T H E EFFECT ON THE CHICK O F SOME GONADOTROPIC HORMONES W. R. RRENEMAN a Zoo'logical Laboratory, University of Wiscomin The work on the bird by Riddle and his co-workers ('31, '33) and the later studies by Schockaert ( '33)' and Evans and Simpson ('34) have led to the formulation of rather definite conclusions concerning the response of avian gonads to various gonadotropic substances. The very marked stimulation of the bird testes by anterior pituitary hormone and the lack of response to pregnancy urine has become almost axiomatic. Evans and Simpson reported that the avian testes (immature squab) do not respond as well to pregnant mare serum as the mammalian ovaries. These investigators also failed to get augmentation in the pigeon when hypophyseal 'synergist' was injected after combination in vitro with pregnancy prolan. This study was initiated in an attempt to analyze the effect on the immature chick of those fractions of the anterior lobe of the pituitary which produce follicle stimulation (F.S.H.) and luteinization (L.H.) in the mammal, and the action of these preparations when combined. A study was also made of the results of treatment with prolactin, unfractionated pregnant mare serum, whole pituitary extract, pregnancy urine (P.U. ), 'Aided in part by a grant from the National Research Council, Committee on Problems of Sex, administered by IF. L. Hiaaw. 'National Research Fellow. The author wishes to express his thanks to Dr. H. L. Fevold f o r the hormone preparations used in these experiments, to Dr. A. A. Hellbaum for the urinary extracts, and to Prof. F. L. Hisaw for the suggestions and helpful criticisms during the work and in preparation of the manuscript. 211 212 normal male urine, preparations. The determine to what nomenon is found in W. R. BEENEMAN and with certain combinations of these object of the combination series was to extent, if any, the augmentation phethe bird. METHODS A total of 209 experimental and 50 control chicks were utilized in this study. Single Comb White Leghorns were used for series A, A,, and L,, and for 10 of the controls. The remainder of the birds were from a barred-non-barred cross of Rhode Island Red males with Barred Plymouth Rock females. All injections were made subcutaneously and, except where otherwise noted, 0.2 cc. of fluid was given daily for 10 days beginning on the fifth day after hatching. The chicks were killed 24 hours after the last injection. Pregnant mare serum and the pituitary preparations, with the exception of prolactin, were extracted by the method of Fevold, et al. ('34). Prolactin was secured from Doctor Hisaw through the courtesy of Dr. Oscar Riddle. Pregnancy urine and normal male urine were precipitated with tannic acid and extracted with pyridine according to the method of Hellbaum, et al. ( '35). A total of seventeen female and twenty-five male chicks were employed as controls for the 10-day experiments, and were killed at the same time as the injected birds. The number of controls for the 5-day series was not as large a a might be desired, since only four females and four males were used, but the range of variation was small and the gonad weights at this age correlated with those of the older animals. The results of the experiments are summarized in the following table. The figures represent the weight in milligrams of the two testes and the left ovary. 213 HORMONE EFFECT O N CHICK GONADS Gonad weight in control and experhentat chicks FEYALE TBEATMEXT Kind 1 Amount Series 10-day (1) (2) (3) (4) L.H. (1) (Hog) (2) (3) (Sheep) (4) (Horse) (5) F.S.H. (Hog) 1 1 gm.eq. gm.eq. 0.5 1 1 0.5 gm. eq. gm. eq. gm.eq. gm.eq. gm. eq. gm. eq. 1 1 J Pit. 'W' Mare serum [ 1 0.25 gm. eq. 50 R.U. 100R.U. Combination series a F.S.H. (1)and L.H. (1) F.S.H. (2) andL.H. (2) F.S.H. (4) and L.H. (3) F.8.H. (3) and L.H. (4) F.S.H. (3) and L.H. (5) F.S.H. (2) Prolac. (1) F.B.H. (2) andP,U. (1) F.S.H. (4) and P.U. (2) L.H. (2) Prolac. (1) L.H. (2) and P.U. (1) L.H. (3) and P.U. (2) 1 Number 1 1 25 4 4 1 - 17 10.8 4 7.1 7 26.1 3 78.2 4 87.3 46.0 4 4 16.5 5 19.1 4 18.9 2 22.9 3 20.3 6 5.8- 13.2 9.8 4 11.1- 18.7 15.7 11.0- 21.8 4 17.7 3 6.3- 13.5 10.8 11.5- 19.8 14.3 3 61.6 36.0- 89.4 4 89.7 5 I54;;4l> 1 .. 17.57.925.157.275.034.116.112.514.820.818.1- 5.1 6.5 27.0 95.5 98.5 55.5 17.0 27.3 21.3 25.0 24.0 6 4 4 3 3 4 21.146.521.545.562.0146.2- 1 5 52.1 20.2- 45.1 16.2- 25.0 11.5- 19.8 115.0- 29.8 4 Lversge Number Range 64.0 71.0 50.1 70.5 73.5 80.0 1 32.6 58.4 37.3 56.3 65.6 60.5 .. 30.6 20.1 16.1 22.4 1 Rnnge kvernge 15.5-10.4 12.5-11.2 16.1-31.5 34.8-35.5 20.1-25.2 20.8-25.8 10.1-12 .o 16.5-27.5 11.0-18.0 16.8-17.7 11.5-22.5 9.2-19.8 10.8-20.5 12.5 11.6 23.6 35.3 22.8 23.5 11.4 21.8 15.7 17.3 16.0 14.4 13.4 16.8 12.5 14.3 12.0-23.8 11.0-14.5 12.5-15.3 16.6-21.2 25.0-9 8.5 243.2 18.1 44.6 3 5 4 3 3 4 17.0-23.6 18.2-39.0 16.0-29.5 21.8-33.1 19.5-21.2 20.7 27.8 23.1 26.4 2 2 22.2-23.0 15.5-20.2 1 1 1 4 .. 21.7 24.8-36.2 18.2-21.8 13.1-19.4 12.5-16.1 1 28.6 22.6 17.8 19.4 16.2 14.3 All weights are in milligrams, the two testes and the left ovary being represented. 'Five-day series. 'Amount of each extract was the same as that given when each was injected separately. Pit. 'W' unfraetionated extract of hog pituitary gland. 214 W. R. BRENEMAN RESTJ'LTS The data on gonad weights indicate that there are several points of difference from findings previously reported for the response of the avian gonad to gonadotropic preparations. These items are briefly discussed in the following paragraphs. Follicle stimzclatirzg hormolze. The small testicular weight increment found in series A is not clear, as the ovarian response was very close to that which occurred in C where testicular increase was marked. The dosage for series A was not standardized in rat units, and it is quite possible that the actual dose was relatively low. The weights, however, in series B, C, and D showed vexy close correlation. Series D was injected for only 5 days and, accordingly, an absolute comparison cannot be made, but it is evident that the gonads after longer treatment would have approximated closely those of the other two experiments. Maximum stimulation of the ovaries occurred in B, with an average increase of nearly 300 per cent the control weight. This is of interest in two respects : first, Evans and Simpson showed that the squab ovary did not increase in weight until the dosage was ten times that which gave a six-times increment in the testes; and second, the extract (F.S.H.2) which gave this stimulation was not the one that gave the maximum testicular growth. Weight increase indicates quite conclusively that the chick ovary at this time can be appreciably stimulated at the same dosage level that may elicit only a slight response in the testes. This will be discussed further in the analysis of some of the other experiments, Whether this ovarian sensitivity is lost or diminishes in older birds remains to be checked. Luteilzizimg preparatiom. Extracts from the pituitaries of hog, sheep and horse were used. There was a definite increase i n the ovaries and testes following the treatment with each of these extracts. The weight variation does not appear to be significantly correlated With the kind of L.H. injected, although the number of animals is too small to permit any generalization as regards species specificity. With the exception of series A,, which gave no response, the ovarian HORMONE EFFECT O N CHICK GONADS 215 weight increased about 50 per cent; the maximum increment, approximately 90 per cent, occurred in B,. All of the groups gave testicular stimulation, with the minimum again falling in A,. The maximum growth in the testes of the 10-day series, a doubling in weight was found in E (sheep L.H.). The greatest stimulation in the 5-day group occurred in D, which had a net increase of more than 160 per cent. Prolacti%. This preparation was standardized in bird units by Doctor Riddle's laboratory. My experiments were made primarily to check two items: first, whether the injection of prolactin would cause a decrease in the gonad weight similar to that described for the ring dove by Riddle ('33) and fowl ('35); and second, what its effect would be when injected in combination with the follicle stimulating principle. I n series GI, which received a, total of 35 B.U. per chick, the average weight of the testes was only 1 mg. less than that of the controls; and the ovaries were 1.9 mg. heavier than the normals. Two of the ovaries had weights of 19.8 mg. and 18.5 mg. respectively ;these, therefore, exceeded the maximum size found for the female gonad in the control series. Group G,, received 70 B.U. per chick, and apparently showed stimulation of the testes but d t h a response in the female less than in (3,. The average was still slightly above the control and one bird had an ovary weighing 20.5 mg. These results seem to indicate clearly that prolactin does not decrease the gonad weight in the chick at this stage, and it is even suggested that slight stimulation of both the testes and ovary may follow this treatment. The average weight and the presence of gonads larger than the control maximum would seem to eliminate the possibility that a subminimal dosage was injected. Uriinary estracts. The response of birds treated f o r 10 days with 2 R.U. of P.U. daily was comparable to that which followed L.H. injection. The average testicular weight increased nearly 7 mg., and the maximum size, 21.8 mg., was significantly in excess of the heaviest control. Two other males had testes weighing 20.2 mg. and 17.3 mg. respectively. 216 W. R. BRENEMAN The ovaries also appeared to have been stimulated, since the average exceeded the control by more than 4 mg. with a maximum of 23.8 mg. for one gonad and 18.1 mg. for a second. Although not as marked, the 5-day series also suggested a response. The testicular increase was 3.7 mg. greater than the control average. The ovarian increment was less in this group and probably is not significant. Male urine was less marked in its action, but the testes again averaged higher than the controls, and one pair weighed 19.8 mg. The number of animals was small, and random sampling could explain the increase observed for the ovaries. The preceding results are difficult to interpret in view of the evidence in the literature concerning the negative effect of P.U. on the bird gonad. The fact that a small number of animals was used, must be considered in any attempt to analyze these data. As in the prolactin series, the presence of birds with gonads which exceeded the controls by an apparently significant amount cannot be discounted readily. Further study is being made of the action of P.U. on the chick gonad, with a comparison of the action of an alcohol-acetone extract against the tannic acid preparation. Until this work is completed it is felt that no positive statement can be made concerning the data presented here. The similarity of P.U. to the action of L.H. when injected with F.S.H. in the combination series, should also be noted in this connection. Unfractionated extract and pregnant mare serum. Whole hog pituitary and pregnant mare serum were injected. The former had a very marked effect on the testes, producing an increment of about 550 per cent in weight; but the ovarian increase was less, with an average of only 50 per cent greater than the controls. The experiment was designed in order to have a control for the combinations in which F.S.H. and L.H. were injected, and will be discussed in greater detail with those series. The rat unit used for the standarization of the mare serum was the minimum amount of hormone which, when injected into immature female rats for 3 days, produced a 100 per HORMONE EFFECT O N CHICK GONADS 217 cent increase in ovarian weight. The dosages used in the bird experiments elicited an increase which was very marked for both the ovary and testes. The maximum increase in testicular weight in the 50 R.U. series was eleven times the control, with an average of eight times, while the gonads of the male that received 100 R.U. was.twelve times the normal size. The maximum increase for the ovaries of the first experiment was eight times the control, having an average of about 3.7 times greater. The female injected with 100 R.U. had an ovary which showed a twenty tim& weight increment. These results indicate definitely, at least as far as weight is concerned, that the chick ovary is capable of being stimulated tremendously-in this instance actually exceeding the testicular response. These fmdings, when compared with the results of Evans and Simpson, demonstrate that the immature fowl is evidently much more sensitive than is the month-old squab to injections of pregnant mare serum. Combinatiom experiments. Hog, sheep, and horse luteinizing fractions, P.U. and prolaction were combined in vitro with the follicle stimulating extracts tested in series A to D inclusive. Extracts from different animals were employed in order to avoid the possibility of using one extract which might contain an inhibitor substance that was absent in the other preparations. It was shown by Leonard, et al. ('35) that the luteinizing fraction from sheep, when injected intraperitoneally into immature rats, inhibits the action of Antuitrin S, while horse L.H. did not exhibit this phenomenon. I n addition to these series, the hog luteinizer was also injected with P.U. and prolactin. With the exception of L,, there was a decrease in the weight of both testes and ovaries when these were compared with the response which followed the injection of the follicle stimulating principle alone. It was also noted previously that A and A, differed from the other experiments; whether this is due to the fact that this was a different strain of chicks or because of the slight response which the extracts gave when injected separately, cannot be determined at present. Series 218 W. R. BRENEMAN L, apparently shows a simple additive effect of the combination treatment. The weight average for all series with the exception of L,, ranged from 56.3 mg. for the testes of L, (sheep L.H. mixture), to 65.6 mg. for those of L, (horse L.H. mixture). The 5-day series gave approximately the same results, as did also the prolactin and P.U. combinations. The latter is not as conclusive since the number of animals was smaller in the 10-day series, although the results in the shorter series closely follow the F.S.H. plus L.H. weights. The ovarian response was more variable, but essentially the same reaction mas given. The maximum increase, 3.6 mg. greater than the control F.S.H. average, occurred in L,; but the horse L.H., all hog L.H., both P.U. series, and prolactin combinations gave ovarian weights which were less than those following the follicle stimulator alone. It is of interest to note in these experiments, the very close correlation in results of the treatment with prolactin and hog L.H. when each was combined with the same preparation (F.S.H. 2). The former gave average weights of 60.5 mg. for the testes and 28.6 mg. for the ovaries j while the latter gave weights of 58.4 mg. and 27.8 mg. respectively. The groups in which prolactin and P.U. were combined with hog L.H. gave a maximum response for the testes of 22.4 mg. which occurred in series Ms, and a maximum for the ovaries, 19.4 mg. in MI. These variations, however, are within the range observed f o r the L.H. injections; and it must be concluded that for these experiments, there was neither an inhibition nor an additive effect as a result of the combinations. A discussion of the histology of the gonads will appear in a subsequent publication in connection with the problem of comb growth. It should be pointed out that the F.S.H. produced a growth of the seminiferous tubules, the L.H., interstitial tissue growth, and a combination of the two produced an increase in the interstitial tissue and the tubules. The situation in the female is more complex, and a definite statement cannot be made at present concerning the factors involved. The histological response of the chicks treated with the unfractionated HORMONE EFFECT O N CHICK GONADS 219 pituitary extract was essentially the same as that of the combination group. It is also noteworthy that the gonad weights of 61.6 mg. for the testes, and 18.1 mg. for the ovaries following the injection of 0.25 gm. equivalent of the whole pituitary powder, are almost identical for the results found in the F.S.H. plus L.H. series. It is evident from these data that there is a definite inhibition of the action of the hog follicle stimulating fraction when injected subcutaneously after combination in vitro with hog, sheep, or horse L.H., prolactin, or P.U. That the injection method is not the important variable in these results is suggested by the evidence of Evans and Simpson, who showed that when prolan was mixed in vitro with their hypophyseal synergist and injected intramuscularly, the gonadotropic effect of the synergist was decreased and no augmentation was given at any of the dosage levels tested. CONCLUSIONS Maximum stimulation of the chick gonads followed the injection of the follicle stimulating principle (F.S.H.) and pregnant mare serum. I n addition, the luteinizing preparations (L.H.) of hog, sheep, and horse gave weight increments in both ovaries and testes. The hormone dosage level giving 160 per cent increase in the testes resulted in ovaries significantly heavier than the normal. It must be concluded from these results plus the stimulation which followed the treatment with F.S.H. and mare serum, that the chick ovary at this time-5 to 15 days after hatching-is readily stimulated. Prolactin did not significantly decrease the gonad weights in either sex, and there was evidence that some of the gonads were stimulated. Since the weight decrease in the ring dove and fowl, reported by Riddle, et al., occurred in older birds, it is suggested that an age factor is probably involved in this reaction. The tannic acid preparations of pregnancy urine used in the P.U. experiments gave increased gonad weights which closely approximated those found in the L.H. series. Normal male T H E ANATOMICAL RECORD, VOL. 64, NO. 2, AND 8UPPLEMENT NO, 2 220 W. R. BRENEMAN urine was less marked in its action, but some stimulation was indicated. I n view of the small number of animals used, it is not felt that a positive statement is justified at this time concerning the action of these extracts. Definite inhibition of the action of the hog follicle stimulating principle followed its injection after admixture in vitro with hog, sheep, or horse luteinizing fractions, P.U. or prolactin. Combination of hog luteinizer with P.U. or prolactin and subsequent injection did not evidence either inhibition or augmentation. The follicle stimulating fraction produced an hypertrophy of the tubules of the testes; the luteinizing fraction, an increase in the interstitial tissue, the combination series and the unfractionated extracts showed tubule and interstitial tissue hypertrophy. The ovaries have not been studied in sufficient detail to permit a statement of the histological modifications involved. LITERATURE CITED W., E. L. LAHE,AND OSCAR RIDDLE 1935 Gross action of prolactin and follicle stimulating hormone on the mature ovary and sex accessories of fowl. Am. J. Physiol., vol. 111, pp. 361-368. EVANS, HEEBERT MCLEAN,AND IV~IRIAX E. SIMPSON1934 The response of the gonads of immature pigeons t o various gonadotropic hormones. Anat. Rec., vol. 60, pp. 405-421. FEVOLD, H. L., AND F. L. HISAW 1934 Interactions of gonad stimulating hormones in ovarian development. Am. J. Physiol., vol. 109, pp. 655-665. HELLBAUY, A. A., H. L. FEVOLD AND F. L. HISAW 1935 Method for concentrating the gonadotropic aotivity in pregnancy urine. Proc. Soe. Exp. Biol. and Med., vol. 32, pp. 1566-1567. LEONARD, 5. L., F. L. HISAWAND H. L. FEVOLD 1935 (In press.) RIDDLE,OSCAR,AND ROBERTW. BATES 1933 Concerning anterior pituitary hormones. Endocrinology, vol. 17, pp. 689-698; AND IRENE POLHEXUS RIDDLE,OSCAR, 1931 Effects of anterior pituitary hormones on gonads and other organ weights i n the pigeon. Am. J. Physiol., vol. 98, pp. 121-130. SCHOCKAERT, JOSEPH A. 1933 Differences between anterior pituitary sex stimulating hormones and pregnancy urine substances as tested in the male mammal and bird. Am. J. Physiol., vol. 105, pp. 497-507. BATES,R.