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Regular Article |
a Institute for Zoo Biology and Wildlife Research, PF 601103, 10252 Berlin, Germany
b Department of Biochemistry, Research Institute for the Biology of Farm Animals, D-18196 Dummerstorf, Germany
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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FSH, LH, seasonal reproduction, spermatogenesis, testes, testosterone
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The optimal timing of the generation of functionally efficacious spermatozoa requires a fine-tuning of the controlling mechanisms. Subsequent steps of spermatogenesis and the seasonally changing activity of regulating factors result in a typical seasonal pattern of cellular composition in roe deer testes, as described by several studies [1–4]. The proliferative activity of testicular cells is intensified early before the rut and is already reduced during the rut. It is important to establish the relationship between the several testicular cell types on the basis of absolute numbers of cells to understand the kinetics of the proliferative processes, as recently emphasized in studies of interstitial cells in rat testis [5].
The initial step for the production of spermatozoa is the proliferation of spermatogonia. Proliferation and survival of spermatogonia depend on interactions of c-kit receptor and its ligand SCF ("steel factor") (for reviews, see [6, 7]). In mice, the c-kit receptor is exclusively expressed in spermatogonia and could be used as a specific marker for the population of type A1 through A4 spermatogonia [8, 9]. Immunostaining of c-kit was found in vitro on the plasma membrane of undifferentiated as well as of differentiating A spermatogonia [10]. The selective labeling of spermatogonia during the seasonally activated and depressed proliferation could provide information regarding the time and role of interrelations with other factors and regarding cellular activities for regulation of spermatogenesis. However, localization and role of the c-kit/SCF system have mainly been studied in mice and rats. Data from ruminants are spare and from roe deer, to our knowledge, not available at all.
Therefore, the purpose of the present study was to use selective labeling of the c-kit receptor on spermatogonia in roe deer and to evaluate the seasonal variation in the quantity and proliferation of those c-kit-positive cells in relation to the serum concentrations of gonadotropins and testosterone as well as to the amount of testicular testosterone.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Thirty adult male roe deer (age 
2 yr) were examined between June 1998 and June 2000 (n = 2–4 per mo). Samples were obtained immediately postmortem from 18 hunted wild animals.
Twelve other bucks were kept under semifree-ranging conditions in large, outside enclosures. Testicular tissue of these animals was obtained by castration. For the surgery, animals were immobilized by i.m. administration of xylazine (2 mg/kg body mass [bm]; Rompun 10%, Bayer, Leverkusen, Germany) in combination with ketamine (4 mg/kg bm; Ketamin 10%, Essex, München, Germany) using a blow pipe. Anesthesia was maintained by application of 1.5%–2% (v/v) isofluran (Forene, Abott, Wiesbaden, Germany) via an endotracheal tube. Before removing the testes, blood was taken to measure the serum level of testosterone, FSH, and LH. After the procedure, the sedative effect of xylazine was reversed with atipamezol (200 µg/kg bm; Antisedan, Pfizer, Karlsruhe, Germany).
After its removal, the testis was dissected into small pieces. Some parts were cooled to 4°C for flow cytometry and estimation of testosterone. The other parts were fixed for histological and immunohistological evaluation.
Five of the roe bucks in captivity were available during 1 annual cycle for repeated blood collection (before castration) and estimation of FSH, LH, and testosterone in blood serum.
Preparation of Testes
The testes and epididymides were separated, and the testes were weighed. To count the number of testicular spermatozoa, 0.2 g of decapsulated testis were carefully pressed through a 28-µm nylon mesh and suspended in 4 ml of medium M199 supplemented with 0.4% (w/v) BSA (Sigma, Deisenhofen, Germany). The germ cell suspension was diluted in an equal volume of water. The osmotic-resistant elongated spermatids and spermatozoa were counted with a hemocytometer. Results are expressed as spermatozoa per gram of testis.
Flow Cytometric DNA Analysis
The proliferation of testicular cells was quantified by one-parameter flow cytometric analysis of the proportion of haploid cells (1C), diploid cells (2C), and cells in the G2/M phase of mitosis (4C).
Testicular cells were prepared according to a procedure described earlier [4]. The cells were dispersed by finely mincing 0.1 g of decapsulated testicular tissue in 1 ml of 100 mM citric acid and 0.5% (v/v) Tween 20. The released nuclei were dispersed by gentle agitation for 20 min and stained with 0.175% (w/v) 4',6-diamidino-2-phenylindol (DAPI; Sigma) in 5 ml of 400 mM Na2HPO4. The flow cytometric analysis was performed in a PAS III flow cytometer (Partec GmbH, Münster, Germany) with a mercury lamp (OSRAM GmbH, Berlin, Germany) using a wavelength of 360 nm for excitation and 420 nm for emission. The cell concentration (cells/ml) was measured in each sample, allowing us to calculate the exact number of cells per gram of testis and per testis, respectively. The DNA histograms obtained were analyzed by DPAC computer software (Partec GmbH) to determine the proportions of cells in each peak. The contents of 1C (spermatids and spermatozoa), 2C (spermatogonia, secondary spermatocytes, and somatic cells), and 4C cells (all cells in the G2/M phase of cell cycle, mainly primary spermatocytes) were expressed as percentages and total amounts (cells/g tissue and cells/testis, respectively).
Flow Cytometric Differentiation of Somatic and Spermatogenic Cells
Somatic and spermatogenic cells were differentiated by dual-parameter flow cytometry after DNA staining and fluorescein-labeling of the vimentin in the somatic cells. Here, the indirect immunofluorescence method was used as previously described by Roelants [11, 12]. In brief, suspended cells were fixed by addition of cold (-20°C) ethanol (96% [v/v]) and stored for 24 h before further preparation. The samples were washed twice with PBS and resuspended in PBS. Then the samples were incubated with a monoclonal antivimentin antibody (clone V9; Sigma) at a dilution of 1:40 for 60 min. Subsequently, the cells were twice washed with PBS (plus 2% [w/v] BSA) and resuspended in PBS. They were next incubated with fluorescein isothiocyanate (FITC)-conjugated anti-mouse antibody (final dilution, 1:50) for 30 min in the dark. After two wash steps with PBS/2% BSA, the pellet was resuspended in a solution of 1 mg/ml of ribonuclease (Sigma) and 50 µg of propidium iodide (Sigma) and incubated at 37°C for 30 min in the dark. The samples were filtered through a 28-µm nylon mesh before analysis with the flow cytometer.
Flow Cytometric Detection of c-kit Receptor-Positive Cells
A method was developed to detect testicular cells bearing the c-kit receptor by flow cytometry, which enables calculation of the amount of c-kit receptor-positive cells in the total spermatogenic 2C and 4C cells. To achieve this, the following indirect immunofluorescence method was used: an aliquot of 0.1 g of testicular tissue was carefully pressed through a 28-µm nylon mesh and suspended in 2 ml of PBS. These suspended cells were washed twice and resuspended in PBS. The sample was incubated in the dark with a monoclonal anti-human c-kit-receptor antibody (clone K44.2; Sigma) at a dilution of 1:2 for 60 min. Subsequently, the cells were washed twice with PBS (plus 2% BSA) and resuspended in PBS. They were incubated with FITC-conjugated anti-mouse antibody (final dilution, 1:50) for 30 min in the dark. After two wash steps with PBS/2% BSA, the pellet was resuspended in PBS and fixed by addition of cold (-20°C) ethanol (96%) at a dilution of 1:2 and stored for 24 h before further preparation. After two wash steps with PBS/2% BSA, the pellet was resuspended in a solution of 1 mg/ml of ribonuclease and 50 µg of propidium iodide and incubated at 37°C for 30 min in the dark. The samples were filtered through a 28-µm nylon mesh before analysis with the flow cytometer.
Fluorescence distributions in both cases were analyzed using the PAS III illuminated with an argon laser at a wavelength of 488 nm. A negative control omitting the primary antibody was included for each sample. Calculation of vimentin-positive cells and of c-kit receptor-positive cells was corrected for the low nonspecific fluorescence. Vimentin-positive and -negative cells represent the somatic and spermatogenic types, respectively. They were calculated both as relative and total amounts by peak analysis using the PAS III software. The same procedure was used for the c-kit receptor-positive cells. Both relative (%) and total amounts (cells/g tissue and cells/testis) of these cells were related to the spermatogenic cells within the 2C and 4C peaks.
Immunohistochemical Evidence of c-kit Receptor
Classification of c-kit-positive cells as spermatogonia was corroborated by immunohistochemical control. The tissue was fixed in Bouin solution, then embedded in paraffin. All sections were cut at a thickness of 3 µm, mounted, and deparaffinized. To unmask the epitopes, we heated the deparaffinized and hydrated sections at 100°C in a 50 mM glycine solution (pH 3.5) for 10 min. To block the endogenous peroxidase activity, the sections were afterward incubated with 3% (v/v) hydrogen peroxide (H2O2) in methanol for 30 min. After rinsing twice with Tris-buffered saline (TBS; 0.1 M Tris [pH 7.4], 0.8% [w/v] NaCl, and 0.0015% [v/v] Triton X-100), the sections were preincubated with blocking buffer (0.1 M Tris [pH 7.4], 0.8% Triton X-100, 0.8% NaCl, and 20% [w/v] normal goat serum; Sigma). The excess blocking buffer was then removed from the slides before incubating them with the primary antibody (1:2 monoclonal anti-human c-kit receptor antibody; clone K44.2) overnight at 4°C. After rinsing twice with TBS, the slides were incubated with the second antibody (1:200 goat anti-mouse IgG peroxidase conjugate; Sigma) for 2 h at 37°C. Then, after again rinsing twice with TBS, the slides were stained with 0.05% (w/v) 3'3-diaminobenzidine tetrahydrochloride in 0.05 M Tris-HCl buffer (pH 7.6) containing 0.01% (v/v) H2O2. After counterstaining with hematoxylin, the sections were dehydrated and mounted. For the negative control of each sample, the primary antibody was omitted. All incubation steps were carried out in a moist chamber.
Estimation of Testosterone by Enzyme Immunoassay
Testicular testosterone was measured by an enzyme immunoassay with a double-antibody technique as described earlier [13]. In short, triplicates of 200 µg of tissue were extracted with 1 ml of ethanol:water (70:30, v/v). The extract was diluted with assay buffer as required, then 2 samples of 20 µl each were analyzed. For testosterone estimation in blood, 0.1 ml of serum was extracted with 2 ml of butyl t-methyl ether:petroleum ether (30:70, v/v) for 30 min. The samples were frozen, and the fluid petroleum ether phase was removed and evaporated at 55°C. The steroids were re-equilibrated with 1 ml of 40% (v/v) methanol, and duplicates of 20 µl each were analyzed.
The assay used a polyclonal antibody raised in rabbits against testosterone-11-hemisuccinate-BSA, and the label was testosterone-3-carboxymethyl-oxime-horse radish peroxidase. The testosterone standard curve ranged from 0.4 pg per 20 µl to 50 pg per 20 µl, and the cross-reactivity with testosterone was 100%, with 5
-dihydrotestosterone 10%, with androstenedione 2%, with estradiol <0.1%, and with progesterone <0.1%. The results are given in nanograms of testosterone per gram of testis and in nanograms per milliliter of serum, respectively. The intra- and interassay coefficients of variation (CVs) were 8.9% and 12.3%, respectively.
Estimation of LH and FSH by Electrochemiluminescence Immunoassay
In contrast to conventional chemiluminescence, with enhanced chemiluminescence, the chemiluminescent species are generated at the surface of an electrode.
For LH, a sandwich test was developed according to the method of Deaver [14]. The electrochemiluminescence immunoassay (ECLIA) was performed in 12 x 75-mm polypropylene tubes. An N-hydroxysuccinimide ester of ruthenium(II)-tris-bipyridine chelate (Ru-ester) was used to label the monoclonal antibody 518 B7 against bovine-LH (b-LH) [15] as recommended by the manufacturer (IGEN, Gaithersburg, MD). The yellow-colored product was fractionated on PD-10 columns prepacked with Sephadex G-25 (Amersham Pharmacia Biotech, Freiburg, Germany) by single elution with buffer. The fraction containing the labeled antibody was stored in the refrigerator; it is stable for more than 1 yr [16]. The standard b-LH (iodination grade) was provided from Biotrend (Köln, Germany). The polyclonal anti-rabbit b-LH was identical with that used in an LH-RIA [17]. The Ru-labeled monoclonal antibody (25 µl) and polyclonal antibody (50 µl) were allowed to bind to the hormone (standard/sample, 20 µl). The standard curve was started at 122 ng/ml and diluted throughout 0.03 ng/ml. After overnight incubation, a "second" antibody, sheep anti-rabbit IgG coupled to magnetic beads (50 ml; Dynal, Oslo, Norway), was added. Finally, the measurement of chemiluminescence was performed in the Origen analyzer (IGEN). It took approximately 1 h per carousel sample changer fitted with 50 tubes. The sensitivity of the method was 0.03 ng/ml. The intra- and interassay CVs were 6.4% and 8.9%, respectively.
The FSH-ECLIA was configured as a competitive assay with a pure ovine standard (oFSH; AFP 7571 A) and a first antibody raised in rabbits (NIDDK-oFSH-1). The labeling of the standard hormone was performed in the same manner as described above for the LH monoclonal antibody. The second antibody coupled to magnetic beads (Dynal) was used to perform the bound/free separation. Serum volumes of 50 µl were used. The sensitivity of the method was 0.1 ng/ml. The intra- and interassay CVs were 8.6% and 9.9%, respectively.
The status of roe deer as a wild animal and our conditions of keeping the animals did not allow us to estimate the daily pulsating course of the gonadotropins without stressing the animals, which is known to influence LH release by a changed GnRH secretion pattern.
Statistical Analysis
For each variable, seasonal mean ± SEM was calculated.
One-way ANOVA was used to investigate the variation among monthly means for testis weight, each of the cellular parameters, and the testicular testosterone concentration. Subsequently, a contrast analysis was performed for each month by testing its mean parameter value against the overall mean of all months. The significance of the resulting P values was evaluated according to the Dunn-Sidak method. Furthermore, each testicular variable was summarized for the breeding (July + August) and the 2 nonbreeding seasons with an intensified (April) and an inhibited (November + December) testicular proliferation and also analyzed via ANOVA. Pairwise post-hoc tests served to compare the seasons.
Because the gonadotropin and testosterone concentrations in serum were measured from the same 5 bucks for the whole year, the repeated-measurements ANOVA was applied to test potential differences between months within the annual cycle. The results of the subsequent pairwise post-hoc tests are shown only for the month with the highest mean (see Figs. 5 and 6). The Kendall coefficient of concordance was used to control the parallel annual course of each serum hormone concentration for the individual bucks.
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Additionally, the seasonal hormone concentrations were compared by calculation of cross-correlation coefficients for 1 yr. These coefficients allow us to detect a potential phase shift of the annual concentration pattern for different hormones. Because this analysis requires a complete data set, missing values for February had to be estimated as the mean of the January and March values.
All calculations were performed using the SPSS 9.0 (SPSS Inc., Chicago, IL) statistical software package. The significance level was always set to 5%. In case of multiple tests, the significance information is provided, but not P values.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The relative and total amounts of both germinal and somatic 2C and 4C cells within the respective total peaks also changed during the annual cycle. The percentages of spermatogenic 2C cells for the breeding (July + August) and the two main nonbreeding seasons (April, November + December) were 60.42% ± 6.42%, 80.36% ± 6.90%, and 40.78% ± 9.86%, respectively. The corresponding proportions of 4C cells were 40.03% ± 7.56%, 58.20% ± 4.79%, and 50.45% ± 11.52%, respectively.
The flow cytometric analysis showed distinct c-kit-positive cells within the populations of 2C and 4C cells after indirect labeling with the monoclonal mouse anti-c-kit receptor-antibody and the FITC-conjugated anti-mouse antibody (Fig. 2). The unspecific FITC-labeling remained at <1% and, therefore, is not shown in Figure 2.
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The immunohistochemical tests performed with the same anti-c-kit-receptor antibody revealed that the cells showing a positive reaction were mainly spermatogonia and some early type I spermatocytes. Among the somatic cells, it seems that some Leydig cells also slightly reacted with the c-kit-receptor antibody (Fig. 3).
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The relative amount of c-kit receptor-positive 2C and 4C cells was the lowest during the rut (July to August) and showed high values from October to April (Fig. 4A). Calculation of the amount of c-kit receptor-positive cells among the 2C cells per gram of testicular tissue shows the highest values from October to February and the lowest value in July. The 4C cells had their maximum from March to June and their minimum in July (Fig. 4B). The highest total amounts of c-kit receptor-positive 2C cells per testis were found during October to November and in the period of February to May. The highest values for the c-kit receptor-positive 4C cells appeared in the prerutting period (April to June), and a slightly increased value was found in September (Fig. 4C). The differences among the months were significant for all variables (percentage of 2C, percentage of 4C, 2C cells/g testis, and 4C cells/g testis; P < 0.001, 2C cells/testis; P = 0.023, 4C cells/testis; P = 0.007).
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These values of c-kit-positive cells were compared for combined data of the breeding (July and August) prebreeding (April), and postbreeding season (November and December). The data show significantly lower percentages of 2C and 4C cells during the rut compared to the nonbreeding periods (Table 2). The differences in numbers of cells per gram of testicular parenchyma were significant between rut and postrut (2C cells) and between rut and prerut (4C cells), respectively. Surprisingly, the total numbers of 2C spermatogonia are significantly higher during nonbreeding seasons than during the rut, in spite of the markedly reduced total mass of testes.
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The annual blood hormone concentration profiles for the 5 bucks can be regarded as parallel (Kendall W: testosterone; W = 0.859, P < 0.001; LH: W = 0.427, P = 0.019; FSH: W = 0.514, P = 0.004). This justifies the calculation of mean annual cycles.
Testosterone concentrations in serum and in the testicular tissue seemed to show two peaks: a minor one in April to May, and a major one during the early rutting period (Fig. 5). The first peak, however, was not statistically significant. The peak in July/August coincides with the period of the most intensive meiotic activity. Thereafter, the testosterone concentration dropped steeply, to low levels in autumn and winter. The differences among the monthly means were significant for both concentration profiles (P < 0.001 for each).
The serum gonadotropins, LH and FSH, showed a maximum in March (Fig. 6) that preceded the testosterone peak. The differences among the monthly means were significant for both gonadotropins (LH: P = 0.006; FSH: P = 0.005). The cross-correlations showed that the curves of LH and FSH are significantly congruent, whereas the serum testosterone showed congruence with LH and FSH only by a significant phase shift of 5 mo.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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It has been shown that c-kit and its ligand SCF play a key function in the formation, development, and function of the male gonad in the mouse and the rat: They are essential for the survival of primordial gonocytes and for the proliferation and survival of the postnatal spermatogonia [6, 7, 19]. The c-kit proto-oncogene encodes the c-kit transmembrane tyrosine kinase receptor. This 150-kDa c-kit protein is localized in the cell membrane of spermatogonia and early type I spermatocytes. Some authors have detected this membrane protein also on Leydig cells [8, 20–22]. Its ligand SCF is expressed by the surrounding Sertoli cells [23–25]. A truncated c-kit protein (24–28 kDa) can be detected in spermatozoa, elongated spermatids, and, rather sparsely, in round spermatids [26, 27]. Until now, FSH induction of the soluble form of SCF in Sertoli cells and its stimulation of DNA synthesis in type A spermatogonia has represented the only example of a link between the hormonal stimulation of testicular somatic cells and the release by these cells of a growth factor that directly promotes germ cell proliferation [7, 24].
Studies on c-kit receptor in male gonads have been performed primarily in the mouse, rat, and human. Little information is available regarding other species, such as the Djungarian hamster [28] and cattle [29]. To our knowledge, no such studies have been carried out for wild-living ruminants.
The typical quantitative fluctuation of the different testicular cells shown by flow cytometric analysis in this study corresponds to that described in several, mostly histological studies of seasonal changes in roe deer testis [1–4, 30, 31]. The main results of these reports showed that, during the nonbreeding period, the testicular tissue contains only somatic cells, with the exception of Leydig cells, and spermatogonia as the sole germinative cells. In February, the spermatogonia start to proliferate, and spermatogenesis begins. In March, the first Leydig cells are found. In May, all spermatogenic cell types are detectable. During the prerutting period, increasing spermatogenesis leads to a high quantity of functional spermatozoa during the rut in July and August. From the end phase of the rut onward, spermatogenesis is turned down, probably to make sure that, during the nonrutting period in winter, the energy-intensive sexual activity is suppressed. Spermatogenesis comes to an end in October to November, and Leydig cells detectable by light microscopy disappear in October.
Our results of DNA flow cytometry reflect this seasonality of sperm production. In winter, we found a high 2C peak and a very low 4C peak, containing more somatic than germinal cells. In spring, the stimulated proliferation can be seen as an increasing 1C peak, 2C peaks of medium height, and high 4C peaks. During this period, the relative amount of spermatogenic cells within the 2C and 4C peaks also increases. The number of spermatozoa and spermatids with a highly condensed nucleus increases rather slowly during the prerut period, but then rapidly from early July to August. The maximum of the 1C peak shows the most intensive meiotic transformation during the rut. The diminishing 4C peak reflects the already decreasing proliferation during the same period. The changes in proportions of the different cell types demonstrate the strongly reduced proliferation and the stop of meiotic activity during the involution of testes in autumn and winter. Through the disappearance of spermatocytes and spermatids, the proportion of somatic cells in the 2C and 4C peaks expands during this period.
The flow cytometric analysis shows that the amount of c-kit receptor-positive 2C cells has its maximum in the nonbreeding period and its minimum in the rut. The c-kit-positive 4C cells show the highest peak before the rut but a minimum also during the rut. Considering that c-kit receptor may act as a marker for spermatogonia [9] and the results of the light microscopic control (i.e., that those c-kit-positive cells are mainly spermatogonia and some early type I spermatocytes), the results of the flow cytometric analysis may be interpreted as follows: During the nonbreeding period in winter, the spermatogenic cells are only spermatogonia. In the time before the rut, the spermatogonia have an intensified proliferation, indicated by the high c-kit receptor-positive 4C peak. During the increasingly active spermatogenesis, the amount of spermatogenic cells other than spermatogonia also increases, which results in decreasing relative amounts of c-kit-positive cells, with a minimum in the rut. Decreasing spermatogenesis leads to the opposite effect. Light microscopy has revealed that a few Leydig cells also show a slight c-kit receptor-positive reaction. These cells also proliferate strongly in the period from March to May. Further studies are necessary to clearly identify the slight amount of somatic cells that show a weakly positive reaction.
The findings of the analysis suggest a specific time pattern of spermatogonial proliferation in the circannual cycle. The expanding abundance of c-kit-positive cells in November and December is caused by the disappearance of spermatocytes and spermatids. This results in a higher relative frequency (percentage) and an increased total quantity of spermatogonia per gram of parenchyma. The number of spermatogonia per testis also evidences an increase in the total testicular content of spermatogonia, with the highest value in December in spite of the distinct reduction in testis mass. Hence, cell division seems to continue on a level sufficient to accumulate spermatogonia during winter, and these form the necessary source for the creation of primary spermatocytes by the strongly stimulated mitosis the next spring.
The amount of testosterone in the testis and serum of roe deer shows a significant maximum at the beginning of the rutting period (end of July). This major peak corresponds with the period of maximal production of spermatozoa. The subsequent drop of testicular androgen level begins at the end of the rut, which is in concordance with observations from other studies [30, 32]. The slight serum testosterone increase in spring is significantly different from the peak value in August and, at best, indicates a tendency toward a biphasic pattern during the annual periods. Such a pattern, however, would be in accordance with our findings from an earlier study [4] and with reports of a biphasic increase in plasma testosterone concentration during the reproductive cycle in adult roe deer [33–35]. The reported first peak occurred in May; this is the period when the spermatogenesis is fully functional, the first spermatozoa are found in the lumen of the seminiferous tubules, and the proportion of cells in the G2/M phase is enhanced. A biphasic seasonal pattern of reproductive hormones was also observed in other cervids [36, 37]. This phenomenon was further described for pubertal testis growth, during which similar cell processes take place, in various species, such as the ram [38], pig [39], and bull [40, 41].
The presented results suggest the important and complex functional role of testosterone in males. Testosterone is a prerequisite for normal spermatogenesis (for reviews, see [42–44]). Testosterone also plays an essential role in preventing apoptotic cell death in androgen-dependent tissues (for reviews, see [45, 46]). The level of apoptosis is inversely related to both the proliferation and the testosterone concentration in roe deer testis during the annual cycle [47]. Thus, the composition of testicular parenchyma seems to be determined by seasonal changes of both proliferation and apoptosis.
The seasonal peaks of both gonadotropins occurred in spring, and it seems to precede the increase in testosterone concentration. Apparently, the LH peak in March causes activation of the differentiated Leydig cells, which start to synthesize and to release testosterone and, probably, specific growth factors. This then results in an increased testosterone production. During the FSH peak, the Sertoli cells are activated, which start to synthesize and to release growth factors such as SCF, the c-kit-receptor ligand. This could explain the highest amount of c-kit-positive cells in the G2/M phase in April: The proliferation of the spermatogonia, as the carrier of the c-kit receptor, is accelerated.
The subtle cooperation between the gonadotropins and testosterone and the seasonal expression of several growth factors [48] seems, therefore, to be very important for the initiation and maintenance of successful spermatogenesis in the roe deer. This was also described by other authors investigating pubertal testis growth [38–41, 49] and adult spermatogenesis [50–54] in various species and is in disagreement with findings that a single hormone, such as FSH, is sufficient to initiate and to maintain a fully "normal" spermatogenesis [55].
In conclusion, this study demonstrates, to our knowledge for the first time, c-kit receptor-positive cells in the testis of roe deer. The method described in this paper makes it possible to gain more detailed information regarding the specific timing of quantitative, seasonal changes in the c-kit receptor-positive cell population, which immunohistological control has identified mainly as spermatogonia. This will open further possibilities to investigate the relationships of regulatory factors, such as gonadotropins, testosterone, and growth factors, with the proliferation of spermatogonia, which play a crucial role in the time hierarchy of the steps in spermatogenesis. Additionally, it could help to shed light on regulation of the "turning-on" and "turning-off" of spermatogenesis in male gonads of seasonal breeders.
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FOOTNOTES |
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1 Supported by grant BL 319/6-1 from the Deutsche Forschungsgemeinschaft. 
2 Correspondence: Hannelore Roelants, Institute for Zoo Biology and Wildlife Research, Alfred-Kowalke-Straße 17, 10315 Berlin, Germany. FAX: 0049 30 5 12 61 04;roelants{at}izw-berlin.de
Accepted: September 13, 2001.
Received: June 20, 2001.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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