Evidence accumulated in recent years tends more orless to support the view that the biosynthetic pathwaysinvolved in the synthesis of the steroid hormones by theendocrine organs are qualitatively similar. One outstanding exception to this is the ability of the adrenal cortexto hydroxylate the steroid molecule at C-il, which phenomenon is not shared by any other normal mammaliantissue. In a very broad sense one can assume also thatthe contoffling mechanisms at the pituitary level are aLsosimilar and that the major difference among these tissuesis their specific responsivity to their respective anteriorpituitary hormones.

Several mechanisms have been suggested over the pastfew years in attempts to explain the action of these protein hormones on their respective target organs.

Hechter and Lester (7) proposed that adrenocorticotropin (ACTH)3 acts on the adrenal cortex by enhancingthe permeability of the cell to glucose. This, in additionto furnishing the energy requirements of the cell, wouldgive rise to increased TPNH (reduced triphosphopyridinenucleotide) as glucose 6-phosphate is made available tothe various TPNH-generating systems. The TPNHwould then be available to satisfy the co-factor requirenients of the hydroxylating enzyme systems involved insteroid hormone biosynthesis.

Konitz amid Peron (10) have suggested that ACTH maydo 2 things : 1) make more TPNH available for crucialhydroxylat.ions involved in steroid biosynthesis; and 2)release hormone precursor material intracellulanly fromsome previously unavailable form. This is based on observations that freezing quartered rat adrenal glands andsubsequently incubating them prevented the stimulatory

1 This investigation has been supported by USPHS Grant

AM-02672.2 Present address: Institute of Hormone Biology, Syntex Re

search Center, Stanford Industrial Park, Palo Alto, Calif.3 The abbreviations used are : ACTH, adrenocorticotropin;

TPNH, reduced triphosphopyridine nucleotide; TPN, triphosphopyridine nucleotide ; 3‘,5,-AMP, adenosine-3' ,5'-monophosphate; LH, luteinizing hormone; FSH, follicle-stimulating hormone; HCG, human chorionic gonadotropin; G-6-P, glucose6-phosphate; G-6-P DHase, glucose 6-phosphate dehydrogenase.

action of ACTH omi corticoid output by the quarteredglands. If they incubated the prefrozen glands withTPN and glucose 6-phosphate, they observed more corticoid production than with ACTH stimulation of unfrozenglands. ACTH addition to TPN and glucose 6-phosphateproduced no further increase in corticoid output. Theysuggested that the results of the freezing mimicked theACTH effect.

Haynes and Berthet (5), from observations obtained inbovine adrenal cortex slices, advanced the suggestion thatACTH acted by activating adrenal phosphorylase andthat the glucose 6-phosphate resulting from this activationwas metabolized via the hexose monophosphate shunt.The TPNH thus generated increased the rate of steroidogenesis. This was modified by further observations (4)which showed that 3' , 5'-AMP (adenosine-3' , 5'-monophosphate) was increased by ACTH and this in turnstimulated phosphorylase activity. These observationswere confirmed by Haynes et al. (6) for rat adrenal quarters. Similarly, Marsh and Savard (13) reported thatluteinizing hormone (LH) stimulated phosphorylase activity in bovine corpus luteum slices concurrently withstimulation of progesterone synthesis.

Ferguson (2) has suggested that ACTH acts by stiniulating the synthesis of a specific protein which is essentialfor the adrenal to increase steroid output. This is basedon in vitro studies in which puromycin, an inhibitor ofprotein synthesis, was shown to cause parallel inhibitionsof both amino acid-'4C incorporation into adrenal proteinand of corticosteroid synthesis in response to ACTH.

McKerns (14) has presented evidence to show that theprincipal mechanism by which ACTH stimulates adrenalcortical function is by the activation of glucose 6-phosphatedehydrogenase which, in the presence of added glucose6-phosphate and TPN, results in the elevated levels ofTPNH essential for steroid hydroxylations and corticoidproduction. One of the most interesting aspects of thiswork was that ACTH could be shown to stimulate corticoidproduction in a cell-free system. In the latter respect itconfirms a report from our laboratory (9), which is thesubject of the discussion to be presented here, demonstrat

1076

A Mechanism of Action of the Gonadotropins'

ENRICO FORCHIELLI,2 RALPH I. DORFMAN, SHOGO ICHII, AND K. MENON

(Worcester Foundation for Experimental Biology, Shrewsbury, Alassachusetis)

SUMMARY

Results are presented from studies with a soluble bovine corpus luteum and a rattestis mitochondnial preparation which have the ability to cleave the sidechain ofcholesterol. These results show that the pituitary hormones which influence steroidogenic organs, exert at least one of their actions by affecting a specific step in the sequence from cholesterol to pregnenolone—namely, 20a-hydroxylation. Furthermore,this action can take place at the subcellular and, presumably, enzymatic level.

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30

FORCHIELLI et al.—MechaniSfli of Action of the Gonadotropins 1077

HO1OI@@@TEROL

MATERIALS AND i\IETHODS

The methods used for enzyme preparation, deterniination of rate of side-chain cleavage, and characterizationof products have been described elsewhere (9) and will beoutlined here only briefly.

The emizyme preparation was obtained from an acetonepowder of a cell-free homogenate of bovine corpora lutea.The acetone powder was suspemided in 0.066 M phosphatebuffer and centrifuged at 105,000 X g for 30 nun to remove insoluble material; the supernatant fluid served asthe enzyme source.

The rate of side-chain cleavage was determined bymeasuring the aniount of radioactivity liberated as isocaproic acid from incubated substrates labeled with ‘@Cin the sidechain at C-22 or C-26. The isocaproic acidwas isolated by steam distillation of incubation extracts.The C-21 products, pregnenolone and progesterone, wereisolated froni extracts of incubations with ring-labeledcholesterol-4-'4C.

In order to establish a nelatiomiship between the steamvolatile product, isocaproic acid, and the C-21 products,progesterone amid pregnenolone, both ring- and side-chainlabeled substrates were incubated as a mixture.

Follicle-stimnulating hormone, FSH (NIH-FSH-Sl), andluteinizing hormone, LH (NIH-LH-Bl), were obtainedfrom the Endocrinology Study Section of the NIH. The

INCUBATION TIME - minutesCHART 2.—The effect of added FSH on the rate of cholesterol

side-chain cleavage. Each flask contained 5.0 ml of acetonepowder extract (prepared by extracting 500mg of acetone powder,batch No. 9—13,with 11ml of 0.066 Mphosphate buffer at pH 7.2),15 mg of TPN, 30 mg of glucose 6-phosphate, 10 units of glucose6-phosphate dehydrogenase dissolved in 0.5 ml of 0.1 M MgCl,,4.5 ml of 0.154 MKC1, and 1.5 X 10@cpm of cholesterol-26-'4C in1.0 ml of propylene glycol. To 1 flask was added 1 mg of solidFSH, and it was incubated at room temperature. Two-ml aliquots of each incubation mixture were taken at the times indicatedfor estimation of isocaproic acid.

60 90

ft20a - HYDROXY

CHOLESTEROL22 - HYDROXY

/ CHOLESTEROL

HOX6@<HOJZ16@<

20a, 22e-DIHYDROXY

CHOLESTEROL20a- HYDROXY,

22-KETOCHOLESTEROL

@, 00

xc6@÷Hk<HO@ ISOCAPRO

PREGNENOLONE ALDEHYDE

CHART 1.—A scheme for the biosynthesis sequence from cholesterol to pregnenolone.

ing that the gonadotropins can exert an action at thesubcellular and, presumably, at the enzymatic level.

Our contribution to the over-all problem—how theprotein hormones act—centers on the enzymatic sequencefrom cholesterol to pregnenolone in gonadal tissue andthe influence of the gonadotropiri3 on this complex of reactions. From studies using a soluble cholesterol sidechain-cleaving enzyme system prepared from bovine corpusluteurn, evidence will be presented which strongly mdicates that the gonadotropins can exert an action at theenzymatic level influencing the rate of cholesterol cleavageto pregnenolone, and that this effect is exerted at a specificstep in the sequence from cholesterol to pregnenolone.

Cleavage of the cholesterol side chain is one of the majorreactions leading to the biosynthesis of the steroid hormones. The cleavage reaction is common to all steroidproducing tissues and, it also seems certain, so are theenzymatic events leading to the cleavage of cholesteroland resulting in the formation of pregnenolone. Thescheme for this biosynthetic sequence is presented inChart 1. Cholesterol is hydroxylated at C-20 to yield 20crhydroxycholesterol, followed by hydroxylation at C-22 toyield the dihydroxycholesterol intermediate, which is thencleaved to pregnenolone and isocaproaldehyde, and thelatter fragment is then rapidly oxidized to isocaproic acid.The possibility exists that the 1st hydroxylation couldtake place at C-22 and be followed by hydroxylat.ion atC-20 and then side-chain cleavage, since the 22-hydroxycholesterol can serve as an intermediate. However, theC-22 hydroxycholesterol has never been isolated and char

HO@L@

ISOCAPROIC

ACID20

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x

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0UiI-.4Ui-i

0

C)4

C)0a-4C)0Cl)

NO

2

8

4

actem-ized from a biologic system.

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Co@.pus Wt. (gm) OFLUTEUM No. cozpus LUTEUMIsoc@paoic

ACIDLIBERATED (cpm)STIMULATION

(%)NoneWithFSH1

23456789

104.0

4.06.53.73.32.92.75.06.84.71,140

6,77016,20034,700

3,760

1,5504,00046,4003,2301,3501,140

9,47022,40043,300

4,140

1,7108,7904,4103,2802,0400

39.737.925.010.010.0

119.800

51.3

1078 Cancer Research Vol.25, August 1965

0xE0.C)

0UiI-4a:Ui—J004C)0a:a.400U)

TABLE 1VARIABILITYIN CLEAVAGEENZYMECONTENT ANDRESPONSE TO

ADDED LH OF limIvIDuAL CORPORA LuvzmA'

C

E.0

4

C

00

I

00In

II—U4

0

ICl)U.C

00

I-J

CCCz

a In addition to 200,000 cpm of cholesterol-26-―C, each incubation flask contained 30 mg of acetone powder suspendedin 2.0 ml of 0.066 M phosphate buffer (pH 7.2), 1.5 mg of TPNand 100 @gof LII each in 0.1 nil of 0.066 M phosphate buffer, and0.1 ml of 0.1 MMgC1,. Incubation time was 1 hr at 37°Cin air.

ence of added FSH. Chart 3 graphically represents resuits which demonstrate that this enzyme system respondsspecifically to gonadotropins. Addition of FSH, LH, andHCG all brought about nearly a 2-fold increase in rate,whereas ACTH produced a questionable 12 % increaseover control values and crystalline plasma albumin hadno effect whatsoever. Chart 4 illustrates the dependencyof the rate of side-chain cleavage on the concentration ofadded gonadotropin. As little as 1 mg/jig of added LHincreased the rate 40 % and, at the maximum concentration of LH used, the rate was increased 150 % or 2.5 timesover the control values.

One of the stumbling blocks in this study was the lackof reproducibility from enzyme preparation to enzymepreparation with respect to both basal levels of cholesterolside-chain-cleaving activity and response to in vitro addition of gonadotropins. This situation is very well illustrated in Table 1, which summarizes the results obtainedwith enzyme preparations made from 10 individual bovinecorpora lutea collected at time of slaughter. It can bereadily seen that the variability in rate of side-chaincleavage and response to added gonadotropin is quitegreat. This can in part explain why an acetone powderprepared from a pool of randomly collected corpora luteamay on the one hand possess an adequate complement ofside-chain-cleaving activity while on the other hand itshows little or no response to added gonadotropin. Undoubtedly, the age of the corpus luteum is an importantfactor and presents a problem which needs to be investigated.

Another factor which complicated the picture furtherwas the influence of the cofactor make-up not only on therate of side-chain cleavage but also on the effect of addedgonadotropin on this rate. Table 2 shows that TPN plusa TPNH-generating system and TPNH alone were, surprisingly, much less effective than TPN alone as cofactorsfor the side-chain cleavage system. Although all 3 systems responded to added FSH, the one with TPNH alone

2

0-

8-

6

4.

2

0@

CHART 3.—Effect of addition of various tropic hormones on therate of side-chain cleavage of cholesterol. Each flask contained50 mg of acetone powder extract, batch No. 9—13,in 2.0 ml of0.066 M phosphate buffer (pH 7.2), 2.3 X 10' cpm of cholesterol26-'4C, 3 mg of TPN, 6 mg of glucose 6-phosphate, and 2 units ofglucose 6-phosphate dehydrogenase in 0.1 ml of 0.066 Mphosphatebuffer (pH 7.2) and 0.1 ml 0.1 MMgCl,. Incubation time was 1 hrat 37°Cin air.

,@__ --- - . - .-

CHART 4.—Effect of increasing concentrations of LH on the rateof cholesterol side-chain cleavage. Each flask contained 28 mgof acetone powder extract, batch No. 10-15, suspended in 2.0 mlof 0.066 Mphosphate buffer. All other additions and conditionswere the same as described for Chart 3.

human chonionic gonadotropin (HCG) used was SquibbFollutein Forte and the adrenocorticotropin (ACTH,a@+ cr2) was obtained from Dr. Paul H. Bell of LederleLaboratories.

EXPERIMENTAL AND RESULTS

Chart 2 illustrates the results obtained in a typicalstudy of the rate of cholesterol cleavage by the enzymepreparation in the presence and absence of added FSH.The rate of reaction is significantly increased in the pres

0

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0UiI.-4a:Ui

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Ca2

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Cofactors addedFSH added(;sg)Isocaproic

acid(cpm) liberated%

Stixnulation3

mg TPN, 6 mg G-6-P,2@None10,600—G-6-PDHase5

255012,400

13,60014,50018

30383mgTPNNone

5255025,400

36,20041,50043,300—

2747

53100@igTPNHNone

5

25509,160

18,40024,70027,600—

102172203

Co@;roaISOCAPROICACID

LIBERATED(cpm)STIMULATION(%)No

FSR50 @gFSHTPNH

1030100200500

10003000

TPN100500100030003,280

7,52011,40016,90021,500

34,10029,300

5,73017,00017,70024,4003,180

15,30027,90028,60028,000

32,60028,300

5,20013,30019,60034,2000

1011547130

00

—9—211241

FORCHIELLI et al.—Mechanism of Action of the Gonad otropins 1079

TABLE 2EFFECT OF FSH ON CHOLESTEROL SIDE-CHAIN CLEAVAGE IN

THE PRESENCE OF TPN ALONE, TPNH ALONE, AND TPNPLus A TPNH-GENERATING SYSTEM@'

TPNH levels are increased, this interaction takes placeby sheer mass action, thus supplanting the role of FSH.

Table 4 summarizes the results obtained in an attemptto correlate the isocaproic acid determined with theamount of ring-labeled products, progesterone and pregnenolone, isolated from incubations with and withoutadded FSH. The increase in isocaproic acid formation inthe presence of added FSH is paralleled by a concomitantincrease in progesterone and pregnenolone formation. Inthe presence of the lower leveLs of added TPNH, there isexcellent agreement, on a molar basis, between the amountof isocaproic acid liberated and the sum of the pregnenolone and progesterone isolated. At the higher leveLs ofTPNH, the sum of the pregnenolone and progesterone is20—25% less than the isocaproic acid formed. This ismost likely due to the formation of other 0-21 productsin the presence of higher levels of TPNH. However, theorder of magnitude of the stimulation by FSH is the samefor both the C-21 products and the isocaproic acid. Theabsolute counts differ because the ring-labeled cholesterol,which would be reflected in the pregnenolone and progesterone isolated, had a specific activity 1.4 times that ofthe side-chain-labeled cholesterol.

Table 5 summarizes the results obtained when the ratesof cleavage of cholesterol and 20cr-hydroxycholesterolwere compared in the presence and absence of added LH,with the most interesting result that the rate of cholesterolside-chain cleavage is stimulated by LH, whereas thehormone had no effect on the rate of 20a-hydroxycholesterol cleavage. Although not indicated here, LH similarly had no influence on the rate of cleavage of the 20,22-dihydroxycholesterol. The amount of radioactivity ofthe respective substrates differed because the 20cr-hydroxycholesterol had a much lower specific activity than thecholesterol. However, the actual concentrations incubated of the 2 substrates were comparable. These datastrongly suggest, then, that the rate-limiting step in theover-all reaction sequence from cholesterol to pregnenolone is 20cr-hydroxylation, and it seems to be preciselythis step that is influenced by the gonadotropins.

Cholesterol side-chain cleavage in rat testis mitochondrialpreparation.—The above observations are supported byanother line of investigations carried out in our laboratorieswith a rat testis enzyme preparation: they attempt todelineate the manner in which the gonadotropins effectthe various steps in the biosynthetic sequence from cholesterol to pregnenolone in this tissue.

The analytic technics used for this part of the studywere essentially the same as those employed in the bovinecorpus luteum studies. The source of cholesterol sidechain-cleaving enzyme was the washed mitochondrialfraction of rat testis homogenate.

Effect of pretreatnwnt of immature mate rats with humanchorionic gonadotropin.—The effect of pretreatment ofimmature male rats with HCG on the in vitro rate ofcholesterol side-chain cleavage is illustrated in Chart 5.It can be seen that with the lowest dose of HCG therewas a 3-fold increase in the rate of cholesterol side-chaincleavage, as indicated by the increase in isocaproic acidformation, and this rate was further augmented with in

a Each incubation flask contained 33 mg of acetone powder(2—25)suspended in 2.0 ml of 0.066 M phosphate buffer (pH 7.2),cofactor and FSH each dissolved in 0.1 ml 0.066 M phosphatebuffer, 0.1 ml 0.1 MMgC12, and 200,000 cpm of cho1esterol-26-'@C.Incubation time was 30 mm at 37°Cin air.

TABLE 3

EFFECT OF FSH ON RATE OF CHOLESTEROL SIDE-CHAIN CLEAVAGE

IN THE PRESENCE OF INCREASING CONCENTRATIONS

OF TPNH AND TPNa

a Each incubation flask contained 33 mg of acetone powder

suspended in 2.0 ml of 0.066 Mphosphate buffer (jll 7.2), cofactor and FSH each dissolved in 0.1 ml of 0.066 M phosphatebuffer, 0.1 ml of 0.1 MMgCl,, and 200,000cpm of cholesterol-26-―C.Incubation time was 30 mm at 37°Cin air.

showed the greatest response, even though the TPNHsystem does not quite approach the maximal leveLs in rateof side-chain cleavage achieved when TPN alone is added.Table 3 shows another interesting relationship betweenFSH and the TPNH system; as the TPNH concentrationis increased, the FSH effect diminishes, although the totalside-chain cleavage rate continues to increase until maxima! leveLs are reached. This seems as if, at limiting levelsof TPNH, the FSH in some way effects an interactionbetween TPNH and the enzyme system, but, as the creasing levels of administered gonadotropins. These re

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Cofactor addedIsocaproic acid (cpm),imoles formed―Pregnenolone@ Progesterone(cpm)@ (cpm)@mo1es

formecV'TPNH,

5O@gTPNH, 50,@g+ FSH, 50i.sgTPNH, 200 @gTPNH, 200 @g+ FSH, 50;hg7,400

12,40012,30018,7005

X 1O@8 X 10@8 X 10@

1.2X 10@8,650

@ 4,10013,600@ 7,7207,@0@ 7,84011,200@ 12,3005.3

X 10@8.4X 10@6.3 X 10@9 X 10@

SUBSTRATEIsocAPRoIc

ACID(cpm) LIBERATEDFROM:Cholesterol

26-'4C (200,000cpm incubated)Hydroxycholesterol

22-'4C-20a-(30,000cpmincubated)TPNH,100,sg

TPNH, 100 @ig+ 200 @zgLHTPNH, 300 @gTPNH, 300 @g+ 200 @sgLH4,000

6,5008,000

10,4001960

217035803600

1080 Cancer Research Vol. 25, August 1965

TABLE 41)ETERMINATION OF PRODUCTS FROM RING-LABELED AND SIDE-CHAIN-LABELED CHOLESTEROL:

INFLUENCE OF ADDED FSH AND TPNH ON YIELD

a Calculated from the sum of progesterone and pregnenolone formed. In addition to 1.96 X 10@ cpm

of cholesterol-26-'4C and 2.1 X 10' cpm of cholesterol-4-'4C, each incubation flask contained 36 mg of

acetone powder (3—25) suspended in 0.066 M phosphate buffer and 0.1 ml of 0.1 M MgCl2. Incubation

timewas 30mm at37°Cinair.

suits suggested a pronounced control by the gonadotropinson the over-all sequence involved in the cholesterol sidechain cleavage. There was no observable effect on thisreaction rate after administration of either LII or FSHto intact immature animals. However, when LH andFSH were mixed in a ratio of 80 :20 and the mixture administered at the rate of 100 .zg for 7 days, there was a33% increase in the in vitro rate of cholesterol side-chaincleavage by the testis mitochondrial preparaton of thetreated rats as compared to the untreated group. Thiscould be explained on the basis of synergisni betweenFSH amid LII (Chart 6).

In contrast to the intact animals, the hypophysectomized rats readily responded to LII and FSH pretreatment. Twenty-one day-old hypophysectomized male ratswere treated daily for 4 days with a total dose of 240j.@gof LII. At the end of the period, the washed testisniitochondrial preparation was prepared and incubatedwith cholesterol-26-'4C arid the rate of side-chain cleavagewas determined. The results show that pretreatmentwith LH enhanced the ability of the rat testis mitochondrial

0 70 140 280 560 700HCG l.U.

CHART 5.—Response of the cholesterol side-chain-cleaving eiszyme of the immature rat testis mitochondrial preparation to in vivo HCG treatment. Each

vessel contained 2 X 10@cpm of cholesterol-26-'4C in 0.05 ml of propylene, 2 mgof TPNH in 0.1 ml of 0.066 M phosphate buffer (pH 7.2), 0.8 ml of buffered 0.25 Msucrose suspension of the mitochondrial enzyme preparation corresponding to 1

gm (wet weight) of tissue, and 0.1 ml of 0.1 M MgCl2. The final volume of theincubation mixture was 2.0 ml, and incubations were carried out in air for 2 hr at

37°Cwith constant shaking.

TABLE 5

Co@IPARIsoN OF LH EFFECT ON RATE OF SIDE-CHAIN CLEAVACE

OF CHOLESTEROL AND 20a@HYDROXYCHOLESTEROLa

a Incubation conditions : each vessel contained 30 mg of acetone powder suspended in 2.0 ml of 0.066 M phosphate buffer

(PH 7.2), 0.1 ml of 0.1 M MgCl2 and TPNH dissolved in 0.1 ml0.066 M phosphate buffer. Incubation time was 30 mm at 37°C

inair.Although the specific activity of cholesterol-26-'4C was 14

times greater than that of 20a-hydroxycholesterol-22-'4C, the

actual concentrations of the 2 substrates used in the incubationswere comparable.

6

5

5R40

C)4

0@

0

5.4o20a)

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FORCHIELLI et al.—Mechanism of Action of the Gonadotropins

C)4C)

0

0.0C)0U,

0LH @ag

FSH @g

1081

Effect of treatment of immaturerats with LH and FSH for 7 dayson the in vitro cleavage ofcholesterol side chain

P

Effect of 7 days treatmentwith combination of LHand FSH on the in vitrocholesterol side chaincleavage in immature

rats

3

2

075000700@g00350LH'FSH(80'20)

CHART 6.—A. Effect of treatment of immature rats with LII and FSH for 7days on the in vitro cleavage of the cholesterol chain. B. Effect of 7 days'treatment with combination of LH and FSH on the in vitro cholesterol chain

cleavage in immature rats. The additions and conditions for incubation werethe same as for Chart 5.

treatment with LH at a dose level of 100 j@gdaily for 4days, the cleavage activity was enhanced 9-fold over theuntreated hypophysectomized control. FSH also supported the cholesterol-cleavage reaction in hypophysectomized animals although much less efficiently than LIIdid (Charts 9, 10).

It was interesting at this point to compare relative ratesof cleavage of cholesterol-26-'4C and 20a-hydroxycholesterol-22-'4C by immature and mature rat testis preparations. The rates of cleavage of cholesterol by the maturerat testis was 3 times greater than that of the immaturerat testis preparations. However, the rates of cleavageof 2Ocr-hydroxycholesterol were essentially the same inboth the mature and immature rat testis preparations.It should be especially noted that the rate of 20a-hydroxycholesterol side-chain cleavage was manyfold greater thanthat for cholesterol (Chart 11). This suggested verystrongly that, as in the case of the corpus luteum, therate-limiting step in the over-all sequence between cholesterol and pregnenolone might well be the 20a-hydroxylation step, and it may be this step that is influenced bythe gonadotropins. To test this hypothesis, 25 day-oldmale rats were treated daily for 7 days with a total doseof 700 IU of HCG. At the end of this period a washedtestis mitochondrial fraction was incubated separatelywith cholesterol-26-'4C and 2Ocx-hydroxycholesterol-22-‘4C,and the rate of cleavage of the respective substrateswas deterniined. The results show that pretreatmentwith HCG enhanced the cleavage of the cholesterol sidechain 6-fold over the saline controls, with no effect fromHCG pretreatment on the rate of 20a-hydroxycholesterolside-chain cleavage (Chart 12). This clearly demonstrates that the gonadotropin is exerting its influence atthe 2Oct-hydroxylation step, which is then the rate-limitation step in the over-all sequence from cholesterol topregmienolone. These data support the results obtainedabove with the bovine corpus luteum preparation.

4@

3.

2

a2•0

C)4C)

0

0.0C)0U,

(Mean of

0Control Hypox LH

LH @g 0 0 60/day

CHART 7.—Effect of 4 days' LH treatment in hypophysectomizedimmature rats on in vitro cholesterol side-chain cleavage. Incu

bation conditions and additions were the same as for Chart 5.

preparatioli to cleave the cholesterol side chain 10-foldover the preparation obtained from untreated controlanimnals. This increased mate brought the cholesterolcleavage reaction to a level comparable to that of normalintact immature animals (Chart 7). Examination of theventral prostate weights revealed a maximumu response tothe goiiadotropin administration indicating increased androgen output (Table 6).

Hypophysectomy had a remarkable effect on the rateof cholesterol side-chain cleavage in the testis preparationfrom mature animals. Three days after hypophysectomythe cleavage reaction had dropped to@ of that of the intact mature animals, and 7 days after hypophysectomythis rate was further reduced (Chart 8). On subsequent

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No. of ratsCortisol (pg)Luteinizing hor-@ Testicular weightmone (jig)@ (mg) E S.D.Ventral

prostate I Seminal vesicles(ing)@ S.D.10

10200 2000@ 186.8 ±23.0

240@ 290.4± 67.29.68± 1.33 I@ ±@

32.44± 8.37@ 7.56± 2.57

1082 Cancer Research

TABLE 6

Vol. 25, August 1965

EFFECT OF 4-DAY LH TREATMENT ON VENTRAL PROSTATE AND SEMINAL VESICLE WEIGHTS INIMMATURE HYPOPHYSECTOMIZED RAT5a

a Luteinizing hormone (NIH-LH-Sl) and cortisol were administered in 4pophysectomized male rats (23 days old) were employed for the assay.

divided doses. fly

7

6

U

•0U

4

U

05.0.0U0U,

Hypox 3 daysFSH /@L9

Effect of hypophysectomy oncholesterol side chain cleavingactivity in mature rat tests.

Substrate cholesterol-26—C14 2

0cr4

@0

C)4C)

0

CC)0U,

@ I+ +

0 100/day

CHART 10.—Effect of 4 days' FSH treatment in hypophysectomized mature rats on the in vitro cholesterol sidechain cleavageactivity of the testis mitochondrial enzyme preparation. Incubation conditions and additions were the same as for Chart 5.

Intact 3 7IMMATURE TESTIS

1

MATURE TESTIS50

40

30

20

10

Days after hypophysectomy

rCHART 8.—Effect of hypophysectomy on the cholesterol sidechain-cleaving activity in mature rat testis. Incubation conditions and additions were the same as for Chart 5.

5@

4

0‘4.

@0

C)4U

0

0.0U0

U1:

(54

(5

05.

0.0U0a)

C-26-'4C I.3X10520a-C-22- ‘4C 0

0 l.3XI05lXlO4 0

0 CPMlXlO4 CPM

2 CHART 11.—Conversion of cholesterol-26-'4C (C@26@14C) and2Ooc-hydroxycholesterol-22-'4C(20aC.22@14C)to isocaproic acid byimmature and mature rat testis mitochondrial enzyme preparations. Incubation conditions and additions were the same as forChart5.

DISCUSSION

Most attempts to explain the action of the proteinhormones influencing the steroidogenic tissues seem tomerge to a common conclusion, which is that these hormones function by making TPNH available for the vanous hydroxylation steps involved in steroid biosynthesis.If this were so, then one would expect the individual

Hypox 7 daysLH @g

+0

+100/day

CHART 9.—Effect of 4 days' LH treatment inhypophysectomizedmature rats on the in vitro cholesterol side-chain cleavage activityof the testis mitochondrial preparation. Incubation conditionsand additions were the same as for Chart 5.

@PL@i@L

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FORCHIELLI et al.—Mechanism of Action of the Gonad otropins 1083

synthesis of 14C-labeled steroids by endocrine tissue invitro is lower than that of the steroids isolated from thesame tissue. Savard and Casey (15) have concludedfrom studies on the effect in vitro of LII and ACTH onbovine corpus luteum and adrenal slices, respectively,that these hormones stimulate steroid biosynthesis fromsmall-molecule precursors and not from cholesterol,whereas TPNH stimulated the conversion of cholesterol toprogesterone in the corpus luteum slices and to cortisoland corticosterone in the adrenal slices. It is interestingto note in the data from their more extensive corpus luteumstudies that the de novo synthesis of progesterone fromcholesterol in the presence of TPNH was in all instancessignificantly greater than from acetate-'4C stimulated by

____@.LII.Onemightfacetiouslyquestiontherelativeilupor700 tance of LH in the biosynthesis of progesterone in face of

0 the much greater effectiveness of TPNH. This may on+ the one hand point to the limiting nature of available

TPNH in the acetate-'4C-LH system, but on the otherhand one cannot ignore the wide divergence in time relationship in the biosynthetic sequence between acetate amidprogesterone as compared to that between cholesterol andprogesterone. In addition one must remember the littleunderstood role of the cholesterol pools which may beavailable under the 2 different sets of conditions. Thesetaken together may have a profound effect on the results,making them more apparent than real.

Then again, the report of Knum et al. (11) lends support to the view that cholesterol is the obligatory intermediate in the biosynthesis of adrenal steroids. They feddogs a low-cholesterol synthetic diet containing cholesterol-4-'4C for periods of 31—45days until the specificactivity of free plasma cholesterol had plateaued. Theythen compared the specific activity of the plasma-freecholesterol with that of adrenal-free cholesterol and cortisol, Reichstein's 5, and corticosterone before and afterACTH stimulation. They found that the specific activityof the plasma cholesterol, adrenal-free cholesterol, and theadrenal steroids were identical, and ACTH did not effectany change on the specific activities of these substances.These data indicated that cholesterol is an obligatoryprecursor in the biosynthetic pathway of adrenocorticalsteroids in the dog. They also indicated that the adrenalfree cholesterol either exchanges very rapidly with plasmacholesterol after synthesis in situ or is derived from plasmacholesterol. It is apparent also from these results thatACTH had not altered the biosynthetic pathway by bringing in nonradioactive, noncholesterol precursors. Hadthis occurred, the specific activities of the isolated hormones would have been lowered after ACTH treatment.

The results from our own studies reported here notonly support the view that these tropic hormones do havean action between cholesterol and pregnenolone, but alsopinpoint this particular action of these hormones at thelevel of a specific enzymatic step, namely 20a-hydroxylation. 1\iost interesting is the fact that this influence takesplace at the subcellular and, presumably, at the enzymaticlevel.

The fact that these tropic hormones can exert an actionat the enzymatic level has certain implications. It should

0‘4.

@0

(5

4 3@•

C)

05.0.0 20C)0.!f!

10,

HCG (l.U.) 0C-26--14C +2OaC-22-14C 0

700+0

CHART 12.—Influence of HCG treatment (1 week) on the invitro sidechain cleavage of cholesterol-26-'@C (C-26-'4C) and20a-hydroxy-cholesterol-22-'@C (20a-C-22-'4C) by the testis mitochondrial enzyme preparation. Incubation conditions and additions were the same as for Chart 5.

hydroxylation steps, such as at C-i 1, C-17, and C-21, tobe stimulated by the tropic hormones. However, thelack of definitive evidence to favor such a conclusionmakes one doubt that this is a primary action of ACTH.Similarly, increasing cell permeability need not necessarily pertain, since McKerns (14) has shown that ACTHdoes stimulate corticoid production in cell-free adrenalhomogenate preparations. This is in agreement with theobservations reported by us (9) on the effect of the gonadotropins on a soluble bovine corpus luteum preparation.

Ferguson (2) proposes the attractive hypothesis thatACTH acts on the adrenal by stimulating the synthesisof a protein which is essential for increased synthesis ofcorticoids. However, stimulation of de novo synthesis ofprotein paralleling increased corticoid production has notbeen established.

There are several lines of evidence which favor the viewthat these tropic hormones stimulate steroid hormonebiosynthesis by acting on steps between cholesterol andpregnenolone. Stone and Hechter (16) postulated thatone of the actions of ACTH was to activate an earlystep in the biosynthetic sequence between cholesterol andpregnenolone. In their studies on the perfusion of radioactive precursors through the isolated bovine adrenalgland, they were able to demonstrate that addition ofACTH to the perfusion media stimulated biosynthesis ofcorticoids from cholesterol but not from progesterone.It has been well established in vivo in the rat (12) thatACTH can reduce the total adrenal cholesterol by 50%.Pretreatment of male rats with gonadotropins results inan enhanced ability of the testis to cleave cholesterol invitro (3). Treatment of female rats with gonadotropinsbrings about a significant drop in ovarian cholesterol (1).

However, the obligatory nature of cholesterol on thebiosynthesis of the steroid hormones has been questioned.Hechter et al. (8) observed that the specific activity ofthe cholesterol formed from acetate-'4C during the bio

00+

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1084 Cancer Research

be possible,then, to isolateand purify the sensitive enzymesystems and carry out detailed studies of the complex interaction between enzyme, substrate, cofactor, and proteinhormone in isolation. Such studies could then providemodel systems for the study of other aspects of hormoneaction.

REFERENCES

1. Bell, E. T., Mukerji, S., arid Loraine, J. A. A New BioassayMethod for Luteinizing Hormone Depending on the Depletionof Rat Ovarian Cholesterol. J. Endocrinol., 58: 321—28,1964.

2. Ferguson, J. J., Jr. Protein Synthesis and Adrenocorticotrophin Responsiveness. J. Biol. Chem., @S8:2754—59,1963.

3. Hall, P. F., and Eik-Nes, K. B. The Influence of Gonadotrophins in Vivo upon the Biosynthesis of Androgens byHomogenate of Rat Testis. Biochim. Biophys. Acta, 71: 438—47, 1964.

4. Haynes, R. C., Jr. The Activation of Adrenal Phosphorylaseby the Adrenocorticotrophic Hormone. J. Biol. Chem., 533:1220-22, 1964.

5. Haynes, R. C., Jr., and Berthet, L. Studies on the Mechanismof Action of the Adrenocorticotrophic Hormone. Thid., 555:115—24,1957.

6. Haynes, R. C., Jr., Koritz, S. B., and Peron, F. G. Influenceof Adenosine 3',S'-Monophosphate on Corticoid Production byRat Adrenal Glands. Ibid., 234: 1421—23,1959.

7. Hechter, 0., and Lester, G. Cell Permeability and HormoneAction. Recent Progr. Hormone lies., 16: 139—86,1960.

8. Hechter, 0., Solomon, M. M., Zaffaroni, A., Pincus, G., Scully,E., and Gobeil, E. Transformation of Cholesterol and Acetateto Adrenal Cortical Hormones. Arch. Biochem. Biophys., 46:201—14, 1953.

9. Ichii, S., Forchielli, E., and I)orfman, R. I. In Vitro Effect ofGonadotrophins on the Soluble Cholesterol Sidechain CleavingEnzyme System of Bovine Corpus Luteum. Steroids, 5: 631-56, 1963.

10. Koritz, S. B., and Peron, F. G. Studies on the Mode of Actionof the Adrenocorticotrophic Hormone. J. Biol. Chem., 280:343—52,1958.

11. Krum, A. A., Morris, M. D., and Bennett, L. L. Role of Cholesterol in the in Vivo Biosynthesis of Adrenal Steroids by theDog. Endocrinology, 74: 643-47, 1964.

12. Long, C. N. H. The Conditions Associated with the Secretionof the Adrenal Cortex. Federation Proc., 6: 461—71,1947.

13. Marsh, J. M., and Savard, K. The Activation of Luteal Phosphorylase by Luteinizing Hormone. J. Biol. Chem., 289: 1—7,1964.

14. McKerns, K. W. Mechanism of Action of AdrenocorticotrophicHormone through Activation of Glucose-ti-phosphate 1)e-hydrogenase. Biochim. Biophys. Acta, 90: 357—71,1964.

15. Savard, K., and Casey, P. J. Effect of Pituitary Hormones andNADPH on Acetate Utilization in Ovarian and AdrenocorticalTissues. Endocrinology, 74: 599—610,1964.

16. Stone, D., and Hechter, 0. Studies on ACTH Action in Perfused Bovine Adrenals : The Site of Action of ACTH in Corticosteroidogenensis. Arch. Biochem. Biophys., 51: 457—69,1954.

Vol. 25, August 1965

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1965;25:1076-1084. Cancer Res   Enrico Forchielli, Ralph I. Dorfman, Shogo Ichii, et al.   A Mechanism of Action of the Gonadotropins

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