BIOLOGY
OF
44, 292-298
REPRODUCTION
Effect
of Aspiration
Function,
(1991)
of the
and Peripheral l(
Section
on Luteinization,
Gonadotropin
Concentrations
W. M.
RAND,2
Health,2
Tufts
de Recherche Centre
School
Agronomique,3 Tours,
Luteum
in the Mare
of Veterinwy 19348
of Medicine,
Station
de
Corpus
E. PALMER3
School Pennsylvania
Square,
University
de Recherches
and
of Pennsylvania
Studies,’ University Center, Kennett
of Community National
Follicle
Bolton
New Institut
Plasma
HINRICHS,’
of Reproductive
Department
Preovulatory
Boston,
de Physiologie
Nouzilly
Medicine
02111
Massachusetts
de Ia Reproduction
37380,
France
an inhibiting
effect
ABSTRACT Follicular cells
fluid
from
small-
This
study
was
in vitro.
gonadotropin follicles C).
to medium.sized conducted
and
concentrations, of mares
Mares
when
in Group
the
ovulation of the
fofficle
and
the
for
B than
other
groups.
control These
luteinization
mares. data
inhibitor,
support
and
in Group
the
indicate
of a normal
for development
Mares
in vitro
that
follicles
mm
findings
Follicular
fluid
induction,
may
be
controls. LH and FSH
to have fluid
and
during follicular maturation. smalland medium-sized teinization, progesterone ceptors
by FSH
follicular hances receptor horse,
fluid from progesterone induction granulosa
regulator
progesterone
cells
in vitro
large follicles (6-12 secretion, estrogen
[3,4]. In cells removed
of recep-
ulosa
cells ovulation,
[71; this may occur or when
follicular fluid. Little work has follicular fluid ers [8) reported
from that
follicles did not the oocyte and
Accepted
September
Received
June
fluid from inhibits luof LH re-
University
done
follicles drainage
mm diameter) ensecretion, and LH
B),
peripheral
the
preovulatory
or 40-44
mm
A had
(Group
a significantly
in duration of progesterone higher for mares in Groups
A
of secondary
ovulations
mares
in
contain
a
to medium-sized
days
did
than
follicles
of fofficular
on
the
effect
of aspiration
Since in the
maturation
may is not
essential
in premature inhibitors due
Road,
[11].
of
during Such
this ocThe ef-
proestrus aspiration
in may
due to removal of luteinfluid or in impaired CL
of follicular
induction have occurred.
fluid
before
optimum
and stimulation of progesterone Aspiration of follicular fluid from may also affect growth of secinhibin and other inhibitors
[9, 10].
an
it
one
or
interesting occasionally
LH concentrations
licular discrete
rise
to evaluate the effect preovulatory follicles ovarian characteristics model two
gradually
phase and peak after ovulation preovulatory LH surge seen
for this follicles
of of of
study. The per cycle
throughout
the
fol-
[12, 13], without the in most other species.
The exact stimulus for ovulation and luteinization in the mare is not known; however ovulation does occur at a precise interval following exogenous W administration [14]. Follicular and luteal characteristics in the mare are easily monitored by ultrasonography per rectum [15]. The pre-
of Medicine, North
fluid known.
luteinization in the follicular
to removal
the mare make mare ovulates
ovulatory Department
200 Westboro
follicular is not
This experiment was conducted aspiration of follicular fluid from varying maturity in the mare. The
whereas removal of result in partial lutein-
Dr. K. Hinrichs.
the rabbit is an induced ovulator, absence of ovulatory LH stimulation.
of folliculogenesis
during follicular maturation are cultured in vitro without
Medicine,
from
in Group
the putative ovulatory follicle ondary follicles by removing
in vivo. El-Fouly and coworkof follicular fluid from rabbit
address:
(Group
of granulosa
luteinization,
no difference significantly
smallfinal
LH receptor production
17, 1990.
of Veterinary
from the
function
[1, 2]. In contrast,
many species, including the from preovulatory follicles
result in luteinization, follicular fluid did
and current School
fluid during
result ization
8, 1990.
‘Correspondence Tufts
been
cells
incidence
on
aspirated
Mares
fect of removal of spontaneous ovulators
luteinize in vitro without gonadotropin stimulation [5,6]. It has been hypothesized that luteinization cannot occur unless a luteinization inhibitor (LI) is removed from the granand
was
were
was
mm
aspirated.
There
ization. curred
secretion
In the pig, follicular (1-5 mm) follicles secretion, and induction
in granulosa
35-39
not
values
a higher
follicular
that
of follicular
A),
fluid
on luteinization
fluid
CL
an important
luteinization,
was
of follicular
Fotlicular
(Group
did
INTRODUCTION
tor
removal
mare.
follicle
than groups.
A tended
presence
in the
30-34
was
to have
of in vivo
preovulatory
earlier rise in peripheral progesterone concentrations secretion or peak progesterone production between and
shown
effect
the
of secondary
as controls
been
has
to investigate
diameter
D served
follicles
before
Grafton,
the
MA 01536.
292
follicle ovulation
preovulatory
reaches [16], follicle
and
an due may
average to the be
of 45 long
identified
mm
follicular as the
diameter phase, largest
ASPIRATION
PREOVtJIATORY
OF
293
FOLLICLES
follicle on the ovary as early as 6 or 7 days before ovulation, at about 30 mm diameter [15, 16]. Aspiration of the follic-
and with the other hand placed against the wall
ular
contents
the
ovary
folliclewas punctured. The follicular fluid was ing gentle suction applied with a 50-cc syringe
needle
can per
placed
be done
without
surgery,
by manipulating follicle
with
a
gust, vania.
through
the
flank
[17, 18].
pump set at -100 mm Hg until the follicle was empty and a small amount of blood was obtained. There were no dif-
AND
METHODS
ferences
in follicular
the two
methods
was
the natural Fourteen
conducted
during
breeding light horse
season mares,
15 yr, were used as subjects. in pasture with supplemental
The hay
daily
with
a stallion
to detect
of the 400-500
pation
and
ultrasound
examination
Au-
mares were kept outdoors and grain, and were teased evaluated
tracts
through
horse in Pennsylkg and aged 6-
were
reproductive
tween height and averaged to obtain
April
When
the
of all follicles over 20 mm, amination, were recorded.
late
estrus.
estrus,
per
mares daily
rectum.
as measured If there was
were
by pal-
Diameters
by ultrasound a discrepancy
width of a follicle, the the recorded diameter
in
exbe-
two values were for the follicle.
44 mm
and
Group
in diameter.
C, the Group
performed.
aspiration was largest follicle
was
first
D served If on than
greater
day
the 34
the
follicle
was
as a control first mm
cycle was not assigned to a group and was not the study. If, after assigning the mare to a group,
40-
and
day of estrus in diameter,
no the that
included in the largest
follicle ovulated before reaching the designated size for aspiration, that cycle also was not included in the study. If the follicle evidenced
was not by presence
completely of 5 mm
emptied
on aspiration,
or greater
diameter
in the follicle on ultrasound the cycle was not included
examination in the study.
After
ovulation,
to be teased
daily
mares
continued
trus. On they
the first day mares were were again randomly assigned
Each
mare
was
used
only
once
after
through
found to be to aspiration
in each
as
of fluid aspiration,
aspiration
volume
or dies-
in estrus, groups.
dissection
microscope.
pation
and
days were
the
scrubbed,
preovulatory
follicle
was
with The located
the mare ovary that with
one
on the flank ipsilateral follicle was shaved
and a small area was locally anesthetized
us-
ing 2% lidocaine. An equine trochar was introduced through the flank and an 8-inch (20.3 cm), 14-gauge needle was passed
retrieval
between
[19]. fluid
recorded,
and
ultrasound
examination
ter aspiration. Changes follicle and development cles were recorded. licles aspirated, were third
day
follicles
per
rectum
over 20 monitored
the
with
monitored
in the appearance and ovulation
a
by pal-
daily
for
7
mm in diameter until 10 days afof the
aspirated
of secondary
folli-
Control mares, which did not have folmonitored daily through ovulation until
of diestrus,
over
of an oocyte
The ovaries were
after aspiration; if follicles present, the ovaries were
the
was
for the presence
20 mm
Hormone
or
for
10 days
in diameter
were
after
ovulation
present
if
on the
ova-
Assays
Heparinized assays were of
was
taken
jugular blood daily from
estrus, continuing monitored, and
sequent estrus. was removed
sampling
changes
the time ovarian times weekly until
activity the sub-
centrifuged for 10 mm and at -20#{176}Cuntil analyzed.
of gonadotropins
for studying
samples (10 ml) for hormone mares starting on the first day
throughout then three
Blood was and stored
has been
in hormone
shown
plasma Daily
to be adequate
concentration
in the
horse
[201. Plasma LH concentrations viously described [21]. The ml;
and
the
intra-
and
were measured sensitivity of the
interassay
were 13% and 33%. Plasma FSH concentrations previously described [211.
by RIA as preassay was 1 ng/
coefficients
of variation
(CVs)
6 ng/ml,
and
23%. Plasma
the
intra-
progesterone
RIA as previously
group.
was performed tranquilization.
hand per rectum. A 10 X 10-cm area to the ovary bearing the preovulatory and
aspirated
examined
or oocyte
and
the
and
interassay
CVs were
concentrations
described intra-
were measured by RIA as sensitivity of the assay was
The
and
[22].
The
were
measured
sensitivity
interassay CVs were
13%
11%
and by
was
0.25
and
5%.
Aspiration
Aspiration of follicles standing in stocks, without contained
of the
fluid was
ng/ml
Follicle
emptying
of aspiration
ries.
On the first day of estrus, mares were randomly assigned to one of four groups. Mares in Group A had the preovulatory follicle (the largest follicle present on the ovary) aspirated on the first day the follicle was 30-34 mm in diameter; Group B, the first day the follicle was 35-39 mm in diameter;
aspirated usor a vacuum
puncturing
The study
the needle was follicle and the
and
Mares The
the needle, designated
rectum
MATERIALS
the
guiding of the
through
ity. By manipulating
the trochar cannula the
ovary
with
into the peritoneal cavthe
hand
in the
rectum
Analysis The
of Data Fisher
exact test was used to compare the numbers ovulations. The Mann-Whitney two-sample test used to compare groups to controls for days from the
of secondary was
time a 30-34-mm follicle progesterone concentrations ple
t-tests
with
significance were gesterone gesterone
the
was found to the first day that were over 1 ng/ml. Two sam-
Bonferroni used
adjustment
to compare
values remained concentrations.
above Repeated
the
of the
level
length of time
of pro-
1 ng/ml and peak promeasures analysis of
294
HINRICHS
variance
was
centrations used
used
to compare
over time,
to identify
icance
level
gesterone quired
using Cruncher
Hintze
The
were analysis
used of
statistical System,
test
with
logarithms
to achieve variance.
con-
range
differences,
natural
the NCSS Statistical
JL, Kaysville,
in hormone
Newman-Keuls
group
0.05.
1 fol-
mm in diameter to the onset of progesterwas 4.6 ± 0.5 days for Group A; 5.5 ± 0.6 B; 5.8 ± 0.8 days for Group C; and 6,25 ±
1.2 for controls. Time to onset of progesterone production was significantly shorter in Group A than in controls. The life span time progesterone
of the CL was concentrations
assessed
remained
by the over
length of I ng/ml.
The average
the effect
of hormone
was
centrations
lengths of CL life span (days) for the four groups Group A, 12.5 ± 3.7; Group B, 12.9 ± 2.7; Group C, ± 1.0; and control, 13.4 ± 1.24. These differences were
were 12.8
Concentrations
ovulation on peripheral cycles in which there
There
concentrations no significant
In Group
higher than for control showed a significant
of ovu-
2/8. There ovulation
individually
trols.
ferent FSH,
giving the appearance of a corpus hem[23]. Follicles that were seen to be collapsed examination did not refill with fluid over the 7 days,
echogenic
with
both
idly in Group A mares than in control LH and FSH values were significantly
The number of cycles A, 11; Group B, 6; Group
1/7.
for
group-by-day
Follicles
cytes
in each group thus used for comparison was as follows: Group A, 5; Group B, 4; Group C, 6; and control, 6. LII, FSH and progesterone concentrations for Groups A, B, and C from the day of aspiration to 6 days afterward are presented in Figure 1. In each case, the hormone concentrations were compared to those for control mares with cycles standardized so that Day 0 = the day that the preovulatory follicle was the designated size (i.e. Group A, 30A, LH and FSH were mares. Progesterone
RESULTS
tion
ET AL.
of secondary
follicle
hormone concentrations, was no secondary ovulation concentrations.
The
number
growth and only those were used of cycles
not
significant.
for
each
B, 6.9 These
group
The
average
(ng/ml)
peak was
2.1; Group C, 8.2 differences were also ±
Group ±
progesterone A, 9.06
4.2; and control, not significant.
production ±
3.3; 8.2
Group ±
2.7.
ASPIRATION
o-o
Group
A
PREOVULATORY
OF
295
FOLLICLES
(n
#{149} 5)
:::
10
(1
0
1
2
I
I
I
I
3
4
5
6
10 0-0
Group
A(n5)
#{149}-.
GroupD(n6)
#{163}.-.i
OroupA2(n6)
00
Group
.-.
GroupD(n.6)
B(n4)
5
I 0
Ii
012
0
1
2
4
3
5
6 0-0 6
0roupC(i6)
#{149}-#{149} Group
0 (n
#{149} 2)
5
4
I
3 2
e 0
1
from
Days
FIG. 1. (Group
mm
30-34mm
Mean (±SEM) A), 35-39 mm
follicle
LH, FSH, and progesterone (Group B), or 40-44 mm
1
2
Days
from
of follicular
DISCUSSION fluid from
follicles (Group A) was luteinization, as determined progesterone
values.
progesterone
production
The
immature
associated by the time
with time
from
in Group
aspiration
sistent (4 or 5 days in all 11 mares versus the time of a 30-34-mm follicle to onset production removal
in control mares). of the follicular fluid
cells able serum. horse
clear. horse
Channing follicles
in vitro, follicles However, granulosa
whereas 2-6 cm the cells
early rise in
to onset
remarkably
of con-
5-8 days from of progesterone
This early luteinization after suggests that LI may be pres-
ent in the follicular fluid at this stage of follicular In vitro evidence on the distribution of LI in horse is not atretic
et al. [24] reported inhibits luteinization
growth. follicles
that fluid from of pig granulosa
the effect of follicular fluid from vidiameter is not different from mare use of pig granulosa cells rather in the culture system may have
founded
the
results.
In
peared
to be
better
than
another plasma
study,
35-39mm
5
6
0
follicle
follicular
in stimulation
fluid
at 30-34 so that
1
2
than conap-
of proges-
I
3
Days from
40-44mm
mm diameter and that had a second Day 0 = the day that the preovulatory
follicle follicle
a
follIcle
was aspirated at a diameter was not aspirated in Group
terone and estrogen production by in vitro, but the assay was performed
preovulatory
significantly to the first
A was
4
concentrations for mares in which the preovulatory follicle (Group C), and that had no secondary ovulations. The follicle
Group A2 represents mares in which the preovulatory follicle was aspirated of aspiration. For each comparison, data for controls have been standardized size for aspiration (e.g. 30-34 mm for comparison to Group A).
Aspiration
3
of 30-34 D mares.
ovulate within 10 days was of the designated
horse granulosa by incubating
cells gran-
cells with fluid from the same follicle [25]. In the pig, to demonstrate the LI activity of follicular fluid, cells from large follicles are incubated with fluid from small follicles; ulosa
to demonstrate LS activity (stimulation of progesterone secretion), cells from small and medium follicles are incubated with fluid from large follicles [26]. In the pig, LI may be found in follicular fluid in small
or mid-size in follicles
follicles (up to 5 mm in size), while LS is found 6-12 mm in size. The difference in luteinizing
activity of follicular to the preponderance categories licles that estrogen at in 12 in
[27]. have production
fluid has been hypothesized of atretic follicles in the However, received
large follicles preovulatory
by preovulatory
to be smaller
due size
may represent folstimulation. In pigs, follicles
rises
sharply
Day 16-17, when follicles are growing from 5 to 10 mm diameter, and the LH peak occurs when follicles are 10mm diameter [28]. Although no discrete LH peak occurs the horse, if we assume that the signal for ovulation is
296
HINRICHS
ET AL.
which
might
have
the follicle, 10
I
‘ A
I
I
I
I
I
0
1
2
3
4
inhibit
that
bined induce
with increased LH stimulation, early luteinization. rise
such
in FSH
receptor
after
5
follicular suppression
fluid has been shown of FSH and follicular
The
in FSH
rise
either
6
tact. after
10
suggests
prevents
the
It is notable aspiration,
could
E C
I
be
rectly
n, -1
#{149}
that
release
to
pre-ovulatory
aspiration of
fol-
a sufficient when the
to produce
[24] and by mare [31,32].
of follicular
these
hormones amount follicular
that the follicles even though those
expected
i
23456
7
Group
A (n
5)
Group
B (n
4)
Group
C (n =6)
A-
A
ml
the
to reduce wall is left
did not aspirated
7-15
fluid into
refill with at 30-34
more
piin-
fluid mm
fluid
before
ovulated, as determined from follicles in Group
A
S
centrations,
I
days
/
from
3
aspiration
4
5
or ovulation
presence in the
received ovulatory gous to mid-size possible include
stimulation and thus follicles in the pig. stimuli for the increased
2 days before would not yet may
early luteinization FSH seen after
that
[9] or depress
directly
followed
suppress
be
aspiration.
are
the
size
mares by hemiovariin plasma FSH con-
of a second
differences follicle
ovuhave
homoloin Group aspiration,
of the follicular wall present study. However,
seen in horse mares control of secondary
ovuand follicle
follicle
about
study, the ovulations following 30-34-mm group occurred 3-8
Major of the
in
[10]. Removal of these compomay have been associated with
by ovulation present in the
of fol-
folliculogenesis
at the
between
these
time
of aspiration
total and
[11, 33], follicular
suggesting growth
two
population the continued
and, subsequently, another important
ence may be that pony mares rarely ovulate [11] and do not show the mid-cycle follicular
of
of the CL differ-
two follicles development
differences in the between horses and
ponies. The
approximately at 30-34 mm
factors
of a pool
or removal, the removal of half of the follicles in the case of hemiovariectomy,
6
FIG. 2. Mean LH, FSH, and progesterone concentrations for mares in which the preovulatory follicle was aspirated at a diameter of 30-34 mm (Group A), 35-39 mm (Group B), or 40-44 mm (Group C), and that had no secondary ovulations. The follicle was not aspirated in Group D mares. The data are standardized so that Day 0 = the day the preovulatory follicle was aspirated (Groups A, B, and C) or the day ovulation was detected (Group D).
by the follicle then the follicles
absence
36.7 mm) in pony in a prolonged rise
later. In the aspiration
after
studies
Days
contain
growth
diameter resulted
15 days follicular
2
may
follicle
(average ectomy
/
I
fluid
in the
the tendency for an increased incidence of secondary lations in Group A mares. In a study by Driancourt Palmer [33], complete removal of the preovulatory
/
GroupD(nz6)
-A
secondary
bloodstream
response to gonadotropins nents, and the rise in FSH,
/
4-
to the
licular fluid. Follicular
.
a 0-0
Other mares
com-
helped
B and Group C. The rise in FSH levels would seem to indicate either that the granulosa cells stopped producing these hormones after aspiration or that they are not released di-
5,
received lation,
compo-
This,
have
by rat bioassay activity in the
the time they would normally have from the amount of fluid recovered
I
[29].
could of the
within
fluid
formation
aspiration
bloodstream, or removes tuitary suppression, even
A
of LH receptors
of the follicular
licle may be due to removal of inhibin or estrogen. Estrogen supresses FSH secretion in the mare [30]. Inhibin has not been isolated in the horse, but its presence in mare
5
-1
formation
nents
The
0#{149}
enhanced
in the absence
B is not removal
cause
of the
immediately of factors
increase
in LH seen
in Groups
A and
apparent, but may be associated with present in the follicular fluid. Estrogen
has been shown to be a stimulator of LH release in the mare [20, 30], so estrogen withdrawal is probably not the cause of the LH rise. Removal of testosterone, another component
of horse
follicular
fluid
[34],
could
be
related
to the
ASPIRATION
increase
in
LH.
Administration
press LH secretion increase in mares
of
OF
testosterone
in mares [35], and passively immunized
may
luteum
gesterone tions, was development.
production and peak progesterone concentranot affected by aspiration at any stage of follicular Ultrasound examination also indicated that
morphologically normal mal luteal function has ture
follicles during
and
mares
[37-39]
granulosa tissue study, aspiration aspiration tubing, piration, resulting
the follicle.
The
as determined
CL formed been found
oocyte and
after after
recovery
has
been
by length
a
Abnorof ma-
in humans,
on FSH values
of pituitary
were
similar,
suppression
probably
by inhibin
amount
reflect-
or estrogen
been
demonstrated
high
in the
aspiration
group;
this
probably
terone
production
from
the
histologically groups
but
is a result with
[42]. LH values
remained ovulation
of the
resulting
of
removed from the follicle. In the present was stopped when blood was seen in the and the follicle was not flushed after asin minimal loss of granulosa tissue from normal CL function in Group A mares sug-
in rapdue has
declined
in the more
rapid
suppression
proges-
of LH release
pituitary. It must be noted that at a definite point (between
while 0800
was performed
monkeys,
to the
loss
both cases. Progesterone values, however, rose more idly after ovulation than after aspiration. This may be to preovulatory luteinization of the follicle wall, which
of pro-
aspiration. aspiration
related
ing
297
FOLLICLES
and ovulation
sup-
LH concentrations against androgens
[36]. Corpus
function,
PREOVULATORY
aspiration h and 1100
h on the morning of the day of aspiration), ovulation may have occurred at any time in the 24 h previous to the day of detection of ovulation. Duplicate analyses were performed Day
assuming
ovulation
had
0, with no significant This study raises many
occurred
change questions
24 h previous
in results. about the
incidence
to and
gests that the follicular fluid was not needed past 30-34 mm diameter to modulate LH receptor formation or stimulation of granulosa cells for proper luteinization.
function of follicular fluid investigation is warranted
Evidence hibition of
on granulosa cell function in vitro. The results of this study also emphasize the need for further work on the origin of LI and its prevalence in atretic versus growing follicles.
gested
has been luteinization the oocyte
that
presented that links [8,40,41], and may be the source
the oocyte to init has been sugof the luteiniza-
from
the
tion inhibitor found in follicular study, few oocytes were recovered ular fluid; this low recovery rate
fluid [41]. In the present in the aspirated follicis expected when aspir-
Because
ating
follicles
granulosa
immature
(nonstimulated)
[17-19].
No
00-
cytes were recovered in the Group A cycles, had significantly earlier onset of progesterone
yet this group production
than
the
did
controls.
It is possible,
however,
procedure disrupted communication and adjacent granulosa cells and thus
that between lowered
aspiration
the oocyte the effective
amount of LI present. In Groups B and C, there were no apparent differences in time from aspiration to onset of progesterone production between the mares from which oocytes were bers are too
and small
were not recovered, although the numto make any meaningful comparisons.
The aspiration size work done by Pierson
groups were and Ginther
chosen on the basis of [16], who reported that
the average diameter of the follicle the day tion was 45 mm. However, the average size
before of the
ovulafollicle
before ovulation in control mares in this study was 39 mm, and 5 of 8 control mares ovulated before the follicle reached 40 mm. For this reason, mares in Group C, with the follicle aspirated in that reaching
at 40-44 mm, constitute an atypical group of mares they were selected for the preovulatory follicle at least 40 mm. This, and the fact that only 2 con-
trol
mares
and pare
had no hormone
make
any
ficult. When
(the
2 control
mares
that
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of the
ovulated were those
results
after
available of Group from
40 mm to comC mares
Group
C dif-
various
of
pared (Fig. 2), did not entirely
effects
of aspiration
it is evident that mimic ovulation.
and
ovulation
are
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