Alterations in follicular estradiol and gonadotropin receptors during development of bovine antral follicles

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: Accepted:

March I5, 199s 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
LH receptors,

FSH receptors,

follicles,

cattle

Acknowledgments This study was supported by the Wisconsin Experiment Station and USDA grant number 9 l37203-6557. 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. 655 Avenue of the Americas, New York, NY 10010

0093-691 W96/$15.00 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 Animal Procedures,

Experimental

AND METHODS

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 lineararray 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 Estradiol

Theriogenology 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

503

Theriogenology

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 Bound (PM)

6

0

10

12

14

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

I

0

lo6

c

104

FSH receptors

10 5

I

10 6

**- 0

hCG receptors

-A+ -c+

Day4LF Day4SF 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.

505

Theriogenology Table 1: Comparisons

of the largest follicles

End point

of the first follicular

Day 2

Follicle diameter

(mm)

Day 4

wave in heifers

Day 6

8.5

f 0.4a

13.0

+ 0.9n

15.2

Day

-+: 0.3uC

16.5

10

-+ 1.5’

Estradiol

(@ml)

373

+ 151a

265

+ 63a

177

k 36a

42

Estradiol

(ng/follicle)

161

f

89ab

305

f

33ab

336

‘-c 86a

127

12.2

+ 2.3a

17.1

+ l.6ab

19.0

k

1.5ab

24.4

f

4.8b

26.1

f

13.9a

15.2

f

9.5

f

l.9ab

2.1

f

0.7b

26

f

5

38

f

24

7793

f

3212

Progesterone

(ng/ml)

EstradioLProgesterone Ratio (ng/ml) Granulosa cells6 per follicle(x 10 ) FSH Receptors

/cell

Receptors

/follicle(xlO’O)

Receptors

/cell

Receptors

/follicle(xlO’O)

601 I f 15.8

30

2.9ab

k 7

50

711

14196

f

8045

7984

3.8

34.9

f

14.6

33.5

+ 1790

7521

+ 3112

7609

f

k 14

rt 2185 f

7.8

15.2

f

12b

k 31b

-e 5.5

LH 2344 6.8

+ 5.4

19.3

f

6.8

31.9

t_ 2267 r

8.2

9046 12.2

+- 6717 * 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 standard plasma membrane pool (39).

in the samples

was similar to that of the

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.

Theriogenology

506

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

End point

Largest follicle

Follicle diameter (mm)

8.5 f

Estradiol (ng/ml)

Largest follicle

Day 4 Second-largest follicle

7.3 * 0.7

13.0 + 0.9

8.9 + 0.5**

373 & 151

42 + 26**

265 f 63

6 rt 2*

Estradiol (ng/foIlicle)

161 zk 89

21 f

305 + 33

2 * 1**

Progesterone (ng/ml)

12.2 f 2.3

EstradiokProgesterone Ratio (ng/ml) Granulosa cells6 per follicle(x 10 ) FSH Receptors /cell Receptors /follicle(xlO1°) LH Receptors /cell Receptors /follicle(xlO’O)

26.07

0.5

Dav 2 Second-largest follicle

2 13.9

26 A 5

6011

+- 711

15.8 f 3.8

2344

f

1790

6.8 + 5.4

18*

20.1 f 8.6

17.1 f

1.6

9.5 * 8.8

15.2 f 2.9

16 f 4

30 + 7

8925 + 2246 14196 + 8045 12.5 + 1.4

21.1 -+ 13.3

0.98 + 0.5

10 * 3**

4322

+ 953

14.6

3.6 +- 0.7’”

3013 + 1234

7521 f 3112

792 + 361*

4.9 f 2.0

19.3 + 6.8

0.97 + 0.5*

34.9 f

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
507

Theriogenoiogy

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.

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