ENDOCRINE INFLUENCES UPON T H E GROWTH O F T H E RUDIAIENTARY GONAD O F FOWL1 WALTER R O R N F E L D Department of Plzysiology and Pharmacology, School of Veterinary Medicine, University of Georgia, A t h e n s , Georgia I n domestic fowl the right female gonad ceases to grow about the ninth day of incubation. At that time cortical development may or may not have occurred. The gonadal rudiment can subsequently be induced to proliferate by removal of the ovary, and Domm ('27) suggested that the extent of embryonic differentiation determines whether the rudiment develops into a testis-like compensatory gonad, an ovotestis, or an ovary. Because the rudimentary gonad always hypertrophies following sinistral ovariectomy, it is obvious that the ovary must exert an inhibitory function even in the immature bird. The mechanism of inhibition is not known. Kornfeld and Nalbandov ('54) have shown that estrogen acts as an inhibitor in. vivo. They demonstrated estrogenic activity in the blood of female chicks 16 to 20 days old and succeeded in preventing the hypertrophy of the right gonad of immature poulards with physiological amounts of an estrogen. Hypophysectomy depressed the established testicular compensatory gonad of poulards and prolactin depressed rudiment hypertrophy. Kornfeld ('57) found no effect of sinistral ovariectomy upon adrenal and thyroid growth when seasonal variations were not considered. I wish t o thank Dr. J. L. Carmon f o r his help with the statistical analysis, Dr. J. Davidson (Upjohn Company) f o r estradiol cyclopentylpropionate, testosterone eyelopentylpropionate, and gonadotropic hormone of pregnant mare serum, and Dr. N. L. Heminway (Schering Corporation) f o r progesterone. 619 T H E ALNATOMICAb RECORD, VOL. APRIG 1958 130, N O . 4 620 WALTER KORKFELD Evidence for the presence of androgens in female fowl is only indirect (Breneman, ?55). Kornfeld and Nalbandov ( '54) and Taber and Salley ('54) inhibited the rudimentary gonad of poulards with massive doses of an androgen. There is no evidence for the presence of progesterone in very young birds and since the right gonad is inhibited from the start, it is doubtful that this hormone plays a role in the development of this gland in vivo. The purpose of this paper is to elucidate further endocrine interrelations affecting the rudimentary gonad of female domestic fowl. MATERIALS AXTD METHODS Female White Leghorn chicks of the same commercial stock were used. The battery-brooded birds were randomly assigned to treatments and to compartinents of finishing batteries; a commercial ration, which contained neither a hormonal nor a coccidiostatic supplement, was fed throughout. Unless otherwise stated, sinistral ovariectomies were performed 1 2 to 21 days after hatching. Intact pullets, selected at random before ovariectomies were performed, were also kept. Corn oil (hfazola) was used a s diluent for the injection of steroids. A11 other substances were made u p in distilled water. Injection site was alternately right and left pcctoral and right and left gluteal region. Doses of steroids will refer to v,g per 100 gm of body weight per bird per day. Dosages were adjusted at 10-day intervals to the mean body weight of each group and injected subcutaneously once daily. When only steroids were administered, the total volume injected was the same for all birds. In the experiment involving the injection of fat and water soluble hormones (table 5), controls were not injected during the last 20 days, but all birds were handled similarly. Pituitaries used for injection were collected locally from 2,100 unsexed broilers (New Hampshires and White Rocks) approximately 10 weeks old. The fresh glands were stored as rapidly as possible in acetone following Breneman's ('45) procedure. Before use the pituitaries were dried over calcium H O R M O N E CONTROL O F GON A D A L R U D I M E N T 621 chloride, and the weight was oLtained of each group of 100 glands (146.85 mg with a standard deviation of the mean of 15.44). The glands were finely ground and suspended in water just prior to administration. One group of randomly chosen pituitaries was assayed according to Breneman’s (’45) technic which determines primarily FSH potency by its effect upon testis weight. The activity was found to be 2.4 chick units per pituitary. The equivalent of 2.5 pituitaries per bird was made u p to 0.25 em3 in distilled water twice daily and injected. Other groups received a total of 5 Cartland-Nelson units of pregnant mares’ seruni gonadotropin ( P M S ) per bird per day i n a n analogous manner. Body weight and estimates of comb size (comb length x comb height) were recorded at the beginning and end of each experiment. Body weights a r e reported only when significantly affected by treatment. All experiments were terminated 12 to 24 hours after the last injection. Pituitaries, thyroids, adrenals, oviducts, and gonadal tissues were fixed in Bouin’s solution, dried superficially, and weighed on an analytical balance. Trends of treatment effect upon thyroid and adrenal weight of poulards were not apparent and data a r e not presented. Adrenal and thyroid weight of intact and ovariectomized controls was published (Kornfeld, ’57). Rudiments and structures suspected of being gonadal regenerations were embedded in paraffin, sectioned a t 7 p, mounted, and stained with Milligan’s ( ’46) trichrome stain. Only data from completely ovariectomized birds were analyzed. The term “ovotestis” will refer to gonads containing follicles a s well as medullary tubules with lumina. Oviduct growth was used as quantitative index of estrogenic activity. Oviducts distended with abnormal amounts of fluid were excluded from the data and oviduct weights were transformed into their logarithms before analysis for reasons discussed by Kornfeld and Nalbandov ( ’54). 622 WALTER KORNFELD RESULTS Inhibition of rudiment growth with estrogen This experiment was designed to establish the niinimal dosage for estrogen inhibition of the hypertrophy of the right gonad in poulards and to compare endocrinologically ponlard and intact female. Poulards were given injections of the following graded doses of the ester estradiol cyclopentylpropionate ( E C P ) : 0, 0.125, 0.25, 0.5, 1, 2, and 4 pg. A t the start of treatment the birds were 34 days old and reddening of combs had not occurred. The latter serves as a sensitive criterion for determining the time of onset of androgen secretion by the coinpensatory gonad. The experiment was terminated after 60 injections. Rudiments which had not hypertrophied into macroscopically definite structures were assigned a weight of zero and discarded. The pituitary weight of poulards (table 1)was increased by the three lowest doses of estrogen. The lowest dose (2 p g E C P ) which inhibited rudiment growth, resulted in oviduct and pituitary weights which did not differ significantly from those found in pullets of the same age, but it depressed comb growth. Growth of the compensatory gonad Reddening of the comb of White Leghorn poulards occurs 30 to 50 days after ovariectomy of two- to three-week old birds. An experiment was designed to determine whether a latent period of similar duration necessarily precedes endocrine activity (table 2). Two groups, ovariectomized when 12 to 17 days old, were injected with 2 p g E C P daily for 15 and 30 days respectivc~ly. Treatment started at the age of 31 days with the intent of replacing the ovarian inhibitor with ECP f o r a specified period. Other groups were ovariectomized at thc age of 45, 60, and 75 days and the compensatory gonad of two poulards HORMONE CONTROL O F GONADAL RUDIMENT c; 623 9 2 5 (4) 75 Poulards No. OF 12-17 16-17 45 60 75 12-17 12-17 12-17 14-17 12-14 12-17 14-17 12-16 AGE ( I N DAYS) AT TIilIE OF OVARIECTOMY 24 za < 0 0 0 0 0 0 0 1.5 3 6 12 0 0 0 0 0 0 0 0 0 1 1 1 1 1 2 ECP gm'day) TCP (M'~OO DOSE < 58 2 3 1 30 t 10 5 2 7' 9 f 7 6t 8 I t 3' 4 f 11 5 2 13 2f 48 46 f 20 38 f 19 51 f 32 Rudimentary gonad ~ 4.5 2 0.8 7.2 f 2.8 fl 8.3 f 6.2 ' 7.6 k 1.9 ' 8.9 & 2.1 ' 5.8 t 2.4 7.1 2 1.3fl 6.1 f 1.2 7.021.7' 7.0 f 2.0 5.2 f 1.5 ' 5.4 t 1.1 7.0 & 1.3 ' 8.7 t 2.0 Pituitary \VEIGHT O F 129 2 47 68 t 14 8 6 2 0 78 f 18 86 f 31 67 t 11 135 f 40 103 t 24 117 f 8 110 t 23 117 f 20 932 5 932 4 97 f 28 Oviduct a < < The conipensatory gonad of thcse two birds xvab complctely reinoved 83 days a f t e r hatchling. Sumher of observations of oviduct weight (distentions omitted). ECP was discontinued after 15 injections when the birds were 45 days old. ' ECP was discontinued after 30 iiijections when the birds were 60 days old. One compensatory gonad was lost. Highly significant difference from intact controls (P 0.01). ' Sigiiificaiit difference from intact controls (P 0.05). ' Highly significant diffcreiice from ovariectomized controls (P 0.01). Significant difference froin ovariectomized controls (P 0.05). 10 Pullets '8' '3' '3' '8' 2 1 0 0 0 0 1 1 0 0 0 1 1 0 1 0 0 0 1 0 1 1 2 3 1 0 0 1 5 6 1 5 4 0 0 Ovary 9 Testis Ovotestis T Y P E O F HYPERTROPHIED R U D I M E N T : 3 7 2 2 93 505 t 253 202 2 2 0 1 225 f 67'1' 277 k 87' 218 2 26 251 2 92'*' 264 f 63 4 1 8 2 79 780 % 147 1633 f 268 183 k 55'3' 332 -t 162 ' 2 8 8 f 70' GROWTH CoMR (Mean organ and tissue weights in mg 2 S D ; comb growth as % increase in comb area f SD) T h e estrogen-androgen synergism u p o n rudiment inhibition and data on the znduction period of rudiment Ikypertrophy following sinistral ovariectomy a t daferent ages TABLE 2 H O R M O N E CONTROL O F GONADAL R U D I M E N T 625 was removed 83 days after hatching. Each comb was inspected a t 10-day intervals and the onset of reddening was recorded. All birds were terminated at 100 days of age. Neither estrogen treatment to the age of 45 and 60 days, nor ovariectomy at the age of 45 days had a significant effect upon gonad size, but the weight of the compensatory gonad was depressed i n the birds which had been ovariectomized when 60 and 75 days old. There was no significant treatment effect on log oviduct and pituitary weights. Because reddening of some combs occurred during E C P treatment and because combs of the birds which were ovariectomized at 75 days of age never lost their color completely, observations of these groups could not be interpreted. F o r the three remaining treatments data a r e listed in the following sequence from left to right: Age (in days) at the time of ovariectomy ; days elapsed between ovariectomy and first observation of comb reddening; days elapsed between ovariectomy and the time when combs of the majority of birds had begun to turn red. 12-17 45 60 34 14 19 53 34 19 The figures suggest that the interval from ovariectomy to onset of androgen secretion by the compensatory gonad is greatest when the ovary is removed from the very young. Regardless of treatment effect upon rudiment hypertrophy and pituitary size, comb growth f or the entire experimental period was significantly less than that of intact pullets in all groups except the one ovariectomized at 60 days of age. Comb growth of two bilaterally ovariectomized birds was similar to that observed after sinistral ovariectomy at the age of 7 5 days. Comb area (in em2) increased during the 17 days after the removal of the compensatory gonad from 5.64 to 5.99 and from 5.39 to 5.45 respectively. No gonadal tissue was found. Estrogen-androgen synergisna in t h e inhibition of r u d i m e n t growth Because E C P doses of 1 p g and above depressed comb growth of poulards below that of the intact controls, the possi- 626 WALTER KORNFELD bility of a n androgen-estrogen synergism in the pullet’s inhibitory mechanism had to be considered. This hypothesis was tested by treating poulards with a dose of estrogen xhich is subminimal for the inhibition of rudiment growth and with combinations of this dose with graded amounts of androgen. Intact and ovariectomized controls were kept as well as a control group of poulards receiving the inhibitory dose of 2 p g E CP. Poulards were treated with 1 p g E C P alone and in combination with the followiiig amounts of testosterone cyclopentylpropionate ( T C P ) : 1.5, 3, 6, and 12 pg. Treatment was started when the birds were 31 days old and before gonadal androgen had begun to stimulate the comb visibly. The esperinient was terminated after 70 injections. Rudiments which had not visibly hypertrophied were again assigned a weight of zero. Several additional experimental groups of poulards which were run with these same controls (table 2) have already been discussed. Estrogen, singly and in combination with androgen, depressed rudiment weight and reduced the number of proliferating compensatory gonads. Analysis of variance which showed no significant effect of this treatment 011 log oviduct weight, showed a highly significant treatment effect on pituit a r y w i g h t . The mean pituitary weight of intact controls was lovrer than ihat of any treated group. Only the comb growth produced by the estrogen-androgen combination of 1 p g E C P plus 3 p g T C P did not differ significantly from that of intact and ovariectomized controls. Regression coefficients of comb a r ea on age were calcnlated (table 3). The three regression coefficients did not differ significantly from each other and a linear regression fitted the data significantly better than a quadratic. The fact that comb growth of pullet and poulard controls does not differ significantly, indicates that endogpilous androgen secretion alone does not prevent rudiment hypertrophy. This proves, according to the experimental design, that the estrogen-androgen effect represents a synergism. 1.40 1.50 Ovariectomized controls Poulards on estrogen and androgen 9 2.26 2.20 2.69 3.20 2.98 3.42 1 pg ECP plus 3 fig TCP per 100 gm of body weight per day. 10 1.66 49 Dqs 39 Days 29 1);~)s Intact controls (pullets) TREATICENT 10 NO. O F BIRDS 4.24 3.81 4.07 5.41 4.60 4.81 At of 59 the age69 Days Days (CM’) 6.55 5.52 5.72 79 Days 7.30 6.51 6.58 7.62 7.96 7.69 99 Days 89 Days physiological” estrogen-androgen dose X P 4 N COMB AREA Comb growth of controls and of poulards receiving a TABLE 3 0.008 0.094 & 0.005 0.095 0.083 k 0.006 (Comb area on age) REGRESSION COEFFICIENT z z H z m L 2 H d w P 6* 0 0 0 H Q 0 2 0 628 WALTER I i O R N F E L D The number of hypertrophied right gonads was also maximally reduced by the synergistic dose of 1p g ECP plus 3 pg TCP. Only one of 10 rudiments appeared to have hypertrophied and even its weight (9 mg) is in line with that of the rudiment weight of intact pullets (tables 4 and 5). TABLE 4 E f f e c t s of a “physiological” estrogen-androgen dose zcpon immature poulards at t w o different intervals a f t e r ovariectomy at 14-17 days of age (Mean organ and tissue weights in mg f SD; comb growth as % increase i n comb area SD) + AGE A T T I X E OF TERMINATION 100 days 60 days Pullets No. of birds Rudimentary gonad weight Pituitary weight Oviduct weight Comb growth Type of rudiment: Testis Ovotestis Ovary Poulards * Pullets Poulards 7 3 (2) 7+3 8-r-7 4.4 -r- 1.0 8 6+2 10 +6 3.4 k 0.7 4.0 + 1.2 3.8 % 0.6 52 f 9 57 +- 5 511 + 635 106 % 40 157 2 19 414 % 213 5 3 5 0 6 0 2 0 0 121 a + 26 557 k 174 3 0 0 ~_____ ~ (1,ug ECP plus 3 fig TCP per 100 gm of body weight per day.) * 30 injections. Treatment was terminate? when the poulards were 59 days old. 40 injections. Treatment was terminated when the poulards were 99 days old. observations of oviduct weight (distentions omitted). Significant difference from pullets ( P 0.05). ‘ Number of < Additional information regarding the “physiological” dose of 1 p g ECP plus 3 p g TCP is presented in table 4. Two groups of poulards receiving a total of 30 and 40 daily injections were compared with pullet controls. The birds were killed at the ages of 60 and 100 days. All rudiments were weighed and sectioned. Data were analyzed by appropriate use of “student’s’’ t-test. 1165 1170 1104 1134 ' ?C 28 1051 1096 75 1197 1145' 35 & 34 Final body weight adjusted f o r weight differences at the ape of 80 days. Final body weight (in Em) 7f 4 46 rtr: 13 Last 20 days of exper. 330 & 154 184t31 322 C 1 7 6 390 k 1 3 0 7.5 t 2.4 117 t 55 3.5 k 0.4 4.1 t 0.6 768 t 175 1074 1 :224 4.2 t 0.4 338 & 445 Oviduct weight Comb growth : Duration of entire exper. 38 C 12 6 37 & 21 36 C 25 1228 1328 1 1 t 3 1 1 t 3 6 t 2 6 C 2 9 7 3 0 1 0 4 10 5 3 0 0 3 Controls + Progest. (last 20 days) PMS 4 0 1 5 Progest. Pituitary weight Type of gonadal rudiment: Testis Ovotestis Ovary Rudimentary gonad weight: All rudiments Testicular rudiments Non-testicular rudiments No. of birds Controls PULLETS 1098 1156 49 t 16 396 & 9 9 1136 11401 41 rt 14 366 5 8 8 96t21 4.7 t 1.0 6.1 t 1.7 98t20 15 l l t 5 1 1 t 5 9 1 0 10 PMS 1087 1101 58 2 2 6 468 2 1 6 6 89t23 7.5 t 1.8 49 17-C-12 1 3 t l 8 1 0 9 0 91 29 7.0 rt 2.1 1203 1167' 21C16 1176 1157' 26t25 312 & 1 1 7 1079 1095 ' 1 7 ~ 2 1 308 & 186 414 8.0 2 1.6 206 -+ 1.6 85 2 2 7 7.5 27 2 35 18 18 1 4 t 6 7 3 0 10 Avian Pit. 48 t 2 9 25 t 24 1 2 t 5 25 +- 1 2 22t10 33 2 14 7 3 1 11 PMS + A to age 80 day8 + (last 20 days) 7 3 0 10 E 286 t 9 4 POULARDS Avian Pit. + A to age 100 days + (last 20 days) 76 20218 1 5 t 5 11 0 1 12 0 E T h e e f f e c t of gonadotropins u p o n the steroid-inhibited rudimentary gonad (Mean organ and tissue weights in mg t SD; comb growth as % increase in comb area f SD. For details about treatment Bee text) TABLE 5 630 WALTER K O R N F E L D Of the 7 poulards killed at 60 days of age, two had ovarian rudiments. They were larger (15 and 22 mg) than the remainder of testicular structures. The rudiments of the poulards in the older group were all testicular and their growth was inhibited even though treatment had been started about 45 days after the removal of the left gonad. Poulard treatment with a hormone dosage calculated on the basis of body weight, resulted in pullet-like growth of pituitary, rudiment, and oviduct a t the ages of 60 and 100 days. At the age of 60 days, but not at 100 days, comb growth of the treated poulards was significantly greater than that of the intact controls and it appears possible that the rate of pullets’ comb growth increases with age. However, in another experiment (table 3) analysis of analogous comb growth data failed to show a significant difference at the age of 59 days. T h e effect of gonadoti-opins upovt, the inhibited rudiment To determine the possible effect of the pituitary upon the development of the compensatory gonad, crude avian pituitary powder or PMS were administered to poulards either concurrently with the injection of an inhibitory dose of sex hormones, or under the waning influence of residual sex hormones. This approach allowed f o r the possibility that the compensatory gonad of untreated poulards might already he maximally stimulated by endogenous gonadotropins o r that it might only respond to avian gonadotropins. TTeatment with the synergistic dose of 1 p g ECP plus 3 p g TCP per 100 gni of body weight per day was started on 30 day old poulards. Of three groups which were treated with estrogen ( E ) plus androgen ( A ) for 70 days, two groups were injected in addition with 5 CNU of PMS o r the equivalent of five avian pituitaries p3cr day during the last 20 days of the experiment. Three other groups were treated in an analogous manner, except that the injection of E plus A was discontinued the day before the administration of €’>‘IS or pituitaries was begun. Intact and ovariectomizcd controls HORMONE CONTROL O F GONADAL R U D I M E N T 631 were maintained and two groups of intact pullets were injected f o r 70 days with 0.5 mg of progesterone per 100 g m of body weight per day with or without the addition of 5 CNU of PAIS during the last 20 days of the experiment. The age selected for the start of gonadotropin injection was 80 days because the compensatory gonads of ovariectomized controls are large enough then to permit an accurate interpretation of differences in size due to treatment. Twenty days was considered adequate for a weight response. All rudiments were weighed and sectioned. Rudiment weights mere converted into logs f o r statistical analysis because of unequal variances. Data are presented in table 5. Neither progesterone nor gonadotropins had a significant effect upon rudiment growth. None of the treatments affected log oviduct weight significantly. The pituitaries of all pullet groups and those of the poulards treated with P M S plus E plus A were lighter than the rest. An analysis of variance for treatment effect upon comb growth was not significant for the first 50 days, but was highly significant for the entire experimental period. During the last 20 days of the experiment, E plus A with or without avian gonadotropin caused comb growth to exceed ( P < 0.01) that of the ovariectomized controls. PMS which accelerated the comb growth of the progesterone treated pullets temporarily (the birds became refractory after about 15 days of treatment), failed to have even a transitory effect on the poulards. Analysis of variance showed a very significant difference in the final body weights of the experimental groups which was, however, shown to be due t o differences existing already before the start of gonadotropin injection. The failure of the gonadotropin to accelerate compensatory gonad growth was, therefore, apparently not due t o a general growth depressing effect of the avian pituitary preparation. Analysis of variance shows that treatment with the synergistic dose of steroids depressed log rudiment weight of poulards very significantly. It remained, however, very significantly larger than log rudiment weight of the intact pullet 632 WALTER K O R N F E L D groups. Weights of the testicular rudiments (their variances were homogeneous) did not differ significantly between the three intact groups and those of poulards which had been treated with E plus A throughout the experimental period. This shows that the synergistic androgen-estrogen dosage level inhibited only testicular proliferation. Non-testicular compensatory gonads (18 out of 85) were fairly evenly distributed among treatments and wherever ovarian and ovotesticular types occurred i n the same group, the ovary was larger. DISCUSSION Breneman ('55) reported that very small doses of estradiol increase the gonadotropic potency of the pituitary of pullets and have no significant effect on piuitary weight. Data presented in this paper show that the pituitary weight of poulards, which is already larger than that of pullets, was further increased significantly following the administration of low doses of ECP (0.125 to 0.5 p g ) . A dose of 1 p g ECP was not consistently effective and pituitary weights of poulards treated with 2 pg ECP or more did not differ significantly from those of the intact pullets. Doses of ECP which were followed by a n increase in pituitary weight did not inhibit the hypertrophy of the right gonad. All pituitary weights which decreased following estrogen treatment, approached the value f o r the intact pullet whose rudiment is also inhibited. This indicates that a depression of pituitary weight occurs in the presence of physiological amounts of estrogen. Breneinan's ('55) above mentioned results suggest that the pituitary of the intact pullet is not very sensitive to additional estrogen after its initial response to endogenous hormone. I n line with these findings are those of Kornfeld ('57) who reports a significant difference in pituit a r y and oviduct weight of pullets and poulards as early as a t 45 days of age. While this difference must be attributed to ovarian secretion, the absence of a significant difference in comb size between pullets and poulards causes this worker HORMONE CONTROL O F GONADAL RUDIMENT 633 to discount the importance of a physiological androgen effect upon the hypertrophy of the right gonad. The dosage combination of 1 y g E C P (which alone is subminimal for the consistent inhibition of rudiment growth in the poulard) and 3 p g T C P per 100 gm of body weight per day definitely depressed the growth of testicular rudiments. On the basis of its effect upon comb growth, oviduct weight, and effect on testicular rudiments, i t seeins that this dose approaches the equivalent of the pullet’s endogenous hormone secretion. The pullet’s actual secretion rate of estrogen must nevertheless lie above the equivalent of 1 p g ECP. This bclief is based upon the apparent lack of effect of the above mentioned synergistic androgen-estrogen dose in thc inhibition of non-testicular compensatory gonads of poulards and upon its inconsistent effect upon pituitary size. The lack of effect of dose 1p g E C P plus 3 p g T C P upon the growth of non-testicular compensatory gonads was difficult to prove. Only in one experiment (table 5) were these structures fairly evenly distributed among treatments. I n the second experiment (table 2 ) only testicular gonads were found in the poulards treated with 1 p g E C P alone or in conjunction with 1.5 and 3 pg TCP. Under the influence of 1 p g E C P plus 6 1-19T C P only one gonad hypertrophied, a n ovary (35 mg), and under the influence of 1 p g E C P plus 1 2 p g T C P two gonads developed, one ovary (38 mg) and one ovotestis (7 mg). When the “physiological” dose of 1 p g E C P plus 3 p g T C P was repeated to the age of 60 days (table 4), the two largest gonads found (22 mg and 15 mg) were ovaries and only testicular gonads were found in the group which was treated from the age of 60 to 100 days of age. The effect of the greater weight of non-testicular rudimentary gonads upon mean gonad weight was more pronounced in the experiments in which all rudimentary gonads were actually weighed (tables 4 and 5). Previously a weight of zero had been assigned t o rudiments which were too small to be macroscopically definite structures and which a r e assumed to be maximally inhibited. While the weight of each gonad must obviously exceed zero, the method 634 WALTER KORNFELD of weighing minute gonadal structures tends to overestimate their size somewhat because unrelated tissue is removed with many of these structures which have poorly defined boundaries. It appears definitely, however, that the inhibitory threshold for rudiments with cortical development must lie above that for testicular rudimentary gonads. Pituitary weights following the daily injection of 1p g E C P alone (per 100 gm of body weight per day) were significantly larger than those of intact controls in one experiment (table a), but not in the other (table l), and the lowest androgenestrogen dose depressed pituitary weight (table 2). The dosage combination of 1~gECP with 3 p g or 6 p g TCP results in pituitaries which are very significantly larger than those of pullets and which fail to differ significantly in size from those of the ovariectomized controls. The dose of 2 p g E C P without the addition of androgen, consistently depressed the poulard's pituitary weight to the point where it no longer differed significantly from that of the intact controls (tables 1 and 2). This dose also inhibits rudiment proliferation consistently. Kornfeld ('57) has shown that in birds, ovariectomized at 14 to 17 days of age, the compensatory gonad is not significantly larger a t the age of 45 days than that of pullets of the same hatch. Hypertrophy of the rudiment in poulards ovariectomixed during the third week of age, becomes apparent between the ages of 45 and 60 days. Neither estrogen treatment of poulards to the age of 45 days, nor ovariectomy at 45 days had a significant effect upon the rudiment weight a t 100 days. Estrogen administration to poulards to the age of 60 days as well as ovariectomy a t 60 or 75 days of age caused the weight of the compensatory gonad a t 100 days to fall below that of controls ovariectomized 12 to 21 days after hatching. The current data fail t o show whether the latent period is increased or whether the growth rate of the compensatory gonad is decreased when pullets are ovariectomized during the third week of age rather than a t 45 days of age. At 100 days of age the comb size of birds ovariectomized at 45 days was, however, significantly smaller than that of poulards whose left gonads H O R M O N E CONTROL O F GONADAL R U D I M E N T 635 had been removed at the age of 12 to 17 days even though the compensatory gonads showed no significant difference in size and appearance. This indicates that gonad size and endocrine activity are not influenced alike by the age at the time of ovariectomy. The last experiment of this series (table 5) was designed to test the effect of gonadotropins upon rudiment growth. Because of the complete lack of response t o avian pituitary and to PMS under these conditions, a working hypothesis seems justified now which holds that the inhibition of the right gonad of domestic fowl is due to a direct inhibitory effect of the secretion of the ovary. This view is not incompatible with the observation of Kornfeld and Nalbandov ('54) that the pituitary is necessary for the maintenance of the compensatory gonad of poulards. Until the successful acceleration of the growth of the compensatory gonad of fowl is accomplished, the possibility that a sub-threshold dose of avian gonadotropin was used cannot be discounted. The prolactin content of the pituitary powder is not known, but if one puts credence in Huble's ('56) hypothesis that prolactin is the avian growth hormone, it does not appear likely that the preparation used contained a high amount of it. It is worth noting that a 10 day treatment of pullets of similar age and breed with 4 mg of dried pituitary powder of similar origin, was shown by Das and Nalbandov ('55) to cause a significant increase in comb size and ovarian weight. I n the experiment which is reported here, about twice this amount (7.5 mg per day) was administered f o r twice the length of time. Breneman ( '55), working with immature Leghorn pullets, has shown that the combination of sex hormones with PMS produces marked growth and secretory activity of the ovary. This response occurs along with a decrease of pituitary gonadotropic potency, but not of pituitary weight. There is no indication that the right gonad of fowl responds similarly. I n the absence of any gonadal response, the combined administra- 636 WALTER KORNEELD tion of sex hormones and PMS caused a significant depression of the pituitary weight of poulards (table 5). CONCLUSION The minimal dose of estrogen which prevented the hypertrophy of the rudimentary right gonad of poulards, ovariectomized 12 to 21 days after hatching, lies above 1 p g and may be as high as 2 p g ECP (estradiol cyclopentylpropionate) per 100 gm of body weight per day. The minimal inhibitory dose of estrogen had no effect upon either pituitary or oviduct weight. Pituitaries of poulards were heavier than those of pullets. The poulards’ pituitary weight was further increased by very small doses of ECP (0.5 p g or less). Doses of 1 ug E C P and above decreased the weight of the poulards’ pituitaries t o a level which did not differ significantly from that of the pullets’. A definite synergism between androgen and estrogen for the inhibition of the growth of testicular compensatory gonads of poulards was shown. The synergistic androgen-estrogen dose of 1p g E C P plus 3 pg T C P (testosterone cyclopentylpropionate) per 100 gm of body weight per day, failed t o inhibit nontesticular compensatory gonads completely even in the early stages of proliferation found in poulards killed at 60 days of age. The inhibitory effect of a physiological dose of estrogen ( 2 p g ECP per day f o r 30 days and discontinued at the age of 60 days) upon rudiment growth is completely overcome by the age of 100 days. It was also shown that the size of the rudiment at 100 days of age is no larger m-hen pullets are ovariectomized during the third week after hatching than when the operation i s pcriormed on 45 day old birds. Ovariectomies performed on birds 60 or 75 days of age resulted in smaller compensatory gonads at the age of 100 days. Regardless of the size of the rudiment at the termination of the experiment, comb growth was greater among untreated poulards which had been ovariectomized during the third week of life than in any of the other groups. H O R M O N E CONTROL O F GOiSADAL EUDIMENT 637 Evidence is presented which suggests that the latent period from ovariectomy until the onset of androgen secretion by the compensatory gonad is greatest when the ovary is removed from the very young bird. Attempts to stimulate growth of the steroid inhibited rudiincntary gonad of poulards with PMS o r avian pituitary gonadotropin have failed. L I T E R A T U R E CITED BRENEXAN, W. 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SaLLEY 1954 The effects of sex hormones on the development of the right gonad in female fowl. Endocrinology, 54: 415-424.