Effect of gonadal hormones upon serum proteins and steroid binding in maturing female rats

Life Sciences VoL 9, Part I, pp . 878-882, 1970 . Printed in Gr~.t Britain

Pergamon Press

EFFECT OF GONADAL HORMONES UPON SERUM PROTEI£iS AID STEROID BINDING IN MATUr~ING FEMALE RATS Judith A . Ramaley Anatomy-Physiology Department s Indiana University Bloomington Indiana x+7401 (Received 25 February 1970; in final form 24 March 1970) Int~odaction It has been known for some time that the gonads are under the control of the brain-pituitary axis in both immature and mature animals.

In addition to the obvious fact that adult females ea

hibit cyclic gonadal function while immature females do note there are some age-related differences in the control of gonadotrophin (GTH) secretion.

The most important of these differences is that

young animals appear more sensitive to steroids than do adults (1 ) .

To explain the onset of puberty it has been suggested that

the neural centers responsive to steroid feedback alter their threshold of response at puberty (2) .

Other changes that occur at

puberty that may be instrumental in some manner in altering the neural control of GTH secretion include increased efficiency of liver degradation of steroids (3~~+) and other compounds (5), and alterations in the amount and type of steroids produced by the gonads (~+) . One factor that has not been considered in explaining puberty is the possibility that changes in the serum protein binding of steroids may occur .

It is known that the concentration of total

protein and of individual groups of proteins incr®ases with age in both humans (6,7,8) and rats (9) .

Alterations in serum proteins

1Supported by NSF Grant No . GB-849+ and an Indiana University Faculty Grant-in-Aid of Research. 87S

874

EFFECT OF GONADAL HORMONES

in adults appear to be hormonally dependent (10) .

Vol. 9, No . 12 Steroids bound

to serum proteins are thought to be protected from liver degrada tion (11) and to be biologically inactive while bound (12) .

Since

changes in the levels of steroid binding have been reported in various hormonal states in adults (10)~ it is of interest to know whether the drastic changes in gonadal function at puberty might influence the levels of serum protein and the ratio of unbound to bound steroid in the blood. Experimental Procedure 1.

Serum Collection .

Blood was collected by cardiac punc-

tore under ether anesthesia and immediately placed on ice . was allowed to coagulate for 2~+ hours at lected by centrifugation.

4°

Where possible

Blood

C and the serum col-

regular cardiac punc-

tares were performed on the same animal to obtain a series of samples at different times .

The protein concentration was deter-

mined by the Autoanalyzer using a modification of the Lowry method Crystalline bovine serum albumin was used as standard .

(13) . 2.

Radioisotopes .

Estradiol-17 beta-6~7- 3x(5.6 Ci/mmol)

was obtained from New England Nuclear .

Testosterone-l~2-T ß7 .3

Ci/mmol) and Progesterone-4-Cl ~(60 mCi/mmol) were obtained from Amersham/Seale .

Each was found to be over

98~

pure as determined

by thin layer chromatography . 3.

Electrophoresis ç~ Serum Proteins .

To determine changes

in the amount of serum proteins and in the overall profile of the different camponents~ 5 ~], samples of rat serum erere applied to 7 .5~ polyacrplamide gels and the proteins separated by electrophoresis in 0.005 M Tris-0 .0~+ M glycine buffer s pIi minutes in an ice bath .

8 .35,

for

~+5

The gels were prepared according to the

method of Davis (h+) .

To permit visualization of the proteins the

gels were stained with

7iß

amido black (14) and scanned on a Gilford

Vol . 9, No. 12

EFFECT OF GONADAL HORMONSB

gel scanner at 550 mgt.

875

Mayor protein bands were identified by the

methods of Clarke (15) . ~+ .

Determination ~f St r

Protein.

d B

in¢ t~ Different 3~ecies ,Qg

To determine binäing of steroids to serum proteins 1 ng

of radioactive steroid was added to a small test tube and dried under nitrogen . steroid for

~+5

Fifty ~tl samples of serum were incubated with the minutes at room temperature.

Five ~]. samples of

the serum were then separated by polyacrylamide disk electrophoresis as already described.

The gels were sliced into 1.5 mm sec-

tions with a wire slicer~ minced with scissors and eluted for 2~+ hours into 0.9~ saline (1 ml) . 5.

Determination ~ Radioactivity.

Aqueous samples (each

0 .5 ml) were counted at room temperature in a Beckman LS-233 for a total of 10000 counts in a liquid scintillation mixture con taining

5

g of 2~5-diphenyloxazole (PPO) and 100 g of naphthalene

(m .p . 79-80 C, Eastman scintillation grade) per liter of p-dioxane (scintillation grade s Eastman Kodak Company) . from Packard Înstrument Corporation .

PPO was obtained

Efficiency of counting was

about 15~~ as indicated by addition of a tritiated toluene standard . 6.

Administration ~ Steroids .

Estradiol benzoate (Sharing

Progynort`Y, courtesy Sharing Corporation) was administered subcutaneously in oil at the nape of the neck . Animals . Harlan Laboratories

Sprague-Dawley derived rats were obtained from Indianapolis

at 21 days of age .

Puberty in

this strain has been found to occur at 37 ± 1 .~+ (standard error) days during the season of the year in which these experiments were conducted (November,- February) . 8.

Statistical Evaluation .

Significance of the data was

calculated according to the methods described by Clarke (15) .

878

EFFECT OF GONADAL HORMONES

Vol . 9, No . 12

Results 1.

C

es ii n Ser

Proteins

it

Awe i~r. Females .

Total

serum protein concentration increased with age (Table 1) with a major shift between 34 days and puberty. day 25 .

A minor peak occurred at

There was a gradual increase in the protein present in TABLE 1 Total Serum Proteins as a Function of Age Aae

No . Rats

Total Serum Proteins mg/ml

23 days

4

35 ; 2.1*

25 days

~

37 ± 0 .8

29 days

12

32 ± 0.8

3~+ days

~+

33 ~ 2.6

adult 7

l8

57 ± l.5

4

48 ~ 0.6

adult castrated 7

Significance n.s . P<0 .01 n.s . P<0.01

Y

P<0.01

* t standard error n.s . -- not significant the alphal peak and beta globulin peak .

As puberty approached

increases were found in the alpha2 region and in the alphal components.

At puberty there was a sudden increase in the alphal

region (Figure 1) . 2.

Changes ~n Steroid B

din

with.

Figure 2 shows a

typical elution pattern of steroids as compared with a duplicate gel stained with amido black.

It can be seen that there was some

uptake of steroid by all the major protein fractions in the adult . Rather than reproduce similar figures for all the age groups data will be presented in tabular form (Table 2) .

the

A large increase

occurred in the binding of estradiol to the a2 globulins . erty less marked changes occurred in the ~ globulins .

At pub-

tdo changes

Vol . 9, No . 12

EFFECT OF GONADAL HORMONES

ao

ä0 ~

3.0 r

22 days of age

24 days of age

E o ,n

30 days of age

F O

o

'~ 1 .5

H 1 .5

N

T

'~ 1 .5 v

û

O

a=

O 0

3.0~

 â,

pbbulins

_~

v

O

a O

~,.~ ~ aIWiNn

KYu u u a= 6 a, dbumln plobulkN 0 10 20 30 Lengih of gel in mm

0~

10 20 30 Length of qel in mm

D. ~S

s.0~

II

w v

ô u ~â O

i

0 10 20 30 Length of qel in mm

10 20 30 Length of qel in mm

Cyclic femole 40 days of age E

w

v ô u ~â O

uuu u a~alGmin OL I a=

~~

a= `'~ â~ abunn plobulirs

F.

.a .0

36 days of age

'~ 1 .5 m

w~

0

I ~

É 0 LS

O~

E.

32 days of age F 0 u~

877

OL i 0

n

T

~~

v ô u a O

d

R

u u clhumlr~ ulNu ~ o 10 20 30 Length of gel in mr~ Q:

1 .5

n

pl 0

0

u ~~ u o16u,-,~,nln ae pçbulin.a~ 10 20 30 Length of qel in mm

F IG . 1 Changes in profile of serum proteins with age. Gels were stained with amido black and scanned at 550 mgt. globulins or albumin. '1 Effect ~ Steroid Treatment Upon Serum Proteins

were nDted in binding to 3.

878

EFFECT OF GONADAL HORMONES

origln

Vol. 9, No . 12

2 3

ax ZONE

4 3

f- I

6

ZONE

E .5

s s _8 lo

al Z°NE

2~

N II 12 13J

-ALBUMIN

al AL~UMIN

14~

-PREALBUMIN -bU}}M

F3

16~ IB-

frOn1

1

I

1

1

100 P00 CPM astradbl - 3FI

300

FIG . 2 Profile of steroid bound to serum proteins in adult female rat. TABLE 2 Changes in the Binding of Estradiol As a Function of Age Serum Proteins Age Dass

No . ~t$ -

a2

C1A~ JC,

7.0 2- dpxu

~

X

10~-

oc dAm

S

102

Albumin dnm x LO?

23

3

202 ± 22*

168 t 26

835 f 28 .

531 f 22

34

3

232 t 35

203 ~ 16

899 ; 14

525 f 50

3

358 t 14 ~

242 f 13~

995 f 20

474 t 19

40°

°cyclic female

t P<0.01

*mean * standard error of the mean

$ Pc0.05

Stero d Bindinir . diol benzoate

Weanling females were treated with 5 hg estra0 .05 estradiol benzoate or oil alone for 4 days

Ng

beginning on day 24 (day of birth is day 0) .

Ovulation occurred

Vol. 9, No . 12 in the 0 .05

EFFECT OF GONADAL HORMONES

i~g

EB animals on daps

oil-injected controls . layed

(~+0-~+2

compared to

32-3~~

Ovulation in the

5

87 9

3~-~+0

daps in

~g EB animals was de-

days) although vaginal opening occurred. in response

to the exogenous steroid at day

30-31 .

When the serum proteins

were examined at day 2~~ there was enhancement of the alphal globulins and a slight elevation of the beta .globulins in the 0.05 ptg EB rats .

There was no increase in alphal globulins in the 5 ftg

EB rats but there was elevation of the beta globulins .

When bind-

ing of estradiol was compared in serum from the 3 treatment groups it was clear that there was an increase in binding of estradiol to both beta globulin and alphal globulin (Figure 3) . Effect ~ Ovariectomv dg Serum Proteins g~$ Steroid ,B~g$,fig "

Ovariectomy at weaning res~xlted in the retention of a serum

protein pattern characteristic of the immature female . Discussion The present experiments indicate that changes 3n the amounts of serum proteins and the amount of steroid bound by serum proteins occur during puberty in the . female rat.

Increases in .serum

proteins with age have been shown before in the rat (9) but in that study, male and female sera were pooled to reduce variations related to sex differences .

During maturation there is a gradual

incréase in all serum protein components followed by a rapid increase in components in the alphal globulin range at puberty.

The

induction of precocious puberty by the administration of a low dose of estradiol benzoate daily for ~+ days (17,18) results in the precocious appearance of the adult serum protein pattern and causes an increase in the amount of steroid bound by the alpha and beta globulins .

removal of endogenous gonadal steroids by ovariectomy

prevents the appearance of the adult pattern.

These results in-

dicate that the changes in serum proteins found during growth are

EFFECT OF GONADAL HORMONES

880

Vol . 9, No . 12

hormonally dependent.

3.Or

3.Or

0.05Y EB 27 days of age

27 days of age

bL

3.0

~ f __-"M ~`~~

°~~ pugb6uli~

0 10 20 30 Length of gel in mm

0

5Y EB 27 days of age

°~ ;

elfin

10 20 Lergth of gal In mm

30

0

~dlni

ID

20

30

Length of gal In mm

280

240

200 O X AS 1 160 O Ô O Â m

E 120 n u

80

40

0

I

2

3

4 S 6 7 8 9 Slice number (2mm/slice)

10

II

12

FIG. 3 Serum protein pattern and steroid uptake after estrogen treatment . (See idethods Section)

Vol. 9, No . 12

EFFECT OF GONADAL HORMONES

88 1

A number of previous studies have indicated the importance of hormonal condition upon both the levels of hormone binding proteins and the percentage of hormone bound by the blood in adult humans as well as a number of other species (10) .

Daily treatment

with contraceptive amounts of estrogen in humans causes a decrease in orosomucoid~ haptoglobin albumin and total serum proteins (10) . This result would correspond roughly to the suppressive effect of

5

i~g EB in the present experiments .

corticosteroid-binding globulin

Thyroxine-binding globulin

testosterone and estrogen binding

globulins~ fibrinogen and a number of other proteins are elevated during pregnancy in humans (10)~ but most proteins are unaltered during pregnancy in the rat (19) . Only large swings in the secretion of gonadal steroids seem to affect steroid-binding s since no distinct changes are detected during the menstrual cycle (20) in humans or during the estrous cycle in rats (21) .

The shifts in gonadal function accompanying

puberty are sufficient to alter the serum proteins and the amount of estrogen-binding in rats . The possible effects of altered steroid-binding upon the reproductive physiology of the rat are unimos~.

It has been re-

ported that progesterone bound to serum albumin is inactive in a uterine growth test (12) .

Clinical conditions in which elevations

of serum proteins have been reported are also associated with a reduced rate of steroid inactivation (11) .

To what extent this is

due to a protective effect of protein-binding and to what extent general changes in liver function may influence both parameters independently is not lmo~ . The experiments reported here suggest that the binding capacity of the blood for estradiol increases during sexual maturation in the female .

These changes may be involved in the onset of

882

EFFECT OF GONADAL HORMONES

Vol. 9, No . 12

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~~

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