differences in the rotation spectra of mouse oocytes and zygotes

Biochimica et Biophysica Acta 930 (1987) 65-71 65 Elsevier

BBA 12072

D i f f e r e n c e s in the rotat ion spectra of m o u s e o o c y t e s and z y g o t e s

G. F u h r a, F . Ge i s s l e r b, T. Mi i l l e r a, R. H a g e d o r n a a n d H . T o r n e r b

a Department of Biology, Humboldt University of Berlin, Berlin and b Academy of Agricultural Sciences, Research Unit of Animal Production, Dummerstorf-Rostock (G.D.R.)

(Received 9 January 1987)

Key words: Fertilization; Electrorotation; Mouse egg

Rotation spectra of mouse oocytes, zygotes and embryos in the two-cell stage under the influence of high-frequency rotating fields were studied. The characteristic frequency (fcl) of cells isolated from superovulated + mated mice is different from that of oocytes. This was attributed to an increase in the membrane resistance and, less probably, to a change in the zona pellucida conductivity. The rotation spectra can be used to differentiate between non-fertilized and fertilized eggs. A theoretical interpretation of the measured spectra and simulation of the changes caused by fertilization is given.

Introduction

As described previously [1-10] electrorotation is a new method to determine the electrical properties of the constituents of single cells, such as membrane, interior or cell wall systems. High- frequency rotating electric fields are applied to induce cell rotation. Each cell shows a particular dependence of its rotation speed on the angular frequency of the external applied field frequency [1,3,5-7,9]. Polarization processes at the interfaces of all cell components determined by their electric properties, i.e., conductivity and dielectric constant, cause cell rotation. The rotation spectra are composed of several characteristic frequencies due to different relaxation processes. Each peak is given by its characteristic frequency fc, i.e., the frequency of the external rotating electric field at which a maximum angular velocity of the cell is observed [5,6]. The height of the peak is the maximum rotation Rma x ( R m a x = Wmax/E 2, where Wma x is the maximum angular velocity of the cell

Correspondence: G. Fuhr, Department of Biology, Humboldt University of Berlin, 1040 Berlin, Invalidenstrasse, 42, G.D.R.

and E the external field strength [11]). In general, the rotation spectra of living cells show two peaks with opposite cell spin direction (co-field and anti-field rotation). Shape, position and amplitude of the peaks, however, are dependent on the electrical properties of the cell. The characteristic frequency (fcl; in the kHz range, [1-3,6,7]) is sensitive to changes in the membrane properties as well as in the surface structures.

In previous papers it has been shown that electrorotation can be used to determine changes in membrane conductivity and capacity induced by pharmaca, ionophores or virus contact [2,4, 5,9-11 ]. On the other hand, it is known, that in many invertebrate and vertebrate species, including mammals, the egg plasma membrane, the egg surface and also the egg interior are changed during and after fertilization [16-18]. Short time changes of the membrane potential and resistance as well as processes such as cortical granule exocytosis, membrane or zona pellucida hardening as well as structuralization pro, cesses, e.g., the development of mosaic membranes, have been reviewed recently [18,19,21]. All these processes should lead to differences in the electrical properties between

0167-4889/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)

66

oocytes and zygotes, and consequently should result in different rotation spectra of this cells. Therefore, we investigated the rotational behaviour of mouse eggs with the aim of differentiat- ing between oocytes and zygotes by means of electrorotation. This could be of practical impor- tance since electrorotation is a fast method and, in contrast to the electrode impalement technique, does not cause cell wounding. Especially in the case of embryo transfer or similar procedures it is important to characterize the vitality and other cell conditions, e.g., the state of fertilization, before cell manipulation procedures or embryo transfer take place.

Materials and Methods

E[ectrorotation measurement Two types of measuring chambers were used

(Fig. 1). Type A was constructed according to the method of Arnold and Zimmermann [7]. The chamber consisted of four electrodes mounted on

/

6

5

I

At5

~ x

Fig. 1. Scheme of the experimental apparatus. 1, 0.28 M mannitol solution; 2, 0.28 M metrizamide solution; 3, cell; 4, electrodes (stainless steel); 5, microscope slide; 6, microscope cover slide; 7, microcapillary for cell removal; 8, microscope; 9, electrode holder; 10, holder for electrode calibration with

micromanipulator; 11, insulating cylinder.

a microscope slide. The electrodes formed the sides of the squared chamber, which was filled with two solutions of different densities. The cells were held at the boundary between these solutions. In some cases the chamber was covered with a cover slip in order to avoid evaporation. After measurement the cells were removed using micro- capillaries (diameter, 300 /~m). The distance between the electrodes was 5 ram.

The experimental chamber, type B, consisted of four needle electrodes fixed by a plastic ring. The distance between the opposite electrodes was 500 /Ltn, which allowed the production of rotating fields with field strengths up to 40 kV/m. The whole chamber was manipulated with a micromanipulator over a fixed microscope slide on which a droplet containing one single cell was placed (0.28 M mannitol solution). After the cells sedi- mented on to the glass surface cell rotation was measured. The chamber was closed by a microscope cover slip and an insulating cylinder. After measurement the electrode system and the microscope slide with the cell were removed. This proce- dure should be performed easily and did not result in loss of the cell during cell manipulation.

To generate the rotating electric fields four 90 ° phase-shifted square-topped pulses were used [12]. The field frequency varied between 100 Hz and 2 MHz and amplitude between 2 and 20 V. Heating and convection of the solution were neglible.

For measurement of the rotational behaviour of oocytes and zygotes field strength between 5 and 8 k V / m were used. At this field strength and frequencies near fca (see Fig. 6) the additional induced peak membrane potential ranges between 5 and 50 mV.

Cell preparation The cells were obtained from 6-10-week-old

mice, Dummerstorf strain. The mice were superovulated by subcutane injection of 5 I.U. pregnant mare serum gonadotropin and 5 I.U. human chorionic gonadotropin 48 h after pregnant mare serum injection. Then a group of the mice was mated. Oocytes and zygotes were released from the oviducts into Dulbecco's medium 20 25 h after human chorionic gonadotropin injection. From the mated mice only those with vaginal plug were used. The cumulus cells were removed with

hyaluronidase (100 I .U . /ml ) in Dulbecco's medium and, additionally, embryos of the two-cell stage were used. They could be flushed from the oviducts of females with Dulbecco's medium [13] 1 day after observation of the vaginal plug. Before measurement the cells were washed four times in 0.28 M mannitol solution and the conductivity was calibrated with CaC12 (5 ,10 -3 S/m).

After measuring the electrorotation the eggs were mounted between a microscope slide and a cover slip, using two strips of vaseline to adjust the cells carefully. A fixing solution of 25% acetic alcohol was added. Staining followed with 2% acetic orcein. By means of a Zeiss phase-contrast microscope cells containing swollen sperm heads or male pronuclei with sperm tails in the egg cytoplasm or male plus female pronuclei were considered to be fertilized. The other cells were classified to be oocytes.

Results

Fig. 2 shows cross-section and rotation of mouse oocytes and zygotes as a scheme.

In the type A experimental chamber the whole cell (cell and zona pellucida) always rotated. In the type B chamber we observed either a rotation of the whole cell or a rotation of the vitellus within the zona pellucida. This could be influenced by changing the external osmotic pres- sure and the glass surface preparation. A small number of cells strongly adhered to glass and were not used for measurements.

Only small differences were observed between the two above-mentioned rotation types Fig. 3, curves 1 and 2). Additionally, the rotation spectra of single zona pellucida and vitelli after removing the zona pellucida is given (Fig. 3, curves 3, 4). For isolation of single zona pellucida the vitellus was destroyed by short field pulses and applica- tion of rotating fields at 40 k V / m and 500 Hz. Under these field conditions the egg membrane was disrupted completely. After this the zona was washed several times.

The zona pellucida itself rotated slowly and its characteristic frequency was shifted toward higher frequencies as compared with whole cells or the vitellus, where it cannot excluded completely that remnants of the viteUus could influence the zona

67

Fig. 2. Mouse oocytes and zygotes, respectively, and direction of cell part and field vector rotation, l, Membrane-enclosed

interior; 2, polar body; 3, zona pellucida.

rotation. This result shows that the properties of both the membrane system and the interior determine the rotation spectrum, but the zona pellucida also influences the rotational behaviour of the whole cell.

At first, we were interested in observing differences in the rotational behaviour of oocytes, zygotes and embryos in the two-cell stage. The results are summarized in Table I. The main result was that the mean characteristic frequency (fc]) of three cell populations was different.

However, the data of Table I referring to cells from superovulated and mated mice are composed of both fertilized and non-fertilized cells, as mi-

R ~ 10-7 Er, ad ~m2/V2.~

A A

10 ~

05

i i L I L l l 3 4 5 6 f¢~ Io§ f

Fig. 3. (O) Spectrum of the whole cell with zona; ( x ) rotation spectrum of the membrane-surrounded interior (vitellus) alone inside the zona; (O) rotation spectrum of the zona pellucida after destruction of the vitellus; (A) rotation spectrum of

oocytes with removed zona.

68

TABLE I

M A X I M U M ROTATION ( R max) A N D C HAR AC T E R IS T IC FREQUENCIES (fcl) OF SEVERAL CELL TYPES

Values are mean_+ S.D. n, number of cells; W~ma x, max imum angular velocity of cells; Rma x = Wcmax/E2; E, field strength.

Cel l R m ~ ( X 1 0 - 7 ) fcl n t y p e ( r a d - m 2 ) / ( V 2. s) ( k H z )

O o c y t e 0 .45 + 0 .15 11 .06 _+ 2 .45 49

C e l l s f r o m

s u p e r o v u l a t e d

+ m a t e d m i c e 0 . 3 9 _ + 0 . 0 7 7 .82_+2 .61 79

T w o - c e l l s t a g e 0 . 4 7 _ + 0 . 0 9 13 .6 _+2.22 26

croscopic observation proved. This encouraged us to investigate oocytes and zygotes more in detail. Therefore, we studied the distribution of fd in both populations, oocytes and cells isolated from superovulated + mated mice, respectively. The results are given in Fig. 4.

There are two groups of cells isolated from superovulated and mated mice which differ in their rotational behaviour: 1. A smaller number of cells shows a characteristic frequency ( f d ) almost identical with that of oocytes. 2. The larger group exhibits a characteristic frequency which is consid- erably smaller. Both populations overlap.

For more than 100 cells from superovulated and mated mice the observed individual fc] frequency was correlated to the microscopic observation. Indeed, approx. 90% of cells with fd < 8 kHz are fertilized cells, and, also 90% of cells exhibiting fc] > 10 kHz were non-fertilized eggs. Cells within fc] > 8 and fc] < 0 kHz were classi-

(o\°)

[--n 2(3 t

i r -~ I i i I i I I I i ' I I ' ! . . . . . i i i

10

i . . . . ]

~ 3 4 5 6 7 B 9 ~0~1 1~13~4 ~5~6~7~'8

f~, [kHq

Fig . 4. D i s t r i b u t i o n o f t h e c h a r a c t e r i s t i c f r e q u e n c y , f c ] , o f

o o c y t e s ( . . . . . . ) a n d ce l l s i s o l a t e d f r o m s u p e r o v u l a t e d + m a t e d mice ( ).

fied to be non-fertilized and fertilized eggs in almost equal percentages. The mean fd frequency of cells microscopically classified cells to be fertilized was 6.7 _+ 1.1 kHz. This is approximately half the corresponding oocytes characteristic frequency.

Discussion

The presented results showed that the rotational behaviour of eggs before and after fertilization was different. The cell rotation was measurable in both types of experimental chambers, whereas the needle chamber was better suited for manipulating cells during and after measurement. A larger number of cellg isolated from superovulated + mated mice differed in their characteristic frequency, fcl, in comparison with oocytes. Microscopically these cells were identified to be cells with swollen sperm heads or male pronuclei with sperm tails in the egg cytoplasm or male + female pronuclei. At the present stage a classifica- tion between two groups can be made with the electrorotation technique. To interprete the rotation data qualitatively in detail it was necessary to develop models for oocytes and zygotes. In all models the polar bodies could be neglected because of their small size.

In the simplest model (Fig. 5A) the zona pellucida also was neglected. Since the isolated zona pellucida showed rotation (Fig. 3), this seems to be an unjustified oversimplification. The electrical properties of the zona must be different of those of the external solution, otherwise no rotation could be observed. The second model, however, was more accurate (Fig. 5B). Here the zona pellucida was considered as a second shell, even a more sophisticated model (Fig. 5C) was theoreti- cally investigated. Here the membrane is the first shell, the liquid between the zona pellucida and the membrane is the second shell, and the zona is a third shell. It can be assumed that the second shell (liquid) is nearly identical in its electrical parameters to the external solution and that it is changed only little or not at all during the fertilization process. The l, heoretical analysis of the rotation spectra corresponding to the different models (Fig. 6) showed that there is not much difference between models B and C. This indicates that oocytes and zygotes could be described at a

first approximat ion with single-shell spheres. However, a quanti tat ively more accurate model should be a two-shell sphere. Therefore, we used bo th models A and B to interpret the shift in the first characteristic frequency after fertilization.

In model B there are six parameters: G 1, the internal conductivi ty; G2, the membrane conductivity; G3, the conduct ivi ty of the zona pellucida; D k 1, D k 2, D k 3, the corresponding dielectric constants. In principle, two or at best three quantities can be calculated f rom rotat ion spectra (see Ref. 5). The dielectric constants are relatively insensi- tive to the posit ion of fcl [1,2].

The compar ison of the calculated torque in model A on the one hand and in models B and C on the other hand demonst ra ted that the main influence of the zona pellucida was a decrease of overall torque. However, the characteristic frequencies remained nearly constant in all models. We assumed; internal D k = 50, membrane D k = 9,

zona D k = 70 and external D k = 80. The internal conduct ivi ty (G1) is higher than all the other conductivities, and therefore should not be looked at critically [14,15]. For getting ideas about the conductivities of the membrane and the zona pellucida, we processed, respectively, the results of the simple zona pellucida and cells with removed zona (Fig. 3) using the single-shell model (Fig. 6A). We obtained a membrane conductivi ty of approximately 10 /~S/m (membrane) and a zona conduct ivi ty of nearly 10 m S / m (variation of the zona D k between 50 and 70 led to no significant changes in the rotat ional spectra of model B and C; therefore, this D k value is noncritical). The

69

TABLE II

CALCULATED CHANGES OF THE CHARACTERISTIC FREQUENCY (fcl) BY VARIATION OF THE MEM- BRANE AND ZONA PELLUCIDA CONDUCTIVITY (B)

fcl values are kHz.

1-ss 2-ss 3-ss

L~ N~¢, LI N~, fcl N, oj

Membrane conductivity (S/m)

10 -s 3.6 -0.62 4.2 -0.42 3.9 -0.35 10 -7 3.7 --0.60 4.3 --0.40 4.1 -0.34 10 -6 5.5 --0.43 5.9 --0.28 5.9 --0.25 10- 5 23.4 - 0.09 22.8 - 0.07 24.2 - 0.06

Zona conductivity 10 -3 4.2 -0.11 4.1 -0.10 5.10 3 5.8 - 0.27 5.6 - 0.23 10- 2 5.9 - 0.29 5.9 - 0.25

experimentally observed decrease after fertiliza- t ion indicated changes in the membrane conductivity, and possibly the zona pellucida conductivity changes toward smaller values (see Refs. 2 and 14). However, it could be anticipated that the influence of the zona conductivi ty on the frequency shift would be very small (see Fig. 6). Therefore, we simulated the cell spectra after fertilization by changing the membrane and the zona conduct ivi ty toward smaller values starting f rom the data given above.

The data in Table II demonst ra te that mainly changes in the membrane conductivi ty influenced the rotat ion spectrum, whereas changes of the

3 ̧

1

e

1 4

e

A 15 C

Fig. 5. Three models to describe oocytes and zygotes (for detailed information see text). 1, cytoplasm; 2, plasma membrane; 3, zona pellucida; 4, solution between the zona and the membrane.

70

N/No

~)~--~, 4" \ \ #. \ \ . x

,Y "K,:,

:'~ ~:t ........................ >;OF ig f

\ ,", ,"7 \ % ,~ / / / \ / \ f . / /

3 - s s ' \ . / 2 - 5 5 / ' ~ x I I

-I

Fig. 6. Simulation of the rotational behaviour of oocytes and zygotes using the models described in Fig. 5. (for calculation, see Refs. 2,6). 1-ss, Model A (Fig. 5), single-shell sphere; 2-ss, model B (Fig. 5), two-shell sphere; 3-ss, model C (Fig. 5), three-shell sphere; . . . . . . . changed zona conductivity, 0.1 S/re.

Parameter set 1-ss 2-ss 3-ss

Internal conductivity (S/m) 0.1 0.1 0.1 Membrane conductivity (S/m) 10-6 10 6 10 6 Water layer (S/m) 5.10 3 Zona conductivity (S/m) 10 - 2 10 - z External conductivity (S/m) 5-10-3 5-10- 3 5.10- 3 Internal Dk 50 50 50 Membrane Dk 9 9 9 Water layer Dk 80 Zona Dk 70 70 external Dk 80 80 80 Radius vitellus (~ m) 41 41 41 Radius zona (ff m) 48 50 Membrane thickness (nm) 8 8 8 Zona thickness (ffm) 7 7

zona conduct ivi ty over two decades, which would be necessary to explain the experimentally observed data, are rather improbable. The electrical parameters of the zona pellucida, especially the conductivity, are comparable to the external ones. The measurement of single zona pellucida showed that, in general, the electrical parameter of the zona cannot differ f rom the electrical parameter of the bulk medium used by more than one order of magnitude. In the same way, changes of the membrane D k or membrane thickness can be used to explain the measured values, but the influence of these quantities is much smaller if realistic values are used.

Therefore, we concluded that during or after fertilization the electrical membrane properties were changed. Especially the membrane conduc-

tance was decreased in almost every order of magnitude.

It is necessary to point out that the electrorotation measurements discussed here do not neces- sarily correlate to short time effects observed during fertilization, e.g., membrane potential changes or processes of cortical granule exocytosis, since our investigations started several hours after fertilization before and during development of pronuclei. The differences between the rotational behaviour of oocytes and cells isolated f rom superovulated + mated mice were nearly constant over several hours, this indicates especially long time changes of the plasma membrane. Direct influences of the t ransmembrane potential on the electrorotation can be excluded, since the membrane potential does not have any effect on the rotation spectra of cells [2].

The definition of fertilization is critical because this term is not defined exactly in the literature [19,20]. Not all cells classificated here as having been fertilized are able to produce blastocysts. At present, we cannot differentiate between fertilized eggs and cells, e.g., those which were in sperma- tozoa contact only or those with spontaneously induced cortical granule exocytosis; therefore, in our opinion, the measurements presented mainly characterize the electrical properties of the stabi- lized new membrane after cortical granule fusion. In several papers changes of membrane properties due to the mixing of the plasma membrane and cortical granule parts are described (see Refs. 17 19). Following exocytosis metabolic changes (protein synthesis, ion channel changes, changes of the egg surface and surface charges) should lead to completely different electrical membrane properties which are internally measurable by electrorotation. It has to be added that changes in the zona pellucide cannot be excluded, but their influence on the rotat ion spectra is less than 20%. Our results, together with data f rom the literature, indicate that several processes are superimposed. In future, detailed investigations using the electrorotat ion technique together with other methods may be useful to explore further single steps in egg development during and after fertilization.

It was demonst ra ted that the electrorotation is a new method of classifying and characterize cells without wounding and with a minimal cell loading

(the rotat ing electric field induces periodic changes in the m e m b r a n e potent ia l in the mV range, see

Materials and Methods). After measurement an egg cul t ivat ion was not done by us, bu t from compar isons with the behaviour of other cells (p lant protoplasts) after electrorotat ion measure-

ments we know that normal cell development is no t disturbed. It should be also possible to opti- mize further the composi t ion of the measur ing

solut ion (see Materials and Methods) in order to avoid undesired changes in the cell. Hence, the observed differences in the characteristic frequen-

cies of mouse oocytes and zygotes open up the possibil i ty of using the electrorotat ion technique in developing new separat ion and characterizat ion procedures dur ing and after fertil ization or egg

development .

Acknowledgements

We thank B~irbel Pletz for technical assistance

and Drs. E. Donath , P. Kauffold and C. Pitra for helpful comments on the manuscript .

References

1 Fuhr, G., Hagedorn, R. and Miiller, T. (1985) Stud. Bio- phys. 107, 109-116

2 Fuhr, G. (1985) Dissertation, Humboldt University, Berlin 3 Fuhr, G., Gimsa, J. and Glaser, R. (1985) Stud. Biophys.

108, 149-164

71

4 Gimsa, J., Fuhr, G. and Glaser, R. (1985) Stud. Biophys. 109, 5-14

5 Glaser, R., Fuhr, G., Gimsa, J. and Hagedorn, R. (1985) Stud. Biophys. 110, 43-50

6 Fuhr, G., Glaser, R. and Hagedorn, R. (1986) Biophys. J. 49, 395-402

7 Arnold, W.M. and Zimmermann, U. (1982) Z. Naturforsch. Sect. C Biosci. 37c, 908-915

8 Zimmermann, U. (1982) Biochim. Biophys. Acta 694, 227-277

9 Lovelace, R.V.E., Stout, P.G. and Steponkus, P.L., (1984) J. Membrane Biol. 82, 157-166

10 Arnold, W.M., Wendt, B., Zimmermann, U. and Koren- stein, R. (1985) Biochim. Biophys. Acta 813, 117 131

11 Glaser, R., Fuhr, G. and Gimsa, J. (1983) Stud. Biophys. 96, 11-20.

12 Fuhr, G., Hagedorn, R. and GOring, H. (1984) Stud. Bio- phys. 102, 221-227

13 Dulbecco, R. and Vogt, M. (1954) J. Exp. Med. 99, 167-182 14 Glaser, R. and Fuhr, G. (1986) in Electric Double Layers in

Biology (Blank, M., ed.), pp. 227-242 Plenum Press, New York

15 Hagedorn, R. and Fuhr, G. (1984) Stud. Biophys. 102, 229-238

16 Gould-Somero, M. and Jaffe, L.A. (1984) in Cell Fusion, Gene Transfer and Transformation (Beers, R.F. Jr. and Bassett, E.G., eds.), pp. 27-38, Raven Press, New York

17 Epel, D., Schmidt, T. and Sasaki, H., (1984) in Cell Fusion, Gene Transfer and Transformation (Beers, R.F. Jr. and Bassett, E.G., eds.), pp. 39-48, Raven Press, New York

18 Hagiwara, S., and Jaffe, L.A., (1979) Annu. Rev. Biophys. Bioenerg. 8, 385-416

19 Scbuel, H., (1978) Gam. Res. 1,299-382 20 Vanagimachi, R. (1981) in Fertilization and Embryonic

Development In Vitro (Mastroianni, L. and Biggers, J.D., eds.), pp. 81-182, Plenum Press, New York

21 Jaffe. L.A. and Cross, N.L. (1984) Biol. Reprod. 30, 50-54

differences in the rotation spectra of mouse oocytes and zygotes

Documents