THE INHIBITION O F OVARY OR TESTIS I N IMMATURE AMBLYSTOMa BY ANOTHER

GONAD HOMOTYPIC AS TO SEX

R. R. HUMPHREY Department of Anatoniv, University of Buffalo, N e w Pork

ONE FIGURE

INTRODUCTION

In a recent paper ('39) dealing with the growth of gonads derived from lieteroplastic transplants of their preprimordia, the writer mentioned briefly the inhibition of the testis of A. punctatum by that of A. tigrinum. This inhibition was observed in A. punctatum hosts bearing tigrinum testes de- rived from grafts as -7ell as in the reciprocal combination (punctatum testis as a graft in the tigrinum male). Study of the ovary in punctatum females carrying tigrinum ovaries as grafts showed that it too was apparently retarded in its growth. The present report is intended to present more fully the data on this type of gonadal inhibition, and to record additional evidence bearing upon its interpretation.

MATERIAL AND METHODS

The material on which this report is based consists in all of several hundred anima1s.l It includes (1) A. punctatum hosts bearing tigrinurn gonads developed from orthotopic implants, (2) A. punctatum controls, and (3) A. punctatum semicastrates (gonadal preprimordium extirpated in em- bryoiiic stages). Each of the three groups includes a number of hypophysectomized animals. I n part this material is the

IMuch of the material used was made available through the kindness of Dr. Cranford Hutehinson and the Morris Biological Farm of The Wistar Institute.

345

THE A X E R I C A X JOUIINAII O F A I A T O M Y , VOL. 70, NO, 3 MAY, 1942

346 R. It,. HUMPHREY

same as that used in the previous study ('39) in which at- tention was directed to the graft rather than to its effects upon the gonad of the host. As in that study, the quantitative data were obtained from serial sections projected at a standard magnification ( x 200) and traced in outline on paper of uni- form thickness, the tracings for any organ then being cut out and weighed.

While the puiictatuni gonads in tigrinum hosts appear to suffer inhibition from gonads of the same sex type, the greater variability in these grafts as well as in the gonads of the host makes a quantitative study more difficult. This report therefore, deals only with the remaining gonad in A. puncta- tuin hosts in which the right gonad has been replaced by one derived from an ,4. tigrinum transplant. The technique of orthotopic traiisplaiitation employed in these experiments has been described elsewhere (Humphrey, '29).

DATA

A . l'ra.nsplniats f r o m A. tigriiauin of Chicago straiiz

Series 1. I n this group the A. punctatuni hosts are of the local (Buffalo, N. P.) strain. Since punctatum carrying trans- plants furnished by A. tigrinuni of the Chicago strain almost invariably die just before the expected oiiset of meta- morphosis this series includes 110 animals over 3 months of age. The grafted animals w-ei-e usually fixed iminediately after death or when death appeared imminent; this has neces- sitated grouping them rather arbitrarily f o r comparison with the puiictatum controls, wliich were killed at regular intervals. Hosts dying between the fifty-sixth and sixty-fifth days after the operation are compared with controls killed at 60 clays, while those dying between the sixty-sixth and seventy-fifth days are compared with 70-day controls. Unfortunately, tlicse animals were not weighed, but their leiigths were measured ;

Death of these aiiimals follows the developmeiit of a reactioii syiidrome resulting from substances produced by the cutaiicous part of the g ra f t ; f o r furthcr details eoiisult Humphrey and Bums, '39.

GONADAL INHIBITION IN AMBLYSTOMA 34i

punctatum control B. Testis of grafted

animal when graf t was a testis

C. Testis of grafted

the average body length for each group of grafted animals closely approximates that of the corresponding control group. A small number of grafted animals and controls were also killed at 40 and 50 days. The relative volumes of control and host testes for all four age groups have been assembled in table 1. The relative volumes of the tigrinurn grafts, which

TABLE 1 Relatixe aoliimes of gonads in animals of series 1.

(4) 19 -C 5 ~ (4) 2i t 2 I

, l l ? 1 ~ (1) 14 ( 2 )

D. Ovary of A. punetaturn control ( 2 ) ‘ 19 (4) 45 i 10

(3) 53 t 17

( 6 ) 44 -t- 5

(10) 29 i 7

( 3 ) 59 -t- 6

(4) 90 t 7

(5) i4 t 17

animal when graf t I

was a testis

These animals are 33 days of age instead of 40.

( 3 ) 19 2 4 I ( 8 ) 18 & 4

70 DAYS

( 8 ) 63 t 13

( 9 ) 33 -c 9

(5) 6 4 5 30

[20) 122 -C 20

( 8 ) 119 2 4 4

(8) 20 t 5 ~ _ _

The number in parentheses indicates the number of gonads upon which the average following it is based. The term following the average is the arerage deriation, a number considerably larger than the probable error. The “volume ’ ’ of any gonad was obtained by projecting the serial sections a t a magnification of 200, tracing their outlines on paper of uniform thickness, and cutting out and weighing these tracings. The arbitrary unit of volume used in the table above is one decigram of such tracings.

are always several times the size of the gonads in punctatum controls, have not been included in this table (see Humphrey,

If one compares the volume of the testis in males carrying testis grafts with the volume of the testis in punctatum con- trols (groups B and A, table l), it is apparent that the former

’39).

348 H. R. HUMPHREY

is significantly smaller; for the 70-day group this testis has a volume only 52% of that of the controls. No testis of a grafted animal in either the 60- or 70-day group exceeds in volume the average of the controls. If on the other hand the testis of the male carrying a graft ovary (C, table 1) is com- pared with that of the control, its volume appears to be increased rather than reduced, except in the 70-day group. I n only one instance was the testis smaller than the average for eontrols. Because of the small number of grafted animals of this type and the range of variation they show, it can not be safely concluded that hypertrophy of the testis has oc- curred when the graft is an ovary; it is quite evident however, that this testis has rarely if ever suffered a reciprocal inhibi- tion by the graft ovary, the growth of which it has markedly reduced (Humphrey, ’39).

The ovary of the host carrying an ovarian graft appears to have suffered no inhibition in the animals of this series (D and E, table 1). When the graft is a testis this ovary is of course, reduced to the usual “freemartin” state; such ovaries show no increase in size after the 50-day stage (I?, table 1).

Series 1 a. This series, reared in a different year than the animals of series 1, is treated separately because of differences in the feeding and size of the animals. As in series 1 the graft resulted in the death of the hosts before the onset of metamorphosis. Some of the grafted animals and controls were hypophysectomized ; the relative volumes of the testes in these and the normal animals are shown in table 2. The volumes of the ovaries were not determined.

I n this series the testis of the host bearing a testis graft is again significantly reduced in volume as compared with that of the normal control (B and A, table 2). When the graft is an ovary, however, the testis of the host is greater in average volume than that of controls (117 vs. 78 units). No control testis of the forty measured exceeds 120 units in volume while three of the seven testes of grafted animals were fa r above this level (148, 161, and 169 units). In the hypophys-

GONADAL INHIBITION I N AMBLYSTOMA 349

DESCRIPTION OF GONAD

A. Testis of normal A. punctatum control a t metamorphosis

B. Testis of host carrying a testis

C. Testis of host with graft an ovary

D. Testis of A. punetaturn con- trol hypophyseetomized

E. Testis of hypophysectomized host carrying a testis graft

graft

ectomized hosts carrying testis grafts, the testis is even more markedly reduced than in non-hypophysectomized hosts ; in the one host found to be a male with a graft ovary the testis was somewhat larger than in hypophysectomized controls (74 vs. 60 units).

TABLE 2

Relative volumes of testes in animals of series 1-a.

NUMBER OF GONADS

40

11

7

6

6

MEAN AQE OF .NIYALS -DAYS

66.5

64.4

65.6

70

70

VOLUME OF QONAJJS

78 2 1 7

38 iz 10

117 _C 37

60-C 8

2 2 2 6

The relative volumes of the gonads were obtained as stated for table 1.

B. Trarzsplarzts from, A. tigrinurn of Tenrzessee strain; series 2 The lethal reaction syndrome encountered in the grafted ani-

mals of series 1 and 1 a precluded study of the grafts or their effects in older animals. When it was discovered that this reaction syndrome did not develop when the donors were tigrinum of the Tennessee strain, the investigation was ex- tended to animals of 4 and 6 months. The hosts in this series were entirely of the Tennessee strain of A. punctatum instead of the local (Buffalo, N. Y.) strain used in series 1 and 1 a. All animals were weighed before being killed and the relative volumes of the gonads given in tables 3 and 4 are expressed in units per gram of body weight. This should eliminate in large part any error arising from size differences in the older animals, which vary more than do those killed before meta- morphosis.

The testis in normal hosts carrying testis grafts, when com- pared with the testes of controls ( B and A, table 3) is again

350

D. Testis of hypophysectoniized

E. Testis of hypophyscctorriized, control (16) 90 i 10 (12) 92 i 8

B. R. HUMPHREY

( 8 ) 6 8 ? 6

TABLE 3

Relative xolztmes of testes in animals of series 2.

I

DESCRIPTION O F GONAD 1 2.5MONTHS 4 A I O N T H S 6 MONTHS -1-

A. Testis of normal punetaturn: 1 control ~ (18) 84 i 8 ~ (18) 108? 13

F. Testis of hypopliysectomized I ( 5 ) 98 ? 16 ~ ( 5 ) 124 t 1 2

I .~ host when graf t was an ovary

The number in parentheses indicates the number of gonads upon whieh tlic average following it is based. The number after the average is the probalJlt. error. The relative volumes of the gonads were obtained a s stated for table 1 ; they are expressed a s arbitrary units per gram of body nciglit of the animal.

TABLE 4

Rclative volumes of ovaries in aniinu1.s of wyies ?.

1 4 MONTHS I 6 X O N T R S 1 Y E t R DESCRIPTION OF ~ 2,5 ~ IoNTHS

GONAD I

I---- A. Ovary of ,

normal punc-1 tntnmcontrol~ (20) 023 ? 31 (28) 897 t 131 (12) 1243 i 153 (4) 1688 i 90

is an ovary (6) 225 +. 33 ( 3 ) 354 t 75 ( 4 ) 512 t 114 ( 3 ) 643 i 143

E. Orary of host^ when grafti

C. Ovary of hy- pophysecto- mized punc- taturncontroll (10) 190 I- 2 5 , ( 1 4 ) 377 i 41 ( 8 ) 648 C 37 (4) 10843Z 31

D. Orary of hy- I

pophysecto- mized host I

when graf t 1 I is an ovarr ( 6 ) 124 31 28 (9) 216 t 44 ( 4 ) 153 t 40

I

I

~

, _ _ ~

The number in parentheses indicates the number of gonads upon which the average following it is based. The number after the average is the probal)lc error. The relative volumes of the gonads were obtained as stated f o r table 1; they are given here as arbitrary units per gram of hod? weight of the animal.

GONADAL INHIBITION I N AMBLYSTOMA 351

found significantly smaller, that in the 4-month group being less than half the volume of the control organs. TJ'hile the testis of the 2.5-month host with a graft ovary is not signifi- cantly larger than that of controls, the testis in the 4-month group is 44% greater in volume and in the 6-month group 77% greater. The number of animals in each group is large enough to eliminate the possibility that this is a chance dif- ference and establish that an actual hypertrophy has occurred.

I n the hypophysectomized animals (D, E, F, table 3) the testis in males with a testis graft is less than half the size of that of controls in both the 2.5 and 4-month age groups. Of the males with graft ovaries, those of tlie 4-month group show the greatest enlargement of the testis as compared with the hypophysectomized controls (3570). Of the two 6-month animals of this type one shows a somewhat larger testis than the controls while the other does not; three others which died at 5 to 5.5 months, however, had testes with an average volume of about 140 units per gram of body weight as compared with 92 and 68 units f o r the liypophysectomized controls of 4 and 6 months.

The relative volumes of the ovaries in animals of series 2 are presented in table 4. The ovaries of those females carrying testis grafts are not included ; these gonads are ovaries of the usual freemartin type, with a considerable number in the advanced age-groups showing a partial reversal to testis.

Comparison of the ovary of the host with that of the normal control (groups A and B) shows that at 2.5 nionths the two do not differ in size. This is in agreement with the findings in series 1 (table 1). In the three older groups, however, the ovaries in the grafted animals are only from 38 t o 41% of the volume of the ovaries of controls. The hypophys- ectomizcd controls of the three older groups show a distinct reduction in size of the ovary as compared with the normal ( A and C, table 4), but the ovary in the hppophysectomized host is still more reduced in volume, that of four animals of 6 months averaging only 24% of the volume found for the hppophysectomized controls.

352 R. R. HUMPHREY

To determine whether the greater size of the testis in males with graft ovaries resulted from some influence of the ovary or was due merely to the absence of another testis, the volume of the remaining gonad in a small number of semicastrates was determined. The volumes of such testes as compared with those of the controls are shown in table 5. The testis of the semicastrate is larger than that of the control in all groups, the increase in volume ranging from 347% to almost 50%. One semicastrate had a testis 98% greater in volume than the average for its control group. The increase in testis volume in the 2.5-month semicastrate is much greater than in the

TABLE 5

Relative volumes of testes in semicastrates and controls of series 8.

DESCRIPTION O F GONAD ' 2 . 5 ~ 0 ~ ~ ~ s 1 4 3 c o N T m ___--_ -

A. Testis of normal A. punetaturn control ~ (18) 84 k 8 (18) 108 rt 15 R. Testis of semicastrate punctatum 1 ( 3 ) 116 f 6 (4) 157 zk 7

I C. Testis of hypophysectomized punetaturn

1 (16) 90 f 10 (12) 92 f 8 control D. Testis of hypophysectomized semi-

castrate (4) 121 & 5 ( 3 ) 134f 23 i

Explanation of data as in table 4.

grafted animal (see table 3), but at 4 months the two groups are in fairly close agreement. The testis increased in volume in the hypophysectomized semicastrate as well as in the normal one.

It is of considerable interest to note in a scattergram the distribution of the testis volumes in 4-month animals, when the actual volume in arbitrary units is placed according to the weight of the animal (see chart 1). Testes of the non- hypophysectomized are here represented by solid symbols, those of the hypophysectomized by symbols in outline. Be- cause of the continued growth of the hypophysectomized ani- mals while the normals are undergoing metamorphosis the

GONADAL INHIBITION I N AMBLYSTO3IA 353

two groups scarcely overlap. It will be noted that in each group the smallest gonads are the testes of hosts with a testis graft (rhombi) while the largest are the testes in semicastrates (triangles) and niales carrying ovarian grafts (squares). The testes of controls (circles) take an intermediate position. I n the two controls (one normal, one hypophysectomized) in which a testis is of unusual size, the second testis in each case has a much smaller volume.

BODY WEIGHT IN GRAMS

Distribution of testis volumes in Amblpstoma piiiictntnm of 4 months according to the body weight of the animal. Animals of series 2. Testes of hypophysectomized animals are represented by symbols in outline, those of now hypophysectomized by symbols in solid black. Testes of controls are represented by circles, testes of semicastrates by triangles, t w t e s of nxrles vi th ovarian grafts by squares, and testes of niales carrying testis grafts by rhombi.

Chart 1

354 R. B. HUMPHREY

DISCUSSION

The data obtained from all three series of experimental animals show clearly that in the A. punctatum host with an A. tigrinum testis which replaces its own right gonad, the remaining testis is significantly reduced in size as compared with that of normal punctatum controls. This reduction is apparent before the animals reach an age of 2 months (table 1) and is demonstrable in all older stages investigated. I n some groups the testis in question is actually less than half the volume of that in controls, and in no group does it even approximate the latter in size. It would appear evident, there- fore, that the graft testis has in some fashion checked or inhibited the normal growth of the homotypic host gonad. By what means or agency has this been effected7

The possibility that the reduction in growth of the host testis results from a reduction in an essential pituitary stimu- lation must be considered. If the graft were able to utilize and withdraw from the circulation a gonadotropic substance essential to the testis of the host, the latter organ in con- sequence would develop at a subnormal rate. Rut comparison of the normal and hypophysectomized controls of series 2 (table 3) shows that the hypophysis exerts little or no effect upon the testis during the period considered (up to 6 months). When computed in units per gram of body weight the testis of the liypophysectomized male at 2.5 months is actually somewhat larger than the testis in the normal. The differ- ence in favor of the normal at 4 and 6 months is small and is probably in part accounted for by the fact that larval structures (gills, dorsal fin) increase the weight of the hy- pophysectomized animal and so reduce the volume of its gonad when this is computed per unit of its body weight. If complete absence of anterior lobe tissue during the first G months of life causes at most only an insignificant reduction in testis volume, a reduction of 50% or more can scarcely be inter- preted as due to a pituitary deficiency. This conclusion is in agreement with the finding of others that the early clevelop-

GONADAL INHIBITION I N AMBLYSTOMA 355

nieiit of the gonad in amphibians occurs independently of pituitary stimulation (Atwell, '33 ; Blount, '36).

The possibility that the graft may check the growth of the host testis by depriving it of essential nutritive substances may also be dismissed. I n the first place, tlie animals con- sidered are usually well nourished individuals with a good reserve of stored material in their fat bodies; secondly, the graft ovary in a niale host, even when of a size equivalent to the graft testis, does not produce an equivalent effect. The possibility that the graft testis may reduce the blood supply to the testis of the liost may also be dismissed, since graft ovaries equivalent in size and positioii fail to inhibit this organ.

There remains the interpretation that the observed inhibi- tory effect is produced by the action of hormones or inductor substances released by the graft testis to act directly upon that of the liost. This explanation is in harmony witl1 the fact that the reduction occurs in normal and hypophps- ectomized hosts alike. It helps to account for the marked enlargement of the liost testis when the graft is an inhibited ovary, or the hypertrophy of the remaining testis in a semi- castrate ; the gonadal hypertrophy in such animals, even in the absence of the hypophysis, is logically interpreted as due to the elimination of a restraining effect normally exerted by a second testis.

From the earlier studies on series 1 (see table 1) it was concluded that the graft ovary exerts no inhibitory influence upon the ovary of the host. Though the 2.5-month group in series 2 supports this conclusion (see table 4), it is apparent that in the older groups the ovary of the grafted animal is iiiarkedly reduced in volume. While the reduced size of the ovaries in hypophysectomized controls of 4 months and older might suggest that the reduction in the grafted animal is due merely to insufficient pituitary stimulation, the much greater reduction in the grafted liypophysectomized female indicates that an ovarian inhibition has been added to the effect of hypophysectomy. I n the 6-month hypophysectomized

356 R. R. HUMPHREY

group, for example, the ovary of the grafted animal is less than one-fourth the size of that of the liypophysectomized control (table 4 ; 153 units vs. 648). The data so far obtained indicate that hypophysectomy affects the ovary at a time when it has little if any effect on the testis; this is perhaps clue to a slowing in growth of the oocytes enlarging during this period. When the effect of hypophysectomy is added to that of the graft ovary the result is a far more striking reduction of the host gonad than is seen in the male. T7Yhile in the latter at 4 months the testis in hypophysectomized ani- mals with testis grafts has a volume 37% of that of normal controls (table 3 ) , in the hypophysectomized female with an ovarian graft the ovary is only 24% the size of that in the normal. In the older hypophysectomized females (6 months) it appears still further reduced, four grafted animals having an average ovarian volume only 12% of that in normal females.

The inhibition of one gonad by another of the same sex type, apparently through the agency of a substance trans- ported by the blood, raises the question of the identity of the hormone or other substance involved. The writer has already ( '39) offered the tentative interpretation that these substances may be the same as those by which the heterotypic gonad is affected. The testis in grafted animals, for example, produces a substance effective in checking the growth of an ovary; there seems no reason why the same substance may not also inhibit another testis, although to a lesser degree. The ques- tion has been discussed at some length in a previous paper

The identification of these inhibitory substances of the immature gonads as the sex hormones found in adults, or the proof that they are chemically different substances, must come from studies of a different type than the present investi- gation. This question is discussed in the papers of Burns, '38; Witschi, '36; Foote and Witschi, '39; and Foote, '40. I t is of interest that the embryonic gonads may be inhibited by homotypic hormones of the adult type in some instances

( '39).

GONADAL INHIBITION I N AMBLYSTOXA 357

(chick, Willier and co-workers, '38 ; opossum, Burns, '39). Too little is known, however, as to whether these effects result from a direct action of hormone upon gonad, or whether a different effect might result from a change in dosage as has been reported by Selye ('41) for the testis in the young rat. Finally, of course, a similar inhibition of gonadal growth by two substances does not of itself prove that the two are identical. Although the inhibition of one gonad by its homo- type in grafted Amblystoma would appear definitely estab- lished by the data presented, the identification of the inhibiting agent must await further study.

SUMMARY AND CONCLUSIONS

1. I n A. punctatum hosts carrying large gonads derived from A. tigrinum preprimordia, both testis and ovary suffer a marked reduction in size as a result of the presence of a homotypic gonad.

2. The reduction in size in the testis occurs alike in normal and hypophysectomized grafted animals and a t a time when hypophysectomy alone produces no significant reduction in testis volume. The reduction in size of the ovary, though it occurs later, and a t a time when the size of the gonad is ap- parently affected by hypophysectomy, is nevertheless indi- cated as a direct action of the graft ovary, since the effect of a graft ovary plus hypophysectomy is fa r greater than the effect of hypophysectomy alone.

3. In males carrying ovarian grafts dominated and inhibited by the testis of the host, the latter organ is commonly in- creased in volume, both in normal and hypophysectomized animals. A similar increase is noted in the remaining testis of semicas tr ates. 4. It is concluded that the growth of one gonad in an im-

mature animal is normally checked o r inhibited by the presence of the other gonad; the effect seen in the grafted animals is the result of an intensification of this inhibition, clue probably to the larger size of the graft.

358 R. R. HUMPHREY

5. It is suggested that inhibition of gonads by their homo- types may be brought about through the agency of the same inductor substances o r hormones which cause inhibition of a heterotypic gonad, the effect, however, being less extreme for the homotypic than for the heterotypic gonad.

LITERATURE CITED

ATU-ELI,, W. J.

RI,ouN,r, R. F.

BURNS, R. K., JR.

1933 Development of the gonads following early removal of

The influence of the immature hypophysis i n development.

Hormonal control of sex differentiation. Am. Naturalist,

1939 Sex differentiation during the early pouch stages of the opossum (Didelphys virginiana) and a comparison of the anatomical changes induced by male and female sex hormones. J. Morph., vol. 65,

Response of gonads and gonaducts of Ainhlpstoina larvae to trentinent nilli sex hormones. Proc. SOP. Exp. Biol. and Jled., vol. 43, pp. 519-523.

Effect of sex hormones on the gonads of frog larvae (Rana clamitans). Sex inversion in feniales; stability in males. Anat. Rec., vol. 75, pp. 75-83.

HUMPHREY, R. R. 1929 Studies on sex reversal in Amblystoina. 11. Sex dif- ferentiation and modification following orthotopic implantation of n gonadic preprimordium. J. Exp. Zool., vol. 53, pp. 171-219.

The growth of gonads derived from heteroplastic transplants of their preprimordia in Amblystoma. Am. J. Anat., rol . 65, pp. 35-67.

1939 An incompatibility manifested in lietcroplastic parabiosis or grafting in Amblystoma due to a toxin of cutaneous origin. J. Exp. Zool., 1701. 81, pp. 1-42.

SELYE, HANS 1941 Effect of dosage on the morphogenetic actions of testo- sterone. Proc. SOC. Exp. Biol. and Med., vol. 46, pp. 142-146.

TYILLIER, R. I$., kf.4RY E. R ~ W L E S A N D F. c. KOCH Biological differenecs in the action of synthetic male liorniones on the differentiation of SCY

in the chick embryo. Proc. Nat. Acad. of Sciences, vol. 24, pp. 176-182. WITSCHI, E. 1936 On the separate chemical control of emhryonie sex dif-

ferentiation and of secondary sex charnctcrs. Bull. dc la SOC. dr Biol. de Lettonie, T. 5, pp. 79-85.

the 1iy)olihysis in Rana sylvatica. h a t . Rec., vol. 55, suppl., p. 45.

Anat. Rec., 1701. 64, suppl. 3, p. 5. 1936

1938 V O ~ . 72, pp. 207-227.

pp. 497-547. FOOTR, C. 1,. 1940

FOOTE, C. I,., BND E. WITSCHI 1939

1939

HUNPHREY, R. R., .4ND R. K. BURNS, JR.

1938