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Biology of Reproduction 63, 1006-1013 (2000)
© 2000 Society for the Study of Reproduction, Inc.

ARTICLES

Insulin-Like Growth Factor-II/Cation-Independent Mannose 6-Phosphate Receptor Mediates Paracrine Interactions During Spermatogonial Development1

James K. Tsuruta2,,a,b, E.M. Eddyd, and Deborah A. O'Briena,b,c

a The Laboratories for Reproductive Biology, b Departments of Pediatrics and c Cell Biology & Anatomy, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599 d Laboratory of Reproductive and Developmental Toxicology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The insulin-like growth factor-II/cation-independent mannose 6-phosphate (IGF-II/M6P) receptor transduces signals after binding IGF-II or M6P-bearing growth factors. We hypothesized that this receptor relays paracrine signals between Sertoli cells and spermatogonia in the basal compartment of the seminiferous epithelium. For these studies spermatogonia were isolated from 8-day-old mice with purity >95% and viability >85% after overnight culture. The IGF-II/M6P receptors were present on the surface of spermatogonia, as detected by indirect immunofluorescence. We determined that both IGF-II and M6P-glycoproteins in Sertoli cell conditioned medium (SCM) modulate gene expression in isolated spermatogonia. The IGF-II produced dose-dependent increases in both rRNA and c-fos mRNA. These effects were mediated specifically by IGF-II/M6P receptors, as shown by studies using IGF-II analogues that are specific agonists for either IGF-I or IGF-II receptors. The SCM treatment also induced dose-dependent increases in rRNA levels, and M6P competition showed that this response required interaction with IGF-II/M6P receptors. The M6P-glycoproteins isolated from SCM by IGF-II/M6P receptor affinity chromatography increased spermatogonial rRNA levels at much lower concentrations than required by SCM treatment, providing further evidence for the paracrine activity of Sertoli M6P-glycoproteins. These results demonstrate that Sertoli cells secrete paracrine factors that modulate spermatogonial gene expression after interacting with cell-surface IGF-II/M6P receptors.

gametogenesis, gene regulation, growth factors, IGF receptor, polypeptide receptors, Sertoli cells, signal transduction, spermatogenesis, testes


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The insulin-like growth factor-II/cation-independent mannose 6-phosphate (IGF-II/M6P) receptor is multifunctional [1]. This receptor, along with the smaller cation-dependent M6P receptor, is responsible for the intracellular targeting of proteins to the lysosomes. In addition, the IGF-II/M6P receptor cycles to the plasma membrane where it binds at least two distinct classes of ligands: M6P-bearing glycoproteins (M6P-glycoproteins) and IGF-II. The M6P-glycoproteins interact with two binding sites in the extracellular domain via their M6P moieties, while IGF-II interacts with a single distinct binding site. Recent reports indicate that the IGF-II/M6P receptor also contains a specific binding site for retinoic acid [2].

The M6P-glycoproteins include growth factors such as proliferin [3–5], leukemia inhibitory factor [6], the precursor forms of transforming growth factor beta (TGF-ß) [7], as well as precursors of lysosomal hydrolases such as the cathepsins (B, D, and L) and ß-hexosaminidase. Interestingly, both cathepsin B and cathepsin L are associated with metastatic or transformed cells [8, 9]. Prosaposin [10] or sulfated glycoprotein-1 (SGP-1) [11], another lysosomal enzyme precursor reported to contain M6P, is also a neurotrophic factor [12–14]. Insulin-like growth factor-II is a potent mitogen and has been shown to be involved in the terminal differentiation of myoblasts by regulating the expression of myogenin [15, 16].

Two signaling mechanisms have been proposed for the IGF-II/M6P receptor. In basolateral kidney membranes binding of IGF-II or M6P-containing growth factors increased the activity of phospholipase C, elevating inositol tris-phosphate and diacylglycerol [17–19]. Binding of IGF-II to the IGF-II/M6P receptor in primed-competent BALB;clc 3T3 cells activated a calcium channel by a guanine nucleotide-binding protein-dependent mechanism, allowing the influx of extracellular calcium [20–22]. A specific 14-amino acid sequence in the cytoplasmic domain of the IGF-II/M6P receptor was identified as the location of the G{alpha}i interaction and activation [23, 24]. Although similar coupling of this receptor to G-proteins was not detected in mouse L-cells [25], key aspects of this IGF-II/M6P receptor-mediated pathway have been confirmed in transfected COS cells [26].

Insulin-like growth factor-II/M6P receptors are abundant in the seminiferous epithelium. In vitro studies indicate that Sertoli cells express high levels of the IGF-II/M6P receptor, predominantly on intracellular membranes. In contrast, spermatogenic cells express a significant proportion of their IGF-II/M6P receptors on the cell surface [27]. During spermatogenesis, IGF-II/M6P receptors are most abundant in spermatogonia and early spermatocytes, as shown by immunohistochemistry [28].

Sertoli cells secrete multiple ligands for the IGF-II/M6P receptor, including approximately 10 M6P-glycoproteins identified by two-dimensional electrophoresis [29]. These cells also express IGF-II mRNA. Both IGF-II and the M6P-glycoproteins secreted by Sertoli cells induce rapid and transient increases in c-fos mRNA and rRNA levels in pachytene spermatocytes and round spermatids [30]. The relative abundance of the IGF-II/M6P receptor during the early phases of germ cell differentiation prompted our investigation of spermatogonia as a signaling target for IGF-II or M6P-glycoproteins secreted by Sertoli cells.

The IGF-II/M6P receptors are present on the surface of isolated spermatogonia. Furthermore, we show that IGF-II or M6P-glycoproteins contained in Sertoli cell-conditioned medium induce increases in both c-fos mRNA and rRNA levels in spermatogonia isolated from 8-day-old mice. The specificity of these responses was demonstrated with receptor-specific analogues of IGF-II and with competition for the binding of M6P-glycoproteins by the M6P monosaccharide. Our studies show that one mechanism for the paracrine control of gene expression during the proliferative phase of spermatogenesis is the binding of IGF-II or M6P-glycoproteins to IGF-II/M6P receptors on developing spermatogonia.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals

Testes were isolated from CD-1 mice obtained from Charles River Laboratories (Raleigh, NC) or from the National Institute of Environmental Health Sciences breeding colony. Procedures involving animals were approved in advance by the appropriate Institutional Animal Care and Use Committee.

Cell Isolation, Culture, and Treatment

Sertoli cells were cultured from single cell suspensions prepared from 17-day-old CD-1 mice as described previously [27]. Sertoli cell-conditioned medium (SCM) was collected on Days 5 and 8 of culture and was stored at -20°C after addition of 20% glycerol, 1 mM PMSF, and 1 mM EDTA. The SCM was concentrated and dialyzed against PBS in either a stirred cell equipped with a YM-10 membrane (10 000 molecular weight cutoff) or a tangential flow concentrator (8000 molecular weight cutoff). Final concentration was performed in a centrifugal concentrator (10 000 molecular weight cutoff, Centricon Plus-80). All concentration devices were from Amicon (Beverly, MA). Protein concentrations were determined by the bicinchoninic acid protein assay (Pierce, Rockford, IL). The M6P-glycoproteins were purified from SCM by IGF-II/M6P receptor affinity chromatography as described previously [29].

Preparations enriched for spermatogonia were isolated by enzymatic dissociation of testes from 8-day-old CD-1 mice as reported previously [31, 32]. Single cell suspensions prepared by this method consisted mainly of Sertoli cells and type A through type B spermatogonia. These cells were cultured overnight in MEM/c (Eagle's minimum essential medium with Earle's salts [Life Technologies, Gaithersburg, MD], 1 mM sodium pyruvate, 6 mM sodium L-lactate, 15 mM Hepes [pH 7.2], and antibiotics) supplemented with 5% fetal bovine serum (ES cell grade, Life Technologies). Cells were plated at a density of 5 x 106 cells/ml in 100 mm Primaria tissue culture dishes (Falcon; Becton Dickinson, Bedford, MA).

Spermatogonia were collected from the layer of adherent Sertoli cells with gentle pipetting. Spermatogonia were then cultured in serum-free MEM/c at ~5 x 106 cells/ml for 1 h prior to treatment with IGFs (Becton Dickinson) or SCM proteins. As indicated in the figure legends, for some experiments the overnight culture was performed with serum-free MEM/c on 100-mm tissue culture dishes (Falcon; Becton Dickinson) coated with a recombinant fibronectin peptide (ProNectin F+; Sigma, St. Louis, MO). Quadruplicate samples in multiwell dishes were treated in each experiment. These values were normalized to values obtained in control samples and then plotted as relative fold-increases. Data are presented as means ± SEM. Statistical analyses (ANOVA and the Tukey-Kramer multiple comparison test) were performed with the InStat software package (Graphpad, San Diego, CA).

Indirect Immunofluorescence

Isolated spermatogonia were immunostained with a monospecific antibody raised against purified IGF-II/M6P receptor isolated from rat liver [33]. Antibody binding to live cells was assayed as described previously [34] under conditions that block endocytosis of antibody complexes on the cell surface. Spermatogonia were collected after overnight culture and incubated for 30 min at 4°C with primary antibody diluted 1:100 in PBS, 1% normal goat serum, 10 mM NaN3 (PBS-NGS). The cells were washed three times with PBS-NGS and then incubated for 30 min at 4°C with affinity-purified fluorescein-conjugated goat anti-rabbit immunoglobulin (1:50 in PBS-NGS; Kirkegaard & Perry Laboratories, Gaithersburg, MD). After three additional washes, fluorescent labeling was observed and photographed using Ektachrome P800/1600 color reversal film at ASA 1600.

Isolation of RNA and Northern Analyses of rRNA Levels

Total RNA was isolated from cells by the acid-phenol guanidine isothiocyanate method [35] with a modified denaturing solution (5 M guanidine isothiocyanate, 0.5% [w/v] sarcosine, 25 mM citrate pH 7) as described previously [30]. The RNA samples were electrophoresed through denaturing agarose gels and were transferred to MagnaGraph nylon membranes (MSI, Westborough, MA) by the downward capillary method [36]. Random-primed cDNA probes or riboprobes were hybridized to the membranes in a 50% formamide solution [30] at 42°C or in 7% SDS, 5 mM EDTA, 0.5 M phosphate, pH 7.2 at 65°C [37]. Washed membranes were exposed to storage phosphor screens and band intensities were analyzed on a Molecular Dynamics PhosphoImager (Sunnyville, CA) with ImageQuant software. Experimental values were normalized to control values and relative fold-increases are presented as the mean of quadruplicate samples ± SEM.

Complementary DNA Probes and Plasmid Constructs

The plasmids pFos-ENDO and pFos-STD contain inserts corresponding to base pairs (bp) 236–630 of murine c-fos as described previously [30]. The pFos-STD differs from pFos-ENDO by containing a 20-bp insertion at the PstI site and is used during quantitative reverse transcriptase polymerase chain reaction (RT-PCR) to assess c-fos mRNA levels. The plasmid p18S contains a cDNA probe for 18S rRNA as described previously [30]. Plasmid templates for transcribing riboprobes for 28S rRNA and glyceraldehyde phosphate dehydrogenase (GAPDH) were purchased from Ambion (Austin, TX).

Quantitative RT-PCR to Determine c-fos mRNA Levels

Quantitative RT-PCR with a homologous competitor cRNA was performed to analyze c-fos mRNA levels as described previously [30]. Briefly, homologous competitor RNA (STD-RNA) was transcribed in vitro from pFos-STD. Fixed amounts of sample RNA were mixed with a fixed amount of STD-RNA and were reverse transcribed with a c-fos-specific primer. The PCR was performed with this cDNA using c-fos primers. These products were then radiolabeled by performing an additional heminested PCR in the presence of [{alpha}-32P]dCTP. The PCR products were resolved on denaturing acrylamide gels and exposed to storage phosphor screens. Band intensities were analyzed as described [30] and changes in c-fos mRNA levels, relative to control values, are presented as the mean of quadruplicate samples ± SEM.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Isolation of Spermatogonia from 8-Day-Old CD-1 Mice

By 8 days of postnatal development, the seminiferous tubules contain only Sertoli cells and type A through type B spermatogonia [38]. Sertoli cells present in single cell suspensions from juvenile testes adhered to Primaria tissue culture plates after 16 h of culture in the presence of fetal bovine serum or to ProNectin F+-coated plates in the absence of serum. Spermatogonia were selectively harvested by gentle pipetting, leaving somatic cells attached to the culture dishes. Spermatogonial preparations were contaminated with less than 5% somatic cells, and trypan blue exclusion demonstrated routine cell viabilites in excess of 85%. The presence or absence of serum did not appear to affect either viabilities or purities of spermatogonia. The number of spermatogonia recovered was ~25 to 35% of the total number of cells plated at the beginning of the culture period, comparable to the ratio of spermatogonia to Sertoli cells in the testes of 8-day-old mice [32].

IGF-II/M6P Receptors Are Present on the Surface of Spermatogonia

To determine if IGF-II/M6P receptors are present on the surface of spermatogonia, live germ cells (94% purity) were immunostained with an antibody that specifically recognizes the IGF-II/M6P receptor [33]. Indirect immunofluorescence assays were conducted at 4°C in the presence of NaN3 to prevent endocytosis of surface antibody complexes. Spermatogonia of various sizes showed bright surface labeling and were clumped together in large aggregates, indicative of antibody crosslinking of surface receptors (Fig. 1). In control samples incubated without primary antiserum, spermatogonia were unstained and did not aggregate (not shown).



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  		FIG. 1. Insulin-like growth factor-II/M6P receptors are present on the surface of live spermatogonia. Spermatogonia isolated from 8-day-old mice with 94% purity were immunostained with a specific antibody to the IGF-II/M6P receptor. Staining was detected with indirect immunofluorescence under conditions that inhibit endocytosis of surface antibody complexes (4°C, in the presence of NaN3). Bright surface staining of spermatogonia was observed. Spermatogonia aggregated, an indication of surface antibody crosslinking, only in the presence of primary antiserum

IGF-II Induces Rapid, Transient Increases in rRNA Levels

Spermatogonia isolated after overnight culture were cultured for 1 h in serum-free medium. Subsequent addition of 25 ng/ml IGF-II induced a rapid, transient increase in 18S rRNA levels (Fig. 2). Ribosomal RNA levels peaked after 60–75 min and approached baseline levels after an additional hour of treatment. The change in rRNA levels after 60 min of treatment was highly significant (P < 0.001). When spermatogonia were treated with 500 µg/ml SCM or 1 µg/ml of the calcium ionophore A23187, responses with similar kinetics and magnitude were observed (not shown). The time course for the increase in rRNA levels in spermatogonia is similar to responses observed previously when isolated pachytene spermatocytes and round spermatids were treated with a protein kinase C activator (1 µM TPA) [30].



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  		FIG. 2. Ribosomal RNA levels increased rapidly and transiently after treating spermatogonia with 25 ng/ml IGF-II. Spermatogonia were cultured for the indicated time periods in the presence of IGF-II and were then harvested for the preparation of RNA. Northern analysis was used to determine relative levels of rRNA per 100 000 spermatogonia. Values plotted represent fold-change ± SEM, after being normalized to control values. Cells were treated in quadruplicate, a representative experiment is shown. Statistical differences from control values were assessed with the Tukey-Kramer multiple comparison test: **P < 0.01; ***P < 0.001

IGF-II Stimulates Increases in Spermatogonial rRNA Levels That Are Dose Dependent and Specific

The IGF-binding protein (IGFBP) family consists of at least seven proteins that can bind IGF-II. The IGFBPs serve to transport IGFs, extend their half-lives and can modulate their biological activities [39]. The des(1–6)-IGF-II is an IGF-II analogue that has reduced affinity for IGFBPs. If IGFBPs were modulating the effect of IGF-II on spermatogonia, we predicted that treatment with des(1–6)-IGF-II would cause a response with a different magnitude. Both IGF-II and des-IGF-II stimulated similar increases in rRNA levels after 1 h of treatment, confirming that IGFBPs are not attenuating the response of spermatogonia during our treatments (not shown).

At high concentrations, IGF-II can bind to the IGF-I receptor. Binding to the IGF-I receptor is unlikely to occur at the IGF-II concentrations used in this study. However, to confirm that the effects observed for IGF-II were mediated by its cognate receptor, we treated spermatogonia with [Leu27]-IGF-II. [Leu27]-IGF-II is a class I analogue of IGF-II with a tyrosine to leucine mutation at position 27 [40]. Because this analogue shows 80–220-fold less affinity for the IGF-I receptor, while retaining wild-type affinity for the IGF-II receptor, any effects observed after treatment with [Leu27]-IGF-II can be attributed to binding to the IGF-II/M6P receptor [40]. Figure 3 demonstrates that [Leu27]-IGF-II induced a saturable, dose-dependent increase in spermatogonial rRNA levels after 1 h of treatment.



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  		FIG. 3. Insulin-like growth factor-II increases rRNA levels by interacting with the IGF-II receptor, not by interacting with the IGF-1 receptor. The IGF-II-receptor-specific analogue [Leu27]-IGF-II (Leu, filled circles) was able to increase rRNA levels in a dose-dependent manner (*P < 0.05; ***P < 0.001). In contrast, the IGF-I-receptor-specific analogue [Arg54,55]-IGF-II (Arg, open squares) did not alter rRNA levels (no statistical difference from control values). Neither analogue affected mRNA levels for GAPDH (open diamonds). Spermatogonia were treated with the indicated concentrations of IGF-II analogues. Northern analyses were used to determine relative levels of rRNA or GAPDH mRNA levels. Data from a representative experiment are plotted to represent fold-change ± SEM, after being normalized to control values for quadruplicate treatments

[Arg54,55]-IGF-II is a class II mutant of IGF-II. This analog does not bind to the IGF-II/M6P receptor but retains its affinity for the IGF-I receptor [40]. When spermatogonia were treated with this IGF-I receptor agonist, no change was observed in rRNA levels after 1 h of treatment (Fig. 3). Thus, activating the IGF-I receptor does not increase rRNA in spermatogonia. Taken together, these results demonstrate that IGF-II specifically acts through the IGF-II receptor to increase rRNA levels in spermatogonia.

Figure 3 also shows that neither the IGF-II receptor agonist [Leu27]-IGF-II nor the IGF-I receptor agonist [Arg54,55]-IGF-II caused any significant change in GAPDH mRNA levels. This control demonstrates that neither of these treatments caused a global change in transcription.

IGF-II Induces Specific Increases in c-fos mRNA Levels

C-fos mRNA levels increased in a saturable, dose-dependent manner when spermatogonia were treated with IGF-II (Fig. 4). In contrast, when spermatogonia were treated with the IGF-I receptor agonist [Arg54,55]-IGF-II, c-fos mRNA levels were unaffected. Thus, increases in c-fos mRNA in response to IGF-II were mediated specifically by the IGF-II/M6P receptor rather than by the IGF-I receptor. Our previous studies showed that IGF-II also stimulates a dose-dependent increase in c-fos mRNA levels in spermatogenic cells, predominantly pachytene spermatocytes and spermatids, isolated from adult mouse testes [30].



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  		FIG. 4. Insulin-like growth factor-II binding to the IGF-II receptor increased c-fos mRNA levels in a dose-dependent manner (closed circles). In contrast, the IGF-I-receptor-specific analogue [Arg54,55]-IGF-II (Arg, open squares) did not alter c-fos mRNA levels. The effects of IGF-II were statistically different than the effects of [Arg54,55]-IGF-II (*P < 0.05; **P < 0.01). Spermatogonia were treated with the indicated concentrations of growth factors for 1 h. Quantitative RT-PCR was used to determine relative fold-change in c-fos mRNA levels. Values plotted represent fold-change ± SEM, after being normalized to control values for quadruplicate treatments

Sertoli Cell-Conditioned Medium Contains Factors That Increase Spermatogonial rRNA Levels by Binding to the IGF-II/M6P Receptor

Because Sertoli cells secrete multiple M6P-glycoproteins [29] that stimulate increases in the steady-state levels of 18S rRNA and c-fos mRNA in isolated pachytene spermatocytes and round spermatids [30], we examined the effects of SCM on spermatogonial rRNA levels. When spermatogonia were treated with SCM, rRNA levels increased in a dose-dependent and saturable manner (Fig. 5). The SCM increased rRNA levels in 8 of 10 independent spermatogonia preparations. One SCM preparation failed to elicit a response with two independent spermatogonia preparations, suggesting some variability in the M6P-glycoprotein content of individual SCM preparations. In seven M6P competition experiments, the M6P monosaccharide was able to abolish (n = 6) or reduce (n = 1) the effect of SCM. This confirmed that the paracrine factors(s) in SCM responsible for the observed changes in rRNA contained M6P moieties that were essential for their activity.



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  		FIG. 5. The increase in rRNA levels caused by SCM is mediated by binding to the IGF-II/M6P receptor. Spermatogonia were treated for 1 h with the indicated concentrations of SCM ± 2.5 mM M6P. The SCM (filled circles) caused dose-dependent increases in rRNA levels, whereas there was no response in the presence of M6P (open squares); these effects were statistically different by the Tukey-Kramer multiple comparison test (**P < 0.01). Ribosomal RNA levels were assessed by Northern analyses. A representative experiment is depicted, and each point represents fold-change ± SEM after being normalized to control values for quadruplicate treatments

Mannose 6-Phosphate Receptor Ligands Purified from SCM Increase rRNA Levels at Doses Much Lower than Those Required for SCM

Figure 6 illustrates the effect of treating spermatogonia with M6P-glycoproteins purified from SCM. The 18S rRNA levels were elevated by treatment with 10–25 µg/ml Sertoli M6P-glycoproteins, concentrations that were substantially lower than the amounts of SCM that elicited similar increases in spermatogonial rRNA levels (Fig. 5). This response was reduced, but not eliminated, when 2.5 mM M6P was added to the medium.



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  		FIG. 6. Affinity-purified ligands for the IGF-II/M6P receptor (receptor ligands, filled circles) caused dose-dependent increases in rRNA levels at concentrations much lower than those required for SCM (*P < 0.05). In the presence of 2.5 mM M6P (open squares), relative rRNA levels were consistently lower and were not statistically different than control values. Spermatogonia were treated with affinity-purified receptor ligands ± 2.5 mM M6P for 1 h. Northern analyses determined relative rRNA levels. A representative experiment is depicted, and each point represents fold-change ± SEM, after being normalized to control values for quadruplicate treatments


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Paracrine interactions between Sertoli cells and germ cells have long been postulated to regulate meiosis and spermiogenesis, the phases of spermatogenesis that occur within the adluminal compartment of the seminiferous epithelium. The presence of the blood-testis barrier requires that signals destined for these developing spermatogenic cells originate from or be transduced through the Sertoli cell. Spermatogonia that reside on the basal side of the blood-testis barrier have greater access to signaling molecules in the interstitial fluid. However, it is likely that Sertoli cells play an important role in regulating spermatogonial proliferation and entry into meiosis.

Several studies indicate that Sertoli cells exert paracrine effects on spermatogonia in the basal compartment of the seminiferous epithelium. Sertoli cells secrete a variety of factors that modulate cell division at different stages of spermatogonial differentiation (reviewed by Jegou [41]). In addition, alterations in both FSH and testosterone levels have effects on spermatogonia. In testicular organ cultures from immature rats, FSH was essential for the progression of differentiating spermatogonia to the pachytene stage of meiotic prophase [41]. Follicle-stimulating hormone also specifically increased the number of type B spermatogonia in adult animals in both rat [42] and primate [43] hormone-replacement models. Testosterone withdrawal in the rat substantially reduced the number of type A and type B spermatogonia, although abolishing the conversion of round to elongating spermatids was the most prominent effect of this treatment [44]. These effects on spermatogonia are indirect and are likely to be mediated via FSH and androgen receptors in Sertoli cells [45].

Our previous studies showed that IGF-II/M6P receptor-mediated signal transduction is one specific mechanism for relaying paracrine signals from Sertoli cells to meiotic and postmeiotic germ cells. Treatment of pachytene spermatocytes and round spermatids with IGF-II or M6P-glycoproteins present in Sertoli cell-conditioned medium stimulated increases in the steady-state levels of both rRNA and c-fos mRNA [30]. Our current study shows that these two classes of ligands for the IGF-II/M6P receptor relay paracrine signals from Sertoli cells to spermatogonia.

Ribosomal RNA levels in spermatogonia were upregulated with kinetics similar to those observed in pachytene spermatocytes and round spermatids, reaching maximal levels after 1 h of treatment with IGF-II or M6P-glycoproteins and returning to baseline levels within 1 h. The c-fos mRNA levels were also increased after 1 h of treatment with IGF-II. C-fos and c-jun are components of the AP-1 transcription factor that controls the transcription of a number of genes. It is likely that multiple genes are upregulated after treating spermatogonia with ligands for the IGF-II/M6P receptor. It is also possible that the transient increases in rRNA caused by IGF-II or M6P-glycoproteins resulted in a transient increase in protein synthesis. Although the ultimate consequences of challenging spermatogonia with ligands for the IGF-II/M6P receptor are yet to be elucidated, it is clear that these ligands change gene expression patterns in spermatogonia as well as in meiotic and postmeiotic germ cells.

Our studies show that IGF-II acts on spermatogonia through its cognate receptor. The effects observed with IGF-II were elicited with doses far below the Kd of IGF-II binding to the IGF-I receptor. In addition, IGF-I was not able to elicit the same effects as IGF-II (not shown). However, the use of specific analogues of IGF-II provided more conclusive evidence that IGF-II signaled specifically through the IGF-II receptor. Both IGF-II and [Leu27]-IGF-II consistently acted to increase c-fos and rRNA levels while IGF-I and [Arg54,55]-IGF-II consistently failed to elicit this response. The binding activities of these two IGF-II analogues have been extensively characterized [40]. [Leu27]-IGF-II, a specific IGF-II receptor agonist, retains its affinity for the IGF-II receptor but has a greatly reduced affinity for the IGF-I receptor. Conversely, [Arg54,55]-IGF-II is a specific IGF-I receptor agonist that retains its ability to bind to and activate the IGF-I receptor but has a greatly diminished affinity for the IGF-II receptor. The data obtained from treating spermatogonia with these IGF-II analogues demonstrated that IGF-II altered gene expression specifically via the IGF-II/M6P receptor. Furthermore, IGF-II is a biologically relevant ligand for spermatogonia because we have shown that Sertoli cells contain the mRNA for IGF-II [30] and that SCM contains the IGF-II protein, as determined with a specific RIA (unpublished results).

Sertoli cell-conditioned medium is a complex mixture of proteins containing a number of different growth and differentiation factors in addition to IGF-II. Because of this complexity, it was essential to this study to demonstrate that changes in gene expression were mediated via binding to the M6P-binding sites of the IGF-II/M6P receptor. In six of seven experiments, competition with M6P was able to eliminate the observed response, confirming that the paracrine factor(s) responsible for these changes in gene expression required M6P moieties for their activity. This confirms that the response was not due to IGF-II in SCM preparations. Insulin-like growth factor-II has a molecular weight below the cutoff values of the ultrafiltration membranes used to concentrate our SCM preparations and does not contain M6P. However, in one experiment, the response was only reduced by the M6P monosaccharide, suggesting some variability in SCM preparations. Our M6P competition experiments clearly show that SCM contains paracrine factors for spermatogonia that bear essential M6P sugars.

Based on our results from treating spermatogonia with SCM, we predicted that M6P-glycoproteins isolated from SCM by affinity chromatography would elicit changes at substantially lower protein doses. As expected, the specific activity of affinity-purified M6P-glycoproteins was much higher than that of SCM, showing that we had enriched factors responsible for increasing rRNA levels. At the highest concentrations of Sertoli M6P-glycoproteins used (20–25 µg/ml), M6P reduced but did not eliminate this response. These results confirm that M6P-glycoproteins in SCM mediate paracrine interactions via the IGF-II/M6P receptor and suggest that the M6P-glycoprotein fraction may contain factors with additional IGF-II/M6P receptor-independent activities.

The specific M6P-glycoproteins that activate the IGF-II/M6P receptor have yet to be identified. However, a number of M6P-glycoproteins associated with proliferating cells are present in the testis. The major excreted protein (MEP) of transformed 3T3 fibroblasts [46–48] and cyclic protein-2 that is expressed in a highly stage-dependent manner by Sertoli cells [49] are known to be procathepsin-L, an M6P-bearing acid hydrolase. Procathepsin L has been shown to have paracrine signaling activity when complexed with the tissue inhibitor of metalloproteinase-1 by increasing steroidogenesis in Leydig cells [50]. The pro-TGFßs are also M6P-glycoproteins [51], and we have shown that at least two pro-TGFßs are secreted by mouse Sertoli cells [52]. It is not clear whether the pro-TGFßs activate the IGF-II/M6P receptor, but this receptor plays an important role in the formation of mature TGFßs by facilitating the proteolysis needed to create mature TGFß [53, 54]. Proliferin interacts with the IGF-II/M6P receptor [55] and was originally described in proliferating cells as the mitogen-regulated protein [56–58]. This M6P-glycoprotein is an angiogenic hormone that induces endothelial cell chemotaxis via a mitogen-activated protein kinase-dependent pathway [59]. Although there may be a specific proliferin receptor [60], the signaling activity of proliferin requires binding to the IGF-II/M6P receptor [61]. Prosaposin is an M6P-glycoprotein that was characterized in the testis as sulfated glycoprotein-1 [11]. Peptides derived from prosaposin/SGP-1 are essential glycosphingolipid activator proteins and some prosaposin peptides have been shown to be neurotrophic factors [12–14, 62–64]. Thus, Sertoli cell M6P-glycoproteins may encompass paracrine factors that act directly through the IGF-II/M6P receptor as well as others, like proliferin and the pro-TGFßs, whose paracrine activities require interaction with the IGF-II/M6P receptor.

In conclusion, we have presented evidence that IGF-II and M6P-glycoproteins secreted by Sertoli cells can act as regulators of gene expression in spermatogonia. Identifying paracrine factors and their receptors is an important step toward understanding the relationship between Sertoli cells and developing spermatogonial cells. Paracrine factors are likely to be produced by Sertoli cells in a stage-specific manner and are likely to be targeted to particular spermatogonia with precise kinetics. Further studies are necessary to determine if this is the case with the IGF-II/M6P receptor system and to determine the ultimate physiological effects of signaling through this system. With the growing importance of spermatogonial transplantation in studies of spermatogenesis, it is invaluable to understand the mechanisms that control spermatogonial gene expression in vivo. This knowledge will extend our understanding of the biology of spermatogonia and may reveal rational strategies for manipulating these cells in vitro.


   ACKNOWLEDGMENTS
 
James dedicates this manuscript to the memory of his father, Kinya Tsuruta. The authors would like to thank K. Michelle Cobb and Pat Magyar for technical assistance and Dr. Karen Swanchara for valuable contributions. The IGF-II analogues were a kind gift of Dr. K. Sakano of the Basic Technology Research Laboratory, Daiichi Pharmaceutical Co., Tokyo and the IGF-II/M6P receptor antibody C3-2 was a kind gift of Dr. Carolyn D. Scott of the Dept. of Endocrinology, Royal Prince Alfred Hospital, Camperdown, Australia.


   FOOTNOTES
 
First decision: 16 March 2000.

1 This research was supported by NICHD/NIH through HD26485 (D.A.O.) and cooperative agreement U54 HD35041 as part of the Specialized Cooperative Centers Program in Reproductive Research. Funding was also provided by NIH CA16086 (UNC Lineberger Comprehensive Cancer Center), and additional salary support for J.K.T. was provided by the Andrew W. Mellon Foundation. Back

2 Correspondence: James K. Tsuruta, Laboratories for Reproductive Biology, Pediatrics Dept. CB#7500, 377 Medical Sciences Research Building, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7500. FAX: 919 966 2204; jtsuruta{at}excite.com Back

Accepted: May 11, 2000.

Received: February 22, 2000.


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 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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