Attempts to examine the mechanisms that control follicular recruitment, growth and atresia have been confounded by the dynamic nature of these interrelated.
BIOLOGY
OF
REPRODUCTION
30,
Compensatory MICHAEL
82-86
(1984)
Responses
W. FLEMING,2
After
RICHARD
Division
of
Unilateral C. RHODES
Animal
West Morgan
Ovariectomy
and
Virginia
town,
III
and
Veterinary
Rabbits’
in
ROBERT
A. DAILEY4
Sciences
University
West
Virginia
26506
ABSTRACT Compensatory
examined
ovarian
in the
rabbit
and
doe,
gonadotropic
responses
to
unilateral
ovariectomy
(ULO)
were
an induced
ovulator. On Days 2, 4, 5, 6, 8, 10, 15 and 20 after ULO, ovaries from 3 hemiovariectomized does and 1 sham-hemiovariectomized doe were examined macroand microscopically for number, size and signs of atresia of follicles. The number of surface follicles increased initially to 7 or 8 follicles 2 days after ULO, followed by an increase to 10 or more follicles by Day 15 (control ovaries had 5.7 ± 0.4 follicles). Total numbers of antral follicles and the proportion of follicles which were atretic did not vary relative to day after ULO. However, distributions of antral follicles in classes of 0.2-mm increments were significantly different between sham-ovariectomized and hemiovariecromized does after Day 2 due to shifts of follicles into larger size classes. Peripheral serum concentrations of follicle-stimulating hormone (FSH), but not luteinizing hormone (LFJ), increased temporarily during the 48 h after ULO. Follicular compensation after ULO in the doe entailed nonlinear increases in numbers of preovulatory follicles, due to increased growth within the antral population of follicles, probably the result of an acute surge of FSH. A period of more than 10 days was necessary to restore the number of preovulatory follicles after ULO. Exogenous human chorionic gonadotropin (hCG) induced ovulation of recruited follicles.
Specifically,
INTRODUCTION
Attempts to control follicular
examine the recruitment,
atresia have been confounded nature of these interrelated ovariectomy (ULO) permits,
mechanisms growth
tissue
by the dynamic processes. Unilateral in part, delineation
sheep, Dufour
1970;
hamster,
et
al.,
1971;
1968). Induced ovulators physiologic system in mechanisms
that
the
a
estrous
August
doe
rabbit.
offers
a system
constant uncomplicated
that
supply of by luteal
cyclicity. Carmichael
and Lipschutz ovarian size
and
(1925) weight
or
Compensatory
Marshall
documented after ULO
ovarian
in
hypertrophy
Greenwald,
1961;
was proportional to the amount of ovarian tissue removed (1/2 to 5/6), and the ovulation rate several months after ULO was essentially
pigs,
et
normal
Short
al.,
follicular
(Asdell,
estimated mapping antral
dynamics.
follicle
interval numbers and the
15, 1983.
March 10, 1983. ‘Published with approval of the Director of the West Virginia Agricultural and Forestry Experiment Station as Scientific Paper No. 1827. 2Present address: Animal Parasitology Institute, Beltsville Agricultural Research Center, U.S.D.A., Beltsville, MD 20705. ‘Present address: Dept. of Animal and Veterinary Sciences, College of Resource Development, University of Rhode Island, Kinston, RI 02881. Reprint requests.
1924).
through of the on
10 days in the The present Accepted Received
rabbit
relatively follicles,
(1906),
(1908), increased
provide an alternative which to explore the
govern
and
Bond
of the chronology of follicular growth and atresia. Detailed examination of these phenomena after ULO has been limited to species that are spontaneous ovulators (e.g., rat, Peppler and Greenwald,
the
maintains preovulatory
that and
the
intact study
nous
82
was
surface
designed
is accomplished. were classified the
after
acute
role
follicular development was the ovulatory competency ULO
White
(1933)
laparotomies the life span
ovarian doe. was
sequentially
experiment,
and
7 to
to define
the
assessed
gonadotropin.
by
Populations by size ULO.
and of an
was
necessary to attain compensation of preovulatory follicles after possible mechanisms by which
compensation antral follicles condition
Hill
sequential ovary that
In
of gonadotropins
in ULO this of and
a second
on
examined. Finally, of follicles after
stimulation
with
exoge-
FOLLICULAR
MATERIALS
AND
virgin New Zealand White a single source (Roy’s Rabbitry, utilized in all experiments.
were
Experiment
1. Follicular
Populations
does
(3 to 4 kg)
Warrenton,
VA)
after
ULO
underwent ULO (N=24) or shamovariectomy (control: N=8) via midventral incision. Anesthesia was induced with sodium pentobarbital and maintained with ether. An ovary was selected at random and
and follicles having On Days 2, 4, 5, 6, reproductive tracts
3 hemiovariectomized and 1 sampled, as well as Days 4 and turnover of surface follicles White (1933). Follicles >1 ovaries were counted, and
Bouin’s
solution,
histology section
and was
processed sectioned stained
a
diameter
>1
8, 10,
15
were
collected
control
and
doe.
6, because predicted mm on
Day
20
were after from
ovaries routinely serially at 8 jm.
with was
Every third and eosin. at 10 magnificacross section of
hematoxylin examined
Each ovarian series tions for the location of the largest each antral follicle; the 2 maximum right-angle diameters for each of these cross sections were measured with an ocular micrometer at 50 magnifications and
averaged. At 400 magnifications, these follicles were classified as undergoing atresia or developing. Follicles were classified as atretic if at least 2 of the following criteria were observed: loss of homogeneity of the granulosal layer, presence of leukocytes within the antrum, pyknosis of nuclei of granulosal cells, or lack of mitotic divisions in the granulosal layer (Brand and de Jong, 1973; Harder and Moorhead, 1980). Each
antral
follicle was categorized by diameter into size of 0.2-mm increments from 0.20 to 1.60 mm. diameter of antral follicles was 0.20 mm. Differences among distributions of size classes were tested with the G test (Sokal and Rohlf, 1969), and differences among proportions of atretic follicles were identified with Kruskal-Wallis tests (Hollander and Wolfe, 1973). classes Minimum
Experiment
2. Gonadotro
pie Profiles
after
ULO
Eight
does were bled (3 ml) via cardiac puncture every 12 h for 2 days before surgery to establish basal concentrations of gonadotropmns. Surgery was performed as in Experiment 1 (5 ULO, 3 control), and blood samples were obtained every 6 h for 2 days after surgery and every 12 h for the subsequent 5 days. Serum was stored at -20#{176}C until analyzed by specific double-antibody
radioimmunoassay
for follicle-stimulating hormone
et al. pins
(LH)
(1981).
(NIAMDD) kits (FS}I) and luteinizing to the procedures of Mills included purified gonadotroand NIAMDD-LH-
according
The
kits
and antisera against rabbit and rabbit LH (AFP-8-1-28)
FSH (AFP-Rproduced in pig gamma
7-21-76) guinea pigs. Antiserum against guinea globulin was purchased (United States Biochemical Corp., Cleveland, OH) and used at 1:25 dilution. Intraassay and interassay coefficients of variation for the FSH assays were 8.9% and 5.0% and for the LH assays,
8.2%
were
analyzed (Fogwell
design
Rate
after
ULO
2 control)
or Day
20
(N=7
5 does under1. On Day 5
ULO,
3 control)
RESULTS
Experiment
1
Because
regression
follicles on nonsignificant,
day
combined
to
form
preovulatory
ing
of
one
follicles
ovary
from
did not compared
number
group. (>1
(Table 5.7 per
10 1) ovary
days
observed only by ULO does contained
Day
alent to the mean of control does).
minus 1 SD No differences
occurred
or
in
in
the
total
the
between 20 (Fig.
20 when each at least 9 follicles
number of
of
follicles
mm)
in
in
of
the
larger
no From
antral
was
follicles
which
size
were
(P1
of ULO,
control
the
these
occurred
inspection Therefore,
number
tion
of
be
small
antral
rather
than
follicles
or
a
The
in
function (Schwartz
the
and
also occurred of follicles. stimulus of
rate
guinea
for
the
to
be
the
acute,
of
a and
transient with
follicular Channing,
the
inhibin-like 1977).
rise
in
Analogous
new gross
White, 1 and
1933). 3) and
rate
IU
this
of
of
11
hCG
treatment.
or
removed from
present
work),
higher
ovulation
Experiment have
latory
stimulus also
were
surge
1974; keys FSH, surge
FSH
50
IU
of
of
hCG ovu-
some
FSH effects
1981),
follicles
Apparently
>1
mm
was
for
a pre-
conservative.
increase
number
spontaneous
LH,
16 does
ovulatory
ovulate.
ULO
in
Kotwica (Clark et of
of
of
McNeilly,
which
of
and
exceeded pregnant
compensatory
long-term
and
of
25
of
to
criterion
follicular
apparent
FSH,
induced
chronic
of
12
21
5 so that
after
induced surge
9 and
rate
injected
supraphysiological
Day
size follicle
The
5, 17,
dose a
on
preovulatory
found
does
at mating (analogous to the the excised ovary in the one would have predicted a rate on Day 20 than on Day 5
provided
arbitrary
surface
in
population
3. The
might
mm
a
of (1968)
12
on
predicted
counts
Because
had
of hCG the
Adams
ovulation
(Baird
substance
from 1.
mean
ULO
hypothesized
8)
the
ovulatory
recruit-
to
Days
Com-
al,,
(7
Days
4) follicles
By Day
changes
follicles
2
by a slight diameter.
follicular
follicles,
result
pensation
1968) ment
in
by
compensatory
significant
of antral
an
the
full
accompanied follicular
shifts
atretic
the
a
of
maximum
greater. in
to
number
the
surge 7-
at surgery (Hill and both an acute (between
follicles
several
increased
and
increase
without
total
the
mm)
was
(>9) was in maximum
Because
to
follicles follicles
complement decrease
days,
does,
a second
preovulatory
involved
15,
that
ovulation
in rabbits
in
a group that was present that was recruited after
by
The
Within
the
diameter after
ULO
shift in basal Days 2 and
tomy.
DISCUSSION
Response
the
7 or 8 follicles
of
pre-
follicles
by
plement
was
of
response
predicted
contrast,
Day
to
to
population, and a group
chronic surface
hemiovariec-
15. No between
span of the preovulatory follicles on surface (Hill and White, 1933),
does.
control
but
number
bimodal
differentially
population
3
Does
rates
means
ovariectomized
(Bast
follicles
the
preovulatory
the
identified
Apparently, the 1- to not only resulted in the
the of to
hamsters
by Day detected
Furthermore,
According
6
been
and
twice
follicles FSH was
affected -2
have
preovulatory
recruit
recruitment 0.5
ULO 1977)
increase
adequate
2.0
CO
FSH (Butcher,
and Greenwald, 2-day elevatation
5.0
C
85
after
is
ULO-
analogous of
the
in
the
to
the
periovulatory
ovulators. cows
and Williams, al., 1979) had
1982)
and
a single
accompanied
the
purportedly
recruiting
Ewes et al.,
(Akbar
surge
monof
preovulatory the
ovu-
86
FLEMING
latory cycle are both
follicles lengths
in
similar to for maximal
after mating compensatory
for the subsequent these species (18 the
time ovulatory
(Adams, response
Sequential
to
cycle. 28
interval response
1968) after
and ULO.
examination
in
for
of
The days)
rabbits to hCG maximal
ovarian
and
gonadotropic identified alterations
components after ULO has qualitative, quantitative and temporal that further delineate the nature of
follicular sequential
dynamics shifts
in the rabbit. in follicular
follicular size, both apparently acute surge of FSH after ULO.
These include numbers and induced
by
an
ACKNOWLEDGMENTS Supported
by National Research NIADDKD AMO 7312
Service
Award,
Institutional and Hatch Project 224. Reagents for assay of rabbit LH and FSH were provided by NIAMDD. We thank Mr. L. D. Jones for assistance with histological evaluations of ovarian slides and radioimmunoassays. REFERENCES Adams,
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