Farmacologisch Laboratorium, Universiteit van Amsterdam, Polderweg 104, Amsterdam, The Netherlands
MAMMOTROPHIC ACTIVITY IN RAT SERUM DURING THE OESTROUS CYCLE, PREGNANCY AND LACTATION
By
J.
M. Peters, I. E.
Uyldert
and A. Th. Ari\l=e"\ns
ABSTRACT The mammotrophic activity of various serum samples was assessed on the mammary gland of pregnant rats, in organ culture. Insulin was added to the medium. Serum samples from virgin rats showed little activity, and variation in mammotrophic activity during the oestrous cycle was slight. During pregnancy a significant increase in proliferative activity was not seen during the first week, but around day 8\p=n-\9mammotrophic activity increased sharply. The activity showed maxima around days 12 and 19. A decrease in the activity between these maxima was not consistently observed. Mammotrophic activity was still present in sera collected during parturition, and 30 min after parturition. It had disappeared completely within 24 h. The mammotrophic factor detectable by organ culture in the serum of pregnant rats could be rat chorionic mammo-
trophin. Activity in the serum on day 0 and 1 of lactation was comparable to that of virgin rats, but some activity appeared on day 2. High activities were found frequently around day 4 to 6 of lactation. On other days the activity showed fluctuations without a definite pattern. Litter size was of minor importance but the combination of a large litter size and fasting-overnight seemed to suppress the presence of mammotrophic activity in the serum. Nursing was important: after weaning the activity has disappeared, while renewed nursing after weaning resulted in the appearance of high levels of activity. The mitotic activity obtained with lactating rat serum in the culture was suppressed by addiPart of this work has been Amsterdam, April, 1972.
presented
at the 13th annual "Federatieve
Vergadering",
tion of rabbit anti-rat prolactin serum to the medium. This suggests that the main mammotrophic factor detectable by organ culture in the serum of lactating rats, is prolactin.
Culture of the mammary
gland in vitro can be used to detect mammotrophic in serum activity samples (Forsyth 1971). In this way activity has been de¬ monstrated in the plasma of pregnant goats (Brumby 8c Forsyth 1969) and in various types of human plasma (Frantz et al. 1972). In this study the mammo¬ trophic activity present in rat serum is assayed using the mammary gland of pregnant rats in organ culture. Rat serum was collected during the oestrous cycle, during pregnancy, and during lactation. MATERIALS AND METHODS Serum samples. Serum was obtained from nulliparous, adult female albino rats (random bred, spf, Cpb: WU (WI) strain, TNO Centraal Proefdieren Bedrijf, Zeist, The Netherlands) of about 200 g in weight. They were routinely fasted overnight and exsanguinated late in the morning under light ether anaesthesia, via the right -
artery under sterile conditions. The blood was allowed to clot at room tem¬ perature for 1 h and then centrifuged for 20 min (1400 g); the serum was separated carotid
centrifuged for another 5 min (1400 g). The final samples were stored at -20°C. Samples contaminated by haemolysis were discarded. Before use each serum sample was tested for sterility. Oestrous cycle. Sera were collected during dioestrus (few nucleated epithelial cells and some leucocytes in the vaginal smear), pro-oestrus (many nucleated cells, some cornified cells), oestrus (few nucleated cells, many cornified cells) and metoestrus (many leucocytes, few cornified cells). Pregnancy. The day on which the vaginal smear showed the presence of sperma¬ tozoa, was taken as day 0 of pregnancy. After collection of the sera, the animals were autopsied to verify pregnancy. Lactation. The rats were allowed to nurse their offspring after parturition (= day 0 of lactation). The offspring were removed just before exsanguination. and
-
-
-
»
Medium and hormones The culture medium consisted of 50 % serum (v/v) and 50 % chemically defined medium (t8), a simplified formula adapted from Trowell's T8 (Paul 1970), i. e. without amino acids, vitamins and phenol red. The final glucose concentration of the medium was approximately 2 mg/ml. No antibiotics were used. Insulin (I; bovine; Organon, Oss, The Netherlands; 25.7 IU/mg; 1 mg/ml aqua dest.; membrane filter sterilized; stored at -20°C) was added to the medium in a concentration of 50 pg/ml.
Culture The on
sera were
added to the medium and the mammotrophic activity was tested size: 2x2x1 mm) obtained from the right inguinal mam-
expiants (approximate
of 13 day pregnant rats. The technique was adapted from Trowell (Paul Three expiants were supported on a stainless steel grid and a piece of nylon gauze in such a way that they remained on the surface of 1 ml medium. The watchglass culture vessel was covered by a glass plate, but this did not exclude the pos¬ sibility of gas exchange. The vessels were stored in an incubator at 37°C in a humidi¬ fied atmosphere (relative humidity about 100 %). The incubator (0.19 m3) was flushed with a mixture of 95 % Og and 5 % C02 (20 1/h). When the medium was changed after 3 days, the nylon gauze supporting the 3 expiants was transferred to fresh medium. This was done with the 6 days cultures.
mary
1970).
gland
Histology At the end of the experiments the expiants were fixed in Bouin solution. Two series of sections were made, of the central part of each expiant and stained with haematoxylin and phloxin. The results were evaluated by a procedure adapted from Elias Se Rivera (1959). One section was chosen at random and evaluated blind by the same person (first author) as to the presence and degree of certain characteristics. A grading system was used, 0 indicating absence, 1 slight degree and so on. The following parameters were evaluated at 100 x magnification: a) degree of alveolar development (graded 1-8); b) cytoplasmic opalescence (i.e. the cytoplasm stains very lightly and the cells appear to be empty except for thin thread-like structures while the nucleus/ cytoplasm ratio of these well-developed cells is low; 0-3); c) vacuolization (0-5); d) eosinophilic material (secretion; 0-5) in lumen of acini and ducts, and e) presence of nuclear remnants in the eosinophilic material; f) development of the ductal epi¬ thelium (1-3); g) presence of necrosis; at 400 x magnification: h) frequency of mitotic figures (0-5) and i) pyknosis were noted. The median values and percentages of the expiants showing a given characteristic are reported. This is restricted, however, to those parameters relevant to the experiments and conclusions. Differences between gradings were tested for statistical significance with the distribution-free test of Wilcoxon (Wabeke Se van Eeden 1970). Differences between incidences were tested with the chi-square test with Yates' correction (Croxton 1959), differences between means by Student's i-test. A level of significance of 5 or 1 % was chosen.
Labelling
index
(exp. 5). [3H]thymidine (5 pCilml, Radiochemical Centre, Amersham, England) was added 3V2 h before the end of the experiment. The mammary gland expiants were fixed in Bouin solution, dehydrated and embedded in paraffin. Serial sections (6 pm) were cut transversely. The slides were dipped in Kodak NTB-2 liquid emulsion and stored at 4°C for a period of 5 days. The exposed autoradiographs were developed for 4 min at 15°C in Kodak D19 solution, washed in aqua dest. and fixed for 5 min in Kodak F5 fixative. The autoradiographs were stained with haematoxylin and phloxin. The percentage of labelled epithelial cells was determined for 6 different expiants. Two sections of each expiant were selected at random. Labelled nuclei were counted (x 300) and the slides micro-photographed. The photographs were printed on Agfa TP6 at a final magnification of x 500. The number of nuclei counted per expiant amounted to
approximately
1500.
Experiments The following serum samples were added to the culture medium in a concentration of 50% and their effect on the 13 days pregnant rat mammary gland in vitro studied:
Exp. 1: Sera of virgin rats, collected at 4 different stages of the oestrous Serum collected from 4 rats at a certain stage of the cycle was pooled and 3 days culture with mammary glands of 6 rats.
cycle. assayed;
-
Exp. 2: Sera of pregnant rats, collected each day during pregnancy and 2 days post-partum. Serum from 1 or 2 rats per day of pregnancy was tested; 3 and 6 days cultures, each with 5 mammary glands. Exp. 3: Serum collected from 9, 13, 15 and 19 days pregnant rats. Sera from pregnant rats were assayed individually. They were added to the medium at 1.6, 3.1, 6.3, 12.5 and 25 %. Pooled serum of virgin rats was added to a final percentage of -
-
days culture on 4 mammary glands. of virgin rats, 19 days pregnant rats and sera collected during and Exp. after parturition. The sera were assayed individually. The rats were not fasted overnight. In the case of the rats exsanguinated during parturition, the first rat had de¬
50%
serum; 3
4: Serum
-
livered 9 young out of 10, the second 2 out of 9, the third 2 out of 16 and the fourth 3 out of 13. The rat, which was exsanguinated 30 min after the end of parturition, had delivered 14 young. Two rats which had delivered during the night and were exsanguinated at midday, had had 7 young each. Serum of 3 individual virgin rats served as control; 3 days culture on 3 mammary glands.
Exp. 5: Sera of virgin rats and of 13 and 19 days pregnant rats. Sera were as¬ sayed in a 3 days culture. [3H]thymidine was added at the end of the culture; 1 mam¬ mary gland was used. -
Morphological changes
Fig. 1. gland
in the mammary
culture:
of
a
13
days pregnant
rat in organ
Appearance of the gland at the beginning of the culture. The alveoli are welldeveloped (grade 4). A few isolated vacuoles are noticeable but no traces of secretion. The acinar lumina are very small. The epithelial lining of the duct is well-developed (grade 2). Scale: 100 pm. b Detail of Fig. 1 a. The well-developed acinar cells are translucent and the cytoplasm has the appearance of foam (cytoplasmic opalescence: grade 2). Scale: 25 pm. c Mammary gland expiant after a 3 days culture in a medium containing 50 % mammotrophic-inactive serum (day 0 of pregnancy) with insulin supplementation. Poor development of the alveoli (grade 2). Connective tissue is prominent. The epithelial lining of the ducts is poorly developed (grade 1). Scale: 100 pm. d Detail of Fig. 1 c. The cells are poorly developed without opalescence. The nucleus/ cytoplasm ratio appears to be high. Some pyknotic nuclei are noticeable. The acinar a
lumina
small and empty. Scale: 25 pm. e Mammary gland expiant after a 3 days culture in a medium containing 50 % mammotrophic-active serum (day 13 of pregnancy) with insulin supplementation. The alveoli are very well developed (grade 7). The upper part of the transverse section through the expiant shows some traces of vacuolization, indicated by an arrow. Secre¬ tion, however, is not prominent. Scale: 100 pm. are
f Detail of Fig.
1
e.
The cells with
cytoplasmic opalescence are very similar in appear¬ (Fig. 1 b). The lumina are small. Vacuolization
to those of the starting material is not prominent. Scale: 25 pm. ance
Fig. 1 a-f.
Exp. 6: Serum collected on days 19, 20 and 21 of pregnancy and on 21 days of lactation following parturition. Serum of virgin rats served as control. Serum col¬ lected from 2-3 rats on a certain day of lactation, was pooled and assayed; 3 days culture with 4 mammary glands. -
Exp. 7: Serum collected on day 22 of pregnancy and days 0 to 7 of lactation. Sera were collected from non-fasted rats and assayed individually in a 3 days culture with 4 mammary glands.
—
Exp.
Serum collected
8:
on
day
6
of lactation from
rats
with
varying numbers of
series samples of 15 non-fasted rats, nursing 3 to 17 young were assayed individually; in another series samples of 12 rats, fasted overnight and nursing 0 to 14 young were assayed individually; 3 days culture with 4 mammary glands.
In
young.
one
-
Exp. 9: Serum collected on 5-6 days following weaning on days 0, 10 or 21 of lactation. Sera of non-weaned rats served as control. Serum collected from 2-4 rats on a certain day after weaning was pooled and assayed in a 3 days culture; 4 mam¬ -
mary
glands.
Exp. 10: Serum collected from rats either weaned for 9 h, or weaned for 8 h fol¬ lowed by 1 h nursing, as well as serum collected from non-weaned rats. Serum was collected on day 6 of lactation from non-fasted rats with 8 young and assayed in¬ dividually in a 3 days culture; 4 mammary glands. -
Exp.
rabbit
11:
Effect of the addition of rabbit anti-rat prolactin Serum
serum
and inactive
day 6 of lactation and assayed individually in a 3 days culture; 4 mammary glands. Final glucose concentration: 2 mg/ml. The sera were tested simultaneously in a medium in which 25 % rabbit serum was present, replacing t8. The serum of the first 6 rats was tested in the pre¬ serum.
was
collected from 7 rats
on
-
sence
of
obtained rabbit serum (membrane filter sterilized, Flow Labora¬ serum of the 7th rat was tested in the presence of rabbit serum (preparation Dr. H. G.Kwa, The Netherlands Cancer Institute,
commercially
tories, Irvine, Scotland), the anti-rat
prolactin
Amsterdam, The Netherlands).
RESULTS 1. The mammary
gland of 13 days pregnant rats (Fig.
1 a,
b)
At the time of explantation the alveoli are fairly well developed (grade 4-5), with a well-developed epithelial lining of the ducts (grade 1-2). The cytoplasm of the acini is opalescent. Traces of secretion or vacuolization, and mitotic
figures
are
infrequent.
Mammotrophic activity during the oestrous cycle (exp. 1) The in vitro response of the expiants to the sera collected at different stages of the oestrous cycle showed a low degree of alveolar development (3-4), mitotic activity in only 17 to 33 % of the explants (n 15 to 18), no vacuoliza¬ tion, and traces of eosinophilic material in the lumen in 6 to 33 % of the 2.
=
explants. The differences between the sera were not significant. Cytoplasmic opalescence occurred less with dioestrous-serum (6 %, n=18) than with metoestrous serum (47 %, n= 15, difference significant at 5 % level). 3.
Mammotrophic activity during pregnancy (exp. 2)
If mammary expiants were incubated with rat serum taken from day 10 to 21 pregnancy for 3 days, a high degree of alveolar development, numerous mitoses in acini and ducts, a well-developed ductal epithelium, acinar opales¬ cence (Fig. 1 e, f) and traces of vacuolization and secretion (Fig. 1 e, arrow) were
Fig. 2. collected on different days during pregnancy and lactation (lact.) on rat mammary gland in vitro in a 3 days (left) and 6 days culture (right). Insulin was added to the medium, which contained 50 % rat serum. Top: degree of development (median value of semiquantitative grading). Middle: % of expiants showing one or more mitotic figures in acini (-) and ducts (-). Bottom: % of expiants showing traces of secretion (-) or vacuolization (-) of the acini. Number of expiants: 15, in some cases 12, 13 or 14. P parturition.
Effect of
serum
=
Fig. 3. increasing concentrations of pregnant rat serum, replacing non-pregnant rat serum (50 %) in the medium, on rat mammary gland in vitro. Three days culture with insulin added to the medium. Top, left: % of expiants showing one or more mitotic figures in the acini, right: degree of development (median value of semiquanti¬ tative grading). Bottom, left: % of expiants showing opalescent cytoplasm of the acini, right: % of expiants showing traces of vacuolization of the acini. 9, 13, 15 and 19: day of pregnancy on which serum was collected; no of expiants: 11-12. Effect of
observed (Fig. 2). Before day 10 the activity of the serum was comparable to that of day 0 of pregnancy (Fig. 1 c, d). Degree of alveolar development and presence of mitotic activity were significantly (at 1 % level) higher on day 12 than on day 0, but the differences between day 12 and day 19 were non¬ significant. After parturition the activity had virtually disappeared on day 1 of lactation and alveolar development and mitotic activity were significantly less than with serum from day 19 of pregnancy (at 1 % level). After a 6 days culture alveolar development seemed generally less well maintained and the mitotic activity had decreased (Fig. 2). Again the main mammotrophic activity was found in the sera collected from day 10 to day 21 of pregnancy. Activity
during this period: alveolar development with 12 days serum high comparison with 10 days serum (significant at 1 % level); 14 days showed less activity than either 12 days or 19 days serum in respect to the incidence of mitotic activity, secretion or vacuolization (significant at 5 % level).
was
not constant
was
in
Table 1. The effect of sera collected from 5 19 days pregnant rats, from 4 rats during parturi¬ tion, from 3 rats after parturition (No. 1: 30 min after, Nos. 2 and 3: parturition during the night, bled at midday) and from 4 virgin rats on rat mammary gland in vitro (exp. 4). Three days culture with insulin added to the medium, which contained 50 % serum.
Serum and No. of rats
Nineteen 1
2 3 4 5
(grade)1)
activity
Mitotic
Degree of development
Acini
Ducts
(%)2) (grade)1) (%)2)
Cytoplasmic opalescence
(%)2)
Vacuolization Secretion
(%)2)
(%)2)
11
33 33 11 0 44
days pregnant 5 5 5 5 5
100 100 100 100 100
2 2 3 2 2
67 67 67 67 56
100 100 100 100 100
22 22 0
89 100 100 78
56 89 89 56
67 100 100 78
0 11 0 0
100 33
67 0 22
89
33
During parturition 1 2 3 4
4 5 5 4
22 22 0 44
After parturition 1 2 3
4 3 3
11
11 0
Virgin 1
2 3 4
11 22 0 0
22 22 0 11
0 Median value of semiquantitative grading of response indicated. 2) % of expiants showing response indicated. No. of expiants: 9 per group.
0 0 11 0
Dose-effect relationship for pregnant rat serum (exp. 3) Judging from the alveolar development, mitotic activity, cytoplasmic opales¬ cence and vacuolization (Fig. 3), the highest activity was found in serum on day 19 of pregnancy, the lowest on day 9 and intermediate activities on days 13 and 15. The results indicate that the activity on day 19 was approximately 4 times that observed on day 13 or 15, while the activity on day 13 was ap¬ proximately twice that on day 9. 4.
Activity of serum collected during and shortly after parturition (exp. 4) In 4 rats, exsanguinated during parturition, the following parameters were significantly increased in comparison with serum of virgin rats: development, mitotic activity and cytoplasmic opalescence (at 5 % levels). In 2 rats (no. 2 and 3) the results were comparable with those obtained with sera of 19 days pregnant rats (Table 1). Activity was still present in the serum of a rat 30 min after the end of parturition. The activity of the sera of 2 rats which had delivered during the night and had been exsanguinated at midday was equal to that of virgin rats. 5.
Labelling index with serum of virgin rats and 13 days and 19 days rats (exp. 5) The epithelial cell labelling index with virgin rat serum was 1.7 ± 0.23% (x ± sem, N 6), with day 13 serum 4.2 ± 0.48% (N 6) and day 19 serum 7.6 ± 0.92 % (N 6). The differences between virgin and day 13 serum, and between day 13 and 19 serum were significant at 1 % levels. 6.
pregnant
=
=
=
Mammotrophic activity during lactation (exp. 6) degree of development was high during the last days of pregnancy, fell abruptly after parturition to rise again during the first five days of lactation. The degree of development fluctuated considerably during lactation (Fig. 4 A). It was significantly increased at the 5 % level, as compared with serum of virgin rats, except on days 0, 1, 13, 16 and 19 of lactation. Taking the entire period of lactation the degree of development with these sera was significantly (at the 1 % level) lower than with serum of day 19 of pregnancy. Mitotic activity showed a similar trend as the degree of development (Fig. 4 A) but in comparison to the serum of virgin rats a significant (at the 5 % level) increase was only found on days 19, 20 and 21 of pregnancy and on days 5, 6 and 8 of lactation. Opalescence of the cytoplasm and traces of secretion were seen only with serum from day 2 to 8 of lactation but to a lower extent than with serum taken on days 19, 20 and 21 of pregnancy. Traces of vacuolization were seen with serum from days 19, 20 and 21 of pregnancy, but not during 7.
The
lactation.
Fig. 4A,B. A. The effect of serum collected on day 19, 20 and 21 of pregnancy (-) and on 21 consecutive days of lactation (-). Medium supplemented with insulin; 3 days culture; no. of expiants: 11-12. The arrows indicate the effect of virgin rat serum. B. The effect of serum collected on day 19, 20 and 21 (-) and on day 0-5 of lacta¬ tion. Serum of nursing, non-weaned rats: (non-w); serum of rats weaned imme¬ diately following parturition:-(w). Medium supplemented with insulin; 3 days culture; no. of expiants: 11-12. The arrows indicate the effect of virgin rat serum. Statistical significance for the difference between nursing, non-weaned rats and weaned rats is indicated by ''"' at the 1 % level, and by * at the 5 % level. -
Activity of serum from lactating rats 7 days after parturition (exp. 7) Table 2 shows that the degree of alveolar development was (significantly at the 5 % level) lower using serum from lactating rats than using serum from day 22 of pregnancy with the exception of serum of rat no. 6, 9 and 14 which produced an equal degree of development. The activities of sera collected on the same day differed significantly (at the 5 % level) from each other on 8.
days 5 and 6 but not on days 0, 1 and 3 of lactation. Mitotic activity and traces of vacuolization and secretion were associated with a high degree of alveolar development. 9. The
effect of fasting overnight and the number of young
on
the
mammotrophic activity (exp. 8) Table 3 shows that no clear-cut correlation is found between the number of young and the mammotrophic activity in serum of fasted or non-fasted animals. Table 3 shows that in comparison with the serum of a rat weaned on
Table 2. The effect of serum collected on day 22 of pregnancy, day of parturition and the first 7 days of lactation. Medium supplemented with insulin; 3 days culture; No. of expiants: 11-12. Acir Rat No.
Serum collected on
day
No. of foetuses or
young
Degree development of
(grade)1)
Mitotic Vacuolization Secretion
activity
(%)2)
(%)2)
(%)2)
Pregnancy 1
5-6
100
92
92
3 2 2 3 5 3 3 6 3-4 4 2 2 5 3-4 4-5
33 18 9 10 83 17 50 100 42 83 0 0 92 67 83
17 0 0 0 75 17 0 83 25 58 0 0 50 42
67
Lactation 2 3 4 5 6 7 8 9 10 11
12 13 14 15 16
?3) ?3) 13 13 15 11 11 11 6 15 1 9 10 13 12
0 Median of semiquantitative grading. 2) % of expiants showing response indicated. 3) Number of young not recorded.
42
0 9 50 100 58 42 100 58 50 0 0 75 50 83
The effect of
Table 3. collected on day 6 of lactation from rats either non-fasted or with a varying number of young per litter. Medium supplemented with insulin; 3 days culture.
serum
fasted-overnight,
Rat No.
Degree of development
Acini: mitotic activity
(grade)1)
(%)2)
2 2 3
8 11 17 50
Non-fasted 1
2 3 4 5 6 7 8 9 10 11 12 13 14 15
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
12 9 6 12 11 12 9 3 12 12 8 9 12 12 12
3 5 6 7 8 9 10 11 12 13 15 17
12 9 12 11 12 12 12 12 12 12 12 12
3** 5**
73** 42 67* 33 17 33 88** 78**
2-3 3** 3* 2-3 3
8** 3** 5** 4** 4**
92** 75»* 100**
Fasted-overnight 16 17 18 19 20 21 22 23 24 25 26 27
17 0 33
2 2
3* 4**
73** 75**
3-4* 2
17 92** 0 8 17 83** 8
4** 2 2 2 3-4 * 2
1) Median of semiquantitative grading. 2) % of expiants showing the response indicated. Statistical significance for the difference with rat No. 1 indicated level, and by * at the 5 % level.
by
**
at the 1 %
0 of lactation (no. 1) the degree of development was significantly increased in 64 % of the sera of non-fasted rats and in 42 % of the sera of fasted rats. However, the median alveolar development and mitotic activity obtained with sera of fasted rats, nursing a standard litter of 8 or more tended to be less than those obtained for non-fasted rats (significant at 5 °/o level).
day
Table 4. collected on day 6 of lactation from rats during 9 h, or weaned for 8 h followed by 1 h nursing as compared with serum of non-weaned rats. Medium supplemented with insulin; 3 days culture; No. of expiants: 12. The effect of either weaned
serum
Rat No.
Weaned 8 h 1 2 3 4 5 6 7
Acini: mitotic activity
Degree of development
(grade)1) nursing
(grade)1)
1 It
5 4 6 5 5 5 5
100 81 100 100 100 100 100
2 3 2 2 2 2-3
17 17 25 33 25
Weaned 9 It 8 9 10 11 12 13
N on-weaned 14 15 16 17 18 19 20
4 3 4-5 3-4 4 4 4
1 0 2 0-1 1-2 1 I
•) Median value of semiquantitative grading. 2) % of expiants showing response indicated.
83 42 100 50 92 92 92
Table 5. The effect of the addition of 25 % rabbit serum on the mammotrophic activity of rat serum collected on day 6 of lactation. The effect of regular rabbit serum was studied using the serum of rats 1 to 6. The rabbit serum tested using serum of rat 7, contained anti-rat prolactin activity. Medium supplemented with insulin; 3 days culture; No. of expiants: 11-12.
Rat No.
Degree of development (grade)1) 25 % rabbit
5 4 4 3-4 3-4 3 5
serum
3 3-4 3 3 2 3 3
Mitotic
Pyknosis
activity
(%)2) 25 % rabbit
73 67 50 50 50 100
(%)2> serum
75 67 50 33 27 42 8
25 % rabbit
36 17 17 42 58 42
serum
75 100 75 82 82 100 100
') Median of semiquantitative grading. 2) % of expiants showing response indicated.
10. The
effect of weaning on different days during lactation (exp. 9)
Fig. 4 B shows that when the rats are not allowed to nurse after parturition, mammotrophic activity does not appear in the serum on day 2-5 of lactation. When the rats were weaned on day 10, the serum obtained during the fol¬ lowing 6 days showed in the culture the activity of virgin rat serum. In the serum of the non-weaned animals, activity (alveolar development and/or mitotic activity) was significantly increased in comparison with the weaned rats on day 1, 3 and 6 after weaning. When the rats were weaned on day 21, the effect was similar to that of weaning on day 10. In comparison with the serum of the weaned animals, activity (alveolar development and/or mitotic activity) was significantly in¬ creased with the serum of the nursing rats on the first, third and sixth day after weaning. 11. The
effect of al h nursing period after weaning for 8 h (exp. 10)
Table 4 shows that in the weaned rats the effect of the serum on the degree of development and on the mitotic activity was low as compared with both other groups. Weaning for 8 h followed by nursing for 1 h invariably
resulted in a marked positive effect on the degree of development and on the mitotic activity. Mammotrophic activity was significantly higher than after weaning. Traces of vacuolization and secretion occurred more frequently. 12. The
effect of rabbit anti-rat prolactin
serum on
the
mammotrophic
activity (exp. 11) Regular rabbit
serum or rabbit serum containing antibodies against rat pro¬ lactin was added to the medium in a concentration of 25 %. In the presence of regular rabbit serum (no. 1-6, Table 5) pyknosis is in¬ creased, while the degree of development and the mitotic activity are depressed. Addition of rabbit serum containing anti-rat prolactin activity (no. 7, Table 5) causes a comparable high degree of pyknosis and a similar depression of the degree of development. However, the mitotic activity is suppressed much more than with the regular rabbit serum.
DISCUSSION
In rats the mammary gland is already well developed at day 13 of pregnancy indicating considerable mammotrophic activity in the serum at this period. When expiants of these glands are cultured in a medium containing 50 % rat serum with the addition of insulin only, the presence of mammotrophic activity in the serum can be detected by grading a number of histological endpoints. If serum of virgin rats is added, the gland returns to a level of development comparable to that of virgin rats. If, however, serum is obtained from rats on day 10 to 21 of pregnancy or on certain days during lactation, the levels of development and cytoplasmic opalescence are maintained, a high mitotic activity is seen and traces of secretion and vacuolization are apparent. This indicates that serum of pregnant rats contains mammotrophic activity since it supports development and maintains in the alveoli the morphological differentiation of the acinar cells. The first effect could be confirmed by the uptake of labelled thymidine. The second effect is suggested by the traces of eosinophilic material and the opalescence, apparently caused by the presence of many small lipid droplets, which are removed by the routine histological procedure leaving tiny vacuoles. Large fat droplets leave large vacuoles (vacuolization). The mammotrophic activity of sera collected at various stages of the oestrous cycle did not vary to any great extent. There was a slight peak in activity during metoestrous, which did not coincide with the peak in the concentration of prolactin reported for pro-oestrous and oestrous (Kwa 8c Verhofstad 1967). During pregnancy mammotrophic activity appears in the serum rather abruptly at day 8-10 of pregnancy. The appearance of this mammotrophic
activity in serum coincides with a period of rapid growth of the mammary gland in vivo (Anderson 1974). Mammotrophic activity reaches a peak or pla¬ teau around day 11-12. A second peak is present around day 19. Mammotrophic activity is still present in appreciable amounts during and shortly after par¬ turition. The time of appearance and disappearance of mammotrophic activity suggests that the placenta may be the site of production. In the rat vascularization of the implantation site develops around day 9 of pregnancy (Holmes 8c Davies 1948). The presence of mammotrophic factors in the rat placenta has been de¬ monstrated by Ray et al. (1955). Shani et al. (1970) reported that such activity was still present in the placenta of 18 days pregnant rats. The presence of mammotrophic activity in rat serum during pregnancy has previously been demonstrated by Matthies (1968), and Cohen 8c Gala (1969), using a different assay system. They failed, however, to demonstrate a high activity in sera collected on day 19. In the present study a high activity was detected during the entire period of pregnancy after day 8-10. These results agree with those reported first by Shiu et al. (1973) and Kelly et al. (1975), who describe very high concentrations of a prolactin-like factor in the serum of pregnant rats. The times of appearance and disappearance of the factor cor¬ respond with the present results, but the temporary decline of the activity from day 14 to 17, which they (Shiu et al. 1973; Kelly et al. 1975) reported, was less evident in our experiments. However, whereas these investigators used a more specific radioreceptor assay method for detecting serum levels, the assay used in the present study measures over-all activity and may be less accurate or specific. It is conceivable, that other factors have played a secondary role in the mammotrophic activity of the serum, but the main factor involved in the increase of the mammotrophic activity in the serum during pregnancy appears to be rat chorionic mammotrophin produced by the placenta. Prolactin is not likely to be the factor involved because rabbit anti-prolactin serum had no effect on the mammotrophic activity of pregnant rat serum (to be published). The mammotrophic activity in the serum varied considerably during the period of lactation. In pooled serum mammotrophic activity was consistently absent or low during the two days following parturition; it was usually present around day 4 to 6 of lactation but thereafter mammotrophic activity was ob¬ served irregularly. If individual sera collected on the same day of lactation were tested, activity was not present in all sera of the same day. Even during the period of days 4 to 6, during which activity seemed to be most consistently present, considerable individual variation in activity occurred. Some of the sera seemed comparable to the serum of virgin rats, other sera showed an activity comparable to that of the serum of pregnant rats. Intermediate activity was not only observed using pooled serum, but also using serum obtained from individual rats.
The
possibility that the presence or absence of mammotrophic activity in the of lactating rats was determined partly by the number of young per taken into consideration. Activity was not found in the serum of the was litter, rats nursing one to three young, but otherwise the presence of activity was not significantly affected by the number of young per litter. Similarly fastingovernight was not a decisive factor by itself, but when it was combined with the nursing of a standard number of young (8 or more), active serum tended to be found less frequently. Weaning immediately after parturition prevented the appearance of activity. After weaning on days 10 and 21 of lactation mammotrophic activity of the serum disappeared. After weaning on day 6 of lactation, the activity had disappeared 9 h later. The activity was restored by nursing the young for 1 h. This erratic presence of mammotrophic activity in serum during lactation corresponds best with the changes in the prolactin level in the serum of lactating rats (Tucker 1974). Although a correlation between the actual prolactin con¬ centration in the serum and the activity measured in the present system, re¬ mains to be established, the suppression of the mitotic activity by rabbit anti-rat prolactin serum suggests that the prolactin may be the main factor involved. The last experiment had to be confined to a single sample of serum because relatively large amounts of anti-rat prolactin serum are necessary in the organ culture and the anti-serum is not readily available. In another experiment the anti-rat prolactin obtained from the same source suppressed completely the proliferative effect of rat prolactin added in a concentration of 0.25 and 0.5 pg/ml to a medium containing virgin rat serum (unpublished results). In this instance the suppression could not satisfactorily be explained by a non¬ specific toxic effect of the heterologous type of serum, because the anti-serum did not suppress the mammotrophic activity of 13 and 19 days pregnant rat serum
serum.
ACKNOWLEDGMENTS The authors wish to express their gratitude to G. T. J. Rüdiger and A. Muts for tech¬ nical assistance, to Miss M. G. Smit, Miss D. M. Miltenburg, Mrs. K. J. de Lind van Wijngaarden-de Groot and Mrs. J. van Weeren-Kramer and A. Lind for the prepara¬ tion of the histological sections, Drs. J. van Marie for the autoradiography and Dr. C. van der Meer and Dr. H. G. Kwa for their professional advice. In particular the authors wish to express their gratitude for the gift of rabbit anti-rat prolactin serum.
REFERENCES Anderson R. R. In: Larson B. L. and Smith V. R., Eds. Lactation. A comprehensive treatise, Vol. /. Academic Press, New York (1974) p. 97. Brumby H. I. Se Forsyth I. A.: J. Endocr. 43 (1969) xxiii.
Cohen R. M. Se Gala R. R.: Proc. Soc. exp. Biol. (N. Y.) 132 (1969) 683. Croxton F. E.: Elementary Statistics with Applications in Medicine and the Biological Sciences, Dover, New York (1959). Elias J. J. 8c Rivera E. M.: Cancer Res. 19 (1959) 505. Forsyth I. A.: J. Dairy Res. 3 (1971) 419. Frantz A. G., Kleinberg D. L. Se Noel G. L.: Recent Progr. Hormone Res. 28 (1972) 527. Holmes R. P. Se Davies D. V.: J. Obstet. Gynaec. Brit. Cwlth 55 (1948) 583. Kelly P. A., Shiu R. P. G, Robertson M. G Se Friesen H. G.: Endocrinology 96 (1975) 1187. Kwa H. G. Se Verhofstad J.: J. Endocr. 39 (1967) 455. Mattkies D. L.: Proc. Soc. exp. Biol. (N.Y.) 127 (1968) 1126. Paul J.: Cell and Tissue Culture, Livingstone, Edinburgh (1970). Ray E. W., Averill S. G, Lyons Wm. R. Se Johnson R. E.: Endocrinology 46 (1955) 15. Shani (Mishkinsky) J., Zanbelman L., Khazen K. Se Sulman F. G.: J. Endocr. 46
(1970)
15.
Shiu R. P. G, Kelly P. A. Se Friesen H. G.: Science 180 (1973) 968. Tucker H. A. In: Larson B. L. and Smith V. R., Eds. Lactation. A comprehensive treatise, Vol. /. Academic Press, New York (1974) p. 277. Wabeke D. Se van Eeden G: Handleiding voor de Toets van Wilcoxon, Stichting Mathematisch Centrum, Amsterdam (1970). Received
on
November 4th, 1975.
MAMMOTROPHIC ACTIVITY IN RAT SERUM DURING THE OESTROUS CYCLE, PREGNANCY AND LACTATION
By
J.
M. Peters, I. E.
Uyldert
and A. Th. Ari\l=e"\ns
ABSTRACT The mammotrophic activity of various serum samples was assessed on the mammary gland of pregnant rats, in organ culture. Insulin was added to the medium. Serum samples from virgin rats showed little activity, and variation in mammotrophic activity during the oestrous cycle was slight. During pregnancy a significant increase in proliferative activity was not seen during the first week, but around day 8\p=n-\9mammotrophic activity increased sharply. The activity showed maxima around days 12 and 19. A decrease in the activity between these maxima was not consistently observed. Mammotrophic activity was still present in sera collected during parturition, and 30 min after parturition. It had disappeared completely within 24 h. The mammotrophic factor detectable by organ culture in the serum of pregnant rats could be rat chorionic mammo-
trophin. Activity in the serum on day 0 and 1 of lactation was comparable to that of virgin rats, but some activity appeared on day 2. High activities were found frequently around day 4 to 6 of lactation. On other days the activity showed fluctuations without a definite pattern. Litter size was of minor importance but the combination of a large litter size and fasting-overnight seemed to suppress the presence of mammotrophic activity in the serum. Nursing was important: after weaning the activity has disappeared, while renewed nursing after weaning resulted in the appearance of high levels of activity. The mitotic activity obtained with lactating rat serum in the culture was suppressed by addiPart of this work has been Amsterdam, April, 1972.
presented
at the 13th annual "Federatieve
Vergadering",
tion of rabbit anti-rat prolactin serum to the medium. This suggests that the main mammotrophic factor detectable by organ culture in the serum of lactating rats, is prolactin.
Culture of the mammary
gland in vitro can be used to detect mammotrophic in serum activity samples (Forsyth 1971). In this way activity has been de¬ monstrated in the plasma of pregnant goats (Brumby 8c Forsyth 1969) and in various types of human plasma (Frantz et al. 1972). In this study the mammo¬ trophic activity present in rat serum is assayed using the mammary gland of pregnant rats in organ culture. Rat serum was collected during the oestrous cycle, during pregnancy, and during lactation. MATERIALS AND METHODS Serum samples. Serum was obtained from nulliparous, adult female albino rats (random bred, spf, Cpb: WU (WI) strain, TNO Centraal Proefdieren Bedrijf, Zeist, The Netherlands) of about 200 g in weight. They were routinely fasted overnight and exsanguinated late in the morning under light ether anaesthesia, via the right -
artery under sterile conditions. The blood was allowed to clot at room tem¬ perature for 1 h and then centrifuged for 20 min (1400 g); the serum was separated carotid
centrifuged for another 5 min (1400 g). The final samples were stored at -20°C. Samples contaminated by haemolysis were discarded. Before use each serum sample was tested for sterility. Oestrous cycle. Sera were collected during dioestrus (few nucleated epithelial cells and some leucocytes in the vaginal smear), pro-oestrus (many nucleated cells, some cornified cells), oestrus (few nucleated cells, many cornified cells) and metoestrus (many leucocytes, few cornified cells). Pregnancy. The day on which the vaginal smear showed the presence of sperma¬ tozoa, was taken as day 0 of pregnancy. After collection of the sera, the animals were autopsied to verify pregnancy. Lactation. The rats were allowed to nurse their offspring after parturition (= day 0 of lactation). The offspring were removed just before exsanguination. and
-
-
-
»
Medium and hormones The culture medium consisted of 50 % serum (v/v) and 50 % chemically defined medium (t8), a simplified formula adapted from Trowell's T8 (Paul 1970), i. e. without amino acids, vitamins and phenol red. The final glucose concentration of the medium was approximately 2 mg/ml. No antibiotics were used. Insulin (I; bovine; Organon, Oss, The Netherlands; 25.7 IU/mg; 1 mg/ml aqua dest.; membrane filter sterilized; stored at -20°C) was added to the medium in a concentration of 50 pg/ml.
Culture The on
sera were
added to the medium and the mammotrophic activity was tested size: 2x2x1 mm) obtained from the right inguinal mam-
expiants (approximate
of 13 day pregnant rats. The technique was adapted from Trowell (Paul Three expiants were supported on a stainless steel grid and a piece of nylon gauze in such a way that they remained on the surface of 1 ml medium. The watchglass culture vessel was covered by a glass plate, but this did not exclude the pos¬ sibility of gas exchange. The vessels were stored in an incubator at 37°C in a humidi¬ fied atmosphere (relative humidity about 100 %). The incubator (0.19 m3) was flushed with a mixture of 95 % Og and 5 % C02 (20 1/h). When the medium was changed after 3 days, the nylon gauze supporting the 3 expiants was transferred to fresh medium. This was done with the 6 days cultures.
mary
1970).
gland
Histology At the end of the experiments the expiants were fixed in Bouin solution. Two series of sections were made, of the central part of each expiant and stained with haematoxylin and phloxin. The results were evaluated by a procedure adapted from Elias Se Rivera (1959). One section was chosen at random and evaluated blind by the same person (first author) as to the presence and degree of certain characteristics. A grading system was used, 0 indicating absence, 1 slight degree and so on. The following parameters were evaluated at 100 x magnification: a) degree of alveolar development (graded 1-8); b) cytoplasmic opalescence (i.e. the cytoplasm stains very lightly and the cells appear to be empty except for thin thread-like structures while the nucleus/ cytoplasm ratio of these well-developed cells is low; 0-3); c) vacuolization (0-5); d) eosinophilic material (secretion; 0-5) in lumen of acini and ducts, and e) presence of nuclear remnants in the eosinophilic material; f) development of the ductal epi¬ thelium (1-3); g) presence of necrosis; at 400 x magnification: h) frequency of mitotic figures (0-5) and i) pyknosis were noted. The median values and percentages of the expiants showing a given characteristic are reported. This is restricted, however, to those parameters relevant to the experiments and conclusions. Differences between gradings were tested for statistical significance with the distribution-free test of Wilcoxon (Wabeke Se van Eeden 1970). Differences between incidences were tested with the chi-square test with Yates' correction (Croxton 1959), differences between means by Student's i-test. A level of significance of 5 or 1 % was chosen.
Labelling
index
(exp. 5). [3H]thymidine (5 pCilml, Radiochemical Centre, Amersham, England) was added 3V2 h before the end of the experiment. The mammary gland expiants were fixed in Bouin solution, dehydrated and embedded in paraffin. Serial sections (6 pm) were cut transversely. The slides were dipped in Kodak NTB-2 liquid emulsion and stored at 4°C for a period of 5 days. The exposed autoradiographs were developed for 4 min at 15°C in Kodak D19 solution, washed in aqua dest. and fixed for 5 min in Kodak F5 fixative. The autoradiographs were stained with haematoxylin and phloxin. The percentage of labelled epithelial cells was determined for 6 different expiants. Two sections of each expiant were selected at random. Labelled nuclei were counted (x 300) and the slides micro-photographed. The photographs were printed on Agfa TP6 at a final magnification of x 500. The number of nuclei counted per expiant amounted to
approximately
1500.
Experiments The following serum samples were added to the culture medium in a concentration of 50% and their effect on the 13 days pregnant rat mammary gland in vitro studied:
Exp. 1: Sera of virgin rats, collected at 4 different stages of the oestrous Serum collected from 4 rats at a certain stage of the cycle was pooled and 3 days culture with mammary glands of 6 rats.
cycle. assayed;
-
Exp. 2: Sera of pregnant rats, collected each day during pregnancy and 2 days post-partum. Serum from 1 or 2 rats per day of pregnancy was tested; 3 and 6 days cultures, each with 5 mammary glands. Exp. 3: Serum collected from 9, 13, 15 and 19 days pregnant rats. Sera from pregnant rats were assayed individually. They were added to the medium at 1.6, 3.1, 6.3, 12.5 and 25 %. Pooled serum of virgin rats was added to a final percentage of -
-
days culture on 4 mammary glands. of virgin rats, 19 days pregnant rats and sera collected during and Exp. after parturition. The sera were assayed individually. The rats were not fasted overnight. In the case of the rats exsanguinated during parturition, the first rat had de¬
50%
serum; 3
4: Serum
-
livered 9 young out of 10, the second 2 out of 9, the third 2 out of 16 and the fourth 3 out of 13. The rat, which was exsanguinated 30 min after the end of parturition, had delivered 14 young. Two rats which had delivered during the night and were exsanguinated at midday, had had 7 young each. Serum of 3 individual virgin rats served as control; 3 days culture on 3 mammary glands.
Exp. 5: Sera of virgin rats and of 13 and 19 days pregnant rats. Sera were as¬ sayed in a 3 days culture. [3H]thymidine was added at the end of the culture; 1 mam¬ mary gland was used. -
Morphological changes
Fig. 1. gland
in the mammary
culture:
of
a
13
days pregnant
rat in organ
Appearance of the gland at the beginning of the culture. The alveoli are welldeveloped (grade 4). A few isolated vacuoles are noticeable but no traces of secretion. The acinar lumina are very small. The epithelial lining of the duct is well-developed (grade 2). Scale: 100 pm. b Detail of Fig. 1 a. The well-developed acinar cells are translucent and the cytoplasm has the appearance of foam (cytoplasmic opalescence: grade 2). Scale: 25 pm. c Mammary gland expiant after a 3 days culture in a medium containing 50 % mammotrophic-inactive serum (day 0 of pregnancy) with insulin supplementation. Poor development of the alveoli (grade 2). Connective tissue is prominent. The epithelial lining of the ducts is poorly developed (grade 1). Scale: 100 pm. d Detail of Fig. 1 c. The cells are poorly developed without opalescence. The nucleus/ cytoplasm ratio appears to be high. Some pyknotic nuclei are noticeable. The acinar a
lumina
small and empty. Scale: 25 pm. e Mammary gland expiant after a 3 days culture in a medium containing 50 % mammotrophic-active serum (day 13 of pregnancy) with insulin supplementation. The alveoli are very well developed (grade 7). The upper part of the transverse section through the expiant shows some traces of vacuolization, indicated by an arrow. Secre¬ tion, however, is not prominent. Scale: 100 pm. are
f Detail of Fig.
1
e.
The cells with
cytoplasmic opalescence are very similar in appear¬ (Fig. 1 b). The lumina are small. Vacuolization
to those of the starting material is not prominent. Scale: 25 pm. ance
Fig. 1 a-f.
Exp. 6: Serum collected on days 19, 20 and 21 of pregnancy and on 21 days of lactation following parturition. Serum of virgin rats served as control. Serum col¬ lected from 2-3 rats on a certain day of lactation, was pooled and assayed; 3 days culture with 4 mammary glands. -
Exp. 7: Serum collected on day 22 of pregnancy and days 0 to 7 of lactation. Sera were collected from non-fasted rats and assayed individually in a 3 days culture with 4 mammary glands.
—
Exp.
Serum collected
8:
on
day
6
of lactation from
rats
with
varying numbers of
series samples of 15 non-fasted rats, nursing 3 to 17 young were assayed individually; in another series samples of 12 rats, fasted overnight and nursing 0 to 14 young were assayed individually; 3 days culture with 4 mammary glands.
In
young.
one
-
Exp. 9: Serum collected on 5-6 days following weaning on days 0, 10 or 21 of lactation. Sera of non-weaned rats served as control. Serum collected from 2-4 rats on a certain day after weaning was pooled and assayed in a 3 days culture; 4 mam¬ -
mary
glands.
Exp. 10: Serum collected from rats either weaned for 9 h, or weaned for 8 h fol¬ lowed by 1 h nursing, as well as serum collected from non-weaned rats. Serum was collected on day 6 of lactation from non-fasted rats with 8 young and assayed in¬ dividually in a 3 days culture; 4 mammary glands. -
Exp.
rabbit
11:
Effect of the addition of rabbit anti-rat prolactin Serum
serum
and inactive
day 6 of lactation and assayed individually in a 3 days culture; 4 mammary glands. Final glucose concentration: 2 mg/ml. The sera were tested simultaneously in a medium in which 25 % rabbit serum was present, replacing t8. The serum of the first 6 rats was tested in the pre¬ serum.
was
collected from 7 rats
on
-
sence
of
obtained rabbit serum (membrane filter sterilized, Flow Labora¬ serum of the 7th rat was tested in the presence of rabbit serum (preparation Dr. H. G.Kwa, The Netherlands Cancer Institute,
commercially
tories, Irvine, Scotland), the anti-rat
prolactin
Amsterdam, The Netherlands).
RESULTS 1. The mammary
gland of 13 days pregnant rats (Fig.
1 a,
b)
At the time of explantation the alveoli are fairly well developed (grade 4-5), with a well-developed epithelial lining of the ducts (grade 1-2). The cytoplasm of the acini is opalescent. Traces of secretion or vacuolization, and mitotic
figures
are
infrequent.
Mammotrophic activity during the oestrous cycle (exp. 1) The in vitro response of the expiants to the sera collected at different stages of the oestrous cycle showed a low degree of alveolar development (3-4), mitotic activity in only 17 to 33 % of the explants (n 15 to 18), no vacuoliza¬ tion, and traces of eosinophilic material in the lumen in 6 to 33 % of the 2.
=
explants. The differences between the sera were not significant. Cytoplasmic opalescence occurred less with dioestrous-serum (6 %, n=18) than with metoestrous serum (47 %, n= 15, difference significant at 5 % level). 3.
Mammotrophic activity during pregnancy (exp. 2)
If mammary expiants were incubated with rat serum taken from day 10 to 21 pregnancy for 3 days, a high degree of alveolar development, numerous mitoses in acini and ducts, a well-developed ductal epithelium, acinar opales¬ cence (Fig. 1 e, f) and traces of vacuolization and secretion (Fig. 1 e, arrow) were
Fig. 2. collected on different days during pregnancy and lactation (lact.) on rat mammary gland in vitro in a 3 days (left) and 6 days culture (right). Insulin was added to the medium, which contained 50 % rat serum. Top: degree of development (median value of semiquantitative grading). Middle: % of expiants showing one or more mitotic figures in acini (-) and ducts (-). Bottom: % of expiants showing traces of secretion (-) or vacuolization (-) of the acini. Number of expiants: 15, in some cases 12, 13 or 14. P parturition.
Effect of
serum
=
Fig. 3. increasing concentrations of pregnant rat serum, replacing non-pregnant rat serum (50 %) in the medium, on rat mammary gland in vitro. Three days culture with insulin added to the medium. Top, left: % of expiants showing one or more mitotic figures in the acini, right: degree of development (median value of semiquanti¬ tative grading). Bottom, left: % of expiants showing opalescent cytoplasm of the acini, right: % of expiants showing traces of vacuolization of the acini. 9, 13, 15 and 19: day of pregnancy on which serum was collected; no of expiants: 11-12. Effect of
observed (Fig. 2). Before day 10 the activity of the serum was comparable to that of day 0 of pregnancy (Fig. 1 c, d). Degree of alveolar development and presence of mitotic activity were significantly (at 1 % level) higher on day 12 than on day 0, but the differences between day 12 and day 19 were non¬ significant. After parturition the activity had virtually disappeared on day 1 of lactation and alveolar development and mitotic activity were significantly less than with serum from day 19 of pregnancy (at 1 % level). After a 6 days culture alveolar development seemed generally less well maintained and the mitotic activity had decreased (Fig. 2). Again the main mammotrophic activity was found in the sera collected from day 10 to day 21 of pregnancy. Activity
during this period: alveolar development with 12 days serum high comparison with 10 days serum (significant at 1 % level); 14 days showed less activity than either 12 days or 19 days serum in respect to the incidence of mitotic activity, secretion or vacuolization (significant at 5 % level).
was
not constant
was
in
Table 1. The effect of sera collected from 5 19 days pregnant rats, from 4 rats during parturi¬ tion, from 3 rats after parturition (No. 1: 30 min after, Nos. 2 and 3: parturition during the night, bled at midday) and from 4 virgin rats on rat mammary gland in vitro (exp. 4). Three days culture with insulin added to the medium, which contained 50 % serum.
Serum and No. of rats
Nineteen 1
2 3 4 5
(grade)1)
activity
Mitotic
Degree of development
Acini
Ducts
(%)2) (grade)1) (%)2)
Cytoplasmic opalescence
(%)2)
Vacuolization Secretion
(%)2)
(%)2)
11
33 33 11 0 44
days pregnant 5 5 5 5 5
100 100 100 100 100
2 2 3 2 2
67 67 67 67 56
100 100 100 100 100
22 22 0
89 100 100 78
56 89 89 56
67 100 100 78
0 11 0 0
100 33
67 0 22
89
33
During parturition 1 2 3 4
4 5 5 4
22 22 0 44
After parturition 1 2 3
4 3 3
11
11 0
Virgin 1
2 3 4
11 22 0 0
22 22 0 11
0 Median value of semiquantitative grading of response indicated. 2) % of expiants showing response indicated. No. of expiants: 9 per group.
0 0 11 0
Dose-effect relationship for pregnant rat serum (exp. 3) Judging from the alveolar development, mitotic activity, cytoplasmic opales¬ cence and vacuolization (Fig. 3), the highest activity was found in serum on day 19 of pregnancy, the lowest on day 9 and intermediate activities on days 13 and 15. The results indicate that the activity on day 19 was approximately 4 times that observed on day 13 or 15, while the activity on day 13 was ap¬ proximately twice that on day 9. 4.
Activity of serum collected during and shortly after parturition (exp. 4) In 4 rats, exsanguinated during parturition, the following parameters were significantly increased in comparison with serum of virgin rats: development, mitotic activity and cytoplasmic opalescence (at 5 % levels). In 2 rats (no. 2 and 3) the results were comparable with those obtained with sera of 19 days pregnant rats (Table 1). Activity was still present in the serum of a rat 30 min after the end of parturition. The activity of the sera of 2 rats which had delivered during the night and had been exsanguinated at midday was equal to that of virgin rats. 5.
Labelling index with serum of virgin rats and 13 days and 19 days rats (exp. 5) The epithelial cell labelling index with virgin rat serum was 1.7 ± 0.23% (x ± sem, N 6), with day 13 serum 4.2 ± 0.48% (N 6) and day 19 serum 7.6 ± 0.92 % (N 6). The differences between virgin and day 13 serum, and between day 13 and 19 serum were significant at 1 % levels. 6.
pregnant
=
=
=
Mammotrophic activity during lactation (exp. 6) degree of development was high during the last days of pregnancy, fell abruptly after parturition to rise again during the first five days of lactation. The degree of development fluctuated considerably during lactation (Fig. 4 A). It was significantly increased at the 5 % level, as compared with serum of virgin rats, except on days 0, 1, 13, 16 and 19 of lactation. Taking the entire period of lactation the degree of development with these sera was significantly (at the 1 % level) lower than with serum of day 19 of pregnancy. Mitotic activity showed a similar trend as the degree of development (Fig. 4 A) but in comparison to the serum of virgin rats a significant (at the 5 % level) increase was only found on days 19, 20 and 21 of pregnancy and on days 5, 6 and 8 of lactation. Opalescence of the cytoplasm and traces of secretion were seen only with serum from day 2 to 8 of lactation but to a lower extent than with serum taken on days 19, 20 and 21 of pregnancy. Traces of vacuolization were seen with serum from days 19, 20 and 21 of pregnancy, but not during 7.
The
lactation.
Fig. 4A,B. A. The effect of serum collected on day 19, 20 and 21 of pregnancy (-) and on 21 consecutive days of lactation (-). Medium supplemented with insulin; 3 days culture; no. of expiants: 11-12. The arrows indicate the effect of virgin rat serum. B. The effect of serum collected on day 19, 20 and 21 (-) and on day 0-5 of lacta¬ tion. Serum of nursing, non-weaned rats: (non-w); serum of rats weaned imme¬ diately following parturition:-(w). Medium supplemented with insulin; 3 days culture; no. of expiants: 11-12. The arrows indicate the effect of virgin rat serum. Statistical significance for the difference between nursing, non-weaned rats and weaned rats is indicated by ''"' at the 1 % level, and by * at the 5 % level. -
Activity of serum from lactating rats 7 days after parturition (exp. 7) Table 2 shows that the degree of alveolar development was (significantly at the 5 % level) lower using serum from lactating rats than using serum from day 22 of pregnancy with the exception of serum of rat no. 6, 9 and 14 which produced an equal degree of development. The activities of sera collected on the same day differed significantly (at the 5 % level) from each other on 8.
days 5 and 6 but not on days 0, 1 and 3 of lactation. Mitotic activity and traces of vacuolization and secretion were associated with a high degree of alveolar development. 9. The
effect of fasting overnight and the number of young
on
the
mammotrophic activity (exp. 8) Table 3 shows that no clear-cut correlation is found between the number of young and the mammotrophic activity in serum of fasted or non-fasted animals. Table 3 shows that in comparison with the serum of a rat weaned on
Table 2. The effect of serum collected on day 22 of pregnancy, day of parturition and the first 7 days of lactation. Medium supplemented with insulin; 3 days culture; No. of expiants: 11-12. Acir Rat No.
Serum collected on
day
No. of foetuses or
young
Degree development of
(grade)1)
Mitotic Vacuolization Secretion
activity
(%)2)
(%)2)
(%)2)
Pregnancy 1
5-6
100
92
92
3 2 2 3 5 3 3 6 3-4 4 2 2 5 3-4 4-5
33 18 9 10 83 17 50 100 42 83 0 0 92 67 83
17 0 0 0 75 17 0 83 25 58 0 0 50 42
67
Lactation 2 3 4 5 6 7 8 9 10 11
12 13 14 15 16
?3) ?3) 13 13 15 11 11 11 6 15 1 9 10 13 12
0 Median of semiquantitative grading. 2) % of expiants showing response indicated. 3) Number of young not recorded.
42
0 9 50 100 58 42 100 58 50 0 0 75 50 83
The effect of
Table 3. collected on day 6 of lactation from rats either non-fasted or with a varying number of young per litter. Medium supplemented with insulin; 3 days culture.
serum
fasted-overnight,
Rat No.
Degree of development
Acini: mitotic activity
(grade)1)
(%)2)
2 2 3
8 11 17 50
Non-fasted 1
2 3 4 5 6 7 8 9 10 11 12 13 14 15
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
12 9 6 12 11 12 9 3 12 12 8 9 12 12 12
3 5 6 7 8 9 10 11 12 13 15 17
12 9 12 11 12 12 12 12 12 12 12 12
3** 5**
73** 42 67* 33 17 33 88** 78**
2-3 3** 3* 2-3 3
8** 3** 5** 4** 4**
92** 75»* 100**
Fasted-overnight 16 17 18 19 20 21 22 23 24 25 26 27
17 0 33
2 2
3* 4**
73** 75**
3-4* 2
17 92** 0 8 17 83** 8
4** 2 2 2 3-4 * 2
1) Median of semiquantitative grading. 2) % of expiants showing the response indicated. Statistical significance for the difference with rat No. 1 indicated level, and by * at the 5 % level.
by
**
at the 1 %
0 of lactation (no. 1) the degree of development was significantly increased in 64 % of the sera of non-fasted rats and in 42 % of the sera of fasted rats. However, the median alveolar development and mitotic activity obtained with sera of fasted rats, nursing a standard litter of 8 or more tended to be less than those obtained for non-fasted rats (significant at 5 °/o level).
day
Table 4. collected on day 6 of lactation from rats during 9 h, or weaned for 8 h followed by 1 h nursing as compared with serum of non-weaned rats. Medium supplemented with insulin; 3 days culture; No. of expiants: 12. The effect of either weaned
serum
Rat No.
Weaned 8 h 1 2 3 4 5 6 7
Acini: mitotic activity
Degree of development
(grade)1) nursing
(grade)1)
1 It
5 4 6 5 5 5 5
100 81 100 100 100 100 100
2 3 2 2 2 2-3
17 17 25 33 25
Weaned 9 It 8 9 10 11 12 13
N on-weaned 14 15 16 17 18 19 20
4 3 4-5 3-4 4 4 4
1 0 2 0-1 1-2 1 I
•) Median value of semiquantitative grading. 2) % of expiants showing response indicated.
83 42 100 50 92 92 92
Table 5. The effect of the addition of 25 % rabbit serum on the mammotrophic activity of rat serum collected on day 6 of lactation. The effect of regular rabbit serum was studied using the serum of rats 1 to 6. The rabbit serum tested using serum of rat 7, contained anti-rat prolactin activity. Medium supplemented with insulin; 3 days culture; No. of expiants: 11-12.
Rat No.
Degree of development (grade)1) 25 % rabbit
5 4 4 3-4 3-4 3 5
serum
3 3-4 3 3 2 3 3
Mitotic
Pyknosis
activity
(%)2) 25 % rabbit
73 67 50 50 50 100
(%)2> serum
75 67 50 33 27 42 8
25 % rabbit
36 17 17 42 58 42
serum
75 100 75 82 82 100 100
') Median of semiquantitative grading. 2) % of expiants showing response indicated.
10. The
effect of weaning on different days during lactation (exp. 9)
Fig. 4 B shows that when the rats are not allowed to nurse after parturition, mammotrophic activity does not appear in the serum on day 2-5 of lactation. When the rats were weaned on day 10, the serum obtained during the fol¬ lowing 6 days showed in the culture the activity of virgin rat serum. In the serum of the non-weaned animals, activity (alveolar development and/or mitotic activity) was significantly increased in comparison with the weaned rats on day 1, 3 and 6 after weaning. When the rats were weaned on day 21, the effect was similar to that of weaning on day 10. In comparison with the serum of the weaned animals, activity (alveolar development and/or mitotic activity) was significantly in¬ creased with the serum of the nursing rats on the first, third and sixth day after weaning. 11. The
effect of al h nursing period after weaning for 8 h (exp. 10)
Table 4 shows that in the weaned rats the effect of the serum on the degree of development and on the mitotic activity was low as compared with both other groups. Weaning for 8 h followed by nursing for 1 h invariably
resulted in a marked positive effect on the degree of development and on the mitotic activity. Mammotrophic activity was significantly higher than after weaning. Traces of vacuolization and secretion occurred more frequently. 12. The
effect of rabbit anti-rat prolactin
serum on
the
mammotrophic
activity (exp. 11) Regular rabbit
serum or rabbit serum containing antibodies against rat pro¬ lactin was added to the medium in a concentration of 25 %. In the presence of regular rabbit serum (no. 1-6, Table 5) pyknosis is in¬ creased, while the degree of development and the mitotic activity are depressed. Addition of rabbit serum containing anti-rat prolactin activity (no. 7, Table 5) causes a comparable high degree of pyknosis and a similar depression of the degree of development. However, the mitotic activity is suppressed much more than with the regular rabbit serum.
DISCUSSION
In rats the mammary gland is already well developed at day 13 of pregnancy indicating considerable mammotrophic activity in the serum at this period. When expiants of these glands are cultured in a medium containing 50 % rat serum with the addition of insulin only, the presence of mammotrophic activity in the serum can be detected by grading a number of histological endpoints. If serum of virgin rats is added, the gland returns to a level of development comparable to that of virgin rats. If, however, serum is obtained from rats on day 10 to 21 of pregnancy or on certain days during lactation, the levels of development and cytoplasmic opalescence are maintained, a high mitotic activity is seen and traces of secretion and vacuolization are apparent. This indicates that serum of pregnant rats contains mammotrophic activity since it supports development and maintains in the alveoli the morphological differentiation of the acinar cells. The first effect could be confirmed by the uptake of labelled thymidine. The second effect is suggested by the traces of eosinophilic material and the opalescence, apparently caused by the presence of many small lipid droplets, which are removed by the routine histological procedure leaving tiny vacuoles. Large fat droplets leave large vacuoles (vacuolization). The mammotrophic activity of sera collected at various stages of the oestrous cycle did not vary to any great extent. There was a slight peak in activity during metoestrous, which did not coincide with the peak in the concentration of prolactin reported for pro-oestrous and oestrous (Kwa 8c Verhofstad 1967). During pregnancy mammotrophic activity appears in the serum rather abruptly at day 8-10 of pregnancy. The appearance of this mammotrophic
activity in serum coincides with a period of rapid growth of the mammary gland in vivo (Anderson 1974). Mammotrophic activity reaches a peak or pla¬ teau around day 11-12. A second peak is present around day 19. Mammotrophic activity is still present in appreciable amounts during and shortly after par¬ turition. The time of appearance and disappearance of mammotrophic activity suggests that the placenta may be the site of production. In the rat vascularization of the implantation site develops around day 9 of pregnancy (Holmes 8c Davies 1948). The presence of mammotrophic factors in the rat placenta has been de¬ monstrated by Ray et al. (1955). Shani et al. (1970) reported that such activity was still present in the placenta of 18 days pregnant rats. The presence of mammotrophic activity in rat serum during pregnancy has previously been demonstrated by Matthies (1968), and Cohen 8c Gala (1969), using a different assay system. They failed, however, to demonstrate a high activity in sera collected on day 19. In the present study a high activity was detected during the entire period of pregnancy after day 8-10. These results agree with those reported first by Shiu et al. (1973) and Kelly et al. (1975), who describe very high concentrations of a prolactin-like factor in the serum of pregnant rats. The times of appearance and disappearance of the factor cor¬ respond with the present results, but the temporary decline of the activity from day 14 to 17, which they (Shiu et al. 1973; Kelly et al. 1975) reported, was less evident in our experiments. However, whereas these investigators used a more specific radioreceptor assay method for detecting serum levels, the assay used in the present study measures over-all activity and may be less accurate or specific. It is conceivable, that other factors have played a secondary role in the mammotrophic activity of the serum, but the main factor involved in the increase of the mammotrophic activity in the serum during pregnancy appears to be rat chorionic mammotrophin produced by the placenta. Prolactin is not likely to be the factor involved because rabbit anti-prolactin serum had no effect on the mammotrophic activity of pregnant rat serum (to be published). The mammotrophic activity in the serum varied considerably during the period of lactation. In pooled serum mammotrophic activity was consistently absent or low during the two days following parturition; it was usually present around day 4 to 6 of lactation but thereafter mammotrophic activity was ob¬ served irregularly. If individual sera collected on the same day of lactation were tested, activity was not present in all sera of the same day. Even during the period of days 4 to 6, during which activity seemed to be most consistently present, considerable individual variation in activity occurred. Some of the sera seemed comparable to the serum of virgin rats, other sera showed an activity comparable to that of the serum of pregnant rats. Intermediate activity was not only observed using pooled serum, but also using serum obtained from individual rats.
The
possibility that the presence or absence of mammotrophic activity in the of lactating rats was determined partly by the number of young per taken into consideration. Activity was not found in the serum of the was litter, rats nursing one to three young, but otherwise the presence of activity was not significantly affected by the number of young per litter. Similarly fastingovernight was not a decisive factor by itself, but when it was combined with the nursing of a standard number of young (8 or more), active serum tended to be found less frequently. Weaning immediately after parturition prevented the appearance of activity. After weaning on days 10 and 21 of lactation mammotrophic activity of the serum disappeared. After weaning on day 6 of lactation, the activity had disappeared 9 h later. The activity was restored by nursing the young for 1 h. This erratic presence of mammotrophic activity in serum during lactation corresponds best with the changes in the prolactin level in the serum of lactating rats (Tucker 1974). Although a correlation between the actual prolactin con¬ centration in the serum and the activity measured in the present system, re¬ mains to be established, the suppression of the mitotic activity by rabbit anti-rat prolactin serum suggests that the prolactin may be the main factor involved. The last experiment had to be confined to a single sample of serum because relatively large amounts of anti-rat prolactin serum are necessary in the organ culture and the anti-serum is not readily available. In another experiment the anti-rat prolactin obtained from the same source suppressed completely the proliferative effect of rat prolactin added in a concentration of 0.25 and 0.5 pg/ml to a medium containing virgin rat serum (unpublished results). In this instance the suppression could not satisfactorily be explained by a non¬ specific toxic effect of the heterologous type of serum, because the anti-serum did not suppress the mammotrophic activity of 13 and 19 days pregnant rat serum
serum.
ACKNOWLEDGMENTS The authors wish to express their gratitude to G. T. J. Rüdiger and A. Muts for tech¬ nical assistance, to Miss M. G. Smit, Miss D. M. Miltenburg, Mrs. K. J. de Lind van Wijngaarden-de Groot and Mrs. J. van Weeren-Kramer and A. Lind for the prepara¬ tion of the histological sections, Drs. J. van Marie for the autoradiography and Dr. C. van der Meer and Dr. H. G. Kwa for their professional advice. In particular the authors wish to express their gratitude for the gift of rabbit anti-rat prolactin serum.
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on
November 4th, 1975.
