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3β-Hydroxysteroid dehydrogenaseΔ5−4isomerase activity in the rhesus monkey placenta and fetal adrenal, testis and ovary during late gestation
757
3125 36-HYDROXYSTEROID DEHYDROGENASE/A5-41SOMERASE ACTIVITY IN THE RHESUS MONKEY PLACENTA AND FETAL ADRENAL, TESTIS AND OVARY DURING LATE GESTATION Samuel A. Sholl Wisconsin Regional Primate Research Center, Madison, WI USA 53715-1299 Received:
6-6-83 ABSTRACT
3$-Hydroxysteroid dehydrogenasela 5-4isomerase (3B-HSDH) was measured in the rhesus monkey (Macaca mulatta) placenta, fetal adrenal (whole organ minus medulla),testis and ovary during late gestation (Days145-162). Activities were evaluated from the conversion of C3H]pregnenolone to [3H]progesterone. The maximum enzyme velocity (Vm) in adrenal microsomes (100,000 g pellet) was significantly higher (146 nmoles progesterone/h x mg-Iprotein) than in microsomes from the other tissues. Testicular Vm was greater than either ovarian or placental Vm which were not different from one another (11.5 versus 1.9, 1.2 nmoles progesterone/h x mg-Iprotein, respectively). Apparent MichaelisMenten constants in the adrenal, placenta, testis and ovary averaged 1.8, 2.5, 0.27 and 0.16 PM, respectively. In some cases, substrate inhibition was noted. Estimated dissociation constants for pregnenolone were 2.3 PM (adrenal), 2.1 uM (placenta), 0.74 PM (testis) and 0.13 1111 (ovary). 3B-HSDH was less active in a crude mitochondrial preparation from the fetal adrenal (10,000 g pellet) than in microsomes, whereas activity in the placenta and testis appeared to be equally distributed between mitochrondria and microsomes. Rate measurements were consistent with the apparent potentials of these organs to synthesize their characteristic hormones. Thus, 38HSDH activity may be an important rate determining step in hormone synthesis. The importance of substrate inhibition in progesterone formation remains to be assessed. INTRODUCTION During late gestation fetal testes and ovaries of rhesus monkeys (Macaca mulatta) synthesize testosterone (17B-hydroxy-4-androsten-3-one) ~(1,2) and estradiol-17B (1,3,5(10)-estratriene-3,17B-diol)(3,4),
re-
spectively; the placenta produces progesterone (4-pregnene-3,20-dione) (5-7), and fetal adrenals synthesize corticoids (8,9).
Several find-
ings suggest that there are substantial differences in the rates at Publication no. 22-034 of the Wisconsin Regional Primate Research Center.
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41, Number
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which these organs secrete their characteristic hormones.
For example,
the sex-difference in testosterone concentration in the fetal circulation (1) is more pronounced than the difference in estradiol-17g (3). In addition, despite the fact that the placenta is much larger in size than the fetal adrenals, the production rate of cortisol in the fetus at Day 140 (~3 mg/day) (9) is higher than the maternal production rate of progesterone at 155 days of pregnancy ('~1mg/day) (10). The A4-pathway is operative in fetal monkey gonads (4) and adrenals (ll), and this being the case, progesterone may be a precursor for both sex and corticoid hormones.
Progesterone is synthesized in turn from
pregnenolone (3B-hydroxy-5-pregnen-ZO-one) which requires the enzyme complex, 36-hydroxysteroid dehydrogenase/a5-4isomerase
(38-HSDH).
The
objective of this study was the evaluation of 38-HSDH activity in the monkey placenta, fetal adrenal, ovary and testis during late gestation to ascertain whether there are organ differences in activity which might account for the apparent dissimilarity in steroidogenic activity. MATERIALS AND METHODS Fetuses were obtained from rhesus monkeys during Caesarean section at 145-162 days of gestation. A segment ($2 g) of the primary placental disk was homogenized in 5 ml cold 0.15 M phosphate buffer (pH 7.4). Activity in the fetal adrenal was assessed in the entire gland (minus medulla), since in a recent report (12) it was suggested that the fetal cortex is remodeled to form a portion of the definitive adult cortex and thus could contain enzymes necessary for corticoid formation. A recent study (13) describing the distribution of ll- and 21-hydroxyFetal adrenals and gonads were lases supports this latter possibility. homogenized in 5-10 and 25 volumes (by weight) of phosphate buffer, respectively. Homogenates were centrifuged at 1,000 g for 20 min. Supernatants were centrifuged at 10,000 g for 15 min, and crude microsomal pellets were obtained from the resulting supernatants by additional centrifugation at 100,000 g for 1 h. Microsomes were suspended in a small volume (~5 ml) of phosphate buffer using a teflon-glass homogenizer. SB-HSDH activity was estimated by incubating microsomes (except where noted) with 0.2 PCi [7-3H]pregnenolone (19.3 Ci/mmol, New England
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TIlROIDS
Nuclear Corp., Boston, MA) in the presence of NAD and different concentrations of unlabeled pregnenolone. [4-14C]Progesterone (~~5,000 disintegrations per min, New England Nuclear Corp.) was also included to estimate the combined metabolic loss of the product, [3H]progesterone, and procedural loss during its isolation. Reactions were carried out at 37°C in air in a shaking metabolic incubator for varying lengths of time up to 1 hour. The total reaction volume was 1 ml. The concentration of NAD was 0.4 mM except where noted. Reactions were terminated by adding 0.1 ml ethanol and freezing in dry ice/acetone. Steroids were extracted once with 4 ml petroleum ether. Fifty micrograms of unlabeled progesterone were added to the petroleum ether phase which was subsequently evaporated under N2. Radioactive progesterone was isolated by silica gel G thin layer chromatography, first in the solvent system chloroform/ethyl acetate (4:1, v/v) followed by a second separation in chloroform/dioxane (94:6, v/v). Radioactivity was measured in a Searle 6892 scintillation spectrophotometer (Searle Radiographics, Des Plaines, IL) using toluene-Liquifluor (New England Nuclear Corp.) as the scintillation fluid. After correcting for counting efficiency, activity was expressed as disintegrations per min (dpm). When no substrate i:lhibition was noted, maximum reaction rates (Vm) and apparent Michaelis-Menten constants (Km) were estimated from the equation (Model 2), v = V,/(l t Km/s), where v was the reactionvelocity and s the concentration of pregnenolone. When substrate inhibition was found Vm, Km and K (substrate dissociation constant) were estimated from the relationship ?Model 1), v = Vm/(l t Km/s t s/K,). Coefficient estimations were made by computer least squares analysis (14). Reactions usually were carried out using three different dilutions of the enzyme. Results were reported only when the estimated Vms from at least two of these dilutions were in agreement (computer fit SDS of Vms overlapped). Protein values were determined by the method of Lowry -2 et al (15). The identity of [3H]progesterone formed during the reaction was confirmed by recr stallization in ethanol/H20 to a constant specific activity (dpm 3H/Y 4C). Significant differences between Vms were detected by analysis of variance and Duncan's new multiple range test. RESULTS In the fetal adrenal 3~-HSDH activity was greater in microsomes (100,000 g pellet) than in mitochondria
(Table 1).
In contrast, enzyme
activity in the placenta and fetal testis appeared to be more uniformly distributed between mitochondrial and microsomal fractions.
These dis-
tributions persisted in all tissues regardless of the extent to which the enzyme was diluted.
Activity was not altered by the omission of
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SB
‘J?EI&OIDS
Table 1. 36-Hydroxysteroid dehydrogeanse/A5'4isomerase activity in different cellular fractions of monkey fetal tissues and placenta. Enzyme activity at dilution: Tissue
Fraction
Adrenal
Testis
Placenta
Protein (uglassay)
1,000 g s* 10,000 g P 100,000 g P
144** 38 2
1,000 g s 10,000 g P 100,000 g P
402 17 9
1,000 g s 10,000 g P 100,000 g P
3354 461 167
1 (nmoles $diesterone/h mg- protein)
149 60 2
1:20 x
;i 59
::
4 14 7
0.17 0.65 0.69
0.19 0.90 0.27
3 7" 0.22 0.36 0.41
*S,P = supernatant and pellet. **Amount of protein in undiluted sample.
[14C]progesterone from the incubation (data not shown). Microsomal enzyme activity in the placenta and fetal ovaries, testes and adrenals was linear throughout the one hour incubation and directly related to the amount of protein that was incubated (Fig. 1).
In all
tissues, the recovery of [14C]progesterone remained fairly con-
stant regardless of the length of the incubation, indicating that the extent of progesterone metabolism was minimal.
In some of the placen-
tal incubations and all of the adrenal and ovarian incubations (Figs. 2-4) 3B-HSDH activity was inhibited by high levels of pregnenolone, while in other placental incubations and all but one testicular incubation, substrate inhibition was not noted (Figs. 5,6). Maximum enzyme velocity (V,) was higher in the adrenal (146 nmoles progesterone/h x mg-' protein) than in the other tissues that were examined (P < 0.01) (Table 2).
Testicular V, (11.5 nmoles progesterone/h
761
(2)
(1) ,$emed @om [7-3fflpegnenoRone FQ. 1. ~mouvLt oa [7-3fflp/rogtintenone an a &wsXon ad the. amouti 06 micnodomal pxoZc.in that WM incuba&d and tie RengZh 04 Xhe incubatiqn. OXha incubaLion conditioti ahe da&bed in the texxt. FLcj. 2. Linweavetr-Bunk plot 05 rretipxocul pl.acc?nW 3$-USVtl acfivktg vm3u.h treciyJhoc& phegnnenolone concevLtha;tion. The dashed fine datiben the. q.&zion (Model 1 I which cua~j &zXed Zo the d.oXa. Tlz~~ne. ww~e 149 Hg ticxonomal pho&zin in the. one. hoti LnincubaLLon. CWWL tiaag cond.LLLonn axe da&bed in Xhe texxt. The datd poinX a.$ 9 x 1O-3 uM YJhQjnenolone & noX nhown. Eh;timatc?d V,, I$,, and Kh (&hornMod&T 1 equation) wehe 3.97 nmolalh x mg-’ pho&&, 3.67 VM and 0.55 NM phiLgnf2no~one, ntipwtivtiy. x mg-’
protein)
was higher than ovarian and placental Vms (P < 0.05)
which were not different from one another (1.9 and 1.2 nmoles progesterone/h x mg-' protein, respectively).
Apparent Kms in the adrenal,
placenta, testis and ovary were 1.8, 2.5, 0.27 and 0.16 PM, respectively, while pregnenolone dissociation constants in these tissues were 2.3, 2.1, 0.74 and 0.13 uM, respectively.
In the computation of
apparent Km and KS, estimates of these parameters were highly correlated. high.
As a consequence, SDS (from computer fits) were relatively Conversely, SDS associated with estimated V,s were considerably
62
762
TRROTDS
I ( LIM
PREGNENOLONE)
i -’
Fkj. 3. tifleweavetr-Bw~bp&A 06 nec@zoc& de_&& a&en& 3B-ff.SZXf ac;tiv.&ty vmti rre&mc~~C phqneno%ne concen;Dration. The. dashed Line de-mulben tithe equation (Mod& 1) whi_ch IWXA$Ltted to Ahe da&. Them WYL~ 2.7 ug micrroboma.Cpkottin in the one hawt incubafion. O;theh annay conditions ahe dencnibed .in Ahe Xext. EhaCima&d V,, Km and K, idhorn Model 7 eyuutlan) wme 330 nmoLu/h x tng-l pha;ttin,7.7 PM and 0.49 PM phegnnen&one, ~e.5p~&k~~y. lower.
For NAD, apparent Kms in the placenta and fetal testis were
0.13 mM and 0.84 mM (Fig. 7). In one animal, the extent of microsomal conversion of C3H]pregnenolone to hormones other than [3H]progesterone was assessed in adrenal and placental samples.
Following incubation and extraction several
times with ethyl ether, very little radioactivity remained in the aqueous phase indicating that the extent of steroid conjugation was minimal.
In addition, after the first TLC separation of the extracted hor-
mones, more than 90% of the radioactivity was located in those areas of the chromatographic plate occupied by pregnenolone and progesterone standards. The identity of the reaction product, [3H]progesterone, was confirmed by recrystallization
(Table 3).
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TElEOIDS
763
4. tine.we_aveh-BuA2 plot 06 necipk0ca-t fjeAi7L ovaLan 3f3-tlSVtl acFig. 4. tiv.iXy vmuh ~ec.ip~~oca.t pxegnenoRone conceWn. The tihed .&net dentiba Zhe eyuatin (Model 71 which utu @LaXed ti the data. Thehe
wene 0.62 pg mitio~omaL pfioXe.& in the one howr LncubaGon. Utheh ahnay conditioYLrlahe dadbed in the. Xext. EhGna;ted V,, t&,, and Kn uwtre 2.48 nmoku fh x mg-l plroZtin, 0.1 B PM and 0.07 L&Ipxegnenotone,
nehpuLiv~y. Fig. 5. tine.weavet-Bwtk p.tof 06 wx+v~ocat p.&weW 3B-HSVH acfi.vi~y veh.uh hetipk0ca.L phegnnenoRone concen&u&ion. The ~ok?.id line denuiben the e-qua&ion (Mod& 2) which UEIA I;L%Xed .to the da&~. &Lima&d Vm and Km (dhom Model 2 qua&ion) WULCZ 0.58 nmoltifh x mg-l phO;tein and 0.31 Thtie woe 72 ug micAohom~ photein in I.IMp&egnenoRone, hchpe&tiv~y. Xhe one howr .Lnc.ubation. &then ahnay conditioti ahe. de.wGbed in the. kxxt. The da& point aX 9 x 1 O- 3 UM phegnenolone AL noR 6 hown. DISCUSSION Among those organs that were examined for 38-HSDH activity, the fetal adrenal exhibited the greatest potential for metabolizing pregnenolone to progesterone.
This activity could support the formation of
corticoid hormones which are important for the normal development of a variety of fetal tissues.
In contrast to the adrenal, the placenta was
relatively deficient in 36-HSDH which may restrict progesterone synthesis in this organ.
In a comparative study of placental 38-HSDH, enzyme
levels were considerably lower in monkeys than in humans (16) offering one explanation as to why circulating progesterone values in these two species are so widely different (17,18). Initially, an attempt was made to assess 36-HSDH activity histo-
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764
TnSOIDLl
ADRENAL
(mM
F&J. 6. tinweavkx-Bwth p,tkt 06 r~ec.LpJ~ocaL 6eXiz.t ac;tivtiq vmw hec.+fiocaX pfiegnenoLone. conceMon. dutibu tithe equation (Mod& 21 wcLic/zuu &iLted mated !I, and Km we're 5.97 nmoCti/ h x mg-l p&o&in oRone, f~e.Apectiv&j. Thene. WA 1.b ug miehoboma.t?
hour incubaLion.
O;the,t ubay
conditiaMn
tie
ttiticti
NAD+
313~tf.SUff
The noMd tine fo the da.&. Eb;tiand 0.70 LIM pfiegnenpkottin in the vnC? duc..Gbed in Xhe test.
Linweaven-l3wrk p.&xt 06 ~~.Lp~ocaX pJ?acer~C& and ,(c.XLLJ? ;tinbue Fig. 7. 3f3-tfSVff acLivLty vWun heciphocd NAZ) conceI&u.uXon. The no&d .&%?A deaxibe ;thc Lineax h~on5hipb (obAained by L&eat .k?abX byucvL~ hegh#bion ana.l!ybi_bl 06 Ahe ;trtanbdomed d&u. fat Xhe pJ?aceti and 6e.ak.t tenti, the appahent Km uuh 0.7 3 and 0.84 MMNAP, ne..&pectiv&t. chemically
(Sholl and Claude, unpublished data).
olone and 3$-hydroxy-5-androsten-17-one
Using both pregnen-
as substrates, no reduction of
Nitro-Blue Tetrazolium (NBT) was noted in placental or gonadal slices and only a very slight reaction was observed in the outer region of the
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TPEIROIDS
765
Table 2. Kinetic parameters of 36-hydroxysteroid dehydrogenase/A5-4isomerase activity in microsones of monkey fetal tissues and placenta. Vm
Km
KS
Tissue
(nmoles/h x mg-l protein
(FM)
(IN
Ovary Testis Adrenal Placenta
1.9 * (2) 11.5 + 0.7* 5.2 (3) 146 t 66 (4) 1.2 + 0.4 (5)
0.16 f. (1) 0.27 0.13 (3) 1.8 + 0.5 (4) 2.5 + 1.7 (5)
:*:; [:I 2:3 + 1.4 (4) 2.1 + 0.8 (2)
*Mean f SE. The number of animals in which parameters were estimated is given in parentheses. fetal adrenal.
In comparison, the histochemical reaction in mature rat
ovaries and testes was quite pronounced.
An earlier examination of
human fetal adrenal 3~-HSDH by means of this histochemical technique revealed a pronounced NBT reduction in the definitive cortex of this gland during late gestation (19).
Thus, the human fetal adrenal along
with the placenta would appear to have a more active 3B-HSDH than corresponding monkey organs. Fetectomy in monkeys does not significantly influence one levels in the mother (20).
progester-
Although this finding appears to be in-
consistent with the tissue differences in St?-HSDH, there may be several reasons why the fetal adrenal does not contribute noticeably to the pool of circulating progesterone,
First, the availability of pregnen-
olone to adrenal 36-HSDH is not known; this may be rate limiting.
Sec-
ond, progesterone may be metabolized rapidly to corticoid hormones to the extent that little progesterone is released into the circulation. This possibility is given greater credence by the high fetal cortisol production rate that has been reported (9).
Finally, the large size of
the placenta, in comparison to the fetal adrenals, may be a primary determinant.
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TDEIROIDS
Table 3. Recrystallization of L3H]progesterone formed from E3H]pregnenolone by placental and fetal testicular and adrenal microsomes. Recrystal.
ri0.
Placenta:
; 2 : Adrenal:
; : 4 Testis:
3
H-Activity (DPM)
14 C-Activity (DPM)
3&,'4C_ Activity
6380 824 628 502 1660
1000 126 103 82 269
6.38 6.54 6.10 6.12 6.17 (5.63)"
4440 671 554 458 1520
1050 148 130 107 374
4.23 4.53 4.26 4.28 4.06 (5.03)
14100 1800 1390 1310 3840
1160 142 109 107 310
12.2 12.7 12.8 12.2 12.4 (12.2)
Recrystallizations were carried out in ethanol/H20 against [14C]and radioinert-progesterone standards. *Mother liquor ratios in parentheses. Previously, it was reported that there is an inhibitor of 38-HSDH within the placenta which associates with microsomes (21). quence, enzyme activity increased as it was diluted.
As a conse-
In other species,
including monkeys, an inhibitor of 3B-HSDH was noted when enzyme activity was evaluated in the 10,000 g supernatant (16).
These investigations
were carried out at enzyme concentrations considerably higher than those used in the current study.
As pointed out (22), the effect of endoge-
nous inhibitors upon the reaction kinetics becomes negligible after substantial dilution which may explain why consistent dilution-activity changes were not found in any of the tissues.
In those cases in which
no pregnenolone inhibition was noted, the linearity of the LineweaverBurk plot also indicates that the effect of endogenous inhibitors was
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TDROIDS
negligible in these preparations (23).
767
The relatively high levels of
pregnenolone that were used may have obscured the action of potential inhibitors. In several incubations, reaction rates were inhibited by high concentrations of pregnenolone.
Traditionally, substrate inhibition is
believed to involve the combination of the enzyme-substrate complex (ES) with another molecule of substrate (ES2).
As a result, enzymatic
activity and consequent product formation are retarded. the dissociation of (ES*), namely, (ES)(S)/(ES2).
KS describes
As KS declines, the
ability of the substrate to inhibit product formation is increased. The present study deals with a crude enzyme preparation which includes both 3e-HSDH and A5-4isomerase activities.
Given this situation, it is
difficult to speculate about the kinetics underlying pregnenolone inhibition, and in particular, whether substrate inhibition involves one or both enzymes or the cofactor, NAD. Although pregnenolone has not been detected by gas liquid chromatography within 100,000 g supernatants from placental and fetal adrenal homogenates (24), it is possible that pregnenolone concentrations are higher within organellae, particularly in mitochondria where pregnenolone is synthesized from cholesterol.
In those organs, such as the
placenta, in which mitochondrial 3B-HSDH appears to be relatively active, substrate inhibition may be an important factor in the regulation of enzyme activity.
In this regard, substrate inhibition may ensure
that a fairly constant rate of progesterone production is maintained if pregnenolone levels increase. It was noted previously in the monkey that C17-201yase is less active in the fetal ovary than in the fetal testis (4).
Results from
768
m
IrIIEOXD~
the current investigation suggest that the fetal ovary also contains a lower level of $-HSDH
activity which may further constrain estrogen
synthesis in this organ. ACKNOWLEDGMENTS This work was supported in part by Grant RR-00167 from the NIH, USPHS. Tissues were made available through NIH Grant l-P50-HL-27358 SCOR. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. ::: 13. ;:: 16. ii: 19. z 22: 23. 24.
Resko, J.A., Malley, A., Begley, D. and Hess, D.L. ENDOCRINOLOGY 93: 156 (1973). i%htaniemi, I.T., Korenbrot, C.C., Se&n-Ferrg, M., Foster, D.B., pR;;;;, ;.TA_ and Jaffe, R.B. ENDOCRINOLOGY 100: 839 (1977). Ploem, J.G. and Stadelman, H.L. ENDOCRINOLOGY -97: 425 (i976):' Sholl, S.A. J. STEROID BIOCHEM. 16: 141 (1982). ENDOCRINOLOGY -84: 1421 Ainsworth, L., Daenen, M. and RyaK K.J. (1969). 95: 1704 Walsh, S.W., Wolf, R.C. and Meyer, R.K. ENDOCRINOLOGY (1974). Sholl, S.A., Anderson, N.G., ColLs, A.E. and Wolf, R.C. STEROIDS 29: 249 (1977). J;iffe, R.B., Se&-Ferr&, M., Parer, J.T. and Lawrence, C.C. AM. J. OBSTET. GYNECOL. 131:~164~(1978). Mitchell, B.F., Serhn-Fer&, M., Hess, D.L. and Jaffe, R.B. ENDOCRINOLOGY 108: 916 (1981). Sholl, S.A. andolf, R.C. ENDOCRINOLOGY 95: 1287 (1974). Sholl, S.A. STEROIDS 38: 221 (1981). McNulty, W.P., Novy, Mrj-. and Walsh, S.W. BIOL. REPROD. 25: 1079 %;).S A STEROIDS 40: 475 (1982) Mar&rdt,'D.W. J. SOC INDUST. APPL. MATH. 11: 431 (1963). Farr, A.L. andRandall, R.J. J. Rosebrough, N.J., k3~“,~?&~ '193: 273 (1951) REPROD. 14: 306 (1976). Wiener, M. - mL. Wiest, W.G. STEROIDS 10: n9 (1967). Neill, J.D., Johanss0nFD.B. and Knobil, E. ENDOCRINOLOGY 84: 45 (1969). Goldman, A.S., Yakovac, W.C. and Bongiovanni, A.M. J. CLIN. ENDOCRINOL. 26: 14 (1966). Tullner, W.Wrand Hodgen, G.D. STEROIDS 24: 887 (1974). Wiener, M. MECHANISMS OF AGEING AND DEVELOPMENT 7: 433 (1978). Wiener, M. and Reiner, J.M. MECHANISMS OF AGEINGAND DEVELOPMENT 7: 445 (1978). Reiner, J.M. BEHAVIOR OF ENZYME SYSTEMS. Van Nostrand/Reinhold, New York (1969). Sholl, S.A. STEROIDS (Submitted).
3125 36-HYDROXYSTEROID DEHYDROGENASE/A5-41SOMERASE ACTIVITY IN THE RHESUS MONKEY PLACENTA AND FETAL ADRENAL, TESTIS AND OVARY DURING LATE GESTATION Samuel A. Sholl Wisconsin Regional Primate Research Center, Madison, WI USA 53715-1299 Received:
6-6-83 ABSTRACT
3$-Hydroxysteroid dehydrogenasela 5-4isomerase (3B-HSDH) was measured in the rhesus monkey (Macaca mulatta) placenta, fetal adrenal (whole organ minus medulla),testis and ovary during late gestation (Days145-162). Activities were evaluated from the conversion of C3H]pregnenolone to [3H]progesterone. The maximum enzyme velocity (Vm) in adrenal microsomes (100,000 g pellet) was significantly higher (146 nmoles progesterone/h x mg-Iprotein) than in microsomes from the other tissues. Testicular Vm was greater than either ovarian or placental Vm which were not different from one another (11.5 versus 1.9, 1.2 nmoles progesterone/h x mg-Iprotein, respectively). Apparent MichaelisMenten constants in the adrenal, placenta, testis and ovary averaged 1.8, 2.5, 0.27 and 0.16 PM, respectively. In some cases, substrate inhibition was noted. Estimated dissociation constants for pregnenolone were 2.3 PM (adrenal), 2.1 uM (placenta), 0.74 PM (testis) and 0.13 1111 (ovary). 3B-HSDH was less active in a crude mitochondrial preparation from the fetal adrenal (10,000 g pellet) than in microsomes, whereas activity in the placenta and testis appeared to be equally distributed between mitochrondria and microsomes. Rate measurements were consistent with the apparent potentials of these organs to synthesize their characteristic hormones. Thus, 38HSDH activity may be an important rate determining step in hormone synthesis. The importance of substrate inhibition in progesterone formation remains to be assessed. INTRODUCTION During late gestation fetal testes and ovaries of rhesus monkeys (Macaca mulatta) synthesize testosterone (17B-hydroxy-4-androsten-3-one) ~(1,2) and estradiol-17B (1,3,5(10)-estratriene-3,17B-diol)(3,4),
re-
spectively; the placenta produces progesterone (4-pregnene-3,20-dione) (5-7), and fetal adrenals synthesize corticoids (8,9).
Several find-
ings suggest that there are substantial differences in the rates at Publication no. 22-034 of the Wisconsin Regional Primate Research Center.
Volume
41, Number
6
S
TZIICOID-
June 1983
which these organs secrete their characteristic hormones.
For example,
the sex-difference in testosterone concentration in the fetal circulation (1) is more pronounced than the difference in estradiol-17g (3). In addition, despite the fact that the placenta is much larger in size than the fetal adrenals, the production rate of cortisol in the fetus at Day 140 (~3 mg/day) (9) is higher than the maternal production rate of progesterone at 155 days of pregnancy ('~1mg/day) (10). The A4-pathway is operative in fetal monkey gonads (4) and adrenals (ll), and this being the case, progesterone may be a precursor for both sex and corticoid hormones.
Progesterone is synthesized in turn from
pregnenolone (3B-hydroxy-5-pregnen-ZO-one) which requires the enzyme complex, 36-hydroxysteroid dehydrogenase/a5-4isomerase
(38-HSDH).
The
objective of this study was the evaluation of 38-HSDH activity in the monkey placenta, fetal adrenal, ovary and testis during late gestation to ascertain whether there are organ differences in activity which might account for the apparent dissimilarity in steroidogenic activity. MATERIALS AND METHODS Fetuses were obtained from rhesus monkeys during Caesarean section at 145-162 days of gestation. A segment ($2 g) of the primary placental disk was homogenized in 5 ml cold 0.15 M phosphate buffer (pH 7.4). Activity in the fetal adrenal was assessed in the entire gland (minus medulla), since in a recent report (12) it was suggested that the fetal cortex is remodeled to form a portion of the definitive adult cortex and thus could contain enzymes necessary for corticoid formation. A recent study (13) describing the distribution of ll- and 21-hydroxyFetal adrenals and gonads were lases supports this latter possibility. homogenized in 5-10 and 25 volumes (by weight) of phosphate buffer, respectively. Homogenates were centrifuged at 1,000 g for 20 min. Supernatants were centrifuged at 10,000 g for 15 min, and crude microsomal pellets were obtained from the resulting supernatants by additional centrifugation at 100,000 g for 1 h. Microsomes were suspended in a small volume (~5 ml) of phosphate buffer using a teflon-glass homogenizer. SB-HSDH activity was estimated by incubating microsomes (except where noted) with 0.2 PCi [7-3H]pregnenolone (19.3 Ci/mmol, New England
S
TIlROIDS
Nuclear Corp., Boston, MA) in the presence of NAD and different concentrations of unlabeled pregnenolone. [4-14C]Progesterone (~~5,000 disintegrations per min, New England Nuclear Corp.) was also included to estimate the combined metabolic loss of the product, [3H]progesterone, and procedural loss during its isolation. Reactions were carried out at 37°C in air in a shaking metabolic incubator for varying lengths of time up to 1 hour. The total reaction volume was 1 ml. The concentration of NAD was 0.4 mM except where noted. Reactions were terminated by adding 0.1 ml ethanol and freezing in dry ice/acetone. Steroids were extracted once with 4 ml petroleum ether. Fifty micrograms of unlabeled progesterone were added to the petroleum ether phase which was subsequently evaporated under N2. Radioactive progesterone was isolated by silica gel G thin layer chromatography, first in the solvent system chloroform/ethyl acetate (4:1, v/v) followed by a second separation in chloroform/dioxane (94:6, v/v). Radioactivity was measured in a Searle 6892 scintillation spectrophotometer (Searle Radiographics, Des Plaines, IL) using toluene-Liquifluor (New England Nuclear Corp.) as the scintillation fluid. After correcting for counting efficiency, activity was expressed as disintegrations per min (dpm). When no substrate i:lhibition was noted, maximum reaction rates (Vm) and apparent Michaelis-Menten constants (Km) were estimated from the equation (Model 2), v = V,/(l t Km/s), where v was the reactionvelocity and s the concentration of pregnenolone. When substrate inhibition was found Vm, Km and K (substrate dissociation constant) were estimated from the relationship ?Model 1), v = Vm/(l t Km/s t s/K,). Coefficient estimations were made by computer least squares analysis (14). Reactions usually were carried out using three different dilutions of the enzyme. Results were reported only when the estimated Vms from at least two of these dilutions were in agreement (computer fit SDS of Vms overlapped). Protein values were determined by the method of Lowry -2 et al (15). The identity of [3H]progesterone formed during the reaction was confirmed by recr stallization in ethanol/H20 to a constant specific activity (dpm 3H/Y 4C). Significant differences between Vms were detected by analysis of variance and Duncan's new multiple range test. RESULTS In the fetal adrenal 3~-HSDH activity was greater in microsomes (100,000 g pellet) than in mitochondria
(Table 1).
In contrast, enzyme
activity in the placenta and fetal testis appeared to be more uniformly distributed between mitochondrial and microsomal fractions.
These dis-
tributions persisted in all tissues regardless of the extent to which the enzyme was diluted.
Activity was not altered by the omission of
760
SB
‘J?EI&OIDS
Table 1. 36-Hydroxysteroid dehydrogeanse/A5'4isomerase activity in different cellular fractions of monkey fetal tissues and placenta. Enzyme activity at dilution: Tissue
Fraction
Adrenal
Testis
Placenta
Protein (uglassay)
1,000 g s* 10,000 g P 100,000 g P
144** 38 2
1,000 g s 10,000 g P 100,000 g P
402 17 9
1,000 g s 10,000 g P 100,000 g P
3354 461 167
1 (nmoles $diesterone/h mg- protein)
149 60 2
1:20 x
;i 59
::
4 14 7
0.17 0.65 0.69
0.19 0.90 0.27
3 7" 0.22 0.36 0.41
*S,P = supernatant and pellet. **Amount of protein in undiluted sample.
[14C]progesterone from the incubation (data not shown). Microsomal enzyme activity in the placenta and fetal ovaries, testes and adrenals was linear throughout the one hour incubation and directly related to the amount of protein that was incubated (Fig. 1).
In all
tissues, the recovery of [14C]progesterone remained fairly con-
stant regardless of the length of the incubation, indicating that the extent of progesterone metabolism was minimal.
In some of the placen-
tal incubations and all of the adrenal and ovarian incubations (Figs. 2-4) 3B-HSDH activity was inhibited by high levels of pregnenolone, while in other placental incubations and all but one testicular incubation, substrate inhibition was not noted (Figs. 5,6). Maximum enzyme velocity (V,) was higher in the adrenal (146 nmoles progesterone/h x mg-' protein) than in the other tissues that were examined (P < 0.01) (Table 2).
Testicular V, (11.5 nmoles progesterone/h
761
(2)
(1) ,$emed @om [7-3fflpegnenoRone FQ. 1. ~mouvLt oa [7-3fflp/rogtintenone an a &wsXon ad the. amouti 06 micnodomal pxoZc.in that WM incuba&d and tie RengZh 04 Xhe incubatiqn. OXha incubaLion conditioti ahe da&bed in the texxt. FLcj. 2. Linweavetr-Bunk plot 05 rretipxocul pl.acc?nW 3$-USVtl acfivktg vm3u.h treciyJhoc& phegnnenolone concevLtha;tion. The dashed fine datiben the. q.&zion (Model 1 I which cua~j &zXed Zo the d.oXa. Tlz~~ne. ww~e 149 Hg ticxonomal pho&zin in the. one. hoti LnincubaLLon. CWWL tiaag cond.LLLonn axe da&bed in Xhe texxt. The datd poinX a.$ 9 x 1O-3 uM YJhQjnenolone & noX nhown. Eh;timatc?d V,, I$,, and Kh (&hornMod&T 1 equation) wehe 3.97 nmolalh x mg-’ pho&&, 3.67 VM and 0.55 NM phiLgnf2no~one, ntipwtivtiy. x mg-’
protein)
was higher than ovarian and placental Vms (P < 0.05)
which were not different from one another (1.9 and 1.2 nmoles progesterone/h x mg-' protein, respectively).
Apparent Kms in the adrenal,
placenta, testis and ovary were 1.8, 2.5, 0.27 and 0.16 PM, respectively, while pregnenolone dissociation constants in these tissues were 2.3, 2.1, 0.74 and 0.13 uM, respectively.
In the computation of
apparent Km and KS, estimates of these parameters were highly correlated. high.
As a consequence, SDS (from computer fits) were relatively Conversely, SDS associated with estimated V,s were considerably
62
762
TRROTDS
I ( LIM
PREGNENOLONE)
i -’
Fkj. 3. tifleweavetr-Bw~bp&A 06 nec@zoc& de_&& a&en& 3B-ff.SZXf ac;tiv.&ty vmti rre&mc~~C phqneno%ne concen;Dration. The. dashed Line de-mulben tithe equation (Mod& 1) whi_ch IWXA$Ltted to Ahe da&. Them WYL~ 2.7 ug micrroboma.Cpkottin in the one hawt incubafion. O;theh annay conditions ahe dencnibed .in Ahe Xext. EhaCima&d V,, Km and K, idhorn Model 7 eyuutlan) wme 330 nmoLu/h x tng-l pha;ttin,7.7 PM and 0.49 PM phegnnen&one, ~e.5p~&k~~y. lower.
For NAD, apparent Kms in the placenta and fetal testis were
0.13 mM and 0.84 mM (Fig. 7). In one animal, the extent of microsomal conversion of C3H]pregnenolone to hormones other than [3H]progesterone was assessed in adrenal and placental samples.
Following incubation and extraction several
times with ethyl ether, very little radioactivity remained in the aqueous phase indicating that the extent of steroid conjugation was minimal.
In addition, after the first TLC separation of the extracted hor-
mones, more than 90% of the radioactivity was located in those areas of the chromatographic plate occupied by pregnenolone and progesterone standards. The identity of the reaction product, [3H]progesterone, was confirmed by recrystallization
(Table 3).
S
TElEOIDS
763
4. tine.we_aveh-BuA2 plot 06 necipk0ca-t fjeAi7L ovaLan 3f3-tlSVtl acFig. 4. tiv.iXy vmuh ~ec.ip~~oca.t pxegnenoRone conceWn. The tihed .&net dentiba Zhe eyuatin (Model 71 which utu @LaXed ti the data. Thehe
wene 0.62 pg mitio~omaL pfioXe.& in the one howr LncubaGon. Utheh ahnay conditioYLrlahe dadbed in the. Xext. EhGna;ted V,, t&,, and Kn uwtre 2.48 nmoku fh x mg-l plroZtin, 0.1 B PM and 0.07 L&Ipxegnenotone,
nehpuLiv~y. Fig. 5. tine.weavet-Bwtk p.tof 06 wx+v~ocat p.&weW 3B-HSVH acfi.vi~y veh.uh hetipk0ca.L phegnnenoRone concen&u&ion. The ~ok?.id line denuiben the e-qua&ion (Mod& 2) which UEIA I;L%Xed .to the da&~. &Lima&d Vm and Km (dhom Model 2 qua&ion) WULCZ 0.58 nmoltifh x mg-l phO;tein and 0.31 Thtie woe 72 ug micAohom~ photein in I.IMp&egnenoRone, hchpe&tiv~y. Xhe one howr .Lnc.ubation. &then ahnay conditioti ahe. de.wGbed in the. kxxt. The da& point aX 9 x 1 O- 3 UM phegnenolone AL noR 6 hown. DISCUSSION Among those organs that were examined for 38-HSDH activity, the fetal adrenal exhibited the greatest potential for metabolizing pregnenolone to progesterone.
This activity could support the formation of
corticoid hormones which are important for the normal development of a variety of fetal tissues.
In contrast to the adrenal, the placenta was
relatively deficient in 36-HSDH which may restrict progesterone synthesis in this organ.
In a comparative study of placental 38-HSDH, enzyme
levels were considerably lower in monkeys than in humans (16) offering one explanation as to why circulating progesterone values in these two species are so widely different (17,18). Initially, an attempt was made to assess 36-HSDH activity histo-
S
764
TnSOIDLl
ADRENAL
(mM
F&J. 6. tinweavkx-Bwth p,tkt 06 r~ec.LpJ~ocaL 6eXiz.t ac;tivtiq vmw hec.+fiocaX pfiegnenoLone. conceMon. dutibu tithe equation (Mod& 21 wcLic/zuu &iLted mated !I, and Km we're 5.97 nmoCti/ h x mg-l p&o&in oRone, f~e.Apectiv&j. Thene. WA 1.b ug miehoboma.t?
hour incubaLion.
O;the,t ubay
conditiaMn
tie
ttiticti
NAD+
313~tf.SUff
The noMd tine fo the da.&. Eb;tiand 0.70 LIM pfiegnenpkottin in the vnC? duc..Gbed in Xhe test.
Linweaven-l3wrk p.&xt 06 ~~.Lp~ocaX pJ?acer~C& and ,(c.XLLJ? ;tinbue Fig. 7. 3f3-tfSVff acLivLty vWun heciphocd NAZ) conceI&u.uXon. The no&d .&%?A deaxibe ;thc Lineax h~on5hipb (obAained by L&eat .k?abX byucvL~ hegh#bion ana.l!ybi_bl 06 Ahe ;trtanbdomed d&u. fat Xhe pJ?aceti and 6e.ak.t tenti, the appahent Km uuh 0.7 3 and 0.84 MMNAP, ne..&pectiv&t. chemically
(Sholl and Claude, unpublished data).
olone and 3$-hydroxy-5-androsten-17-one
Using both pregnen-
as substrates, no reduction of
Nitro-Blue Tetrazolium (NBT) was noted in placental or gonadal slices and only a very slight reaction was observed in the outer region of the
S
TPEIROIDS
765
Table 2. Kinetic parameters of 36-hydroxysteroid dehydrogenase/A5-4isomerase activity in microsones of monkey fetal tissues and placenta. Vm
Km
KS
Tissue
(nmoles/h x mg-l protein
(FM)
(IN
Ovary Testis Adrenal Placenta
1.9 * (2) 11.5 + 0.7* 5.2 (3) 146 t 66 (4) 1.2 + 0.4 (5)
0.16 f. (1) 0.27 0.13 (3) 1.8 + 0.5 (4) 2.5 + 1.7 (5)
:*:; [:I 2:3 + 1.4 (4) 2.1 + 0.8 (2)
*Mean f SE. The number of animals in which parameters were estimated is given in parentheses. fetal adrenal.
In comparison, the histochemical reaction in mature rat
ovaries and testes was quite pronounced.
An earlier examination of
human fetal adrenal 3~-HSDH by means of this histochemical technique revealed a pronounced NBT reduction in the definitive cortex of this gland during late gestation (19).
Thus, the human fetal adrenal along
with the placenta would appear to have a more active 3B-HSDH than corresponding monkey organs. Fetectomy in monkeys does not significantly influence one levels in the mother (20).
progester-
Although this finding appears to be in-
consistent with the tissue differences in St?-HSDH, there may be several reasons why the fetal adrenal does not contribute noticeably to the pool of circulating progesterone,
First, the availability of pregnen-
olone to adrenal 36-HSDH is not known; this may be rate limiting.
Sec-
ond, progesterone may be metabolized rapidly to corticoid hormones to the extent that little progesterone is released into the circulation. This possibility is given greater credence by the high fetal cortisol production rate that has been reported (9).
Finally, the large size of
the placenta, in comparison to the fetal adrenals, may be a primary determinant.
S
766
TDEIROIDS
Table 3. Recrystallization of L3H]progesterone formed from E3H]pregnenolone by placental and fetal testicular and adrenal microsomes. Recrystal.
ri0.
Placenta:
; 2 : Adrenal:
; : 4 Testis:
3
H-Activity (DPM)
14 C-Activity (DPM)
3&,'4C_ Activity
6380 824 628 502 1660
1000 126 103 82 269
6.38 6.54 6.10 6.12 6.17 (5.63)"
4440 671 554 458 1520
1050 148 130 107 374
4.23 4.53 4.26 4.28 4.06 (5.03)
14100 1800 1390 1310 3840
1160 142 109 107 310
12.2 12.7 12.8 12.2 12.4 (12.2)
Recrystallizations were carried out in ethanol/H20 against [14C]and radioinert-progesterone standards. *Mother liquor ratios in parentheses. Previously, it was reported that there is an inhibitor of 38-HSDH within the placenta which associates with microsomes (21). quence, enzyme activity increased as it was diluted.
As a conse-
In other species,
including monkeys, an inhibitor of 3B-HSDH was noted when enzyme activity was evaluated in the 10,000 g supernatant (16).
These investigations
were carried out at enzyme concentrations considerably higher than those used in the current study.
As pointed out (22), the effect of endoge-
nous inhibitors upon the reaction kinetics becomes negligible after substantial dilution which may explain why consistent dilution-activity changes were not found in any of the tissues.
In those cases in which
no pregnenolone inhibition was noted, the linearity of the LineweaverBurk plot also indicates that the effect of endogenous inhibitors was
S
TDROIDS
negligible in these preparations (23).
767
The relatively high levels of
pregnenolone that were used may have obscured the action of potential inhibitors. In several incubations, reaction rates were inhibited by high concentrations of pregnenolone.
Traditionally, substrate inhibition is
believed to involve the combination of the enzyme-substrate complex (ES) with another molecule of substrate (ES2).
As a result, enzymatic
activity and consequent product formation are retarded. the dissociation of (ES*), namely, (ES)(S)/(ES2).
KS describes
As KS declines, the
ability of the substrate to inhibit product formation is increased. The present study deals with a crude enzyme preparation which includes both 3e-HSDH and A5-4isomerase activities.
Given this situation, it is
difficult to speculate about the kinetics underlying pregnenolone inhibition, and in particular, whether substrate inhibition involves one or both enzymes or the cofactor, NAD. Although pregnenolone has not been detected by gas liquid chromatography within 100,000 g supernatants from placental and fetal adrenal homogenates (24), it is possible that pregnenolone concentrations are higher within organellae, particularly in mitochondria where pregnenolone is synthesized from cholesterol.
In those organs, such as the
placenta, in which mitochondrial 3B-HSDH appears to be relatively active, substrate inhibition may be an important factor in the regulation of enzyme activity.
In this regard, substrate inhibition may ensure
that a fairly constant rate of progesterone production is maintained if pregnenolone levels increase. It was noted previously in the monkey that C17-201yase is less active in the fetal ovary than in the fetal testis (4).
Results from
768
m
IrIIEOXD~
the current investigation suggest that the fetal ovary also contains a lower level of $-HSDH
activity which may further constrain estrogen
synthesis in this organ. ACKNOWLEDGMENTS This work was supported in part by Grant RR-00167 from the NIH, USPHS. Tissues were made available through NIH Grant l-P50-HL-27358 SCOR. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. ::: 13. ;:: 16. ii: 19. z 22: 23. 24.
Resko, J.A., Malley, A., Begley, D. and Hess, D.L. ENDOCRINOLOGY 93: 156 (1973). i%htaniemi, I.T., Korenbrot, C.C., Se&n-Ferrg, M., Foster, D.B., pR;;;;, ;.TA_ and Jaffe, R.B. ENDOCRINOLOGY 100: 839 (1977). Ploem, J.G. and Stadelman, H.L. ENDOCRINOLOGY -97: 425 (i976):' Sholl, S.A. J. STEROID BIOCHEM. 16: 141 (1982). ENDOCRINOLOGY -84: 1421 Ainsworth, L., Daenen, M. and RyaK K.J. (1969). 95: 1704 Walsh, S.W., Wolf, R.C. and Meyer, R.K. ENDOCRINOLOGY (1974). Sholl, S.A., Anderson, N.G., ColLs, A.E. and Wolf, R.C. STEROIDS 29: 249 (1977). J;iffe, R.B., Se&-Ferr&, M., Parer, J.T. and Lawrence, C.C. AM. J. OBSTET. GYNECOL. 131:~164~(1978). Mitchell, B.F., Serhn-Fer&, M., Hess, D.L. and Jaffe, R.B. ENDOCRINOLOGY 108: 916 (1981). Sholl, S.A. andolf, R.C. ENDOCRINOLOGY 95: 1287 (1974). Sholl, S.A. STEROIDS 38: 221 (1981). McNulty, W.P., Novy, Mrj-. and Walsh, S.W. BIOL. REPROD. 25: 1079 %;).S A STEROIDS 40: 475 (1982) Mar&rdt,'D.W. J. SOC INDUST. APPL. MATH. 11: 431 (1963). Farr, A.L. andRandall, R.J. J. Rosebrough, N.J., k3~“,~?&~ '193: 273 (1951) REPROD. 14: 306 (1976). Wiener, M. - mL. Wiest, W.G. STEROIDS 10: n9 (1967). Neill, J.D., Johanss0nFD.B. and Knobil, E. ENDOCRINOLOGY 84: 45 (1969). Goldman, A.S., Yakovac, W.C. and Bongiovanni, A.M. J. CLIN. ENDOCRINOL. 26: 14 (1966). Tullner, W.Wrand Hodgen, G.D. STEROIDS 24: 887 (1974). Wiener, M. MECHANISMS OF AGEING AND DEVELOPMENT 7: 433 (1978). Wiener, M. and Reiner, J.M. MECHANISMS OF AGEINGAND DEVELOPMENT 7: 445 (1978). Reiner, J.M. BEHAVIOR OF ENZYME SYSTEMS. Van Nostrand/Reinhold, New York (1969). Sholl, S.A. STEROIDS (Submitted).