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BOR - Papers in Press, published online ahead of print July 12, 2006.
Biol Reprod 2006, 10.1095/biolreprod.105.048702
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BIOLOGY OF REPRODUCTION 75, 633–641 (2006)
DOI: 10.1095/biolreprod.105.048702
© 2006 by the Society for the Study of Reproduction, Inc.

Research Article

Suppression of Follicle Wave Emergence in Cyclic Ewes by Supraphysiologic Concentrations of Estradiol-17beta and Induction with a Physiologic Dose of Exogenous Ovine Follicle-Stimulating Hormone1

David M.W. Barrett , Pawel M. Bartlewski 3, Rajesha Duggavathi 4, Kate L. Davies 5, and Norman C. Rawlings 2

Department of Veterinary Biomedical Sciences, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, Saskatchewan, Canada S7N 5B4

ABSTRACT

Follicle waves are preceded by follicle-stimulating hormone (FSH) peaks in ewes. The purpose of the present study was to see whether estradiol implant treatment would block FSH peaks to create a model in which the effect of the timing and mode of FSH peaks could be studied by ovine FSH (oFSH) injection. In Experiment 1, 10 ewes received estradiol-17beta implants on Day 4 after ovulation (Day 0, day of ovulation); five ewes received large implants, and five ewes received small implants. Five control ewes received empty implants. In Experiment 2, 12 ewes received large implants on Day 4. On Day 9, six ewes received oFSH twice, 8 h apart (0.5 µg/kg; s.c.). Implants were left in place for 10 days in both experiments. In both studies, ovarian ultrasonography and blood sampling was done daily. In Experiment 1, estradiol concentrations were significantly higher in ewes with large implants (10.4 ± 0.7 pg/ml) compared with controls (3.9 ± 0.7 pg/ml) and ewes with small implants (5.4 ± 0.7 pg/ml; P < 0.001). A significant reduction was found in mean FSH peak concentration (31%; P < 0.05) and FSH peak amplitude (45%; P < 0.05) in ewes with large implants compared with controls. Mean and basal FSH concentrations were unaffected by the large implants. The large implants halted follicle-wave emergence between Day 0 and 8 after implant insertion. The small follicle pool (2–3 mm in diameter) was unaffected by the large implants. When oFSH was injected into ewes with large implants, a follicle wave emerged 1.5 ± 0.5 days after injection; however, in ewes given saline alone, a follicle wave emerged 4.8 ± 0.8 days after injection (P < 0.01). We concluded that truncation of FSH peaks by estradiol implants prevented follicle-wave emergence, but injection of physiologic concentrations of oFSH reinitiated follicle-wave emergence.

estradiol, follicle-stimulating hormone, follicular development, luteinizing hormone, ovulatory cycle

INTRODUCTION

Before the use of ultrasonography, earlier studies on the endocrine regulation of the growth of antral follicles, ≥2–3 mm in diameter in sheep, led to the conclusion that these follicles are gonadotropin dependent [1–4]. It was generally concluded that growth of these follicles is largely FSH dependent, but some level of basal luteinizing hormone (LH) secretion appears essential, and if pulsatile LH secretion is present, final growth and maturation could become LH dependent [1–5].

With the use of ultrasonography, it was found that during the ovine estrous cycle, one to three antral follicles emerge or grow from a pool of follicles, 2–3 mm in diameter, every 4 to 5 days [6–9]. These follicles grow to a diameter of ≥5 mm in diameter before regression or ovulation [6–9]. Each wave of follicle growth is preceded by a transient peak of FSH secretion [6–10]. The role of basal FSH secretion in ovine antral follicle-wave dynamics is unknown, as is the precise role of basal or pulsed LH secretion. The frequency of LH pulses changes across the estrous cycle, largely regulated by the pattern of progesterone secretion during the genesis and regression of the corpus luteum (CL) [1, 11–17], but changes in LH pulse frequency do not appear to be correlated with or functionally related to specific phases of the growth or regression of follicle waves [10, 18]. This is interesting, as changes in LH pulse frequency occur around the time of follicular deviation in a follicular wave in cattle [19, 20]. Suppression of LH pulse frequency decreases growth of the dominant follicle after deviation, but does not require this increase in pulsed LH secretion [21]. Dominance is the process whereby one antral follicle growing in a wave exceeds others in growth rate and size and suppresses the growth of other subordinate follicles, probably by way of suppressive follicular secretory products [22]. During each follicular wave in sheep, serum concentrations of estradiol peak at the end of the growth phase of the largest follicle of the wave [7, 23]. Inhibin tends to be produced by a wider size range of antral follicles [24–28]. However, ovarian secretory products (i.e., ovarian steroids and inhibin) may not regulate the peaks in serum concentrations of FSH that precede antral follicular waves, and follicular dominance would not appear to be as marked in the ewe as in cattle [9, 29, 30].

It would be useful to develop specific experimental models in which we could examine the individual roles of basal gonadotropin secretion, FSH peaks, and pulsatile LH secretion in governing follicular wave development. The gonadotropin-releasing hormone (GnRH1) agonists and antagonists affect both LH and FSH secretion [31, 32]. Although inhibin specifically suppresses FSH secretion, it is not clear whether this is an effect on basal secretion, peaks, or both. In addition, the maintenance of a constant inhibitory affect of inhibin would be experimentally challenging. Steroid-releasing implants have been widely used in ovariectomized ewes to study estradiol and progesterone feedback regulation of gonadotropin secretion [33–35]. The effects of steroids on LH secretion were dose dependent, but on FSH secretion, they were variable; the possibility of differential effects on basal gonadotropin secretion versus LH pulses and FSH peaks have not been addressed. We hypothesized that varying the dose of estradiol released from subcutaneous implants in cyclic ewes, in the presence of progesterone from the CL, could result in differential regulation of LH and FSH secretion. This approach may also allow targeted manipulation of basal gonadotropin secretion, LH pulses, and/or FSH peaks.

The purpose of the first experiment was to see whether different doses of exogenous estradiol, administered during the luteal phase in the ewe, would differentially affect pulsatile LH secretion, FSH peaks, or baseline serum gonadotropin concentrations and to study the effects of this treatment on antral follicle development. Initial inspection of results revealed that the height of periodic FSH peaks was reduced and follicle-wave emergence was suppressed in cyclic ewes that received high doses of exogenous estradiol (i.e., large implants resulting in an ~2.7-fold increase in circulating concentrations of estradiol). Therefore, the objective of the second experiment was to see whether an FSH peak, created by administration of oFSH, could cause a follicle wave to emerge when endogenous FSH peaks were partially suppressed by large estradiol-releasing implants. This would confirm that a FSH peak is the primary signal for follicle-wave emergence in the ewe and that small follicles (2–3 mm in diameter) remain responsive to administered FSH in the absence of regular endogenous peaks in FSH secretion. Exogenous ovine FSH was given to induce a FSH peak, similar in height to endogenous FSH peaks; such a treatment has been shown to result in follicular wave emergence within 12–24 h, in both cyclic and seasonally anestrous ewes experiencing normal follicular waves [9].

MATERIALS AND METHODS

Animals

Care and handling of experimental animals was done according to the published guidelines of the Canadian Council on Animal Care. Sexually mature, clinically healthy, cyclic Western White Face (WWF) ewes were kept outdoors in sheltered paddocks. Ewes were fed a maintenance diet of hay; cobalt iodized saltlicks and water were freely available. The WWF is a cross between the Columbia and Rambouillet breeds. The mean ovulation rate of WWF ewes during the mid-breeding season is 1.8 ± 0.2 [7].

Ultrasound Technique

Ovarian antral follicular dynamics were monitored in all ewes by transrectal ovarian ultrasonography (scanning) by using a 7.5-MHz transducer stiffened with a hollow plastic rod and connected to a B-mode, real-time echo camera (Aloka SSD-900; Overseas Monitor, Richmond, BC, Canada). This technique has been validated for monitoring ovarian follicular dynamics and CL detection in sheep [36–38]. All images were viewed at a magnification x1.5 with constant gain and focal point settings. Ovarian images were recorded (Panasonic AG 1978; Matsushita Electric, Mississauga, ON, Canada) on high-grade video tapes (Fuji S-VHS, ST-120 N) for later examination. The relative position and dimension of follicles and luteal structures were also sketched on ovarian charts.

Experimental Design

Experiment 1 Fifteen ewes were treated with intravaginal progestogen sponges (medroxyprogesterone acetate [MAP], 60 mg; Veramix; Upjohn, Orangeville, ON, Canada) for 12 days, to synchronize estrus. After a time equivalent to two estrous cycles, after the synchronized estrus, ewes were monitored daily for estrus with a vaginal impedometer (Electronic Estrus Detector; Firma Draminski, Olsztyn, Poland [39]) and with vasectomized, crayon-marker-harnessed rams. Ten ewes received subcutaneous silicone rubber (Silastic) rubber implants containing 10% estradiol-17ß (wt:wt; Sigma Chemical Company, St. Louis, MO) on Day 4 after ovulation (Day 0, day of ovulation); five ewes received large implants (10 x 0.34 cm), and five ewes received small implants (5 x 0.34 cm). Five control ewes received implants without estradiol. To make the implants, liquid Silastic rubber (A-101 medical grade silicone elastomer; Factor II, Inc., Lakeside, AZ) was mixed with the steroid, and a curing catalyst was added (Catalyst; Factor II, Inc.). The mixture was injected into Silastic tubing (Silastic laboratory grade tubing; 0.34 cm i.d. x 0.47 cm o.d.; Dow Corning, Midland, MI) and, once cured, the tubing was cut into the desired lengths. Implants were soaked in sterile 0.9% (wt:vol) saline for 36–48 h at room temperature before insertion. Lidocaine hydrochloride (2%; Xylocaine; AstraZeneca Canada Inc., Mississauga, ON, Canada) was used as a local anesthetic. A 1.5-cm incision was made in the axillary region with a scalpel, the implant was inserted by using a trochar, and the incision was closed with wound clips (9 mm MikRon AUTOCLIP; Becton Dickinson Primary Care Diagnostics, MD). All implants were removed from all ewes on Day 14 after ovulation. Blood samples were collected every 12 min for 6 h (intensive sampling) on Days 10 and 14 from all ewes, to determine the secretory pattern of LH. To characterize the preovulatory gonadotropin surge, blood samples were collected every 6 h from implant removal to 6 h after ovulation from all ewes. Daily blood samples were taken at every period of scanning, and this continued for all ewes until ovulations were detected after treatment. Daily scanning started on the day of estrus at the beginning of the ovulatory cycle in which treatments were given and continued for all ewes until 8 days after the post-treatment ovulations.

Experiment 2 Twelve randomly selected ewes were monitored daily for estrus with vasectomized, crayon-harnessed rams. All ewes received large Silastic rubber implants containing 10% estradiol-17ß (wt:wt; 10 x 0.34 cm; s.c.) on Day 4 after ovulation. Implant handling and insertion procedures were the same as in Experiment 1. On the fifth day after implant insertion (Day 9 after ovulation), six of 12 ewes were injected with oFSH (0.5 µg/kg, s.c.; NIDDK-oFSH-18; 1 mg has a biologic FSH potency equal to 65.6 x NIH-oFSH-S1 or 1 640 IU and biologic LH potency equal to 0.1 x NIH-oLH-S1 or 106 IU) prepared in saline with 0.05% BSA and 50% PVP; control ewes received only the vehicle. The same dose of oFSH was injected 8 h after the initial injection. Ewes were bled every 6 h for 36 h after the first oFSH injection. Implants were removed from all ewes on Day 14 after ovulation. For both groups, daily scanning and blood sampling started on the day of estrus at the beginning of the ovulatory cycle in which treatments were given and continued until ovulations were detected after treatment.

Follicular Data Analyses

A follicular wave was defined as one or more graafian follicles that increased in size from 2 or 3 mm in diameter to ≥5 mm in diameter, and that emerged from the pool of 2- to 3-mm follicles within a maximum period of 48 h [40]. The growth, static, and regression phases of a follicular wave have been defined previously [40]. Ovulation was detected with ultrasonography as the collapse of a large follicle (≥5 mm in diameter). Follicular data were integrated for both ovaries of each animal.

Blood Sampling and Hormone Analysis

Blood samples (10 ml) taken daily and every 6 h were collected by jugular venipuncture into vacutainers (Becton Dickinson, Franklin Lakes, NJ). For intensive sampling, blood was collected via indwelling jugular catheters (5 ml/sample; vinyl tubing, 1.0 mm inside diameter x 1.5 mm outside diameter; SV70; Critchley Electrical Products Pty Ltd., Auburn, NSW, Australia). All samples were permitted to clot at room temperature for 18 to 24 h. These samples were then centrifuged for 10 min at 1500 x g, and serum was removed and kept at –20°C until assayed.

Progesterone [41], estradiol [42], FSH [43], and LH [44] concentrations were measured in serum samples by validated radioimmunoassay (RIA) procedures. Gonadotropin concentrations are expressed in terms of NIAMDD-oFSH-1 and NIAMDD-oLH-24. The assay sensitivities (defined as the lowest concentration of a hormone capable of significantly displacing radiolabeled hormone from the antibody) were as follows: 0.03 ng/ml (progesterone), 1.0 pg/ml (estradiol), and 0.1 ng/ml (FSH and LH). The ranges of standards were as follows: 0.1 to 10 ng/ml, 1.0 to 100 pg/ml, 0.12 to 16.0 ng/ml, and 0.06 to 8.0 ng/ml in the progesterone, estradiol, FSH, and LH assays, respectively. A concentration equivalent to the sensitivity of the assay was assigned to serum samples with hormone concentrations lower than the assay sensitivity. Serum samples collected daily, before and during the period implants were in place, were analyzed for concentrations of estradiol, FSH, and progesterone. All serum samples collected every 6 h and 12 min were analyzed for concentrations of FSH and LH.

The intra- and interassay coefficients of variation (CVs) were 9.0% and 15.4% or 4.9% and 7.2% for reference sera with mean estradiol concentrations of 5.4 or 15.3 pg/ml, respectively. The intra- and interassay CVs were 8.2% and 9.5% or 3.9% and 9.0% for reference sera with mean FSH concentrations of 0.36 or 1.50 ng/ml, respectively. The intra- and interassay CVs were 11.1% and 14.8% or 6.3% and 13.1% for reference sera with mean progesterone concentrations of 0.364 or 0.824 ng/ml, respectively. The intra- and interassay CVs were 7.1% and 13.8% or 3.3% and 11.6% for reference sera with mean LH concentrations of 0.17 or 0.86 ng/ml, respectively.

The PC-PULSAR program [45] was used to assess mean and basal serum LH concentrations as well as LH pulse frequency and amplitude in blood samples collected every 12 min for 6 h.

Peaks of FSH in blood samples taken daily were identified by using cycle-detection software [46]. A fluctuation or cycle was defined as a progressive rise and fall in hormone concentrations that encapsulated a peak concentration (nadir-to-peak-to-nadir [46]). Mean basal FSH concentrations were determined by averaging the lowest points between peaks (nadirs). FSH peak concentration was defined as the concentration of FSH observed at the apex of the FSH peak. FSH peak amplitude was defined as the difference between the FSH peak concentration and the nadir before the peak concentration.

Statistical Analyses

Daily hormone concentrations, maximal follicle diameter, and numbers of follicles in size classes were normalized to the day of implant insertion and analyzed for the period of implant treatment (Days 4 to 14 after ovulation). In some instances, hormone concentrations were also normalized to the peak of the preovulatory LH surge and analyzed for the period from 24 h before to 24 h after this peak. Two-way repeated measures ANOVA (SigmaStat7 for Windows Version 2.03, 1997, SPSS Inc.; Chicago, IL) was used to assess differences in daily hormone concentrations, maximal follicle diameter, and numbers of follicles in size classes over time and among the groups of ewes (i.e., Experiment 1: treated with large, small, or control implants; Experiment 2: with or without exogenous oFSH). Two-way ANOVA was used to assess differences in LH secretory characteristics from blood samples collected every 12 min (i.e., mean and basal concentrations, pulse frequency, and pulse amplitude) among treated and control ewes and between intensive sampling days. One-way ANOVA was used to assess differences in characteristics of FSH peaks (see Table 2 and 4) and ovarian parameters (i.e., interovulatory interval, ovulation rate, interval from implant removal to ovulation, number of follicle waves during the interovulatory interval and period of implant treatment, and interval from implant removal to the emergence of the ovulatory follicle wave) among treated and control ewes. If the main effects, or their interactions, were significant (P < 0.05), Fisher's protected least significant difference (LSD) was used as a post-ANOVA test to detect differences between individual means. Data are expressed as mean ± SEM.


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  		TABLE 2. Characteristics of endogenous FSH peaks during the implant period in cyclic Western White Face ewes treated for 10 days (Day 4 to 14 after ovulation) with silastic rubber implants (s.c.) with no additives (control) or containing 10% estradiol-17ß (small [5 x 0.34 cm] and large [10 x 0.34 cm]).*


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  		TABLE 4. Characteristics of endogenous FSH peaks in cyclic Western White Face ewes treated for 10 days (Day 4 to 14 after ovulation) with large silastic rubber implants (s.c.; 10 x 0.34 cm) containing 10% estradiol-17ß with or without an oFSH treatment on Day 9 after ovulation.*

RESULTS

Experiment 1

Mean daily serum estradiol concentrations When daily serum estradiol concentrations were normalized to the day of implant insertion and analyzed for the period of implant treatment, a treatment effect (P < 0.001) and interaction of treatment by time (P < 0.001) were observed, but no time effect was seen (P > 0.05; Fig. 1). Mean serum estradiol concentrations were significantly higher in ewes treated with large implants (10.4 ± 0.7 pg/ml) compared with control ewes (3.9 ± 0.7 pg/ml) and ewes treated with small implants (5.4 ± 0.7 pg/ml). Comparison of individual means showed that mean serum estradiol concentrations were significantly higher in ewes treated with large implants compared with the control ewes and ewes treated with small implants from 1 to 9 days after implant insertion (Fig. 1).


Figure 1
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  		FIG. 1. Mean (±SEM) daily serum estradiol concentrations during the implant period in cyclic Western White Face ewes treated for 10 days (Day 4 to 14 after ovulation) with Silastic rubber implants (s.c.) with no additives (control [black hexagons; n = 5]) or containing 10% estradiol-17ß (small [5 x 0.34 cm; light gray hexagons; n = 5] and large [10 x 0.34 cm; gray hexagons; n = 5]). Data were normalized to the day of implant insertion (Day 0) in all ewes. Asterisks (*) indicate differences between ewes with large implants and all other groups (P < 0.005)

Mean daily serum progesterone concentrations When mean daily serum progesterone concentrations were normalized to the day of implant insertion and analyzed for the period of implant treatment, a time effect (P < 0.001) was noted, but no treatment effect and interaction of treatment by time occurred (P > 0.05). Serum progesterone concentrations were highest at 6 to 8 days after implant insertion. Mean daily serum progesterone concentrations were decreasing in the control ewes and ewes treated with small implants by 8 days after implant insertion, whereas mean daily serum progesterone concentrations were decreasing in the ewes treated with large implants by 9 days after implant insertion.

Ovulations The ovulation rate, before and after implant treatment, and mean duration of the interovulatory interval did not differ among the treatment and control groups (P > 0.05; Table 1). Two ewes treated with large implants each ovulated a follicle 2 days after the implants were inserted, and one of these ovulations resulted in a CL.


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  		TABLE 1. Characteristics of follicle waves and ovulations in cyclic Western White Face ewes treated for 10 days (Day 4 to 14 after ovulation) with silastic rubber implants (s.c.) with no additives (control) or containing 10% estradiol-17ß (small [5 x 0.34 cm] and large [10 x 0.34 cm]).*

Antral follicle development The number of follicle waves emerging per animal during the period of implant treatment was lower in ewes treated with large estradiol implants compared with ewes treated with small implants and control ewes (P < 0.001; Table 1). This is illustrated in Figure 2, in which follicle-wave emergence is normalized to the average day of maximal FSH concentration of the FSH peaks preceding each follicle wave. In three of five ewes given large estradiol-releasing implants, no follicle waves emerged during the treatment period. In one ewe, a wave emerged on Day 9 of treatment, and the FSH peak associated with this wave reached its zenith about 6 h after implant removal. In one ewe, a wave emerged after the second FSH peak of the treatment period. Follicle waves emerged around the end of treatment in all ewes treated with large implants, with an average day of emergence of 11.6 ± 1.3 days from implant insertion.


Figure 2
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  		FIG. 2. Peaks in serum concentrations of FSH (outlined with shading) and their associated emerging follicle waves in cyclic Western White Face ewes treated for 10 days (Day 4 to 14 after ovulation) with Silastic rubber implants (s.c.) with no additives (control [top panel; n = 5]) or containing 10% estradiol-17ß (small [5 x 0.34 cm; middle panel; n = 5] and large [10 x 0.34 cm; bottom panel; n = 5]). Only one ewe given a large implant had a follicle wave emerge, associated with the second FSH peak during the implant period (indicated by {dagger}). Concentrations of FSH and follicle diameters are expressed as mean ± SEM. The average curves representing the growth, static, and regression phases of all follicles for all ewes in a group were normalized for each follicle wave to the day of wave emergence (indicated by the arrow). All FSH peaks for all ewes are shown normalized to the mean day of occurrence of the apex of the FSH peak for each wave. For every FSH peak, serum-concentration profiles were delimited by the encompassing nadirs in FSH concentrations (hence the overlap of data for adjacent peaks in some cases). Because of the normal variability in the length of the growth, static, and regression phases of individual follicles of a wave and FSH peak profiles, some data points at the extremities of the peaks and waves may represent an individual animal. As follicle waves were normalized to the point of emergence and because of variance in the length of the various phases of individual follicle waves, the greatest follicle diameter for each wave is not a true reflection of the greatest follicle diameter for each ewe. Data were normalized to the day of implant insertion (Day 0) in all ewes

When the numbers of follicles in the 2-, 3-, or 4-mm size classes were normalized to the day of implant insertion and analyzed for the period of implant treatment, no effects of treatment or time for 2- and 3-mm follicles were seen (P > 0.05), but only an interaction of treatment by time for the 3-mm follicles (P < 0.05). Over the last 4 days of treatment, comparison of individual means revealed fewer 3-mm follicles in ewes given large estradiol-releasing implants compared with ewes with small implants on Days 7, 8, and 10 (P < 0.05) and control ewes on Day 9 (P < 0.05). For the 4-mm follicles, the only significant trend was a time effect (P < 0.05). The treatment effect and interaction of treatment by time approached significance (P < 0.1, but >0.05). Numbers of 4-mm follicles probably reflected follicle-wave growth and regression in control ewes and ewes given small estradiol-releasing implants, but numbers of 4-mm follicles appeared to decline from Day 3 of treatment in ewes given large estradiol-releasing implants.

The interval from the end of treatment to emergence of the ovulatory wave was delayed in ewes treated with large implants compared with ewes treated with small implants and control ewes (P < 0.05; Table 1).

Characteristics of serum LH concentrations When mean and basal serum LH concentrations were analyzed for samples taken every 12 min for 6 h, no difference was found among treatments within each intensive sampling day (P > 0.05; Fig. 3). Mean and basal serum LH concentrations were higher during the second intensive sampling period (Day 14) compared with the first (Day 10) in ewes treated with small or large estradiol implants (P < 0.05; Fig. 3). LH pulse amplitude was not affected by the treatment or intensive sampling period (overall mean, 0.59 ± 0.11 ng/ml; Fig. 3). LH pulse frequency did not differ between control ewes and ewes treated with small or large implants within each intensive sampling day (P > 0.05; Fig. 3). However, during the second intensive sampling day (Day 14), the number of LH pulses was higher in ewes treated with small estradiol implants compared with ewes treated with large estradiol implants (P < 0.005; Fig. 3). The number of LH pulses was higher during the second intensive sampling period (Day 14) compared with the first (Day 10) in the control ewes and ewes treated with small estradiol implants (P < 0.05; Fig. 3). In summary, during the period of treatment, the implants had no affect on LH secretory patterns.


Figure 3
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  		FIG. 3. The characteristics of pulsatile LH secretion (mean and basal serum LH concentrations and LH pulse amplitude and frequency; mean ± SEM) determined from serum samples collected every 12 min for 6 h on Days 10 (black bars; n = 5) and 14 (gray bars; n = 4) after ovulation in cyclic Western White Face ewes treated for 10 days (Days 4 to 14 after ovulation) with Silastic rubber implants (s.c.) with no additives (control) or containing 10% estradiol-17ß (small [5 x 0.34 cm] and large [10 x 0.34 cm]). Asterisks (*) indicate differences between sampling days within treatment group (P < 0.05). Letters (a, b) indicate differences between treatment groups within sampling day (P < 0.005)

When serum LH concentrations in blood samples taken every 6 h were normalized to the peak of the preovulatory LH surge after implant treatment and analyzed for the period from 24 h before to 24 h after this peak, a time effect (P < 0.001) and interaction of treatment by time (P < 0.05) were found, but no treatment effect was present (P > 0.05). Comparison of individual means showed that serum LH concentrations were significantly higher in ewes treated with small implants (9.67 ± 1.78 ng/ml) compared with control ewes (1.51 ± 1.78 ng/ml) and ewes treated with large implants (1.93 ± 1.78 ng/ml) at 6 h before the peak in the preovulatory LH surge. Mean serum LH concentrations were significantly higher in control ewes (19.59 ± 1.78 ng/ml) compared with ewes treated with small (11.26 ± 1.78 ng/ml) and large (9.77 ± 1.78 ng/ml) implants at the time of the peak in the preovulatory LH surge.

Characteristics of serum FSH When mean daily serum FSH concentrations were normalized to the day of implant insertion and analyzed for the period of implant treatment, a time effect (P < 0.05) was found, but no treatment effect and interaction of treatment by time (P > 0.05). The mean serum FSH concentration for the period of implant treatment was 1.92 ± 0.06 ng/ml. Mean basal FSH concentrations were higher in ewes treated with small implants (1.91 ± 0.13 ng/ml) compared with ewes treated with large implants (1.29 ± 0.11 ng/ml; P < 0.005) and control ewes (1.48 ± 0.17 ng/ml; P < 0.05); however, no difference was noted between control ewes and ewes treated with large implants (P > 0.05).

During the implant treatment period, and as identified by the cycle-detection software, the number of FSH peaks, FSH peak duration, and interpeak interval did not differ among treatments (P > 0.05; Table 2). Mean FSH peak concentration was lower in ewes treated with large implants compared with control ewes and ewes treated with small implants (P < 0.05); however, no difference was seen between ewes treated with small implants compared with control ewes (P > 0.05; Table 2). A tendency was noted for a reduced mean FSH peak amplitude in ewes treated with large implants compared with control ewes and ewes treated with small implants (P < 0.07; Table 2). A 31% and 45% reduction occurred in mean FSH peak concentration and FSH peak amplitude, respectively, compared with control ewes. The reduction in FSH peak concentration and amplitude, during the treatment period in ewes given large estradiol releasing implants, is illustrated in Figure 2. In Figure 2, the pretreatment FSH peak and the first two peaks during implant treatment are shown, normalized to the average day of the apex of each peak. A third FSH peak was seen only during the treatment period in eight of the 15 ewes; only two of the ewes were in the large estradiol-releasing implant group. We therefore did not attempt to add the third FSH peak of the treatment period to Figure 2.

When serum FSH concentrations in blood samples taken every 6 h were normalized to the peak of the preovulatory LH surge after implant treatment and analyzed from 24 h before to 24 h after this peak, a time effect (P < 0.001) was found, but no treatment effect or interaction of treatment by time was seen (P > 0.05). The peak in mean serum FSH concentrations occurred at the time of the peak of the preovulatory LH surge.

Experiment 2

Mean daily serum estradiol concentrations When mean daily serum estradiol concentrations were normalized to the day of implant insertion and analyzed for the period of implant treatment, a time effect was seen (P < 0.001), but no treatment effect and interaction of treatment by time (P > 0.05). For the ewes treated with estradiol only, serum estradiol concentrations increased from Day 0 to 1 after implant insertion, decreased from Day 1 to 4 after implant insertion, and then remained relatively constant until the end of the implant period. For the ewes given estradiol and oFSH, serum estradiol concentrations increased from Day 0 to 1 after implant insertion and then remained relatively constant until the end of the implant period. The mean serum estradiol concentration for the period of implant treatment was 9.4 ± 0.4 pg/ml.

Mean daily serum progesterone concentrations When mean daily serum progesterone concentrations were normalized to the day of implant insertion and analyzed for the period of implant treatment, a time effect was noted (P < 0.001), but no treatment effect and interaction of treatment by time (P > 0.05). Serum progesterone concentrations were at a maximum at 5 days after implant insertion. Mean daily serum progesterone concentrations were decreasing in all groups by 8 days after implant insertion.

Ovulations The ovulation rate, before and after implant treatment, mean duration of the interovulatory interval, and the interval from implant removal to ovulation did not differ between treatment groups (P > 0.05; Table 3). The ovulation rate before the implant treatment was higher than after the implant treatment in all ewes (P < 0.001; Table 3). One animal treated with estradiol ovulated a follicle 1 day after the implant was inserted and formed a CL.


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  		TABLE 3. Characteristics of follicle waves and ovulations in cyclic Western White Face ewes treated for 10 days (Day 4 to 14 after ovulation) with large silastic rubber implants (s.c.; 10 x 0.34 cm) containing 10% estradiol-17ß with or without an oFSH treatment on Day 9 after ovulation.*

Antral follicle development The number of follicle waves per animal during the period of implant treatment was higher in ewes given estradiol implants and oFSH compared with ewes treated only with estradiol implants (P < 0.005; Table 3). When FSH was injected, a new follicle wave emerged 1.5 ± 0.5 days (range, 1–4 days) after injection; however, in ewes treated with estradiol implants alone, a follicle wave did not emerge until 4.8 ± 0.8 days after injection of saline or around the time of implant removal (P < 0.01). In ewes given estradiol implants and oFSH, only one of six ewes had a follicle wave emerge during the treatment period and before the administration of oFSH; this wave emerged on Day 4 after insertion of implants. Follicle waves emerged around the end of the implant treatment period in all ewes given estradiol implants and oFSH, with an average day of emergence of 9.3 ± 0.3 days from implant insertion. In the implant treatment period, in ewes given estradiol implants only, follicle waves only emerged around the end of the implant treatment period. The average day of emergence for these follicles was 10.4 ± 0.6 days from implant insertion. The interval from implant removal to ovulatory wave emergence did not differ between treatment groups (P > 0.05; Table 3).

When daily maximal follicle diameter was normalized to the day of implant insertion and analyzed for the period of implant treatment, no treatment effect was noted (P > 0.05), but a time effect (P < 0.001) and an interaction of treatment by time were present (P < 0.005; Figure 4). In the ewes treated with estradiol only, maximal follicle diameter was greatest on 0 and 1 day after implant insertion, declined from 1 day to 6 days after implant insertion, and then remained low to the end of the implant period. Maximal follicle diameter was significantly larger in ewes given estradiol and oFSH compared with the ewes treated with estradiol alone from 7 to 9 days after implant insertion (Fig. 4), confirming the induction of emergence of a follicular wave by oFSH administration.


Figure 4
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  		FIG. 4. Mean (±SEM) daily maximal follicle diameter during the implant period in cyclic Western White Face ewes treated for 10 days (Days 4 to 14 after ovulation) with large Silastic rubber implants (s.c.; 10 x 0.34 cm) containing 10% estradiol-17ß, with (open circles; n = 6) or without (solid circles; n = 6) an oFSH treatment on Day 9 after ovulation. Data were normalized to the day of implant insertion (Day 0) in all ewes. Asterisks (*) indicate differences between groups (P < 0.05)

Characteristics of serum FSH The mean peak concentration and amplitude for endogenous FSH peaks were reduced during the implant treatment period compared with the period before implant treatment (P < 0.05; Table 4). A 25% and 46% reduction in mean FSH peak concentration and FSH peak amplitude was found, respectively.

When mean daily serum FSH concentrations were normalized to the day of implant insertion and analyzed for the period of implant treatment, a tendency was noted for a treatment effect (P = 0.05), but a time effect and interaction of treatment by time were present (P < 0.001; Fig. 5). Serum FSH concentrations were highest at 5.5 days after implant insertion in ewes given estradiol and oFSH. Comparison of individual means showed that serum FSH concentrations were significantly higher in ewes given estradiol and oFSH, compared with the ewes treated with estradiol, from 5.25 to 6.5 days after implant insertion (Fig. 5).


Figure 5
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  		FIG. 5. Mean (±SEM) daily serum FSH concentrations during the implant period in cyclic Western White Face ewes treated for 10 days (Days 4 to 14 after ovulation) with large Silastic rubber implants (s.c.; 10 x 0.34 cm) containing 10% estradiol-17ß, with (open circles; n = 6) or without (solid circles; n = 6) an oFSH treatment on Day 9 after ovulation. Data were normalized to the day of implant insertion (Day 0) in all ewes. Asterisks (*) indicate differences between groups (P < 0.05)

Mean basal FSH concentrations appeared to be unaffected by the implant treatment (1.73 ± 0.07 ng/ml; P > 0.05).

DISCUSSION

In the first study, the animals treated with large and small estradiol implants had mean serum estradiol concentrations that were 2.7- and 1.4-fold higher than the control ewes. The large implants used in the second study produced serum estradiol concentrations that were 2.4-fold higher than the control ewes of the first study.

In Experiment 1, the estradiol implants had no effect on the number of FSH peaks, peak duration, and interpeak interval; in both studies, peaks were objectively identified by cycle-detection software. However, in Experiments 1 and 2, the large implants clearly truncated the FSH peaks; both peak concentration and amplitude were reduced. In previous studies on the negative-feedback regulation of FSH secretion by estradiol, only mean serum FSH concentrations were measured [27, 42, 47]. It is intriguing that in the present study, supraphysiologic concentrations of estradiol suppressed the amplitude of the transient peaks in serum FSH concentrations that precede ovarian follicular waves in sheep without significantly affecting basal or mean serum FSH concentrations. Bartlewski et al. [7] showed that Finn ewes have significantly higher mean and peak serum concentrations of estradiol compared with WWF ewes throughout the estrous cycle. However, the majority of FSH peak characteristics examined in that study did not differ between breeds, with the exception that the FSH peak concentration of the first peak of the cycle was higher in Finn ewes compared with WWF ewes. However, in the current study, when serum estradiol concentrations were increased in WWF ewes to those seen in Finn ewes, FSH peaks were truncated. In Experiment 2 in the present study, similar to our previous results [9], treatment with exogenous oFSH created a physiologic FSH peak in every animal. The model developed in the present study, in which only the amplitude and peak concentration of peaks in serum FSH concentrations were altered through the use of large estradiol implants, without an effect on basal serum FSH concentrations, will be useful for studying the effect of altering the characteristics of FSH peaks, created by injection of oFSH, on ovarian follicular wave emergence.

Cyclic ewes treated daily from Days 3 to 21 after ovulation with injections of estradiol (0.5 to 2.5 mg/d) had suppressed follicle development [48, 49]. Concentrations of FSH were not measured in these two studies. FSH is required for follicles to develop to an ovulatory size [1, 3–5] and, at higher than physiologic FSH concentrations, both the length of FSH exposure and the FSH concentration determine the number of follicles that grow [5]. However, in the studies above, only gross follicle numbers and sizes were recorded, based on postmortem or one-time observations. In the present study, one of the most profound effects of the large estradiol implant treatment was on follicle development. In both experiments, the high estradiol concentrations created by the large implants significantly blocked follicular wave emergence. Because the FSH peaks were truncated, follicles present in the 2- to 3-mm follicle pool failed to receive the appropriate FSH signal to stimulate their entry into a follicle wave. In other words, a threshold FSH concentration exists that a FSH peak must reach or exceed to induce the emergence of a follicle wave. However, it is interesting that the large implants had no effect on the number of follicles in the small follicle pool (2–3 mm in diameter). In the present study, when a physiologic FSH peak was created in ewes with suppressed follicle-wave emergence, a new follicle wave emerged 1.5 days after injection. When a physiologic FSH peak is created during the interwave interval in normal anestrous ewes, a follicle wave emerges within ~24 h after injection [9]. In the present study, by using ultrasonography, we were able to manipulate the endogenous pattern of FSH peaks to show that FSH peak concentration and amplitude are critical elements in stimulating antral follicle development into a follicular wave. In summary, the large estradiol implants truncated FSH peaks to a point at which follicle-wave emergence was completely suppressed, without affecting the small follicle pool, and it was only through the injection of a physiologic dose of oFSH that follicle-wave emergence could be restored. It would appear that periodic peaks in serum concentrations of FSH are not required to maintain the pool of small follicles that can respond to a peak in FSH to produce a follicular wave; basal serum concentrations of FSH would appear sufficient for maintaining the growth of small antral follicles.

Another interesting observation from these studies is that FSH peaks continued to occur in the absence of follicle-wave development. These peaks were of reduced concentration, but nevertheless identified by the objective cycle-detection software. As can be seen in Figure 2, at least two successive truncated FSH peaks occurred during the treatment period of ewes from Experiment 1 given large steroid-releasing implants. These truncated FSH peaks occurred at the expected interpeak interval. None of these peaks was followed by emergence of a follicular wave, except in one ewe, in which a wave followed the second truncated peak. The amplitude of that second peak was in the range of the second peak for the other ewes in that group. It is feasible that the first truncated peak of the treatment period could have been signaled or induced by some altered secretory feedback product from the previous follicle wave. However, no follicular wave heralded the second truncated peak of the treatment period.

It has been shown that in the ewe, follicle waves can be induced by injection of oFSH to create physiologic peaks. Such treatment is effective if given at any phase of the previous follicular wave [9, 30]. In addition, such induced follicle waves have the expected normal growth and life-span characteristics as well as estradiol production [9, 30]. Induction of follicle waves does not disrupt the normal train of endogenously induced waves, and endogenous wave emergence, with the concurrent FSH peak, can occur at any phase of the exogenously induced follicular wave [9, 30].

It appears that the FSH peaks may be entrained independent of the growth and regression of previously developing follicular waves in the ewe. If secretory products from follicular waves were important for entraining the FSH peaks, then obviously, based on the present results, estradiol could not be a candidate. The other most obvious candidate would be inhibin [50]. Both inhibin and estradiol do suppress FSH secretion in the ewe, but the effects of inhibin on FSH peaks have not been studied directly. Most inhibin is produced from the larger follicles of a wave (88% by follicles ≥4 mm in diameter) (26, 51); however, up to 12% could be produced by follicles <4 mm in diameter [26, 51]. Inhibin secretion does increase as the follicles of a wave grow during the breeding season [6, 52], but not during anestrus [53]. The regular follicular waves and antecedent FSH peaks must occur independent of any influence of inhibin in anestrous ewes [53]. With no follicular waves emerging during the treatment period in the present study, in ewes given the large estradiol-releasing implants, it is difficult to see how changes in inhibin secretion, or any other secretory products of the follicles of a wave, could have entrained the second truncated FSH peak. The present and previous findings do beg the question as to whether some mechanisms other than ovarian follicular feedback govern the timing of the FSH peaks preceding follicle-wave emergence in the ewe. It is interesting that ovariectomized ewes appear to maintain the generation of FSH peaks with a periodicity similar to that of the intact ewe [30].

In both of the present studies, ovulatory wave emergence was delayed in ewes treated with large implants. This is probably a consequence of the lack of an adequate FSH signal for follicle-wave emergence until removal of the implants. In both experiments, no difference was found in the ovulation rate between treatments, but the ovulation rate was lower after implant removal as compared with before implant insertion in Experiment 2. However, the ovulation rates were all in the range seen for the WWF ewe. Cyclic ewes treated from Days 3 to 21 with daily injections of very high estradiol concentrations fail to ovulate [48]. Cyclic ewes treated with estradiol implants have lower ovulation rates [54]. However, Finn ewes have a higher ovulation rate with higher circulating estradiol concentrations compared with WWF ewes [7]. Atkinson et al. [47] found that the ovulation rate of cyclic ewes is unaffected by implants releasing estradiol (0.57 µg/day for 19 days and 1.90 µg/day for 2, 4, and 6 days). In the present studies, the large implants delayed ovulatory wave emergence without having a significant affect on ovulation rate.

During the implant treatment period of the present experiments, the first and second periods of intensive blood sampling were conducted during the mid-luteal and follicular phases, respectively. In normal cycling ewes, mean and basal LH concentrations and LH pulse frequency during the mid-luteal phase should be quite low compared with those in the follicular phase [17]. Regardless of treatment group, most of these trends in the characteristics of LH concentrations were observed in this present study. During the follicular phase, the LH pulse frequency was higher in ewes treated with small implants and control ewes compared with ewes treated with large implants. This difference may have arisen because the pulse frequency of LH was measured after 3 days of declining progesterone concentrations in the control ewes and ewes treated with small implants, whereas LH pulse frequency was measured after 2 days of declining progesterone concentrations in ewes treated with large implants. Overall, during the period of implant treatment, the implant treatments had no effect on LH secretory patterns. This is an important feature of this model because we affected only the endogenous FSH pattern and not the endogenous LH secretory pattern.

In summary, we created an experimental model in which only the amplitude and peak concentration of the periodic peaks in mean serum FSH concentrations that precede follicular waves were altered by using large estradiol implants; pulsed LH secretion was not affected. The large estradiol implants had little effect on mean and basal serum FSH concentrations. Supraphysiologic concentrations of estradiol-17ß suppressed follicle-wave development by truncating FSH peaks in cyclic ewes; in addition, injection of physiologic concentrations of oFSH reinitiated follicle emergence. This will be a useful model with which to study the characteristics of FSH peaks required to induce follicle waves in the ewe. Generally, the supraphysiologic concentrations of estradiol (2.7-fold higher than control ewes) did not change the small follicle pool (2–3 mm in diameter). Because the FSH peaks were truncated, follicles present in the 2- to 3-mm follicle pool failed to receive the appropriate FSH signal to stimulate their entry into a follicle wave. Maintenance of the small follicle pool would not appear to require regular secretion of FSH peaks. In this study, we clearly showed that the FSH peaks that precede follicular waves in the ewe are critical for the genesis of these waves. A threshold for the amplitude of these peaks exists to stimulate emergence of a wave. The data presented also bring into question the involvement of secretory products from the follicles of a wave in entraining the FSH peak that initiates the subsequent follicular wave.

ACKNOWLEDGMENTS

The authors thank Dr. A. F. Parlow of NIDDK/NHPP for the provision of reagents for the gonadotropin assays; Ms. Susan J. Cook for her help in blood sampling and radioimmunoassay; and Dr. Edward T. Bagu for his help in blood sampling.

FOOTNOTES

1 Supported by the Natural Sciences and Engineering Research Council, Canada (N.C.R.). D.M.W.B., P.M.B., R.D., and K.L.D. were recipients of University of Saskatchewan Graduate Student Scholarships. Back

2 Correspondence: FAX: 306 966 7376; norman.rawlings{at}usask.ca Back

3 Current address: Department of Biomedical Sciences, Ontario Veterinary College, University of Guelph, Guelph, ON, Canada N1G 2W1. Back

4 Current address: Institut de Génétique et de Biologie Moléculaire et Cellulaire, BP 10142, 67404 Illkirch CEDEX, France. Back

5 Current address: Department of Animal and Poultry Science, College of Agriculture, University of Saskatchewan, Saskatoon, SK, Canada S7N 5A8. Back

Received: 21 October 2005.

First decision: 1 December 2005.

Accepted: 12 July 2006.

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