Transport of naturally occurring amino acids and alpha-amino isobutyric acid by normal and diapausing mouse blastocysts

Vol. 78, No. 1, 1977

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

TRANSPORT OF NATURALLY OCCURRING AMINO ACIDS AND ALPHAAMINO ISOBUTYRIC ACID BY NORMAL AND DIAPAUSING MOUSE BLASTOCYSTS

Biochemistry

Lon J.

VanWinkle*

Dept.,

Wayne State Detroit,

Received

July

and Danica

Dabich

University

Michigan,

School

of Medicine

U.S.A.

8, 1977 SUMMARY

Uptake of 14C-labelled a-amino isobutyric acid and a mixture of naturally occurring amino acids by normal mouse preimplantation blastocysts was found to be twice that of delayed implantation blastocysts. With both groups of embryos concentration of the amino acids against a gradient occurred. The results may explain, in part, the lower amino acid incorporation rates found with diapausing vs. normal blastocysts and open the question of “metabolic dormancy" of the diapausing embryo. INTRODUCTION When diapausing vitro

with

found

in proteins

than

radioactive

in normal

factors

of labeling gradation,

contribute

rate

correlation

rates

and rates

of embryos

(2,3),

mine whether *Current

0

1977

of reproduction

of

b.v Academic in any

similarly

rates

it

appears

protein

synthesis.

between

is,

and de-

specific

at this indeed,

activity

Since

amino acid

of the precursors

Dept. of Natural Michigan-Dearborn, Press, Inc. form reserved.

synthesis and the

transport

Among

and the efficiency

transport,

necessary,

less

(1,2,3).

difference

to exist

-in

label

significantly

of protein

incorporation

amino acid

address:

treated

used for

appears

amount of

is

of amino acid pool

the

embryos

to this

absolute

amino acid

close

Copyright All rights

of these

are:

are incubated

amino acids,

blastocysts

which

of the

mouse blastocysts

a

transport

into point,

protein to deter-

diminished

in

Sciences, University Dearborn, Michigan

of

Vol. 78, No. 1, 1977

diapausing uptake acid

BIOCHEMICAL

embryos.

This

of naturally (AIB)

by both

study,

occurring normal

acids port

and/or

by the rates

utilization

two classes

therefore,

amino acids

and diapausing

compound was used to obviate metabolism

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

possible of

concerns and a-amino blastocysts.

differences

the naturally

of embryos

and,

itself

thus,

with

isobutyric The latter

associated occurring evaluate

with

amino trans-

specifically. MATERIALS AND METHODS

The method of Edwards and Gates (4) was used to synchronize estrus and superovulate sexually mature, random-bred Swiss-Webster mice. The presence of a vaginal plug the morning following mating ascertained mating and denotes Day 1 post coitus (p-c.). To obtain delayed blastocysts, bilateral ovariectomy was performed on pregnant mice ca. 80 hours p.c. (5). Normal mothers and those in implantation delay were sacrificed on the same day between 0700 and 1600 hours at the times p.c. indicated in Tables I and II. Blastocysts were flushed from excised uteri with modified Brinster's medium, MBM-2, which incorporated the following changes: 68.94 mM NaC1,29.93 mM sodium lactate, 0.49 mM sodium pyruvate and 11.11 mM glucose (6). Blastocysts were washed once in MBM-2, then transferred in groups of 5 to 50 embryos to Maximov slides containing 0.20 to 0.50 ml of MBM-2 plus 4.4 uCi/ml of a 14C-amino acid mixture (54 mCi/Milliatom: CFB 104; Amersham-Searle) or 5.0 yCi/ml of l- 14C-AIB (60 mCi/mMole; Amersham-Searle). After labeling, each experimental group was washed twice in MBM-2 before solubilization of groups of 2 to 10 embryos in microtiter wells containing 0.10 ml MBM-2 and 0.10 ml 4% (w/v) Each solution was quantitatively transsodium dodecyl sulfate. ferred to a 3 MM filter paper disc which was air dried. The dried discs were either counted, as described below, for total uptake or processed according to Mans and Novelli (7) for measurement of incorporation. The scintillation fluid used for uptake measurements was like that used for incorporation determinations (7) except that the solvent system was toluene: cellosolve (l:l, v/v) Cellosolve reduced the to accommodate water in the samples. to a maximum efficiency counting efficiency by 15% in contrast were processed identically to of 90%. Discs used as controls experimental samples. To assess acid soluble uptake, the average value for incorporation was substracted from each individual total uptake determination. Data is presented as the mean value for cpm/embryo after subtraction of appropriate control values. The 95% confidence limits (c.1.) are also reported. Means of groups where the embryo type was the only variable were compared by a group comparison test (8). RESULTS With

both

normal

and diapausing

358

embryos,

incorporation

Vol.

78,

No.

1, 1977

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Vol. 78, No. 1, 1977

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Table

II

Total uptake of l-14C-a-amino isobutyric after 1.7 or 3.0 hours. In each case there experiments and a total of 12 samples.

Type of embryo

acid by blastocysts were 2 independent

Day p.c.

Labeling period (hours)

normal

5

1.7

43 + 13

delayed

8

1.7

18k

normal

5

3.0

74 i

18

delayed

8

3.0

32?

7

values into

parallel acid

soluble

accumulate

I and II. of utilization

of

increased

does not

the it

might

ported

more rapidly

and take

be argued for

case.

occurring

by normal

An alteration,

cysts

therefore,

as a consequence

transport

with

at

in part,

least

diapausing

in terms

Tables

the

rate

protein

enhanced

synthesis

shares

this

transport (91,

is

blastocysts

or incorporated

is diminished diminished

normal

into

rates.

360

systems also

trans-

although protein

of blastoamino acid

incorporation

blastocysts

of differences

caused

blastocysts,

diapausing

diapause

vs.

that

radioactive

in the biochemistry

Moreover,

capability.

observed

of

day 5 embryos

blastocysts,

amino acids

vs.

5

precursors

up exogenous

which

is not known to be metabolized,

(10).

normal

by normal AIB,

CPM/blastocyst up _t 95% c.1.

radioactive

of diapausing

uptake

naturally

of

However,

the precursors

seem to be the

neutral

I.

rate

amino acid

of the

it

Table protein

at twice Although

of appearance

pool,

radioactive

amino acids

the

the rate

Total taken

rates

are explicable,

in precursor

transport

Vol. 78, No. 1, 1977

BIOCHEMICAL

Less than associated

with

non-specific

both

a gradient

is believed than

and day 5 normal respectively: "C-AIB

blastocysts

is

and delayed

of

a protein accumulate

(11)

then

following

AIB cal-

of delayed

and 0.45

nl

(12),

the maximum radio-

blastocysts if

average after

Table

II.

must have occurred. amino acids

in

precursor.

would be 10 and

equilibration amount of

three

hours

with

14C-AIB

the

in

in 99 dpm

cpm per dpm) and 43 dpm (32 ? 7 cpm/O.75

respectively,

occurring

of the

2.2 c lo6 dpm; and (I=) the

respectively, the

One

enzyme(s)

the volumes

0.91

pCi/ml,

blastocysts

(74 + 18 cpm/0.75

a gradient

5.0

However,

medium occurred.

(a)

and normal

5.0 dpm per blastocyst,

per dpm),

transfer

blastocysts

are ca.

(b) one PCi equals

in diapausing

normal

that

in formation

that:

of

per se. --

can be shown by means of the

concentration

activity

to be the result

incorporation

and diapausing

When one considers

culation.

up was found

of AIB by an unidentified

and resulted

normal

taken

the possibility

group

embryos occurred

14C-AIB

rather

discount

carboxyl

That

total this

adsorption

radioactive

against

the

protein:

however,

cannot,

the

10% of

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Thus concentration Concentration

cpm

against

of the naturally

can be shown similarly. DISCUSSION

The biological amino acid cysts the

is

protein

transport

that

steps

these

involved

to absolute

(a)

results:

during of

delineate

since

synthetic

delayed

rates.

incorporation rates

vs.

implantation, transport The latter rates

diminished normal

blasto-

a change in one of

of exogenous

the diminished

of incorporation

importance

in diapausing

in utilization

the effect

measurements

of the observed

activity

biosynthesis

monstrate

cular

significance

precursors

and (b) deactivity is

on

of parti-

have been equated

and have been used as indicators

361

for

of

Vol. 78, No. 1, 1977

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

120-

120 j

iI0 loo-

100

il

f cpmlO-L

mm--~-, cpm10-L

'

-d

L?

Fig.

3.

Effect of lipoxygenase concentrations luminescence. Curve a) 5.2 x IO-*M; d) Noise level. No luminol.

Fig.

4.

Effect of superoxide dismutase (SOD) on the chemiluminescence. Curve a) no SOD; b) 1.2 x IOm8M SOD; c) 6 x 10d8Y SOD; d) 1.2 x 10m7M SOD. No luminol.

linoleic

acid

increase

and the sharp

the

anaerobic Fig.

substrate

starts

2 shows light well

a sufficiently

emission

just

linoleic

the oxygen in contrast

240 nM. Increasing

substrate

decrease

5 PM. Thus, precede

gradual

the onset

of

in time

concentrations.

At

concentration

(240 uM) there

to the effect

of a substrate

the enzyme concentration

concentration required

acid

(> 240 uM) brings to attain

about

the luminescence

is at

a maximum

3). In Fig.

4 the effect

of superoxide

the lipoxygenase/linoleic

(70 units/ml) which

of varying

below

above

high

proportional

below

of luminescence

the effect

concentrations

concentration

from

decline

concentration

reaction.

no significant

(Fig.

at an oxygen

on the time course of b) 2.6 x 10T8M; c) 1.3 X 10e8M;

is

already indicative

The addition

cause

acid

dismutase

system

an almost

complete

of the involvement of catalase

is

on the light

shown.

quenching

of 0'2 in while

had no effect

426

Small

emission

amounts of

the luminescence

the light-emitting carbonate

process.

(up to 0.25

M)

Vol. 78, No. 1, 1977

12. 13. 14. 15. 16. 17.

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Cole, R. J., (1967) J. Embrol. Exp. Morph. (1973) Regulation of Mammalian McLaren, A., 321-334, Thomas, Springfield, Ill. Psychoyos, A. (1973) Handbook of Physiology, part 2, 187-215. Weitlauf, H. M. (1974) J. Reprod. Fert. 39, R. M. and Tasca, R. J. (1974) Dev. Borland, Epstein, C. J. (1975) Biol. of Reprod. 12,

363

17, 481-490. Reproduction Section 213-224. Biol. 36, 82-105.

pp.

7, vol. 169-182.

2,