Electrophoretic mobility of the rat blastocyst

CONTRACEPTION

ELECTROPHORETIC

MOBILITY

OF THE RAT BIASTOCYST

C. Alan B. Clemetson Mahammad M. Moshfeghi Vaiyachery R. Mallikarjuneswara Department of Obstetrics and Gynecology Methodist Hospital of Brooklyn Brooklyn, New York 112 15

ABSTRACT The five-day rat blastocyst does not move in a charged field while covered by the aolemma, but does behave as a negatively charged body after loss of this coat; the charge is believed to be due to acidic groups on the surface of the ovum.

INTRODUCTION Mammalian implantation may be better understood through increasing knowledge of factors that influence the time when the egg attaches to the uterus in each species. Implantation delay is known to occur in the rat, and some of the chemical influences are well understood (1,2,3), but physical factors are not. In particular, the electric charge on the ovum is not known for the rat or any other mammal, so we decided to study it,

METHODS Conventional equipment, such as the “Zeta Meter”, could not be used, as this requires a multitude of particles and blastocysts con be obtained only in limited numbers. After many trials, a samll plexiglas open-top box was devised with Iron wire mesh two cellophane partitions dividing it into three chambers (Figure I.). electrodes were placed in the two outer chambers adjacent to the cellophane membranes and an aqueous solution of 8.1 % Dextran in 5% dextrose, adjusted to pH 7.4 with THAM, was placed in all three chambers; they were over-filled with the solution, so as to give a convex meniscus at the top of the center chamber (as shown in the diagram): this convexity allowed the ova, which were less dense than the chosen medium, to float in the center of the chamber instead of drifting to the side as they do when there is a concave meniscus. The usual physiological media could not be used owing to their high conductivity, which resulted in heating and convection currents.

Accepted

May

for publication

1970 Vol.

1

April

No. 5

14,

1970

357

CONTRACEPTION

BAB

FIGURE Three

views

microscope

of the slide

microscopy.

Iron wire

Cellophane sealed are

of the the

membranes

in place

shown

in the

center

position

Plexiglas

(E) for

with

observation of the

cell;

of illumination

and

mesh electrodes are

paraffin

center

electorphoretic

ease (A)

supported wax

diagram. cell.

blastocyst

I.

(D)

mounted

during

indicated

in the

Plexiglas

dividers

leakage. wax

spot

meniscus

(C)

on o

phase

are

to prevent

A black

is shown

by the

Poraffin

under

it

control

contrast

upper

Cellophane

windows

(F) was used to reduce in the

of the

upper

aqueous

diagram.

(B); these

diagram

medium

the

are (Al) volume

indicates

in the

upper

diagram.

358

May 1970 Vol. 1 No. 5

CONTRACEPTION

Rat blastocysts dextrose surface

five

days after

of the central

electrodes

A 32-Volt

chamber.

blastocysts

in the eiectrical

was always

and connected

wax particles saline

electrodes

field

of this

which

was applied

microscopy.

The electric

mobility

mm apart

field

5%

across the two

A reversal

switch

of the in the two

strength

was

in the line of the electric field Freedom from interfering factors,

was ensured by identical

due to convection

currents

in the low conductivity

studies

Paraffin

as the blastocysts.

chamber when high conductivity

field

horns with

just under the

the mean of the velocities

potentiometer.

presumably

but do not move in the electric mixture

I

set

of about the same size

are used,

potential

value.

or eiectroendosmosis,

do move in the electric as isotonic

couid be studied;

to a high resistance

such as heat convection

the uterine

to a position

so that the electrophoretic

taken as the final

measured by two platinum

D.C.

by phase contrast

circuit

in both directions

directions

by washing

the ova were transferred

and the ova were studied

was provided

paraffin

were collected

mating;

with

particles

soiutions

such

caused by heating,

Dextran-dextrose

was used for these experiments.

RESULTS At pH 7.4, in a charged

field,

blastocysts

with

their

bodies and moved consistently

towards

the positive

gradient

exceeded

3 V/cm

(or 30 mv/lOO microns).

mobility

at pH 7.4

was i.l

microns/set/V/cm.

Studies markedly reversai

covering

pole at pH 7.4 Their

of charge on lowering

superficiai

did not move

surface

acidic

charged

when the voltage

mean electrophoretic

at pH 5 showed the charge on the naked blastocysts

reduced and the charge was absent at pH3.

argues against

of oolemma,,

but those that had lost this coat behaved as negatively

the pH below

protein

to be

The fact that there was no

the usuai

isoelectric

point

as the source of the charge and suggests

of proteins, the presence

of

groups.

DISCUSSION Our calculations

reveai

the oolemma,

possesses a negative

or an electron

excess of about

l/4-million

Calculations

of the Zeta

about

that the five-day

rat blastocyst,

charge of approximateiy

after

loss of

47 x IO-15 couiombs

electrons. potential

suggests

a surface

potential

of

100 millivolts. Full

publication Wilderness, early

surface

details

of these experiments

of the proceedings Washington

(July

of the Blastocyst 10-13,

and calculations Biology

Symposium

1969) by the University

will

be given

in a

held at Lake

of Chicago

Press,

expected

in 1970.

May 197OVol.1No. 5

359

CONTRACEPTION

REFERENCES I.

Conivenc,

R. and Laffargue,

M.,

2.

Cochrone,

R.

L. and Meyer,

R. K.,

3.

Barnes,

E.

and Meyer,

360

L.

R. K.,

C.

J.

R.

Acad.

Sci.

(Paris)

Proc.

Sot.

Exp.

Biol.

Reprod.

Fe&l.

242:

2857

Med.

96:

(1956). 155 (1957).

7: 139 (1964).

May 1970 Vol. 1 No. 5