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Reproductive organ DNA and RNA of male and female rats from birth to 100 days of age.

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Reproductive Organ DNA and RNA of Male and
Female Rats from Birth to 100 Days of Age'
C. DESJARDINS,z K. L. MACMILLAN? AND H. D. HAFS
Animal Reproduction Laboratory, Dairy Department, Michigan State
University, East Lansing, Michigan
ABSTRACT
Male and female rats were adjusted to six pups per litter and weaned at
20 days of age. Groups of ten males were killed at five- to ten-day intervals between
one and 100 days of age and groups of 12 to 45 females were killed between ten and 100
days. Accumulation with age of gonadal, seminal vesicular, and uterine weight, DNA,
and RNA, were compared with body growth in relative growth analyses to delimit
puberty and quantify reproductive growth.
Males and females possessed similar body growth to 40 days, after which age females
grew more slowly while males continued rapid growth to 60 days. Relative to body
growth, testes grew more rapidly from ten days whereas ovarian growth generally paralleled body growth until 50 days of age. A shift i n gonadal RNA/DNA ratio from less
than to greater than unity occurred early in puberty in both sexes. Nucleic acid contents
of accessory reproductive organs indicated that effective levels of gonadal steroids promote pubertal growth beginning at about 40 days in males and about 33 days of age in
females. These marked pubertal growths ended at 70 days i n males and 60 days of age
in females.
The weight and nucleic acid content of
reproductive organs decline following gonadectomy, but can be restored and even
elevated above pre-operational levels with
injected sex steroids (Velardo, '59; Kochakian and Harrison, '62; Williams-Ashman,
'65). The restorational changes in the nucleic acids are rapid and may be detected
before significant increases occur in organ
weight. Telfer ('53) demonstrated that a
single injection of 5 pg estradiol almost
doubles RNA content of ovariectomized
rat uteri within 48 hours, but responses
in DNA and organ weight are neither as
rapid nor as dramatic. When testosterone
propionate pellets were implanted into
castrated male mice, weights of the ventral prostates and seminal vesicles were
increased twenty-fold within 30-40 days
(Kochakian and Harrison, '62). Levels of
RNA and DNA were similar to those in
the organs of intact control mice within
five days after testosterone implantation.
This sensitivity of nucleic acid levels
in reproductive organs to the sex steroids
was the basis for the following experiment
which was designed to measure changes
in the nucleic acid contents of gonads
and main accessory reproductive organs
during reproductive development. These
changes, especially those for RNA in accessory organs, may reflect fluctuations
ANAT. REC., 161: 17-22.
in steroidal titers during puberty more precisely than organ weight or histological
criteria previously used (e.g., Moore, '39).
An additional object was to provide bases
for comparism of future treatments designed to alter reproductive growth.
METHODS
Male and female Sprague-Dawley rats
were adjusted at birth to six pups per litter and weaned at 20 days of age. After
weaning, males and females were reared
separately with standardized numbers per
cage, with feed and water ad libitum, in
a room at 24"
1°C and provided with a
14-hour photoperiod daily. Ten male rats
were killed by cervical dislocation at 1, 10,
20, 25, 30, 35, 40, 45, 50, 60, 80, 90 and
100 days of age. Forty-five females were
killed at ten days, 24 females at 15, 20,
and 25 days and 12 females at 30, 31, 32,
33, 34, 35, 40, 50, 60, 70, 80, 90, and 100
days of age. The testes, seminal vesicles,
ovaries and uteri were removed, isolated
from extraneous tissue, weighed, placed
in 0.25M sucrose at 5"C, and stored at
1 Published with the approval of the Director of the
Michigan Agricultural Experlment Station as paper
4101.
This
.~.
_ research was suuuorted by U.S.P.H.S. grant
HD-01374.
2,Present address: Jackson Laboratory, Bar Harbor,
Maine.
3 Present address: New Zealand Dairy Board, Newstead, Hamilton, N. Z .
~~
~
~~~~
_-
17
18
C. DESJARDINS, K. L . MACMILLAN A N D H. D. HAFS
-20". Seminal vesicles were too small to 20-day level was less than the comparable
measure reliably at one and ten days of increase in organ weight; suggesting tesage. Tissue DNA and RNA were deter- ticular hypertrophy coincidental with hymined by the procedures of Schmidt and perplasia. DNA concentration (mg DNA/
Thannhauser ('45) as modified by Tucker gm testis) declined at a diminishing rate
('64). In some younger age groups, or- after 20 days of age.
Seminal vesicular weight, DNA, and
gans from several animals were pooled to
obtain sufficient tissue for assay purposes. RNA increased most rapidly from 40 to
70 days of age (table 2). Increases in
RESULTS
RNA with age were greater than comparThe growth rate of male rats (table 1 ) able increases in DNA and, consequently,
was not reduced until 60 days of age (256 the RNA/DNA ratio increased from 0.57
gm body weight), but standard errors at 30 days of age to 3.46 at 100 days of
associated with mean body weights in- age. Standard errors associated with mean
creased considerably from 70 to 100 days organ weights and nucleic acid contents
of age. Average testes weight (table 1 ) increased dramatically beyond 60 days of
increased most rapidly between 20 (206 age.
The body growth rate of females (table
mg) and 70 (3,291 mg) days and declined
slightly thereafter. Although total testicu- 3 ) was similar to that for males to 40 days
lar DNA also increased from 20 (1.77 mg) of age. Thereafter, females grew less rapdays of age, the increase relative to the idly and their body weight apparently staTABLE 1
Body weight and testicular weight, D N A and R N A / D N A ratio
Age
dags
1
10
20
25
30
35
40
45
50
60
70
80
90
100
Bqdv
weight
m
720.1
2010.1
43t-1
58% 1
8011
11512
12622
16422
174t-4
25626
27519
265212
274k12
309t-15
Testicular
weight
m g
72:1
5922
20625
370213
600% 13
945237
1,194*78
1,911260
2,167255
3,009r72
3,291247
2,712279
2,828287
2,9092116
DNA
RNA/DNA
qng
0.01~0.00
0.32-CO.03
1.77t-0.05
2.4510.08
3.50*0.14
4.55k0.25
5.5220.24
6.72a0.32
9.1520.26
9.40*0.37
9.7020.33
8.2710.44
7.7510.59
7.0520.32
1.78
0.84
0.77
0.80
1.01
1.26
1.27
1.60
1.40
1.27
1.47
1.43
1.70
1.93
TABLE 2
Seminal vesicular weight, D N A , R N A and R N A / D N A ratio
Age
Weight
DNA
dags
m g
nw
20
25
30
35
40
45
50
60
70
80
90
100
8*l
821
6* 1
16t-1
2022
52*3
6517
174210
330230
278528
308*21
449245
0.05*0.01
0.06*0.01
0.07t-0.01
0.10~0.01
0.11*0.01
0.2410.02
0.34W.03
0.63k0.04
0.9Ot-0.12
0.6710.12
0.6320.05
0.6320.05
RNA
RNAiDNA
m g
0.03t0.01
0.05-CO.01
0.04-CO.01
0.1220.01
0.12r0.01
0.48r0.06
0.54r0.06
1.1420.07
1.9920.19
1.28-CO.24
1.43-CO.10
2.2020.3
1
0.65
0.79
0.57
1.13
1.17
1.94
1.63
I.84
2.40
1.90
2.33
3.46
19
REPRODUCTIVE GROWTH O F RATS
bilized after 80 days of age (189 gm body
weight). Ovarian weight increased at a
constant rate from 15 (8 mg) to 80 (83
mg) days of age (table 3 ) . Although
ovarian DNA content showed considerable
variation throughout, the generally linear
increase in DNA with advancing age was
interrupted by a plateau between 30 and
50 days. There was little change in either
total DNA or total RNA after 60 days of
age. Uterine criteria (table 4 ) continued
to increase to 90 days of age.
Both testicular and ovarian RNA/DNA
ratios generally changed from values of
about 0.8 before 30 days to values over
1.0 after 35 days of age. Greatest changes
in seminal vesicular and uterine weights
and nucleic acid contents also occurred
after 35 days of age (tables 2, 4), but the
elevation in gonadal RNA/DNA ratios apparently preceded increments in accessory
organ criteria.
DISCUSSION
The decline in growth rate of female rats
occurs soon after puberty; vaginal opening
occurred at 38 days of age. Similar sexual
differences in rate of body growth were
TABLE 3
Body weight and ovarian weight, DNA and RNA/DNA ratio
Age
Body
weight
days
10
15
20
25
30
31
32
33
34
35
40
50
60
70
80
90
100
Ovarian
weight
DNA
gm
mg
mg
2020.3
3221
4421
5222
7423
84fl
8723
9023
9522
12523
14725
16725
18023
18926
19925
19124
720.1
820.2
1721
2221
2921
2922
28k2
3021
3021
3322
3922
4822
6423
7023
8326
72*3
6623
4421
5524
16126
142i8
252213
256211
236-22
236-24
182211
186214
194-13
295233
459530
385-27
538-38
437516
419-20
RNA/DNA
0.87
0.83
0.82
0.88
0.75
0.74
0.75
0.93
1.06
1.03
1.06
1.24
1.07
1.25
1.06
1.05
0.95
TABLE 4
Changes in the weight, DNA and RNA contents and RNA/DNA ratio o f the uterus
Age
Weight
DNA Content
RNA Content
days
mg
mg
mg
10
15
20
25
30
31
32
33
34
35
40
50
60
70
80
90
100
18a0
3921
5021
4721
7324
8223
77c7
7923
127215
141218
234226
289217
348227
384223
413218
493222
435221
10528
190213
25829
236213
39341
399*46
435*41
393228
539254
560260
981299
1,326258
1,918291
1,633296
1,980296
2,036297
1,925*70
60C1
125C8
162*5
16225
304k22
311k24
419k67
282-t21
605290
611295
1,136f182
1,3642123
2,3782247
2,2232178
2,628f172
2,8102193
2,266-C153
RNA/DNA
0.64
0.65
0.64
0.74
0.77
0.89
0.96
0.73
1.07
1.09
1.12
1.01
1.21
1.38
1.32
1.37
1.18
20
C . DESJARDINS, K. L. MACMILLAN AND H. D. HAFS
published by Donaldson (’24) and Jack- starting as early as day 20. While size
of testes, as measured by weight, DNA
son (’13).
Because growth of reproductive organs content or RNA content, increased until
is selectively stimulated at puberty, ex- 70 days, the nucleic acid changes were
pression of their sizes either in absolute not large beyond 50 days. In fact, DNA
values or on body weight bases as has decreased appreciably beyond 70 days.
been previously done may be misleading. Jackson (’13) also noted a decline in tesConsequently, we have chosen to express ticular weight, but at a later age than we
increments in weight, DNA, and RNA of observed and Drori and Folman (’64) reeach reproductive organ relative to its re- ported a marked reduction in rate of inspective weight, DNA, or RNA at a base crease of testicular size beyond 67 days.
age. Comparison of these measures of reo n e may compute, from the data in
productive “growth with similarly ex- table 1, that the concentration of testicupressed body growth aids interpretation of lar DNA declines nearly continuously but
the tabular data because units are elimi- at a declining rate from a high of 8.65
nated with this method and the different mg/gm at 20 days to 2.45 mg/gm at 110
criteria, organs, and sexes may be com- days, similarly to that reported by Fujii
pared directly rather than on body weight and Koyama (’62). However, our data rebases. Size at age ten days was arbitrarily veal a dramatic increase from the lowest
selected as the base for growth compari- value observed (1.57 mg/gm) at birth to
son in all criteria except seminal vesicular that at 20 days of age. The decline in
criteria where 20 days was selected be- DNA concentration beyond 20 days is no
cause this organ was too small for accu- doubt due to increased diameter of semirate evaluation at earlier ages.
niferous tubules. Since the growth of
Size of the testes with advancing age these tubules is under the influence of
(fig. 1 ) relative to that at ten days of FSH (Nelson, ’52), we interpret the obage increased at rates more than double served decline in DNA concentration to
that for body weight, indicating that the reflect increased FSH secretion.
testes may be stimulated by gonadotropins
The relative growth analysis of seminal
vesicles (fig. 2) revealed no marked dif60 0 Body Weight
ferences between rate of seminal vesicular
0 Testes Weight
growth and rate of body growth between
20 and 30 days. Beyond these ages,
OTestes DNA
growth of seminal vesicles became pro50
A T e s t e s RNA
gressively greater than body growth to 70
ZI
IrJ
0
days. RNA content at 35 days is more
-0
than double that at 30 days, but greatest
040 increments in each seminal vesicular pac
0
rameter occur between 40 and 70 days of
age. The large changes in RNA and
weight relative to DNA undoubtedly reflect accumulated seminal vesicular secretion. Thus, although androgens may affect seminal vesicular activity (RNA) as
early as 30 days of age, marked pubertal
stimulation occurs beginning at 40 days
of age, about ten days later than testicular
pubertal growth. Both testicular and seminal vesicular parameters peaked at 70
days of age. These responses are reminiscent of responses to exogenous testosterone propionate implants which produce
Age (days)
dramatic increases in the RNA contents
of seminal vesicles, prostates and kidneys
Fig. 1 Changes in testes weight, DNA and
of castrate mice followed by declines in
RNA relative to ten days of age.
21
REPRODUCTIVE GROWTH O F RATS
A
'O
a
-
Body Weight
Seminal Vesicle weigw
0 Seminal Vesicle DNA
60
-
uted to the variation among rats within
an age for ovarian and uterine criteria.
In conclusion, relative to body growth,
testes grow more rapidly from ten days of
A Seminal Vesicle RNA
i
14
50
0 Body Weight
-
i"
Ovary Weight
0 Ovary DNA
u
h
l
12
A O v a r y RNA
Q
0
0
cu 40
10
+
0
0
30
0
0
+
9.
0
/\
1
8 -
+
0
.c
4-
o
e!
t
0)
6-
al
>
.t
0
5
I
30
50
70
4 -
N
Q)
I
.-
90
v)
2-
Age (days)
Fig. 2 Changes in seminal vesicular weight,
DNA and RNA relative to 20 days of age.
0
I
,
,
,
,
50
90
10
each after the maximum response was
produced (Kochakian and Harrison, '62).
In contrast to testes, relative growth of
ovaries is similar to that for the body, at
least until age 50 days (fig. 3 ) . In fact,
even beyond 50 days, ovarian weight
changes with advancing age do not differ
greatly from those for the body. Each
measured ovarian criterion attains maximal value at 80 days of age.
The relative growth analysis of uteri
(fig. 4 ) revealed that until 30 days of
age, its rate of growth paralIeled body
growth. Beyond that age, the uterus grew
much more rapidly than the body and increases in uterine weight and especially
RNA were more rapid than increases in
uterine DNA. In fact, DNA changed little
after 60 days while weight and RNA continued to accumulate at least until 90
days.
Because this experiment was designed
to measure the rats at given ages, it was
not possible to standardize the stage of
estrous cycle at slaughter of the post-pubertal females. This undoubtedly contrib-
70
30
Age (days)
Fig. 3 Changes in ovarian weight, DNA and
RNA relative to ten days of age.
50
-
Body Weight
0 Uterine Weight
0 Uterine DNA
u)
g40 U
AUUterine RNA
0
c
4-
30 -
0
f
220al
>
_c
-0
E
l0-
0)
N
.cn
r
OW
I0
30
50
70
90
Fig. 4 Changes in uterine weight, DNA and
RNA relative to ten days of age.
22
C . DESJARDINS, K. L. MACMILLAN AND H. D. HAFS
age whereas ovarian growth generally
parallels body growth until 50 days of age.
A marked shift in gonadal RNA/DNA
ratio of both males and females from less
than unity to greater than unity, occurred
early during puberty. This gonadal hypertrophy apparently preceded sex steroid
secretion. Based upon nucleic acid content of rat accessory reproductive organs,
gonadal sex steroids begin to promote
marked pubertal development at about 40
days of age in males and about 33 days
of age in females. Terminal ages for these
marked pubertal changes are about 70
days in males and 60 days in femalesages a t which rats may be considered to
be postpubertal.
LITERATURE CITED
Donaldson, H. H. 1924 The Rat. Memoirs Wistar Inst. Anat. Biol., Philadelphia, p. 301.
Drori, D., and Y. Folman 1964 Effects of cohabitation on the reproductive system, kidneys,
and body composition of male rats. J. Reprod.
Fertil., 8: 351-359.
Fujii, T., and R. Koyama 1962 Nucleic acid
contents of various organs i n rats during postnatal growth. Endocrinol. Japon., 9: 66-73.
Jackson, C. M. 1913 Postnatal growth and variability of the body and of the various organs in
the albino rat. Am. J. Anat., 15: 1-68.
Kochakian, C. D., and D. G. Harrison 1962
Regulation of nucleic acid synthesis by androgens. Endocrinology, 70: 99-108.
Moore, C. R. 1939 Biology of the testes. In:
Sex and Internal Secretions. 2nd Ed., edited by
A. D. Doisy. Williams and Wilkins Co., Baltimore. chapt. 7.
Nelson, W. 0. 1952 Interrelations of gonadotrophic and gonadal hormones in the regulation
of testicular functions. Ciba Found. Colloquia
Endocrinol., 4: 271-281.
Schmidt, G., and S. J. Thannhauser 1945 A
method for the determination of desoxyribonucleic acid, ribonucleic acid, and phosphoproteins in animal tissues. J. Biol. Chem., 161:
83-89.
Telfer, M. A. 1953 Influence of estradiol on
nucleic acids, respiratory enzymes and the distribution of nitrogen in the rat uterus. Arch.
Biochem., 44: 111-119.
Tucker, H. A. 1964 Influence of number of
suckling young on nucleic acid content of lactating rat mammary gland. Proc. SOC.Exptl.
Biol. Med., 116 : 218-220.
Velardo, J. T. 1959 Steroid hormones and uterine growth. Ann. New York Acad. Sc., 75: 441462.
Williams-Ashman, H. G. 1965 Ribonucleic acid
and protein synthesis in male accessory reproductive glands and its control by testosterone.
In: Mechanisms of Hormone Action. Edited by
P. Karlson. Academic Press, New York. p. 214221.
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