Serum changes in developing rats

Con@. Biochem. Pkyti*oL, 1972, Vol. 43A, pp. 897 to 903. Pergamon Press. Printed in Great Britain

SERUM CHANGES PETER

C. READEl

IN DEVELOPING and KEVEN

RATS*

J. TURNER2

l Department a Clinical

of Dental Medicine and Surgery, University of Melbourne; Immunology Unit, Princess Margaret Hospital, Subiaco, Western Australia (Received 21 march 1972)

Abstract-l. Serum levels of complement, amylase and bactericidal activity were shown to increase with age from foetal to adult rats. 2. Foetal liquor was shown to contain similar proteins to the serum of the foetus. 3. Differences in the electrophoretic separation of serum albumin and prealbumin in an age range of animals is described. INTRODUCTION

STWDIESdesigned to investigate the functional development of the reticulo-endothelial system in rats have demonstrated that both cellular and humoral aspects of this system are present in the foetus of these animals (Karthigasu et al,, 1965; Reade & Jenkin, 1965 ; Reade, 1968). These studies showed that some of the manifestations of host-defence mechanisms such as phagocytosis of foreign particles, bactericidal ability of fixed and free macrophages and circulating antibody increase quantitatively as foetuses mature to adults. Investi~tions leading from these studies indicated other changes which occur in the serum of developing rats. Some of these such as the bactericidal capacity of serum and complement levels give addditional information concerning the increasing immunological stature of growing rats while others such as serum protein changes indicate more general and apparently parallel aspects of development (Wise et al.) 1963; Kithier & Prokes, 1966). It is the purpose of this co~uni~tion to present findings which demonstrate a progressively changing pattern in serum proteins from foetal to adult rats. These show that the development of some serum fractions associated with host-defence mechanisms like other developmental changes are progressive and frequently evident in foetal animals. MATERIALS

AND METHODS

Animals Hooded Wistar rats (Rattus norvegicus) of known ages and determined stages of pregnancy were used throughout the study. e Supported by a grant from the National Health and MedicaI Research Council of Australia.

PETER C. REAJNZAND

898

KEVEN

J. TURNER

Bacteria Escherichia coli (Lilly) (Wardlaw, 1963) cultures were grown at 37°C in minimal medium for 18 hr with constant shaking and used in the bactericidal experiments. Serum Blood from foetal rats at 16, 18, 20 and 22 days of gestation and natal animals of various ages was collected by cardiac puncture and the serum prepared and stored according to the method of Borsos 8z Cooper (1961). In essence this method makes use of collection in the cold, rapid centrifugation in the cold and storage at - 50°C and is designed essentially for the maximum preservation of complement. Foetal liquor The extra foetal fluids enclosed by the placental membranes comprise exocoelomic and amniotic fluids. These were collected together by detaching each conceptus from the uterine wall, washing in sterile isotonic saline, mopping dry with absorbent paper and incising the placental membranes to collect the fluid in bottles for immediate use or storage at -50°C. Antiserum Antiserum to normal adult rat serum proteins was produced in adult rabbits by two intramuscular injections of 1.0 ml rat serum in complete Fruend’s adjuvant at a week’s interval and then three intraperitoneal injections each separated by 1 week. Immunoelectrophoresis Immunoelectrophoresis of undiluted sera from foetal and natal rats was carried out as described in a previous study (Reade et al., 1965). Starch-gel

electrophoresis

Sera from foetal and natal rats, diluted to a concentration of 10 mg protein/ml, were examined by electrophoresis in starch gel using the discontinuous buffer system of Poulik (1957) at a potential gradient of 200 V/cm. Bactericidul assay The bactericidal activity of serum from foetal and natal rats was determined in a system using E. coli (Lilly) as the test bacteria. Aliquots of 0.1 ml of the test serum, 0.4 ml of basal medium (to give a serum dilution of one-fifth) and approximately lo3 organisms were placed in carefully washed small test-tubes which were subsequently covered with aluminium caps. A blank of 0.5 ml of basal medium to which the same number of organisms were added was used as a control. The samples were incubated in a water-bath at 37°C during the experiment and duplicate O-04-ml samples were taken from each tube at times 0, 10, 20, 40 and 60 min and placed on to dried nutrient agar plates. These were incubated at 37°C for 18 hr and the resulting colonies were then counted. The count resulting from each sample was expressed as a percentage of the zero time count. Complement assay The haemolytic method used was essentially that of McGhee (1952) gaining its sensitivity from using one-tenth the usual number of sheep erythrocytes. The highest dilution giving 100 per cent haemolysis was taken as the end point, titres being expressed as units per ml of undiluted serum. Amylase assay A standard starch-iodine titration based on the method of van Loon et al. (19.52) was used to obtain a value expressed in arbitrary units/ml obtained from the expression:

SERUM

CHANCES

IN

O.D. blank - O.D. test O.D. blank Optical densitiesI(0.D.)

DEVELOPING

899

RATS

x 800 = units/100 ml.

were measured at 620 mm. RESULTS

Bactericidal activity The serum titres of bactericidal activity increased as the rats matured from foetus to adult (Fig. 1). This increase could be recorded over the range spanning the period of 16-day foetal life to 7-day neonatal life but from this latter age to 84 days the serum samples rapidly reduced the number of viable bacteria to a level which could not be counted (Fig. 1).

b-day

L

0

SO.

22.doy

toetus

____ 7- doy

neonate

foetus

i

::

40.

: 30. 20. IO.

FIG.

1. The percentage survival of E. colt' (Lilly) at times 0, 10, 20, 40 and 60 min after incubation with 1 : 5 dilutions of foetal and 7-day post natal serum.

Serum complement activity The serum complement activity was constant throughout uterine life but showed a marked increase shortly after birth. Thereafter the complement titres increased with age of the natal animal (Table 1). Serum amylase activity Investigation into the amylase activity of rat serum and foetal prompted by the observation that, following electrophoresis of such starch gel, the gel was damaged in the location of the application damage was particularly apparent on the cathodic side of the well.

liquor was samples in well. This The results

900

PRTRRC.RRADEAI~DKRVI~J.~IIURNW

TABLE l-COMPLEMBNT

TITRR WPRESSEDAS I-IAI~IOLYTICUNITS/ANIMALS FOR GROUPSOF RATSOF VARIOUSAGES Age (days)

Foetal

16 18 20 22

Natal

5 10 20 30 60 90

Units of complement/ ml of serum

125 125 125 170 250 300

obtained by standard iodine-starch titrations of foetal and natal sera and foetal liquor are shown in Table 2 and illustrate the high concentration of starch-reducing activity which was presumed to be due to smylase. A marked increase in serum amylase activity was observed towards the end of foetal life. This was thought to TABLE ~--AMYLASE CONTENTIN UNITS/l00 Ink OF RAT SERUMAND FORTALLIQUOR

Age (days)

Serum amylase (units/l 00 ml)

Foetal

16 18 20 22

1950 1400 1420 5370

Natal

7 28 56 84

1610 1800 2510 2230

Foetal liquor amylase (units/100 ml) 3400 3800 3900 -

be due to imbibition by the foetus of foetal liquor which is reduced considerably in volume in the last days before birth and which can be seen from Table 2 to contain relatively large amounts of amylase Immwwekctrophoresis Foetal sera when developed against rabbit-anti-adult rat serum exhibited a simple pattern which was highlighted by a pronounced, somewhat linear precipitin arc indicated by an arrow in Fig. 2 in the LX-~region. Similar observations have also been reported by Kelleher & Villee (1962). This protein was particularly apparent

FIG. 2. Immunoelectrophoretic patterns of foetal and natal rat sera. Troughs contained rabbit anti-adult rat serum and wells the following: (a) 14-day foetal rat serum; (b) 16-day foetal rat serum, (c) 1%day foetal rat serum; (d) 20-day foetal rat serum; (e) 22-day foetal rat serum; (f) 5-day natal rat serum; (g) IO-day natal rat serum, (h) 20-day natal rat serum, (i) 30-day natal rat serum, and (j) adult rat serum

foetal

f

natal

A3 A2 Al

FIG. 3. S tarch gel electrophoretic patterns of I, XI and Ill, 18” foetal rat serum respectively, IV, V, and VI, 30-, 30-day and respectively.

and 22 . :rum rat SC

SERUM CHANGES IN DEVELOPING RATS

901

in sera from foetuses in the 14-18 day periods of gestation (Fig. 2, a-c), was barely detectable at day 20 (Fig. 2d) but, under the conditions examined, was not noted in serum from normal adult rats. The appearance of discrete proteins in the 012region as the animal matures is apparent and mirrors the development shown in the starch gel electrophoresis pattern in Fig. 3. In this aspect the serum from foetuses of 22-day gestation (Fig. 2e) is almost identical to that of adult animals (Fig. 2j). Starch-gel electrophoresis

The most striking observation which was noticed when sera from foetal and natal rats was examined by starch-gel electrophoresis was the appearance of three distinct albumin components referred to as Al, A2 and A3 in Fig. 3. The latter (A3), the post albumin of Wise et al. (1963) and Kithier & Prokes (1966) was easily discernible in foetal serum but decreased in relative concentration following parturition. Concomitant with this decrease in the concentration of the A3 component there was a corresponding increase in the relative concentration of the Al component. However, at all stages of foetal and natal development the three albumin components were clearly detectable. The prealbumin components were shown to increase in relative concentration as foetal age increased and to remain at approximately the same concentration from 22 day foetus to adult animals. Foetal liquor was also subjected to starch gel electrophoresis and the pattern of protein distribution was found to be similar to that seen following electrophoresis of serum from foetal animals of the same age. DISCUSSION

Previous studies have shown that serum IgG (Reade et al., 1965) and intracellular factors which are thought to play a part in host defence (Reade, 1968) are evident in foetal animals and increase quantitatively during foetal and early post natal life. Furthermore, when rats reach the age of approximately 10 days they attain adult-like maturity in terms of their immunological development. The current study supports this general concept of maturation and indicates that other serum factors such as bactericidal activity, complement activity, amylase activity and protein synthesis in general increase during the development from foetal to natal life. The picture presented by the complex albumin pattern has interesting aspects for it is doubtful whether the albumin components A, or As are simple polymers of the A, component since their mobilities relative to A, are higher than those generally found for dimers and trimers of serum albumin (Marinio & Ott, 1964). They seem rather to be a genuine example of paralbuminaemia which as proposed by Earle et al. (1959) may be the heterozygous condition for two co-dominant alleles. Such cases have been reported in birds (Haley, 1965), cattle (Ashton, 1964) and horses (Stormont & Susuki, 1963). It is of interest that the mobility relationship of various albumin bands in Fig. 3 are very similar to the corresponding values found for albumin phenotypes in horses (Stormont et al., 1963). Clearly these variants

902

F%TERC.READBANDKEVENJ.TURNER

of albumin in rat sera do not show immunochemical differences since they give rise on immunoelectrophoresis to a single undulating bow (Fig. 2). The reasons why the variants A, and A, should decrease in concentration relative to A, during development from foetus to adult is not clear. If they are phenotypes as proposed it may be that during foetal development there is a temporary activation of genes conditioning the synthesis of albumin variants which are produced only in small amounts in adult life. Alternatively since the serum samples were pooled from a much larger number of animals than were the adult samples there would be a greater likelihood of finding all variants of albumin in the foetal rather than in the adult sample. These studies were designed to find if any fundamental differences exist between the serum components of foetal, neonatal and adult rats. It is apparent that certain differences do exist in the bactericidal properties and the complement titres of the serum and in the relative quantities of various serum proteins, particularly in the region of the 01globulins and albumins. It is to be expected that differences should exist between foetal and adult sera because even though rats belong to the group of “full transmitters” of serum proteins across the placenta some selectivity applies (Morgan, 1964). E ven in full transmitters homologous yG is transmitted more readily than homologous albumin (Brambell & Hemmings, 1960; Stormont & Susuki, 1963). Moreover, foetal rats are able to synthesize a wide range of serum proteins including the immunoglobulin-like proteins (Fishel & Pearlman, 1961: Reade et al., 1965) and it follows, therefore, that the operation of these two factors would lead to differences in the relative concentrations of serum proteins in foetal and maternal sera. REFERENCES G. C. (1964) Serum albumin polymorphism in cattle. Genetics 51,1421-1426. BOR~OST. & COOPERM. (1961) On the hemolytic activity of mouse complement. Proc. Sot. exp. Biol. Med. 107, 227-232. BRAMBELLF. W. R. & HEMMINGSW. A. (1960) The transmission of antibodies from mother to fetus. In The Placenta and Fetal Membranes (Edited by VILLEE C. A.), pp. 71-84. Williams & Wilkins, Baltimore. EARLE D. P., HUTT M. P., SCHMIDK. & GITHIN D. (1959) Observations on double albumin: a genetically transmitted serum protein anomaly. J. clin. Inwest. 38, 1412-1420. FISHEL C. W. & PEARLMAND. S. (1961) Complement components of paired mother-cord sera. Proc. Sot. exp. Biol. Med. 107, 695-699. HALEY L. E. (1965) Serum albumin polymorphism in quail and chicken-quail hybrids. Genetics 51, 983-986. KARTHIGASU K., READE P. C. & JENKIN C. R. (1965) The functional development of the reticuloendothelial system-III. The bactericidal capacity of fixed macrophages of foetal and neonatal chicks and rats. Immunology 9, 67-73. KELLEHERP. C. & VILLEE C. A. (1962) A protein present in fetal but not in maternal rat serum. Science, N.Y. 138, 510-511. KITHIER K. & PROKESJ. (1966) Fetal ol,-globulin of rat serum. Biochim. biophys. Acta 127, 390-399. MCGHEE R. B. (1952) Presence of complement in serum of the mouse. PYOC.Sot. exp. Biol. Med. 80, 419420.

ASHTON

sBRUMCHANGES IN DEVELOPING RATS

903

MARINIS S. & OTT H. (1964) Naturliche und kunstliche Polymeren von Plasmaproteinen (Untersuchungen in Acrylamid-gel). In Protides of the Biological Fluids, Proceedings of the 12th Colloquium, Bruges (Edited by PIETERSH.), pp. 420-422. Elsevier, Amsterdam. MORGANE. H. (1964) Passage of transferrin, albumin and gamma golbulin from maternal plasma to foetus in the rat and rabbit. r. Physiol., Land. 171,264l. POULIKM. D. (1957) Starch gel electrophoresis in a discontinuous system of buffer. Nature, Lond. 180,1477-1479. READEP. C. & JENKIN C. R. (1965) The functional development of the reticuloendothelial system-I. The uptake of intravenously injected particles by foetal rats. Immunology 9, 53-60. READEP. C., JENKIN C. R. & TURNERK. J. (1965) The synthesis by foetal chicks and rats of serum proteins having some properties of the immunoglobulins. Aust.J. exp. Biol. med. Sci. 43, 699-712. REAnE P. C. (1968) The development of bacetericidal activity in rat peritoneal macrophages. Aust.J. exp. Biol. med. Sci. 46, 231-247. STORMONTC. & SUSUKIY. (1963) Genetic control of albumin phenotypes in horses. Proc. Sot. exp. Biol. Med. 114, 673-675. VAN LOON E. J., LIKINS M. R. & SEGERA. J. (1952) Photometric method for blood amylase by use of starch-iodine colour. Am.J. clin. Path. 22, 1134-l 136. WISE R. W., BALLARDF. J. & EZEKIEL E. (1963) Developmental changes in the plasma protein pattern of the rat. Camp. Biochem. Physiol. 9, 23-30. Key Word Index--Serum; amylase.

developing rats; bactericidal activity of serum; complement;