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Biology of Reproduction 63, 730-735 (2000)
© 2000 Society for the Study of Reproduction, Inc.

Regular Article

Alteration of Follicle-Stimulating Hormone and Testosterone Regulation of Messenger Ribonucleic Acid for Sertoli Cell Proteins in the Rat During the Acute Phase of Spinal Cord Injury1

John E. Ottenweller5,a, Ming-Tang Lib, William Giglioa, Robert Anesettib, Leonard M. Pogacha, and Hosea F.S. Huangc

a Veterans Affair Medical Center, East Orange, New Jersey 07019 b Departments of Surgery Section of Urology and c Neuroscience, UMD-New Jersey Medical School, Newark, New Jersey 07103

ABSTRACT

The detrimental effects of spinal cord injury (SCI) on spermatogenesis in the rat can be attenuated by exogenous testosterone (T) but enhanced by exogenous follicle-stimulating hormone (FSH). These results suggest that T-dependent cellular events may be involved in testicular injury after SCI and that such events may be associated with modification of FSH effects on Sertoli cell function. The current study compared the responses of Sertoli cells to exogenous T and FSH after SCI or sham surgery using steady-state levels of Sertoli cell protein mRNA transcripts as markers of responsiveness. Rats underwent sham surgery or SCI and then were treated for 7 or 14 days with T-filled silastic capsules (2 x 5 cm) and/or daily injections of 0.1 units of porcine FSH. Vehicle-treated control rats received 5-cm empty capsules and daily injections of saline vehicle. Two weeks after sham surgery, levels of mRNA for the androgen receptor (AR), FSH receptor (FSHR), androgen-binding protein (ABP), or sulfated glycoprotein (SGP)-2 in the testis were unaffected by T or FSH alone. Testosterone alone, however, significantly decreased transferrin (Trf) mRNA levels in the testis (P < 0.01). The combination of T and FSH treatments resulted in significant decreases in levels of the above transcripts (P < 0.05; P < 0.01). Seven days after SCI, the testes of vehicle-treated SCI rats had higher levels of AR and SGP-2 mRNA than did those of sham control rats (P < 0.01); such effects were transient and disappeared by Day 14 post-SCI. Testosterone treatment of SCI rats for 7 days resulted in decreases in mRNA levels for AR and Trf in the testes (P < 0.01) but increased testicular levels of mRNAs for FSHR and SGP-2 in SCI rats. Follicle-stimulating hormone treatment for 7 days prevented the increase in AR mRNA that was seen in the testis of untreated SCI rats and increased levels of ABP and SGP-2 mRNAs in SCI rats (P < 0.01). Follicle-stimulating hormone treatment of SCI rats did not affect FSHR mRNA levels by itself, but it blocked the stimulatory effect of T on FSHR and SGP-2 mRNAs. Fourteen days after SCI, testicular AR mRNA levels were not affected by T alone, but they increased in those rats that received FSH with or without concurrent T treatments (P < 0.05). In contrast to their effects in sham control rats, T or FSH alone or in combination resulted in significant increases in testicular levels of ABP, SGP-2, and FSHR mRNAs (P < 0.05). At this time, Trf mRNA in the testis of SCI rats was also suppressed by T (P < 0.05), as it did in sham control rats, but Trf mRNA was increased by the FSH (P < 0.01) that had inhibited this transcript in the testes of sham control rats. The effects of FSH on the Sertoli cell transcripts in SCI rats were either attenuated or blocked when T was given concurrently. In addition, testicular and serum T levels in those SCI rats that received FSH (alone or in combination with T) for 14 days were significantly increased, an effect that was not seen after sham surgery. These findings demonstrate that hormonal regulation of both Sertoli and Leydig cells was altered during the acute phase of SCI. Such changes may modify the functions of both cell types, thereby affecting the endocrine and/or paracrine microenvironment within the seminiferous epithelium. These effects could impair the functional capacity of Sertoli cells and contribute to impairment of spermatogenesis after SCI.

androgen receptor, FSH, Leydig cells, LH, Sertoli cells, sperm, testes, testosterone

INTRODUCTION

Male infertility resulting from spinal cord injury (SCI) is associated with abnormal semen parameters, including a general decrease in sperm production and fewer sperm with normal morphology and progressive motility [1–4]. Examination of testicular biopsies of SCI men also revealed a wide spectrum of changes in the seminiferous epithelium [2, 3]. These observations suggest that multiple mechanisms, perhaps in different phases of spermatogenic differentiation, might be involved in the deleterious changes in sperm quality after SCI. Spermatogenesis is supported and regulated by Sertoli cells, the functions of which are modulated by testosterone (T) and follicle-stimulating hormone (FSH) [5]. Thus, understanding the effects that SCI has on Sertoli cells and on their hormonal regulation is the first prerequisite in preserving spermatogenic function and, by extension, the fertility of SCI men.

In the rat, impairment of spermatogenesis during the acute phase of surgically induced SCI is preceded by transient but significant decreases in serum FSH, luteinizing hormone (LH), and testicular T concentrations [6, 7], which suggests a possible cause-and-effect relationship between these changes. However, deleterious changes in spermatogenesis continue during the chronic phase of SCI (a month or more after injury in the rat), even after normal function of the pituitary-testis hormone axis has been restored, and these changes are associated with abnormal Sertoli cell morphology [7, 8]. Recently we reported that regression of spermatogenesis in SCI rats was partially reversed by exogenous T treatment but paradoxically enhanced by exogenous FSH [9]. These spermatogenic effects of T and FSH were associated with up-regulation of mRNA transcripts for several Sertoli cell proteins, including androgen-binding protein (ABP), transferrin (Trf), and FSH receptor (FSHR) [9]. In addition, mRNA transcripts for these Sertoli cell proteins were also overexpressed in rats with Sertoli cell-enriched testes during the chronic phase of SCI [10]. These results indicate that the responsiveness of Sertoli cells to FSH and T might be altered after SCI, and such effects may mediate the effects of SCI on spermatogenesis. To test this thesis, we initiated a series of experiments to determine the effects of SCI on Sertoli cell and germ cell functions. We report our findings on the effects of exogenous T and FSH on the expression of mRNAs for various Sertoli cell proteins during the acute phase of SCI.

MATERIALS AND METHODS

Animals

Mature Sprague-Dawley rats (300–350 g; Taconic Farm, Taconic, NY) were caged individually in an air-conditioned, light-controlled animal room for 2 wk prior to the experiment. They were fed Purina rat chow and had access to water ad libitum. The rats were then randomly assigned to undergo either SCI or a sham operation. A total of 60 rats underwent surgical transection of the spinal cord at the level of the ninth thoracic vertebra, as described previously [7]. The SCI procedures were approved by the Institutional Animal Care and Use committees at both the East Orange V.A. Medical Center and the UMDNJ-New Jersey Medical School.

Beginning immediately after the surgery, SCI rats were given daily injections of 0.1 units of porcine FSH (Sigma Chemical Co., St. Louis, MO), s.c. implantation of 2 x 5-cm testosterone-filled silastic capsules, or a combination of the two treatments for 7 and 14 days (n = 6–8 rats/group). Control SCI rats received saline vehicle injections and 5-cm empty capsules. Sham control rats underwent sham surgeries without laminectomy, as described previously [7], and they received identical hormone regimens for 14 days. The animals were killed by decapitation at the end of the treatments, and trunk blood was collected for measurement of serum hormones. One testis from each rat was frozen immediately in isohexane immersed in a mixture of methanol and dry ice and was then stored at -80°C.

Northern Blot cDNA Hybridization

The procedures for isolation of testicular RNA [11], purification of poly(A)+ RNA by oligo(dT) cellulose chromatography [12], electrophoresis, and Northern blotting of RNA [13] have been described previously [9, 10]. The cDNA probes for the FSHR [14], androgen receptor (AR) [15], ABP [16], Trf [17], and sulfated glycoprotein (SGP)-2 [18] were isolated by agarose electrophoresis after appropriate endonuclease digestion. These probes were radiolabeled with [32P]dCTP using a random priming kit (Boehringer-Mannheim, Indianapolis, IN) and were used within 24 h for hybridization according to the procedures described previously [9, 10]. The autoradiographs were developed after 1–6 days of exposure using an intensifying screen. The membranes were subsequently stripped and rehybridized with 32P-labeled cDNA for 18S ribosomal RNA. The relative abundance of each mRNA transcript (FSHR: 2.6 kilobases [kb]; AR: 9.4 kb; ABP: 1.7 kb; Trf: 2.7 kb, hemiferrin: 0.9 kb; and SGP-2: 2.1 kb) were estimated by densitometry and normalized against that of the 18S ribosomal RNA in each sample. The ratios between the densities of the target mRNAs and the 18S ribosomal RNA for each lane containing samples from the control animals were averaged for each blot. Then, for each blot, the target mRNA:18S ratios for all the lanes were divided by this average and converted into percentages, which were used in the statistical analyses.

Hormone Measurement

Serum FSH, LH, and T and intratesticular T (ITT) concentrations were determined by radioimmunoassay, as previously described [19].

Statistics

All data were evaluated to determine that they were normally distributed. Then the hormone and mRNA levels were analyzed using three (sham, 7-day SCI, and 14-day SCI) by four (control, T, FSH, and T+FSH) ANOVAs. When the treatment effects were significant (P < 0.05), planned a priori comparisons were made using Dunn tests to determine the statistical significance of differences among treatment groups.

RESULTS

Hormone Concentrations

Serum and testicular hormone levels are presented in Figure 1. In sham animals, serum FSH levels were suppressed by T alone (P < 0.05) or in combination with FSH (P < 0.01), whereas only the combination suppressed LH (P < 0.05). Treatment with T alone did not affect serum T levels, but it decreased testicular T with or without daily FSH injections (P < 0.01). FSH alone did not affect serum or testicular T.



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  		FIG. 1. Serum FSH, LH, testosterone, and intratesticular testosterone concentrations in sham control and SCI rats after FSH and/or T treatment. Results are expressed as means ± SEM. N = 5–8 rats/group. Significant differences from sham controls: * P < 0.05; ** P < 0.01

There were no significant changes in serum levels of FSH, LH, T, and testicular T concentrations, nor were there changes in serum FSH and LH responses to T treatments. However, treatment of SCI rats with FSH alone or in combination with T for 14 days resulted in increases in serum and testicular T levels that were above those of vehicle-treated SCI rats and sham control rats (P < 0.01).

Messenger RNA for Sertoli Cell Proteins

Table 1 summarizes the effects of FSH and T treatments upon the levels of various Sertoli cell mRNA transcripts in sham control rats and SCI rats.


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  		TABLE 1. Comparison of the effects of FSH and T on the levels of Sertoli cell transcripts between sham-operated control rats and SCI rats.*

Androgen receptor In sham control rats, testicular AR mRNA levels were unaffected by FSH alone but were 20%–30% lower in those rats given T with or without FSH, despite the lack of statistical significance. Seven days after SCI, AR mRNA levels in vehicle-treated SCI rats were 60% higher than those in sham control rats (P < 0.01), but this increase was blocked by all three treatments (Fig. 2, B and D). Fourteen days after SCI, AR mRNA levels in vehicle-treated SCI rats had recovered to control levels and were not affected by T treatment (Fig. 2, C and D). However, AR mRNA levels in those SCI rats that received FSH alone or in combination with T were higher than those in sham controls and vehicle-treated SCI rats (P < 0.05), a response that is opposite to that observed in sham control rats that received identical treatments.



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  		FIG. 2. Northern blot analysis of mRNA transcripts for AR and FSHR. Each lane contained 15 µg poly(A)+ RNA, and the radiographs were developed after 6 days of exposure. A) Sham control rats after 14 days of treatment. B and C) SCI rats after 7 and 14 days of treatment, respectively. D and E) Quantitative analyses of AR mRNA and FSH-R mRNA levels, respectively. These results are expressed as mean (±SEM) percent of sham controls in each blot. N = 5–6 rats/group. Significant differences from sham controls: * P < 0.05; ** P < 0.01

Follicle-stimulating hormone receptor Testicular FSHR mRNA levels in sham control rats were suppressed by the combined treatment with T and FSH (P < 0.05) but not by either hormone alone (Fig. 2, A and E). Unlike those associated with AR mRNA, FSHR mRNA levels in vehicle-treated SCI rats 7 and 14 days post-SCI were not different from those observed in sham controls. However, unlike the responses in sham control rats, T treatment of SCI rats increased FSHR mRNA both 7 and 14 days post-SCI (P < 0.05; Fig. 2, B, C, and E). The responses of FSHR mRNA to FSH alone or FSH plus T in SCI rats were also different from those observed in sham control rats. These treatments increased FSHR mRNA 14 days after SCI when compared with vehicle-treated SCI rats and sham controls (P < 0.05; P < 0.01).

Androgen-binding protein Similar to those associated with FSHR mRNA, ABP mRNA levels in sham controls were not affected by FSH or T alone, but they were suppressed by the combined treatment (P < 0.05; Fig. 3, A and D). Spinal cord injury per se did not affect ABP mRNA levels either 7 or 14 days post-SCI, but it did alter the responses of ABP mRNA levels to FSH and T. At both times, testicular ABP mRNA levels in SCI rats that received either hormone alone or both hormones in combination were higher than those of sham controls, vehicle-treated SCI rats, and sham control rats that received the same treatments for 14 days (P < 0.01; Fig. 3, B–D).



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  		FIG. 3. Northern blot analysis of the effects of SCI and hormone treatment on mRNA transcripts for ABP, Trf, Hemiferrin, and SGP-2. A) Sham control rats after 14 days of hormone treatment. B and C) SCI rats after 7 and 14 days of hormone treatment, respectively. D–G) Quantitative analysis of ABP, Trf, Hemiferrin, and SGP-2 mRNA levels, respectively. Results are expressed as means (±SEM) for the percent of sham controls in each blot. N = 5–6 rats/group. Significant differences from sham controls: * P < 0.05; ** P < 0.01

Transferrin In sham control rats, testicular Trf mRNA levels were 60% lower after treatment with T alone or when FSH was administered concurrently (P < 0.01), but they were not affected by FSH treatment alone (Fig. 3, A and E). Spinal cord injury did not alter the inhibitory effect of T on Trf mRNA at either time point. However, unlike the response in sham control rats, there was a twofold increase in Trf mRNA in SCI rats given FSH alone for 14 days (P < 0.01; Fig. 3, C and E). In addition, FSH attenuated the T-induced decrease of Trf mRNA in SCI rats at both times, whereas no such effect was seen in sham control rats.

Hemiferrin Levels of the 0.9-kb germ cell-specific hemiferrin mRNA were unaffected by T or FSH in sham controls when administered alone, but they were reduced by more than 50% in those rats given the combination of both (P < 0.01; Fig. 3, A and F). Hemiferrin mRNA was unaffected by SCI, but there was a transient increase in its responsiveness to T treatment 7 days post-SCI (P < 0.05; Fig. 3, B and F); this increase disappeared by Day 14 (Fig. 3, C and F). In addition, the drop in hemiferrin mRNA levels seen in sham control rats given both T and FSH did not occur in SCI rats. As with Trf mRNA, levels of hemiferrin mRNA in SCI rats were significantly increased after 14 days of FSH treatment, as compared to those levels in vehicle-treated SCI rats and sham control rats (P < 0.01; Fig. 3, C and F).

Sulfated glycoprotein 2 The level of SGP-2 mRNA in sham controls was not affected by FSH or T alone, but it was suppressed by the combined treatment (P < 0.01; Fig. 3, A and G). The SGP-2 level was transiently increased 7 days after SCI compared to levels in sham controls (P < 0.01; Fig. 3, B and G), but it recovered by Day 14 after SCI (Fig. 3, C and G). Sulfated glycoprotein 2 mRNA levels in SCI rats were stimulated by T or FSH alone both 7 and 14 days post-SCI (P < 0.01); such effects were no longer present when both hormones were used.

DISCUSSION

Both FSH and T can modulate spermatogenesis independently or synergistically through their effects on Sertoli cells [20, 21]. Testosterone is absolutely required for the initiation, maintenance, and restoration of qualitatively complete spermatogenesis in both immature and adult animals [19, 22], whereas FSH is only required for the initiation of spermatogenesis in postnatal animals [23]. Nevertheless, FSH has been found to augment the efficacy of testosterone in both maintaining and restoring spermatogenesis after hypophysectomy [19, 24]. Previously showed that regression of the seminiferous epithelium in SCI rats was attenuated by exogenous T but was unexpectedly and paradoxically enhanced when the SCI rats were given exogenous FSH [9]. These results and the up-regulation of transcripts for Sertoli cell-specific proteins by FSH [9] suggest that the T-dependent cellular events might be involved in SCI-related spermatogenic regression, and such effects might be associated with alteration in the signal events that mediate the effects of FSH and T on Sertoli cells. The current results provide evidence to support the above thesis.

Decreases in testicular mRNA levels for ABP and Trf in sham control rats after 2 wk of T and/or FSH treatments are different from those observed in cultured immature Sertoli cells, in which FSH and T stimulation of ABP and Trf secretion lasts for more than 10 days [25, 26]. Such differences could be related to experimental conditions (in vivo versus in vitro), or they could reflect age-related differences in Sertoli cell responsiveness to FSH or T [27, 28]. The decreases in testicular levels of AR and FSHR mRNAs in sham control rats following T or FSH treatment, respectively, are consistent with the feedback regulation of AR and FSHR genes by their respective ligands [14, 15]. Such decreases were accompanied by similar changes in ABP, Trf, and SGP-2 mRNA levels, thus suggesting that changes in the latter transcripts might be related to the availability of AR and FSHR and downstream signal events. Finally, the level of the germ cell-specific hemiferrin mRNA was significantly reduced in those rats that received combined FSH and T treatment. This effect cannot be accounted for by the loss of spermatogenic cells, as testis weights did not change significantly after FSH and T treatment (data not shown). The decrease in hemiferrin mRNA levels likely reflects changes in spermatocytes and/or spermatids, changes that are secondary to the effects of FSH and T on Sertoli cells.

The presence of normal FSHR mRNA levels in the testes of untreated SCI rats during the first 2 wk after injury is consistent with normal serum FSH levels in these rats and with what we have observed in Sertoli cell-enriched testes during the acute phase of SCI [10]. However, in contrast to those in sham control rats, hypophysectomized prepubertal rats [14], and cultured Sertoli cells [14], FSHR mRNA levels in SCI rats were significantly elevated after 14 days of FSH and/or T treatments. A transient but significant increase in testicular AR mRNA levels 7 days after SCI is also consistent with that observed in SCI rats with Sertoli cell-enriched testes [10]. Such an increase cannot be accounted for by the lack of a negative feedback signal, because serum and testicular T levels were normal in these SCI rats. Furthermore, AR mRNA levels were normal in those T-treated SCI rats that had less than 10% of normal ITT concentrations, and those levels in SCI rats that received FSH treatment for 14 days with or without concurrent T treatment were also elevated in the presence of increased levels of testicular T. Together, these results suggest that the responsiveness of AR and FSHR genes to their respective ligands might have been altered after SCI.

Suppression of Trf mRNA levels in SCI rats by T resembles that observed in T-treated sham control rats, indicating that SCI does not alter the responsiveness of the Trf gene to a reduced ITT environment. The twofold increase in Trf mRNA in SCI rats after 14 days of FSH treatment may have resulted from synergism between FSH and T on the Trf gene, as levels of FSHR and AR mRNA and testicular T were all increased in these SCI rats. These effects may also account for the lack of a fall in Trf mRNA levels in T-treated SCI rats that were also given FSH for 14 days. This proposition is corroborated by concomitant increases in levels of FSHR, AR, and Trf mRNA in Sertoli cell-enriched testes during the chronic stage of SCI [10]. In contrast, ABP mRNA levels were significantly increased in SCI rats that received FSH and T alone or in combination. In general, such increases paralleled those observed in FSHR and AR mRNA, and thus, they may reflect stimulation of the ABP gene by FSH and T.

In the prostate, SGP-2 (testosterone repressed prostate message—or TRPM-2) mRNA levels increase during organ involution after androgen ablation, leading to the belief that this protein might be associated with apoptosis [29]. However, others have demonstrated that SGP-2 also increases during organ/tissue regeneration [30–32], and the gene that encodes SGP-2 is homologous to that of human complement cytolysis inhibitor SP-40 [33]. These results indicate that SGP-2 might have a role in protecting cells against cellular damage. Consequently, an increase in SGP-2 mRNA levels in the testis 7 days after SCI perhaps represents a response of Sertoli cells to the acute effect of SCI on the testis. Such increases may result from enhanced Sertoli cell responses to FSH or T, as SGP-2 mRNA levels in the testes of SCI rats increased significantly after FSH and T treatment, a phenomenon not seen in sham control rats. Modification of androgen regulation of TRPM-2 mRNA in rat prostate after SCI has been reported previously [34]. Finally, the transient increase in hemiferrin mRNA 7 days after SCI suggests that the functions of spermatocytes and spermatids might have been altered, which is consistent with our previous histological findings [6]. Unlike those levels for Sertoli cell-specific Trf, levels of hemiferrin mRNA were stimulated by T and FSH alone 7 and 14 days post-SCI, respectively. Such effects may thus reflect changes in spermatogenic cells, changes that are secondary to the effects of hormones on Sertoli cells.

In addition to changes in Sertoli and germ cell function, the current study also found changes in Leydig cell steroidogenic function. Despite normal serum levels of FSH, LH, and T and normal testicular T concentrations in SCI rats at both time points, FSH alone or in combination with T significantly increased testicular and serum T concentrations 14 days after SCI. These increases cannot be correlated with serum LH nor with the status of LH receptors, since LH-receptor mRNA levels, measured by Northern blot cDNA hybridization [35], were not affected by current hormone treatments (data not shown). Such increases in Leydig cell T production in SCI rats given FSH might have been mediated by local paracrine factors [36] as a result of changes in Sertoli cell functions.

In addition to the neuroendocrine effects, SCI may also affect testicular function by disrupting normal neural input to the testis [37]. Previous studies have demonstrated that Sertoli cell function can be modulated by interactions between FSH and neurotransmitters or neuropeptides. In cultured immature Sertoli cell, the effects of FSH on cAMP production and protein kinase activity can be modulated by the adrenergic agonist isoproterenol [38, 39]. In addition, stimulatory effects of FSH on Sertoli cell proliferation [40] and on ABP and inhibin production were decreased by beta-endorphin [41, 42]. The Leydig cells are the major source of testicular beta-endorphin [43] that modulates Leydig cell T responses to LH [44], and they may serve as a paracrine feedback signal between Leydig and Sertoli cells [42]. It is postulated that SCI might perturb neural-hormonal interactions in the testis and result in up-regulation of AR and FSHR genes by their respective ligands. Consequently, Sertoli cell functions might be overstimulated by T and FSH, resulting in an intraepithelial environment that is not optimal for normal spermatogenesis. In addition, changes in FSH-Sertoli cell interactions could also disrupt paracrine feedback mechanisms between Sertoli and Leydig cells. Such effects could further alter the function of Leydig cells, resulting in increased T production of testosterone, and perhaps of other paracrine factors, such as beta-endorphin, which could further stimulate Sertoli cells to alter their normal function.

In summary, in non-SCI adult rats, levels of mRNA transcripts for most Sertoli cell proteins were not affected by FSH and T when each was administered alone, but these levels were reduced when both hormones were used. In contrast, identical hormone treatments resulted in increases in the same Sertoli cell transcripts in SCI rats. These results indicate that the signal pathways mediating the effects of FSH and T might have been compromised after SCI. The impact that such changes may have on spermatogenesis is suggested by the changes that occurred in germ cell-specific hemiferrin mRNA after the hormone treatments. These results are consistent with the hypothesis that altered FSH and AR signal events are involved in SCI-related abnormal Sertoli cell function and spermatogenic regression. In addition, Leydig cell steroidogenic function was also compromised in SCI rats. Further investigation into the signal events that mediate the changes in Sertoli and Leydig cell functions after SCI will provide new insights into the mechanisms that lead to the deleterious effects of SCI on spermatogenesis and will form the basis for the development of therapeutic regimens to prevent and treat SCI-related male infertility.

ACKNOWLEDGMENTS

We would like to thank the following investigators for their generous gift of the cDNA that was used in the current experiment: Drs. M. Griswold (transferrin and FSH-R), D. Joseph (ABP), S.C. Liao (androgen receptor), and D. Segaloff (LH receptor). The FSH and LH RIA kits were kindly provided by the National Institutes of Health Pituitary Hormone Program and Dr. A.F. Parlow. The editorial assistance of Randi Rutan is also appreciated.

FOOTNOTES

First decision: 30 March 2000.

1 Supported by V.A. Rehabilitation R&D Services (B885-2RA). Back

2 Correspondence: H.F.S. Huang, Department of Surgery Section of Urology, UMD-New Jersey Medical School, 185 S. Orange Avenue, Newark, NJ 07103. FAX: 973 972 6803; huanghf{at}umdnj.edu Back

Accepted: April 11, 2000.

Received: February 25, 2000.

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