Partial characterization of a unique mitogenic activity secreted by rat Sertoli cells

Molecular and Cellular Endocrinology, 19 (1991) 1- 12 0 1991 Elsevier Scientific Publishers Ireland, Ltd. 0303-7207/91/$03.50

MOLCEL

02540

Partial characterization

of a unique mitogenic activity secreted by rat Sertoli cells

Dolores J. Lamb *TV, Gerald S. Spotts ‘, Sankararaman and Kelty R. Baker ’

Shubhada

’

’ The Scott Department of Urology, and 2 Department of Ceil Biology, Baylor College of Medicine, Houston, TX, U.S.A (Received

Key words: Growth

factor;

Characterization;

7 December

A431 cell; (Sertoli

1990; accepted

5 April

1991)

cell)

Summary

Sertoli cell conditioned medium (SCCM) contains a potent mitogen, Sertoli cell secreted growth factor (SCSGF). A431 cells, derived from a human epidermoid carcinoma have provided an excellent model cell line for the study of this apparently unique activity secreted by rat Sertoli cells in vitro. Previously, it was shown that SCCM contained an epidermal growth factor (EGF)-like activity which was thought to be the mitogen for A431 cells. The present study showed that these two factors are distinct entities. The secretion of the EGF-like activity decreased with increasing number of culture days, while that of SCSGF and of another Sertoli cell specific protein, transferrin remained constant. The addition of SCCM stimulated whereas 2.5 ng/ml EGF inhibited the A431 cell growth. The proliferative response of A431 cells to a wide variety of growth factors and known Sertoli cell secretions was investigated. SCSGF was the only growth factor of known Sertoli cell secretions tested (transforming growth factors (TGF,, TGFp), EGF, bombesin, fibroblast growth factor (FGF), platelet derived growth factor (PDGF), insulinlike growth factors 1 and 2 (IGF-1 and IGF-21, prostaglandins E-l and E-2, insulin, transferrin and lactate) which stimulated A431 cell proliferation. SCSGF was mitogenic for A431 cells even in the presence of serum in the culture medium. The partially purified SCSGF was heat- and acid-stable, protease-sensitive with a molecular weight of 14,000. It did not bind to heparin or concanavalin A-Sepharose. The secretion of a mitogenic activity by the Sertoli cell which is different from other previously identified growth factors and which coincides with active spermatogenesis could have important implications in the regulation of spermatogenesis.

Introduction Address partment of Plaza/Room Supported 39719 to Dr.

for correspondence: Dr. Dolores J. Lamb, DeUrology, Baylor College of Medicine, One Baylor 440E, Houston, TX 77030, U.S.A. in part by USPHS RR-05425 and NIH-DKLamb.

A mitogen, secreted by rat and human Sertoli cells in vitro, Sertoli cell secreted growth factor (SCSGFI appears to be unique when compared

with other growth factors (Holmes et al., 1986; Buch et al., 1988). Sertoli cell conditioned medium (SCCM), also contained a factor which displaced the binding of ‘251-epidermal growth factor (EGF) from its receptor. At the time the identity of these factors and whether both activities were the result of a single growth factor were unknown. In early studies, A431 cell line was used to study these factors. A431 cells were originally isolated from an epidermoid carcinoma from an 85year-old female (Giard et al., 1973). These cells were found to have very high concentrations of the EGF receptor on their surface (nearly 3 million receptor sites per cell) (Fabricant et al., 1977; Haigler et al., 1978) and amplification of the EGF receptor gene (Merlin0 et al., 1985). Interestingly, EGF has been shown to inhibit the cell growth of A431 cells (Barnes et al., 1982; Kamata et al., 1986), and paradoxically to stimulate A431 cell proliferation at low concentrations of EGF (Kawamoto et al., 1983). Holmes et al. (1986) previously demonstrated that Sertoli cell conditioned medium contained an activity(s) which could displace 12”1-EGF from its receptor. Although the conditioned medium contained EGF-like activity based upon the radioreceptor assay, unlike authentic EGF, the conditioned medium did not inhibit A431 cell proliferation, and was mitogenic for A431 cells (Holmes et al., 1986; Buch et al., 1988). A number of growth factors have been reported to be secreted by Sertoli cells in culture in addition to SCSGF, including immunoreactive somatomedin A (Hall et al., 1983), somatomedin C-like activity (Chatelain et al., 1987; Smith et al., 19X7), a transforming growth factor beta (TGFp)like activity (Morera et al., 1987; Benahmed et al., 1988; Skinner and Moses, 1989), TGF, (Skinner et al., 1989) and basic fibroblast growth factor (bFGF) (Smith et al., 1989). Seminiferous tubule growth factor (SGF) is an FGF-like growth factor which is present in homogenates of isolated Sertoli cells (Feig et al., 1980, 1983; Bellve and Feig, 1984). Based upon preliminary biochemical characterization of the unfractionated Sertoli cell conditioned medium (Holmes et al., 1986; Lamb et al., 1987) the factor did not have the characteristics of any of these growth factors.

The present study provides important information concerning the response of the model celi line, A431 cells (the cell line which we have chosen to study SCSGF) to a number of known growth factors and Sertoli cell secretions in an attempt to further characterize this growth factor. The growth factor has been partially purified and its characteristics compared with other growth factors. This factor may play an important role in the regulation of germ cell proliferation and/or meiosis in the testis. Materials

and methods

Materials Male Sprague-Dawley rats were obtained from Harlan (Houston, TX, U.S.A.). Dulbecco’s modified Eagle’s medium (DME), Hanks’ balanced salt solution (HBSS), trypsin and fetal bovine serum (FBS) were obtained from Gibco (Grand Island, NY, U.S.A.). Insulin, lactate, hyaluronidase I, DNAse and prostaglandins E-l and E-2 were purchased from Sigma (St. Louis, MO, U.S.A.). EGF, TGFol, TGFp, bombesin, FGF and platelet derived growth factor (PDGF) were from Collaborative Research (Bedford, MA, U.S.A.). As a control, these growth factors were tested for mitogenicity in Swiss 3T3 cells and the DDT,-MF2 cell lines (data not shown). Transferrin and collagenase were obtained from Cooper Biomedical (Malvern, PA, U.S.A.) and follicle stimulating hormone (FSH) was a gift from the National Hormone and Pituitary Program (NIADDK, NIH-FSH-O-14). Cell lines The A431 cells (Giard et al., 1973) were obtained from the American Type Culture Collection (ATCC, Rockville, MD, U.S.A.). The cell has a rapid doubling time which makes it useful for studies on proliferation and high concentrations of EGF receptors (Fabricant et al., 1977; Haigler et al., 1978). The Swiss 3T3 cells and the DDT,-MF2 cell lines are available from the ATCC. These cell lines are well established. The cells were tested for the presence of mycoplasma contamination using the Mycoplasma T.C. test kit obtained from Genprobe. Measurement of the EGF receptor levels by a radioreceptor assay

3

(Todaro et al., 1980) confirmed that the cell line contained approximately the reported levels of EGF receptors for A431 cells (Fabricant et al., 1977; Haigler et al., 1978). The Sertoli cells were primary cultures. The isolation and culture procedures are described below. Rat Sertoli cell isolation and culture

Sertoli cells were isolated from the testes of 19-21-day, 35-day, and 45-day Sprague-Dawley rats according to the method described by Steinberger et al. (1975) and modified by Tung et al. (1984). Testes were removed aseptically, decapsulated and cut into 12-14 fragments of approximately equal size and incubated at 37°C for 15 min in 0.25% trypsin containing 2 pg/ml of DNase I in HBSS with agitation. The tubule fragments were washed 3 times with HBSS, incubated at 37°C for 20-30 min with 0.1% collagenase (type II), 0.1% hyaluronidase, then with DNAse (5 pug/ml), and finally with 0.1% hyaluronidase for 30 min. After washing 3 times with HBSS, the Sertoli cells were plated in 100 mm dishes (22-25 X lo6 cells/dish) cultured in 7 ml DME, which in some experiments contained insulin (5 pgs/ml> and FSH (200 ng/ml> for 2 days. The cells were cultured for an additional 6-9 days with medium changes at 3-day intervals and the media harvested for further study. Although our cultures from 21-day-old rats routinely contain greater than 90% Sertoli cells, it is more difficult to achieve this degree of purity with cultures from older rats. The cultures from 35 and 45-day-old rats were treated on day 3 with 20 mM Tris-HCI, pH 7.4 to remove residual germ cells (Galdieri et al., 1981). Sertoli cell specific parameters appear to be unaffected by this treatment (Galdieri et al., 1981; Wagle et al., 1986). Assay of mitogenic activity

The growth assay was performed exactly as described by Holmes et al. (19861, unless otherwise specified. Subconfluent A431 cells were plated (lo-20 x lo3 cells) in DME with 5% FBS on 30 x 10 mm Petri dishes. After 24 h, the medium was replaced with 1 ml of DME and 0.5-1.0 ml of Sertoli cell conditioned medium.

Control dishes received DME only with the same concentrations of hormones used in the Sertoli cell cultures. The cells were collected by trypsinization and counted in a Coulter counter. All samples were assayed in triplicate. EGF binding assay

Subconfluent monolayer cultures of A431 cells were fixed in 10% formalin as described by Todaro et al. (1980). “‘1-EGF was obtained from New England Nuclear and binding assays were performed in DME supplemented with 1 mg/ml bovine serum albumin, 20 mM Hepes (pH 7.4) and 0.2 ng “‘1-EGF for 90 min at 37°C. Nonspecific binding was measured in the presence of 500 ng EGF. The standard curve ranged from 0.2 to 12.8 ng EGF. The samples were measured in triplicate. Transferrin assay

Transferrin was measured by radioimmunoassay (Holmes et al., 1982). The samples were assayed in triplicate. Column chromatography of SCCM SCCM was concen(a) Size exclusion HPLC.

trated 5-lo-fold using an Amicon YM-2 membrane with a molecular weight exclusion limit of 1000 Da. The medium was chromatographed on a TSK SW2000 column (7.5 mm X 60 cm; Beckman Instruments, Houston, TX, U.S.A.) in DME buffered with 25 mM Hepes, pH 7.2. 1 ml volumes of concentrated SCCM were fractionated with a flow rate of 0.5 ml/min and 1 ml fractions collected. The high performance liquid chromatography (HPLC) fractions were immediately passed through a 0.2 Frn filter for sterilization and added to the A431 growth assay at 33% concentration. To provide sufficient sample for analysis, three replicate HPLC column runs were pooled for each treatment (control, heated, trypsin treated). Fractions 24-34 which contained the activity previously described by Holmes et al. (1986) as SCSGF were subjected to heat (100°C water bath, 10 min) or trypsin (500 pg/ml for 3 h, 37°C) and the reaction stopped with the addition of 2 mg/ml soybean trypsin inhibitor. Control DME column fractions were treated identically (100°C; trypsin treated).

4

(b) Column chromatography. SCCM was diluted 1: 5 with dH,O to lower the salt concentration of the medium to about 25 mM prior to application of 20 ml onto a 1 ml QAE-Sephadex column. 20 ml of the diluted SCCM was retained to measure mitogenic activity applied onto the column. The flow-through (unbound fraction) was collected following extensive washing of the column. The diluted SCCM and flow-through fractions (representing non-bound substances) were lyophilized and brought to their original starting volume with dH,O. The samples were tested in the A431 cell growth assay at 10, 2.5, and 50% concentration. For chromatography on a heparin agarose column (1.5 ml), the same protocol was used as described for QAE-Sephadex. 100 ml SCCM was lyophilized and reconstituted in 20 ml water (concentrated 5-fold). Hepes was added to 20 mM concentration and 15 ml loaded on concanavalin A (ConA)-Sepharose 4B (5 ml column) equilibrated with 5 X DME containing 20 mM Hepes. An additional 5 ml 5 X SCCM was retained to be used as a control in the growth assay. The column was washed with 10 ml of 5 X DME with 20 mM Hepes. The flowthrough and water wash were then diluted with water 5 times.

iment has been repeated a minimum of two times. Data were analyzed by Duncan’s multiple range test and one-way analysis of variance (ANOVA).

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Fig. 1. Sertoli cell secretion of SCSGF, EGF-like factor and transferrin during culture. Sertoh cells were isolated from ?l-day-old rats and cultured as described in Materials and Methods. SCCM was collected from days 3, 7 and 10 of culture and aliquoted for measurement of mitogenic activity, transferrin, and EGF-displacing activity. The concentration of EGF-like factor was measured using a radioreceptor assay using formalin-fixed A431 cells (as described in Materials and Methods); results are expressed as ng equivalents of EGF (ng eq EGF) since the factor was not authentic EGF. Mitogenic activity was measured using the A431 cell growth assay with 33% SCCM and the secretion of transferrin, a protein known to be secreted by rat Sertoli cells in vitro (Skinner and Griswold, 1980) was measured by radioimmunoassay. The data was normalized to reflect secretion of EGF and transferrin per day of Sertoli cell culture and per 10’ Sertoli cells. Each sample was tested in triplicate and results expressed as mean f SD.

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Results

The EGF displacing acticity and Sertoli cell secreted growth factor are different factors Fig. 1 shows that the pattern of secretion of the EGF-like factor (based upon the EGF radioreceptor assay) is not similar to the secretion of SCSGF or transferrin during various times in culture. Over a lo-day culture period, the secretion of EGF displacing activity by rat Sertoli cells decreased from about 12 ng equivalents of EGF to almost non-detectable levels. In contrast the mitogenic activity, as measured using the A431 cell growth assay of the same SCCM, remained elevated. Similarly, the secretion of transferrin (a Sertoli cell secretion) approximately paralleled the secretion of the mitogenic activity. Thus, the mitogenic activity and the EGF-like activity are different. Stimulation of A431 cell proliferation in ritro: comparison of Sertoli cell secreted growth factor with other mitogens We asked whether other classes of growth factors, in addition to SCSGF will stimulate the

SCCM

1

TABLE GROWTH

FACTOR

STIMULATION

OF A431 CELLS

A431 cells were plated in DME+S% FBS, followed by a 5-day incubation with the growth factors in DME+0.5% FBS. Fresh conditions were added on the third day. Cells were collected by trypsinization for counting and each sample was tested in triplicate. Fold stimulation corresponds to ratio of number of cells from the dishes treated with the growth factor to the number of cells in the control dishes. Growth

factor

Control Mouse EGF SCCM TGFa SCCM SCCM TGFfl SCCM SCCM

+ EGF + EGF + EGF + TGFB +TGFn + EGF

Concentration

Cell growth (fold stimulation)

_

1.0 kO.01 0.51 * 0.04 1.11+0.11 4.89 * 0.07 1.06 kO.05 2.27 + 0.16 4.84 + 0.05 0.47 f 0.05 4.39*0.19 1.63 f 0.07

2.5 ng/ml 0.01 ng/ml 25% 1 ng/ml 2S%, 2.5 ng/ml 25%. 0.01 ng/ml 2.5 ng/ml 25%, 2 ng/ml 25%, 2 ng/ml, 2.5 ng/ml

* p < 0.05; * * p < 0.001.

* ** ** ** * *

Fig. 2. Growth factors and A431 cell growth. A431 cells were incubated with TGF,,. IGF-1, IGF-2, FGF. EGF, prostaglandins E-l and E-2 (PGE-I and PGE-2) and increasing concentrations of SCCM for 3 days. The cells were then trypsinized and counted. The results are expressed as the mean + SD of triplicate samples. Data was analyzed by Duncan’s multiple range test and one-way analysis of variance.

growth of A431 cells. Table 1 and Fig. 2 summarize different growth studies carried out with various growth factors. Mouse EGF at a concentration of 0.01 ng/ml had no effect, whereas at a higher concentration (2.5 ng/ml) was inhibitory. SCCM (25%) increased growth by 4.9-fold. TGF, was ineffective at 2 ng/ml and did not potentiate the effect of SCCM when used in combination. The addition of EGF (2.5 ng/ml) partially inhibited the proliferative effect of 25% SCCM (Table 1). Similarly, 10 ng/ml bombesin, 10 ng/ml fibroblast growth factor (FGF), platelet derived growth factor (PDGF; tested at 2.5, 5.0 and 12.2

6

ng/ml), somatomedin C, and 74 pg/ml insulin did not stimulate the proliferation of A431 cells. The non-mitogenic Sertoli cell secretions transferrin and lactate also did not have any effect on the A431 cell growth (not shown). Pharmacologic concentrations of TGF,, TGFp, insulin-like growth factor 1 (IGF-11, IGF-2, acidic (a) FGF, bFGF, and prostaglandins E-l and E-2 (PGE-1 and PGE-2) did not stimulate A431 cell growth (Fig. 2). EGF, when tested at pharmacologic concentrations similarly had no effect. Since SCCM has also been reported to contain an interleukin-l-like factor (IL-I), IL-lfi was also tested over a concentration range of l-10 IU. Significantly, the addition of these factors did not stimulate A431 cell growth. It is clear from these studies that the Sertoli cell growth factor is distinct from TGF,, TGFp, aFGF, bFGF, PDGF, bombesin, IGF-1 (somatomedin C), IGF-2, IL-lo, prostaglandins and EGF.

TABLE

2

CHARACTERIZATION

OF SCSGF

ACTIVITY

IN SCCM

Sertoli cell conditioned medium (SCCM) was obtained from cells cultured from 18-21-day-old rats. A portion of the SCCM was frozen and thawed, another portion was treated with RNase A (100 Kg/ml, 2 h room temperature) and a third portion (10 ml) of SCCM was incubated at pH 3.0 with the addition of HCI for 1 h at room temperature, followed by the addition of 240 nl 2.5 N NaOH to neutralize the acidity. 5 ml SCCM was placed in a boiling water bath for 2 min and 10 min. For each treatment, portions of DME were treated identically as the SCCM for the controls. The SCCM or DME after treatment was added in 33% concentration to the A431 cells. Treatment

Growth promoting (A431 cell number

Control DME SCCM SCCM-freeze thaw SCCM-heated at 100 ’ C for 2 min SCCM-heated at 100 o C for 10 min

122’, 2 225k23 ** 233k 7 ** 220*

5 **

191+

8 **

RNAse A DME RNAse A SCCM

76+ 230+

2 8 **

Acid-treated Acid-treated

54+ 166k

7 6 *

Results

DME SCCM

were analysed

by ANOVA;

activity x lo-‘)

* I, I 0.05; * * p < 0.001.

D”E

CONTROL SCCM

CONDITIONS

FLOW-THRU

ADDED

Fig. 3. Chromatography of SCCM. SCCM was chromatographed on (A) ConA-Sepharose, (B) QAE-Sephadex, and (C) heparin-agarose as described in detail in Materials and Methods. To account for the dilution during the processing of SCCM, the flow-through was lyophilized and reconstituted in water to the original volume. The flow-through and the SCCM were analyzed in the A431 growth study. A431 cells were plated in DME + 5% FBS in 24-well plates (7500 cells/well). Next day the medium was replaced with DME containing different concentrations of SCCM or flow-through. The cells were cultured for 3 more days and then trypsinized and counted.

This series of experiments emphasizes the uniqueness of the Sertoli cell secreted growth factor relative to a variety of known growth factors. SCCM was the only factor to stimulate the proliferation of A431 cells (Table 1, Fig. 2). While the addition of EGF with SCCM partially inhibited the proliferative effect of SCCM, the mitogenie effect of SCCM is not necessarily mediated via the EGF-like activity. In other words, these activities may not act through the same receptor. Characterization

of SCSGF

activity in SCCM

In order to study the biochemical characteristics of the mitogenic activity in SCCM, we divided the medium into equal portions, treated the SCCM and then tested the medium for prohferative activity in the A431 cell growth assay (Table 2). The mitogenic activity in SCCM was stable to freeze-thaw, as well as to boiling for 2 or 10 min.

Similarly, the mitogenic activity was unaffected by RNAse A, or treatment with acid (pH 3.0 for I h). Since heparin-agarose has been used to bind and purify fibroblast growth factors (Gospodarowicz, 19841, we tested whether the mitogenic activity in SCCM will bind to heparin-agarose. Fig. 3C shows that the flow-through retained almost ail the growth promoting activity of the SCCM. Similarly, the mitogenic activity did not bind to ConA-Sepharose (Fig. 3A). About 50% of the activity was bound to QAE-Sephadex (Fig. 38). To further characterize the mitogen, we used partially purified SCSGF. SCSGF was purified by size exclusion chromatography on a TSK SW2000 column. A major peak of mitogenic activity eluted just prior to the lysozyme standard (14,300 MW) with a calculated apparent molecular weight of 14,000 (Fig. 4). Fractions 24-34 were treated with heat, or trypsin or stored at 4°C prior to use in the A431 cell growth assay. Trypsin treatment (Fig. 4, Iower panel) of these fractions resulted in the loss of the majority of the mitogenic activity; however, the factor was unaffected by heat (Fig. 4, upper panel). In this particular experiment,

Fig. 4. Stability of the purified SCSGF. The SCCM was concentrated 5-fold with Amicon YM-2 membrane prior to HPLC fractionation on a TSK 2000SW column. The column was eluted in DME and 1.0 ml fractions collected. Fractions 24-34, representing the peak of SCSGF activity were heated (lOWC, 10 min, panel A, A ), treated with trypsin (500 pg/ml, panel 6, VI for 3 h at 37°C and the reaction was stopped by addition of soybean trypsin inhibitor (2 mg/ml). Control DME column &tents were similarly treated with heat and trypsin. The untreated SCCM, heat and trypsin treated fractions of DME and SCCM were then added to the A431 growth assay. The results reflect different HPLC column runs and each treatment contains fractions pooled from three column runs. The fractions were added to the A431 cells at 33% concentration. The percent of control represents the percent of number of A431 cells in dishes treated with SCCM fractions as compared to the number of A431 cells in dishes treated with the corresponding DME fractions. Results are expressed as -fold stimulation above control and represent the mean + SD for triplicate determinations. Standard deviations for some points are not evident since they are smaller than the symbol size. This experiment was performed twice.

there was some broadening of the elution of the SCSGF (untreated) sampIe7due to pooling repetitive sample runs; however, the total activity (area under the curve) was similar for the SCCM (3.92 mm*> and heat-treated (4.3 mm’) fractions. We have compared the biochemical characteristics of SCSGF with other factors known to be secreted and expressed by testicular cell types. Although SCSGF shares characteristics in common with a number of growth factors, it has properties which distinguish it from others (Table

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No

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[4,5,73

Yes SC PT

None

No

No No

6.8

Yes

[6,x-101

None Yes SC PT

YfS

No

Yes No

Yes

Yes

Yes

No?

12.5 Dimer 25 kDa

TGF@

7.4 No

TGF,,

N.D Yes SC

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Yes

ND.

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None ?

Yes

YCS

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None Yes SC

Yes

No No Yes Yes Yes

4.8-5.8

No

Large aggregates No

15.7

SGF

None

Ml

None

NO Yes

Yes

Yes Yes

5-6

No

t7

IL-1 testis

None

None Yes

No

No

Y ‘0

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Yes 10.2

Yes

NO

No

14-17 Dimer

PDGF

15.5

bFGF

1

None Yes SC

No

NU

No No

X.3 Yes No

No

5.8

Yes

No

15.5 NO

7

aFGF

NO

IGF-2

7.5 cm

Yes

7 No

IGF-I

[22]

N.D. Yes

Yes No

No

Yes

45 N.D.

RTFGF

1231

N.D Yes SC

Yes

Yes

20-30

SC TCFfilike

a References: [I] Holmes et al., 1986; [2] Lamb et al., 1987; [3] Cohen, 1962; [4] Marq~lardt and Todaro, 1982; [5] Skinner et al., 1989; [h] Assoian et al., 1983; [7] Frotick et al., 1983; [8] Massaque, 1484; [9] Skinner and Moses, 1989; [lo] Frolick and DeLarco, 1987; [I l] Chatelain et al., 1987; [12] Closset et al., 1989; [13] Hansson et al., 1989; [14] Humbel, 1984; [I51 Smith et al., 1987; 1161Gospodarowicz, 1984; 1171Kahn et at., 1987; [18] Bellve and Feig, I984; [19] Feig et al., 1980; [ZO]Feig et al., 1983; [21] Braunhut et al., 1990; [22] Brown et al., 1982; [23] Benahmed et al., 1988; [24] Hannick and Donoghue, 1989; [25] Westmark et al., 1983. SC = Sertoli ceil: PT = peritubular cell; pl = isoelectric point.

t

Yes NU ? Yes SC

Yes No Yes Yes

Yes No

Yes No Yes No

FSH regulation of Sertoli ceit secretion Testicular secretion during sexual maturation

Inactivated by reducing~agents Carbohydrate Trypsin sensitive Heparin binding Binds to anion exchange resin Binds to cation exchange resin Effect on A431 cell growth Expressed or secreted by testicular cell types

Yes

Acid stable PI 4.6

Yes

Ye5

Heat stable

N.D.

6.4 No

14 ‘>

M~~iecularweight (kDa) Subunits

Yes

EGF

SCSGF

Characteristics

COMPARISON OF GROWTH FACTOR CHARACTERISTICS

TABLE 3

P

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Days

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Fig. 5. Effect of SCCM on A431 cell growth in presence of different concentrations of FBS. A431 cells were plated in 5% FBS-DME for 24 h. The cells were incubated with either 50% SCCM or DME and O%, OS%, 2%, and 5% FBS with medium changes on days 2, 3, and 6. Cells were collected by trypsinization and counted in a Coulter counter. Standard deviations were too small to be seen on a log scale.

Time course of A431 cell growth: effect of SCCM and FBS

The temporal effect of repeated application of SCCM on the growth of A431 cells was studied (Fig. 5). Sertoli cells were isolated from 19-21day-old rats and cultured for 3 days in DME and the media collected and stored at 4°C. A431 cells were plated in 5% FBS-DME for 24 h (20 X 10” cells/30 mm dish). The cells were then incubated with 50% SCCM and DME with (a> no serum; (b) 0.5% FBS; (c) 2% FBS; (d) 5% FBS or medium a, b, c, and d in the absence of SCCM and the media changed at 2-day intervals with duplicate culture dishes harvested and counted at each time point (Fig. 5). In the absence of serum by day 4 of culture, there was a 3.7-fold stimulation of growth of A431 cells by SCCM with little further proliferation by day 6. However, in the presence of 0.5% FBS and SCCM, the A431 cells were maximally stimulated to grow with a 7.2-fold stimulation by day 6. The addition of 2% or 5% FBS increased the rate of proliferation in the absence of SCCM to nearly the maximal rate observed with SCCM. However, most significantly, even in the presence of 2% or 5% FBS, there was a 2.3-fold increase in A431 cell number in the SCCM treated cultures as compared with DME with serum treated cells. Thus, the mitogenie effect of SCCM could be observed even in the presence of 5% FBS.

Recently, the identification of growth factors of testicular origin has been an active area of research. Bellve and co-workers (Feig et al., 1980, 1983; Bellve and Feig, 1984; Braunhut et al., 1990) have partially characterized an FGF-like growth factor from the seminiferous epithelium of the testis. This activity is present in seminiferous tubule homogenates from both prepubertal (mouse and calf) and adult (mouse, rat and guinea pig> and the concentration of growth promoting activity is greater in homogenates from the prepubertal tubules. The SCSGF has distinctly different characteristics when compared with SGF. Brown et al. (1982) have shown the presence of a growth factor in rete testis fluid from the ram testis. Based upon its biochemical characteristics, this activity is distinct from the growth factor described by Feig et al. (1983). However, homogenates of ram testis showed evidence of an SGF-like activity (Brown et al., 1982). More recently, Sertoli cells have been reported to secrete TGF, (Skinner et al., 1989) and TGF,like proteins (Benahmed et al., 1988; Skinner and Moses, 19891, as well as a somatomedin-/ insulin-like growth factor (Hall et al., 1983; Chatelain et al., 1987; Smith et al., 1987). Smith et al. (1989) have shown that rat testis, cultured Sertoli cells and peritubular cell extracts contain a basic FGF-like factor which stimulates the expression of c-fos in cultured Sertoli cells. In view of the wide variety of growth factors which have been reported to be secreted by rat Sertoli cells, it becomes imperative that the uniqueness of the SCSGF be described. In the present report, we have concentrated on the functional evidence of a unique growth factor using a model cell line, A431, which is either non-responsive or exhibits growth inhibition when exposed to other factors reported to be secreted by rat Sertoli cells. Although the initial observation by Holmes et al. (1986) suggested that the SCSGF was similar to EGF/TGF,, Fig. 1 clearly demonstrates the disparity between the secretion of the mitogen and the secretion of EGF-displacing activity. Based upon the findings of Skinner et al. (1989) the EGF-displacing activity is likely to be TGF,.

Our data demonstrate the presence of at least two factors, one mitogenic in the A431 cell growth assay and one which displaces EGF binding. Importantly, even though a number of growth factors are secreted by Sertoli cells, the A431 cell growth assay is specific for SCSGF, since these cells do not respond to other growth factors. In order to classify SCSGF into a group or subset of mitogens, we screened a variety of reagents for their ability to stimulate the proliferation of A431 cells. Since there were no published reports of any known mitogens for A431 cells, except for a relatively uncharacterized factor from bovine kidney (Halper and Moses, 19871, it was important to test whether other growth factors or Sertoli cell secretions were stimulatory. SCCM contained the only growth stimulatory activity and it did not potentiate the action of any of the reagents tested. Other known Sertoli cell secreted products tested, such as transferrin (Skinner and Griswold, 1980; Holmes et al., 1984; Skinner et al., 19841, and lactate (Jutte et al., 1982) did not stimulate A431 cell proliferation. EGF has previously been demonstrated to inhibit the growth of A431 cells (Barnes, 1982; Kamata et al., 1986). Similarly, TGFp has been reported to be growth inhibitory for the soft agar growth of A431 cells (Marquardt and Todaro, 1982). In contrast, Masuda et al. (1988) reported that although A431 cells secreted a heparin-binding growth factor and a TGFp factor, their growth was not affected by these factors or bFGF, insulin, human transferrin, or bovine serum albumin alone or in combination. The lack of inhibition may reflect a difference in the culture conditions, since in our studies, as well as those of Masuda et al. (1988), the A431 cells were not cultured on soft agar. bFGF does not stimulate A431 cell growth (Fig. 2; Halper and Moses, 1987; Masuda et al., 1988). Since A431 cells lack PDGF receptors (Westermark et al., 1983) it is not surprising that PDGF did not stimulate cell proliferation (Fig. 2). Based on preliminary characterization of SCSGF it may be similar to the apparently novel transforming growth factor isolated from bovine kidney by Halper and Moses (1987), which also stimulated A431 cell growth.

After comparison of the characteristics of the SCSGF and other growth factors (Tables 1 and 3; Fig. 2), it is concluded that SCSGF is not EGF, TGF,, TGF,, IGF-1, IGF-2, IL-lp, aFGF, bFGF, PDGF, or SGF, although most of these factors are known to be either secreted or expressed by testicular cell types. SCSGF is heat- and acid-stable, does not bind to heparin, is not a glycoprotein (does not bind to ConA-Sepharose), is sensitive to reducing agents with an approximate molecular weight of 14,000 (Massaque, 1984; Froelick and DeLarco, 1987). Some of our findings conflict with the earlier observations of Holmes et al. (1986) who reported that the mitogenic activity in SCCM was heat-sensitive. The reason for this discrepancy is unclear. Since a flocculent precipitate forms after boiling the SCCM, at higher protein concentration some of the mitogenic activity may have been trapped in the precipitate in the studies of Holmes et al. (1986). Buch et al. (1988) observed that the human SCSGF was heat-stable and trypsin-stable, which may reflect a species difference in the SCSGF amino acid sequence. Our results have been highly consistent and reproducible. Although the apparent molecular weight (14,000) is higher than previous estimates, the chromatography conditions (column length, elution buffer), which can have dramatic effects on the elution of various proteins, were different. Since no single method yields an absolute determination of molecular weight and because knowledge of the sedimentation coefficient of SCSGF would be required for a more precise estimate, the apparent molecular weight described differs slightly from previous reports (Holmes et al., 1986; Buch et al., 1988). The exact molecular weight will eventually be determined following amino acid analysis of the purified factor. In conclusion, the Sertoli cell secreted growth factor is a potent mitogen, capable of stimulating the growth of cells even in the presence of 5% FBS. The transforming growth factor isolated from bovine kidney by Halper and Moses (1987) and the growth factor in SCCM were the only mitogens reported which stimulate A431 cells. The SCSGF may regulate germ cell proliferation or stem cell renewal. Further studies on this

11

growth factor will provide an important key to the understanding of the regulation of spermatogenesis.

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