Serum protein fractions in C57BL mice during growth and senescence

Exp.

Geront.

Vol.

I. pp.

SERUM

105-I

16. Pergamon

Press

1965.

Printed

in Great

Britain

PROTEIN FRACTIONS IN ChBL MICE GROWTH AND SENESCENCE

DURING

J. H. DAAMS* Department

of Experimental

Pathology,

The

Netherlands

(Receizwd 16 June

Cancer

Institute,

Amsterdam

1964)

INTRODUCTION WHILE

total serum protein level remains fairly constant throughout life, relative amounts of different fractions change markedly with age, presumably reflecting age-dependent shifts in functions and requirements. In many cases descriptive data only are available, the reasons for changes in serum protein fractions remaining unclarified. In general, the number of electrophoretically discernible fractions increases during embryonic life and early childhood, while the albumin to globulin ratio gradually rises. Data on early changes are extant for various species, including man (Hitzig, 1960). Specific embryonic proteins have been described in chicks (Sofsky et al., 1962), rats (Kelleher, 1964; Heim, 1961), mice (Pantelouris and Hale, 1962; Pantelouris, 1963) and pigs (Lecce et al., 1962). In chickens, these embryonic proteins show a low mobility on starch gel electrophoresis, while in mice, rats, and pigs they migrate nearly as fast as the albumin fraction. Heim (1961) describes a prealbumin in the serum of 17- and l&da> old rat embryos. These embryonic proteins, which in mammals were not demonstrable in the serum of pregnant females, disappear from the serum within the first few weeks of life. In man, no such specific embryonic proteins have been found, although the newborn child has a markedly elevated concentration of acid ai glycoprotein (Hitzig, 1960). In other instances, causal factors behind age dependent changes in serum proteins have been identified. In mammals, immune globulins may pass from mother to child through the placenta or through the milk. The serum level of these globulins decreases during early life until endogenous production is established (Hitzig, 1960). Endocrine factors are known to influence other changes in serum proteins. In man, lipoproteins are extensivelv studied in relation to atherosclerosis, and sex hormones have been found to influence the relative amounts of these fractions. Women have a higher level of a lipoproteins than men, and oestrogens have been found to increase the ratio of a lipoproteins to /3 lipoproteins in patients with vascular disease (Furman et al., 1958; Rivin, 1959; Danemann et al., 1960). When conventional protein stains are used, results of oestrogen treatment include increase of al globulins as found in women (Studnitz and Berezin, 1956). Androgens also influence the relative amounts of different serum protein fractions. In mice, a prealbumin has been described, the level of which waxes and wanes parallel to testicular or exogenous male hormone activity (Riimke and Thung, 1964), although in the serum this protein is demonstrable by immunological methods only. Androgens have been reported to cause an increase of as globulin in women (Studnitz and Berezin, 1956) while a rising albumin level has been described in rats (Hoch-Ligeti and Irvine, 1954). * With the support

of the Organisation

for Health 105

Research

T.N.O.

106

J. It. DAAMS

These effects of sex hormones may reasonably be related to sex differences found in various animals. Besides the human sex difference in ~ lipoprotein level cited above, mention may be made of the lower level of/3 glycoprotein in adult bovine males as compared with females. (Dimopoullos et al., 1959). Data from Heim's (1961) study on developmental changes indicate a higher level of all globulins in adult male than in female rats. Recently, sex-associated differences in serum proteins of inbred strains of mice have been described by Espinosa et al. (1964), using agar electrophoresis. The nomenclature used by these authors differs somewhat from that observed in the present report, but their results correspond in various respects with our findings. Sex hormones may similarly be involved in some of the senile changes in serum protein fractions described in various animals, although in most cases the mechanisms concerned have not been analysed. A decreasing albumin to globulin ratio with increasing age has been found in man (Appleton and Pritham, 1963; Acheson and Jessup, 1962) and in various other species (Ringle and Dellenback, 1963). In women/3 lipoproteins rise with increasing age until the male level is reached (Burstein and Samaille, 1958; Cram6r, 1962). For shifts in lipoprotein fractions with increasing age in the golden hamster, cf. House et al. (1961). It should be noted that some reports deny specific sex or age-related differences in human serum fractions. Thus NScker (1956) found no sex differences in serum proteins, while Goldbloom et al. (1959) failed to find either sex or age influences on lipid fractions. According to Edel (1962) and Eckerstrom (1958) changes in serum proteins in old people are indicative of diseases such as atherosclerosis or neoplasms, or of protein-deficient diets. In the absence of disease, these authors found normal adult serum protein values in old age. The present observations deal with age-related changes in one specific strain of mice and were made as part of a programme of gerontological experiments on inbred strains of mice. Besides descriptive data, experiments are reported by which we tried to identify causes for some changes in specific serum protein fractions. M E T H O D S AND PROCEDURE Mice of the inbred strain CsvBL were fed commercial pellets and given tap water ad libitum.

Oestrone was either administered subcutaneously in pellets of about 2 mg each, containing ½ of oestrone and ~ of cholesterol, or given in drinking water in a concentration of 2 mg/1. Androgen was given subcutaneously in pellets containing 2 mg of testosterone propionate each.* Blood was obtained by decapitation and bleeding of the animals. The blood volume of very young animals was usually insufficient for serum separation by centrifugation. In these cases clot formation and retraction proceeded in a paraffin coated and moisture saturated Petri disk. The serum was subsequently taken up into a capillary for introduction into the starch gel. Starch gel electrophoresis was carried out with the micromethod described previously (Daams, 1963). For two dimensional analysis, electrophoresis was first carried out in 0.9 per cent agar gel containing 0.06 M veronal buffer (pH 8-6), using a potential of 50 V/cm and pentane cooling. The agar strip was then divided longitudinally, one * H o r m o n e s were kindly s u p p l i e d by N, V. O r g a n o n , Oss.

prealbwmin

ALBUMIN

alglobulin fast a2 globulin fast haptoglobul

in

@Ag obulin pB g I obulin /SC globulin slow haptoglobul slow a2globul

,~.~_~.~.‘.~.~.~.~.~.~.‘.~.~ . . . . . . . . . . ~.~.‘.~.~.~.~.~.~.~.~.~.~.~~ ~.~.l.~.~.~.~.~.~.~.~.~.~.~r

.

. .

.

in

in

ori in yg 3 obulin

FIG. 1. Serum protein fractions of adult female CBTBL mouse after micro starch gel electrophoresis for 15 min in borate buffer 0.03 M pH 8.4, 80 V/ cm. Length of the electropherogram 2 cm; staining with Amido Black 10 B. Nomenclature after Duke (1963).

SERUM

PROTEIN

FRACTIONS

IN C57BL MICE

DURING

GROWTH

AND SENESCENCE

107

half being fixed and coloured and the other half being introduced in a starch gel layer of 1 mm thickness on a 5 x 5 cm glass support. In other respects, this second electrophoresis run was carried out as prescribed for normal micro starch gel electrophoresis. Stained starch gel strips were scanned with a Chromoscan reflecting and integrating densitometer (Joyce, Loebl & Co). For quantitating the relative concentrations of protein fractions, the readings of the integrator of this instrument may be used. In our opinion, however, the data thus obtained are highly inaccurate, since separation of adjacent protein bands is usually incomplete, the peaks not being separated by a base line interval (cf. Figs. 2, 4, and 5). From the emerging peaks, we therefore extrapolated the intersections of ascending and descending slopes with the base line and evaluated the resulting ideal fraction surface planimetrically. The quantitated data often give different results from those obtained by judging the scans visually.

For a description

Albumin

of this method

see Ogston

(1956).

Embryonol a, globulin

AA Origin

FIG. 2. Scans of micro starch gel electropherograms of newborn and young adult male CsiBL serum. Note the slightly increased mobility and lower concentration of albumin and the high concentration of embryonic a~ globulin, and the low concentration of /IHglobulin in the neonatal serum. For nomenclature cf. Fig. 1. RESULTS Nomenclature The nomenclature for serum protein fractions is based upon the sequence found in moving boundary and in paper and agar electrophoresis and cannot be directly applied to starch gel electrophoresis strips because the fraction sequence in these is slightly different. Using two dimensional electrophoresis as described above, we first correlated the sequence of mouse serum protein fractions after starch gel electrophoresis with that after agar gel electrophoresis. Since our results were in agreement with those of Duke (1963), who used CaH mice, we have adopted the nomenclature used by this author (Fig. 1). In mice, as in man (Smithies, 1959) and in rats (Reaton et al., 1961; Espinosa, 1961), aa globulin in starch gel splits into two fractions called slowaa and fast a2 globulin. This

108

J. H. DAAMS

split probably indicates differences in molecular size of these proteins. The prealbumin fraction observed in the agar electrophoresis strip could not be located in the starch gel. A prealbumin seen after starch gel electrophoresis, on the other hand, appeared to originate from the albumin zone in the agar gel and may perhaps represent an al globulin. The embryonic protein which we have previously called a “second albumin” (Daams, 1964), similarly seems to be an al globulin (cf. Pantelouris, 1963).

be,

Age,

days

Adult values

days

FIG. 3. Changes in serum protein fractions during the first 2 weeks of life. Values of individual samples from fifteen CSBL mice, ranging in age from 0 to 14 days, were calculated from scans of micro starch gel electropherograms. Upper part of the figure demonstrates an increase in albumin level and the diminishing of embryonic al globulin. Lower part of the figure shows a marked rise in ps globulin as main characteristic. At the right, mean values and standard deviations are shown of /3globulins in serum of adult mice (6&7 months age).

Changes

during early life

Forty-two mice, ranging in age from 0 to 14 days after birth, three animals for each interval of 1 day, were studied. No differences were found between male and female sera; a representative scan of neonatal serum, as compared to young adult male serum,

SERUM PROTEIN

FRACTIONS

IN C57BL MICE DURING

GROWTH

AND SENESCENCE

109

is shown in Fig. 2. For one series of mice between 0 and 14 days of age, the relative amounts of various protein fractions were quantitated, as shown in Fig. 3. The conclusions drawn from these data are in good agreement with the scans from the remaining twenty-eight mice and may be summarized as follows. In the albumin region, the embryonic ai globulin described by Pantelouris (1963) is distinct in neonatal mice (Fig. 2) and gradually decreases in amount during the next 2 weeks. The albumin fraction itself originally has a slightly greater mobility and is relatively smaller than in adult mice. During the first 2 weeks of life, the mobility of the albumin decreases and its relative concentration increases, until the adult condition is approximated. Another characteristic of neonatal mice is a low pn globulin value which later increases until, around 14 days of age, it equals the /3cglobulin fraction. Albumin

FIG. 4. Scans of micro starch gel electropherograms of adult male and female C~TBL serum. .4ge of both animals was 7 months. Most striking differences are the higher p B globulin and the lower albumin concentration in the male.

Sex di#erences

Serum samples taken after the first 2 weeks of life showed no major changes until, around 8 weeks of age, the animals attained sexual maturity. Around this time, a striking difference between male and female mice occurred in the /3globulin region. In females, the fin globulin value becomes lower than that of pc, and in the scan this may lead to the impression of a single p globulin peak. In males, these two fractions are about equal and two peaks are easily distinguished, see Fig. 4. For quantitative data on this and other sex differences, which have been observed in animals up to 1 year of age, see Table 1. The scans give the impression of a higher /3cvalue in females as compared to males. Because of a higher relative amount of albumin in females, however, the ,Bc fraction in females, as percentage of total protein, equals the /3n and pc values in males. Other and minor differences are a slightly higher prealbumin and y-globulin value in males. Some fractions in the region between /3globulins and albumin perhaps are somewhat elevated in the male animals, but the resolution of our method did not permit quantitation of these fractions. In Fig. 4, representative scans of male and female sera are shown.

110

J. 11.DAAMS

Changes

in old age

At 12 months of age, our CsiBL mice have a life expectancy of about 11 months, and at 24 months of age this has decreased to about 3 months (for survival curves cf. ICIiihlbock, 1959). Studying the sera of a number of female and male mice, between the ages of 18 and 24 months, we noticed few changes in old age as compared with the TABLE

Values

1. SERUM

are given

FRACTIONS

OF 6&-7

MOKTHS

as mean percentages with standard prealbumin, albumin, ,& globulin

Sex

Number of animals

3

10 10

PROTEIN

prealb.

Albumin

(1I

2.7~0.5 1.7 iO.4

54.2~14.3 61.6 ‘1.8

2.2 -0.3 2.2 : 0.6

OLD

MALE

A~-D FEMALE

&BL

MICE

deviations. Sote the sex differences and y globulin fractions

Pn 6.711.3 3.6 +W3

San

i?‘(‘ 6.1 il.1 6.1 il.6

4.6 5.0

~1.7 1.4

in the

>’ 4.0 2.1

1.0 0.4

findings

in young adult animals. Minor sex differences, such as the higher prealbumin and y globulin values in males, remained unchanged. In the p globulin region, however, the sex difference had disappeared. In females as well as in males the pn and pc fractions are now represented by two peaks of about equal height. In Fig. 5, representative scans

FIG. 5. Scans of micro starch gel electropherograms of senescent male and female Cs7BL mice. Age of both animals was 23 months. In the /3 globulin region the female serum has a male aspect due to the increase of /3n globulin.

from old animals are shown, while Fig. 8. gives the quantitative data from these observations as well as from other experiments with old mice described further on.

111

SERUM PROTEIN FRACTIONS IN C5iBL %IICE DURING GROWTH AND SENESCENCE

TABLE 2. INFLUENCE OF SEX HORMONES ON SERUM PROTEIN FRACTIONS OF CASTRATED MALE Cs;BL MICE Values are given as mean percentages with standard deviations. The animals were castrated at 6 months of age. Treatment was started 2 weeks after castration. Oestrone was given in drinking water and testosterone propionate was given subcutaneously as indicated under Methods and Procedure. The animals were killed after 3 weeks treatment. Note lower values for prealbumin, PD globulin and y globulin in the oestrone treated animals, and the lower albumin level in the testosterone treated group, as compared to untreated castrates

Treatment

Number of animals

prealb.

Xlbumin

a1

~__ 1 >*

2 oestrone ,’ testosterone

6 6

3.9 + 0.9 1.8 50.7

60.4 63.6

.,3.2 2.0

6

4.1 10.9

50.4 _:3,9

h3 __-

Sq

PC ------

Y

2.1 iO.8 1.7 =0.7

5.9 51.5 3.5 +0.9

6.2 ~~1.3 6.3 + 1.4

5.4 -1 1.8 5.8 +1,9

4.3 : 1.2 2.5 10.5

2.1 -10.6

6.6 _ 1.6

6.1 I 1.4

4.9

4.2 2 1.1

11.7

The role of sex hormones To evaluate the role of sex hormones in the sex differences observed in young adult mice, 6-months old male mice were castrated and the effect of castration and subsequent hormone treatment on the serum proteins was studied. Detailed data concerning these experiments are given in Table 2. The results may be summarized as follows and are illustrated by the representative scans shown in Fig. 6.

-

-

,

bumIn

\

FIG. 6. Scans of micro starch gel electropherograms of serum of three castrate 6-month-old males with and without hormonal treatment, taken from the animals of Table 1. Note peaks for albumin in the testosterone-treated castrate and for ,GDglobulin in the oestrone animal.

Cs7BL smaller treated

112

J.

H. DAAMS

7 Albumin

FIG. 7. Scans of micro starch gel electropherograms of serum of three castrate 24-month-old CsiBL females, treated with oestrone (in drinking water), untreated and treated with testosterone propionate respectively. Treatment was started 2 weeks after castration, duration was 3 weeks. Note smaller peaks in the testosterone-treated animal for albumin and in the oestrone-treated animal for j5’~ globulin.

q

se

Globulin

apt 1

9r

Globulin Standard

deviation

8

T

7 8

T

6

.F- 5 aI $4 a F3

:2 Sex 0:

Number of o-2 days old

ienescent 23 months

Castrates [C.F: table II]

Senescel castrates

1t

animals

24months

FIG. 8. Values of p globulins during the first 2 days of life, and in adult (6$-7 months) and senescent mice, as compared to hormone-treated castrate animals. Of the senescent female castrates, five received oestrone in drinking water (cf. Methods and Procedure for doses), while the other five received subcutaneous pellets. Testosterone propionate was given in subcutaneous pellets. Treatment was started 2 weeks after castration, and was continued during 3 weeks. The old castrate mice were killed at 24 months of age. Values are means with standard deviations. Note low ,!?B globulin values in neonatal, female and oestrone-treated animals.

SERUM PROTEIN

FRACTIONS

IN C57BL MICE DURING GROWTH AND SENESCENCE

113

While castrate serum differed little from normal male serum of the same age, testosterone propionate in the doses used in this experiment (Table 2) gave a slight increase of the Ps globulin fraction. The prealbumins were similarly increased, and this latter effect was also found after oestrone treatment although less pronounced. The main effect of oestrone, which in this experiment was given in drinking water, however, was the marked lowering of the PB globulin value resulting in a typical female aspect of this region (Fig. 6). Although no definite conclusions could be drawn concerning the y globulin, it is clear that the main sex differences observed in young adult mice can be explained from the effects of the sex hormones, of which the female hormone seems to be the most important. The next question was whether the change in the /3 globulin fractions in old females, leading to a male aspect of this region, is ascribable to changes in female hormone level. In our Cs?BL mice, after 17 months of age the frequency of normal ovarian cycles decreases and at 25 months no normal cycles occur, although intervals of irregular oestrogenic stimulation may occur (Thung et al., 1956). On the actual amount of oestrogens secreted in old as compared to young mice, little is known although indirect evidence suggests a decline of this function (Thung, 1962). If this decline is involved in senile changes in serum proteins, exogenous hormones should restore the young adult pattern. This was actually observed in experiments using old female mice, intact as well as castrated. Oestrone was administered in drinking water as well as subcutaneously and in other animals testosterone propionate was given subcutaneously. Both hormones led to identical changes in serum protein fractions as observed in young castrates (see Fig. 7 and Fig. 8). From these observations it is concluded that, in principle, the senile Cs;BL organism may respond to sex hormones as at younger ages, the main point of interest being the lowering of the Pn globulin value by the female hormone.

DISCUSSION Our findings concerning the high al globulin values in neonatal mice, agree with the results of Pantelouris and Hale (1962) and Pantelouris (1963). It is of interest that a similar perinatal al globulin peak has been described in rats (Kelleher, 1964) and in pigs (Lecce et al., 1962). The origin and significance of this fraction, however, have not been clarified. For the low fls globulin fraction of neonatal mouse serum, on the other hand, a tentative explanation may be put forward. In adult mice, this fraction was found to be specifically decreased in females and this phenomenon was reproduced in castrates by administering oestrogens. It may, therefore, be suggested that the low neonatal level is due to the influence of maternal oestrogens during intrauterine life. Further experiments should of course be done to check this hypothesis. The specific decrease of the fin globulin fraction by oestrogens, as found in our experiments, may well correspond with the sex-associated differences described by Espinosa et al., (1964) in his “az" fractions. These fractions probably are identical with our /3 globulin fractions. The differences in electrophoretic technique, however, impede detailed comparison. This also applies to the findings of other authors concerning the effects of sex hormones on serum proteins in man and in rats. Attention may be drawn, however, to the apparent differences between rats and man on the one hand and mice on the other. In man, as in the rat, a globulins were specifically

J.

114

H.

DAAMS

increased by oestrogens and in man this leads to changes in lipoprotein distribution (Rivin, 1959 ; Furman et al., 1958 ; Danemann et al., 1960). In mice, however, lipoproteins are mainly found in the /3 globulin region (Goranson et al., 1960). The fact that in mice oestrogens may selectively influence the /3 globulin region thus, perhaps, reflects functional similarities between the two species. Other aspects of sex hormonal influences on serum proteins seem of minor importance. The CQfraction in oestrone-treated and in intact female mice sometimes showed a double band in this region, as has also been noted by Hoch-Ligeti and Irvine (1954). A higher relative level of albumins in female as compared to male mice, has been reported These authors, however, also by Espinosa et al. (1964) and agrees with our findings. found an increased y globulin level in female mice, which we cannot confirm. The increased ,/3s globulin fraction in females is the most striking aspect of senile changes in serum proteins. Since experimentally this fraction could be decreased to young adult levels in old mice, this phenomenon may be related to ovarian involution. Whether the PR globulin fraction would react similarly, in senile mice, to physiological oestrone levels has not been ascertained in the present experiments. Besides serving as a starting point for further observations, however, our findings may draw attention to serum proteins as an aspect of the many ageing changes related to the female sex hormones.

REFEREKCES ACHESON, R. M. and JESSUP,W. J. E. (1962) Gerontologiu 6, 193. APPLETON, M. D. and PRITHAM, G. H. (1963) Amer. J. ment. Dejic. 67, 521. BEATON, G. H., SELBY, A. E. and WRI(;HT, A. M. (1961) J. biol. Chem. 236, 2001. BURSTEIN,M. and SAMAILLE, J. (1958) S’u~z,~29, 312. CRAT&R, K. (1962) Acta med. Stand. 171, 429. D.-mm, J. I-I. (1963) 3’. Chromatog. 10, 450. DAA~IS, J. H. (1964) Pvoc. 6th int. Congr. Gerontology Copenhagen 1963, p. 184. Munksgaard,

Copenhagen. DANE~IANN, H. PICK, R. and KATZ, L. N. (1960) J. lab. c&z. Med. 55, 682. DmoPoumos, G. l’., SCHRADER,G. T. and FI.ET(~HER,B. H. (1959) Poor. Sot. esp. Biol. JZed. 102, 704. DUKE, E. J. (1963) Nature, Lond. 197, 288. ECKERSTROM,S. (1958) Geriatrics 13, 744. EDIX, H. (1962) Z. Ges. inn. Med. 17, 373. ESPINOSA, E. (1961) Biochim. Biophys. Acta 48, 445. ESPINOSA, E., CANELO, E. BRAVO, M. and GONZ~~LEZ,0. (1964) Science 144, 417. FURMAN, R. H., HOU~ARD,D. P., NORCIA, L. N. and KEATY, E. C. (1958) Amer. J. Med. 24, 80. GOLDBLOOM, A. A., BOYD, L. J., WOLF, J. L. E. and BENTIVEGNA,S. (1959) Amer. J. Gastroent. 31,

171. GORAIVSON,E. S., MCCULLOCH, E. A. and CINITS; E. A. (1960) Radiat. Hes. 12, 560. HEN, W. G. (1961)J. Embryol. exp. Morph. 9, 52. HITZIG, W. H. (1960) In Analyse Immztno-i/ectrophor&ique (GRABAR, P. and BURTIN, P., eds.) p. 121. Masson et Cie, Paris. HOCH-LIGETI, C. and IRVINE, K. (1954) Proc. Sot. exp. Biol. Med. 87, 324. HOUSE, E. L., PANSKY, B. and JACOBS, M. S. (1961) Amer. J. Physiol. 200, 1015. KELLEHER,P. C. (I 964) Fed. Proc. 23, 556. LECCE, J. G., MORGAN, D. 0. and MATRONE, G. (1962) J. Nzrtr. 77, 349. M~~HLBOCK,0. (1959) Gerontologia 3, 177. N~~CKER,J. (1956) Experentia Suppl. IV, 188. OGSTON, A. G. (1956) In Physical Techniques in Biological Research, (OSTER, G. and POLLISTER, A. W. eds.) Vol. II, p. 124. Academic Press, New York.

SERUM PROTEIN FRACTIONS IN C57BL MICE DURING GROWTH AND SENESCENCE PANTELOURIS, E. M. (1963) Brit. J. Cancer 17, 179. PANTELOURIS, E. M. and HALE, P. A. (1962) Nature, Lond. 195,79. RINGLE, D. A. and DELLENBACK, R. J. (1963) Amer. J. Physiol. 204, 275. RIVIN, A. U. (1959) Metabolism 8, 704. R~;TMKE, PH. and THUNG, P. J. (1964) Acta Endocr. in press. SMITHIES, 0. (1959) Adz?. Protein Chem. 14, 65. QOFSKY, R. A., TRNKA, Z. and THORBECKE, G. J. (1962) PYOC. Sot. exp. Biol. Med. STUDNIZ, W. v. and BEREZIN, D. (1956) Klin. W~chr. 34, 1239. THUNG, P. J. (1962) Gerontologiu 6, 41. THUNC, P. J., BOOT, L. M. and M~HLBOCK, 0. (1956) Actu Endow. 23, 8.

111, 497

Summary-Serum protein fractions have been studied quantitatively, using a micromethod for starch gel electrophoresis combined with densitometric scans of the gel strips. Observations were made on C57BL male and female mice during the first 2 weeks of life, as well as on young adult and on senile animals. The main findings include : (i) a specific embryonic aI globulin, the origin of which is unexplained and which disappears within the first 2 weeks of life; (ii) a low pB globulin value in neonatal mice which increases during the first 2 weeks and which may tentatively be ascribed to the influence of maternal oestrogens during intrauterine life (cf. iii); (iii) some differences between inale and female sera, which occur as the animals attain sexual maturity, were experimentally shown to be caused by sex hormones. Most striking of these sex differences is the decreased pB globulin level caused by oestrogens ; (iv) in senile mice, few changes in serum protein fractions were found, except a marked elevation in the PB globulin fraction in females. This change perhaps is ascribable to decreased oestrogen production in old female mice. It was found experimentally that high oestrone doses may decrease this fraction to the young adult level. R&urn&-On a Ctudie quantitativement des fractions de protide scrique, en utilisant une micro-methode en usage pour l’electrophorese du gel d’amidon combinee a l’exploration densimetrique des stries du gel. Les observations se firent sur des souris males et femelles C57BL au tours des deux premieres semaines de la vie, aussi bien que sur des adultes jeunes et sur des animaus seniles. Les principales constatations que l’on fit concernent les sujets suivants: (1) une globuline embryonnaire specifique aI, d’origine inexpliquee et qui disparait avant la fin de la deusieme semaine; (2) le taux peu i-leve de Pi3 globuline chez les souris nouvelles-n&es, qui augmente pendant les deux premieres semaines et qu’on peut provisoirement attribuer a l’influence des oestrogenes maternels au tours de la vie intra-uterine (cf. 3); (3) certaines differences entre le serum des males et des femelles, qui surviennent quand les animaux atteignent la maturite sexuelle, et qu’on demontra experimentalement relever des hormones sexuelles. Plus frappante que ces differences likes au sexe, est la baisse du niveau de la Pn globuline provoqude par les oestrogtines; (4) chez les souris seniles, peu de transformations dans les fractions de la protidc strique, a part une elevation marquee de la fraction p II globuline chez lcs femelles. Ce changement est peut-Ctre imputable a la baissc de la production oestrogenique chez les souris femelles &&es. On trouva experimentalement que de hautes doses d’oestrogene pourraient abaisser cette fraction jusqu’au niveau trouve chez l’adulte jeune. Zusammenfassung-Serumeiweififraktionen wurden quantitativ untersucht, \vozu eine Mikromethode zur Starkekolloidelektrophorese zusammen mit densitometrischen Bestimmungen der Kolloidstreifen angewandt wurde. Beobachtungen

115

116

J. H. DAAMS

wurden an C57BL mannlichen und weiblichen Miiusen wahrend der ersten zwei Lebenswochen, an jungen ausgereiften und an senilen Tieren angestellt. Zu den wichtigsten Untersuchungsergebnissen gehoren: (1) ein spezifisch embryonales “1 Globulin, dessen Entstehung noch ungeklart ist und das in den ersten zwei Lebenswochen verschwindet; (2) ein niedriger /3~ Globulinwert bei neugeborenen Mausen, der sich in den ersten zwei Wochen erhijht und der-als Versuch einer Erkllrung-dem Einflufl der mutterlichen &trogene wahrend des intrauterinen Stadiums zugeschrieben werden kiinnte. (cf. 3); (3) einige Unterschiede zwischen mannlichen und weiblichen Sera, die auftreten sobald die Tiere geschlechtsreif werden, konnten, durch Geschlechtshormone verursacht, experimentell nachgewiesen werden. Besonders auffallend ist bei diesen geschlechtlich bedingten Unterschieden der durch &trogene verringerte & Globulinwert. (4) bei senilen MHusen wurden nur geringfiigige Veranderungen der Serumeiweiljfraktionen gefunden, ausgenommen jedoch ein deutlicher Anstieg der pa Globulinfraktion bei weiblichen Tieren. Dieser Ansteig kann vielleicht auf die verminderte Zjstrogenausschiittung der alten weiblichen Mause zurtickgefiihrt werden. Im Experiment wurde nachgewiesen, dafl hohe ijstrondosen diese Eiweinfraktion auf den Wert senken konnen, der den jungen ausgereiften Tieren entspricht. Pe3JOMe

-

I;LIZJIi

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