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a Department of Animal Science, College of Veterinary Medicine, The University of Tennessee, Knoxville, Tennessee 37996
b University Department of Reproductive and Developmental Sciences, University of Edinburgh, Royal Infirmary of Edinburgh, Edinburgh EH3 9YW, United Kingdom
ABSTRACT
Leydig cells in the adult rat testis differentiate during the neonatal-prepubertal period. However, the stimulus for the initiation of their differentiation is still not clear. In the present study our objectives were to test the effects of thyroid hormone and LH on the initiation of precursor cell differentiation into Leydig cells in the prepubertal rat testis. Four groups of Sprague-Dawley rats were used. All treatments began at postnatal Day 1. Rats in groups I, II, and III received daily s.c. injections of saline (200 µl, controls), triiodothyronine (T3, 50 µg/kg body weight, hyperthyroid), and LH (ovine LH 10 µg/rat/day), respectively. Rats in group IV were made hypothyroid from postnatal Day 1 by adding 0.1% propylthiouracil (PTU) to their mother's drinking water. Testes of rats were collected at 7, 8, 9, 10, 11, 12, 16, and 21 days of age, fixed in Bouin's solution, and embedded in paraffin for immunocytochemical studies. Immunoexpression of 3ß-hydroxysteroid dehydrogenase (3ß-HSD) and LH receptors (LHR) in testicular interstitial cells (other than the fetal Leydig cells) was observed using the avidin-biotin method. In control rats, out of all spindle-shaped cell types in the testis interstitium, only the peritubular mesenchymal cells showed positive immunolabeling for 3ß-HSD, beginning from the postnatal Day 11. However, positive immunolabeling for LHR was first detected in these cells at Day 12, i.e., after acquiring the steroidogenic enzyme activity. In T3-treated rats 3ß-HSD positive spindle-shaped cells were first observed at Day 9 (i.e., 2 days earlier than controls), and LHR-positive cells were first observed on Day 11 (2 days later than obtaining 3ß-HSD immunoactivity); they were exclusively the peritubular mesenchymal cells. The 3ß-HSD- and LHR-positive spindle-shaped cells were absent in the testis interstitium of LH-injected rats from Days 7 through 12 but were present at postnatal Day 16. In addition, more fetal Leydig cell clusters and fetal Leydig cells in mitosis were present in LH-treated rats compared to rats in all other treatment groups. Following their first detection, the number of positive cells for each protein continued to increase at each subsequent age in controls, T3-, and LH-injected groups. In PTU rats, 3ß-HSD and LHR-positive spindle-shaped cells were absent throughout the experimental period. From these observations, it is possible to suggest the following regarding the developing rat testis interstitium. 1) The precursor cells for the adult generation of Leydig cells in the postnatal rat testis are the peritubular mesenchymal cells. 2) Luteinizing hormone does not initiate the onset of mesenchymal cell differentiation into Leydig cells, instead it delays this process. However, daily LH treatment causes mitosis in fetal Leydig cells and increase in fetal Leydig cell clusters. 3) Thyroid hormone is critical to initiate the onset of mesenchymal cell differentiation into adult Leydig cells.
developmental biology, interstitial cells, Leydig cells, puberty, testes, testosterone
INTRODUCTION
The mammalian male needs androgen during all stages of life. In the neonatal period androgens are required for the activation of the hypothalamo-hypophyseal-testicular axis, which is essential for the completion of the testicular descent, masculinization of the brain, control of Sertoli cell number, initiation of spermatogenesis, and sexual behavior [1]. In the adult, androgens are needed for many functions associated with reproduction as well as for the maintenance of androgen-dependent tissues and organs that include bone, muscle, skin, spleen, and kidney. Therefore, the presence of functional Leydig cells is essential for the well being of the adult mammalian male at all stages of life, because they are the primary source of androgens for the males of all mammalian species.
Two distinct populations of Leydig cells (i.e., fetal Leydig and adult Leydig cells) are present in mammals. Fetal Leydig cells are differentiated from primitive mesenchymal cells in the rat testis around Day 14 of gestation [2] and continue to be present at birth [3, 4] and up to sexual maturity [5–7]. However, it is established that the adult population of Leydig cells is the most abundant and the primary source of androgens in the sexually mature rat testis. These adult Leydig cells differentiate postnatally from the spindle-shaped cells in the testis interstitium [8–12], and it is important to determine what stimuli are involved in initiating the nonsteroidogenic spindle-shaped precursor cells in the testis interstitium to begin the process of Leydig cell differentiation required for steroidogenic activity.
In testes interstitium, there are several types of cells that appear as spindle-shaped in section, namely the endothelial cells, pericytes, myoid cells, and fibroblasts; collectively these cells will be referred to as spindle-shaped cells in the present study. Moreover, as in many other studies fibroblasts in the testis interstitium of the present investigation are identified as mesenchymal cells; they are found in the peritubular region as well as scattered randomly in the rest of the testis interstitium. At present, various opinions exist on which of these cells are the precursors to Leydig cells, but the majority of studies performed throughout the years in different laboratories suggest that peritubular mesenchymal cells are the main cell type (if not the only cell type) that gives rise to Leydig cells [10–12]. In contrast to this view, it is also reported that the precursor cells for the Leydig cells in the prepubertal rat testis are the mesenchymal cells in the central part of the testis interstitium but not the ones in the peritubular region [13].
Luteinizing hormone has been suggested by some investigators [14–16] as the triggering hormone for the process of Leydig cell differentiation. Other observations, however, question the validity of this view. First, it is known that the adult population of Leydig cells initially emerge in the neonatal rat testis during the second week of postnatal life [3, 4] when the circulating LH is at a very low level [17, 18]. Second, after transient neonatal hypothyroidism, when the circulating LH is at very low levels [18], Leydig cells still differentiate [19–21]. It is also interesting to note that in the latter situation, Leydig cell differentiation takes place under euthyroid status, i.e., after the withdrawal of propylthiouracil (PTU) treatment used to induce the hypothyroid status in these rats. However, during the hypothyroid status, adult Leydig cell differentiation is observed to be arrested [4, 22]. Teerds et al. [22] also reported that daily injections of triiodothyronine (T3) induced precocious differentiation of adult Leydig cells in the prepubertal rat testis. These observations suggest that thyroid hormone has an important regulatory role in the initiation of precursor cell differentiation into Leydig cells. In contrast to this view, Hardy et al. [23] have reported that neonatal hypothyroidism induces, instead of arrests, adult Leydig cell differentiation and proliferation in the neonatal rat testis.
Our recent data revealed that the onset of the differentiation of the adult generation of Leydig cells in the prepubertal rat testis is at the end of postnatal Day 10, and the precursors were exclusively the peritubular mesenchymal cells [24]. This was determined by the detection of the steroidogenic enzyme activity (3ß-hydroxysteroid dehydrogenase [3ß-HSD], cytochrome P450 side-chain cleavage [P450scc], and 17
-hydroxylase cytochrome P450 [P450c17]) in testicular interstitial cells (other than fetal Leydig cells [24, 25]) using adjacent testicular tissue sections. At this stage these differentiated cells were still spindle-shaped and did not show positive labeling for LH receptors (LHR) [24, 25]. This observation suggested that the onset of peritubular mesenchymal cell differentiation into Leydig progenitor cells does not require LH. In the present investigation, we tested the effects of thyroid hormone and LH on the onset of precursor cell differentiation into Leydig progenitor cells. The experiment was specifically designed to monitor precursor cells gaining LHR and 3ß-HSD (steroidogenic potential) activity in the prepubertal rat testes (7 through 21 days) in response to LH, T3, and PTU treatments using immunocytochemistry.
MATERIALS AND METHODS
Animals
Female Sprague-Dawley rats in midpregnancy were purchased from Harlan Industries (Madison, WI) and were housed in the animal facility of The University of Tennessee, College of Veterinary Medicine, one rat per cage, under conditions of controlled temperature (25°C) and lighting (14L:10D). They were provided food (Agway Prolab rat formula, Syracuse, NY) and water ad libitum and were examined for litters twice (morning and evening) daily. The day of birth of pups was considered as Day 1 of postnatal life.
Experimental Design
Four groups of 1-day-old rats were treated as follows. Rats in group I were given daily s.c. injections of saline (200 µl/rat). Rats in group II received a daily s.c. injection of T3 at a dosage of 50 µg/kg body weight (hyperthyroid group). Rats in group III were subjected to daily s.c. injections of LH (ovine LH, 10 µg/animal/day), and rats in group IV were made hypothyroid by adding 0.1% PTU to their mother's drinking water. Rats in each group were killed at 7, 8, 9, 10, 11, 12, 16, and 21 days of age (n = 4 rats per group at each age). Testes were collected and processed for immunocytochemistry and immunoexpression of 3ß-HSD and LHR was performed as described below.
Tissue Preparation
Rats were killed daily during postnatal Days 7 through 21 by CO2 inhalation. Both testes of each rat were removed from the body and fixed by immersing in Bouin's fluid for 5–6 h. Fixed testicles were then washed in 70% ethyl alcohol for several days until the picric acid was removed, processed, and embedded in low melting Paraplast (Oxford Labware, St. Louis, MO). Serial sections of 5 µm in thickness were cut from the testis blocks and adhered on ProbeOn Plus glass slides (Fisher Scientific, Pittsburgh, PA) to use for immunocytochemistry.
Antibodies
The polyclonal 3ß-HSD antibody was a rabbit IgG antibody against purified human placental 3ß-HSD [26]. This antibody has been previously used in immunolocalization of 3ß-HSD antigen in rat testis in many studies, including Leydig cell differentiation studies during the fetal ages [2, 27]. Moreover, it yields a single band on immunoblots with Leydig cells [28]. A biotinylated goat anti-rabbit IgG (StrAviGen supersensitive; BioGenex, San Raman, CA) was used as the second antibody for immunolocalization of 3ß-HSD. The purified LHR antibody used in the present study was a monoclonal antibody (P1B4) raised in mice [29]. It has been used in other studies to immunolocalize LHRs in the rat testis [2, 30]; however, to our knowledge, this antibody has not been tested in immunoblots for testis tissue. Because this is an antibody of the IgM class, a biotinylated goat-anti-mouse IgM antibody (Sigma, St. Louis, MO) was used as the second antibody.
Immunocytochemistry
3ß-Hydroxysteroid dehydrogenase Tissue sections were deparaffinized in xylene, rehydrated in a series of graded ethanol, and brought into distilled water. To detect 3ß-HSD enzyme activity, tissue sections were washed in PBS (pH 7.6) and incubated in 3% H2O2 in absolute methanol for 30 min at room temperature to block endogenous peroxidase activity. Slides were then protein blocked by immersing them in a solution of 10% normal goat serum plus 1% BSA (fraction V; Sigma) for 6 h at 4°C. The primary antibody against 3ß-HSD was tested at various dilutions in protein-blocking solution, and the optimum was decided to be 1:2000. It was applied on to the sections and incubated overnight at 4°C. Slides were washed in PBS on the following day, and the bound antibody was detected by biotin-streptavidin method using a commercially available supersensitive detection kit (BioGenex) according to the manufacturer's instructions. This kit uses 3'3-diaminobenzidine hydrochloride as a chromogen. The slides were counterstained with Mayer's hematoxylin (Sigma), dehydrated in a series of increasing concentrations of ethanol, and coverslipped under Permount (Fisher Scientific, Fair Lawn, NJ).
Luteinizing hormone receptor The rehydrated sections were washed in Tris-buffered saline (TBS: 0.05 M Tris-HCl, 0.85% NaCl, pH 7.4), and tissue peroxidase activity was inactivated by incubating the sections in 3% H2O2 in water at room temperature for 30 min. Other steps of the procedure were the same as those described for 3ß-HSD, except that instead of PBS, TBS was used as washing, diluting, and incubating medium. A 1:2000 dilution of primary antibody was employed. For each antibody, the control slides were incubated in preimmune serum instead of primary antibody. Immunolabeling experiments were repeated three times for each antibody to establish reproducibility of these results.
RESULTS
Fetal Leydig cells showed 3ß-HSD and LHR immunoactivity at all tested ages, in all three treatment groups. However, LH-treated rats contained more fetal Leydig cells with large clusters compared to all other treatment groups at all tested time points; Figure 1, A and B, show fetal Leydig cell clusters immunolabeled for 3ß-HSD enzyme in control and LH-injected rats at postnatal Day 16 for comparison. Some fetal Leydig cells were observed to be undergoing mitosis throughout the tested ages, especially in LH-treated rats (Fig. 1B). In PTU rats, no other interstitial cell type was immunolabeled for either 3ß-HSD or LHRs up to 21 days (Fig. 1, C and D).
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At Day 9, 3ß-HSD-positive cells other than the fetal Leydig cells were absent in the testis interstitium of both control and LH-treated rats (Fig. 2, A and B), but few were observed for the first time in T3-injected rats (Fig. 2C). They were exclusively the peritubular mesenchymal cells as shown in Figure 2C. However, at Day 9, LHR-positive cells (other than the fetal Leydig cells) were absent in the testis interstitium of rats in all three treatment groups (Fig. 2, D–F). At Day 11, 3ß-HSD-positive peritubular mesenchymal cells were detected for the first time in control rats of this experiment; however, they were not yet positive for LHRs (results not shown). Such cells (i.e., 3ß-HSD- and LHR-positive testicular interstitial cells other than the fetal Leydig cells) were still absent in the LH-treated rats. At Day 12, 3ß-HSD- and LHR-positive cells other than the fetal Leydig cells were present in control (Fig. 2, G and J) and T3-treated rats (Fig. 2, I and L) relatively more in numbers compared to those at Day 9 of each treatment group (Fig. 2, A and D, for control, Fig. 2, C and F, for T3-treated); however, such cells were still absent in LH-treated rats at this stage (Fig. 2, H and K). It also appeared that T3-treated rats contained more 3ß-HSD- and LHR-positive testicular interstitial cells (other than the fetal Leydig cells) than control rats at this stage (Fig. 2, G and I, J and L).
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At Days 16 and 21, 3ß-HSD- and LHR-positive cells were present in all three treatment groups (Fig. 3, A–L). However, such cells appeared to be in abundance in LH-treated rats compared to the age-matching controls and T3-treated rats (Fig. 3, A–L). Also, such cell profiles appeared larger in LH-treated rats compared to the other two treatment groups. Comparison between control (Fig. 3, A, D, G, and J) and T3-treated rats (Fig. 3, C, F, I, and L) showed that the testes interstitium of T3-treated rats contained relatively more immunolabeled cells than those in controls.
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The immunoexpression of LHRs and 3ß-HSD in progenitor cells in control, T3-treated, LH-treated, and PTU-treated rats are summarized in Figure 4.
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DISCUSSION
Our most recent observations showed that the first appearance of 3ß-HSD-positive testicular interstitial cells other than the fetal Leydig cells in the postnatal rat testis is at the end of the postnatal Day 10 [24]. Therefore, the observation of 3ß-HSD-positive mesenchymal cells exclusively in the peritubular region at postnatal Day 11 in the present investigation is in agreement with these earlier immunocytochemical findings [24, 25]. Moreover, these results compare favorably with the previous findings on the earliest detection of the adult Leydig cells in the postnatal rat testis, which is on postnatal Day 10 [3].
The results of the present investigation further support the view on peritubular origin of Leydig cell progenitors as suggested earlier by many investigators including de Kretser [10], Christensen [11], and Van Straaten and Wensing [12]. Similar findings have been observed in the human cryptorchid testis [31] and adult rat testes following transient neonatal hypothyroidism [20]. By contrast, Hardy et al. [13] suggested that the fusiform cells in the central region of the interstitial space are the precursors for the adult Leydig cells in the rat testis. The basis for this suggestion is not clear in the latter study [13], especially because no markers have been used to identify the Leydig cell precursors; it appears that this conclusion has been made without a proper justification.
Although we did not observe 3ß-HSD-positive vascular endothelial cells in the prepubertal rat testis interstitium in the present investigation, studies of Haider et al. [32] and Haider and Servo [33] on the prepubertal rat interstitium detected some 3ß-HSD enzyme activity in vascular endothelial cells. However, in their report [32] they mentioned that the observed 3ß-HSD activity in these vascular endothelial cells appears to be due to a paracrine effect of adjacent Leydig cells rather than these vascular endothelial cells being differentiated into Leydig cell progenitors. Moreover, Misrahi et al. [34] reported the detection of LHR activity in vascular endothelial cells in the testis interstitium. We were unable however to reproduce these observations.
Luteinizing hormone has been implicated as the main stimulus for the initiation of the development of the adult type Leydig cells, both in prepubertal animals [14–16] and in ethane dimethane sulfonate (EDS)-treated adult rats [35]. However, the present investigation showed that the mesenchymal cell precursors differentiated into Leydig progenitor cells (gained 3ß-HSD activity) prior to acquiring LHRs. In a recent study [24], using adjacent testis tissue sections, we also showed that the steroidogenic enzymes 3ß-HSD, P450scc, and P450c17 were simultaneously immunoexpressed in peritubular mesenchymal cells (for the first time) prior to the immunoexpression of LHRs in the prepubertal rat testis. These findings suggest that the onset of mesenchymal cell differentiation into Leydig cells is independent of LH. Alternatively, the absence of LHRs in progenitors at Day 11 may be due to its presence in very low levels than the other steroidogenic enzymes (3ß-HSD, P450scc, and P450c17) to be biologically active, and therefore the method of detection (i.e., immunocytochemistry) was insensitive. Although this view is still a possibility, in the present investigation we also observed that the initiation of mesenchymal cell differentiation into Leydig cells was not triggered by daily s.c. LH injections. Instead, initiation of this process was delayed with LH treatment. This observation adds strength to the view that LH does not stimulate the onset of mesenchymal cell differentiation into Leydig cells in the prepubertal rat testis. Therefore, the conclusion of Benton et al. [14], which is that LH has a critical role in Leydig cell differentiation and is one of the three key regulatory factors in Leydig cell differentiation, appears not applicable to the stage of the onset of Leydig cell differentiation.
By contrast to the effects of LH on mesenchymal cell differentiation to adult Leydig cells, daily injections of LH caused expansion of the population of fetal Leydig cells as evidenced by the obvious enlargement of the size of fetal Leydig cell clusters and numerous mitotic divisions in these cells. These observations compare favorably with the other studies that reported fetal Leydig cell hyperplasia in neonatal rats following hCG treatment [36, 37]. Moreover, as shown previously, fetal Leydig cell atrophy is observed when adult Leydig cells differentiate in significant numbers [3, 4], and fetal Leydig cell atrophy is prevented when there is an arrest in the adult Leydig cell differentiation [4]. These observations prompted a suggestion of an interdependent relationship between the two populations of Leydig cells in the rat testis. Results of the present investigation on delay in the differentiation of adult Leydig cells in the presence of increased fetal Leydig cells further add strength to this view. Previous studies of Gaytan et al. [37] also showed such a relationship as a long-term effect of hCG on fetal and adult Leydig cells in the neonatal rat testis (hCG injection to prepubertal rats during 2–4 days of their age) and add support to this concept. Studies of Kerr et al. [38] also demonstrated premature differentiation of adult Leydig cells in the neonatal rat testis when fetal Leydig cells in the neonatal testis were destroyed by EDS treatment. These findings suggest that factors emanating from the fetal Leydig cells may also have a regulatory role on the differentiation of adult Leydig cells in the prepubertal rat testis.
The present study also detected an increase in the number of hypertrophied adult Leydig cells at Days 16 and 21 in rats given LH and many mitotic divisions among these Leydig cells. This observation suggests that LH regulates Leydig cell number and their function at a step beyond the initial differentiation stage of these cells from their mesenchymal precursors. In support of this suggestion, enhanced Leydig cell proliferation [39–41] and hypertrophy [40–42] have been observed when LH/hCG was given chronically to prepubertal or adult rats. Moreover, under conditions of LH deficiency, Leydig cells undergo cell hypotrophy [43] and under chronic LH deficiency Leydig cell numbers are also reduced [43–45].
The effect of thyroid hormone on the development of the adult population of Leydig cells has been previously investigated using a transiently hypothyroid neonatal rat model [19–21]. A twofold increase in adult Leydig cell numbers at adulthood has been reported in rats who were transiently hypothyroid during the neonatal period [19–21]. We suggested that this unprecedented increase of Leydig cell number is due to the continuous proliferation of mesenchymal precursor cells in the absence of their differentiation into Leydig cells during the hypothyroid period, providing a larger pool of precursor cells that can be transformed into Leydig cells when the animal becomes euthyroid [19, 20]. In agreement with this suggestion, morphometric studies of the neonatal rat testicular interstitium, under a hypothyroid status, demonstrated a significantly increase in number of mesenchymal cells and the absence of adult Leydig cells [4]. In contrast to this finding, Hardy et al. [23] reported proliferation of immature adult Leydig cells rather than increased proliferation of their mesenchymal precursors to be the principal mechanism responsible for the increase in the Leydig cell number in the adult rat testis after neonatal hypothyroidism. As adult Leydig cell differentiation is arrested under hypothyroid conditions [4, 22], the statement made by Hardy et al. [23] is not possible to justify. In addition, the increase in the labeling index of Leydig cells in hypothyroid rats during the neonatal period by Hardy et al. [23] implies that fetal Leydig cells (i.e., the only Leydig cell type present) but not immature Leydig cells are undergoing mitosis. However, Teerds et al. [22] could not observe an increase in the labeling index for Leydig cells in PTU rats and quantification studies performed by us [4] using a state-of-the-art stereological method (i.e., the disector method [46]) showed that the fetal Leydig cell number per testis in PTU rats did not change up to 21 days [4]. Therefore, it is difficult to understand how Hardy et al. [23] could observe a high labeling index for Leydig cells in hypothyroid neonatal rats. Moreover, although Hardy et al. [23] showed an absence of Leydig cell proliferation in control neonatal rats of the latter study [23], they reported just the opposite for control rats of the same age in a previous study [13]. Because these contradictory results are produced by the same group of investigators, using similar techniques and animals, it is difficult to understand the reasons for these discrepancies.
In the present study, the immunocytochemical data of the hypothyroid rats confirm the morphological observations of Mendis-Handagama et al. [4] and support the suggestion that hypothyroidism inhibits Leydig cell development in prepubertal rats. The absence of Leydig cell progenitors in hypothyroid rats as detected by 3ß-HSD immunocytochemistry indicated that the inhibitory effect of hypothyroidism on Leydig cell development was at least partly due to the inhibition of the differentiation of mesenchymal cells into Leydig cell progenitors. The extent of this inhibitory effect of thyroid hormone deficiency on adult Leydig cell differentiation appears to be dependent on the degree of thyroid hormone suppression, because 0.006% PTU in mother's drinking water, which was the lowest effective dose for maximum suppression of Sertoli cell differentiation [47], was only partially suppressive for Leydig cell differentiation (results not shown) in comparison to the 0.1% used in the present investigation. By contrast, hyperthyroidism promoted precocious differentiation of the adult population of Leydig cells as evident by 3ß-HSD-positive progenitor cells at Day 9, also stimulated transformation of more precursor cells into Leydig cells in subsequent age groups. Stimulated proliferation of newly formed Leydig cells under hyperthyroid conditions as observed by mitotic figures in these newly formed Leydig cells may also have contributed to the significantly higher numbers of adult Leydig cells present in these animals in comparison to the age-matched controls.
In summary, the results of the present study suggest that the onset of adult Leydig cell differentiation in the prepubertal rat testis does not require LH, similar to what occurs in the prenatal rat [2, 27] and mouse [48] testes. In addition, the present study demonstrates that daily LH treatment delays the onset of mesenchymal cell differentiation into Leydig cells, probably via the fetal Leydig cell factors that need to be identified in future studies. Therefore, these findings do not support the view that LH stimulates mesenchymal cell differentiation into progenitor cells in the postnatal rat testis as proposed by others [14–16]. Additionally, the present investigation further confirms that hyperthyroidism stimulated and hypothyroidism prevented the differentiation of adult Leydig cells in the prepubertal rat testis [4, 22] and supports the view that thyroid hormone, but not LH, plays an important role in the onset of the differentiation of the adult Leydig cell population in the prepubertal rat testis.
ACKNOWLEDGMENTS
We thank Dr. J. Wimalasena (The University of Tennessee, Knoxville) for providing us the LHR antibody used in our study. We also appreciate the assistance of Phil Snow and Tom Jordan (Photography Laboratory, Institute of Agriculture, The University of Tennessee) in preparation of the color plates. Ovine LH used in this study was provided by the National Pituitary Hormone Distribution program.
FOOTNOTES
First decision: 10 March 2000.
1 Supported by CMH grants from COE R180101-08 and UT Minkel; JIM-MRC. 
2 Correspondence: C. Mendis-Handagama, Department of Animal Science, College of Veterinary Medicine, The University of Tennessee, 2407 River Drive, Knoxville, TN 37996. FAX: 423 974 2215; mendisc{at}utk.edu
Accepted: May 1, 2000.
Received: February 10, 2000.
REFERENCES
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