MAMMARY NUCLEIC ACIDS AND PITUITARY PROLACTIN SECRETION DURING PROLONGED LACTATION IN MICE H. NAGASAWA
Pharmacology Division,
AND
R. YANAI
National Cancer Center Research Institute,
Tokyo 104, Japan
(Received 17
October
Tsukiji 5-1-1, Chuo-ku,
1975)
SUMMARY
Lactation was prolonged until 61 days by repeated renewal of litters every week after day 12 in primiparous C3H/He strain mice. On days 12, 19, 40 and 61 of lactation, litters were removed for 5 h and after 1 h of resuckling the synthesis of DNA and RNA in the mammary gland was estimated by the incorporation of [3H]thymidine and [14C]uridine into mammary DNA and RNA in vitro respectively. Mammary nucleic acid content and pituitary and plasma levels of prolactin were also assayed. Nulliparous mice were similarly treated on day 19 of pregnancy. The percentage gain in litter weight per week was highest between days 5 and 12 of lactation, declined until days 26\p=n-\33and became steady thereafter. Mammary DNA synthesis was extremely high on day 19 of pregnancy, decreased on day 12 of lactation to less than one\x=req-\ fifteenth of that on day 19 of pregnancy and increased linearly thereafter. Changes in mammary DNA content were not so marked, but DNA content was high on days 12 and 19 of lactation. RNA synthesis was highest on day 19 of pregnancy, abruptly decreased on days 12 and 19 of lactation and increased again with the advance of lactation. Mammary RNA content, RNA:DNA and 14C:3H ratios increased from day 19 of pregnancy to days 12\p=n-\19 of lactation and decreased on days 40 and 61. While the pituitary levels of prolactin were almost constant during lactation, they were significantly higher than those on day 19 of pregnancy. There were only slight differences in plasma prolactin levels at any stage.
INTRODUCTION
It is well recognized that lactation can be prolonged for several months by replacement of pups in mice (Parkes, 1926; Selye & McKeown, 1934; Fekete, 1940; Zeilmaker, 1968) and in rats (Bruce, 1958, 1961); however, information on changes in the hormonal milieu and mammary gland function during prolonged lactation is scanty. Normal or prolonged lactation results in a decrease in the incidence of spontaneous mam¬ mary tumours and an increase in tumour age when compared with forced breeding without subsequent lactation in mice (Fekete, 1940; Mühlbock & Boot, 1959; Mühlbock 8c van Ebbenhorst Tengbergen, 1961 ; Zeilmaker, 1968). Similar inhibition by lactation of carcino¬ gen-induced mammary tumorigenesis was observed in mice (Marchant, 1958, 1964) and rats (Dao, 1971; Dao & Sunderland, 1959; Dao, Bock & Greiner, 1960). However, the precise mechanism of the inhibitory effect of lactation on mammary tumorigenesis is not understood. Recently Nagasawa & Yanai (1974 a) suggested that DNA synthesis in the mammary gland around the time when a carcinogen acts on the gland is an important factor for mammary tumour induction. In this paper, we report changes in the synthetic activity and content of nucleic acids
in the mammary mice.
gland
and
pituitary prolactin secretion during prolonged lactation
in
MATERIALS AND METHODS
Female mice of a highly inbred C3H/He strain, bred in our laboratory, were used. They were mated at about 70 days of age. The number of pups was adjusted to six (three females and three males) on the day of parturition (day 0 of lactation) and this number was main¬ tained throughout the experimental period. Litters of 5 days of age and weighing 21-23 g were exchanged on day 12 of lactation. The renewal of pups was repeated every 7 days and lactation was prolonged to a maximum of 61 days. Vaginal smears were checked every day and body weights of mothers and litters were measured at every replacement. Throughout the experiment, mice were kept in an air-conditioned (24 + 0-5 °C and 65-70% relative humidity) and artificially illuminated room (light from 07.00 to 21.00 h) and provided with a commercial diet (CA-1 : CLE A Japan Inc., Tokyo, Japan) and tap water ad libitum. On the mornings of days 12, 19, 40 and 61 of lactation, mothers were separated from their pups by wire nets for 5 h and then resuckled for 1 h. Immediately after resuckling, blood was collected from the vena cava of the mother with a heparinized syringe under ether anaesthesia. Anterior pituitary, adrenals, ovaries and uterus were rapidly removed and weighed. Anterior pituitaries and plasma samples were frozen at —20 °C for assay of pro¬ lactin by radioimmunoassay (Sinha, Selby, Lewis & VanderLaan, 1972). The right third thoracic and bilateral inguinal mammary glands were used for the measurement of the incorporation of [3H]thymidine and [14C]uridine into mammary DNA and RNA in vitro as indices of the synthetic activity of DNA and RNA respectively. Total content of DNA and RNA was assayed using the left third thoracic gland defatted with hot alcohol-ether. All procedures were the same as described previously by Nagasawa & Yanai (19740). Some nulliparous pregnant mice were similarly treated on day 19 of pregnancy, one day before parturition in this strain. RESULTS
Oestrous cycle 15 and 19 of lactation and then at intervals of 9-21 days days until 61 14-9 of lactation. These results are in good agreement with those days) day (average of Parkes (1926) and Selye & McKeown (1934).
Oestrus re-occurred between
Litter and maternal weights and organ weights Table 1 shows the litter, maternal and organ weights throughout the experiment. The per¬ centage gain in litter weight per week was highest on day 12 of lactation, declined until day 33 of lactation and became steady thereafter. There were no significant differences between days 12 and 19 of lactation or between any periods after day 33 of lactation. The percentage gain in litter weight in the former two periods was significantly higher than that of the late periods (P < 0-01). Maternal weight increased gradually with the advance of lactation. While there was little difference between any stage examined in the weight of either pituitary or adrenals, the weights of ovaries and uterus increased with duration of lactation. The ovarian weight was significantly greater on days 40 and 61 of lactation than on day 19 of pregnancy and days 12 and 19 of lactation (P < 0-05 or 0-01). Uterine weight on day 12 of lactation was significantly lower than on days 40 and 61 of lactation (P < 005 or 0-01).
Table 1. Litter and maternal
weights and organ weights
lactation in mice
gain
No. of in litter estima- weight Stage tions per week
the end
of pregnancy
and
during
No.
Percentage
at
(means + s.e.m.)
Organ weight (mg)
Maternal
of esti-
weight (g)
ma-
Anterior
tions
pituitary
Adrenals
Ovaries
11
2-26 + 013
6-29 + 0-31
15-6 + 0-5
Uterus
Day of pregnancy 19 —
—
—
—
Day of lactation 12 19 26 33 40 47 54 61
35 26 17 17 17 9 9 9
95 + 3 83 + 5 69 + 5
53±3 50±3 41 + 8 43 + 4 48 + 7
32-6 + 0-4 33-3 + 0-5 33-6 + 0-4 34-4 + 0-5 34-2 + 0-4 33-6 + 0-7 35-4 + 0-7 36-9 + 0-7
9 9
2-69 + 0-15 2-38 + 0-12
6-31+0-19 6-21+0-17
14-3 + 0-7 16-3 + 1-2
81-3 + 4-3 89-2 + 61
8
2-59 + 0-09
6-71+0-22
20-9+1-1
1020 + 61
9
2-66 + 0-08
6-42 + 0-31
23-1 + 1-3
120-0+11-7
Synthesis and levels of mammary nucleic acids Results are shown in Fig. 1. [3H]Thymidine incorporation into mammary DNA was ex¬ tremely high on day 19 of pregnancy and decreased on day 12 of lactation to less than onefifteenth ofthat on day 19 of pregnancy. It increased during lactation and the value on day 61 was more than sixfold that on day 12, but still less than half of that on day 19 of preg¬ nancy.
The
change
in mammary DNA content
marked as that in DNA synthesis, 19 12 and of lactation than on day 19 of significantly higher days 0-01) and days 40 and 61 of lactation (P < 0-05). pregnancy (P The rate of mammary RNA synthesis estimated by [14C]uridine incorporation was sig¬ nificantly higher on day 19 of pregnancy than at any other stage (P < 0-01). The values on days 12 and 19 of lactation were approximately one-fourth ofthat on day 19 of pregnancy. RNA synthesis was significantly increased on days 40 and 61 when compared with days 12 and 19 of lactation (P< 0-05 or 001). There was no significant difference between days 40 and 61. Mammary RNA content increased from day 19 of pregnancy to day 12 of lactation and then gradually decreased. All values during lactation were significantly higher than that'on day 19 of pregnancy (P < 0-05 or 0-01) except for that on day 61 of lactation. The values on days 12 and 19 of lactation were also significantly higher than those on days 40 and 61 (P < 0-01), although there was little difference between these two stages. The ratio of [14C]uridine to [3H]thymidine incorporated (14C:3H) increased more than threefold from day 19 of pregnancy to day 12 of lactation. This ratio was maintained until day 19 of lactation and then decreased linearly. The change in RNA:DNA ratio showed a similar trend to that of 14C: 3H, except that on day 61 of lactation the ratio was not signifi¬ cantly different from that on day 40 of lactation.
although it
was
Pharmacology Division,
AND
R. YANAI
National Cancer Center Research Institute,
Tokyo 104, Japan
(Received 17
October
Tsukiji 5-1-1, Chuo-ku,
1975)
SUMMARY
Lactation was prolonged until 61 days by repeated renewal of litters every week after day 12 in primiparous C3H/He strain mice. On days 12, 19, 40 and 61 of lactation, litters were removed for 5 h and after 1 h of resuckling the synthesis of DNA and RNA in the mammary gland was estimated by the incorporation of [3H]thymidine and [14C]uridine into mammary DNA and RNA in vitro respectively. Mammary nucleic acid content and pituitary and plasma levels of prolactin were also assayed. Nulliparous mice were similarly treated on day 19 of pregnancy. The percentage gain in litter weight per week was highest between days 5 and 12 of lactation, declined until days 26\p=n-\33and became steady thereafter. Mammary DNA synthesis was extremely high on day 19 of pregnancy, decreased on day 12 of lactation to less than one\x=req-\ fifteenth of that on day 19 of pregnancy and increased linearly thereafter. Changes in mammary DNA content were not so marked, but DNA content was high on days 12 and 19 of lactation. RNA synthesis was highest on day 19 of pregnancy, abruptly decreased on days 12 and 19 of lactation and increased again with the advance of lactation. Mammary RNA content, RNA:DNA and 14C:3H ratios increased from day 19 of pregnancy to days 12\p=n-\19 of lactation and decreased on days 40 and 61. While the pituitary levels of prolactin were almost constant during lactation, they were significantly higher than those on day 19 of pregnancy. There were only slight differences in plasma prolactin levels at any stage.
INTRODUCTION
It is well recognized that lactation can be prolonged for several months by replacement of pups in mice (Parkes, 1926; Selye & McKeown, 1934; Fekete, 1940; Zeilmaker, 1968) and in rats (Bruce, 1958, 1961); however, information on changes in the hormonal milieu and mammary gland function during prolonged lactation is scanty. Normal or prolonged lactation results in a decrease in the incidence of spontaneous mam¬ mary tumours and an increase in tumour age when compared with forced breeding without subsequent lactation in mice (Fekete, 1940; Mühlbock & Boot, 1959; Mühlbock 8c van Ebbenhorst Tengbergen, 1961 ; Zeilmaker, 1968). Similar inhibition by lactation of carcino¬ gen-induced mammary tumorigenesis was observed in mice (Marchant, 1958, 1964) and rats (Dao, 1971; Dao & Sunderland, 1959; Dao, Bock & Greiner, 1960). However, the precise mechanism of the inhibitory effect of lactation on mammary tumorigenesis is not understood. Recently Nagasawa & Yanai (1974 a) suggested that DNA synthesis in the mammary gland around the time when a carcinogen acts on the gland is an important factor for mammary tumour induction. In this paper, we report changes in the synthetic activity and content of nucleic acids
in the mammary mice.
gland
and
pituitary prolactin secretion during prolonged lactation
in
MATERIALS AND METHODS
Female mice of a highly inbred C3H/He strain, bred in our laboratory, were used. They were mated at about 70 days of age. The number of pups was adjusted to six (three females and three males) on the day of parturition (day 0 of lactation) and this number was main¬ tained throughout the experimental period. Litters of 5 days of age and weighing 21-23 g were exchanged on day 12 of lactation. The renewal of pups was repeated every 7 days and lactation was prolonged to a maximum of 61 days. Vaginal smears were checked every day and body weights of mothers and litters were measured at every replacement. Throughout the experiment, mice were kept in an air-conditioned (24 + 0-5 °C and 65-70% relative humidity) and artificially illuminated room (light from 07.00 to 21.00 h) and provided with a commercial diet (CA-1 : CLE A Japan Inc., Tokyo, Japan) and tap water ad libitum. On the mornings of days 12, 19, 40 and 61 of lactation, mothers were separated from their pups by wire nets for 5 h and then resuckled for 1 h. Immediately after resuckling, blood was collected from the vena cava of the mother with a heparinized syringe under ether anaesthesia. Anterior pituitary, adrenals, ovaries and uterus were rapidly removed and weighed. Anterior pituitaries and plasma samples were frozen at —20 °C for assay of pro¬ lactin by radioimmunoassay (Sinha, Selby, Lewis & VanderLaan, 1972). The right third thoracic and bilateral inguinal mammary glands were used for the measurement of the incorporation of [3H]thymidine and [14C]uridine into mammary DNA and RNA in vitro as indices of the synthetic activity of DNA and RNA respectively. Total content of DNA and RNA was assayed using the left third thoracic gland defatted with hot alcohol-ether. All procedures were the same as described previously by Nagasawa & Yanai (19740). Some nulliparous pregnant mice were similarly treated on day 19 of pregnancy, one day before parturition in this strain. RESULTS
Oestrous cycle 15 and 19 of lactation and then at intervals of 9-21 days days until 61 14-9 of lactation. These results are in good agreement with those days) day (average of Parkes (1926) and Selye & McKeown (1934).
Oestrus re-occurred between
Litter and maternal weights and organ weights Table 1 shows the litter, maternal and organ weights throughout the experiment. The per¬ centage gain in litter weight per week was highest on day 12 of lactation, declined until day 33 of lactation and became steady thereafter. There were no significant differences between days 12 and 19 of lactation or between any periods after day 33 of lactation. The percentage gain in litter weight in the former two periods was significantly higher than that of the late periods (P < 0-01). Maternal weight increased gradually with the advance of lactation. While there was little difference between any stage examined in the weight of either pituitary or adrenals, the weights of ovaries and uterus increased with duration of lactation. The ovarian weight was significantly greater on days 40 and 61 of lactation than on day 19 of pregnancy and days 12 and 19 of lactation (P < 0-05 or 0-01). Uterine weight on day 12 of lactation was significantly lower than on days 40 and 61 of lactation (P < 005 or 0-01).
Table 1. Litter and maternal
weights and organ weights
lactation in mice
gain
No. of in litter estima- weight Stage tions per week
the end
of pregnancy
and
during
No.
Percentage
at
(means + s.e.m.)
Organ weight (mg)
Maternal
of esti-
weight (g)
ma-
Anterior
tions
pituitary
Adrenals
Ovaries
11
2-26 + 013
6-29 + 0-31
15-6 + 0-5
Uterus
Day of pregnancy 19 —
—
—
—
Day of lactation 12 19 26 33 40 47 54 61
35 26 17 17 17 9 9 9
95 + 3 83 + 5 69 + 5
53±3 50±3 41 + 8 43 + 4 48 + 7
32-6 + 0-4 33-3 + 0-5 33-6 + 0-4 34-4 + 0-5 34-2 + 0-4 33-6 + 0-7 35-4 + 0-7 36-9 + 0-7
9 9
2-69 + 0-15 2-38 + 0-12
6-31+0-19 6-21+0-17
14-3 + 0-7 16-3 + 1-2
81-3 + 4-3 89-2 + 61
8
2-59 + 0-09
6-71+0-22
20-9+1-1
1020 + 61
9
2-66 + 0-08
6-42 + 0-31
23-1 + 1-3
120-0+11-7
Synthesis and levels of mammary nucleic acids Results are shown in Fig. 1. [3H]Thymidine incorporation into mammary DNA was ex¬ tremely high on day 19 of pregnancy and decreased on day 12 of lactation to less than onefifteenth ofthat on day 19 of pregnancy. It increased during lactation and the value on day 61 was more than sixfold that on day 12, but still less than half of that on day 19 of preg¬ nancy.
The
change
in mammary DNA content
marked as that in DNA synthesis, 19 12 and of lactation than on day 19 of significantly higher days 0-01) and days 40 and 61 of lactation (P < 0-05). pregnancy (P The rate of mammary RNA synthesis estimated by [14C]uridine incorporation was sig¬ nificantly higher on day 19 of pregnancy than at any other stage (P < 0-01). The values on days 12 and 19 of lactation were approximately one-fourth ofthat on day 19 of pregnancy. RNA synthesis was significantly increased on days 40 and 61 when compared with days 12 and 19 of lactation (P< 0-05 or 001). There was no significant difference between days 40 and 61. Mammary RNA content increased from day 19 of pregnancy to day 12 of lactation and then gradually decreased. All values during lactation were significantly higher than that'on day 19 of pregnancy (P < 0-05 or 0-01) except for that on day 61 of lactation. The values on days 12 and 19 of lactation were also significantly higher than those on days 40 and 61 (P < 0-01), although there was little difference between these two stages. The ratio of [14C]uridine to [3H]thymidine incorporated (14C:3H) increased more than threefold from day 19 of pregnancy to day 12 of lactation. This ratio was maintained until day 19 of lactation and then decreased linearly. The change in RNA:DNA ratio showed a similar trend to that of 14C: 3H, except that on day 61 of lactation the ratio was not signifi¬ cantly different from that on day 40 of lactation.
although it
was
