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Biology of Reproduction 61, 154-161 (1999)
©Copyright 1999 Society for the Study of Reproduction, Inc.

Articles

Improvement of Spermatogenesis in Adult Cryptorchid Rat Testis by Intratesticular Infusion of Lactate1

J.L. Courtens2,a, and L. Plöenb

a Institut National de la Recherche Agronomique, Department Physiologie de la Reproduction des Mammifères domestiques, F-37380 Nouzilly, France b Swedish University of Agricultural Sciences, Centre for Reproductive Biology, Department Anatomy and Histology, S-750 07 Uppsala, Sweden


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In order to test the hypothesis that a lack of energy could be a cause of germ cell death at high temperatures, cryptorchid rats testes were infused with lactate, delivered by osmotic pumps over 3–15 days. In cryptorchid testes, the spermatids and spermatocytes were lost between 3 and 8 days. In cryptorchid testes supplemented with lactate, elongated spermatids persisted in a few seminiferous tubules at Day 15. Elimination of round spermatids occurred progressively between 3 and 15 days, mostly at stage VIII. The loss of spermatocytes increased after 8 days, and 30% of seminiferous tubules still contained meiotic or meiotic plus spermiogenetic cells at Day 15. After 8 days, the chromatin of step 8 round spermatids was abnormal and nuclear elongation did not commence. The Sertoli cell cytoplasm that was retracted toward the basal compartment of the seminiferous epithelium could not hold the germ cells of the adluminal compartment. Therefore, attachment of germ cells to Sertoli cells and the supply of lactate seem necessary for the development of germ cells at high temperatures. The improvement in spermatogenesis in cryptorchid supplemented testes for several days is a new finding.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In most mammals, the testis is kept 3–5°C below body temperature. A slight increase of temperature for a short time or experimental cryptorchidism, which is more convenient for longer periods, results in a rapid loss of mature germ cells [1–3]. While the population of Sertoli cells is generally not affected, the number of primary spermatocytes and spermatids drops rapidly [4]. In many species, including the rat, the populations of spermatogonia are not modified immediately [5], allowing new spermatogenetic cycles to start again if a normal temperature is restored within a reasonable time period [6]. High temperatures induce an increased synthesis of several proteins [7, 8] and a decrease of many others [9, 10]. Several enzymatic activities are affected [5, 11–13], and the lipid composition of plasma membranes is modified [14]. The profound modifications of primary spermatocyte and spermatid cell structures [15] generate, or are a consequence of, rapid and massive apoptosis [1, 16, 17]. Recently, the p53 inducer of apoptosis was proposed as a main factor occurring in cryptorchidism [18]. However, it was rapidly shown that the effects of heat are only delayed in mice lacking p53 [19]. The apparent multiple-point actions of elevated temperature on the testis, and the rapid reversal of deleterious effects on germ cells, are intriguing. Taking into account that ischemia leads to the same troubles in a few hours [20] and that some animal species are not strongly affected by cryptorchidism [21], the multiple-point actions could be more easily understood if high temperature actually acted on a few biological key functions. Among these, nutrition drives all aspects of cell synthesis.

Testis energy is provided mostly by glucose, which is the preferred substrate in mammals [22, 23]; and most germ cell protein synthesis is under control of glucose metabolism [24, 25]. Glycolytic enzymes are present in germ cells of the adluminal compartment of the seminiferous tubules, but they are probably not active in the normal testis [26–28]. Moreover, glucose is present in very low concentration in the lumen of the seminiferous tubules [29]. Most spermatid ATP is therefore synthesized through the degradation of lactate and pyruvate produced massively by Sertoli cells [28, 30–32]. Pharmacological lactate deprivation induces cell death in isolated rat germ cells [33]. However, the testicular lactate concentration is not well established in cryptorchid animals. According to the literature, it could be low [34] or high [35]. Thus, the situation in cryptorchid testes is the following: the inter-Sertoli cell junctions are not modified [36]; Sertoli cells, glucose, and/or other energy substrates are present. Either the germ cells located in the adluminal compartment of the seminiferous tubules could be unable to use lactate produced by the Sertoli cells, or the production could be lowered at high temperatures.

To test these hypotheses, we measured intratesticular lactate and supplemented the cryptorchid testes of adult rats to maintain the testicular concentration. In order to avoid systemic side effects of lactate, we used direct supplementation by internal osmotic pumps in ambulatory animals. Rodents were chosen because the continuous lymphatic space surrounding the seminiferous tubules allows a rapid diffusion to the entire testis. Rats were selected for their convenient testis size.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals and Treatments

Adult Wistar male rats weighing between 480 and 540 g and aged 3–6 mo were made unilaterally cryptorchid by surgery for 3, 4, 6, 8, 10, and 15 days (n = 3 for each duration); or they were made unilaterally cryptorchid and the cryptorchid testes were supplemented with potassium lactate for similar durations (n = 3 for each duration). Supplementation started during the surgery and was achieved by intraperitoneal osmotic pumps connected to the testes through a silicon catheter. The contralateral scrotal testes, as well as testes from six intact animals, were used as controls. Additional controls included the lactate infusion of scrotal testes for 6 and 8 days. The testes were processed for qualitative and semiquantitative histological evaluation of spermatogenesis.

The investigations were conducted in accordance with the Guiding Principles for the Care and Use of Research Animals promulgated by the Society for the Study of Reproduction.

Preparation of Osmotic Pumps

The amount of lactate to be delivered per testis (1.66 µM/h) was calculated from the mean number of Sertoli cells per testis [37] and their daily lactate production in vitro [38]. Lactate solutions were prepared by neutralization of lactic acid (60% syrup, equal amount of D- and L-isomers; Sigma Chimie, Saint Quentin Fallavier, France) to pH 7.2 with 2 M potassium hydroxide. After neutralization, distilled water was added to obtain a final lactate concentration of 36%. Osmotic pumps (Alzet; Phymep, Paris, France; sterile, 2-ml capacity, 1-wk constant delivery at 1 µl/h) were filled with the lactate solution, filtered through 0.22-µm sterile filters. A silicon catheter (0.76/1.65-mm internal/external diameters; Sigma Medical France, Nanterre, France), filled with the same solution, was attached to the pumps. To start the pumps, they were incubated in 0.9% sterile NaCl at 37°C for 4 h before surgery.

Surgery

The rats were anesthetized with gaseous ethyl ether. The abdomen was incised on the median line, and the right testis was gently pushed upward into the abdominal cavity. The gubernaculum either was sewn to the abdominal wall or was cut. With the cutting edge of a fine needle, a small hole was made in the tunica albuginea in a place free of apparent blood vessels. Care was taken not to damage the seminiferous tubules lying underneath. The pump was placed in the abdomen, and the catheter was inserted slowly into the testicular parenchyma through the hole before being glued to the tunica albuginea with one drop of histoacryl surgical glue. When the gubernaculum was cut, the osmotic pump was placed in the empty scrotum, thus avoiding further testicular descent. This location was used to avoid reopening of the abdomen when the pumps had to be replaced after 1 wk. In that case, the scrotum was incised under general anesthesia, and the new pump was connected to the catheter.

In 2 controls, the left scrotal testis was injected as well. The pump was connected as described above, and the testis was replaced down in the scrotum through the inguinal canal.

Rats were returned to their room, under a constant 14L:10D light schedule, and were watered and fed ad libitum with a commercial rat food.

Histology

Rats were killed by lethal anesthesia on Days 3, 4, 6, 8, 10, and 15 following the induction of cryptorchidism ± supplementation. The testes were weighed and cut into two parts. One half was frozen for further analysis of lactate, and the second was fixed by immersion for 1–3 days at 4°C with 4% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4. The best-fixed equatorial part was cut into small pieces, which were postfixed in 2% osmium tetroxide in cacodylate buffer for 1 h at room temperature, dehydrated in an alcohol series, and embedded in Epon. Semithin sections (2 µm) stained with toluidine blue (pH 9.0, 60°C, 1 min) were observed under a light microscope equipped with a digital video camera.

Evaluation of Spermatogenesis

For each treatment, 70–165 cross sections of seminiferous tubules were observed and staged according to Clermont and Leblond [39]. Since staging was not possible in all tubules, additional patterns were counted: seminiferous tubules (ST) with Sertoli cells plus spermatogonia only; ST with Sertoli cells, spermatogonia, and spermatocytes only; ST with massive loss of spermatocytes; and ST with massive degeneration or loss of round spermatids. Nuclei of Sertoli and germ cells present in cross sections of seminiferous tubules were counted on magnified digital pictures. Since no modification was noticed in the number of Sertoli cells per tubule cross-area, ratios of germ cells/Sertoli cells were used to compare the tubules active in spermatogenesis.

Estimation of Lactate

Testicular D- and L-lactate concentrations were measured using a commercial enzymatic kit and the procedure recommended by the manufacturer for tissues (Boehringer Mannheim, Mannheim, Germany). The results are expressed as the sum of both isomers.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Testis Weight

The weights of the contralateral control testes were not significantly different from those of intact rats during the treatment (Fig. 1). When scrotal testes were supplemented with lactate for 4 and 6 days, the weights did not vary (not shown). The weights of cryptorchid testes were significantly lower after 4 days, and after 15 days, they were only 26% of the control value.



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  		FIG. 1. Testis weights (mean ± SD, n = 3) in control, cryptorchid, and lactate-supplemented rats.

In cryptorchid supplemented testes, the weights were significantly different from those of the controls and from those of cryptorchid testes at 8 days. After 15 days, the mean weight diminished by 50% (Fig. 1).

Lactate in Testes

The lactate concentrations were close to 8 mM in control rat testes, in the scrotal testes of hemi-cryptorchid animals, and in scrotal testes supplemented with lactate. The lactate concentration decreased in cryptorchid testes to 3 mM at 4 days; and from 4 to 15 days, it re-increased progressively up to 6 mM. In supplemented testes, the lactate concentration remained close to 6–7 mM from Day 3 to 15 (Fig. 2).



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  		FIG. 2. Lactate (D+L) concentration in control, cryptorchid, and supplemented testes, mean mM/L of testis ± SD, n = 3. The value at Day 0 is from intact animals. Significant differences between treatments occurred at 3 and 4 days. The lactate concentration did not vary in supplemented testes. It became rapidly low in cryptorchid testes, but increased after 4 days, reflecting a decrease in testis weight (Fig. 1).

The total content of lactate/testis was calculated from the concentrations and testis weights (Fig. 3). It the intact rats it was close to 1.9 mg, while it was remarkably low and stable (0.4–0.6 mg) in cryptorchid testes. It decreased progressively to 0.7 mg in supplemented testes during the 15-day experimental period.



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  		FIG. 3. Calculated mean lactate (D+L) mg/testis. The value at Day 0 is the mean from testes in intact control animals. The points correspond to calculations from mean data presented in Figures 1 and 2. The amount of lactate was low and constant in cryptorchid testes and decreased in the supplemented testes. This indicates that a basal production is maintained in the cryptorchid testes but that the concentration (Fig. 2) is probably not regulated. In contrast, in the supplemented testes, the amount diminished, due to lower testis weights, but the concentration (Fig. 2) did not vary, suggesting regulation.

Quantitative and Qualitative Spermatogenesis

In the cryptorchid testes The number of Sertoli cells per transversal cross section of seminiferous tubule was not modified. Sertoli cells displayed normal indented nuclei. Their cytoplasms were punctuated with some holes at Day 3, and many more at Day 4 (Fig. 4, a–d). From Day 6 to 15, the Sertoli cells were highly vacuolated, displayed both small and large holes in sections, and contained many lipid droplets (Fig. 4, e, f, and h). The numbers and morphology of spermatogonia were similar to those for controls.



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  		FIG. 4. Semithin sections through seminiferous tubules of cryptorchid testes (bars = 20 µm). a, b) 3 days. a) Spermatids at step 9 (beginning of nuclear elongation) were still present, but elongation ceased rapidly and was not observed after 4 days (e–h). Few vacuoles in Sertoli cell cytoplasm were present around the primary spermatocytes. b) Stage II seminiferous tubule with normal appearance. c, d) 4 days. c) Spermiation. All germ cells usually present at this stage were present and displayed a normal appearance. However, large vacuoles (arrow) were present at the limit of the basal/adluminal compartments of the seminiferous tubule. d) Stage VI. Clear areas surrounded most germ cells present in the adluminal compartment of the seminiferous tubule. Round germ cells were released in the lumen (arrow). e, f) 8 days. The seminiferous tubules contained mostly Sertoli cells and spermatogonia. Numerous lipid droplets accumulated. Flagella, not linked to spermatid heads, were present (arrow). g, h) 15 days. Same situation as for e and f.

Complete spermatogenesis was present in most seminiferous tubules 3 days after the induction of cryptorchidism (Fig. 4, a and b). Later, elongating spermatids were not observed (Fig. 4, c–h). Primary spermatocytes and round spermatids were massively lost into the lumen of the seminiferous tubules at Days 4–6 (Fig. 4d). Elongated spermatids degenerated after 6 days, and most of their nuclei were absent after 8 days. However, many flagella were still present at Day 15 (Fig. 4, f and h). Stage VIII seminiferous tubules were more frequent (18% and 31%, respectively, at 3 and 6 days) than in intact testes (9.85%), suggesting an arrest of spermatogenesis at that point (Fig. 5a). A massive degeneration of step 8 spermatids was observed in 8% of tubules at Day 3 and proceeded until Day 8 (Fig. 5a). The elimination of primary spermatocytes increased from Day 6 to 15 (respectively, in 4% and 20% of the seminiferous tubules at Days 6 and 8). The last remaining primary spermatocytes were lost in 30% of the seminiferous tubules at Day 15 (Fig. 5a). About 50% of them displayed distended cytoplasm poor in organelles and/or compacted chromatin suggesting apoptosis (Fig. 4, d and f).



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  		FIG. 5. Percentages of seminiferous tubules observed at a given stage in controls, cryptorchid testes, and cryptorchid testes supplemented with lactate. I–VII: Seminiferous tubules containing round spermatids. VIII: Spermiation. IX–XIV: Seminiferous tubules with elongating spermatids. Ser + Gon: Tubules with only Sertoli cells and spermatogonia. Ser + Gon + SC1: Tubules also containing primary spermatocytes. All SC1 lost: Tubules depicting massive loss of primary spermatocytes. Stide 8 lost: Massive loss of spermatids at stage VIII. In cryptorchid testes (a), the proportion of stage I–VII seminiferous tubules decreased from Day 0 to Day 8, while stages IX–XIV were not observed after 3 days. The proportion of tubules at stage VIII increased from 0 to 6 days, suggesting an arrest of spermatogenesis at this point. Abnormal seminiferous tubules appeared at Day 3 and their proportion increased afterward. In lactate-supplemented testes (b), the proportions of seminiferous tubules at stages I–VII and IX–XIV decreased progressively during 15 days. The proportion of tubules at stage VIII only increased at Day 3 (with a corresponding release of round spermatids in the lumen) and decreased afterward. The most important defects in seminiferous tubule cell contents were delayed as compared to observations in the cryptorchid testes.

In cryptorchid supplemented testes Sertoli cell nuclei were not different from those in controls. The Sertoli cell cytoplasm displayed some vacuoles at Day 3, and more holes from Days 8 to 15 (Fig. 6, a–h). However, the cells were comparatively less vacuolized than in the unsupplemented testes (compare Figs. 4 and 6). A few lipid droplets appeared at Day 8, and their number increased mostly at Day 15 (Fig. 6, f and g). The spermatogonia in the cryptorchid supplemented testes were similar to those in controls.



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  		FIG. 6. Semithin sections through seminiferous tubules of cryptorchid testes supplemented with lactate (bars = 20 µm). a, b) 4 days. a) Stage III, b) early stage IX. No difference from normal testis was noticed. c, d) 6 days. c) Stage IX showing a normal cell content and beginning of elongation of spermatids. Residual bodies (rb) are normally phagocytosed by Sertoli cells. d) Stage III. Elongation of old spermatids proceeds normally. e, f) 8 days. e) Stage IX. Normal cell types are present, but the spermatids have not started elongation (compare with c) and look more like step 8; but since there are no older (step 19) spermatids, the stage is IX and the spermatids are arrested in step 8. f) Stage >= IX. The chromatin of spermatids arrested at step 8 displays vacuoles (double arrow). The Sertoli cell cytoplasm contains small lipid droplets (arrow). g, h) 15 days. g) Stage XIV. Meiotic divisions (arrow) occur in a seminiferous tubule devoid of elongated spermatids. h) Stage III. Spermatids step 16 are still present, while the round spermatid nuclei contain vacuoles (arrow) and the basal compartment of the seminiferous tubule is highly vacuolated and contains dead cells.

Primary spermatocyte number was not modified until Day 6 (Fig. 5b). Most primary spermatocytes still had a normal appearance at 8–10 days. Few were apoptotic as judged from the scarcity of images with chromatin compaction or fragmentation and of cytoplasm poor in organelles (Fig. 6, e and g). Morphologically intact spermatocytes were shed into the lumen in 3%, 5%, and 6% of the seminiferous tubules, respectively, at Days 6, 8, and 15. At Day 15 (Figs. 5b and 6g), 28% of seminiferous tubules still contained intact primary spermatocytes.

Most round spermatids younger than step 8 were normal until Days 10–15. Elongated spermatids were still present in 4% of seminiferous tubules at Day 15 vs. 21% in the controls (Fig. 5b). Elimination of round spermatids occurred progressively between 3 and 15 days, mostly at stage VIII. However, the percentage of tubules observed at stage VIII was only slightly elevated at Day 3 as compared with that in the testes of control and cryptorchid animals (Fig. 5). After 8 days, holes in the chromatin of step 8 spermatids appeared (Fig. 6, f and h), and many heads were lost into the lumen of the seminiferous tubules, together with spermatocytes (Fig. 6g). Elongated spermatids were morphologically normal, and spermiation occurred until Day 15 (Fig. 6h). Thirty percent of the seminiferous tubules were still active in spermiogenesis and/or meiosis prophase at Day 15 (Figs. 5b and 6, g and h).

Yield of Spermatogenesis in Fairly Intact Seminiferous Tubules Containing Most Spermatogenetic Cell Types

In control testes, the ratio of germ cells/Sertoli cells was comparable to classical data in the rat [40]. In cryptorchid supplemented testes, the ratio of spermatogonia/Sertoli cells did not vary, while the yield for primary spermatocytes was stable for 8 days before decreasing to 60% at 15 days. Round spermatids/Sertoli cells diminished to 60% of the control value at 6–8 days and remained stable thereafter. Elongating spermatids/Sertoli cells diminished dramatically at Days 8 and 15 (50% and 30% of the control value). The elongated spermatids/Sertoli cells diminished to 60% of normal values at Day 15.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the cryptorchid testes, lactate dropped to about 25% of the normal content after 3 days, and the level thereafter was fairly constant. Loss of the germ cells located in the adluminal compartment of the seminiferous tubules started at Days 3–4 and developed rapidly.

In the supplemented testes, lactate concentration was maintained at close to the normal level, and several observations were made: 1) the weights dropped less rapidly than in nonsupplemented cryptorchid controls; 2) germ cell loss did not commence as early as in the nonsupplemented testes; 3) many primary spermatocytes developed through the meiotic divisions, and some spermatids could differentiate up to spermiation; 4) the elongation of spermatids and the Sertoli cell architecture were maintained for 1 wk.

The weight curve in cryptorchid testes showed a rapid decrease starting at Day 3, and a parallel slope of decrease was observed after lactate supplementation starting at Day 6 (Fig. 1). The differences in weight should be due to the loss of spermatocytes and spermatids, which persisted in higher numbers in supplemented testes, at any given time. It has been demonstrated that in culture, 95% of available glucose is degraded to lactate and pyruvate by Sertoli cells, which also reduce their production when exogenous lactate or pyruvate is added to the milieu [24]. Such regulation probably exists in vivo as well, since no increase in lactate occurred in the scrotal supplemented testes. Lactate is known as the preferred energy substrate for spermatocytes and spermatids [23, 25, 41, 42]. In these cells, no ATP is generated through lipid degradation [32], and the low glucose concentration in the adluminal compartment of the seminiferous tubules [29] is not sufficient to feed the glycolytic pathway.

The ATP production by germ cells depends mostly upon the mitochondrial metabolism and the regular provision of usable "fuel" [32]. The synthesis of lactate and pyruvate, necessary for germ cell protein synthesis, is partly regulated by FSH and insulin [24]. None of these regulators are supposed to vary notably after cryptorchidism for few days. It is conceivable that any lowering in energy substrate could have the deleterious effects observed here. The addition of exogenous lactate, which limits the loss of germ cells for several days and also lowers the number of degenerating germ cells for 2 wk, favors the hypothesis that energy provision is one of the important parameters for survival of testicular germ cells at high temperature.

In the present experiments we interpret the low lactate concentration in the cryptorchid testis as maintenance of a basal production by the interstitial tissue, the Sertoli cells, and the spermatogonia, because the total amounts per testis were not modified when germ cells were lost. This excludes the possibility of a lowering of lactate due to increased germ cell consumption. Our results demonstrate that the lactate production was rapidly lowered at high temperature, before any morphological alterations in the Sertoli cells and in germ cells were observed. No experimental explanation can be offered now, but heat susceptibility of enzymes cannot be disregarded, since many testicular processes are sensitive to temperature [5, 11–13].

Nakamura et al. [41] demonstrated that lactate degradation by isolated spermatids in vitro at high temperatures, while being effective, was not coupled to oxidative phosphorylation. However, they used 20 mM lactate, a concentration that, being above the normal range, could also alter the pyruvate/lactate ratio, which must be close to or lower than 1/10 to favor ATP production through the lactate-to-acetate pathway [27].

Under our in vivo conditions, the provision of exogenous lactate maintained adluminal germ cells for some days, but it was not sufficient to support full spermatogenesis for more than 1 wk. The behavior and aspects of spermatocytes suggest that apoptosis was less prominent under lactate treatment. Most of the released spermatocytes were morphologically intact but were not correctly attached to the Sertoli cells. Some of them underwent meiotic divisions after 2 wk, suggesting that the microenvironment was correct for their development. Young spermatids that had formed before the treatment were also able to develop up to spermiation at high temperature, whereas spermatids formed during the treatment did not elongate and displayed abnormal chromatin patterns. This means that the maintenance of spermiogenesis at body temperature requires both an adequate extracellular milieu and some additional factors. Among them, the relations between spermatids and Sertoli cells through specialized contacts [43–56] are very important. These were modified after 1 wk, and spermatids at step 8 not embedded in Sertoli cytoplasm accumulated, a phenomenon observed earlier in the heated ram testis [4].

Concomitantly with the problems of cell contacts, vacuoles appeared in the cytoplasm of Sertoli cells. These are generally considered the first signs of dysfunction. Because no deleterious effects were noted in the scrotal testis infused with lactate, and since vacuoles were also observed in the cryptorchid testes, it seems that lactate addition was not involved in the formation of vacuoles but instead could have delayed their appearance. Moreover, lactate is necessary for the normal expression of at least one Sertoli cell gene involved in the control of spermatogenesis [57].

At this point in our work, we suggest that the initiation of specialized cell contacts between Sertoli cells and germ cells is one of the main factors impaired by high temperature that is not corrected by lactate provision.

In conclusion, it seems that lowered lactate is not the only parameter involved in germ cell degeneration at high temperature, but that supplementation supports spermatocyte development and delays the degeneration of spermatids.


   ACKNOWLEDGMENTS
 
We sincerely thank Dr. J.C. Thierry for suggesting the use of osmotic pumps; Ms. O. Moulin and Mr. A. Begué for photographic works; and Drs. M.T. Hochereau de Reviers, B.P. Setchell, J.L. Dacheux, and U. Kvist for their comments, suggestions, and critical reading of the manuscript.


   FOOTNOTES
 
1 This work was supported through a scientific exchange program between France (INRA) and Sweden (SJFR). Back

2 Correspondence: FAX: 33 2 47 42 77 43; courtens{at}tours.inra.fr Back

Accepted: February 22, 1999.

Received: September 15, 1998.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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