SELECTIVE BREEDING FOR THYROID 1311 UPTAKE IN MICEIJ
C. K. CHAI
The Jackson Laboratory,3 Bar Harbor, Maine
Received June 25, 1969
LTHOUGH it has long been known that the activity of endocrine systems is
important to growth, development, and behavior, and that deviation or imbalance beyond a threshold limit can cause various diseases or disorders, the
mechanism of hormonal regulation involved in gene-controlled protein synthesis
has been recognized only recently (KARLSON 1968). There is ample evidence that
differences in endocrine funotioning vary between and within species, and between inbred strains of mice (CHAI and DICKIE1967). This would suggest that
hormoeal activity itself is also under gene control; but studies on the inheritance
of hormonal activity in any endocrine system are lacking.
A genetic approach encounters difficulties ordinarily nonexistent in studies
dealing with morphologic and anatomic traits. First, hormone secretions are available in only minute amounts, and some are unstable, so that accurate quantitative
determinaltion is difficult; secondly, endocrine activity varies in individuals of
many animal species diurnally, seasonally, and cyclically; and thirdly, endocrine
systems are interlocked, and the body tends to homeostasis through feedback
mechanisms. As inherent trait characteristics, eaich or all of the above could contribute to experimental errors.
We have undertaken to study the inheritance of thyroid activity in mice by
selective breeding. We chose this endocrine system because the technique, u s i n g
radioactive iodine as a tracer, provides relatively reliable quantitative determinations. Of the various methods of measuring thyroid activity we used the thyroid
1311uptake because of its simplicity in determination without sacrifice of the animals used for breeding. Though the results reported here are preliminary, they
show not only genetic influences but also a characteristic pattern of responses to
selection and variations believed to be the consequences of genetic changes in
thyroid functioning due to selection.
A
MATERIALS A N D METHODS
Mass selection for high and low 1311 uptake in the thyroids of mice began with the F, generation of a hybrid stock produced by intercrossing six inbred strains (CHAI 1960). This F, generation is referred to in the experiment as the zero generation. In general, 8-12 matings were seThis paper is dedicated to the late MARGARET
M. DICXIEwhose work constitutes a great
contribution to mouse genetics.
* This investigation was supported by National Institutes of Health Research Grant CA 03108 from the National Cancer
P. MORANG,
N. A.
Institute. The author wishes to thank the following for their technical assistance: J. MORRISON,
JENKINSON and the summer students L. R . MOORE
and D. S. MATCHAR.
The principles of laboratory animal care as promulgated by the National Society of Medical Research are observed in
this Laboratory.
Genetics 64: 2 9 4 0 January 1970
30
C . K. CHAI
TABLE 1
The actual number of breeders and the weighted selection differential (percentage
in each generation of selection for 24-hour lSlZ uptake
High line
Generation
2
3
4
5
6
7
8
9
10
16
14
14
20
14
18
24
18
24
uptake)
Low line
Selection differential
Number
of breeders
1311
0
d
40
14
24
12
14
1
15
18
20
29
21
14
9
16
4
16
19
8
Selection differential
Number
of breeders
18
14
14
26
22
22
26
26
28
0
d
18
15
12
5
8
5
4
8
2
15
13
1
4
5
5
4
7
4
lected in each generation. We often supplemented with more matings when poor matings were
discovered. The actual number of breeders is given in Table 1 for the second part of the experiment. With the restricted numbers of breeders, there was a resulting increment of inbreeding of
coefficient of inbreeding. A
approximately .02 to .03 in each generation in terms of WRIGHT'S
maximum of eight mice was kept in a litter and the first and second litters were used. They were
weaned a t three weeks of age and the males and females kept in separate pens. Individual thyroid
I3lI uptake was determined at ages between 90 and 100 days, and the matings were set immediately following the determination.
Zn uiuo l3II uptake was measured according to the method of CHAI, MORRISONand LENZ
(1964), with slight modifications. Five pc of 1311 carrier-free, in 0.5 m l of 0.9% saline was injected
subcutaneously to each mouse between 12:30 and 1:30 P.M. External thyroid and body radiation
counts were made on succeeding days, beginning at 12:30 P.M. and usually completed before 3:OO
P.M. To measure radiation, a scintillation detector with a 2.54 cm diameter crystal was connected
to a Tracerlab rate meter from which the signal was transferred to a recorder. The mice were
restrained in tightly fitting, individual .6 cm mesh wire chambers and placed over the crystal
with the thyroid region of the neck facing the window of the collimator. After completion of the
neck count the mouse was moved forward and the radioactivity of the epigastric region determined.
This latter count was called the body background count (BBC), 0.65 of which was deducted from
the neck count (NC) to correct for nonthyroid radiation in the neck region. Room back'gomd
counts (RBC) were made before and after the daily counts, and averaged to further correct the
net thyroid count. Two 0.2 ml samples of the injected solution were made before each injection
and put in vials. The thickness of the bottoms of the vials approximated the distance of the thyroid
from the detector window when the mouse was counted. The 1311 uptake was computed as a
percentage of the dose injected according to the following formula:
Percentage
1311 uptake
=
[ (NC - RBC)
- 0.65
(BBC - RBC)] X 100
counts of total dose injected
To eliminate possible errors in 1311 calibration, the percentage of 1311 uptake instead of the actual
counts was used for analysis.
In the first four generations of selection, 1311 counts were made on the third day following
injection. This procedure, while allowing for the excretion of extra isotope from the body, caused
the counts to be so reduced that responses were poor. In the following generations, therefore, we
made the 1311 counts on the first day after injection.
We used other methods for testing directly or indirectly variations in thyroid iodine metabo-
SELECTION FOR THYROID UPTAKE
31
lism: (1 ) T/S ratio, the ratio of iodine content per milligram of thyroid tissue to that per milliet al. (1%0). After a dose of
liter of serum. The general procedure follows that of SII~VERSTEIN
trapazo had been given to block the release of iodine from the thyroids, five fic of 1311 was injected into each mouse, and 5 hr later blood was obtained by heart puncture and the thyroid was
dissected. Radioactivity counts were made immediately for the thyroid and serum. (2) Urinary
iodine concentration. Total urine amounts were collected after the injection of 1311 for 3 consecutive days. Three to four mice of each line were grouped in one apparatus, sexes separated.
Radiation counts were made for the urine collections of 0-21; 2 4 4 8 , and 48-72 h r a t the end of
each hour period. We used 34 mice from the high uptake line and 60 from the low line, all being
in the 7th generation. Since amounts of urine excretion differ between mice, an average urinary
13x1 excretion was computed per day per mouse. (3) PBI determination. Protein-bound iodine
was determined in the plasma of periphwal blood obtained by tail bleeding. For each sample,
blood of three mice was pooled. The determination was made according to the cuvette PBI determinations of Hycel, Inc., Houston, Texas (1967). (4) Thyroid weight. Thyroids of &e 7th
and 8th generation mice, including both lobes and asethma, were dissected. They were weighed
immediately on a Roller-Smith &lance with sensitivity to one-hundredth of one milligram.
RESULTS
The results given here include, in addition to the direct response to selection in
thyroid iodine uptake, other indices of thyroid activity such as thyroid and plasma
iodine (T/S) ,urinary iodine excretion, protein bound iodine (PBI) and thyroid
weight. Characteristics considered to be associated with changes in thyroid activity between the two lines included basal metabolic rate (BMR),body growth,
biological markers for growth and sexual maturation, and response to X irradiation.
l S 1 l uptake: Mean percentages of thyroid 1311
uptake were given for each generation of the high (HIU) and low (LIU) lines, sexes separate (Table e), and
for males (Figure 1). Responses based on third day counts were small, as previously mentioned, while those based on first day uptake showed good separation
due essentially to the large HIU responses. A sudden percentage drop in both
lines at the 4th generation reflected a food change from one low in iodine content
(Purina Laboratory Chow) to one high in iodine (Old Guilford).
The selection differential (Table 1 ) for each generation was weighted by the
number of offspring produced by each mating. The coefficients of regression of
generation means on the cumulative selection differentials were computed, and
1960). In the present
they are an estimate of realized heritability (FALCONER
experiment, the change of counting procedures and of food complicate the estimation, and appropriate corrections for effects are difficult. We therefore used
only the 5th to 10th genenation data of the first day counts. Since there was a
slight sex difference in thyroid activity, and since thyroid function possibly interacts with sex hormones, we computed the coefficient of regression of generation
means for females on the selection differentials of females and means of males
on the differentials of males. The estimates of h2for HIU mice were 0.42 * .I6 for
females and 0.56 * . I 7 for males. Those for the LIU were practically zero.
T / S ratio: T/S determinations are given in Table 3. As expected, the TJS ratio
of high-uptake mice was about five times that of low-uptake mice. We tested for
response to thyroid-stimulating hormone (TSH) by injecting one internat. unit
32
C. K. CHAI
TABLE 2
Percentages of
1311
uptake in the thyroids of mice selected for high (HIU) and low (LIU)
uptake in different generations
Females
Males
LIU
Generation*
1
2
3
4
1
2
3
4
5
6
7
8
9
10
HIU
No.:
mean
32
38
48
62
41
44
39
35
31
7
8
8
64
39
39
29
10
12
13
14
18
11
9
16
13
12
5
6
5
5
8
4
5
60
30
76
75
74
60
58
68
79
No. mean
SD
35
49
38
52
41
25
23
29
81
30
51
50
17
29
38
39
59
41
64
48
27
62
66
37
65
58
57
63
LIU
SD
No.
16
10
6
12
29
36
40
65
23
58
56
30
96
87
73
82
65
81
79
13
6
8
17
11
16
17
19
16
HIU
mean
SD
No.
mean
SD
37
19
13
17
13
3
5
7
38
52
35
44
26
24
20
26
6
8
6
11
32
11
10
6
5
5
6
3
8
4
5
69
50
36
67
65
27
70
63
48
66
25
39
36
16
22
31
37
59
18
13
10
6
11
8
13
16
16
14
28
16
10
11
11
11
15
10
9
44
63
* Generations 1 4 in the upper part of the table were based on third day counts, and 1-10 in
the lower part were based on first day counts.
No. = number of individual observations.
+
I
70
I
0
I
1
2
3
4
\ \
'
1
I
1
2
3
G E N E RAT1ON
4
5
I
6
7
8
9
1
I0
FIGURE
1.-Plots of mean percentages of total dose 1 3 1 1 injected present in the thyroids against
generations of selection for mice of the high (HIU) and low (LIU) 1311 uptake lines. Generations
0 to 4 in the left part of the graph were based on counts obtained the third day after injection;
generations 1 to 10 in the right were based on the first day counts. The large drop at the third
and fourth generations was due to food change from low iodine to high iodine content.
33
SELECTION FOR THYROID UPTAKE
TABLE 3
T/S ratios of the high l j l l uptake (HIU) and low uptake (LIU) mice and T / S ratios
after thyroid stimulating hormone (TSH) administration
Line
Treatment
LIU
HIU
LIU
HIU
Significance of differences:
LIU us. HIU
LIU (TSH) us. HIU (TSH)
P
P
T/S C SE
Number of mice
None
None
TSH
TSH
20
20
15
16
55.6
303.2
61.4
416.6
f 3.7
f 30.1
f 6.4
t- 42.3
< 0.001
< 0.001
TSH at 24 and 12 hr prior to the administration of trapazo. There was a slightly
greater response in HIU than in LIU mice and the difference in T/S between the
two lines persisted. The differences in both cases are highly sigmficant.
Urinary lS1I concentration: The averages of urinary 1311concentration are
plotted in Figure 2. The high-uptake mice excreted less 1311in the urine than did
the low-uptake mice, suggesting that there was more iodine in both the thyroid
and circulation of the HIU mice. Iodine in the circulation is assumed to be partially in the form of proteindbound iodine (PBI) or thyroid hormones.
PBI determination: The results in Table 4 are expressed in micrograms percent
per milliliter plasma. Higher mean PBI values were obtained in the high-uptake
line than in the low, but the order of magnitude for the difference was less than
that for the T/S ratio.
Thyroid weight: Thyroid weights are directly correlated with iodine uptake
and thyroid function. Although thyroid weights varied more among individuals
in the high line than in the low, the HIU mice had much larger thyroids than the
LIU mice and the differences between the two lines were highly significant
(Table 5 ) .
Growth and maturation: Birth and 60-day body weights were taken for all mice
in each generation of each line. There was no difference at generation one, but
60-day weights varied increasingly with the advance of selection. Growth curves
(Figure 3 ) were constructed for 8th generation mice from three litters, eight in
a litter, for each line and were based on weights taken weekly from birth to 60
TABLE 4
Protein bound iodine (PBI) and basal mitabolic rate ( B M R ) of mice
of the high 1 3 1 I uptake and low 1 J l I uptake lines
~
BMR ( CC OJmin)
HIU
LIU
PBI (mg %)
-
NUlIlba
MeanzkSE
12
12
3.08 f 0.6
1.86 & 0.6
Number’
24
27
(8)
(9)
* Peripheral blood from three mice pooled for each PBI determination.
M e a n 2 SE
3.76 +. 0.31
2.24 & 0.26
34
C . K. CHAI
\
1
Low
----- High 1'"
2
DAYS AFTER INJECTION
Uptake
Uptake
3
FIGURE
&.-Plots of percentages of dose 1311 injected present in the urine of the first, second,
and third day following the injection, showing the difference in amount of 1311 excretion between
the HIU and LIU mice.
TABLE 5
Thyroid weight of mice selected for high ( H I U ) and low l s l l
uptake (LIU) in the seventh generation
~~
Thyroid weight (mg)
SD
Lnes
Sex
Number of thyroids
Mean fSD
HIU
LIU
HIU
LIU
0
0
8
8
27
8.9 f 3.0
3.6 f 1.0
7.3 f 2.4
3.4 f 1.0
25
19
26
= standard deviation of individual thyroid weights.
35
SELECTION FOR THYROID UPTAKE
0
1
2
3
4
5
6
7
0
13-
AGE ( W E E K S )
FIGURE
3.-Growth curves for mice of the high (HIU) and low (LIU) 1311 uptake lines. The
body weights were taken once a week for eight weeks and once at three months old.
days and at 90 days. High-uptake mice showed more rapid growth than lowuptake mice, and the departure became greater with the advance of age.
We checked for birth and maturation characteristics daily at 7:30 AM and
4:00 PM, observing appearance of hair coat and the opening of external ears, eyes,
and vaginas. The criterion for each type of observation was based on that outlined by SNELL(1941). The results are given in Table 6. Except for the first
appearance of coat hair, all characteristics appeared sooner in the l q h line than
in the low, and the differences were all significant.
TABLE 6
Mean ages in days of first appearance of the external ears, coat hair, and nipples, and of opening of
the eyes and u a g i m in mice of the high 131I uptake and low uptake lines
External ears*
No. mean
HIU
1 1 1 2.9 0.60
107
LIU
140 2.6 0.62
134 5.1
SD
Nipples'
Coat hair
Line
No. mean
5.2
SD
No. mean
Eyes'
SD
1.2 57
8.0 0.66
1.1 37
8.6 0.55
No.
Q 50
8 56
mean
Vagina*
SD
53
26.0 0.3
1.0
44
28.5 0.3
Q 44 13.7
* P <O.Ol for the significance of difference between HIU and LIU.
= standard deviation of individual observations.
No. = number of individual observations.
SD
12.9 1.4
13.1 1.1
8 58 14.0 1.5
SD
No. mean
36
C. K. C H A I
DAYS A F T E R X - I R R A D I A T I O N ( 7 0 0 R )
FIGURE
4.-Percentages of accumulated deaths of mice of the high (HIU) and low (LIU) 1311
uptake lines 30 days after 70Or X irradiation.
Basal metabolic rate and X irradiation: Basal metabolic rate was tested in mice
of the 7th generation by means of a spirometer (Model 160, Custom Engineering
and Development Co.) connected to a chamber containing an individual mouse.
A total of 24 mice (12 HIU and 12 LIU) were tested, each for at least 1 hr. The
O2uptake was recorded on a strip chart and the consumption converted to cc/min.
The results (Table 4) showed that HIU mice consume 0, more rapidly than LIU
mice, and that the difference is significant.
Whole-body irradiation of 700r delivered at a rate af 60r per min was administered to mice of the 7th generation, and daily observations for death were made
for 30 days. The cumulative percentages plotted in Figure 4 show that a total of
15 out of 26 mice died within this period in the HIU line, and 1 7 out of 33 in the
LIU line. When the percentages were transformed to probits and the regression
coefficients computed, the differences between the two lines were not significant,
though they approached significance (0.10 > P > 0.05). High line mice appear
more sensitive to X irradiation than those of the low line. The results agree with
et al. ( 1963) with inbred strains of mice differing in
those reported by TSUCHIYA
thyroid activity.
DISCUSSION
The early part of the experiment was hampered by irregular procedures. Old
SELECTION FOR THYROID U P T A K E
37
Guilford chow contains (about0.1 ppm af iodine and Purina Mouse Chow 0.05.
The time of sudden drop of 1311uptake corresponds well with the time of food
change. Before the fifth generation the mice were housed in a wooden building
where the room temperature could not be well controlled. Mice of the later
generations were housed in an isolation unit where temperature was regulated
to
1.5"C, with light (IO hr) and dark durations (14 hr) automatically controlled.
In spite of all these difficulties, two lines of mice with 4- to 5-fold differences
in thyroid iodine uptake have been produced. Apparently, selection for low I3lI
uptake was less effective than that for high uptake. The sharp rise of the uptake
curve for the high line, the lack of selection response in the low line, and the
sterility frequently found in mice of the same line, indicate that selection for low
iodine uptake is much more limited biologically than selection for high uptake.
Perhaps natural selection was in favor of high thyroidal iodine uptake in the
present stock.
Directional selection generally has effects counter to natural selection. The
latter tends to maintain the population in its best adapted norm whereas the
former tends to move the population away from it. The responses to directional
selection may be nearly symmetrical in some traits but not in others. This depends
on the degree of fitness which a trait confers and the departure of the initial population from its optimum under natural selection for the chosen trait. Nevertheless,
the asymmetrical response in the selection for thyroid iodine metabolism, which
is of vital importance in development and basal metabolism, is apparently a case
in sharp contrast to symmetrical responses to selection on a trait such as Drosophila bristle number, which confers no recognizable fitness.
Changes in thyroid function are expected to affect the secretion rate of the
thyroid-stimulating hormone by the pituitary. A lower rate of thyroid hormone
secretion causes a higher rate of TSH and vice versa, as a homeostatic adjustment
of the body. The height of follicle cells of the thyroids of the low line mice is in
general taller and the colloid in the follicles is less than in the high line mice,
which suggests that a negative feedback mechanism is in operation. We frequently found that mice in the LIU line were extremely small in body size, and
that many had fatty degeneration of the thyroid at the center of the gland
(Figure 5). This degenerative change may be the result of overstimulation by a
high level of TSH on a small thyroid gland. We do not know, however, whether
there were associated genetic changes directly affecting TSH secretion.
Various physiological parameters expressing thyroid activity were used-such
as, percentage of iodine uptake, T/S ratio, PBI level in the plasma, etc. Rate of
iodine uptake, and release from the thyroid, and the thyroid hormone synthesis
and degradation, involve many intermediate biochemical reactions which are not
well known. The proportion of different thyroid hormones varies between species
and between individuals. Neither measurement alone may be a good expression
of thyroid activity. The overall evidence in the present experiment indicates that
thyroid activity in HIU mice is much greater than that in LIU mice.
There was not much difference between the lines in the rate of very early
*
38
C. K. CHAI
P
FIGURE
5.-Photomicrographs of thyroid sections (upper) of a HIU mouse with large follicles
and droplets at the apices of the follicle cells, and (lower) of a LIU mouse with fatty degeneration
and few follicles appearing to be active.
development as indicated by the opening of the external ears, appearance of coat
hair, and increase in body weight. However, as postnatal development proceeded
differences became apparent in the age at which eyes and vaginas first opened,
SELECTION FOR THYROID UPTAKE
39
and nipples first appeared. High line mice matured earlier than those in the low
line, and the age differences, though not large, were significant. General body
growth differences increased with the advance of age (Figure 3). Mouse thyroid
begins to function a few days before birth and reaches its peak activity at about
2 months old (CHAI, MORRISON
and LENZ1964). The gradual divergence in
growth and maturation characteristics between the two lines corresponds well
with the development of thyroid €unction.
The much greater basal metabolic rate of the high line mice is expected. Mice
in the low line show more resistance to lethal doses of X irradiation. The difference, although not significant, is in the same direction with respect to thyroid
activity as that observed by TSUCHIYA
et al. ( 1963) in different inbred strains of
mice.
A number of behavioral tests were applied to the mice by STEWART
(unpublished) , including responses to air puff, forepaw grasping, righting responses,
pivoting, responses to click, etc. Although the relationship between thyroid
activity and emotional development remains to be established, many of these tests
revealed differences between the high and low uptake lines of mice. Differences
in thyroid function were found between mice selectively bred for emotional
characteristics ( FEUER
and BROADHURST
1962).
In multicellular organisms internal harmony in the development of an organ
and the integration of the organism as a whole are of primary importance. Thus,
the genetic alteration of a trait by either artificial or natural selection may involve
(1954) has shown that selection for
changes in other traits. For instance, LERNER
increased shank length in chickens alters the entire conformation of the bird. It
is difficult to know whether these correlated changes are due to genes which
control the development of the traits selected or to other genes necessary to
counterbalance the imposed genetic changes. In the present case, it is very likely
that changes in thyroid activity caused changes in other endocrines and that both
these are in part responsible for some of the associated variations.
SUMMARY
Selectivebreeding for high and low thyroid 1311uptake in mice was undertaken,
beginning from a hybrid population. After ten generations of selection, these was
a 4-fold average difference in 1311uptake between the high and low lines. The
heritability estimate was 0.56 for males and 0.42 for females in the high line and
practically zero in the low line. Thyroid function was tested accordingto different
techniques such as thyroid to serum iodine ratio, protein-bound iodine, thyroid
weight and histology, and the results were in general agreement with the difference in 1311uptake. High line mice had a higher basal metabolism, were more
sensitive to X irradiation, and showed earlier maturation and faster body growth
than low line mice. These differences are considered to be consequences of the
selection for l3IIuptake, and of the pleiotropic effects of genes.
LITERATURE CITED
CHAI,C . K., 1960 Endocrine variation: heritability of iodine metabolism in the thyroids of mice.
40
C . K. CHAI
In: Biometrical Genetics. Edited by 0. KEMPTHORNE.Pergamon Press, New York.
CHAI, C. K. and M. M. DICKIE, 1966 Endocrine variations. Pp. 387-403 in: Biology of the
Laboratory Mouse, 2nd edition. Edited by E. L. GREEN.McGraw-Hill, New York.
CHAI, C. K., J. L. MORRISON
and J. L. LENZ,1964 Changes in thyroid during lifespan of mice.
J. Heredity 55 : 270-275.
FALCONER,
D. S., 1960 Introduction to Quantitative Genetics. Ronald Press, New York.
1962 Thyroid function in rats selectively bred for emotional
FEUER,G. and P. L. BROADHURST,
elimination. 1. Differences in thyroid hormones. J. Endocrinol. 24: 127-136.
KARLSON,P., 1968 Regulation of gene activity by hormones. Humangenetik 6:99-109.
LERNER,
J. M., 1954. Genetic Homeostmis. Wiley, New York.
SILVERSTEIN,
E., L. SOKOIBFP,0. MICKEUENand G. E. JAY,JR., 1960 Thyroid function in
various strains of mice: T/S ratio, PBI and thyroid weight. Amer. J. Physiol. 199: 203-208.
SNELL,G. D., 1941 Reproduction. Pp. 55-88 in: Biology of the Laboratory Mouse. Edited by
G. D. SNELL,Dover, New York.
TSUCHIYA,
T., J. I. HAYAKAWA,
S. MURAMATSU
and H. ETO,1963 Radiosensitivity and thyroid
function in mice. Radiation Res. 19: 316-323.