Regulation of ₂-Macroglobulin Expression in Rat Sertoli Cells and Hepatocytes by Germ Cells In Vitro¹

^a The Population Council, Center for Biomedical Research, New York, New York 10021 ^b Institute of Pharmacology and Pharmacognosy, University of Rome "La Sapienza", 00185 Rome, Italy

	ABSTRACT

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Germ cells isolated from rat testes by trypsinization have been shown to yield unwanted artifacts in biological assays, since conditioned media derived from these germ cells (germ cell-conditioned media [GCCM]) can modulate Sertoli cell secretory function because of the presence of residual trypsin. To determine whether germ cells themselves can modulate Sertoli cell function, we isolated germ cells from tubules by a mechanical procedure and assessed the effect of these cells on Sertoli cell ₂-macroglobulin (₂-MG) steady-state mRNA level. It was found that germ cells indeed could stimulate Sertoli cell ₂-MG expression. This effect is probably mediated by a soluble factor(s) released from germ cells, since GCCM fractionated by HPLC contained multiple fractions that can stimulate Sertoli cell ₂-MG expression dose-dependently. These results illustrate that germ cells play a role in regulating testicular ₂-MG expression. Since Sertoli cells synthesize and secrete many of the serum proteins behind the blood-testis barrier that are also produced by hepatocytes, we sought to ascertain whether germ cells can affect hepatic ₂-MG expression. When germ cells were cocultured with hepatocytes isolated from adult rats, the hepatocyte ₂-MG steady-state mRNA level was shown to be stimulated by germ cells dose-dependently. Using different pools of fractions derived from GCCM after their fractionation by a preparative anion-exchange HPLC column, GCCM was found to contain a factor(s) that stimulated hepatocyte ₂-MG expression dose-dependently. More importantly, the fractions that stimulated hepatocyte ₂-MG expression had a retention time different from that of the factor(s) that affected Sertoli cell ₂-MG expression. These data illustrate that germ cells secrete multiple biological factors capable of regulating ₂-MG expression in the testis and the liver. In summary, this study reveals a possible physiological link between the testis and the liver in that germ cells may release a factor(s) capable of modulating ₂-MG expression in both organs.

	INTRODUCTION

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₂-Macroglobulin (₂-MG) is a 720-kDa tetrameric glycoprotein consisting of four identical monomeric subunits of 180 kDa each. Two of these monomers are disulfide-bonded, and the two dimers are in turn assembled noncovalently to yield a tetramer [1, 2]. ₂-MG is capable of inhibiting a wide range of proteases and is the only known naturally occurring inhibitor of aspartic proteases [3]. The mechanism of action of ₂-MG is different from that of other known protease inhibitors, which exert their action via site-directed inhibition: ₂-MG acts by sterically hindering and trapping proteases in such a way that the resultant complexes are rapidly cleared from the systemic circulation by cells of the reticuloendothelial system in the liver [4, 5]. Another unusual feature of ₂-MG is its extraordinary ability to bind a wide range of physiologically important molecules: e.g., cytokines [6, 7]; endopeptidases [5]; mitogens such as concanavalin A, phytohemagglutin, and lipopolysaccharide; histones; and ions such as zinc and nickel [8]. ₂-MG is also one of the major proteins in serum. Although it is predominantly produced in the liver, its synthesis in other organs—including the ovary [9], testis [10], placenta [11], uterus [12], fibroblasts, macrophages, and astrocytes [13]—has been reported.

In the rat, ₂-MG belongs to a group of serum proteins designated acute-phase proteins, whose concentration increases by several orders of magnitude in response to inflammatory conditions [14, 15]. During acute inflammation, the serum ₂-MG level increases by up to 100 times the basal level and is modulated by cytokines and glucocorticoids [12, 15]. ₂-MG is also known to stimulate the growth of a variety of cells in vitro [16, 17]. This activity may be explained in part by the findings that ₂-MG binds a variety of growth factors, which include transforming growth factor ß (TGFß) [18], basic fibroblast growth factor [19], and platelet-derived growth factor [20], as well as cytokines such as interleukins (IL)-1 and -6 [7, 21].

In the rat testis, ₂-MG is secreted by Sertoli cells (for review, see [22]). The synthesis of ₂-MG by Sertoli cells, unlike hepatocytes, does not increase in response to inflammation in vivo nor is it regulated by IL-6 in vitro, suggesting that ₂-MG is not an acute-phase protein in the testis [15, 23, 24]. The production of ₂-MG by Sertoli cells is likely to be regulated by a mechanism(s) distinctly different from that of the liver [24]. The physiological function of ₂-MG in the testis is not clear. On the basis of immunohistochemical studies, it was postulated that ₂-MG secreted by Sertoli cells in the seminiferous epithelium may inactivate proteases that are released by degenerating germ cells; it also limits the action of growth factors and proteases that are involved in tissue remodeling, thereby limiting tissue damage in the epithelium throughout spermatogenesis [25]. Therefore, we thought it pertinent to examine whether germ cells can indeed secrete a factor(s) that modulates Sertoli cell ₂-MG expression. In view of the findings that the ₂-MG regulation pathway in the testis appears to be different from that of the liver, we also sought to determine, using hepatocytes cultured in vitro, whether such a factor(s) regulates ₂-MG expression in the liver.

	MATERIALS AND METHODS

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Biochemicals

Serum-free culture medium was prepared by mixing Ham's F–12 nutrient mixture (F12) and Dulbecco's modified Eagle medium (DMEM) in a ratio of 1:1 (v:v) supplemented with sodium bicarbonate (1.2 g/L), Hepes (15 mM), and gentamycin (20 mg/L). Collagenase (types I and VIII), collagenase-dispase, trypsin, hyaluronidase, bacitracin, bovine insulin, epidermal growth factor, human transferrin, prolactin, glucagon, dexamethasone, EDTA, sodium lactate, sodium pyruvate, soybean trypsin inhibitor, Hanks' Ca²⁺- and Mg²⁺-free balanced salt solution (BSS), boric acid, formamide, 3-[N-morpholino] propanesulfonic acid (MOPS), sodium acetate, dextran sulfate, salmon sperm DNA, and tRNA were obtained from Sigma Chemical Company (St. Louis, MO). Glycine, Tris, SDS, ammonium persulfate, N,N'-methylene bis-acrylamide, N,N,N',N'-tetramethylethylenediamine (TEMED), 2-mercaptoethanol, N,N'-diallyltartardiamide (DATD), and low molecular-weight standards were from Bio-Rad (Richmond, CA). Silver nitrate and citric acid were from Aldrich Chemical Co. (Milwaukee, WI). Collagen solution (Vitrogen 100; 3 mg/ml) was obtained from Collagen Corp. (Palo Alto, CA). Matrigel (basement membrane matrix) was obtained from Becton Dickinson (Bedford, MA). Methanol and formaldehyde (37%, v:v) were from Fisher Scientific Co. (Fair Lawn, NJ). Recombinant human IL-6 and DNA markers VI were obtained from Boehringer-Mannheim (Indianapolis, IN). Nitrocellulose paper (0.45-µm pore size) was obtained from Schleicher and Schuell, Inc. (Keene, NH). RNA-STAT 60 was obtained from Tel-Test "B", Inc. (Friendswood, TX). Avian myeloblastosis virus (AMV)-reverse transcriptase, RNase inhibitor, T4-polynucleotide kinase, and Taq-DNA polymerase were obtained from Promega (Madison, WI). [-³²P]ATP and [-³²P]deoxycytidine triphosphate (dCTP) were obtained from Amersham (Arlington Heights, IL). Glycerol was obtained from Life Technologies, Inc. (Gaithersburg, MD). Metofane (methoxyflurane) was obtained from Mallinckrodt Veterinary, Inc. (Mundelein, IL).

Animals

Sprague-Dawley rats at 20 days of age were used for the preparation of Sertoli cell-enriched cultures. Sprague-Dawley rats at 250–300 g BW were used for the preparation of germ cell and hepatocyte cultures. All Harlan Sprague-Dawley rats were purchased from Charles River (Kingston, MA) and were housed at the Rockefeller University Laboratory Animal Research Center with a 12L:12D cycle and had free access to standard rat chow and water. Twenty-day-old rats arrived with foster mothers. The studies described in this paper were approved by the Rockefeller University Institutional Animal Care and Use Committee with Protocol Numbers 94132, 95129, 95129-R, and 97101.

Preparation of Sertoli Cell Cultures

Sertoli cells were isolated from the testicles of 20-day-old Sprague-Dawley rats, and primary cultures were prepared essentially as previously described [26]. Briefly, decapsulated testes were subjected to three sequential enzymatic treatments of trypsin (1 mg/ml), collagenase (1 mg/ml), and hyaluronidase (1 mg/ml) for 30 min each at 37°C with gentle oscillation. The resulting Sertoli cells were plated on 100-mm Petri dishes previously coated with 50 µl/cm² Matrigel (diluted 1:9 with F12/DMEM) at 4.5 x 10⁶ cells per 9 ml of F12/DMEM supplemented with sodium bicarbonate (1.2 mg/ml), Hepes (15 mM), gentamycin (20 µg/ml), insulin (10 µg/ml), human transferrin (5 µg/ml), epidermal growth factor (2.5 ng/ml), and bacitracin (5 µg/ml). Cells were maintained in a humidified atmosphere of 95% air:5% CO₂ (v:v) at 35°C for 48 h. Thereafter, contaminating germ cells were lysed by a brief hypotonic treatment with 20 mM Tris, pH 7.4, at 22°C for 2.5 min [27]. Sertoli cells at 4.5 x 10⁶ cells per 9 ml F12/DMEM were then cultured for an additional 5 days before their use for any experiments, to eliminate the possible influence of germ cells on their basal steady-state ₂-MG level.

Isolation of Sertoli Cells from Adult Rat Testes

Sertoli cells were isolated from testes of 35- and 90-day-old rats as previously described [28]. Briefly, decapsulated testes were incubated with 0.1% collagenase (w:v), 0.2% hyaluronidase (w:v), 0.03% DNase (w:v), and 0.03% soybean trypsin inhibitor (w:v) at 35°C for 25 min at 80 oscillations/min. This digestive step was repeated three times. Clumps of Sertoli cells that were heavily contaminated with germ cells together with tubular fragments were removed after sedimentation at unit gravity. A six-step washing procedure by sedimentation under unit gravity was then used to separate the Sertoli cell clumps from the tubule fragments and germ cells [28]. The resulting Sertoli cells were plated on 100-mm Petri dishes at 4.5 x 10⁶ cells per 9 ml F12/DMEM. Cells were maintained in a humidified atmosphere of 95% air:5% CO₂ (v:v) at 35°C for 48 h, after which contaminating germ cells were lysed by a brief hypotonic treatment with 20 mM Tris, pH 7.4, at 22°C for 2.5 min [27]. Cells were then cultured for an additional 24 h before their use for RNA extraction. The purity of these cell preparations was about 85% when examined microscopically, whereas the immature Sertoli cell preparation had a purity higher than 90% [26].

Preparation of Germ Cell Cultures

Germ cells from 90-day-old rats were isolated from seminiferous tubules by a mechanical procedure [29]. Briefly, rats were killed by CO₂ asphyxiation, testes were immediately removed and decapsulated, and blood vessels were carefully removed. Tubules suspended in F12/DMEM were minced using scalpels and then centrifuged at 100 x g for 1 min. Supernatant was collected, and the loose pellet was washed to recover additional germ cells. Supernatants from several washes were pooled to increase the yield. The cell suspension was filtered successively—through miracloth (cat. no. 475855; Calbiochem, La Jolla, CA) to remove tissue debris, through 100-µm and 20-µm nylon meshes to eliminate cell clumps, and finally through glass wool to remove elongated spermatids and spermatozoa. Cells, largely spermatogonia, pachytene spermatocytes, and round spermatids [29], were suspended in F12/DMEM supplemented with sodium bicarbonate (1.2 mg/ml), Hepes (15 mM), gentamycin (20 µg/ml), 6 mM sodium DL-lactate, 2 mM sodium pyruvate, and bacitracin (10 µg/ml); counted; and plated in culture dishes. For coculture experiments, germ cells were used within 3 h after their isolation. To obtain GCCM, freshly isolated germ cells were cultured for 20 h at 35°C at 2.5 x 10⁶ cells/9 ml per 100-mm dish with a surface area of 78.5 cm², as previously described [29]. The cell viability was higher than 90% within 20 h after the initial isolation as judged by the erythrosine red dye exclusion test. Media (GCCM) were collected from dishes and centrifuged successively at 800 x g (30 min) and 5000 x g (1 h) to remove residual germ cells and were stored at -20°C until use. To obtain nonviable germ cells, freshly isolated germ cells were cultured as described above for 27–30 h at 35°C. The viability of these cells was shown to be lower than 5% by the erythrosine red dye exclusion test.

Sertoli-Germ Cell Cocultures

For coculture experiments, highly purified Sertoli cells were cultured for 5 days to allow the establishment of monolayers. Germ cells were plated at 13.5 x 10⁶ cells/9 ml onto the Sertoli cell monolayer at a germ cell:Sertoli cell ratio of 3:1 in 100-mm dishes and were cultured for an additional 6 days at 35°C in F12/DMEM. Replicate cultures were terminated at specified time points as noted in figure legends. Total RNA was extracted using RNA STAT–60 as previously described [24, 30].

Preparation of Hepatocyte Monolayer Cultures

Hepatocytes were isolated from adult Sprague-Dawley rat liver by a modification of an established procedure [31]. Rats were anesthetized with Metofane. A catheter was inserted into the portal vein in situ, and perfusion was carried out for 5 min with BSS at a flow rate of 20 ml/min using a peristaltic pump. After perfusion, the liver was immediately excised and placed onto a nylon mesh. Rats were killed by cervical dislocation under anesthesia. The liver was rinsed twice in BSS and sliced into 2- to 3-mm fragments, and hepatocytes were isolated by a two-step collagenase treatment (1 mg/ml). The cell suspension was filtered through a 100-µm nylon filter and washed by four consecutive centrifugations (2 min each at 290 x g). Isolated hepatocytes were plated in 100-mm Petri dishes precoated with collagen at 9 x 10⁶ cells/9 ml in F12/DMEM supplemented with sodium bicarbonate (1.2 mg/ml), Hepes (15 mM), gentamycin (20 µg/ml), insulin (10 µg/ml), glucagon (10 µg/ml), epidermal growth factor (50 ng/ml), prolactin (20 mU/ml), and bacitracin (5 µg/ml). Cells were maintained in a humidified atmosphere of 95% air:5% CO₂ (v:v) at 37°C for 24 h before their use in any experiments.

Hepatocyte-Germ Cell Cocultures

Freshly isolated germ cells were added onto hepatocytes (9 x 10⁶ cells/9 ml per 100-mm dish) using a germ cell:hepatocyte ratio of 1:1 and were cultured for an additional 24 h at 35°C in F12/DMEM. Replicate cultures were terminated at specified time points. Total RNA was extracted using RNA STAT–60. To distinguish whether the effect of germ cells on the hepatocyte ₂-MG steady-state mRNA level was a result of a soluble biological factor(s) or direct cell-cell contact, germ cells were plated at 9 x 10⁶/9 ml in F12/DMEM and maintained at 35°C for 30 h to obtain nonviable cells. These cells were scraped, centrifuged, and added to the hepatocyte cultures at a ratio of 1:1. The viability of the germ cells after 30 h of incubation at 35°C was lower than 2% as judged by the trypan blue dye exclusion test. Replicate cultures were terminated at specified time points. Total RNA was extracted using RNA STAT–60.

Fractionation of Proteins in GCCM

About 2 L of GCCM prepared as described above were concentrated and equilibrated against solvent A (20 mM Tris, pH 7.4, at 22°C) using a Millipore Minitan tangential unit equipped with eight Minitan plates (Millipore Corp., Bedford, MA; molecular weight cut-off at 10 kDa). The resulting sample was loaded onto a preparative anion-exchange HPLC column (HR 10/10, Mono-Q; i.d.: 10 x 100 mm; particle size: 10 µm; Pharmacia Biotech, Piscataway, NJ) at a flow rate of 4 ml/min as previously described [32]. Bound proteins were eluted using a linear gradient of 0–80% solvent B (20 mM Tris, pH 7.4, at 22°C, containing 600 mM NaCl) at a flow rate of 4 ml/min over a period of 45 min. Fractions (4 ml each) were collected, and the eluents were monitored by UV absorbance at 280 nm. An aliquot from selected fractions was then resolved by SDS-PAGE under reducing conditions onto 12.5% T (T is total acrylamide concentration [g/100 ml], acrylamide + methylene-bisacrylamide) SDS-polyacrylamide gels [33], and proteins were visualized by silver staining [34]. Different pools of fractions were prepared on the basis of the protein patterns on the silver-stained gels.

Bioassay of Germ Cell Factors That Modulate the Sertoli Cell or Hepatocyte ₂-MG Steady-State mRNA Level

Protein estimation for each pool of fractions from the Mono-Q run was performed by the Coomassie blue dye binding assay [35]. These samples were then added onto either the Sertoli cell or hepatocyte monolayer cultures to examine their effect on the steady-state ₂-MG mRNA level at specific time points. Each pool of samples was bioassayed in duplicate.

Partial Hepatectomy

Animals were anesthetized by Metofane (methoxyflurane). After a small abdominal incision, two-thirds of the liver was removed as described by Higgins and Anderson [36]. After surgery, the abdomen was closed by a suture. A sham operation was performed by making a midline incision and manipulating the liver lobes without resection before closing the abdomen. Animals were killed at 6 h, 18 h, 24 h, and 48 h after the operation. About 200 mg of liver tissue was removed and stored at -70°C until it was used for RNA extraction.

RNA Extraction and Reverse Transcription-Polymerase Chain Reaction (RT-PCR) Analysis

Total RNA was extracted from cells and tissues using RNA STAT–60 as previously described [24, 30]. Approximately 1 µg of total RNA from each cell or tissue preparation was reverse-transcribed to cDNAs using an AMV Reverse Transcriptase kit (Promega). About 1/5 of the RT product was then used as template for PCR to amplify the ₂-MG cDNA. ß-Actin was coamplified in these experiments and used as an internal control. RT-PCR was performed as detailed elsewhere [30]. The primers used for ₂-MG [37] and ß-actin [38] amplifications were as follows: 5'-AACCAATCTACATGGTGATGGTTC–3' (₂-MG, sense, nucleotides 158–181); 5'-TTTGGGATCCT-GAATGTACAGTAG–3' (₂-MG, antisense, nucleotides 562–585); 5'-TCACCGAGGCCCCTCTGAACCCTA–3' (ß-actin, sense, nucleotides 314–337); 5'-GGCAGTAATCTCCTTCTGCATCCT–3' (ß-actin, antisense, nucleotides 931–954). The cycling parameters for PCR were as follows: denaturation at 94°C for 1 min, annealing at 58°C for 2 min, and extension at 72°C for 3 min. A total number of 18–25 cycles were performed. The cycles were followed by a 15-min extension at 72°C. Under these conditions, the production of both ₂-MG and ß-actin was at the linear phase as demonstrated in preliminary experiments. PCR products were analyzed by resolving a 5- to 10-µl aliquot from the final reaction mixture onto 5% T polyacrylamide gels in 0.5-strength TBE running buffer (45 mM Tris-borate/1 mM EDTA, pH 8.0 at 22°C) and then were visualized with ethidium bromide. To increase the detection sensitivity, the sense probe was 5' end-labeled with [-³²P]ATP using T4 polynucleotide kinase, and about 1 x 10⁶ cpm was used for PCR. Autoradiography was performed as described [30]. X-ray films were densitometrically scanned at 600 nm, and results were normalized against ß-actin.

Northern Blot Analysis

For Northern blot analysis, total RNA between 10 and 30 µg was denatured in 6% formaldehyde/50% formamide in single-strength MOPS buffer (40 mM MOPS, 10 mM sodium acetate, 1 mM EDTA, pH 7.0, at 22°C) at 65°C for 15 min. RNA was then fractionated on a 1% agarose gel in 0.5-strength MOPS buffer and transferred onto a nylon membrane. RNA was cross-linked to nylon membranes by UV. Prehybridization was performed in 50% deionized formamide, 10% dextran sulfate, 1% SDS, 100 µg/ml sheared and denatured salmon sperm DNA, 0.5 µg/ml tRNA, and 1 M sodium chloride at 42°C for 3–5 h. Hybridization, using about 0.5–1 x 10⁶ cpm of an -³²P-labeled ₂-MG cDNA (428 base pairs [bp])/ml hybridization mix, was carried out at 42°C for 16–20 h. The blots were washed twice in double-strength SSC (0.3 sodium chloride, 30 mM sodium citrate) at room temperature for 5 min, twice in double-strength SSC/1% SDS at 65°C for 30 min, and twice in 0.1-strength SSC at room temperature for 30 min. The blots were exposed to x-ray film at -70°C.

Statistical Analysis

Statistical analyses to assess the effects of 1) GCCM or its HPLC fractions and 2) germ cells, on Sertoli cell or hepatocyte ₂-MG expression were performed by Student's t-test using the GB-STAT Statistical Analysis Software Package (Version 3.0) from Dynamic Microsystems, Inc. (Silver Spring, MD).

	RESULTS

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Modulation of Sertoli Cell Steady-State ₂-MG mRNA Level by Germ Cells

When Sertoli cells (4.5 x 10⁶ cells/9 ml per dish) were cocultured with germ cells (13.5 x 10⁶ cells/9 ml per dish), RT-PCR using primers specific for ₂-MG showed that the steady-state ₂-MG mRNA level in Sertoli cells increased steadily, peaked by Day 4 (Fig. 1, A and B), and declined thereafter. Similar results were obtained when these samples were examined by Northern blots (Fig. 2, A and B) using a 428-bp ₂-MG cDNA probe [24, 37]. However, when Sertoli cells were cultured under the same conditions for the specified time points as those shown in Figures 1 and 2, A and B, but in the absence of germ cells, no changes in the Sertoli cell ₂-MG expression were detected (Fig. 2, C and D), indicating that the increase in the Sertoli cell ₂-MG expression shown in Figures 1 and 2, A and B, was mediated by germ cells since germ cells were shown not to express nor secrete ₂-MG in vitro [10, 15, 24]. These results also illustrate a time-dependent stimulation of Sertoli cell ₂-MG expression by germ cells.

View larger version (57K): [in this window] [in a new window]   FIG. 1. A and B) Changes in Sertoli cell 2-MG steady-state mRNA level in vitro in Sertoli-germ cell cocultures. A) RT-PCR was used to analyze changes in Sertoli cell 2-MG mRNA level after coculture with germ cells. Sertoli cells were isolated from 20-day-old rats and cultured at 4.5 x 106 cells/9 ml per 100-mm dish in F12/DMEM as described in Materials and Methods. Germ cells isolated from adult rats were cocultured with Sertoli cells at a ratio of 3:1 in F12/DMEM. At 30 min, 1 h, 6 h, 24 h, 4 days (d), and 6 days, cultures were terminated, and total RNA was extracted for hot-nested RT-PCR coamplified with ß-actin. B) Three autoradiographs, such as the one shown in A, were densitometrically scanned at 600 nm. Data were normalized against ß-actin. Each time point represents the mean ± SD of 3 experiments. ns, Not significantly different from control at Time 0 by Student's t-test; *p < 0.05; **p < 0.01.

View larger version (49K): [in this window] [in a new window]   FIG. 2. A–D) Northern blot analysis to examine the effect of germ cells on the Sertoli cell 2-MG steady-state mRNA level. The experimental conditions were the same as described in Figure 1. A) Each lane contained about 10 µg of total RNA hybridized with an -32P-labeled 2-MG cDNA probe of 428 bp. B) Ethidium bromide staining of the same blot shown in A, indicating that similar amounts of RNA were used for each lane. To see if the changes in Sertoli cell 2-MG expression in Sertoli-germ cell cocultures shown above and in Figure 1 were indeed mediated by germ cells, Sertoli cells were cultured alone without germ cells under the same experimental conditions. C) Northern blot using about 10 µg of total RNA hybridized with an 2-MG cDNA probe. D) The same blot as shown in C but stained with ethidium bromide.

Fractionation of Proteins in GCCM by Anion Exchange HPLC

To further investigate whether GCCM indeed contains a biological factor(s) that can modulate Sertoli cell ₂-MG expression, GCCM was prepared from germ cells isolated from adult rat testes by a mechanical procedure [29] without using trypsin or collagenase, and was fractionated by a preparative anion-exchange HPLC column. A total of 10 distinctive protein peaks were resolved when the eluents were monitored by UV absorbance at 280 nm (Fig. 3A). When an aliquot from selected fractions was resolved by SDS-PAGE under reducing conditions, and the gels were silver-stained, different groups of proteins were identified (Fig. 3B). These proteins were divided into pools A through H (Fig. 3B) according to their apparent molecular masses and were used for subsequent studies.

View larger version (61K): [in this window] [in a new window]   FIG. 3. A and B) Fractionation of proteins in GCCM by preparative anion-exchange HPLC. A) GCCM (2 L) was concentrated, equilibrated, and fractionated on a preparative Mono Q column as described in Materials and Methods. A total of 10 major protein peaks were noted when the eluents were monitored by UV absorbance at 280 nm. B) An aliquot of 50 µl from selected fractions was withdrawn for SDS-PAGE, resolved onto 12.5% T polyacrylamide gels under reducing conditions, and silver-stained. Lane S, molecular weight markers (x 10-3) containing 0.2 µg protein each of phosphorylase b, 97 kDa; BSA, 68 kDa; ovalbumin, 45 kDa; carbonic anhydrase, 31 kDa; soybean trypsin inhibitor, 21.5 kDa; and lysozyme, 14.4 kDa. A total of 8 pools (A–H) were tested for their effect on the Sertoli cell 2-MG mRNA level. Each pool was dialyzed against double distilled water, lyophilized, and resuspended in F12/DMEM, and protein estimation was performed by Coomassie blue-dye binding assay [35]. Pool A (lanes 5–7), pool B (lanes 8–12), pool C (lanes 13–18), pool D (lanes 19–25), pool E (lanes 26–29), pool F (lanes 30–35), pool G (lanes 36–41), and pool H (lanes 42–50).

Regulation of Sertoli Cell ₂-MG Expression by Pools of GCCM Fractions

Pools of GCCM fractions (about 20 µg protein/dish) derived from the anion-exchange HPLC as shown in Figure 3, A and B, were tested for their effect on Sertoli cell ₂-MG expression by incubating Sertoli cells with different pools of proteins (Fig. 3B) for 6 h at 35°C in vitro. Using RT-PCR coamplified with ß-actin to assess the changes in Sertoli cell ₂-MG expression, pools A, B, C, and H were shown to increase the Sertoli cell ₂-MG steady-state mRNA level by about 80%, 60%, 50%, and 180%, respectively, when compared to control cultures without any GCCM proteins (Fig. 4, A and B). Pools D–G had no statistically significant effect on Sertoli cell ₂-MG steady-state mRNA level (Fig. 4, A and B). These results further support the observations shown in Figure 1, A and B, and that there may be more than one factor in GCCM that can modulate Sertoli cell ₂-MG expression. When increasing concentrations of either pool A or pool H at 25, 50, and 100 µg protein/9 ml/100-mm dish were tested for their effect on Sertoli cell ₂-MG expression by incubating these samples with Sertoli cells for 6 h at 35°C, both pools A and H induced a dose-dependent stimulation of Sertoli cell ₂-MG expression (Fig. 5, A and B), which is consistent with the results shown in Figure 4, A and B. Pool H was more potent than pool A in stimulating Sertoli cell ₂-MG expression (Fig. 5, A and B). Pool B showed a dose-dependent response similar to that of pool A, and pools D–G did not show any effects (data not shown).

View larger version (49K): [in this window] [in a new window]   FIG. 4. A and B) Effects of GCCM pools after preparative anion-exchange HPLC on the Sertoli cell 2-MG steady-state mRNA level in vitro. A) RT-PCR analysis of the Sertoli cell 2-MG steady-state mRNA level after Sertoli cells (4.5 x 106 cells/9 ml per 100-mm dish) were incubated with different pools of GCCM (about 20 µg protein/dish) for 6 h in vitro. CTRL, Sertoli cells cultured alone without the addition of any GCCM fractions. Pools A–H correspond to the same pools shown in Figure 3B. Pools A, B, C, and H induced a stimulation on Sertoli cell 2-MG expression. In this RT-PCR, 2-MG was coamplified with ß-actin to ensure that a similar amount of RNA from each sample was used for RT and PCR. B) Autoradiographs, such as the one shown in A, were densitometrically scanned at 600 nm. Data were normalized against ß-actin. Each data point represents the mean ± SD of four experiments. ns, Not significantly different from control at Time 0 by Student's t-test; *p < 0.05; **p < 0.01.

View larger version (49K): [in this window] [in a new window]   FIG. 5. A and B) Dose-dependent stimulation of Sertoli cell 2-MG steady-state mRNA level by active pools of GCCM after anion-exchange HPLC. A) RT-PCR analysis of Sertoli cell 2-MG mRNA: Sertoli cells (4.5 x 106 cells/9 ml per 100-mm dish) were incubated for 6 h with different concentrations (25, 50, and 100 µg total protein/dish) of pools A and H from the GCCM fractionated by Mono Q (see Fig. 3). CTRL, Sertoli cells cultured alone without the addition of GCCM. Pools A and H derived from lanes 5–7 and lanes 42–50, respectively, as shown in Fig. 3B. B) Autoradiographs, such as the one shown in A, were densitometrically scanned at 600 nm. Data were normalized against ß-actin. Each time point represents the mean ± SD of two experiments. *p < 0.05; **p < 0.01.

Developmental Regulation of Sertoli Cell ₂-MG Expression during Aging

During postnatal development in the testis, the ratio of germ cells:Sertoli cells increases drastically. Estimations by morphometric analysis using serial sections of adult rat testes indicated that a single Sertoli cell has direct contact with at least 30–50 developing germ cells [39, 40]. Since germ cells play a significant role in modulating Sertoli cell ₂-MG expression, we sought to determine whether Sertoli cells derived from rats at 20, 35, and 90 days of age have similar levels of ₂-MG mRNA and whether their expression is largely regulated by germ cells. Surprisingly, the steady-state ₂-MG mRNA level in Sertoli cells when cultured in the absence of germ cells was found to be drastically lower in mature rats than in immature rats (Fig. 6, A and B). When these results were normalized against ß-actin, there was almost a 100-fold reduction in ₂-MG expression in adult rats at 90 days of age compared to rats at 20 days of age (Fig. 6B). It is likely that a higher level of ₂-MG in immature Sertoli cells is needed because there is extensive tissue restructuring, such as organ growth and formation of the blood-testis barrier, in the developing testis. Moreover, germ cells, the positive modulator of ₂-MG as shown in the present study, are not present in sufficient quantity in immature rats to maintain a high level of ₂-MG expression. This alternative pathway may safeguard the seminiferous epithelium from tissue damage in immature rats during testicular maturation. It must be noted that the basal level of ₂-MG expression in the testis in adult rats was still higher than that of the liver and brain [24], which may be related to its function in limiting tissue damage during spermatogenesis due to the release of proteases from degenerating spermatids.

View larger version (42K): [in this window] [in a new window]   FIG. 6. A and B) Developmental regulation of Sertoli cell 2-MG steady-state mRNA level during aging. Sertoli cells were isolated from rats at 20, 35, and 90 days (D) of age as described under Materials and Methods. These cells were cultured at 4.5 x 106 cells/9 ml per 100-mm dish in F12/DMEM for 2 days before cultures were terminated for RNA extraction. A) RT-PCR analysis of 2-MG mRNA in Sertoli cells from the same rats. Germ cells were separated from Sertoli cells by successive sedimentation washing and hypotonic treatment. The Sertoli cells isolated from 35- and 90-day-old rats were about 80–85% pure while those from 20-day-old rats were more than 90% pure when examined microscopically. ß-actin was coamplified with 2-MG in this experiment. B) Autoradiographs, such as the one shown in A, were densitometrically scanned at 600 nm. Data were normalized against ß-actin. Each time point represents the mean ± SD of 3 experiments. **p < 0.01.

Regulation of Hepatocyte ₂-MG Expression by IL-6, Dexamethasone, and Germ Cells

Previous studies have demonstrated that the expression of ₂-MG in rat hepatocytes is regulated by dexamethasone and IL-6 (for reviews, see [5, 13, 41]). We investigated whether germ cells can also affect hepatocyte ₂-MG expression. To determine whether the hepatocytes to be used for the subsequent studies were responsive to hormonal treatment, dexamethasone at 10^-6 M and IL-6 at 2 and 10 ng/dish were tested for their effects on hepatocyte ₂-MG expression. Both dexamethasone and IL-6 were shown to stimulate hepatocyte ₂-MG expression (data not shown). When hepatocytes (9 x 10⁶ cells/9 ml/dish) were cocultured with germ cells using a hepatocyte:germ cell ratio of 1:1 for 0, 1, 2, 6, 12, and 24 h in vitro, it was noted that germ cells induced an observable effect on hepatocyte ₂-MG expression by as early as 2 h (Fig. 7, A and B). A drastic increase as high as 50- and 80-fold was noted after these cells were cocultured for 12 and 24 h, respectively (Fig. 7, A and B). However, a temporal decline in ₂-MG expression in these cocultures at 6 h versus 2 h was consistently noted (Fig. 7, A and B), and the immediate explanation for this observation is not known. To ascertain whether the stimulation of hepatocyte ₂-MG steady-state mRNA level by germ cells was the result of cell-cell contact or via soluble factor(s) released from germ cells, hepatocytes were cultured with nonviable germ cells with a hepatocyte:germ cell ratio of 1:1. After 2 h of coculture, an 8-fold increase in the hepatocyte ₂-MG level was noted when hepatocytes were cocultured with nonviable germ cells (Fig. 7, C and D), consistent with the results shown in Figure 7, A and B. By 6 h the effect had begun to diminish, and by 12 h no observable effect was noted (Fig. 7, C and D). While cell-cell contact between nonviable germ cells and hepatocytes can stimulate hepatocyte ₂-MG expression, these results seem to suggest that viable germ cells are required to sustain the continual stimulation via soluble factors. To confirm that the effects of germ cells on hepatocyte ₂-MG expression shown in Figure 7, A–D, indeed were mediated by germ cells instead of reflecting a time-dependent change in ₂-MG expression, hepatocytes were cultured under the same conditions as those shown in Figure 7, A–D, without the addition of any germ cells. No detectable change in ₂-MG expression was noted (Fig. 7, E and F), illustrating that germ cells can modulate hepatocyte ₂-MG expression.

View larger version (44K): [in this window] [in a new window]   FIG. 7. A–F) Changes in the 2-MG steady-state mRNA level in hepatocytes in the presence or absence of viable or nonviable germ cells in vitro. A) RT-PCR was used to assess the changes in steady-state 2-MG mRNA level in hepatocytes cocultured with viable germ cells. Hepatocytes were isolated from adult rats and cultured at 9 x 106/9 ml per 100-mm dish in F12/DMEM for 24 h. Germ cells isolated from adult rats were cocultured with hepatocytes at a cell ratio of 1:1 at 35°C. At 0, 1, 2, 6, 12, and 24 h, cultures were terminated and total RNA was extracted. ß-Actin was coamplified with 2-MG in this experiment. B) Autoradiographs such as the one shown in A were densitometrically scanned at 600 nm. Data were normalized against ß-actin. Each time point represents the mean ± SD of 3 experiments. C) RT-PCR analysis of steady-state 2-MG mRNA level in hepatocytes cocultured with nonviable germ cells. Freshly isolated hepatocytes were cultured at 9 x 106/9 ml per 100 mm-dish in F12/DMEM for 24 h at 37°C. Germ cells were isolated from adult rats and cultured at 9 x 106 cells/9 ml per dish in F12/DMEM for 27–30 h at 35°C. After 27 h, the viability of germ cells was lower than 5% when judged by erythrosine red dye exclusion test. These nonviable germ cells were cocultured with hepatocytes at a cell ratio of 1:1. At 0, 1, 2, 6, 12, and 24 h, cultures were terminated and total RNA was extracted for RT-PCR. ß-Actin was coamplified with 2-MG in this experiment. D) Autoradiographs, such as the one shown in C, were densitometrically scanned at 600 nm. Data were normalized against ß-actin. Each time point represents the mean ± SD of 3 experiments. E and F) Controls corresponding to A–D, which consisted of hepatocytes cultured alone without germ cells. E) RT-PCR results of 2-MG coamplified with ß-actin. F) Densitometrically scanned data from three separate experiments indicating that hepatocytes cultured alone did not show changes in 2-MG expression. ns, not significantly different from control at Time 0 by Student's t-test; *p < 0.05; **p < 0.01.

Regulation of Hepatocyte ₂-MG Expression by Pools of GCCM Fractions In Vitro

Different pools of GCCM (200 µg protein/dish), derived from an anion-exchange HPLC as shown in Figure 3B, were tested for their effect on hepatocyte ₂-MG expression after a 24-h incubation at 37°C (Fig. 8, A and B). Pools B–F were shown to stimulate hepatocyte ₂-MG expression. It was clear that the pools that regulated Sertoli cell ₂-MG expression were different from those that regulated hepatocyte ₂-MG expression (Fig. 8, A and B, vs. Fig. 4, A and B). To confirm the observations shown in Figure 8, A and B, we next tested different concentrations of pools B and E at 100, 200, and 400 µg protein/9 ml per dish to see if these selected pools could induce a dose-dependent effect on hepatocyte ₂-MG expression when incubated with primary cultures of hepatocytes for 24 h at 37°C. Both pools B and E showed a dose-dependent effect in stimulating hepatocyte ₂-MG expression (Fig. 9, A and B). Pools C, D, and F showed a similar dose-dependent effect whereas other fractions such as pools A, G, and H had no significant effect in stimulating hepatocyte ₂-MG expression (data not shown).

View larger version (50K): [in this window] [in a new window]   FIG. 8. A and B) Effect of different pools of GCCM, after fractionation of GCCM by Mono Q, on the hepatocyte 2-MG steady-state mRNA level in vitro. A) RT-PCR analysis of 2-MG mRNA in hepatocytes, in which cells were incubated for 24 h with different pools of GCCM (about 200 µg protein/dish) such as those shown in Figure 3B. CTRL, hepatocytes cultured alone without GCCM. B) Autoradiographs, such as the one shown in A, were densitometrically scanned at 600 nm. Data were normalized against ß-actin. Each data point represents the mean ± SD of two experiments. Pools C, D, and E were the most potent fractions that stimulated the hepatocyte 2-MG steady-state mRNA level. ns, Not significantly different from controls at Time 0 without GCCM proteins; *p < 0.05; **p < 0.01.

View larger version (47K): [in this window] [in a new window]   FIG. 9. A and B) Dose-dependent stimulation of hepatocyte 2-MG steady-state mRNA level by active pools of GCCM. A) RT-PCR analysis of 2-MG mRNA in hepatocytes: cells were incubated for 24 h with different concentrations (100, 200, 400 µg protein/dish) of active pools of GCCM (pools B and E). CTRL, hepatocytes cultured alone without the addition of any GCCM. Pools B (lanes 8–12) and E (lanes 26–29) were derived from the preparative Mono Q fractionation as shown in Figure 3B. ß-Actin was coamplified with 2-MG in this experiment. B) Autoradiographs, such as the one shown in A, were densitometrically scanned at 600 nm. Data were normalized against ß-actin. Each data point represents the mean ± SD of two experiments. ns, Not significantly different from control at Time 0 without GCCM proteins; *p < 0.05; **p < 0.01.

Developmental Regulation of ₂-MG in the Liver

We also sought to determine whether the expression of ₂-MG in the liver also showed an age-dependent regulation similar to the results shown for Sertoli cells (Fig. 6, A and B). When the level of ₂-MG steady-state mRNA in liver derived from rats at 20, 35, 60, and 90 days of age was examined (Fig. 10, A and B), an age-dependent reduction in ₂-MG expression in the liver was noted (Fig. 10, A and B). However, its magnitude of decline was not as drastic as that found in Sertoli cells (compare Fig. 10, A and B, with Fig. 6, A and B). At present, it is not known if such a difference is related to the presence of germ cells in the testis or is mediated by a factor(s) as yet undefined.

View larger version (58K): [in this window] [in a new window]   FIG. 10. A and B) Developmental regulation of liver 2-MG steady-state mRNA level during aging. A) RT-PCR analysis of the liver steady-state 2-MG mRNA level in rats at 20, 35, 60, 90 days of age. About 1 µg of total RNA was used for RT, and PCR was performed as described in Materials and Methods. B) Autoradiographs, such as the one shown in A, were densitometrically scanned and normalized against ß-actin. Each data point represents the mean ± SD of two experiments. ns, Not significantly different from rats at 20 days of age; *p < 0.05; **p < 0.01.

Involvement of ₂-MG in Liver Regeneration

In the rat, the liver has a remarkable ability to regenerate after partial hepatectomy. After removal of as much as 70% of the liver mass, the liver can be restored to its original size in about 3–4 wk, involving extensive tissue restructuring and remodeling [36]. Since ₂-MG is a major acute-phase protein in the liver, we investigated the possibility of a correlation between its expression and liver regeneration after partial hepatectomy. In the normal liver, the ₂-MG steady-state mRNA level was almost undetectable (Fig. 11, A and B). After partial hepatectomy, the ₂-MG steady-state mRNA level had begun to increase by 6 h post-operation, peaked at 18 h, and declined rapidly thereafter (Fig. 11, A and B). By 48 h, the ₂-MG mRNA level had returned to its basal level. The sham-operated rats, in comparison to controls, showed an increase in ₂-MG steady-state mRNA level by 6 h after the operation, but this increase was significantly lower than that in the hepatectomized rats by 6 h (Fig. 11, A and B), illustrating that the response observed in regenerating liver is not simply an acute-phase response.

View larger version (33K): [in this window] [in a new window]   FIG. 11. A and B) Changes in 2-MG steady-state mRNA level during liver regeneration. A) RT-PCR analysis of the 2-MG steady-state mRNA level in regenerating and sham-operated liver. After partial hepatectomy, animals were killed at 6, 18, 24, and 48 h, and about 0.1 g liver tissue was removed for RNA extraction. CTRL, normal liver. SHAM, liver from sham operated rats 6 h after surgery. RT-CR was performed as described in Materials and Methods. B) Autoradiographs, such as the one shown in A, were densitometrically scanned and normalized against ß-actin. Each data point represents the mean ± SD of two experiments. ns, Not significantly different from rats without receiving hepatectomy; *p < 0.05; **p < 0.01.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
In the seminiferous epithelium, germ cells associate closely with Sertoli cells via specialized intercellular junctions such as desmosome-like junctions, ectoplasmic specializations, tubulobulbar complexes, gap junctions, and cell surface receptors (for reviews, see [42–44]). Furthermore, it has been demonstrated that germ cells actively regulate Sertoli cell activities, which include protein secretion, number of hormone receptors, and the expression of selected genes (for reviews, see [42–44]). Numerous studies strongly suggest that germ cells release biological factors that modulate Sertoli cell secretory function [29, 45–47]. In a recent study from this laboratory, a factor isolated from GCCM that was able to affect Sertoli cell clusterin and testin secretion was subsequently shown to be the residual trypsin originally used in isolating germ cells from the seminiferous tubules [29]. Thus, it is likely that some of the earlier reports on the effects of germ cells on Sertoli cells may be the result of an artifact due to the presence of contaminating trypsin. To determine whether germ cells indeed modulate Sertoli cell ₂-MG expression, germ cells were isolated from tubules by a mechanical procedure without any enzymatic treatments. Interestingly, these non-enzymatically treated germ cells were still found to affect the Sertoli cell ₂-MG steady-state mRNA level in vitro. When Sertoli cells were cultured in monolayers at about 5 x 10⁴ cells/cm² in the absence of germ cells, which had been removed by a hypotonic treatment [27], it was found that the ₂-MG steady-state mRNA level in these Sertoli cells declined slightly with time in culture, as shown in Figure 2, C and D, compared to Figure 2, A and B; this substantiates our observation that germ cells can stimulate Sertoli cell ₂-MG expression. The stimulatory effect of germ cells on Sertoli cells is likely to be mediated via a soluble factor(s) since GCCM fractionated by HPLC contained fractions that could modulate Sertoli cell ₂-MG expression dose-dependently. Moreover, nonviable germ cells failed to sustain the enhanced Sertoli cell ₂-MG expression. These results provide relatively convincing evidence that germ cells cultured in vitro can indeed regulate Sertoli cell function.

When germ cells were cocultured with hepatocytes in vitro, the hepatocyte ₂-MG steady-state mRNA level was also stimulated by germ cells. Such a stimulation could be maintained for up to only 6 h when nonviable germ cells were used, suggesting that a soluble factor(s) released by viable germ cells is needed to maintain a sustained stimulation. When selected pools of GCCM fractions derived from an anion-exchange HPLC were used to screen their effect on hepatocyte ₂-MG expression, several pools that were not effective in stimulating Sertoli cell ₂-MG expression were able to stimulate hepatocyte ₂-MG expression. These results show that a factor(s) derived from germ cells that affects Sertoli cell ₂-MG expression may be a different entity from the one that affects hepatocytes. Even though the significance of these data may not be immediately known, these results do illustrate that a physiological link may exist between the liver and testis. It is also noteworthy to mention that the Sertoli cell in the testis has long been regarded as the hepatic counterpart of the systemic circulation because this cell type produces many hepatic proteins, such as androgen binding protein, ceruloplasmin, transferrin, ₂-MG, clusterin, and others (for reviews, see [22, 41, 44, 47]).

In the liver, IL-6 is the most potent inductor of ₂-MG, and its induction is associated with the phosphorylation of the acute-phase response factor, which is also known as STAT3 (signal transducers and activator of transcription–3) [48–50]. The ability of germ cells to stimulate ₂-MG mRNA expression in the hepatocyte and Sertoli cell cultures could be due to the activation of one or more of these transcription-activating factors, such as STAT-1, STAT-3, and STAT-5. Since, in the Sertoli cell, IL-6 is unable to induce its ₂-MG expression [24], it is possible that either IL-6 receptors are absent in Sertoli cells, the signaling components required to induce IL-6-dependent responses are absent in Sertoli cells, or an alternative signaling pathway is present in Sertoli cells. Thus Sertoli cell ₂-MG expression may be regulated by different STAT proteins instead of those modulating liver ₂-MG. The fact that GCCM fractions that stimulated Sertoli cell ₂-MG were different from those affecting hepatocyte ₂-MG suggests the presence of a different signaling pathway for the activation of ₂-MG gene expression in these two organs. The finding that germ cells stimulate ₂-MG expression in the liver suggests that they secrete factors belonging to the STAT protein family, or factors able to stimulate the transcription-activating proteins present in the liver cells. Since germ cells release several growth factor-like proteins and cytokines, such as nerve-growth factor [51, 52], TGFß [53], basic fibroblast growth factor [45], IL-1 [54], and interferon- and - [55], these proteins may be used to activate ₂-MG gene expression.

A specific RIA for ₂-MG showed that the concentration of ₂-MG in the testis during maturation increased with aging, whereas in other organs such as the liver, spleen, brain, and kidney, the ₂-MG level declined during aging [15, 41]. Surprisingly, the expression of ₂-MG in Sertoli cells isolated from immature rat testes revealed a drastic reduction in expression compared to that from adult rat testes, suggesting that the expression of ₂-MG by Sertoli cells per se without germ cells is likely to be regulated by the differentiation status of the cell. This postulate appears to be valid since the expression of ₂-MG in the aging liver displayed a similar trend, illustrating that the cellular differentiation status may be an important regulator of ₂-MG expression. The high level of ₂-MG mRNA in Sertoli cells from immature rats suggests that this protein may be used to protect the tissue from damage throughout testicular development, such as the formation of the blood-testis barrier. Its enhanced expression in adult rats is likely to be regulated by germ cells. This high level of ₂-MG expression in adult rats is possibly required to maintain the tissue integrity during spermatogenesis, which is accompanied by extensive tissue restructuring and remodeling (for review, see [56]). These results suggest that testicular ₂-MG expression needs to be maintained at a relatively high level throughout the life span of the rat, either by the cellular differentiation status or by germ cells. Furthermore, our data support the findings of the involvement of ₂-MG in liver regeneration [57, 58], suggesting that this acute-phase protein plays an important role in remodeling and restructuring of damaged tissue. Since ₂-MG is known to bind a wide range of growth factors and cytokines, it is possible that ₂-MG binds some of these factors during liver regeneration. It is suggested that ₂-MG plays a role in inactivating TGF1ß, a known inhibitor of cell proliferation in hepatocyte cultures [59] that appears to be involved in the mechanism that terminates liver regeneration [58]. TGF1ß was shown to increase in expression shortly after hepatectomy, to remain moderately elevated until 18–20 h, and then to rise, sharply peaking at 72 h [60]. The results of our study demonstrating that the expression of ₂-MG increased within a few hours after hepatectomy suggest its effect in modulating the action of TGF1ß and its association with the proliferation and differentiation of the liver mass [61, 62]. The rapid decline in ₂-MG expression after 18 h post-hepatectomy is likely to allow TGF1ß to exert its action when its expression is greatly enhanced, which occurs at 72 h [60]. In this way, ₂-MG may be involved in triggering the regenerative response in the liver.

	FOOTNOTES

 
¹ This work was supported in part by grants from the Noopolis Foundation, Rockefeller Foundation (PS9528, PS9601, PS9721), National Institutes of Health (HD–13541), and CONRAD Program (CICCR/CIG–96–05). L.B. was supported by a fellowship from the Sovena Foundation.

² Correspondence: C. Yan Cheng, The Population Council, 1230 York Avenue, New York, NY 10021. FAX: (212) 327–7678; yan{at}popcbr.rockefeller.edu

Accepted: February 25, 1998.

Received: September 8, 1997.

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