OF REPRODUCTION

BIOLOGY

45, 466-470

Nuclear

(1991)

Maturation

L. GOODROWE,23

KAREN

Metro Department

of Domestic

Toronto

of Biomedical

Cat Ovarian

MARGERY

Zoo,3

West

Oocytes W.

HAY,#{176}and

Hill,

Ontario,

KING4

ALLAN

Canada

Sciences,4 Ontario Veterinaiy Guelph, Ontario, Canada NIG

In Vitro1

MIE

College, 2W1

4R5 University

of Guelpi,

ABSTRACT Using

domestic

the

cat as a model

for salvaging

genetic

material

from

rare

Felidae,

we

collected

from

oocytes

ovarian

tissue

placed

them in I of 3 treatments to observe time-related, meiotic changes of in vitro oocyte maturation. Oocytes obtained from ovaries collected at ovario-hysterectomy were assigned to I of 3 treatment groups: 1) modifIed Krebs-Ringer bicarbonate buffer (mKRB) + 4% BSA and 5 g/m1 FSH (+FSH, n = 499); 2) mKRB + 4% BSA (-FSH, n = 502); or 3) mKRB + 5% natural estrus cat serum (NE, n = 873). They were placed in the respective media in a 5% Co2 humidified environment at 38#{176}C. Beginning at 16 h, oocytes were removed at 4-h intervals through 48 h, and the meiotic status was evaluated by means of cytogenetic analysis. On the basis of chromosomal analysis, each cell was placed into one of the following categories: metaphase II (MU); metaphase I (MI); pre-MI (germinal vesicle IGVI, GV breakdown, or diakinesis); degenerate or unidentifiable. The percentage of oocytes with degenerate chromatin increased over time in all culture treatments, but was always greatest (p < 0.05) in the NE group. In the +FSH and -FSH treatments, the proportion of oocytes with nuclear material reaching ML! increased with time in culture to 32 h and was equal to or greater than the proportion of oocytes with pre-MI + MI chromatin at this time interval (-FSH, 55%; +FSH, 38%). After 32 h, the number of -ESH oocytes reaching MU status was not sustained, whereas in the +FSH group, the proportion of ova with MI! material remained constant (45-48%, p > 0.05). While Mu chromosomes were observed at all times in the NE treatment and peaked at 36 h, levels never became greater than those with pre-MI + ML chromatin, indicating a lack of support for nuclear maturation in this treatment. These results indicate that although oocytes in the -FSH and NE treatments can achieve nuclear maturation in vitro, this process generally is not supported under the described and

conditions.

In contrast,

to be maintained

supplementation

throughout

the

20-48-h

of culture

medium

culture

interval.

with

INTRODUCTION Collection their

of immature

subsequent

opment

oocytes

development

of sophisticated,

enhanced our clear maturation for successful Subsequently, matured and

defined

understanding processes

of pregnancies transfer in mice

or endangered

(IVM)

through

the

devel-

culture

systems

has

greatly

uration

wildlife.

Collection

and

fertilization

or

at lost

(IVF)

for

of oocytes

of such

oocytes

mation tissue

May

Received

September

‘This Fellowship NSERC

work

was

from

of the Zoological

requirements

poten-

in

pan

by the

Max

of Metropolitan

Bell

Foundation,

Toronto

(K.L.G.

the

Blackstock

and

M.H.)

allows

MIl

chromosomes

maintenance of genetic diversity within be accomplished by genetic input from number of individuals.

because ovarian readily available, threatened

tissue detailed

species

then rare

from nondomestic studies of oocyte

usually

be used on an species becomes

[15],

there

is still

in this

Johnston et al. [15] have cytes will spontaneously

are

not

anmat-

permissible.

opportunistic available.

basis

when

a paucity

species.

Byers

of information and

Hunter

for

IVM

[16]

and

demonstrated that domestic cat 00resume meiosis in vitro either with

or without hormonal supplementation, but they did not observe the effects of serum on NM, a factor recently shown to enhance the proportion of oocytes reaching metaphase II in cattle [17]. In addition, the time course of meiotic events for domestic cat oocytes previously has not been defined.

2-i, 1990. Society

can from

in vitro

mat-

1, 1991. supported

with

and

In the Felidae family, the domestic cat serves as the model for reproductive studies of nondomestic species [13]. Although recent efforts in the domestic cat have led to advances such as the birth of offspring following 1W of oocytes matured in vivo [14] and fertilization of oocytes matured

tially could lead to the production of offspring posthumously or from animals no longer capable of reproducing. This is important for species that exist only in limited num-

Accepted

maturation

Therefore, a domestic animal counterpart is necessary to define conditions for maturation and fertilization that may be unique to each family or genera of animals. This infor-

oofor

ovario-hysterectomy gametes. In vitro

nuclear

However, imals is not

nu-

of offspring fol[3) cows [4-10],

of the requirements offers unique opportunities

enhances

bers: by this means a population would the greatest possible

and

in vitro

or birth [1,2), rats

obtained postmortem allow salvage of otherwise

uration

follicles

of the cytoplasmic and that are necessary prerequisites

sheep [11] and pigs [12]. Increasing our knowledge cyte maturation in vitro also ovaries would

ovarian

fertilization and embryonic development. the developmental competence of oocytes fertilized in vitro has been demonstrated by

the establishment lowing embryo

threatened

from

FSH

Therefore, the objectives of the present study were 1) to study the time-related meiotic changes of domestic cat oocytes collected from ovarian tissue and matured in vitro and

and

(WA.l()

correspondence.

466

NUCLEAR

2) to evaluate plementation

the effects on nuclear

collected

OF

of FSH or estrual cat serum maturation in vitro.

MATERIALS Ovaries

MATURATION

AND

from

germinal

sup-

cats

In

at ovario-hyster-

10 ml of PB1 prevent blood by repeatedly

needles then 16],

until

the

tissue

searched and, those oocytes

tached

cumulus

supplemented

cells from puncturing was

with

with 0.3% h of collec-

20 U heparin/

coagulating). the ovaries

finely

minced.

Oocytes

with The

were 22-gauge

dishes

and

cytoplasm

to homogeneously

dark

(without

heparin)

and

carbonate collection, numbers

(mKRB) medium [14, 18]. the gametes were assigned to 1 of 3 treatment groups:

then

removed,

washed

in a modified

once

Kreb’s

in PBI

Ringer

bi-

On

each day of ovary at random in equal 1) mKRB + 4% BSA

ON) 502); 873).

(+FSH, n = 499); 2) mKRB + 4% BSA (-FSH, n or 3) mKRB + 5% natural estrus cat serum (NE, n Gametes were placed in 100-pA drops of respective

38#{176}C (no

Vetrepharm,

oil in a 5% CO2

more

than

blood samples (3-5 Day 2 or 3 of estrus. pooled, heat-treated

pipetted into Beginning

20-pA aliquots with a culture

removed 40,

at 4-h

44,

48 h)

intervals

and

humidified

15 oocytes/drop).

estrus serum, 5 queens on were decanted,

placement

with

oocytes with

natural

ml) were collected from After centrifugation, sera (57#{176}C for 30 mm), and

up to 48 h (16, for

a small-bore,

the cells and blowing After air drying, the acid-methanol stained with tilled with

obtain

20,

cytogenetic

24, 28, 32, 36, analysis

as de-

involved removing of exposure to 0.2% and mechanical dis-

hand-pulled

glass

pipette.

subsequently

fixed

to cause dispersion slides were placed

of the chromatin. into a 1:3 acetic fixation, with dis-

water. Each oocyte was then identified and observed light microscopy (400x or 1 000X) to evaluate meiotic

status. On was placed

the basis into one

II (MIl,

second

meiotic

metaphase);

meiotic

of chromosomal of the following metaphase); pre-MI

(intact

analysis, categories: metaphase germinal

of oocytes

I (MI,

(n

immediately after before culture.

not

identifiable chrofor all treatments. 117)

=

was

collection

fixed

(as

to determine

Between treatments and at all time intervals, differences in the proportion of oocytes in each category were evaluated by Chi-square analysis. Within treatments, differences in the proportion of oocytes in each category at each time interval were also evaluated by Chi-square analysis.

Of the prior

31 oocytes

to culture,

with

six (73.5%)

of the

unidentifiable were subjected

identifiable

19 (61.3%)

onstrated GVB, 1 (3.2%) was at the MI stage, and

chromatin

contained

oocytes

examined

chromatin. to fixation

of 20 oocytes interval. The chromatin ranged

contained

in

each

82% (NE),

on the timing

within the +FSH group between the proportion

oocytes

demonstrating

groups (Fig. 1). level of degenerate

-FSH>

In-

+FSH.

of meiotic

events

indicated

there was an inverse relationof pre-MI + MI chromatin and

MIl chromosomes

(Fig.

2A). This

re-

lationship is best defined by a linear decrease in pre-MI + MI chromatin (y = 132.53 2.54x; r2 = 0.95) coincident with a gradual rise and plateau of MIl chromosomes (y = -

-70.66 portion terial

5.81x of pre-MI (38%), while +

centage of MIl than the number analysis

-

+

0.071x2; r2 = 0.95). At 32 h, the proMI (43%) was equivalent to MIl maat each subsequent interval, the per-

oocytes (approximately of pre-MI + MI oocytes

demonstrated

that

between

45%) (Fig.

16 and

was 2A).

greater Further

32 h, the

pro-

ET AL.

portion percentage

of

pre-MI oocytes of MI chromatin

Overall

dynamics

for

above

the

FSH group

-

through

0

0

+

0.05x2

ever, were

r2

0

0. 0 0.

16

20

24

28

32

36

40

44

48

(y

=

predominated,

an

(Fig.

increase 2B).

gradually

decreased

24 and

through

32 h, the

the

material declined

(Fig. 2B). As observed

constant declined

MI

through to a low

24-h

interval

became

greater.

of ova with MII in Figure

2C, the

in the

NE

group

28 h (71-83%), of 24% (y = 817.66

then

progres135.31x

+

oocytes

replaced

of MI oocytes

while those with MIl chromatin

of pre-MI

was

number

intervals, the percentage

How-

3 categories pre-MI chro-

and

In all later time

mained sively

the

declined,

in MI chromatmn

Between

+ 2.51x

0.64) of MIl chromosomes.

=

more complex relationships between observed. At the onset of observations,

matin by

rise then decline

0.0014x3;

-

-

re+

0.00002x5, r2 = 0.83). While at all time intervals, lev-

0.0046x4 were observed

+

-

never became greater than those for pre-MI + (p > 0.05). Two apparent waves of Mu material, by a fifth-degree polynomial equation (y =

-2209.3 + 398.57x 0.00008x5, r2 = 0.75), first peaking at 16-20

C 0

+

-44.81

-

els (30%) MI oocytes represented

(I, U. +

similar to those

2B, 32 h: pre-MI

with a concomitant

-

C) 0

while the (Fig. 2A).

a linear decline + MI chromatin

0.093x2 0.0003x3 MIl chromosomes

a’

were

32 h (Fig.

0, 0,

constant, declined

MI, 45%; MIl, 55%) and also demonstrated (y = 124.35 2.22x; r2 = 0.90) of pre-MI

percentage Pre-M1

for the group

remained progressively

27.19x2 + 0.88x3 0.014x4 + were observed in this treatment; the h and the second occurring between -

-

36 and 44 h. In all treatments, a decline or plateau in viable chromatin was observed after 36 h, with a concomitant rise in the proportion of oocytes with degenerate chromatin. Intra-treatment statistical peaks (p < 0.05) for MII chromosomes occurred at 24, 32, and 36 h for the +FSH, -FSH, and NE groups, respectively. Inter-treatment comparison of nuclear maturation demonstrated that at 24 h, the proportion (33%)

of MIl

oocytes

compared

was to the

greater -FSH

while at 32 h, the proportion did not differ between the 28

32

36

40

44

48

groups 0.005). oocytes group These

100

80

and was greater At each subsequent with Mil chromatin (p data