The effects of hypophysectomy and human chorionic gonadotropin administration on the in vitro metabolism of progesterone by the rat testis

351

THE EFFECTS OF HYPOPHYSECTOMYAND HUMAN CHORIONIC GONADOTROPIN ADMINISTRATIONON THE IN VITRO METABOLISM OF PROGESTERONE BY THE RAT TESTIS-

RODNEY A. APPELLI

Jefferson Medical College Philadelphia,Pa. 19107 Received:

7112173

ABSTRACT Changes In the In vitro capacity to convert progcstcrone to its metabol-eswere studled in testes of adult rats hypophyscetomizcdfor varying lengths of time. After 30 days of hypophysectomyrats were injected for periods of 10 and 20 days with 100 1.u. of HCG daily to observe what changes could be Induced in the testicular conversion of progesterone. HyponhysectomyIncreased the formation of-20*hydroxy-4-pregnen-3-oneand deoreased the formation of testosterone. In hypophyseotomlzedanimals inJected with HCG there was an immediate decrease in the 20Arhydroxy-4pregnen-3-one formation, but no appreciable accumulations of testosterone,as the animals demonstrated an immature pattern of testicular function. The results Indicate that 20&hydroxy-4-pregnen-3-onemay act as a positive feedback agent to prolong and heighten gonadotropfndischarge, and confirm the importance of metabolitcs of testosteroneprior to adulthood. INTRODUCTION Removal of the pars dlstalls results In atrophy of the germinal elements of the testes as well as arrest of Leydlg cell hormonal actlvltles. The life cycle of the Leydig cell has been shown to be biphaslc (2). Steinberger and Flcher (3) strated a biphasic

clearly dcmon-

curve in the oapacity of the testes to

convert progesterone to testosterone that Is related to the age of the animal. Rat testes have demonstrated a capability ff converting progesterone to testosteroneduring the later Now of Geo. Washington Univ. Medial Center, Wash., D.C. 20037

352

stage

22:3

STEROIDS

of fetal development (4,s) and the first day after

birth (3). After two weeks of age the rate of testosterone accumulation decreases rapidly in the rat and remains minimal between 20 days (3) and 50 days (6).

It has been reported

(6,~) that the testis is oapable of the oonverslon of proges-

terone to testosteroneduring this time period but that the testosterone Is further metabolized. Inano and Tamaokl (8) demonstrated the presence of h4-a-hydrogenase in rat testes as the enzyme responsible for the conversion of testosterone to Its a-hydrogenated metabolltes. Inano, &

&

(9) then

demon&rated that the amount of this enzyme was dramatloally decreased by day 60 after birth. HCG has been &own to act on t‘hfsenzyme to lnorease the formation of C-19 %-hydrogenated steroids and to decrease the formation of C-21 uhydrogenated compoqnds (10). HCG has also been shown to stimulate the 20rbhydroxylasesystem (11), steroid q-01dehydrogenase (12). and the desmolase system (13). Considering the above findings it seemed necessary to determine the effects

of HCG in ohanging the pattern of uti-

lization of progesterone by hypophyseotomizedrat testes. HATERIAIS AND METHODS Tbe testicular tissue used for this study was obtained from 5, 15, 30, and 40 day post-hypophyseotomyadult SpragueDawley rats and from Sprague-Dawleyrats Injected suboutaneously with 100 i.u. Human Chorionic Gonadotropin (HCG) per day for periods of 10 and 20 days after they had been experiencing the effects of hypophysectomyfor a perio of 30 d ys. The testicular tissue was Incubated with 20 x 10B dpm W- 3Hprogesterone by the method of Stelnberger and Ficher (2). The ?&H-progesterone was supplied by the manufacturerat a opeolflc activity of l*Cl/m)lM; unlabeled progesterone was added in order to lower the specific activity to 2.@aCi/~M. Testloular tlssue was Incubated for a period of 3 hours.

Sept. 1973

STEROIDS

353

After the Incubation 40 x 103 dpm each of 14C labeled tracers of androstsnedlone,progesterone, 17-hydroxyprogesterone, and testosterone were added to the incubate In order to correct for losses during subsequent manlpulatlons. Final ldentiflcatlon of testosterone and certain other steroids obtained on chromatogramswas made by using the technique of so plc dilution and recrystallizationto constant ratio of 3H/1sC. Composition of the Incubation Medium (for total 1.5 ml) 1 ml Hanks' buffered salt solution with glucose (14) 25 ul ethanol 175 1.0 NADP, Na solution (3.3 uM/ml) 175 ul G-6-P, Na2 solution (36 uM/ml) 125 ul 1.3% sodium bicarbonate solution Extraotlon Procedure The Stelnberger-Flchermodiflcatlon (2) of the techg (4) was followed Involving repeated extraction of the Incubate with ethyl acetate followed by methanol-hexanerrartitlonand chloroform extraction, with 50 ug each of androstenedione,progesterone, 17-hydroxyprogesterone,and testosteroneunlabeled carriers added to the dry residue. nique described by Noumara, 3

Paper Chromatography The procedure used was that described by Steinberger, et& (15). Whatman No. I filter paper (washed with methanol) was spotted with the extracts and reference steroids and developed In systems Bush A (heptanetmethanoltwater, 100;80;20); Bush 3 (heptane:benzeneimethanol:water, 66:34:80~2o);T-4 (lso-octane:methanoltwates, 1019tl). The dried papers were stanned In the ultraviolet lamp at 254 nm which located the A -30ketosterolds. Sample and reference channels were separated and the former were scanned for radioactivity. The location of each steroid on the radioactive strips was determined with the help of the reference strips treated at various tlmes with Zlmmermann's reagent to locate 17-ketosteroldsand 5% ethanollc phosphomolybdlcacid to locate other steroids. Acetylation The dry material was dissolved In 0.1 ml pyrldlne, to which 0.1 ml acetic anhydrlde was added, leaving the reaction mixture overnight at room temperature;0.2 ml methanol was added to stop the reactlon followed by evaporation to dryness. Chemicals Analytical grade solvents were redistilled before use. Steroids (Steralolds,Inc., Pawllng, N.Y.) were purified on silica gel column and recrystallizedto the correct melting

354

STEROIDS

22~3

point. Where lndlca ed, the radlolabeled steroids of 4-14CC-progesterots,7ti-3H-progesterone, 17-hyandrostenedlone,4-l); droxy-4.14C-progesterone,and 4-l C-testosterone (N5w England Nuclear Corp., Boston, Mass.) were purified by paper and thin layer chromatography. Human Chorlonlc Gonadotropin (HCG) was obtained from The Upjohn Company, Kalamazoo, Michigan. Measurement of Radioactivity Paper chromatogramswere scanned in a Packard model 7200 radlochromatogramscanner. The chromatogramswere then dlvlded into radioactive zones and eluted with a descending capillary flow of methanol. Duplicated 1 ml. samples were then counted In Packard model 3003 liquid sclntll ator spectrometer, set for simultaneous c unts of 3H and 1tC ,114 efflclencles of 21% and 0.02% for sIIand 56% and 6% for C In the respective channels. The samples were prepared by evaporation in vacua and dlsso ved in 10 ml. solntlllatlonsolution (0.4$-i-f 2.+bls- t2w(5~tert-butylbenzoxazo~yl)l thlophene In toluene. The radioactivityof the Incubation medium containing the 3H-progesteronesubstrate and of the tracers were measured y setting for s ngle oounts with efficiencies of 37% for BH and 861% for lb Counts were expressed as dlslntegratlonsper minute (dim). Quenching was usually absent, but in certain cases It was necessary to correot for quenching by using an internal standard and the automatic external standardizationprovided In the instrument; both methods gave the same correction factor. Calculations The absolute percentage conversion of progesterone to testosteronewas obtained by the following formula: $ Conversion = ,3H dpm in last orystals X dpm_14C-testosteroneadded X 100 dpm In last crystals X dpm JH-progesteroneIn substrate

*T

RESULTS The body and reproductive organ weights of the animals used in this study are summarized In Table 1. The relative percentage radioactivityrecovered In the respective zones for specific steroids derived from Drogesterone after the first chromatographyIn Bush A system ban be found in Table 2. Testes froa rats hypophysectomlzedfor 5 days Chromatographyof the chloroform extract in Bush A

Sept. 1973

355

STEROIDS

TABLE 1. Relation of the Duration of Hypophyseotomyand HCG Administration to Body and ReproductiveOrgan Weights Days After Hypophysectomy 5

15

30

40

40*

so**

Body Weight (g)

183

204

205

212

218

224

Testes (mg)

2238

1309

589

549

945

1144

Prostate (mg)

78

22

6

9

114

222

Seminal Vesicle (one) pull(mg) 0 Empty bud 20

56

;9

;8

:90;

695 208

Tissue (mg)/incubate67

74

65

73

74

72

* Received 100 1.~. RCG for last 10 days ** Received 100 i.u. HCG for last 20 days TABLE 2, Percentage Conversion of Progesterone by Rat Testes at Various Times after Hypophysectomyand BCG Injeatlon Days After Hypouhysectomy 15

30

40

4oa

sob

Progesterone (unconverted Substrate) 17

27

37

33

24

12

Testosterone and/or other comnounds

29

19

9

6

?

15

2O&-hydro;r;4?;;:--

3

21

23

24

2

5

Androsterone and/or other compounds

12

-

-

-

12

20

3

3

Metabolltes*

5

Testosterone (by recrystalllzatlon)**23

9

-

3

* Radloactlvlty recovered In the respective zones after the

first ohromatography In Bush A system +* % conversion obtained by formula In Methods Section t Received 100 I.u. HCG for last 10 days Received 100 1.~. HCG for last 20 days system (Fig. 1) revealed a pattern of radioactivitythat places the age of the rat between 40 and 60 days. Important

356

2213

STEROIDS

la the large peak

of

unconverted progesterone substrate (17s

of the total radioactivity reoovered) and a peak In Zone 2 The corresponding In moblllty to 204rhydroxy;4tiprtgden-3-one. material from Zone 2 was acetylated and rechrom8tographed with a standard acetate of 20Ghydroxy-4+pregnen-3-onewith which It ran correopondlnglywell. Zone 1 (29% of radioactivityof first chromatograph)was rechromatographedIn reveral systems, acetglated, and finally Identified to be testosterone, In part, by recrystallizationto ooncstantratio 3H/14C. Upon acetylatlon Zone 1 gave two radioactive peaks, the first corresponding to testosterone acetate and the second to the dlacetate of s-atirostane-X,13$-dlol, again demonstrating that the animal was not as yet sexually mature. Testes from rats hypoDhysectomlzedfor 15 days Chromatographyof the chloroform extract In Bush A 8ystem (Fig. 2) revealed the adult pattern with the exception of a large peak in Zone 4 corresponding in mobility to a standard of 2O&-hydroxy-4;pregnen-3-one,The amount of progesterone converted to testosterone (as determined by formula In Methods section) wa8 smaller (9%) when compared with the conversion

observed with tissue from rat8 5 days hypo-

physeotomlzed (23%). Aoetylation of Zone 4 of Fig. 2 gave a peak correspondingto the acetate of aM+ydroxy-&preener+3one and a second less polar peak which was shown to be pragesterone due to reoxldstlon of the compound 8econdPry to the te&nlqus of acetglatlon. Testes from rats hrDophy8ectoalzedfor 30 days Chromatographyof the chloroform extract In Bush

A

STEROIDS

Sept. 1973

357

CHROMATOGRAMS OF RAT TESTICULAR METABOLIS!YOF PROGESTERONE (Scanned at sensitlvtty 3X10 ) FIG. It 5 Days Hypophyseotomlzed

FIG. 21

-30

15

Days Hypophysectomized

Days Hypophysectomized

FIG. 41 40 Days Hypophysectomized

-40

Daya Hypophysectomized 10 Days HCG Admlnlstratlon

FIG. 6~ 50 Days Hypophysectomized 20 Days HCG Administration

STANDARDS:

22:3

STEROIDS

358

system

(Pig.

terone

substrate

percentage

mueh less

3) revealed

(37% in Table

recovery

of

from this

crystalllzatlon, percentage substrate

At the same time the high to

20duhjdroHy-4rgreg4enn3-one

15 day experiment but were lost

conversion

in mobility

was found as In the S$ day hypophy-

The aaetglated

animals. and the

2).

of the progss-

Zone 2 correspondLng

a0Jrhydroryn4cgTegnen-3-~e seotomlzed

conversion

of

had been pooled

due to technical

testosterone

for

error.

reThe

from the progesterone

wa8 OS, as no teetosterone

could

be recrystallized

from Zone 1. Testes

from rats

hyDoDhgsectomieed

Chromatography system

(Fig.

tlcular with

the ohloroform

4) was qualitatively

tissue 33% of

of

from rat8

the total

ted progesterone

for

(Pig.

5) dlffered

day non-injected (corresponding reduced

the chloroform qualitatively

hyr>ox,hysedtomized

extract

animals

with

(Pig.

the oorreeponding

reoovered

ted

In Bush A

from that

total

2% corresponded

greater

as unoonver-

40 days and receivlna

40 day hypophyseatomlzed

radloaotivity

percentage

to metabolltes

after

the flrst

of 4).

the progesterone

region animals.

Zone 2

(Zone

3)

Of the

ohromatogram

substrate

than in the non-injected

the 40

was stgnifiogntly

to the 2O&+ydroxy-4~pregnen-3-one of

tecr-

30 days,

to 2O&-hydraxy+4-pregnen-3-one)

when compared

of

and 24% aa W#rhydroxy-4-plr&.gnen-3~one.

in the non-lnjeoted

only

for

reoovered

Testes from rats hypOphy8eOtOmlZ8d for 100 t.u. of HCG for the laet 10 dasr

system

In Bush A

to that

hypophyrectomleed

subetrate

of

extraot

rlmilar

radloaotlvity

Chromatography

40 days

and a

was oonver-

anlmals,

espeolally

Sept. 1973

STEROIDS

to a compound In Zone 4.

359

Rechromatographyand acttylatlon

of Zone 4 revealed a comDound corresponding In mobility in various systems to androsterone and dlhydratestosterone. The separation of these two compounds requires a gas-liquid chromatographlc rystem described by Unhjem (16) not available to this lab. Regardless of which of the two steroids the cornpound actually Is, It helps to explain the fact that the accumlatlon of testosterone was not elgnlflcantlydifferent from the non-inJected rat8 after recry8talllzatlon(Table 2) as both of these compounds are metabolltes of progesterone through a testosterone Intermediate. Testes from rats hsx,Dhysectomlzed for 50 days and receiving 100 I.u. of HCG for the last 20 days Chromatographyof the chloroform extract In Bush A system (Pig. 6) qualitatively revealed a great deal of conversion of substrate to compound8 correspondingIn mobility to testosterone (Zone 1). Subsequent chromatographyand acetylstlon revealed two major peaks, one corresponding in moblllty to testosterone acetate and the second to the dlacetate of ~-anbrostant-3~,17ij-dlol~ In fact, the percentage conversion of progesterone accumulatingas testosterone remained the same as In rats Injected for 10 days (3%). Zone 4 corresponded to the same compound In Zone 4 of Flg. 5, but there was a greeter amount of the compound than in the animals lnjetted for only 10 days. DISCUSSION Effects of hypophysectomyon the metabolism of progesterone With cessation of productlon of ICSH there 18 a pro-

360

STEROIDS

22~3

nounced atrophy of the testes with an asmoclated decreased rate of manufacture and release of androgena into the clrculatlon as demonstrated by the atrophy of the secondary sex organs (Table 1). The Important features of hypophysectomy include the lowered percentage conversion of progesterone and the formation of a~hydroxy-4eigragn~n-3~ona. The slgn~flcence of the 2O+hydFoxysteFold dshydrogenasemay be Interpretedas a mean8 of adjusting ths biological potencies of the endocrine rsecretlonsto the changing requirements of the animal. Put another way, It may be considered a genetically Instituted protective positive feedback device. In fact, work by Hllllard, et $_ (17) has indicated the ~-hydcoxy-4-pregnen~3-one acts as a positive feedback agent to prolong and heighten LH discharge In the mated rabbit. ~0d-~droxg=4~regnunu3_cms has been shown to be reversibly Interrelatedwith progesterone through a metabolic reaction catalyzed by a NADP dependent enzyme,

thus permitting the reutilizationof progesterone

(18,19), and the 2O+hydroxystbrold dehydrogenase has been located In the soluble fraction of the cell while the majority of testicular enzymes related to steroid transformationare bound to the mltochondrla and mlcrosomes (20). Inano, -et al (21) have demonstrated that the action of the 20drhydroxysterold dehydrogenase Is a competitive lnhlbltlon of the desmolase system, thus preventing androgen formation. Testosterone 1s recognized as a negative feedback device in controlling the secretion of ICSH. Continued testosterone fonmatlon after hypophysectomywould have an ln-

Sept. 1973

361

STEROIDS

creased

detrimental

effect

on the

2a=hgdroxg-~.pregnen~~-one

By rhunting

organism.

the organham

attempt8

into

to enhance

the

secretion of ICSH by conserving the progerterone In a form that will not produce time be reconverted production

testosterone,

to progesterone

of testosterone

and/or

whloh oan at a later

but

for reutlllzrtlon

In the

androgens.

other

Effects of HCG treatment to hypophyaeotomlzedrats on the metabolism of progesterone Shlklta and Hall (22) observed a decrease In 2W-hydroxysteroid.dehydrogenaseactivity -In vlvo when mlcrosomal enzymes were etlmulated by HCG. This work shows that the LO#hydroxy-4-pegnen-J-one was rerronverted to progesterone more progesterone However,

aould

be utilized

the hypophysectomy

of ade-dlfferentlatlng*

In androgen

In these animals

the Leydlg

cell8

so that

formation.

had the effect

and thU8 the entire

maturation cycle was begun again with InJectIons of HCG, In this work it was observed that HCG administrationdecreased the amount of ~O~~droxy-4~.pregnQnc3-oneand lncrearsedth8 csleavage of the 21 oarbon oompounds to the C-19 androgens, pecially

steroids.

For this reason not lm-

OOnVerSlOn

Of

substrate

as the S&-hydrogenated

steroid8

s-hydrogenated

orease In the percentage terone assumed

appeared,

es-

to be formed

through

the Intermediate

to te8tO8formed

formation

are of

testosterone,a8 oonflrmed In the normal rat by Nayfeh, ct & (6) and Coffey, Qt al_(7). The pOS8lblllty exists that testoeterons oould be transformed to other steroids more promptly than progesterone Is converted to testo8tsroneand, as a result, the amount of testosteronedetected would be small.

STEROIDS

362

22:s

During the 20 days of HCG admlnlstratlona great lncrease In the weights of the secondary sex organs took place (Table 1). Previous experiments by Bruchavsky and Wilson (23) are highly suggestive that androgenlc aotlons on secondary sex organs are due to the formation of s-hydrogenated metabolltes of testosterone. Thus, at the time of completed sexual maturation the Increase In testosterone secretion may be due to a decrease In testicular catabolism of testosterone rather than to an Increased rate of Its formation. Styllanou, et &

(24) have postulated that this capacity to metabolize

testosterone may be of homeostatlc significance since a change in enzyme activity with age could function as a regulatory mechanism Involved ln the onset of puberty in the male. The results In this work demonstrate that changes in the metabolism of testosteroneare most probably dependent on both gonadotroplns and the testicular tissue, with control over the qualitative nature of the final product probably residing In the testes themselves. ACKNOWLEDGEMENTS I would like to express my appreciation to Doctors E. Stelnberger and W. Flcher for their time, teeohlngs, encouragement, and laboratoriesto perform lndlvldual research under the ausplaes of the National Science Foundation. REFERENCES

(1)

The followlng trlvlal names and abbreviations have been used in this paper:

Androetenedlone (A) Androsterone (An) Dehydroeplandrosterone(D) Dlhydrotestosterone Epltestosterone (E) 170hydroxyprogesterone(17)

4-androstenr3,17-dlone J+hydroxy-s-androstan-17-one s-hydroxy-5-androsten-170one 17&hydroxy-5A_androstan-3-one 1x0hydroxy-4-androsten-Y-one 17.hydroxy-4-pregnen_3,2O=dlone

STER

Sept. 1973

If:!ndrosterone (I) Progesterone (P) Testosterone (T) ilCG

LH ICSH G-6-P NADP

OIDS

363

2O&hydroxy-4-pregnen-j-one 3$-hydroxy-5&-androstan-l7-one 4. regnen-3,200dlone 17s -hydroxg-4-androsten-j-one Human Chorlonlc Gonadotropin Lutelnlzlng Hormone InterstltlalCell Stimulating Hormone Glucose-6-Phosphate Nlcotlnam~deAdenlne Dlnucleotide Phosphate

(2)

Nfeml, M. and Kormano, M., ENDOCRINOLOGY 74, 996 (1964).

(3)

Stelnberger, E. and Flcher, M., STEROIDS 11, 351 (1968).

(4)

Noumara, T., Welsz, J., and Lloyd, C.W., ENDOCRINOLOGY 78, 245 (1966).

(5)

Bloch, E., STEROIDS 9, 415 (1967).

(6)

Jr., and Baggett, B., ENDONayfeh, S.N., Barefoot, S.'rl. CRINOLOGY 78, 1041 (1966).

(7)

Coffey, J.C., French, F.S., and Nayfeh, S.N., ENDOCRINOLOGY 89, 865 (1971).

(8)

Inano, H. and Tamaokl, B., ENDOCRINOLOGY79, 579 (1966).

(9)

Inano, H., Horl, Y., and Tamaokl, B., CIBA FOUNDATION COLLOQUIA ON ENDOCRINOLOGY 16, 105 (1967).

(10) Oshlma, H., Sarada, T., Ochlai, K., ENDOCRINOLOGY86, 1215 (1970). (11)

and

‘l’amaoki,

Be,

Drosdonsky Dorfman R.I., and ForMenon' K*"*J*iTEROID3 3up;l";; 95 (196;). chlelll, E.,

(12) Samuels, L.T. and Helmrelch, M.L., ENDOCRINOLOGY 58, 435 (1956). (13) Hall, P.F. and Elk-Nes, K.B., BIOCHIM. BIOPHYS. ACTA 71, 438 (1963). (14) Hanks, J.H. and Wallace, R.E., PROC. SOC. EXPER. BIOL. MED. 71, 196 (1949). (15) Stelnberger, E., Stelnberger A., Vllar, O., Salamon, I.I., and Sud, B.N., CIBA FOUNDATION COLL,OQUIAON ENDOCRINOLOGY 16, 56 (1967). (16) UnhJem, 0,, ACTA ENDOCRINOLOGICA63, 69 (1970). (17) Hllllard, J., Penardl, R., Sawyer, C.H., ENDOCRINOLOGY

STEROIDS

364

22:3

80, 901 (1967). M. and Ikemoto, M., ENDOCRINOL. JAPON. 14, 232

(18) y-y .

(19) Weist, W.C., J. BIOL. CHEM. 234, 3115 (1959). (20) Inano, H., Egusa, M., and Tamaokl, B., BIOCHIM. BIOPHYS. ACTA 144, 165 (1967). (21) Inano, H., Nakano, H., Shlklta, M., and Tamaokl, B., BIOCHIH. BIOPHYS. ACTA 137, 540 (1967). (22) Shiklta, M. and Hall, P.F., BIOCHIM. BIOPHYS. ACTA 136, 484 (1967). (23) Bruohovsky N. and Wilson, J.D., J. BIOL. CHEM. 243, 2012 (19683. (24) Styllanou, M., Forohielll, E., and Dorfman, R.I., J. BIOL. CHEM. 236, 1318 (1961).