Retinol Processing by the Peritubular Cell from Rat Testis - CiteSeerX

BIOLOGY OF REPRODUCTION 52, 356-364 (1995)

Retinol Processing by the Peritubular Cell from Rat Testis' JAMES T. DAVIS and DAVID E. ONG 2 Department of Biochemistr,

Vanderbilt University, Nashville, Tennessee 37232 ABSTRACT

The cells that surround and support the seminiferous epithelium of the tubules in the testis, i.e., the peritubular or myoid cells, are known to contain relatively high amounts of cellular retinol-binding protein (CRBP). This suggests that they may play an important role in the movement or metabolism of retinol (vitamin A alcohol), which is required for the maintenance of spermatogenesis. Peritubular cells in culture, isolated from the testes of the 20-day-old rat, maintained high levels of CRBP and had the ability to internalize retinol from retinol-binding protein (RBP), the blood transport protein for retinol, in a manner suggesting a receptor-mediated process. Little of the internalized retinol was esterified, in contrast to what occurs in other cell types that contain high amounts of CRBPs, and very little, if any, lecithin-retinol acyltransferase activity was present in microsomes obtained from the cultured cells. The cells did, however, have the ability to synthesize and release their own RBP to the medium. This suggests that retinol from the blood may actually reach the seminiferous epithelium by passing across the peritubular cell, released on a new molecule of RBP, rather than by entering into the tubule bound to the preexisting RBP present in the interstitial fluid.

INTRODUCTION The cells that surround the seminiferous tubules of the testis are referred to as peritubular or myoid cells [1-3]. The widespread occurrence of peritubular cells among various species suggests that these cells are an integral functional component of the mammalian testis [4-7]. Not only do they provide structural integrity for the tubule [1, 4], but they have also been implicated in several other testicular functions. The peritubular cells cooperate with the Sertoli cells in secreting the intercellular matrix (basement membrane) [8-10]. Junctional complexes are formed between most of the peritubular cells; this limits free access to the space between them and the seminiferous epithelium. This restriction, coupled with the intercellular matrix and the better-appreciated junctional complexes between neighboring Sertoli cells, provides the tubule with an efficient blood-testis barrier [11-14]. Peritubular cells also synthesize several secretory products including transforming growth factor-alpha [15], transforming growth factor-beta [16], insulin like growth factor-I [17], P-Mod-S [18-201, and plasminogen activator inhibitor [21]. These factors presumably have regulatory roles important in the coordination of the complex process of spermatogenesis. Retinol (vitamin A alcohol) and its derivatives, the retinoids, are essential for maintenance of the germinal epithelium and the process of spermatogenesis (reviewed in [22]). Retinol circulates in the plasma bound to retinolbinding protein (RBP), largely secreted from the liver. In the plasma, retinol-RBP forms a high-affinity 1:1 complex with transthyretin (TTR). Because of the relatively small size of RBP (21 kDa), formation of this complex prevents exAccepted September 21, 1994. Received August 15, 1994. 'Supported by NIH grant RO1 HD25206. 2 Correspondence. FAX: (615) 343-0704.

cessive loss of retinol-RBP by glomerular filtration in the kidney. This plasma complex delivers retinol to most retinol-requiring cells of the body but is restricted from reaching certain sequestered cells such as the late germ cells in the testis. Sertoli cells contain cellular retinol-binding protein CRBP [23-25] and the retinol-esterifying enzyme, lecithin-retinol acyltransferase, LRAT, [26], and are able to synthesize and release RBP, presumably for delivery of retinol to those late germ cells [27]. Interestingly, peritubular cells also contain CRBP, at levels even greater than in Sertoli cells [23, 25]. This suggests that peritubular cells might also be involved in retinoid processing for the tubule, but little is known about their possible contribution. We report here that cultured peritubular cells from rat testis can internalize retinol from its complex with RBP. In contrast to Sertoli cells, these cells synthesize little retinyl ester but do share the ability to synthesize and release RBP. This suggests that retinol may reach the seminiferous epithelium, in part, by passing through the peritubular cell, which would release retinol (internalized from the RBP found in the interstitium) to the peritubular space on a newly synthesized molecule of RBP. MATERIALS AND METHODS Cell Culture Peritubular and Sertoli cells were isolated from the testis of 20-day-old Sprague-Dawley rats by a sequential enzymatic digestion 128] as modified by Tung et al. [29] and Skinner and Fritz [18, 19]. Decapsulated testis fragments were digested first at 37C for 30 min with trypsin (2.0 mg/ml; Sigma Chemical Co., St. Louis, MO) and DNAse (type 1, 0.13 mg/ml; Sigma) to remove the interstitial (and other) cells. To inactivate the trypsin, soybean trypsin inhibitor (Sigma) was added to the tubular suspension. After each digestion the tubular fragments were allowed to settle by gravity and 356

PERITUBULAR CELL RETINOL PROCESSING

washed twice with Hanks' Balanced Salt Solution (HBSS, Ca 2 + and Mg 2+ free). This was followed by a collagenase (type I, 1.0 mg/ml; Sigma) digestion that included DNAse (0.13 mg/ml) and finally by hyaluronidase (1.0 mg/ml, Sigma) including DNAse (0.13 mg/ml). Sertoli cell clumps were isolated at this point and put into culture. The peritubular cells were isolated from the collagenase supernatant after the tubular fragments had sedimented by gravity. The supernatant liquid was centrifuged at 50 x g for 3 min and the pelleted material discarded. The supernatant liquid was then centrifuged at 500 x g for 10 min. The pellet, containing the peritubular cells, was suspended in medium and plated in 100-mm-diameter tissue culture plates containing 15 ml of Ham's F-12 medium containing 10% new-born calf serum (Sigma) and 100000 units of penicillin/L, 100 mg streptomycin/L, and 12.5 ,ug amphotericin B/L (Sigma). The cells were cultured at 32°C in a 5% CO 2 atmosphere until confluent. Microscopically, the cells appeared to be homogeneous without any obvious contamination by fibroblasts. At this time they were washed with HBSS (Sigma), and the various experiments were performed. Some preparations were plated in 24-well plates and for certain experiments were treated the same as the others. Other preparations were taken through five passages before use. No fibroblastic contamination was noted. Preparationof PeritubularCell Cytosol and Microsomes Primary cultures of peritubular cells grown to confluency as described above were washed with PBS three times and then sonicated in PBS + 1 uM dithiothreitol (DIT) for 10 sec through the use of a Branson Sonifier (Ultrasonics Inc., Plainview, NY) at a power output of 7. The cell lysate was centrifuged at 15 000 rpm for 30 min in a Tomy MTX-150 centrifuge (Pennisula Labs., Belmont, CA). The supernatant liquid was collected on ice. The pellet was then resuspended in PBS + 1 uM DTT, sonicated, and centrifuged again as above. This supernatant liquid was pooled with the first and then centrifuged at 40 000 rpm (100 000 x g) for 1 h in a Beckman Ultra Centrifuge L5-65 (Beckman Instr., Palo Alto, CA). The cytosol, containing CRBP, was aliquoted into small Eppendorf tubes and quick frozen in methanol and dry ice. The microsomes, containing LRAT, were suspended in PBS + 1 .M DTT and quick frozen. Samples were stored at -80 0C. Determinationof CRBP Concentration An ELISA was developed to determine the concentration of CRBP in the cultured peritubular cells; purified recombinant CRBP [30] was used as the standard. The standard (1-25 ng) and the cytosol (5-20 dl) were dried under a small fan overnight in a 96-well titer plate (Immulon-2; Dynatech Labs., Alexandria, VA). Each coated well was filled with the blocking buffer (PBS, 0.05% Tween 20, 5.0% dried milk, pH 7.5), and incubation was performed for 1 h at

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37°C. Each well was washed with the washing buffer (PBS and 0.05% Tween 20) three times. The primary rabbit polyclonal antibodies to rat CRBP (affinity purified on a recombinant CRBP-Sepharose-4B column) were added in optimal quantities to each well, and incubation was carried out for 1 h at 37°C. The plate was then washed again at least five times with the washing buffer. A biotinylated secondary antibody (Vector Labs., Burlingame, CA) was added to each well, and a 1-h incubation was performed at 37°C. The plate was again washed at least five times. Avidin-biotinylated horseradish peroxidase (ABC reagent; Vector Labs.) was then added to each well; this was followed by incubation for 30 min at 370C. After the wells were washed at least three times, substrate (0.4 mg/ml orthophenylene diamine and 0.002% H2 02 in 50 mM Citrate buffer, pH 5.0) was added and the

enzyme reaction stopped at 30 min by the addition of 3 M HCl. The absorbance at 492 nm was read on a Titertel Multiskan (Flow Labs., McLean, VA) plate reader. The quantity of CRBP in the cytosol was calculated from the standard curve. Western Blot Analysis of CRBP in PeritubularCytosol

Aliquots of peritubular cytosol and pure CRBP were submitted to SDS-gel electrophoresis. The resolved proteins were transferred to nitrocellulose paper and examined for immunoreactivity with the affinity-purified CRBP antibody. The ECL system (Amersham, Arlington Heights, IL)was employed to visualize the antibody-CRBP complex. Uptake of Retinol by PeritubularCells

Peritubular cells cultured on 24-well plates (Falcon 3047; Falcon Plastics, Los Angeles, CA) were allowed to reach confluency (7-10 days). The cells were then washed three times with serum-free Ham's F-12 medium and incubated for 2 h with medium containing 0.1% BSA. They were then washed once and incubated with 250 p1 medium containing varying amounts of [ 3H]retinol (15.1 Ci/mmol), prepared as described previously [31]. The cells were incubated at 32°C for 1 h. The reaction was stopped by adding ice-cold PBS and evacuating the medium as quickly as possible. The cells were washed three times with cold PBS and then solubilized in 0.5 ml of 1% SDS per well. The radioactivity was determined and normalized to the protein content as measured by the BCA method (Pierce Chemical Co., Rockford, IL). For competition studies, 1.0 ,uM [3 H]retinol and varying amounts of nonradioactive retinol were incubated with peritubular cells for 1 h at 320C. The cells were washed and solubilized, and the radioactivity and protein were determined as described above. Uptake of Retinol by PeritubularCellsfrom [3H]RetinolRBP and Competition by Holo-RBP and Apo-RBP

Apo-RBP was prepared by extracting 4 mg of lyophilized RBP with ethanol until the extracts showed no retinol flu-

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DAVIS AND ONG absence of PMSF, washed, and sonicated in 2 ml of PBS (with 1 mM DTT). One half of the sonicate was extracted with ethanol/water/hexane (1:1:8) and then chromatographed on 10% deactivated alumina; radioactivity in the ester fraction, which elutes with 2% ether in hexane, was determined. The LRAT activity of microsomes prepared from cultured peritubular cells was determined as previously described [33]. Each assay tube contained 100 RIg microsomal protein, 1.0 mM DTT, 40 iLM BSA, and 3 jiM [3H]retinol-CRBP in 0.2 M phosphate buffer, pH 7.2.

FIG. 1. Western blot analysis of peritubular cytosol of 20-day-old rats. Lane 1 is authentic rat CRBP. Lane 2 is the Coomassie blue staining of the molecular mass standards (BSA, 69.0 kDa; ovalbumin, 46.0 kDa; carbonic anhydrase, 30.0 kDa; trypsin inhibitor, 21.5 kDa; lysozyme, 14.3 kDa). Lane 3 is a photoautographic image on x-ray film of CRBP in peritubular cytosol, separated by SDS-PAGE and transferred to nitrocellulose paper. CRBP was detected after exposure to affinity-purified antibody by the ECL procedure. Lane 4 is the Coomassie blue staining of the transferred protein of the peritubular cytosol.

orescence. The pellet of protein and salts was dried under nitrogen and brought to the desired concentration with PBS. Scanning between 240 and 440 nm showed no absorbance attributable to retinol. Tritiated retinol-RBP was prepared by a modification of the method described by Futterman and Heller [32]. Three milligrams of apo-RBP was provided with a 10% molar excess of [3 H]retinol in dimethylsulfoxide during vortexing. Then BSA (2 mg) and nonradioactive retinol (100 jig), to displace any nonspecifically bound [3 H]retinol, were added to the [3H]retinol-RBP, and the preparation was immediately passed over a transthyretin-Sepharose affinity column. The 3 column was washed with 20 ml of PBS and the [ H]retinolRBP was eluted with deionized water (> 18 M fl). The peritubular cells were plated on 24-well plates (Falcon 3047) and incubated until confluency (7-10 days). Cells were washed and preincubated as indicated above. In the first twelve wells, graded amounts of holo-RBP were added; then 1 aM [3H]retinol-RBP added. In the second twelve wells, graded amounts of apo-RBP were added and the plate was incubated for 1 h at 32 0C. At that time the cells were washed and solubilized as before, and radioactivity and protein concentration were determined. 3 A time-dependent uptake was also performed. [ H]RetinolRBP was added in a timed fashion from 0 to 24 h. The reactions were stopped as described above and the cells were washed, solubilized, and counted. Retinyl Ester Isolation and LRAT Quantitation Primary confluent cultures of peritubular cells were incubated with [3 H]retinol (1 jiM) for 1 h in the presence and

Isolation of Newly Synthesized RBP and TTR by Affinity Chromatography Affinity columns of TTR-Sepharose 4B and RBP-Sepharose 4B were prepared as previously described [34]. After incubation of the peritubular cells with 100 jCi [3 5S]methionine (Amersham Corp., Arlington Heights, IL) in serum-free, methionine-deficient medium (Sigma M-3911; 10 ml) for 72 h, the medium was passed through a 0.22jim filter (Acrodisc; Gelman Science, Ann Arbor, MI). The medium was applied to the TTR-Sepharose-4B affinity column after the addition of 100 jig affinity-purified human RBP and 0.1 volume of 10-strength PBS (0.05 M potassium phosphate, 0.15 M NaCI, pH 7.2). After the medium was applied, the column was washed with PBS + 0.5 M additional NaCl (10 ml) and PBS (10 ml). The column was then eluted with deionized water (> 18 megaohms), and the radioactivity and fluorescence were measured for each fraction (600 jil). The medium that had passed through the TTR column was then put over an RBP-Sepharose-4B column, washed, and eluted as above. The radioactivity was measured in each fraction by liquid scintillation. SDS-PAGE and Radioautography The radioactive peak isolated from a TTR-Sepharose-4B column was subjected to SDS-PAGE as described previously [34]. The minigel was stained with Coomassie Blue R-250, destained, dried, and exposed to a PhosphorImager (Molecular Dynamics, Sunnyvale, CA) phosphor-screen overnight and scanned. Rat RBP Antibody Production Rat RBP was isolated from rat serum (Pel Freez, Rogers, AK) by the method of Berni et al. [35]. It was further purified by collection on a human TTR affinity column and then eluted with deionized water; this procedure was repeated once. Before the RBP was passed over the TTR affinity column, its buffer was changed to PBS (pH 7.5) by diafiltration (Amicon Corp., Lexington, MA). A New Zealand White rabbit (4 lb) was immunized with 100 jig (250 j1d) of the purified rat RBP, which was mixed with an equal volume of adjuvant (Titer Max; Vaxcel Inc., Norcross, GA) and homogenized with a sonicator. The ho-


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Time (hrs) FIG. 2. Uptake of [3H]retinol from the retinol-RBP complex by peritubular cells. Incorporation was monitored with time (solid circles) when 1.0 i~M [3 H]retinol-RBP was incubated with peritubular cells for periods from 0 to 24 h. When 25 RiM unlabeled retinol-RBP was incubated with each time course the incorporation of radioactivity was significantly reduced (open circles).

mogenate was injected intradermally over a wide area of the back. The antibody production was followed by immunodetection of pure RBP on dot blots. The rabbit received three booster injections at intervals of approximately 1 to 1 1/2 mo. After the third booster, at the height of antibody production, the animal was killed and the blood taken. The IgG fraction of the preimmune and immune serum was isolated by a protein A column (15 x 40 mm), neutralized, and frozen at -70°C. Antibody Titration of [35S]RBP

The radioactive peak isolated by a TTR-Sepharose column from the medium of peritubular cells grown in the presence of [3 5S]methionine was titrated with the anti-rat antibody (IgG fraction) described above. A constant amount of radioactivity (approximately 15 000 cpm) was titrated against an increasing amount of IgG. The antibody-bound radioactivity was precipitated with Pansorbin cells (Calbiochem-Behring Corp., San Diego, CA) and counted by liquid scintillation spectrometry. RESULTS Presence of CRBP in Cultured PeritubularCells

To determine whether or not cultured peritubular cells would be appropriate for the study of retinoid processing,

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we determined the levels of CRBP that these cells were able to maintain under our culture conditions. The presence of CRBP in the cultured cells was demonstrated by Western blot analysis of the soluble proteins prepared from the cells (Fig. 1). A single strong immunoreactive band was observed at the same migration position as that of pure recombinant CRBP and corresponded to a band detectable by Coomassie staining. This suggested that CRBP was quite abundant in the cultured cells and also indicated the specificity of the immune reagent. The abundance of CRBP was confirmed by ELISA, which was developed through use of the same antibody preparation. Four separate preparations of cytosol were analyzed and found to contain CRBP in the range from 100 to 360 pmol/mg protein (average of 230 pmol/mg); this was similar to the average level of 95 pmol/ mg reported previously for peritubular cells cultured under similar conditions but then kept for 24 h in serum-free medium before collection [25]. These levels of CRBP are high compared to those in most in other tissues or isolated cells and are consistent with an active role in retinol processing for the peritubular cell. Immunolocalization of CRBP in the cultured cells showed a uniform brown staining of essentially all cells (results not shown), indicating that there was little contamination of the cultures with fibroblasts, which do not show CRBP immunostaining in our hands. Uptake of Retinolfrom RBP by Cultured PeritubularCells

Peritubular cells are constantly in the presence of plasma retinol-RBP in the interstitial fluid of the testis [36]. When the cultured peritubular cells were incubated with 1.0 JIM [3H]retinol-RBP, a time-dependent uptake of radioactivity was observed (Fig. 2). The rate of uptake was relatively constant for the first hour and then declined. A similar pattern was previously observed for uptake of retinol from RBP by Sertoli cells [37]. Inclusion of a 25-fold excess of retinol-RBP substantially decreased the amount of radioactivity accumulated at all time periods examined, indicating a specific and saturable process. It has been argued that cellular uptake of retinol from RBP may actually require prior dissociation of the complex, with the free retinol subsequently partitioning into the cell membrane and the uptake regulated, in part, by the amount of free CRBP present in the receiving cell (e.g., [38]). Because of the considerable abundance of CRBP in these cells, it is unlikely that the CRBP was saturated with retinol at the early time points; but competition for uptake was still observed, consistent with the presence of a membrane receptor for the retinol-RBP complex. Further, we found no evidence for specific accumulation of retinol by peritubular cells when the retinol was provided as the free compound. A substantial amount of the added free retinol became associated with the cells, with that amount directly related to the concentration added, over the range examined (Fig. 3a). There was no decrease in this association when increasing amounts of unlabeled retinol

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FIG. 4. Competition by holo-RBP and apo-RBP for uptake of [3 Hlretinol from RBP. When increasing concentrations of either holo-RBP (solid circles) or apo-RBP (open circles) were incubated with 1 pM [3H]retinol-RBP and peritubular cells, both reduced cell-associated radioactivity.

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were included (Fig. 3b), indicating that the association was nonspecific. In contrast, similarly increasing the retinol-RBP concentration in the presence of a constant amount of [3 H]retinol-RBP clearly decreased [3H]retinol accumulation (Fig. 4), suggesting an uptake process that required recognition of the complex rather than recognition of free retinol. Over the concentration range examined, complete inhibition of uptake was not achieved. Because it is expected

that a certain amount of dissociation of the complex will occur, undoubtedly some of the radioactivity became associated with the cells in a nonspecific manner due to the presence of some free [3 H]retinol. This is consistent with the fact that apo-RBP was even more effective than holoRBP at reducing the amount of [3H]retinol that became cell associated (Fig. 4). Because the structures of apo- and holoRBP are essentially identical [39], an ability of apo-RBP to compete with holo-RBP for recognition by a cell-surface receptor and reduce specific uptake was not surprising. Because apo-RBP would also reduce the amount of free retinol present, nonspecific association would be reduced as well. While we find this evidence consistent with the presence of a cell-surface receptor for retinol-RBP, definitive demonstration will require the isolation and characteriza-

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LRAT Activity and Retinyl Ester Synthesis in Peritubular Cells Cells that contain abundant CRBP are usually found to synthesize considerable retinyl ester and to be relatively TABLE 1. The formation of retinyl esters by peritubular myoid cells in culture. %Total cell-associated

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PERITUBULAR CELL RETINOL PROCESSING I

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Fraction (No.) FIG. 5. Synthesis of RBP and TTR by peritubular cells. Presence of [S1labeled RBP (solid circles) and TTR (solid squares) in peritubular cell-conditioned medium was determined as described in Materials and Methods after cells were incubated with 100 Ci [3 5 S]methionine for 72 h. Authentic TTR was found to elute from the RBP-Sepharose column at fraction 5, but no radioactivity was observed. Authentic RBP eluted from the TTR-Sepharose column coincident with the radioactive peak shown.

rich in LRAT activity. This includes such cells as the liver stellate cell [40], the retinal pigment epithelial (RPE) cell of the eye [41], and the Sertoli cell [26, 42]. Even though the level of LRAT in the Sertoli cell is considerably less than that found in the other two examples, 20-40% of newly internalized retinol is recovered as ester from cultured cells after several hours of incubation [26,43]. However, the peritubular cells synthesized relatively little retinyl ester. After incubation of confluent cells with 1 IM [3 H]retinol (15.1 Ci/mmol) for 1 h, only 1.6% of the cell-associated radioactivity was recovered as ester (Table 1). In the presence of PMSF, an inhibitor of LRAT, this decreased to 0.9%, suggesting that less than half of this synthesis was attributable to LRAT. Consistent with this low conversion, LRAT activity in microsomes isolated from cultured peritubular cells was essentially undetectable under our assay conditions. We estimate that a specific activity as low as 0.08 pmol/mg protein/ min would have been detectable. This is in contrast to a specific activity of 0.3 -+ 0.1 pmol/mg protein/min (n = 3) for microsomes from cultured Sertoli cells [42]. This finding indicated that the peritubular cell is not serving as a local storage site for retinyl ester. Synthesis of RBP and 77TR by PeritubularCells Certain cells that are essential elements of blood-organ barriers contain high levels of CRBP and have been shown

FIG. 6. Examination of material isolated by TTR affinity chromatography by SDS-PAGE. Lane 1 contains Coomassie-stained molecular mass markers; in descending order are phosphorylase (96 kDa), BSA (69 kDa), ovalbumin (46 kDa), carbonic anhydrase (30 kDa), and trypsin inhibitor (21.5 kDa). Lane 3 is Coomassie-stained authentic human RBP that was added to the medium before isolation of the radioactive material by the TTR affinity column. Lane 4 is Coomassie-stained authentic human RBP. Lane 2 is the Phosphorlmager scan of lane 3. Lane 2 superimposes on lane 3.

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IgG Protein (ng) FIG. 7. Antibody titration of the radioactive material isolated by the TTR affinity column. A constant amount of radioactivity (approximately 15 000 cpm) was titrated with an increasing amount of antibody (gG fraction). The radioactivity that bound to the antibody was precipitated with Pansorbin and counted (solid circles). Little activity (solid squares) was bound when preimmune IgG was substituted for the specific IgG fraction.

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DAVIS AND ONG

FIG. 8. Model for the delivery of retinol to the cells of the seminiferous tubule. The retinol-RBP-TTR would reach the peritubular cell after escape from a testicular capillary, shown with associated interstitial cells in the lower left of the figure.

either to secrete RBP or to contain its mRNA [44]. These cells include the RPE cells, which form the barrier between the blood and the retina proper, and the Sertoli cell ([27,34] and references therein). The RPE cell, but not the Sertoli cell, also synthesizes and secretes TTR [34]. When cultured peritubular cells from 20-day-old rats were incubated with [ 3 5S]methionine, radioactivity was found in RBP recovered from the medium by chromatography on TTR-Sepharose; but there was no evidence for the presence of radioactive TTR as assessed by chromatography on an RBP-Sepharose column (Fig. 5). The identity of the radioactive peak from the TTR affinity column was confirmed as RBP by its comigration with authentic RBP during SDS-PAGE (Fig. 6) and its precipitation by an IgG preparation from the serum from a rabbit immunized with rat RBP (Fig. 7). Peritubular cells from 35-day-old rats synthesized and secreted approximately the same quantity of RBP as did the 20-day-old rats. Peritubular cells were also passed a total of five times and allowed to come to confluency each time. The synthesis of RBP was monitored in the first and fifth passes. RBP synthesis was approximately the same as in the primary cells (data not shown). Rat skin fibroblasts (FR[F907]) showed no ability to synthesize and release either RBP or TTR when cultured in the same way. DISCUSSION It has been pointed out [13-14] that most (85-90%) of the junctions between the peritubular cells are occluded,

as shown by the inability of lanthanum nitrate to penetrate to the germinal epithelium above such junctions, for example. The junctions that are open, with a continuous interspace of about 200 A [14], do permit passage of molecules as large as ferritin (450 kDa). However, extracellular material of appreciable density often occupies the intercellular cleft, and in rare instances this may be concentrated in dense plaques between the outer leaflets of the cell membranes ([13] and references). This material may obstruct the flow of large molecules. For example, both ferritin and horseradish peroxidase (44 kDa) were observed only sporadically in the peritubular space and in the clefts between Sertoli cells 1 h after interstitial injection [13]. The TTR-RBP complex (76 kDa), which is present in the interstitial fluid, has an estimated diameter of 110 A and undoubtedly would be similarly slow in diffusing into the peritubular space. Since so few junctions between peritubular cells are open, it might be difficult for the retinolRBP-TTR complex to penetrate sufficiently rapidly to meet the needs of the seminiferous epithelium in areas of the basal compartment not directly above an open junction. The characteristics of the peritubular cell described here suggest that retinol reaches the seminiferous epithelium by passing through the peritubular cell rather than through the few open junctions between cells. The low LRAT activity and small quantity of retinyl ester found in the cultured cells suggest that storage of vitamin A is not an important


role of the peritubular cell. But of particular interest was the fact that the cultured peritubular cell, like the Sertoli cell [25], synthesized and secreted RBP, but not TTR, into the medium. The RPE cell synthesizes and secretes both RBP and TTR [34]. Furthermore, the RPE cell synthesizes 30-50-fold greater amounts of TTR than RBP. In the testis, if the uncomplexed TTR present in the interstitium is also somewhat restricted from the peritubular space, as might be expected from the observation of restriction of the smaller horseradish peroxidase, then the newly synthesized and secreted RBP might exist as the free molecule within this space rather than forming a complex with TTR as occurs in the plasma. Since RBP is a relatively small protein (40 A in diameter), it would diffuse more freely. In addition, it would presumably be released uniformly by the peritubular cells to supply all areas of the seminiferous epithelium. Thus, our proposed model (Fig. 8) for retinol delivery to the cells of the seminiferous tubule involves uptake of retinol from the interstitial retinol-RBP complex; movement through the peritubular cell, with release of a newly synthesized retinol-RBP complex into the peritubular space; and then uptake of the retinol by the Sertoli cell [37] (and perhaps the spermatogonia and early spermatocytes) with further delivery to germ cells by release of newly synthesized retinol-RBP from the Sertoli cell [27], presumably from the apical face, to supply the late germ cells. The spermatids, rich in LRAT [42], would then synthesize the retinyl ester found in testicular spermatozoa [45]. This model would explain why injected 125I-RBP was observed in a previous study only in the interstitium and was found not to have penetrated to the Sertoli cells [46]. TTR is present in a 2:1 excess over RBP in the plasma, and the injected RBP would thus have been found in complex with TTR-too large to move freely into the peritubular space. It had been thought for many years that the peritubular cells served only as support for the tubule. Clermont [1] was perhaps the first to suggest that the boundary tissue contained fibrous elements that seemed to bear some resemblance to smooth muscle cells; Clermont suggested, together with others [2-7], that these cells were responsible for the movements of sperm down the tubule, thus indicating a function other than architectural. Then it was discovered that both the peritubular and Sertoli cells cooperatively produced the components of the extracellular matrix [8-10]. Studies [15-21] have indicated that peritubular cells secrete products that can regulate Sertoli cell function. The results presented here indicate that the peritubular cells may also play an important role in delivery of an essential nutrient, necessary because these cells create a significant barrier to free movement of larger molecules to the seminiferous epithelium. REFERENCES 1. Clermont Y. Contractile elements in the limiting membranes of the seminiferous tubules of the rat. Exp Cell Res 1958; 15:438-440.

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