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Regular Article |
a Department of Biomedical Sciences, Cornell University, Ithaca, New York 14853
ABSTRACT
Low molecular weight insulin-like growth factor binding proteins (IGFBPs), particularly IGFBP-4, are believed to inhibit the actions of insulin-like growth factors (IGFs). We showed previously that ovarian follicular dominance in cattle is associated with the presence of a protease that degrades IGFBP-4. To test the hypothesis that specific IGFBP-4 proteolysis is associated with selection of the dominant follicle, we induced codominant follicles (co-DFs) during the first follicular wave of the estrous cycle. The ovaries of Holstein heifers were examined twice daily by ultrasonography; when the largest follicle reached 6 mm in diameter, saline (control, n = 5) or 2 mg of recombinant bovine (rb) FSH (FSH, n = 5) was injected i.m. every 12 h for 48 h. Follicular fluid was collected by aspiration from the two largest follicles/heifer 12 h after the last injection. IGFBPs in follicular fluid were quantified by Western ligand blotting/phosphorimaging. IGFBP-4 protease activity was measured by incubating follicular fluid with recombinant human (rh) IGFBP-4 substrate, followed by ligand blotting/phosphorimaging to quantify the percent of substrate loss and Western immunoblotting to detect specific proteolytic fragments. Co-DFs of FSH heifers did not differ (P > 0.05) from the single dominant follicle of controls in size, or in concentration of progesterone or level of IGFBP-4 in follicular fluid. In contrast, the largest subordinate follicle of control heifers was smaller, with lower progesterone and higher IGFBP-4 in the follicular fluid (P < 0.05). Concentrations of estradiol in follicular fluid were high in dominant follicles, intermediate in co-DFs, and low in subordinate follicles (P < 0.05). IGFBP-4 protease activity in co-DFs was similar (P > 0.05) to that of dominant follicles, but fourfold higher (P < 0.05) than that of subordinate follicles. The results strongly suggest that an FSH-dependent IGFBP-4 protease is associated with selection of the dominant follicle in cattle.
follicle, follicular development, FSH, growth factors, ovary
INTRODUCTION
A critical transition in ovarian follicular development is the selection of a dominant follicle, capable of ovulating, from a cohort of synchronously growing antral follicles. However, little is known about mechanisms and factors that regulate the selection and further differentiation of dominant follicles. The growth of large follicles in cattle occurs in a wave-like fashion, with two or three successive waves per estrous cycle [1–4]. Follicular waves consist of the contemporaneous emergence of six to seven follicles 5 mm in diameter, in association with small elevations in circulating FSH [5]. Newly recruited follicles grow in parallel for a brief period of time, until the largest follicle in the wave reaches a critical size (about 8.5 mm in diameter), and then deviation in growth rates between the two largest follicles begins [6, 7]. At deviation, the largest follicle continues to grow and becomes dominant, whereas the remaining follicles in the cohort cease growing and become subordinate follicles.
Careful examination of follicular dynamics in conjunction with endocrine profiles has shown a close temporal association between deviation in growth rates and declining levels of circulating FSH [8]. Although this observation suggests an increased sensitivity of the dominant follicle to FSH, the molecular and cellular mechanisms that allow the largest follicle to thrive in an environment of decreasing concentrations of FSH remain elusive. As soon as the presumptive dominant follicle is detected as slightly larger (size difference 
1 mm) than the largest subordinate follicle, it has a greater capacity to produce estradiol [9, 10]. However, the increased estradiol production is not accompanied by parallel increases in gonadotropin receptors [9] or in levels of mRNA for gonadotropin receptors, P450 17
-hydroxylase or P450 aromatase [10]. Therefore, the putative increased sensitivity of the future dominant follicle to FSH could be due to enhanced availability of growth factors that are known to increase the responsiveness to FSH. The central role of insulin-like growth factors (IGFs) as amplifiers of gonadotropin actions is well documented [11, 12]. The actions of IGFs are exerted through their receptors and are modulated by the presence of IGF-binding proteins (IGFBPs). Follicular growth is associated with low intrafollicular concentrations of low molecular weight IGFBPs (IGFBP-2, -4, and -5) and, conversely, follicular atresia is associated with high concentrations of these IGFBPs [13, 14]. In addition, FSH treatment in vivo causes an increase in the number of large (
8 mm in diameter), estrogen-active follicles that have little or barely detectable levels of low molecular weight IGFBPs, including IGFBP-4 [15]. These observations lend support to the hypothesis that either timely and highly localized expression, or post-translational modifications (namely proteolytic degradation), or both, of the low molecular weight IGFBPs regulate the bioavailability of IGFs, and ultimately determine follicular fate (i.e., continued growth or atresia). Indeed, we have shown that in cattle, IGFBP-4 proteolytic activity is higher in dominant, estrogen-active follicles than in subordinate follicles of the same cohort, as early as Day 2 of the follicular wave [16, 17].
To begin to explore the role of proteolytic processing of the low molecular weight IGFBPs in follicular selection, we tested the hypothesis that selection of the dominant follicle from a cohort of growing follicles involves an FSH-dependent IGFBP-4 protease. We used an experimental model of codominant follicles induced by low doses of FSH to assess the FSH-dependence of intrafollicular IGFBP-4 proteolytic activity and its association with follicular selection for dominance. A preliminary report of these data has been presented elsewhere [18].
MATERIALS AND METHODS
Animals and Experimental Protocols
Holstein heifers with regular estrous cycles were used in accordance with procedures approved by the Cornell University Animal Care and Use Committee (protocol 86-214-99). Luteolysis, a follicular phase, and ovulation were induced by injecting heifers with prostaglandin F2 and the ovaries of each heifer were examined twice daily by ultrasonography, as described in the companion paper [17]. Animals were observed in estrus (Day 0 of the cycle) within 3–5 days after prostaglandin F2, and the experiment was carried out during the first wave of follicular development of the next estrous cycle. The day of emergence of the first follicular wave (on average, Day 1 of the cycle) was designated as Day 0 of the wave and was retrospectively identified as the last day on which the dominant follicle was 4 or 5 mm in diameter.
Codominant follicles (co-DFs) were induced during the first follicular wave by injecting small amounts of FSH [19] to prevent the spontaneous decline in serum FSH concentrations that occurs during selection and the establishment of follicular dominance [20]. When the largest follicle of the first follicular wave reached 6 mm in diameter, saline (control, n = 5) or 2 mg recombinant bovine (rb) FSH (lot number IG-07-11088; Granada Biosciences, Marquez, TX; FSH, n = 5) was injected i.m. every 12 h for 48 h. Follicular fluid was collected by ultrasound-guided follicular aspiration from the two largest follicles/heifer 12 h after the last injection, as previously described [17]. Samples were centrifuged and stored at -80°C for later determinations. The interval between PGF2 injection and sample collection was not different (P > 0.05) between control and FSH-treated animals (mean ± SEM; control group = 196 ± 12 h; FSH group = 185 ± 12 h).
Analysis of IGFBP-4 Proteolytic Activity
Levels of an intrafollicular protease activity for IGFBP-4 were assessed by incubating 5 µl of follicular fluid plus substrate for 18 h at 37°C in a solution of 20 mM Tris (pH 7.5) containing 137 mM NaCl (TBS) and 0.1% BSA (final volume, 15 µl). Recombinant human (rh) IGFBP-4 (Austral Biologicals, San Ramon, CA) was used as substrate. After incubation, the extent of substrate loss in the assay samples was determined by SDS-PAGE, followed by Western ligand blotting/phosphorimaging to quantify the percent of substrate loss, and specific proteolytic fragments were detected by Western immunoblotting.
Western Ligand and Immunoblot Analysis
Western ligand blot analysis was performed as described in the companion paper [17]. Immunoblot analysis to detect the presence of specific proteolytic fragments of IGFBP-4 was performed on the same blots previously subjected to ligand blot analysis, according to the procedure described in the companion paper [17].
Phosphorimaging and Autoradiography
Phosphor screen autoradiography of ligand blots (72–96 h) was performed as described in the companion paper [17]. For each follicular fluid sample, proteolytic activity was expressed as percent of substrate loss after 18 h of incubation relative to the same sample without incubation. Relative abundance (%) of IGFBPs was expressed as the ratio 100 x IIGFBPx/IIGFBP, where IIGFBPx = intensity (arbitrary units) of the particular IGFBP (either IGFBP-2, -3, -4, or -5) band, and IIGFBP = sum of intensity (arbitrary units) for all IGFBPs (-2, -3, -4, and -5) for samples incubated for 0 h of each individual follicle.
Radioimmunoassays and Protein Determinations
Duplicate aliquots (0.1–5.0 µl) of follicular fluid from each dominant and subordinate follicle were assayed without extraction for estradiol and progesterone as described previously [10]. Total protein concentrations in samples of follicular fluid were measured in duplicate according to the Bradford method [21] using reagents purchased from Bio-Rad (Melville, NY).
Statistical Analysis
Follicular diameters were compared by repeated measures ANOVA using the MIXED procedure of SAS (SAS Institute, Cary, NC). Concentrations of steroids and protein in follicular fluid, relative abundance of IGFBP-4, and IGFBP-4 degradation were analyzed by ANOVA with a nested design to evaluate the effects of treatment, animal (nested within treatment), follicle type (largest or second largest), and the interaction of treatment by follicle type using the GLM procedure of SAS. Bartlett test was used to test for heterogeneity of variance. When appropriate, logarithmic or square root transformations were used to yield variance homogeneity. After ANOVA, individual means were compared by Scheffé multiple comparison test. Values are presented as means ± SEM of untransformed variables.
RESULTS
Follicular Dynamics and Intrafollicular Steroid Concentrations in Control and rbFSH-Treated Heifers
Follicular dynamics in control and rbFSH-treated heifers are shown in Figure 1. As expected, the injection of small doses of rbFSH induced co-DFs, as evidenced by the similar growth rates and maximum diameters at the time of aspiration (P > 0.05) of co-DFs in rbFSH-treated heifers and the single dominant follicle in control heifers. In contrast, the second largest follicle in controls stopped growing by 60 h after the emergence of the follicular wave and started regressing by 84 h, becoming a clearly subordinate follicle (Fig. 1). Concentrations of estradiol in follicular fluid were high in the single dominant follicle and low in subordinate follicles from controls; intermediate concentrations were observed in co-DFs from rbFSH-treated heifers (P < 0.05; Fig. 2). Concentrations of progesterone were similar (P > 0.05; Fig. 2) in dominant follicles and co-DFs, but lower (P < 0.05) in subordinate follicles. Protein concentration was measured in follicular fluid to verify that potential differences in proteolytic activity in samples obtained by ultrasound-guided follicular aspirations were not due to unequal protein concentrations in the samples. No differences (P > 0.05) were observed in concentrations of protein in follicular fluid among the various follicle categories (controls: dominant follicles = 101 ± 10, subordinate follicles = 95 ± 9; FSH: co-DF1 = 100 ± 13, co-DF2 = 96 ± 6 mg/ml, mean ± SEM).
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IGFBPs and IGFBP-4 Proteolytic Activity in Follicular Fluid of Follicles from Control and rbFSH-Treated Heifers
Relative abundance of IGFBPs Relative abundance of IGFBPs in follicular fluid was assessed by ligand blot analysis. Figs. 3A and 4A show representative ligand blots of follicular fluid samples from the dominant and subordinate follicles of controls and from co-DFs of rbFSH-treated heifers, respectively. Follicular fluid from the single dominant follicle of controls or the co-DFs of rbFSH-treated heifers had high levels of IGFBP-3, but low or barely detectable levels of low molecular weight IGFBPs, including IGFBP-4 (Fig. 3A, lanes 4–5; Fig. 4A, lanes 4–5 and 9–10, respectively). In contrast, follicular fluid from subordinate follicles of controls contained readily detectable levels of low molecular weight IGFBPs, including IGFBP-4 (Fig. 3A, lanes 9–10). Quantitative results derived from phosphorimaging of ligand blots revealed higher (P < 0.05) abundance of low molecular weight IGFBPs (IGFBP-2, -4, -5) and lower (P < 0.05) abundance of IGFBP-3 in follicular fluid from subordinate follicles vs. the single dominant follicle of controls (Fig. 5 and Fig. 6, top panel). In contrast, the co-DFs of FSH-treated heifers had low levels of low molecular weight IGFBPs, similar (P > 0.05) to those of the single dominant follicle of controls (Fig. 5 and Fig. 6, top panel). The most pronounced difference was observed, interestingly, in IGFBP-4; subordinate follicles of controls had about a sixfold higher (P < 0.05) abundance of IGFBP-4 than the single dominant follicle of controls or the co-DFs of FSH-treated heifers (Fig. 6, top panel).
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IGFBP-4 proteolytic activity IGFBP-4 proteolytic activity in follicular fluid was assessed by incubating 5 µl of sample with 50 ng rhIGFBP-4, followed by Western ligand blotting/phosphorimaging to determine substrate loss and Western immunoblotting to detect specific proteolytic fragments. Figures 3 and 4 show representative ligand (panel A) and immunoblots (panel B) of protease assays run on follicular fluid samples from the dominant and subordinate follicles of controls and from co-DFs of rbFSH-treated heifers, respectively. The single dominant follicle of controls and co-DFs of FSH-treated heifers contained higher levels of IGFBP-4 proteolytic activity than subordinate follicles of controls, as evidenced by the significant reduction or almost complete disappearance of the band corresponding to the intact rhIGFBP-4 after 18 h of incubation with follicular fluid (Fig. 3A, lane 2 vs. 3; Fig. 4A, lanes 2 vs. 3 and 7 vs. 8). In addition, the reduction in the intensity of the IGFBP-4 band detected by ligand blot analysis was accompanied by the generation of specific proteolytic fragments, as determined by Western immunoblotting (Fig. 3B, lane 2 vs. 3; Fig. 4B, lanes 2 vs. 3 and 7 vs. 8). In contrast, follicular fluid from subordinate follicles of controls failed to degrade exogenous rhIGFBP-4 or endogenous IGFBP-4 to any significant extent (Fig. 3A, lanes 7 vs. 8 and 9 vs. 10, respectively). As expected, specific proteolytic fragments were barely detectable when rhIGFBP-4 was incubated with follicular fluid from subordinate follicles (Fig. 3B, lane 7 vs. 8). Quantitative results, derived from phosphorimaging of ligand blots and expressed as percent of rhIGFBP-4 degradation, are shown in Figure 6 (bottom panel). IGFBP-4 proteolytic activity in follicular fluid from co-DFs of FSH-treated heifers was similar (P > 0.05) to that of single dominant follicles of controls, but fourfold higher (P < 0.01) than that of subordinate follicles of control heifers.
DISCUSSION
The results support the hypothesis that the acquisition of an IGFBP-4 proteolytic activity is an integral component of the biochemical mechanisms underlying selection of a dominant follicle. In agreement with the present results, we have shown previously that IGFBP-4 proteolytic activity is higher in dominant, estrogen-active follicles than in subordinate follicles of the same cohort, as early as Day 2 of the first follicular wave [16, 17]. To our knowledge, this and the companion paper are the first reports of an association between the acquisition of intrafollicular IGFBP-4 proteolytic activity and selection of the dominant follicle in mammals. The current results show further that this proteolytic activity is regulated by FSH, because injection of low doses of rbFSH before selection of the dominant follicle successfully induced co-DFs with intrafollicular IGFBP-4 proteolytic activity similar to that of single dominant follicles obtained from control estrous cycles.
Injection of low doses of rbFSH before the expected time of follicular selection is useful in studying the hormonal dependence of morphological [19] and functional [22] follicular selection. As shown previously [19, 22] and in the present study, the injection of small doses of FSH overrides the spontaneous decline in circulating FSH that occurs around the time of follicle growth deviation and widens the window of time during which gonadotropin-dependent follicles remain viable. Consequently, follicles otherwise destined to become subordinate escape from atresia and develop further to a stage of functional codominance [23]. That codominance occurs, rather than a delay in the selection process or maintenance of cohort-type follicles, is supported by several observations. From the cohort of growing follicles, only two to three follicles/heifer grew synchronously to 10 mm in diameter after FSH treatment in the current experiment and in a previous study [19], whereas a follicular cohort comprises a mean of six to seven recruited follicles 6 mm in diameter [6]. In FSH-treated heifers, the two largest follicles continued growing at a similar rate well after selection had occurred in saline-treated heifers and they did not differ in size. FSH-induced co-DFs had intrafollicular IGFBP-4 proteolytic activity similar to that of single dominant follicles of control heifers, but significantly higher than control subordinate follicles. When the estradiol:progesterone ratio was used to classify follicles as estrogen-active (ratio >1) or atretic (ratio <1) in this and a previous study [22], dominant follicles from controls and co-DFs from rbFSH-treated animals were clearly estrogen-active, whereas subordinate follicles from controls were not.
The intrafollicular mechanisms associated with selection of a dominant follicle from among a cohort of recruited follicles are not understood. Because selected, dominant follicles have the distinct characteristic of highly increased intrafollicular estradiol concentration [9, 10, 24, 25], it has been hypothesized that differential expression of gonadotropin receptors, steroidogenic enzymes, or both, may be part of the mechanisms leading to the establishment of follicular dominance [5, 26, 27]. Although expression of mRNA for gonadotropin receptors, key steroidogenic enzymes, and steroidogenic acute regulatory protein (StAR) was greater in dominant than in recruited follicles of the first follicular wave [28], such differences were not apparent when the dominant and the two largest subordinate follicles of the first wave were compared directly around the time of follicular selection [10]. On Day 2 of the follicular wave, when the two largest follicles differed in size by just 1 mm, mRNA for LH receptor was negligible in granulosa cells of both dominant and subordinate follicles and no differences were detected in the levels of mRNAs for P450 17-hydroxylase and LH receptor in theca cells or FSH receptor and P450 aromatase in granulosa cells. In those experiments [10], the only differences observed between dominant and subordinate follicles collected on Day 2 of the follicular wave were that dominant follicles had higher concentrations of estradiol in follicular fluid and that their granulosa cells secreted more estradiol in culture. Similarly, Bodensteiner et al. [9] reported that increased estradiol production preceded, rather than followed, an increase in the number of gonadotropin receptors detected by radioreceptor assays. Therefore, differential expression of steroidogenic enzymes and acquisition of LH receptors by granulosa cells do not appear to be key components of follicular selection.
Because an increase in the capacity to secrete estradiol is highly coupled to selection for dominance [8, 10, 29, 30], in the absence of changes in mRNAs for steroidogenic enzymes and gonadotropin receptors [10], it is logical to hypothesize that changes in components of the intrafollicular IGF system, known to synergize with FSH [12], may heighten the response of the future dominant follicle to the decreasing levels of FSH encompassing follicular selection. The decline in FSH concentration at the time of follicular growth deviation has been shown previously to be associated with some changes in intrafollicular factors (including IGFBPs) believed to be critical for follicle survival [22]. A slight reduction in total IGF-I levels in follicular fluid was observed in FSH-induced vs. control dominant follicles in one study [22]; whereas no differences were detected in follicular fluid concentration of IGF-I between control and FSH-induced dominant follicles collected at 0 or 48 h after prostaglandin F2 in an earlier report [15]. In addition, several groups have shown no differences in follicular fluid IGF-I [31, 32] or IGF-II [32] concentrations between dominant and subordinate follicles of the first follicular wave of the bovine estrous cycle. The lack of changes in intrafollicular concentration of IGFs underscores the importance of changes in the levels of IGFBPs. Indeed, we observed higher intrafollicular levels of low molecular weight IGFBPs (IGFBP-2, -4, and -5) in subordinate follicles just around the time of selection of the dominant follicle [17]. Likewise, follicular fluid concentrations of low molecular weight IGFBPs were higher in subordinate and atretic follicles compared to estrogen-active, dominant bovine follicles, assessed well after selection had occurred [15, 22, 31, 32]. Furthermore, the results of the present (Fig. 5 and Fig. 6, top panel) and previous studies [15, 22] suggest that the levels of these IGFBPs are down-regulated by FSH in vivo. Therefore, it is hypothesized [33] that an increase in the intrafollicular ratio of IGFs:IGFBPs could be one differential change that allows the future dominant follicle to continue growing and producing more estradiol than the future subordinate follicles. This notion is supported by the recent study of Mihm et al. [30], who demonstrated that intrafollicular levels of IGFBP-4 were lower, and levels of estradiol were higher, in the first-wave follicle destined to become dominant compared with follicles destined to become subordinates. In the present experiment, FSH-induced co-DFs and single dominant follicles from saline treated-animals had IGFBP-4 protease activity in follicular fluid that was similar, but about fourfold higher than subordinate follicles from control heifers. We propose that FSH acts on the future dominant follicle to increase IGFBP-4 protease activity, which by reducing the intrafollicular levels of IGFBP-4, provides more free IGF to interact with FSH to increase estradiol secretion.
The lower intrafollicular levels of low molecular weight IGFBPs observed in dominant vs. subordinate follicles, and the apparent down-regulation by FSH, could result from alterations in transcriptional regulation, proteolytic processing, or both, of the secreted IGFBPs. Although a paucity of information exists in this regard, we observed in preliminary studies that follicular fluid from bovine preovulatory follicles did not degrade rhIGFBP-2 to any significant extent; in contrast, rhIGFBP-5 was strongly degraded (data not shown). The latter observations suggest an association of IGFBP-5 proteolysis with follicular dominance; however, a role for IGFBP-5/IGFBP-5 degradation in follicular selection seems unlikely in light of recent findings that intrafollicular levels of IGFBP-5 (as well as IGFBP-2) were similar among cohort follicles of the first follicular wave destined to become dominant or subordinate [30]. Although expression of mRNA for IGFBP-4 is higher in atretic than in healthy follicles, mRNA expression is similar in healthy follicles of various size classes [34–37]. The latter finding led us to hypothesize that post-translational proteolytic degradation, rather than decreased IGFBP-4 gene expression, is the operative mechanism that produces decreased levels of IGFBP-4 associated with follicular growth and selection for dominance. Although decreased intrafollicular levels of IGFBP-4 [30], increased IGFBP-4 proteolytic activity, or both, as shown in this and our previous reports [16, 17], appear closely coupled with selection for dominance, the physiological role of the intrafollicular IGFBP-4/IGFBP-4 proteolytic system in vivo remains to be determined. However, in vitro studies have shown that intact IGFBP-4 [38–40], but not IGFBP-4 proteolytic fragments [39], inhibits estradiol production by isolated granulosa cells.
Little is known about the regulation of the intrafollicular IGFBP-4 protease. In the ovary, granulosa cells are believed to be the source of the IGFBP-4 protease present in follicular fluid of several species [12]. Interestingly, two peptides known to inhibit ovarian steroidogenesis, interferon-
and activin A, have been recently shown to increase IGFBP-4 and decrease IGFBP-4 protease activity in culture medium of human granulosa cells [41]. Of potential physiological relevance to the regulation of the IGFBP-4 protease present in follicular fluid are the recent observations that IGFBP-2 and -5, which are readily detectable in atretic but not in dominant, estrogen-active follicles, inhibit IGFBP-4 degradation [42, 43]. In contrast, IGFs and FSH, potent stimulators of ovarian steroidogenesis, have been shown to increase IGFBP-4 protease activity in cultures of human [44] and rat [38] granulosa cells. In addition, the close association between low or undetectable levels of IGFBP-4 and high levels of estradiol in follicular fluid [15, 22, 30], together with increased IGFBP-4 proteolysis in dominant, estrogen-active, but not in subordinate, atretic follicles as observed in the present and previous [17, 43] studies, points to estradiol as a potential regulator of the IGFBP-4 protease activity. Estradiol can reverse the parathyroid hormone-induced suppression of the IGFBP-4 protease activity secreted by SaOS-2 osteoblastic-like cells [45] and higher levels of immunoreactive pregnancy-associated plasma protein A (IGFBP-4 protease) were observed in estrogen-active than in androgen-active human follicles [46]. However, studies designed to directly address the role of steroids in the regulation of the intraovarian IGFBP-4/IGFBP-4 proteolytic system are lacking.
In conclusion, the present results, showing that FSH-induced codominant follicles have IGFBP-4 proteolytic activity similar to that of single dominant follicles from control nonstimulated cycles, provide the first evidence that follicular selection involves an FSH-dependent IGFBP-4 protease.
ACKNOWLEDGMENTS
We thank Granada Biosciences for the rbFSH, Dr. G.D. Niswender for the estradiol antiserum, B.S. Hansen for her excellent technical assistance, Dr. E. Zambrano for assistance in ultrasound-guided follicular aspirations, D. Bianchi for care of the animals, and Drs. M.S. Roberson and P.J. Bridges for critical reading of the manuscript.
FOOTNOTES
First decision: 13 November 2000.
1 Supported by National Institutes of Health grant HD38276 to J.E.F. and a Fulbright-Universidad Nacional de Río Cuarto (Argentina) Fellowship to G.M.R. 
2 Correspondence. FAX: 607 253 3476; jf11{at}cornell.edu
Accepted: February 14, 2001.
Received: September 26, 2000.
REFERENCES
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