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a Departments of Animal Health and Biomedical Science and
b Dairy Science, University of Wisconsin-Madison, Madison, Wisconsin 53706
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(PGF2; n = 6) or a control group (n = 5). Twenty-four hours later, all detectable follicles (
2 mm) were ablated (Hour 0). Follicular development was monitored Hours 0, 3, 6, 9, 12, and 16, at 8-h intervals thereafter until Hour 112. To monitor FSH concentrations, blood was sampled at Hours -24, 0, 3, 6, 9, 12, and 16, and at 8-h intervals thereafter until Hour 104. There were no differences (p > 0.05) between the PGF2-treated group and controls in the patterns of the FSH surge or follicular development.
Experiment 2 tested the hypothesis that 3-mm follicles do not have FSH-suppressing capacity and that suppression increases as follicles grow beyond 5 mm. Twenty-four hours after an injection of PGF2 (Days 6–8), heifers were subjected to either ablation of follicles 2 mm or ovariectomy. Intact heifers were allocated into four groups (n = 5) in which all follicles of the new wave were ablated upon reaching either 3, 5, or 7 mm or were not ablated (controls). Blood was sampled at 8-h intervals to monitor FSH and estradiol-17ß. Averaged over Hours 8–120, FSH concentrations (ng/ml) were higher (p < 0.05) in the ovariectomized (2.02 ± 0.05) and the 3-mm groups (1.91 ± 0.05) than in the 5-mm (1.52 ± 0.05), 7-mm (1.35 ± 0.04), and control groups (1.33 ± 0.05); and estradiol concentrations (pg/ml) were lower (p < 0.05) in the ovariectomized group (0.19 ± 0.03) than in the 3-mm (1.48 ± 0.16), 5-mm (1.56 ± 0.15), 7-mm (2.22 ± 0.27), and control groups (2.55 ± 0.49).
In conclusion, the presence of endogenous progesterone did not affect FSH patterns or follicular development. Follicles 
3 mm had no detectable capacity to suppress FSH. As follicles grew from 3 to 5 mm, they gained the capacity to suppress FSH; however, as follicles grew beyond 5 mm, FSH-suppressing capacity did not increase. The FSH decline was not attributable to an increase in circulating estradiol.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In heifers, progesterone can affect follicular development. In three-wave cycles, dominant follicles of the second anovulatory follicular wave, when progesterone is elevated, had smaller maximum diameters than dominant follicles from the first anovulatory follicular wave [16]. A decreasing dose of exogenous progesterone over the first 5 days of the first follicular wave suppressed the maximum diameter of the dominant follicle; however, the effect was not detected until 6 days after emergence of the wave, and the mechanism apparently did not act through circulating FSH concentrations [17]. Different progesterone environments early in follicular waves may alter the maximum diameter of the future dominant follicle, although these effects are not detected until well after follicle deviation. Growth rates of follicles were not different between anovulatory and ovulatory follicular waves for the first 5 days of the follicular wave [18], which encompassed the time from wave emergence through deviation. In pregnant cows, follicular waves became less pronounced during the last 5 mo of gestation as the maximum diameters of the dominant follicle and largest subordinate follicle were suppressed, but FSH surges remained rhythmic and periodic [19]. It is possible that prolonged exposure to elevated progesterone from the corpus luteum and developing conceptus may also be responsible for suppressing follicular development directly or by selectively altering LH concentrations, during late gestation.
The purpose of experiment 1 was to test the hypothesis that progesterone alters FSH patterns and early follicular growth profiles. The purpose of experiment 2 was to test the hypothesis that 3-mm follicles do not suppress FSH and that the FSH-suppressing capacity of follicles increases as they grow beyond 5 mm.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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On Day 7 (Day 0 = ovulation), Holstein heifers (University of Wisconsin research herd) weighing 300–450 kg were randomly allocated into a group (n = 6) receiving 25 mg of prostaglandin F2 (PGF2) at two separate injections (i.m.) 12 h apart (Lutalyse; Pharmacia and Upjohn Co., Kalamazoo, MI) and a group (n = 5) not receiving PGF2 (controls). Follicles were monitored transrectally with an ultrasound scanner equipped with a 7.5-MHz transducer (Aloka 500-V; Corometrics Medical Systems, Inc., Wallingford, CT). Twenty-four hours after the first PGF2 injection or at a corresponding time in the controls, all detected follicles ( 2 mm) were ablated using a transvaginal ultrasound-guided (5-MHz transducer) approach. A subsequent transrectal ultrasound ovarian examination was performed approximately 20 min after the ablation to ensure that all detected follicles had been destroyed. Ultrasound examinations were performed at Hours 0, 3, 6, 9, 12, and 16, and at 8-h intervals thereafter until Hour 112 (Hour 0 = initial ablation) to monitor follicular development [20]. Blood samples were taken at Hours -24, 0, 3, 6, 9, 12, 16, and at 8-h intervals thereafter until Hour 104 to monitor progesterone and FSH concentrations. Follicular wave emergence was defined as occurring during the untrasound examination during which the retrospectively identified dominant follicle reached 4 mm. Follicular deviation was defined as occurring during the retrospectively identified ultrasound examination at the beginning of the greatest difference in growth rates between the two largest follicles [3, 4].
Experiment 2
On Days 6–8, Holstein heifers (University of Wisconsin research herd) weighing 300–450 kg received 25 mg of PGF2 at two separate injections (i.m.) 12 h apart. Twenty-four hours after the first PGF2 injection, heifers (n = 5 per group) were subjected to either transvaginal follicular ablation (four groups) or ovariectomy (one group) via colpotomy. After the initial ablation (Hour 0), ovary-intact heifers were randomly allocated into four groups in which follicles of the new wave were ablated upon reaching 3, 5, or 7 mm in diameter or were not ablated (controls). Ultrasound examinations were performed to monitor follicular development in the ovary-intact heifers at 8-h intervals from Hour 0 until Hour 120. Ablation of new follicles was performed at each 8-h scanning session (Hours 8–112) during which the follicles reached the designated diameter (3, 5, or 7 mm) or larger. This technique allowed follicular development to be stopped at diameters that are consistent with time periods just prior to the peak of the FSH surge (3-mm group), just after the FSH peak (5-mm group), and just prior to deviation (7-mm group) for comparison to ovariectomized and control groups. Blood samples were taken from all animals at 8-h intervals from Hour 0 until Hour 120 to monitor FSH and estradiol concentrations.
RIAs
Serum concentrations of progesterone were determined using a solid-phase 125I RIA kit (Coat-a-Count Progesterone; Diagnostic Products Corporation, Los Angeles, CA). The kit was modified using charcoal-stripped bovine serum plus the appropriate amount of progesterone (40, 20, 10, 5, 1, 0.5, and 0.25 ng/ml) for assay standards or for maximum binding tubes (no progesterone added). Serum samples (25 ml) from 15 heifers at random stages of the interovulatory interval were pooled to serve as a quality control to determine intra- and interassay coefficients of variation, which were 2.3% and 5.6%, respectively.
Serum concentrations of FSH were determined using a solid-phase 125I RIA previously validated in this laboratory [2, 5]. Intra- and interassay coefficients of variation were 9.1% and 14.2%, respectively.
Serum concentrations of estradiol-17ß were determined using a commercially available solid phase 125I RIA kit (Ultra sensitive oestradiol assay; DSL-4800, Diagnostic Systems Laboratory, Webster, TX), which was modified by the addition of two extraction steps. Samples (500 µl) were combined with 3 ml of diethyl ether and vortexed (2 min). Samples were then frozen, and the diethyl ether fraction was decanted and saved. After a second similar extraction step, the decanted diethyl ether plus the organic portion of the sample was dried and resuspended in 100 µl of assay buffer for determination of estradiol concentration. All samples were extracted and assayed in duplicate, and the mean intraassay coefficient of variation was 16.8%. To have enough serum for the double-extraction estradiol assay procedure and for FSH assays from the same samples, 750 µl of each of three consecutive 8-h samples were pooled together. Additionally, estradiol concentrations were determined from the pretreatment Hour 0 samples.
Statistical Analysis
Single point values (growth rates, maximum diameters, peak FSH concentrations, and the time required to reach various follicular and FSH endpoints) were compared using either a Student's t-test (experiment 1) or an ANOVA (experiment 2). Repeated measurements over time (progesterone, FSH, and estradiol concentrations; follicle diameters) were compared using a split-plot ANOVA to determine main effects of group and hour and their interaction. The maximum follicular diameters for the dominant and subordinate follicles were retrospectively determined, and the growth rates for the dominant and subordinate follicles were calculated (diameter at follicular deviation minus diameter on the day of follicular wave emergence divided by the number of days required from emergence to deviation).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The diameters of the dominant and largest subordinate follicles at wave emergence and follicular deviation, the growth rates and maximum diameters of the two follicles, and the time from ablation until wave emergence or deviation were not different between controls and PGF2-treated heifers (Table 1). However, the diameter of the largest subordinate follicle at wave emergence tended to be larger (p = 0.053) in the controls than in the PGF2-treated group (Table 1). The mean and peak FSH concentrations, and the time required for the FSH surge to reach the peak and the subsequent nadir, were similar between the controls and PGF2-treated heifers (Table 1).
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The main effects of group and hour and the interaction were significant for the concentrations of progesterone (Fig. 1). Progesterone concentrations declined within 24 h after PGF2 injection and remained below 1 ng/ml in PGF2-treated heifers, whereas progesterone concentrations remained above 4 ng/ml in controls throughout the experiment. For the FSH surge and the growth profiles of the dominant and largest subordinate follicles, the main effects of hour were significant (p < 0.001), but the main effects of group and the group-by-hour interactions were not (p > 0.10; Fig. 2).
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Experiment 2
There was a significant main effect of group (p < 0.05) and hour (p < 0.001) and a group-by-hour-interaction (p < 0.02) for FSH concentrations (Fig. 3). For the main effects of group (Hours 8–120), the ovariectomized and 3-mm groups had greater (p < 0.05) circulating FSH concentrations than each of the other three groups (Table 2). The interaction seemed primarily due to greater FSH concentrations in the ovariectomized and 3-mm groups than in any of the other three groups at Hours 52, 88, 96, and 112 (Fig. 3). Because the time from the initial ablation until the peak of the FSH concentrations was delayed in the ovariectomized and 3-mm groups compared to the other groups, the times required to reach the FSH peak and nadir for those two groups were not statistically analyzed (Table 2). In the control heifers, there was an increase (p < 0.05) in FSH concentrations from Hour -40 until Hour 0 and then a decrease (p < 0.05) from Hour 0 until Hour 40, relative to the emergence of the follicular wave (Fig. 4). Follicular wave emergence occurred, on average, 30.4 h after the initial ablation of all follicles. All controls underwent follicular deviation during the experimental period (96.0 ± 8.8 h after the initial ablation or 65.6 ± 6.2 h after wave emergence). For estradiol concentrations (Hours 8–120), the main effects of group were significant (p < 0.01); the ovariectomized group had lower estradiol than the 3-mm, 5-mm, 7-mm, and control groups (Table 2). Further, the 3- and 5-mm groups had lower (p < 0.05) estradiol concentrations than the control group, whereas the 7-mm group was intermediate (Table 2).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-induced luteolysis). The presence or absence of a corpus luteum and endogenous progesterone did not affect the FSH surge or the growth profiles of the dominant or largest subordinate follicles of the post-ablation follicular wave. The largest subordinate follicles tended to be larger at follicular wave emergence in controls than in PGF2-treated heifers. Although not statistically different, the time of wave emergence was 2 h later in the controls, which might account for the slightly larger diameter at wave emergence. The FSH patterns were similar between controls and PGF2-treated heifers. Similarly, a report in ewes indicated that the presence or absence of endogenous progesterone had no effect on FSH secretion [21]. The number of hours from follicular ablation until the peak of the FSH surge, the nadir of the FSH surge, or deviation was not related to the progesterone environment, and the values for these endpoints were consistent between experiments 1 and 2 and with the literature [2]. In previous studies, in an apparent contrast to the results of experiment 1, the maximum diameter of the dominant follicle of the first postovulatory follicular wave was reduced by exogenous progesterone [17] and apparently by endogenous progesterone [16]. However, these differences were not detected until 5 or 6 days after follicular wave emergence, which is well after deviation and beyond the experimental period for the present experiment. Similar FSH patterns and follicular growth profiles in experiment 1 minimized the likelihood of bias in experiment 2 associated with the presence or absence of a corpus luteum and endogenous progesterone following ovariectomy. Control heifers in experiments 1 and 2 exhibited a surge in FSH that was followed closely by the emergence of a follicular wave. The temporal association between the FSH surge and follicular development for the controls in these experiments was similar to that reported for spontaneous [1] or post-ablation [2] follicular waves.
The FSH concentrations were not suppressed in the 3-mm group compared to the ovariectomized group, indicating that the substance(s) responsible for suppressing FSH were not produced in sufficient quantities from follicles 3 mm. Estradiol has been shown to be effective in suppressing FSH concentrations either alone or synergistically with inhibin [22]. Estradiol concentrations were similar in pooled follicular fluid from medium-sized follicles (5–7 mm) and small follicles ( 4 mm) [6]; however, both of these groups had considerably lower estradiol concentrations than the follicular fluid of large (approximately 17.5 mm), estrogen-active follicles [6]. Estradiol production seems to be more closely associated with early follicular development, as there was a 6-fold increase, although the difference was not significant, in follicular fluid estradiol concentrations in follicles recovered 1 day compared to 3 days after estrus as follicles grew from approximately 6 to 8 mm in diameter [23]. The synergistic FSH-suppressive effects of estradiol and immunoreactive inhibin have also been shown to be associated with early follicular development, as both substances increased in the follicular fluid from 1 to 6 days after estrus, a period that corresponded to a subtle decline in systemic FSH [23]. Additionally, Brahman heifers had higher estradiol concentrations (caudal vena cava and jugular vein) during the growth phase (approximately 7 mm) than during the plateau phase (no daily increase in size) of the dominant follicle [9]. In a previous report, intrafollicular concentrations of estradiol recovered in situ were similar between the future dominant and largest subordinate follicles on the day before and the day at the beginning of follicle deviation [7]. There was a dramatic increase in the intrafollicular estradiol concentrations on the day after the beginning of follicle deviation, suggesting that estradiol from the dominant follicle may not be produced in sufficient quantities to suppress systemic FSH until approximately the time of deviation [7]. In another report, the estradiol concentration from the utero-ovarian vein draining the ovary with the dominant follicle reached 1500-fold above that of the contralateral ovary, accounting for the transient increase in estradiol from vena cava samples that was temporally associated with a decrease in systemic FSH [9]. Heifers in the 5-mm group in experiment 2 exhibited suppressed FSH compared to those in the 3-mm group, but this suppression was not attributable to an increase in estradiol, supporting the concept that estradiol alone may not be responsible for FSH suppression during the first 2–3 days of a follicular wave.
In experiment 2, serum estradiol concentrations in the controls and the 7-mm group were also similar; this result seems to conflict with the concept that estradiol secretion increases with increasing follicular diameter [9]. The level of estradiol was higher during the growing phase (approximately 2.5 days after ovulation) of the dominant follicle than during the plateau phase (approximately 5.6 days after ovulation); however, the dominant follicle had already reached 7 mm in the growing phase of that experiment [10]. In nonmanipulated heifers, follicles reach 7 mm just prior to deviation [3], perhaps explaining the similarity in estradiol concentrations between the 7-mm group and controls (experiment 2) that had only recently gone through deviation. Also, because the cessation of the experiment (Hour 120) was only about 24 h after deviation (on average, 96 h from ablation), the estradiol concentrations may not yet have reached maximal values in the controls. Additionally, because serum samples were pooled for estradiol assay, some effects may have been masked, and jugular samples may have lower detectable estradiol concentrations than do corresponding vena cava samples [9–11].
Inhibin, found in follicles of various sizes and physiological states [24], has been shown to be a powerful inhibitor of FSH secretion [22, 25] and is likely involved in FSH suppression [13]; however, the isoform of inhibin responsible for the FSH suppression [23] has not been fully elucidated and seems to be confounded with the effects of estradiol [26] in growing and regressing follicles [27]. Intrafollicular concentrations of dimeric inhibin were similar in follicles recovered 1 and 3 days after estrus, but they were decreased 6 days after estrus [23]. The ability of the 5-mm follicles in the current study (experiment 2) to suppress FSH, whereas the 3-mm follicles were unable to do so, may have been partially due to the effects of inhibin. Dimeric inhibin has been found in pooled follicular fluid of small growing follicles (2–7 mm; [28]) and in follicles as small as 6 mm in diameter [23]; however, little information is available in the literature on inhibin concentrations from individual, small follicles shortly after wave emergence.
Results from experiment 2 failed to support the hypothesis that the FSH-suppressing capacity of follicles increases as they grow beyond 5 mm. Instead the results indicated that as follicles grew from 3 to 5 mm, they gained the capacity to suppress FSH; however, as follicles grew 5 mm, this FSH-suppressing capacity did not increase. Control heifers had higher estradiol and lower FSH concentrations than either the ovariectomized or 3-mm groups; however, the capacity of the 5-mm follicles to suppress FSH whereas the 3-mm follicles were unable to do so was not attributable to an increase in systemic estradiol. The growth of follicles from follicular wave emergence through deviation is temporally associated with FSH suppression as shown in these experiments and in the literature [3, 4]. Clearly the deviation process and a dominant follicle are not required in order for FSH suppression to occur.
In conclusion, in heifers, systemic FSH patterns and follicular growth profiles from wave emergence through follicle deviation of a post-ablation follicular wave were not altered by the absence of a corpus luteum and endogenous progesterone. Follicles growing from 3 to 5 mm acquired the capacity to suppress systemic FSH concentrations through an unknown mechanism; however, FSH concentrations were not suppressed when the follicles were halted at 3 mm. As follicles grew (> 3 mm), FSH declined at a similar rate regardless of whether follicles were eliminated upon reaching 5 mm. That is, growth of follicles beyond 5 mm apparently did not enhance the capacity for FSH suppression. The suppression of FSH is temporally associated with follicle growth from wave emergence to deviation; however, the deviation process is not a requirement for suppression of FSH. Serum estradiol likely plays a role in keeping FSH suppressed to nadir levels, although on a temporal basis the present study did not indicate that FSH suppression from the peak of the surge to the nadir was attributable to an increase in circulating estradiol.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Accepted: August 25, 1998.
Received: July 6, 1998.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, ßA and ßB subunit messenger ribonucleic acids following increase in size and during different stages of differentiation or atresia of non-ovulatory follicles in cows. Biol Reprod 1994; 50:492–501.[Abstract] subunit and -ß dimer) in morphologically dominant follicles during their growing and regression phases of development in cattle. Biol Reprod 1993; 48:268–276.[Abstract]This article has been cited by other articles:
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  H. Lopez, R. Sartori, and M. C. Wiltbank Reproductive Hormones and Follicular Growth During Development of One or Multiple Dominant Follicles in Cattle Biol Reprod, April 1, 2005; 72(4): 788 - 795. [Abstract] [Full Text] [PDF]
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 B. Sisco, L. J. Hagemann, A. N. Shelling, and P. L. Pfeffer Isolation of Genes Differentially Expressed in Dominant and Subordinate Bovine Follicles Endocrinology, September 1, 2003; 144(9): 3904 - 3913. [Abstract] [Full Text] [PDF]
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O.J. Ginther, M.A. Beg, D.R. Bergfelt, F.X. Donadeu, and K. Kot Follicle Selection in Monovular Species Biol Reprod, September 1, 2001; 65(3): 638 - 647. [Abstract] [Full Text] [PDF]
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E. C.L. Bleach, R. G. Glencross, S. A. Feist, N. P. Groome, and P. G. Knight Plasma Inhibin A in Heifers: Relationship with Follicle Dynamics, Gonadotropins, and Steroids During the Estrous Cycle and after Treatment with Bovine Follicular Fluid Biol Reprod, March 1, 2001; 64(3): 743 - 752. [Abstract] [Full Text]
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O.J. Ginther, D.R. Bergfelt, L.J. Kulick, and K. Kot Selection of the Dominant Follicle in Cattle: Role of Estradiol Biol Reprod, August 1, 2000; 63(2): 383 - 389. [Abstract] [Full Text]
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O.J. Ginther, D.R. Bergfelt, L.J. Kulick, and K. Kot Selection of the Dominant Follicle in Cattle: Role of Two-Way Functional Coupling Between Follicle-Stimulating Hormone and the Follicles Biol Reprod, April 1, 2000; 62(4): 920 - 927. [Abstract] [Full Text]
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E.L. Gastal, M.O. Gastal, and O.J. Ginther Experimental Assumption of Dominance by a Smaller Follicle and Associated Hormonal Changes in Mares Biol Reprod, September 1, 1999; 61(3): 724 - 730. [Abstract] [Full Text]
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