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Biology of Reproduction 63, 544-550 (2000)
© 2000 Society for the Study of Reproduction, Inc.

Regular Article

Retinoid Modulation of Plasminogen Activator Production in Rat Sertoli Cells1

Rita Canipari2,,a, and Michela Galdieria,b

a Department of Histology and Medical Embryology, University of Rome "La Sapienza," Rome, Italy b Institute of Histology and Embryology, II University of Naples, Naples, Italy

ABSTRACT

Tissue type (t) and urokinase type (u) plasminogen activators (PAs) have been shown to be secreted by Sertoli cells in the seminiferous tubules in a cyclic fashion and to be dependent upon FSH stimulation or upon the presence of adjacent spermatogenic cells. In the present study we have analyzed the production of PAs by retinoid-treated rat Sertoli cells. In addition, because retinoids modulate the response of Sertoli cells to FSH either potentiating or antagonizing its action, we have investigated a possible modulation of FSH-stimulated PA production. Under basal conditions, Sertoli cells, isolated from prepubertal rats, secrete predominantly uPA. A significant dose-dependent inhibition of uPA activity was observed after treatment with retinol, while no significant effect was detected upon tPA secretion. When Sertoli cells were cultured in the presence of 0.25 µM retinol, a significant inhibition of uPA activity was evident after 16 h of treatment and reached approximately 80% after 48 h of treatment. The analysis of the mRNA levels revealed that retinol induces an inhibition of the steady-state levels of uPA mRNA without affecting those of tPA. Moreover, retinol affected uPA mRNA levels by increasing mRNA turnover. The effect of retinoids on Sertoli cells isolated from older animals was less evident, possibly due to the reduced production of uPA with the increase of age of the donor animals. Our results on the effect of retinoids upon Sertoli cell uPA production reinforce the importance of retinoids in the control of postnatal testis development.

gene regulation, Sertoli cells

INTRODUCTION

In mammals, spermatogenesis takes place in the seminiferous tubules where Sertoli cells create the unique microenvironment necessary for normal germ cell development [1]. Sertoli cells are subjected to a complex control by different molecules such as hormones, growth factors, and vitamins. Vitamin A is one of the molecules regulating spermatogenesis, and vitamin A deficiency causes loss of developing germ cells and sterility [2, 3]. Sertoli cells are target cells for retinol as indicated by the presence in their cytoplasm of high levels of retinol and retinoic acid (RA) binding proteins [4, 5]. In addition, different isoforms of RA nuclear receptors have been found in these cells [6–8]. This vitamin regulates a variety of cellular metabolic activities. In particular, retinol strongly inhibits Sertoli cell protein kinases, either calcium-phospholipid dependent (PKC) or cAMP dependent (PKA) [9, 10], and antagonizes the stimulatory effect of FSH on Sertoli cell cAMP production and aromatase activity [11]. Moreover, it has been demonstrated that secretory activity of Sertoli cells is under retinoid control. Addition of retinol in the medium doubles the total amount of secreted glycoproteins [12, 13] and the production of specific proteins like androgen binding protein and transferrin is also modulated by vitamin A [14–17]. In addition, we have recently demonstrated that Sertoli cells are the main sites of RA production in the seminiferous tubules because Sertoli cells subcellular fractions actively convert retinol into RA [18].

The plasminogen activator (PA) system is a general proteolytic system involved in many biological processes such as spermatogenesis, oogenesis, embryo implantation, fibrinolysis, angiogenesis, inflammation, and tumor metastasis [19–22]. Two forms of PA, urokinase type (uPA) and tissue type (tPA), have been characterized in mammals. They are encoded by two different genes but share the ability to cleave plasminogen to form the active protease plasmin. Both enzymes have been detected in rat testis where Sertoli cells are believed to be the main source of PA production. The two different PAs are secreted by Sertoli cells; uPA is secreted when the cells are cultured under basal conditions, whereas the tPA is secreted in large amount in response to FSH stimulation [23]. Other identified proteins of the PA system are two specific PA inhibitors: PAI-1 and PAI-2 [24]. In the testis PAI-1 is secreted either by peritubular cells [25, 26] and by Sertoli cells [27].

The function of these proteases and antiproteases in the testis is not yet understood. Because of their stage-specific production and their different regulation by gonadotropins and growth factors, it has been hypothesized that each of these molecules can be associated with different functions. A role for these proteases has been suggested in the continuous remodeling of the seminiferous epithelium taking place during the release of the preleptotene spermatocytes from basement membrane [28], spermiation [29], the detachment of residual bodies from the mature spermatids [30], and their phagocytosis by Sertoli cells [31]. In addition, they can open the tight junctions between neighboring Sertoli cells allowing migration of zygotene spermatocytes to the adluminal compartment of the seminiferous epithelium [32]. Conversely, the production of protease inhibitors by Sertoli cells in the adluminal compartment can modulate the net protease activity within the seminiferous tubule at defined stages, such as during the process of translocation and spermiation, therefore maintaining the integrity of the Sertoli cell barrier [27].

It has already been demonstrated that uPA production is modulated by RA in rat Sertoli cells, but contrasting data appear in the literature. While Rosselli and Skinner [33] showed a significant suppression of uPA production in midpubertal Sertoli cells, Vihko et al. [34] showed a stage-specific stimulation of the same enzyme. To characterize further the regulation of PA production in the testis, we have investigated the effect of retinoids on Sertoli cell secretion of either uPA or tPA in basal conditions and after FSH stimulation. Moreover, the age dependency of PA regulation has also been studied.

MATERIALS AND METHODS

Materials

Eagles minimum essential medium (MEM) was obtained from Gibco (Grand Island, NY). Chromogenic plasmin substrate S-2251 (D-val L-leu L-lys, p-nitroanilide·2HCl) was obtained from Bachem Feinchemikalien AG (Basel, Switzerland); calphostin C was obtained from Calbiochem (La Jolla, CA). [32P]GTP was from New England Nuclear Corp. (Boston, MA). Hyaluronidase, amiloride, casein, dibutyryl cAMP, fibrinogen, plasminogen, and urokinase were purchased from Sigma Chemical Co. (St. Louis, MO). Nylon membranes and films were from Amersham Italia (Milano, Italy). Enzymes were purchased from Boehringer-Mannheim (Mannheim, West Germany) or Promega (Madison, WI). All other chemicals used were analytical grade and purchased from Sigma or Bio-Rad. Ovine FSH-S17 was supplied by the NIDDK rat Pituitary Hormone Distribution Program (NIH, Bethesda, MD). The mouse uPA and tPA cDNA clones were kindly provided by Dr. D. Belin (Genève, Switzerland).

Sertoli Cell Preparation and Culture

Primary Sertoli cell cultures from 10-, 15-, 20-, and 30-day-old Wistar rats were prepared as previously described [23]. Briefly, detunicated testis fragments, obtained from pools of 8–12 rats of the same age, were subjected to sequential enzymatic digestions with trypsin (0.25%), collagenase (0.1%), and hyaluronidase (1 mg/ml) at 32°C for 30 min each. Between enzymatic treatment and washes, tubules sedimented by gravity. At the end of the enzymatic digestions the tubules were fragmented by gentle pipetting and centrifugation for 2 min at 75 x g. The pellet was resuspended in MEM at a ratio of 0.1 ml of fragments to 1 ml of serum-free MEM supplemented with glutamine, nonessential amino acids, gentamicin, penicillin, and streptomycin. Equal amounts of the suspension were plated and incubated at 32°C in a controlled atmosphere of 95% air and 5% CO2. On the third day of culture, a monolayer of Sertoli cells was usually formed and the cells were treated with a hypotonic solution to remove contaminating germ cells [35]. All treatments were performed in medium supplemented with 0.1% BSA and started on the fourth day of culture. Retinoids were usually added to the cells at a concentration of 0.25 µM (final concentration). Retinoid stock solutions were made up in ethanol (0.1% final concentration in the medium). At the end of incubation, culture fluids were collected and stored frozen until assayed for PA activity. The cells were washed extensively with fresh medium and further processed.

The assessment of contamination due to peritubular myoid cells was performed routinely on every culture. Myoid cells were detected by histochemical localization of alkaline phosphatase activity [36]. Cultures with more than 2% myoid cell contamination were not used in the present study.

PA Assay

Enzymatic activity of PA was assessed by the method of Shimada et al. [37] using a chromogenic substrate assay. In this assay, the absorbance generated at 405 nm is related to PA activity that was expressed in milli-international units (mIU) by comparison with a standard preparation of urokinase and normalized to the milligrams of protein present in the samples. To characterize the type of PA present in the sample, the assay was performed in the presence or in the absence of 125 µM amiloride, a specific uPA inhibitor that does not affect tPA activity [38]. Values obtained in the presence of amiloride were considered to reflect tPA activity, while uPA activity was calculated by subtracting tPA activity from total activity. Protein content was measured by the method of Lowry et al. [39], using BSA as a standard.

Gel Electrophoresis and Zymography

For zymography of PA, culture fluids or cell homogenates were separated by electrophoresis in 8% polyacrylamide slab gels in the presence of SDS (SDS-PAGE) under nonreducing conditions according to the procedure of Laemmli [40]. The PA was then visualized by placing the Triton X-100-washed gel on a casein-agar-plasminogen underlay as previously described [41].

Probe Synthesis and Hybridization Conditions

32P-Radiolabeled uPA (pDB4501) [42] antisense probe was generated from plasmids with SP6 polymerase according to the protocol enclosed in the Promega kit. Filters were prehybridized, hybridized and washed as previously described [23]. After autoradiography, the filters were then probed with a random-primed cDNA for mouse 18S rRNA [43] to account for any variability in the amount of RNA present in the filters.

Isolation and Analysis of Total Cellular RNA

Total RNA was prepared from Sertoli cells [44] and analyzed for relative abundance of specific mRNAs by Northern blot and/or slot-blot hybridization. For Northern blot analysis, RNAs (30 µg/lane) were denatured with formaldehyde, fractionated on a 1.2% agarose gel containing 6% formaldehyde and transferred to Hybond nylon membrane (Amersham) by capillary blotting with 20x standard saline citrate (SSC) for 24 h [45]. For slot-blot analysis, RNA (10 µg/slot) was resuspended in 100 µl of a solution containing 10% formaldehyde and 50% formamide. The mixture was incubated for 30 min at 60°C, placed on ice, and then applied with suction to a nylon membrane by means of an IBI slot-blot apparatus. Each slot was washed twice with 1x SSC. Each specific mRNA was quantified by densitometry of the films after autoradiography and was normalized to the amount of rRNA. Densitometric data are reported as arbitrary units.

Statistical Analysis

Statistical analysis was performed by ANOVA followed by the Tukey-Kramer test for comparison of multiple groups.

Experimental Animals

Animals used for the required experiments in this report were treated in accordance with NIH "Guidelines for Care and Use of Experimental Animals."

RESULTS

Effects of Retinol on PA Activity in Cultured Rat Sertoli Cells

Based on the fact that Sertoli cells have age-related declines in uPA production [23] and in their sensitivity to FSH [46], we selected younger, 15-day-old animals for the present series of experiments.

To evaluate the effect of retinoids on Sertoli cell PA secretion, the cells were incubated for 16 h in MEM supplemented with 0.1% BSA in the presence of different concentrations of retinol. The enzymatic activity released in the culture medium was then quantified by a chromogenic substrate assay. As shown in Figure 1, retinol inhibited uPA secretion in a dose-dependent manner. For the subsequent experiments, the dose of 0.25 µM retinol was used. Similar results were obtained utilizing RA (data not shown).



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  		FIG. 1. Dose-dependent inhibition of uPA secretion by Sertoli cells treated with retinol. Cells obtained from 15-day-old rats were cultured for 16 h in medium alone (C) or with increasing doses of retinol (R) (0.005–1.0 µM). Each value represents the mean ± SEM of four independent experiments each done in triplicate. Results are expressed as percentage of control set equal to 100. ***P < 0.001, **P < 0.01, *P < 0.05 versus control values

The effect of retinol on PA secretion was evaluated also at different culture times. Sertoli cells were cultured in medium alone (C) or with 0.25 µM retinol (R) for the times indicated in Figure 2. In control cultures there was an increase in uPA production that reached a plateau at 24 h, whereas in the presence of the vitamin there was a statistically significant inhibition of uPA secretion that became more evident after 2 days of culture in the presence of retinol (Fig. 2).



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  		FIG. 2. Time course of uPA production by Sertoli cells in response to retinol. Sertoli cells obtained from 15-day-old rats were cultured in medium alone (closed circles) or supplemented with 0.25 µM retinol (open circles). At the indicated times, conditioned media were collected and assayed for PA activity by chromogenic substrate assay. Results represent the mean ± SEM of three independent experiments each done in triplicate. **P < 0.001, *P < 0.05 versus control values

To characterize the type of PA modulated by retinoids, conditioned media were analyzed for PA activity by zymography. Sertoli cells treated with 100 ng/ml FSH were utilized as a positive control. As already described, unstimulated Sertoli cells essentially produced uPA and treatment with FSH inhibited such a production, whereas it stimulated tPA secretion. The addition of retinol to the culture medium inhibited uPA activity as well as FSH but did not stimulate tPA secretion (Fig. 3). Because retinol has been demonstrated to inhibit the amount of cAMP produced by Sertoli cells in response to FSH [12], the effect of retinol on the FSH-stimulated tPA secretion was also evaluated. By administering retinol and FSH at the same time, a slight decrease in the amount of both uPA and FSH-stimulated tPA was observed (Fig. 3).



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  		FIG. 3. Zymography of PA secreted by Sertoli cells in response to retinol and FSH. Sertoli cells from 15-day-old rats were cultured in medium alone (C) or supplemented with 0.25 µM retinol (R) with 100 ng/ml FSH or with retinol and FSH (R-FSH). After 16 h, conditioned media were collected and analyzed by casein-agar underlay. A representative zymography of two performed is reported. Aliquots (20 µl) of conditioned medium were loaded onto each lane. The photograph was taken after 24 h of incubation at 37°C

Effect of Retinol and FSH on the Steady-State Levels of uPA and tPA mRNAs

To determine if retinoids modulate PA mRNA levels, total RNA was extracted from Sertoli cells that were incubated for 16 h in medium alone (C) or supplemented with 0.25 µM retinol (R) or 100 ng/ml FSH, and analyzed by Northern blot. The stimulation with retinol decreases uPA mRNA levels, whereas it did not affect tPA mRNA levels. Consistent with earlier reports [23], the stimulation of tPA mRNA and the inhibition of uPA mRNA induced by FSH were also detected (Fig. 4A). A densitometric analysis of the bands (Fig. 4B) showed that after retinol treatment, uPA mRNA comprised 42% of the amount found in basal conditions. Thus, these results correlated well with the values of PA activity obtained by enzymatic assay.



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  		FIG. 4. Effect of retinol and FSH on PA mRNA levels in Sertoli cell cultures. Sertoli cells from 15-day-old rats were stimulated with 0.25 µM retinol (R) or FSH (100 ng/ml) for 16 h. At the end of the incubation period, cells were collected and relative abundance of mRNAs for uPA and tPA was determined by Northern blot analysis. A) Autoradiography of Northern blot. B) Averaged data from densitometry of Northern blots from two independent experiments. Optical density values of mRNA in each lane were normalized by respective optical density values of 18s rRNA signals. Results are expressed relative to control set equal to 100 (uPA) and equal to 1 (tPA)

Effect of Actinomycin D on uPA mRNA Decay

In order to determine a possible effect of retinol and FSH on uPA mRNA stability, Sertoli cells were cultured with retinol and FSH in the presence or absence of the transcriptional inhibitor actinomycin D. Sertoli cells from 15-day-old rats were cultured with 5 µg/ml actinomycin alone or combined with retinol or FSH. At the concentration used, actinomycin inhibited 90% of the RNA synthesis (data not shown). Cells were collected 10 h later, and total RNA was analyzed by Northern and slot-blot analyses. As shown in Figure 5, retinol and FSH significantly decreased uPA mRNA levels when compared either to untreated or actinomycin-treated cells. In actinomycin-treated cells, FSH had no effect on the half-life of uPA mRNA, whereas actinomycin did not disrupt the effects of retinol.



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  		FIG. 5. Effect of retinol and FSH on uPA mRNA stability. Sertoli cells from 15-day-old rats were stimulated with 0.25 µM retinol (R) or FSH (100 ng/ml) for 10 h in the presence or absence of 5 µg/ml actinomycin D. At the end of the incubation period, cells were collected and relative abundance of mRNA for uPA was determined by slot-blot analysis. A) Autoradiography of slot blot. B) Averaged data from densitometry of slot blots from three independent experiments. Optical density values of mRNA in each slot were normalized by respective optical density values of 18s rRNA signals. Results are expressed relative to control set equal to 1.

Effect of Phorbol 12-Myristate-13-Acetate and PKC Inhibitors on Retinol Inhibition of uPA

In order to investigate if PKC was involved in the regulation of uPA activity by retinoids we have evaluated the effect of phorbol 12-myristate-13-acetate (PMA), a potent tumor promoter known to stimulate PKC [47, 48], on the Sertoli cell response to retinol. Fifteen-day-old Sertoli cells were treated for 15 h with 10-7 M PMA, 0.25 µM retinol, or 100 ng/ml FSH. The enzymatic activity released in the medium was analyzed by zymography. PMA alone or in combination with retinol did not have a significant effect on uPA activity, whereas it had a slight antagonizing effect on FSH action (Fig. 6). The inhibitory effect upon FSH action was expected because it is known that in Sertoli cells PMA inhibits the FSH-dependent cAMP response and estrogen accumulation [49].



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  		FIG. 6. Effect of PMA on uPA secretion. Sertoli cells from 15-day-old rats were stimulated with 0.25 µM retinol (R) or 100 ng/ml FSH in the presence or absence of PMA (10-7 M). PMA was added 30 min before retinol or FSH. At the end of the culture period conditioned media were collected and analyzed by casein-agar underlay. A representative zymography of two performed is reported. Aliquots (20 µl) of conditioned medium were loaded onto each lane. The photograph was taken after 24 h of incubation at 37°C

To determine if inhibition of PKC could affect retinol action, Sertoli cells were incubated for 15 h with retinol or FSH in the presence or absence of a PKC inhibitor, calphostin C (50 and 100 nM). The inhibition of PKC did not affect retinol and FSH inhibition of uPA activity (data not shown).

PA Production at Different Ages

The effect of both retinol and RA on PA production was also analyzed at different stages of testis development. Sertoli cells isolated from rats of different ages (10-, 15-, 20-, and 30-day-old rats) were treated with both retinol and RA for 16 h. Conditioned medium was collected and analyzed for PA activity by chromogenic substrate assay. As previously shown [23] the amount of uPA secreted under basal conditions decreased with increase in age. As with FSH, both retinoids caused a significant reduction in enzyme activity in 10-, 15-, and 20-day-old rats while no statistically reproducible effect was observed in 30-day-old rats (Fig. 7). Conversely, FSH caused a significant induction of tPA at all ages [23], but no induction was observed after retinol stimulation at the same ages (data not shown).



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  		FIG. 7. Effect of retinol and RA on uPA production in Sertoli cells isolated from the testes at various stages of pubertal development. Sertoli cells, prepared from rats of different ages, were cultured in medium alone (closed circles) or supplemented with 0.25 µM retinol (triangles) or 0.25 µM RA (open circles). After 16 h, conditioned media were collected and analyzed for PA activity by chromogenic substrate assay. Results represent the mean ± SEM of three independent experiments each done in triplicate. **P < 0.001, *P < 0.01 versus control values

DISCUSSION

It has previously been shown that in rat Sertoli cells tPA and uPA production is under gonadotropin control, with tPA being stimulated and uPA inhibited by FSH [23]. Moreover PAs are secreted in a cyclic fashion during the cycle of the seminiferous epithelium [34, 50] and also regulated in a stage-specific manner by two factors: FSH and RA [34].

The data obtained in the present study show that, like FSH, retinol inhibits uPA activity in a dose- and time-dependent manner but, unlike FSH, does not stimulate tPA production and actually antagonizes FSH action on tPA stimulation. Moreover, in Sertoli cells, FSH action on PA production is mediated by cAMP [23], but basal cAMP levels are not affected by vitamin A [11]. This suggests that retinoids and FSH have different mechanisms of action. In addition, PKA activity is inhibited by retinol [10], and it has previously been shown that pretreatment with retinol antagonizes the intracellular cAMP increase induced by FSH [11]. Therefore, the observed inhibitory effect of retinoids on the FSH induction of tPA can be ascribed to an effect of retinol upon cAMP levels.

Retinoids have been shown to exert an inhibitory action on PKC activity [9] but we can exclude that this is the pathway utilized by retinol to inhibit uPA production, because neither stimulation nor inhibition of PKC activity is able to modulate uPA activity.

Vitamin A and its derivatives transcriptionally regulate many genes by acting through two distinct families of nuclear receptors the RA receptors (RARs) and the retinoid X receptors that are also present in Sertoli cells [51]. However, in addition to transcriptional regulation, other mechanisms of action of the vitamin have been described. In fact, vitamin A posttranscriptionally downregulates the expression of the spr1 gene in airway epithelia [52] and the adipsin gene in adipocytes [53] by decreasing the stability of the message. In both studies, inhibition did not require protein synthesis.

The regulation of mRNA stability plays an important role in the modulation of the levels of expression of many eukaryotic genes, and instability-determining sequences have been identified in the 5' or 3' untranslated regions (UTRs) of many RNAs [54]. At least three independent instability determinants have been found in the 3'-UTR of uPA mRNA. One is an AU-rich sequence (ARE), one is a sequence containing a stem structure, and one seems to require ongoing RNA synthesis for its activity [55, 56].

Our experiments demonstrate that the observed effect of retinol on PA activity is exerted via modulation of steady-state levels of the specific mRNAs. To verify if changes in mRNA levels could be attributed to modifications in the mRNA stability, we performed experiments with the transcription inhibitor, actinomycin D. The results obtained suggest that retinol post-trancriptionally downregulates uPA gene expression, at least in part, by decreasing the stability of the message. Moreover, the reduction in uPA production observed in response to retinol occurs in the absence of transcription, thus suggesting that retinol exerts its effect without the need of new protein synthesis. Conversely, FSH action appears to be mediated through different instability determinants. In fact, the FSH effect requires transcription and production of one or more factors involved in uPA mRNA degradation. Retinoid receptors modulate gene expression directly by binding to a response element (RARE) in the gene promoter or indirectly by binding other transcription factors. No canonical RARE has been found in the human, porcine, or bovine uPA gene, and this is consistent with our results that indicate a post-trancriptional control of uPA mRNA. At present, the mechanism involved in the downregulation of uPA mRNA by retinoids is unknown. Further studies on the metabolic pathways utilized by retinoids and gonadotropins and their interactions will give more insight upon the role played by these substances in the complex regulation of the seminiferous epithelium.

We also studied retinol regulation of PA in Sertoli cells isolated from rats of different ages, chosen as an example of specific stages of postnatal development [57]. As already shown, uPA was produced under basal conditions. Retinoid treatment of Sertoli cells from animals of different ages caused a decrease in uPA production that was more pronounced in younger rats and not statistically significant in 30-day-old rats. Our data contrast with previous data showing inhibition of basal uPA by retinol only in midpubertal Sertoli cells [33] or stimulation of uPA production in cultures of seminiferous tubules at stages VII–IX [34]. A possible explanation for these differences can be the purity of cell culture used in the different studies. Rosselli and Skinner [33] showed a 40% myoid cell contamination in their cultures from 10-day-old rats, whereas in our cultures, a myoid cell contamination lower than 2% was present. In the other study [34], cultures of seminiferous tubule segments were used; therefore many different cell types were present and a retinoid effect might be ascribed to other cell types different from Sertoli cells. In fact, other tubular cells, such as myoid cells, are also able to produce uPA (manuscript in preparation).

Sertoli cell responsiveness to retinoids decreases with increasing age of the donor animals. Our findings could be ascribed to an age-dependent decrease in the expression of retinoid receptors in Sertoli cells. In fact, it has been recently demonstrated that in Sertoli cells the levels of RAR{alpha} decrease with increasing age of the donor animals. However, other retinoid receptors increase their expression levels in parallel with increasing age [58]. Alternatively, the lack of response to retinoids in our cultures obtained from 30-day-old rats could be due to the presence of Sertoli cells derived from nonresponsive segments. In fact, it has been shown that RA treatment selectively affects specific stages of the spermatogenetic cycle [34].

In conclusion, our results on the effect of retinoids on PA production reinforce the importance of retinoids in modulating Sertoli cell functions and therefore the role of retinoids in the control of development of the mammalian testis.

ACKNOWLEDGMENTS

We thank Dr. Lauren Pecorino for helpful discussion; Mrs. Salvatore Greci and Roberto Pezzotti for excellent technical assistance; and the National Hormone and Pituitary Distribution Program, NIDDK, for providing the ovine FSH.

FOOTNOTES

First decision: 10 December 1999.

1 This work was supported by grants from the Ministero per l'Università e la Ricerca Scientifica (MURST) to R.C. and M.G. Back

2 Correspondence: Rita Canipari, Dipartimento di Istologia ed Embriologia Medica, Università di Roma "La Sapienza," Via A. Scarpa 14, 00161 Rome, Italy. FAX: 39 06 4462854; canipari{at}uniroma1.it Back

Accepted: March 28, 2000.

Received: October 27, 1999.

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