It has been established for some time that a normal female guinea pig given testicular substances will show behavioral and somatic masculinization (1). The pregnant guinea pig, however, possesses a natural protection against androgen-induced masculinization which is not available to its nonpregnant control. The behavioral protection has been found to be progesterone dependent; the mechanism of the somatic protection is unknown (2). The protective effect afforded the pregnant animal is evidenced by a lack of male-like mounting behavior and by minimal clitoral hypertrophy in response to daily injections of testosterone propionate; this is in contrast to the nonpregnant female given the same treatment. Further investigation has shown that this anti-androgenic protection is observed only in true pregnancy, even despite early ovariectomy, but is not available to the pseudopregnant (hysterectomized) guinea pig (3).

It was found previously (4) that testosterone injections depressed corticosteroid-binding globulin (CBG, transcortin) activity in the intact female rat while progesterone administration increased the CBG level. These observations raised the general question of whether the antiandrogenic protective processes are related to the protein binding of steroids, especially of testosterone, progesterone and corticosteroids. Such relationships appear possible when one considers that in the guinea pig the CBG activity rises considerably during pregnancy (5, 6), as does the progesterone level (7-9).

While much information on steroid binding to serum proteins is available for various species, no systematic study has been reported on such binding in the cycling, pregnant or lactating guinea pig, under normal conditions and after androgen treatment. The present study was undertaken to test whether the antiandrogenic protective processes seen in the pregnant guinea pig and not in the non-pregnant one might be related to steroid-binding parameters.

Materials and Methods

Animal material

Heterozygous English short-hair guinea pigs bred in our own laboratory from a Louisville stock were used for the experiments. Daily vaginal checks of all animals were conducted for at least 3 estrous cycles to insure use of only regularly cycling females and to determine the date of conception as judged from the presence of sperm. All experimentally treated animals received testosterone propionate (TP, Upjohn; 50 mg/ml in sesame oil) intramuscularly between 7 and 8 a.m. daily; the dose was 5 mg on the first day of treatment, and 1 mg daily thereafter until the day of sacrifice. Control females received corresponding volumes of the oil vehicle. Treatment started for the cycling females on the first day of the estrous cycle (first day of vaginal rupture) and for the pregnant females on the 18th day of pregnancy.

Samples were collected on days 1, 6, 11 and 15 of the standard 16-day estrous cycle; on days 22, 44, 55 and 66 of pregnancy, which usually lasts for 68 ± 2 days; and on days 1, 3 and 20–22 of the puerperium. The blood was drawn between 1 and 2 p.m. by cardiac puncture while the animals were under Nembutal anesthesia. The blood was allowed to clot and stand overnight at 4 C; serum was then obtained by centrifugation. The serum samples were pooled, quick-frozen and kept below –20 C until analyzed. Each pool consisted of serum from at least 5 animals.

Biochemical procedures

The unlabeled steroids used were commercial preparations which were recrystallized to the melting points reported in the literature. All radiolabeled steroids were obtained from New England Nuclear Corporation; their radiochemical purity was periodically checked by chromatographic techniques and found to be 95% or higher. The specific activities were for cortisol-4-14C, 40 or 45 mCi/mmole; for testosterone-1,2-3H, 42.3 Ci/mmole; and for progesterone-4-14C, 45.8 mCi/mmole.

Peripheral cortisol concentrations were determined by the colorimetric method of Peterson et al. (10). Since these levels were found to be unusually high in the pregnant guinea pig, further verification was sought by comparative analysis of a few serum samples by alternate means. The fluorometric technique of Silber (11) substantiated the values within ± 4%. The colorimetric data were also confirmed by isotope dilution analysis which agreed within ±7%. The isotope dilution procedure used was essentially that of Peterson (12); the chromatographic separation of the cortisol was carried out by the method of Vogt (13), modified by application of longer (34 cm) paper strips. The result of the isotope dilution experiment provided further evidence that the corticosteroid measured was indeed cortisol. The range of error involved in the colorimetric measurement of cortisol will be discussed below. Protein concentrations of the sera were determined by the biuret technique of Gornall et al. (14).

The binding activities of the sera for the 3 steroid hormones investigated were determined in duplicate by multiple equilibrium dialysis (15) at 37 C essentially as previously described (16). Four ml aliquots of 1:10 diluted sera were used as inside solutions; the protein concentrations ranged from approximately 4.5 to 5.5 mg/mi. In the measurement of the binding parameters for cortisol, testosterone and progesterone, a total of 32, 30 and 28 bags, respectively, was included in each of the duplicate multiple equilibrium dialysis systems; the total inside solutions were therefore 128, 120 and 112 ml, and the outside solutions 256, 240 and 224 ml, respectively. The radiolabeled steroids were added to the outside solutions at a level of 2 ng/ml. Streptomycin and penicillin were added to the system to give final concentrations of 20 μg and 500 U/ml, respectively. These concentrations have been found not to influence the binding activity while providing effective inhibition of bacterial growth. Both gas flow and scintillation counting were used for determination of radioactivity as previously reported (16, 17). Correction factors were applied for any hemolyzed serum to compensate for color quenching.

The steroid-binding activity was expressed as “combining affinity” or C-value (18), by calculating

[Formula displayed as image]

where [Sbd] and [S] are the concentrations of bound steroid and unbound steroid, respectively; [P] is the total protein concentration in g/l. The application, limitations and significance of the C-value for measurement of steroid-binding activity of sera or other protein mixtures have been discussed in detail (19, 20).

The range of error of the C-values for the 3 different steroids as well as that of the cortisol assays was determined by standard formulas (21) and expressed as fiducial limits for percentage deviations from the mean, according to

[Formula displayed as image]

The standard error values, [Formula displayed as image] , were calculated from the duplicates of 24 observations. At a 0.01 probability level, the fiducial limits for the C-values obtained with cortisol, testosterone and progesterone were found to be ±5.8%, ±1.5% and ±3.2%, respectively. The fiducial limits for the cortisol determinations at the same level of probability were ± 2.5%. These calculations of error were made in order to check the internal consistency of the analytical methods as performed in our laboratory. Application of the fiducial statement permitted assessment of the significance of differences among the experimental sera. It should be emphasized that the technique of multiple equilibrium dialysis itself assures determination of steroid-binding activity under well-controlled and strictly identical conditions so that the results obtained for the different sera are truly comparable (20).

As has been pointed out (20), the C-value as determined by equilibrium dialysis to measure steroid-binding activity is the resultant of binding affinity and binding capacity. Any steroid-binding serum protein, such as CBG or albumin, will contribute to the C-value according to its binding affinity and its concentration. In the case of corticosterone binding in normal rat serum, the C-value is given essentially by CBG; the approximate contribution of albumin was found to be normally about 8% (19). For interaction of guinea pig albumin with cortisol we found in other experiments a C-value of 0.03 at 4 C, which would correspond, according to our experience with other steroid-albumin complexes, to a C-value of about 0.02 at 37 C. This figure may be used for an assessment of the approximate albumin contribution to the C-value for cortisol of the guinea pig sera analyzed; for most of these sera, this contribution is a small fraction of the C-values obtained.

To further assess, by an independent procedure, the validity of the C-values obtained in the present study, the corticosteroid-binding capacity was determined by gel filtration (22) in a few guinea pig samples. The method used was essentially that of Doe et al. (23) with slight modification (20). For example, a normal serum pool was compared with the sample that gave the highest CBG activity observed among the sera investigated. The C-values were 0.05 and 1.12, respectively, whereas the corresponding cortisol-binding capacities were found to be 26 and 530 μg/100 ml. Although the values obtained by the 2 methods cannot be directly related to each other, they show clearly that CBG activity can be measured by the multiple equilibrium dialysis technique as well as by gel filtration. Other comparative examples have been reported (20).

For a correct interpretation of the C-values observed with testosterone and progesterone, it is realized that these hormones are bound to albumin with greater affinity than are the corticosteroids. The contribution of the albumin interaction to the C-value, therefore, is higher than in the case of cortisol. This must be considered in the evaluation of small differences between C -values for testosterone and between those for progesterone. Clearly, the contribution of albumin cannot result in a C-value higher than the lowest one obtained among all subjects studied, since no gross variations in albumin concentration are assumed.

Results

All results are reported in Table 1 and depicted in Fig. 1 and 2.

Table 1. Steroid-binding activities (C-value) at 37 C and cortisol concentrations in guinea pig sera under various reproductive conditions
(F = cortisol; T = testosterone; P = progesterone)

Sample
 Cortisol
μg/100 ml 		C-value
F		 C-value
T		 C-value
P
Normal females

Estrous cycle
   day 1
   day 6
   day 11
   day 15

Pregnancy
   day 22
   day 44
   day 55
   day 66

Post partum
   day 1
   day 3
   day 20-22



133
111
99
118


163
224
364
502


323
271
78


0.14
0.17
0.20
0.19


0.45
0.69
0.76
0.84


0.50
0.42
0.13


0.41
0.43
0.36
0.33


1.63
6.5
5.2
4.8


3.4
1.62
0.46


0.25
0.25
0.26
0.23


9.2
22.0
20.2
20.3


27.7
16.8
0.46
Androgen-treated

Estrous cycle
   day 1
   day 6
   day 11
   day 15

Pregnancy
   day 22
   day 44
   day 55
   day 66

Post partum
   day 1



71
60
53
60


118
188
292
432


232


0.04
0.07
0.08
0.03


0.13
0.47
0.76
1.12


0.79


0.35
0.37
—
0.27


2.0
5.6
6.6
8.1


3.8


0.20
0.25
—
0.29


16.7
26.6
26.2
32.1


31.8
Others

   ♀ Guinea pigs, castrated over 90 days
   ♀ Guinea pigs, TP for 100 days
   ♂ Guinea pigs, adult control
   ♂Guinea pigs,  castrated over 90 days


70
64
48
95

0.24
0.07
0.07
0.10

0.35
0.36
0.37
0.28

0.32
0.41
0.32
0.19

Peripheral cortisol levels of guinea pig serum appeared to vary systematically with the 16-day estrous cycle, being highest on day 1 and lowest on day 11. The cortisol-binding activity of the sera, however, varied inversely with these cortisol levels; the greatest activity was seen on day 11 and the lowest on day 1. The binding activities toward testosterone or progesterone did not seem to be affected similarly (Fig. 1; Table 1). The binding activity for testosterone was found highest on day 6 and significantly lower on days 11 and 15. In normal females the binding activity for progesterone did not seem related to the stage of the estrous cycle. The cortisol level as well as the cortisol-binding and testosterone-binding activities in the TP-treated cycling animals paralleled the normal values but were significantly lower in every case.

      
Fig. 1. Steroid-binding activities and total serum cortisol levels in the normal and androgen-treated female guinea pig during the estrous cycle.
  Fig. 2. Steroid-binding activities and total serum cortisol levels in the normal and androgen-treated female guinea pig during pregnancy and puerperium.

The decrease in cortisol-binding levels in response to the larger dose of TP (5 mg) was seen within a day; androgen treatment for 100 days (1 mg daily) did not seem significantly more effective in lowering cortisol-binding values than did treatment for one or six days (Table 1). Long-term androgen treatment did not lead to a marked change in the serum-binding activity for testosterone.

During pregnancy, the serum cortisol concentrations and the binding activities for cortisol, testosterone and progesterone were progressively and markedly elevated over normal estrous cycle levels (Fig. 2; Table 1). The 100-fold increase in the binding activity (C-value) toward progesterone was particularly noteworthy, as was the 15-fold increase in testosterone-binding activity (Fig. 2).

In the untreated pregnant animals the greatest progesterone and testosterone-binding activities were found on day 44. These values remained high but decreased significantly on days 55 and 66. The CBG values, in contrast, continued to rise throughout pregnancy as did all binding values in the animals treated with TP. The steroid-binding activity of serum in the treated animals during pregnancy was even greater than in normals. Progesterone binding increased over 150 times normal cycling levels and testosterone binding rose 20 times.

Within 24 hours after normal parturition, cortisol concentrations and the serum-binding activity for cortisol and testosterone decreased markedly. In contrast, progesterone binding remained high for some time after parturition, with or without androgen treatment. By three weeks post partum, CBG and testosterone-binding activities approximated pregestational levels, while the progesterone-binding activity of the serum was still elevated (Fig. 2; Table 1).

Females showed at least twice the cortisol concentrations and cortisol-binding values seen in males (Table 1). The binding activity for testosterone, however, was about the same in both sexes. Androgen administration to the female reduced the cortisol-binding values toward that of the male; ovariectomy did not have a similar effect. Orchidectomy resulted in an increase of peripheral cortisol to approach female levels, while the CBG activity appeared little affected; the C-values for both testosterone and progesterone were decreased by castration.

Discussion

Fig. 1 shows an inverse fluctuation between the peripheral cortisol levels and the CBG activities during the estrous cycle of the guinea pig; either of these parameters changes in parallel when normal and androgen-treated animals are compared. This may indicate a relationship of these properties to physiological phases of the estrous cycle. Cyclicity of corticosterone levels in the normal rat has been previously reported (16, 24); the highest corticosteroid concentrations were seen during estrus in rat and guinea pig (Table 1). In contrast to the rat, however, the cortisol-binding values of the guinea pig appeared to be highest during diestrus.

Previous observations (4) have indicated that daily administration of estrogen to female rats did not affect CBG activity. Daily administration of progesterone, however, induced a significant increase in CBG activity and decrease in resting corticosterone levels (4). This inverse relationship of rising corticosteroid-binding values and lowered corticosteroid levels in response to progesterone was also observed in the present study in the cycling guinea pig. The CBG values fluctuated in parallel with the peripheral progesterone concentrations which Feder et al. (25) reported to have maximal values at days 9 and 11 of the estrous cycle; the cortisol values changed inversely to the CBG and progesterone levels (Table 1). The binding of progesterone was apparently unaltered during the estrous cycle. When androgen was given, the progesterone-binding activity was increased significantly by day 15. The fluctuations in testosterone binding were consistent and parallel in both normal animals and those treated with testosterone propionate but their relation to the estrous cycle was not evident from the present data.

The mechanisms regulating steroid binding during pregnancy appear to be different from those operating during the estrous cycle. During pregnancy both CBG activities and cortisol levels increase considerably, as does the binding capacity for progesterone and testosterone. The extremely high binding activities for progesterone as well as for testosterone found in the pregnant guinea pig are the highest reported for any species. Even more noteworthy is the further increase of testosterone and progesterone binding in the pregnant animals treated with androgen. TP administration tended to lower the binding values during the estrous cycle but increased most values during pregnancy. Elevated cortisol-binding activity has been reported previously (5, 6, 26) for the pregnant guinea pig. Increased testosterone binding has also been observed in pregnant women (27-30).

Our values for progesterone and testosterone binding rise to a high about day 44 and decline thereafter. The values for plasma progesterone given by Heap and Deanesly (7) also rise to their highest values about this stage and decline thereafter. A relationship between plasma progesterone levels, metabolism and binding has been suggested by these investigators (8).

Following parturition an immediate return toward cycling levels of cortisol and cortisol-binding capacity is seen but pregestational levels are not attained within three days. Progesterone-binding activity in the normal animal is highest immediately after parturition and does not return to normal within 21 days. This slow return of all measures may possibly be related to lactational processes as postulated by Gala and Westphal (6) but the high postpartum CBG values obtained in our present study would not be in keeping with a theory which required low CBG values and high levels of unbound cortisol to collaborate with progesterone and estrogen in the initiation of lactation. To be sure, the original theory is primarily aimed at functions in the rat and the discrepancies and reservations associated with data from the guinea pig have previously been noted (6).

Recently, Pearlman et al. (27, 28), Mercier et al. (29) and Rivarola et al. (30) have described a testosterone-binding protein which becomes markedly elevated in pregnancy. The physiological role of this protein and the relationship to other proteins binding steroids with high affinity has not yet been clarified. Chemical separation from CBG has been reported (31).

The determinations of the steroid-binding activities of the present guinea pig sera by multiple equilibrium dialysis allow an assessment of the uniqueness of the proteins binding testosterone, progesterone and cortisol. For each experimental condition, the three binding activities were measured with one and the same serum pool. If the binding affinity for different steroids, e.g., for cortisol and progesterone, is given by one binding protein, and if interference by binding inhibitors is excluded, the C-values for the two steroids should parallel each other in the various experimental samples analyzed. The present results show that this is not the case for any two of the three steroids tested. The curves in Fig. 1 demonstrate this for the sera obtained during the cycle, where the C-values are generally low and not too different from one serum to the other. It becomes more evident in Fig. 2, where the differences between sera are considerably greater. These results then would suggest that the high-affinity binding activities for progesterone, testosterone and cortisol in guinea pig serum are given by three molecular entities which are independent of each other. Conclusive evidence for this assumption awaits isolation of the binding proteins.

It should be pointed out in connection with the interpretation of the C-values that the basic results for the individual sera, as obtained by multiple equilibrium dialysis at 37 C, are independent of the question of binding competition between the various steroids. This is a consequence of the different sera being measured under conditions which equalize any effects of competing steroids among all sera present in the same multiple equilibrium dialysis system. It is the unique and important feature of the multiple equilibrium dialysis technique at 37 C that the steroids are being equally distributed throughout all dialysis bags containing the different sera. Differences in binding activity (C-values) found between different sera are therefore the result of differences of binding affinity and binding capacity in the macromolecular components (binding proteins) and not of the concentrations of potentially competing steroids which may have been originally present in different quantities in the different sera.

Even if competition for the macromolecular binding sites occurs, for example, between cortisol and progesterone, such competition would equally affect all serum samples. It would, therefore, not be the cause of an artifactual decrease of binding activity for the test steroid in those serum samples in which the original concentration of competing steroid was particularly high. This does not exclude the possibility that he general level of the C-values of all samples of the multiple dialysis system may be somewhat lower or higher, depending on the presence of greater or smaller total amounts of competing steroids; however, the C-values measured for the individual serum samples will correctly represent the binding activity of the given macromolecular systems. Experience over the years has shown that the general level of C-values is closely comparable when large numbers of different serum samples are used in a multiple equilibrium dialysis system; this is being monitored in our laboratory by inclusion of the same normal control sample in the different multiple systems. It should be added that as far as the authors are aware the multiple equilibrium dialysis method at 37 C is the only procedure in which interference with the measurement of binding activity of competing steroids is compensated for by providing equal distribution of all potential binding competitors throughout the system. Moreover, as indicated in the Materials and Methods section, the validity of the results obtained by the multiple equilibrium dialysis method at 37 C has been confirmed in numerous cases by other techniques.

Gonadectomy in the female guinea pig resulted in a significant decrease in resting cortisol levels and an increase in cortisol binding, whereas orchidectomy raised both the peripheral cortisol levels and the cortisol-binding activity. These findings are at variance with those in the rat, where castration increased CBG values in the male while the effect on the female rat was uncertain (4, 32). This difference may indicate a species variation since the rat has a four-or five-day estrous cycle with a relatively short luteal period compared with the 16-day cycle of the guinea pig characterized by an extensive luteal phase. Testosterone administration to the cycling female guinea pig depresses cortisol and cortisol-binding values toward levels seen in the intact male. In the rat this has been previously noted for corticoids (4, 33, 34) and CBG (4). In both the rat and the guinea pig, however, elimination of testosterone by orchidectomy did not result in a CBG activity as high as that of intact females. These findings indicate that, in addition to the presence of androgens, other factors are responsible for the sex differences in CBG levels.

It has been pointed out (35, 36) that reversible association between steroids and plasma proteins (e.g., transcortin, albumin) may provide a storage and buffer system rather than merely a transport mechanism. The complex formation then would regulate the function and metabolism of the steroid, shield the extravascular spaces from inundation, and possibly protect the organism from the effects of excess steroid concentrations. It might be considered that these mechanisms also provide protection to genital tissues and the (behavior-mediating) nervous system. it is postulated that the steroid binding factors in conjunction with high progesterone levels (2, 3) may play a role in protecting the female from androgenic influences. Our present data taken with those of others (8) show parallel changes and dramatic increases in both progesterone and sex steroid-binding factors; and these factors are seen to be sensitive to androgen treatment. Such a physiological process would not be unusual when it is realized that high androgen levels may be present in the maternal organism from aberrant metabolism or from the sexually differentiating male fetus. Rivarola et al. (30) have reported that plasma of pregnant women contains significantly greater levels of androgens than plasma from nonpregnant females. They report that most of the testosterone is protein bound. Unbound androgens would be detrimental (2).

It is realized that a similar protective mechanism has yet to be demonstrated for the fetus. It is known that androgens may be administered to the pregnant female without effect to her while permanently affecting the female fetuses she carries (37). Seal and Doe (5) have reported CBG levels in the fetal guinea pig to be approximately nine times that seen in the normal female but still about that of the mother. These investigators on the basis of cross-species studies found positive correlation between the differences in normal and pregnancy cortisol binding and the type of placental vascular relationship in which, at the same stage in pregnancy, the maternal blood directly bathes the fetal trophoblast (hemochorial) (5). In such a case a mechanism to protect mother and fetus from each other’s negative influences would be valuable.

We postulate that the binding capacity of the pregnant serum serves to prevent active androgenic steroids from acting on the normally sensitive genital tissues. It is further postulated that, when reaching the blood-brain and placental barriers, the macromolecular carrier proteins do not readily pass, whereas the smaller steroid molecules do. Within the CNS of the pregnant female the comparatively large retained levels of progesterone are sufficient to antagonize the androgenic action of testosterone and thus protect against behavioral masculinization.

In the fetus, while some similar protection might normally be available, progesterone and testosterone-binding proteins may not be available in quantities sufficient for protection when abnormally large amounts of androgen are present. Dorfman (38) has reviewed extensive data suggesting that progesterone, in pregnancy, may serve for the anti-androgenic defense of the fetus and this has been suggested also by Diamond (3).

Recently Goldman and Yakovac (39) have demonstrated that corticosteroid administered to pregnant rats could suppress clitoral hypertrophy (masculinization) of female fetuses treated with an inhibitor of 33-hydroxysteroid dehydrogenase. Raised levels of corticosteroids might also offer a behavioral protection of a related nature since it has recently been demonstrated in the cat that corticosteroids in the nervous system are 30 to 800 times as high as in the peripheral blood and following adrenalectomy these concentrations fall 90 to 95 % (40). Henkin et al. (40) suggest this is related to the findings that many nervous system arousal functions (taste, smell and hearing) are significantly increased in patients with untreated adrenal cortical insufficiency and these conditions are remediable by treatment with glucocorticosteroids. By further extrapolation we might suggest added CNS corticoids, as in pregnancy, would depress sexual behavior either directly or via related arousal systems.

Acknowledgments

The authors are indebted to Mr. Calvin Wong, Mrs. Judy Elkins and Mr. Robert Trigg for their assistance, and to Mr. George B. Harding for the chromatographic analyses of the radiolabeled steroid used.

 

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