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RESEARCH |
Department of Life Sciences, Second University of Naples, Via Vivaldi 43, 81100 Caserta, Italy
Correspondence should be addressed to M M Di Fiore; Email: mariam.difiore{at}unina2.it
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Abstract |
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Introduction |
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In rodent testis, the c-kit expression starts on post-natal day 5 and is restricted to differentiating type A spermatogonia (SPG), type B SPG, primary spermatocytes (SPC; Yoshinaga et al. 1991, Manova et al. 1993, Vincent et al. 1998, Sette et al. 2000) and Leydig cells (Manova et al. 1990, Motro et al. 1991, Rossi et al. 1991). For its exclusive expression, c-kit receptor could be used as a specific marker for these germinal cell populations (Dym et al. 1995, Morena et al. 1996). The c-kit mRNA was found to be expressed at high levels in SPG, and at lower levels in meiotic pachytene SPC (Sorrentino et al. 1991). A truncated version of c-kit was detected in spermatids and spermatozoa (SPZ; Albanesi et al. 1996). Moreover, the immunohistochemical analysis demonstrated the presence of c-kit in acrosomial granules of round spermatids and the acrosomial region of SPZ (Sandlow et al. 1996a, 1996b).
In the testis, the ligand of c-kit receptor stem cell factor (SCF) is uniquely expressed by Sertoli cells (Motro et al. 1991, Rossi et al. 1991, Tajima et al. 1991, Yan et al. 1999). The signalling cascade involving SCF and c-kit-receptor system has been demonstrated to be indispensable for spermatogenesis and spermatogonial proliferation/differentiation/survival (Vincent et al. 1998, Blume-Jensen et al. 2000, Sette et al. 2000). Binding of SCF to c-kit receptor activates the tyrosine kinase receptor, followed by phosphorinositide 3-kinase and p70 S6 kinase activation to promote spermatogonial cell proliferation (Feng et al. 2000). In addition, i.v. injection of blocking anti-c-kit MAB activated Cdc42-associated tyrosine-kinase 2 (ACK2) caused a depletion of differentiating type A SPG (Yoshinaga et al. 1991) with an increased rate of apoptotic death in SPG and in dividing SPC (Packer et al. 1995). In mutant mice, abnormalities of the SCF/c-kit gene expression, such as gene deletions, point mutations, alternative splicing defects, lead to different changes in spermatogenesis (e.g. decreased primordial germ cell migration, decreased in spermatogonial proliferation; for reviews, see Morrison & Takahashi 1993, Loveland & Schlatt 1997).
However, the literature has mainly been focused on the localization and on the role of SCF/c-kit system in mice and rodents, species characterized by continuous spermatogenesis. Only recently were c-kit receptor positive SPG and SPC found in the testis of a seasonal breeder, roe deer, Capreolus capreolus and the changes of c-kit expression correlated to spermatogenetic activity and the cyclic fluctuation of steroid hormones (Roleans et al. 2002). The release of testosterone is known to be a prerequisite for normal sperm production (see, for reviews, Zirkin 1993, Sharpe 1994). On the other hand, SCF is expressed in the testis by Sertoli cells under testosterone stimulation (Mi et al. 2004) and Leydig cells are a target for SCF (Rothschild et al. 2003). The SCF stimulates DNA synthesis in type A SPG cultured in vitro (Rossi et al. 1993, Hakovirta et al. 1999) and the injection of c-kit antibodies blocks their proliferation in vivo (Yoshinaga et al. 1991, Packer et al. 1995). Furthermore, there are several lines of in vitro data that indicate FSH regulates SCF expression in isolated rat Sertoli cells (McLean et al. 2002). In addition, the protein proliferating cell nuclear antigen (PCNA) is essential for the proliferation of SPG. It is also utilized in cell-cycle control through direct interaction with cyclin and cdk complex, where it allows progression through the G1/s boundary (Zhang et al. 1993). For these considerations, PCNA is utilized as an endogenous and molecular marker for mitotic and testicular epithelium proliferation (Chieffi et al. 2000).
Excluding the data reported for mammalian species, there are no studies yet available on c-kit receptor and on its putative involvement in the spermatogenesis of lower vertebrates. Regarding gonadal activity, seasonal breeders offer a good model to study basic mechanisms on the regulation of spermatogenesis without any manipulations. The purpose of this study was to investigate the expression of c-kit receptor in the testis of the amphibian Rana esculenta, which exhibits a well-known seasonality of reproduction (Rastogi et al. 1976, 2005, Di Fiore et al. 2005). The selective labelling of SPG to c-kit receptor protein and its expression during the seasonally activated and reduced proliferation could provide information on the time and the role of interrelationships withotherfactors and cellular activities regulating spermatogenesis. Therefore, to better understand the control mechanisms regulating spermatogenesis, we evaluated the seasonal fluctuations of c-kit expression with both testicular and plasma profiles of testosterone during the reproductive cycle. Finally, the expression of c-kit receptor is assessed and related to mitotic activity of the testis of R. esculenta, tested with both immunolocalization and western blot analyses for PCNA.
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Materials and Methods |
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Seasonal changes in the weight of the testes reflect the state of spermatogenesis. In fact, the seasonal maximum of testicular weight occurs when the testes are in an advanced stage of spermatogenesis and therefore in reproductive period (0.30–0.35 g). In post-reproductive period, the testicular weight decreases until 0.21–0.25 g and the seasonal minimum corresponds to the stage of pre-reproductive period (0.14–0.19 g).
Animals
Taking into account the reproductive characteristics, adult males of R. esculenta were captured in the countryside of Caserta (Italy) during the pre-reproductive (January–February), reproductive (March–June) and post-reproductive (October–December) phases. To evaluate a possible stressing cause that can impair the reproductive processes of male frogs, some animals were killed in the field and the others were transferred to a laboratory terrarium with a photothermal regimen consistent with the period of the year. They were fed on mealworms and Drosophila fruit flies ad libitum. Mortality rates were low (<5%).
The technical procedures illustrated below have been carried out either on group of field animals or on group of animals captured and transferred in laboratory terrarium. No differences have been registered between two groups.
Collection of samples
Soon after capture, animals for each phase of the reproductive cycle were anaesthetized by immersion in a ratio of 1:1000 tap water solution of MS-222 (tricaine methylsulphonate; Sigma). Blood samples were centrifuged at 800 g for 15 min and the resulting plasma was stored at –80 °C for testosterone assay as reported below. The testes were rapidly dissected out. From each animal, one testis was frozen in liquid nitrogen for testosterone assay and western blot analyses, while the other testis was fixed by immersion in Bouin’s fluid and processed for histology and immunohistochemistry (IHC).
The methods of capture and dissection and the captive rearing conditions were in accordance with Italian law (D. L.vo 116/92) and authorized by the appropriate Italian government administrative office (Servizio Veterinario della A.S.L. 44, Prot. Vet. 22/95).
Protein extract preparation
A pool of three frozen frog testes coming from three different animals were homogenized directly into lysis buffer containing 5 mM Tris–HCl, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 10% glycerol, 1% Triton X-100, 1 mM phenylmethylsulphonyl fluoride, 1 µg leupteptin, 0.5 mM sodium orthovanadate, 20 mM sodium pyrophosphate and clarified by centrifugation at 14 000 r.p.m. for 10 min. Protein concentrations were estimated using a modified Bradford assay (Bio-Rad). This procedure has been carried out for all the three phases of the sexual cycle.
Western blot analysis
Forty micrograms of proteins were boiled in Laemmli buffer for 5 min before electrophoresis. The sample was subjected to SDS-PAGE (10% polyacrylamide) under reducing conditions. After electrophoresis, proteins were transferred to nitrocellulose membrane (Immobilion Millipore Corporation, Bedford, MA, USA); completeness of transfer was assessed using prestained protein standards (Bio-Rad). The membrane was treated for 2 h with blocking solution (TBS/T: 5% nonfat powdered milk in 25 mM Tris (pH 7.4); 200 mM NaCl; 0.5% Triton X-100) and was incubated overnight with rabbit-anti c-kit receptor antibody (Santa Cruz, CA, USA) diluted in the ratio of 1:500. After washing with the TBS/T and TBS, membrane was incubated with a horseradish peroxidase-conjugated secondary antibody (1:2000; Chemicon, Temecula, CA, USA) for 60 min and washed again. Indeed, for PCNA detection, a mouse MAB against recombinant PCNA (Dako Corp., Hamburg, Germany) diluted in the ratio of 1:2000 was used. Then the membranes were incubated for 60 min with the anti-mouse peroxidase conjugated (1:5000; Chemicon). Reactions were detected using the ECL system (Amersham Life Science).
The quantification of the bands was obtained by SCAN densitometry. Data are expressed as percentage relative to the maximum value obtained for reproductive period (taken as 100%). The protein levels were determined and quantified in three separate determinations.
Immunohistochemical localization of c-KIT receptor
To investigate the presence of c-kit receptor protein in the germinal epithelium of testis, c-kit immunolabelling was carried out. Five randomly chosen sections of the testis, for each frog from each phase of the sexual cycle (three frogs for phase), were examined. Testicular sections fixed in Bouin’s solution and embedded in paraffin wax were used. The slides were washed in PBS 0.1 M (pH 7.4) for 15 min, treated with 3% H2O2 for 45 min to inhibit endogenous peroxidase and rinsed in PBS. The slides were then incubated in the blocking solution containing 20% normal goat serum (Sigma) with 10% BSA in PBS for 1 h at room temperature. After blocking, the slides were rinsed in PBS–BSA 10% and incubated overnight with polyclonal rabbit anti-c-kit antibody (1:200; Santa Cruz). After three rinses in PBS–BSA 10%, the slides were incubated sequentially with biotinylated goat anti-rabbit antibody (1:500; Sigma) diluted in PBS–BSA 10% and streptavidin (1:200, Pierce, IL, Rockford, USA). Bound antibody was visualized using 3,3'-diaminobenzidine tetrahydrochloride solution (Dako Corp.) according to the manufacturer’s instructions. Sections were counterstained with haematoxylin, dehydrated, mounted and visualized by light microscope.
The scoring of immunoreaction was performed on five randomly chosen sections from each animal testis on the total of five different fields for each slide.
Immunohistochemical localization of PCNA protein
To evaluate germ cells, the PCNA IHC was performed according to a recent study reported by Raucci et al.(2004). Serial deparaffinized sections of frog testes were sequentially incubated in normal goat serum (1:20; Sigma) for 45 min to reduce undesired background staining, primary antiserum (monoclonal mouse anti-PCNA diluted in the ratio of 1:400 with 10% BSA; Dako Corp.) overnight at 4 °C in a dark moist chamber, biotinylated secondary antibody (goat anti-mouse IgG diluted in the ratio of 1:500; Sigma), and streptavidin (1:200; Pierce). The immunopositivity was visualized by exposure to 3,3'-diaminobenzidine tetrahydrochloride in Tris buffer (Dako Corp.). Sections were mounted with a synthetic medium.
Controls
Specificity of immunostaining for both c-kit receptor and PCNA was determined by (a) omission of primary antiserum, (b) substitution of the primary antiserum with 0.1 M PBS or no immune serum (1:200) and (c) primary antiserum preabsorbed overnight with synthetic antigen (only for c-kit receptor). These controls completely eliminated immunostaining.
Histology
After dissection, frog testes were rapidly removed and fixed in Bouin’s fluid for 48 h at room temperature. The fixative was then extracted with 70% ethanol and the tissues were embedded in paraffin wax. The histological exam of the testes was carried out in deparaffinized sections (7 µm), rehydrated and stained with haematoxylin and eosin reaction, as described in Mazzi (1977).
Testosterone assays in plasma and testis
Testosterone determinations in the plasma were conducted utilizing the Enzyme Immunoassay Kits (Adaltis, Bologna, Italy). The following limits of detection were sensitivity 50 pg/ml (intra-assay variability 4.0%, inter-assay variability 9.0%). Plasma samples (100 µl) were vortexed with ethyl ether (1:10, v/v) for 5 min and centrifuged at 3000 g for 10 min. The upper phase (ethyl ether) was transferred to a glass tube. Two extractions were performed. The pooled ether phases were left to evaporate on a hot plate at 40–50 °C under a hood. The residue was dissolved in a 0.5 ml sodium phosphate buffer 0.05 M (pH 7.5), containing BSA at a concentration of 10 mg/ml, and then utilized for the assay. Tissue samples (testis) were homogenized in the ratio of 1:10 (w/v) with distilled water. The homogenate was then mixed vigorously with ethyl ether (1:10, v/v) and the ether phase was withdrawn after centrifugation at 3000 g for 10 min. Three extractions were performed. Pooled ether extracts were dried and then utilized for testosterone immunoassay as previously reported. Testosterone recovery was 85% from plasma and 80% from tissues.
Statistical analysis
Data were compared by ANOVA followed by Duncan’s test for multi-group comparison and the Student’s t-test for between-group comparison. All data were expressed as mean±S.D. The level of significance was taken at P<0.01 and P<0.05.
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Results |
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Expression of c-kit receptor protein during the seasonal cycle
The c-kit receptor protein was characterized by western blot and the specific band of about 150 kDa was detected during the annual cycle with a different expression status (Fig. 1
). Strong expression of c-kit receptor was present in the reproductive period (Fig. 1, lane 2). By contrast, during the pre- and post-reproductive periods, the testis showed a lower expression of c-kit (Fig. 1, lanes 1 and 3 respectively).
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). The cytological elements of germinal epithelium show a positive reaction localized in both plasma membrane and cytoplasm. They were mainly I and II SPG, I and II SPC, elongating spermatids and SPZ. Among the somatic cells, Leydig cells also reacted with the c-kit receptor antibody (Fig. 2). The intensity of immunolabelling for c-kit receptor protein varied depending on the phase of the reproductive cycle. In the reproductive phase (Fig. 2C), the positivity was higher than the pre- (Fig. 2B) and post-reproductive (Fig. 2D) phases. The antibody used for c-kit detection was subjected to immunopreabsorption, revealing that the labelling was totally blocked by preincubation with the cognate peptide (Fig. 2A).
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shows the immunopositivity of different cytological elements of the testis during the sexual cycle. In the reproductive period, strong c-kit positive staining was detected in I SPG (Fig. 3B1); the immunopositivity continued to be seen on I SPG at the pre- (Fig. 3A1) and post-reproductive (Fig. 3C1) periods, but its intensity was less than the reproductive phase. In the pre-reproductive period, II SPG were slightly reacted with the c-kit receptor antibody (Fig. 3A2). In the reproductive and post-reproductive periods, II SPG were intensely positive (Fig. 3B2 and C2 respectively). Moreover, the staining of I and II SPC of pre-reproductive testis (Fig. 3A3 and A4 respectively) was less intense than the reproductive (Fig. 3B3 and B4 respectively) and post-reproductive periods (Fig. 3C3 and C4 respectively). Strong immunopositivity can be seen also in both elongating spermatids and SPZ during reproductive period (Fig. 3B5), although it decreases in post-reproductive period (Fig. 3C5). In the pre-reproductive period, the reaction in SPZ is the lowest observed during all the phases of the sexual cycle(Fig. 3A5).
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reports the seasonal fluctuation of testosterone levels detected in testis and plasma of green frog R. esculenta during the reproductive cycle. In all the major phases of the sexual cycle, substantial amounts of testosterone were found in both testicular extracts and plasma. The androgen level showed significant variation depending on the phase of reproductive cycle and reaching its higher content during the reproductive period (115±8.0 ng/ml testis and 17.4±1.5 ng/g plasma respectively). In the pre- and, particularly, post-reproductive periods, the testosterone content detected was significantly lower (48.6±2.5 ng/g testis, 7.5±0.7 ng/ml plasma and 25.3±1.4 ng/g testis, 2.5±0.4 ng/ml plasma respectively) than the reproductive period (P<0.01). Figure 4 also reports the modification of germinal epithelium of testis through the sexual cycle. The testis of green frog R. esculenta shows a cystic pattern, i.e. the germ cells proliferate in coordinated clusters enclosed in membranous cyst walls (ampoules). In the pre-reproductive period, seminiferous ampoules of testis contained predominantly I and II SPG. Although I and II SPC and SPT were present, their number was low because they underwent degeneration. Some SPZ were present in the lumen. In the reproductive period, testicular epithelium contained the maximal number of I SPG and appeared highly differentiated. The highest number of II SPG was present in the cysts and their massive transformation into I SPC occurred; II SPC was also evident and the lumen was full of SPZ. In the post-reproductive period, high numbers of cysts still contained I and II SPC although their decrease was closely associated with the formation of SPT. Not many SPZ were present in the lumen.
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). The expression of PCNA underwent fluctuation depending on the phase of the cycle. A higher expression of PCNA was seen in the reproductive period (Fig. 5, lane 2) than pre- and post-reproductive periods (Fig. 5, lanes 1 and 3 respectively).
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In the pre-reproductive period, seminiferous ampoules of the testis showed fair immunoreactive material in I SPG multiplying. Moreover, II SPG and I SPC were moderately positive to PCNA (Fig. 6A). In the reproductive period, testicular epithelium showed the highest intensity of immunoreaction for PCNA. The positivity was localized in I and II SPG and in I SPC (Fig. 6B). In the post-reproductive period, the immunopositivity for PCNA was contained in identical cell types observed in the other two periods; but in this phase, the intensity of the immunolabelling was visibly lower than the pre- and post-reproductive phases (Fig. 6C).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Results of western blot analysis demonstrated the expression of c-kit receptor in the testis of this species and indicated the polyclonal antibody recognized a single protein of ~150 kDa. Two forms of c-kit receptor with an apparent molecular size of 124 and 160 kDa have been detected in the rat testis (Dym et al. 1995) and results indicate that the 124 kDa protein could be an immature protein, while the 160 kDa protein is the mature fully glycosylated form of c-kit (Nocka et al. 1989). On the other hand, there may be three isoforms of c-kit receptor proteins in mouse sperm, one isomatic isoform (33 kDa) that is expressed in the sperm tail and two testis-specific isoforms (150 and 48 kDa) that are expressed in the acrosomial region (Feng et al. 1998). It is also possible that the 33 kDa protein is generated by proteolytic cleavage of 150 kDa c-kit receptor protein during sample preparation or represents a cross-reaction with a somatic protein. In addition, more recent reports propose that the binding of c-kit ligand to receptor induces c-kit dimerization autophosphorylation so that the c-kit receptor is present as both membrane-associated and soluble (Kierszenbaum 2006).
Using a rabbit polyclonal anti-c-kit, we also carried out the IHC for c-kit receptor protein in the germinal epithelium of testis. The finding showed that the c-kit receptor protein was predominantly localized on the membrane of I and II SPG, I and II SPC, elongated spermatids and SPZ in all the phases of the yearly cycle. Particularly, strong c-kit positivity staining was observed in the testis during the reproductive period, when the spermatogenesis was strongly activated (Rastogi et al. 1976, 2005, Di Fiore et al. 2005). Since the antibody was raised against the c-terminal domain of the c-kit receptor, which is known to be the intracellular domain of the receptor (Yarden et al. 1987, Qiu et al. 1988), it may be assumed that the signal was membrane associated (possibly intercellular). Previously, Yoshinaga et al.(1991) showed high expression of c-kit receptor in mouse testis in type A SPG, low expression in intermediate and type B SPG and in the earliest preleptotene SPC. Later, it was demonstrated that the c-kit receptor also is expressed in the acrosomal granules of round spermatids and the acrosomic region of SPZ (Albanesi et al. 1996, Sandlow et al. 1996a, 1996b, 1997, Feng et al. 1998), suggesting the receptor’s involvement in some crucial step(s) for capacitation and/or the acrosome reaction (Feng et al. 2005).
C-kit receptor is a conservative molecule and the cDNA has been isolated in lower vertebrates, such as chicken (Sasaki et al. 1993), Xenopus (Baker et al. 1995, Kao & Bernstein 1995) and zebrafish (Parichy et al. 1999). Addition of human recombinant SCF to newt testicular organ cultures stimulates the SPG proliferation and they progressed to the seventh generation, the penultimate stage before the primary spermatocyte stage. However, the SPG do not differentiate into SPC and do not die by apoptosis (Abè et al. 2002). These data are consistent with the previous results in mice where added SCF to cultured male germ cells stimulates DNA synthesis in type A SPG (Rossi et al. 1991, Hakovirta et al. 1999). Moreover, the i.v. injection of ACK-2, a MAB that blocks the binding of SCF to c-kit, causes a loss of differentiating type A SPG, but has no effect on the transition of intermediate SPG to type B SPG or primary SPC (Yoshinaga et al. 1991).
The expression of c-kit receptor protein in the testis of frog R. esculenta evaluated by western blot data parallels cyclic changes of germinal epithelium and is well correlated with both plasma and testicular levels of testosterone. In fact, the highest expression of c-kit receptor occurred during the reproductive period, when seminiferous ampoules were completely differentiated and both testicular and plasma content of T was at the maximum peak. Instead, during the pre- and post-reproductive periods, the expression of c-kit as well as testosterone levels was lower. The fluctuation of c-kit receptor protein was also correlated to mitotic activity of the testis, evaluated by both western blot and immunohistochemical detection of PCNA during the sexual cycle. In the reproductive period, in fact, PCNA expression was the highest as well as the immunoreactivity of germinal epithelium to this mitotic factor. These findings suggest that c-kit receptor could be implied in the proliferation of the germinal epithelium of R. esculenta. On the another hand, Roleans et al.(2002) observed that in roe deer, Capriolus capriolus, the testis showed a specific pattern of spermatogonial c-kit receptor positive cell, in relation to testosterone levels.
In the amphibian R. esculenta, the highest level of testosterone during the reproductive phase is beneficial for spermatogenesis and the development and maturation of the androgen-dependent target organ (Rastogi et al. 1976, Delrio et al. 1980, Varriale & Serino 1994, Di Fiore et al. 2005). On the other hand, Raucci et al.(2004) demonstrated that the increase of testosterone titres causes and increase of seminiferous ampulae area and their spermatid and sperm contents. In addition, in vitro experiments carried out on cultured testicular germ cells of embryonic chickens demonstrated that the testosterone treatment induced stronger c-kit and PCNA expressions, indicating that this hormone promoted testicular germ cell proliferation probably through indirect effects on Sertoli cells (Mi et al. 2004). Furthermore, in cultured cells of mouse SPG, SCF enhanced c-kit expression of type A SPG by stimulating their progression into the mitotic cell cycle (Dolci et al. 2001).
In our study, light microscopy revealed that Leydig cells show a c-kit immunopositive reaction. c-kit localization in Leydig cells has already been known (Sandlow et al. 1996a, 1996b, 1997) and its expression is connected to a paracrine regulation of Leydig cell steroidogenetic function (Rothschild et al. 2003). However, soluble SCF might participate in the regulation of formation of Leydig cell population during testicular development. These data indicate that there is a reciprocal control between SCF production by Sertoli cells, testosterone production by Leydig cells and c-kit expression (Yan et al. 2000).
In conclusion, our study demonstrates for first time in non-mammalian vertebrate testis, the presence of c-kit receptor protein and suggests a direct subtle cooperation between the testosterone and the expression of c-kit during the seasonal cycle of R. esculenta. In addition, mitotic activity of germinal epithelium is well correlated with plasma and testicular levels of the androgen hormone.
As demonstrated in mammals, in R. esculenta, c-kit could play a pivotal role in the timing of the steps of the spermatogenesis. Future investigations should address the precise function of c-kit receptor in the processes of spermatogenesis. This will open further investigation on the involvement of regulatory factors in the proliferation of the SPG.
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Footnotes |
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References |
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, I SPC;&
, II SPC; asterisk, spermatozoa (x1000).
) and plasma (
) of green frog Rana esculenta, during the reproductive cycle. Results are expressed as mean±S.D. obtained from five individual animals. Histological sections of testis of Rana esculenta during pre-reproductive, reproductive and post-reproductive periods. Arrowhead, I SPG; arrow, II SPG; 
