In the postnatal testis, the c-kit transmembrane tyrosine-kinase receptor is expressed in type A spermatogonia, and its transcription ceases at the meiotic phase of spermato-genesis. Alternative, shorter c-kit transcripts are expressed in post-meiotic germ cells. These transcripts should encode a truncated version of the c-kit protein, lacking the extra-cellular, the transmembrane and part of the intracellular tyrosine-kinase domains. The 5′ end of the alternative c-kit transcripts maps within an intron of the mouse c-kit gene. We now show that this intron contains a promoter active in nuclear extracts of round spermatids, and that two discrete sequences upstream of the transcriptional start site bind spermatid-specific nuclear factors. Deletion of both these sequences abolishes activity of the promoter in vitro. We have also established that this promoter is functional in vivo, in a tissue- and cell-specific fashion, since intronic sequences drive the expression of the E. coli lacZ reporter gene in transgenic mice specifically in the testis. Transgene expression is confined to haploid germ cells of seminiferous tubules, starting from spermatids at step 9, and disappearing at step 13, indicating that a cryptic promoter within the 16th intron of the mouse c-kit gene is active in a short temporal window at the end of the transcriptional phase of spermiogenesis. In agreement with these data, western blot experiments using an antibody directed against the carboxy-terminal portion of the mouse c-kit protein showed that a polypeptide, of the size predicted by the open reading frame of the spermatid-specific c-kit cDNA, accumulates in the latest stages of spermatogenesis and in epididymal spermatozoa. An immunoreactive protein of the same size can be produced in both eukaryotic and prokaryotic artificial expression systems.

In both W and Sl mutant mice, one major symptom, together with anemia and pigmentation defects, is sterility (Russell, 1979). W locus encodes the c-kit transmembrane receptor tyrosine-kinase (Chabot et al., 1988). The c-kit receptor is a protein of approx. 150×103Mr consisting of an immunoglobulin-like extracellular domain, a transmembrane domain, and an intracellular tyrosine kinase domain, characteristically split by an intervening protein sequence into an ATP-binding site and a phosphotransferase catalytic site (Qiu et al., 1988). c-kit mRNA is expressed in primordial germ cells of the embryonal gonad (Manova and Bachvarova, 1991), and it is also expressed, at high levels, in type A spermatogonia of the post-natal testis (Sorrentino et al., 1991).

The Sl locus encodes the c-kit ligand (Besmer, 1991), which can exist in either a soluble or a transmembrane form (Flanagan et al., 1991). Both forms of the c-kit ligand are essential for survival of primordial germ cells in culture (Dolci et al., 1991; Godin et al., 1991), and are expressed in the postnatal testis by Sertoli cells under cAMP and/or FSH stimulation (Rossi et al., 1991, 1993b). After birth, the soluble form of the ligand stimulates DNA synthesis selectively in type A spermatogonia (Rossi et al., 1993b). The relevance of c-kit in male gametogenesis after birth has been also established in vivo (Yoshinaga et al., 1991; Brinster and Avarbock, 1994).

In the meiotic phase of spermatogenesis c-kit expression ceases, but in the subsequent haploid phase alternative shorter transcripts are present (Sorrentino et al., 1991). These transcripts should encode a truncated form of the receptor, with a predicted molecular mass of approx. 23 ×103, in which only the second box of the split kinase is present, whereas the extra-cellular and transmembrane domain, and the first box of the split kinase domain, are missing (Rossi et al., 1992). The 5′ end of the alternative c-kit transcripts maps within an intron that separates the exon encoding the interkinase domain from the first exon encoding the phosphotransferase domain (Rossi et al., 1992). According to the published structure of the murine c-kit gene (Gokkel et al., 1992), these sequences correspond to the 16th intron.

During the haploid phase of spermatogenesis, together with the expression of germ cell-specific genes with a known function, such as protamine (for a review, see Hecht, 1990) or PGK2 (McCarrey and Thomas, 1987), a peculiar pattern of expression of additional genes with no obvious function is observed. In many cases, expression of RNA transcripts has not been accompanied by demonstration of the presence of the corresponding protein products. This pattern ranges from expression of regulatory genes normally involved in embryonal development, such as Hoxa-4 (Behringer et al., 1993) or Sry (Rossi et al., 1993a), to the presence of alternative transcripts for genes normally expressed in somatic cells. These alternative transcripts are generated either by utilization of alternative promoters, such as in the case of ACE (Langford et al., 1991), or by alternative splicing of precursor RNAs, as in the case of many protooncogenes (Propst et al., 1988; Sor-rentino et al., 1988).

In order to establish the function of the alternative c-kit gene products in the haploid phase of spermatogenesis, in the present work, as an initial approach, we have addressed two major questions: (1) whether a transcriptional promoter specifically activated during spermiogenesis is present within the 16th intron of the mouse c-kit gene; and (2) whether the truncated c-kit mRNAs are translated into the predicted polypeptide. We show that a testis-specific promoter is actually present within the 16th intron of the murine c-kit gene, and that this promoter is specifically active in a short temporal window during spermiogenesis. Moreover, we show that this intronic promoter is responsible for the accumulation in elongating spermatids and epididymal spermatozoa of a truncated c-kit protein of the size predicted through molecular cloning of the alternative c-kit transcripts.

Testicular cell preparation

Spermatogonia from 7-to 8-day old mice and primary Sertoli cell-enriched cultures from 18-day old mice were prepared as previously described (Rossi et al., 1993b). Germ cell-Sertoli cell cocultures were prepared from 13-day old mice using the same procedure that was utilized to obtain Sertoli cell-enriched monolayers, but omitting the hypotonic treatment, which selectively eliminates germ cells. Germ cells at pachytene spermatocyte, round spermatid and elongating spermatid steps were obtained by elutriation of unfractionated sus-pensions of germ cells from testes of adult mice, as previously described for the rat (Meistrich, 1977). Homogeneity of cell populations ranged between 90% (pachytene spermatocytes) and more than 95% (spermatids at various steps of differentiation), and was routinely controlled morphologically. To obtain spermatozoa, cauda epididymis of adult mice were squeezed using a syringe needle. Spermatozoa were allowed to disperse for 30 minutes at 37 °C in Minimum Essential Medium Eagle’s supplemented with 2 mM sodium lactate, 1 mM sodium pyruvate and 1 mg/ml bovine serum albumin (BSA) under a 5% CO2 atmosphere, and collected by centrifugation.

DNA constructions for in vitro transcription and transgenic analysis

Plasmid p-kit-int was constructed by subcloning in the SmaI site of pBluescript KS M13+ (Stratagene) a PCR-amplified 1161 bp genomic fragment from the mouse c-kit gene. This fragment contains the last 47 bp from the 16th exon, the entire 16th intron, and the first 87 bp from the 17th exon. These sequences span from −623 to +538, with respect to the presumptive transcriptional start site utilized for the generation of the alternative c-kit transcripts expressed in round sper-matids (Rossi et al., 1992). Plasmid p-kit-int-Gal was constructed by subcloning a 1.16 kb SalI/BamHI fragment from p-kit-int (containing sequences between −623 and +526 with respect to the presumptive spermatid-specific transcriptional start site). After addition of synthetic BamHI-XhoI adaptors, the fragment was cloned in the XhoI site of plasmid pNASSβ (Clontech), in the direct orientation in front of the E. coli lacZ gene. All constructs were made by using standard recombinant methods (Sambrook et al., 1989). Synthetic oligonu-cleotides utilized in DNA binding experiments and PCR analysis were produced through standard techniques with a 391 DNA synthesizer (PCR-MATE EP; Applied Biosystems-Perkin Elmer).

In vitro transcription

Nuclear extracts from purified testicular cells were prepared as previously described (Bunick et al., 1990), with modifications. Pellets from freshly prepared cells were resuspended in two volumes of a homogenization buffer containing 25 mM KCl, 10 mM Hepes, pH 7.6, 1 mM EDTA, 0.5 mM dithiothreitol, 10% (v/v) glycerol, 0.5 mM phenylmethylsulphonylfluoride (PMSF), 10 μg/ml pepstatin A and 10 μg/ml antipain. After homogenization with 40 strokes in a glass Dounce homogenizer, nuclei were pelleted by centrifugation and washed with two volumes of nuclei buffer, containing 10 mM Hepes pH 7.9, 1.5 mM MgCl2, 0.1 mM EDTA, 0.5 mM dithiothreitol, 10% (v/v) glycerol, 0.5 mM PMSF, 10 μg/ml pepstatin A and 10 μg/ml antipain. Nuclei were resuspended in 2.2 volumes of nuclei buffer with the addition of KCl to a final concentration of 0.3 M. Samples were gently rocked for 1 hour at 4 °C, and then clarified by centrifu-gation at 100,000 g for 30 minutes. Supernatants were diluted with two volumes of nuclei buffer and concentrated through Centricon-10 (Amicon) to reach a final volume of about one half of the original volume of the nuclear pellet (protein concentration 10-15 mg/ml), dispensed into samples and stored at −75 °C. Run-off transcription reactions were performed for 60 minutes at 25 °C using 10 μl of nuclear extracts, 1 μl of a 1 μg/μl linearized plasmid DNA template, 10 μl of a buffer containing 14% glycerol, 20 mM KCl, 6.5 mM MgCl2, 14 mM Hepes pH 7.9, 1.2 mM of each ATP, CTP and UTP, 0.05 mM GTP, 0.5 μl RNAsin (60 U/μl, Amersham), 0.5 μl [α-32P]GTP (Amersham, 400 Ci/mmol, 10 mCi/ml). Reactions were stopped with 380 μl of a buffer containing 50 mM Tris-HCl, pH 7.5, 1% SDS, 5 mM EDTA and 25 μg/ml tRNA, extracted twice with phenol-chloroform, precipitated, dessiccated and electrophoresed on 4% acrylamide gels containing 7 M urea.

DNA binding assays

Nuclear extracts (protein concentration approx. 15 mg/ml) from testicular cells for DNA binding assays were prepared as previously described (Grimaldi et al., 1993). DNA restriction fragments for electrophoretic mobility shift assays (EMSA) were labeled at the 5′ end with [γ-32P]ATP using sequential alkaline phosphatase and T4 polynucleotide kinase treatment, whereas synthetic oligonucleotides for both EMSA experiments and southwestern analysis were filled-in with [α-32P]dATP and Klenow enzyme (Sambrook et al., 1989). Labeled restriction fragments were purified by non-denaturing poly-acrylamide gel electrophoresis, whereas labeled oligonucleotides were purified by gel filtration on Sephadex G50. Conditions utilized for EMSA and southwestern experiments have been described previously (Grimaldi et al., 1993).

Generation and identification of transgenic mice

A 5 kb PstI restriction fragment from plasmid p-kit-int-Gal, containing the 16th intron of the mouse c-kit gene linked to the E. coli lacZ gene, was purified by electroelution and microinjected into male pronuclei of one-cell (C57/6J×DBA/2)F2 mouse zygotes. Transgenic mice were produced by standard techniques (Brinster et al., 1985). Identification of transgenic animals was performed by standard Southern blot analysis (Sambrook et al., 1989) of tail DNA digested with EcoRI, using as a probe a 3 kb EcoRI-SacI fragment from plasmid p-kit-int-Gal labeled with [α-32P]dATP by random priming (Boehringer). This probe detects a 5 kb band corresponding to the transgene, and a 2 kb band from the endogenous c-kit gene. Verifica-tion of heterozygosity or homozygosity for the transgene was performed by PCR analysis of genomic DNA from the offspring of the transgenic animals mated with non-transgenic partners. The E. coli lacZ gene was detected with GCATCGAGCTGGGTAATAAGGGT-TGGCAAT and GACACCAGACCAACTGGTAATGGTAGCGAC (amplifying a 822 bp fragment), and the endogenous RapSyn gene with AGGACTGGGTGGCTTCCAACTCCCAGACAC and AGC-TTCTCATTGCTGCGCGCCAGGTTCAGG (amplifying a 590 bp fragment).

Analysis of lacZ protein (β-galactosidase) and RNA expression

Fresh tissues were fixed for 2 hours at 4 °C in 0.1 M phosphate buffer, pH 7.3, 0.01% sodium deoxycholate, 0.02% Nonidet-P40, 0.2% glu-taraldehyde and 2% formaldehyde. β-galactosidase staining was performed, after three rinses at room temperature in 0.1 M phosphate buffer, by overnight immersion of tissues at 37 °C in 0.1 M sodium phosphate pH 7.3, 1.3 mM MgCl2, 5 mM K3Fe(CN)6, 5 mM K4Fe(CN)6, 1 mg/ml X-gal (Sigma). After a brief rinsing with 0.1 M phosphate buffer, pH 7.3, tissues were prepared for sectioning in frozen blocks of OCT (Tissue Tek). 30 μm cryostatic sections were washed with 0.1 M phosphate buffer, pH 7.3, dehydrated with ethanol and mounted with Eukitt (Kindler Gmbh). Alternatively, seminifer-ous tubules were isolated mechanically in PBS, layered on a slide, covered with a coverslip and directly observed through transillumi-nation. For more detailed morphological analysis of stage-specific expression, after β-galactosidase staining, tissues were fixed again in buffered formalin, and paraffin-embedded. 5 μm sections were prepared, stained with PAS and counterstained with hematoxylin. Total RNAs for northern blot analysis were prepared by homoge-nization in a Dounce glass homogenizer of frozen tissues with RNAzol (Biotecx Laboratories). Equal amounts of RNA from each sample were electrophoresed through denaturing agarose gels and transferred to Hybond-N membranes (Amersham). Blots were hybridized with a 2.2 kb SacI fragment from plasmid pNASSβ (Clontech), containing sequences from the E. coli lacZ gene, which had been labeled with [α-32P]dATP by random priming (Boehringer). Blots were washed under highly stringent conditions (Sambrook et al., 1989) and subjected to autoradiography at −75 °C with intensifier screens for 60 hours.

Immunofluorescence and western blot analysis

Polyclonal antibodies were raised in rabbits using a synthetic peptide corresponding to the last 13 amino acids encoded by the mouse c-kit open reading frame (ORF): Ala-Ser-Ser-Thr-Gln-Pro-Leu-Leu-Val-His-Glu-Asp-Ala.

For immunofluorescence experiments, Sertoli cell-mitotic germ cell cocultures were performed on sterile glass coverslips, whereas germ cell suspensions in advanced stages of spermatogenesis were spotted on glass slides previously treated with poly-L-lysine. Samples were fixed for 15 minutes at room temperature with 4% paraformalde-hyde in phosphate-buffered saline (PBS), and treated with 0.1% Triton X-100 in PBS for a further 15 minutes. Slides were dipped in a blocking solution containing 0.5% BSA in PBS overnight at 4 °C. Samples were then incubated at 37 °C for 1 hour with protein A-Sepharose affinity-purified preimmune or immune IgG fractions (10 mg/ml stock solutions) that had been diluted 1:100 in PBS containing 0.5% BSA. After three washes for 5 minutes at room temperature in the same buffer, samples were incubated for 30 minutes at 37 °C with a goat anti-rabbit IgG polyclonal antibody conjugated with rhodamine (Calbiochem), diluted 1:75. After two 5 minute washes at room temperature, slides were mounted with 50% glycerol in PBS.

For western blot analysis, frozen pellets of germ cells at different stages of spermatogenesis and epididymal spermatozoa were directly resuspended in SDS-PAGE sample buffer, homogenized first in a glass Dounce homogenizer, and then sheared through an insulin syringe. After boiling for 5 minutes, samples containing similar amount of proteins were electrophoresed on 13% SDS-polyacry-lamide gels. Proteins from gels were transferred to Hybond-C nitro-cellulose membranes (Amersham) overnight at 4 °C. Filters were blocked for 1 hour at room temperature with a buffer containing 10% non-fat dry milk, 0.1% Tween-20 in PBS, and then incubated for 1 hour at room temperature in a solution of total preimmune or immune serum diluted 1:200 in the same buffer. Filters were developed with a second anti-rabbit IgG antibody conjugated with peroxidase (1:20,000), using the ECL immunodetection system (Amersham) followed by autoradiography, according to the manufacturer’s instructions.

Expression of the truncated c-kit protein in COS cells and in E. coli

For eukaryotic expression, a 1.8 kb HindIII fragment from plasmid pSTK3b, containing part of the 5′ and 3′ untranslated regions and the entire ORF of spermatid-specific c-kit cDNA clone 3b (Rossi et al., 1992), was subcloned in the pCMV5 expression vector, in the direct orientation downstream from the cytomegalovirus promoter. For protein expression, subconfluent cultures of COS cells were trans-fected using the standard CaPO4 coprecipitation method (Gorman et al., 1982) with 20 μg of PCMV5-c-kit hybrid construct, for 5 hours at 37 °C. At the end of the incubation cells were shocked for 2 minutes with 10% glycerol in growth medium, rinsed twice with PBS, and grown for an additional 24 hours. Cells were then harvested and lysed in 50 mM Hepes, pH 7.4, 150 mM NaCl, 1.5 mM MgCl2, 1 Mm EGTA, 10% glycerol, 1% Triton X-100, 50 mM benzamydine, 0.5 μg/ml leupeptin, 0.7 μg/ml pepstatin, 4 μg/ml aprotinin and 2 mM PMSF. Cell extracts were centrifuged for 10 minutes at 15,000 g. The resulting supernatants were analyzed by SDS-PAGE and western blotting.

For prokaryotic expression, the ORF deduced from the spermatid-specific c-kit cDNA (Rossi et al., 1992) was amplified by RT-PCR and subcloned into a pQE-60 expression vector. E. coli strain M15 was used for transformation. Selection was performed with kanamycin and ampicillin. For protein expression, positive colonies or wild-type bacteria were grown to OD595nm=0.8. Growth was continued for a further 3 hours in the absence or presence of 2 mM IPTG. Cells were collected by centrifugation and lysed by sonication. The bacterial lysates were analyzed by SDS-PAGE and western blot analysis.

Identification of a presumptive promoter for the expression of the truncated c-kit

In vitro run-off transcription experiments, using nuclear extracts from round spermatids (Fig. 1), suggested that the alternative c-kit transcripts during spermiogenesis are generated by a cryptic promoter present within the 16th intron of the mouse c-kit gene. We used nuclear extracts from mouse round spermatids (steps 1-9 of spermiogenesis) and, as a template for in vitro RNA synthesis, plasmid p-kit-int, containing sequences between −622 and +538, with respect to the presumptive transcriptional start site utilized in spermatids. The plasmid had been digested with restriction enzymes, which cut at different distances downstream from the presumptive transcriptional (CAP) site. Three labeled RNA fragments were generated, of 540 (BamHI), 700 (PvuII) and 730 (BglI) nucleotides (n.) respectively, as expected if transcription started at the predicted point. Transcription is RNA polymerase II-dependent, since it is completely abolished by using α-amanitin as a specific inhibitor. Using as a template intronic sequences placed in different vectors, we obtained similar results with nuclear extracts from round spermatids, whereas no specific in vitro transcription products were generated with nuclear extracts from Sertoli cells or pachytene spermatocytes (data not shown).

Fig. 1.

Sequences within the 16th intron of the mouse c-kit gene act as a transcriptional promoter in vitro. Run-off transcription experiments using nuclear extracts from mouse round spermatids (steps 1–9 of spermiogenesis) in the presence of labeled ribonucleotide precursors, using as a template for in vitro RNA synthesis plasmid p-kit-int, which is schematically shown on the right side of the picture. Restriction enzymes used to linearize the template, and the position of the presumptive spermatid-specific transcriptional (CAP) site present within the c-kit genomic sequences, are indicated. Arrows on the left side of the autoradiograph indicate the RNA bands of expected size, as schematically represented by thick bars adjacent to the scheme. Other bands represent aspecific transcription starting from the M13 F1 intergenic region, indicated by thin bars adjacent to the scheme, or products derived from premature termination or degradation. Transcription is RNA polymerase II-dependent, since it is completely abolished by using as a specific inhibitor α-amanitin (+ lanes).

Fig. 1.

Sequences within the 16th intron of the mouse c-kit gene act as a transcriptional promoter in vitro. Run-off transcription experiments using nuclear extracts from mouse round spermatids (steps 1–9 of spermiogenesis) in the presence of labeled ribonucleotide precursors, using as a template for in vitro RNA synthesis plasmid p-kit-int, which is schematically shown on the right side of the picture. Restriction enzymes used to linearize the template, and the position of the presumptive spermatid-specific transcriptional (CAP) site present within the c-kit genomic sequences, are indicated. Arrows on the left side of the autoradiograph indicate the RNA bands of expected size, as schematically represented by thick bars adjacent to the scheme. Other bands represent aspecific transcription starting from the M13 F1 intergenic region, indicated by thin bars adjacent to the scheme, or products derived from premature termination or degradation. Transcription is RNA polymerase II-dependent, since it is completely abolished by using as a specific inhibitor α-amanitin (+ lanes).

The presumptive promoter has binding sites for spermatid-specific nuclear proteins

DNA-binding experiments show that nuclear factors present in extracts from spermatids and from spermatocytes, but not from Sertoli cells, bind to c-kit intronic sequences between −622 and −289 from the presumptive CAP site (Fig. 2). However, specific DNA-protein complexes were observed only with round spermatid nuclear extracts when adding 8 μg of protein, and the electrophoretic mobility of DNA-protein complexes formed with meiotic germ cells was different from that observed in postmeiotic germ cells at any tested concentration of nuclear extracts, suggesting that haploid cell-specific nuclear factors were present in these extracts. Fig. 3 schematically shows that, using EMSA experiments with 8 μg of protein, we have been able to map two small regions within this presumptive promoter, which are essential for binding of spermatid-specific nuclear factors: a 82 bp sequence at position −485/−404, and a 19 bp sequence between −68/−50 from the presumptive transcriptional start site. EMSA experiments using as probes an oligonucleotide spanning from −471 to −437, or another oligonucleotide spanning from −456 to −423 (Fig. 4), show that only the −471/−437 oligonucleotide was able efficiently to form specific DNA-protein complexes. In the −464/−456 area a perfect ‘enhancer core’ element (GTGTGGTAA) is found. The ‘enhancer core’ element can bind transcription factors such as AP-3 and C/EBP (Faisst and Meyer, 1992), and the importance of these sequences is strengthened by the observation that formation of the complex can be inhibited in the presence of a SV40 enhancer fragment, which contains a similar sequence element. Fig. 5 shows that the −471/−437 oligonucleotide, but not an unrelated oligonucleotide, recognizes in southwestern experiments a series of polypeptides present in nuclear extracts from round spermatids, but not from spermatocytes or Sertoli cells.

Fig. 2.

Nuclear factors present in meiotic and postmeiotic germ cells, but not in Sertoli cells, bind sequences within the 16th intron of the mouse c-kit gene. EMSA experiments with increasing concentrations of nuclear extracts from purified testicular cells, using as a probe a PstI-XbaI fragment from plasmid p-kit-int, containing sequences from −623 to −289 from the presumptive spermatid-specific c-kit transcriptional start site.

Fig. 2.

Nuclear factors present in meiotic and postmeiotic germ cells, but not in Sertoli cells, bind sequences within the 16th intron of the mouse c-kit gene. EMSA experiments with increasing concentrations of nuclear extracts from purified testicular cells, using as a probe a PstI-XbaI fragment from plasmid p-kit-int, containing sequences from −623 to −289 from the presumptive spermatid-specific c-kit transcriptional start site.

Fig. 3.

Discrete sequences within the 16th intron of the mouse c-kit gene specifically bind nuclear factors present in round spermatids. The top scheme represents a summary of the results of EMSA experiments with 8 μg of nuclear extracts from mouse round spermatids (steps 1-9 of spermiogenesis) using as probes several labeled restriction fragments of the DNA region spanning the 16th intron of the mouse c-kit gene (indicated by bars). The restriction map of the intron is shown above the scheme, whereas a schematic representation of the two areas which are critical for specific binding of nuclear factors is drawn below. CAP site indicates the presumptive start of transcription of the truncated c-kit mRNAs expressed during mouse spermiogenesis. Restriction fragments numbered in the scheme correspond to those utilized in the representative example of EMSA experiments, whose autoradiograph is shown at the bottom of the figure.

Fig. 3.

Discrete sequences within the 16th intron of the mouse c-kit gene specifically bind nuclear factors present in round spermatids. The top scheme represents a summary of the results of EMSA experiments with 8 μg of nuclear extracts from mouse round spermatids (steps 1-9 of spermiogenesis) using as probes several labeled restriction fragments of the DNA region spanning the 16th intron of the mouse c-kit gene (indicated by bars). The restriction map of the intron is shown above the scheme, whereas a schematic representation of the two areas which are critical for specific binding of nuclear factors is drawn below. CAP site indicates the presumptive start of transcription of the truncated c-kit mRNAs expressed during mouse spermiogenesis. Restriction fragments numbered in the scheme correspond to those utilized in the representative example of EMSA experiments, whose autoradiograph is shown at the bottom of the figure.

Fig. 4.

Spermatid-specific nuclear factor/s binding between −471 and −456 from the presumptive spermatid-specific c-kit transcriptional start site recognize an enhancer core element. EMSA experiments with nuclear extracts from round spermatids using as probes synthetic oligonucleotides. Sequences within the 16th intron of the c-kit gene in the region between −471 and −423 from the presumptive spermatid-specific transcriptional start site are shown above, with the oligonucleotides used indicated. The ‘enhancer core’ (potential AP3 binding site) between −464 and −456 is boxed. Molar excess of competitors was 100-fold. The SV40 enhancer fragment used as a competitor is a 72 bp SphI restriction fragment from plasmid PSV2CAT (Gorman et al., 1982).

Fig. 4.

Spermatid-specific nuclear factor/s binding between −471 and −456 from the presumptive spermatid-specific c-kit transcriptional start site recognize an enhancer core element. EMSA experiments with nuclear extracts from round spermatids using as probes synthetic oligonucleotides. Sequences within the 16th intron of the c-kit gene in the region between −471 and −423 from the presumptive spermatid-specific transcriptional start site are shown above, with the oligonucleotides used indicated. The ‘enhancer core’ (potential AP3 binding site) between −464 and −456 is boxed. Molar excess of competitors was 100-fold. The SV40 enhancer fragment used as a competitor is a 72 bp SphI restriction fragment from plasmid PSV2CAT (Gorman et al., 1982).

Fig. 5.

Multiple factors present in nuclear extracts from round spermatids, but not from pachytene spermatocytes (P.Sc.) nor from Sertoli cells (S.C.), are specifically recognized by sequences between −471 and −437 from the presumptive spermatid-specific transcriptional start site. Southwestern experiments with the indicated amounts of nuclear extracts from different testicular cell types, used as a probe oligonucleotide 1, indicated in Fig. 4, or an unrelated oligonucleotide of the same size containing a random sequence (control).

Fig. 5.

Multiple factors present in nuclear extracts from round spermatids, but not from pachytene spermatocytes (P.Sc.) nor from Sertoli cells (S.C.), are specifically recognized by sequences between −471 and −437 from the presumptive spermatid-specific transcriptional start site. Southwestern experiments with the indicated amounts of nuclear extracts from different testicular cell types, used as a probe oligonucleotide 1, indicated in Fig. 4, or an unrelated oligonucleotide of the same size containing a random sequence (control).

Deletion of the two binding sites for spermatid-specific nuclear proteins abolishes in vitro transcription from the presumptive intronic c-kit promoter

We performed progressive deletions 5′ with respect to the spermatid-specific CAP site within the 16th c-kit intron in plasmid p-kit-int, and we tested these templates in in vitro run-off transcription experiments with nuclear extracts from round spermatids, after linearization with PvuII (Fig. 6). A specific 700 n. RNA band indicates correct transcriptional initiation from the presumptive CAP site. Deletion up to −460, just within the ‘enhancer core’ element, provokes a twofold reduction in the intensity of the 700 n. specific RNA band, and a threefold reduction is achieved with a deletion up to −404, which completely removes the first of the two regions essential for binding of spermatid-specific nuclear factors (see Fig. 3). Even though the area between −404 and −68 does not contain major binding sites for spermatid-specific nuclear factors (see Fig. 3), at least in the conditions of gel-shift analysis that we used, a further reduction of run-off transcription is obtained with a deletion up to −313, suggesting that other sequences in this area contribute to overall specific transcription. Deletion up to −50 completely abolishes correct transcriptional initiation of the 700 n. specific RNA band, indicating that the −68/−50 region, essential for binding of spermatid-specific nuclear factors (see Fig. 3), is required for the full functionality of the intronic c-kit promoter in haploid germ cells. This region contains the sequence TGAAAGTG, which is present in the binding site of transcription factors IRF-1, IRF-2 (Tanaka et al., 1993) and PRDI-BF1 (Keller and Maniatis, 1991), which are involved in the regulation of interferons and interferon-regulated genes. We also found that deletion of 400 bp at the 3′ end of the intronic sequence (from +138 to +538) resulted in generation of the predicted RNA bands with nuclear extracts of round spermatids (data not shown). Thus deletion analysis suggests that sequences between −464 and −50 from the presumptive CAP site are required for efficient transcription of the alternative c-kit mRNA in spermatids.

Fig. 6.

Deletion of the two binding sites for spermatid-specific nuclear proteins completely abolishes in vitro transcription from the presumptive intronic c-kit promoter. Autoradiograph of run-off transcription experiments using nuclear extracts from round spermatids (steps 1-9 of spermiogenesis) in the presence of labeled ribonucleotide precursors, using as a template for in vitro RNA synthesis plasmid p-kit-int linearized with PvuII, or the same template with the indicated progressive deletions 5′ with respect to the presumptive transcriptional start site, obtained with the indicated restriction enzymes. S.D. and S.A. indicate splice donor and acceptor sites, respectively.

Fig. 6.

Deletion of the two binding sites for spermatid-specific nuclear proteins completely abolishes in vitro transcription from the presumptive intronic c-kit promoter. Autoradiograph of run-off transcription experiments using nuclear extracts from round spermatids (steps 1-9 of spermiogenesis) in the presence of labeled ribonucleotide precursors, using as a template for in vitro RNA synthesis plasmid p-kit-int linearized with PvuII, or the same template with the indicated progressive deletions 5′ with respect to the presumptive transcriptional start site, obtained with the indicated restriction enzymes. S.D. and S.A. indicate splice donor and acceptor sites, respectively.

The intronic c-kit promoter is active in spermatids of transgenic mice in seminiferous tubules at stages IX-XI

The observation that spermatid-specific nuclear proteins specifically recognize two discrete sequence elements within the 16th intron of the mouse c-kit gene, upstream from the presumptive transcriptional start site, and that deletion of both these sequences abolishes in vitro transcription with spermatid nuclear extracts, strongly supports the hypothesis that a promoter specifically activated in the haploid phase of spermatogenesis is present within this intron.

In order to confirm in vivo the activity of the intronic promoter, we have generated five independent lines of trans-genic mice in which approx. 1.1 kb of these sequences (from −622 to +538 with respect to the presumptive CAP site) are linked to the reporter E. coli lacZ gene (Fig. 7). The transgene was correctly integrated into the mouse genome and transmit-ted in a Mendelian fashion. Selective expression of the transgene in the testis was observed through northern blot analysis. Fig. 8 shows that strong RNA signals of the predicted size were observed in the testis of most members of the ‘blue’ and ‘orange’ families. Expression of the lacZ gene in the testis was evident at the RNA level, even though the signal was of lower intensity, also in members of the ‘violet’ and ‘green’ lines. Members of the ‘red’ family did not show any detectable lacZ RNA signal. RNA signals in the four expressing families were of the predicted size (approx. 4.0 kb, including approx. 0.5 kb of c-kit sequences and approx. 3.4 kb of lacZ sequences), indicating that the CAP site of the endogenous gene, utilized for generation of the spermatid-specific c-kit transcripts both in vivo (Rossi et al. 1992) and in vitro (see Fig. 1 and 6), corresponds to the transcriptional start site within the transgene. The less intense RNA smears of larger and smaller size probably represent different degrees of polyadenylation and/or incomplete removal of the SV40 small T intron sequences (see Fig. 7).

Fig. 7.

Schematic representation of recombinant p-kit-int-Gal plasmid used for generation of transgenic mice. Mouse c-kit sequences between −623 and +538 from the presumptive spermatid-specific transcriptional start site within the 16th intron were placed 5′ to the E. coli lacZ gene in plasmid pNASSβ, containing SV40 splice signals (small T intron) 5′ with respect to lacZ, and SV40 polyadenylation signals at the 3′ end of the reporter gene. A 5 kb restriction fragment between the two PstI sites was used for microinjection of mouse one-cell stage zygotes, whereas a 3 kb fragment between EcoRI and SacI was used as a probe in Southern blot analysis of tail DNA for identification of transgenic animals.

Fig. 7.

Schematic representation of recombinant p-kit-int-Gal plasmid used for generation of transgenic mice. Mouse c-kit sequences between −623 and +538 from the presumptive spermatid-specific transcriptional start site within the 16th intron were placed 5′ to the E. coli lacZ gene in plasmid pNASSβ, containing SV40 splice signals (small T intron) 5′ with respect to lacZ, and SV40 polyadenylation signals at the 3′ end of the reporter gene. A 5 kb restriction fragment between the two PstI sites was used for microinjection of mouse one-cell stage zygotes, whereas a 3 kb fragment between EcoRI and SacI was used as a probe in Southern blot analysis of tail DNA for identification of transgenic animals.

Fig. 8.

Four out of five families of transgenic mice express E. coli lacZ RNA under the control of the 16th intron of the mouse c-kit gene specifically in the testis. Northern blot analysis of 15 μg of total RNAs from testis and/or other indicated tissues of wild-type (WT) and transgenic mice from the five different lines (‘blue’, ‘orange’, ‘violet’, ‘green’, and ‘red’) that we established. Additional members of the ‘orange’ family are indicated by asterisks. The generation of transgenic animals (F2, F3, or F4) is indicated. RNA samples were similar for both quality and quantity, as judged after ethidium bromide staining before blotting, by evaluating the intensity of the 28 S and 18 S ribosomal RNA bands. Blots were hybridized using as a probe a fragment of the E. coli lacZ gene (see Materials and methods).

Fig. 8.

Four out of five families of transgenic mice express E. coli lacZ RNA under the control of the 16th intron of the mouse c-kit gene specifically in the testis. Northern blot analysis of 15 μg of total RNAs from testis and/or other indicated tissues of wild-type (WT) and transgenic mice from the five different lines (‘blue’, ‘orange’, ‘violet’, ‘green’, and ‘red’) that we established. Additional members of the ‘orange’ family are indicated by asterisks. The generation of transgenic animals (F2, F3, or F4) is indicated. RNA samples were similar for both quality and quantity, as judged after ethidium bromide staining before blotting, by evaluating the intensity of the 28 S and 18 S ribosomal RNA bands. Blots were hybridized using as a probe a fragment of the E. coli lacZ gene (see Materials and methods).

Histochemical analysis revealed that β-galactosidase activity in the testis was specifically expressed inside seminiferous tubules, within the cytoplasm of haploid germ cells (Fig. 9). Transillumination analysis clearly indicated that specific β-galactosidase staining was much more evident in some areas of the seminiferous tubules than in others, thus suggesting a stage-specific expression of this promoter (Fig. 9, top). This inter-pretation was confirmed through the analysis of frozen sections, in which positivity was very evident in some seminiferous tubules, whereas in other tubules lacZ activity was less intense or absent (Fig. 9, bottom). Accurate analysis of paraffin-embedded sections (Fig. 10) indicated that specific β-galactosi-dase staining was evident in haploid germ cells starting at stage IX, when spermiation has just occurred and round spermatids at steps 8-9 of spermiogenesis cease their transcriptional activity. Positivity progressively moved toward the lumen of the seminiferous tubules: it was clearly evident in stage X and XI, and it declined in stage XII. In stage I some positivity was still evident only in the second layer of haploid germ cells immediately surrounding the lumen (elongating spermatids at step 13 of spermiogenesis), and it was absent from stages II to VII (round spermatids at steps 1-7, and elongating spermatids at steps 14-16). No β-galactosidase activity was detected in seminiferous tubules of non-transgenic animals. β-galactosidase staining observed in interstitial cells, both in control and in transgenic animals, is due to endogenous activity, as already observed by others (Behringer et al., 1993). No expression was found in other organs of transgenic animals (such as brain, liver, kidney, skeletal muscle and heart). The copy number of the integrated transgene did not appear to affect the degree of its expression in spermatids, since one of the families (‘orange’) had multiple copies integrated into the genome, whereas the second one (‘blue’) had only two copies, but expression of lacZ in haploid cells appeared to be of the same intensity. Intensity of lacZ expression appeared to be similar in mice homozygous for the transgene and in heterozygous mice. The two other expressing lines of transgenic animals (‘violet’ and ‘green’) showed a weaker histochemical detection of β-galactosidase activity, again exclusively in seminiferous tubules, whereas members of the fifth family (‘red’) were negative, in perfect agreement with the results of expression at the RNA level.

Fig. 9.

Sequences within the 16th intron of the mouse c-kit gene drive specific expression of the E. coli lacZ reporter gene in male haploid germ cells in vivo. Top: two transilluminated whole seminiferous tubules isolated from the testis of an adult F4 heterozygous transgenic mouse (‘orange’ line), after staining of the whole fixed organ with X-gal. Expression of β-galactosidase is revealed by a green precipitate in the central part of the tubules, which is clearly evident in some areas, but reduced or absent in others. Bottom: two photographs of 30 μm frozen sections of testis from another transgenic animal. The blue-green precipitate, due to β-galactosidase, is evident in haploid germ cells immediately surrounding the lumen of some, but not all, sections of seminiferous tubules. The scale bar equals 75 μm for the two top pictures, 50 μm for the left bottom picture, and 25 μm for the right bottom picture.

Fig. 9.

Sequences within the 16th intron of the mouse c-kit gene drive specific expression of the E. coli lacZ reporter gene in male haploid germ cells in vivo. Top: two transilluminated whole seminiferous tubules isolated from the testis of an adult F4 heterozygous transgenic mouse (‘orange’ line), after staining of the whole fixed organ with X-gal. Expression of β-galactosidase is revealed by a green precipitate in the central part of the tubules, which is clearly evident in some areas, but reduced or absent in others. Bottom: two photographs of 30 μm frozen sections of testis from another transgenic animal. The blue-green precipitate, due to β-galactosidase, is evident in haploid germ cells immediately surrounding the lumen of some, but not all, sections of seminiferous tubules. The scale bar equals 75 μm for the two top pictures, 50 μm for the left bottom picture, and 25 μm for the right bottom picture.

Fig. 10.

Stage-specific expression of the c-kit-lacZ reporter gene during spermiogenesis in transgenic mice. 5 μm sections of paraffin-embedded testis of an adult F4 heterozygous transgenic mouse (‘orange’ line), after staining of the whole fixed organ with X-gal. Sections were stained with PAS and counterstained with hematoxylin. The different pictures represent segments of seminiferous tubules representative of 8 of the 12 stages of the mouse seminiferous epithelium. In each picture, the bottom part represents the periphery of seminiferous tubules, whereas the top part corresponds to the central lumen, with the exception of stage X, in which the lumen is in the middle of the picture (this section being longitudinal instead of a cross-section). Expression of β-galactosidase is revealed by a blue-green precipitate, which is clearly evident in haploid germ cells in stages IX-XI, reduced in stages XII-I, and absent from stages II to VII. Specific testicular cell types: S.C., Sertoli cell; Sg, spermatogonium; Sc I, primary spermatocyte; Sc II, secondary spermatocytes at metaphase; Sd1-Sd16, spermatids from steps 1 to 16 of spermiogenesis. The scale bar equals 6.5 μm.

Fig. 10.

Stage-specific expression of the c-kit-lacZ reporter gene during spermiogenesis in transgenic mice. 5 μm sections of paraffin-embedded testis of an adult F4 heterozygous transgenic mouse (‘orange’ line), after staining of the whole fixed organ with X-gal. Sections were stained with PAS and counterstained with hematoxylin. The different pictures represent segments of seminiferous tubules representative of 8 of the 12 stages of the mouse seminiferous epithelium. In each picture, the bottom part represents the periphery of seminiferous tubules, whereas the top part corresponds to the central lumen, with the exception of stage X, in which the lumen is in the middle of the picture (this section being longitudinal instead of a cross-section). Expression of β-galactosidase is revealed by a blue-green precipitate, which is clearly evident in haploid germ cells in stages IX-XI, reduced in stages XII-I, and absent from stages II to VII. Specific testicular cell types: S.C., Sertoli cell; Sg, spermatogonium; Sc I, primary spermatocyte; Sc II, secondary spermatocytes at metaphase; Sd1-Sd16, spermatids from steps 1 to 16 of spermiogenesis. The scale bar equals 6.5 μm.

Thus, the 16th intron of the mouse c-kit gene contains sufficient information for tissue- and developmental stage-specific transcription of the reporter gene in haploid germ cells, and it is active only in the latest transcriptional steps of round sper-matids (steps 8-9). These data suggest that production of the truncated form of the c-kit receptor should start during the steps of elongation of haploid cells (from step 9 of spermiogenesis), when tail and acrosome formation, and nuclear elongation and condensation occur.

The truncated form of c-kit accumulates in elongating spermatids

A polyclonal antibody, raised against the last 13 amino acids of the cytoplasmic carboxyterminal part of the mouse c-kit receptor, detects a clear membrane staining in mitotic germ cells cocultured with Sertoli cells in tissue explants, whereas no staining is detectable in the underlying somatic monolayer (Fig. 11). Membrane staining in mitotic germ cells is due to the specific expression in type A spermatogonia of the normal approx. 140-160×103Mr receptor, confirmed by western blot analysis (data not shown). Antibodies against the extracellular domain of the c-kit receptor stain only spermatogonia and early spermatocytes, but not spermatids (Yoshinaga et al., 1991). To establish whether a truncated c-kit protein of the size predicted on the basis of molecular cloning of the alternative c-kit transcripts is actually present during the haploid phase of spermatogenesis, we performed western blot experiments using our polyclonal antibody, raised against the cytoplasmic car-boxyterminal part of the mouse c-kit receptor (Fig. 12). A protein of approx. 23×103Mr (A), corresponding to the size predicted by the ORF (606 bp = 202 aa) of the alternative c-kit cDNA (Rossi et al., 1992), can be identified in haploid cells, but not in meiotic spermatocytes (P.Sc.), only with the immune serum. The intensity of the band progressively increases during spermiogenesis (from steps 1–8 to steps 9–12), reaching a maximum in the final steps of spermatid elongation (steps 13-16). The same approx. 23×103Mr protein is also present in mature spermatozoa obtained from the epididymis (S.zoa).

Fig. 11.

The transmembrane c-kit receptor present in spermatogonia from young mice is recognized by antibodies directed against the last 13 amino acids of the mouse c-kit ORF. Immunofluorescence staining with a polyclonal antibody, raised against the cytoplasmic carboxy-terminal domain of mouse c-kit protein, or preimmune serum, of seminiferous tubule explants from 13-day old mice after 4 days of culture. Positive cells are spermatogonia lying on negative Sertoli cell monolayers. The scale bar equals 10 μm.

Fig. 11.

The transmembrane c-kit receptor present in spermatogonia from young mice is recognized by antibodies directed against the last 13 amino acids of the mouse c-kit ORF. Immunofluorescence staining with a polyclonal antibody, raised against the cytoplasmic carboxy-terminal domain of mouse c-kit protein, or preimmune serum, of seminiferous tubule explants from 13-day old mice after 4 days of culture. Positive cells are spermatogonia lying on negative Sertoli cell monolayers. The scale bar equals 10 μm.

Fig. 12.

The truncated form of the c-kit protein translated from the alternative c-kit mRNAs transcribed in round spermatids is accumulated in elongating spermatids, and is also present in epididymal spermatozoa. The first nine lanes show western blot analyses of cell extracts from meiotic (P.Sc., pachytene spermatocytes) and postmeiotic germ cells at the indicated phase of maturation using a polyclonal antibody against the cytoplasmic carboxyterminal domain of mouse c-kit protein or preimmune serum. The lane indicated by an asterisk represents the sample of spermatids at steps 13–16 after a threefold increase in the amount of 2-mercaptoethanol before loading (from 50 mM to 150 mM). The last three lanes show a different experiment, in which cell extracts from epididymal spermatozoa (S.zoa), and from a different preparation of elongating spermatids at steps 9–12, were analyzed with the immune serum. The last lane (C), was treated with the antibody that had been preincubated for specific competition with approx. 100-fold molar excess of the oligopeptide utilized to immunize rabbits. A and B on the right side of the picture indicate the position of the bands specifically recognized by the immune serum.

Fig. 12.

The truncated form of the c-kit protein translated from the alternative c-kit mRNAs transcribed in round spermatids is accumulated in elongating spermatids, and is also present in epididymal spermatozoa. The first nine lanes show western blot analyses of cell extracts from meiotic (P.Sc., pachytene spermatocytes) and postmeiotic germ cells at the indicated phase of maturation using a polyclonal antibody against the cytoplasmic carboxyterminal domain of mouse c-kit protein or preimmune serum. The lane indicated by an asterisk represents the sample of spermatids at steps 13–16 after a threefold increase in the amount of 2-mercaptoethanol before loading (from 50 mM to 150 mM). The last three lanes show a different experiment, in which cell extracts from epididymal spermatozoa (S.zoa), and from a different preparation of elongating spermatids at steps 9–12, were analyzed with the immune serum. The last lane (C), was treated with the antibody that had been preincubated for specific competition with approx. 100-fold molar excess of the oligopeptide utilized to immunize rabbits. A and B on the right side of the picture indicate the position of the bands specifically recognized by the immune serum.

Transfection of COS cells with a eukaryotic expression vector containing the ORF of the spermatid-specific c-kit transcript resulted in expression of an immunoreactive protein of similar size (Fig. 13, left panel). The other aspecific bands, present in both transfected and control COS cells, were also detected by the preimmune serum (not shown). A protein of similar size was specifically detected by the immune serum after expression of the spermatid-specific c-kit ORF in E. coli, using a prokaryotic expression vector (Fig. 13, right panel).

Fig. 13.

A protein of the same size predicted by the ORF of the spermatid-specific c-kit cDNA can be produced in artificial eukaryotic or prokaryotic expression systems. Left panel: western blot analysis of transfected eukaryotic cells using the polyclonal antibody against the cytoplasmic carboxy-terminal domain of mouse c-kit protein. Expression of the truncated c-kit receptor approximating the predicted size of approx. 23×103Mr is evident in COS cells transfected with an eukaryotic expression vector containing the spermatid-specific c-kit ORF, but not in mock-transfected COS cells. Right panel: western blot analysis of transformed prokaryotic cells using the polyclonal antibody against the cytoplasmic carboxy-terminal domain of mouse c-kit protein. Expression of the truncated c-kit receptor of the predicted size (approx. 23×103Mr) is evident in E. coli cells transformed with a prokaryotic expression vector containing the spermatid-specific c-kit ORF, but not in wild-type E. coli cells, nor in transformed bacteria without IPTG induction.

Fig. 13.

A protein of the same size predicted by the ORF of the spermatid-specific c-kit cDNA can be produced in artificial eukaryotic or prokaryotic expression systems. Left panel: western blot analysis of transfected eukaryotic cells using the polyclonal antibody against the cytoplasmic carboxy-terminal domain of mouse c-kit protein. Expression of the truncated c-kit receptor approximating the predicted size of approx. 23×103Mr is evident in COS cells transfected with an eukaryotic expression vector containing the spermatid-specific c-kit ORF, but not in mock-transfected COS cells. Right panel: western blot analysis of transformed prokaryotic cells using the polyclonal antibody against the cytoplasmic carboxy-terminal domain of mouse c-kit protein. Expression of the truncated c-kit receptor of the predicted size (approx. 23×103Mr) is evident in E. coli cells transformed with a prokaryotic expression vector containing the spermatid-specific c-kit ORF, but not in wild-type E. coli cells, nor in transformed bacteria without IPTG induction.

An additional band of approx. 48×103Mr is also specifically recognized by the immune serum in haploid germ cells and spermatozoa (B in Fig. 12), but not in COS cells or in E. coli (Fig. 13). Detection of both the A and B bands in elongating spermatids is suppressed after preincubation of the immune serum with an excess of the peptide that had been utilized to immunize rabbits (lane labelled C in Fig. 12), whereas aspecific bands are still present, thus confirming that the A and B signals should correspond to specific c-kit gene products. We do not know the nature of the approx. 48×103Mr B signal in haploid cells, but a reasonable interpretation is that it reflects a covalent interaction of the approx. 23×103Mr c-kit protein with other proteins present only in haploid cells, due to formation of disulfide bonds between cysteine residues. Indeed its intensity varies according to the sample preparation, it is greatly reduced by increasing threefold the concentration of 2-mercaptoethanol in the samples before SDS-gel electrophore-sis, whereas a corresponding increase in the level of the approx. 23×103Mr band is observed in this condition (see the lane labeled with an asterisk in Fig. 12) and, finally, the progressive accumulation of this band during spermiogenesis parallels exactly that of the approx. 23×103Mr band.

Preliminary immunofluorescence experiments with the same antibody showed that specific immunostaining can be observed in the elongating steps of spermiogenesis. With respect to the background fluorescence observed with anti-bodies from the preimmune serum, fluorescence with the antibody from the immune serum is constantly very strong in the region of the head, and can be observed occasionally in the middle piece of the spermatid tail (Fig. 14). Strong positivity is also constantly observed in residual bodies, which might account for the reduction in the intensity of c-kit immunoreactive bands observed in western blot analysis of epididymal spermatozoa with respect to elongating spermatids at steps 13-16 (see Fig. 12).

Fig. 14.

Elongating spermatids and residual bodies are strongly positive to anti-c-kit antibodies in immunofluorescence experiments. Immunofluorescence staining of postmeiotic germ cells in advanced phases of maturation, with a polyclonal antibody against the cytoplasmic carboxy-terminal domain of mouse c-kit protein or preimmune serum. Spermatids at steps 13-16 and residual bodies had been obtained from adult mouse testes through elutriation techniques. The scale bar equals 10 μm.

Fig. 14.

Elongating spermatids and residual bodies are strongly positive to anti-c-kit antibodies in immunofluorescence experiments. Immunofluorescence staining of postmeiotic germ cells in advanced phases of maturation, with a polyclonal antibody against the cytoplasmic carboxy-terminal domain of mouse c-kit protein or preimmune serum. Spermatids at steps 13-16 and residual bodies had been obtained from adult mouse testes through elutriation techniques. The scale bar equals 10 μm.

The cryptic promoter present within the c-kit gene may be viewed as a further example of haploid-specific transcription;, however it appears to be not only tissue- and cell-specific, but also stage-specific. Indeed, transgenic experiments indicate that β-galactosidase activity starts to be evident in spermatids at steps 8-9, suggesting that a cryptic haploid-specific promoter present within the 16th intron of the c-kit gene is selectively active at the end of the transcriptional phase of spermiogene-sis.

Other promoters active in adult male germ cells identified up to now, such as those for protamine-1 (Peschon et al., 1987), protamine 2 (Stewart et al., 1988), PGK2 (Robinson et al., 1989), ACE (Langford et al., 1991), proenkephalin (Zinn et al., 1991), proacrosin (Nayernia et al., 1992), Hoxa-4 (Behringer et al., 1993), Tcp-10bt (Ewulonu et al., 1993), hst70 (Wis-niewski et al., 1993), p53 (Almon et al., 1993), β-actin (Sands et al., 1993), Zfy-1 (Zambrowicz et al., 1994) and calmodulin II (Ikeshima et al., 1994), have been shown to be active in earlier stages of spermatogenesis (meiosis and/or early spermiogenesis) with respect to what we observe with the cryptic intronic c-kit promoter.

Deletion analysis in both DNA-binding and in vitro transcription experiments suggests that at least two cis-acting elements are essential for stage-specific activation of this promoter during spermiogenesis: the ‘enhancer core’ element between −464 and −456, and the region between −68 and −50, which contains sequences similar to those essential for promoter regulation of interferon and interferon-regulated genes. This observation raises the possibility that related cytokines, which might be locally produced in the microenvironment of the seminiferous epithelium, could influence the expression of this or similar stage-specific promoters during spermiogenesis.

In agreement with the stages of activation of the cryptic c-kit promoter in transgenic mice, immunoblotting and immuno-fluorescence experiments show that a truncated c-kit protein starts to accumulate at the same maturative steps, reaching a maximum in the elongating phase of spermiogenesis, and that the protein is also present in epididymal spermatozoa.

None of the mutations in the W locus identified up to now can lead us to propose a role for the truncated c-kit protein expressed in late spermiogenesis. Deletion of the transmembrane domain of the c-kit receptor in the classical W mutation (Nocka et al., 1991) is accompanied by complete absence of germ cells within the postnatal testis, due to lack of migration and/or proliferation of primordial germ cells in the embryonal gonad (Coulombre and Russell, 1954). A point mutation in the ATP-binding site of the intracellular domain of c-kit in Wv mutants (Nocka et al., 1991) is accompanied by presence of spermatogonia and few meiotic germ cells after birth, but sper-matogenesis is completely arrested after these phases (Coulombre and Russell, 1954), raising the hypothesis of a possible function for the c-kit gene not only in germ cells in the early stages of development, but also in early meiotic stages of spermatogenesis. Mutations in the phosphotransferase domain of the c-kit protein (corresponding to the truncated c-kit protein expressed in postmeiotic germ cells), probably due to a different degree of impairment of tyrosine-kinase function (Nocka et al., 1991), are associated to either a severe phenotype, such as in W42, which is deleterious for gameto-genesis also in the het-erozygous condition (Tan et al., 1990), or to mild effects on male fertility, such as in W41. It cannot be excluded that other, still unknown, mutations in the carboxyterminal domain of the c-kit receptor selectively impair spermiogenesis or sperm cell function, without affecting mitotic germ cells or stem cells of other lineages. Such mutants, not presenting pigmentation defects, would not be easy to identify phenotypically. Since the truncated c-kit protein should lack intrinsic kinase activity, its expression after the beginning of spermatid elongation could imply a tyrosine-kinase-independent role for the c-kit carboxy terminus during sperm cell morphogenesis, as has been proposed for the carboxyterminal domain of Drosophila c-abl protooncogene in axonal outgrowth during neurogenesis (Henkemeyer et al., 1990).

Rather than in sperm cell morphogenesis, the truncated c-kit protein might play a role in mature sperm cell function, during or after fertilization. Indeed, assuming that it has not an intra-acrosomal location, it might be transferred by the spermatozoon into the oocyte. In this case, a functional interaction between the truncated c-kit protein eventually transferred by the fertilizing sperm cell and the full-length c-kit receptor expressed in mature oocytes (Manova et al., 1990; Horie et al., 1991; Yoshinaga et al., 1991) might be possible locally, at the point of membrane fusion. The functional significance of the presence of the full length c-kit receptor in mature oocytes and early embryogenesis is unknown, since the c-kit ligand expression in follicle cells surrounding growing oocytes (Motro et al., 1991) ceases at the end of maturation of the female gamete (Manova et al., 1993). A functional interaction between a full-length tyrosine kinase receptor and a truncated receptor containing only the intracellular kinase domain, with consequent receptor activation, independent of ligand binding, has been recently demonstrated in cotransfection experiments in the case of the EGF receptor (Chantry, 1995).

In the very recent literature, some answers to the functional meaning of haploid specific transcription from alternative promoters of genes normally expressed in diploid cells are coming from gene knock-out experiments, such as the case of ACE (Krege et al., 1995), which has been shown to have an unexpected, important role in male fertility. Promoter sequences within the 16th intron of the mouse c-kit gene could be an ideal target for homologous recombination in order to establish the functional meaning of the truncated receptor in spermatids, without altering the structure of the normal receptor expressed in the mitotic phase of spermatogenesis and in stem cells of the hematopoietic and melanoblastic lineages.

This work was supported by CNR targeted project ‘FATMA’ n. 94.00590.PF41, Telethon-Italy grant n. D.2, grants from AIRC, and by the WHO special program for Research Development and Research Training in Human Reproduction. M.G. was the recipient of an AIRC fellowship. We are indebted to Prof. Vincenzo Sorrentino and Dr Giovanna Marziali for the generous gift of plasmid p-kit-int, and for the preparation of polyclonal antibodies utilized in this work at the EMBL facilities (Heidelberg, Germany). We especially thank Dr Laura Pozzi for her collaboration in the generation of transgenic mice. We also thank Drs Domenica Piscitelli, Maria Laura Giustizieri and Ms Naomi De Luca for their help in the initial stages of this work.

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