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Endocrinology Vol. 142, No. 3 1024-1032
Copyright © 2001 by The Endocrine Society

ARTICLES

Follicle-Stimulating Hormone and Leukemia Inhibitory Factor Regulate Sertoli Cell Retinol Metabolism1

Xiaojia Guo, Patricia L. Morris and Lorraine J. Gudas

Department of Pharmacology, Weill Medical College of Cornell University (X.G., L.J.G.); The Center for Biomedical Research, The Population Council (P.L.M.); and Rockefeller University (P.L.M.), New York, New York 10021

Address all correspondence and requests for reprints to: Dr. Lorraine J. Gudas, Department of Pharmacology, Weill Medical College of Cornell University, 1300 York Avenue, New York, New York 10021. E-mail: ljgudas{at}mail.med.cornell.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Sertoli cells, the somatic epithelial cells of the seminiferous tubules, provide both structural and biochemical support for developing male germ cells. The Sertoli cells are targets of retinoid action in the testis. We have found that FSH, (Bu)2cAMP, and leukemia inhibitory factor elicit substantial changes in the metabolism of [3H]retinol (vitamin A) in primary cultures of purified rat Sertoli cells. Addition of (Bu)2cAMP for 2 h or FSH for 6 h results in a 3-fold increase in the metabolism of [3H]retinol to [3H]retinoic acid ([3H]RA); the esterification of [3H]retinol to [3H]retinyl esters, especially [3H]retinyl palmitate, is also increased by approximately 5-fold. The addition of 1 µM all-trans-RA also elicits changes in [3H]retinol metabolism, but in this case the metabolism of [3H]retinol to [3H]RA is inhibited, whereas the metabolism of [3H]retinol to [3H]retinyl esters is increased by over 50-fold. Leukemia inhibitory factor increases the esterification of [3H]retinol by 2- to 3-fold. FSH leads to a reduction in the level of cellular retinol binding protein I transcripts, whereas RA increases the cellular retinol binding protein I messenger RNA level by about 2-fold at approximately 24 h. Levels of AHD-2 (aldehyde dehydrogenase-2) and RALDH-2 (retinaldehyde dehydrogenase-2) messenger RNAs, which encode enzymes that convert [3H]retinaldehyde to [3H]RA, are increased by about 2-fold by FSH, whereas no change in CYP26 (RA hydroxylase) expression is seen. Our results suggest that one function of FSH (and/or (Bu)2cAMP) in Sertoli cells is to increase the metabolism of retinol to the biologically active metabolite RA and to retinyl esters.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
RETINOIDS (vitamin A and its metabolites and analogs) play a number of critical roles during mouse development (for review, see Refs. 1 and 2). For example, it is known that retinol (vitamin A) is required for normal spermatogenesis and that retinol-deficient animals are infertile (3, 4). In retinoid-deficient rodents (rats and mice), retinol itself, retinoic acid (RA), and 4-oxoretinoic acid can support spermatogenesis (5, 6). During the process of spermatogenesis, the Sertoli cells of the testis provide both physical and biochemical support for the differentiating spermatogonia. Sertoli cells, which are closely associated with the developing germ cells, synthesize and secrete several proteins involved in the process of spermatogenesis (7). It is thought that many of the actions of retinoids on the process of spermatogenesis are mediated via the Sertoli cells.

Numerous retinoid-binding proteins and receptors are expressed in the testis. The cellular retinol-binding protein I (CRBP-I) and one of the cellular RA-binding proteins (CRABP-II) are expressed in the Sertoli cells of the testis (8, 9, 10, 11, 12, 13, 14, 15, 16, 17). CRABP-II was detected in both Sertoli and fetal Leydig cells, whereas CRABP-I was primarily expressed in gonocyte and gonocyte-derived spermatogonia (17). The nuclear RA receptors RAR{alpha}, -{beta}, and -{gamma} and the retinoid X receptor {alpha} are also expressed in the testis. Vitamin A status as well as the administration of retinoids have been shown to influence the expression of some of these genes (18, 19, 20, 21, 22, 23, 24, 25, 26). In addition, RAR{alpha} and RAR{gamma} null mouse mutants exhibit male sterility, among various other defects, suggesting roles for these receptors in the testis (27, 28). The functions of these retinoid-binding proteins and receptors in the testis are not fully understood. Only a few specific target genes for these receptors in the testis have been identified to date (29).

Retinol can be metabolized to many different retinoids in various cell types. Both esterification of retinol to retinyl esters and conversion of retinol to retinaldehyde and then to RA can occur, depending on the cell type (for review, see Ref. 30). With respect to retinol metabolism, it was shown that cultured peritubular cells from the testis of 20-day-old rats did not esterify much retinol, and very little lecithin-retinol acyltransferase (LRAT) activity was present in these cells (31). In contrast, cultured Sertoli cells can esterify retinol and possess acyl-coenzyme A acyltransferase (ARAT) and LRAT activities (32, 33). Mature sperm were also found to possess high levels of retinyl esters (34). Several enzymes, including mouse AHD-2 (aldehyde dehydrogenase-2) (35, 36) and RALDH-2 (retinaldehyde dehydrogenase-2) (37, 38), have been shown to be capable of metabolizing retinaldehyde to RA, and RALDH-2 message has been reported to be present in adult testes by Northern or ribonuclease protection assays (38). The mechanism(s) by which these metabolic enzymes are regulated is not well understood. There is evidence that the testis can synthesize RA from retinol (39, 40), although more information is needed with respect to the enzymes that mediate this metabolism in Sertoli cells.

One of the major endocrine hormones that influences the onset of Sertoli cell differentiation at puberty in males and maintains Sertoli cell differentiation in the adult testis is FSH (41, 42, 43). FSH and LH, released by the gonadotrophs in the pituitary gland, control the process of spermatogenesis in part by regulating androgen production and the functions of Leydig and Sertoli cells. The actions of FSH result in an increase in intracellular cAMP and subsequent changes in gene expression in the Sertoli cells. That there is some interaction between the retinoid and FSH signaling pathways is indicated by the report that retinol can influence the Sertoli cell’s response to FSH; exogenous retinol in the culture medium exerts an inhibitory effect on FSH-induced cAMP production by rat Sertoli cells (44). In Sertoli-derived cell lines, FSH inhibits RAR{alpha} nuclear localization (45).

The differentiated functions of Sertoli cells, including their response to one testicular cytokine, leukemia inhibitory factor (LIF), enhance the survival of Sertoli cells and gonocytes in a co-culture system (46). LIF mediates the phosphorylation of STAT-3 (signal transducer and activator of transcription-3) and STAT-1 as well as protooncogene expression in rat Sertoli cells (47). FSH responsiveness is also affected by growth factors (48).

In this report we demonstrate that the synthesis of retinyl esters and RA from retinol by the Sertoli cells is greatly enhanced by the administration of FSH or cAMP analogs. In addition, we show that LIF administration to Sertoli cells results in a reduction in the total rate of retinol metabolism while increasing retinyl ester formation from retinol.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Preparation of Sertoli cells and Sertoli cell culture
Rat Sertoli cells were isolated as previously described (47, 49). Primary cultures of Sertoli cells were isolated and purified from 18-day-old Wistar rats (Charles River Laboratories, Inc., Kingston, NY) and incubated at 32 C in 5% CO2 at a density of 1 x 107 cells/100-mm polystyrene dish in phenol red-free, serum-free, and endotoxin-free DMEM/Ham’s F-12 medium (Irvine Scientific, Santa Ana, CA) as described previously (47, 49). The purity of the Sertoli cells was 95%, as previously reported (48). The medium was supplemented with 2.5 µg/ml bovine insulin (Sigma, St. Louis, MO), 1 µg/ml transferrin (Calbiochem, La Jolla, CA), and 10 µg/ml bacitracin (Sigma). On day 2 of culture, duplicate or triplicate culture dishes were used for each drug treatment, and each treatment was performed at least twice, with two different preparations of Sertoli cells. Duplicate wells of cells were counted; the various drug treatments did not alter the cell number. Cells were then cultured in the presence of insulin and transferrin in F-12/DMEM medium plus 1% BSA or 5% FBS (to stabilize the [3H]retinoids from oxidation) in the presence of [3H]all-trans-retinol or [3H]all-trans-RA for various lengths of time at 32 C in 5% CO2. All-trans-RA (Sigma), LIF (Life Technologies, Inc., Grand Island, NY), (Bu)2cAMP (Sigma), 8-bromo-cAMP (Sigma), and FSH [ovine FSH (BIO), USDA (SIAFP), provided by Dr. S. Raiti, National Hormone and Pituitary Program and NIDDK] were added to the cells at the times indicated in the figure legends. [3H]All-trans-retinol and [3H]all-trans-RA were purchased from NEN Life Science Products (Boston, MA). The numbers of cells were determined using a Coulter counter (Coulter Corp., Hialeah, FL). The retinyl ester standards were a gift from Dr. Marcia Simon (State University of New York, Stony Brook, NY).

Extraction of retinoids and HPLC
The retinoids were extracted as previously described (50, 51). Certain samples were dried using a SpeedVac (Savant Instrument Co., Farmingdale, NY) and resuspended in 130 µl methanol and 5 µl trimethylsilyl diazomethane (Aldrich, Milwaukee, WI; 2 M solution in hexane) to derivatize retinoids. Retinoid standards were added to samples before extraction.

The HPLC analysis was performed using a Waters Millenium system (Waters Corp., Milford, MA) to separate the various retinoids. Samples were applied to an analytical 5-µm reverse phase C18 column (Vydac, Hesperia, CA) at a flow rate of 1.5 ml/min. Two mobile phase gradient systems were used as previously described (50, 51). These two gradient mobile phases were chosen for their ability to resolve the acids, alcohols, and esters of the retinoid classes. Nonradiolabeled retinoid standards were run concurrently and monitored at a wavelength of 340 nm, and a Packard A-500 radiochromatography detector (Packard Instruments, Downers Grove, IL) was employed to monitor the radiolabeled retinoids (51).

Retinoids were identified by HPLC based on at least two criteria: an exact match of the retention time of an unknown peak with that of an authentic retinoid standard and identical UV spectra (220–400 nm) of unknowns against spectra from authentic retinoid standards during HPLC by the use of the photodiode array detector. RA was also identified by the shift of the retention time of the methylated RA derivative to the same position as the corresponding methyl ester of the RA standard. The methyl ester of RA was synthesized by reaction with trimethylsilyl diazomethane. The quantitation of [3H]retinoids was performed as previously described (50).

RNA isolation and Northern blot analysis
Total cellular RNA was isolated from Sertoli cells cultured in the presence or absence of appropriate factors for 48 h using RNA Stat-60 (Tel-Test, Inc., Friendswood, TX) according to the manufacturer’s instructions. RNA was electrophoretically fractionated by size on 1% agarose/2.2 M formaldehyde gels, transferred to nylon filters by blotting, and attached to the filters using a UV Stratalinker 1800. The complementary DNA (cDNA) probes used in the analysis were labeled with [32P]deoxy-CTP using a random primer labeling kit (Roche Molecular Biochemicals, Indianapolis, IN) according to the manufacturer’s directions. The following murine cDNAs were used as probes for Northern blots, as described previously (50): RA hydroxylase (CYP26) was obtained from the EST database (EST AA239785), the mouse AHD-2 cDNA was previously cloned in this laboratory (36), and the RALDH-2 probe was obtained from the EST database (EST177263) from the American Type Culture Collection Institute for Genomic Research (Manassas, VA). An 800-bp PstI fragment containing the entire human CRBP I cDNA was excised from pSG-CRBP and was also used as a probe (51).

Blots were prehybridized and hybridized at 42 C in 50% (wt/vol) formamide/5 x SSC (standard saline citrate), 50 mM NaH2PO4 (pH 7.4), 5 mM EDTA, 0.08% polyvinylpyrrolidone, 10% (wt/vol) BSA, and 10% (wt/vol) salmon sperm DNA. After 10–16 h of hybridization, blots were washed twice in 2 x SSC/0.1% SDS for 20 min at room temperature and twice in 0.2 x SSC/0.1% SDS at 50–60 C for 30 min. Autoradiographs were quantitated using a PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Sertoli cells metabolize [3H]retinol to [3H]retinoic acid and [3H]retinyl palmitate
Primary cultures of Sertoli cells exhibit metabolism of exogenously added [3H]retinol (Fig. 1Go, A–C); by 12 h after the addition of 50 nM [3H]retinol to the culture medium, approximately 50% of the [3H]retinol had been metabolized. Some of the [3H]retinol was metabolized to [3H]RA (Fig. 1Go, A–C). [The [3H]RA peak was identified by coelution with nonradiolabeled all-trans-retinoic acid and by the fact that this peak could be shifted to a different elution position by diazomethane, as could the nonradiolabeled RA standard (see below).] The [3H]RA was not significantly metabolized further to more polar derivatives, such as [3H]4-oxoretinoic acid, under these culture conditions (Fig. 1Go, A–C).



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  		Figure 1. The HPLC profiles of [3H]retinol metabolites in Sertoli cells cultured in the presence or absence of various drugs or hormones. Cells were cultured in the presence or absence of 250 µM (Bu)2cAMP and theophylline for 24 h. The medium was then changed, and these drugs were added again, along with 50 nM [3H]retinol. Samples were then harvested at 2, 6, or 12 h, when retinoids were extracted, and reverse phase HPLC analysis was performed. Only the intracellular retinoids are shown. The data for each sample are plotted as 3H counts per min vs. HPLC retention times, normalized to 1 x 107 cells. Arrows point to the [3H]all-trans-retinol (ROH) peaks, to the [3H]all-trans-RA peaks, and to the [3H]retinyl palmitate (RP) peaks in A–F. A–C, Untreated Sertoli cells cultured in the presence of 50 nM [3H]retinol for 2, 6, or 12 h, respectively. D, E, and F, Cells first cultured in the presence of (Bu)2cAMP and theophylline (to inhibit cAMP phosphodiesterase) were then cultured in the presence of 50 nM [3H]retinol for 2, 6, or 12 h, respectively. Nonradiolabeled retinoids were included with each sample as standards to determine the elution time of the various retinoids (G). The standards are: 4-oxoretinoic acid (lane 1), 7.5 min; 4-oxoretinol (lane 2), 17 min; 13-cis-RA (lane 3), 19.3 min; all-trans-RA (lane 4), 20.5 min; retinaldehyde (lane 5), 34 min; retinyl acetate (lane 6), 37.5 min; anhydroretinol (lane 7), three peaks at 39.5, 40.5, and 41.4 min; and retinyl palmitate (lane 8), 55.5 min. Not shown in the standards is all-trans-retinol; all-trans-retinol is eluted at 31.8 min and can be seen as the major peak in A–F. The peak at 41 min in E is an artifact of the small amount of ethanol added to the cultures along with the [3H]retinol. This peak is an ethyl ester of [3H]RA, [3H]ethyl retinoate (Guo, X., and L. J. Gudas, unpublished). This experiment was performed three times with very similar results. One experiment is shown here.

 
In addition to the metabolism of [3H]retinol to [3H]RA by the untreated Sertoli cells, a small amount of the [3H]retinol was esterified to [3H]retinyl palmitate (Fig. 1Go, A–C). Small amounts of other [3H]retinyl esters were synthesized from [3H]retinol by the cells, but [3H]retinyl palmitate was the predominant [3H]retinyl ester present in the cells.

The primary cultures of Sertoli cells contained extremely low levels of endogenous retinol and retinyl esters, as determined by HPLC photodioarray analysis (<75 nM retinol and <5 nM retinyl palmitate; data not shown). Thus, the measurements of metabolism of [3H]retinol are not subject to errors of isotype dilution resulting from equilibration of the isotope with an endogenous unlabeled retinol pool.

Culture of Sertoli cells in the presence of FSH or cAMP analogs enhances [3H]retinol esterification to [3H]retinyl palmitate
Sertoli cells were cultured in the presence of 250 µM (Bu)2cAMP and 500 µM theophylline (to inhibit cAMP phosphodiesterases) for 24 h. (Bu)2cAMP and theophylline were then added again, and the cells were cultured in the presence of 50 nM [3H]retinol for 2, 6, or 12 h (Fig. 1Go, D–F). Alternatively, the cells were cultured in the presence of 100 ng/ml FSH for 30 min, followed by the readdition of FSH and culture in the presence of [3H]retinol for 6 h (Fig. 2Go). All of these treatments with either FSH or cAMP analogs increased the metabolism of [3H]retinol to [3H]retinyl palmitate by 4- to 7.5-fold relative to that in untreated controls (Figs. 1Go, 2Go, and 3A).



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  		Figure 2. The HPLC profiles of [3H]retinol metabolites in Sertoli cells cultured in the presence or absence of 100 ng/ml FSH. FSH was added for 30 min, and then the medium was changed, FSH was added again, and cells were cultured for 6 h in the presence of 50 nM [3H]retinol. Cells were harvested, retinoids were extracted, and reverse phase HPLC analysis was performed. The data shown are those for intracellular retinoids. Medium samples were also run, but are not shown here. The data for each sample are plotted as 3H counts per min vs. HPLC retention times. A, Untreated Sertoli cells harvested at 6 h after 50 nM [3H]retinol addition. B, FSH (100 ng/ml) treated Sertoli cells harvested 6 h after [3H]retinol addition. A sample of the [3H]retinol starting material, incubated at 37 C for 6 h in the absence of cells, is shown in C; the asterisks surround a contaminant of the [3H]retinol label that is not taken up by cells. Nonradiolabeled retinoids were included with each sample as standards to determine the elution of the various retinoids (D): 4-oxoRA (lane 1), 4-oxoretinol (lane 2), all-trans-RA (lane 3), retinyl acetate (lane 4), and retinyl palmitate (lane 5). This experiment was performed three times with very similar results. One representative experiment is shown here.

 
Culture of Sertoli cells in FSH or cAMP analogs enhances [3H]retinol metabolism to [3H]RA
The rate of [3H]retinol metabolism to [3H]RA was also more rapid in the presence of these cAMP analogs than in control cultures (Fig. 1Go, D and E; Fig. 2Go, A and B; and Fig. 3BGo). Within 2 h after (Bu)2cAMP (250 µM) addition, the intracellular concentration of [3H]RA derived from [3H]retinol metabolism was 3 times that in control, untreated Sertoli cells [Fig. 1Go, D vs. A; 37 nM at 2 h for the control Sertoli cells; 121 nM at 2 h for the (Bu)2cAMP-treated cells]. This increase in [3H]RA synthesis from [3H]retinol was transient, and by 12 h after (Bu)2cAMP addition no difference was seen from the control (Fig. 3BGo). This may be due to the instability of (Bu)2cAMP in the culture system. At 6 h, FSH treatment also increased the rate of metabolism of [3H]retinol to [3H]RA by 3-fold over that in control, untreated Sertoli cells (Fig. 2Go, B vs. A; and Fig. 3BGo).



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  		Figure 3. Effects of cAMP analogs and FSH on retinol metabolism. Cells were cultured with or without (Bu)2cAMP or FSH for 24 h, followed by incubation with 50 nM [3H]retinol. A quarter of the medium was collected, and cells were washed once with PBS, [3H]retinoids were extracted and separated by HPLC. The amount of each [3H]retinoid was calculated as described previously (50 ). A, Total [3H]retinyl palmitate synthesized from 50 nM [3H]retinol by 1 x 107 cells at various times. B, Total [3H]RA synthesized from 50 nM [3H]retinol at various times.

 
It is of interest to note that concomitant with the increase in metabolism of [3H]retinol to [3H]RA observed after (Bu)2cAMP and theophylline or FSH treatment, there was a large increase in the concentration of [3H]retinaldehyde (Figs. 1Go and 2Go, peak at 33–34 min). These data indicate that [3H]retinol is first metabolized to [3H]retinaldehyde, and then the [3H]retinaldehyde is metabolized to [3H]RA. The rate-limiting step appears to be the conversion of [3H]retinaldehyde to [3H]RA under these conditions, as [3H]retinaldehyde levels increase greatly after FSH or (Bu)2cAMP and theophylline addition (Fig. 1Go, D vs. A; and Fig. 2Go, B vs. A).

Sertoli cells metabolize [3H]retinol to [3H]retinoic acid: proof of the identity of the RA peak by a diazomethane shift
A Sertoli cell sample from cells first cultured in the presence of 250 µM (Bu)2cAMP and 500 µM theophylline and then cultured in 50 nM [3H]retinol was used for the diazomethane shift experiments. Diazomethane converts RA to methyl retinoate, a more lipophilic compound, which exhibits a different retention time than RA (50, 51). When the sample of Sertoli cells shown in Fig. 4AGo was treated with diazomethane, the putative RA peak that eluted at 20.8 min was shifted to 40 min (Fig. 4CGo). A nonradiolabeled control RA standard, which was included in the same sample treated with diazomethane, was also shifted to 40.2 min [Fig. 4Go, B (before diazomethane) vs. D (after diazomethane)]. This experiment proves that the peak that elutes at approximately 20.8 min in Figs. 1Go and 2Go is [3H]RA synthesized from [3H]retinol.



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  		Figure 4. HPLC profiles of cells treated with (Bu)2cAMP and theophylline, cultured in 50 nM [3H]retinol, and then harvested and treated with diazomethane. The [3H]retinoids were extracted, nonradiolabeled retinol and retinoic acid standards were added, and the samples were run on HPLC to separate the retinoids, either with or without treatment with diazomethane for 5 min. A, (Bu)2cAMP and theophylline sample, not treated with diazomethane. B, Nonlabeled all-trans-RA standard, not treated with diazomethane. C, (Bu)2cAMP and theophylline sample, treated for 5 min with diazomethane before HPLC analysis. Note the loss of the [3H]retinoic acid peak and the presence of a new peak at 40.1 min; also note that the all-trans-retinol peak is not affected by the diazomethane treatment. D, All-trans-RA nonradiolabeled standard, treated with diazomethane for 5 min before HPLC analysis. Arrows indicate the positions of [3H]retinol (ROH) and [3H]RA in A, and the arrowhead in C indicates the position of the methyl retinoate.

 
Metabolism of 1 µM[3H]retinol in Sertoli cells
In the experiments described above, only small amounts of [3H]retinol (50 nM) were used, so that the retinol itself would not perturb the cells. In a variation of the experiment described above, Sertoli cells were cultured in the presence of 250 µM (Bu)2cAMP and theophylline for 24 h, followed by culture in the presence of a higher final concentration of exogenous [3H]retinol (1 µM, approximately to the concentration of retinol in 100% serum) for either 6 or 12 h. In the presence of the higher exogenous concentration of retinol, both control cultures of Sertoli cells and the (Bu)2cAMP- and theophylline-treated Sertoli cells exhibited higher internal concentrations of [3H]retinol than they did when incubated in the presence of 50 nM exogenous [3H]retinol. More [3H]retinyl palmitate was synthesized from 1 µM [3H]retinol in the (Bu)2cAMP- and theophylline-treated cultures than in the control cultures, consistent with what was observed in the experiments using "tracer" 50 nM [3H]retinol (Figs. 1Go and 3Go), but in addition, many other types of [3H]retinyl esters (retinyl oleate, etc.) were synthesized from 1 µM [3H]retinol by both the control cultures of Sertoli cells and the (Bu)2cAMP-treated cultures (Fig. 5Go).



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  		Figure 5. Total [3H]retinyl palmitate made from 1 µM [3H]retinol. Cells were cultured, and samples were prepared as described in Fig. 3Go. Samples are plotted vs. culture times. This experiment was performed twice with very similar results; one experiment is shown because the time points for data analysis were slightly different in the two experiments.

 
When cultured for 6 h in the presence of 1 µM exogenous [3H]retinol, the (Bu)2cAMP- and theophylline-treated cells did not synthesize more [3H]retinoic acid from [3H]retinol than control cells (data not shown). This data indicates that when Sertoli cells are cultured in high but physiological concentrations of retinol (i.e. 1 µM), the esterification reaction is favored over the metabolism of retinol to RA. Thus, our data suggest that the concentration of retinol in the Sertoli cells can influence whether retinol is metabolized to RA or to retinyl esters by these cells in response to (Bu)2cAMP.

LIF (leukemia inhibitory factor) and RA enhance the rate of [3H]retinol esterification
Sertoli cells were cultured in the presence of 1000 U/ml (total, 2 ml/dish) LIF or in the presence of 1 µM RA for 24 h, followed by the readdition of LIF or RA and the addition of 50 nM [3H]retinol for 2, 6, or 12 h. In the presence of LIF, the metabolism of [3H]retinol to [3H]retinyl esters increased 2- to 3-fold (Fig. 6Go), whereas the metabolism by the cells of [3H]retinol to [3H]RA and to other metabolites of [3H]retinol decreased (data not shown).



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  		Figure 6. Effect of RA and LIF on retinol metabolism. Cells were cultured with or without RA and LIF for 24 h, followed by incubation with 50 nM [3H]retinol. A quarter of the medium was collected, cells were washed once with PBS, and [3H]retinoids were extracted, and separated by HPLC. The amount of [3H]retinyl palmitate was calculated as described previously (50 ) and plotted here.

 
Culture in the presence of 1 µM retinoic acid resulted in an enormous increase in [3H]retinol esterification to [3H]retinyl esters; in particular, [3H]retinyl palmitate levels were extremely high (>50-fold higher than in control Sertoli cells; Fig. 6Go). It was of interest that treatment with RA inhibited the metabolism of [3H]retinol to [3H]retinoic acid (data not shown) while at the same time markedly enhancing the metabolism of [3H]retinol to [3H]retinyl esters (Fig. 6Go). This result suggests a feedback mechanism whereby high concentrations of RA, when added exogenously, inhibit the conversion of retinol to RA by the Sertoli cells and concomitantly enhance retinol esterification.

Metabolism of exogenously added [3H]RA by cultured Sertoli cells
Up to this point we have discussed the results of experiments focused on the metabolism of [3H]retinol by Sertoli cells. We also performed experiments in which Sertoli cells were cultured in the presence or absence of (Bu)2cAMP and theophylline or LIF for 24 h, and then cultured in the presence of 50 nM [3H]all-trans-RA to assess whether [3H]RA was further metabolized to [3H]4-oxo-RA under these conditions. We found that neither (Bu)2cAMP and theophylline nor LIF resulted in an increase in polar metabolites of [3H]RA (Fig. 7Go), consistent with the lack of detectable expression of CYP26 messenger RNA (mRNA) in these cells (see below). This indicates that the FSH- and (Bu)2cAMP-associated increase in [3H]RA from [3H]retinol in Sertoli cells (Figs. 1Go and 2Go) does not result from an FSH- and cAMP-associated inhibition of further RA metabolism.



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  		Figure 7. RA metabolism in Sertoli cells. Cells were cultured in the presence or absence of 250 µM (Bu)2cAMP and theophylline or in the presence of 1000 U of LIF for 24 h. The medium was then replaced with medium containing the drugs and 50 nM [3H]RA. Samples were harvested, and retinoids were extracted and separated by HPLC. The total amount of [3H]RA remaining was plotted vs. the incubation time. This experiment was performed twice with very similar results; one experiment is shown because the time points for data analysis were slightly different in the two experiments.

 
Analysis of gene expression in Sertoli cells treated with FSH or (Bu)2cAMP
We examined the expression of genes encoding some of the proteins that have been reported to be involved in retinol metabolism. CRBP-I mRNA levels decrease when cells are cultured in FSH or (Bu)2cAMP and theophylline, and increase when cells are cultured in RA (Fig. 8AGo). The levels of mRNAs that encode two of the enzymes involved in the metabolism of retinaldehyde to RA, AHD-2 and RALDH-2, are shown (Fig. 8BGo). The RALDH-2 and AHD-2 mRNAs were expressed at very low levels, and their expression levels increased slightly upon FSH treatment (Fig. 8BGo). The CYP26 (RA hydroxylase) gene, which encodes an enzyme that metabolizes RA to more polar RA derivatives (52, 53, 54), was not expressed at detectable levels by the Sertoli cells, even after RA addition (Fig. 8CGo).



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  		Figure 8. Northern analysis of mRNA expression in Sertoli cells cultured under various conditions. Ten micrograms of total RNA were loaded per lane. The concentrations of drugs are given in Materials and Methods [CT, (Bu)2cAMP, and theophylline]. RNA from F9 cells (A), liver (B), and CCE ES cells (C) was loaded as a positive control. Autoradiograms of the blots were hybridized to 32P-labeled cDNA probes as indicated. The blots were quantitated using the PhosphorImager, and the mRNAs were normalized for loading by comparison with glyceraldehyde phosphate dehydrogenase mRNA. This experiment was performed three times with similar results; one experiment is shown. A, The messages shown are CRBP-I and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Exposure times were 10 h for CRBP-I and 2 h for GAPDH. B, The mRNAs shown are AHD-2, RALDH-2, and GAPDH. Exposure times were 3 h for AHD-2, 48 h for RALDH-2, and 2 h for GAPDH. C, The mRNAs for CYP26 and GAPDH. Exposure times were 48 h for CYP26 and 2 h for GAPDH.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have shown that in primary cultures of Sertoli cells both FSH and (Bu)2cAMP can increase the rate of [3H]retinol metabolism to [3H]RA and to [3H]retinyl esters by severalfold ( Figs. 1–3GoGoGo; see Fig. 9Go for summary diagram). RA is already known to be a potent, bioactive member of the retinoid signaling family that can interact with the RAR/RXR family of nuclear receptors (for review, see Ref. 55). Retinyl esters, such as retinyl palmitate, are thought to be a storage form of retinol (for review, see Ref. 30); under certain conditions, these retinyl esters are hydrolyzed by cells to produce retinol and bioactive retinoids such as RA. Although it has already been shown that retinol can be esterified by Sertoli cells (32, 33), it has not been reported previously that the hormone FSH can influence this esterification reaction. Additionally, that FSH and/or (Bu)2cAMP can increase the intracellular concentration of RA in Sertoli cells has not been shown before (Figs. 1Go and 2Go). Because FSH is important for the regulation of spermatogenesis, the ability of FSH and one of its intracellular signaling molecules, cAMP, to modulate the levels of RA and retinyl esters within the Sertoli cells may represent an important function of FSH in these cells. The RA synthesized from retinol may then be secreted from the Sertoli cells and delivered to the developing germ cells.



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  		Figure 9. Schematic diagram of some of the enzymes involved in retinol metabolism and proposed regulation by FSH, (Bu)2cAMP, and RA.

 
The enzymes involved in the metabolism of retinol to RA have not been fully characterized, but it is known that enzymes of the alcohol dehydrogenase/short chain dehydrogenase/reductase family can carry out the metabolism of retinol to retinaldehyde (40, 56, 57, 58). The metabolism of retinaldehyde to all-trans-RA is mediated by retinaldehyde dehydrogenases, some of which are members of the class I aldehyde dehydrogenase family (35, 36 ; for review, see Ref. 59). Enzymes of this class, which can metabolize retinaldehyde to RA, include murine AHD-2 and human aldehyde dehydrogenase-1 (35, 36). RALDH-2 is a retinaldehyde dehydrogenase that exhibits approximately 72% amino acid identity with the AHD-2 and ALDH-1 enzymes (37, 38). We found that in the Sertoli cells, AHD-2 (ALDH-1) mRNA is expressed at a higher level than RALDH-2 (Fig. 8BGo). We do not know whether either AHD-2 or RALDH-2 is responsible for the increase in RA synthesis following FSH addition.

We also did not detect expression of cytochrome P450 CYP26 (RA hydroxylase) mRNA (52, 53, 54) (Fig. 8CGo), consistent with the fact that we did not observe much [3H]RA metabolism to [3H]4-oxo-RA and other more polar RA metabolites in the Sertoli cells, either untreated or after (Bu)2cAMP and theophylline or FSH addition (Fig. 7Go).

Treatment of the Sertoli cells with 1 µM nonradiolabeled RA for 24 h also resulted in an enormous increase in [3H]retinol esterification, including increased [3H]retinyl palmitate. These data indicate that RA can up-regulate the enzymes involved in the esterification process, LRAT and/or ARAT, but the mechanism by which this is accomplished is not understood. As the human LRAT partial cDNA has recently been cloned (60) and the ARAT cDNA has not been cloned, it is not clear whether this increase in [3H]retinol esterification after RA treatment results from increased transcription of either or both of these genes or from an increase in the activity and/or level of the LRAT and/or ARAT proteins. Treatment of the Sertoli cells with RA also inhibited the metabolism of [3H]retinol to [3H]RA, suggesting that RA, when present in the Sertoli cells, can inhibit its synthesis from retinol. It is not known whether the RA is acting at the retinol dehydrogenase or retinaldehyde dehydrogenase step (see Fig. 9Go for diagram).

LIF has been reported to enhance the survival of neonatal rat Sertoli cells and gonocytes in a 3- to 6-day coculture system (46). No effect of LIF was seen on the mitotic activity of proliferating gonocytes or Sertoli cells. We previously demonstrated that LIF treatment of these Sertoli cells results in activation of the JAK-STAT pathway, STAT-3 and -1 phosphorylation and DNA binding, and induction of c-fos transcription and AP-1 activation (47). In this study using more mature, nondividing, and differentiated Sertoli cells, we show that LIF administration resulted in a reduction in retinol metabolism to RA and other metabolites while increasing retinol esterification (Fig. 6Go).

It has been shown in a wide variety of cell types that bioactive retinoids such as RA can regulate the expression of a number of different genes (for review, see Refs. 1 and 2). Pharmacological doses of RA can cause cell growth arrest, cell differentiation, and changes in gene expression in a wide variety of cell types (1, 2). However, less information is available concerning how the endogenous RA signal is generated in various cell types from retinol, the retinoid that circulates in the blood of animals and is the form of the vitamin delivered to cells from the blood. In this report we show that the metabolism of retinol to both RA and retinyl esters can be regulated by cAMP analogs such as (Bu)2cAMP and by FSH, at least in the context of these primary rat Sertoli cells cultured in serum-free conditions. Therefore, these data provide new insights into the mechanisms by which the production of the endogenous RA-signaling molecule is regulated. This information is important if we are to understand retinoid signaling in its entirety in the context of the animal.


   Acknowledgments
 
We thank Lyann Mitchell for technical assistance, and Taryn Resnick for editorial assistance.


   Footnotes
 
1 This work was supported by Grant R01-CA-43796 (to L.J.G.) and Grants R01-HD-29428 and U54-HD-13541 (to P.L.M.). Back

Received September 25, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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