ELSEVIER

ALTERATIONS IN FOLLICULAR ESTRADIOL AND GONADOTROPIN RECEPTORS

DURING DEVELOPMENT OF BOVINE ANTRAL FOLLICLES

K.J. Bodensteiner, M.C. Wiltbank,] D.R. Bergfelt and O.J. Ginther’

I
Departments of Dairy Science
Animal Health and Biomedical Sciences2
University of Wisconsin-Madison
Madison, WI 53706 USA

Received for publication: March I5, 199s

Accepted: June 30, 1995

ABSTRACT

It was hypothesized that growth divergence of dominant and subordinate follicles during
Wave 1 and growth termination of the dominant follicle would be associated with changes in the
number of gonadotropin receptors on granulosa cells and estradiol in follicular fluid. To test this
hypothesis, follicular development of 16 Holstein heifers was monitored by ultrasound, and follicles
were collected on Days 2,4,6 and 10 (Day 0 = ovulation). Dominant follicles were compared across
days, whereas dominant and largest subordinate follicles were compared on Days 2 and 4 only. The
numbers of LH and FSH receptors on the granulosa cells of dominant follicles did not differ
significantly over Days 2. 4, 6 and 10. In contrast, concentrations of estradiol in follicular fluid
decreased (PcO.05) from Days 2 to 10 (373 f 150 to 42 f 12 ng/ml) and concentrations of
progesterone in follicular fluid increased (P<O.OS) from Days 2 to 10 (12.2 + 2.3 to 24.4 & 4.8
nglml). Correspondingly, the ratio of estradiol:progesterone in the dominant follicles decreased
(PcO.05) from Days 2 to 10. Comparisons between dominant and subordinate follicles indicated
greater (P<O.O5) estradiol concentrations in the dominant follicle on Day 2, but the number of
gonadotropin receptors was not different until Day 4. Thus, differences in concentrations of
follicular fluid estradiol. but not numbers of granulosa cell gonadotropin receptors, were associated
with the early growth divergence of dominant and subordinate follicles (Day 2) and the eventual
growth termination of the dominant follicle (Day IO). Late divergence (Day 4) was associated with
higher gonadotropin receptor numbers and follicular estradiol concentrations in the dominant than
in the subordinate follicles. These results indicate that an increase in estradiol productivity of the
selected dominant follicle occurred before an increase in the number of gonadotropin receptors.

Key words: estradiol, LH receptors, FSH receptors, follicles, cattle

Acknowledgments
This study was supported by the Wisconsin Experiment Station and USDA grant number 9 l-
37203-6557. We would like to thank Lisa Kulick for assistance with graphs and Steve Tomasko
for iodination of hormones. Please address correspondence to M.C. Wiltbank.

Theriogenology 45:499-512, 1996

0 1996 by Elsevier Science Inc. 0093-691 W96/$15.00
655 Avenue of the Americas, New York, NY 10010 SSDI 0093-691X(95)00387-8
500 Theriogenology

INTRODUCTION

Follicular development in cattle is characterized by distinct waves of growth and atresia (for
reviews see 9, 11, 2 1). Under the influence of increasing plasma concentrations of FSH, a cohort
of follicles begins to grow and a follicular wave emerges (1, 38). During the subsequent decreases
in FSH concentrations, the dominant follicle of the wave begins to exceed the diameter of the
subordinate follicles. Under basal concentrations of FSH, the dominant follicle continues
development while the subordinate follicles cease growth and eventually regress. The dominant
follicle normally reaches a diameter of 11 mm or greater approximately 6 d after follicular wave
emergence and in nonovulatory waves will then stop growing and ultimately undergo atresia (10).
In this report, selection of the dominant follicle refers to the mechanisms that determine which
follicle of a wave is physiologically designated to become the dominant follicle; divergence refers
to the days when the dominant and subordinate follicles grow at different rates, but both follicle
types are viable.

The cellular mechanisms regulating the selection, growth divergence, growth termination and
eventual regression of follicles are incompletely defined. However, a primary role of gonadotropins
in the regulation of follicular development has been well established (1, 13). The ability of the
dominant follicle to continue growth under decreased FSH concentrations suggests that cellular
responsiveness to FSH may be altered during follicular development. Exogenous FSH treatment for
2 d at the expected time of follicular divergence delayed the growth divergence between dominant
and subordinate follicles and delayed regression of the subordinate follicles (3). The same treatment
given after divergence did not alter follicular development. Therefore, the difference in growth rate
at the time of follicular divergence may be due to differences between dominant and subordinate
follicles in number of FSH receptors, responsiveness to FSH, or decreased dependency of the
dominant follicle on FSH.

Correspondingly, as plasma FSH decreases, the dominant follicle may increase its reliance
on LH for continued development. Increased LH pulse frequency has been shown to be associated
with maintenance of a dominant follicle (32, 33, 37). In addition, the high progesterone levels
associated with the luteal phase of the bovine estrous cycle correspond to a decrease in LH pulse
frequency, which leads to turnover of the dominant follicle (32-34). Hence, the difference in growth
rate at the time of follicular divergence may be explained, in part, by LH receptors activating cellular
pathways similar to those that are activated by FSH receptors.

It has been demonstrated (15) that the ability of bovine granulosa cells to bind gonadotropins
differs according to the physiologic state of the follicle; dominant versus subordinate status was
unknown. In follicles defined as estrogen-active, the mean capacity of granulosa cells to bind FSH
increased from Day 3 to Day 5 postestrus, but then decreased on Day 7. In contrast, the mean
capacity of granulosa cells to bind hCG increased significantly from Day 3 to Day 7. The mean
capacity of granulosa cells from estrogen-inactive follicles to bind FSH or hCG was less than that
for estrogen-active follicles on Days 5 and 7 postestrus. These results are consistent with the
assumption that as FSH levels decrease the dominant follicle assures itself of continued development
by acquiring granulosa cell LH receptors, whereas the subordinate follicles may lack this ability.
Theriogenology 501

The present study was designed to characterize gonadotropin receptor concentrations of

granulosa cells and concentrations of estradiol in follicular fluid from dominant and subordinate
follicles at defined stages of development. The hypothesis was that growth divergence of dominant
and subordinate follicles and eventual growth termination of the dominant follicle were associated
with alterations in concentrations of gonadotropin receptors on granulosa cells and estradiol in
follicular fluid.

MATERIALS AND METHODS

Animal Procedures, Experimental Groups and Tissue Collection

Sixteen nulliparous Holstein heifers 2.5 to 3 yr of age and weighing 500 to 700 kg were used
during October to December 1992. The heifers were kept in outdoor paddocks and had free access
to shelter. They were maintained on grass hay with grain supplement, and fresh water was available
ad libitum. Ultrasound examinations were done with a scanner equipped with a 7.5 MHz linear-
array transducer (210-DXII; Corometrics Medical Systems Inc., Wallingford, CT). Daily
examinations were initiated during the estrous cycle preceding the one to be studied and were
continued until the day of slaughter. Individual follicles were identified and serially monitored as
previously described (11, 18).

On the day of detected ovulation (Day 0), heifers were randomized into 1 of 4 groups (n =
4 per group). One animal was assigned to each group before proceeding to the next replicate.
Groups were chosen to represent various stages of antral follicular development (11). Ovaries were
collected at slaughter on the following days after ovulation: Group 1) Day 2-- beginning of follicular
divergence; Group 2) Day 4-- end of follicular divergence; Group 3) Day 6-- large, active dominant
follicle; and Group 4) Day lo-- large follicle has become nondominant as indicated by emergence
of a new follicular wave.

Ovaries were collected within 15 min of slaughter and immediately placed on ice for
transport to the laboratory. At the laboratory, individual follicles that were identified in vivo by the
daily examinations were further identified by ultrasound of the ovary in a water bath as follows:
Days 2 and 4-- largest and second largest growing follicles; Days 6 and lo-- dominant follicle.
Follicular fluid and granulosa cells were collected from individual follicles similar to procedures
previously described (14, 36). The volume of follicular fluid was estimated by aspirating the
follicular fluid into a graduated syringe. Follicular fluid was then centrifuged at 500 x g for 10 min
to remove any granulosa cells from the fluid. The supernatant was decanted and stored at -80 “C
until used for hormone assay. To remove granulosa cells, follicles were washed with
homogenization buffer (0.25 M sucrose; 10 mM Tris-HCL; 1 mM CaCL2; 1 mM MgCL2; 0.02%
NaN3; pH 7.4) and vacuumed with a blunted small-gauge needle attached to a water faucet vacuum
apparatus. The washing procedure was repeated 3 times for each follicle. Granulosa cells from
follicular fluid were combined with respective aliquots of granulosa cells from follicle aspirates and
stored at -80 “C in homogenization buffer until used for receptor assays. One heifer from Group 1
was removed from the study because the second and third largest follicles were mistakenly pooled.
502 Theriogenology

Estradiol and Progesterone Assays

fluid concentrations of estradiol-17P were determined by using a modification of

asolid_~~~f~“’
I-radiotmmunoassay kit for estradiol (Diagnostic Products Corp., Los Angeles, CA).
Since the kit provided estradiol standards in human serum, the kit was modified using estradiol
(Diosynth, Inc., Chicago, IL) standards prepared in 100 pl of steroid-reduced bovine follicular fluid
(25). Steroid-reduced bovine follicular fluid was also used for maximum and nonspecific binding
tubes as well as for dilution of follicular fluid. Pooled follicular fluid of various dilutions in a total
volume of 100 pl yielded a curve that was not different (P > 0.05) from the estradiol standard curve.
Follicular fluid samples of individual follicles were initially diluted 1:200. Samples in which
radiolabelled estradiol bound < 10% or > 90% of the antibody relative to maximum binding were
assayed again with an adjusted dilution. The intra- and inter-assay coefficients of variation were 6
and 12%, respectively, with an assay sensitivity of 0.03 ng/ml. Concentrations of estradiol were
expressed both as ng/ml of follicular fluid and ng/follicle (follicular fluid volume of each follicle).

Concentrations of follicular fluid progesterone were determined by using a specific

progesterone ELISA modified to allow direct determination of progesterone in follicular fluid.
Procedures were similar to those previously described for the determination of serum progesterone
(28). Follicular fluid samples were diluted 1: 100 in assay buffer (0.04 M MOPS, 0.12M NaCI, 0.0 1
M EDTA, 0.05% Tween 20, 0.005% Chlorhexidine-digluconate, 0.1% gelatin). Standards were
diluted 1: 100 in assay buffer containing steroid-reduced bovine follicular fluid at a dilution of 1: 100
(25 p1 follicular fluid in 25 ml assay buffer).

Radioreceptor Assays

A standard pool of plasma membranes from bovine granulosa cells was obtained for use in
validation of the radioreceptor assays. The pool was obtained by aspirating all follicles 2 4 mm and
I 20 mm in diameter from approximately 500 slaughterhouse ovaries using procedures described
above for experimental follicles. The pool of granulosa cells was homogenized in a small ground
glass homogenizer (Dual1 20, Kontes Glass Co.) and stored at -20 “C until used in the radioreceptor
assay. After homogenization, samples of the heterogenous pool were taken for protein (Bio-Rad
Laboratory; Richmond. CA) and DNA (20) determination. To determine a constant for the number
of granulosa cells in 1 pg of DNA, 3 random follicles were selected from slaughterhouse ovaries and
the granulosa cells were isolated as described above. A hemocytometer was then used to determine
cell number per follicle. The gra ulosa cell numbers were compared to the DNA standard curve,
6
yielding a constant of 3.078 x 10 cells per pg DNA.

Four micrograms of hCG (CR-127) were radioiodinated using chloramine-T (12) and 4 ug
ovine FSH (USDA-oFSH-19-SIAFP-I-l) were radioiodinated using iodogen (23). The maximum
amount of the iodinated hormone preparations that specifically bound the granulosa cell pool was
33 and 49.7% for hCG and FSH, respectively. On the day of the assay, homogenates were
centrifuged for 20 min at 30,000 x g and 4°C to remove the homogenization buffer, and the pellet
was resuspended in assay buffer (PBS with 0.02% NaN3 and 0.1% BSA). Aliquots of the granulosa
cell pool and individual samples were prepared in assay buffer containing 1 mg DNAase/ml to
eliminate clumping of the plasma membrane fractions. Assay buffer was also used to prepare
Theriogenology 503

concentrations of nonradiolabeled and radiolabeled hCG (100,000 cpm 25/pl) or oFSH (20,000 cpm
25/ul). The specific activity of each radioiodinated preparation of hormone was determined by
displacement analysis of both radioactive and nonradioactive hormone as previously described (8).
All radioreceptor assays were performed in duplicate in 96-well microtiter plates with a total assay
volume of 100 yl. Membrane fractions were incubated with hormone at room temperature for 18
to 24 h. The time and temperature dependencies of the radioreceptor assays were evaluated in
preliminary studies and were similar for bovine luteal cells (hCG binding) or granulosa cells (FSH
binding). The separation of bound hormone from unbound hormone was done by filtration through
glass-fiber filter paper (#32 glass; Schleicher and Schuell, Keene, NH) that had been presoaked in
0.6% polyethylenimine (7). The filtration apparatus used was a multiwell cell harvester (Mini Mass
II; Whittaker Bioproducts, Walkersville, MD). The number and affinity of receptors in the plasma
membrane pools were determined by Scatchard analysis of the displacement of radioactive hormone
by nonradioactive hormone (35). The specificity of radioactive hCG and oFSH binding was
determined by incubating the granulosa cell pool at a l/S dilution with a lOO-fold excess of the
nonradioiodinated homologous or heterologous ligand. It was found that the hCG did not displace
oFSH nor did oFSH displace hCG, indicating specificity of binding.

Numbers of unoccupied LH and FSH receptors on granulosa cells from experimental follicles
were determined using the standard curve method (6,26). Differing amounts of plasma membrane
were incubated with a constant amount of radioactive hormone with or without an excess of
nonradioactive hormone. Two different levels (20 and 40 pl) of plasma-membrane from individual
follicles were also incubated with this amount of radioactive hormone with or without an excess of
nonradioactive hormone. A comparison of counts specifically bound in the experimental samples
to counts bound in the characterized plasma membrane pool allowed determination of receptor
numbers in the experimental samples. The evaluation of 2 different amounts of tissue allowed
comparison of the relationship between binding and tissue quantity in the experimental samples and
the plasma membrane pool. Parallelism between the experimental sample and the standard curve
indicated that the affinity of receptors in the samples was similar to that in the pool (39).

Statistical Analysis

Differences in parameters between the largest and second-largest growing follicles on Days
2 and 4 were examined using split-plot analysis of variance. The largest follicles on Days 2,4,6 and
10 were analyzed using one-way analysis of variance after square-root transformation of the data
(31). Residuals for each analysis were checked for violation of assumptions (equal variance,
normality of errors). If a significant difference (PcO.05) among follicles was indicated, a least
squared means test was used as an indicator of differences among means.

RESULTS

Scatchard analysis of the displacement of homologous hormone yielded a Kd for oFSH of

82.6 pM with a Bmax of 185.3 pM and a Kd for hCG of 32.1 pM with a Bmax of 12.7 pM (Figure
1). Standard curves for FSH and hCG were linear (Figure 2). The 2 amounts of granulosa cells in
the samples displayed a parallel increase in binding when compared to the standard curves for FSH
504 Theriogenology

3.0 0.3
Kd=62.56 pM
FSH receptors
2.5- Smax=165.33pM

0.2
$
5
e
9
g
0.1

Q.0
14 6 0 10 12 14
Bound (PM)

Figure 1. Scatchard analysis of the displacement of homologous hormone. Iodinated oFSH or hCG
was incubated with increasing concentrations of unlabelled homologous hormone. Affinity of the
ligand for the receptor is expressed as Kd and the number of receptors is expressed as Bmax.

Number of granulosa cells

10 4 10 5 lo6 104 10 5 10 6
0
I c I
**- 0
FSH receptors hCG receptors

-A- Day4LF
+ Day4SF
-c- Day6DF
+ DaylODF

- -6

Number of receptors in standard curve

Figure 2. Standard curves (n= 3) for iodinated oFSH and hCG binding. Data were transformed to
logit (% Bound) and log and fitted with regression equations for calculating numbers of receptors
in the samples. Samples represent the mean values for each group of follicles studied. LF= largest
follicle; SF= second largest follicle; DF= dominant follicle.
Theriogenology 505

Table 1: Comparisons of the largest follicles of the first follicular wave in heifers

End point Day 2 Day 4 Day 6 Day 10

Follicle diameter (mm) 8.5 f 0.4a 13.0 + 0.9n 15.2 -+: 0.3uC 16.5 -+ 1.5’

Estradiol (@ml) 373 + 151a 265 + 63a 177 k 36a 42 f 12b

Estradiol (ng/follicle) 161 f 89ab 305 f 33ab 336 ‘-c 86a 127 k 31b

Progesterone (ng/ml) 12.2 + 2.3a 17.1 + l.6ab 19.0 k 1.5ab 24.4 f 4.8b

EstradioLProgesterone
Ratio (ng/ml) 26.1 f 13.9a 15.2 f 2.9ab 9.5 f l.9ab 2.1 f 0.7b

Granulosa cells6
per follicle(x 10 ) 26 f 5 30 k 7 50 k 14 38 f 24

FSH
Receptors /cell 601 I f 711 14196 f 8045 7984 rt 2185 7793 f 3212

Receptors /follicle(xlO’O) 15.8 f 3.8 34.9 f 14.6 33.5 f 7.8 15.2 -e 5.5

LH
Receptors /cell 2344 + 1790 7521 + 3112 7609 t_ 2267 9046 +- 6717

Receptors /follicle(xlO’O) 6.8 + 5.4 19.3 f 6.8 31.9 r 8.2 12.2 * 4.9

lZ&~ are means f SEM (n= 3 heifers for Day 2 and n= 4 heifers for Days 4,6, and 10).
Values with different superscripts within rows are different (P < 0.05).

and hCG suggesting that the affinity (Kd) of receptors in the samples was similar to that of the
standard plasma membrane pool (39).

Table 1 shows the comparisons of the largest follicle among the 4 d for each end point.
Follicle diameter increased (P&.05) from Day 2 to 6, but it did not change between Days 6 and 10.
In contrast, estradiol concentration in follicular fluid decreased over days and was significantly lower
on Day 10 than on Day 2,4 or 6. Follicular fluid progesterone concentration was significantly higher
on Day 10 than on Day 2. In addition, the estradiol to progesterone ratio was significantly lower on
Day 10 than on Day 2. Numbers of gonadotropin receptors per granulosa cells and per follicle did
not change over days.
506 Theriogenology

Table 2: Comparison of the 2 largest follicles on Days 2 and 4 of the first follicular wave

Dav 2 Day 4
Largest Second-largest Largest Second-largest
End point follicle follicle follicle follicle

Follicle diameter (mm) 8.5 f 0.5 7.3 * 0.7 13.0 + 0.9 8.9 + 0.5**

Estradiol (ng/ml) 373 & 151 42 + 26** 265 f 63 6 rt 2*

Estradiol (ng/foIlicle) 161 zk 89 21 f 18* 305 + 33 2 * 1**

Progesterone (ng/ml) 12.2 f 2.3 20.1 f 8.6 17.1 f 1.6 21.1 -+ 13.3

EstradiokProgesterone
Ratio (ng/ml) 26.07 2 13.9 9.5 * 8.8 15.2 f 2.9 0.98 + 0.5

Granulosa cells6
per follicle(x 10 ) 26 A 5 16 f 4 30 + 7 10 * 3**

FSH
Receptors /cell 6011 +- 711 8925 + 2246 14196 + 8045 4322 + 953

Receptors /follicle(xlO1°) 15.8 f 3.8 12.5 + 1.4 34.9 f 14.6 3.6 +- 0.7’”

LH
Receptors /cell 2344 f 1790 3013 + 1234 7521 f 3112 792 + 361*

Receptors /follicle(xlO’O) 6.8 + 5.4 4.9 f 2.0 19.3 + 6.8 0.97 + 0.5*

Data are means + SEM (n= 3 heifers for Day 2 and n= 4 heifers for Day 4).
** = Values compared within days are different (P < 0.05).
* = Values compared within days tend to be different (P < 0.1).

Table 2 shows the values for end points for the largest and second-largest follicles on Day
2 and Day 4. The estradiol concentration in follicular fluid was greater (PcO.05) for the largest
follicle on Day 2; no other end point was different between follicles on Day 2. In contrast, on Day
4 follicle diameter, estradiol concentration per follicle, number of granulosa cells per follicle, and
number of FSH receptors per follicle were greater (P<O.O5)in the largest follicle than in the second-
largest follicle. Follicular fluid progesterone and the estradiol to progesterone ratio were not
different between the largest and second largest follicles on Day 4. There was a tendency (P<O. 1)
for fewer LH receptors per cell and per follicle for the second-largest follicle on Day 4. A
comparison of the second-largest follicles between Day 2 and Day 4 demonstrated that estradiol
concentrations and number of FSH receptors per cell were lower (PcO.05) on Day 4 than on Day 2.
Theriogenoiogy 507

Other end points including follicular diameter, granulosa cell number, and LH receptor number were
not different between Days 2 and 4 for the second-largest follicles.

DISCUSSION

In earlier studies, the day of estrus was often used as the reference day, whereas in the present
study, and in many studies which have used ultrasonography as a follicular monitoring tool, the day
of ovulation was used. To minimize confusion in this discussion, 1 d will be subtracted from the day
designations of reports that used estrus as a reference so that the days will be approximately
equivalent among reports.

In a previous study (lo), the day-to-day identity of follicles of the first wave of the estrous
cycle was monitored by ultrasound. The follicle that became dominant did not differ in mean
diameter from the next largest follicle on the first day of detection of the wave (mean, Day 0), but
was significantly larger on Day 1. These changing diameter relationships were confirmed in an
examination of the composite data from all studies in our laboratories (n = 71 follicular waves;
unpublished study). The frequency (percentage of waves) in which the retrospectively identified
dominant follicle was larger than any subordinate follicle on the indicated days was as follows: Day
0, 37%; Day 1, 40%; Day 2, 75%; Day 3, 90%; Day 4, 99%; and Day 6, 100%. Thus, the
identification of the dominant follicle without information from subsequent days did not exceed the
reliability of a guess until Day 2. The largest subordinate was at maximum mean diameter on Days
3 and 4 and then decreased in diameter. In the present study, follicle identity was maintained until
the ovaries were removed to assure that only growing follicles of a new wave were processed. On
the basis of the unpublished study, the largest follicle on Days 4, 6, and 10 in the present study was
the dominant follicle, and the largest follicle on Day 2 had a 75% probability of being the dominant
follicle. However, the levels of estradiol in the follicular fluid of the largest follicle of Day 2 were
much higher than in the second-largest follicle in all three heifers. Thus, it seems likely that the
largest follicle was the dominant follicle on Day 2 as well as on the other days. In the following
discussion, therefore, the largest follicle will be described as the dominant follicle for all test days,
but for Day 2 the caveat of presumption will be retained. In addition, on the basis of the unpublished
study, Day 2 represents the early portion of divergence and Day 4 represents the end of divergence;
divergence is characterized by more rapid growth of the dominant follicle as compared to the
subordinates. During this phase, however, both follicle types are viable as indicated by the
following: 1) continued growth of the subordinates when the dominant follicle is removed during
divergence (Day 3) but not when removed after divergence (Day 5; 19), and 2) stimulation of the
subordinates when FSH is given during divergence (Days 0.5 to 2) but not when given after
divergence (Days 5 to 6.5; 3).

Selection of the dominant follicle and the subsequent growth divergence between dominant
and subordinate follicles are intriguing aspects of follicular development. The mechanisms
responsible for the designation of a dominant follicle and the timing of selection relative to wave
emergence are not known. Presumably, physiologic selection of the dominant follicle occurred
before Day 2 in this study since the largest follicle contained almost a lo-fold greater concentration
of estradiol in follicular fluid than did the next largest follicle.
508 Theriogenology

The mechanisms of divergence may involve an interaction of multiple factors. For example,
FSH and estrogens act synergistically to enhance follicular growth, follicular differentiation, and
steroid production (39). Estradiol and FSH also stimulate CAMP production and CAMP dependent
FSH and LH receptor formation by rat granulosa cells (17). Divergence of the follicles could involve
other intrafollicular factors, such as activin, inhibin, or growth factors that may enhance the FSH
responsiveness and/or growth potential of the selected dominant follicle (5, 22).

Decreases in plasma FSH concentration are associated temporally with follicular wave
divergence (2, 37). indicating that the dominant follicle may grow more readily than subordinate
follicles under reduced FSH concentrations. Therefore, it was hypothesized in the present study that
differences in gonadotropin receptor numbers on the granulosa cells of dominant and subordinate
follicles would correspond to the time of follicular wave divergence. The FSH receptor numbers
were significantly lower in the subordinate follicles on Day 4, but not on Day 2. Therefore, the
hypothesis was not supported since divergence was expected to begin before Day 2 (IO). It is
possible that there may be differences in gonadotropin receptors that we were not able to detect with
our measurement techniques, such as localization of receptors on the plasma membrane as compared
to the intracellular membrane or binding affinity of the receptors under in vivo conditions. In
contrast, substantially higher levels of follicular fluid estradiol was associated with the first portion
of wave divergence; greater estradiol concentrations were detected in the presumptive dominant
follicle versus the subordinate on Day 2. These results suggest that estradiol concentrations, but not
gonadotropin receptor numbers, play a role in the divergence in growth rate between the dominant
and subordinate follicles and the apparent decrease in responsiveness of the subordinate follicles to
basal levels of FSH.

Diminished physiologic capabilities of subordinate follicles by Day 4 were indicated by

decreased granulosa cell numbers, LH and FSH receptor concentrations, and estradiol
concentrations. These results are consistent with previous reports. Reduced follicular fluid estradiol
concentration and granulosa cell aromatase activity have been reported to occur in subordinate
follicles on Day 4 (5). In addition, the ability of granulosa cell gonadotropin receptors to bind FSH
and hCG was reduced in estrogen-inactive follicles as compared with the estrogen-active follicles
on Days 4 and 6 (15). A decrease in intrafollicular estradiol concentrations in the subordinate
follicles between Day 2 and Day 4 was observed in the present study, in agreement with Badinga et
al. (5), who reported a decrease in the ability of subordinate follicles to synthesize estradiol from Day
4 to Day 7. Although not significant, the decrease in estradiol:progesterone ratio in subordinate
follicles on Day 4 may be an indication of early follicular atresia (15). Thus, by the end of growth
divergence, a number of physiologic and biochemical differences between the dominant and the
subordinate follicles become apparent.

Follicular fluid progesterone concentrations were not different between the dominant and
subordinate follicles on Days 2 or 4. This is consistent with the finding that progesterone
concentrations of growing and static follicles are not different and that concentrations of follicular
fluid progesterone do not increase until the follicle starts to regress (27). The estradiol:progesterone
ratio was numerically lower in subordinate follicles on Days 2 and 4. Thus, in the present study, it
appears that the alterations in estradiol to progesterone ratio are primarily due to changes in
intrafollicular estradiol concentration and not to changes in follicular fluid progesterone.
Theriogenology 509

At the end of follicular divergence, the subordinate follicles cease to grow, whereas the
dominant follicle continues to grow until approximately Day 6 (11). The period of continued growth
of the dominant follicle is associated temporally with basal levels of FSH in the blood (1, 37). In
the present study, it was postulated that the concentrations of gonadotropin receptors on the
granulosa cells of the dominant follicles would reflect the continued growth of the dominant follicle
despite the reported reduced circulating concentrations of FSH. A numerical but not statistically
significant decrease in FSH receptor number per cell was noted between Days 4 and 6; however,
FSH receptor number per follicle and LH receptor number per follicle or per cell did not decrease.
In addition, there was no difference in intrafollicular estradiol concentration, intrafollicular
progesterone concentration, or estradiokprogesterone ratio between Day 2 and Day 6. Thus, it
appears that the biochemical characteristics measured in this study were not altered during the
continued growth of the dominant follicle.

Loss of functional dominance occurs by Day 10 as indicated by the emergence of a new

follicular wave (11). A plateau in diameter of the dominant follicle was described for approximately
Days 6 to 12. A plateau in follicle diameter is consistent with the similar diameter between Days
6 and 10 in the present study. However, during this period, there was a numerical, but not
statistically significant, decrease in both FSH and LH receptor numbers per follicle and a significant
decrease in concentrations of estradiol in follicular fluid. The decrease in estradiol concentration
may indicate a decrease in aromatase activity in the dominant follicle. It has been reported that
dominant follicles on Days 4 and 7 have more aromatase activity than on Day 11 (5). Additionally,
it was shown that the fall in follicular fluid estradiol concentration on Days 7 and 11 corresponded
to a parallel decrease in intrafollicular androstenedione concentration. Therefore, a decrease in
androgen as a substrate for estrogen biosynthesis may contribute to the regression of the first-wave
dominant follicle (4).

Intrafollicular progesterone concentrations were higher in the dominant follicle on Day 10

than on Day 2. In addition, the estradiol to progesterone ratio decreased in the dominant follicle
from Day 2 to Day 10. It has been demonstrated that decreased estradiol concentrations and
increased progesterone concentrations are associated with follicular atresia (27, 5, 24, IS).
Therefore, atresia of the dominant follicle may have begun by Day 10 in the present study.

The extent to which estradiol directly regulates granulosa cell function and follicular
development is not fully understood. Data from this study and others indicate that estradiol is
associated with growth divergence, growth termination, and eventual regression of follicles. It is
known that estradiol is obligatory for granulosa cell differentiation in rats (29). Furthermore, it has
been demonstrated that the synergistic actions of estradiol and FSH cause an increase in the number
of LH receptor binding sites on granulosa cells (28) and an increase in the activity of the adenylate
cyclase enzyme system in rats (16). It also appears that decreased androgen availability or decreased
steroid biosynthesis capabilities may be an integral component in the mechanisms of growth
termination and follicular atresia (4). Although most of the data suggesting an important role for
estradiol in follicular development comes from research in rats, it appears that similar mechanisms
may exist in the bovine.
510 Theriogenology

In summary, higher concentrations of estradiol in the follicular fluid were present in the
presumptive dominant follicle by the beginning of divergence (Day 2), but the estradiol differences
were not associated with differences in numbers of receptors for FSH or LH on the granulosa cells.
By the end of growth divergence (Day 4), the 2 types of follicles (dominant and subordinate) differed
in numbers of both FSH and LH receptors as well as in granulosa cell numbers and estradiol
concentrations. The growing phase of the dominant follicle (Days 2 to 6) was not associated with
a significant alteration in either gonadotropin receptor number or estradiol concentration in follicular
fluid. However, just before the expected regression of the dominant follicle (Day 10) estradiol
concentrations and gonadotropin receptor numbers did decrease. Thus, estradiol concentration in
follicular fluid, but not alterations in numbers of gonadotropin receptors on granulosa cells, appears
to be closely associated with physiologic changes in the bovine antral follicle during development
of follicular waves.

REFERENCES

1. Adams, GP, RL Matteri, JP Kastelic, JCH Ko and OJ Ginther. Association between surges of
follicle-stimulating hormone and the emergence of follicular waves in heifers. J Reprod 1992a;
94: 177- 188.
2.. Adams, GP, RL Matteri and OJ Ginther. Effect of progesterone on ovarian follicles, emergence
of follicular waves and circulating follicle-stimulating hormone in heifers. J Reprod Fertil 1992b;
95: 627-640.
3. Adams, GP, K Kot, CA Smith and OJ Ginther. Selection of a dominant follicle and suppression
of follicular growth in heifers. Anim Reprod Sci 1993; 30: 259-27 1.
4. Badinga, L, MA Driancourt, JD Savio, D Wolfenson, and WW Thatcher. Changes in follicular
development, aromatase activity, and follicular steroids in dominant and subordinate follicles
at Day 5, 8, and 12 of the estrous cycle in cattle. Biol Reprod 1991; 44 (Suppl 1): 71 abstr.
5. Badinga, L, MA Driancourt, JD Savio, D Wolfenson, M Drost, RL de la Sota, and WW
Thatcher. Endocrine and ovarian responses associated with the first-wave dominant follicle in
cattle. Biol Reprod 1992; 47: 871-883.
6. Braden, TD, JG Manns, DL Cermak. TM Nett, and GD Niswender. Follicular development
following parturition and during the estrous cycle in beef cows. Theriogenology 1986; 25: 833-
843.
7. Bruns, RF, K Lawson-Wendhng, TA Pugsley. A rapid filtration assay for soluble receptors using
polyethylenimine-treated filters. Anal Biochem 1983; 132: 74-8 1.
8. Diekman, MA, P O’Catlaghan, TM Nett, and GD Niswender. Validation of methods and
quantification of luteal receptors for LH throughout the estrous cycle and early pregnancy in
ewes. Biol Reprod 1978;19: 999-1009.
9. Fortune, JE. Ovarian follicular growth and development in mammals. Biol Reprod 1994; 50:
225-232.
10. Ginther, OJ, JP Kastelic and L Knopf. Composition and characteristics of follicular waves during
the bovine estrous cycle. Anim Reprod Sci 1989a,20: 187-200.
11. Ginther, OJ, L Knopf, and JP Kastelic. Temporal associations among ovarian events in cattle
during oestrus cycles with two and three follicular waves. J Reprod Fertil 1989b87: 223-230.
12. Hunter, WM and FC Greenwood. Preparation of iodine- 13 1 labeled human growth hormone of
high specific activity. Nature (London) 1962; 194: 495-496.
Theriogenology 511

13. Ireland, JJ. Control of follicular growth and development. J Reprod Fertil 1987;34 (Sup@): 39-
54.
14. Ireland, JJ and JF Roche. Development of antral follicles in cattle after prostaglandin-induced
luteolysis: changes in serum hormones, steroids in follicular fluid, and gonadotropin receptors.
Endocrinology 1982; 111: 2077-2086.
15. Ireland. JJ and JF Roche. Development of nonovulatory antral follicles in heifers: changes in
steroids in follicular fluid and receptors for gonadotropins. Endocrinology 1983; 122: I50- 156.
16. Jonassen, JA, K Bose, and JS Richards. Enhancement and desensitization of hormone-responsive
adenylate cyclase in granulosa cells of preantral ovarian follicles: effects of estradiol and follicle-
stimulating hormone. Endocrinology 1982; 111: 74-79.
17. Knecht, M, JM Darbon, T Ranta, AJ Baukal, and KJ Catt. Estrogens enhance the adenosine 3’,5’-
monophosphate-mediated induction of follicle-stimulating hormone and luteinizing hormone
receptors in rat granulosa cells. Endocrinology 1984; 115: 41-49.
18. Knopf, L, J Kastelic, E Schallenberger and OJ Ginther. Ovarian follicular dynamics in heifers:
Test of the two-wave hypothesis by ultrasonically monitoring individual follicles. Dom Anim
Endocrinol 1989;6: 11 l- 119.
19. Ko, JCH, JP Kastelic, MR Del Campo and OJ Ginther. Effects of a dominant follicle on ovarian
follicular dynamics during the oestrous cycle in heifers. J Reprod Fertil 1991;91: 5 11-5 19.
20. Labarca, C and K Paigen. A simple, rapid and sensitive DNA assay procedure. Anal Biochem
1980; 102: 344-352.
2 I. Lucy, MC, JD Savio, L Badinga, RL de la Sota and WW Thatcher. Factors that effect ovarian
dynamics in cattle. J Anim Sci 1992;70: 36153626.
22. Martin, TL, RL Fogwell, and JJ Ireland. Concentrations of inhibins and steroids in follicular
fluid during development of dominant follicles in heifers. Biol Reprod 1991;44: 673-700.
23. Matteri, RL, JF Roser, DM Baldwin, V Lipovetshy, H Papkoff. Characterization of a monoclonal
antibody which detects luteinizing hormone from diverse mammalian species. Dom Anim
Endocrinol 1987;4: 157-165.
24. McNatty, KP, DA Heath, KM Henderson, S Lun, PR Hurst, LM Ellis, GW Montgomery, L
Morrison, and DC Thurley. Some aspects of thecal and granulosa cell function during follicular
development in the bovine ovary. J Reprod Fertil 1984;72: 39-53.
25. Miller, KF. JK Critser, RF Rowe, and OJ Ginther. Ovarian effects of bovine follicular fluid
treatment in sheep and cattle. Biol Reprod 1979;2 1: 537-544.
26. Nett, TM, ME Crowder, GE Mass and TM Duello. GnRH receptor interaction. V. Down
regulation of pituitary receptors for GnRH in ovariectomized ewes by infusion of homologous
hormones. Biol Reprod 1981;24: 1145-l 155.
27. Price, CA, PD Carriere, B Bhatia. and NP Groome. Comparison of hormonal and histological
changes during follicular growth, as measured by ultrdsonography, in cattle. J Reprod Fertil
1995; 103: 63-68.
28. Pursley. JR, MO Mee. and MC Wiltbank. Synchronization of ovulation in dairy cows using
PGF2e and GnRH. Theriogenology 1995; in press,
29. Richards, JS, JJ Ireland, MC Rao, GA Bernath, AR Midgley Jr, and LE Reichert Jr. Ovarian
follicular development in the rat: hormone receptor regulation by estradiol, follicle stimulating
hormone, and luteinizing hormone. Endocrinology 1976:99: 198.
30. Richards. JS. Maturation of ovarian follicles: actions and interactions of pituitary and ovarian
hormones on follicular cell differentiation. Endocrinology 1980; 115: 4 l-49.
512 Theriogenology

31. SAS. SAS User’s Guide: Statistics. SAS Institute, Inc., Cary, NC 1985.
32. Savio, JD, WW Thatcher, L Badinga, RL de la Sota and D Wolfenson. Turnover of dominant
ovarian follicles is regulated by progestins and dynamics of LH secretion in cattle. J Reprod
Fertil Abstr Ser 1980;6: 23.
33. Savio, JD, WW Thatcher, L Badinga, RL de la Sota and D Wolfenson. Regulation of dominant
follicle turnover during the estrous cycle in cows. J Reprod Fertil 1993a; 97: 197-203.
34. Savio, JD, WW Thatcher, GR Morris, K Entwistle, M Drost, and MR Mattiacci. Effects of low
plasma progesterone concentrations with a progesterone-releasing intravaginal device on
follicular turnover and fertility in cattle. J Reprod Fertil 1993b;98: 77-84.
35. Scatchard, G. The attraction of proteins for small molecules and ions. Annal New York Acad Sci
1949;5 1: 660-672.
36. Spicer, LJ, EM Convey, K Leung, RE Short,&4 Tucker. Anovulation in postpartum suckled
beef cows. II. Associations among binding of I-labeled gonadotropins to granulosa and thecal
cells, and concentrations of steroids in serum and various sized ovarian follicles. J Anim Sci
1986;62: 742-750.
37. Stock, AE and JE Fortune. Ovarian follicular dominance in cattle: Relationship between
prolonged growth of the ovulatory follicle and endocrine parameters. Endocrinology 1993; 132:
1108-l 114.
38. Sunderland, SJ, MA Crowe, MP Boland, JF Roche and JJ Ireland. Selection, dominance, and
atresia of follicles during the oestrus cycle of heifers. J Reprod Fertil 1994;lOl: 547-555.
39. Suter, DE. Regulation of Receptors for Luteinizing Hormone in the Ovine Corpus Luteum. Fort
Collins: Colorado State University; Ph.D. Dissertation 1981.
40. Wang, XN and GS Greenwald. Synergistic effects of steroids with FSH on folliculogenesis,
steroidogenesis and FSH- and hCG- receptors in hypophysectomized mice. J Reprod Fertil
1993;99: 403-413.