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C21 steroids and transcortin-type protein during delayed implantation in the european badger Meles meles L.
J. steroid Biochem.Vol. 20, No. 2, pp. 575-580,1984
0022-4731/84 $3.00+0.00
Copyright cm 1984 Pergamon Press Ltd
Printed in Great Britain. All rights reserved
C,, STEROIDS
AND TRANSCORTIN-TYPE
PROTEIN DURING IN THE EUROPEAN
DELAYED IMPLANTATION BADGER MELES MELES L.
M. BONNIN, B. MARTIN*, G. CHARRON, M. C. AUDY and R. CANIVENC Laboratoire d’Endocrinologie Experimentale, Universitt de Bordeaux II, 146, rue L&o Saignat, 33076 Bordeaux Cedex and *Laboratoire de Physiologie de la Reproduction, Groupe des Sttro’ides, Universitir Pierre et Marie Curie, 75005 Paris, France (Received 21 December 1982) Summary-A high-affinity corticosteroid-binding protein (CBG) roughly resembling a transcortin-type protein is present in badger plasma. Plasma CBG, corticosteroid and progesterone (P) concentratior,s were measured in relation to delayed implantation, true progestation and gestation. Two significant CBG increases were observed during pregnancy. The first, in the second half of embryonic diapause coincides with an increase in plasma corticosteroid concentration and the second, during true progestation and gestation, with an increase in P concentration. Relationship of CBG increases with pregnancy in badger are discussed.
INTRODUCTION Pregnancy occurs in the female European badger (Meles meles L.) in February-March, just after parturition [ 11. Ovulation and fertilization are followed by a prolonged period of embryonic diapause at the blastocyst stage, lasting for 30&310 days. Implantation is delayed until mid-December or early January. Marked changes occur in circulating levels of ovarian steroids during the delayed implantation period. Low plasma concentration of progesterone (P) indicates diminished luteal activity during the major part of the diapause. Just before blastocyst implantation, increased P level testifies to luteal reactivation during true progestation [3]. The concentration of estrogens is generally low, except for episodic releases occurring during diapause [4]. During the true progestation and gestation a biphasic P profile [5] is observed, with a first peak at time of implantation and another during the placental phase. Because a sharp increase in P concentration is observed just before implantation, the latter has been thought to be progesterone-dependent. However administration of exogenous P fails to induce implantation during diapause [6]. Because luteal reactivation and implantation always occur in late autumn or early winter, when daylength is short, it is obvious that delayed implantation in the badger is influenced by environmental factors such as photoperiod and temperature [7, 8,9]. The variations in plasma P concentration suggest possible related variations in steroid-protein binding capacity [lo]. Three plasma proteins with high affinity, limited capacity and great ligand specificity for steroids have been described in vertebrate plasma:
then in several other mammals [lo, 13, 14, 15). (2) A corticosteroid-binding globulin (CBG) or transcortin, first discovered in man [16, 171 and also widely distributed in nature [lo, 18, 19,201. (3) A progesterone-binding globulin (PBG) distinguishable from CBG, found in the plasma of pregnant guineapigs [21,22,23] and other hystricomorph rodents (241 such as the casiragua, the cuis, the degu and the plains viscacha. The biological function of these binding proteins could well be to regulate the physiologically-effective level of circulating hormones and maintain a store of the hormone as a readily-dissociable steroid-protein complex. We studied the binding of corticosteroids and P to plasma protein(s) in the female badger and then quantified the Cz, steroid binding capacities during embryonic diapause, true progestation and gestation in order to establish a possible relationship between plasma binder(s) and variations in peripheral corticosteroid and P levels during the annual cycle and particularly at different stages of pregnancy. MATERIAL AND METHODS Animals and blood samples
From fertilization in February-March to parturition in the following February-March of the next year, 30 female badgers were bled on arrival at the laboratory just after capture. Plasma samples from these animals gave data for monthly means. Four females were bled twice a month onwards for horizontal studies.
from
September
Hormonal determinations
P was assayed by a radioimmunoassay already described [3]. Total corticosteroid levels were measured by a radio-competitive assay [25] using human
(1) A sex-steroid-binding protein (SBP) which binds C,, and C,, steroids first identified in man [l 1, 121and 575
516
M. BONNINet ul.
transcortin as binding protein. The measurements were made on 200 p 1plasma samples after extraction by methylene dichloride which extracted less than 20% of P but 95% of corticosteronc and cortisol. Human transcortin binds corticosterone and cortisol strongly and with the same affinity while proaffinity. gesterone is bound at 113 the [‘Hlcorticosterone (107 ci/mM C.E.A. France) served as the standard hormone. Measurement of steroid-binding protein concentration
High affinity steroid-binding protein concentration from plasma was assayed by the equilibrium dialysis method already described [26]. The isotope-labeled steroids purchased from Amersham (France) used were: [ 1,2,6,7-3H]Progesterone (99 ci/mM) and [ 1,2,6, 7 (n)-3H]corticosterone (91 ci/mM). All binding experiments were performed on plasma stored for less than 2 weeks, at 4°C with stirring for 48 h. Statistical analysis
All values are indicated as means +SEM. An analysis of variance (F-test) was performed followed by a non parametric test (Scheffe test or MannWhitney U-test) giving statistical significance. RESULTS
C,, steroid levels during pregnancy Plasma P levels during pregnancy. After postpartum fertilization and during the delayed implantation period (February, to December) the monthly means of P level fluctuated between 6.47 + 0.80 nM and 16.42 + 1.16 nM (Table 1). At the time of implantation (January) this level rose significantly (P < 0.001) to 38.92 f 4.37 nM and remained high during the gestation (47.66 & 10.23 nM) in February,. The individual profiles of plasma P (Fig. 1) from mid-diapause to parturition showed: (i) an increase at the end of diapause, the first peak
Table I. Plasma concentrations
Months February, March April June July September October November December January February,
state
coinciding with implantation; (ii) a second peak during the gestation. These results are confirmed when data are normalized to the day of implantation (Table 2). Plasma corticosteroid levels during pregnancy. After fertilization, in early diapause (February,) the mean corticosteroids in plasma value of was 64.66 + 9.35 nM. There was no significant variation in this level during the first half of diapause (83.48 f 11.93 nM for March, April, June, July) but during the second half (September to December) a significant increase of corticosteroids was observed (mean value 115.88 k 14.40 nM P < O.Ol), this level being maintained the during gestation (114.76 f 13.73 nM). The individual profile given in Fig. 1 (female badger No. 2) is representative of seasonal means. Steroid protein interaction-Spec$city
A high-affinity protein system was found to bind corticosterone. The binding constants at 4°C and pH 7.4 in plasma diluted 40 times were calculated from Scatchard plots as shown in Fig. 2. For all 60 determinations, the mean values of binding capacities and of dissociation constants (&) were: N = 76.19 + 8.34 nM; K,, = 0.95 & 0.081 nM. Competition analysis (Fig. 3) at different concentrations of competitive steroids revealed that a variety of corticosteroids (corticosterone, cortisol, and cortisone) progestins (progesterone, 17%-hydroxyprogesterone) inhibit [3H]corticosterone binding to a transcortin-type protein. Testosterone and 17c(-epitestosterone were poor inhibitors. Estradiol. 178 -dihydrotestosterone and dexamethasone were not inhibitors. Variation of‘ transcortin -type protein concentration during pregnancy.
Monthly means *SEM of transcortin concentration are given in Table 1. Variance analysis shows
of CBG, corticosteroid and progesterone during delayed implantation, progestation and gestation in female badger CBG (“M)
of pregnancy
Fertilization P 2 4 a .o K
True “roeestation ImplantaEon Gestation
70.5 63.5 42.7 53.8 33.9 86.2 86.5 88.7 86.9
+ f + f f f f + _;
studies
0.1 (6) 0.3 (4) 0.1 (3) 0.9 (5) 0.4 (2) O.l** (5) 0.5;’ (6) 0.4;’ (6) 0.3** (3)
114.0 * 0.7’ (14) 120.5 f 0.1’ (6)
Corticosteroids (“M) 64.7 & 9.3 (6)
83.5 k 11.9 (6)t
115.9 _+ 14.4** (6):
114.8 _ + 13.7** (6)5
true
Progesterone (“M) 7.0 f 0.8 (8) 7.3 f 0.8 (1 I) 6.5 f 1.7 (4) 7.9 + 3.2 (6) 8.2 * 1.9 (3) 23.1 + 4.2 (9) 11.2kO.3(14) 10.3 + 1.3 (9) 16.4_+ 1.2(15) 38.9 + 4.4*** (23) 47.6: 10.2*** (9)
Results are presented as the mean f SEM, number of animals in parentheses. For CBG and P concentrations, monthly means are given. For corticosteroid concentration, means are calculated for February,, tfrom March to July, Sfrom September to December, $January-February, The mean between corticosterone and cortisol molecular weights was used for corticosteroid results. ‘P < 0.025, **p < 0.01, ***p < 0.001. Because pregnancy lasts for one year, two mea” values are given for February: February, = females at the beginning of pregnancy. February, = females in gestation.
Female badger C,,-steroid-plasma-protein
511
interaction
.
100
90
i
.
60
:
S
0
N
D
J
F
M
I
S
I
0
Month
I
N
I
I
D
I
J
I
F
M
Month
Fig. I. Individual profiles of plasma progesterone (O-O), corticosteroid binding capacities (CBG) (O---O) and corticosteroid (O-.-O) levels in two females from mid diapause to parturition.
these means to be significantly different (P < 0.001). During embryonic diapause, a significant increase was observed during September, October, November, December compared with March, April, June and July (P < 0.01). A second significant increase occurred in January and February, (P < 0.025). The 2 mean values obtained for February (February, and February,) were significantly different (P < 0.025). CBG concentration was 1.7 times higher in February, than February,. Individual profiles of CBG concentration in two females from September to February of the following year are given in Fig. 1, together with
steroid profiles. They show an increase in CBG during the last months of pregnancy. In each animal the first CBG peak occurs just before the progesterone peak. Data given in Table 2 are normalized to the day of implantation (day 0). They confirm an increase in CBG capacities just before implantation. DISCUSSION
Plasma P concentrations confirmed those previously observed [3,5]. The maximal values at the end of autumn and in winter show the resumption of
Table 2. Plasma concentrations of CBG, corticosteroid and progesterone during the end of embryonic diapause, the true progestation and the gestation State of pregnancy End of embryonic diapause (days -20 to - 10) True progestation (days -7 to-l) Implantation (day 0) Gestation days 1 to 10 days 10 to 30 days 30 to 45
CBG (nW
Corticosteroids (nM)
Progesterone (nM)
85.9 f 0.3 (3)
98.9 (1)
14.7 & 1.2 (15)
121.3 f 11.2*(7)
101.3 i 6.8 (4)
35.5 + _ 6.1** (7)
114.5 k 1.6’ (3)
106.9 (1)
64.4 _ + 12.9** (3)
117.4*11.5*(4) 120.8 + 11.8’ (6) 92.9 + 7.6 (3)
123.2 (1) 95.2 f 7.2 (6)
39.6 + 4.0’* (4) 58.3 + 10.7** (6) 13.0 + 2.8 (10)
Results are normalized to the implantation day (day 0) determined by the first P peak in animals bled twice a week. Mean values + SEM, number of animals are given in parentheses. *Significantly different P < 0.01. **Significantly different P < 0.001.
578
M.
BONNIN et al.
lb)
I / 2
(a)
I
3
4
5
6
7
8
B
(nM)
9 10.14 15
Fig. 2. Scatchard [38]plots and Rosenthal’s [39]corrections for the binding of corticosterone with plasma proteins from one female badger at two periods of the year. Equilibrium dialysis experiments with 40-fold diluted plasma. B: concentration of bound corticosterone; U: concentration of unbound corticosterone. a: IS/IO - second half of diapause: N=76.80*0.24nM. k,=1.09fO.l5nM. b: 16/l-true progestation: N = 153.60k 1.42nM. kd = 1.15f 0.45 nM. luteal activity coinciding with true progestation, implantation and gestation. Corticosteroid concentrations have not previously been given for the female badger. Among carnivores, peripheral adrenal steroid levels have been given for the fox and the dog but with no relation to sex or physiological state [27]. In these two species, cortisol is the major corticosteroid in circulation at a concentration four times higher than that of corticosterone. Total corticosteroid concentrations found in badger plasma are of the same order as in fox plasma. Our results reveal a circannual rhythm in corticosteroid plasma concentration in the pregnant female badger. Maximal values occur with resumption of endocrine events in autumn and winter [7]. The circannual changes in the luteal function are under the sole control of short photoperiods [9] but it is still not known whether the photoperiod alone and/or other environmental factors (such as temperature, locomotor activity, food availability etc.) are involved in the control of adrenal activity. As regards increases in corticosteroid and P in plasma, it might be thought that the increase in adrenal activity precedes the resumption of luteal activity. However a previous study of the morphological aspect and P content of luteal tissue [28] has demonstrated that luteal activity is resumed at the beginning of autumn, but that this is not reflected in plasma P concentrations until 15 days before implantation [3]. No change in total corticosteroid concentrations is observed during the gestative phase, as in humans (where total cortisol concentration rises above that of the non-pregnant control after the 11th week of gestation [29]) and other mammal species such as the guinea pig [30]. However in pregnant detected [lo].
rats and mice no changes
are
As in other mammals [18], a high-affinity corticosteroid-binding protein, roughly resembling a transcortin-type protein, is present in badger plasma. Our specificity studies on this protein in female plasma are more detailed than those previously reported giving transcortin variation in male badger plasma [26]. Cortisol is bound more strongly than corticosterone. Progestins are bound with high affinity. Although 17aOH progesterone (17%OHP) is bound more strongly than progesterone, its competition with CBG is negligible since 17ctOHP plasma concentration in female plasma is low: 3 nM during diapause, 3 to 6nM during gestation (from unpublished results in collaboration with M. MondainMonval). Transcortin concentrations measured in females over one year reveal a circannual rhythm. Maximal values are observed in autumn and winter as is the case for corticosteroids and P. The first increase occurs from September to December during diapause, with a further increase in January-February, during true progestation and gestation. In September, plasma corticosteroid level and P content of the luteal tissue evolve similarly to CBG, unlike the plasma P level. Both P tissue and P plasma levels increase again with CBG, in January-February,. A summary of these variations is given in Fig. 4. Increased CBG levels during pregnancy have been shown to exist in conjunction with hemo-chorial placentation, which occurs in primates and some other mammals [18]. With other types of placentation, no CBG changes have been observed (10, 18, 311. The results reported here in pregnant female badger show two significant changes in CBG concentration over the year, which may or may not be related to pregnancy. The first increase during the diapause, from September to December must be considered a seasonal variation since it has also been observed during the same period in nonpregnant females (unpublished observations) and in males [26]. However the second increase in January and February, seems to be related to true progestation and gestation. We sought to determine the type of regulation is involved in CBG changes. I
.-.
Corrlcasterone
100
d &
50
Fig. 3. Specificity cortin. B/B,,: ratio unlabeled steroid unlabeled steroid.
of [jH] corticosterone binding to transof labeled steroid bound in presence of to the labeled steroid in absence of Equilibrium dialysis with 40-fold diluted plasma.
Female badger C,,-steroid-plasma-protein
interaction
579
100
:”
Plasma
CBG
/
d 3 Luteal tissue progesterone
3
Plosmo progesterone
, , , , , , )\
j4,
/ I
4 I
I
I
I
FMAMJJASONDJFMA Month
Fig. 4. Schematic variations of mean values of plasma corticosteroids, plasma corticosteroid binding globulin, luteal tissue progesterone and plasma progesterone during pregnancy. P + F: parturition and fertilization. ED: embryonic diapause. TP + G phases: true progestation and gestation.
High estrogen levels are known to be effective in inducing CBG increase in man [lo, 32,33,34] and in some other primates [ 181.In other mammals estrogen treatment has given varied results [18,35]. Circulating estrogen concentrations have been demonstrated to rise during embryonic diapause in late May late July when we have shown there is no increase in CBG. Thus endogenous estrogen activity cannot be related to CBG increases. A biosynthetic stimulus other than estrogen must be responsible for the rise in CBG concentration, as has already been suggested in the case of the prepuberal increase of both CBG and SBP in man [34,36]. A pituitary factor cannot be excluded. In the rat, CBG activity is diminished in the hypophysectomized female [37]. In the badger, increases in adrenal and luteal activity suggest pituitary stimulation from September onwards. This has been demonstrated for LH secretion [7]. The strict parallelism between P and CBG profiles (Fig. 1) during the true progestation and gestation tends to suggest that the stimulatory mechanism involved is the same. A pituitary
stimulation
is thought
to account
for the
first P peak. It has been shown that the second P peak is controlled by the presence of placenta [5]. One possible explanation is that the placenta produces a stimulating factor which acts upon both CBG synthesis and P level either directly or indirectly by means of pituitary. Thus, whatever the type of regulation in CBG changes in the badger, it is clear that
the increases of P and CBG concentrations during the true progestation and gestation are related. Ac/cnowledgement-This research was supported in part by grant No. 2D 1557 from E.D.F.
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4. Mondain-Monval M., Bonnin M., Canivenc R. and Scholler R.: Plasma oestrogen level during delayed implantation in the European badger (Meles meies L.) Gen. Camp. Endocr. 41 (1980) 143-149. 5. Bonnin M., Canivenc R. and Charron G.: Variations du taux de la progestk-one plasmatique chez la femelle de Blaireau apres ablation de l’uterus gravide. C.r. Acad. sci. 293 (1981) 143-14s. 6. Canivenc R. and Laffargue M: Action de diffkents Bquilibres hormonaux sur la phase de vie libre de I’oeuf fkcond6 chez le Blaireau europken (Me/es meles L.) C.r. Sot. Biol. 152 (1958) 58-61. 7. Canivenc R. and Bonnin M.: Environmental control of delayed implantation in the European badger (Meles meles L.). Symposium on “Delayed implantation”
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8. Canivenc R. and Bonnin M.: Delayed implantation
is under environmental control in the Badger (Me/es meles L.) Nature 278 (1979) 849-850. 9. Canivec R., Bonnin M. and Ribes C.: Declenchement de l’ovo-implantation par allongement de la phase sombre de la photoperiode chez le Blaireau europeen (Meles meles L.). C.r. Acad. Sci. 292 (1981) 1009-1012. 10. Westphal Il.: Steroid-Protein Interactions. Springer, Berlin (1971). Il. Daughaday W. H.: Binding of corticosteroids by plasma proteins. III-the binding of corticosteroid and related hormones by human plasma and plasma protein fractions as measured by equilibrium dialysis. J. clin. Invest. 37 (1958) 511-518. 12. Mercier C., Alfsen A. and Baulieu E. E. : Testosterone binding globulin in human plasma. In Proceedings Zhd Symposium on Steroid Hormones. Excerpta Medica Int. Cong. Ser. 101 (1966) 212.
13. Wenn R. U., Kamberti I. A., Keyvanjah M. and Johannes A.: Distribution of testosterone estradiol binding globulin (TeBG) in higher vertebrates. Endokrinologic 69 (1977) 151-156. 14. Sernia C., Bradley A. J. and McDonald I. R.: High affinity binding of adrenocortical and gonadal steroids by plasma proteins of Australian marsupials, Gen.
protein
(PBP). Nature 228 (1970)
24. Heap R. B., Ackland N. and Weir B. J.: Progesteronebinding proteins in plasma of guinea-pigs and other hystricomorph rodents. J. Reprod. Fert. 63 (1981) 477489.
25. Murphy B. E. P.: Some studies of the protein-binding of steroids and their application of the routine micro and ultramicro measurement of various steroids in body fluids by competitive protein-binding radio-assay. J. clin. Endocr. 27 (1967) 973-990.
26. Audy M-C., Martin B., Charron G. and Bonnin M.: Steroid-binding proteins and testosterone level in the badger plasma during the annual cycle. Gen. camp. Endocr. 48 (1982) 239-246.
27. Oddie C. J., Blaine E. H., Bradshaw S. D., Coghlan J. P., Denton D. A., Nelson J. F. and Scoggins B. A.: Blood corticosteroids in Australian marsupial and placental mammals and one monotreme. J. Endocr. 69 (1976) 341-348.
28. Canivenc R., Short R. V. and Bonnin-LaBargue M.: Etude histologique et biochimique du corps jaune du Blaireau europeen (Meles meles L.) Ann. Endocr. 27 (1966) 401413.
29. Demey-Ponsart E., Foidart J. M., Sulon J. and Sodoyez J. C.: Serum CBG, free and total cortisol and circadian patterns of adrenal function in normal pregnancy. J. steroid Biochem. 16 (1982) 165-169.
camp. Endocr. 38 (1979) 496503.
15. Renoir J. M., Mercier-Bodard C. and Baulieu E. E.: Hormonal and immunological aspects on the phylogeny of sex steroid binding plasma protein. Proc. natn. Acad. Sci. 77 (8) (1980) 45784582.
16. Daughaday W. H.: Evidence for two corticosteroid binding systems in human plasma. H. Lab. Clin. Med. 48 (1956) 799-808. 17. Bush J. E.: The physicochemical state of cortisol in blood. In Hormones in Blood (CIBA Found. Colloq. Endocrinol. Vol 11) (Edited by G. E. W. Wolstenhollme and E. C. P. Millar). Little Brown, Boston (1957) p. 263. 18. Seal U. S. and Doe R. P.: The role of corticosteroidbinding globulin in mammalian pregnancy. Proc. 2hd Intern. Congr. on Hormonal Steroids. Excerpta Intern Congr. Series No. 132 (1967) 697-706.
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19. Sandberg A. A., Rosenthal H., Schneider S. L. and Slaunwhite W. R. Jr: Protein steroid interactions and their role in the transport and metabolism of steroids. In Steroid Dynamics (Edited by G. Pincus, T. Nakao and J. F. Tait) (1966). 20. Martin B.: Steroid protein interactions in non mammalian Vertebrates: distribution, origin, regulation and physiological significance of plasma steroid binding proteins, In Steroids and their Mechanism of Action in Non Mammalian Vertebrates. Raven Press (1980) pp. 63-73. 21. Heap R. B.: The binding of plasma progesterone in pregnancy J. Reprod. Fert. 18 (1969) 546548. 22. Heap R. B. and Illingworth D. V.: The maintenance of gestation in the guinea-pig and other hystricomorph rodents: Changes in the dynamics of progesterone metabolism and the occurrence of progesterone binding globulin (PBG). Symp. zool. Sot. Lond. 34 (1974) 385415.
23. Milgrom E., Atger M. and Baulieu E. E.: Progesterone
30. Gala R. R. and Westphal U.: Corticosteroid-binding activity in serum of mouse, rabbit and guinea-pig during pregnancy and lactation: possible involvment in the initiation of lactation. Acta endocr., Copenh. 55 (1967) 47-61.
31. Savu L., Nunez E. and Jayle M. F.: Corticosterone binding by mouse sera during foetal and post-natal development. Acta endocr., Copenh. 84 (1977) 177-190. 32. Sandberg A. A. and Slaunwhite W. R.: Transcortin: a corticosteroid-binding protein of plasma. II-Levels in various conditions and the effects of estrogens. J. clin. Invest. 38 (1959) 1290-1297.
33. Musa B. U., Seal U. S. and Doe R. P.: Elevation of certain plasma proteins in man following estrogen administration: a dose response relationship. J. clin. Endoer. 25 (1965) 1163-I 166. 34 Wagner R. K.: Extracellular and intracellular steroid binding proteins. Properties, discrimination, assay and clinical applications. Acta Endocr., Copenh. suppl. 218 (1978) 73. 35 Gala R. R. and Westphal U.: Corticosteroid binding globulin in the rat: studies on the sex difference, Endocrinology 77 (1965) 84 1-85 1. 36. Falman C. and Winter J. S. D.: The Control of the Onset of Puberty (Edited by M. M. Grumbach, G. D. Grave and F. E. Meyer). Wiley, New York (1974) p. 32. 37. Gala R. R. and Westphal U.: Relationship between the pituitary gland and the corticosteroid-binding globulin in the rat. Endocrinology 78 (1966) 277. 38. Scatchard G.: The attraction of proteins for small molecules and ions. Ann. N. Y. Acad. Sci. 51 (1949) 6tW672.
39. Rosenthal H. E.: A graphic method for the determination and presentation of binding parameters in a complex system. Analyt. Biochem. 20 (1967) 525-532.
0022-4731/84 $3.00+0.00
Copyright cm 1984 Pergamon Press Ltd
Printed in Great Britain. All rights reserved
C,, STEROIDS
AND TRANSCORTIN-TYPE
PROTEIN DURING IN THE EUROPEAN
DELAYED IMPLANTATION BADGER MELES MELES L.
M. BONNIN, B. MARTIN*, G. CHARRON, M. C. AUDY and R. CANIVENC Laboratoire d’Endocrinologie Experimentale, Universitt de Bordeaux II, 146, rue L&o Saignat, 33076 Bordeaux Cedex and *Laboratoire de Physiologie de la Reproduction, Groupe des Sttro’ides, Universitir Pierre et Marie Curie, 75005 Paris, France (Received 21 December 1982) Summary-A high-affinity corticosteroid-binding protein (CBG) roughly resembling a transcortin-type protein is present in badger plasma. Plasma CBG, corticosteroid and progesterone (P) concentratior,s were measured in relation to delayed implantation, true progestation and gestation. Two significant CBG increases were observed during pregnancy. The first, in the second half of embryonic diapause coincides with an increase in plasma corticosteroid concentration and the second, during true progestation and gestation, with an increase in P concentration. Relationship of CBG increases with pregnancy in badger are discussed.
INTRODUCTION Pregnancy occurs in the female European badger (Meles meles L.) in February-March, just after parturition [ 11. Ovulation and fertilization are followed by a prolonged period of embryonic diapause at the blastocyst stage, lasting for 30&310 days. Implantation is delayed until mid-December or early January. Marked changes occur in circulating levels of ovarian steroids during the delayed implantation period. Low plasma concentration of progesterone (P) indicates diminished luteal activity during the major part of the diapause. Just before blastocyst implantation, increased P level testifies to luteal reactivation during true progestation [3]. The concentration of estrogens is generally low, except for episodic releases occurring during diapause [4]. During the true progestation and gestation a biphasic P profile [5] is observed, with a first peak at time of implantation and another during the placental phase. Because a sharp increase in P concentration is observed just before implantation, the latter has been thought to be progesterone-dependent. However administration of exogenous P fails to induce implantation during diapause [6]. Because luteal reactivation and implantation always occur in late autumn or early winter, when daylength is short, it is obvious that delayed implantation in the badger is influenced by environmental factors such as photoperiod and temperature [7, 8,9]. The variations in plasma P concentration suggest possible related variations in steroid-protein binding capacity [lo]. Three plasma proteins with high affinity, limited capacity and great ligand specificity for steroids have been described in vertebrate plasma:
then in several other mammals [lo, 13, 14, 15). (2) A corticosteroid-binding globulin (CBG) or transcortin, first discovered in man [16, 171 and also widely distributed in nature [lo, 18, 19,201. (3) A progesterone-binding globulin (PBG) distinguishable from CBG, found in the plasma of pregnant guineapigs [21,22,23] and other hystricomorph rodents (241 such as the casiragua, the cuis, the degu and the plains viscacha. The biological function of these binding proteins could well be to regulate the physiologically-effective level of circulating hormones and maintain a store of the hormone as a readily-dissociable steroid-protein complex. We studied the binding of corticosteroids and P to plasma protein(s) in the female badger and then quantified the Cz, steroid binding capacities during embryonic diapause, true progestation and gestation in order to establish a possible relationship between plasma binder(s) and variations in peripheral corticosteroid and P levels during the annual cycle and particularly at different stages of pregnancy. MATERIAL AND METHODS Animals and blood samples
From fertilization in February-March to parturition in the following February-March of the next year, 30 female badgers were bled on arrival at the laboratory just after capture. Plasma samples from these animals gave data for monthly means. Four females were bled twice a month onwards for horizontal studies.
from
September
Hormonal determinations
P was assayed by a radioimmunoassay already described [3]. Total corticosteroid levels were measured by a radio-competitive assay [25] using human
(1) A sex-steroid-binding protein (SBP) which binds C,, and C,, steroids first identified in man [l 1, 121and 575
516
M. BONNINet ul.
transcortin as binding protein. The measurements were made on 200 p 1plasma samples after extraction by methylene dichloride which extracted less than 20% of P but 95% of corticosteronc and cortisol. Human transcortin binds corticosterone and cortisol strongly and with the same affinity while proaffinity. gesterone is bound at 113 the [‘Hlcorticosterone (107 ci/mM C.E.A. France) served as the standard hormone. Measurement of steroid-binding protein concentration
High affinity steroid-binding protein concentration from plasma was assayed by the equilibrium dialysis method already described [26]. The isotope-labeled steroids purchased from Amersham (France) used were: [ 1,2,6,7-3H]Progesterone (99 ci/mM) and [ 1,2,6, 7 (n)-3H]corticosterone (91 ci/mM). All binding experiments were performed on plasma stored for less than 2 weeks, at 4°C with stirring for 48 h. Statistical analysis
All values are indicated as means +SEM. An analysis of variance (F-test) was performed followed by a non parametric test (Scheffe test or MannWhitney U-test) giving statistical significance. RESULTS
C,, steroid levels during pregnancy Plasma P levels during pregnancy. After postpartum fertilization and during the delayed implantation period (February, to December) the monthly means of P level fluctuated between 6.47 + 0.80 nM and 16.42 + 1.16 nM (Table 1). At the time of implantation (January) this level rose significantly (P < 0.001) to 38.92 f 4.37 nM and remained high during the gestation (47.66 & 10.23 nM) in February,. The individual profiles of plasma P (Fig. 1) from mid-diapause to parturition showed: (i) an increase at the end of diapause, the first peak
Table I. Plasma concentrations
Months February, March April June July September October November December January February,
state
coinciding with implantation; (ii) a second peak during the gestation. These results are confirmed when data are normalized to the day of implantation (Table 2). Plasma corticosteroid levels during pregnancy. After fertilization, in early diapause (February,) the mean corticosteroids in plasma value of was 64.66 + 9.35 nM. There was no significant variation in this level during the first half of diapause (83.48 f 11.93 nM for March, April, June, July) but during the second half (September to December) a significant increase of corticosteroids was observed (mean value 115.88 k 14.40 nM P < O.Ol), this level being maintained the during gestation (114.76 f 13.73 nM). The individual profile given in Fig. 1 (female badger No. 2) is representative of seasonal means. Steroid protein interaction-Spec$city
A high-affinity protein system was found to bind corticosterone. The binding constants at 4°C and pH 7.4 in plasma diluted 40 times were calculated from Scatchard plots as shown in Fig. 2. For all 60 determinations, the mean values of binding capacities and of dissociation constants (&) were: N = 76.19 + 8.34 nM; K,, = 0.95 & 0.081 nM. Competition analysis (Fig. 3) at different concentrations of competitive steroids revealed that a variety of corticosteroids (corticosterone, cortisol, and cortisone) progestins (progesterone, 17%-hydroxyprogesterone) inhibit [3H]corticosterone binding to a transcortin-type protein. Testosterone and 17c(-epitestosterone were poor inhibitors. Estradiol. 178 -dihydrotestosterone and dexamethasone were not inhibitors. Variation of‘ transcortin -type protein concentration during pregnancy.
Monthly means *SEM of transcortin concentration are given in Table 1. Variance analysis shows
of CBG, corticosteroid and progesterone during delayed implantation, progestation and gestation in female badger CBG (“M)
of pregnancy
Fertilization P 2 4 a .o K
True “roeestation ImplantaEon Gestation
70.5 63.5 42.7 53.8 33.9 86.2 86.5 88.7 86.9
+ f + f f f f + _;
studies
0.1 (6) 0.3 (4) 0.1 (3) 0.9 (5) 0.4 (2) O.l** (5) 0.5;’ (6) 0.4;’ (6) 0.3** (3)
114.0 * 0.7’ (14) 120.5 f 0.1’ (6)
Corticosteroids (“M) 64.7 & 9.3 (6)
83.5 k 11.9 (6)t
115.9 _+ 14.4** (6):
114.8 _ + 13.7** (6)5
true
Progesterone (“M) 7.0 f 0.8 (8) 7.3 f 0.8 (1 I) 6.5 f 1.7 (4) 7.9 + 3.2 (6) 8.2 * 1.9 (3) 23.1 + 4.2 (9) 11.2kO.3(14) 10.3 + 1.3 (9) 16.4_+ 1.2(15) 38.9 + 4.4*** (23) 47.6: 10.2*** (9)
Results are presented as the mean f SEM, number of animals in parentheses. For CBG and P concentrations, monthly means are given. For corticosteroid concentration, means are calculated for February,, tfrom March to July, Sfrom September to December, $January-February, The mean between corticosterone and cortisol molecular weights was used for corticosteroid results. ‘P < 0.025, **p < 0.01, ***p < 0.001. Because pregnancy lasts for one year, two mea” values are given for February: February, = females at the beginning of pregnancy. February, = females in gestation.
Female badger C,,-steroid-plasma-protein
511
interaction
.
100
90
i
.
60
:
S
0
N
D
J
F
M
I
S
I
0
Month
I
N
I
I
D
I
J
I
F
M
Month
Fig. I. Individual profiles of plasma progesterone (O-O), corticosteroid binding capacities (CBG) (O---O) and corticosteroid (O-.-O) levels in two females from mid diapause to parturition.
these means to be significantly different (P < 0.001). During embryonic diapause, a significant increase was observed during September, October, November, December compared with March, April, June and July (P < 0.01). A second significant increase occurred in January and February, (P < 0.025). The 2 mean values obtained for February (February, and February,) were significantly different (P < 0.025). CBG concentration was 1.7 times higher in February, than February,. Individual profiles of CBG concentration in two females from September to February of the following year are given in Fig. 1, together with
steroid profiles. They show an increase in CBG during the last months of pregnancy. In each animal the first CBG peak occurs just before the progesterone peak. Data given in Table 2 are normalized to the day of implantation (day 0). They confirm an increase in CBG capacities just before implantation. DISCUSSION
Plasma P concentrations confirmed those previously observed [3,5]. The maximal values at the end of autumn and in winter show the resumption of
Table 2. Plasma concentrations of CBG, corticosteroid and progesterone during the end of embryonic diapause, the true progestation and the gestation State of pregnancy End of embryonic diapause (days -20 to - 10) True progestation (days -7 to-l) Implantation (day 0) Gestation days 1 to 10 days 10 to 30 days 30 to 45
CBG (nW
Corticosteroids (nM)
Progesterone (nM)
85.9 f 0.3 (3)
98.9 (1)
14.7 & 1.2 (15)
121.3 f 11.2*(7)
101.3 i 6.8 (4)
35.5 + _ 6.1** (7)
114.5 k 1.6’ (3)
106.9 (1)
64.4 _ + 12.9** (3)
117.4*11.5*(4) 120.8 + 11.8’ (6) 92.9 + 7.6 (3)
123.2 (1) 95.2 f 7.2 (6)
39.6 + 4.0’* (4) 58.3 + 10.7** (6) 13.0 + 2.8 (10)
Results are normalized to the implantation day (day 0) determined by the first P peak in animals bled twice a week. Mean values + SEM, number of animals are given in parentheses. *Significantly different P < 0.01. **Significantly different P < 0.001.
578
M.
BONNIN et al.
lb)
I / 2
(a)
I
3
4
5
6
7
8
B
(nM)
9 10.14 15
Fig. 2. Scatchard [38]plots and Rosenthal’s [39]corrections for the binding of corticosterone with plasma proteins from one female badger at two periods of the year. Equilibrium dialysis experiments with 40-fold diluted plasma. B: concentration of bound corticosterone; U: concentration of unbound corticosterone. a: IS/IO - second half of diapause: N=76.80*0.24nM. k,=1.09fO.l5nM. b: 16/l-true progestation: N = 153.60k 1.42nM. kd = 1.15f 0.45 nM. luteal activity coinciding with true progestation, implantation and gestation. Corticosteroid concentrations have not previously been given for the female badger. Among carnivores, peripheral adrenal steroid levels have been given for the fox and the dog but with no relation to sex or physiological state [27]. In these two species, cortisol is the major corticosteroid in circulation at a concentration four times higher than that of corticosterone. Total corticosteroid concentrations found in badger plasma are of the same order as in fox plasma. Our results reveal a circannual rhythm in corticosteroid plasma concentration in the pregnant female badger. Maximal values occur with resumption of endocrine events in autumn and winter [7]. The circannual changes in the luteal function are under the sole control of short photoperiods [9] but it is still not known whether the photoperiod alone and/or other environmental factors (such as temperature, locomotor activity, food availability etc.) are involved in the control of adrenal activity. As regards increases in corticosteroid and P in plasma, it might be thought that the increase in adrenal activity precedes the resumption of luteal activity. However a previous study of the morphological aspect and P content of luteal tissue [28] has demonstrated that luteal activity is resumed at the beginning of autumn, but that this is not reflected in plasma P concentrations until 15 days before implantation [3]. No change in total corticosteroid concentrations is observed during the gestative phase, as in humans (where total cortisol concentration rises above that of the non-pregnant control after the 11th week of gestation [29]) and other mammal species such as the guinea pig [30]. However in pregnant detected [lo].
rats and mice no changes
are
As in other mammals [18], a high-affinity corticosteroid-binding protein, roughly resembling a transcortin-type protein, is present in badger plasma. Our specificity studies on this protein in female plasma are more detailed than those previously reported giving transcortin variation in male badger plasma [26]. Cortisol is bound more strongly than corticosterone. Progestins are bound with high affinity. Although 17aOH progesterone (17%OHP) is bound more strongly than progesterone, its competition with CBG is negligible since 17ctOHP plasma concentration in female plasma is low: 3 nM during diapause, 3 to 6nM during gestation (from unpublished results in collaboration with M. MondainMonval). Transcortin concentrations measured in females over one year reveal a circannual rhythm. Maximal values are observed in autumn and winter as is the case for corticosteroids and P. The first increase occurs from September to December during diapause, with a further increase in January-February, during true progestation and gestation. In September, plasma corticosteroid level and P content of the luteal tissue evolve similarly to CBG, unlike the plasma P level. Both P tissue and P plasma levels increase again with CBG, in January-February,. A summary of these variations is given in Fig. 4. Increased CBG levels during pregnancy have been shown to exist in conjunction with hemo-chorial placentation, which occurs in primates and some other mammals [18]. With other types of placentation, no CBG changes have been observed (10, 18, 311. The results reported here in pregnant female badger show two significant changes in CBG concentration over the year, which may or may not be related to pregnancy. The first increase during the diapause, from September to December must be considered a seasonal variation since it has also been observed during the same period in nonpregnant females (unpublished observations) and in males [26]. However the second increase in January and February, seems to be related to true progestation and gestation. We sought to determine the type of regulation is involved in CBG changes. I
.-.
Corrlcasterone
100
d &
50
Fig. 3. Specificity cortin. B/B,,: ratio unlabeled steroid unlabeled steroid.
of [jH] corticosterone binding to transof labeled steroid bound in presence of to the labeled steroid in absence of Equilibrium dialysis with 40-fold diluted plasma.
Female badger C,,-steroid-plasma-protein
interaction
579
100
:”
Plasma
CBG
/
d 3 Luteal tissue progesterone
3
Plosmo progesterone
, , , , , , )\
j4,
/ I
4 I
I
I
I
FMAMJJASONDJFMA Month
Fig. 4. Schematic variations of mean values of plasma corticosteroids, plasma corticosteroid binding globulin, luteal tissue progesterone and plasma progesterone during pregnancy. P + F: parturition and fertilization. ED: embryonic diapause. TP + G phases: true progestation and gestation.
High estrogen levels are known to be effective in inducing CBG increase in man [lo, 32,33,34] and in some other primates [ 181.In other mammals estrogen treatment has given varied results [18,35]. Circulating estrogen concentrations have been demonstrated to rise during embryonic diapause in late May late July when we have shown there is no increase in CBG. Thus endogenous estrogen activity cannot be related to CBG increases. A biosynthetic stimulus other than estrogen must be responsible for the rise in CBG concentration, as has already been suggested in the case of the prepuberal increase of both CBG and SBP in man [34,36]. A pituitary factor cannot be excluded. In the rat, CBG activity is diminished in the hypophysectomized female [37]. In the badger, increases in adrenal and luteal activity suggest pituitary stimulation from September onwards. This has been demonstrated for LH secretion [7]. The strict parallelism between P and CBG profiles (Fig. 1) during the true progestation and gestation tends to suggest that the stimulatory mechanism involved is the same. A pituitary
stimulation
is thought
to account
for the
first P peak. It has been shown that the second P peak is controlled by the presence of placenta [5]. One possible explanation is that the placenta produces a stimulating factor which acts upon both CBG synthesis and P level either directly or indirectly by means of pituitary. Thus, whatever the type of regulation in CBG changes in the badger, it is clear that
the increases of P and CBG concentrations during the true progestation and gestation are related. Ac/cnowledgement-This research was supported in part by grant No. 2D 1557 from E.D.F.
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