Biphasic effect of extracts of Lithospermum ruderale on the uptake of 32P by testes and thyroids of cockerels

Vol. 67, No. 3, 1975

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

BIPHASIC EFFECT OF EXTRACTS OF LITHOSPERMUM RUDERALE ON THE UPTAKE OF 32P BY TESTES AND THYROIDS OF COCKERELS Frank

Received

J. Zeller, W. R. Breneman, Marvin Carmack, C. J. Kelley Departments of Zoology1 and Chemistry Indiana University, Bloomington, Indiana

October

9,1975

Summary - A polyphenolic fraction of the plant Lithospermm ruder&e was injected into White Leghorn cockerels at various time intervals and the effect on testes and thyroids was measured by the uptake of 32P. The 32~ uptake 8 hours after lithosperm2 injection was markedly depressed and 12 hours after administration was notably increased. It is suggested that the hypothalamic regulation of the testes and thyroids is altered by lithosperm interference of the hypothalamic feedback system and/or the binding of receptor sites in the two glands. The synthesis been shown fractions exist

and release

to be inhibited (2)

by cold

of the plant

concerning

of chick

the site

hormones

of the anterior

pituitary

cells

administered ments were

analyzed

for

from

the anterior

thyrotropin the

testes

their

action

and/or

(1)

effects

the inhibition of the sensitivity

hormones.

before

on the

pituitary

including

possibilities

This

of Lithospermum

times

autopsy

release

has

and polyphenolic

Several

the altering

fractions

at various

and thyrotropin

ruderale.

to releasing

polyphenolic

to cockerels

extracts

2

of lithosperm

releasing

in which

water

Lithospermum

of hypothalamic

experiments

gonadotropins

paper

reports

ruderale and the

were treat-

of gonadotropins

as assayed

by the uptake

and

of 32P by

and thyroids.

Materials and Methods. Single-comb White Leghorn cockerels aged 2, 9 or lldays were used in the experiments. The chicks were removed from feed 12 hours prior to being killed at which time the glands were quickly removed and weighed to the nearest 0.1 mg. The thyroids and testes were dried and then counted on a Nuclear Chicago gas flow counter. The end-point of the assays was the uptake of 32 P in terms of wet-weights of the glands. Counts were expressed as Antilog Llo CPM Cl,3 X \r Body weight (gm) gland (mg) Analysis of variance (ANOVA) was employed for the evaluation of data and the

1

Waterman

Contribution Institute.

2. inhibiting

The term lithosperm is used principle, or principles,

1.

Cop.vright AN rights

number

o 19 75 b-v Academic Press, Inc. in anv form reserved.

of reproduction

1019

from

the Department

as a generic in the genus 1234

of Zoology,

designation Lithospermum.

for

115 from

the

the hormone

Vol. 67, No. 3, 1975

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

t-test was used when two groups were compared. The anterior pituitary glands were stored in acetone, then air-dried, ground, suspended in distilled water and assayed for their gonadotropin and thyrotropin content. Injections were made subcutaneously in a volume of 0.2 ml. Carrier free 32P was administered one hour before autopsy. The lithosperm fractions were prepared from water-soluble extracts of the dried roots of Lithospermum ruderale The polyphenolic fraction Ea used in these experiments was prepared from a (3). combination of chromatographic separations on Sephadex and a chemical fractionation (3, Table 1, Footnote a>. Three extraneous thyroids of different in body weights. No either analysis. (b) grams of wet-weights groups were comparable morning and continued the pineal gland was ment was performed in spanned the duration ferences were observed Results.

Fraction

eleven-day-old

variables were analyzed: (a) The weights of the testes and groups were compared and also were corrected for variations significant differences between groups were observed by The uptake of 32P by the two glands was expressed in milliand of dry-weight and related differences between treatment by either method. injections began in early (c) Finally, until early afternoon, therefore, the light stimulation via considered as a possible source of variation (4). An experi which cockerels received only 32P at time intervals which of the lithosperm experiments. No significant group difin the 32P uptake by either the testes or thyroids.

Ea (3) was administered

cockerels

four

hours

before

were

bimodal.

beginning

autopsy

There

initiated

at 10 or 12 hours ranges

and a very

at the

The effects related

(2)

treatment, The total of both one-hour 7.52,

treatment the

injection P < 0.007

5.10,

in

in

before

autopsy.

the

overlapped

three

groups

Notable for

were

12 hours

had the maximum deviation

fact

which

increases and the from

P uptake

the

testes

6 and &hour

when treatment

was

overlapped

the con-

examined by the

testes

that

the range range.

received

combined control

1235

responses value;

of the

uptake

of 8 hours

occurred

testes:

F = 106.75,

F = 0.4 mg

of 32P duration;

following

of these

1B).

The combined

of the means of the A lowered

of

the influence

of lithosperm.

treatment

in 32 P uptake

(Table

reflected

32 P uptake

the

to be dose

by the duration

simultaneously

elevated

control

are known

to be modified

and of the dosage

the

of the

in the

and thyroids

shown

were

slightly of the

and terminating

responses

uptake

of

periods.

on ')LP uptake

periods

32

to groups

autopsy

The 32 P counts

on the testes

of treatment

in spite

P ~0.025.

to lithosperm

increase

variables

effects

duration

and 0.8 mg dosages occurred

marked

experiment both

injection

before

in 'LP

of lithosperm

therefore,

hours

decrease

4, 14 and 16-hour

and in this

a single

The 32 P uptake

1A).

was a notable

groups

trol

sixteen

(Table

treatment

in

three

F =

exposure groups

P ~8.0 -10 .

The

Vol. 67, No. 3, 1975

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

TABLE

Testes Uptake of 32 P in Eleven-Day-Old Cockerels Which Ea. 2.0 u Ci 32P Was Injected One Hour Before Autopsy

A.

Duration -Testes: X

Range

iz Range ANOVA

B.

1

Treatment

Control 180.57 203.98-159.86

4 Hours 188.36 210.23-168.77

6 Hours 138.77* 154.91-124.30

8 Hours 128.30*** 145.00-112.75

10 Hours 378.07*** 412.74-346.32

12 Hours 263.11*** 290.78-238.09

14 Hours 196.92 216.92-178.77

16 Hours 218.99 243.26-181.60

Treatments 7 Error 104

Testes

of Lithosperm

Received 0.4 mg of Fraction To All Animals. f = 14.

and Thyroid

F MsQ 0.3745 47.38 S2 = 0.0066 Gland Amount

Uptake

<2t)o-29

of 32 P in Nine-Day-Old

and Duration

of Lithosperm

Cockerels

f = 14.

Treatment

1 Hour Testes: - X Range

Control 176.84 198.18-157.79

0.2 mg 227.14** 255.91-201.60

0.4 mg 210.00 235.57-187.20

0.8 mg 198.09 221.83-176.87

154.07 172.40-137.69

134.36** 150.88-119.64

330.37*** 365.95-298.26

291.60*** 319.11-266.43

8 Hours

z

171.85 191.97-153.83

Range

12 Hours 432.16*** 523.92-356.49

z Range ANOVA (Excluding

Controls)

MsQ 0.3774

Treatments 8 Error 117

F 44.93 S2 = 0.0084

< 4:2-32

1 Hour Thyroids: z Range

Control 203.10 223.01-184.98

0.2 mg 266.25*** 305.70-231.89

0.4 mg 297.90*** 341.53-259.82

0.8 mg 241.70 276.59-211.20

157.29** 173.68-142.27

149.84** 164.56-136.44

305.59*** 350.21-266.65

236.98 265.35-211.65

8 Hours

z

181.40 203.59-161.64

Range

12 Hours

f

348.68*** 425.49-285.71

Range ANOVA (Excluding

Controls)

Range = 95% Confidence

Limits.

Treatments 8 Error 117 *P = ~0.05,

**P = < 0.01,

1236

WQ 0.2402

F 24.06 S2 = 0.010

***P=
< 25-21

by t - test.

Vol. 67, No. 3, 1975

responses those

BIOCHEMICAL

of testes

described

for

The data those

for

that

very

three

similar

with

fore

autopsy

treatments thyroid

F = 15.08,

had a substantial which

were

glands;

F = 26.78,

was the most inhibitory

dosage

for Lower

dosages

ment effects

were

groups,

groups

or when all

nificance

being

the

0.02

but

the 0.06

than

similar

mg dosage

that

dosage

levels.

clined

as the dosage The data

the

three

P co.04. an interaction

but

which which

There

was a

were

treated

one-

began

8 hours

be-

P co.003 32

augmented

whereas

P uptake

in all

three

the responses

tested

next

(Table

of the

testes

for

by the time-

at this

the control

The 0.18

to the 0.2 mg dosage 8-hour

group

mg dosage

did

the

either

mg dosage

Total three

treat-

one-hour

as individual did

approach

sig-

in Table

1B.

The 32P uptake

differ

from

the

not

groups

The animals

2).

had substantially

which

received

control lower

lithosperm

at

average uptakes 12 hours

had 32 P counts

higher than those of the controls at all three 32 the P uptakes in the 8-hour and 12-hour series de-

Once again

for

of lithosperm

32 P uptake

were

one-hour This

with

testes.

with

from

combined.

animals.

autopsy

testes

not differ

mg and the 0.18

of control

suggested

similarities

The 0.8 mg dosage

than

of each of the

F = 9.96,

markedly

Ea (3) were

were

in the

before

the

did

effects

the

groups

effect;

The 32 P uptake

groups

for

Treatments

duration

more variable

testes.

of Fraction

however,

showed

and was comparable

marked.

dosage

P ~1.5~'~.

paralleled

treatment

values

inhibitory

by the

were

Comparisons

the three

P ~6.4~'~.

groups

for

observed

in

of 12 hours

32P uptake

glands

present.

effects

by lithosperm

autopsy;

thyroid

thyroid

time-dose

stimulation

before

control

therefore,

1A.

the ANOVA F-value

the

to the relative

in Table

were

RESEARCH COMMUNICATIONS

experiment,

by the

differences

periods

hour

treatments

testes,however,

significant

pronounced

in this

the 32 P uptake

for

the

time

respect

to lithosperm

AND BIOPHYSICAL

less dosages

was increased.

by the

affected

of dose x time.

is

in general

by the lithosperm.

differed

significance

thyroids

from

the control

paralleled The combined at an F value

questionable,

however,

After

of treatment

8 hours

1237

those

because

of

effects

of

of 4.46,

ANOVA indicated

the mean for

32P

Vol. 67, No. 3, 1975

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

TABLE 2 Testes and Thyroid Gland Uptake of 32 P in Nine-Day-Old Cockerels Fraction Ea. 2.0 u Ci 32P Was Injected One Hour Before Autopsy f = 13. Treatment Amount and Duration of Lithosperm

Which To All

Received Animals.

1 Hour Testes: z Range

0.02 mg 185.09 213.58-160.39

Control 181.23 205.50-170.60

0.06 mg 190.34 212.89-170.19

0.18 mg 206.10 218.94-194.30

139.27*** 154.85-125.24

129.40*** 145.75-114.

242.10*** 278.04-210.81

225.14** 243.46-208.19

8 Hours

i Range 12 Hours z Range ANOVA (Excluding

291.02*** 327.96-258.24

P -31
F MSQ 0.1610 25.56 S2 = 0.0063

Treatments 8 Error 108

Controls)

1 Hour Thyroids: z Range

0.02 mg 156.73 169.77-144.70

Control 174.40 191.14-159.12

0.06 mg 150.05 168.75-133.42

0.18 mg 153.87 168.05-140.88

106.30*** 117.60-96.08

105.84*** 118.53-94.50

182.45 201.91-164.87

161.72 192.97-135.54

8 Hours

z

145.70 161.35-131.57

Range

12 Hours 225.10*** 253.14-200.14

ii Range ANOVA (Excluding

Range = 95% Confidence

uptake

at 0.02

The 12-hour

mg dosage

series

elevated

of time

in

however,

gland

to determine hormone

with

except

**P

lower

at the 0.06

contrast

the effect

In order

*P = < 0.05,

was slightly

32 P uptake

x dosage,

of variance,

pituitary

Limi.ts.

of 32 P uptake

depression

cantly

Treatments 8 Error 108

Controls)

when the interaction of time was apparent; the effect content,

than

that

1238

= < 0.001

of the control

data,

not

There

cockerels

but

by t - test.

the

show signifiwas interaction

mean was employed F = 17.57,

< A-10

was more marked.

did

mg dosage.

of the lithosperm

U-day-old

***P

mg dosages

testicular

at the 0.02

F 19.78 = 0.0065

= < 0.01,

mg and 0.18 the

MSQ 0.12862 S

as an estimate

P
were

injected

on anterior with

0.4 mg

Vol. 67, No. 3, 1975

of Ea (3) on the

BIOCHEMICAL

at 1, 2, 8 and 12 hours

thyroids

and testes

those

previously

described,

animal

received

itary clearly hour,

the

a-hour

noteworthy

that

in

donor

the one-hour

whereas

gonadotropin.

of the

uptake

and 12-hour

gonadotropin

mals were

“P

"donor"

birds these

The effects were

data

groups

differed

are

were

32

group

P uptake

those

not

fresh

chicks

received

(Table

glands

groups

Each assay anterior

groups.

had a much higher

testes

the one-

had a lower

thyroids

pitu-

3A) indicate

from

the control

by the

of the

the same as

tabulated.

of donor

from

recipient

of the lithosperm

essentially

the assay

which

recipient

in the than

in

the groups

the 12-hour

striking

testes

and a-hour

Differences less

autopsy.

dose of 1.0 mg equivalent

The responses

that

before

therefore,

a single

gland.

of the

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

is

titer

of

level

of the

and there

It

were

of

assay

ani-

two varia-

TABLE 3 Testes and Thyroid Uptake of “P in Two-Day-Old Cockerels Given Anterior Pituitary from Donor Birds Which Received 0.4 mg Lithosperm Fraction Ea for One, Two, Four, Eight or Twelve Hours. 1.0 u Ci 32P Was Administered Hour Before Autopsy. 1.0 mg Equivalent of Fresh Anterior Pituitary Gland Injected Five Hours Before Autopsy. f = 15. A. Donors:

x

Range

ANOVA

Treatments Error

1 Hour LSPM 149.21*** 160.55-138.68

2 Hours LSPM 166.95 184.59-151.00

Donors: j; Range

8 Hours LSPM 124.39*** 131.32-117.84

12 Hours LSPM 240.99*** 276.16-210.28

MSQ

0.3832

S2

F 58.95 = 0.0065

ANOVA

Treatments Error

Thyroids

Control AP 100.20 110.51-90.86

1 Hour LSPM 73.10*x* 82.91-64.45

2 Hours LSPM 93.01 103.62-83.49

Donors: z Range

8 Hours LSPM 67.91*** 76.06-60.64

12 Hours LSPM 100.00 110.61-90.41

5 a4

Range = 95% Confidence

=Q 0.079Z2 s Limits.

F 11.65 = 0.0068

4 Hours LSPM 173.97 195.84-154.55

< 3Po-25 B.

Donors: x Range

Testes

Control AP 179.78 191.21-169.02

5 a4

One Was

4 Hours LSPM 87.28x 94.46-80.66

< lPawoa

*P = < 0.05,

**P = < 0.01,

1239

***P

= < 0.001

by t - test.

Vol. 67, No. 3, 1975

in

tions

the responses

pituitaries uptake

from in

the

which

Discussion.

the

elevated

variable.

The thyroids than

increased

did

after

therefore,

as well

are

similar

rat

(5,6,7,8)

observed the rat

in which

following

(LH-RF)

the response terior

but,

by endogenous

responsiveness

The timing cockerels

of these but

assay

responses

parallels.

titers

of both

gonadotropins

animals

synthesis

had marked

and release

of

32

control.

P uptake

It

was suggested

prior

the anterior

but

the

;IL

P was

of lithoof

hour

the

pituitary

gland

was

following

injection

hormone

releasing

an enhancement cells

to LH-RF,

that

of the rat the cells

secretion,

may be

and that feedback effects

treated testes It

hormones

is

group

pitui-

axis. on

had low

and thyroids evident

that

was inhibited.

of an-

of lithosperm-treated

8-hour

P uptake.

data

in the female

the

glands

lithosperm

pituitary

that

and the

1240

had

to a lesser

to the duration

of the lithosperm

pituitary

32

more

administration

the reverse

of

somewhat

The analysis

One

estrogen

and thyrotropin depression

autopsy

The thyroidal

in a hypothalamic

from

of "P,

injection

respect

sensitivity

by variations are

received

before

to luteinizing

estradiol

The glands

suggest

were

on the anterior

after

their

which

have been reported

sensitivity

of the anterior

chicks

these

in

caused

from

had a

12 hours

of estradiol.

LH-RF following is

and the

differ

uptakes

not known

which

effect

hours

cyclic

are

received

employed. is

to results

decreased

eight

with

of lithosperm

to LH-RP occurred.

pituitary

"primed" tary

showed

depressed

was similar.

of the dosages

a biphasic

animals

in one experiment.

the administration

pituitary

factor

the pattern

terms

in some respects

not

to the lithosperm

treatment

of action

which

the control

donors

intervals

must be evaluated

as in

The mechanism

time

responded

but

that

lithosperm

Other

glands

the one-hour

sperm effects, treatment

administered

testes

did

the S-hour

had markedly

'LP uptakes.

the

chicks

than

glands

demonstrate

autopsy

of birds

lower

donor from

in the

the control.

responses

strikingly

degree

than

P uptake

was slightly

the glands

before

testes

32

The

donors

given lower

8 hours

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of the 12-hour

Testicular

lithosperm

3B).

4-hour

were

was markedly

whereas

(Table

the recipients

The birds which

BIOCHEMICAL

of both The

Vol. 67, No. 3, 1975

pituitary

glands

12-hour

of the assay

treated

and thyrotropin "P

BIOCHEMICAL

uptakes.

on the other

and the

testes

at this

time

releasing

factors

is

release

leasing

factors

and decrease tion

in the

and release

has only

effective

had broken

or if

suggests

pituitary

either

additional

the

by the

may not

occur

cells

of treatment

could

The occurrence

of either

or both

the sensitivity

of the pituitary

cells

to stimulation;

would

be explained

if

synthesized

in

would

in pituitary

events

secre-

lithosperm

factors

during

of these

the re-

the lithosperm

increase

complex

of inhibition.

effect

Secretion for

with

of hypothalamic

had been

greatly

of hypothalamic

The marked

a receptor-lithosperm receptors

were

treatment.

may complex

release

had enhanced

hormones

8 hour

the

gonadotropins

donors

action

from

or the receptors

to respond.

A sudden

of both

of the 12-hour

in some fashion

action.

material

titers

that

12 hours

if

had high

This

of the cells

a short-term

pituitary

of the pituitary

the anterior

after

especially

hand,

the hypothalamus

ability

received

and release

period.

blocked

from

which

and thyroids

synthesis

augmented

and/or

birds

donors,

Both

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

be

cells

the interval would

essentially

increase

a "priming"

occur. References

1.

W. R. Breneman and Frank 1049-1053, 1975.

2.

W. R. Breneman; Comp. Endocrinol.

3.

Charles J. Kelley; J. R. Mahajan; R. Breneman and Marvin Carmack.

4.

Jerome

5.

G. Zaballose

and S. M. McCann.

6.

C. Libertun;

R. Orias

7. 8.

A. Castro-Vasquez K. J. 1975.

Zeller.

Biochem.

Frank 3. Zeller; Marvin (In Press) 1975.

M. Yochim

Cooper;

J.

and Edward

Lucille J. Org.

P. Wallen.

and C. J. Kelley.

94: 1094-1099, Endocrinol.

and S. M. McCann.

Comm. 65: Gen. and

A. Neubert; 1975.

of Rep. 11: 125-132,

Endocrinol.

1241

Res.

C. Brooks; Leonard Chem. 40: 1804-1815, Biol.

and S. M. McCann.

C. D. Fawcett

Carmack

Endocrinol.

and S. M. McCann.

and Biophys.

96:

1974. 1377-1385,

97: 13-19, Endocrinol.

1974. 1975.

1975. 96:

1123-1129,

W.