Tripronuclear Human Oocytes: Altered Cleavage Patterns and ...

BIOLOGY

OF

REPRODUCTION

37,

395-401

(1987)

Tripronuclear Human Oocytes: Subsequent Karyotypic ISMAIL

Altered Cleavage Patterns Analysis of Embryos

KOLA,”2

GAREY

ALAN

DAWSON,3

and

and

TROUNSON,2 PETER

ROGERS4

Centre for Early Human Development2 Cytogenetics Laboratory, Division of Laboratory

Medicine3

and Department

of Obstetrics

Monash Melbourne,

& Gynecology4

University Australia

3000

ABSTRACT Between

1 and

4%

of

human

oocytes

fertilized

tripronuclear oocytes can develop to grossly could be triploid, diploid, or severely depleted. embiyos deriving from tripronuclear that most (18 of 29) tripronuclear

human human

embryos

(but

have

tripronuclear (7 of 29)

a severely

abnormal

human oocytes cleave cleave to 2-cells, and these

in vitro

normal-appearing The etiology

are

oocytes oocytes

is unknown. cleave directly

triploid)

chromosomal

not

to 2-cells embryos

trip ronuclear.

morulae and and proportion

plus an extrusion, are triploid after

This study to 3-cells

Although

into

the

1-4%

mechanisms

by which

these

oocytes

fail

to develop

50.5

(Mahadevan

severely

Trounson,

1984)

into

h

after

about

subsequent

chromosomal

nuclear

oocytes

embryos

and

with

4,6-diamidino-2-phenylindole

blastomere Rudak

contained et

Accepted Received 1 Reprint ment,

Monash

3168,

Australia.

al. (1984)

February December requests: University,

(DAPI),

46

chromosomes.

reported

that

25, 1987. 5, 1986. Dr. Ismail Monash

Kola,

Centre

Medical

that

for Early Centre,

Human Clayton,

some

(4 of 29)

one

complement

blastomere of

33

et al. (1986) evaluated of 5 embryos derived and

two

found

1 diploid,

embryos

with

two

near

triploid

and

39

chromo-

per

evaluated by DNA microfluorimetry, Most of the embryos in the Angell

We apparent

oocytes,

evaluated

respectively.

after

Two

the second

a

the chromofrom tripro-

bryos, diploid.

were

blastomere,

38

had

chromosomes.

somes

study

other

em-

were near et a!. (1986)

cleavage

division.

were interested in the derivation of the diploidy and aneuploidies in tripronuclear especially as electron microscopic observa-

tions of tripronuclear oocytes had confirmed the presence of three pronuclei arising through dispermy

one

(Sathananthan and Trounson, 1985). Zenzes et a!. (1985) have shown that oocyte was hypotriploid (had 64

In addition,

in an embryo

these

is also provided.

insemination,

Recently, Angell somal constitution

composition of preimplantation embryos that derive from these oocytes, and the data that does exist suggest a complex picture. We (Trounson, 1982) previously found that three of five cleaving embryos from tripronuclear human oocytes were triploid, and two were near diploid. Van Blerkom et al. (1984) showed that two such embryos may develop to at least the 8-cell stage and demonstrated, by serialsectioning a 12-cell embryo and fluorescent staining

embryos

abnormal

tripronuclear information

and

Furthermore,

triploid

oocytes fertilized in vitro are 3 pronuclei), there is little development

that

these embryos and aneuploid

pattern at the first cleavage division, that into triploid embryos, and that a correlasubsequent karyotype of these embryos.

of human (contain the

reported

provides evidence for the first time at the first cleavage division. These

complement.

INTRODUCTION

and

been

and these embryos are diploids, whereas some the first cleavage division. These findings demon-

strate that most tripronuclear human oocytes have an altered cleavage most tripronuclear human oocytes (76% in this study) do not develop tion exists between the pattern of the first cleavage division and the Insight

It has

that chromosomally, of apparently diploid

fixed

before the tripronuclear chromosomal

Develop-

division occurs

Victoria,

395

first

Furthermore, a tripronuclear chromosomes)

cleavage division. Thus, it appears that oocytes have a triploid or near triploid complement before the first cleavage

and that if deviation from at later stages of embryogenesis.

triploidy occurs, Theoretically,

it

396

KOLA

for

a tripronuclear

oocyte

(consisting

of 69

chromo-

or

cleavage vestigated

the

oocyte

cleaves

to

3 cells

tripronuclear

human

constitution

oocytes

these

of

cleavage

division

trip!oid

and

the

embryos both

oocytes to gain

MATERIALS

Oocytes undergoing

obtained treatment

number

of

microscopy pronuclear and/or

12-20 oocyte interference

pronuclei

were

develop into the

an

of

oocytes

and fertilized and Mohr, 1983).

scored

which

under

as The

dissecting

In addition, indicates

state of these to dispermy.

human

investigation,

4

initial

human

being with with

all oocytes that the

oocytes

is almost

oocytes

tripronuclear three clearly colcemid (0.1

that

(Fig. visible pg/mI)

were

la

shows a pronuc!ei) just before

the first cleavage division (24 h after insemination). The oocytes were assessed for chromosomal constitution by using a modification of the technique previously described (Kola et a!., 1986). Briefly, embryos

were

incubated

in hypotonic

solution

(1%

trisodium citrate) for 3-5 mm at room temperature. The embryos were then transferred onto clean glass slides One 3:1)

in

a minimal

drop was

amount

of

hypotonic

of fixative (methanol/glacial dropped onto the embryo

from

solution. acetic acid, behind it.

Immediately drop of

after adherence to the slide, a second fixative was dropped onto the embryo. Finally, 3 more drops of fixative were added 1 at a time to the side of the embryo. Chromosomes were stained with 10% Giemsa (BDH, Poole, England) in buffer,

pH

6.8,

In subsequent were monitored and

cleavage.

for

10 mm.

experiments, tripronuclear constantly for dissolution Three

trip ronuclear

oocytes

oocytes of pronuclei were

time-lapse incubated

dissolution

filmed

and

cinematography. with colcemid

first

cleavage

fixed

The

the

first

of embryos did fixed earlier.

conducted

8

embryos division

not have any Chromosome

independently

by at least

using

embryos were 12 h after the

remaining cleavage

by

embryos were 6-8 h after the

The first constitutions

division.

14 h after

cleavage

The beginning

first cleavage division. assessed for chromosomal

into mech-

h after insemination. Each tnwas reexamined under phase contrast microscopy and

to be tripronuclear. polar bodies,

evaluated as human oocyte were incubated

second

from superovulated patients of infertility by in vitro fertil-

(IVF) were collected described (Trounson

tripronuclear certainly due

the

pronuclear

number when

METHODS

ization previously

confirmed had two

inof

proportion

that insight

AND

first

chromosomal

the

anism by which these tripronuclear fail to develop into triploid embryos.

to to is

we division

before

to determine

human embryos and

tripronuclear

In

at the

division. Against this background, the pattern of the first cleavage

AL.

for

somes before the first cleavage division) to develop a diploid embryo, one of two mechanisms have operate. Either a haploid set of chromosomes ejected,

ET

were

because

a

cells in mitosis scoring was

two

investigators.

RESULTS

The cleavage

4

oocytes division

that each

had

were fixed before 69 chromosomes.

the

first

Of 29 tripronuclear oocytes allowed to cleave, (62%) cleaved directly to 3-cells (Table 1, Fig. We found that the dissolution of the pronuclei cleavage both

to 3-cells microscopic

was

cinematography). varied; 6 embryos whereas smaller embryo,

The had

synchronous evaluation regularity 3 equally

18 ib). and

(as determined and time

by lapse

of the blastomeres sized blastomeres,

the remaining 12 embryos had 1 large and 2 blastomeres. Figure 1(b) shows a 3-cell human with one large and two smaller blastomeres

that had resulted from cleavage of a tripronuclear blastomere

size

was

the not

direct oocyte. related

and synchronous The variation

of

to

of

synchrony

cleavage, since those embryos that had 1 large and 2 smaller blastomeres had also cleaved synchronously to 3-cells. Chromosoma! analysis of 10 of these 3-cell embryos just before the second cleavage division revealed that all of these embryos had a severely abnormal (Table

1).

an embryo

but near-diploid Furthermore, different

of chromosomes.

It in

that

cleave directly to 3-cells. a 3-cell embryo, but

numbers

constitution 3-cells within

also appeared that the number of chromosomes individual cells was random. This is the first demonstrating

had

chromosomal in all cases the

tripronuclear

human

study oocytes

Angell et al. (1986) observed were unable to differentiate

between direct cleavage to 3-cells or cleavage to 2-cells and a rapid second division. We found that 14% (4 of 29) of tripronuclear human oocytes cleaved to 2-cells plus an extrusion (a mass that is extruded from the embryo simultaneously with cleavage of the embryo) (Table 1). Figure 2a shows a 2-cell-plus-extrusion embryo, with the extrusion indicated by an arrow. Three of these 4 embryos were successfully karyotyped, the cells of these embryos were found Figure 2b shows a chromosomal spread

and both to be diploid. of 1-cell of a

TRIPRONUCLEAR

HUMAN

397

OOCYTES

#{149}.

‘..

$

.

a FIG.

1. (a)

to the permy.

Photomicrograph

of

center

of the oocyte. (b) Photomicrograph

2-cell-plus-extrusion diploid shown

that

two

interphase

somes,

had

not

been

nuclei,

possibly

Twenty-four oocytes in normal

(46XY) 2b. The

this

2-cell

Figure

one

from

(7 of cleaved (Fig.

(X 200).

2c shows

the

spread in this

karyotyped,

haploid the

oocyte

bodies are also seen, 2 smaller blastomeres)

metaphase embryo

successfully

percent study

embryos

and of the remaining

and

derived

human

and second polar embryo (1 large,

embryo,

karyotype in Figure

group

a tripronuclear

The first of a 3-cell

had

set of chromo-

extrusion. 29) to

Figure the

near-triploid embryos (Table a metaphase spread of a 2-cell 3c shows the triploid karyotype

spread

or

shown

in Figure

1). Figure

3a is

1). Figure embryo, (69XXX)

3b and of

3b.

DISCUSSION

The findings large majority develop cleavage

in this study of tripronuclear

have demonstrated human oocytes

into triploid embryos, division is a critical stage

and that

photomicrograph

that a do not

that the influences

subsequent chromosomal constitution of tripronuclear human oocytes. We have shown that three types

first the

clearly

demonstrating derived from

that the the direct

events occur at pronuclear human cells,

2)

they

the

the

the

three

first oocytes:

cleave

to

pattern

cleavage 1) they

triploid

For spindle

an

that

have

oocyte oocyte

that of

migrated

is due to dis(X200).

division cleave

of to

2-cells-plus-extrusion,

at the

chromosomal

tnthree

and

triploid embryos. a correlation

cleavage

first

3)

Our exists division

composition

the embryos that abnormal chromosomal

the 2-cell-plus-extrusion whereas the morphologically were

pronuclei

2-cell

subsequent

these embryos-all had a severely

of

cleaved to 3-cells composition;

embryos were regular-looking

diploid embryos

or near-tniploid. embryo

formed

to at the

cleave first

from

cleavage

1- to

3-cells,

division

should

the be

tripolar.

The data in this study indicate that the movement of chromosomes on such a tripolar spindle is disorganized, resulting in the cells in an embryo having different numbers of chromosomes in each cell, and the cells thus having an abnormal chromosomal constitution. Tn- and/or quadripolar spindles have been observed in the sea urchin (Wilson, 1925) and

in rat The

oocytes

finding

plus-extrusion,

of

shows

tripronuclear state of the cleavage of a tripronuclear

they cleave to regular findings also demonstrate and

a photomicrograph of a morphologically normal 2-cell embryo. Chromosomal analysis of 4 of these embryos at the second cleavage division revealed triploid shows

The

between

of tripronuclear morphologically

3a, Table

b

suggest

that

(Austin

and

Walton,

1960).

that some embryos cleave to 2-cellswhich are karyotypically diploid, in this situation one haploid set of

398

KOLA

TABLE

1. Cleavage

details

and

Number of cells after first cleavage division

Oocyte

3-cell

+

3 fragments

2-cells

+ extrusion

2-cells

*ND *

tThe

=

Not

chromosome

findings

of tripronuclear

human

ET

AL. oocytes.

Time of first cleavage division (h after insemination)

code

Number

of

metaphase

Chromosome findings

cells

57,51

MCK MUL DEM7 DEM1 MIT JAN CHR SHA4 TS1 BAL GAN SUM COP FRA3 HOL COA OTT MAR

29 32 28 27 30 28 33 26 28 26 33 28 28 33 33 27 29 30

2 0 1 0 0 1 ND* 3 1 ND ND ND 1 2 1 ND

COC THE DOM COR

30 30 27 27

2 2 2 2 nuclei

DEM4 SM1 BRO HOO SHAI AT!’ FRA4

34 24 33 31 36 27 29

2 0 0 ND I 1 1

-

57 -

54 ND 42,44,5 2 34 ND ND ND 51 52,39 38 ND 34 34,42,32,9,12,6**

-

3

46,46 46,46 46,46 + 23 chromosomes 69,69 -

ND 69 72 69

done.

derivation

chromosomes at the first

of these

6 groups

of chromosomes

is unclear.

is excluded from the metaphase cleavage division, and that this

plate division

participate

in

mitosis.

the

only involves 46 chromosomes and a normal bipolar spindle. It may be that the chromosomes of one pronucleus has a different degree of condensation

triploid

chromosomal

complement.

study,

two of the embryos fixed at the 2-cell

from the other two; thus, a haploid set of chromosomes do not gather on the metaphase plate. However, we have not yet elucidated whether the subsequent development of these embryos is normal nor deter-

found to be triploid. This study has

mined the fate of the haploid set of chromosomes. Van Blerkom et al. (1984) found that one tnipronuclear oocyte that cleaved to 2-cells was diploid at the 8-cell stage, but overlooked. The situation

logically these

it is possible

data

in

this

that study

(tripronuclear

extrusion

suggest

2-cell are

tniploid),

was

that in the third cleaving to morphoembryos; chromosomally,

oocytes

normal embryos

a nucleated

all

the

chromosomes

a!. (1986) 2-cells

and

tnipronuclear

production

These embryos have a regular of daughter cells having a full

division,

were

human

In

and

recently

a!., 1986).

one

that

cleaved to were also

that

only

develop

into

tniploid

division. One conceptions

percent in man

thus demonstrating do develop to have also been survived from

that stage

et

oocytes

are tniploid (Jacobs et a!., 1978). (1985) have reported the live

There have

Angel!

demonstrated

embryos after the first cleavage of all in vivo clinically recognized

infants, embryos

the

Uchida birth that stages

later

24%

of

and Freeman of 7 triploid some tniploid of pregnancy.

reports of 5 tniploid infants who 2 to 7 mo (Borgaonkar, 1984), survived

to

10#{189} mo

(Sherard

et

TRIPRONUCLEAR

HUMAN

OOCYTES

399

‘I

a

B

a

1W

-‘-I-i-’F

b FIG. 2. (a) Photomicrograph of 2-cells-plus-extrusion meres are clearly visible. The extrusion is indicated embryo (X 1000). These photomicrographs demonstrate

by

G

C

embryo, derived an arrow. (b) that the cell had

from the direct cleavage of a tripronuclear Metaphase spread and (c) karyotype of a diploid (46 XY) karyotype.

oocyte (X 200). The two blasto1-cell from a 2-cell-plus-extrusion

400

KOLA

ET

AL.

a b

icu’

*r3MV E

2-s-I--

F

FIG. logically that the

The

3. (a) Photomicrograph normal 2-cell embryo. cell was triploid (69

findings

in this

-$r*-j

C of

2-cell

(b) Metaphase

xxx

embryo spread

derived from the and (c) karyotype

and

the

direct cleavage of 1-cell from

of a tripronuclear a 2-cell embryo

oocyte (X 1000).

(X200). This embryo These photomicrographs

is a morphodemonstrate

karyotype).

study

have

demonstrated

that

the vast majority of tripronuclear human oocytes do not develop into triploid embryos and that a correlation exists between the pattern of the first cleavage division

C

subsequent

karyotype

of

these

embryos. This study has also provided mechanisms by which tnipronuclear to develop into triploid embryos

insight into the oocytes fail in the human.

However, microtubules

on staining the 1986), centro-

research is being (Schatten et

focused a!., 1985,

TRIPRONUCLEAR

somes,

and

mechanisms fail

chromosomes whereby

to develop

into

to

further

tnipronuclear triploid

HUMAN

investigate human

the

Rudak

oocytes

OOCYTES

E, Dor J, Mashiach S, Nebel L, Goldman B, 1984. Chromosome analysis of multipronuclear human oocytes fertilized in vitro. Fertil Steril 41:538-45

Sathananthan AH, fine structure

embryos.

401

Trounson AO, of monospermic

1985. and

The human polyspermic

pronuclear ovum: fertilization in

Gamete Res 12:385-98 H, Schatten G, Mazin D, Balezan R, Simerly C, 1986. Behavior of centrosomes during fertilization and cell division in mouse oocytes and in sea urchin eggs. Proc NatI Acad Sci (USA) 83:105-09 Schatten G, Simerly C, Schatten H, 1985. Microtubule configurations during fertilization, mitosis, and early development in the mouse and the requirement for egg microtubule-mediated motility during mammalian fertilization. Proc NatI Acad Sci (USA) 82:4152-56 Sherard J, Bean C, Bove B, Delduca V, Esterly KL, Kavish HJ, Munshi G, Reamer iF, Suazo G, Wilmoth D, Dahlke MB, Weiss C, Borgaonkar DS, 1986. Long survival in a 69XXY triploid male. AmJ Med Gen 25:307-12 Trounson AO, 1982. Discussion on risks of triploidy arising through polyspermy in vitro. In: Edwards RG, Purdy JM (eds.), Human Conception in Vitro. London: Academic Press, p. 227 vitro.

ACKNOWLEDGMENTS We would like to thank C. King, du Plessis, G. Jones, and all the members program for support and encouragement; the film; Ian Barker for photographic manuscript; and Dr. Jill Shaw for critical

Schatten L.

Chiappazzo, C. Thomas, V of the Monash-Epworth IVF Chris Hale for assistance with work; Jan Ryan for typing the comments on the manuscript.

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RR, spermy

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AA, Cytogenet

Messinis Cell

IE, Genet

1986. 42:1-7

Austin

CR, Walton A, 1960. Fertilisation. In: Physiology of Reproduction. London:

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poly-

(ed.), Marshall’s Green & Co.,

pp. 310-416 Borgaonkar DS, 1984. Chromosomal Variation in Man-A Catalog of Chromosomal Variants and Anomalies, 4th Ed. New York: Alan R. Liss, pp. 778-85, 953 Jacobs PA, Angell RR, Buchanan JM, Hassold TJ, Matsuyama AM, Manuel B, 1978. The origins of human triploids. Ann Hum Genet 42:49-57 Kola I, FoIb P1, Parker Ml, 1986. Maternal administration of cyclophosphamide induces chromosomal aberrations and inhibits cell number, histone synthesis, and DNA synthesis in preimplantation mouse embryos. Teratog Carcinog Mutagen 6:115-27 Mahadevan MM, Trounson AO, 1984. The influence of seminal characteristics on the success rate of human in vitro fertilization. Fertil Steril 42:400-05

Trounson AO, Mohr LR, 1983. Human pregnancy following cryopreservation, thawing and transfer of an eight-cell embryo. Nature 305:707-09 Iichida IA, Freeman VC, 1985. Triploidy and chromosomes. Am J Obstet Gynecol 151:65-69 Van Blerkom J, Henry G, Porreco R, 1984. Preimplantation human embryonic development from polypronuclear eggs after in vitro fertilization. Feral Steril 41:686-96 Wilson EB, 1925. The Cell in Development and Heredity. New York: Macmillan, pp. 416 zenzes MT, Belkien L, Bordt J, Kan I, Schneider HPG, Nieschlag E, 1985. Cytologic investigation of human in vitro fertilization failures. Fertil Steril 43:883-91