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RESEARCH |
1 Centre for Reproduction and Development, Monash Institute of Medical Research, Monash University, Clayton, Victoria 3168, Australia, 2 Department of Experimental Radiation Oncology, University of Texas M D Anderson Cancer Center, Houston, Texas 77030, USA, 3 Reproductive Science Group, Discipline of Biological Sciences, University of Newcastle, Callaghan, New South Wales, Australia and 4 Australian Research Council Centre of Excellence in Biotechnology and Development
Correspondence should be addressed to K L Loveland; Email: Kate.loveland{at}med.monash.edu.au
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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In the mouse, c-Kit mRNA is detected at 7.5–12.0 days postcoitum in primordial germ cells, during their lineage specification and migration to the developing gonadal ridge (Manova & Bachvarova 1991), and animals with mutations in the genes encoding either SCF or c-Kit exhibit aberrant germ cell migration and proliferation during this period (for review see Mauduit et al. 1999, Zama et al. 2005). The c-Kit mRNA was also detected by in situ hybridization within clones grown in vitro from the germ cells of the newborn mouse testis, which are termed gonocytes, although the functional significance of this observation is uncertain, as no protein expression data were provided (Hasthorpe et al. 1999). In addition, lack of in situ protein expression has been previously reported in gonocytes (Yoshinaga et al. 1991). In contrast to the mouse, rat gonocytes have been shown to express both c-Kit mRNA and protein (Orth et al. 1996) which was indicated to be required for their normal migration from the centre to the perimeter of the seminiferous cord from day-1 to day-5 postpartum in vitro (Orth et al. 1997). Various studies employing in situ hybridization, immunohistochemistry and Northern and Western blotting at time-points concordant with spermatogonial differentiation have reported relatively high levels of c-Kit expression in the cytoplasm of the differentiating spermatogonia and persisting low levels in the meiotic pachytene spermatocytes (Manova et al. 1990, Sorrentino et al. 1991, Dym et al. 1995). The requirement for SCF/c-Kit interactions for progression to and survival in the meiotic pachytene stage of spermatogenesis has also been indicated (Packer et al. 1995, Vincent et al. 1998). However, amongst these studies persist discordant conclusions about the timing and expression pattern of c-Kit mRNA and protein at various stages in the juvenile and adult rodent testis, and no single study has addressed this issue in rats and mice simultaneously. An alternative c-Kit transcript, the tr-kit, lacking the extracellular, trans-membrane and part of the intracellular kinase domain of the c-Kit receptor protein, is expressed in post-meiotic male germ cells (Albanesi et al. 1996).
More recently, the elevation of c-Kit expression has been linked with the commitment of spermatogonial stem cells to differentiate (Schrans-Stassen et al. 1999). It has also been reported that c-Kit-positive germ cells among the prospermatogonia population of the juvenile testis (termed undifferentiated spermatogonia by Schrans-Stassen et al. 1999) have less repopulating activity than the c-Kit-negative corresponding population (Ohbo et al. 2003). This is in general agreement with studies showing that only c-Kit-negative stem spermatogonia exhibit regenerative activity when transplanted into the adult mouse testis (Shinohara et al. 1999). Thus, knowledge of the timing of c-Kit expression and its regulation is essential for further studies regarding establishment and maintenance of the stem cell population during the first wave of spermatogenesis.
In this study we interrogated the expression profile of c-Kit mRNA and protein in two independent systems, purified spermatogonia derived from 8-day-old mice testes and irradiated adult rat testis. The irradiated testis model provides a powerful avenue for investigating the regulation of c-Kit expression spermatogonia, as irradiation destroys the radiosensitive differentiating spermatogonia, leaving the testis populated only by undifferentiated spermatogonia (Kangasniemi et al. 1996). Gonadotrophin-releasing hormone (GnRH) antagonists stimulate recovery of spermatogenesis when applied at 15 weeks after irradiation and recovery is assessed by scoring the percentage of tubules containing germ cells that have reached the B spermatogonial stage or later, termed the repopulation index. By 4 weeks of treatment, the repopulation index displays a significant increase relative to the 3-week treatment group, and this index increases steadily in subsequent weeks (Shuttlesworth et al. 2000). In order to examine whether the stimulated recovery of spermatogonial differentiation with GnRH antagonist treatment is linked to changes in the SCF/c-Kit signalling system, we studied the mRNA and protein expression profiles for c-Kit in this model. Surprisingly, in both of these systems we observed that c-Kit mRNA could be detected in the absence of protein expression.
This report is the first comprehensive comparison of the timing and expression of c-Kit mRNA and protein cellular expression in developing and adult mouse and rat testes. From examination of purified spermatogonial populations and adult irradiated rat testes we have provided evidence that, in purified juvenile mouse germ cells and recovering adult rat testes, c-Kit mRNA and protein are differentially regulated in spermatogonia. Our observations are in agreement with previous findings that, based on the age of the postnatal testes, spermatogonial differentiation is indeed concordant with the expression of c-Kit protein.
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Materials and Methods |
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For studies on irradiated testes, adult LBNF1 male rats (hybrids between Lewis and Brown–Norway) were purchased from Harlan Sprague–Dawley, Inc. (Indianapolis, IN, USA). Animals were allowed to acclimatize for 1 week before initiation of the experiment. All procedures were approved by the M D Anderson Cancer Institutional Animal Care and Use Committee.
Irradiation and hormone treatment
Animals were anaesthetized with 0.72 mg ketamine/kg and 0.022 mg acepromazine/kg (i.m.), and then a single 6 Gy dose was delivered to the lower part of the abdomen by a 60Co 
-ray unit (Eldorado 8; Atomic Energy of Canada, Ltd, Ottawa, Canada) as described previously (Shuttlesworth et al. 2000). GnRH antagonist treatment was also performed as previously described (Shuttlesworth et al. 2000). Briefly, 15 weeks after irradiation animals were given simultaneous injections of 1.5 mg Cetrorelix pamoate and 1.5 mg Cetrorelix acetate (ASTA Medica, AG Frankfurt, Germany), each at a different site in the upper portion of the dorsal region. A second dose of Cetrorelix pamoate injection was given 3.3 weeks after the first. This dose continuously suppresses intratesticular testosterone levels by at least 80% for 5 weeks (Shuttlesworth et al. 2000).
Preparation of testes sections
Tissue samples for in situ hybridization and immunohistochemistry were placed in Bouin’s fixative for 5 h immediately after collection, then dehydrated through a graded ethanol series and embedded in paraffin. Sections of 3–5µm were dried onto Superfrost Plus II slides (Menzel-Glaser, Braunswchweig, Germany).
Isolation of spermatogonial fractions
For isolation of spermatogonia, decapsulated testes from 20–30 8-day postnatal Swiss mice were pooled and incubated for 15 min each in 0.5 mg/ml collagenase (Sigma)/Dulbecco’s modified Eagles’ medium (DMEM; Invitrogen Australia Pty Ltd, Mount Waverley, Victoria, Australia) with agitation and then in 0.25% (v/v) trypsin/EDTA in DMEM. Tubules were dissociated manually by pipetting and washed in 0.5% (v/v) bovine serum albumin (BSA) in DMEM by centrifugation. Cell pellets were resuspended in DMEM and filtered twice through a 70µm membrane, then separated over a 2–4% BSA gradient as described (Baleato et al. 2005). Fractions were collected based on pilot experiments in which labelling with the germ cell marker, GCNA (Wang & Enders 1996), morphological characteristics and lectin labelling (Arya & Vanha-Perttula 1986) were used to identify spermatogonia. Cell numbers were quantified (typically 3–6 x 106 cells/ml), and the cells were washed and resuspended in DMEM supplemented with 10% (v/v) fetal calf serum (FCS).
Laminin (Sigma; 10µg in 500µl) in phosphate-buffered saline (PBS) was used to coat the bottom of six-well tissue culture plate wells by overnight incubation at 37°C. Laminin was removed, the wells were washed three times with 2 ml PBS, and then the dishes were treated with 2 ml 0.5 mg/ml BSA in PBS for 1 h at 37°C. The wells were then rewashed in PBS three times. Washed, isolated germ cells were resuspended in 2 ml DMEM/10% (v/v) FCS in wells at a final concentration of 4 x 105/well and incubated for 1 or 2 h at 32°C. Unbound cells were removed and centrifuged at 1500 r.p.m. for 5 min at 4°C, washed twice in PBS and then fixed in 4% paraformaldehyde for 30 min. Cells bound to laminin-coated wells were removed by incubation with 0.25% (v/v) trypsin/1 mM EDTA at room temperature for 5 min followed by vigorous pipetting. Cells were transferred to centrifuge tubes and washed and fixed as described above. Isolated laminin-bound and -unbound spermatogonia were placed onto slides and probed for expression of c-Kit mRNA and protein expression.
Quantitative mRNA analysis
For real-time PCR analysis of each postpartum time-point, total RNA from Swiss mice (two animals pooled per sample, with duplicate samples) and Sprague–Dawley rats (one individual animal per sample, three samples per time-point) were prepared using the Qiagen RNeasy (Qiagen, Victoria, Australia) following the manufacturer’s instructions for mouse samples and the acid–phenol extraction method for rat samples (Chomczynski & Sacchi 1987) Each total RNA sample was treated with DNA-free (Ambion, Austin, TX, USA) according to the manufacturer’s specifications. Five hundred nanograms of this DNA-free total RNA was used for each 20µl reverse transcription reaction with 100 U Superscript III reverse transcriptase (Life Technologies, Grand Island, NY, USA) and oligo-dT primer, according to the enzyme manufacturer’s guidelines. Negative control reverse transcription samples were included and these did not have any Superscript III.
PCR samples were prepared in a final volume of 10µl using Roche Diagnostics (Castle Hill, NSW, Australia) SYBR-Green PCR master mix containing 500 nM each forward (F) and reverse (R) primers for mouse (accession number: NM_021099 [GenBank] c-Kit; F, 5'-tcatcgagtgtgatgggaaa-3'; R, 5'-ggtgacttgtttcaggcaca-3') and rat (accession number: NM_022264 [GenBank] c-Kit; F, 5'-ctggtggttcagagtttccatagac-3'; R, 5'-tcaacgaccttcccgaagg-cacca-3') and 1µl reverse-transcribed template. PCR was performed in the LightCycler 2.0 Instrument (Roche Molecular Biochemicals, Mannheim, Germany) using the following light cycler conditions: denaturation 95°C for 10 min, amplification 95°C for 15 s, 60°C for 5 s, 72°C for 10 s and 72°C for 7 min for 48 cycles. Melting curve analysis and agarose gel electrophoresis were used to monitor accumulation of the PCR products.
The PCR reaction for each sample was performed in duplicate. Negative controls, in which water was used in place of the reverse-transcribed template, were included for each primer pair to detect PCR amplification of any contaminating DNA. The amounts of Actb mRNA (accession number: NM_007393 [GenBank] ; F, 5'-aggctgtgctgtccctgtat-3'; R, 5'-aaggaaggctggaaaagagc- 3') for the mouse samples and GAPDH mRNA (accession number: BC013852 [GenBank] ; F, 5'-ttggtatcgtggaaggactca-3'; R, 5'-tgtcatcatatttggcaggttt-3') for the rat samples were measured in each sample template to enable normalization for sample loading of target gene values between samples. The selection of an appropriate housekeeping standard involved comparing abundant mRNAs in the testis over the entire age series. When an analysis of both GAPDH and Actb was performed to validate those genes as standards, they each demonstrated constant abundance over the different ages. Actb for the mouse and GAPDH for the rat was chosen to reflect the alignment of their amplification pattern with the number of cycles required for the c-Kit cDNA crossing points. To correlate the crossing points from the sample amplification plot with target mRNA copy number, a standard curve was produced for each product in every experiment using cDNA containing the target gene. The data were calculated as the means ± S.D.
In situ hybridization on tissue sections
In situ hybridization was used to localize c-Kit mRNAs in mouse and rat testis sections using digoxygenin (DIG)-labelled sense and anti-sense c-Kit probes. The probes were initially generated by producing a PCR product from mouse testis cDNA corresponding to base pairs (bp) 89 through 448 of the mouse clone accession number Y00864
[GenBank]
. The PCR product of 454 bp was cloned into the pGEM T-Easy plasmid (Promega, Madison, WI, USA) following the manufacturer’s instructions. The product was verified by sequencing, and it was shown by Northern blot analysis to hybridize to a single band of 5.5 kilobase pairs, as expected for c-Kit mRNA (Qiu et al. 1988, data not shown)
The plasmid containing the c-Kit cDNA fragment was used to produce sense and antisense DIG-cRNAs using SP6 and T7 RNA polymerases in separate reactions containing DIG-UTP (Roche Molecular Biochemicals) and probe labelling was quantified according to the specifications of the DIG-UTP manufacturer. Hybridization was performed using 150 ng/µl of each DIG-cRNA, with sections incubated overnight at 55°C. Washes were performed in decreasing concentrations of SSC (at 2 x , 1 x and 0.1 x ) at the hybridization temperature. Single strength SSC (1 x ) is 0.15 M sodium chloride and 0.015 M sodium citrate. Treatment with anti-DIG antibody (Roche Molecular Biochemicals; 1:1000 in Roche 1 x blocking solution) and then final development with 5-bromo-4-chloro-3-iodylphosphate/nitro blue tetrazolium (BCIP/NBT; Astral Scientific, Sydney, Australia) at room temperature in a light-restricted box was performed to visualize the cell types containing c-Kit mRNA. When sufficient signal had developed, the sections were stained with Harris haematoxylin (Sigma) and mounted in GVA aqueous mounting solution (Zymed Laboratories Inc., San Francisco, USA). Both anti-sense and sense (negative control) cRNAs were used at the same concentration on each sample, in every experiment, for each set of conditions tested.
Immunohistochemistry on tissue sections
Immunohistochemistry with anti-c-Kit antibodies was undertaken to characterize the cellular c-Kit protein expression profile in tissue sections. Antigen retrieval was performed in 50 mM glycine (pH 3.5; >90°C maintained for 8 min). For detection of c-Kit protein in rat and mouse testes sections, anti-CD117, a rabbit anti-human antibody (DAKO, Botany, NSW, Australia; used at 0.5–2.0µg/ml) and C-19, a rabbit anti-peptide antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA, used at 0.1– 0.5µg/ml) were used as a primary antibodies diluted in 5% (v/v) of normal sheep serum in 0.1% BSA/TBS and sections were incubated overnight at room temperature. Subsequent TBS washes were performed at room temperature between incubations. Primary antibody binding was detected using a biotinylated anti-rabbit antibody (DAKO; biotin anti-rabbit raised in sheep; 1:500 dilution in TBS, 1 h) and then the Vectastain Elite ABC kit according to the manufacturer’s instructions (Vector Laboratories, Burlingame, CA, USA). Antibody binding was detected as a brown precipitate following development with 3,3'-diaminobenzidine tetrahydrochloride (DAB; Sigma), and Harris haematoxylin was used as the counter-stain. The sections were mounted under glass coverslips in Depex (BDH Laboratories, Poole, Dorset, UK).
The two different antibodies used were independently verified by Western blots and recognized proteins of the expected sizes (Nocka et al. 1989, data not shown).
Detection of c-Kit mRNA and protein in isolated spermatogonia
For detection of mRNA in isolated cell preparations, the c-Kit probe was prepared and quantified as described above. Washed spermatogonia were air-dried onto wells in glass slides and then washed (2 x 5 min in PBS, 1 x 5 min in PBS/0.02% (v/v) Triton X-100, 2 x 5 min in PBS). Cells were then incubated in proteinase K for 5 min at 37°C (5µg proteinase K/ml) and then incubated in 4% (v/v) paraformaldehyde at 4°C for 15 min. Cell were washed (2 x 5 min in PBS) and hybridization was performed overnight at 52°C (25 ng probe/well) in a humid chamber. Slides were washed (5 min in 2 x SSC/50% (v/v) formamide) at 55°C twice and then 5 min in 1 x SSC twice. This was followed by treatment as described above with anti-DIG antibody and then final development with BCIP/NBT (Astral Scientific) at room temperature to visualize the cells containing c-Kit mRNA.
For immunohistochemical detection of c-Kit protein, following fixation and washing (2 x ) in PBS/0.02% (v/v) Triton X-100, spermatogonia were allowed to settle and adhere to 12-well slides overnight at 4°C. The slides were washed once in PBS, permeablized with 100% methanol for 5 min and washed again in PBS. The slides were blocked using 3% (v/v) BSA/PBS for 1 h at room temperature, then treated with peroxidase (3% H2O2 in PBS) for 10 min at room temperature, washed twice in PBS and incubated with the C-19 primary antibody at 1:100 in 5% (v/v) normal rabbit serum in PBS overnight at 4°C. The slides were washed twice in PBS, incubated with a secondary antibody labelled with horseradish peroxidase (sheep anti-rabbit; DAKO) at 1:100 for 1 h at room temperature, and antibody binding was detected as a brown precipitate following development with DAB for 5 min, followed by treatment with Harris haematoxylin counterstain. The cells were mounted under glass coverslips in Depex.
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Results |
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). In situ hybridization analysis of the mouse testis indicated no discernable staining at day 2 (Fig. 1B) and day 5 (data not shown). The c-Kit mRNA signal was first evident at day 7 in the cytoplasm of some spermatogonia (data not shown). This timing is in accord with the appearance of differentiating spermatogonia (McCarrey 1993). The signals in the day-10 (Fig. 1C) testis samples were confined to the cytoplasm of the differentiating spermatogonia. In addition to differentiating spermatogonia cytoplasmic staining, there was also spermatocyte staining with a relatively lesser intensity and interstitial staining in the adult mouse testis (Fig. 1D).
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’) or at day 5 (data not shown); however, staining was evident in some interstitial cells associated with the persistence of some fetal-type Leydig cells at these ages (Fig. 1B’). Signals were first evident in some spermatogonia in the day7 testis, consistent with the in situ hybridization results (data not shown), and these were distinct in spermatogonia in the day-10 (Fig. 1C’), day-15 (data not shown) and adult testis samples (Fig. 1D’). However, protein was observed in some spermatocytes from day 10 and higher ages at a lesser intensity. The adult testis also displayed protein expression of elongated spermatids (Fig. 1D’). This staining could be attributed to the expression of tr-kit in these specific germ cell types since the antibodies used recognize an intracellular sequence in the protein (Albanesi et al. 1996). tr-kit mRNA expression was, however, not detected because the probe was designed to correspond to a region encoding the extra-cellular domain of the c-Kit protein.
Expression of c-Kit mRNA and protein in isolated mouse spermatogonia
Both the laminin-bound and -unbound populations were probed for the presence of c-Kit mRNA and protein. This separation was intended to discriminate between spermatogonia with stem cell properties (laminin bound) and those that lack stem cell properties (laminin unbound) which can be expected to have differentiated based on the transplantation studies of Shinohara et al.(1999). We predicted that the latter population would be more likely to contain c-Kit mRNA and protein, while the former would not. In situ hybridization examination of isolated spermatogonial cells (>95% GCNA-1 positive) showed that c-Kit mRNA expression was detectable in the cytoplasm of 100% of spermatogonia from both the laminin-bound and -unbound populations (Fig. 2A). In contrast to the in situ hybridization data, protein detection in the cytoplasm of spermatogonia occurred in 54 ± 2% of laminin-bound and 69 ± 4% of the laminin-unbound cells (means ± S.E.M.) (Fig. 2B). These experiments were performed at least three times for both mRNA and protein expression. Cells with obvious red cytoplasmic staining in the presence of primary antibody were scored as positive for c-Kit protein.
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). To more precisely assess the pattern of change in mRNA levels, day-12 testis samples were included in a subsequent analysis (data not shown) with samples spanning day 9 through 26, and the day-12 sample had a higher relative level of c-Kit mRNA compared with either day 9 or day 15. Thus the peak in expression of this mRNA most likely occurs between day 9 and day 15. In situ hybridization analysis of the 4-day postnatal rat testes did not show any detectable signal within gonocytes or in any other cell type (Fig. 3B). At day 13 and day 15, a weak to moderate signal for c-Kit mRNA was observed in the cytoplasm of spermatogonia and spermatocytes (data not shown). The signal appeared to be more intense and distinct in spermatogonia of day 19, with staining in only some spermatocyte (Fig. 3C). Adult rat testes also exhibited similar staining patterns (Fig. 3D). Both day-19 and adult testes had detectable interstitial staining.
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’) and day-6 rat testis (data not shown). Since no mRNA signal was detected in gonocytes it is possible that the c-Kit protein is produced in male germ cells of the fetal testis, as previously documented (Manova & Bachvarova 1991) and the protein remains stable within these cells for several days. A similar observation has been made for the activin ßA subunit in the fetal and newborn rat testis (Meehan et al. 2000). A relatively weak signal was observed in differentiating spermatogonial subtypes at day 13 (data not shown), while at days 15 (data not shown) and 19 (Fig. 3C’) most of the differentiating spermatogonia had readily detectable brown cytoplasmic staining, as did some pachytene spermatocytes. In the adult testis, c-Kit protein was observed in differentiated spermatogonia, pachytene spermatocytes and elongated spermatids (Fig. 3D’). As in the case of the in situ hybridization localization of c-Kit mRNA, the protein signal was not evident in all pachytene spermatocytes. The antibody staining of pachytene spermatocytes was more readily detectable in the day-15 and day-19 testis sections than in those from the adult testes, giving the impression that the protein was present in more of these cells and at a higher level. Staining of a distinct intracellular vesicle was also noted within some pachytene spermatocytes. Interstitial staining was present in the day-19 and adult testes sections (Fig. 3C’ and D’). Tr-kit staining in the elongating spermatids was evident in the adult testis (Fig. 3D’).
Irradiated adult rat testis: a comparison of mRNA and protein expression
In situ hybridization analysis of irradiated rat testis samples showed that c-Kit mRNA was detectable in the cytoplasm of spermatogonial cells from rats that received no GNRH antagonist treatment (Fig. 4A), 1 week of treatment (Fig. 4B), 2 weeks of treatment (Fig. 4C) and 4 weeks of treatment (Fig. 4D). Staining in interstitial cells, including Leydig and endothelial cells was particularly prominent in the samples from animals treated with GNRH antagonist for 4 weeks.
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’), 3 days (data not shown), 1 week (Fig. 4B’) and 2 weeks (Fig. 4C’) of treatment. The signal was, however, readily evident in cells with the appearance of differentiating spermatogonia that were present in the 4-week GnRH antagonist treatment group (Fig. 4D’). Leydig cell staining was present in all samples, in agreement with the in situ hybridization data. These data were consistently observed using two different antibodies (CD117 and C-19 (data not shown)) that were verified as recognizing c-Kit protein by Western blot. These results were identical in triplicate experiments performed using testis sections from three individual animals. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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. In essence, this study has illustrated the relationship between c-Kit mRNA and protein expression and highlights the key developmental time-points for the onset of differentiation of both rat and mouse germ cells during the first wave of spermatogenesis. Published descriptions of c-Kit localization and expression do not come to a common conclusion about the expression of c-Kit mRNA and protein in pachytene spermatocytes (Manova et al. 1990, Sorrentino et al. 1991, Dym et al. 1995). In this study, both mRNA and protein expression data identified c-Kit in pachytene spermatocytes and showed that it is undetectable in round spermatids. The functional significance of expression in pachytene spermatocytes could relate to the survival of these cells during meiosis (Yan et al. 2000). The full-length c-Kit is, therefore, present both in the cytoplasm, and presumably the plasma membrane, of differentiating spermatogonia and pachytene spermatocytes in both rat and mouse. Comparison of mRNA levels measures in the total rat and mouse testis RNA by real-time PCR analysis with the appearance of c-Kit mRNA within specific cell types by in situ hybridization, in both rodents, revealed that expression levels increase at time-points concordant with the appearance of differentiating spermatogonia containing this transcript (McCarrey 1993). In comparison with the spermatogonial and spermatocyte staining, there was weak to no staining in the Leydig cells, a finding which suggests that Leydig cells contribute little to the total level of c-Kit mRNA in the testis at all ages tested. To complete this study, we were required to undertake a comparison of two commercially available antibodies that recognize the intracellular domain of the rodent c-Kit protein, and similarities and differences in their target recognition became apparent from this analysis. Though both antibodies (CD117 and C-19) gave similar outcomes when applied to Bouin-fixed sections of rat testis, only the CD117 antibody gave a clear signal on mouse testes sections.
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Our examination of the irradiated rat testis model provides a clue that factors regulating c-Kit protein synthesis in the testis are influenced by its exposure to hormonal stimulation. Coupling the present data with previous observations of spermatogonial morphology in these testes, we now understand that c-Kit protein expression occurs only when differentiating spermatogonia emerge in the testes of irradiated rats treated with the GnRH antagonist for 4 weeks and more (Shuttlesworth et al. 2000). In a study of W/W v mutant mice, which produce non-functional c-Kit receptor protein, spermatogonia expressing c-Kit are present in 2-week-old W/W v mutant mice but are absent in 10-week-old animals (Ohta et al. 2003). Administration of a GNRH antagonist for 4 weeks to reduce intratesticular testosterone in 10-week-old W/W v mice resulted in the emergence of c-Kit-positive spermatogonia. While production of c-Kit mRNA was not assessed in the latter study, it appears that reduction of the intratesticular testosterone concentration stimulates differentiation of spermatogonia in a manner independent of c-Kit function, but leading to the onset of c-Kit protein expression. Another known factor to stimulate the expression of surface c-Kit protein is BMP4, as shown by the expression of surface c-kit protein on 4-day postnatal spermatogonia after exposure to BMP4 (bone morphogenetic protein-4; Pellegrini et al. 2003). Microarray data for BMP4 expression in the irradiated rat testes indicate an elevation in this mRNA after 2 weeks of GnRH antagonist treatment group (O U Bolden-Tiller & M L Meistrich, unpublished results). These data coupled with our localization studies on c-Kit expression suggest that there could be translational regulation of c-Kit by BMP4. Current studies are underway to assess BMP4 protein expression at this stage.
In conclusion, the results of our study identify the key time-points and cell types for c-Kit mRNA production in the developing rodent testis and indicate that mRNA is present in the absence of detectable levels of protein. It also indicates that a spermatogonial stem cell could have c-Kit mRNA expression and exhibit regenerative capacity, while corresponding protein expression in the spermatogonia results in lesser potential for regeneration. This observation is particularly intriguing, as it provides the opportunity to design appropriate studies for analysis of the regulation of both c-Kit mRNA transcription and its translation into protein, the latter of which is a key determinant of germ cell survival, proliferation and migration.
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Acknowledgements |
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Footnotes |
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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Albanesi C, Geremia R, Giorgio M, Dolci S, Sette C & Rossi P 1996 A cell- and developmental stage-specific promoter drives the expression of a truncated c-kit protein during mouse spermatid elongation. Development 122 1291–1302.[Abstract]
Arya M & Vanha-Perttula T 1986 Comparison of lectin-staining pattern in testis and epididymis of gerbil, guinea pig, mouse, and nutria. American Journal of Anatomy 175 449–469.[CrossRef][Web of Science][Medline]
Baleato RM, Aitken RJ & Roman SD 2005 Vitamin A regulation of BMP4 expression in the male germ line. Developmental Biology 286 78–90.[CrossRef][Web of Science][Medline]
Chomczynski P & Sacchi N 1987 Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Analytical Biochemistry 162 156–159.[Web of Science][Medline]
Dym M, Jia MC, Dirami G, Price JM, Rabin SJ, Mocchetti I & Ravindranath N 1995 Expression of c-kit receptor and its autophosphorylation in immature rat type A spermatogonia. Biology of Reproduction 52 8–19.[Abstract]
Hasthorpe S, Barbic S, Farmer PJ & Hutson JM 1999 Neonatal mouse gonocyte proliferation assayed by an in vitro clonogenic method. Journal of Reproduction and Fertility 116 335–344.
Kangasniemi M, Huhtaniemi I & Meistrich ML 1996 Failure of spermatogenesis to recover despite the presence of spermatogonia in the irradiated LBNF1 rat. Biology of Reproduction 54 1200–1208.[Abstract]
Loveland KL & Schlatt S 1997 Stem cell factor and c-kit in the mammalian testis: lessons originating from Mother Nature’s gene knockouts. Journal of Endocrinology 153 337–344.
McCarrey JR 1993 Development of the germ cell. In Cell and Molecular Biology of the Testis, pp 58–89. Eds C Desjardins & LL Ewing. Oxford: Oxford University Press, Inc.
Manova K, Nocka K, Besmer P & Bachvarova RF 1990 Gonadal expression of c-kit encoded at the W locus of the mouse. Development 110 1057–1069.
Manova K & Bachvarova RF 1991 Expression of c-kit encoded at the W locus of mice in developing embryonic germ cells and presumptive melanoblasts. Developmental Biology 146 312–324.[CrossRef][Web of Science][Medline]
Mauduit C, Hamamah S & Benahmed M 1999 Stem cell factor/c-kit system in spermatogenesis. Human Reproduction Update 5 535–545.
Meehan T, Schlatt S, O’Bryan MK, de Kretser DM & Loveland KL 2000 Regulation of germ cell and Sertoli cell development by activin, follistatin, and FSH. Developmental Biology 220 225–237.[CrossRef][Web of Science][Medline]
Nocka K, Majumder S, Chabot B, Ray P, Cervone M, Bernstein A & Besmer P 1989 Expression of c-kit gene products in known cellular targets of W mutations in normal and W mutant mice, evidence for an impaired c-kit kinase in mutant mice. Genes and Development 3 816–826.
Ohbo K, Yoshida S, Ohmura M, Ohneda O, Ogawa T, Tsuchiya H, Kuwana T, Kehler J, Abe K, Scholer HR & Suda T 2003 Identification and characterization of stem cells in prepubertal spermatogenesis in mice. Developmental Biology 258 209–225.[CrossRef][Web of Science][Medline]
Ohta H, Tohda A & Nishimune Y 2003 Proliferation and differentiation of spermatogonial stem cells in the w/wv mutant mouse testis. Biology of Reproduction 69 1815–1821.
Orth JM, Jester WF Jr & Qiu J 1996 Gonocytes in testes of neonatal rats express the c-kit gene. Molecular Reproduction and Development 45 123–131.[CrossRef][Web of Science][Medline]
Orth JM, Qiu J, Jester WF Jr & Pilder S 1997 Expression of c-kit gene is critical for migration of neonatal rat gonocytes in vitro. Biology of Reproduciton 57 676–683.[CrossRef]
Packer AI, Besmer P & Bachvarova RF 1995 Kit ligand mediates survival of type A spermatogonia and dividing spermatocytes in post-natal mouse testes. Molecular Reproduction and Development 42 303–310.[CrossRef][Web of Science][Medline]
Pellegrini M, Grimaldi P, Rossi P, Geremia R & Dolci S 2003 Developmental expression of BMP4/ALK3/SMAD5 signaling pathway in the mouse testis: a potential role of BMP4 in spermatogonia differentiation. Journal of Cell Science 116 3363–3372.
Qiu FH, Ray P, Brown K, Barker PE, Jhanwar S, Ruddle FH & Besmer P 1988 Primary structure of c-kit: relationship with the CSF-1/PDGF receptor kinase family – oncogenic activation of v-kit involves deletion of extracellular domain and C terminus. EMBO Journal 7 1003–1011.[Web of Science][Medline]
de Rooij DG & Grootegoed JA 1998 Spermatogonial stem cells. Current Opinion in Cell Biology 10 694–701.[CrossRef][Web of Science][Medline]
Rothschild G, Sottas CM, Kissel H, Agosti V, Manova K, Hardy MP & Besmer P 2003 A role for kit receptor signaling in Leydig cell steroidogenesis. Biology of Reproduction 69 925–932.
Schrans-Stassen BH, van de Kant HJ, de Rooij DG & van Pelt AM 1999 Differential expression of c-kit in mouse undifferentiated and differentiating type A spermatogonia. Endocrinology 140 5894–5900.
Shinohara T, Avarbock MR & Brinster RL 1999 beta1- and alpha6-integrin are surface markers on mouse spermatogonial stem cells. PNAS 96 5504–5509.
Shuttlesworth GA, de Rooij DG, Huhtaniemi I, Reissmann T, Russell LD, Shetty G, Wilson G & Meistrich ML 2000 Enhancement of A spermatogonial proliferation and differentiation in irradiated rats by gonadotropin-releasing hormone antagonist administration. Endocrinology 141 37–49.
Sorrentino V, Giorgi M, Geremia R, Besmer P & Rossi P 1991 Expression of the c-kit proto-oncogene in the murine male germ cells. Oncogene 6 149–151.[Web of Science][Medline]
Vincent S, Segretain D, Nishikawa S, Nishikawa SI, Sage J, Cuzin F & Rassoulzadegan M 1998 Stage-specific expression of the Kit receptor and its ligand (KL) during male gametogenesis in the mouse: a Kit-KL interaction critical for meiosis. Development 125 4585–4593.[Abstract]
Wang & Enders 1996 Expression of a specific mouse germ cell nuclear antigen (GCNA1) by early embryonic testicular teratoma cells in 129/Sv-Sl/+ mice. Cancer Letters 100 31–36.[CrossRef][Web of Science][Medline]
Yan W, Suominen J & Toppari J 2000 Stem cell factor protects germ cells from apoptosis in vitro. Journal of Cell Science 113 161–168.[Abstract]
Yoshinaga K, Nishikawa S, Ogawa M, Hayashi S, Kunisada T, Fujimoto T & Nishikawa S 1991 Role of c-kit in mouse spermatogenesis: identification of spermatogonia as a specific site of c-kit expression and function. Development 113 689–699.[Abstract]
Zama AM, Hudson FP & Bedell MA 2005 Analysis of hypomorphic kitlsl mutants suggests different requirements for KITL in proliferation and migration of mouse primordial germ cells. Biology of Reproduction 73 639–647 Epub 2005 May 25.
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