chromatography of extractive alkylation of T:fm (0+) and T:fm (oh V ) hypoth~amusand cortex '~='lo.."'+";o-- _.:..~,.,...r:..-...ost=rone...:...::. ~..._ ,..- "'- """I;O~'" III;;; xii 95, )

14 >: ~ I /!,3,,.:"", ~ " 3 " Figure 24 Title -Classical- technique :for studying, aromatization. Page 142 i ~ " 25 Extractive alkylation as a,method :for studying aromatization Mass :fragmentation pattern o:f ),17adimethoxyes~iol-D-ring ',- ",; '1 -, ;' '., xih

15 J, i ABBREVIATIONS AND TRIVIAL NAMES The :following abbreviations and trivial name~ are used in this thesis: testosterone (T) progesterone (p) androstenedione (A) dehydroepiandrosterone (DHA) androstenediol (AEOH 2 ) 4-androsten-17S~J-one 4-pregnen-J.20-dione 4-androsten-3.17-dione 5-androsten-~-ol-17-one 4-androsten-Jtt.17~-diol " ".: Sd-dihydro1;estosterone (DHT) Sd-androstanediol (AOH 2 ) choles"terol pregnenolone androsterone J.17S-dimethoxyestriol 2.J-dimethoxyes"trone (2.J-diOMeEI) Sd-androstan-17~-ol-J-one Sd-androstan-Jtt.17S-diol 5-cholesten-J$-ol / 5-pregnen-JS-ol-20-one 5a-androstan-Jtt-ol-17~one. i.... ~,,,.,.' estradiol-17~(ezi7~) estrad~ol-17~(ezi7s) estriol (EJ) estrone (EI) 2-hydroxyestrone (2-0HE I ) J-methoxyestradiol-17~ (E20Me-17~) I.J.5(IO)-estratrien-J.17~-diol I.J.5(IO)-estratrien-J.17S-diol I.J.5(IO)-estratrien-J.I6a.17S-triol 3-hydroxy-I.J.5(IO)-estratrien-17~onE /J.17S-dimethoxy-I.J.5(IO)-estra "trien-17s-01 ' 2.J-dimethoxy-I.J.5(IO)-estratrien-17-one 2.J-dihydroxy-I.J.5(IO)-estratrien- 17-one J-methoxy-I.J.5(IO)-estratrien 17~-ol J ; J 4,.~. xiv

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Complete. Male Hormones SAMPLE PATIENT. Patient: Order Number: Age: 47 Sex: M MRN:

Complete. Male Hormones SAMPLE PATIENT. Patient: Order Number: Age: 47 Sex: M MRN: Patient: Age: 47 Sex: M MRN: SAMPLE PATIENT Order Number: 81200334 Completed: September 20, 2007 Received: September 20, 2007 Collected: September 20, 2007 Complete Male Hormones Patient: SAMPLE PATIENT