The apparent molecular weight of androgen-binding protein (ABP) in the blood of immature rats differs from that of ABP in the epididymis

J. steroid Biochem.Vol. 28. No. 4, pp.411-419, 1987 Printed in Great Britain. All rights reserved

0022-4731/87$3.00f 0.00 Copyright 6 1987Pergamon Journals Ltd

THE APPARENT MOLECULAR WEIGHT OF ANDROGEN-BINDING PROTEIN (ABP) IN THE BLOOD OF IMMATURE RATS DIFFERS FROM THAT OF ABP IN THE EPIDIDYMIS B~JAMIN J. DANZO, BARBARA C. ELLW and BEVERLYW. BIILL Departments of Obstetrics and Gynecology, and Biochemistry, and the Center for Reproductive Biology Research, Vanderbilt University School of Medicine, Nashville, TN 37232, U.S.A. (Received 29 January 1987) ~-~en andro~n-binding protein (ABP) in unf~ctionat~ immature (20&y old) male rat serum was covalently labeled with the site-specific photoaflinity l&and [3H]17~-hydroxy-4,~~drostadien3-one and analyzed on 5.6% polyacrylamide tube gels containing SDS (SDS-PAGE), a protein of M, 33,700 f 1200 was shown to be specifically labeled. Rat epididymal ABP from unfractionated cytosol analyzed under identical conditions exhibited two androgen-specific peaks of radioactivity, M, 49,900 f 600 and M, 44,100 & 800, which correspond to the previously described subunits of ABP. The apparent molecular weight differences between serum and epididymal ABP were further assessed on preparations of serum ABP that had been partially purified by chromato~aphy on A&Gel blue (to remove albumin) and on Sephadex G-150 (to remove other proteins). When these preparations of ABP were photolabeled and analyzed by SDS-PAGE as above, two subunits of M, 61,700 -+ 1300 and M, 47,100 f 700 were resolved. Serum and epididymal ABP were further purified by androgen affinity chromatography. When these preparations were subjected to SDS-PAGE on slab gels containing 10% polyacrylamide and identified by fluorography of photolabeled ABP or by immunochemical localization following electrophoretic transfer to nitrocellulose, differences in the apparent molecular weight of ABP from the two sources persisted. Immunochemicai localization studies on ABPs that had been desialylated with neura~~d~ indicated that there was an increased mobility of the subunits, as one would anticipate from removal of carbohydrate. Differences in apparent mofecular weight of ABPs from the two sources are likely due to differences in glycosylation.

~RODU~ION

Chemicals

Androgen-binding protein (ABP) is a Sertoli cell product [l-2] that has been identified in the testis, epididymis, and male reproductive tract fluids of several species including man [2-g]. In addition, ABP has been identified in the blood stream of the rat by both immunologioal [IO] and steroid binding [ll] techniques. Both of these methods indicate that the levels of ABP in blood are age-dependent. Low levels

of ABP are present before a postnatal age of 10-15 days, levels peak at 20-25 days of age, and decline to very low or nondectable levels at 30-40 days of age [lO-111. There are clearly similarities between ABP in the blood and that in the epididymis regarding atBnity for Concanavalin A (Con A [12]), and mobility on polyacrylamide gels under non-denaturing conditions [l 11.We had previously noticed, however, that the apparent molecular weight of partiaily purified plasma ABP that had been covalently labeled with the site-specific photoaffinity ligand [13-141 [3H]178-hydroxy-4,6-androstadien-3one was greater than that of epididymal ABP from the same age group 1111.The studies presented in this communication were designed to investigate this phenomenon further. 411

rH] 17/?-hydroxy-4,6-androstadien-3-one (60 Ci/ mmol) was prepared by New England Nuclear Corp. (Boston, MA) according to our synthetic scheme [15]. [3H]17~-hydroxy-5~-androstan-3-one (13H].5cr-DHT, 50.6 Ci/mmol) was also purchased from New England Nuclear Corp. Unlabeled steroids were purchased from Steraloids (Wilton, NH); electrophoresis and electroblotting supplies, Affi-Gel blue and Bio-Gel P-6DG were purchased from Bio-Rad Laboratories (~chmond, CA). Sephadex G-150 and G-50 were from Pharmacia (Piscataway, NJ). Spectrafluor was from Amersham (Arlington Heights, IL). A polyclonal antiserum to rat epididymal ABP was obtained from the NICHHD. Neuraminidase, type VI was obtained from Sigma (St Louis, MO).

Twenty-day old male Sprague-Dawley rats were used as the source of serum ABP since its blood level is highest in this age group [l 11. Cytosol was prepared

from epididymides of retired breeder rats by homogenixing the trimmed organs 1:2 (w/v) in 0.01 M

412

BENJAMINJ. DANZO P/ rd.

sodium phosphate buffer, pH 7.4, and centrifuging the homogenate for 30 min at 247,000 g in a Beckman 70.1 Ti rotor (Palo Alto, CA). The supernatant fluid (cytosol) was the source of epididymal ABP. All procedures were conducted at 04 “C unless otherwise noted. Two methods to partially purify serum ABP were used. The first-method consisted of desalting the serum by chromatography on a 1.5 x 28 cm column of Bio-Gel P-6DG using phosphate buffer as the eluant. The fractions containing ABP were detected by the charcoal assay procedure (see below), pooled, concentrated to the original serum volume using an Amicon (Danvers, MA) stirred cell and a PM-10 or YM-10 membrane applied to a column, 1.5 x 15 cm, of A&Gel blue and eluted with phosphate buffer. The fractions from this column that contained ABP (as determined by the charcoal assay) were pooled, concentrated and applied to a 1 x 50 cm Sephadex G-150 column. The sample was eluted from the G-150 column with phosphate buffer. The fractions containing ABP were detected using the charcoal assay; the absorbance of the fractions at 280 nm was also determined. Aliquots of the unfractionated serum, of the concentrated Affi-Gel blue pool, and of the Sephadex G-150 pool were used immediately, or were frozen for further analysis. The second method involved purification by chromatography on a column containing 6-(5a-androStan- 17/l-01- 17~ -yl) hexanoic acid (synthesized by modifications of the method of Musto[l7]) linked to Sepharose CL4B by the method of Mickelson and Petra[lS]. Crude preparations of serum or partially purified epididymal ABP were applied to the column, the matrix was washed with T.E. Buffer [IOmM Tris-HCl (pH 7.5) 1 mM EDTA] containing 10% dimethysulfoxide and 2 M KC1 (TDMK) until no absorbance at 280nm could be detected. ABP was eluted from the column with TDMK containing 60pg/ml of unlabeled Sc(-DHT and 20% dimethylsulfoxide (TDMKD) following overnight incubation of the matrix with TDMKD at room temperature. The affinity column eluate was diafiltrated against water on an Amicon stirred cell. An aliquot of the sample was incubated overnight with 40 nM [3H]5~-DHT followed by chromatography on Sephadex G-50 to quantitate macromolecular-bound [‘H]Scr-DHT, i.e. ABP. The remainder of the sample was lyophylized for future use. Photolysis The concentration of androgen-specific binding sites in unfractionated and partially purified preparations of serum or epididymal cytosol was determined using a 6-s charcoal assay procedure [3, 111. Other samples were incubated with a concentration of [3H]17/?-hydroxy-4,6-androstadien-3-one equivalent to 5 times the concentration of specific binding sites or with [3H] 178 -hydroxy-4,6-androstadien-3-one and

a lOOO-fold excess of unlabeled Sa-DHT (to determine non-specific binding) for at least I h on ice. The samples were then subjected to photolysis for 60 min and processed as previously described [14]. Analytical PAGE Non-denaturing [ 161 polyacrylamide tube gels containing 10% glycerol and [3H]5c(-DHT (4 nM) alone or together with a IOO-fold excess of unlabeled Sc(-DHT were run under steady-state conditions (SSPAGE) as described by Ritzen et a/.[191 and modified by us [20]. Two systems were used for electrophoresis under denaturing conditions. One was that of Fairbanks and Avruch[21] in which the photolabeled samples were subjected to electrophoresis on 5.6% polyacrylamide tube gels exactly as we have described previously [20]. The other system was that of Laemmli[22] in which the photolabeled samples were subjected to electrophoresis on slab gels in which the resolving gel contained 10% acrylamide and the stacking gel contained 4% acrylamide. Electrophoresis was allowed to proceed at 15 mA until the sample had entered the resolving gel, the amperage was then increased to 30mA. Electrophoresis was stopped when the tracking dye was about 2 cm from the end of the gel. Electrophoretic transfer and immunochemical staining After SDS-PAGE, proteins from the slab gels were transferred to nitrocellulose sheets (Millipore, Bradford MA) as described by Towbin et a/.[231 using a Bio-Rad transblot apparatus. Transfers were routinely done at 30V for approx 16 h followed by 60 V for 1.5-2 h. After the transfer was completed, the nitrocellulose sheet was removed from the transblot chamber and rinsed with water. The sheet was then incubated on a shaker for 1 h at 40°C in the blocking solution [IO mM sodium phosphate (pH 7.2), 0.84% NaCl, 10% horse serum 3% bovine serum albumin]; this solution was also used for making the antiserum dilutions. The blocking solution was discarded, replaced by a 1:2000 dilution of a rabbit anti-rat-ABP antiserum, and the sheet was incubated on a shaker overnight at 4°C. After the incubation, the sheet was rinsed 4 times with PBS. Goat anti-rabbit-IgG (Research Biogenics, Bastrop, TX), diluted 1 :200 was added to the nitrocellulose sheet. The sheet was incubated at room temperature for 1 h, and then rinsed with PBS (10 mM sodium phosphate, pH 7.4, 0.84% NaCl). Peroxidase anti-peroxidase (PAP) (Miles Scientific, Naperville, IL) diluted 1:200 was added to the sheet, it was further incubated for 1 h at room temperature, and then rinsed with PBS. A diaminobenzadine (DAB) solution [9 mg DAB, 30 ml 50 mM Tris (pH 7.5), 5 ~130% H,O,] was then added to the sheet to stain the ABP. After staining was achieved, the excess DAB solution was washed away with water.

Serum ABP Fluorography

After SDS-PAGE of photolabeled samples, the gels were stained overnight in a solution consisting of 0.05% Coomassie blue, destained, treated with Fluoro-hance (RPI, Inc., Mt Prospect, IL), dried, exposed to Kodak X-Omat AR film at -80°C for various periods of time, and the film was developed. Desialylation

Approximately 410 ng of affinity purified serum ABP and approx. 130ng of affinity purified epididymal ABP were incubated with 1 unit of neuraminidase in 25 ~1 of deionized water at 37°C for 60min. The samples were solubilized, subjected to SDS-PAGE, electrophoretically transferred to nitrocellulose, and immunochemically localized.

RESULTS

Analysis of ABP in unfractionated immature male rat serum

Unfractionated serum from immature male rats was photoaffinity labeled with [3H]178 -hydroxy4,6-androstadien-3-one and analyzed on SDS PAGE [21]. When the gels were sliced and counted, two peaks of radioactivity were detected. One peak migrated with proteins having a M, of 3 1,000 and the other peak migrated near the tracking dye (Fig. 1A). The fact that labeling of the 31,000 dalton peak was decreased when photolysis was conducted in the presence of unlabeled 5a-DHT indicated that androgen-specific sites were present on the protein. Since photolabeled ABP from immature rat Sertoli cell culture media [2] and from immature and adult [ 11,201 rat epididymides was shown to be composed of two subunits having M,‘s of approx 50,000 and approx 42,000, we decided to determine if the

6c

lower molecular weight exhibited by ABP from the serum represented an inherent property of the molecule or if it was a result of interactions with serum components. To explore these possibilities, partially purified and affinity purified serum ABP was prepared, photoaffinity labeled, and compared to ABP in unfractionated serum and to epididymal ABP. Analysis of ABP in partially purijied preparations of serum from immature male rats

The first step used to partially purify serum ABP was chromatography of serum on Bio-Gel P-6DG to remove salts from the sample. The fractions containing ABP (as determined by charcoal assay) were pooled, concentrated, and a portion was applied to a column of Al&Gel blue. The fractions from this column were assayed by the charcoal method to detect ABP and a portion of the pooled and concentrated fractions containing ABP were applied to a column of G-l 50. The fractions from the G-l 50 column that contained ABP were concentrated to the original serum volume. SS-PAGE

Aliquots of unfractionated serum (Fig. 2A), and of the pools from the Affi-Gel column (Fig. 2B) and the Sephadex G-150 column (Fig. 2C) were analyzed for the presence of ABP under non-denaturing conditions. Androgen-specific peaks of ABP could be detected in all three preparations. The large nonspecific albumin peak detected in crude plasma (Fig. 2A) was greatly reduced after chromatography on Affi-Gel blue (Fig. 2B) and it was eliminated after chromatography on Sephadex G-150 (Fig. 2C). Thus, our objective of reducing or eliminating this major contaminating steroid-binding protein from the serum samples was achieved.

T.D.

SLICE

413

NUMBER

Fig. 1. SDS-PAGE of various photolabeled immature male rat serum ABP preparations. Crude (Fig. 1A), Affi-Gel blue treated (Fig. 1B) and At&Gel blue plus Sephadex G-150 treated serum samples (1C) were photolabeled with 17,!7-hydroxy-4,6-androstadien-3-one alone (0) or together with unlabeled Sa-DHT (0). The samples were dialyzed, lyophylized, and the lyophylized material was then subjected to electrophoresis [20]. Molecular weight markers were: phosphorylase b, 97,000; bovine serum albumin, 68,000; ovalbumin, 45,000; carbonic anhydrase, 31,000; and soybean trypsin inhibitor, 21,500; lactoglobulin A, 18,367. T.D., tracking dye; Kd, kilodaltons.

BENJAMINJ. DANZO cr at.

414

16

lH oH+C

14 12 IO i

I 0

I

0

20

40

40

20

0

I

I

20

40

I

SLICE NUMBER

Fig. 2. SDS-PAGE of crude and partially purified 20-day old male rat sera. Aliquots of ~fmctionated serum (Fig. 2A), Affi-Gel blue treated serum (Fig. 2B) and serum that had been chromatographed on Affi-Gel blue and Sephadex G-l 50 (Fig. 2C) were incubated with 4 nM [3H]5a-DHT alone (a) or together with 400 nM Sa-DHT (0) and subjected to electrophoresis (3 mA/gel) on tube gels containing the same concentrations of steroid as were present in the incubations. The gels were sliced and counted.

SDS-PAGE of~hotolabeled Fart~a~~ypw$ied serum ABP and ABP in epididymal cytosol The electrophoretic patterns of the photolabeled proteins in crude and partially purified serum samples are shown in Fig. 1. As noted previously, androgen

specific photola~ling of a low molecular weight protein in crude serum is detected (Fig. IA). Both the serum sample that had been chromatographed on Affi-Gel blue (Fig. 1B) and that which had been chromatographed on A&Gel blue and on Sephadex G-150 (Fig. 1C) had two androgen-specific peaks. One peak had a molecular weight larger than, and one had a molecular weight similar to, the subunits of ABP from Sertoli cell culture media [2] and from the epididymis [20], strengthening our earlier observations [ 111 that the heavy subunit of ABP from the serum has an apparent molecular weight greater than that of the heavy subunit of ABP from the

reproductive tract. These data suggested that the partial purification scheme, in addition in removing albumin, removed serum factors capable of degrading ABP. Table 1 summarizes the results of several experiments that show differences in the molecular weight of ABP isolated from the serum and from the epididymis. Subunit molecular weights obtained after Affi-Gel alone or in combination with Sephadex G-150 have been combined to obtain the data shown in Table 1. SDS-PAGE of photolabeled ~~~ity-p~rl~ed and e~ididymai ABP

To further assess possible differences in the molecular weight of ABP from the two sources, aliquots of lyophylized affinity purified ABP from serum and epididymal cytosol were reconstituted in phosphate buffer, photolabeled, subjected to SDS-PAGE 124,

Table 1. The molecular weiaht of ABP oreuarations --

serum

from serum and eoididvmis

serum

Epididymis .--______.-.

Type of analysis

Crude serum

Peak I

Peak II

SDS-PAGE* 5.6% Tube gets

33,700 + 1200 (16)t

61,700+ 13OOf19)

SDS-PAGE2 10% Slab gels

N.D.

56,000 + 1900 (5)

^_

Peak I

Peak II

47,100 * 7OOfl9)

49,9~~~(1~)

~.~~~8~(~5)

49,800 * 1200 (5)

50,200 * 1900 (5)

43,700 + 2200(S)

N.D. = Not determined. ‘These analyses were conducted on material obtained from four separate partial (A&Gel blue and Affi-Gel blue plus Sephadex G-150) purifications of serum ABP and on unfractionated serum and epididymal cytosol. ?The number in parentheses indicates the number of separate analyses conducted. fThese analyses were done on affinity-purified ABP from serum and epididymis.

Serum ABP

415

MW Kd

66

31)

Fig. 3. Fluaorography of serum and epididymal ABP purified by androgen. affinity column chromatography. Samples of ABP from serum, lanes 1 and 3, and of ABP from epididymal cytosol lanes 2 and 4 which had been purified by chromatography on the androgen affinity column and photoaffinity labeled were subjected to SDS-PAGE. The gel was processed for fluorography, exposed to X-ray film and developled. The arrows indicate the migration of the molecular weight standards-see Fig. 1.

and fluorography was performed. The results of one such experiment are shown in Fig. 3. ABP from both sources exhibited two bands of radioactivity, a broad band representing the higher molecular weight species and a narrow lower molecular weight band. There was little sign of other labeled proteins in the preparations. These androgen-specific bands correspond to the subunits of ABP. Approximately 80 ng of ABP were loaded on the left hand pair of lanes and approx 29 ng of ABP were loaded on the right hand pair of lanes. Exposure time was 4 days. ABP from the serum, lanes 1 and 3, migrates slower than ABP from the epididymis, lanes 2 and 4, indicating that even in the highly purified preparations there are differences in apparent molecular weight between the ABPs from the two sources. The molecular weights obtained in this experiment were: serum ABP, 5 1,400 and 45,600; epididymal ABP, 49,700 and 44,600 for the heavy and light subunits, respectively. Furthermore, these data substantiate that both subunits of

ABP from both sources bind [‘HISa-DHT. Densitometric scans of the fluorogram indicated that 67% of the radioactivity was associated with the heavy subunit and 28% was associated with the light subunit. Electrophoretic transfer and immunochemical ization of ABP

local-

Affinity-purified serum and epididymal ABP were evaluated by SDS-PAGE followed by electrophoretic transfer to nitrocellulose sheets and immunochemical localization. As can be noted (Fig. 4), the broad and narrow bands corresponding to the high and low molecular weight subunits of ABP were localized with an antiserum to rat epididymal ABP as they were with the photolabeling method (Fig. 3). These data indicate that both subunits of ABP from both sources bind [‘HISa-DHT and possess epitopes recognized by antibodies in the antiserum. As with previous methods, immunochemical localization indi-

416

BENJAMIN J. DANZO et al.

MW

Kd

sponsible for the observed molecular weight heterogeneity of ABP from the serum and epididymis, ABPs from the two sources were treated with neuraminidase to remove terminal sialic acid moieties that might be associated with the proteins. The desialylated and untreated proteins were then subjected to SDS-PAGE, transferred to nitrocellulose and immunochemically localized. Figure 6 shows the results obtained. Untreated serum ABP, lane 2, exhibited two bands of M, 55,100 and 46,300, while untreated epididymal ABP, lane 4, exhibited two bands of M, 48,000 and 44,000. When serum ABP was treated with neuraminidase, lane 1, there was an increase in the mobility of the subunits; the heavy subunit now had an apparent M, of 48,000, and the light subunit had an apparent M, of 44,000. A similar phenomenon was observed with epididymal ABP: with the neuraminidase treated heavy and light subunits having apparent M,‘s of 46,400 and 42,400 respectively (lane 3). These data indicate that both subunits of ABP from both sources contain terminal sialic acid residues. The presence of high molecular weight immunoreactive bands in the neuraminidase treated samples is noted. but currently unexplained.

,

97r-

66,

31)

21,

,

jFig. 4. Immunochemical localization of ABP. Androgen affinity column purification of serum and epididymal ABP was followed by SDS-PAGE of the samples, electrophoretic transfer of the proteins to nitrocellulose, and immunochemical localization of ABP using an antiserum to ABP and PAP. The left lane is serum ABP and the right lane is epididymal ABP. The arrows indicate the migration of the molecular weight standards-see Fig. 1.

)-

.pABP oeABP ,-

cated the ABP from serum, left lane, migrated more slowly than ABP from the epididymis, right lane, indicating that it has a greater apparent molecular weight. The molecular weights obtained in this experiment were: serum ABP, 52,100 and 45,600; epididymal ABP, 48,400 and 42,900 for the heavy and light subunits, respectively. In further studies photolabeled ABP from the two sources was subjected to SDS-PAGE, blotted, and localized immunochemically. The nitrocellulose strips were then divided into segments and counted in a scintillation fluid containing toluene and Spectrafluor (2460: 100, v/v). Two major peaks of radioactivity were present in each ABP preparation (Fig. 5). The peaks of radioactivity coincided with the stained ABP bands verifying that the photolabeling and immunochemical methods identified the same proteins. Desialylation

of ABPs

To determine whether glycosylation

might be re-

?

2

4

6

STRIP

8

IO

12

14

NUMBER

Fig. 5. Scintillation counting of strips from an electrophoretic transfer of photolabeled ABPs. Androgen affinity column purified serum ABP (0, pABP) and epididymal ABP (0, eABP) were photoaffinity labeled, subjected to SDS-PAGE and electrophoretically transferred to nitrocellulose. The ABP’s were localized immunochemically, and the nitrocellulose was cut into strips. The strips were cut so that each immunochemically stained band and the area between each band was placed into a separate vial, the remainder of the nitrocellulose sheet was divided into equal segments. All of the segments were then subjected to liquid scintillation counting.

Serum ABP DISCUSSION

In an earlier study [ 1l] we noted that photolabeled partially purified serum ABP had subunits of 60,000 and 48,000 daltons, whereas those of epididymal ABP were 47,000 and 41,000 daltons, as determined using tube gels composed of 5.6% polyacrylamide. The current studies confirm and extend the earlier observations. On every occasion in which serum ABP and epididymal ABP were analyzed by SDS-PAGE in parallel lanes, both subunits of serum ABP migrated slower than the subunits of epididymal ABP, resulting in higher calculated molecular weights. The molecular weights that we have obtained are considered to be estimates since glycoproteins are known to migrate anomolously under SDS-conditions [21]. Molecular weights for the protein backbone of ABP are currently being obtained using completely deglycosylated preparations. To demonstrate the large molecular weight subunit of serum ABP, the serum must be fractionated prior to SDS-PAGE. If this is not done, only a single low molecular weight (N 34,000) androgen-specific protein is detectable. Although the low molecular weight species may be attributable to an electrophoresis artifact, our experience indicates that the shorter the exposure time of ABP to whole serum is prior to purification, the greater is the recovery of the high molecular weight species. This suggests that enzymatic activity in the serum is capable of reducing the amount of the high molecular weight species of serum ABP. This appraisal is strengthened by our previous observation [24] that an injected bolus of photolabeled ABP is degraded in the circulation. Such degradative processes may be responsible for the differences in the labeling ratios of the serum ABP subunits seen in partially purified and affinity purified preparations. It is possible that the purification procedures eliminate the low molecular weight species from the preparation, thus enabling us to detect only high molecular weight species. An examination of Figs 3, 4 and 6 indicates that the heavy band of both serum and epididymal ABP is quite broad, even at different protein loadings (Fig. 3), which is not what one would expect if the band contained a homogeneous protein. Therefore, we postulated that the band might contain heterogeneous forms of ABP possibly arising from differences in the degree of glycosylation of the protein backbone. Enzymatic cleavage of sialic acid residues resulted in a narrowing of the broad band which contained the higher molecular weight forms of the ABP subunits and in an increased mobility of the subunits (Fig. 6). These data indicate that the subunits of ABP isolated from both the epididyrnis and serum contain terminal sialic acid residues. Desialylation did not result in the appearance of one and molecular weight homogeneous subunit, differences in the subunits isolated from the two

sources persisted. These data are interpreted as indi-

417

eating that the difference in the molecular weight of the subunits from the two sources is only partly due to differences in the degree of sialylation and that other post-translational modifications such as other types of glycosylation, phosphorylation [25], or other processes are responsible for the observed differences. Several investigators have provided evidence that ABP is a glycoprotein. Hsu and Troen [9] first showed that ABP was partially retained on a Con A column, suggesting that terminal mannose and/or glucose residues are present on the molecule. This observation was extended by Cheng er af.[12] who showed that there were age- and tissue-dependent differences in the retention of ABP by Con A. Both of these observations indicate that differences in glycosylation exist among ABP species. Feldman et a1.[26] showed that rat epididymal ABP is composed of 25% carbohydrate, and Larrea et a/.[271 showed that [3H]fucose was selectively incorporated into the heavy ABP subunit by rat Sertoli cells in culture. All of these data substantiate that ABP is a glycoprotein and strengthen the postulate that differences in glycosylation account for the differences in the apparent

1

2

3

4

Fig. 6. Analysis of desialylated ABPs. Affinity purified ABP from the serum and epididymis were either untreated (lanes 2 and 4, respectively) or treated with one unit of neuraminidase for 1 h at 37°C (lanes 1 and 3). The samples were subjected to SDS-PAGE on 10% slab gels, transferred to nitrocellulose, and localized immunochemically. The arrows indicate the migration of molecular weight markers--see Fig. 1).

418

BENJAMINJ. DANZO EIof.

weights of the ABP subunits in serum and in the epididymis. The data of Feldman et al.[26] indicating that ABP can be localized by immunochemical means at the basal and adluminal regions of the seminiferous tubules of 28-day old hypophesectomized rats, suggest that there may be a bi-directional release of ABP from the Sertoli cells. The ABP at the base of the cells would be secreted into the blood stream and that at the apex of the cells would be secreted into the reproductive tract. If this is the case, and if the difference in the apparent molecular weight of serum and epididymal ABP is due to differences in postmechanisms translational modifications, for differential processing of ABP destined for release into each of the two compartments must be invoked. However, the existence of such mechanisms is not currently known. Alternatively, the same form of ABP may be secreted in both directions and degradative processes may be responsible for the different forms that we detect. Whether these putative degradative processes are of a specific or non-specific nature remains to be ascertained. It is of interest with regard to the preceding discussion that ABP secreted into the media by cultured Sertoil cells from immature rats has a subunit molecular weight, as determined by SDS-PACE, identical to that from the epididymis [2]. Since, under our culture conditions the Sertoli cells did not assume their normal physiological orientation, it is possible that proper orientation, and its attendant cell associations, are essential for normal synthesis and processing of ABP. To date, the physiological function of ABP is unknown. Whether the heterogeneity of ABP forms that we have demonstrated is involved in modulating its function remains to be explored.

Androgen-binding proteins (ABP) in fluids collected from the rete testis and cauda epididymidis of sexually mature and immature rabbits and observations on morphologic changes in the epididymis following ligation of the ductuli efferents. Viol. Reprod. I7 (1977)

molecular

64-77. 6. Jegou B.

and Gac-Jegou F.: Androgen-binding protein in the seminal plasma of some mammalian species. J. Endow. 17 (1978) 267-268.

I

(1982) 513-527. 8 Holland M. K., Rogers B. J., Orgebin-Crist M.-C. and

9

10.

11.

12.

13.

14.

15.

16. Acknowledgements-This research was supported by NIH grant HD13813 and a grant from the Andrew W. Mellon Foundation. The Center for Reproductive Biology Research is supported By NIH grant HD 0.5797.

REFERENCES 1. Steinberger A., Heindel J. J., Lindsey J. N., Elkington

J. S. H., Sanborn B. M. and Steinberger E.: Isolation and culture of FSH responsive Sertoli cells. Endocr. Res. Commun. 2 (1975) 261-267. 2. Schmidt W. N., Taylor C. A. Jr and Danzo B. J.: The use of a photoaffinity ligand to compare androgenbinding protein (ABP) present in rat Sertoli cell culture media with ABP present in rat epididymal cytosol. Endocrinology 108 (198 1) 786-794.

3, Danzo B. J., Orgebin-Crist M.-C. and Toft D. 0.: Characterization of a cytoplasmic receptor for Sa-dihydrotestosterone in the caput epididymidis of intact rabbits. Endocrinology 92 (i973) %f%j17. 4. Carreau S., Drosdowskv M. A. and Courot M.: Age related effects on androgen binding protein (ABP) in sheep testis and epididymis. Int. J. Androl. 2 (1979) 49-61.

5. Danzo B. J., Cooper T. G. and Orgebin-Crist M.-C.:

Danzo B. J., Dunn J. C. and Davies J.: The presence of androgen-binding protein in the guinea-pig testis. epididymis and epididymal fluid. Molec. cell. Endoer. 28

Danzo B. J.: The effects of photoperiod on androgenbinding protein and sperm fertilizing capacity in the hamster. J. Reprod. Fertl. 81 (1987). In press. Hsu A. F. and Troen P.: An androgen-binding protein in testicular cytosol of human testis: Comparison with human testosterone estrogen binding globulin. 1. c/in, Inaest. 61 (1978) 1611-1619. Gunsalus G. L., Musto N. A. and Bardin C. W.: Immunoassay of androgen-binding protein in blood: a new approach for the study of the seminiferous tubule. Science 200 (1978) 65-66. Danzo B. J. and Eller B. C.: The ontogeny of biologically active androgen-binding protein in rat plasma, testis, and epididymis. ~ndocr~o~ogy 117 (1985) 138%.1388. Cheng S. Y., Gunsalus G. L., Musto N. A. and Bardin C. W.: The heterogeneity of rat androgen-binding protein in serum differs from that in testis and epididymis. Endocrinology 114 (1984) 1386.1394. Danzo B. J., Taylor C. A. Jr and Schmidt W. N.: Binding of the photoa~nity ligand 17~-hydroxy-Soandrostadien-3-one to rat androgen-binding protein: comparison with the binding of 178-hydroxy-5aandrostan-3-one. Endocrinology 107 (1980) 1169-I 175. Taylor C. A. Jr, Smith II. E. and Danzo B. J.: Photoaffinity labeling of rat androgen-binding protein. Proc. natn. Acad. Sci. U.S.A. 77 119801 234-238. Taylor C. A. Jr., Danzo B. J.‘ and’ Smith H. E.: Preparation of 17~-hydroxy-[lt, 2f-3H,]4, 6-androstadien-3-one. J. Lube/. camp. Rad~op~larmacellt. 17 (1980) 627-634. Davis B. J.: Disc electrophoresis. II. Method and application to serum proteins. Ann. N. Y. Acad. Sci. 121 (1964) 404.-427.

17. Musto N. A., Cunsalus G. L., Miijkovic M. and Bardin C. W.: A novel affinity column for isolating androgen binding protein from rat epididymis. Endocr. Res. Commun. 4 (1977) 147-158. 18. Mickelson K. E. and Petra P. H.: Purification and characterization of sex steroid-binding protein of rabbit serum. Comparison with the human protein. J. hiol. Chem. 253 (1978) 529335298. 19. Ritzen E. M., French F. S., Weddington S. C. and Nayfeh S. N.: Steroid binding on polyac~lamide gels, quantitation at steady state conditions. J. bio/. Chem. 249 (1974) 6597-6604.

20. Taylor C. A. Jr, Smith H. E. and Danzo B. J.: Characterization of androgen-binding in rat epididymal cytosol using a photoaffinity ligand. J. biol. Chem. 255 (1980) 7769-7773.

21. Fairbanks G. and Avruch J.: Four gel systems for electrophoretic fractionation of membrane proteins using ionic detergents. J. Supramot. Sfr~ct. I (1970) 680-685.

22. Laemmli U. K.: Cleavage of structural oroteins durine the assembly of the heath of bacteriophage T4. Nature 227 (1970) 680-685. 23. Towbin H., Staehelin T. and Gordon J.: Elcctrophoretic transfer of proteins from polyacrylamide gels to nitro-

Serum ASP cellulose sheets: procedure and some applications. Proc. nam. Acad. Scf. U.S.A. 76 (1979) 435&4354. 24. Danzo Et. J. and F&r Ft. C.: Clearance, metabolic fate and tissue ~t~~~~5~ of an injected boIus of phot~ity-Ia~l~ rat androgen binding protein. Sol. Reprod. 31 (1984) 259-270. 25. Steinberg R. A., O’Farrell P. M., Friedrich U. and Coffin0 P.: Mutations causing charge alterations in regulatory subunits of the CAMP-dependent protein kinase of cultured S49 lymphoma cells. Ceff 10 (1977) 381.

419

26. Feldman M., Lea 0. A., Pertrusz P., Tres L. L., Kierszenbaum A. L. and French F. S.: Androgenbinding protein purgation from rat e~djd~id~, characterization and ~~un~h~~ Iocafization, f. biof. Citem. 256 (f981) 5170-5175. 27. Larrea F., Musto N. A., Gunsalus G. L., Mather J. P. and Bar&n C. W.: Origin of the heavy and light protomers of ~drogen-binding protein from the rat testis. J. biol. Chem. 256 (1981) 12566-12573,