- Email: [email protected]
Pulsatile LH secretion and ovarian follicular wave emergence and growth in anestrous ewes
Available online at www.sciencedirect.com
Theriogenology 74 (2010) 912–921 www.theriojournal.com
Pulsatile LH secretion and ovarian follicular wave emergence and growth in anestrous ewes Srinivas V. Seekallua,b, David M.W. Barretta,c, Behzad M. Toosia, Kelsey Clarkea, Kirk A. Ewena, Rajesha Duggavathia,d, Kate L. Daviesa,e, Kim M. Pattulloa, Edward T. Bagua,f, Norman C. Rawlingsa,* a
Department of Veterinary Biomedical Sciences, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, SK, Canada S7N 5B4 b Manitoba Institute of Cell Biology, CancerCare Manitoba, 675 McDermot Avenue, Winnipeg, MB, Canada R3E 0V9 c Department of Plant and Animal Sciences, Haley Institute, Nova Scotia Agricultural College, Truro, NS, Canada, B2N 5E3 d Department of Animal Science, McGill University, 21, 111 Lakeshore Road, Ste-Anne-de-Bellevue, Québec, Canada, H9X 3V9 e Department of Animal and Poultry Science, College of Agriculture, University of Saskatchewan, Saskatoon, SK, Canada, S7N 5A8 f Centre de Recherche-CHUM Hospital Notre-Dame, Y-5625-2099 Rue Alexandre de Séve, Montreal, Quebec, Canada, H2L 2W5 Received 15 September 2009; received in revised form 8 April 2010; accepted 11 April 2010
Abstract The objective of this study was to determine if pulsatile LH secretion was needed for ovarian follicular wave emergence and growth in the anestrous ewe. In Experiment 1, ewes were either large or small (10 ⫻ 0.47 or 5 ⫻ 0.47 cm, respectively; n ⫽ 5/group) sc implants releasing estradiol-17 beta for 10 d (Day 0 ⫽ day of implant insertion), to suppress pulsed LH secretion, but not FSH secretion. Five sham-operated control ewes received no implants. In Experiment 2, 12 ewes received large estradiol-releasing implants for 12 d (Day 0 ⫽ day of implant insertion); six were given GnRH (200 ng IV) every 4 h for the last 6 d that the implants were in place (to reinitiate pulsed LH secretion) whereas six Control ewes were given saline. Ovarian ultrasonography and blood sampling were done daily; blood samples were also taken every 12 min for 6 h on Days 5 and 9, and on Days 6 and 12 of the treatment period in Experiments 1 and 2, respectively. Treatment with estradiol blocked pulsatile LH secretion (P ⬍ 0.001). In Experiment 1, implant treatment halted follicular wave emergence between Days 2 and 10. In Experiment 2, follicular waves were suppressed during treatment with estradiol, but resumed following GnRH treatment. In both experiments, the range of peaks in serum FSH concentrations that preceded and triggered follicular wave emergence was almost the same as control ewes and those given estradiol implants alone or with GnRH; mean concentrations did not differ (P ⬍ 0.05). We concluded that some level of pulsatile LH secretion was required for the emergence of follicular waves that were triggered by peaks in serum FSH concentrations in the anestrous ewe. © 2010 Elsevier Inc. All rights reserved. Keywords: Anestrous; Estradiol; FSH; Ovarian follicular waves; Sheep
1. Introduction With the recent use of transrectal ultrasonography for imaging ovaries in both cyclic and anestrous ewes, * Corresponding author. Tel.: 306-966-7068; fax: 306-966-7314. E-mail address: [email protected] (N.C. Rawlings). 0093-691X/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2010.04.016
it was found that 1–3 ovarian antral follicles emerged or grew from a pool of small follicles, 1–3 mm in diameter, every 4 –5 d [1– 4]. These follicles grow to ⱖ5 mm in diameter before regression or ovulation [1– 4]. Each wave of follicular growth was preceded by a transient peak of FSH secretion that lasted 3 or 4 d and was characterized by blood samples taken daily [3,5,6].
S.V. Seekallu et al. / Theriogenology 74 (2010) 912–921
This peak in FSH secretion was the essential trigger for the follicular wave [3,5,6]; however, the maximum concentration that peaks achieved and the nadirs in serum FSH concentrations between peaks, varied considerably among follicular waves [3,4,7]. When cyclic ewes were treated for 10 d with large estradiol-17 releasing implants (10 ⫻ 0.47 cm), that created supraphysiological serum concentrations of estradiol-17 (10.4 ⫾ 0.7 pg/mL vs. 3.9 ⫾ 0.7 pg/mL for treated vs. control ewes, respectively), the amplitude of the FSH peaks that preceded follicular waves was reduced and follicular wave emergence was blocked [7]. Injection of ovine FSH (oFSH), to recreate FSH peaks, re-initiated follicular waves [7]. In that study, pulsed LH secretion was not affected. Pulses of LH were released at a frequency of ⬃1 every 1– 6 h in the ewe and lasted no more than ⬃2 h [8,9]. Although peaks in FSH secretion initiated follicular waves, the role of pulsed LH secretion in the emergence, growth and regression of ovine antral follicles was unclear. Changes in LH pulse frequency during the estrous cycle did not appear to be correlated with or functionally related to specific phases of the growth or regression of follicular waves in the ewe [8,9]. Profiles of FSH in blood samples collected from the jugular vein in ewes were not pulsatile [10,11]. Compared to ewes during the breeding season, seasonally anestrous ewes had lower circulating concentrations of LH, FSH, and estradiol, little or no progesterone, and serum concentrations of estradiol and inhibin which were not correlated with follicular wave development [8,12–15]. The frequency of LH secretory pulses was very low and fluctuated very little across seasonal anestrus [13–17], with no apparent association to stages of follicular wave development and regression. During seasonal anestrus, estradiol exerted a more powerful negative feedback effect on pulsatile LH secretion than during the breeding season, but the effects on FSH secretion appeared to be minimal [18 –20]. We hypothesized that although the frequency of pulses of LH secretion were very low in anestrous ewes, they were still essential for emergence and growth of ovarian follicular waves. The objective of the present study was to examine the need for pulsed LH secretion for the emergence and growth of ovarian follicular waves in anestrous ewes. Two experiments were conducted. The purpose of the first experiment was to determine if treatment of anestrous ewes with large estradiol releasing implants (10 ⫻ 0.47 cm) that created supraphysiological concentrations of estradiol [7] would suppress the pulsed secretion of LH, but leave FSH peaks
913
with a concentration at their zenith similar to control ewes and in a range that could still initiate a follicular wave. The purpose of the second experiment was to replace LH pulses with frequent GnRH injections, in the experimental model above, to see if restored LH pulsatility would in fact allow restoration of follicular waves. This would confirm that, although peaks in FSH secretion initiated follicular waves in the anestrous ewe, some level of pulsed LH secretion was also required. 2. Materials and methods 2.1. Animals Care and handling of experimental animals was done according to the Canadian Council on Animal Care’s published guidelines. Sexually mature, clinically healthy, seasonally anestrous, 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. 2.2. Ultrasound technique The growth and regression of ovarian antral follicles were monitored in all ewes by transrectal ovarian ultrasonography (scanning), 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 [1,21,22]. All images were viewed at a magnification of ⫻ 1.5 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, Fujifilm, Tokyo, Japan) for later examination. The relative position and dimensions of follicles and luteal structures were also sketched on ovarian charts. 2.3. Experimental design 2.3.1. Experiment 1 Fifteen seasonally anestrous (June) WWF ewes (mean body weight of 76.9 ⫾ 2.9 kg) were allocated into three groups of five ewes each. The experimental design is summarized in Figure 1. Five ewes received large subcutaneous silastic rubber implants (10 ⫻ 0.47 cm) and five ewes received small subcutaneous silastic rubber implants (5 ⫻ 0.47 cm) containing 10% estradiol17 w/w (Sigma Chemical Company, St. Louis, MO, USA [7]. Five sham operated control ewes received no
914
S.V. Seekallu et al. / Theriogenology 74 (2010) 912–921
Fig. 1. Schematic representation of the experimental design of the two studies conducted in ewes during the anestrous season. Estradiol implants were inserted subcutaneously on Day 0 and left in place for 10 or 12 d in Experiments 1 and 2, respectively. In Experiment 1, ewes received 5 cm (n ⫽ 5) or 10 cm (n ⫽ 5) implants; control ewes (n ⫽ 5) underwent sham surgery only. In Experiment 2, all ewes received 10 cm implants. Injections of GnRH (200 ng IV; 6 ewes) or saline (IV; 6 ewes) were given every 4 h from Days 6 to 12 after implant insertion in Experiment 2. Daily ultrasonography and blood sampling were done from 4 or 5 d before to 20 or 22 d after implant insertion in Experiment 1 and 2, respectively. Blood samples were also taken every 12 min for 6 h (intensive bleeds) on Days 5 and 9 in Experiment 1, and on Days 6 and 12 in Experiment 2 to characterize the secretory patterns of LH and FSH. In Experiment 2, intensive bleeds started 36 min prior to the first and penultimate injection of GnRH or saline, and ended 2 h after the second and last injections respectively.
implants. All implants were removed 10 d after insertion (Day 0 ⫽ day of insertion). Mean serum estradiol concentrations for the implant treatment period, were 3.69 ⫾ 0.4, 9.82 ⫾ 1.1, and 11.84 ⫾ 1.0 pg/mL for control ewes, and those given small or large implants, respectively. Daily scanning and blood sampling were done from 4 d before to 20 d after implant insertion. Blood samples were collected in evacuated tubes (10 mL; Becton Dickinson, Franklin Lakes, NJ, USA) by jugular venipuncture, starting at 0800 h, and followed immediately by scanning. Blood samples (5 mL) were also collected every 12 min for 6 h (intensive sampling) on Day 5 and 9 starting at 0800 h to determine the secretory patterns of LH and FSH at the middle and end of implant treatments. Intensive sampling was via catheter (vinyl tubing, 1.0 mm id ⫻ 1.5 mm od; 530070 Biocorp Australia Pty Ltd., Auburn, NSW, Australia) inserted into the jugular vein. 2.3.2. Experiment 2 Twelve seasonally anestrous (May/June) WWF ewes (mean body weights of 83.5 ⫾ 3.0 kg) received large subcutaneous silastic rubber implants (10 ⫻ 0.47 cm) containing 10% estradiol-17 w/w for 12 d (Day 0 ⫽ day of implant insertion; Fig. 1). Six of the twelve were given GnRH (200 ng IV in saline; Sigma Chemical Com-
pany) every 4 h for the last 6 d that the implants were in place, starting at 0800 h on Day 6 (Fig. 1). The dose of GnRH was designed to create LH pulses within a physiological range. Six control ewes received only saline (IV). Mean serum estradiol concentrations for the implant treatment period were 12.08 ⫾ 0.7 and 10.81 ⫾ 0.7 pg/mL in control ewes (ewes treated with only estradiol implants) and ewes treated with GnRH, respectively. Daily scanning and blood sampling were done from 5 d before to 22 d after implant insertion, as detailed for Experiment 1. Blood samples also were collected every 12 min, by a catheter inserted in the jugular vein, starting 36 min before the first and before the penultimate GnRH/ saline injections, and ending 2 h after the second and after the last GnRH/ saline injections, respectively, in order to characterize the secretory patterns of LH and FSH and their responses to GnRH at the onset and end of the period of treatment with GnRH. 2.4. Analysis of follicular data An ovarian follicular wave consisted of an ovarian follicle or a group of follicles that emerged and grew from 2 or 3 mm in diameter to ⱖ5 mm (growth phase)
S.V. Seekallu et al. / Theriogenology 74 (2010) 912–921
before regressing to 2 or 3 mm in diameter (regression phase); time spent at ⱖ5 mm was regarded as the static phase [4]. Emergence into a wave was restricted to a period of 24 h [22]. Data were analyzed for the number of follicular waves in each group of ewes. The number of small follicles (ⱖ1 to ⱕ3 mm in diameter) each day of the study also was analyzed. Ovulation was detected with ultrasonography as the collapse of a large follicle (ⱖ5 mm in diameter) that had been followed in its growth/ static phase for several days. Follicular data were combined for both ovaries of each ewe. 2.5. Hormone analysis All blood samples were permitted to clot at room temperature for 18 –24 h. Samples were then centrifuged for 10 min at 1500 ⫻ g, and serum was removed and kept at ⫺20 oC until assayed. Serum concentrations of progesterone [23], estradiol [20,24], LH, and FSH [25] were measured by validated radioimmunoassays (RIA). The assay sensitivities (defined as the lowest concentration of a hormone capable of significantly displacing radio-labeled hormone from the antibody) were: 0.03 ng/mL for progesterone, 1.0 pg/mL for estradiol and 0.1 ng/mL for FSH and LH. The ranges of standards were: 0.1–10 ng/mL, 1.0 –100 pg/mL, 0.12–16.0 ng/mL, and 0.06 – 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, throughout the experimental period, were analyzed for concentrations of progesterone, estradiol, and FSH. All serum samples collected every 12 min were analyzed for concentrations of LH and FSH. For Experiment 1, the intra- and inter-assay coefficients of variation (CVs) were 9.8 and 15.2%, or 6.7 and 14.2% for reference sera with mean progesterone concentrations of 0.24 or 1.14 ng/mL, respectively. The intraand inter-assay coefficients of variation were 9.5 and 10.5%, or 7.8 and 16.2% for reference sera with mean estradiol concentrations of 3.6 or 31.0 pg/mL, respectively. The intra-assay CVs were 5.4 or 8.4% for reference sera with mean FSH concentrations of 1.10 or 5.71 ng/mL, respectively. The intra- and inter-assay CVs were 10.0 and 10.3%, or 8.5 and 9.0% for reference sera with mean LH concentrations of 0.11 or 2.62 ng/mL, respectively. For Experiment 2, the intra- and inter-assay coefficients of variation (CVs) were 15.9 and 6.5%, or 7.2 and 12.8% for reference sera with mean progesterone concentrations of 0.21 or 1.13 ng/mL, respectively. The intra- and inter-assay coefficients of variation were 7.4
915
and 9.0%, or 5.5 and 6.8% for reference sera with mean estradiol concentrations of 7.97 or 25.17 pg/mL, respectively. The intra-assay CVs were 4.2 or 4.2% for reference sera with mean FSH concentrations of 1.12 or 3.34 ng/ mL, respectively. The intra- and inter-assay CVs were 4.7 and 5.4%, or 9.6 and 10.0% for reference sera with mean LH concentrations of 0.4 or 2.97 ng/mL, respectively. The PC-PULSAR program [26] was used to assess mean serum LH and FSH concentrations as well as LH and FSH pulse frequency and amplitude in blood samples collected every 12 min for 6 h. Standard deviation criteria (G and Baxter parameters) were used for pulse detection. Peaks of FSH in blood samples taken daily were identified using cycle-detection software [27]. 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; [27]). Follicle stimulating hormone peak concentration was defined as the concentration of FSH observed at the apex of the FSH peak. 2.6. Statistical analyses Observations on hormone concentrations and ovarian follicles measured daily were normalized to the day of implant insertion for analysis and presentation. Twoway repeated measures ANOVA (Sigma Stat 7 for Windows Version 2.03, 1997, SPSS Inc., Chicago, IL, USA) was used to assess differences in hormone concentrations and observations on ovarian follicles over time and among groups of ewes (i.e., treated with large, small, or no implants in Experiment 1, and with or without exogenous GnRH injection in Experiment 2). Two-way repeated measures ANOVA was used to assess differences in LH and FSH secretory characteristics from blood samples collected every 12 min for 6 h (i.e., mean concentrations, pulse frequency, and pulse amplitude) among groups of ewes and between intensive sampling days. If the main effects, or their interactions, were significant (P ⬍ 0.05), Fisher’s protected least significant difference (LSD) was used as a postANOVA test to detect differences between individual means (P ⬍ 0.05). The Chi-square test was used to analyze for the proportion of missing FSH peaks. Data were expressed as mean ⫾ SEM. 3. Results 3.1. Experiment 1 3.1.1. Serum hormone concentrations Pulses of LH were blocked in ewes treated with implants, but control ewes had mean LH pulse frequency
916
S.V. Seekallu et al. / Theriogenology 74 (2010) 912–921
of 1.5 ⫾ 0.2 per 6 h. Profiles of FSH were not pulsatile, and mean serum concentrations of FSH (1.43 ⫾ 0.2 ng/ mL) did not differ with treatment or day. However, in ewes treated with large estradiol implants, mean serum FSH concentrations were lower (P ⬍ 0.05) during intensive sampling on Day 9 (1.00 ⫾ 0.1 ng/mL) compared to Day 5 (1.38 ⫾ 0.2 ng/mL). Apart from a high peak serum FSH concentration on Day 1 in ewes treated with small implants (P ⬍ 0.05; Fig. 2B), neither treatment nor day affected FSH peak concentrations (Fig. 2) in daily samples. Whereas four of five ewes given small implants and all control ewes had an increase in FSH concentrations during the fourth FSH peak, only two of five ewes treated with high estradiol had an increase (P ⬍ 0.05; Fig. 2C). In ewes given large or small estradiol implants, peak serum FSH concentrations during treatment and when follicular waves were blocked, had a range of 0.9 –2.6 ng/mL (mean, 1.7 ⫾ 0.18 ng/mL). In control ewes, FSH peaks occurring at a similar time and that were accompanied by wave emergence, covered a range of 0.9 –3.3 ng/mL (mean, 2.0 ⫾ 0.46 ng/mL). 3.1.2. Antral follicle development No ewe given large estradiol implants, and only two of five ewes given small estradiol implants, had follicular waves emergence during treatment (emergence on Day 1). Follicular waves emerged after the end of treatment in all ewes given implants, with an average day of emergence of 13.9 ⫾ 0.5 d from implant insertion. Treatment did not affect the number of small follicles (16.40 ⫾ 2.5; P ⬎ 0.05). 3.2. Experiment 2 3.2.1. Serum hormone concentrations Each GnRH injection led to a pulse in LH secretion, whereas LH pulses were absent in ewes after implant insertion alone. Mean serum LH concentrations did not differ between Days 6 and 12 in ewes treated with GnRH (0.24 ⫾ 0.1 ng/mL). There was no difference in mean serum FSH concentrations amongst days of intensive blood sampling (Day 6 or 12), treatment or its interaction with day (mean, 1.26 ⫾ 0.2 ng/ml). Within days of intensive sampling, there was a time effect and an interaction of treatment by time (P ⬍ 0.05): serum FSH concentrations were greater in ewes given GnRH compared to control ewes from 12– 60 min (1.40 ⫾ 0.2 vs. 1.10 ⫾ 0.1 ng/mL) after the first GnRH injection, and from 24 –36 min (1.60 ⫾ 0.1 vs. 1.20 ⫾ 0.1 ng/mL) after the second GnRH injection on Day 12 (P ⬍ 0.05). Mean peak concentrations did not differ between control ewes and ewes treated with GnRH, but peak serum
Fig. 2. Peaks in serum concentrations of FSH (outlined with shading) and their associated emerging follicle waves in anestrous Western White Face ewes treated for 10 d (Days 0 –10; open rectangle on X-axis) with no sc implants (control; Panel A) or small (Panel B) or large (Panel C) silastic rubber implants containing 10% estradiol17. Data were normalized to the day of implant insertion (Day 0) in all ewes. The average curves representing the growth, static, and regression phases of follicular waves for all ewes in a group were normalized for each follicle wave to the mean day of wave emergence occurring either before or after implant insertion (Day 0; arrows indicates days of wave emergence; [7]). All FSH peaks for all ewes in a group are shown normalized to the mean day of occurrence of the apex of the FSH peak for each wave. The day of the apex of FSH peak was then plotted according to the mean day it occurred before or after implant insertion. Wave emergence may occur on the day of the zenith of the FSH peak or 1 d before or after the zenith [3]. For every FSH peak, serum concentration profiles were delimited by the encompassing nadirs of the FSH concentrations (hence the overlap of the data for adjacent peaks in some cases). Numbers in parenthesis are the number of ewes in a group with an FSH peak at that time. In ewes given small implants, 2 of 5 ewes had a follicular wave emergence on Day 1 from implant insertion (Day 0; indicated by †). Concentrations of FSH and follicle diameters are expressed as mean ⫾ SEM.
S.V. Seekallu et al. / Theriogenology 74 (2010) 912–921
FSH concentration on Day 10 was less than on Day 4 of the treatment in ewes given only estradiol implants (P ⬍ 0.05; Fig. 3). One control ewe and three ewes
917
given GnRH did not have a detectable increase in FSH at the time of the 2nd FSH peak seen for other ewes in these groups, and two control ewes and one ewe given GnRH did not have a detectable increase in FSH at the time of the 4th FSH peak detected for other ewes in these groups (Fig. 3).The proportion of ewes having peaks did not differ between groups (P ⬎ 0.05). In ewes given only estradiol implants, peak serum concentrations during treatment and during the time when follicular waves were blocked, had a range of 0.7–2.1 ng/mL (mean of 1.3 ⫾ 0.17 ng/mL). In ewes given GnRH, FSH peaks occurring at a similar time and that were accompanied by wave emergence, covered a range of 0.8 –2.4 ng/mL (mean of 1.5 ⫾ 0.17 ng/mL). 3.2.2. Antral follicle development No ewe given only estradiol implants had follicular waves emerge during treatment (Fig. 3). In ewes treated with GnRH, four ewes on Day 5 and five ewes on Day 8 had follicular wave emergence during the implant period. All ewes had normal follicular waves before implant insertion and after implant removal. The number of small follicles did not differ between control ewes and ewes treated with GnRH (25.81 ⫾ 2.0; P ⬎ 0.05). 3.3. Ovulations, cystic follicles, corpora lutea, and mean daily serum progesterone concentrations
Fig. 3. Peaks in serum concentrations of FSH (outlined with shading) and their associated emerging follicle waves in anestrous Western White Face ewes treated for 12 d (Days 0 –12; open rectangle on X-axis) with large silastic rubber sc implants containing 10% estradiol-17 (10 ⫻ 0.47 cm) without (panel A; n ⫽ 6) or with (panel B; n ⫽ 6) GnRH treatment for 6 d (Days 6 –12; checked rectangle on X-axis), starting on Day 6 of implant insertion (200 ng given IV every 4 h for 6 d). Data were normalized to the day of implant insertion (Day 0) in all ewes. The average curves representing the growth, static, and regression phases of follicular waves for all ewes in a group were normalized for each follicle wave to the mean day of wave emergence occurring either before or after implant insertion (Day 0; arrows indicates days of wave emergence; [7]). All FSH peaks for all ewes in a group are shown normalized to the mean day of occurrence of the apex of the FSH peak for each wave. The day of the apex of FSH peak was then plotted according to the mean day it occurred before or after implant insertion. For every FSH peak, serum concentration profiles were delimited by the encompassing nadirs of the FSH concentrations (hence the overlap of the data for adjacent peaks in some cases). In the control group, a follicular wave emerged and grew in only one ewe the day before implant insertion (indicated by †). In ewes treated with GnRH, four of six had a follicular wave emergence on Day 5 after implant insertion, and five of six on Day 8 (indicated by $). Numbers in parenthesis give the number of ewes in a group with an FSH peak at that time. Concentrations of FSH and follicle diameters are expressed as mean ⫾ SEM.
Cystic or luteinized follicles were detected in estradiol-treated ewes in both experiments. In Experiment 1, one ewe with a large implant formed a cystic follicle 3 d after implant insertion which luteinized and remained until the end of the experiment. Two ewes, treated with large implants, formed luteal structures with a lifespan similar to a normal CL. A short lifespan luteal structure (present for ⬍9 d) was developed by one ewe treated with a large implant and one ewe treated with a small implant. In Experiment 2, three ewes from each of the treatment groups formed a cystic follicle 3 d after implant insertion. These cystic follicles luteinized and remained until the end of the experiment. In Experiment 1, mean serum progesterone concentrations were higher in ewes that had luteal structures or cystic follicles compared to ewes that had no such structures (0.22 ⫾ 0.03 vs. 0.06 ⫾ 0.03 ng/mL; P ⬍ 0.05). Serum progesterone concentrations increased to a peak of 0.38 ⫾ 0.17 ng/mL on Day 9 after implant insertion in those ewes that had luteal structures or cystic follicles (n ⫽ 5). In Experiment 2, mean serum progesterone concentrations were higher in ewes that had cystic follicles compared to ewes that had no cystic follicles (0.21 ⫾ 0.05 vs. 0.08 ⫾ 0.05 ng/mL; P ⬍
918
S.V. Seekallu et al. / Theriogenology 74 (2010) 912–921
0.05). Serum progesterone concentrations increased significantly to a peak of 0.44 ⫾ 0.10 ng/mL on Day 9 after implant insertion in ewes that had cystic follicles (n ⫽ 6). 4. Discussion In the present study, the most profound effects of both sizes of estradiol implant were to block follicular wave emergence and the pulsatile secretion of LH. Clearly, both sizes of implant released sufficient estradiol to block LH pulses. There was some indication that the implants releasing estradiol used in this study caused some suppression of FSH secretion, particularly toward the end of the period of the implant treatment. However, perhaps the most salient finding for serum FSH concentrations from blood samples taken daily was that the range in peak concentrations in ewes given estradiol implants and in which follicular waves were blocked was the same as for the peaks in control ewes occurring at the same time, and that were followed by emergence of waves. It has been clearly established that the discrete FSH peaks that precede follicular waves in the ewe are the essential triggers for the orderly, rhythmic initiation of the waves, even though the concentration of FSH at the zenith of these peaks, and the nadirs in serum FSH concentrations between peaks, can vary widely [3,4,7]. Without these discrete peaks, follicular waves do not occur [7]. The findings in the present study supported our hypothesis that in the anestrous ewe the presence of pulsed LH secretion was required for the generation of the follicular waves that are triggered by peaks of FSH secretion. Restoration of LH pulses, by the GnRH treatment, reinitiated follicular wave emergence at the expected intervals and rhythm. Even though GnRH injections transiently increased serum FSH concentrations up to 1 h, as noted in blood samples collected every 12 min for 6 h, this increase was not enough to cause changes in peak serum FSH concentrations measured daily over the GnRH treatment period, in comparison to control ewes. The FSH peaks that precede follicular waves lasted 3– 4 d from nadir to nadir [3,28]. The dose of GnRH was very low and designed to cause physiological pulses of LH. In Experiment 2 of the present study, the FSH peaks, that were able to initiate follicular waves in the presence of restored pulsatile secretion of LH, were in the same range in concentration as those occurring in control ewes at a similar time, and in which follicular waves were blocked during the period of implant treat-
ment and absence of pulsed LH secretion. It is particularly intriguing that the peaks in serum concentrations of FSH at Day 4 in GnRH treated ewes gave rise to follicular wave emergence on Day 5, even though pulsatile LH (GnRH) support was not restored until Day 6. With the ultrasound equipment used, we were able to track follicular waves back to their emergence from the pool of small follicles, 2–3 mm in diameter. The size of this pool of follicles was not affected by treatment with estradiol 17-, as growth of antral follicles to a size of 2–3 mm is largely believed to be gonadotropin independent [29,30]. In the waves mentioned above, the FSH peak on Day 4 could have stimulated wave emergence from 2 mm follicles on Day 5 and follicles would have transitioned from 3 to 4 mm on Days 6 and 7 during the start of GnRH treatment and restored pulsed secretion of LH. Follicular waves normally emerge on the day of the zenith of the peak in serum FSH concentration, or 1 d before or after [3,5,6]. It should be noted that the peak concentration of FSH on Day 4 in control and GnRH treated ewes in Experiment 2 were virtually identical, whereas the FSH peaks on Day 4 were followed by follicular waves only in GnRH treated ewes. In Experiment 1, two ewes given small implants experienced wave emergence on Day 1 of treatment. We speculate that perhaps pulsed LH secretion was not immediately abolished in these ewes and supported wave emergence in response to the FSH peak on Day 1. In another recent study, the final growth of antral follicles destined to luteinize following prostaglandin and hCG treatment of ewes with ovarian autotransplants, did not appear to require pulsatile LH secretion [31]. In the latter study, LH secretion was suppressed with a GnRH antagonist. Final antral follicle growth appeared to have occurred in ewes given constant or pulsed infusion of LH, as well as in ewes receiving no supplemental LH. However, in the latter study, the growth of follicles during treatment was significant only at the higher of two doses of LH given by constant infusion. All ewes formed a CL in response to prostaglandin and hCG treatment. In another study, in prepubertal ewes, LH given as pulses or constant infusion was equally effective in inducing an LH surge [32]. The need for LH to initiate maturation of potentially ovulatory follicles was similar to that observed in pigs [33]. However, in contrast, in GnRH-antagonist treated ewes, ovulations were detected following infusions of only FSH [34]. In the present study, removal of pulsed LH secretion failed to allow emergence of follicular waves, but res-
S.V. Seekallu et al. / Theriogenology 74 (2010) 912–921
toration of LH pulses by GnRH treatment allowed follicular waves to occur in response to FSH peaks. Interestingly, Lacker [35] developed a mathematical model to predict follicle growth and ovulation rate in mammals and the role of estradiol as a regulatory agent [36]. As for rabbits [36], the present results support Lacker’s model of a role for estradiol in controlling the development of ovulatory sized follicles by way of regulation of gonadotropin (LH) secretion. Another interesting observation from the present study was that FSH peaks continued to occur in the absence of follicular wave development. If changes in the temporal pattern of secretion of products from follicular waves were important for regulating FSH peaks, based on the present results, estradiol could not be the hormone, as relatively constant serum concentrations resulted from the implant treatment. Inhibin also suppresses FSH secretion in the ewe; however, the specific role of inhibin in regulating FSH peaks has not been studied [37]. Peaks of FSH and the associated follicular waves are not regulated by inhibin in anestrous ewes, as serum concentrations of inhibin do not change with the pattern of follicular waves in the non-cycling ewe [12]. As no follicular waves emerged during the treatment period, in 8 of 10 ewes given estradiol releasing implants in Experiment 1, and all the control ewes in Experiment 2 of the present study, it is unlikely that secretory products of the follicles of a wave, could have entrained the second, third, and fourth FSH peaks during the period of implant treatment and immediately after treatment. We speculate that ovarian follicular feedback may not regulate the rhythm of the FSH peaks that precede follicular wave emergence in the ewe. In ovariectomized ewes, FSH peaks had a rhythm similar to the intact ewe [28]. Also, artificial peaks created by FSH injections between two endogenously driven peaks did not alter the rythmicity of FSH peaks [5]. It is intriguing that in our previous study in cyclic ewes [7], when similar sized estradiol implants were used, supra-physiological concentrations of estradiol had no significant effect on the LH secretory pattern. However, in cyclic ewes, estradiol implants producing higher concentrations of estradiol abolished pulsed LH secretion [38]. It has been shown that estradiol exerted a stronger negative feedback effect on LH secretion during seasonal anestrous compared to the breeding season [20,39 – 42]. There was no evidence of regular pulsed FSH secretion in blood samples taken from the jugular vein in the present study [10,11]. We inferred that the ovary does not need FSH pulses to generate follicular waves. The
919
large second FSH peak, in ewes given small implants in Experiment 1, was due to high concentrations of FSH in two ewes. We have no explanation for this, but FSH peak concentrations can be quite variable [4,6]. Progesterone concentrations produced by the cystic follicles or luteal structures formed after ovulation in Experiment 1 and 2, were very low compared to peak serum concentrations of progesterone during the physiologic luteal phase of cyclic ewes (3.58 ⫾ 0.18 ng/mL; [42]). Furthermore, there was no evidence that the presence of cystic follicles affected the pattern of emergence and growth of follicular waves in response to GnRH. We concluded that the supra-physiological concentrations of estradiol-17 created by estradiol-releasing implants suppressed follicular wave development by abolishing LH pulses in anestrous ewes. Restoration of LH pulses in implant treated ewes by frequent injections of GnRH allowed the re-establishment of follicular waves that were stimulated by peaks in secretion of FSH. In this study, we showed that LH pulses in the anestrous ewe were critical for the development of follicular waves. It is unclear whether secretory products from the follicles of a wave entrained the FSH peaks that initiated follicular waves in anestrous ewes.
Acknowledgements This research was supported by the Natural Sciences and Engineering Research Council of Canada to N.C.R. The authors thank Dr. A.F. Parlow of NIDDK/NHPP for the provision of reagents for the gonadotropin assays, and Ms. Susan J. Cook and Ms. Kim Tran for their help in blood sampling and radioimmunoassay. S.V.S., D.M.W.B., B.M.T., R.D., K.L.D., and E.T.B. were recipients of University of Saskatchewan Graduate Student Scholarships. K.C., K.A.E., and K.M.P. were recipients of summer undergraduate research studentships from NSERC (KC), and the Western College of Veterinary Medicine (KAE and KMP).
References [1] Schrick FN, Surface RA, Pritchard JY, Dailey RA, Townsend EC, Inskeep EK. Ovarian structures during the estrous cycle and early pregnancy in ewes. Biol Reprod 1993;49:1133– 40. [2] Souza CJ, Campbell BK, Baird DT. Follicular dynamics and ovarian steroid secretion in sheep during anoestrus. J Reprod Fertil 1996;108:101– 6. [3] Bartlewski PM, Beard AP, Cook SJ, Chandolia RK, Honaramooz A, Rawlings NC. Ovarian antral follicular dynamics and their relationships with endocrine variables throughout the oes-
920
[4]
[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
[15] [16]
[17]
[18]
S.V. Seekallu et al. / Theriogenology 74 (2010) 912–921 trous cycle in breeds of sheep differing in prolificacy. J Reprod Fertil 1999;115:111–24. Bartlewski PM, Beard AP, Cook SJ, Rawlings NC. Ovarian follicular dynamics during anoestrus in ewes. J Reprod Fertil 1998;113:275– 85. Duggavathi R, Bartlewski PM, Barrett DM, Gratton C, Bagu ET, Rawlings NC. Patterns of antral follicular wave dynamics and accompanying endocrine changes in cyclic and seasonally anestrous ewes treated with exogenous ovine follicle-stimulating hormone during the inter-wave interval. Biol Reprod 2004; 70:821–7. Bartlewski PM, Vanderpol J, Beard AP, Cook SJ, Rawlings NC. Ovarian antral follicular dynamics and their associations with peripheral concentrations of gonadotropins and ovarian steroids in anoestrous Finnish Landrace ewes. Anim Reprod Sci 2000; 58:273–91. Barrett DM, Bartlewski PM, Duggavathi R, Davies KL, Rawlings NC. Suppression of follicle wave emergence in cyclic ewes by supraphysiologic concentrations of estradiol-17beta and induction with a physiologic dose of exogenous ovine folliclestimulating hormone. Biol Reprod 2006;75:633– 41. Bartlewski PM, Beard AP, Rawlings NC. An ultrasound-aided study of temporal relationships between the patterns of LH/FSH secretion, development of ovulatory-sized antral follicles and formation of corpora lutea in ewes. Theriogenology 2000;54: 229 – 45. Duggavathi R, Bartlewski PM, Barrett DM, Rawlings NC. The temporal relationship between patterns of LH and FSH secretion, and development of ovulatory-sized follicles during the mid- to late-luteal phase of sheep. Theriogenology 2005;64: 393– 407. Wallace JM, McNeilly AS. Changes in FSH and the pulsatile secretion of LH during treatment of ewes with bovine follicular fluid throughout the luteal phase of the oestrous cycle. J Endocrinol 1986;111:317–27. Wheaton JE, Mullett MA, Cornelius SG. Plasma follicle stimulating hormone and luteinizing hormone patterns during the estrous cycle of ewes. Theriogenology 1984;21:989 –99. Evans ACO, Duffy P, Quinn KM, Knight PG, Boland MP. Follicular waves are associated with transient fluctuations in FSH but not oestradiol or inhibin-A concentrations in anoestrous ewes. Animal Science 2001;72:547–54. McNatty KP, Hudson NL, Henderson KM, Lun S, Heath DA, Gibb M, Ball K, McDiarmid JM, Thurley DC. Changes in gonadotrophin secretion and ovarian antral follicular activity in seasonally breeding sheep throughout the year. J Reprod Fertil 1984;70:309 –21. Walton JS, Evins JD, Fitzgerald BP, Cunningham FJ. Abrupt decrease in daylength and short-term changes in the plasma concentrations of FSH, LH and prolactin in anoestrous ewes. J Reprod Fertil 1980;59:163–71. Yuthasastrakosol P, Palmer WM, Howland BE. Release of LH in anoestrous and cyclic ewes. J Reprod Fertil 1977;50:319 –321. Jackson GL, Davis SL. Comparison of luteinizing hormone and prolactin levels in cycling and anestrous ewes. Neuroendocrinology 1979;28:256 – 63. McNatty KP, Gibb M, Dobson C, Thurley DC. Evidence that changes in luteinizing hormone secretion regulate the growth of the preovulatory follicle in the ewe. J Endocrinol 1981;90:375– 89. Karsch FJ, Foster DL. Sexual differentiation of the mechanism controlling the preovulatory discharge of luteinizing hormone in sheep. Endocrinology 1975;97:373–9.
[19] Karsch FJ, Cummins JT, Thomas GB, Clarke IJ. Steroid feedback inhibition of pulsatile secretion of gonadotropin-releasing hormone in the ewe. Biol Reprod 1987;36:1207–18. [20] Joseph IB, Currie WD, Rawlings NC. Effects of time after ovariectomy, season and oestradiol on luteinizing hormone and follicle-stimulating hormone secretion in ovariectomized ewes. J Reprod Fertil 1992;94:511–23. [21] Ravindra JP, Rawlings NC, Evans AC, Adams GP. Ultrasonographic study of ovarian follicular dynamics in ewes during the oestrous cycle. J Reprod Fertil 1994;101:501–9. [22] Duggavathi R, Bartlewski PM, Barrett DM, Rawlings NC. Use of high-resolution transrectal ultrasonography to assess changes in numbers of small ovarian antral follicles and their relationships to the emergence of follicular waves in cyclic ewes. Theriogenology 2003;60:495–510. [23] Rawlings NC, Jeffcoate IA, Rieger DL. The influence of estradiol-17beta and progesterone on peripheral serum concentrations of luteinizing hormone and follicle stimulating hormone in the ovariectomized ewe. Theriogenology 1984;22:473– 88. [24] Currie WD, Rawlings NC. Fluctuation in responsiveness of LH and lack of responsiveness of FSH to prolonged infusion of morphine and naloxone in the ewe. J Reprod Fertil 1989;86: 359 – 66. [25] Rawlings NC, Jeffcoate IA, Currie WD, Cook SJ. Control of the surge release of LH and FSH in oestradiol and progesterone treated ovariectomized ewes. Can J Anim Sci 1988;68:1089 –96. [26] Gitzen JF, Ramirez VD. PC-Pulsar-Pulsar for the IBM-PC: Operating instructions distributed with the PC-Pulsar program. 1988. [27] Clifton DK, Steiner RA. Cycle detection: a technique for estimating the frequency and amplitude of episodic fluctuations in blood hormone and substrate concentrations. Endocrinology 1983;112:1057– 64. [28] Duggavathi R, Bartlewski PM, Agg E, Flint S, Barrett DM, Rawlings NC. The effect of the manipulation of follicle-stimulating hormone (FSH)-peak characteristics on follicular wave dynamics in sheep: does an ovarian-independent endogenous rhythm in FSH secretion exist? Biol Reprod 2005;72:1466 –74. [29] Driancourt MA, Fry RC, Clarke IJ, Cahill LP. Follicular growth and regression during the 8 days after hypophysectomy in sheep. J Reprod Fertil 1987;79:635– 41. [30] Dufour J, Cahill LP, Mauleon P. Short- and long-term effects of hypophysectomy and unilateral ovariectomy on ovarian follicular populations in sheep. J Reprod Fertil 1979;57:301–9. [31] Campbell BK, Kendall NR, Baird DT. The effect of the presence and pattern of luteinizing hormone stimulation on ovulatory follicle development in sheep. Biol Reprod 2007;76:719 –27. [32] Keisler DH, Inskeep EK, Dailey RA. Roles of pattern of secretion of luteinizing hormone and the ovary in attainment of puberty in ewe lambs. Domest Anim Endocrinol 1985;2:123–32. [33] Guthrie HD, Bolt DJ, Cooper BS. Effects of gonadotropin treatment on ovarian follicle growth and granulosal cell aromatase activity in prepuberal gilts. J Anim Sci 1990;68:3719 –26. [34] Campbell BK, Dobson H, Baird DT, Scaramuzzi RJ. Examination of the relative role of FSH and LH in the mechanism of ovulatory follicle selection in sheep. J Reprod Fertil 1999;117: 355– 67. [35] Lacker HM. Regulation of ovulation number in mammals. A follicle interaction law that controls maturation. Biophys J 1981;35:433–54.
S.V. Seekallu et al. / Theriogenology 74 (2010) 912–921 [36] Meuli LE, Lacker HM, Thau RB. Experimental evidence supporting a mathematical theory of the physiological mechanism regulating follicle development and ovulation number. Biol Reprod 1987;37:589 –94. [37] Mann GE, McNeilly AS, Baird DT. Hormone production in vivo and in vitro from follicles at different stages of the oestrous cycle in the sheep. J Endocrinol 1992;132:225–34. [38] Barrett DM, Duggavathi R, Davies KL, Bartlewski PM, Bagu ET, Rawlings NC. Differential effects of various estradiol17beta treatments on follicle-stimulating hormone peaks, luteinizing hormone pulses, basal gonadotropin concentrations, and antral follicle and luteal development in cyclic ewes. Biol Reprod 2007;77:252– 62.
921
[39] Goodman RL, Bittman EL, Foster DL, Karsch FJ. The endocrine basis of the synergistic suppression of luteinizing hormone by estradiol and progesterone. Endocrinology 1981;109:1414 –7. [40] Karsch FJ, Legan SJ, Ryan KD, Foster DL. Importance of estradiol and progesterone in regulating LH secretion and estrous behavior during the sheep estrous cycle. Biol Reprod 1980;23:404–13. [41] Martin GB, Scaramuzzi RJ, Henstridge JD. Effects of oestradiol, progesterone and androstenedione on the pulsatile secretion of luteinizing hormone in ovariectomized ewes during spring and autumn. J Endocrinol 1983;96:181–93. [42] Bartlewski PM, Beard AP, Rawlings NC. An ultrasonographic study of luteal function in breeds of sheep with different ovulation rates. Theriogenology 1999;52:115–30.
Theriogenology 74 (2010) 912–921 www.theriojournal.com
Pulsatile LH secretion and ovarian follicular wave emergence and growth in anestrous ewes Srinivas V. Seekallua,b, David M.W. Barretta,c, Behzad M. Toosia, Kelsey Clarkea, Kirk A. Ewena, Rajesha Duggavathia,d, Kate L. Daviesa,e, Kim M. Pattulloa, Edward T. Bagua,f, Norman C. Rawlingsa,* a
Department of Veterinary Biomedical Sciences, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, SK, Canada S7N 5B4 b Manitoba Institute of Cell Biology, CancerCare Manitoba, 675 McDermot Avenue, Winnipeg, MB, Canada R3E 0V9 c Department of Plant and Animal Sciences, Haley Institute, Nova Scotia Agricultural College, Truro, NS, Canada, B2N 5E3 d Department of Animal Science, McGill University, 21, 111 Lakeshore Road, Ste-Anne-de-Bellevue, Québec, Canada, H9X 3V9 e Department of Animal and Poultry Science, College of Agriculture, University of Saskatchewan, Saskatoon, SK, Canada, S7N 5A8 f Centre de Recherche-CHUM Hospital Notre-Dame, Y-5625-2099 Rue Alexandre de Séve, Montreal, Quebec, Canada, H2L 2W5 Received 15 September 2009; received in revised form 8 April 2010; accepted 11 April 2010
Abstract The objective of this study was to determine if pulsatile LH secretion was needed for ovarian follicular wave emergence and growth in the anestrous ewe. In Experiment 1, ewes were either large or small (10 ⫻ 0.47 or 5 ⫻ 0.47 cm, respectively; n ⫽ 5/group) sc implants releasing estradiol-17 beta for 10 d (Day 0 ⫽ day of implant insertion), to suppress pulsed LH secretion, but not FSH secretion. Five sham-operated control ewes received no implants. In Experiment 2, 12 ewes received large estradiol-releasing implants for 12 d (Day 0 ⫽ day of implant insertion); six were given GnRH (200 ng IV) every 4 h for the last 6 d that the implants were in place (to reinitiate pulsed LH secretion) whereas six Control ewes were given saline. Ovarian ultrasonography and blood sampling were done daily; blood samples were also taken every 12 min for 6 h on Days 5 and 9, and on Days 6 and 12 of the treatment period in Experiments 1 and 2, respectively. Treatment with estradiol blocked pulsatile LH secretion (P ⬍ 0.001). In Experiment 1, implant treatment halted follicular wave emergence between Days 2 and 10. In Experiment 2, follicular waves were suppressed during treatment with estradiol, but resumed following GnRH treatment. In both experiments, the range of peaks in serum FSH concentrations that preceded and triggered follicular wave emergence was almost the same as control ewes and those given estradiol implants alone or with GnRH; mean concentrations did not differ (P ⬍ 0.05). We concluded that some level of pulsatile LH secretion was required for the emergence of follicular waves that were triggered by peaks in serum FSH concentrations in the anestrous ewe. © 2010 Elsevier Inc. All rights reserved. Keywords: Anestrous; Estradiol; FSH; Ovarian follicular waves; Sheep
1. Introduction With the recent use of transrectal ultrasonography for imaging ovaries in both cyclic and anestrous ewes, * Corresponding author. Tel.: 306-966-7068; fax: 306-966-7314. E-mail address: [email protected] (N.C. Rawlings). 0093-691X/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2010.04.016
it was found that 1–3 ovarian antral follicles emerged or grew from a pool of small follicles, 1–3 mm in diameter, every 4 –5 d [1– 4]. These follicles grow to ⱖ5 mm in diameter before regression or ovulation [1– 4]. Each wave of follicular growth was preceded by a transient peak of FSH secretion that lasted 3 or 4 d and was characterized by blood samples taken daily [3,5,6].
S.V. Seekallu et al. / Theriogenology 74 (2010) 912–921
This peak in FSH secretion was the essential trigger for the follicular wave [3,5,6]; however, the maximum concentration that peaks achieved and the nadirs in serum FSH concentrations between peaks, varied considerably among follicular waves [3,4,7]. When cyclic ewes were treated for 10 d with large estradiol-17 releasing implants (10 ⫻ 0.47 cm), that created supraphysiological serum concentrations of estradiol-17 (10.4 ⫾ 0.7 pg/mL vs. 3.9 ⫾ 0.7 pg/mL for treated vs. control ewes, respectively), the amplitude of the FSH peaks that preceded follicular waves was reduced and follicular wave emergence was blocked [7]. Injection of ovine FSH (oFSH), to recreate FSH peaks, re-initiated follicular waves [7]. In that study, pulsed LH secretion was not affected. Pulses of LH were released at a frequency of ⬃1 every 1– 6 h in the ewe and lasted no more than ⬃2 h [8,9]. Although peaks in FSH secretion initiated follicular waves, the role of pulsed LH secretion in the emergence, growth and regression of ovine antral follicles was unclear. Changes in LH pulse frequency during the estrous cycle did not appear to be correlated with or functionally related to specific phases of the growth or regression of follicular waves in the ewe [8,9]. Profiles of FSH in blood samples collected from the jugular vein in ewes were not pulsatile [10,11]. Compared to ewes during the breeding season, seasonally anestrous ewes had lower circulating concentrations of LH, FSH, and estradiol, little or no progesterone, and serum concentrations of estradiol and inhibin which were not correlated with follicular wave development [8,12–15]. The frequency of LH secretory pulses was very low and fluctuated very little across seasonal anestrus [13–17], with no apparent association to stages of follicular wave development and regression. During seasonal anestrus, estradiol exerted a more powerful negative feedback effect on pulsatile LH secretion than during the breeding season, but the effects on FSH secretion appeared to be minimal [18 –20]. We hypothesized that although the frequency of pulses of LH secretion were very low in anestrous ewes, they were still essential for emergence and growth of ovarian follicular waves. The objective of the present study was to examine the need for pulsed LH secretion for the emergence and growth of ovarian follicular waves in anestrous ewes. Two experiments were conducted. The purpose of the first experiment was to determine if treatment of anestrous ewes with large estradiol releasing implants (10 ⫻ 0.47 cm) that created supraphysiological concentrations of estradiol [7] would suppress the pulsed secretion of LH, but leave FSH peaks
913
with a concentration at their zenith similar to control ewes and in a range that could still initiate a follicular wave. The purpose of the second experiment was to replace LH pulses with frequent GnRH injections, in the experimental model above, to see if restored LH pulsatility would in fact allow restoration of follicular waves. This would confirm that, although peaks in FSH secretion initiated follicular waves in the anestrous ewe, some level of pulsed LH secretion was also required. 2. Materials and methods 2.1. Animals Care and handling of experimental animals was done according to the Canadian Council on Animal Care’s published guidelines. Sexually mature, clinically healthy, seasonally anestrous, 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. 2.2. Ultrasound technique The growth and regression of ovarian antral follicles were monitored in all ewes by transrectal ovarian ultrasonography (scanning), 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 [1,21,22]. All images were viewed at a magnification of ⫻ 1.5 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, Fujifilm, Tokyo, Japan) for later examination. The relative position and dimensions of follicles and luteal structures were also sketched on ovarian charts. 2.3. Experimental design 2.3.1. Experiment 1 Fifteen seasonally anestrous (June) WWF ewes (mean body weight of 76.9 ⫾ 2.9 kg) were allocated into three groups of five ewes each. The experimental design is summarized in Figure 1. Five ewes received large subcutaneous silastic rubber implants (10 ⫻ 0.47 cm) and five ewes received small subcutaneous silastic rubber implants (5 ⫻ 0.47 cm) containing 10% estradiol17 w/w (Sigma Chemical Company, St. Louis, MO, USA [7]. Five sham operated control ewes received no
914
S.V. Seekallu et al. / Theriogenology 74 (2010) 912–921
Fig. 1. Schematic representation of the experimental design of the two studies conducted in ewes during the anestrous season. Estradiol implants were inserted subcutaneously on Day 0 and left in place for 10 or 12 d in Experiments 1 and 2, respectively. In Experiment 1, ewes received 5 cm (n ⫽ 5) or 10 cm (n ⫽ 5) implants; control ewes (n ⫽ 5) underwent sham surgery only. In Experiment 2, all ewes received 10 cm implants. Injections of GnRH (200 ng IV; 6 ewes) or saline (IV; 6 ewes) were given every 4 h from Days 6 to 12 after implant insertion in Experiment 2. Daily ultrasonography and blood sampling were done from 4 or 5 d before to 20 or 22 d after implant insertion in Experiment 1 and 2, respectively. Blood samples were also taken every 12 min for 6 h (intensive bleeds) on Days 5 and 9 in Experiment 1, and on Days 6 and 12 in Experiment 2 to characterize the secretory patterns of LH and FSH. In Experiment 2, intensive bleeds started 36 min prior to the first and penultimate injection of GnRH or saline, and ended 2 h after the second and last injections respectively.
implants. All implants were removed 10 d after insertion (Day 0 ⫽ day of insertion). Mean serum estradiol concentrations for the implant treatment period, were 3.69 ⫾ 0.4, 9.82 ⫾ 1.1, and 11.84 ⫾ 1.0 pg/mL for control ewes, and those given small or large implants, respectively. Daily scanning and blood sampling were done from 4 d before to 20 d after implant insertion. Blood samples were collected in evacuated tubes (10 mL; Becton Dickinson, Franklin Lakes, NJ, USA) by jugular venipuncture, starting at 0800 h, and followed immediately by scanning. Blood samples (5 mL) were also collected every 12 min for 6 h (intensive sampling) on Day 5 and 9 starting at 0800 h to determine the secretory patterns of LH and FSH at the middle and end of implant treatments. Intensive sampling was via catheter (vinyl tubing, 1.0 mm id ⫻ 1.5 mm od; 530070 Biocorp Australia Pty Ltd., Auburn, NSW, Australia) inserted into the jugular vein. 2.3.2. Experiment 2 Twelve seasonally anestrous (May/June) WWF ewes (mean body weights of 83.5 ⫾ 3.0 kg) received large subcutaneous silastic rubber implants (10 ⫻ 0.47 cm) containing 10% estradiol-17 w/w for 12 d (Day 0 ⫽ day of implant insertion; Fig. 1). Six of the twelve were given GnRH (200 ng IV in saline; Sigma Chemical Com-
pany) every 4 h for the last 6 d that the implants were in place, starting at 0800 h on Day 6 (Fig. 1). The dose of GnRH was designed to create LH pulses within a physiological range. Six control ewes received only saline (IV). Mean serum estradiol concentrations for the implant treatment period were 12.08 ⫾ 0.7 and 10.81 ⫾ 0.7 pg/mL in control ewes (ewes treated with only estradiol implants) and ewes treated with GnRH, respectively. Daily scanning and blood sampling were done from 5 d before to 22 d after implant insertion, as detailed for Experiment 1. Blood samples also were collected every 12 min, by a catheter inserted in the jugular vein, starting 36 min before the first and before the penultimate GnRH/ saline injections, and ending 2 h after the second and after the last GnRH/ saline injections, respectively, in order to characterize the secretory patterns of LH and FSH and their responses to GnRH at the onset and end of the period of treatment with GnRH. 2.4. Analysis of follicular data An ovarian follicular wave consisted of an ovarian follicle or a group of follicles that emerged and grew from 2 or 3 mm in diameter to ⱖ5 mm (growth phase)
S.V. Seekallu et al. / Theriogenology 74 (2010) 912–921
before regressing to 2 or 3 mm in diameter (regression phase); time spent at ⱖ5 mm was regarded as the static phase [4]. Emergence into a wave was restricted to a period of 24 h [22]. Data were analyzed for the number of follicular waves in each group of ewes. The number of small follicles (ⱖ1 to ⱕ3 mm in diameter) each day of the study also was analyzed. Ovulation was detected with ultrasonography as the collapse of a large follicle (ⱖ5 mm in diameter) that had been followed in its growth/ static phase for several days. Follicular data were combined for both ovaries of each ewe. 2.5. Hormone analysis All blood samples were permitted to clot at room temperature for 18 –24 h. Samples were then centrifuged for 10 min at 1500 ⫻ g, and serum was removed and kept at ⫺20 oC until assayed. Serum concentrations of progesterone [23], estradiol [20,24], LH, and FSH [25] were measured by validated radioimmunoassays (RIA). The assay sensitivities (defined as the lowest concentration of a hormone capable of significantly displacing radio-labeled hormone from the antibody) were: 0.03 ng/mL for progesterone, 1.0 pg/mL for estradiol and 0.1 ng/mL for FSH and LH. The ranges of standards were: 0.1–10 ng/mL, 1.0 –100 pg/mL, 0.12–16.0 ng/mL, and 0.06 – 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, throughout the experimental period, were analyzed for concentrations of progesterone, estradiol, and FSH. All serum samples collected every 12 min were analyzed for concentrations of LH and FSH. For Experiment 1, the intra- and inter-assay coefficients of variation (CVs) were 9.8 and 15.2%, or 6.7 and 14.2% for reference sera with mean progesterone concentrations of 0.24 or 1.14 ng/mL, respectively. The intraand inter-assay coefficients of variation were 9.5 and 10.5%, or 7.8 and 16.2% for reference sera with mean estradiol concentrations of 3.6 or 31.0 pg/mL, respectively. The intra-assay CVs were 5.4 or 8.4% for reference sera with mean FSH concentrations of 1.10 or 5.71 ng/mL, respectively. The intra- and inter-assay CVs were 10.0 and 10.3%, or 8.5 and 9.0% for reference sera with mean LH concentrations of 0.11 or 2.62 ng/mL, respectively. For Experiment 2, the intra- and inter-assay coefficients of variation (CVs) were 15.9 and 6.5%, or 7.2 and 12.8% for reference sera with mean progesterone concentrations of 0.21 or 1.13 ng/mL, respectively. The intra- and inter-assay coefficients of variation were 7.4
915
and 9.0%, or 5.5 and 6.8% for reference sera with mean estradiol concentrations of 7.97 or 25.17 pg/mL, respectively. The intra-assay CVs were 4.2 or 4.2% for reference sera with mean FSH concentrations of 1.12 or 3.34 ng/ mL, respectively. The intra- and inter-assay CVs were 4.7 and 5.4%, or 9.6 and 10.0% for reference sera with mean LH concentrations of 0.4 or 2.97 ng/mL, respectively. The PC-PULSAR program [26] was used to assess mean serum LH and FSH concentrations as well as LH and FSH pulse frequency and amplitude in blood samples collected every 12 min for 6 h. Standard deviation criteria (G and Baxter parameters) were used for pulse detection. Peaks of FSH in blood samples taken daily were identified using cycle-detection software [27]. 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; [27]). Follicle stimulating hormone peak concentration was defined as the concentration of FSH observed at the apex of the FSH peak. 2.6. Statistical analyses Observations on hormone concentrations and ovarian follicles measured daily were normalized to the day of implant insertion for analysis and presentation. Twoway repeated measures ANOVA (Sigma Stat 7 for Windows Version 2.03, 1997, SPSS Inc., Chicago, IL, USA) was used to assess differences in hormone concentrations and observations on ovarian follicles over time and among groups of ewes (i.e., treated with large, small, or no implants in Experiment 1, and with or without exogenous GnRH injection in Experiment 2). Two-way repeated measures ANOVA was used to assess differences in LH and FSH secretory characteristics from blood samples collected every 12 min for 6 h (i.e., mean concentrations, pulse frequency, and pulse amplitude) among groups of ewes and between intensive sampling days. If the main effects, or their interactions, were significant (P ⬍ 0.05), Fisher’s protected least significant difference (LSD) was used as a postANOVA test to detect differences between individual means (P ⬍ 0.05). The Chi-square test was used to analyze for the proportion of missing FSH peaks. Data were expressed as mean ⫾ SEM. 3. Results 3.1. Experiment 1 3.1.1. Serum hormone concentrations Pulses of LH were blocked in ewes treated with implants, but control ewes had mean LH pulse frequency
916
S.V. Seekallu et al. / Theriogenology 74 (2010) 912–921
of 1.5 ⫾ 0.2 per 6 h. Profiles of FSH were not pulsatile, and mean serum concentrations of FSH (1.43 ⫾ 0.2 ng/ mL) did not differ with treatment or day. However, in ewes treated with large estradiol implants, mean serum FSH concentrations were lower (P ⬍ 0.05) during intensive sampling on Day 9 (1.00 ⫾ 0.1 ng/mL) compared to Day 5 (1.38 ⫾ 0.2 ng/mL). Apart from a high peak serum FSH concentration on Day 1 in ewes treated with small implants (P ⬍ 0.05; Fig. 2B), neither treatment nor day affected FSH peak concentrations (Fig. 2) in daily samples. Whereas four of five ewes given small implants and all control ewes had an increase in FSH concentrations during the fourth FSH peak, only two of five ewes treated with high estradiol had an increase (P ⬍ 0.05; Fig. 2C). In ewes given large or small estradiol implants, peak serum FSH concentrations during treatment and when follicular waves were blocked, had a range of 0.9 –2.6 ng/mL (mean, 1.7 ⫾ 0.18 ng/mL). In control ewes, FSH peaks occurring at a similar time and that were accompanied by wave emergence, covered a range of 0.9 –3.3 ng/mL (mean, 2.0 ⫾ 0.46 ng/mL). 3.1.2. Antral follicle development No ewe given large estradiol implants, and only two of five ewes given small estradiol implants, had follicular waves emergence during treatment (emergence on Day 1). Follicular waves emerged after the end of treatment in all ewes given implants, with an average day of emergence of 13.9 ⫾ 0.5 d from implant insertion. Treatment did not affect the number of small follicles (16.40 ⫾ 2.5; P ⬎ 0.05). 3.2. Experiment 2 3.2.1. Serum hormone concentrations Each GnRH injection led to a pulse in LH secretion, whereas LH pulses were absent in ewes after implant insertion alone. Mean serum LH concentrations did not differ between Days 6 and 12 in ewes treated with GnRH (0.24 ⫾ 0.1 ng/mL). There was no difference in mean serum FSH concentrations amongst days of intensive blood sampling (Day 6 or 12), treatment or its interaction with day (mean, 1.26 ⫾ 0.2 ng/ml). Within days of intensive sampling, there was a time effect and an interaction of treatment by time (P ⬍ 0.05): serum FSH concentrations were greater in ewes given GnRH compared to control ewes from 12– 60 min (1.40 ⫾ 0.2 vs. 1.10 ⫾ 0.1 ng/mL) after the first GnRH injection, and from 24 –36 min (1.60 ⫾ 0.1 vs. 1.20 ⫾ 0.1 ng/mL) after the second GnRH injection on Day 12 (P ⬍ 0.05). Mean peak concentrations did not differ between control ewes and ewes treated with GnRH, but peak serum
Fig. 2. Peaks in serum concentrations of FSH (outlined with shading) and their associated emerging follicle waves in anestrous Western White Face ewes treated for 10 d (Days 0 –10; open rectangle on X-axis) with no sc implants (control; Panel A) or small (Panel B) or large (Panel C) silastic rubber implants containing 10% estradiol17. Data were normalized to the day of implant insertion (Day 0) in all ewes. The average curves representing the growth, static, and regression phases of follicular waves for all ewes in a group were normalized for each follicle wave to the mean day of wave emergence occurring either before or after implant insertion (Day 0; arrows indicates days of wave emergence; [7]). All FSH peaks for all ewes in a group are shown normalized to the mean day of occurrence of the apex of the FSH peak for each wave. The day of the apex of FSH peak was then plotted according to the mean day it occurred before or after implant insertion. Wave emergence may occur on the day of the zenith of the FSH peak or 1 d before or after the zenith [3]. For every FSH peak, serum concentration profiles were delimited by the encompassing nadirs of the FSH concentrations (hence the overlap of the data for adjacent peaks in some cases). Numbers in parenthesis are the number of ewes in a group with an FSH peak at that time. In ewes given small implants, 2 of 5 ewes had a follicular wave emergence on Day 1 from implant insertion (Day 0; indicated by †). Concentrations of FSH and follicle diameters are expressed as mean ⫾ SEM.
S.V. Seekallu et al. / Theriogenology 74 (2010) 912–921
FSH concentration on Day 10 was less than on Day 4 of the treatment in ewes given only estradiol implants (P ⬍ 0.05; Fig. 3). One control ewe and three ewes
917
given GnRH did not have a detectable increase in FSH at the time of the 2nd FSH peak seen for other ewes in these groups, and two control ewes and one ewe given GnRH did not have a detectable increase in FSH at the time of the 4th FSH peak detected for other ewes in these groups (Fig. 3).The proportion of ewes having peaks did not differ between groups (P ⬎ 0.05). In ewes given only estradiol implants, peak serum concentrations during treatment and during the time when follicular waves were blocked, had a range of 0.7–2.1 ng/mL (mean of 1.3 ⫾ 0.17 ng/mL). In ewes given GnRH, FSH peaks occurring at a similar time and that were accompanied by wave emergence, covered a range of 0.8 –2.4 ng/mL (mean of 1.5 ⫾ 0.17 ng/mL). 3.2.2. Antral follicle development No ewe given only estradiol implants had follicular waves emerge during treatment (Fig. 3). In ewes treated with GnRH, four ewes on Day 5 and five ewes on Day 8 had follicular wave emergence during the implant period. All ewes had normal follicular waves before implant insertion and after implant removal. The number of small follicles did not differ between control ewes and ewes treated with GnRH (25.81 ⫾ 2.0; P ⬎ 0.05). 3.3. Ovulations, cystic follicles, corpora lutea, and mean daily serum progesterone concentrations
Fig. 3. Peaks in serum concentrations of FSH (outlined with shading) and their associated emerging follicle waves in anestrous Western White Face ewes treated for 12 d (Days 0 –12; open rectangle on X-axis) with large silastic rubber sc implants containing 10% estradiol-17 (10 ⫻ 0.47 cm) without (panel A; n ⫽ 6) or with (panel B; n ⫽ 6) GnRH treatment for 6 d (Days 6 –12; checked rectangle on X-axis), starting on Day 6 of implant insertion (200 ng given IV every 4 h for 6 d). Data were normalized to the day of implant insertion (Day 0) in all ewes. The average curves representing the growth, static, and regression phases of follicular waves for all ewes in a group were normalized for each follicle wave to the mean day of wave emergence occurring either before or after implant insertion (Day 0; arrows indicates days of wave emergence; [7]). All FSH peaks for all ewes in a group are shown normalized to the mean day of occurrence of the apex of the FSH peak for each wave. The day of the apex of FSH peak was then plotted according to the mean day it occurred before or after implant insertion. For every FSH peak, serum concentration profiles were delimited by the encompassing nadirs of the FSH concentrations (hence the overlap of the data for adjacent peaks in some cases). In the control group, a follicular wave emerged and grew in only one ewe the day before implant insertion (indicated by †). In ewes treated with GnRH, four of six had a follicular wave emergence on Day 5 after implant insertion, and five of six on Day 8 (indicated by $). Numbers in parenthesis give the number of ewes in a group with an FSH peak at that time. Concentrations of FSH and follicle diameters are expressed as mean ⫾ SEM.
Cystic or luteinized follicles were detected in estradiol-treated ewes in both experiments. In Experiment 1, one ewe with a large implant formed a cystic follicle 3 d after implant insertion which luteinized and remained until the end of the experiment. Two ewes, treated with large implants, formed luteal structures with a lifespan similar to a normal CL. A short lifespan luteal structure (present for ⬍9 d) was developed by one ewe treated with a large implant and one ewe treated with a small implant. In Experiment 2, three ewes from each of the treatment groups formed a cystic follicle 3 d after implant insertion. These cystic follicles luteinized and remained until the end of the experiment. In Experiment 1, mean serum progesterone concentrations were higher in ewes that had luteal structures or cystic follicles compared to ewes that had no such structures (0.22 ⫾ 0.03 vs. 0.06 ⫾ 0.03 ng/mL; P ⬍ 0.05). Serum progesterone concentrations increased to a peak of 0.38 ⫾ 0.17 ng/mL on Day 9 after implant insertion in those ewes that had luteal structures or cystic follicles (n ⫽ 5). In Experiment 2, mean serum progesterone concentrations were higher in ewes that had cystic follicles compared to ewes that had no cystic follicles (0.21 ⫾ 0.05 vs. 0.08 ⫾ 0.05 ng/mL; P ⬍
918
S.V. Seekallu et al. / Theriogenology 74 (2010) 912–921
0.05). Serum progesterone concentrations increased significantly to a peak of 0.44 ⫾ 0.10 ng/mL on Day 9 after implant insertion in ewes that had cystic follicles (n ⫽ 6). 4. Discussion In the present study, the most profound effects of both sizes of estradiol implant were to block follicular wave emergence and the pulsatile secretion of LH. Clearly, both sizes of implant released sufficient estradiol to block LH pulses. There was some indication that the implants releasing estradiol used in this study caused some suppression of FSH secretion, particularly toward the end of the period of the implant treatment. However, perhaps the most salient finding for serum FSH concentrations from blood samples taken daily was that the range in peak concentrations in ewes given estradiol implants and in which follicular waves were blocked was the same as for the peaks in control ewes occurring at the same time, and that were followed by emergence of waves. It has been clearly established that the discrete FSH peaks that precede follicular waves in the ewe are the essential triggers for the orderly, rhythmic initiation of the waves, even though the concentration of FSH at the zenith of these peaks, and the nadirs in serum FSH concentrations between peaks, can vary widely [3,4,7]. Without these discrete peaks, follicular waves do not occur [7]. The findings in the present study supported our hypothesis that in the anestrous ewe the presence of pulsed LH secretion was required for the generation of the follicular waves that are triggered by peaks of FSH secretion. Restoration of LH pulses, by the GnRH treatment, reinitiated follicular wave emergence at the expected intervals and rhythm. Even though GnRH injections transiently increased serum FSH concentrations up to 1 h, as noted in blood samples collected every 12 min for 6 h, this increase was not enough to cause changes in peak serum FSH concentrations measured daily over the GnRH treatment period, in comparison to control ewes. The FSH peaks that precede follicular waves lasted 3– 4 d from nadir to nadir [3,28]. The dose of GnRH was very low and designed to cause physiological pulses of LH. In Experiment 2 of the present study, the FSH peaks, that were able to initiate follicular waves in the presence of restored pulsatile secretion of LH, were in the same range in concentration as those occurring in control ewes at a similar time, and in which follicular waves were blocked during the period of implant treat-
ment and absence of pulsed LH secretion. It is particularly intriguing that the peaks in serum concentrations of FSH at Day 4 in GnRH treated ewes gave rise to follicular wave emergence on Day 5, even though pulsatile LH (GnRH) support was not restored until Day 6. With the ultrasound equipment used, we were able to track follicular waves back to their emergence from the pool of small follicles, 2–3 mm in diameter. The size of this pool of follicles was not affected by treatment with estradiol 17-, as growth of antral follicles to a size of 2–3 mm is largely believed to be gonadotropin independent [29,30]. In the waves mentioned above, the FSH peak on Day 4 could have stimulated wave emergence from 2 mm follicles on Day 5 and follicles would have transitioned from 3 to 4 mm on Days 6 and 7 during the start of GnRH treatment and restored pulsed secretion of LH. Follicular waves normally emerge on the day of the zenith of the peak in serum FSH concentration, or 1 d before or after [3,5,6]. It should be noted that the peak concentration of FSH on Day 4 in control and GnRH treated ewes in Experiment 2 were virtually identical, whereas the FSH peaks on Day 4 were followed by follicular waves only in GnRH treated ewes. In Experiment 1, two ewes given small implants experienced wave emergence on Day 1 of treatment. We speculate that perhaps pulsed LH secretion was not immediately abolished in these ewes and supported wave emergence in response to the FSH peak on Day 1. In another recent study, the final growth of antral follicles destined to luteinize following prostaglandin and hCG treatment of ewes with ovarian autotransplants, did not appear to require pulsatile LH secretion [31]. In the latter study, LH secretion was suppressed with a GnRH antagonist. Final antral follicle growth appeared to have occurred in ewes given constant or pulsed infusion of LH, as well as in ewes receiving no supplemental LH. However, in the latter study, the growth of follicles during treatment was significant only at the higher of two doses of LH given by constant infusion. All ewes formed a CL in response to prostaglandin and hCG treatment. In another study, in prepubertal ewes, LH given as pulses or constant infusion was equally effective in inducing an LH surge [32]. The need for LH to initiate maturation of potentially ovulatory follicles was similar to that observed in pigs [33]. However, in contrast, in GnRH-antagonist treated ewes, ovulations were detected following infusions of only FSH [34]. In the present study, removal of pulsed LH secretion failed to allow emergence of follicular waves, but res-
S.V. Seekallu et al. / Theriogenology 74 (2010) 912–921
toration of LH pulses by GnRH treatment allowed follicular waves to occur in response to FSH peaks. Interestingly, Lacker [35] developed a mathematical model to predict follicle growth and ovulation rate in mammals and the role of estradiol as a regulatory agent [36]. As for rabbits [36], the present results support Lacker’s model of a role for estradiol in controlling the development of ovulatory sized follicles by way of regulation of gonadotropin (LH) secretion. Another interesting observation from the present study was that FSH peaks continued to occur in the absence of follicular wave development. If changes in the temporal pattern of secretion of products from follicular waves were important for regulating FSH peaks, based on the present results, estradiol could not be the hormone, as relatively constant serum concentrations resulted from the implant treatment. Inhibin also suppresses FSH secretion in the ewe; however, the specific role of inhibin in regulating FSH peaks has not been studied [37]. Peaks of FSH and the associated follicular waves are not regulated by inhibin in anestrous ewes, as serum concentrations of inhibin do not change with the pattern of follicular waves in the non-cycling ewe [12]. As no follicular waves emerged during the treatment period, in 8 of 10 ewes given estradiol releasing implants in Experiment 1, and all the control ewes in Experiment 2 of the present study, it is unlikely that secretory products of the follicles of a wave, could have entrained the second, third, and fourth FSH peaks during the period of implant treatment and immediately after treatment. We speculate that ovarian follicular feedback may not regulate the rhythm of the FSH peaks that precede follicular wave emergence in the ewe. In ovariectomized ewes, FSH peaks had a rhythm similar to the intact ewe [28]. Also, artificial peaks created by FSH injections between two endogenously driven peaks did not alter the rythmicity of FSH peaks [5]. It is intriguing that in our previous study in cyclic ewes [7], when similar sized estradiol implants were used, supra-physiological concentrations of estradiol had no significant effect on the LH secretory pattern. However, in cyclic ewes, estradiol implants producing higher concentrations of estradiol abolished pulsed LH secretion [38]. It has been shown that estradiol exerted a stronger negative feedback effect on LH secretion during seasonal anestrous compared to the breeding season [20,39 – 42]. There was no evidence of regular pulsed FSH secretion in blood samples taken from the jugular vein in the present study [10,11]. We inferred that the ovary does not need FSH pulses to generate follicular waves. The
919
large second FSH peak, in ewes given small implants in Experiment 1, was due to high concentrations of FSH in two ewes. We have no explanation for this, but FSH peak concentrations can be quite variable [4,6]. Progesterone concentrations produced by the cystic follicles or luteal structures formed after ovulation in Experiment 1 and 2, were very low compared to peak serum concentrations of progesterone during the physiologic luteal phase of cyclic ewes (3.58 ⫾ 0.18 ng/mL; [42]). Furthermore, there was no evidence that the presence of cystic follicles affected the pattern of emergence and growth of follicular waves in response to GnRH. We concluded that the supra-physiological concentrations of estradiol-17 created by estradiol-releasing implants suppressed follicular wave development by abolishing LH pulses in anestrous ewes. Restoration of LH pulses in implant treated ewes by frequent injections of GnRH allowed the re-establishment of follicular waves that were stimulated by peaks in secretion of FSH. In this study, we showed that LH pulses in the anestrous ewe were critical for the development of follicular waves. It is unclear whether secretory products from the follicles of a wave entrained the FSH peaks that initiated follicular waves in anestrous ewes.
Acknowledgements This research was supported by the Natural Sciences and Engineering Research Council of Canada to N.C.R. The authors thank Dr. A.F. Parlow of NIDDK/NHPP for the provision of reagents for the gonadotropin assays, and Ms. Susan J. Cook and Ms. Kim Tran for their help in blood sampling and radioimmunoassay. S.V.S., D.M.W.B., B.M.T., R.D., K.L.D., and E.T.B. were recipients of University of Saskatchewan Graduate Student Scholarships. K.C., K.A.E., and K.M.P. were recipients of summer undergraduate research studentships from NSERC (KC), and the Western College of Veterinary Medicine (KAE and KMP).
References [1] Schrick FN, Surface RA, Pritchard JY, Dailey RA, Townsend EC, Inskeep EK. Ovarian structures during the estrous cycle and early pregnancy in ewes. Biol Reprod 1993;49:1133– 40. [2] Souza CJ, Campbell BK, Baird DT. Follicular dynamics and ovarian steroid secretion in sheep during anoestrus. J Reprod Fertil 1996;108:101– 6. [3] Bartlewski PM, Beard AP, Cook SJ, Chandolia RK, Honaramooz A, Rawlings NC. Ovarian antral follicular dynamics and their relationships with endocrine variables throughout the oes-
920
[4]
[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
[15] [16]
[17]
[18]
S.V. Seekallu et al. / Theriogenology 74 (2010) 912–921 trous cycle in breeds of sheep differing in prolificacy. J Reprod Fertil 1999;115:111–24. Bartlewski PM, Beard AP, Cook SJ, Rawlings NC. Ovarian follicular dynamics during anoestrus in ewes. J Reprod Fertil 1998;113:275– 85. Duggavathi R, Bartlewski PM, Barrett DM, Gratton C, Bagu ET, Rawlings NC. Patterns of antral follicular wave dynamics and accompanying endocrine changes in cyclic and seasonally anestrous ewes treated with exogenous ovine follicle-stimulating hormone during the inter-wave interval. Biol Reprod 2004; 70:821–7. Bartlewski PM, Vanderpol J, Beard AP, Cook SJ, Rawlings NC. Ovarian antral follicular dynamics and their associations with peripheral concentrations of gonadotropins and ovarian steroids in anoestrous Finnish Landrace ewes. Anim Reprod Sci 2000; 58:273–91. Barrett DM, Bartlewski PM, Duggavathi R, Davies KL, Rawlings NC. Suppression of follicle wave emergence in cyclic ewes by supraphysiologic concentrations of estradiol-17beta and induction with a physiologic dose of exogenous ovine folliclestimulating hormone. Biol Reprod 2006;75:633– 41. Bartlewski PM, Beard AP, Rawlings NC. An ultrasound-aided study of temporal relationships between the patterns of LH/FSH secretion, development of ovulatory-sized antral follicles and formation of corpora lutea in ewes. Theriogenology 2000;54: 229 – 45. Duggavathi R, Bartlewski PM, Barrett DM, Rawlings NC. The temporal relationship between patterns of LH and FSH secretion, and development of ovulatory-sized follicles during the mid- to late-luteal phase of sheep. Theriogenology 2005;64: 393– 407. Wallace JM, McNeilly AS. Changes in FSH and the pulsatile secretion of LH during treatment of ewes with bovine follicular fluid throughout the luteal phase of the oestrous cycle. J Endocrinol 1986;111:317–27. Wheaton JE, Mullett MA, Cornelius SG. Plasma follicle stimulating hormone and luteinizing hormone patterns during the estrous cycle of ewes. Theriogenology 1984;21:989 –99. Evans ACO, Duffy P, Quinn KM, Knight PG, Boland MP. Follicular waves are associated with transient fluctuations in FSH but not oestradiol or inhibin-A concentrations in anoestrous ewes. Animal Science 2001;72:547–54. McNatty KP, Hudson NL, Henderson KM, Lun S, Heath DA, Gibb M, Ball K, McDiarmid JM, Thurley DC. Changes in gonadotrophin secretion and ovarian antral follicular activity in seasonally breeding sheep throughout the year. J Reprod Fertil 1984;70:309 –21. Walton JS, Evins JD, Fitzgerald BP, Cunningham FJ. Abrupt decrease in daylength and short-term changes in the plasma concentrations of FSH, LH and prolactin in anoestrous ewes. J Reprod Fertil 1980;59:163–71. Yuthasastrakosol P, Palmer WM, Howland BE. Release of LH in anoestrous and cyclic ewes. J Reprod Fertil 1977;50:319 –321. Jackson GL, Davis SL. Comparison of luteinizing hormone and prolactin levels in cycling and anestrous ewes. Neuroendocrinology 1979;28:256 – 63. McNatty KP, Gibb M, Dobson C, Thurley DC. Evidence that changes in luteinizing hormone secretion regulate the growth of the preovulatory follicle in the ewe. J Endocrinol 1981;90:375– 89. Karsch FJ, Foster DL. Sexual differentiation of the mechanism controlling the preovulatory discharge of luteinizing hormone in sheep. Endocrinology 1975;97:373–9.
[19] Karsch FJ, Cummins JT, Thomas GB, Clarke IJ. Steroid feedback inhibition of pulsatile secretion of gonadotropin-releasing hormone in the ewe. Biol Reprod 1987;36:1207–18. [20] Joseph IB, Currie WD, Rawlings NC. Effects of time after ovariectomy, season and oestradiol on luteinizing hormone and follicle-stimulating hormone secretion in ovariectomized ewes. J Reprod Fertil 1992;94:511–23. [21] Ravindra JP, Rawlings NC, Evans AC, Adams GP. Ultrasonographic study of ovarian follicular dynamics in ewes during the oestrous cycle. J Reprod Fertil 1994;101:501–9. [22] Duggavathi R, Bartlewski PM, Barrett DM, Rawlings NC. Use of high-resolution transrectal ultrasonography to assess changes in numbers of small ovarian antral follicles and their relationships to the emergence of follicular waves in cyclic ewes. Theriogenology 2003;60:495–510. [23] Rawlings NC, Jeffcoate IA, Rieger DL. The influence of estradiol-17beta and progesterone on peripheral serum concentrations of luteinizing hormone and follicle stimulating hormone in the ovariectomized ewe. Theriogenology 1984;22:473– 88. [24] Currie WD, Rawlings NC. Fluctuation in responsiveness of LH and lack of responsiveness of FSH to prolonged infusion of morphine and naloxone in the ewe. J Reprod Fertil 1989;86: 359 – 66. [25] Rawlings NC, Jeffcoate IA, Currie WD, Cook SJ. Control of the surge release of LH and FSH in oestradiol and progesterone treated ovariectomized ewes. Can J Anim Sci 1988;68:1089 –96. [26] Gitzen JF, Ramirez VD. PC-Pulsar-Pulsar for the IBM-PC: Operating instructions distributed with the PC-Pulsar program. 1988. [27] Clifton DK, Steiner RA. Cycle detection: a technique for estimating the frequency and amplitude of episodic fluctuations in blood hormone and substrate concentrations. Endocrinology 1983;112:1057– 64. [28] Duggavathi R, Bartlewski PM, Agg E, Flint S, Barrett DM, Rawlings NC. The effect of the manipulation of follicle-stimulating hormone (FSH)-peak characteristics on follicular wave dynamics in sheep: does an ovarian-independent endogenous rhythm in FSH secretion exist? Biol Reprod 2005;72:1466 –74. [29] Driancourt MA, Fry RC, Clarke IJ, Cahill LP. Follicular growth and regression during the 8 days after hypophysectomy in sheep. J Reprod Fertil 1987;79:635– 41. [30] Dufour J, Cahill LP, Mauleon P. Short- and long-term effects of hypophysectomy and unilateral ovariectomy on ovarian follicular populations in sheep. J Reprod Fertil 1979;57:301–9. [31] Campbell BK, Kendall NR, Baird DT. The effect of the presence and pattern of luteinizing hormone stimulation on ovulatory follicle development in sheep. Biol Reprod 2007;76:719 –27. [32] Keisler DH, Inskeep EK, Dailey RA. Roles of pattern of secretion of luteinizing hormone and the ovary in attainment of puberty in ewe lambs. Domest Anim Endocrinol 1985;2:123–32. [33] Guthrie HD, Bolt DJ, Cooper BS. Effects of gonadotropin treatment on ovarian follicle growth and granulosal cell aromatase activity in prepuberal gilts. J Anim Sci 1990;68:3719 –26. [34] Campbell BK, Dobson H, Baird DT, Scaramuzzi RJ. Examination of the relative role of FSH and LH in the mechanism of ovulatory follicle selection in sheep. J Reprod Fertil 1999;117: 355– 67. [35] Lacker HM. Regulation of ovulation number in mammals. A follicle interaction law that controls maturation. Biophys J 1981;35:433–54.
S.V. Seekallu et al. / Theriogenology 74 (2010) 912–921 [36] Meuli LE, Lacker HM, Thau RB. Experimental evidence supporting a mathematical theory of the physiological mechanism regulating follicle development and ovulation number. Biol Reprod 1987;37:589 –94. [37] Mann GE, McNeilly AS, Baird DT. Hormone production in vivo and in vitro from follicles at different stages of the oestrous cycle in the sheep. J Endocrinol 1992;132:225–34. [38] Barrett DM, Duggavathi R, Davies KL, Bartlewski PM, Bagu ET, Rawlings NC. Differential effects of various estradiol17beta treatments on follicle-stimulating hormone peaks, luteinizing hormone pulses, basal gonadotropin concentrations, and antral follicle and luteal development in cyclic ewes. Biol Reprod 2007;77:252– 62.
921
[39] Goodman RL, Bittman EL, Foster DL, Karsch FJ. The endocrine basis of the synergistic suppression of luteinizing hormone by estradiol and progesterone. Endocrinology 1981;109:1414 –7. [40] Karsch FJ, Legan SJ, Ryan KD, Foster DL. Importance of estradiol and progesterone in regulating LH secretion and estrous behavior during the sheep estrous cycle. Biol Reprod 1980;23:404–13. [41] Martin GB, Scaramuzzi RJ, Henstridge JD. Effects of oestradiol, progesterone and androstenedione on the pulsatile secretion of luteinizing hormone in ovariectomized ewes during spring and autumn. J Endocrinol 1983;96:181–93. [42] Bartlewski PM, Beard AP, Rawlings NC. An ultrasonographic study of luteal function in breeds of sheep with different ovulation rates. Theriogenology 1999;52:115–30.