BIOLOGY
OF
REPRODUCTION
19,
959-964
(1978)
Ovarian Development in Hypopituitary Snell Dwarf Mice. The Size and Composition of the Follicle Population EFFIE
HOWE,
G.P.M.
SUE
MOORE1
and
LINTERN-MOORE, JENNIFER
HAWKINS
School of Biological Sciences, Mac quarie University, North Ryde, N.S. W. 2113, Australia and Ian
Clunies
Ross
Division
Animal
of Animal
Research
Laboratory1,
Production,
CSIRO,
P.O. Box 239, N.S. W. 2148,
Blacktown,
Australia
ABSTRACT The size and composition of the follicle population in the ovaries of sterile Snell dwarf (div/die) strain mice differ from that in phenorypically normal contemporaries from 21 days of age onwards. The number of small (primordial) and growing follicles in the ovaries of dwarfs and phenotypically normal contemporaries was not statistically different during the infant and juvenile periods. At 21 days of age, ovaries from dwarf mice contained significantly more primordial follicles due to a reduction
in the rate of atresia in the nongrowing pool of oocytes. This difference was maintained the adult period studied. The dwarf mice used in this study did not ovulate. Nevertheless, antral follicles were present and the developmental sequence of follicular growth followed that of controls. From 92 to 432 days of age, however, the total number of growing follicles was reduced in dwarfs. Furthermore,
throughout
specific compartments of the growing follicular population were affected much earlier. These data are discussed in relation to the reportedly normal concentrations of follicle stimulating hormone and luteinizing hormone and the reduced concentrations of prolactin and growth hormone in the pituitary gland and blood plasma of the dwarf female.
INTRODUCTION Mice
homozygous
mutation
dwarf
growth
(for
for
and
review,
cause
to
dwarfism
specifically
an
lactotropes
resulting
of
(Francis, ductive
are occur,
in
sterile. but
amount
of
However,
Bartke
June April
days
ovaries tissue absent
and
(1964,
are
1965a,
b)
of
proceed
and with
Der
that
antral
contrast
is in
Van
showed
present of
the
The
females
ranging
show
We
dwarfs
follicles aries,
the
reduced
(Kemp,
the effect
population
adult
(Mac-
small,
not
follicle
age.
the
mating
ensues
is
life
This
study
function.
repro-
that
opening never
In
the
of of
indicate
Vaginal
interstitial reportedly
Accepted Received
dwarfs
The
ovulation
of
occur.
which
did
contain ovulation
Kroon
ovarian
beyond
the
and
follicular multilamin-
stage.
the
1960)
investigations
pregnancy
1931).
and
growth
5-12
Early
(1974)
1930;
Rennels,
of
cessation
function
Dowell,
and
histological
Buis
females
spontaneous
may
the
ar
dwarf
that
estrus
growth
deficiency;
MacDowell,
between 1944).
size
primary
somatotropes
and
a
The
from and
cyclic
retarded adult
pituitary
absence
adenohypophysis
females
a
Carsner
from
show normal
1952).
is
(Smith 1938;
may
1929) reach
ovaries
follicles
recessive
Mendelian
see Griinenberg,
of
Kemp,
the
(Snell, fail
that
that
we
dwarf
mutation
size
and
from
that normal the
7
the
have
reexamined on
ovarian
composition
have
contain
than but
study,
been days
to
ovaries
of
of
15
littermates
growing
of and
more or
of
in
months
prepuberal
significantly
number
the
quantitated
small
contemporfollicles
is reduced.
and 1938).
MATERIALS
observed
AND
METHODS
Female Snell dwarf mice (genetic symbol div/die) and their phenorypically normal littermates or contemporaries of the same age were obtained from a colony maintained by CSIRO Division of Animal Production, Blacktown, N.S.W. The colony originated
5, 1978 21, 1978
959
HOWE
ET AL.
from a #/dw female which was imported by the Genetics Research Laboratory, CSIRO, Ryde, N.S.W. from the Institute of Animal Genetics, Edinburgh, U.K. during 1952. All mice were raised in an air conditioned room at 21#{176}C(12 h light:12 h dark) and fed commercial pelletted food and water ad libitum. Mice were weaned at 28 days of age. High mortality among dwarf mice has been observed (Dung, 1975). This is thought to be related to a deficiency in cell mediated immunity resulting from
(Tables
pathological changes (Dung and Swigart, a!., 1970). Although routinely observed mortality of dwarf
because
960
observed in the thymus gland 1970; Baroni, 1967; Duquesnoy et premature thymic involution was at autopsy in the present study, females was sufficiently low to
permit a significant proportion to more than 1 year of age. Fifty-seven dwarf and 68
of the mice
to survive
females.
phenotypically
normal
group,
control
age
dwarfs
from
1966).
was as
dwarf
changes
in
homozygous uteri
were
throughout
mutation the
reproductive
for
the
trait.
infantile the
and period
in
system of In all dwarfs,
mice
the
small
the
ovaries
studied.
Puberty,
the
similar at
in the
ovaries
7 and
14
days
of
of
2).
growing
mice
dwarf
were
(Table
was
control
In
follicles
significantly
mice
at
92
days
of age and at subsequent sampling times (Table 1). Prior to 92 days, however, growing follicles in specific
growth
compartments
(Table 2). This was age with a reduction laminar in
and
dwarf
antral ovaries.
days, ovaries cantly more phenotypically
follicles
(Types
At
age
the
of dwarf Type 7 normal
days
were
there
of
days
of multi5b,
of
6 and
7)
puberty
mice contained follicles than counterparts,
of follicular reduced
age,
affected
21
in
of antral follicles of Type 4 follicles in 28-dayand Paradoxically, at 92
at all other stages were significantly of
were
first evident at in the numbers
control mice, the number (Type 7) and the number were reduced, respectively, 35-day-old dwarf ovaries.
no
signifidid their although
growth, numbers (Table 2). At 135 differences
between
the numbers of multilaminar and antral follicles in dwarfs and controls, but in dwarfs the initial stages of growth were reduced. By 432 days of all
antral
ovaries
follicles and
stages
than
of
control
had
the
disappeared of follicles
numbers
growth
values.
of yellow tissue had region of the ovary.
were
At
significantly
this
developed
from in all
stage, in the
deposits medullary
DISCUSSION
growth, testes
animals
dimensions mice
number
ovaries
had
1).
as
age onwards (Table 1). The size and of the small and growing follicular were
of in
control
numbers
(Table
dwarf
total
ovaries than
28 days of composition control
was
in ovaries groups as
than
or
preovulatory in
the
lower
dwarf
populations
values
of
addition,
defined by the first ovulation, was not attained in dwarf mice, whereas corpora lutea were present in the ovaries of control animals from
and
this
follicles distinct
dwarfs
control
observed
less
defined
of
follicles
significantly
greater
younger
Follicles rarely
remaining resulted
not
of small into two
fell
small
However,
significantly
in to
dwarf
The
was
and days
in the high standard error of the mean 1). Either the number of primordial
follicles
age, RESULTS
mice
432
of
days.
28-daydwarf
until
controls
number
dwarf
the
between
number
432
dwarf
reflected (Table
of
maintained
and
significantly
21-day-old
exception
The
at the
were in
difference
was
1).
different
in
the
this
(Table
there
follicles
mice
declined
every tenth section. Growing follicles were counted in every fifth section using the oocyte nucleolus as a marker. The classification of follicles employed was modified from that of Pedersen and Peters (1968) and is based on the size of the oocyte and the number of granulose cells in the largest cross section of the follicle. 1) Small (primordial) follicles, Type 2: nongrowing oocytes surrounded by up to 7 granulosa cells. 2) Medium follicles,s Types 3a, 3b, 4 and 5a: growing oocytes surrounded by 8-20, 21-60, 61100 and 101-200 granulosa cells, respectively. 3) Large follicles, Types Sb, 6, 7 and 8: fully grown oocytes surrounded by more than 200 granulosa cells. 4) Type 5b follicles: 201-400 granulose cells. 5) Type 6 follicles: 401-600 granulosa cells with pockets of fluid in the follicle envelope. 6) Type 7 follicles: fully grown oocytes surrounded by more than 600 granulosa cells with a well defined antrum. 7) Type 8 follicles: preovulatory antral follicles in which the stalk of the cumulus oophorus has formed. Statistical significance was tested using Student’s
However,
With
old
in
2).
primordial
values
females were killed at 7, 14, 21, 28, 35, 92, 135, 183 or 432 days of age. Both ovaries were removed from each mouse and fixed in Bouin’s solution. The fixed tissues were embedded in wax, serially sectioned at 7 m and stained with Harris’ haematoxylin and eosin. The size and composition of the follicular population was quantitated in each ovary using the method of Zuckerman (1951). Small follicles were counted in
test (Miller,
1,
more
of dwarf of
age
Hypoplasia
of
the
adenohypophysis
in
the
mouse is associated with a retardation of abnormalities in the thymus, thyroid, and ovaries and sterility (Bartke, 1964;
Baroni, observations
1967;
Duquesnoy have
all
et led
al., to
1970). the
These
suggestion
OVARIAN
1. Number
TABLE their
of small
phenotypically
(primordial)
normal
Age (days)
7
14
21
and
littermates
IN SNELL
growing follicles same age.
(mean
MICE
961 per ovary
SEM)
±
(die)
in dwarf
mice
Small follicles
Growing follicles
N
C
10(5)
5,383
385
409
32
die
15(8)
6,076
319
381
22
0 0
C
17(9)
5,584
375
472
die
12(6)
5,983
576
529
26 52
0 0
C
19(10) 11(6)
3,919 5,428
218
491
32
0
sloe
502
29
0
14(7) 12(6)
2,892 4,718
353 312
320 280
20 36
1/7 0/6
12(6) 12(6)
3,419
242
4,476
247c
327 298
32 13
2/6 0/6
27(14) 19(10)
2,405 3,262
207 132c
1,094 584
79 43
14/14 0/10
4(2) 6(3)
752 3,182
55 246d
493 194
77 21C
2/2 0/3
20(10) 14(7)
933 1,501
129 192b
955 613
96 48b
10/10 0/7
10(5) 10(5)
395 1,184
62 425
323 84
34 22d
5/5 0/5
C
die
and
of the
(C)
Group
die 28
DEVELOPMENT
Corpora
lutea
35 C
die 92
C
die 135
C die
183
C
die 432
C
die aN
of ovaries
number
=
counted;
( )
number
=
of animals.
cp
OF
REPRODUCTION
19,
959-964
(1978)
Ovarian Development in Hypopituitary Snell Dwarf Mice. The Size and Composition of the Follicle Population EFFIE
HOWE,
G.P.M.
SUE
MOORE1
and
LINTERN-MOORE, JENNIFER
HAWKINS
School of Biological Sciences, Mac quarie University, North Ryde, N.S. W. 2113, Australia and Ian
Clunies
Ross
Division
Animal
of Animal
Research
Laboratory1,
Production,
CSIRO,
P.O. Box 239, N.S. W. 2148,
Blacktown,
Australia
ABSTRACT The size and composition of the follicle population in the ovaries of sterile Snell dwarf (div/die) strain mice differ from that in phenorypically normal contemporaries from 21 days of age onwards. The number of small (primordial) and growing follicles in the ovaries of dwarfs and phenotypically normal contemporaries was not statistically different during the infant and juvenile periods. At 21 days of age, ovaries from dwarf mice contained significantly more primordial follicles due to a reduction
in the rate of atresia in the nongrowing pool of oocytes. This difference was maintained the adult period studied. The dwarf mice used in this study did not ovulate. Nevertheless, antral follicles were present and the developmental sequence of follicular growth followed that of controls. From 92 to 432 days of age, however, the total number of growing follicles was reduced in dwarfs. Furthermore,
throughout
specific compartments of the growing follicular population were affected much earlier. These data are discussed in relation to the reportedly normal concentrations of follicle stimulating hormone and luteinizing hormone and the reduced concentrations of prolactin and growth hormone in the pituitary gland and blood plasma of the dwarf female.
INTRODUCTION Mice
homozygous
mutation
dwarf
growth
(for
for
and
review,
cause
to
dwarfism
specifically
an
lactotropes
resulting
of
(Francis, ductive
are occur,
in
sterile. but
amount
of
However,
Bartke
June April
days
ovaries tissue absent
and
(1964,
are
1965a,
b)
of
proceed
and with
Der
that
antral
contrast
is in
Van
showed
present of
the
The
females
ranging
show
We
dwarfs
follicles aries,
the
reduced
(Kemp,
the effect
population
adult
(Mac-
small,
not
follicle
age.
the
mating
ensues
is
life
This
study
function.
repro-
that
opening never
In
the
of of
indicate
Vaginal
interstitial reportedly
Accepted Received
dwarfs
The
ovulation
of
occur.
which
did
contain ovulation
Kroon
ovarian
beyond
the
and
follicular multilamin-
stage.
the
1960)
investigations
pregnancy
1931).
and
growth
5-12
Early
(1974)
1930;
Rennels,
of
cessation
function
Dowell,
and
histological
Buis
females
spontaneous
may
the
ar
dwarf
that
estrus
growth
deficiency;
MacDowell,
between 1944).
size
primary
somatotropes
and
a
The
from and
cyclic
retarded adult
pituitary
absence
adenohypophysis
females
a
Carsner
from
show normal
1952).
is
(Smith 1938;
may
1929) reach
ovaries
follicles
recessive
Mendelian
see Griinenberg,
of
Kemp,
the
(Snell, fail
that
that
we
dwarf
mutation
size
and
from
that normal the
7
the
have
reexamined on
ovarian
composition
have
contain
than but
study,
been days
to
ovaries
of
of
15
littermates
growing
of and
more or
of
in
months
prepuberal
significantly
number
the
quantitated
small
contemporfollicles
is reduced.
and 1938).
MATERIALS
observed
AND
METHODS
Female Snell dwarf mice (genetic symbol div/die) and their phenorypically normal littermates or contemporaries of the same age were obtained from a colony maintained by CSIRO Division of Animal Production, Blacktown, N.S.W. The colony originated
5, 1978 21, 1978
959
HOWE
ET AL.
from a #/dw female which was imported by the Genetics Research Laboratory, CSIRO, Ryde, N.S.W. from the Institute of Animal Genetics, Edinburgh, U.K. during 1952. All mice were raised in an air conditioned room at 21#{176}C(12 h light:12 h dark) and fed commercial pelletted food and water ad libitum. Mice were weaned at 28 days of age. High mortality among dwarf mice has been observed (Dung, 1975). This is thought to be related to a deficiency in cell mediated immunity resulting from
(Tables
pathological changes (Dung and Swigart, a!., 1970). Although routinely observed mortality of dwarf
because
960
observed in the thymus gland 1970; Baroni, 1967; Duquesnoy et premature thymic involution was at autopsy in the present study, females was sufficiently low to
permit a significant proportion to more than 1 year of age. Fifty-seven dwarf and 68
of the mice
to survive
females.
phenotypically
normal
group,
control
age
dwarfs
from
1966).
was as
dwarf
changes
in
homozygous uteri
were
throughout
mutation the
reproductive
for
the
trait.
infantile the
and period
in
system of In all dwarfs,
mice
the
small
the
ovaries
studied.
Puberty,
the
similar at
in the
ovaries
7 and
14
days
of
of
2).
growing
mice
dwarf
were
(Table
was
control
In
follicles
significantly
mice
at
92
days
of age and at subsequent sampling times (Table 1). Prior to 92 days, however, growing follicles in specific
growth
compartments
(Table 2). This was age with a reduction laminar in
and
dwarf
antral ovaries.
days, ovaries cantly more phenotypically
follicles
(Types
At
age
the
of dwarf Type 7 normal
days
were
there
of
days
of multi5b,
of
6 and
7)
puberty
mice contained follicles than counterparts,
of follicular reduced
age,
affected
21
in
of antral follicles of Type 4 follicles in 28-dayand Paradoxically, at 92
at all other stages were significantly of
were
first evident at in the numbers
control mice, the number (Type 7) and the number were reduced, respectively, 35-day-old dwarf ovaries.
no
signifidid their although
growth, numbers (Table 2). At 135 differences
between
the numbers of multilaminar and antral follicles in dwarfs and controls, but in dwarfs the initial stages of growth were reduced. By 432 days of all
antral
ovaries
follicles and
stages
than
of
control
had
the
disappeared of follicles
numbers
growth
values.
of yellow tissue had region of the ovary.
were
At
significantly
this
developed
from in all
stage, in the
deposits medullary
DISCUSSION
growth, testes
animals
dimensions mice
number
ovaries
had
1).
as
age onwards (Table 1). The size and of the small and growing follicular were
of in
control
numbers
(Table
dwarf
total
ovaries than
28 days of composition control
was
in ovaries groups as
than
or
preovulatory in
the
lower
dwarf
populations
values
of
addition,
defined by the first ovulation, was not attained in dwarf mice, whereas corpora lutea were present in the ovaries of control animals from
and
this
follicles distinct
dwarfs
control
observed
less
defined
of
follicles
significantly
greater
younger
Follicles rarely
remaining resulted
not
of small into two
fell
small
However,
significantly
in to
dwarf
The
was
and days
in the high standard error of the mean 1). Either the number of primordial
follicles
age, RESULTS
mice
432
of
days.
28-daydwarf
until
controls
number
dwarf
the
between
number
432
dwarf
reflected (Table
of
maintained
and
significantly
21-day-old
exception
The
at the
were in
difference
was
1).
different
in
the
this
(Table
there
follicles
mice
declined
every tenth section. Growing follicles were counted in every fifth section using the oocyte nucleolus as a marker. The classification of follicles employed was modified from that of Pedersen and Peters (1968) and is based on the size of the oocyte and the number of granulose cells in the largest cross section of the follicle. 1) Small (primordial) follicles, Type 2: nongrowing oocytes surrounded by up to 7 granulosa cells. 2) Medium follicles,s Types 3a, 3b, 4 and 5a: growing oocytes surrounded by 8-20, 21-60, 61100 and 101-200 granulosa cells, respectively. 3) Large follicles, Types Sb, 6, 7 and 8: fully grown oocytes surrounded by more than 200 granulosa cells. 4) Type 5b follicles: 201-400 granulose cells. 5) Type 6 follicles: 401-600 granulosa cells with pockets of fluid in the follicle envelope. 6) Type 7 follicles: fully grown oocytes surrounded by more than 600 granulosa cells with a well defined antrum. 7) Type 8 follicles: preovulatory antral follicles in which the stalk of the cumulus oophorus has formed. Statistical significance was tested using Student’s
However,
With
old
in
2).
primordial
values
females were killed at 7, 14, 21, 28, 35, 92, 135, 183 or 432 days of age. Both ovaries were removed from each mouse and fixed in Bouin’s solution. The fixed tissues were embedded in wax, serially sectioned at 7 m and stained with Harris’ haematoxylin and eosin. The size and composition of the follicular population was quantitated in each ovary using the method of Zuckerman (1951). Small follicles were counted in
test (Miller,
1,
more
of dwarf of
age
Hypoplasia
of
the
adenohypophysis
in
the
mouse is associated with a retardation of abnormalities in the thymus, thyroid, and ovaries and sterility (Bartke, 1964;
Baroni, observations
1967;
Duquesnoy have
all
et led
al., to
1970). the
These
suggestion
OVARIAN
1. Number
TABLE their
of small
phenotypically
(primordial)
normal
Age (days)
7
14
21
and
littermates
IN SNELL
growing follicles same age.
(mean
MICE
961 per ovary
SEM)
±
(die)
in dwarf
mice
Small follicles
Growing follicles
N
C
10(5)
5,383
385
409
32
die
15(8)
6,076
319
381
22
0 0
C
17(9)
5,584
375
472
die
12(6)
5,983
576
529
26 52
0 0
C
19(10) 11(6)
3,919 5,428
218
491
32
0
sloe
502
29
0
14(7) 12(6)
2,892 4,718
353 312
320 280
20 36
1/7 0/6
12(6) 12(6)
3,419
242
4,476
247c
327 298
32 13
2/6 0/6
27(14) 19(10)
2,405 3,262
207 132c
1,094 584
79 43
14/14 0/10
4(2) 6(3)
752 3,182
55 246d
493 194
77 21C
2/2 0/3
20(10) 14(7)
933 1,501
129 192b
955 613
96 48b
10/10 0/7
10(5) 10(5)
395 1,184
62 425
323 84
34 22d
5/5 0/5
C
die
and
of the
(C)
Group
die 28
DEVELOPMENT
Corpora
lutea
35 C
die 92
C
die 135
C die
183
C
die 432
C
die aN
of ovaries
number
=
counted;
( )
number
=
of animals.
cp
