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The effect on the chick of some gonadotropic hormones.

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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.
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