Proc. Nati. Acad. Sci. USAVol. 76, No. 4, pp. 2027-2031, April 1979Medical Sciences

Human placental immunoreactive corticotropin, lipotropin,and f3-endorphin: Evidence for a common precursor

(radioimmunoassay/gel exclusion chromatography/gel electrophoresis/immune affinity chromatography)

EMI ODAGIRI, BARBARA J. SHERRELL, CHARLES D. MOUNT, WENDELL E. NICHOLSON, ANDDAVID N. ORTH*Department of Medicine and Cancer Research Center, Vanderbilt University School of Medicine, Nashville, Tennessee 37232

Communicated by Sidney P. Colowick, January 18, 1979

ABSTRACT The concentrations and molecular sizes ofimmunoreactive corticotropin (ACTH), lipotropin (LPH, /BLPHplus yLPH), 7LPH, and (B-endorphin (END) were determinedin human placental extracts. Serial dilutions of a water extractof placenta generated competitive binding curves parallel withthat of the standard in each assay. The concentrations of ACTH,LPH, yLPH, and SEND were 3.3, 0.8, 0.7, and 1.1 ng/g wetweight of tissue, respectively. A partially purified extract appliedto a Sephadex G-50 column contained high Mr components withACTIH, LPH, 'yLPH, and MEND immunoreactivities. The ex-tract was applied to an immune affinity chromatography col-umn consisting of affinity-purified (1-24)ACTIH antiserum co-valently bound to agarose. The material that adsorbed to thecolumn and eluted with buffer containing sodium dodecyl sul-fate had ACTH, LPH, and #END immunoreactivities, indi-cating that there was a component or components containingantigenic determinants for all of these peptides. Sodium dodecylsulfate/polyacrylamide gel electrophoresis of the affinity-pu-rified placental extract revealed at least two high Mr compo-nents (Mr - 48,000 and 36,000) with all three immunoreactivi-ties. These data suggest, but do not prove, that the placentasynthesizes ACTH, the LPHs, and SEND from a common pre-cursor molecule.

Normal human pregnancy is associated with maternal adre-nocortical hyperfunction that apparently is secondary to in-creased corticotropin (ACTH) secretion, since concentrationsof maternal plasma ACTH increase progressively duringpregnancy; urinary excretion of free cortisol is relatively re-sistant to glucocorticoid negative-feedback suppression (1, 2).It has been suggested that the placenta is involved in this in-creased secretion of ACTH because several workers have re-ported the presence of bioactive (1-8) or immunoreactiveACTH (1-3) in placental extracts.The ACTH extracted from placenta appears to consist, in

addition to ACTH, of a high Mr form of the hormone (3). Inmouse pituitary tumor cells (9, 10) and human nonpituitarycarcinoma cells (11, 12), ACTH appears to be formed by pro-cessing of a high Mr precursor molecule. This ACTH precursorcontains the 13-lipotropin (f3LPH) sequence and, therefore, mayalso be processed to form f3LPH and its related peptides, yLPH[(1-58)/3LPH] and f3-endorphin [BEND, (61-91)/3LPH] (9, 11,12). The high Mr ACTH precursor molecule thus contains an-tigenic determinants for ACTH, the LPHs, and BEND.

In the present study, we have used radioimmunoassays forACTH, LPH (J3LPH plus "yLPH), 'yLPH, and fiEND, gel ex-clusion chromatography, affinity chromatography, sodiumdodecyl sulfate (NaDodSO4)/polyacrylamide gel electropho-resis to determine if all of these hormones are present in thehuman placenta and if they are parts of a common high Mrmolecule which might represent a precursor.

MATERIALS AND METHODSExtraction of Peptides from Placenta. Freshly obtained

human placentas from women who had normal term deliveriesand one from a woman whose pregnancy was terminated at 16weeks by hysterotomy were trimmed of cord and membranes,cut into 5- to 10-g pieces, and washed exhaustively with coldisotonic saline. Several extraction procedures were studied todetermine the most efficient extraction method.

Water. Placental tissue was homogenized in 9 ml of waterper g of wet tissue at 4VC for 90 sec, immediately heated in awater bath at 1000C for 15 min, cooled to 250C, and centri-fuged at 10,000 X g for 20 min. The supernate was lyophilizedand stored at -70'C.

Acetic acid. Placental tissue was homogenized in 5 ml ofglacial acetic acid or 5 ml of 1 M acetic acid per g of wet tissueat 40C for 90 sec, immediately heated in a water bath at 1000Cfor 15 min, cooled to 250C, and centrifuged at 10,000 X g for20 min. The supernate was lyophilized and stored at -700C.

Glacial acetic acid/acetone. Placental tissue was homoge-nized in 9 ml of glacial acetic acid per g of wet tissue and cen-trifuged for 30 min at 10,000 X g. An equal volume of acetonewas added to the supernate, which was allowed to stand over-night at 40C and was centrifuged at 10,000 X g for 30 min. Theacetone in the supernate was evaporated in a water bath at50'C. The remaining supernate was lyophilized and stored at-700C.

Payne's method. A modification of the method of Payne etal. (13) was used. The tissue was homogenized in 18 ml of gla-cial acetic acid and 2 ml of acetone per g of wet tissue. Thehomogenate was heated in a water bath at 70'C for 30 min,cooled to 250C, and centrifuged at 10,000 X g for 30 min. Fivemilliliters of acetone/0.5 M NaCI was added to the supernate;the mixture was allowed to stand overnight at 4"C and wascentrifuged at 5000 X g for 30 min. The ACTH-containingfraction was precipitated by adding an equal volume of etherand allowing it to stand for 5 hr at 40C. The precipitate wasrinsed three times with acetone, dried under reduced pressure,and stored at -700C.

Preparation of Extracts for Radioimmunoassay. Samplesof placental powder equivalent to 2 g of tissue were dissolvedin 2 ml of buffer A (50mM sodium phosphate, pH 7.4/77 mMNaCI containing 100 mg of Merthiolate per liter), after whichthe pH was readjusted to 7.4 with 1 M NaOH, if necessary.

Abbreviations: ACTH, adrenocorticotropic hormone (corticotropin);fiLPH, (P-lipotropin; 'yLPH, y-lipotropin; (iMSH, 03-melanocyte-stimulating hormone (ft-melanotropin); #END, fl-endorphin; Na-DodSO4, sodium dodecyl sulfate. Fragments of ACTH and otherpeptides are indicated by a prefix listing their inclusive amino acidsin parentheses [e.g., (1-24)ACTH is the NH2-terminal 24-amino-acidfragment of ACTH].* To whom reprint requests should be addressed.

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2028 Medical Sciences: Odagiri et al.

After centrifugation at 5000 X g for 15 min, the supernate wasassayed immediately or stored at -700C until assayed.

Radioimmunoassays. Radioimmunoassay of ACTH wasperformed as described (12, 14) except that antiserum R1543was used. Antiserum R1543 was raised to commercial porcineACTH (Duracton, Nordic Pharmaceuticals, Laval, Quebec) andreacts equally with all available analogues containing the (5-18)ACTH sequence, but not with synthetic a-melanotropin(aMSH), #END, or (37-58)LPH (all <0.1%) or with purifiedfLPH (0.3%) or yLPH (3%), both of which appear to haveminor ACTH contamination. The radioimmunoassays of LPHwith antiserum R3, which is specific for (37-58)LPH and,therefore, reacts with both f3LPH and yLPH, of 'yLPH withantiserum R1547, which does not react with f3LPH, and offiEND with antiserum RB100-11/15 (kindly provided by R.Guillemin), which also reacts with f3LPH, were performed asdescribed (12).

Partial Purification of Placental Extracts by Adsorptionto Silicic Acid. Placental powder (11.4 g, equivalent to 200 gof wet placental tissue), obtained by water extraction, wasdissolved in 456 ml of buffer A and centrifuged at 5000 X g for20 min. The supernate from each 16-ml aliquot was adsorbedwith 1.6 g of silicic acid for 30 min by end-over-end mixing (14)and was centrifuged at 5000 X g for 15 min. The silicic acidpellet was washed twice with water, recentrifuging after eachwash, and was mixed with 40 ml of acetone/glacial aceticacid/water, 15:1:80 (vol/vol) for 30 min. After centrifugation,the supernates were pooled, lyophilized, and stored at -70'C.The lyophilized, partially purified extract was reconstitutedin 10 ml of water; 1 ml was equivalent to 20 g of wet placentaltissue.

Gel Exclusion Chromatography. A 1.5 X 90 cm column ofSephadex G-50 Fine gel (Pharmacia) was equilibrated anddeveloped with buffer A containing 100 mg of lysozyme and10 ml of plasma (obtained from a normal individual whoseACTH and LPH secretion had been suppressed by adminis-tration of dexamethasone) per liter. A 5.5-ml aliquot of the si-licic acid-purified water extract corresponding to 110 g ofplacenta was applied and eluted at a flow rate of 0.3 ml/min;2.1-ml fractions were collected. The column was calibrated withthe following materials: bovine serum albumin (Sigma), puri-fied human f3LPH and yLPH (kindly provided by C. H. Li andP. J. Lowry, respectively), synthetic human ACTH (Ciba-Geigy, Summit, NJ), synthetic human (37-58)LPH ("f3MSH,"Ciba-Geigy), and NaCI.Immune Affinity Chromatography. Affinity-purified an-

tiserum R1549 against (1-24)ACTH, coupled to CNBr-Seph-arose 4B (Pharmacia), was used as described (12). An 8.5-mlaliquot of silicic acid-purified water extract corresponding to170 g of placenta'was applied to the column. Immunoadsorbedmaterials were eluted with 4 ml of 0.1 M ammonium acetatecontaining 1 g of NaDodSO4 per liter and were lyophilized.NaDodSO4/Polyacrylamide Gel Electrophoresis. The

apparent molecular size of immune affinity-purified placentalACTH was analyzed by the method of Laemmli (15) with a10% acrylamide/0.54% N,N-methylenebisacrylamide gel witha 3% acrylamide/0.16% bisacrylamide stacking gel. The fol-lowing dansyl-labeled proteins were used as Mr markers onparallel gels: bovine serum albumin (Mr 67,000), human'y-globulin heavy and light chains (Mr 50,000 and 23,500, re-spectively), ovalbumin (Mr 45,000), myoglobin (Mr 17,200),and cytochrome c (Mr 12,400) (all from Sigma). Synthetichuman ACTH and purified ACTH of Mr 31,000 and Mr 23,000from mouse pituitary tumor cell line AtT-20/D-16v (kindlyprovided by B. A. Eipper and R. E. Mains) were also run inparallel gels and located by ACTH radioimmunoassay. Dan-

sylated cytochrome c and ovalbumin were included with eachsample of placental ACTH as internal markers. Gels were cutinto 1-mm slices, crushed, and eluted with 1 ml of buffer B for8 hr at 370C. After centrifugation, the supernates were aspi-rated and assayed.

RESULTSExtraction Procedures. Placental extracts obtained with each

procedure had activity in each of the four radioimmunoassays(Table 1), and there was no apparent difference in the con-centrations of any of the immunoreactive peptides at 16 weeksof gestation and at term. Glacial acetic acid extracted moreimmunoreactive ACTH than other procedures. The results ofSephadex G-50 chromatography of the acetic acid and waterextracts (Fig. 1) revealed that the difference was due to moreefficient extraction of immunoreactive material the size of(1-39)ACTH by acetic acid. With the exception of this and theslightly less efficient extraction of immunoreactive fEND(Table 1), water extraction appeared to be as efficient as theother procedures, was clearly the easiest, and had the potentialadvantage of avoiding damage to acid-labile material. In ad-dition, the water extract generated competitive binding curvesparallel to those of standards in all radioimmunoassays, whereassome other extracts generated nonparallel curves in some assays(Fig. 2). Because of this, and because our primary interest wasto characterize further the high Mr material, water was usedto extract placental peptides for additional studies.

Silicic acid partial purification of water extracts of placentaltissue yielded recoveries of 81.5, 83.5, 93.7, and 75.0% for im-munoreactive ACTH, LPH, yLPH, and fiEND, respec-tively.

Gel Exclusion Chromatography. A silicic acid-purifiedwater extract was applied to a column of Sephadex G-50 Fineresin. The results of the four radioimmunoassays of the columneluate fractions are shown in Fig. 3. A high Mr component(s)that had ACTH, LPH, yLPH, and ,BEND immunoreactivitieseluted as a broad peak (Kd = 0.00-0.30) in and after the columnvoid volume. A small peak of immunoreactive ACTH coelutedwith synthetic human ACTH (Kd = 0.51), immunoreactiveLPH eluted as a broad peak from the void volume through theelution position of purified human flLPH (Kd = 0.19), and, inaddition to a peak of immunoreactive fEND coeluting withpurified human flLPH, a small peak of immunoreactive fiENDcoeluted with synthetic human ,BEND (Kd = 0.44). A smallamount of immunoreactive human yLPH eluted in the voidvolume, a similar small peak coeluted with human yLPH (Kd= 0.37), and another small peak eluted between synthetichuman (37-58)LPH and the salt peak.

Thus, the major immunoreactive materials appeared to bea high Mr component(s) with ACTH, LPH, fEND, and some'yLPH immunoreactivity; smaller amounts of materials similarin size and immunoreactivity to human ,BLPH, yLPH, ,BEND,and ACTH, and a small amount of material with yLPH im-munoreactivity eluting after "fMSH".Immune Affinity Chromatography. A sample of water

extract partially purified with silicic acid was applied to theanti-ACTH Sepharose column (Fig. 4). The excluded fractioncontained 26, 54, and 48% of the applied immunoreactiveACTH, LPH, and fEND, respectively. Less than 2.5% of theimmunoreactive peptides was recovered in the washes. How-ever, the fraction eluted with NaDodSO4/ammonium acetatecontained 68.5, 18.5, and 40.6% of the applied immunoreactiveACTH, LPH, and fiEND, respectively. Thus, it appeared thatthere was material that adsorbed to and eluted from the ACTHantibody column that had, in addition to an immunoreactiveACTH sequence, LPH and fEND immunoreactive se-quences.

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Proc. Natl. Acad. Sci. USA 76 (1979) 2029

Table 1. Concentrations of immunoreactive peptides (ng/g of wet tissue) in 16-wk and term placentas extracted by various methods

ACTH LPH yLPH SENDMethod 16-wk Term 16-wk Term 16-wk Term 16-wk Term

Water 1.9 3.3 1.6 0.8 0.7 0.4 0.8 1.1(1.8-5.6)* (0.6-1.1)* (0.3-0.5)t (0.9-1.3)1

1 M Acetic acid 1.3 4.5 0.9 1.4 0.7 0.4 2.8 3.0(1.3-12.2)* (0.9-2.4)* (0.2-0.7)t

Glacial acetic acid 13.9 19.5 1.3 1.7 0.1 0.1 1.0 3.0Glacial acetic acid/acetone <0.3 0.7 0.7 0.9 0.1 0.1 5.3 2.9Payne's method 0.6 0.4 0.7 0.7 0.2 0.2 1.1 1.5* Range; n = 7.t Range; n = 4.I Range; n = 3.

NaDodSO4/Polyacrylamide Gel Electrophoresis. Thematerial purified by anti-ACTH immune affinity chroma-tography was lyophilized and analyzed by NaDodSO4/poly-acrylamide gel electrophoresis (Fig. 5). Presumably, only thosematerials that possessed an ACTH antigenic sequence wereadsorbed to the antibody column and eluted with Na-DodSO4-ammonium acetate. A high Mr component (Mr ;

48,000) was observed that had ACTH, LPH, and BEND im-munoreactivities. A second immunoreactive ACTH component(Mr ' 36,000) also appeared to have LPH and, possibly, BENDimmunoreactivity. A third peak of immunoreactive ACTH was

d X X

0 Z g.Z16.0_ + * *4+ + +

14.0 4

12.0-

10.0 X

0

0.. .4 06 0. .

E 1.2

C

3~ ~ ~ ~~K

0.--

0.4-0

EE

8.0

4.0

0 0 ~~0.2 0.4 0.6 0.8 1.0Kd

FIG. 1. Sephadex G-50 Fine gel exclusion chromatography ofplacental extracts. The elution profiles of immunoreactive ACTH inglacial acetic acid (Upper) and water (Lower) extracts of differentamounts (10 and 110 g of wet tissue, respectively) of the same termplacenta are shown. Alternate fractions (Upper) or each fraction(Lower) was analyzed. Kd, fractional elution volume. 0, Undetectableimmunoreactive hormone at the concentration plotted. BSA, bovineserum albumin; prefix h, human.

broad and may have consisted of three components, one thatmigrated between ACTH of Mr 23,000 and myoglobin (Mr18,000), one in the position of ACTH (Mr t 4500), and onebeyond ACTH. In addition to the high Mr components, im-munoreactive LPH was found in a peak (Mr 18,000) whichalso appeared to have ACTH immunoreactivity. Immuno-

100 lo0 102 i13pg of peptide or p1 of extract/tube

FIG. 2. Dilution curves of placental extracts in radioimmunoas-says. Curves generated by serial dilutions ofwater (0), 1 M acetic acid(A), glacial acetic acid (a), glacial acetic acid/acetone (-), and Payne's(1) extraction procedures are compared with those of referencestandard hormones (0) in each radioimmunoassay. Data are plottedas B/Bo (fraction of initial binding) against log pg of peptide or Al ofplacental extract added per tube. (A) ACTH; (B) human LPH; (C)human yLPH; (D) human #END.

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0

0JCC0.

1-C001 B

E

EC

21

1 ~~~~~~DI0 0.2 0.4 0.6 0.8 1.0

Kd

FIG. 3. Sephadex G-50 Fine gel exclusion chromatography of awater extract of placenta. Concentrations of immunoreactive ACTH(A), human LPH (B), human -yLPH (C), and human REND (D) weredetermined in alternate fractions. Data are plotted in the samemanner as in Fig. 1.

reactive #END migrated as a broad peak, the major componenthaving an apparent Mr z 48,000; no immunoreactive #ENDcomponents with a Mr of less than about 23,000 were ob-served.

Since it was not clear whether the peaks of immunoreactiveACTH represented three or more components, the five pooledsamples shown in Fig. 5 were desalted on a column of SephadexG-10 (0.9 X 50 cm, equilibrated and developed with 0.1 Macetic acid). They were then lyophilized and reconstituted, afterwhich NaDodSO4 gel electrophoresis was again performed. Thepresence of at least four immunoreactive ACTH components(Mr - 48,000, 36,000, 18,000, and 4500) was confirmed (datanot shown).

>

0

:2 60

40.0.

40-

co

E0 20E s01 Ig

Excluded Buffer NaDodSO4wash eluate

FIG. 4. Anti-(1-24)ACTH immune affinity chromatography ofwater extract of term placenta partially purified with silicic acid.Percent of total recovered immunoreactive ACTH (1), human LPH(a), and human #tEND (0) in the nonadsorbed materials excludedfrom the column, in the buffer wash, and in the material eluted with4 ml of 0.1 M ammonium acetate containing 1 g ofNaDodSO4 per literis plotted. Recovery of applied immunoreactive ACTH, human LPH,and human BEND was 111, 103, and 81%, respectively.

U*F 1 U V V

E~~~~~ 11 -Of IN. M4 .

:2 B

C t

0.2

0

E

C0.4

0.2

10 30 50Slice no.

FIG. 5. Polyacrylamide gel electrophoresis of anti-(1-24)ACTHimmune affinity-purified placental extract. The concentrations ofimmunoreactive ACTH (A), human LPH (B), and human BEND (C)were determined in each 1-mm gel slice after NaDodSO4/polyacryl-amide gel electrophoresis of an aliquot of the immune affinity eluate(Fig. 4). Five pools (I-V, A) were desalted, lyophilized, reconstituted,and again analyzed by NaDodSO4/polyacrylamide gel electrophoresis.Ovalb., ovalbumin; Myoglob., myoglobin; Cyto. c, cytochrome c; BPB,bromphenol blue.

DISCUSSIONThe results of the present study indicate the presence, not onlyof immunoreactive ACTH, but also of immunoreactive LPHsand #END in human placental extracts. The finding of im-munoreactive f3LPH or 'yLPH or both in placenta is not sur-prising, since (i) human flMSH is the (37-58) sequence of flLPHand yLPH (16), (ii) immunoreactive flMSH has been reportedto be increased in the plasma of pregnant women (17, 18), and(iii) bioactive MSH (19) and immunoreactive fMSH (20) havebeen described in placental extracts. It is now thought thathuman flMSH may have been artifactually produced duringits original isolation and may not normally exist in humans (21).Thus, the "flMSH" bioactivity or immunoreactivity or bothdescribed in plasma from pregnant women or placenta byearlier workers (17-20) may have been flLPH, yLPH, arti-factually produced "flMSH," or a combination of these pep-tides. In addition to immunoreactive ACTH and LPHs, theplacental extracts also contained immunoreactive BEND, whichis the COOH-terminal 31-amino-acid sequence of flLPH (16).Immunoreactive BEND has recently been reported by othersin placental extracts (22).

Recently, several investigators have suggested that ACTH,LPHs, and fEND are derived from a common biosynthetic

Proc. Natl. Acad. Sci. USA 76 (1979)

Proc. Natl. Acad. Sci. USA 76 (1979) 2031

precursor molecule (9-12). The results of the present studysuggest that this may also be true in the human placenta. Whena water extract of placenta was partially purified with silicicacid and applied to a Sephadex G-50 column, high Mr com-ponents with ACTH, LPH, yLPH, and OEND immunoreac-tivities were found. Smaller immunoreactive materials, prob-ably representing ACTH, yLPH, and OEND, were also de-tected, as well as an immunoreactive yLPH component thateluted between synthetic human (37-58)LPH and the salt peak.The nature of this last material is not clear, but the possibilitythat it was artifactually derived by cleavage of yLPH orR3LPHhas not been excluded.

Liotta et al. (3) recently reported that 54% of the immuno-reactive ACTH in a 0.1 M HCl extract of placenta eluted froma column of Sephadex G-50 in the region of (1-39)ACTH and29% in the void volume region. We obtained similar results witha glacial acetic acid extract and found that high Mr ACTHcomponents were extracted with similar efficiency by bothwater and glacial acetic acid (2.8 and 0.9 ng/g of wet tissue,respectively), but that small Mr ACTH was extracted much lessefficiently by water than by glacial acetic acid (0.8 and 17.0ng/g of wet tissue, respectively) (Table 1 and Fig. 1). Thus, therelative amounts of high and small Mr ACTHs observed inextracts of the placenta-and, presumably, of other tissues-may depend upon the extraction method used.

In order to determine whether the high Mr ACTH, LPH, and3END immunoreactivities observed after Sephadex G-50chromatography were possessed by the same or differentmolecules, we performed anti-ACTH immune affinity chro-matography. Since immunoreactive ACTH, LPH, and fENDwere all found in the eluate, it appears that the placenta containsa molecule(s) that possesses in its sequence the antigenic de-terminants of LPH, SEND, and ACTH, and may, therefore,represent a common biosynthetic precursor for all of thesepeptides.

In order to determine the molecular size of the high Mrsubstance, we performed NaDodSO4/polyacrylamide gelelectrophoresis on the immune affinity-purified placental ex-tract. It appeared that there were at least two high Mr com-ponents, Mr 48,000 and 36,000, both of which had ACTH,LPH, and 3END immunoreactivities. In addition, at least oneintermediate-sized component (Mr 18,000) that containedthe antigenic determinants for ACTH and LPH, but not forBEND, was observed. High Mr ACTHs appear to be glyco-proteins (10, 23); thus, Mr values determined by NaDodSO4/polyacrylamide gel electrophoresis may be overestimates.However, the two largest placental immunoreactive ACTHsmigrated more slowly than did the ACTH glycoprotein of Mr31,000 purified from mouse AtT-20/D-16v pituitary tumorcells applied to parallel gels, suggesting that the human pla-cental proteins are larger than this mouse ACTH precursormolecule.

Three lines of evidence suggest that ACTH is synthesized bythe placenta: (i) ACTH, LPHs, and BEND, which are pre-sumably derived from the same precursor molecule, are allfound in placental extracts; (ii) high Mr molecules that pre-sumably represent common biosynthetic precursors of ACTH,LPHs, and fEND and are not thought normally to circulate inplasma are also found in placental extracts; and (iii) placentalACTH concentrations (3) and the increased cortisol excretionobserved during pregnancy (1) are both resistant to glucocor-ticoid suppression. unlike pituitary ACTH and urinary cortisolexcretion in nounipregnant subjects. In addition, preliminary

results have suggested that immunoreactive ACTH materialmay be produced by acutely dispersed placental cells (3) orplacental fragments (24) in vitro. Nevertheless, final proof willrequire demonstration of labeled amino acid incorporation intoACTH, LPH, or 3END by human placental cells in culture orby placental polysomes or mRNA in a cell-free translationsystem. The possible roles of these putative placental peptidesin the maintenance or termination of normal human pregnancyremain to be elucidated.

We thank Dr. R. Guillemin for the generous gift of 3END antiserumRB100-11/15, Dr. C. H. Li for highly purified human 3LPH, Dr. P.J. Lowry for highly purified human yLPH, Ciba-Geigy for synthetichuman (1-39)ACTH and (37-58)LPH, Drs. B. A. Eipper and R. E.Mains for purified AtT-20/D-16v ACTH of Mr 23,000 and 31,000 usedin these studies, Ms. Yvonne S. Brown for technical assistance, and Ms.Doris Harris for secretarial assistance. These studies were supportedin part by National Institutes of Health Grants 5-P30-AM17026, 5-P17-HL14214, and 5-RO1-AM05318 and by National Cancer InstituteResearch Grants 5-ROl-CA11685 and 5-R25-CA19429.

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