Dec 3, 1985 - influence the initiation of meiosis since germ cells closest to the medulla or the rete cells of the ovary were found to be the first to enter meiosis.
BIOLOGY
OF
35,
REPRODUCTION
Role
191-197
(1986)
of the Pituitary
in Controlling
M. MAZUR
and
Department
Oogenesis
of Obstetrics Health
Rabbit’
E. V. YOUNGLAI2
McMaster Hamilton,
in the
and
Gynecology
University Sciences
Ontario,
Center L8N
3Z5
Canada
ABSTRACT The
involvement
experimental pituitaries
of
the
model.
Fetal
or as half
cranial
pituitary and
and
neonatal
mesonephros ovaries
or caudal
in
were
portions.
oogenesis
cultured
Progression
was
for
examined
8 days
of meiosis
with
with
and
the
without
was followed
rabbit their
histologically
and
graphically by the uptake of tritiated thymidine. Pulse labeling for 24 b occurred on Days 0, 2, 4 ture. No difference was noted in labeling or progression of meiosis in neonatal ovaries with or without tary or in sectioned ovaries. By contrast, index (positive cells/1000 im2 of cortex)
fetal ovaries with of 282.1 ± 32.9
without logically,
7 showed the in percentage
2.9%
pituitary. there
was
compared
ducing
Those labeled a threefold
on Day difference
to 7.1 ± 2.8%
meio tic prophase
without).
These
data suggest
is dependent resolved.
pophysectomy of pregnant monkeys 58 and 71 of gestation had no adverse an morphology of term young, whereas between
Days
in disordered
atresia
and
114
1977).
that this
placental gonadotropins time (Hodgen et al.,
Higher
These
findings,
is essential for and the development levels
Reviers,
1969)
and
of gestation cords
combined
with
could not 1975) suggest the
3, 1985. 23, 1985. supported by
(263.7 ± 46.0 vs. 160.2 pituitary undergoing
the
has
± 20.8). meiosis
an important
to al.,
hormone male 1976),
newborn
re-
the
ating (Stein ovaries
early
ones, (Fajer
(FSH)
tissue
have been found sheep (Maulon female
pitui-
Histo(21.2 ±
role
in in-
Medical
Research
rabbits
Council
cultured (Burns,
LH),
premeiotic 1961;
as well
as sex
ovaries, Mauleon,
failed 1975;
1975).
rat
ovary
induction et al.,
coincided
with 1979). were
the
meiosis-inducing media model
cell
favorable
provides
a
of
older with
of meiosis was observed 1979). It is believed that
produces
the proliferathe differenti-
onset
When cultured
which is present in culture Evidence using the rat contact
the initiation of the medulla or the to be the first to
1975). Moreover, epithelium into
and Anderson, of the hamster
meiosis meiotic younger
in the latter mesonephric
substance (Byskov, suggests
(MIS),
1975). that somatic
environment
for
meiosis. Using cultured gonads on a collagenated nucleopore filter, which prevented cell-to-cell contact between the pairs, Stein and Anderson (1981) showed that each gonad proceeded along its specified genetic meiotic pathway regardless of the underlying tissue. The presence no difference
the
hormone,
enter meiosis (Byskov, tion of the mesonephric
et fact
of
luteinizing
The rete system may influence meiosis since germ cells closest to rete cells of the ovary were found
oocyte (Gulyas
and
Challoner,
(YoungLai et al., 1981; Berger et al., 1982; deTurckheim et al., 1983), further implicating gonadotropins in oogenesis. By contrast, gonadotropic hormones
Accepted December Received September ‘This work was Canada. 2 Reprint requests.
with
steroids added to to induce meiosis
be detected at that the fetal
progression of rete ovarii.
of follicle-stimulating
in female fetuses compared in the human (Reyes et and
117 rete
on Hy-
between Days effects on ovarifetal hypophy-
of oogenesis,
of the
al.,
pituitary oogenesis
and
progression
degeneration
autoradio-
and 7 of cul-
were labeled on Day 4 had a labeling compared to 107.1 ± 24.9 for ovaries
that the fetal pituitary
(FSH
The question whether oogenesis extraovarian factors is not completely
sulted
same differences of germ cells
as an
in the rabbit.
INTRODUCTtON
sectomy
pituitary that (mean ± SEM)
used
corresponding
or absence of mesonephric tissue in the morphological differentiation
the ovary. No MIS matrix, even though
of
system 191
were
intact.
was found in the ovarian
the collagen cords and
caused of gel rete
192
MAZUR
Many
questions
oogenesis
regarding
remain
model,
we
tested
factors
from
the
the
control
unanswered. the
Using
pituitary
rabbit
that
are involved
as our
extraovarian
in the
YOUNGLAI
used
of mammalian the
hypothesis
AND
per
and
induction
dish.
The
heat-inactivated streptomycin
MEM
fetal solution
streptomycin
was
base
with
10,000
pg/ml)
and progression of meiosis. In addition, we sought to determine whether the cranial end of the ovary with its developing rete system would have a higher percentage to the because completed
Days 0, 2, 4 and 7. After 24 h, medium the label was replaced with fresh medium [3 H] thymidine because the minimum
and
of germ cells in meiosis in culture compared caudal end. The rabbit is a unique model oogenesis does not begin until birth and is within the first three weeks of life (Teplitz
Ohno,
1963;
Peters
et al.,
1965).
MATERIALS
AND
of
(Crone every
New Zealand White rabbits were obtained from local breeders. Adult females were housed individually with
food
mated on Day provided still
and
with
water
a fertile
available buck.
28 of pregnancy fetal (premeiotic)
dividing
within neonatal
by mitosis.
12-30 h of birth (when meiosis
Culture Biologicals, activity
media NY. 76.6
libitum
and
females
(Day 28 ovaries,
were
were
killed
post coitum), when oogonia
Newborn
female
(Day starts)
1 postpartum) ovaries and
rabbits
and are killed
provided pituitaries.
were purchased from Grand Island Methyl [3 H] thymidine, specific
Ci/mmole,
gland Nuclear autoradiography
ad
These
was
Corp., were
obtained
Boston, MA. performed
from
New
En-
Histology and using standard
procedures. Organ
Culture
Methods were similar to those of Baker and Neal (1969). Ovaries without external rete were washed in sterile Hank’s balanced salt solution before transfer to Pyrex culture dishes. Cultures were maintained at 37#{176}Cin an incubator and 100% humidity.
containing Two half
95% or whole
placed in each culture dish with or pituitary placed adjacent to the ovaries. was made to separate the anterior from
CO2 /5% CO2 ovaries were without the No attempt the posterior
pituitary. Two different protocols were Protocol 1, fetal (Day 28 postcoitum) or (Day 1 postpartum) ovaries were cultured without pituitary. In Protocol 2, fetal or ovaries were sectioned into cranial and caudal and cultured sterile lens Five ml without
separately. paper supported
used. In neonatal with or neonatal halves
The organs were placed on on a stainless steel tripod.
of sterile Minimum ribonucleases and
Essential Medium deoxyribonucleases
(MEM) were
and
the germ cells was accomplished HI thymidine, 25 pCi per dish,
[3
terminated fixed
was
3 separate
at
(DNA)
from
is
14
h
of
8 days
for
of
histology
were serially sectioned from each ovary were Fetal tissues from at
were
selected
end fixative
Tissues sections analysis.
litters
obtained
the
in Davidson’s
and autoradiography. at 8 pm, and 6-17 subjected to further least
acid
on
containing without time for
1965). Thereafter, media were changed Regardless of labeling date, all organ
were
culture
of
Deoxyribonucleic
et al., 3 days.
cultures METHODS
0.5%
U/mI).
Pulse labeling adding sterile
synthesis
10%
and
Nystatin by
(10,000
supplemented
bovine serum, 1% penicillin(penicillin base 10,000 U/mi
cultured.
Neonatal
randomly
different
tissue litters.
Analysis Classification of germ cells from haematoxylinand eosin-stained sections was carried out as described by Peters et al. (1965) for the rabbit ovary. Further definitions Challoner
were
provided
by
the
descriptions
of
(1975).
The autoradiographic analysis consisted the number of positive germ cells, i.e., silver grains in a cell per 1000 pm2 live
of counting more than 4 cortical area,
since background per cell. In this differences in
than 4 grains be based on germ cells
between
radioactivity was less way comparisons could percentages of labeled
groups
and
between
quantified morphometric
Nashville error was
TN) with a Wild-Leitz less than 0.1%). Figure
tive germ
autoradiographs cells in fetal
by
days
area was digitizing
of and
means system
of
labeling.
The
of a BIOQUANT (R & M Biometrics,
II
microscope (user 1 shows representa-
[3 H] thymidine neonatal ovaries.
labeling Germ
of cells
were distinguished from interstitial or rete cells by their large size and round shape. The rete cells were small and surrounded the germ cells in nests, or were confined to the hilar region of the ovary. The interstitial cells were oblong, and meiotic cells could be easily distinguished (Fig. 2). Prior to analysis, all slides were coded and randomly ing the various stages of meiosis ages of statistical
labeled analysis
germ cells. of results.
selected and areas Data
were
for quantifyand percentdecoded
for
CONTROL
FIG.
1. Autoradiographs
of
3
H] thymidine
uptake
by
OF
representative
cultured rabbit ovaries. (A) Fetal ovary cultured with fetal pituitary. Note integrity of medulla and arrangement of germ cells in cell nests in cortex (X110). (B) Neonatal ovary. Note necrosis of medulla (X110). (C) Enlargement of autoradiograph. Note: Two germ cell nests with interstitial mitosis cell (arrow) (X660).
OOGENESIS
FIG. 2. Photomicrographs of representative cultured rabbit (A) Fetal ovary showing germ cells in interphase (I), preleptotene and leptotene (L) (X 1050). (B) Neonatal ovary showing germ zygotene (Z) and pachytene (P) (X 1050).
193
ovaries. (Pr) cells in
194
MAZUR
AND
YOUNGLAI
With
Statistics
test
Statistical analyses for nonparametric
one-way Duncan’s level was
included data
and two-way multiple range taken as 5%.
the and
the
Mann-Whitney unpaired
analysis of test, wherein
regard to (p
