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A Factorial Analysis of Pituitary-Gonad-Comb Relationships in the Chick1
A Factorial Analysis of Pituitary-GonadComb Relationships in the Chick1 2. ACTION OF PREGNANT-MARE-SERUM AND TESTOSTERONEPROPIONATE ON THE GROWTH OF THE PITUITARY AND GONAD W. R. BRENEMAN Department of Zoology and the Waterman Institute, Indiana University2 (Received for publication September 28, 1950)
T
HE application of the statistical technique of factorial analysis to endocrine experiments was outlined in the first paper of this series, Breneman (1951). The factorial experiment was developed by the administration of pregnant-mare-serum (P.M.S.) and testosterone-propionate (T.P.) separately and in combination to unoperated chicks, unilaterally-castrated chicks, and capons. The data previously considered were those related to the factors which influence androgen production in chicks, and several important features of testicular secretion in the chick were demonstrated by the factorial analysis. First, the single testis of the unilaterally-castrated fowl secretes considerable androgen, but much less than secreted by the two gonads of unoperated chick. Second, the secretory activity of the single testis is increased only slightly by P.M.S. Third, there is an inhibition of the effect of the P.M.S. as a result of simultaneous administration of T.P. Fourth, the capon combs demonstrated a relatively greater response to T.P. than did those of unilaterals or normals.
terone-propionate and the stimulating effect of the pregnant-mare-serum on androgen secretion make it important to analyze the action of these two preparations on the weight of the gonads and the pituitary, since the action of the hormones must be mediated through these glands. The factorial experiments accordingly were planned in such a fashion that comparisons could be made between the unilateral and normals, the unilateral and capons, and the normals and capons. The hormones were administered both alone and in combination in each group.
The inhibitory effect of the testos1 This research was supported in part by a grant from the National Institute of Health, Grant R. G. 882 (C). 2 Contribution No. 433 from the Department of Zoology and No. 101 from the Waterman Institute of Indiana University.
399
MATERIALS AND METHODS Single Comb White Leghorn chicks were used in all experiments and unilateral castrations or caponizations were performed under ether anesthesia before the 5th day of age. The left testis was removed in the unilateral castration. When the animals were 40 days of age, they were divided into experimental groups. The body weights and comb measurements were taken at that time and were used to separate the birds into series as nearly uniform as possible. Daily injections were made from the 40th to the 49th day and the animals were autopsied on the 50th day. Comb weights and gonad weights were taken at autopsy and the two gonads of the unoperated animals were weighed separately in order that each gonad could be compared with the single remaining
400
W. R. BRENEMAN
testis' (right) of the unilateral-castrate. The anterior lobes of the pituitary glands were removed immediately and were placed in a moist chamber. These glands were weighed in groups. This gave greater accuracy but, unfortunately, did not permit an adequate analysis of variance. The pregnant-mare-serum3 was administered subcutaneo'usly in daily dosages of 3 I.U., a total dosage of 30 I.U.; and the testosterone-propionate3 was injected subcutaneously in daily amounts of 0.05 mg.; a total amount of 0.50 mg. When an animal received both preparations one was administered on the right side, the other on the left. Control animals received both 0.1 cc. of sesame oil and 0.1 cc. of distilled water which were the carriers used for the hormones in the other experiments. The number of animals in the series varied from eleven to fifteen and a total of 165 chicks was used. Group means were used for comparisons and the total sum of squares due to error was divided by the harmonic mean in order to compensate for unequal group sizes. The " t " test, Snedecor (1946), was used to estimate the 5% and 1% levels of significance.
TABLE 1.—Gonad weight averages DesignaI a b ab
c ac be abc
Treatment
No.
Average
Male (control) Unilaterally-castrated Male+P.M.S. Unilaterally-castrated +P.M.S. Male+T.P. Unilaterally-castrated Male+P.M.S.+T.P. Unilaterally-castrated +P.M.S.+T.P.
IS 15 IS 15
224.73 83.60 382.31 188.93
15
191.63 127.27 203.87 133.07
chick chick
chick+T.P. 15 chick
15 15
mg.
The designations in the table are those used for the calculation of total effects in the left histogram of Graph I. A distinction should be made between the small and capital letters. A, represents the total effects of the removal of one gonad, for example: a—\-{-ab—b-Yac—c-\-abc—bc. B, the total effects of administration of 30 I.U. of P.M.S. C, the total effects of administration of 0.50 mg. T.P. AB, the total effects of administration of P.M.S. to unilateral. AC, the total effects of administration of T.P. to unilaterals. BC, the total effects of administration of P.M.S. and T.P. to males. ABC, the total effects of administration of P.M.S. and T.P. to unilaterals. I represents the control.
primary interest only in showing the basic data, whereas the total effects and the interactions are illustrated in Graph 1. It should be pointed out that a capital letter, for example A, represents the total effects of a given treatment (in this case a, ab, ac, and abc) minus the sum of the effects in the series which did not receive the treatment (in this case I, b, c, and be). Likewise, the interactions, for example BC or AC, can be determined in a similar manner. If an interaction is positive it is concluded that one treatment has augmented the effect of another; if the interaction is negative, one treatment has RESULTS AND DISCUSSION inhibited the effect of another; if no signifiThe factorial analysis affords an excel- cant increase or decrease occurs, the lent opportunity to evaluate the inhibi- effects of the combined treatments are tory action of testosterone-propionate and additive only. A more detailed discussion the stimulating effect of pregnant-mare- of the factorial experiment was given serum, especially since gonad and pitui- in the first paper of this series, Breneman tary weights changes could be correlated (1951). The first (left histogram) of Graph with the previously described effects of 1 illustrates the effect of various treatthese hormones on endogeneous androgen ments on gonad weights in the unilateralsecretion. The data for gonad weights will normal series in which the weights of the be considered first and are summarized in two gonads of the unoperated animal Table 1. and Graph 1. The former is of served as controls. Treatment a was the effect of the removal of the left gonad, therefore, the weight of the remaining * The author wishes to thank Dr. M. H. Kuizenga, The Upjohn Company, for the pregnantright testis was used as an estimate of mare-serum and Dr. Erwin Schwenk, Schering an effect of unilateral castration. Corporation, for the testosterone-propionate used in this investigation.
The sum of the effects of the removal of
401
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EXPLANATION OF GRAPH 1
Left histogram—the weights of the two gonads of the unoperated male were used as control. A—Total effects of the removal of one gonad. B—Total effects of 30 I.U. P.M.S. in unoperated chicks. C—Total effects of 0.50 mg. T.P. in unoperated chicks. AB—Total effects of 30 I.U. P.M.S. in unilaterals. AC—Total effects of 0.50 mg. T.P. in unilaterals. BC—Total effects of P.M.S. and T.P. in unoperated chicks. A B C Total effects of P.M.S. and T.P. in unilaterals. Second histogram—The weight of the left testis of the male was used as control. A—Represented the effect of the removal of one gonad and all combinations were according to the pattern outlined for the left histogram. Third histogram—The weight of the right testis of the male was used as control. A—Represented the effect of the removal of one gonad and all combinations were according to pattern outlined for left histogram. Right histogram—Comparison of the weight changes in right and left testes of unoperated. The right testis was used as control and A—Represented weight of left testis, B—The effect of P.M.S. on right testis, C—The effect of T.P. on right testis, AB—the effect of P.M.S. on left testis, AC—The effect of T.P. on left testis, BC—The effect of P.M.S. and T.P. on right testis, ABC—The effect of P.M.S. and T.P.' on left testis. X indicates significance at the 5% level and XX indicates significance at the 1% level according to the " t " test.
one gonad (A) on the weight of the remaining gonad demonstrates that the single testis was significantly lighter than the two gonads of the control. These data are in agreement with those in our previous study, Breneman (1950), on gonad weight relationships of normal and unilaterallycastrated chicks over a period of 140 days. The left testis of the chick was reported to be heavier than the right in approximately two-thirds of the animals examined, and it seemed important,
therefore, to compare the gonad of the unilateral with each of the two gonads of the control. Furthermore, the testis of the operated chick is slightly heavier than the corresponding right testis of the normal chick though not significantly so. Since the left testis of control chicks is consistently heavier than the right, these data would indicate that a slight hypertrophy occurred in the single testis of the unilateral-castrate. Arai (1920), and others have reported
402
W. R. BRENEMAN
that in female rats a marked compensatory hypertrophy of the ovary resulted after unilateral-ovariectomy. It must be concluded that such hypertrophy is not characteristic of the chick testis and this offers an explanation for the comb growth data previously reported, Breneman (1951). The fact that the single testis secretes much less androgen than the two testes can be explained as a result of a smaller amount of gonad tissue in the operated birds. The total effect of pregnant-mare-serum on the gonads (B) is very marked. It was observed previously, Breneman (1951), that daily comb growth and, to a more limited degree, comb weights were increased following administration of P.M.S. to the unoperated chicks but that this growth increment was slight in the unilaterals. It is interesting to note, therefore, that the gonad weights were increased significantly when the hormone was administered (B). This increase was also present in the unilaterals (ab of Table 1) but was not reflected in the total effects involving one gonad (AB of Graph 1). This supports the idea expressed by Selye (1940), and by our own work, Breneman (1950), that the single gonad is definitely limited in its capacity to secrete and to increase in size. It must be concluded from these facts that the testis of the unilateral-castrate is unable to utilize the gonadotropins either for growth or for secretion of androgens as effectively as are the two testes of the unoperated chick. It was observed in the earlier paper that testosterone-propionate apparently inhibited the secretion of androgen and again it is interesting to note the total effect of the T.P. on the gonads (C) and on the testis of the operated chicks (AC). The former demonstrates that there was a definite inhibition of the gonads and although data are significant at only the
5% level they are consistent in the comparisons shown in the other histograms. Likewise, it will be observed that the stimulating effect of the P.M.S. on gonad growth in the unoperated animals was inhibited by the T.P. (BC). This combination of hormones also was previously reported to result in a marked inhibitory interaction on androgen secretion when comb growth was studied. The unilaterals responded differently, however, when the T.P. was given in combination with the P.M.S. (ABC), as is demonstrated by the fact that there was no interaction. This result is not surprising, however, because the effect of the P.M.S. in the unilateral (AB) was not significant and the testis weight was less affected by the T.P. (AC). Because of the inhibitory effect of the T.P. on gonad weights (C), it is interesting to note the action of this hormone on the gonad of the unilateral (AC). The testis weight average was greater in all the AC comparisons involving the unilateral. The data are not statistically significant, but the estimates in each instance approached a 5% " t " value, and it will be noted later that this unexpected result is also reflected in the pituitary weights. This suggests that there may be a certain minimum level of androgen necessary for normal growth of the gonads and that this level is not supplied by the endogenous androgen in the unilateral, but was supplemented adequately by the injected T.P. It should be possible to check this hypothesis by additional factorial experiments in which slightly lower and higher dosages of androgens are used. The fact that the total effect (C) demonstrates an inhibition of gonads whereas the interaction with the single gonad does not (AC) emphasizes that the normal gonads are inhibited. This is further shown in Table 1 in which the normal gonad weights are decreased after administration of T.P. (c) but the
PlTUITARY-GONAD RELATIONSHIPS
testis of the unilateral is actually heavier (ac). The two middle histograms of Graph 1 were briefly considered when the weight of the testis of the unilateral was compared with that of each of the two gonads of the unoperated animals. The remaining comparisons are very similar to those shown in the left histogram and differ primarily only in the degree of response. This would be expected, however, since single gonads are being compared with each other; this comparison also is important because of the previously demonstrated asymmetry of the gonads of the chick. The right histogram illustrates an extension of the factorial method which is of value. The data shown in this histogram are for comparison of the left and right testes of the unoperated chick. The weight of the left gonad (A) is greater than that of the right and although this difference is not significant it is in harmony with previous observations. The responses of the gonads to P.M.S. (B) and to T.P. (C) are the significant features of these comparisons. The pregnant-mare-serum had little effect on the weight of the left gonad (AB in this histogram). Since the total effect of P.M.S. (B) was significant, although only at the 5% level, the response must have been due largely to the effect on the right gonad and this observation answers a very important question which could be raised concerning the corresponding interaction in the unilateralnormal comparisons (AB in left histogram). Since the right gonad of the normal is smaller than the left, an explanation for the lack of an AB interaction could have been that the right testis is less responsive to gonadotropin. The data in the right histogram would indicate that such is not the case. Also, the T.P. produced a marked suppression of the weight of the gonads (C) which was significant at 1%,
403
but this was not evident in the interaction which involved the left gonad (AC), further indicating a greater responsiveness of the right testis. The effects of the P.M.S. (AB) and T.P. (AC) on the left testis are not significant and the final point to be considered in the action of the hormones is the effect of the simultaneous administration of P.M.S. and T.P. on the right testis (BC) and on the left (ABC). The BC interaction was inhibitory, and it must be concluded that the inhibiting action of T.P. was great enough to overcome the stimulating effect of the P.M.S. However, this was not true of the action on the left gonad in which the ABC interaction was negligible. These observations may be extended to point out important differences in the behavior of the right testis in a normal environment in contrast with its response in the unilateral. The C effect in the right histogram is comparable to the AC interaction in the left histogram, namely, the effect of T.P. on the right testis. The physiological conditions in the two situations are very different, however, because in the former (C of right histogram) there is a larger amount of endogenous androgen present. The question may again be raised, as it was in connection with the AC interaction of the unilateral-normal data, whether there is a minimum amount of androgen necessary for normal gonad growth and that above this level inhibition will occur. There are obviously fundamental differences in the reactions of the right and left testes and furthermore, the physiological differences between the unoperated and unilaterally-castrated chicks add additional variables to these responses. The effects of the various treatments on the anterior pituitary are important for the interpretation of the growth and secretory responses of the gonads. These data
404
W. R. BRENEMAN
are summarized in Table 2 and Graph 2. It was noted earlier that individual pituitary weights were not taken, hence analysis of variance is not adequate. It is possible on the basis of past experience, however, to make approximations concerning the 1% and 5% value for the " t " test. Previous data have shown that pituitary weights are less variable than TABLE 2.—Pituitary weight averages Treatment
Male (Unoperated) Unilaterally-castrated chicks Capon Male+P.M.S. Unilaterally-castrated chick +P.M.S. Capon+P.M.S. Male+T.P. Uniaterally-castrated chick +T.P. Capon+T.P. Male+P.M.S.+T.P. Unilaterally-castrated chick +P.M.S.+T.P. Capon+P.M.S.+T.P.
No.
Av
e age A
;
15 15 11 15
4.92 4.25 7.33 5.93
15 11 15
4.93 7.00 5.13
15 12 15
4.42 6.11 4.93
15 11
5.40 6.45
The P.M.S. was designated treatment b in all experiments. The T.P. was designated treatment c in all experiments. The removal of one gonad in the unilaterals was designated as treatment a when males or capons served as controls. The effect of two gonads of the normal was designated as treatment a when males were compared with capons as controls. The calculation of total effects (A, B, AB etc.) were made in the manner indicated in the text and under Table 1.
are those of the combs and gonads, accordingly standard errors of the means are lower; it is possible to approximate standard errors and assign values for "t". It must be emphasized, however, that the figures represent only approximations. A study of the middle and right histograms of Graph 2, in which comparisons are made between the pituitary weights of unilaterals-capons, and males-capons respectively, demonstrates that the capon pituitary is much heavier, and these data even by inspection are clearly significant. The left histogram in which comparisons are made between unilaterals and normals
shows that the pituitaries in the operated animals are lighter than those in the normal. It is estimated that the significance would be at least at the 5% level. Confirmation for these conclusions may be found in similar results of pituitary weight changes reported previously, Breneman (1950). The fact that the capons with an absence of gonadal androgen have the heaviest pituitaries, whereas, the unilaterallycastrated chicks with a diminished amount of androgen have the lightest pituitaries, emphasized the importance of understanding the effects of T.P. and P.M.S. Attention will be directed, first of all to the action of the P.M.S. The comparisons between the unilaterals and normals (left histogram) show that the administration of P.M.S. was accompanied (B) by an increase in pituitary weight judged to be significant at about the 1% level. The treatment of the unilaterals with the same preparation (AB), however, did not result in any significant difference in pituitary weights. Thus, the increase must have been largely due to the response of the normal males and is borne out by comparing the male with the male plus P.M.S. in Table 2. It may be recalled also that the P.M.S. was accompanied by increased gonad size and secretion in males but that in the unilaterals these effects were negligible. Pregnant-mareserum had no effect on the weight of pituitaries when comparisons are made with capons (B of the middle and right histogram). The total effect of T.P. on the pituitary was not significant (C) but in the unilaterals the pituitary weight was slightly increased (AC). When a comparison of pituitary weights is made between the unilaterals and capons (middle histogram), the AC interaction is more evident with the weight increased to an estimated 5% significance. A comparable
PlTUITARY-GONAD RELATIONSHIPS
GRAPH
405
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\BA B
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ABA CCB C C
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PITUITARIES
6 EXPLANATION or
GRAPH 2
Left histogram—The weight of the pituitaries of unoperated males was used as control. A—Total effects of unilateral castration on pituitary weight. B—Total effects of 30 I.U. P.M.S. on pituitary weights of unoperated males. C—Total effects of 0.50 mg. T.P. on pituitary weights of unoperated males. AB—Total effects of 30 I.U. of P.M.S. on pituitary weights of unilaterals. AC—Total effects of 0.50 mg. T.P. on pituitary weights of unilaterals. BC—Total effects of P.M.S. and T.P. on pituitary weights of unoperated chicks. ABC—Total effects of P.M.S. and T.P. on pituitary weights of unilaterals. Middle histogram—The weight of the pituitaries of capons was used as control. A—Total effects of the removal of one gonad on pituitary weights and all combinations followed the pattern as outlined for left histogram. Right histogram—The weight of the pituitaries of capons was used as control. A—Total effects of presence of two gonads of unoperated male and all combinations followed the pattern as outlined for left histogram.
interaction, however, is not present in the. male-capon series (AC of right histogram). Although the data are suggestive only, the possibility that a minimum amount of androgen is necessary for normal pituitary growth, but that this minimum is not present in the unilaterally-castrated chick, should be further investigated. This problem was also considered in the previous discussion of gonad growth.
There were no significant interactions involving the simultaneous action of P.M.S. and T.P. in any of the comparisons with the possible exception of the ABC interaction in the male-capon series (right histogram). Since the later interaction is significant at the best at only the 5% level it is questionable if much weight should be given this result. The final important information in the data con-
406
W. R. BRENEMAN
cerns the effects of testosterone-propionate on the capon pituitaries. It was previously suggested that T.P. had no effect on the pituitary weights of the normals and iii the comparison of unilaterals with capons there was no marked difference from expected weight. The study of the total effect of T.P. in the normalcapons series, however, shows that the pituitaries were markedly suppressed (C of right histogram). It appears that the capon pituitary is much more sensitive to variation in androgen level than is the normal pituitary. Reference should again be made to Table 2 in which it will be noted that the capon pituitary weight was reduced from 7.33. mg. to 6.11 mg. following the injection of T.P.
theories is whether the amount of testosterone-propionate was a "physiological" dosage. The amount of T.P. administered was much lower than that frequently used in androgen studies and the dosage was chosen because on the basis of previous experiments it was considered to be about a threshold amount. for chicks, Breneman (1938, 1940, 1942). It was not anticipated that the factorial analysis would be so efficient in demonstrating the effect of the androgen or the dosage could have been substantially reduced. Although the amount used was above threshold level it does not seem justifiable to consider that it was either excessive or non-physiological.
The fluctuations in pituitary weights and the related variations in gonad weight and secretion reported in these factorial experiments are interesting relative to the current theories of pituitary-gonad interaction. Jungck, Heller and Nelson (1947), and Heller and Jungck (1947), advanced the interesting hypothesis that estrogens in physiological dosages have only a minor inhibitory effect on the secretion of gonadotropin by the pituitary. They further suggested that estrogen has a direct inhibitory effect on the ovary. The pituitary enlargement which follows the removal of the gonads is interpreted by them to be a result of the absence of target-organs to metabolize the hormone with a consequent accumulation of gonadotropin in the pituitary. This "target-organ" hypothesis is in marked contrast with the more widely accepted theory that the gonadal hormones have an inhibitory effect on the pituitary, Meyer et al. (1932), Moore and Price (1932), and that the increased size and potency of the castrate pituitary is a result of the absence of the inhibitory substances.
Several features of the data appear to be in accord with the target-organ hypothesis. It was noted that the testosteronepropionate had no inhibitory effect on the pituitary weights of either the unoperated or unilaterally-castrated chicks. Actually the pituitary weights were slightly increased in the latter group. Gonad weights, on the other hand, were decreased in the chicks which received T.P., or T.P. in combination with P.M.S. and this fact suggests that the gonads in the normal animal are very responsive to slight elevations in androgen level. The single testis of the unilateral was not inhibited by T.P., in fact, was slightly heavier.- It is evident that the total amount of male hormone in the unilateral represented by the endogenous androgen plus the injected androgen did not reach an inhibitory level. It was also observed in the first paper of this series, that T.P. inhibited the stimulating effect of P.M.S. on androgen secretion when the two preparations were administered simultaneously. It is improbable that this effect could have been mediated at any other site than the gonad.
The first problem to be considered in the relation of these experiments to the
Certain aspects of the study, however, do not support the target-organ hypothe-
PlTUITARY-GONAD RELATIONSHIPS
sis and some appear more in line with the idea of pituitary inhibition. The enlargement of the castrate pituitary is explained by Jungck, Heller and Nelson (1947) on the assumption that since gonads are not present, pituitary gonadotropin would not be metabolized and would, therefore, accumulate (backlog) in the gland. It was hoped that the administration of P.M.S. would give information on this point by supplying additional material which might backlog. When the P.M.S. was administered to capons, however, the pituitary weights were not increased. (Table 2 and the consideration of the C plus AC effects in the capon comparisons.) Two objections to the supposition that P.M.S. might accumulate in the pituitary can be raised. The hormone was mammalian and not avian, therefore, might not behave as would the endogenous gonadotropin. Also, it was suggested in the first paper of this series that P.M.S. may stimulate the adrenal cortex of the capon to produce androgen, in which case the adrenal would be a target-organ and the P.M.S. would be metabolized. Although the effect of P.M.S. on the capon pituitary was not significant, it was accompanied by a size increase in the pituitary of the unoperated chicks. Since both the secretory activity of the gonads and the gonad weights were increased, it is evident that gonadotropin was being utilized, but in spite of this, the pituitaries were larger. It is difficult to see how this could be explained as an accumulation of gonadotropin. The marked decrease in the pituitary weights of the capons which received T.P. is clear evidence that male hormone will inhibit the pituitary. It is probable that the capon pituitary is more sensitive to the inhibitory effect of the androgen because it is growing more rapidly than the pituitary of either the unoperated or unilateral. In summary it may be said that the data reported here appear to
407
support two important features of the target-organ hypothesis, namely: that physiological dosages of androgen do not inhibit the pituitaries of normal animals nor of unilaterally-castrated chicks, but, that androgen has an inhibitory effect on the testis. The observations that the capon pituitary is inhibited by androgen and that there is no evidence of extra accumulation of gonadotropin when P.M.S. in administered are not in harmony with the target-organ hypothesis but more in accord with the idea of an inhibitory mechanism. Certain features of both hypotheses appear to be necessary to explain the pituitary-gonad interactions in the chick. SUMMARY
Factorial analysis of the effects of pregnant-mare-serum and testosteronepropionate on capons, unilaterally-castrated chicks, and unoperated chicks has afforded an opportunity to analyze the interaction between the gonad and pituitary in chicks. The single (right) testis of the unilateral was slightly larger than either of the testes of the unoperated chicks, but there was no marked compensatory hypertrophy such as that which follows the removal of one ovary in mammals. Pregnant-mare-serum produced a size increase in the weight of the two gonads of normal chicks but the weight increase was very slight in the testis of the unilateral. Data..previously reported on the quantity of androgen secreted by the gonads following treatment with P.M.S. were observed to be directly correlated with the amount of gonad tissue present. It is concluded, however, that the testis of the unilateral is limited in its ability to grow and secrete androgen in response to gonadotropin. Testosterone-propionate inhibited testicular growth in the chicks but the weight of the testis of the unilateral was actually
408
W. R. BRENEMAN
slightly increased. It is suggested that a minimal amount of androgen may be necessary for normal testicular growth. The stimulating effect of P.M.S. on the gonads was counteracted by the simultaneous injection of T.P., but there was no significant interaction between P.M.S. and T.P. in the unilaterally-castrated birds. When the right and left gonads of the male were compared factorially it was observed that the right testis was much more responsive than the left testis to P.M.S. and T.P. The responses of the right testis to hormones was different in the normal animal than it was in the unilateral which indicates important hormonal differences in the two series. The capon pituitary was much heavier than the pituitary of either the male or the unilateral. The injection of P.M.S. was not accompanied by pituitary weight changes in any series except the unoperated chicks in which weights were increased. There was no evidence for the accumulation (backlog) of this gonadotropin in capons. Testosterone-propionate did not decrease pituitary weights in either males or unilateral-castrates, but decreased the weight of capon pituitaries. There was actually a slight increase in the pituitary weights of the unilateral after T.P. injection and this was correlated with the greater testicular weight in those animals. Simultaneous administration of P.M.S. and T.P. produced no significant interactions on pituitary weights. The data which demonstrate that low dosages of T.P. inhibit the gonads but have little or no effect on the pituitary, appear to support the "target-organ" hypothesis of Jungck, Heller and Nelson concerning pituitary-gonad interaction,
but it must be concluded that this hypothesis does not explain either the pituitary size increase which occurred in unoperated chicks which received P.M.S., the inhibition of the capon pituitary which followed the injection of testosterone-propionate, or the failure of the P.M.S. to "backlog" in the capon pituitary. REFERENCES Arai, H., 1920. On the cause of hypertrophy of the surviving ovary after spaying (albino rat) and on the number of ova in it. Am. J. Anat. 28: 59-78. Breneman, W. R., 1938. Relative effectiveness of testosterone-propionate and dihydroandrosterone-benzoate in the chick as indicated by comb growth. Endocrinology 23: 44-52. Breneman, W. R., 1940. Response of pullets to androgens. Poultry Sci. 19: 147-153. Breneman, W. R., 1942. The chick comb for androgen assay. Endocrinology 30: 277-285. Breneman, W. R., 1950. A study of the pituitarygonad-comb relationship in normal, unilateralcastrate, and caponized chicks. J. Exp. Zool. 114: 115-136. Breneman, W. R., 1951. A factorial analysis of pituitary-gonad-comb relationship in the chick—I. Poultry Sci. 30: 231-239. Heller, C. G., and E. Jungck, 1947. Regulation of ovarian growth: inhibition by estrogen or stimulation by gonadotropin? Proc. Soc. Exp. Biol. Med. 65: 152-154. Jungck, E. C , G. Heller and W. O. Nelson, 1947. Regulation of pituitary gonadotropic secretion: Inhibition by estrogen or inactivation by ovaries? Proc. Exp. Biol. Med. 65: 148-152. Meyer, R. K., S. L. Leonard, F. L. Hisaw and S. J. Martin, 1932. The influence of oestrin on the gonad-stimulating complex of the anterior pituitary of castrated male and female rats. Endocrinology 16: 655-665. Moore, C. R., and D. Price, 1932. Gonad hormone functions and the reciprocal influence between gonads and hypophysis with bearing on the problem of sex hormone antagonisms. Am. J. Anat. 50: 13-71. Selye, H., 1940. Are gonadotropic hormones destroyed while they exert their action on the ovary? Proc. Soc. Exp. Biol. Med. 43: 404-406. Snedecor, G. W., 1946. Statistical methods. Iowa State Coll. Press.
T
HE application of the statistical technique of factorial analysis to endocrine experiments was outlined in the first paper of this series, Breneman (1951). The factorial experiment was developed by the administration of pregnant-mare-serum (P.M.S.) and testosterone-propionate (T.P.) separately and in combination to unoperated chicks, unilaterally-castrated chicks, and capons. The data previously considered were those related to the factors which influence androgen production in chicks, and several important features of testicular secretion in the chick were demonstrated by the factorial analysis. First, the single testis of the unilaterally-castrated fowl secretes considerable androgen, but much less than secreted by the two gonads of unoperated chick. Second, the secretory activity of the single testis is increased only slightly by P.M.S. Third, there is an inhibition of the effect of the P.M.S. as a result of simultaneous administration of T.P. Fourth, the capon combs demonstrated a relatively greater response to T.P. than did those of unilaterals or normals.
terone-propionate and the stimulating effect of the pregnant-mare-serum on androgen secretion make it important to analyze the action of these two preparations on the weight of the gonads and the pituitary, since the action of the hormones must be mediated through these glands. The factorial experiments accordingly were planned in such a fashion that comparisons could be made between the unilateral and normals, the unilateral and capons, and the normals and capons. The hormones were administered both alone and in combination in each group.
The inhibitory effect of the testos1 This research was supported in part by a grant from the National Institute of Health, Grant R. G. 882 (C). 2 Contribution No. 433 from the Department of Zoology and No. 101 from the Waterman Institute of Indiana University.
399
MATERIALS AND METHODS Single Comb White Leghorn chicks were used in all experiments and unilateral castrations or caponizations were performed under ether anesthesia before the 5th day of age. The left testis was removed in the unilateral castration. When the animals were 40 days of age, they were divided into experimental groups. The body weights and comb measurements were taken at that time and were used to separate the birds into series as nearly uniform as possible. Daily injections were made from the 40th to the 49th day and the animals were autopsied on the 50th day. Comb weights and gonad weights were taken at autopsy and the two gonads of the unoperated animals were weighed separately in order that each gonad could be compared with the single remaining
400
W. R. BRENEMAN
testis' (right) of the unilateral-castrate. The anterior lobes of the pituitary glands were removed immediately and were placed in a moist chamber. These glands were weighed in groups. This gave greater accuracy but, unfortunately, did not permit an adequate analysis of variance. The pregnant-mare-serum3 was administered subcutaneo'usly in daily dosages of 3 I.U., a total dosage of 30 I.U.; and the testosterone-propionate3 was injected subcutaneously in daily amounts of 0.05 mg.; a total amount of 0.50 mg. When an animal received both preparations one was administered on the right side, the other on the left. Control animals received both 0.1 cc. of sesame oil and 0.1 cc. of distilled water which were the carriers used for the hormones in the other experiments. The number of animals in the series varied from eleven to fifteen and a total of 165 chicks was used. Group means were used for comparisons and the total sum of squares due to error was divided by the harmonic mean in order to compensate for unequal group sizes. The " t " test, Snedecor (1946), was used to estimate the 5% and 1% levels of significance.
TABLE 1.—Gonad weight averages DesignaI a b ab
c ac be abc
Treatment
No.
Average
Male (control) Unilaterally-castrated Male+P.M.S. Unilaterally-castrated +P.M.S. Male+T.P. Unilaterally-castrated Male+P.M.S.+T.P. Unilaterally-castrated +P.M.S.+T.P.
IS 15 IS 15
224.73 83.60 382.31 188.93
15
191.63 127.27 203.87 133.07
chick chick
chick+T.P. 15 chick
15 15
mg.
The designations in the table are those used for the calculation of total effects in the left histogram of Graph I. A distinction should be made between the small and capital letters. A, represents the total effects of the removal of one gonad, for example: a—\-{-ab—b-Yac—c-\-abc—bc. B, the total effects of administration of 30 I.U. of P.M.S. C, the total effects of administration of 0.50 mg. T.P. AB, the total effects of administration of P.M.S. to unilateral. AC, the total effects of administration of T.P. to unilaterals. BC, the total effects of administration of P.M.S. and T.P. to males. ABC, the total effects of administration of P.M.S. and T.P. to unilaterals. I represents the control.
primary interest only in showing the basic data, whereas the total effects and the interactions are illustrated in Graph 1. It should be pointed out that a capital letter, for example A, represents the total effects of a given treatment (in this case a, ab, ac, and abc) minus the sum of the effects in the series which did not receive the treatment (in this case I, b, c, and be). Likewise, the interactions, for example BC or AC, can be determined in a similar manner. If an interaction is positive it is concluded that one treatment has augmented the effect of another; if the interaction is negative, one treatment has RESULTS AND DISCUSSION inhibited the effect of another; if no signifiThe factorial analysis affords an excel- cant increase or decrease occurs, the lent opportunity to evaluate the inhibi- effects of the combined treatments are tory action of testosterone-propionate and additive only. A more detailed discussion the stimulating effect of pregnant-mare- of the factorial experiment was given serum, especially since gonad and pitui- in the first paper of this series, Breneman tary weights changes could be correlated (1951). The first (left histogram) of Graph with the previously described effects of 1 illustrates the effect of various treatthese hormones on endogeneous androgen ments on gonad weights in the unilateralsecretion. The data for gonad weights will normal series in which the weights of the be considered first and are summarized in two gonads of the unoperated animal Table 1. and Graph 1. The former is of served as controls. Treatment a was the effect of the removal of the left gonad, therefore, the weight of the remaining * The author wishes to thank Dr. M. H. Kuizenga, The Upjohn Company, for the pregnantright testis was used as an estimate of mare-serum and Dr. Erwin Schwenk, Schering an effect of unilateral castration. Corporation, for the testosterone-propionate used in this investigation.
The sum of the effects of the removal of
401
PITUITARY-GONAD RELATIONSHIPS
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EXPLANATION OF GRAPH 1
Left histogram—the weights of the two gonads of the unoperated male were used as control. A—Total effects of the removal of one gonad. B—Total effects of 30 I.U. P.M.S. in unoperated chicks. C—Total effects of 0.50 mg. T.P. in unoperated chicks. AB—Total effects of 30 I.U. P.M.S. in unilaterals. AC—Total effects of 0.50 mg. T.P. in unilaterals. BC—Total effects of P.M.S. and T.P. in unoperated chicks. A B C Total effects of P.M.S. and T.P. in unilaterals. Second histogram—The weight of the left testis of the male was used as control. A—Represented the effect of the removal of one gonad and all combinations were according to the pattern outlined for the left histogram. Third histogram—The weight of the right testis of the male was used as control. A—Represented the effect of the removal of one gonad and all combinations were according to pattern outlined for left histogram. Right histogram—Comparison of the weight changes in right and left testes of unoperated. The right testis was used as control and A—Represented weight of left testis, B—The effect of P.M.S. on right testis, C—The effect of T.P. on right testis, AB—the effect of P.M.S. on left testis, AC—The effect of T.P. on left testis, BC—The effect of P.M.S. and T.P. on right testis, ABC—The effect of P.M.S. and T.P.' on left testis. X indicates significance at the 5% level and XX indicates significance at the 1% level according to the " t " test.
one gonad (A) on the weight of the remaining gonad demonstrates that the single testis was significantly lighter than the two gonads of the control. These data are in agreement with those in our previous study, Breneman (1950), on gonad weight relationships of normal and unilaterallycastrated chicks over a period of 140 days. The left testis of the chick was reported to be heavier than the right in approximately two-thirds of the animals examined, and it seemed important,
therefore, to compare the gonad of the unilateral with each of the two gonads of the control. Furthermore, the testis of the operated chick is slightly heavier than the corresponding right testis of the normal chick though not significantly so. Since the left testis of control chicks is consistently heavier than the right, these data would indicate that a slight hypertrophy occurred in the single testis of the unilateral-castrate. Arai (1920), and others have reported
402
W. R. BRENEMAN
that in female rats a marked compensatory hypertrophy of the ovary resulted after unilateral-ovariectomy. It must be concluded that such hypertrophy is not characteristic of the chick testis and this offers an explanation for the comb growth data previously reported, Breneman (1951). The fact that the single testis secretes much less androgen than the two testes can be explained as a result of a smaller amount of gonad tissue in the operated birds. The total effect of pregnant-mare-serum on the gonads (B) is very marked. It was observed previously, Breneman (1951), that daily comb growth and, to a more limited degree, comb weights were increased following administration of P.M.S. to the unoperated chicks but that this growth increment was slight in the unilaterals. It is interesting to note, therefore, that the gonad weights were increased significantly when the hormone was administered (B). This increase was also present in the unilaterals (ab of Table 1) but was not reflected in the total effects involving one gonad (AB of Graph 1). This supports the idea expressed by Selye (1940), and by our own work, Breneman (1950), that the single gonad is definitely limited in its capacity to secrete and to increase in size. It must be concluded from these facts that the testis of the unilateral-castrate is unable to utilize the gonadotropins either for growth or for secretion of androgens as effectively as are the two testes of the unoperated chick. It was observed in the earlier paper that testosterone-propionate apparently inhibited the secretion of androgen and again it is interesting to note the total effect of the T.P. on the gonads (C) and on the testis of the operated chicks (AC). The former demonstrates that there was a definite inhibition of the gonads and although data are significant at only the
5% level they are consistent in the comparisons shown in the other histograms. Likewise, it will be observed that the stimulating effect of the P.M.S. on gonad growth in the unoperated animals was inhibited by the T.P. (BC). This combination of hormones also was previously reported to result in a marked inhibitory interaction on androgen secretion when comb growth was studied. The unilaterals responded differently, however, when the T.P. was given in combination with the P.M.S. (ABC), as is demonstrated by the fact that there was no interaction. This result is not surprising, however, because the effect of the P.M.S. in the unilateral (AB) was not significant and the testis weight was less affected by the T.P. (AC). Because of the inhibitory effect of the T.P. on gonad weights (C), it is interesting to note the action of this hormone on the gonad of the unilateral (AC). The testis weight average was greater in all the AC comparisons involving the unilateral. The data are not statistically significant, but the estimates in each instance approached a 5% " t " value, and it will be noted later that this unexpected result is also reflected in the pituitary weights. This suggests that there may be a certain minimum level of androgen necessary for normal growth of the gonads and that this level is not supplied by the endogenous androgen in the unilateral, but was supplemented adequately by the injected T.P. It should be possible to check this hypothesis by additional factorial experiments in which slightly lower and higher dosages of androgens are used. The fact that the total effect (C) demonstrates an inhibition of gonads whereas the interaction with the single gonad does not (AC) emphasizes that the normal gonads are inhibited. This is further shown in Table 1 in which the normal gonad weights are decreased after administration of T.P. (c) but the
PlTUITARY-GONAD RELATIONSHIPS
testis of the unilateral is actually heavier (ac). The two middle histograms of Graph 1 were briefly considered when the weight of the testis of the unilateral was compared with that of each of the two gonads of the unoperated animals. The remaining comparisons are very similar to those shown in the left histogram and differ primarily only in the degree of response. This would be expected, however, since single gonads are being compared with each other; this comparison also is important because of the previously demonstrated asymmetry of the gonads of the chick. The right histogram illustrates an extension of the factorial method which is of value. The data shown in this histogram are for comparison of the left and right testes of the unoperated chick. The weight of the left gonad (A) is greater than that of the right and although this difference is not significant it is in harmony with previous observations. The responses of the gonads to P.M.S. (B) and to T.P. (C) are the significant features of these comparisons. The pregnant-mare-serum had little effect on the weight of the left gonad (AB in this histogram). Since the total effect of P.M.S. (B) was significant, although only at the 5% level, the response must have been due largely to the effect on the right gonad and this observation answers a very important question which could be raised concerning the corresponding interaction in the unilateralnormal comparisons (AB in left histogram). Since the right gonad of the normal is smaller than the left, an explanation for the lack of an AB interaction could have been that the right testis is less responsive to gonadotropin. The data in the right histogram would indicate that such is not the case. Also, the T.P. produced a marked suppression of the weight of the gonads (C) which was significant at 1%,
403
but this was not evident in the interaction which involved the left gonad (AC), further indicating a greater responsiveness of the right testis. The effects of the P.M.S. (AB) and T.P. (AC) on the left testis are not significant and the final point to be considered in the action of the hormones is the effect of the simultaneous administration of P.M.S. and T.P. on the right testis (BC) and on the left (ABC). The BC interaction was inhibitory, and it must be concluded that the inhibiting action of T.P. was great enough to overcome the stimulating effect of the P.M.S. However, this was not true of the action on the left gonad in which the ABC interaction was negligible. These observations may be extended to point out important differences in the behavior of the right testis in a normal environment in contrast with its response in the unilateral. The C effect in the right histogram is comparable to the AC interaction in the left histogram, namely, the effect of T.P. on the right testis. The physiological conditions in the two situations are very different, however, because in the former (C of right histogram) there is a larger amount of endogenous androgen present. The question may again be raised, as it was in connection with the AC interaction of the unilateral-normal data, whether there is a minimum amount of androgen necessary for normal gonad growth and that above this level inhibition will occur. There are obviously fundamental differences in the reactions of the right and left testes and furthermore, the physiological differences between the unoperated and unilaterally-castrated chicks add additional variables to these responses. The effects of the various treatments on the anterior pituitary are important for the interpretation of the growth and secretory responses of the gonads. These data
404
W. R. BRENEMAN
are summarized in Table 2 and Graph 2. It was noted earlier that individual pituitary weights were not taken, hence analysis of variance is not adequate. It is possible on the basis of past experience, however, to make approximations concerning the 1% and 5% value for the " t " test. Previous data have shown that pituitary weights are less variable than TABLE 2.—Pituitary weight averages Treatment
Male (Unoperated) Unilaterally-castrated chicks Capon Male+P.M.S. Unilaterally-castrated chick +P.M.S. Capon+P.M.S. Male+T.P. Uniaterally-castrated chick +T.P. Capon+T.P. Male+P.M.S.+T.P. Unilaterally-castrated chick +P.M.S.+T.P. Capon+P.M.S.+T.P.
No.
Av
e age A
;
15 15 11 15
4.92 4.25 7.33 5.93
15 11 15
4.93 7.00 5.13
15 12 15
4.42 6.11 4.93
15 11
5.40 6.45
The P.M.S. was designated treatment b in all experiments. The T.P. was designated treatment c in all experiments. The removal of one gonad in the unilaterals was designated as treatment a when males or capons served as controls. The effect of two gonads of the normal was designated as treatment a when males were compared with capons as controls. The calculation of total effects (A, B, AB etc.) were made in the manner indicated in the text and under Table 1.
are those of the combs and gonads, accordingly standard errors of the means are lower; it is possible to approximate standard errors and assign values for "t". It must be emphasized, however, that the figures represent only approximations. A study of the middle and right histograms of Graph 2, in which comparisons are made between the pituitary weights of unilaterals-capons, and males-capons respectively, demonstrates that the capon pituitary is much heavier, and these data even by inspection are clearly significant. The left histogram in which comparisons are made between unilaterals and normals
shows that the pituitaries in the operated animals are lighter than those in the normal. It is estimated that the significance would be at least at the 5% level. Confirmation for these conclusions may be found in similar results of pituitary weight changes reported previously, Breneman (1950). The fact that the capons with an absence of gonadal androgen have the heaviest pituitaries, whereas, the unilaterallycastrated chicks with a diminished amount of androgen have the lightest pituitaries, emphasized the importance of understanding the effects of T.P. and P.M.S. Attention will be directed, first of all to the action of the P.M.S. The comparisons between the unilaterals and normals (left histogram) show that the administration of P.M.S. was accompanied (B) by an increase in pituitary weight judged to be significant at about the 1% level. The treatment of the unilaterals with the same preparation (AB), however, did not result in any significant difference in pituitary weights. Thus, the increase must have been largely due to the response of the normal males and is borne out by comparing the male with the male plus P.M.S. in Table 2. It may be recalled also that the P.M.S. was accompanied by increased gonad size and secretion in males but that in the unilaterals these effects were negligible. Pregnant-mareserum had no effect on the weight of pituitaries when comparisons are made with capons (B of the middle and right histogram). The total effect of T.P. on the pituitary was not significant (C) but in the unilaterals the pituitary weight was slightly increased (AC). When a comparison of pituitary weights is made between the unilaterals and capons (middle histogram), the AC interaction is more evident with the weight increased to an estimated 5% significance. A comparable
PlTUITARY-GONAD RELATIONSHIPS
GRAPH
405
2
4 isT-^
c?-tf
2 0
\BA B
•2
ABA CCB C C
•4
-6 WOT.
-8
A
XX
Of
PITUITARIES
6 EXPLANATION or
GRAPH 2
Left histogram—The weight of the pituitaries of unoperated males was used as control. A—Total effects of unilateral castration on pituitary weight. B—Total effects of 30 I.U. P.M.S. on pituitary weights of unoperated males. C—Total effects of 0.50 mg. T.P. on pituitary weights of unoperated males. AB—Total effects of 30 I.U. of P.M.S. on pituitary weights of unilaterals. AC—Total effects of 0.50 mg. T.P. on pituitary weights of unilaterals. BC—Total effects of P.M.S. and T.P. on pituitary weights of unoperated chicks. ABC—Total effects of P.M.S. and T.P. on pituitary weights of unilaterals. Middle histogram—The weight of the pituitaries of capons was used as control. A—Total effects of the removal of one gonad on pituitary weights and all combinations followed the pattern as outlined for left histogram. Right histogram—The weight of the pituitaries of capons was used as control. A—Total effects of presence of two gonads of unoperated male and all combinations followed the pattern as outlined for left histogram.
interaction, however, is not present in the. male-capon series (AC of right histogram). Although the data are suggestive only, the possibility that a minimum amount of androgen is necessary for normal pituitary growth, but that this minimum is not present in the unilaterally-castrated chick, should be further investigated. This problem was also considered in the previous discussion of gonad growth.
There were no significant interactions involving the simultaneous action of P.M.S. and T.P. in any of the comparisons with the possible exception of the ABC interaction in the male-capon series (right histogram). Since the later interaction is significant at the best at only the 5% level it is questionable if much weight should be given this result. The final important information in the data con-
406
W. R. BRENEMAN
cerns the effects of testosterone-propionate on the capon pituitaries. It was previously suggested that T.P. had no effect on the pituitary weights of the normals and iii the comparison of unilaterals with capons there was no marked difference from expected weight. The study of the total effect of T.P. in the normalcapons series, however, shows that the pituitaries were markedly suppressed (C of right histogram). It appears that the capon pituitary is much more sensitive to variation in androgen level than is the normal pituitary. Reference should again be made to Table 2 in which it will be noted that the capon pituitary weight was reduced from 7.33. mg. to 6.11 mg. following the injection of T.P.
theories is whether the amount of testosterone-propionate was a "physiological" dosage. The amount of T.P. administered was much lower than that frequently used in androgen studies and the dosage was chosen because on the basis of previous experiments it was considered to be about a threshold amount. for chicks, Breneman (1938, 1940, 1942). It was not anticipated that the factorial analysis would be so efficient in demonstrating the effect of the androgen or the dosage could have been substantially reduced. Although the amount used was above threshold level it does not seem justifiable to consider that it was either excessive or non-physiological.
The fluctuations in pituitary weights and the related variations in gonad weight and secretion reported in these factorial experiments are interesting relative to the current theories of pituitary-gonad interaction. Jungck, Heller and Nelson (1947), and Heller and Jungck (1947), advanced the interesting hypothesis that estrogens in physiological dosages have only a minor inhibitory effect on the secretion of gonadotropin by the pituitary. They further suggested that estrogen has a direct inhibitory effect on the ovary. The pituitary enlargement which follows the removal of the gonads is interpreted by them to be a result of the absence of target-organs to metabolize the hormone with a consequent accumulation of gonadotropin in the pituitary. This "target-organ" hypothesis is in marked contrast with the more widely accepted theory that the gonadal hormones have an inhibitory effect on the pituitary, Meyer et al. (1932), Moore and Price (1932), and that the increased size and potency of the castrate pituitary is a result of the absence of the inhibitory substances.
Several features of the data appear to be in accord with the target-organ hypothesis. It was noted that the testosteronepropionate had no inhibitory effect on the pituitary weights of either the unoperated or unilaterally-castrated chicks. Actually the pituitary weights were slightly increased in the latter group. Gonad weights, on the other hand, were decreased in the chicks which received T.P., or T.P. in combination with P.M.S. and this fact suggests that the gonads in the normal animal are very responsive to slight elevations in androgen level. The single testis of the unilateral was not inhibited by T.P., in fact, was slightly heavier.- It is evident that the total amount of male hormone in the unilateral represented by the endogenous androgen plus the injected androgen did not reach an inhibitory level. It was also observed in the first paper of this series, that T.P. inhibited the stimulating effect of P.M.S. on androgen secretion when the two preparations were administered simultaneously. It is improbable that this effect could have been mediated at any other site than the gonad.
The first problem to be considered in the relation of these experiments to the
Certain aspects of the study, however, do not support the target-organ hypothe-
PlTUITARY-GONAD RELATIONSHIPS
sis and some appear more in line with the idea of pituitary inhibition. The enlargement of the castrate pituitary is explained by Jungck, Heller and Nelson (1947) on the assumption that since gonads are not present, pituitary gonadotropin would not be metabolized and would, therefore, accumulate (backlog) in the gland. It was hoped that the administration of P.M.S. would give information on this point by supplying additional material which might backlog. When the P.M.S. was administered to capons, however, the pituitary weights were not increased. (Table 2 and the consideration of the C plus AC effects in the capon comparisons.) Two objections to the supposition that P.M.S. might accumulate in the pituitary can be raised. The hormone was mammalian and not avian, therefore, might not behave as would the endogenous gonadotropin. Also, it was suggested in the first paper of this series that P.M.S. may stimulate the adrenal cortex of the capon to produce androgen, in which case the adrenal would be a target-organ and the P.M.S. would be metabolized. Although the effect of P.M.S. on the capon pituitary was not significant, it was accompanied by a size increase in the pituitary of the unoperated chicks. Since both the secretory activity of the gonads and the gonad weights were increased, it is evident that gonadotropin was being utilized, but in spite of this, the pituitaries were larger. It is difficult to see how this could be explained as an accumulation of gonadotropin. The marked decrease in the pituitary weights of the capons which received T.P. is clear evidence that male hormone will inhibit the pituitary. It is probable that the capon pituitary is more sensitive to the inhibitory effect of the androgen because it is growing more rapidly than the pituitary of either the unoperated or unilateral. In summary it may be said that the data reported here appear to
407
support two important features of the target-organ hypothesis, namely: that physiological dosages of androgen do not inhibit the pituitaries of normal animals nor of unilaterally-castrated chicks, but, that androgen has an inhibitory effect on the testis. The observations that the capon pituitary is inhibited by androgen and that there is no evidence of extra accumulation of gonadotropin when P.M.S. in administered are not in harmony with the target-organ hypothesis but more in accord with the idea of an inhibitory mechanism. Certain features of both hypotheses appear to be necessary to explain the pituitary-gonad interactions in the chick. SUMMARY
Factorial analysis of the effects of pregnant-mare-serum and testosteronepropionate on capons, unilaterally-castrated chicks, and unoperated chicks has afforded an opportunity to analyze the interaction between the gonad and pituitary in chicks. The single (right) testis of the unilateral was slightly larger than either of the testes of the unoperated chicks, but there was no marked compensatory hypertrophy such as that which follows the removal of one ovary in mammals. Pregnant-mare-serum produced a size increase in the weight of the two gonads of normal chicks but the weight increase was very slight in the testis of the unilateral. Data..previously reported on the quantity of androgen secreted by the gonads following treatment with P.M.S. were observed to be directly correlated with the amount of gonad tissue present. It is concluded, however, that the testis of the unilateral is limited in its ability to grow and secrete androgen in response to gonadotropin. Testosterone-propionate inhibited testicular growth in the chicks but the weight of the testis of the unilateral was actually
408
W. R. BRENEMAN
slightly increased. It is suggested that a minimal amount of androgen may be necessary for normal testicular growth. The stimulating effect of P.M.S. on the gonads was counteracted by the simultaneous injection of T.P., but there was no significant interaction between P.M.S. and T.P. in the unilaterally-castrated birds. When the right and left gonads of the male were compared factorially it was observed that the right testis was much more responsive than the left testis to P.M.S. and T.P. The responses of the right testis to hormones was different in the normal animal than it was in the unilateral which indicates important hormonal differences in the two series. The capon pituitary was much heavier than the pituitary of either the male or the unilateral. The injection of P.M.S. was not accompanied by pituitary weight changes in any series except the unoperated chicks in which weights were increased. There was no evidence for the accumulation (backlog) of this gonadotropin in capons. Testosterone-propionate did not decrease pituitary weights in either males or unilateral-castrates, but decreased the weight of capon pituitaries. There was actually a slight increase in the pituitary weights of the unilateral after T.P. injection and this was correlated with the greater testicular weight in those animals. Simultaneous administration of P.M.S. and T.P. produced no significant interactions on pituitary weights. The data which demonstrate that low dosages of T.P. inhibit the gonads but have little or no effect on the pituitary, appear to support the "target-organ" hypothesis of Jungck, Heller and Nelson concerning pituitary-gonad interaction,
but it must be concluded that this hypothesis does not explain either the pituitary size increase which occurred in unoperated chicks which received P.M.S., the inhibition of the capon pituitary which followed the injection of testosterone-propionate, or the failure of the P.M.S. to "backlog" in the capon pituitary. REFERENCES Arai, H., 1920. On the cause of hypertrophy of the surviving ovary after spaying (albino rat) and on the number of ova in it. Am. J. Anat. 28: 59-78. Breneman, W. R., 1938. Relative effectiveness of testosterone-propionate and dihydroandrosterone-benzoate in the chick as indicated by comb growth. Endocrinology 23: 44-52. Breneman, W. R., 1940. Response of pullets to androgens. Poultry Sci. 19: 147-153. Breneman, W. R., 1942. The chick comb for androgen assay. Endocrinology 30: 277-285. Breneman, W. R., 1950. A study of the pituitarygonad-comb relationship in normal, unilateralcastrate, and caponized chicks. J. Exp. Zool. 114: 115-136. Breneman, W. R., 1951. A factorial analysis of pituitary-gonad-comb relationship in the chick—I. Poultry Sci. 30: 231-239. Heller, C. G., and E. Jungck, 1947. Regulation of ovarian growth: inhibition by estrogen or stimulation by gonadotropin? Proc. Soc. Exp. Biol. Med. 65: 152-154. Jungck, E. C , G. Heller and W. O. Nelson, 1947. Regulation of pituitary gonadotropic secretion: Inhibition by estrogen or inactivation by ovaries? Proc. Exp. Biol. Med. 65: 148-152. Meyer, R. K., S. L. Leonard, F. L. Hisaw and S. J. Martin, 1932. The influence of oestrin on the gonad-stimulating complex of the anterior pituitary of castrated male and female rats. Endocrinology 16: 655-665. Moore, C. R., and D. Price, 1932. Gonad hormone functions and the reciprocal influence between gonads and hypophysis with bearing on the problem of sex hormone antagonisms. Am. J. Anat. 50: 13-71. Selye, H., 1940. Are gonadotropic hormones destroyed while they exert their action on the ovary? Proc. Soc. Exp. Biol. Med. 43: 404-406. Snedecor, G. W., 1946. Statistical methods. Iowa State Coll. Press.