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Ovary; |
Department of Veterinary Biomedical Sciences, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, Saskatchewan, Canada S7N 5B4
ABSTRACT
The mechanisms of ovulatory compensation following unilateral ovariectomy (ULO) are still not understood. In the present study, we investigated the short- and long-term effects of ULO in sheep using transrectal ovarian ultrasonography and hormone estimations made during the estrous cycle in which surgery was done, the estrous cycle 2 mo after surgery, and the 17-day period during the subsequent anestrus. The ULOs were done when a follicle in the first follicular wave of the cycle reached a diameter
5 mm, leaving at least one corpus luteum and one ovulatory-sized follicle in the remaining ovary. Ovulation rate per ewe was 50% higher in the ULO ewes compared with the control ewes at the end of the cycle during which surgery was performed, but it did not differ between groups at the end of the cycle, 2 mo later. This compensation of ovulation rate in ULO ewes was due to ovulation of follicles from the penultimate follicular wave in addition to those from the final wave of the cycle. Ovulation from multiple follicular waves appeared to be due to a prolongation of the static phase of the largest follicle of the penultimate wave of the cycle. Interestingly, the length of the static phase of waves was prolonged in ULO ewes compared with control ewes in every instance where the length of the static phase could be determined. Changes in follicular dynamics due to ULO were not associated with alterations in FSH and LH secretion. In conclusion, ovulatory compensation in ULO sheep involves ovulation from multiple follicular waves due to the lengthened static phase of ovulatory-sized follicles. These altered antral follicular dynamics do not appear to be FSH or LH dependent. Further studies are required to examine the potential role of the nervous system in the enhancement of the life span of the ovulatory-sized follicles leading to ovulatory compensation by the unpaired ovary in ULO sheep.
follicle, follicle-stimulating hormone, follicular wave, FSH, ovulation, ovulation compensation, sheep, ultrasonography, unilateral ovariectomy
After John Hunter's first demonstration in pigs in 1787, the phenomenon of compensation of litter size (ovulation rate) after unilateral ovariectomy (ULO) has been demonstrated in other species, including the mouse, rat, hamster, cow, and sheep [1]. ULO continues to be an important model for studying ovarian follicular development [2]. In a study by Martin et al. [3], it was shown that a transient increase in FSH secretion after ULO in suckled sows was responsible for ovarian compensatory hypertrophy. In contrast, Fry et al. [1] concluded that the transient increase in serum FSH concentration after ULO (reaching a peak by 12 h after surgery) is unlikely to be the causative factor for subsequent ovulation compensation. In a study in cattle, ovarian compensation was attributed to a transient increase in circulating FSH concentrations [4], whereas in another report no compensation [5] was noted following ULO. The inconsistencies in the effects of ULO and the possible involvement of altered FSH secretion could be due to the timing of surgeries relative to the stage of the estrous cycle. In addition, most of the studies on ULO in sheep were performed before the concept of follicular waves was proposed [6, 7].
The use of transrectal ovarian ultrasonography in sheep has allowed the description of three to four waves of antral follicular growth in each estrous cycle [6–8]. Each wave comprises one to three antral follicles that emerge or grow from a pool of follicles 2–3 mm in diameter to reach diameters 5 mm before regression or ovulation (last wave of the cycle only) [6–8]. The emergence of each follicular wave is induced by a transient increase in serum concentration of FSH [9].
In light of these recent data, questions such as whether changes in the dynamics of follicular waves and the preceding FSH peaks are involved in the ovulatory compensation in response to ULO remain to be answered. Thus, the objectives of the present study were 1) to follow, by ultrasonography, the ovarian antral follicular dynamics immediately after ULO; 2) to determine changes in the circulating concentrations of FSH throughout the experiment; and 3) to examine in sheep the long-term effects of ULO on the remaining ovary.
All of the experimental procedures were approved by the University of Saskatchewan Committee on Animal Care. Twelve adult, clinically healthy, Western White Face ewes (2–3 yr old, average body weight: 89 ± 8 kg) were used in the present study, which was conducted during the breeding (October to January) and the nonbreeding (June to July) seasons. All ewes received daily maintenance rations of alfalfa pellets, and water, hay, and cobalt iodized salt bars were available ad libitum. All ewes were checked for estrus daily using vasectomized, crayon-harnessed rams.
Transrectal Ultrasonography and Blood Sampling
Daily transrectal ultrasonography (scanning) was done in the early morning with high-resolution, real-time, B-mode ultrasonographic equipment (Aloka SSD-900; Aloka Co. Ltd.) connected to a 7.5-MHz transducer (UST-5821; Aloka). During each scanning session, the number, diameter, and relative position of all follicles 1 mm in diameter and corpora lutea (CL) were sketched onto ovarian charts, and all ovarian images also were recorded on high-grade video tapes (Fuji S-VHS, ST-120 N) using a super VHS-VCR (Panasonic AG-1978; Matsushita Electric, Mississauga, ON, Canada) equipped with digital frame memory. The initial period of transrectal scanning was done for the one ovulatory cycle in which surgery was performed. Scanning began on the day the ewes were marked by the rams and was terminated on the day when the ovulation at the end of the cycle was confirmed by ultrasonography. Scanning also was done for a second ovulatory cycle 2 mo after surgery. In the subsequent anestrous season, scanning was performed for a 17-day period. Ovulation was regarded as the disappearance of a large ovulatory-sized follicle (e.g., 5–7 mm in diameter) [10] that had been detected and followed by ultrasonography from just before estrus. Ovulation also was confirmed by the appearance of CL [11]. A follicular wave consists of one or more follicles that emerge and grow from 2–3 mm in diameter and reach 5 mm (growth phase). Follicles remain at their maximum diameter (static phase) from a period of time before regression to 2 or 3 mm in diameter (regression phase) or ovulation [7]. Emergence is restricted to a 24-h period [8]. The interwave interval is defined as the interval between the time of wave emergence (i.e., time at which the largest follicles of a wave were 2–3 mm in diameter) of two consecutive follicular waves.
Blood samples (10 ml) were collected by jugular venipuncture using vacutainers (Becton Dickinson, Rutherford, NJ) prior to each ultrasonographic examination. Additional blood samples (5 ml) were also collected every 6 h from 1 day before to 1 day after surgery. Further, one session of intensive blood sampling every 12 min for 6 h (between 0900 and 1500 h) via indwelling jugular catheters (vinyl tubing; 1.00 mm inner diameter x 1.50 mm outer diameter; 530070; Biocorp Australia Propriety Ltd., Huntingdale, Australia) was done on Day 10 after ovulation in the cycle in which surgery was performed. Blood samples were incubated at room temperature 12–16 h, and serum was harvested by centrifuging blood samples at 1500 x g (3000 rpm) for 10 min. All serum samples were stored at –20°C until radioimmunoassay (RIAs).
The ewes were divided into two groups (n = 6 per group) for ULO or sham operation. They were taken off food and water for 12 h when one or more 4-mm follicles, which grew from 2–3 mm in diameter, were observed by ultrasonography during the first follicular wave of the cycle. Surgeries were performed when a follicle in the first follicular wave of the cycle reached a diameter 5 mm. Surgery in the ULO ewes was designed such that the remaining ovary had at least a corpus luteum and one antral follicle 5 mm in diameter; this ensured that the dynamics of the first follicular wave and corpus luteum of the cycle were not affected by treatment in the ULO ewes. Ewes were housed inside with lighting set to match ambient day length for 24 h before the scheduled surgery, and they were returned to paddocks 48 h after surgery. All surgeries were performed within 3 h of ultrasonographic examination, as described previously [11]. Briefly, general anesthesia was induced by intravenous injection of 2.5% thiopental sodium (25 mg/kg; Pentothal; Abbott Laboratories Ltd.) in sterile water and maintained by 3%–5% halothane (Halothane; Halocarbon Laboratories). Ovaries were exteriorized by midventral laparotomy. In the ULO ewes, the ovary designated for removal in accordance with the criteria above was excised. In the control ewes, both ovaries were left intact. After closing the incision, ewes were kept under close observation until they recovered from the general anesthesia.
FSH concentrations were measured in blood samples collected daily, every 6 h (from 1 day before to 1 day after surgery) and by intensive bleeding (on day 10 after ovulation). Progesterone and LH concentrations were measured in blood samples collected daily and intensively, respectively. The double-antibody RIAs used to determine serum concentrations of FSH, LH, and progesterone have been described elsewhere [12–14]. The sensitivities of the assays, defined as the lowest concentrations of standard capable of significantly displacing labeled hormones from the antiserum (unpaired t-test), were as follows: FSH and LH, 0.1 ng/ml; progesterone, 0.03 ng/ml. The ranges of standards used in the FSH, LH, and progesterone assays were 0.13–16.0 ng/ml, 0.06–8.0 ng/ml, and 0.10–10 ng/ml, respectively. For FSH reference sera with mean concentrations of 1.27 or 3.43 ng/ml, the intraassay and interassay coefficients of variation (CVs) were 5.6% and 6.3% or 6.9% and 4.8%, respectively. For reference sera with mean LH concentrations of 0.13 or 0.99 ng/ml, the intraassay and interassay CVs were 9.3% and 8.8% or 10.1% and 7.0%, respectively. For reference sera with mean progesterone concentrations of 0.22 or 0.63 ng/ml, the intraassay and interassay CVs were 7.1% and 9.2% or 5.5% and 7.9%, respectively. Peaks in daily serum concentrations of FSH were determined using a cycle detection computer program [15]. The PC-PULSAR program [16] was used to estimate LH pulse frequency and amplitude as well as mean and basal serum concentration, as described previously [17].
All statistical analyses were done using SigmaStat Statistical Software (Version 2.0 for Windows, 1997; Chicago, IL). Differences between the two groups of ewes in single point observations, such as ovulation rate, were analyzed by Student t-test. Comparisons of multiple time point observations, such as hormonal profiles and follicular dynamics, were analyzed by two-way repeated-measures ANOVA. Multiple comparisons were made by the method of Fisher least significant difference (LSD). All results are expressed as means ± SEM.
Estrous Cycle and Ovulation in ULO and Control Ewes
At the beginning of the estrous cycle during which the surgery was performed, the ewes ovulated at a rate (2.0 ± 0.2 follicles) that is normal for the breed [7]. Surgery was performed on Days 2.2 ± 0.2 and 2.5 ± 0.2 of the estrous cycles of the ULO and sham-operated (control) ewes, respectively (Day 0 = day of ovulation). The length of the estrous cycle did not differ between the ULO and control ewes (17.2 ± 0.3 days and 18.0 ± 0.5 days, respectively; P > 0.05). However, the mean number of ovulations per ewe at the end of the cycle following surgery was 50% higher in the ULO ewes with one intact ovary compared with control ewes (3.2 ± 0.2 and 2.2 ± 0.2, respectively; P < 0.01).
During the estrous cycle 2 mo after ULO, the length of the estrous cycle (17.6 ± 0.4 days and 18.2 ± 0.4 days in ULO and control ewes, respectively) and the ovulation rate per ewe at the end of the cycle (2.0 ± 0.3 follicles and 1.8 ± 0.2 follicles in ULO and control ewes, respectively) did not differ between the groups.
Antral Follicular Dynamics in ULO and Control Ewes
Analysis of the ultrasonographic data revealed that the mean daily numbers of small (1 mm but 
3 mm in diameter) and medium (4 mm in diameter) follicles per ovary did not differ between ULO and control ewes (Fig. 1, A and B) throughout the estrous cycle during which the surgery was performed. However, the ULO ewes had consistently greater numbers of large (5 mm in diameter) follicles per ovary compared with control ewes starting from Day 3 after surgery until ovulation at the end of the cycle (Fig. 1C).
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Likewise, during the estrous cycle 2 mo after surgery, the mean daily numbers of small and medium follicles per ovary did not differ between ULO and control ewes (Fig. 2, A and B). The mean daily numbers of large follicles per ovary were higher in ULO ewes than control ewes from Days 2–16 of the estrous cycle (Day 0 = day of ovulation; P < 0.05; Fig. 2C).
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Similarly, during the 17-day period during midanestrus following surgery, the mean daily numbers of small and medium follicles per ovary did not differ (Fig. 3, A and B; P > 0.05) between the two groups of ewes, whereas the ULO ewes had a greater number of large follicles per ovary compared with control ewes (Fig. 3C; P < 0.05).
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Growth Characteristics of the Follicular Waves in ULO and Control Ewes
The mean length of the growth and regression phases of the largest follicle of the waves and mean days of emergence of follicular waves during the estrous cycle in which surgeries were performed did not vary between ULO and control ewes (Table 1). However, the static phase (the period during which the follicles in a follicular wave maintain ovulatory diameter 5 mm) of the largest follicle of follicular waves was longer in the ULO ewes compared with that in control ewes (Table 1). Intriguingly, this characteristic of a prolonged static phase was maintained in the ULO ewes even during the estrous cycle 2 mo after surgery (Table 1) and also during the subsequent anestrous season (Table 1). Additionally, the number of antral follicles growing to reach an ovulatory diameter (5 mm) in each wave did not differ (P > 0.05) between the two groups of ewes in all three periods of the study. The mean numbers of follicles of ovulatory diameter for all waves in ULO and control ewes, respectively, were: 2.1 ± 0.6 vs. 2.7 ± 0.7 during the estrous cycle during which surgery was performed; 1.6 ± 0.3 vs. 1.7 ± 0.5 during the estrous cycle 2 mo after surgery; and 1.3 ± 0.4 vs. 1.3 ± 0.6 during anestrus subsequent to surgery.
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Follicles Ovulating from the Penultimate and Final Follicular Wave of the Cycle
In ULO ewes, in the cycle in which surgery was performed, 86% of the ovulatory-sized follicles from the penultimate wave of the cycle ovulated with 92% of such follicles from the final wave. However, none of the penultimate wave follicles ovulated in control ewes, but 100% of ovulatory-sized follicles from the final wave ovulated. Surprisingly, even 2 mo following surgery, when the ovulation rate in the ULO ewes was equal to that of control ewes, 50% of the ovulatory-sized follicles from the penultimate wave ovulated with 70% of such follicles from the final wave of the cycle in ULO ewes. In control ewes, no follicles ovulated from the penultimate wave, but 90% of ovulatory-sized follicles from the final wave ovulated.
Serum Concentrations of FSH, LH, and Progesterone in ULO and Control Ewes
During the estrous cycle in which surgery was performed, the mean daily serum concentrations of FSH did not differ between ULO and control ewes (Fig. 4A; P > 0.05). Blood samples collected every 6 h around the time of surgery revealed a sharp but nonsignificant increase in the serum FSH concentration at 12 h after surgery in the ULO ewes (Fig. 4A, inset), which declined to control levels by 18 h after surgery. Application of the cycle detection algorithm revealed that there were transient peaks in serum FSH concentrations preceding the emergence of follicular waves. However, both interpeak interval (in days; waves 1 to 2: 4.5 ± 0.4 vs. 4.9 ± 0.3; waves 2 to 3: 4.1 ± 0.3 vs. 3.8 ± 0.5; waves 3 to 4: 4.0 ± 0.6 vs. 4.9 ± 0.3) and peak amplitude (in ng/ml; wave 1: 2.2 ± 0.3 vs. 2.1 ± 0.3; wave 2: 1.6 ± 0.3 vs. 1.8 ± 0.3; wave 3: 1.0 ± 0.3 vs. 0.9 ± 0.3; and wave 4: 1.0 ± 0.3 vs. 1.1 ± 0.3) did not differ between ULO and control ewes (P > 0.05). There was no difference in the mean (1.8 ± 0.2 ng/ml vs. 1.9 ± 0.2 ng/ml) and basal (1.5 ± 0.2 ng/ml vs. 1.6 ± 0.2 ng/ml) concentrations of FSH in the serum samples collected intensively on Day 10 after ovulation between the control and ULO ewes. Likewise, the mean (0.25 ± 0.1 ng/ml vs. 0.29 ± 0.2 ng/ml) and basal (0.20 ± 0.20 ng/ml vs. 0.21 ± 0.2 ng/ml) concentrations of LH, and the frequency (1.85 ± 0.5 vs. 1.57 ± 0.3 pulses per 6 h) and amplitude (0.58 ± 0.5 ng/ml vs. 0.57 ± 0.3 ng/ml) of LH pulses did not differ between the two groups. However, the mean daily serum concentration of progesterone showed significant differences between ULO and control ewes (Fig. 5A). ULO ewes had lower progesterone concentrations from Days 7 to 11 of the estrous cycle in which surgery was done compared with control ewes (P < 0.05). During the estrous cycle 2 mo after the surgeries, the mean daily serum concentrations of FSH (Fig. 4B) and progesterone (Fig. 5B) did not differ between ULO and control ewes. Similarly, during the subsequent anestrus, the mean daily serum concentrations of FSH (Fig. 4C) did not differ between ULO and control ewes.
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Unilateral ovariectomy followed by ovarian compensation, in terms of ovulation rate and fecundity, is an intriguing phenomenon for which the regulatory mechanisms remain to be understood. In the present study, we investigated both immediate and long-term dynamic changes in the numbers and growth patterns of ovarian antral follicles in the unpaired ovary after ULO. Lack of homogeneity in the time of the estrous cycle and ovarian follicular status at which ULO was performed could affect ovarian compensation. To rule out this possibility, we chose to perform surgeries on the day when the follicle of the first wave of the cycle reached an ostensibly ovulatory diameter 5 mm. In addition, we were careful to have at least one CL and an ovulatory-sized follicle in the remaining ovary to avoid disruption of follicular wave dynamics and the estrous cycle. On the day we chose to do surgery, we expected serum FSH concentration to be basal [7–9], which would enable us to pick up subtle changes in FSH secretion due to ULO.
Compared with the control ewes, ULO in the present study resulted in a 50% increase in ovulation rate at the end of the cycle during which surgery was performed. This overcompensation is in agreement with the previous data from sheep [18], rats [19], and hamsters [20].
We used transrectal ovarian ultrasonography to characterize changes in antral follicles. The lack of difference in mean daily numbers of small (1 mm but 3 mm in diameter) and medium (4 mm in diameter) sized follicles between the control and ULO ewes led us to conclude that follicular growth up to medium size was not affected by the removal of one of the ovaries. This is in contrast to data from the rat, where it was suggested that decreased atresia or increased follicular recruitment into the growth pool explained ovarian compensation after ULO [21, 22]. However, in mice it was shown that even though follicular recruitment was not altered by ULO, the entry of medium-sized follicles to the larger size category appeared to be enhanced by surgery [2]. In the present study, the daily numbers of large ovulatory-sized follicles were increased by 2-fold in the ULO ewes relative to the control ewes. This increased number of large follicles did not appear to be due to increased numbers of growing follicles, as the recruitment of antral follicles into follicular waves was not affected in the ULO ewes. In this respect, the growth rate of the largest follicle of the waves did not differ between the control and ULO ewes. However, the static phase, during which a follicle is maintained at maximum diameter, was significantly longer in the ULO ewes compared with the control ewes. The prolonged static phase of ovulatory-sized follicles resulted in the ovulation of follicles from the penultimate wave along with those from the final wave of the cycle. Enhanced survival of follicles has been previously inferred in rodents, based on the increased numbers of large follicles [2, 21, 22]. Our data provide evidence for the exclusive enhancement of the survival of large ovulatory-sized follicles, as we monitored individual follicles on a daily basis by transrectal ultrasonography.
Additionally, we studied the long-term effects of ULO on the follicular dynamics of cyclic ewes by reexamining both control and ULO ewes 2 mo after surgery. Although the numbers of follicles in different categories during the cycle 2 mo after surgery were similar to those during the surgery cycle, the ovulation rate in the ULO ewes declined back to what is normal for the breed and comparable to the control ewes. The phenomenon of additional ovulations from the penultimate wave of the cycle was sustained even at 2 mo after surgery in the ULO ewes. The growth characteristics of the follicles growing into waves in the ULO ewes were similar to those in control ewes, with the exception of the prolonged static phase. This feature of a prolonged static phase of the largest follicle of the wave was maintained even during the subsequent anestrous period in the ULO ewes, suggesting that the unpaired ovary tries to maintain the number of ovulatory-sized follicles for the species by prolonging the lifespan of such follicles.
There appears to be disagreement in the literature with regards to the role of FSH in the compensation of the ovulation rate following ULO. Although most studies from rodents [23] appear to suggest that a transient rise in FSH concentrations is enough to increase the ovulation rate in the unpaired ovary upon ULO, the studies from large ruminants like sheep contradict this assumption [1]. In the present study, we demonstrated that there was no difference between the ULO and control ewes in FSH concentrations, except for a distinct and short-lived but nonsignificant increase in the FSH concentration by 6 h after surgery. Such a transient elevation of FSH concentrations, albeit lower than ULO ewes, also was observed in the control ewes. This observation indicated that the transient elevation in FSH concentrations might have been due to handling of the ovaries and/or anesthesia. A similar conclusion was made in a study on ULO in prepubertal gilts [24]. In the present study, none of the characteristics of the transient FSH peaks associated with follicular wave emergence differed between the two groups of ewes. These data are consistent with the data from previous reports in ruminants [25], except that none of them addressed the transient FSH peaks. It has been suggested that increased gonadotropin secretion due to decreased negative feedback from estrogen and inhibin [26] is responsible for ovarian compensation after ULO. However, the FSH concentrations remained largely unaltered following ULO in the present study. In addition, in a previous study, it was shown that hypophysectomized ULO ewes supplemented with physiological amounts of exogenous gonadotropins showed ovarian compensation [1]. The possibility of a greater utilization of a fixed amount of gonadotropins by the unpaired ovary was ruled out by a study in mice [23]. In the present study, the static phase, during which follicles are dependent on LH [27], was prolonged in the ULO ewes, which resulted in ovulations from both penultimate and final follicular waves. However, there were no changes in LH secretion in the ULO ewes, as determined by intensive blood sampling in the present study. Several previous studies also reported that ULO was not followed by any changes in serum LH concentrations in rodents and large ruminants alike [1, 3, 5, 18, 23, 26]. Thus, altered gonadotropin secretion as the mechanism for ovulatory compensation following ULO is not supported by the results of the present study. However, the possibility of the existence of an isoform of FSH that is not detected by our RIA can not be completely ruled out. Ovulations from penultimate and final follicular waves have been described previously for normal cycles in prolific Finn ewes and also for the nonprolific breed used in the present study when treated with medroxyprogesterone acetate and prostaglanding F2
[7, 28, 29]. In all these studies, the prolonged life span of the ovulatory-sized follicles and the increased ovulation rate were attributed to reduced serum concentrations of progesterone [30]. In the present study, the ULO ewes had lower levels of progesterone than control ewes during the cycle in which surgery was performed, but not during the estrous cycle 2 mo later. With these data, it is plausible that reduced progesterone concentration in the ULO ewes immediately after surgery was responsible for the overcompensation by the remaining ovary. However, the idea of reduced progesterone concentration is not sufficient to explain how ULO ewes continue to ovulate from multiple follicular waves to maintain the ovulation rate normal for the breed.
An additional possible mechanism could be that ovulatory-sized follicles in the unpaired ovary survived longer through some direct neural mechanisms [23]. It is not outside the realm of possibility that neuronal connections may help one ovary know what is happening on the other ovary. Neuronal components could be involved in regulating the ovulation rate for a species. Afferent and efferent adrenergic neural elements appear to be involved in ovarian compensatory hypertrophy in rats [31]. Adrenergic nerves appear to play a role in follicular dynamics in ovaries undergoing compensatory hypertrophy but are not necessary for the increase in the number of healthy ovulatory follicles in guinea pigs [32]. On the contrary, Wylie et al. [33] indicated that changes in neural signals between the hypothalamus and ovaries were not involved in compensatory ovarian response to ULO in rats. Further studies with concomitant ULO and ablation of the nervous supply to the ovary are necessary to resolve the mechanisms of the enhanced survival of ovulatory-sized follicles in ULO ewes without elevated serum FSH concentrations.
The removal of 50% of follicles resulted in the reduction of the expected number of estrous cycles by 25% in mice [2]. The premature cessation of estrous cycles in ULO mice supports the view that the number of cycles throughout the lifespan is dependent on the available follicular reserve [2]. However, the present study was not extended long enough to test this hypothesis in the sheep model. In conclusion, the present study provides evidence that the increased length of the static phase of ovulatory-sized follicles, leading to ovulations from multiple follicular waves, provides a mechanistic explanation for ovulatory compensation in the ewe following ULO. This mechanism did not appear to be dependent on the elevation of FSH secretion. The role of potential direct neuronal mechanisms at the ovary needs to be tested further.
ACKNOWLEDGMENTS
The authors thank Dr. Parlow and the National Institute of Diabetes and Digestive and Kidney Diseases for hormones for RIA; Ms. Susan Cook and Mr. Tim Hegan for excellent technical support; and the Animal Care Crew for care and management of the sheep.
FOOTNOTES
1Supported by the Natural Sciences and Engineering Research Council, Canada (N.C.R.). R.D., D.M.W.B., and E.B. were supported by a University of Saskatchewan graduate student scholarship; P.M.B. was supported by a Saskatchewan Health Services Utilization and Research Commission postdoctoral fellowship. 
Correspondence: 2FAX: 306 966 7376; e-mail: norman.rawlings{at}usask.ca
3Current address: Institut de Génétique et de Biologie Moléculaire et Cellulaire, 1 rue Laurent Fries, 67404 Illkirch, France
Received: 11 July 2007.
First decision: 10 September 2007.
Accepted: 16 November 2007.
REFERENCES
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