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Ontogeny of pituitary β-endorphin and related peptides in the human embryo and fetus
Ontogeny of pituitary ~-endorphin and related peptides 1n the human embryo and fetus F. Facchinetti, M.D., A. R. Storchi, M.D., F. Petraglia, M.D., G. Garuti, M.D., and A. R. Genazzani, M.D. Modena, Italy In this study we evaluated the presence of proopiomelanocortin-related peptides (13-lipotropin, 13-endorphin, and '1-endorphin) in five embryos (5 to 10 weeks of pregnancy) and 11 fetal pituitaries (15 to 25 weeks) by means of high-performance liquid chromatography coupled with specific radioimmunoassays. Tissues were collected at laparotomy for ectopic pregnancy (five embryos) or after spontaneous (seven) or prostaglandin-induced (four) abortion. 13-Endorphin and 13-lipotropin were present starting at the seventh week of pregnancy while '1-endorphin appeared ·only in the second trimester. During embryonic life opioid activity was limited to the cephalic portion. The three peptides, but little, if any, acetylated 1-31 13-endorphin, were recognized in the fetal pituitary throughout the second trimester, at which time 13-lipotropin and 13-endorphin showed constant values in spite of increasing '1-endorphin concentrations. 13-Lipotropin was the predominant peptide in both embryonic and fetal life. In conclusion, the three peptides related to proopiomelanocortin were expressed from the precursor at different times throughout development. By the beginning of the second trimester the pituitary processing of proopiomelanocortin is similar to that of adult life and the functional activity of the anterior lobe seems to prevail over that of the "fetus-related" neurointermediate lobe around the twenty-fifth week of pregnancy. (AM J OssrET GYNECOL 1987;156:735-9.)
Key words: Embryo, anterior pituitary, neurointermediate lobe, 13-endorphin, 13-lipotropin, y-endorphin The activity of the proopiomelanocortin-related opioid system undergoes important changes during pregnancy as suggested by the increased adrenocorticotropic hormone and 13-endorphin levels in the maternal circulation' and by the changing number and affinity of naloxone binding sites in uterine rat membranes.' In addition to the maternal pituitary and placenta, whose opioid secretory ability has been demonstrated," the fetal pituitary is believed to contribute to the increase in opioid peptide levels. Immunohistochemical data indicate the presence of adrenocorticotropic activity from the seventh week of pregnancy' while no 13-lipotropin, another proopiomelanocortin-related peptide, was found. On the other hand, Begeot and Dubois' recently reported the coexistence of several proopiomelanocortin-related peptides in fetal pituitary corticotropes. Biochemical studies of opioid ontogeny in human material are scanty and contradictory. Some authors 6 reported a large amount of 13-endorphin in the fetal pituitary, and others 7 described the presence of 13-li-
From the Department of Obstetrics and Gynecology, University of Modena. This work has been partly supported by CNR P.F. "Medicina Preventiva e Riabilitativa" SP7 and SP8. Received for publication June 12, 1986; revised July 18, 1986; accepted September 2, 1986. Reprint requests: Dr. F. Facchinetti, Clinica Ostetrica e Ginecologica, Via del Pozzo 71, 41100 Modena, Italy.
potropin but were unable to demonstrate authentic 13-endorphin in the 11 to I 9 weeks' gestational age range. Moreover, with the use of sophisticated techniques it has been ascertained that the fetal pituitary does not contain true a-melanocyte-stimulating hormone, as reported several years ago by Silman et al.," but its desacetylated form, adrenocorticotropic hormone 1-13 amide. 9 More recently, the presence of a-endorphin and 'Y-endorphin, two N-terminal derivatives of 13-endorphin ( 1-16 and 1-17, respectively), has been verified in the human pituitary gland.'° In this study therefore we evaluated 13-lipotropin, 13-endorphin, and 'Y-endorphin in embryos and fetal pituitaries up to the beginning of the third trimester by means of high-performance liquid chromatography coupled with specific radioimmunoassays.
Material and methods Tissue collection. Five embryos at 5 to I 0 weeks of pregnancy were obtained at operation in ectopic pregnancies. The smallest embryo had a rostral-caudal length of 10 mm, ascribed to a gestational age of 4.7 weeks. In two specimens, the caudal portion was divided from the rostral one and processed separately. Eleven whole pituitaries were collected from fetuses between 15 and 25 weeks of pregnancy. Seven of the 11 fetuses were spontaneously aborted, and four were delivered after a prostaglandin induction of labor performed because of rubella infection. Every tissue was 735
736
Facchinetti et al.
March 1987
Am
EMBRYO (CAUDl'l POOTJON)
J-EP
B-L?JI
B-F.P
J Obstet Gynecol
B-EP
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jf-tP ("~~ il-LPH
lOOl :
•.
'
'
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~ •• hl 5
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15
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Fig. 1. Chromatogram of 13-endorphin-like immunoreactivity and specific )'-endorphin (y-EP), 13-lipotropin (B-LPH), and 13-endorphin (B-EP) immunoreactivity in caudal and rostral portions of a 7-week-old embryo. Arrows indicate elution of respective standard peptide. In the small panel, solid line on the left refers to y-EP immunoreactivity; those on the right, to B-EP immunoreactivity; and dotted line refers to B-LPH imm unoreactivity.
boiled for 10 minutes in 0.05 mol/L acetic acid in order to destroy proteases and then homogenized with a Polytron. After two successive centrifugations at 4000 x g for 15 minutes, clear supernatants were stored at - 20° C until assayed. The pellets were treated with 1 mol/L sodium hydroxyde and submitted to a protein content evaluation with the use of the Folin phenol reagent. High-performance liquid chromatography. Homogenates were injected in a high-performance liquid chromatography apparatus (Waters, Milford, Massachusetts) consisting of two pumps (Model 510), an ultraviolet detector (Model 440), and a gradient controller (Model 680). A reverse phase C-18 column (µBondapack, 39 by 300 mm, 10 µm size) was eluted in a linear gradient from 25% to 40% acetonitrile in 0.01 mol/L hydrochloric acid, for 15 minutes, at a flow rate of 1.5 ml/min. For each sample, 30 fractions were collected (each for 30 seconds), evaporated to dryness, and redissolved in 1 ml of 0.12 mol/L phosphate buffer, pH 7.4, and 0.1% bovine serum albumin. In order to evaluate the amount of material sticking on the column, 1 ng of each of the three peptides was injected into the system and processed as described above. This experiment was replicated five times. Recoveries were 88.1 % ± 10.4% for 13-endor-
F~ACTIOllS
Fig. 2. Chromatogram of 13-endorphin-like immunoreactivity and specific )'-endorphin (y-EP), 13-lipotropin (B-LPH), and 13-endorphin (B-EP) immunoreactivity in two whole pituitaries obtained from a fetus at the fifteenth week and a fetus at the twenty-fifth week of pregnancy.
phin, 91.5% ± 17.3% for 13-lipotropin, and 79.4% ± 12.8% for -y-endorphin. Radioimmunoassays. Each fraction was tested for its .13-endorphin-like immunoreactivity with the use of the anti-13-endorphin serum B4 (kindly donated by Dr. V. M. Wiegant, Utrecht, The Netherlands), which cross reacts with many 13-endorphin-related peptides including 1-16 13-endorphin (260% on a molar basis), 1-17 13-endorphin (450%), 1-26 13-endorphin (11.5%), 1-27 13-endorphin (9.5%), and acetyl 13-endorphin (100%). Thus, according to this 13-endorphin immunoreactivity and in view of the retention times (Table I) of -y-endorphin, 13-lipotropin, and 13-endorphin, fractions were retested for their immunoreactivity with the use of appropriate iodinated peptides and specific antisera kindly donated by Dr. V. M. Wiegant (-y-endorphin) and by Professor C. H. Li (13-endorphin and 13-lipotropin, San Francisco, California). Details of the different radioimmunoassays were previously reported. 2 · "
Results In the embryo of 4.7 weeks' gestation neither 13-endorphin-like immunoreactivity nor specific peptides were detected. 13-Endorphin (195.9 ± 57.7 fmol, mean ± SE) and 13-lipotropin (569.6 ± 64.9 fmol)
Ontogeny of pituitary opioids
Volume 156 Number 3
737
2"[ 201
'"
" D-EP
B-L.PH
1!5-:LB
G-EP
2m-2s
Fig. 3. Total content (mean ± SD) of r:l-endorphin (B-EP), -y-endorphin (G-EP), and r:l-lipotropin (B-LPH) in embryos and fetal pituitaries from 5 to 25 weeks of pregnancy.
were found but no '{-endorphin was present in embryos until the tenth week of pregnancy with one exception. This latter sample was the only one showing a poor ~-lipotropiri content. During the embryonic period the peptides were measured only in the cephalic portion and no immunoreactivity was found in the caudal one (Fig. I). High amounts of the three proopiomelanocortin-related peptides were present in the fetal pituitaries; immunoreactive 13-lipotropin, 13-endorphin, and '{-endorphin elute at the same retention times as standard molecules (Fig: 2). The total content of 13-endorphin, 13-lipotropin, and -y-endorphin in embryos and fetal pituitaries is shown in Fig. 3. There is a progressive increase of the three peptides throughout pregnancy, but the rates of such increases are different. This is reflected by the absence of correlation between 13-lipotropin and 13-endorphin, ~-lipotropin and '{-endorphin, and '{-endorphin and ~-endorphin during pituitary development. Interestingly enough, the 13-lipotropin/13-endorphin ratio increases from 1.41 ± 0.37 at the beginning of the second trimester to 4.4 ± 1.40 (p < 0.5) in the pituitaries of22 to 25 weeks' gestational age. The opposite occurs in the 13-endorphin/'{-endorphin ratio, which decreases from 1.64 ± 0.58 to 1.08 ± 0.29. In fetal pituitary extracts (Fig. 2), no 13-endorphin-like immunoreactivity (lower part of each panel) was detected in fractions 24 to 30, corresponding to the elution pattern of acetyl 13-endorphin. Comment
These data provide evidence that 13-endorphin, and '{-endorphin are present in the fetal pituitary throughout development. Time-related dif-
~-lipotropin,
Table I. Retention times, in minutes (mean ± SD of at least 10 determinations) with the use of a µBondapack C-18 RP column eluted from 25% to 40% acetonitrile in 0.01 mol/L hydrochloric acid, in 15 minutes at a flow rate of 1.5 ml/min Peptide
1-16 Endorphin 1-17 Endorphin r:l-Lipotropin r:l-Endorphin Acetyl 1-31 endorphin 1-26 Endorphin 1-27 Endorphin Adrenocorticotropin 4-10 a-Melanocyte-stimulating hormone* Adrenocorticotropin 1-39 Met-enkephalin
Retention time (min, mean ± SD)
3.12 6.5 9.4 11.9 13.6 14.4 14.8 2.9 3.62
± 0.07 ± 0.14 ± 0.48
± 0.69 ± 0.58 ± 0.34 ± 0.48
± 0.31 ± 0.1
8.1 ± 0.13 3.6 ± 0.0
*Acetyl 1-13 adrenocorticotropin.
ferences in the appearance of the three peptides in the pituitary exist. 13-Endorphin and 13-lipotropin appeared in the seventh week when they were present in the cephalic portion, indicating that at that moment proopiomelanocortin activity was confined to pituitary and central nervous system structures. Few studies exist about the ontogeny of 13-lipotropin and 13-endorphin in the human embryo. Some authors reported the presence of 13-endorphin6 and 13-lipotropin7 in the pituitary in the eleventh week of pregnancy when bioassayable adrenocorticotropic hormone is also demonstrable. 11 lmmunohistochemical data have indicated positive staining for 13-endorphin early in development, that is, in the seventh week, 4 and the present
738
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Facchinetti et al.
biochemical characterization agrees completely with such a study. -y-Endorphin, a product off3-endorphin cleavage that is present in brain synaptosomal membranes and in the adult pituitary, 11 is undetectable in all but one embryo, thus suggesting that the proopiomelanoc ortin-enzymatic system needs further maturational processes in intrauterine life in order to be fully expressed. It was assumed that during fetal life pituitary proopiomelanocorti n undergoes two successive cleavage steps: the first one to produce adrenocorticotro pic hormone and (3-lipotropin and the second one further cleaving these two peptides to give o:-melanocyte-st imulating hormone and (3-endorphin, respectively." The second step of proopiomelanoc ortin processing is typical of the intermediate lobe of the rat pituitary," a structure lacking in the human adult pituitary but well defined during fetal life. However, in whole fetal pituitaries and embryos, f3-lipotropin was the predominant peptide and the f3-lipotropin/f3-e ndorphin ratio increased until the end of the second trimester, suggesting that the first proopiomelanoc ortin cleavage step is the most active in this period of life. Gibbs et al. 14 superfused the fetal pituitary and demonstrated that the anterior lobe acquires its responsiveness to corticotropin-rel easing factor just after the twentieth week of pregnancy. On the other hand, the neurointermedia te lobe releases (3-endorphin only after a potassium chloride-induce d cell depolarization, and no changes occurred with corticotropin-rel easing factor or dopamine. Thus the major peak of f3-lipotropin obtained from whole pituitaries until the end of the second trimester can be interpreted as originating from the anterior lobe. We suggest that as early as the embryo the enzy!lle pattern for proopiomelanoc ortin cleavage is similar to that existing in the adult pituitary. Other authors, 15 studying human adrenocorticotro pic hormone ontogeny, also concluded that the anterior lobe matures before the intermediate one. Another feature of the neurointermedia te lobe (in the rat) is the acetylation of proopiomelanoc ortin and its related peptides, a process that inactivates the ability to bind opiate receptors. 16 It is uncertain if acetylation occurs in the human being and we found little immunoreactivit y, if any, related to acetyl f3-endorphin. In conclusion proopiomelanoc ortin-related peptides are evident early in development and both (3-endorphin and (3-lipotropin were present in embryonic life, between 5 and 7 weeks, while smaller fragments of (3-endorphin, such as -y-endorphin, have a delayed appearance. By the beginning of the second trimester the pituitary processing of proopiomelanoc ortin is similar to that of the adult and the functional activity of the
Am
J Obstet Gynecol
anterior lobe seems to prevail over that of the "fetusrelated" neurointermedia te lobe around the twentyfifth week of pregnancy. We are indebted to Dr. V. Wiegant and Dr. P. Burbach of the Rudolf Magnus Institute for Pharmacology, University of Utrecht, The Netherlands, for their advice about high-performan ce liquid chromatography techniques and for the supply of the quoted materials. We also acknowledge Prof. A. E. Panerai, of the Istituto di Farmacologia, Universita di Milano, for his help in performing our first high-performan ce liquid chromatography runs. Finally, the generous supply of anti(3-endorphin and anti-(3-lipotropi n sera from Prof. C. H. Li, of the Hormone Research Laboratory, University of California, San Francisco, is also acknowledged. REFERENCES I. Genazzani AR, Facchinetti F, Parrini D. Beta-lipotropin
2.
3.
4.
5.
6. 7. 8. 9.
I 0. 11.
12. 13.
and beta-endorphin plasma levels during pregnancy. Clin Endocrinol 1981; 14:409. Baraldi M, Giarre G, Santi M, Facchinetti F, Petraglia F, Genazzani AR. Pregnancy-related changes of opiate re3 ceptors identified in rat uterine membranes by H naloxone binding. Peptides 1985;6:97 I. Liotta A, Krieger DT. Biosynthetic pathway of ACTH and related peptides of the human placenta: in vitro demonstration. In: Klopper A, Genazzani AR, Crosignani PG, eds. Human placenta-proteins and hormones. London: Academic Press, 1980:267. Dubois P, Vargues-Regairaz H, Dubois MP. Human foetal anterior pituitary immunofiuorescen t evidence for corticotropin and melanotropin activities. Zellforschung 1973; . 45:131. Begeot M, Dubois MP. Immunologic localization of alphaand beta-lipotropin in corticotropic cells of the normal and anencephalic fetal pituitaries. Cell Tissue Res 1978; 193:413. Holland D, Silman RE, Chard T, et al. Corticotropin and lipotropin "family tree" of the human pituitary gland. J Endoc;rinol I 979;83:488. Brubaker PL, Baird AC, Bennett HPJ, Browne CA, Solomon S. Corticotropic peptides in the human fetal pituitary. Endocrinology 1982; 111: 1150. Silman RE, Chard T, Lowry PJ, Smith I, Young IM. Human foetal pituitary peptides and parturition. Nature 1976;260:716. Tilders FJH, Parker CR, Barnea A, Porter JC. The major immunoreactive a-melanocyte-stim ulating hormone (a-MSH)-like substance found in human fetal pituitary tissue is not a-MSH but may be desacetyl a-MSH (adrenocorticotropin 1-13 NH2). J Clin Endocrinol Metab 1981;52:319. Verfoef J, Wiegant VM, De Wied D. Regional distribution of alpha- and gamma-type endorphins in rat brain. Brain Res I 982;23 l :454. Kaplan SL, Grumbach MM, Aubert ML. The ontogenesis of pituitary hormones and hypothalamic factors in the human fetus: maturation of central nervous system regulation of anterior pituitary function. Recent Prog Horm Res 1976;32:161. Burbach JPH, Wiegant VM. Isolation and characterization of a-endorphin and 'Y-endorphin from single human pituitary glands. FEBS Lett 1984;166:267. Przewlocki R, Hollt V, Voigt KH, Herz A. Distinctive
Ontogeny of pituitary opioids
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in vitro release of J3-endorphin from the anterior compared to the posterior/intermediate lobe of rat pituitary. Life Sci 1979;24:1601. Gibbs DM, Stewart RD, LiuJH, Vale W, Rivier J, Yen SSC. Effects of synthetic corticotropin-releasing factor and dopamine on the release of immunoreactive J3-endorphin/J3-lipotropin and a-melanocyte-stimulating hormone from human fetal pituitaries in vitro. J Clin Endocrinol Metab 1982;55: 1149.
15. Allen RG, Pintar JE, Stack J, Kendall JW. Biosynthesis and processing of pro-opiomelanocortin-derived peptides during fetal pituitary development. Dev Biol 1985; 102:43. 16. Smyth DG, Massey DE, Zakarian S, Finnie MDA. Endorphins are stored in biologically active and inactive form: isolation of a-N-acetyl peptides. Nature I 979;279:252.
Key words: Embryo, anterior pituitary, neurointermediate lobe, 13-endorphin, 13-lipotropin, y-endorphin The activity of the proopiomelanocortin-related opioid system undergoes important changes during pregnancy as suggested by the increased adrenocorticotropic hormone and 13-endorphin levels in the maternal circulation' and by the changing number and affinity of naloxone binding sites in uterine rat membranes.' In addition to the maternal pituitary and placenta, whose opioid secretory ability has been demonstrated," the fetal pituitary is believed to contribute to the increase in opioid peptide levels. Immunohistochemical data indicate the presence of adrenocorticotropic activity from the seventh week of pregnancy' while no 13-lipotropin, another proopiomelanocortin-related peptide, was found. On the other hand, Begeot and Dubois' recently reported the coexistence of several proopiomelanocortin-related peptides in fetal pituitary corticotropes. Biochemical studies of opioid ontogeny in human material are scanty and contradictory. Some authors 6 reported a large amount of 13-endorphin in the fetal pituitary, and others 7 described the presence of 13-li-
From the Department of Obstetrics and Gynecology, University of Modena. This work has been partly supported by CNR P.F. "Medicina Preventiva e Riabilitativa" SP7 and SP8. Received for publication June 12, 1986; revised July 18, 1986; accepted September 2, 1986. Reprint requests: Dr. F. Facchinetti, Clinica Ostetrica e Ginecologica, Via del Pozzo 71, 41100 Modena, Italy.
potropin but were unable to demonstrate authentic 13-endorphin in the 11 to I 9 weeks' gestational age range. Moreover, with the use of sophisticated techniques it has been ascertained that the fetal pituitary does not contain true a-melanocyte-stimulating hormone, as reported several years ago by Silman et al.," but its desacetylated form, adrenocorticotropic hormone 1-13 amide. 9 More recently, the presence of a-endorphin and 'Y-endorphin, two N-terminal derivatives of 13-endorphin ( 1-16 and 1-17, respectively), has been verified in the human pituitary gland.'° In this study therefore we evaluated 13-lipotropin, 13-endorphin, and 'Y-endorphin in embryos and fetal pituitaries up to the beginning of the third trimester by means of high-performance liquid chromatography coupled with specific radioimmunoassays.
Material and methods Tissue collection. Five embryos at 5 to I 0 weeks of pregnancy were obtained at operation in ectopic pregnancies. The smallest embryo had a rostral-caudal length of 10 mm, ascribed to a gestational age of 4.7 weeks. In two specimens, the caudal portion was divided from the rostral one and processed separately. Eleven whole pituitaries were collected from fetuses between 15 and 25 weeks of pregnancy. Seven of the 11 fetuses were spontaneously aborted, and four were delivered after a prostaglandin induction of labor performed because of rubella infection. Every tissue was 735
736
Facchinetti et al.
March 1987
Am
EMBRYO (CAUDl'l POOTJON)
J-EP
B-L?JI
B-F.P
J Obstet Gynecol
B-EP
PIT 15Tlt WEEK
jf-tP ("~~ il-LPH
lOOl :
•.
'
'
B-EP
~ •• hl 5
ElmYO
'
10
15
20
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jQ
f.
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J
_
100
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0
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•
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10
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15
20
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J
J
25
3ll
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......
2.2 20 1.1 lU
15
20
i5
Fig. 1. Chromatogram of 13-endorphin-like immunoreactivity and specific )'-endorphin (y-EP), 13-lipotropin (B-LPH), and 13-endorphin (B-EP) immunoreactivity in caudal and rostral portions of a 7-week-old embryo. Arrows indicate elution of respective standard peptide. In the small panel, solid line on the left refers to y-EP immunoreactivity; those on the right, to B-EP immunoreactivity; and dotted line refers to B-LPH imm unoreactivity.
boiled for 10 minutes in 0.05 mol/L acetic acid in order to destroy proteases and then homogenized with a Polytron. After two successive centrifugations at 4000 x g for 15 minutes, clear supernatants were stored at - 20° C until assayed. The pellets were treated with 1 mol/L sodium hydroxyde and submitted to a protein content evaluation with the use of the Folin phenol reagent. High-performance liquid chromatography. Homogenates were injected in a high-performance liquid chromatography apparatus (Waters, Milford, Massachusetts) consisting of two pumps (Model 510), an ultraviolet detector (Model 440), and a gradient controller (Model 680). A reverse phase C-18 column (µBondapack, 39 by 300 mm, 10 µm size) was eluted in a linear gradient from 25% to 40% acetonitrile in 0.01 mol/L hydrochloric acid, for 15 minutes, at a flow rate of 1.5 ml/min. For each sample, 30 fractions were collected (each for 30 seconds), evaporated to dryness, and redissolved in 1 ml of 0.12 mol/L phosphate buffer, pH 7.4, and 0.1% bovine serum albumin. In order to evaluate the amount of material sticking on the column, 1 ng of each of the three peptides was injected into the system and processed as described above. This experiment was replicated five times. Recoveries were 88.1 % ± 10.4% for 13-endor-
F~ACTIOllS
Fig. 2. Chromatogram of 13-endorphin-like immunoreactivity and specific )'-endorphin (y-EP), 13-lipotropin (B-LPH), and 13-endorphin (B-EP) immunoreactivity in two whole pituitaries obtained from a fetus at the fifteenth week and a fetus at the twenty-fifth week of pregnancy.
phin, 91.5% ± 17.3% for 13-lipotropin, and 79.4% ± 12.8% for -y-endorphin. Radioimmunoassays. Each fraction was tested for its .13-endorphin-like immunoreactivity with the use of the anti-13-endorphin serum B4 (kindly donated by Dr. V. M. Wiegant, Utrecht, The Netherlands), which cross reacts with many 13-endorphin-related peptides including 1-16 13-endorphin (260% on a molar basis), 1-17 13-endorphin (450%), 1-26 13-endorphin (11.5%), 1-27 13-endorphin (9.5%), and acetyl 13-endorphin (100%). Thus, according to this 13-endorphin immunoreactivity and in view of the retention times (Table I) of -y-endorphin, 13-lipotropin, and 13-endorphin, fractions were retested for their immunoreactivity with the use of appropriate iodinated peptides and specific antisera kindly donated by Dr. V. M. Wiegant (-y-endorphin) and by Professor C. H. Li (13-endorphin and 13-lipotropin, San Francisco, California). Details of the different radioimmunoassays were previously reported. 2 · "
Results In the embryo of 4.7 weeks' gestation neither 13-endorphin-like immunoreactivity nor specific peptides were detected. 13-Endorphin (195.9 ± 57.7 fmol, mean ± SE) and 13-lipotropin (569.6 ± 64.9 fmol)
Ontogeny of pituitary opioids
Volume 156 Number 3
737
2"[ 201
'"
" D-EP
B-L.PH
1!5-:LB
G-EP
2m-2s
Fig. 3. Total content (mean ± SD) of r:l-endorphin (B-EP), -y-endorphin (G-EP), and r:l-lipotropin (B-LPH) in embryos and fetal pituitaries from 5 to 25 weeks of pregnancy.
were found but no '{-endorphin was present in embryos until the tenth week of pregnancy with one exception. This latter sample was the only one showing a poor ~-lipotropiri content. During the embryonic period the peptides were measured only in the cephalic portion and no immunoreactivity was found in the caudal one (Fig. I). High amounts of the three proopiomelanocortin-related peptides were present in the fetal pituitaries; immunoreactive 13-lipotropin, 13-endorphin, and '{-endorphin elute at the same retention times as standard molecules (Fig: 2). The total content of 13-endorphin, 13-lipotropin, and -y-endorphin in embryos and fetal pituitaries is shown in Fig. 3. There is a progressive increase of the three peptides throughout pregnancy, but the rates of such increases are different. This is reflected by the absence of correlation between 13-lipotropin and 13-endorphin, ~-lipotropin and '{-endorphin, and '{-endorphin and ~-endorphin during pituitary development. Interestingly enough, the 13-lipotropin/13-endorphin ratio increases from 1.41 ± 0.37 at the beginning of the second trimester to 4.4 ± 1.40 (p < 0.5) in the pituitaries of22 to 25 weeks' gestational age. The opposite occurs in the 13-endorphin/'{-endorphin ratio, which decreases from 1.64 ± 0.58 to 1.08 ± 0.29. In fetal pituitary extracts (Fig. 2), no 13-endorphin-like immunoreactivity (lower part of each panel) was detected in fractions 24 to 30, corresponding to the elution pattern of acetyl 13-endorphin. Comment
These data provide evidence that 13-endorphin, and '{-endorphin are present in the fetal pituitary throughout development. Time-related dif-
~-lipotropin,
Table I. Retention times, in minutes (mean ± SD of at least 10 determinations) with the use of a µBondapack C-18 RP column eluted from 25% to 40% acetonitrile in 0.01 mol/L hydrochloric acid, in 15 minutes at a flow rate of 1.5 ml/min Peptide
1-16 Endorphin 1-17 Endorphin r:l-Lipotropin r:l-Endorphin Acetyl 1-31 endorphin 1-26 Endorphin 1-27 Endorphin Adrenocorticotropin 4-10 a-Melanocyte-stimulating hormone* Adrenocorticotropin 1-39 Met-enkephalin
Retention time (min, mean ± SD)
3.12 6.5 9.4 11.9 13.6 14.4 14.8 2.9 3.62
± 0.07 ± 0.14 ± 0.48
± 0.69 ± 0.58 ± 0.34 ± 0.48
± 0.31 ± 0.1
8.1 ± 0.13 3.6 ± 0.0
*Acetyl 1-13 adrenocorticotropin.
ferences in the appearance of the three peptides in the pituitary exist. 13-Endorphin and 13-lipotropin appeared in the seventh week when they were present in the cephalic portion, indicating that at that moment proopiomelanocortin activity was confined to pituitary and central nervous system structures. Few studies exist about the ontogeny of 13-lipotropin and 13-endorphin in the human embryo. Some authors reported the presence of 13-endorphin6 and 13-lipotropin7 in the pituitary in the eleventh week of pregnancy when bioassayable adrenocorticotropic hormone is also demonstrable. 11 lmmunohistochemical data have indicated positive staining for 13-endorphin early in development, that is, in the seventh week, 4 and the present
738
March 1987
Facchinetti et al.
biochemical characterization agrees completely with such a study. -y-Endorphin, a product off3-endorphin cleavage that is present in brain synaptosomal membranes and in the adult pituitary, 11 is undetectable in all but one embryo, thus suggesting that the proopiomelanoc ortin-enzymatic system needs further maturational processes in intrauterine life in order to be fully expressed. It was assumed that during fetal life pituitary proopiomelanocorti n undergoes two successive cleavage steps: the first one to produce adrenocorticotro pic hormone and (3-lipotropin and the second one further cleaving these two peptides to give o:-melanocyte-st imulating hormone and (3-endorphin, respectively." The second step of proopiomelanoc ortin processing is typical of the intermediate lobe of the rat pituitary," a structure lacking in the human adult pituitary but well defined during fetal life. However, in whole fetal pituitaries and embryos, f3-lipotropin was the predominant peptide and the f3-lipotropin/f3-e ndorphin ratio increased until the end of the second trimester, suggesting that the first proopiomelanoc ortin cleavage step is the most active in this period of life. Gibbs et al. 14 superfused the fetal pituitary and demonstrated that the anterior lobe acquires its responsiveness to corticotropin-rel easing factor just after the twentieth week of pregnancy. On the other hand, the neurointermedia te lobe releases (3-endorphin only after a potassium chloride-induce d cell depolarization, and no changes occurred with corticotropin-rel easing factor or dopamine. Thus the major peak of f3-lipotropin obtained from whole pituitaries until the end of the second trimester can be interpreted as originating from the anterior lobe. We suggest that as early as the embryo the enzy!lle pattern for proopiomelanoc ortin cleavage is similar to that existing in the adult pituitary. Other authors, 15 studying human adrenocorticotro pic hormone ontogeny, also concluded that the anterior lobe matures before the intermediate one. Another feature of the neurointermedia te lobe (in the rat) is the acetylation of proopiomelanoc ortin and its related peptides, a process that inactivates the ability to bind opiate receptors. 16 It is uncertain if acetylation occurs in the human being and we found little immunoreactivit y, if any, related to acetyl f3-endorphin. In conclusion proopiomelanoc ortin-related peptides are evident early in development and both (3-endorphin and (3-lipotropin were present in embryonic life, between 5 and 7 weeks, while smaller fragments of (3-endorphin, such as -y-endorphin, have a delayed appearance. By the beginning of the second trimester the pituitary processing of proopiomelanoc ortin is similar to that of the adult and the functional activity of the
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anterior lobe seems to prevail over that of the "fetusrelated" neurointermedia te lobe around the twentyfifth week of pregnancy. We are indebted to Dr. V. Wiegant and Dr. P. Burbach of the Rudolf Magnus Institute for Pharmacology, University of Utrecht, The Netherlands, for their advice about high-performan ce liquid chromatography techniques and for the supply of the quoted materials. We also acknowledge Prof. A. E. Panerai, of the Istituto di Farmacologia, Universita di Milano, for his help in performing our first high-performan ce liquid chromatography runs. Finally, the generous supply of anti(3-endorphin and anti-(3-lipotropi n sera from Prof. C. H. Li, of the Hormone Research Laboratory, University of California, San Francisco, is also acknowledged. REFERENCES I. Genazzani AR, Facchinetti F, Parrini D. Beta-lipotropin
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and beta-endorphin plasma levels during pregnancy. Clin Endocrinol 1981; 14:409. Baraldi M, Giarre G, Santi M, Facchinetti F, Petraglia F, Genazzani AR. Pregnancy-related changes of opiate re3 ceptors identified in rat uterine membranes by H naloxone binding. Peptides 1985;6:97 I. Liotta A, Krieger DT. Biosynthetic pathway of ACTH and related peptides of the human placenta: in vitro demonstration. In: Klopper A, Genazzani AR, Crosignani PG, eds. Human placenta-proteins and hormones. London: Academic Press, 1980:267. Dubois P, Vargues-Regairaz H, Dubois MP. Human foetal anterior pituitary immunofiuorescen t evidence for corticotropin and melanotropin activities. Zellforschung 1973; . 45:131. Begeot M, Dubois MP. Immunologic localization of alphaand beta-lipotropin in corticotropic cells of the normal and anencephalic fetal pituitaries. Cell Tissue Res 1978; 193:413. Holland D, Silman RE, Chard T, et al. Corticotropin and lipotropin "family tree" of the human pituitary gland. J Endoc;rinol I 979;83:488. Brubaker PL, Baird AC, Bennett HPJ, Browne CA, Solomon S. Corticotropic peptides in the human fetal pituitary. Endocrinology 1982; 111: 1150. Silman RE, Chard T, Lowry PJ, Smith I, Young IM. Human foetal pituitary peptides and parturition. Nature 1976;260:716. Tilders FJH, Parker CR, Barnea A, Porter JC. The major immunoreactive a-melanocyte-stim ulating hormone (a-MSH)-like substance found in human fetal pituitary tissue is not a-MSH but may be desacetyl a-MSH (adrenocorticotropin 1-13 NH2). J Clin Endocrinol Metab 1981;52:319. Verfoef J, Wiegant VM, De Wied D. Regional distribution of alpha- and gamma-type endorphins in rat brain. Brain Res I 982;23 l :454. Kaplan SL, Grumbach MM, Aubert ML. The ontogenesis of pituitary hormones and hypothalamic factors in the human fetus: maturation of central nervous system regulation of anterior pituitary function. Recent Prog Horm Res 1976;32:161. Burbach JPH, Wiegant VM. Isolation and characterization of a-endorphin and 'Y-endorphin from single human pituitary glands. FEBS Lett 1984;166:267. Przewlocki R, Hollt V, Voigt KH, Herz A. Distinctive
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in vitro release of J3-endorphin from the anterior compared to the posterior/intermediate lobe of rat pituitary. Life Sci 1979;24:1601. Gibbs DM, Stewart RD, LiuJH, Vale W, Rivier J, Yen SSC. Effects of synthetic corticotropin-releasing factor and dopamine on the release of immunoreactive J3-endorphin/J3-lipotropin and a-melanocyte-stimulating hormone from human fetal pituitaries in vitro. J Clin Endocrinol Metab 1982;55: 1149.
15. Allen RG, Pintar JE, Stack J, Kendall JW. Biosynthesis and processing of pro-opiomelanocortin-derived peptides during fetal pituitary development. Dev Biol 1985; 102:43. 16. Smyth DG, Massey DE, Zakarian S, Finnie MDA. Endorphins are stored in biologically active and inactive form: isolation of a-N-acetyl peptides. Nature I 979;279:252.