BIOLOGY OF REPRODUCTION 57, 1066-1073 (1997)
Functional Interrelationships between Follicles Greater Than 4 mm
and the Follicle-Stimulating Hormone Surge in Heifers'
J.R. Gibbons, 3 M.C. Wiltbank,4 and O.J. Ginther 2,3
Departments of Animal Health and Biomedical Sciences3 and Dairy Science, 4 University of Wisconsin,
Madison, Wisconsin 53706
ABSTRACT
The interrelationships between the FSH surge that initiates a
follicular wave and the follicles in the wave were examined in
heifers. In experiment 1, - 5-mm follicles were ablated 5 days
after ovulation and heifers (n = 6/group) received a total dosage
of 0, 37.5, 75, or 150 units of porcine FSH. Half of the FSH
dosage was administered 24 h after ablation followed by the
other half 12 h later. Blood samples were taken after the initial
FSH injection for FSH assay, and ovaries were examined daily
with ultrasound to monitor follicle growth. There were progressively higher FSH concentrations at the mean peak (8 h after
initial injection in all groups) as the dosage increased (interaction of dose and time; p < 0.001). Compared to values in controls, the highest dosage (150 units) approximately doubled the
number of 5- and 6-mm follicles; this then progressed into a 4to 7-fold increase in the number of 7- and 8-mm follicles.
In experiment 2, either all (controls; n = 6), two (n = 11),
one (n = 6), or zero (n = 6) follicles of the first wave of an
estrous cycle were retained and the remaining were ablated
upon reaching 5 mm. Scanning and blood sampling were performed every 8 h for 72 h after the initial ablation. Mean FSH
concentrations during 0 to 72 h decreased (p < 0.004) as the
number of retained follicles increased. In heifers in the onefollicle group, the randomly chosen 5-mm follicle developed the
characteristics of a dominant follicle.
The following conclusions were made: 1) the number of follicles that advanced into a follicular wave was increased by exaggerating the height of the FSH surge, 2) all 5-mm follicles
of a wave contributed to the declining portion of the FSH surge,
and 3) any 5-mm follicles at the emergence of a wave were
capable of becoming the dominant follicle.
INTRODUCTION
The initiation of a follicular wave in cattle is temporally
associated with a surge in FSH concentrations [1]. The largest follicles of the wave are approximately 4 mm in diameter at the peak concentration of the FSH surge [2, 3].
As the FSH concentrations decline, the follicles of a wave
continue to grow. Approximately 3 days after the FSH
peak, when the largest follicles reach approximately 8 mm,
one follicle continues to grow and becomes the dominant
follicle, and the remaining follicles regress and become
subordinate follicles [4]; this event is termed deviation [3].
Although the declining portion of the FSH surge is temporally associated with follicle growth beyond 4 mm, it is
not known whether these growing follicles play a direct role
Accepted June 20, 1997.
Received April 16, 1997.
'Research supported by College of Agricultural and Life Sciences, University of Wisconsin-Madison, and USDA Grant No. 9401480. Some of
the data were presented at the 1996 Midwestern Section of the American
Society of Animal Science meeting or the 1997 Society for the Study of
Reproduction meeting.
'Correspondence: O.J. Ginther, Animal Health and Biomedical Sciences, University of Wisconsin-Madison, 1655 Linden Drive, Madison,
WI 53706. FAX: (608) 262-7420; e-mail: [email protected]
in the initial decline in FSH concentrations. The final decline in the FSH surge may be part of the deviation mechanism, since the FSH nadir is reached about the time of
deviation [4]. The growth of the selected dominant follicle
likely plays a role in the continued suppression of FSH after
deviation.
Multiple ovulations can be induced effectively by administration of exogenous FSH on the day of or the day
before follicular wave emergence at 5 mm [5]. A 4-day
regimen of recombinant bovine FSH increased the number
of ovulatory follicles when treatment was initiated on the
day after ovulation but not when it was initiated 5 days
after ovulation [6]. Multi-day FSH administration protocols
rely on increasing the width of the FSH surge during wave
emergence and advancing more follicles into the preovulatory pool. The protocol may delay deviation and allow
many follicles to advance and assume the characteristics
that only a single dominant has in an unstimulated wave.
In this regard, a 2-day FSH regimen begun when the follicles of a wave reached 6 mm did not increase the number
of follicles per wave but did delay deviation, as indicated
by greater maximum diameter and delayed regression of
subordinate follicles [6].
The presence of a viable dominant follicle inhibits emergence of a new follicular wave by an unknown mechanism
but likely involves the continued suppression of FSH by
the dominant follicle. Experimental destruction of the dominant follicle results in an immediate increase in FSH concentrations, and a new follicular wave emerges within 48
h [1, 2, 7-9]. Similarly, the inclining portion of a new FSH
surge may reflect the loss of the dominant follicle's ability
to suppress FSH concentrations [3].
In experiment 1, the height and width of the FSH surge
were manipulated by administration of porcine FSH, and
developing follicles from the new wave were examined.
The hypothesis that the number of follicles in the pre-deviation pool is a function of the height and width of the
FSH surge was tested. In addition, growth profiles of dominant and subordinate follicles, the time required to reach
deviation and ovulation, and the diameter of ovulatory follicles were analyzed.
The purpose of experiment 2 was to separate the FSHsuppressing actions of all follicles acting in concert from
the suppressing actions of one or two follicles. In this experiment, follicles were manipulated and FSH was monitored to test the following hypotheses: 1) the declining portion of the FSH surge (prior to deviation) is a function of
the pool of growing 5-mm follicles acting in concert and
2) any 5-mm follicle of a wave has the capacity to become
dominant. In addition, analyses were made of the interrelationships among follicle numbers, types, diameters, and
locations (ipsilateral and contralateral ovaries) and between
the follicles and corpora lutea; no hypotheses were under
test in these comparisons.
1066
INTERRELATIONSHIPS BETWEEN FOLLICLES AND FSH
MATERIALS AND METHODS
Ultrasound-guided transvaginal ablation of follicles was
performed as previously described [2, 9]. Briefly, an Aloka
500-V (Wallingford, CT) ultrasound scanner with a 5-MHz
curvilinear transvaginal probe was used to image the follicles during ovarian manipulation per rectum. A 17-gauge
needle (RAM IVF Supply, Madison, WI) was inserted
through the transvaginal probe and into the follicle to be
ablated, and the contents were removed under vacuum suction (-100 mm Hg). The ovary was reexamined with a
7.5-MHz transrectal probe to assess whether all targeted
follicles were ablated successfully.
Experiment 1
Follicles (- 5 mm) of nulliparous, 2-yr-old Holstein
heifers (approximately 400 kg) were ablated 5 days after
ovulation to initiate synchronized follicular wave emergence [2, 9]. Heifers (n = 6 per group) were randomized
by replicates into groups receiving s.c. injections of 10 ml
of saline (controls) or a total dosage of 37.5, 75, or 150
units of porcine FSH (SuperOv; Ausa International Inc.,
Tyler, TX). Half of the dosage was administered 24 h after
ablation followed by the other half 12 h later. The day of
ablation was designated as Day 0, and the 24-h injection
was given on Day 1. A fifth group received 75 units of
FSH in a similar schedule on Day 2 (48 h after ablation).
Luteolysis was induced with a total dosage of 50 mg prostaglandin 2 (two injections of 25 mg each; 12 h apart) 48
h after the initial FSH injection [10] to allow study of the
number of ovulations. Blood samples were taken 24 h before ablation, just prior to ablation, 12 h after ablation, and
0, 1, 2, 4, 8, 12, 24, 48, and 72 h after the initial FSH
injection for analysis of FSH concentrations. Ovaries were
scanned daily to monitor follicular development from the
day before ablation until ovulation. Beginning on the day
of FSH treatment, the diameter of individual follicles was
recorded by maintaining their day-to-day identity as previously described [11, 12]. The number of growing 5- and
6-, 7- and 8-, and - 9-mm follicles on each day was determined retrospectively. Once a follicle began to decrease
in diameter it was no longer considered for the endpoints
involving number of follicles. Deviation was defined as the
beginning of the greatest difference in growth rates between
the largest ovulatory and nonovulatory follicles [4]. The
diameter of the ovulatory follicles on the day before ovulation was considered the preovulatory diameter.
Experiment 2
Nulliparous, 2-yr-old Holstein heifers (approximately
400 kg) were examined daily with transrectal ultrasound
beginning 17 days after ovulation. When the corpus luteum
regressed to c 18 mm and the suspected preovulatory follicle reached - 14 mm, heifers were scanned every 8 h to
detect the emergence of the first follicular wave of the next
estrous cycle (wave 1). Emergence of the wave was defined
as occurring at the examination when a follicle first reached
5 mm. Heifers were randomized by replicates into groups
in which either all (controls), two, one, or zero follicles of
wave 1 were retained and all others ablated upon reaching
5 mm in diameter. Each group was assigned 6 heifers, except that the group with two retained follicles was assigned
12 heifers so that ipsilateral and contralateral relationships
between the two follicles could be examined. Only heifers
with a single pretreatment ovulation, emergence of the first
1067
5-mm follicle within 48 h after ovulation, and more than
one 5-mm follicle in wave 1 were used.
Follicle ablation sessions began at the time of emergence
of the first 5-mm follicle of wave 1 (Hour = 0). When
indicated by the group assignment, follicles to be retained
at 5 mm were chosen by randomization. After Hour 0, ultrasound scanning to monitor individual follicle growth
[11, 12] and blood sampling for FSH assay were performed
at 8-h intervals until Hour 72. An additional sample was
collected at Hour 96. Ablations were performed on new
follicles when they reached a diameter of 5 mm at any
scanning session between Hour 0 and Hour 72.
FSH Assay
Analysis of sera samples for FSH concentration was performed using a solid-phase, double-antibody RIA [2]. Briefly, 100 txl of a 1:10 000 dilution of anti-FSH (primary antibody; USDA anti-ovine FSH) was added to plastic wells
(Immulon; Dynatech Laboratories, Inc., Chantilly, VA) previously coated with 10 RIg/ml anti-rabbit antibody and were
incubated for 1 h. After removal of the antibody solution
and rinsing of the wells, 100 1l of sample serum or standard (USDA-bFSH -1-2; AFP-5318C) was added to the
wells and incubated at room temperature for 24 h. Radiolabeled bovine FSH (50 000 cpm) was then added and coincubated with the sample for an additional 2 h. Wells were
rinsed and counts per minute established. Inter- and intraassay coefficients of variation were 12.6% and 9.7%, respectively, for experiment 1 and 15.8% and 10.6%, respectively,
for experiment 2.
Statistical Methods
Sequential FSH concentrations, numbers of follicles, and
follicle diameters were analyzed by factorial (time, group)
analyses of variance using a nested classification for time.
Differences among groups within time were further analyzed by Student's t-tests or analyses of variance. Endpoints
with a single value per animal were examined by one-way
analyses of variance. Growth rates for follicles and corpora
lutea were calculated as follows: (maximum diameter diameter at Time 0)/(number of days required to reach the
maximum diameter). In experiment 2, the frequency at
which the randomly chosen, 5-mm retained follicle became
the dominant follicle in the one-follicle group was compared to the frequency at which a 5-mm follicle present at
Hour 0 in controls became the dominant follicle, using a
chi-square analysis. The frequency of occurrence of the two
follicles (ipsilateral versus contralateral) for the two-follicle
group was also analyzed using chi-square.
RESULTS
Experiment 1
The group effects for maximum FSH concentrations (Table 1) and overall FSH concentrations per group (Fig. 1)
were not significant. A significant group-by-time interaction
apparently was due in part to a tendency (p < 0.08) for a
higher FSH concentration 8 h after the initial FSH treatment
in the group given 150 units of FSH compared to controls.
All groups, including the group treated 48 h after ablation,
showed the mean peak of the FSH surge 8 h after the initial
injection. The group effect and interaction for the number
of 5- and 6-mm follicles were not significant (Fig. 2A),
although there were tendencies for differences between the
group given 150 units and controls on Days 2 (p < 0.06),
GIBBONS ET AL.
1068
TABLE 1. Experiment 1: Effects (means + SEM) of exogenous FSH on circulating FSH concentrations and follicles.
Amount of FSH given after ablation of all follicles - 5 mm*
24 h after ablation
Endpoint
Maximum FSH
Concentration (ng/ml)
Percentage of controls
Largest ovulatory follicle
Growth rate (mm/d)
Maximum diameter (mm)
Largest nonovulatory follicle
Growth rate (mm/d)
Maximum diameter (mm)
Time (d)from ablation to:
Deviation'
Ovulation
Diameter of largest follicle at deviation (mm)
Number of ovulations
48 h after ablation
0 (controls)
37.5 units
75 units
150 units
1.68 0.16
100%
1.98 0.07
118'o%
2.08 + 0.21
124%
2.22
0.27
132%/
1.77
1.4 + 0.2
11.7 + 0.8
1.4 + 0.1
13.5 + 1.0
1.5 + 0.1
13.7 + 0.8
1.1 + 0.2
12.2 + 0.7
1.0 + 0.1
12.0 + 0.8
1.2 + 0.2
6.7 + 0.6
1.0 + 0.2
7.3 + 0.5
1.2 + 0.2
8.2 + 0.5
1.3 + 0.2
7.8 + 0.7
1.4 + 0.1
8.3 + 0.6
3.6 + 0.4'
4.3 + 0.5"b
5.0 + 0.5, b
5.8 + 0.71"
6.7 + 0.2"
7.6 + 0.9
1.5 + 0.2
7.0 + 0.4'
8.8 + 0.5
1.3 + 0.2
6.8 + 0.8"
10.2 + 1.0"
8.3 + 0.8
1.2 + 0.2
8.0 + 0.8ab
9.4 + 0.7
1.5 + 0.2
9.7 + 1.0b
8.3 + 0.5
1.8 + 0.3
75 units
+
0.07
NA
* Controls received saline, and blood was sampled frequently 24 h after ablation; NA, not applicable.
Deviation is defined as the beginning of the greatest difference in growth rates between the largest ovulatory and nonovulatory follicles.
. b Means with different superscripts within an endpoint are different (p < 0.05); n = 6 heifers/group.
3 (p < 0.06), and 4 (p < 0.09) and there was a significant
difference between these two groups on Day 6 (p < 0.02).
Both main effects and the interaction were significant for
the number of 7- and 8-mm follicles. On Days 4, 5, and 6,
heifers given 150 units 24 h after ablation and heifers given
75 units 48 h after ablation had more (p < 0.05) 7- and
8-mm follicles than heifers given 0, 37.5, or 75 units of
FSH 24 h after ablation (Fig. 2B). The group effect and the
9 mm
interaction were not significant for the number of
follicles (Fig. 2C), although there were more (p < 0.05) 9 mm follicles on Day 4 in the group receiving 75 units 48
h after ablation than in the controls.
The main effects and interaction were significant for
changes in diameter of the largest ovulatory follicle (Fig.
3A). The group-by-time interaction apparently was due in
part to smaller mean diameter on Day 7 for the groups
given 150 units 24 h after ablation or 75 units 48 h after
E
C)
_r
ablation than for the groups given 37.5 or 75 units 24 h
after ablation. On Day 8, the diameter of the largest ovulatory follicle was larger (p < 0.05) in the groups given
37.5 or 75 units at 24 h than in the group given 75 units
at 48 h after ablation; because of ovulations and truncating
data, values were not included for the other two groups.
The growth profile of the largest nonovulatory follicle was
not affected by treatment (Fig. 3B).
The amount and time of exogenous FSH treatment did
not affect the growth rate or maximum diameter of the largest ovulatory follicle or the largest nonovulatory follicle
(Table 1). Deviation and ovulation were delayed (p < 0.05)
in heifers given 150 units of FSH 24 h after ablation and
in heifers given 75 units 48 h after ablation compared to
the controls (Table 1). The diameter of the largest ovulatory
follicle at the time of deviation was not affected by treatment (Table 1). Totals for all groups show that heifers with
multiple ovulations (2 or 3) had a smaller (p < 0.05) maximum diameter of the preovulatory follicles on the day before ovulation than did heifers with a single ovulation (10.6
0.7 mm). The group given 150 units of
± 0.3 vs. 12.8
FSH had smaller (p = 0.05) preovulatory follicles than the
group given 75 units of FSH (10.4
0.4 vs. 12.4 + 0.9
mm). There were no differences in the diameter of the preovulatory follicles in any other group, and ovulation number was not different among groups.
I
Experiment 2
LL
The FSH concentrations for Hours 0-96 showed significant main effects and an interaction (Fig. 4A). For the main
effect of group, the mean FSH concentrations were lower
(p < 0.05) in controls than in any of the other groups (all
0.07;
follicles retained, 0.61
0.07; two follicles, 0.82
one follicle, 0.93
0.08; zero follicles, 1.09 + 0.10) and
were lower (p < 0.05) in the two-follicle group than in the
zero-follicle group. The interaction was represented by a
follicle-group effect on FSH concentrations within each of
Hours 16-96 as shown (Fig. 4A).
The mean total number of 5-mm follicles (sum of ablated plus retained) was similar among all groups at Hour 0
(Table 2), and for the four groups that had at least one
follicle retained, but was less (p < 0.05) in these groups
than in the zero-follicle group during Hours 8-96. The
U,
-24
0
24
48
72
96
Hours after ablation
FIG. 1. Mean FSH concentrations relative to ablation of all follicles 5 mm in heifers (n = 6 per group) given a total dosage of 0 (open circles),
37.5 (solid circles), 75 (open squares), or 150 (solid squares) units of
porcine FSH initiated 24 h after ablation (indicated by *)or 75 (triangles)
units initiated 48 h after ablation (indicated by #). Main effect and interaction probabilities are shown. Pooled mean square error is 0.47. Experiment 1.
1069
INTERRELATIONSHIPS BETWEEN FOLLICLES AND FSH
A
12-
18
Group: NS
Time: P<0.001
GT: NS
.o 10-
16a
E 8E
CD
-
ab
ab
b
b
4-
E 2z
0-
1086-
.
I
=
I
I
I
w
I
.
E 4E
B
8-
8
E
E
o 6-
2
E .-
._u 1lu
r-4
0
3
.
.
I
.
.
.
.
Group: NS
Time: P<0.001
GT: NS
Largest non-ovulatory follicle
B
8'
2-
E
z 0-
Li
12-
D 6a1)
14-
Group: P=0.012
J
ICropN
4-
Group: NS
C
a,
0.
34'
E
E
2-
Al
.0
1-
2
E
z
0
I
I
*
#
2
after
4
5
6
Days after ablation
FIG. 2. Mean ( SEM) number of 5- and 6- (A), 7- and 8- (B), and 9-mm (C) follicles relative to ablation of all > 5-mm follicles in heifers
(n = 6 per group) given a total dosage of 0 (open circles), 37.5 (solid
circles), 75 (open squares), or 150 (solid squares) units of porcine FSH
initiated 24 h after ablation (indicated by *)or 75 (triangles) units initiated
48 h after ablation (indicated by #). Main effect and interaction probabilities are shown for each panel. Different superscripts indicate differences (p < 0.05) among groups within a time period. Experiment 1.
group effect for the mean number of ablated plus retained
5-mm follicles was due primarily to the zero-follicle group
having more 5-mm follicles (p < 0.05) than the other
groups for Hours 48, 64, and 96 (Fig. 4B). More > 5-mm
follicles developed at Hour 16 in the group with all follicles
retained than in the groups with two or one follicle retained.
All heifers with all, two, or one follicle retained formed a
single dominant follicle during wave 1. The mean total
number of 5-mm follicles ablated during Hours 8 to 72 in
the group with zero follicles retained was more (p < 0.05)
than for any of the other groups (Table 2).
Four heifers in the one-follicle group had two to four
5-mm follicles present at Hour 0. In all heifers in the onefollicle group, the 5-mm follicle chosen at random became
the dominant follicle. This frequency (4 dominant follicles
from 4 follicles chosen at random) was higher (p < 0.03)
4
6
Days after ablation
8
FIG. 3. Mean (+ SEM) diameters of the largest ovulatory (A) and nonovulatory follicles (B) relative to ablation of all 5-mm follicles in heifers
(n = 6 per group) given a total dosage of 0 (open circles), 37.5 (solid
circles), 75 (open squares), or 150 (solid squares) units of porcine FSH
initiated 24 h after ablation (indicated by *)or 75 (triangles) units initiated
48 h after ablation (indicated by #). Main effects and interaction probabilities are shown for each panel. Different superscripts indicate differences (p < 0.05) among groups within a time period. Experiment 1.
than the frequency at which a randomly chosen 5-mm follicle at Hour 0 became the dominant follicle in controls (5
dominant follicles from 13 follicles).
The growth rates and maximum diameters of the dominant and subordinate follicles did not differ among groups
with all, two, or one follicle retained (Fig. 4; Table 2). The
growth rate and maximum diameter of the corpora lutea for
the group with all follicles retained were greater (p < 0.05)
than for the group with zero follicles retained (Table 2).
The length of the interovulatory interval was not affected
by treatment.
In the two-follicle group, the subordinate follicle was
more frequently (p < 0.05) ipsilateral (8 of 11 heifers) than
contralateral (3 of 11 heifers) to the dominant follicle. The
subordinate follicle located ipsilateral to the dominant follicle reached maximum diameter later (p < 0.05) than the
subordinate follicle located contralateral to the dominant
follicle (Table 2). The interval (d) from Hour 0 to the regression of the subordinate follicle to 4 mm was longer (p
GIBBONS ET AL.
1070
Group: P<0.004
1- -~ -R
1.4-
A
1.2
E 1.0I 0.80.6. .
.
.
. . I
6- B
c)
a,
. . I
.
3
54-
E
E
3
0
2
.0
E
1
z
0
. . . . *.
.
.
.
.
. *.
* .
.
.
.
.
E 14- C
E
12
so
c
8-
o(1)cm8
10o
6'0 66· 4
E
co
._
'
'6 '2
4 48
72
96
120'
Hours from appearance of first 5-mm follicle
FIG. 4. Mean ( SEM) FSH concentrations (A), number of follicles
emerging to - 5 mm (ablated plus retained) (B), and
diameters of the
largest follicles (C) in heifers with either all (open
(solid circles; n= 11), one (open squares; n = 6), orcircles; n = 6), two
zero (solid squares;
n = 6) follicles retained and the others ablated upon
reaching 5 mm.
Data are shown for Hours 0-72 and for Hour 96. Main
effects and interaction probabilities are shown for each panel. Different
superscripts indicate differences< (p
0.05) among groups within a time period. There
were not differences in diameter or growth rates of
the largest follicles
among the three groups with retained follicles (C. Experiment
2.
< 0.05) when the two follicles were ipsilateral (8.1
± 0.8)
as compared to contralateral (4.9
0.8).
DISCUSSION
In experiment 1, the exogenous FSH increased the
mean
peak FSH peripheral concentrations at a time corresponding
to the mean peak concentrations of the post-ablation
FSH
surge in controls (8 h after FSH treatment). Maintaining
high FSH concentrations in the group given 75 units 48
h
after ablation was successful in that a secondary surge occurred with mean peak values 24 h after the mean peak
values in controls. Thus, the timing for the injections
was
proper for test of the hypothesis on the follicular-stimulatory effects of the height and width of the FSH peak.
The
highest dosage given in this experiment (150 U) approximately doubled the number of 5- and 6-mm follicles
beginning 1 day after the FSH injection; this then progressed
into a 4- to 7-fold increase in the number of 7- and
8-mm
follicles beginning the next day. The intermediate dosages
(37.5 or 75 U) given 24 h after ablation did not
increase
the number of follicles; the height of the FSH surge
was
increased but apparently was below the threshold
needed
for stimulating more follicles. In another study, FSH
given
for 2 days, beginning when the follicles were 6 mm,
not increase the number of follicles [6]; perhaps the did
was started too late or the dosage was too low. TheFSH
increased number of 7- and 8-mm follicles was similar
for
the group given 75 units of FSH 48 h after ablation
and
the group given 150 units at 24 h. The results demonstrated
that the number of follicles that progress to larger diameters
can be increased both by increasing the height of the
FSH
surge in synchrony with the endogenous peak or by
an
exogenous peak 24 h after the endogenous peak.
Data from experiment 2 were also consistent with a positive relationship between the FSH surge and the number
of follicles emerging at 5 mm. The heifers with zero
licles retained had sustained high concentrations of folFSH
and more 5-mm follicles (all ablated) than the other groups
(ablated plus retained). The greater number of follicles
ablated during Hours 8 to 72 in the zero-follicle group
may
have been due to the elevated FSH and a resulting ongoing
attempt to initiate a new follicular wave. Heifers in the
twoor one-follicle groups had FSH concentrations intermediate
between those of the controls and the zero-follicle
group
but did not have an increase in the number of emerging
5-mm follicles. These results, and those of experiment
1,
are not consistent with a dose response of FSH on the
number of follicles of a wave but do suggest that when
FSH
concentrations reach a certain height threshold, an
increased number of follicles are stimulated to grow
into a
follicular wave. These studies were not designed to
determine whether increasing dosages of FSH, above the
threshold, will continue to increase the number of follicles.
spite of the increase in the number of follicles that grew In
to
a larger size, high concentrations of FSH did not alter
the
growth rate of the two largest follicles in either experiment.
Results of experiment 2 supported the hypothesis that
follicles of a wave contribute to the declining portion all
the FSH surge following emergence of the first 5-mm of
follicle. The specific results supporting this conclusion
are as
follows: 1) there was a main effect of group in the
factorial
analysis with the overall mean FSH concentration per group
increasing as the number of retained follicles decreased;
2)
at several times during Hours 0 to 72, the FSH concentrations for the one- or two-follicle groups were intermediate
between the concentrations for controls and concentrations
for the zero-follicle group; and 3) the one-follicle
group
showed greater depression in FSH concentrations than
the
zero-follicle group at Hours 48, 64, and 72. It appears,
therefore, that cattle follicles in the initial portion of a wave
act in concert to suppress FSH concentrations soon
after
the peak of the FSH surge is reached. This conclusion
consistent with the reported temporal relationships betweenis
the declining portion of the FSH surge and growth
of follicles of the resulting wave
[1
].
Two secretions of follicles, estradiol and inhibin, have
been suggested as FSH-suppressing substances. Inhibin
is
produced by the granulosa cells of growing and regressing
1071
INTERRELATIONSHIPS BETWEEN FOLLICLES AND FSH
TABLE 2.
Experiment 2: Mean (SEM)
for follicular and luteal endpoints in heifers with all, two, one, or zero follicles retained upon reaching 5 mm.
Number of follicles retained upon reaching 5 mm*
Two
Endpoints
Dominant follicle
Growth rate (mm/d)
Maximum diameter (mm)
No. days to maximum
Largest subordinate follicle
Growth rate (mm/d)
Maximum diameter (mm)
No. days to maximum
Corpus luteum
Growth rate (mm/d)
Maximum diameter (mm)
No. days to maximum
Number of 5-mm follicles (ablated
plus retained)
At Hour 0
Total for Hours 8 to 72
Number of 5-mm follicles ablated
At Hour 0
Total for Hours 8 to 72
Interovulatory interval (d)
All
(controls; n = 6)
Ipsilateral
(n = 8)
Contralateral
(n = 3)
One
(n = 6)
Zero
(n = 6)
1.7 + 0.2
15.2 + 0.6
7.0 + 0.5
1.8 ± 0.1
14.9 + 1.0
6.4 + 0.6
1.5 + 0.2
15.0 + 1.2
7.0 + 0.6
1.7 + 0.2
16.2 + 0.7
6.8 + 0.7
NA
NA
NA
1.2 + 0.3
8.0 + 0.6
3.3
0.4
0.8 + 0.4
6.3 + 0.9
NA
NA
NA
NA
1.9
NA
NA
2.0 ± 0.2
ab
24.0 + 1 1
1.3 + 0.3b
20.0 ± 2 .1 b
1.5 + 0.1
7.5 + 0.4
2.0
0.4b
2.2 + 0.3
26.2 + 1 .4
a
a
1.7 ± 0 .2 b
ab
23.2
1.1
0 .2b
2.2 + 0 .4ab
25.7 + 0 .7ab
7.0 + 0.7
8.4 + 0.4
6.7
±
0.3
7.3
0.4
7.7 + 0.8
4.3 + 0.6
3.0
0.8
4.9 + 0.6
2.4
0.6a
5.6
2.3
±
±
1.2
0.9a
4.2
2.3
1.1
1.7
5.2
7.2
NA
NA
21.7 - 0.7
3.1 + 0.5
2.1 + 0.5a
23.6
2.1
3.6
1.2
2.3
0.9
21.3 + 0.3
3.2
2.3
21.3
1.1
1.7
0.3
± 0.3
± 0 .4 b
5.2 + 0.3
7.2
04b
20.3
0.8
*One-follicle group had no subordinate follicle and zero-follicle group had no dominant or subordinate follicles, and no follicles were ablated in
controls; NA, not applicable.
a b Means with different superscripts within an endpoint are different (p < 0.05).
tertiary follicles and is found in similar concentrations in
anovulatory dominant and atretic follicles as early as 3 days
after estrus [13], before follicles would be expected to deviate into dominant and subordinate follicles. Active immunization of heifers against a portion of the oa subunit of
inhibin 48 h after the induction of luteolysis increases FSH
concentrations immediately following injection, with peak
FSH concentrations reaching approximately 3 times that of
the controls [14]. Concentrations of estradiol-17[3 in plasma
and follicular fluid increase early in the follicular wave [3].
On the day after deviation when FSH is at the lowest concentration, follicular fluid from the dominant follicle exhibited estradiol-173 concentrations that were several times
those of the largest subordinate [15]. Although inhibin and
estradiol can suppress FSH concentrations individually, it
is likely that they act synergistically with each other [14]
and with other follicular fluid substances [16] to suppress
FSH or its effects. Recombinant bovine somatotropin has
been shown to increase the number of small follicles (2-5
mm) without a change in peripheral FSH concentrations
[17, 18], supporting the premise that factors in addition to
FSH may affect growth of early antral follicles.
Results supported the second hypothesis of experiment
2, that any 5-mm follicle of the wave has the capacity to
become the dominant follicle. The randomly chosen retained follicle in the one-follicle group always became the
dominant follicle as indicated by its diameter. In contrast,
when the largest follicles of a wave are 5 mm in diameter
in nonmanipulated heifers, the chance of a given follicle's
becoming the future dominant follicle is only about 20%
as indicated by the presence of approximately five follicles
at the initiation of the experiment and as supported by the
literature [2]. The dominant follicles of the one-follicle
group were indistinguishable from the dominant follicles of
any other group on the basis of diameters and growth rates.
Many follicles early in a wave are capable of responding
to a single s.c. injection of exogenous FSH [10], further
suggesting that follicles are functionally similar early in the
wave. In addition, prior to deviation, follicles produce similar quantities of estradiol commensurate with diameter but
independent of whether a follicle later becomes dominant
or subordinate [15]. The concept that all follicles are functionally similar prior to deviation is consistent with the proposal of a rapid deviation mechanism that abruptly halts
the growth of future subordinate follicles while allowing a
single dominant follicle (usually) to continue growing [3,
4]. It is unknown whether the subordinates play a role after
deviation; however, in vitro data from pigs suggest that anovulatory follicles produce high quantities of androstenedione, which may aid estradiol production by the dominant
follicles [19].
The discussion above concerns functional interrelationships between the FSH surge and the number of 5- to 8-mm
follicles in the follicular wave. These interrelationships
continue for only a few days between the peak of the FSH
surge and the deviation in growth rates of the two largest
follicles. During this time, FSH is declining and reaches a
nadir at the time of deviation [4]. Deviation occurred a
mean of 2.6 days after the peak of FSH surge in the controls
of experiment 1, which is similar to the reported peak-todeviation interval [4]. Even though manipulation of FSH
profiles by exogenous FSH (experiment 1) altered the number of follicles, there were no indications of an effect on
the growth profile of the largest and second-largest follicles
prior to deviation. Diameter of the largest follicle at the
beginning of deviation was approximately 8 mm, a value
consistent with previous findings [4]. It has been suggested
that the cessation of growth of the second-largest follicle
and the other subordinate follicles at deviation is a function
of the suppressed FSH concentrations, which are at a nadir
at this time [4]. The selected dominant follicle may continue to grow after deviation by a change in gonadotropin
dependency to LH [3].
Consistent with the need for low concentrations of FSH
at the time of deviation, the day of deviation between the
largest ovulatory and nonovulatory follicles was delayed
1072
GIBBONS ET AL.
by 2 days in the groups given 150 units of FSH 24 h after
ablation or 75 units at 48 h. A similar delay was reported
when FSH was given for 2 days beginning when the largest
follicle was 6 mm [7]. Although this was not studied in the
present experiment, the high concentrations probably
caused a later FSH nadir, as indicated by the declining portion of the surge. The intermediate concentrations were intermediate in the time of deviation, but not significantly.
The delay in deviation likely accounts for the corresponding delay in ovulation. The delay in deviation was accompanied by more 7- and 8-mm follicles, as noted above. An
unexpected finding was the larger diameter of the dominant
follicles 7 and 8 days after the injection of intermediate
dosages of FSH. Apparently, concentrations of exogenous
FSH that are inadequate for superstimulation of the number
of follicles can cause increased diameter of dominant follicles after deviation, but this observation requires confirmation.
The presence of the subordinate follicle in the same ovary as the dominant follicle resulted in a positive effect on
the subordinate follicle; the follicle continued to grow so
that maximum diameter was reached more than 1 day later
than when the two follicles were in opposite ovaries. This
effect was unexpected and requires confirmation. In a previous study [20], no unilateral relationships were found between dominant and subordinate follicles. In another unexpected finding, the corpus luteum grew at a slower rate
and reached a smaller maximum diameter in the zero-follicle group than in the control group. Destruction of 5-mm
follicles during Hours 0 to 72 may have resulted in suppressed estradiol concentrations; estradiol has been shown
to be necessary for normal luteal growth and function in
some species [21, 22]. In ewes, suppression of estradiol1713 with a nonsteroidal aromatase inhibitor has been shown
to delay the onset of the luteal phase, as evidenced by lower
progesterone concentrations in the treated animals [23].
In experiment 1, there were 12 double ovulations (13 of
30 heifers, 43%) and one triple ovulation over all groups,
but the ovulation rate was similar among groups. In controls, the rate of multiple ovulations (3 of 6; 50%) was
higher than the reported incidence of 13% [24] or the incidence of double corpora lutea at the time of ablation 5
days after ovulation (0 of 30). The reason for the high incidence of double ovulations in the controls is not known,
but the finding may be a result of the ablation procedure
or the post-ablation FSH surge. In a previous study [9],
double ovulations occurred in 2 of 7 beef heifers after ablation but did not occur in any of the 8 nonablated controls.
Also, it is not clear whether a post-ablation FSH surge is
similar to an FSH surge in intact heifers. The possibilities
of an increased double ovulation rate and altered characteristics of the FSH surge after ablation will require additional study. Previous results have demonstrated that a single s.c. injection of FSH can enhance the number of ovulations [10]; however, the age and type of cattle (beef vs.
dairy), the purity of the FSH product, and whether or not
follicles were ablated prior to FSH injection may explain
why the number of ovulations in this study was unaffected
by treatment. The smaller diameter of preovulatory follicles
associated with multiple ovulations has been reported for
mares [25], another single-ovulatory species, and for sheep
[26], but confirmation of this phenomenon in cattle was not
found in the literature.
In conclusion, a functional follicle-FSH interrelationship
prior to deviation was indicated by the following: 1) the
number of follicles that were advanced into a follicular
wave was increased when the height or width of the FSH
surge at the time of wave emergence was increased, and 2)
all - 5 mm follicles of a wave contributed to the declining
portion of the FSH surge. It was also demonstrated that any
5 mm follicle at the emergence of a wave is capable of
becoming the dominant follicle. Also, exogenous FSH given near the time of the endogenous FSH peak affected not
only the number of follicles, but also the time to reach
deviation and the diameter of the largest ovulatory follicle.
ACKNOWLEDGMENTS
The authors wish to thank Ausa International Inc. for the donation of
FSH (Super Ov), Upjohn Company for the donation of prostaglandin 2a
(Lutalyse), Zeki Beyhan for technical support, and Travis Clary and Lisa
Kulick for preparation of figures.
REFERENCES
I. Adams GP, Matteri RL, Kastelic JP, Ko JCH, Ginther OJ. Association
between surges of follicle-stimulating hormone and the emergence of
follicular waves in heifers. J Reprod Fertil 1992; 94:177-188.
2. Bodensteiner KJ, Kot K, Wiltbank MC, Ginther OJ. Synchronization
of emergence of follicular waves in cattle. Theriogenology 1996; 45:
1115-1128.
3. Ginther OJ, Wiltbank MC, Fricke PM, Gibbons JR, Kot K. Selection
of the dominant follicle in cattle. Biol Reprod 1996; 55:1187-1194.
4. Ginther OJ, Kot K, Kulick LJ, Wiltbank MC. Emergence and deviation of follicles during the development of follicular waves in cattle.
Theriogenology 1997; 48:75-87.
5. Nasser LF, Adams GP, Bo GA, Mapletoft RJ. Ovarian superstimulatory response relative to follicular wave emergence in heifers. Theriogenology 1993; 40:713-724.
6. Adams GP, Kot K, Smith CA, Ginther OJ. Selection of a dominant
follicle and suppression of follicular growth in heifers. Anim Reprod
Sci 1993: 30:259-271.
7. Adams GP, Kot K, Smith CA, Ginther OJ. Effect of the dominant
follicle on regression of its subordinates in heifers. Can J Anim Sci
1993; 73:267-275.
8. Ko JCH, Kastelic JP, Del Campo MR, Ginther OJ. Effects of a dominant follicle on ovarian follicular dynamics during the oestrous cycle
in heifers. J Reprod Fertil 1991; 91:511-519.
9. Bergfelt DR, Lightfoot KC, Adams GP Ovarian synchronization following ultrasound-guided transvaginal follicle ablation in heifers.
Theriogenology 1994; 42:895-907.
10. Bo GA, Hockley DK, Nasser LF, Mapletoft RJ. Superovulatory response to a single subcutaneous injection of Folltropin-V in beef cattle. Theriogenology 1994; 42:963-975.
11II.Pierson RA, Ginther OJ. Ultrasonic imaging of the ovaries and uterus
in cattle. Theriogenology 1988; 29:21-37.
12. Sirois J, Fortune JE. Ovarian follicular dynamics during the estrous
cycle in heifers monitored by real-time ultrasonography. Biol Reprod
1988; 39:308-317.
13. Martin TL, Fogwell RL, Ireland JJ. Concentrations of inhibins and
steroids in follicular fluid during development of dominant follicles in
heifers. Biol Reprod 1991; 44:693-700.
14. Kaneko H, Nakanishi Y, Akagi S, Taya K, Watanabe G, Sasamoto S.
Hasegawa Y. Immunoneutralization of inhibin and estradiol during the
follicular phase of the estrous cycle in cows. Biol Reprod 1995: 53:
931-939.
15. Ginther OJ, Kot K, Kulick LJ, Wiltbank MC. Sampling follicular fluid
without altering follicular status in cattle: oestradiol concentrations
early in a follicular wave. J Reprod Fertil 1997; 109:181-186.
16. Law AS, Baxter G, Logue DN, O'Shea T. Webb R. Evidence for the
action of bovine follicular fluid factor(s) other than inhibin in suppressing follicular development and delaying oestrus in heifers. J Reprod Fertil 1992; 96:603-616.
17. Gong JG, Bramley T, Webb R. The effect of recombinant bovine somatotropin on ovarian function in heifers: follicular populations and
peripheral hormones. Biol Reprod 1991; 45:941-949.
18. Gong JG, Bramley T, Webb R. The effect of recombinant bovine somatotropin on ovarian follicular growth and development in heifers.
J Reprod Fertil 1993; 97:247-254.
19. Westof G, Westof KE Braendle WL, diZerega GS. Differential steroid
secretion and gonadotropin response by individual tertiary porcine fol-
INTERRELATIONSHIPS BETWEEN FOLLICLES AND FSH
licles in vitro. Possible physiologic role of atretic follicles. Biol Reprod 1991; 44:461-468.
20. Ginther OJ, Kastelic JP, Knopf L. Intraovarian relationships among
dominant and subordinate follicles and the corpus luteum in heifers.
Theriogenology 1989; 32:787-795.
21. Gadsby JE, Keyes PL, Bill CH. Control of corpus luteum function in
the pregnant rabbit: role of estrogen and lack of a direct luteotropic
role of the placenta. Endocrinology 1983; 113:2255-2262.
22. Gibori G, Keyes PL. Role of intraluteal estrogen in the regulation of
the rat corpus luteum during pregnancy. Endocrinology 1978; 102:
1176-1182.
1073
23. Benoit AM, Inskeep EK, Dailey RA. Effect of a nonsteroidal aromatase inhibitor on in vitro and in vivo secretion of estradiol and on
the estrous cycle in ewes. Domest Anim Endocrinol 1992; 9:313-327.
24. Roberts SJ. Veterinary obstetrics and genital diseases (Theriogenology). Woodstock, Vermont: Stephen J Roberts; 1986: 96.
25. Ginther OJ. Reproductive biology of the mare. Cross Plains, WI:
Equiservices; 1992: 221.
26. Gibbons JR, Kot K, Thomas DL, Wiltbank MC, Ginther OJ. Influence
b
of the Booroola FEC allele on follicular dynamics. Theriogenology
1996; 45:252 (abstract).