Comp. Biochem. Physiol., 1976, [,Iol. 55B, pp. 109 to 115. Pergamon Press. Printed in Great Britain

DEHYDROGENASES, NUCLEIC ACIDS AND SOLUBLE PROTEINS IN MAMMARY GLAND OF THE MONGOLIAN GERBIL DURING PREGNANCY, LACTATION AND INVOLUTION KERSTIN H. C. WALDEMARSONAND BORJE W. KARLSSON Institute of Zoophysiology, University of Lund, Lurid, Sweden

(Received 31 Ocwber 1975) Abstract--1. The activities of lactate dehydrogenase (LDH), malate dehydrogenase (MDH), glucose-6phosphate dehydrogenase (G-6-PDH) and glycerol-3-phosphate dehydrogenase (G-3-PDH), as well as the isoenzymes of LDH and the two molecular forms of MDH have been assayed in mammary tissue of the Mongolian gerbil, Meriones u~uiculatus, during pregnancy, lactation and involution. In addition, the amounts of DNA, RNA and soluble proteins were determined. 2. The results differed in some respects from the principal pattern seen in other small rodents. The activity of LDH dominated over the activity of MDH during all three stages, in contrast to e.g. the rat. Moreover, the distribution of the LDH isoenzymes, in contrast to the rat, showed a pattern with a pronounced dominance of LDH 5. The relative activities of S-MDH and M-MDH were almost equal during pregnancy, lactation and involution. As regards the nucleic acids, the peak value of the quotient RNA/DNA obtained during lactation was higher than the quotient from the mammary gland of the rat, mouse or hamster. 3. Mammary glands from the anterior and posterior parts of the body were analysed separately, and in general, the activities of the dehydrogenases as well as the amount of protein and RNA were higher in the anterior glands. The reason for these divergencies is not known, but may be due to differences in structure and function between the glands. 4. During late involution, there was an increase in the activities of LDH, MDH and G-3-PDH, while G-6-PDH remained at a low level. This difference was supposed to be a result of hormonal influence on the mammary tissue.

INTRODUCTION

opment in Meriones. In this report results are preNumerous biochemical studies, including e.g. sented on dehydrogenases, nucleic acids and soluble proteins during normal pregnancy, lactation and inenzymes, nucleic acids, proteins and lipids, have been volution. The dehydrogenases include L D H as a performed on the mammary tissue of several mammalian species (reviewed by Reynolds & Folley, 1969; representative of anaerobic glycolysis, M D H representing citric acid cycle activity, glycerol-3-phosphate Cowie & Tindal, 1971; Falconer, 1971; Carlsson, 1972). The studies on domestic animals are generally dehydrogenase, G-3-PDH, (EC 1.1.1.8) representing fat metabolism and glucose-6-phosphate dehydrogenmotivated from a practical point of view while studies ase, G-6-PDH, (EC 1.1.1.49) representing pentoseon laboratory animals such as rat, mouse, rabbit and shunt activity. Moreover, the isoenzymes of L D H and guinea-pig are of particular interest to investigators concerned with the basic processes of differentiation the two molecular forms of M D H (S-MDH and as well as synthesis and secretion of macromolecules M - M D H ) are included in the study, since these enzymes, especially LDH, can be used as indicators at the cellular level. In a study on lactate dehydrogenase, L D H (EC of the stage of differentiation (Karlsson & Carlsson, 1.1.1.27) and malate dehydrogenase, M D H , (EC 1968; Simpson & Schmidt, 1970; Richards & Hilf, 1.1.1.37) in rat, mouse, rabbit and Mongolian gerbil 1972b) and their activities may change due to hor(Meriones uncduiculatus) some preliminary data were monal influence (Richards & Hilf, 1972a, 1972b). presented on L D H and M D H in the mammary gland of the latter species (Karlsson & Larsson, 1971). As MATERIAL AND METHODS revealed by L D H / M D H quotients and by L D H and M D H isoenzyme patterns the metabolism of Mer- Experimental animals iones may differ in some respects from that of mouse, The study included a total of 63 young female Mongorat and rabbit. Of special interest is the ability of lian gerbils (Meriones unouiculatus) from the same strain Meriones to exist on low water consumption (Sch- as used by Karlsson & Larsson, 1971. The animals were wentker, 1963, 1966; Rich, 1968; Arrington & bred on a constant supply of water and dry pellets (Ewos Ammerman, 1969). It may be questioned as to how standard food for rats and mice. Each animal was kept this species solves the problem of water conservation in a separate cage after fertilization. The variations found in enzyme activities and content of DNA, RNA and produring lactation when large quantities of water are tein were not correlated to litter size. The litters generally lost via milk to the young. consisted of 4 young. The animals were killed by decapiThe first step in studying this problem comprised tation after light anaesthesia with ethyl ether. Mammary a detailed investigation of the mammary gland devel- glands from the anterior and posterior part of the body 109

110

KERSTIN H. C. WALDEMARSON AND BORJE W.

were rapidly excised and analysed separately. The mammary gland tissues were immediately frozen and stored at - 2 0 ° C until analysis. The glands were finely divided and homogenized in ice-cold 0.9~ NaCI solution (weight:volume proportions 1:10) using a glass homogenizer with a teflon pestle. The supernatants obtained after centrifugation of the homogenates at +4°C for 1 hr at 3000 x g were used for enzyme and protein assay. Mammary glands from three functionally different stages were examined; late pregnancy, lactation and involution. Involution was considered to start when the litters were removed from the female on the 21st day of lactation. RNA and DNA assay The method described by Schmidt and Tannhaiiser was used with the modification recommended by Munro & Fleck (1966). The tissue homogenates were treated with ice-cold 0.4M perchloric acid to inactivate interfering enzymes and to precipitate DNA and RNA. After alkaline hydrolysis in 0.3 M potassium hydroxide at 37°C for 1 hr, the samples were again treated with perchloric acid. The soluble digestion products of RNA were then separated from the precipitated DNA by centrifugation. The RNA in the supernatant was measured by its uv-absorbance at 260nm. Burton's diphenylamine method (1956) was used to measure the DNA content of the sample. Enzyme and isoenzyme assay The activities of LDH (lactate dehydrogenase EC 1.1.1.27), MDH (malate dehydrogenase EC 1.1.1.37), G-3-PDH (glycerol-3-phosphate dehydrogenase EC 1.1.1.8) and G-6-PDH (glucose-6-phosphate dehydrogenase EC 1.1.1.49) were determined spectrophotometrically at 340 nm by registering the rate of conversion of NAD or NADP at 30°C to NADH and NADPH respectively. The details of these determinations have been published earlier (for LDH and MDH by Karlsson & Carlsson, 1968, and for G-3-PDH and G-6-PDH by Bergelin & Karlsson, 1973). The relative isoenzyme activities for LDH and MDH were determined by electrophoresis in agarose gels (citrate phosphate buffer, pH 7.0) on microscope slides, as described by Karlsson & Larsson (1971). Evaluation of the Nitro BT stained isoenzymes was performed using a Chromoscan densitometer. An isoenzyme index (IE, ~o A-subunits) was calculated according to Karlsson & Palmer (1971). Protein and .[at content The protein content of the supernatants of the centrifuged homogenates was determined using Folin Ciocalteau reagent, according to the method described by Lowry

KARLSSON

et al. (1951). The results indicate the amounl of stllublc proteins. In order to obtain an approximate value of the lipid content, the fat obtained in the upper part of the centrifuge tubes was estimated subjectively and calculated as !'i~ of the total homogenate volume.

RESt LTS

DNA

The values for D N A expressed as mg/g tissue are somewhat higher in the anterior than in the posterior gland (Table 1). There are no significant changes in D N A content during lactation but there is a tendency for gradual decrease from pregnancy t h r o u g h o u t lactation. RNA

The R N A values expressed as mg/g tissue are presented in Table 1. The values are higher in the anterior than in the posterior gland. The content of RNA gradually increases to a m a x i m u m at the 13th 15th day of lactation in both the anterior and in the posterior glands. W h e n expressed in relation to D N A (Fig. 2) maximal values are obtained on the 16th 18th day for the anterior gland a n d on the 13th-15th day for the posterior gland. During involution the R N A content of the glands rapidly decreases a n d 50?; of the maximal values is obtained within 1 3 days. At the beginning of lactation, the R N A / D N A ratios from the two types of glands are similar. During late lactation and early involution, the values from the posterior gland are 60-70°,; of those from the anterior gland. Enzyme and isoenzyme activities

In order to obtain some correlation for uncontrollable individual and methodological variations, the enzyme activities are expressed in relation to D N A content. The four enzyme systems, L D H , M D H , G - 3 - P D H and G - 6 - P D H , behave similarly during lactation. They all increase in activity as compared to D N A after the onset of lactation and reach maximal values in the latter part of lactation. During involution, however, the enzymes behave in different ways (Fig. 1). The e n z y m e / D N A ratios are higher in the

Table I. Soluble proteins, RNA and DNA (mg/g wet wt) in anterior and posterior gerbil mammary glands during pregnancy lactation and involution. Number of individuals analysed is indicated by n. The values are means +_ S.EM. Soluble protein

Pregnancy (n} 22-24 days (21 Lactation I 3 days (11} 4 6 days (4) 7 9 days (5) 10 12 days (6) 13-15 days 14) 16~18 days 13) 19 21 days (3) Involution 1 3 days (41 4 6 days (3) 7 9 days (1) 10-12 days (4) 13-15 days (51

RNA

Anterior glands

Posterior glands

Anterior glands

~98 + 7 0

27.2 ± 13.0

6.8 + 17

50.0 57.9 60.0 629 65.6 68.0 61.0

+ 18 ± 5.7 _+ 31 + 2.7 ± 2.0 ± 3,5 ± 3.~

39.7 49,4 52.6 61.8 58.1 65.9 574

51.0 58.1 40.2 287 270

_+ 1.7 ± 2.4

449 + 50.2 ± 29.2 21.2 ± I¢~N ~

± 36 + ~

_+ 3.6 4- 4,5 ± 1.0 + 1.7 ± 5.0 + 2.9 ± 1.9 1.7 3.9 3.2 31

DNA Posterior glands

Posterior glands

468 ± 133

2.14 ± 0.74

0.5 0.4 1.0 0.9 1.5 1.4 1.3

213 + 2.74 + 2.(}7 ± 177 ± 164 + I 12 + 1.12 ±

11.22 0.16 0.21 0.12 0.28 (I.18 (tl3

203 ÷ (119 1.7/I + 0.ll 172 +_ 019 178 ± (1 t8 1.65 + 0 5 0 157 + 0,23 142 + 0 1 4

_+ 1.2 ± 0,3

1.0N ± 190 ± 2 36 I1~ + I 4q ~

(I.22 019

162 ,+ 191 + I 13 199 + (17(I •

3.9 ± 0.8

8.1 15.2 16.8 17.4 19.9 17,6 17.3

± 0.8 ± 0.8 _+ 0.8 + 0.9 + 1.3 + 1.0 ,+ 1.3

5.8 12.7 14.2 16.4 17.0 16.2 15.2

7.5 4,0 3.1 t.7 1.9

,+ I.I + 0.2

74 39 hi 14 II

± I).3 + 04

Anterior glands

+ + ± ± ± ± .±

± I).3 + l)

0.19 11~4

026 t)41 I).37 I} I(~

Dehydrogenases in mammary gland of the Mongolian gerbil 50

(o)

111

r\ 4O



(b)

Z

E 3c

i

g c

Z

E 20

[

.J

\

I/

I0

\

I

z

I

• ,o

".,,V! ',,,'___]

o

I I I I I I I I I I I I 13-15 19-21 1-3 7-9 13-15 ~2-a~1-3 7-9 L I

I

~ -~

I i I I-3 7-9

L

Days 50

I

I I I I I I I I 13-15 19-2q I-3 7-9 13-15 I

De/s

(c)

40

(d) 3

,< z

z r-t

[

E2

%.

~a . 2D

/,,,

i

//

./!

CL I rO

\/ "

1

~ -~

-3

-

L

-5

19-21 t-3

?-91

,

I

~.,@- --o

t,~ [ I I I I I ':I I I I 1 ~ - ~ t ,-3 ,-~ ~-~ ,0-,~,~-~,~-= ,~-~, ~-~ ~-~ ~-9 ,o-,~,~-,~ I L P|

13-15

Days

Days

Fig. 1. Changes in (a) LDH, (b) MDH, (c) G-6-PDH and (d) G-3-PDH in the mammary gland of the gerbil during pregnancy (P), lactation (L) and involution (I). The values are expressed as enzyme units per mg DNA and originate from the anterior ( ) and posterior (. . . . ) mammary glands and represent means of 2 11 determinations. Tabular data is available direct from the author. anterior than in the posterior gland, especially midway through lactation. LDH

The activity of L D H increases 4.5-fold in the anterior gland, and 3.5-fold in the posterior gland within 16-18 days after onset of lactation. Following a de-

crease at the beginning of involution the L D H activities again increase, at first in the anterior gland (Fig.

1). L D H isoenzymes

The predominant form of L D H isoenzymes during pregnancy, lactation and involution is L D H 5, which

112

KERSTIN

7o

H.

C.

WALDEMARSON

(o)

/

o, 4 0

E

...:7

._¢

3c

:,-z,/

\/,,/.

,

~' 20

"V

t

tO

I

KARLSSON

reaches its highest value midway through lactation constituting 92.6'!~, of the total LDH activity in the anterior gland, and 93.1,; in the posterior gland (Table 2). Within 13 15 days after onset of involution LDH 5 falls about 10'!i, in the anterior gland and about 5% in the posterior gland. During the same period LDH 4, LDH 3 and, in a few cases LDH 2, increase in activity. Isoenzyme index (i'; of subunits AI is represented in Fig. 3. It is notable that during pregnancy and early lactation the values of these isoenzyme indicies are higher in the anterior than in the posterior gland while at the end of involution the reverse is true.

6O

Z

AND B~)RJE W.

MDH )

I

t

I

I

I

1

I

I

I

L

I

-24 I-3 4-6 7-9 10-1213-1516-18 19-21 I-3 4- 6 Y-9 10-1213-15

L

I

I

Doys

(b)

\.

The increase in MDH activity during involution is not as striking as that of LDH. The activity of M D H is, with very few exceptions, lower than the activity of LDH, indicating a possible higher rate of metabolism in the glycolytic pathway than in Krebs cycle. MDH isoenzymes

/, /

z,g, l0

g Z

S-MDH and M - M D H exhibit almost equal values during the whole period of lactation. During involution, however, the mitochondrial form becomes more dominating in the anterior gland, while the two forms remain in the same relationship as during lactation in the posterior gland (Fig. 3).

\

t/I

/

Io

G-6-PDH

i

E I 1 I I I I I l l l l l 2-24 I-3 4 - 6 7-9 10-1213-1516"18 19-21 I-3 4-6 7-9 10-12 13-15 L

Of the enzyme systems examined, G-6-PDH shows the greatest increase during lactation. In the anterior gland the activity increases 15-fold within 16-18 days while in the posterior gland it increases I l-fold within 13 15 days. 4-6 days after the onset of involution the activity is of the same low level as at the beginning of lactation. There is no increase in activity during involution, in contrast to LDH, M D H and G-3-PDH (Fig. 1).

I Doys

Fig. 2. Changes in (a) soluble proteins and (b) RNA, using DNA (mg per gram tissue) as a reference, in the anterior ( - - - ) and posterior (....... ) mammary glands of the gerbil during pregnancy (P), lactation (L) and involution (I). The values represent means from 2-11 determinations.

G-3-PDH The total activity of G-3-PDH is much lower than the activities of LDH, M D H and G - 6 - P D H The G-3-PDH activity increases to a maximum within 16--18 days for both the anterior and posterior glands. The increase in the anterior gland is 17-fold and in

Table 2. Relative activities of LDH isoenzymes (LDH I - LDH 5) in anterior and posterior mammar~ glands of the gerbil during pregnancy, lactation and involution. The values are means _+ S.E.M. Number of individuals is indicated by n LDH

I

LDH 2

L[Mt 3

LDH 4

LDH 5

Anterior glands

Posterior glands

Anterior glands

Posterior glands

Anterior glands

Posterior glands

Antcrim glands

Postcrim glands

Anteriol glands

Pregnancy (n) 22 24 days (2)

0.0

0.0

(10

0.fl

5.0 ± 0 4

7,5 + 0 7

12.3 + 0 4

129 + 0 4

827 + 04

7 9 6 + 11.5

Lactation I 3 days (101 4 6 days (6) 7 9 days (5) 1G 12 days 16) 13--15 days (4) 16 Ig day's 131 19 21 days (5)

0./) 11.(] 0.0 0.0 O.0 0.0 I1f)

0.0 0.0 0.0 0.0 011 00 11./)

0It 00 ().l} 11.0 0.0 fl,(I 0.0

1111 1}.0 1t./I 0.0 00 00 0p

3,1 ± (iS 2.2 ± 0 6 2.2 ~ Ill 10±05 ~.1 ± 1.1 0(I 11.9 ± o.(,

4.1 f ll) 2.4 + 0 9 2.11 + 0 9 i f l + 0.4 x 4 ~: 1 :~ 12 "+" 0 7 21 ~ 1.4

81 +. 116 7 7 + 1/6 6.9 + 0.6 6.4±11.5 6.8 4 115 7 6 + 0N t,,6 ± I)~,

73 * 06 7 7 ± 114 6 5 z (16 59+/14 7 3 + 113 h 7 -+ 0 3 t, 6 ~ 0 ~,

Sg.7 +- I1 90 ] ~- ]l) 91.0 + 1.0 92.6+_0.7 8 9 8 4 1.7 9 2 4 + 11.8 9 2 5 + (!.S

S8 I~ f 1.5 8 9 9 ± 11 91.4 ± 1.2 93.1 + 0 . 6 89.3 ± 1.3 92.1 _+ 0.7 9 1 3 ± 19

Involution I 3 days 17~ 4 6 days 14) 10- 12 days (4} 13 15 days (51

0.0 0.0 0.0 It.1/

{).0 0.0 0.0 0.0

11.0 0.0 0.6 ± 0.3 0.7+05

0.0 0.0 00 0.0

311 ~ (1,8 3 5 ± 1.3 5. t ± 1.0 5~+07

4.:~ + (I.9 3.9 2 l.I 2.3 + I11 1.5±06

7.6 9.7 13.0 131

7s+(.4 1/I.4 ± 12 134 + 0.5 I l l , ± 15

~ 9 4 3; o.9 8 6 8 + 2.5 8 1 3 +_ 1 4 804+ 15

882±t.I 8 5 7 + 2.~ 84.3 + 0.7 8(,9+ 19

+ 0,5 + 1.3 ± 0.6 +11.7

Posterim glands

Dehydrogenases in mammary gland of the Mongolian gerbil tOO

-

,,5,.f -'~

.~

beginning of involution, the values from the anterior gland are higher than those from the posterior gland (Fig. 2).

1~)

--

ed~//

113

, ~~_-.' /- .e

Fat Because of the inexact method used, the values fluctuate but there is a tendency for the posterior gland to contain more fat than the anterior gland during involution.

9C

r~ m

DISCUSSION

g

a'

;

I I I 2-L~ I-5 4-6

I I I I I 7-910-1213-1516-1819-21

I I I I I I-3 4-6 7-910-1213-15

L

I

I~ys -x-

(b)

:SS0 o

T :Z

"6 E 40

o

"1" r~

± g I I I I I I I I I I I I I z,a-a~ I-3 4-6 7-9 IO-la I V , 16-~ 19-a~ 1-3 4-6 7-9 t0-1213-15 I P L

Doys

I

Fig. 3. Changes in (a) the percentage of A subunits of LDH (isoenzyme index) and (b) relative percentage of the mitochondrial form of MDH (M-MDH) in the anterior ( ) and posterior ( - - - - ) mammary glands of the gerbil during pregnancy (P), lactation (L) and involution (I). The values represent means from 2-11 determinations.

the posterior gland 8-fold. After onset of involution, the activity initially decreases but thereafter increases (Fig. 1). Soluble proteins The protein content of the mammary gland reaches its highest value 16-18 days after onset of lactation, and then decreases. The values from the anterior gland are higher than for the posterior gland. This is most pronounced during pregnancy and involution. However, the values from the posterior gland are never less than 60% of the values from the anterior gland (Table I). In principle the values of protein/ DNA show the same development as the values of protein/g wet tissue. At the end of lactation and the ( ~.1, 55 IB

]l

When the mammary gland of the Mongolian gerbil passes from the condition of pregnancy, through lactation to involution there are pronounced changes in the activities of LDH, MDH, G-6-PDH and G-3-PDH, and in the content of soluble protein and RNA. On the other hand, only minor changes are noted in isoenzyme patterns and DNA content. The results obtained, indicate functional, structural and metabolic alterations in the mammary gland cells, especially at the onset of lactation and involution. Similar results have been reported for many other mammals such as rat, mouse and hamster (Glock & McLean, 1954; Baldwin & Milligan, 1966; Karlsson & Carlsson, 1968; Sinah et al., 1970; Rivera & Cummins, 1972; Carlsson et al., 1973). However, the results obtained on the Mongoliam gerbil show some differences, which may depend on the unusual mode of water metabolism of this species. Comparing the amounts of nucleic acids in the mammary glands of the gerbil with those of the rat (Baldwin & Milligan, 1966; Richards & Hilt', 1972b) and mouse (Rivera & Cummins, 1972), differences are found, especially during lactation. DNA, expressed as mg/g wet tissue, is thus lower in the gerbil than in the rat and mouse, whereas the content of RNA is higher. The quotient RNA/DNA, which can be used as an index of metabolic activity in the mammary gland, is consequently higher than in the mouse and rat. Investigations on the mammary gland of the hamster also show that the RNA/DNA quotient is lower than that in the gerbil (Sinah et al., 1970). In fact, just before the onset of lactation the quotient RNA/DNA shows a value of 1-2 for both the rat, mouse, hamster and gerbil, whereas during lactation the maximal value obtained in the gerbil is considerably higher than that of the rat, mouse and hamster, their maximal values not exceeding 5. This indicates that the mammary gland of the gerbil, from a metabolic point of view, is more activated during lactation than the mammary gland of the other species mentioned. However, it must be kept in mind, that these species differences may partly depend upon the fact that various investigators have used different methods for RNA and DNA assays. The anterior glands exhibited slightly higher RNA values than the posterior ones. The reason for this might be sought in e.g. a structural disparity between the glands, with more glandular tissue in the anterior ones. It might also be due to a more effective removal of milk from the anterior glands, resulting in an increased stimulation of protein synthesis. As regards the levels of DNA, no such difference was found between the anterior and posterior glands. In both glands, the peak values appear around onset

114

KERSTIN H. C. WALDEMARSONAND BORJE W. KARLSSON

of lactation as reported in other species (Traurig, 1967; Tucker, 1969). A great problem in biochemical studies on mammary gland tissue is to obtain a reliable basis for expressing the content of DNA, RNA, protein and enzyme activities (Slater, 1962; Jones, 1967; Gul & Dils, 1969). Varying amounts of milk are retained in the tissue during lactation and early involution, and therefore the weight of the gland is an unreliable reference. In this study, no corrections for retained fluid have been made, due to difficulties in obtaining sufficiently large amounts of milk to estimate lactose content. It is true that tissue strips of the mammary gland of the gerbil were shown to contract upon addition of oxytocin in vitro, but after administration of oxytocin in vivo, the efforts of milking the animals resulted in only a few drops of milk. In choosing between expressing the enzyme activities in relation to DNA, protein or tissue weight, we preferred the first alternative, which has also been used in other investigations (Tucker & Reece, 1963; Ota, 1964: Jones, 1967; Gul & Dils, 1969). However, we are aware of the fact, that the amount of DNA per nucleus is probably not constant during the different stages of development in the gland, judging from experiments on other species (Simpson & Schmidt, 1969). The activities of the enzymes examined in the mammary gland of the gerbil showed a similar pattern as those of other small rodents during pregnancy, lactation and early involution, The enzyme activities increased after onset of lactation of the gerbil, and reached a peak value when the litters were 16-18 days old. At this time the young had opened their eyes and started to gnaw on pellets, thus beginning to use another source of nutrition than the secretion of the mammary glands of the dams. During late involution, there were some interesting results on the activities of the enzymes LDH, MDH and G-3-PDH. About one week after the beginning of involution an increase was noted in the activities of these enzymes, most pronounced for LDH. Contrary to this, G-6-PDH remained at a constant low level throughout involution. These findings confirm the close relationship between secretion from mammary glands and increasing G-6-PDH activity as also found by Glock & McLean (1954) in their investigation on lactating rats. These differences in enzyme activities can hardly be explained as being due to new metabolic needs arising during late involution. One possible explanation is that the enzymes react in different ways to hormonal influence. Ovulation occurs in the gerbil within a few days after onset of lactation, and is then probably inhibited, as in the rat (Crighton, 1971), until a few days after the cessation of lactation. It thus seems reasonable that the complex hormonal interactions during the ovulation cycle may even influence the activity of the cells in the mammary tissue. One of the reasons for this suggestion is the study on the rat by Patterson & Master, 1970, showing that during the different stages of the normal oestrus cycle of the reproductive tract, the activity of LDH varies during the different stages. Moreover, several authors have reported that enzyme activities of mammary cells react to hormonal stimulation (Goodfriend & Kaplan, 1964; Heitzman, 1969; Raineri & Levy, 1970:

Green et al., 1971 ; Rivera & Cummins. 1971; Korsrud & Baldwin, 1972b; Richards & Hilf, 1972a). It has also been reported, that hormonal changes may not necessarily affect all enzymes in the same matter. For example a decrease in the activity of G-6-PDH was noted in the lactating rat after adrenalectomy, but the activities of LDH, MDH and G-3-PDH were unaffected (Korsrud & Baldwin, 1972a). The activities of the enzymes investigated are higher in the anterior glands than in the posterior ones, especially towards the end of lactation. RNA activity behaves similarly. The differences in enzyme activities between the glands remain to some extent during involution. Comparing the levels of enzyme activities expressed per g wet tissue from the posterior glands of the gerbil with those of the rat and mouse (unpublished), there are no striking species differences for LDH, G-6-PDH or G-3-PDH during corresponding stages of late pregnancy, lactation and early involution. For MDH the activity is somewhat lower in the mammary glands of the gerbil. Consequently, LDH dominates over MDH to a greater extent in the gerbil than in the rat and mouse. This may indicate a higher level of anaerobic metabolism in the mammary tissue of the gerbil. More evidence for this theory is provided by the distribution of LDH isoenzymes. LDH 5 is by far the predominating form in the mammary glands of the gerbil during pregnancy, lactation and involution, in contrast to the rat. It is notable that LDH 5 and other LDH isoenzymes containing A subunits, only increase slightly to a broad maximum during lactation of the gerbil. In comparison a radical increase in the activity of LDH 5 occurs during lactation of the rat. In order to draw further conclusions on anaerobic metabolism during the lactation cycle. more research must be done on the balance between lactate and pyruvate. The number of mitochondria has been shown to increase in mammary tissue at the beginning of lactation in the mouse (Sekhri et al., 1967; Mills & Topper, 1970), an observation confirmed from a more biochemical point of view by an increase in activity of the mitochondrial form of MDH in the rat (Karlsson & Carlsson, 1968). During involution of the mammary gland in the rat, the number of mitochondria decrease (Sekhri et al., 1967) and there is accordingly a decrease in the mitochondrial form of MDH (Karlsson & Carlsson, 1968). In this investigation on the gerbil, the two forms of MDH exhibit almost equal relative activities throughout lactation and involution. Taking into consideration that there is an absolute decrease in the activity of mitochondrial MDH of the gerbil as the mammary glands pass from lactation to involution, these changes are not as substantial as in the rat. Summarising all these findings on the rat and gerbil, it is evident that there are some differences between these species as regards function and biochemical characteristics of the mammary gland. It is possible that the gerbil, although bred in the laboratory with a free supply of water, still utilizes those metabolic pathways developed to overcome the water deficiency in its natural habitat. Acknowledgements--Our sincere thanks are due lo Mrs Marie Adler-Maihofer and Mrs Marianne Andersson for

Dehydrogenases in mammary gland of the Mongolian gerbil excellent technical assistance and secretarial help. The investigation was supported by grants from the foundation of Magnus Bergwall and the Physiographic Society of Lund.

REFERENCES ARRINGTON L. R. & AMMERMANC. B. (1969) Water requirements of gerbils. Lab. Anita. Care 19, 503-505. BALDWIN R. L. & MILLIGAN L. P. (1966) Enzymatic changes associated with the initiation and maintenance of lactation in the rat. J. Biol. Chem. 241, 2058-2066. BERGELIN I. S. S. & KARLSSON B. W. (1973) Dehydrogenases in the kidney of developing neonatal pigs as compared with the foetal and adult pig. Int. J. Biochem. 4. 51-61. BURTON K. (1956) A study of the conditions and mechanisms of the diphenylamine reaction for the colorimetric estimation of deoxyribonucleic acid. Biochem. J. 66, 315-323. CARLSSON E. I. (1972) On mammary gland development and milk secretion. Enzymological and electron microscopic studies on rat during pregnancy, lactation and involution. Thesis, Lund. CARLSSON E. I., KARLSSON B. W. & WALDEMARSON K. H. C. (1973) Dehydrogenases and nucleic acids in rat mammary gland during involution initiated at various stages of lactation. Comp. Biochem. Physiol. ,liB, 93-108. COWIE A. T. & TINDAL J. S. (1971) The Physiology of Lactation. Arnold, London. CRIGHTON D. B. (1971) Lactation anoestrus and the effects on lactation of the induction of varying levels of ovarian and uterine activity. In Lactation. (Edited by FALCONER I.R.) pp. 105-122. Butterworths, London. FALCONER I. R. (1971) Lactation. Butterworths, London. GLOCK G. E. & MCLEAN P. (1954) Levels of enzymes of the direct oxidative pathways of carbohydrate metabolism in mammalian tissue and tumors. Biochem. J. 56, 171-175. GOODFRIEND T. L. & KAPLAN N. O. (1964) Effects of hormone administration on lactic dehydrogenase. J. Biol. Chem. 239, 130-135. GREEN C. D., SKARDAJ. & BARRY J. M. (1971) Regulation of glucose 6-phosphate dehydrogenase formation in mammary organ culture. Biochim. biophys. Acta 244, 377-387. GUL B. & DILS R. (1969) Enzymic changes in rabbit and rat mammary gland during the lactation cycle. Biochem. J. 112, 293-301. HEITZMAN R. J. (1969) The induction by exogenous hormones of enzymes metabolising glucose 6-phosphate in the mammary gland of the pseudopregnant rabbit. J. Dairy Res. 36, 47-52. JONES E. A. (1967) Changes in the enzyme pattern of the mammary gland of the lactating rat after hypophysectomy and weaning. Biochem. J. 103, 420-427. KARLS~SON B. W. & CARLSSON E. I. (1968) Levels of lactic and malic dehydrogenase isoenzymes in mammary gland, milk and blood serum of the rat during pregnancy, lactation and involution. Comp. Biochem. Physiol. 25, 949-971. KARLSSON B. W. t~z LARSSON G. B. (1971) Lactic and malic dehydrogenases and their multiple molecular forms in the Mongolian gerbil as compared with the rat, mouse and rabbit. Comp. Biochera. Physiol. 40B, 93-108. KARLSSON B. W. & PALMERL. S. (1971) Lactic dehydrogenase isozyme distribution in various tissue fractions of the developing mammalian liver. Comp. Biochem. Physiol. 38B, 299-308. KORSRUD G. O. & BALDWIN R. L. (1972a) Effects of adrenalectomy, adrenalectomy-ovariectomy, and cortisol and estrogen therapies upon enzyme activites in lac-

115

tating rat mammary glands. Can. J. Biochem. 50. 366-376. KORSRUD G. O. & BALDWINR. L. (1972b) Hormonal regulation of rat mammary gland enzyme activities and metabolite patters. Can. J. Biochem. 50, 377-385. LOWRY O. H., ROSENBROUGHN. J., FARR A. L. & RANDALL R. J. (1951) Protein measurements with the Folin-phenol reagent. J. Biol. Chem. 193, 265-275. MILLS E. S. & TOPPER Y. J. (1970) Some ultrastructural effects of insulin, hydrocortisone, and prolactin on mammary gland explants. J. Cell Biol. 44, 310-328. MUNRO H. N. & FLECK A. (1966) Recent developments in the measurements of nucleic acids in biological materials. Analysts 91, 78-88. OTA K. (1964) Mammary involution and engorgement after arrest of suckling in lactating rats indicated by the contents of nucleic acids and milk protein of the gland. Endocr. Jap. 11, 146-152. PATTERSON C. t~ MASTERS C. J. (1970) The influence of progesterone on the lactate dehydrogenase isoenzymes of the rat reproductive tract. FEBS Lett. 12, 69-71. RAINERI R. t~ LEVY H. R. (1970) On the specificity of steroid interaction with mammary glucose 6-phosphate dehydrogenase. Biochemistry 9, 2233-2243. REYNOLDS M. & FOLLEY S. J. (1969) Lactogenesis: The Initiation of Milk Secretions at Parturition. University of Pennsylvania Press, Philadelphia. RICH S. T. (1968) The Mongolian gerbil (Meriones unguiculatus) in research. Lab. Anita. Care 18, 235-243. RICHARDS A. H. & HmF R. (1972a) Effect of estrogen administration on glucose 6-phosphate dehydrogenase and lactate dehydrogenase isoenzymes in rodent mammary tumours and normal mammary glands. Cancer Res. 32, 611-616. RICHARDS A. H. & HILF R. (1972b) Influence of pregnancy, lactation and involution on glucose-6-phosphate dehydrogenase and lactate dehydrogenase isoenzymes in the rat mammary gland. Endocrinology 91, 287-295. RIVERA E. M. & CUMMINSE. P. (1971) Hormonal induction of dehydrogenase enzymes in mammary gland in vitro. Gen comp. Endocr. 17, 319-326. RIVERA E. M. & CUMMINS E. P. (1972) Nucleic acid levels during functional development of mouse mammary gland. Proc. Soc. exp. Biol. Med. 140, 502-504. SCHWENTKER V. (1963) The gerbils, a new laboratory animal. Illinois Vet. 6. 5-9. SCHWENTKER V. (1966) The Gerbil. Tumblebrook Farm, Brant Lake, New York. SEKHRI K. K., PITELKA D. R. & DEOME K. B. (1967) Studies of mouse mammary glands--I. Cytomorphology of the normal mammary gland. J. natn. Cancer Inst. 39, 459-490. SIMPSON A. A. & SCHMIDT G. H. (1969) Nucleic acid content of rat mammary gland nuclei during pregnancy, lactation and involution. Proc. Soc. exp. Biol. Med. 132. 978-983. SIMPSON A. A. & SCHMIDT G. H. (1970) Lactate dehydrogenase in the rat mammary gland. Proc. Soc. exp. Biol. Med. 133, 897-900. SINHA K. N., ANDERSON R. R. & TURNER C. W. (1970) Growth of the mammary glands of the golden hamster, Mesocricetus auratus. Biol. Reprod. 2, 185-188. SLATER T. F. (1962) Studies on mammary involution--I. Chemical changes. Archs int. Physiol. Biochim. 70. 167-178. TRAURIG H. H. (1967) Cell proliferation in the mammary gland during late pregnancy and lactation. Anal. Rec. 157, 489-504. TUCKER H. A. (1969) Factors affecting mammary gland cell numbers. J. Dairy Sci. 52, 720--729. TUCKER H. A. & REECE R. P. (1963) Nucleic acid content of rat mammary glands during postlactational involution. Proc. Soc. exp. Biol. Med. 112, 1002-1004.

Dehydrogenases, nucleic acids and soluble proteins in mammary gland of the Mongolian gerbil during pregnancy, lactation and involution.

Comp. Biochem. Physiol., 1976, [,Iol. 55B, pp. 109 to 115. Pergamon Press. Printed in Great Britain DEHYDROGENASES, NUCLEIC ACIDS AND SOLUBLE PROTEIN...
629KB Sizes 0 Downloads 0 Views