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a Institute of Anatomy, University Hospital, Hamburg-Eppendorf, 20246 Hamburg, Germany
b Institute for Hormone and Fertility Research, University of Hamburg, 22529 Hamburg, Germany
c Institute for Reproductive Medicine, University of Münster, 48129 Münster, Germany
ABSTRACT
The structure of the endozepine-like peptide (ELP) gene is closely related to the intracellular acyl-CoA binding protein (ACBP), but unlike the generalized distribution of the latter, it is restricted to the male germ cells of the testis. In the present study, a combination of nonradioactive in situ mRNA hybridization and immunohistochemistry was used to precisely determine the cellular expression patterns of ELP mRNA and protein in control and methoxyacetic acid (MAA)-treated rat testes. ELP transcripts are first detectable in late stages (step 6) of round spermatids, with transcription increasing through late-elongating steps. Translation of the ELP mRNA is delayed, with first immunohistochemical staining occurring in elongated spermatids at step 16, and protein accumulating through step 19. ELP immunoreactivity proves to be an excellent marker for late spermatid stages and highlights the presumably clonal recovery of spermatids following MAA treatment.
spermatid, spermatogenesis, testis
INTRODUCTION
The endozepine-like peptide (ELP) was identified from a differential cloning project in the mouse testis as a germ cell-specific cDNA clone, which was absent in the w/wv azoospermic mouse [1]. Subsequently, we also cloned the equivalent cDNA from testes of the rat [2] and bull (R. Bathgate and R. Ivell, unpublished results). The encoded ELP protein shares approximately 50% identity with the ubiquitous intracellular acyl-CoA binding protein (ACBP), also known as endozepine or diazepam-binding inhibitor, because of its ability to compete with diazepam for binding to the peripheral benzodiazepine receptor [3–6]. ELP retains the highly conserved acyl-CoA binding motif of ACBP, and in preliminary experiments (J. Knudsen, personal communication), a recombinant ELP is indeed able to bind mid- to long-chain acyl-CoA. It therefore appears likely that ELP shares some of the functions characterized for ACBP, such as transport of mid- to long-chain fatty acids to the mitochondrion for ß-oxidation, stimulation of steroidogenesis [7–9], as well as modulation of intracellular signaling mediated by acyl-CoA [10].
Earlier studies indicated that ACBP in testis was expressed almost exclusively in Sertoli and Leydig cells [11–13], although a more recent study also suggests possible low level ACBP expression in male germ cells [14]. The more or less exclusive distribution of ACBP in testicular somatic cells and ELP in male germ cells strongly suggests an important role for ELP in performing ACBP-like functions in maturing spermatozoa.
A preliminary study in the mouse [1] suggested that both ELP mRNA and protein were postmeiotically expressed in haploid germ cells, although a precise characterization of this expression was not made. The present study was carried out using improved techniques and materials in order to provide an accurate cellular and developmental characterization of ELP expression in rat testis, where the differentiation status of spermatogenesis can be reliably determined from tubule cross sections.
MATERIALS AND METHODS
Animals and Treatments
Testes were collected from adult Wistar rats (Charles River, Sulzfeld, Germany), which had been killed by cervical dislocation. Some rats were additionally treated with methoxyacetic acid (MAA) in order to specifically destroy pachytene spermatocytes and follow the subsequent recovery of the seminiferous epithelium [15–18]. MAA (650 mg/kg; Aldrich-Chemie, Steinheim, Germany), adjusted to pH 7.4 with NaOH, was administered i.p. to six groups of six animals each (in 0.9% saline and a volume of 5 ml/kg). Animals were killed in groups on Days 1, 3, 7, 14, 21, and 28 after treatment. Control rats (n = 18) received an equivalent volume of vehicle. Time points were chosen on the basis of elimination of pachytene spermatocytes and round spermatids, but which still permitted an accurate staging of the seminiferous epithelium. Testes were fixed in Bouin's solution for subsequent histological evaluation.
In Situ Hybridization
After passage through ascending ethanols, Bouin's-fixed testis tissue was finally embedded in paraffin wax. Sections (10 µm) were dewaxed in xylol and descending ethanols, then treated sequentially in 0.2 N HCl (20 min, room temperature [RT]) to inhibit endogenous alkaline phosphatase in 0.3% Triton X-100 in PBS (15 min, RT), and then in 0.1 M Tris-HCl (pH 7.5) with 5 mM EDTA (15 min, RT). Sections were then incubated in 20 µg/ml proteinase K in the same buffer (30 min, 37°C). Digestion was stopped in 0.2% glycine (1 min, 4°C), sections were postfixed in 3% paraformaldehyde (5 min, 4°C), then rinsed in diethyl pyrocarbonate (DEPC)-treated water (5 min, RT) before equilibrating in 0.1 M triethanolamine (pH 8.0; 3 min, RT), acetylating in 0.5% acetic anhydride in the same buffer (10 min, RT), and rinsing in 2x SSC (5 min, RT). Sections were then dried for 1 h at 50°C and stored at -20°C.
Sense and antisense cRNA, representing the full-length rat ELP cDNA [2], were transcribed in vitro in the presence of digoxigenin-UTP (Boehringer-Mannheim, Mannheim, Germany) according to the manufacturer's instructions. Prehybridization was performed for 2–3 h at 52°C in the following mix: 20 mM Tris-HCl (pH 7.5), 0.3 M NaCl, 1 mM EDTA, 100 mM dithiothreitol, 50% deionized formamide, 1x Denhardts solution, 100 µg/ml poly(A), 500 µg/ml denatured herring sperm DNA, and 500 µg/ml calf thymus tRNA. For hybridization, this mix was exchanged with 20 µl fresh mix containing 10 ng digoxigenin-labeled probe. Coverslips were placed over the sections, which were then incubated overnight at 52°C. After hybridization, coverslips were removed by washing twice in 2x SSC (15 min, RT), and sections were stringently washed in hybridization buffer at 60°C (10 min) followed by RNase A (Sigma, Deisenhofen; 50 µg/ml in 0.5 M NaCl, 10 mM Tris-HCl [pH 7.5], 1 mM EDTA) digestion (30 min, 37°C). Sections were then washed in the same buffer without RNase A (30 min, 37°C), twice in 2x SSC (3 min, RT), once in 0.1x SSC (15 min, 52°C), followed by equilibration in 0.1x SSC at RT. For signal detection, sections were washed in antibody buffer (0.1 M Tris-HCl [pH 7.5], 0.15 M NaCl; 5 min RT), blocked with 20% normal sheep serum in the same buffer (30 min RT), and briefly washed in buffer without serum. Sections were then incubated with sheep anti-digoxigenin antibody alkaline phosphatase conjugate (Boehringer-Mannheim) diluted 1:5000 in antibody buffer containing 1% sheep serum and 0.3% Triton X-100 (2 h, RT); rinsed twice in antibody buffer (15 min RT); and once in phosphatase buffer (0.1 M Tris-HCl [pH 9.5], 0.1 M NaCl, 5 mM MgCl2). Color development proceeded in the dark in the same buffer, also containing 0.3% Triton X-100, 2.4 mg/ml levamisole, 0.18 mg/ml 5-bromo-4-chloro-3-indolyl-phosphate (BCIP; Boehringer-Mannheim), and 0.34 mg/ml nitroblue tetrazolium chloride (NBT; Boehringer-Mannheim) and stopped in 10 mM Tris-HCl (pH 9.5) with 1 mM EDTA (10 min RT). Finally, sections were mounted in Faramount medium (Dako Diagnostica, Hamburg, Germany).
Immunohistochemistry
New polyclonal antibodies were raised in rabbits against a recombinant mouse ELP protein exactly as previously described for antibodies raised in rats [1]. Immunohistochemistry was performed using a double PAP-ABC combination described in detail elsewhere [19]. In addition, sections of normal adult rat testis were analyzed for the presence of ACBP epitopes using a rabbit polyclonal antiserum raised against recombinant rat ACBP (courtesy of Professor J. Knudsen). This antiserum had been tested by Western blot analysis of rat testis extracts and shown to interact at high affinity with only a single protein of ~10 kDa (data not shown).
Testis sections were staged according to the method of Leblond and Clermont [20] as elaborated by Russell et al. [21].
Northern Hybridization
Total RNA from rat testis and from the mouse germ cell-derived cell lines GC1 and GC2 was prepared according to the single-step method of Chomczynski and Sacchi [22] and electrophoresed (20 µg per lane) on a MOPS/formaldehyde gel [23]. The separated RNA was capillary transferred to Hybond-N+ (Amersham-Pharmacia, Freiburg, Germany) nylon membranes and hybridized to a double-stranded probe of the full-length rat ELP cDNA [2] labeled with fluorescein using the GENE-IMAGES system (Amersham-Pharmacia). Hybridization was performed in 5x SSC, 0.1% SDS, and 5% Liquid Block (Amersham-Pharmacia) at 65°C overnight. Washing was performed at hybridization temperature, with the last wash under stringent conditions (0.1x SSC/0.1% SDS). The membrane was then blocked, incubated with antifluorescein-alkaline phosphatase conjugate, and washed in accordance with the manufacturer's instructions (Amersham-Pharmacia). Finally, the membrane was incubated with CDP-star detection substrate (Amersham-Pharmacia) for 5 min and exposed to autoradiographic film for 1 h.
RESULTS
Distribution of ELP Gene Transcripts in the Rat Testis
ELP mRNA expression was distinctly germ cell-specific and stage-specific. Positive signals were observed for the antisense probes only, first and with low intensity in round spermatids during spermatogenic stages VI-VIII (Fig. 1, C and E, arrows). Signal intensity increased in subsequent steps up to step 18 elongated spermatids (Fig. 1E). After spermiation, the residual bodies retained by the Sertoli cells at stages VII-IX showed persistent strong signal intensity for ELP mRNA (data not shown). There was no evidence for any ELP gene transcription in any of the somatic cells of the testis, nor in any premeiotic or meiotic germ cell stages. All hybridizations with the sense-strand probe as control were negative (Fig. 1). The latter was supported by Northern blot hybridization (Fig. 2), which showed that RNA from the two mouse spermatogonial cell lines, GC1 and GC2, both with diploid chromosome complement, was negative for ELP transcripts.
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ELP Immunohistochemistry in the Rat Testis
ELP-specific immunohistochemical signals were seen only in germ cells, but not in any other cellular compartment (Fig. 3). Negative controls using preimmune serum were free of any signal (Fig. 3G). ELP protein expression appears delayed by comparison with the mRNA, with the first specific signals being detectable during spermatid elongation (steps 17 to 19). Also, the residual bodies showed positive staining (Fig. 3, B and C). Specific staining was restricted to the cytoplasm of cells only, where it was homogeneous across a cell and not restricted to any subcellular structures.
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Effect of MAA Treatment on ELP Expression in the Rat Testis
MAA treatment caused extensive degeneration of pachytene spermatocytes, so that by Day 7, all such spermatocytes were either missing or showed abnormal morphology (Fig. 3K). Round spermatids were also absent in stages I-II and IV of the seminiferous epithelium. By Day 21 after MAA treatment, a loss of elongated spermatids was evident (Fig. 3M). The sequence of germ cell degeneration and subsequent recovery was thus as follows: Day 3, pachytene spermatocytes missing in stages XII-VII; Day 7, pachytene spermatocytes missing in stages VII-XIV and round spermatids missing in stages I-VI; Day 14, round spermatids missing in stages I-VI and elongating spermatids missing in stages IX-III; Day 21, only elongating spermatids were missing in stages IV-VIII. Despite these profound alterations to the architecture of the germinal epithelium, ELP expression remained unaffected at the mRNA and protein levels in the remaining cells. No qualitative differences could be detected in testicular ELP expression between vehicle- and MAA-treated animals. During the recovery of spermatogenesis on Day 28 following MAA exposure, the appearance of spermatids in steps 15-19 could be easily recognized on the basis of their ELP expression (Fig. 3, N–P). Interestingly, the restoration of spermatogenesis and the stage-dependent cellular associations occurred focally within a cross section of the seminiferous tubule (Fig. 3, N–P).
Parallel treatment of rat testis tissue sections with antibodies specific for rat ACBP (Fig. 3H) showed no specific immunostaining of germ cells. Only Sertoli and Leydig cells indicated clear positive reactions for ACBP epitopes.
DISCUSSION
Within rat testis, the ELP gene appears to be actively and uniquely transcribed in postmeiotic germ cell stages. The first detectable specific mRNA signals are seen in round spermatids (Fig. 4A). Thereafter, mRNA concentrations markedly increase during spermatid elongation, showing that the ELP gene is still being actively transcribed at a time when histones are being replaced in the spermatid nucleus by transition proteins and protamines [24]. ELP protein is first detectable in elongating spermatids approximately 10 days into the spermatogenic cycle after the first detection of ELP mRNA in late round spermatids (Fig. 4B). Thus, ELP belongs to the category of haploid sperm proteins that are subjected to translational delay [24]. Maximal immunostaining for ELP was observed in the latest stages of spermatogenesis, immediately prior to spermiation. Indeed, the cytoplasmic droplets, which are retained by the Sertoli cells in the form of residual bodies following their removal from the mature spermatozoa, show most intense staining both for ELP mRNA and protein.
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In the rat testis, the structurally related molecule, ACBP, appears to be expressed mostly in Leydig and Sertoli cells (Fig. 3H), a role that is compatible with the involvement of ACBP in the steroidogenic process via its interaction with the peripheral benzodiazepam receptor [8]. It seems likely, therefore, that ELP has evolved as a sperm-specific variant of ACBP, with functions adapted to sperm metabolism. The high degree of sequence conservation among the known ELP molecules from rat, mouse, and bull, particularly in the region of the acyl-CoA binding motif, together with the preliminary observation that recombinant ELP can bind acyl-CoA with high affinity (J. Knudsen, personal communication), lends support to this view. ACBP has been described not only as a transport protein delivering acyl-CoA to the mitochondrion for energy metabolism via lipid ß-oxidation, but also as a ligand (acyl-CoA)-dependent intracellular signaling molecule [10]. It is to be expected, therefore, that ELP subserves similar important functions in relation to sperm metabolism. The fact that extratesticular sperm have little if any ELP, as judged from immunohistochemistry of cauda epididymal sections [1], reflects the loss of most soluble cytoplasmic proteins in the cytoplasmic droplet. Mature sperm retain only a small percentage of their original cytoplasm in the neck region of the cell. This does not therefore signify that ELP does not have a function in free spermatozoa. This issue is confused somewhat by a recent article by Kolmer et al. [14] that claimed that rat spermatozoa also contain low levels of ACBP (diazepam binding inhibitor), and that mRNA and protein are expressed with a similar cellular distribution as ELP in haploid germ cells. The concentrations appear to be much lower, however, than either ACBP levels in Leydig and Sertoli cells or ELP levels in spermatids. This gametic ACBP is thus possibly a remnant of an ancestral situation.
Treatment of rats with MAA had no influence on the basic pattern of ELP expression. Thus, the loss of complete cohorts of germ cells did not exert a paracrine influence on ELP gene expression in neighboring cells, affirming the general hypothesis that most postmeiotic spermatogenic events, like ELP gene expression, are preprogrammed and operate independently of the immediate cellular milieu within the seminiferous tubule. In this context, however, ELP immunoreactivity, because of its very restricted cell-type specificity, makes an ideal marker to follow the fate of elongated spermatids. This is excellently seen in the recovery of germ cells 28 days after MAA treatment (Fig. 3, N–P). For the first time, it can be clearly seen that elongated spermatids focally reappear within a tubule cross section. Although this may represent a local paracrine effect, it is more likely to indicate the clonal nature of the ELP-positive cohorts, whose precursor spermatocytes presumably differentiated through the pachytene stage faster than other neighboring germ cells.
In summary, therefore, ELP gene expression, at both the mRNA and protein levels, offers an excellent marker system to follow the fate of very late haploid stages of spermatogenesis. The structural and biochemical similarity of ELP to the closely related ACBP, and their more or less mutually exclusive cellular distribution within the testis, strongly implies that ELP is playing an important function in the context of sperm metabolism, possibly related to the specialized complex mitochondrion of the mature spermatozoon. Experiments are currently in progress to generate mice with a deleted ELP gene in order to test this hypothesis.
ACKNOWLEDGMENTS
We are particularly grateful to Professor J. Knudsen, Odense University, Denmark, for permission to mention unpublished results, and for the kind gift of the specific anti-ACBP antibodies. We also thank Ms. M. Valentin, Odense; and Dr. H. Lauke, Hamburg, for helpful discussions; Professors A.F. Holstein and F. Leidenberger for their generous support of this project; and Dr. A. Meinhardt, Marburg, for the kind gift of RNA samples from the germ cell lines, GC1 and GC2.
FOOTNOTES
First decision: 28 March 2000.
1 This project has been supported by the Hamburg-Münster Confocal Research Group of the Deutsche Forschungsgemeinschaft (Iv7/4-2, Ni130/15-4). 
2 Correspondence: Richard Ivell, Institute for Hormone and Fertility Research, University of Hamburg, Grandweg 64, 22529 Hamburg, Germany. FAX: 49 40 56190864; ivell{at}ihf.de
Accepted: April 17, 2000.
Received: February 23, 2000.
REFERENCES
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