University of Pittsburgh School of Medicine, Center for Research in Reproductive Physiology, Department of Cell Biology and Physiology, Pittsburgh, PA 15261, USA
1 To whom correspondence should be addressed to: University of Pittsburgh School of Medicine, Department of Cell Biology and Physiology, W952 BST, 3500 Terrace Street, 15261 Pittsburgh, PA, USA. Email: schlatt{at}pitt.edu
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Abstract |
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Key words: Macaca mulatta/non-human primates/proliferation/rhesus monkey/spermatogonial stem cells
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Introduction |
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In the present study we extended our novel bio-imaging approach with additional scientific tools including organ culture and detection of label-retaining cells to functionally define several features of spermatogonial stem cells and their differentiating progeny in the primate testis. The aims of the present study are (i) to unequivocally determine that the starting point of spermatogenesis in the monkey testis is a division of Apale spermatogonia at stage VII of the seminiferous epithelial cycle, (ii) to substantiate the proposed model for the kinetics of spermatogonial expansion (Ehmcke et al., 2005) and (iii) to determine whether Adark spermatogonia represent a population of true stem cells with a very low rate of proliferative activity. Our studies are highly relevant to extend our very limited knowledge of human spermatogonial stem cells since the human testis contains the same two subtypes of spermatogonia. It is quite likely that human spermatogonial expansion follows similar patterns as observed in non-human primate testes.
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Materials and methods |
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Tissue preparation
For preparation of tissue sections testicular tissue samples were fixed overnight at room temperature (RT) in fresh Bouin's fixative, washed with 70% ethanol, dehydrated and embedded in paraffin. Five-micrometer serial sections were cut.
For preparation of tissue culture, testicular tissue from one adult rhesus monkey (which had not received an iv bolus injection of BrdU) was teased in sterile PBS to obtain fragments of seminiferous tubules between several millimeters and 2 cm in length. The fragments were incubated for 2 h at 37 °C in Dulbecco's Modified Eagle's Medium (DMEM; 4.5 g/l glucose; Mediatech, Herndon, VA) supplemented with nonessential amino acids (NAA; Cambrex Bio Science, Walkerville, MD; dilution following manufacturer's instructions), glutamine (365 mg/l; Sigma, St Louis, MO), antibiotics (penicillin 100 IU/ml, streptomycin 100 µg/ml; Mediatech, Herndon, VA) and BrdU (100 µM; Sigma, St Louis, MO) to allow cells in S-phase to incorporate BrdU. Some of these fragments were then fixed immediately in fresh Bouin's fixative and were transferred into 70% ethanol for storage until further processing. Other fragments were subjected to organ culture. Seminiferous tubules were placed on top of 25 mm tissue culture inserts (polycarbonate membrane, 8 µm pores; Nalge Nunc, Naperville, IL) floating upside down in 35 mm cell culture wells (6-well plates; Greiner Bio-One, Frickenhausen, Germany) filled with DMEM (1.0 g/l glucose; Mediatech, Herndon, VA) supplemented with nonessential amino acids (NAA; Cambrex Bio Science, Walkerville, MD; dilution following manufacturer's instructions), glutamine and antibiotics. The tissue, thus placed at the interphase between cell culture medium and surrounding atmosphere, was cultured at 35 °C in 5% CO2 in air and saturated humidity for 48 and 72 h. Finally, the tissue fragments were fixed overnight at 4 °C in fresh Bouin's fixative and stored in 70% ethanol.
Testicular tissue retrieved from the macaque which had received a bolus injection of BrdU 40 days before castration was teased in cold PBS, fixed overnight at 4 °C in fresh Bouin's fixative and stored in 70% ethanol.
Immunohistochemical staining procedure
The staining procedures of sections and whole mounts were quite similar and have been described previously (Ehmcke et al., 2005). In brief, tissue sections were deparaffinized, rehydrated, incubated in 1M hydrochloric acid for 10 min at RT, washed in distilled water, incubated for 5 min at RT with Trypsin solution [0.1% in Tris-buffered saline (TBS); Sigma, St Louis, MO], washed with distilled water followed by TBS, incubated for 30 min at RT with blocking solution [5% goat serum and 0.1% bovine serum albumine (BSA) in TBS; goat serum and BSA from Sigma, St Louis, MO] and incubated overnight (ON) at 4 °C with the primary antibody (monoclonal anti-BrdU, clone BU-33; either from Sigma, St Louis, MO or from Biomeda, Foster City, CA; diluted 1:50 in TBS containing 0.1% BSA). Then the sections were washed with TBS, incubated for 1 h at RT with the secondary antibody (goat anti-mouse, biotinylated; Sigma, St Louis, MO; diluted 1:100 in TBS containing 0.1% BSA), washed with TBS, incubated for 1 h at RT with a mix of a second primary antibody and a streptavidin-conjugated fluorescent dye (monoclonal anti-Acrosin, clone Acr-C5F10; Biosonda, Miami, FL, and fluorescent dye AlexaFluor 488, streptavidin-conjugated; Molecular Probes, Eugene, OR; both diluted 1:100 in TBS containing 0.1% BSA), washed in TBS, incubated for 1 h at RT with a secondary antibody (goat anti-mouse, fluorescent dye AlexaFluor 546-conjugated; Molecular Probes, Eugene, OR; diluted 1:100 in TBS containing 0.1% BSA), washed with TBS and mounted using VectaShield Mounting Medium (Vector, Burlingame, CA) containing 4,6-diamidino-2-phenylindole (DAPI; 1.5 µg/ml). When whole mounts were stained the incubation times for hydrochloric acid and trypsin were prolonged (15 min at RT for both). Whole mounts were mounted on microscope slides using VectaShield Mounting medium without DAPI (Vector, Burlingame, CA).
Intense haematoxylin staining of the nuclei is necessary to enable unequivocal morphological identification of the two spermatogonial subtypes. To achieve this goal, tissue sections were deparaffinized, rehydrated, incubated in 1 M hydrochloric acid for 15 min at RT, washed in distilled water, stained with hematoxylin solution (Sigma, St Louis, MO), incubated in tap water followed by distilled water, incubated with Trypsin solution (10 min; 0.1 % in TBS; see above) and washed with distilled water followed by TBS. The next steps of the immunohistochemical staining was performed following the haematoxylin staining as described above, starting with the incubation in blocking solution.
Tissue analysis
Sections and wholemounts were analyzed using a Nikon Eclipse E800 fluorescence microscope (Nikon, Melville, NY) with attached digital camera (Olympus, Melville, NY) and Nikon CI confocal scanning system. All images were acquired digitally using MagnaFire Software (Optronics, Goleta, CA).
Determination of labeling indices
A total of 18 sections derived from the testis of four adult male rhesus monkeys (five sections each for monkeys #2509, #2518 and #2556, three sections for monkey #2699) were used for the determination of the labeling indices of Apale and Adark spermatogonia. A total of randomly selected 332 fields of view (78 for monkey #2509, 71 for monkey #2518, 130 for monkey #2556 and 53 for monkey # 2669) were evaluated at x40 magnification in brightfield mode. All Adark spermatogonia (regardless of the stage of the cycle of the seminiferous epithelium) and all Apale spermatogonia (only in stage VII of the cycle of the seminiferous epithelium) encountered in each evaluated field of view were viewed at x100 magnification in both brightfield mode (to confirm cell type) and in fluorescent mode (to determine BrdU labeling). The percentage of labeled cells (labeling index) was calculated.
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Results |
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Detection of label-retaining cells 40 days after BrdU incorporation
Whole mounts of seminiferous tubules of the monkey, which had received an iv bolus injection of BrdU 40 days prior to castration, were stained immunohistochemically for BrdU and Acrosin as described above. Since 40 days after labeling, all differentiating germ cells should have been eliminated from the testis as mature sperm, we expect to see only those cells that remain as positive progeny, which had not differentiated and have rarely divided in the meantime. In other stem cell systems these label-retaining cells have been recognized as adult stem cells (Cotsarelis et al., 1990). In the tissue of this monkey we observe occasionally BrdU-positive single cells and very rarely small clones of BrdU-positive cells (Figure 1D).
BrdU label in Adark spermatogonia after acute labeling
We used tissue sections to determine whether any BrdU-positive Adark spermatogonia were present in the seminiferous tubules and to analyze whether they are preferentially observed at specific stages of the seminiferous epithelial cycle. We noted that very few Adark spermatogonia show an intense label for BrdU and that these cells were distributed among different stages of the seminiferous epithelial cycle (Figure 2DF). When we determined the ratio of BrdU-labeled Adark spermatogonia across all stages of the seminiferous epithelial cycle, <1% of these cells were intensively BrdU-positive representing the intense staining of cells in S-phase of the cell cycle (Table III). Surprisingly we encountered a large number (19%, Table III) of Adark spermatogonia showing a weak, but clearly visible granular nuclear label for BrdU which was not observed in control sections after omission of the primary antibody or when the tissue had not been exposed to BrdU (Figure 3A and B). A similar weak and granular staining pattern for BrdU was observed in elongating spermatids at stages VII and VIII of the cycle of the seminiferous epithelium (Figure 2D F). Apart from Adark spermatogonia and elongating step 7 and 8 spermatids, no other cell type showed this type of weak granular BrdU label.
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Discussion |
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In addition, in the present study we demonstrate the presence of BrdU-positive spermatogonia at stage VII of the cycle of the seminiferous epithelium not only after acute labeling (Ehmcke et al., 2005) but also 11 days after BrdU injection. It is important to consider that the intensity of the BrdU label diminishes by 50% with each mitotic division, as unlabeled nucleotides are incorporated into the newly synthesized DNA. In Figure 6 we present a calculation of the progressive dilution of the BrdU label. The startpoint is set at stage VII and the presumptive changes over one full spermatogenic epithelial cycle (10.5 days, de Rooij et al., 1986) are indicated. Following this calculation, the presence of strongly BrdU-positive (Apale) spermatogonia (25% of the incorporated BrdU is still present, Figure 6A) in stage VII of the seminiferous epithelium 11 days after an iv bolus injection of BrdU revealed that at least some of the Apale spermatogonia, which proliferated at stage VII in the previous cycle, did not undergo differentiation. We conclude that the persistence of non-differentiating Apale spermatogonia is the mechanism to maintain a stable Apale spermatogonial population under steady-state conditions. The same cross-sections contained weakly labeled pre-leptotene spermatocytes (dilution of the original BrdU label to <2%, Figure 6B). These cells derived from the differentiating Apale spermatogonia of the previous cycle. The label is significantly diluted since these cells had gone through five subsequent mitotic divisions. In addition, we found a strong BrdU label in all pachytene spermatocytes which had developed without further divisions from pre-leptotene spermatocytes of the previous cycle and therefore maintain 100% of the incorporated BrdU label (Figure 6C).
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We can only speculate which (if any) of the two A-spermatogonial subtypes in the monkey testis corresponds to the single undifferentiated spermatogonium acting as male germline stem cell in the rodent (Huckins, 1971). Adark spermatogonia only very occasionally proliferate in healthy adult rhesus monkeys (Clermont and Antar, 1973
; Fouquet and Dadoune, 1986
). Therefore, Adark spermatogonia are considered to be quiescent or reserve stem cells which only proliferate after the significant loss of Apale spermatogonia due to X-ray irradiation or cytotoxic exposure. Then, the Apale population is restored from the Adark pool (Dym and Clermont, 1970
; van Alphen et al., 1988a
,b
). When such repopulation occurs, long chains of Adark and Apale spermatogonia are observed, but
2040% of both types of A-spermatogonia are single cells (van Alphen et al., 1988b
). Unfortunately, no analysis of the proliferation indices has been performed in the radiation study. Here we show that under normal conditions proliferating single spermatogonia are extremely rare. Taken together, this might indicate that the monkey testis contains a significant number of single A-spermatogonia which very rarely divide. The latter is true for the Adark spermatogonial population. However, we do not know whether Adark spermatogonia would more intensively proliferate during recolonization or whether they would be replenished from the Apale pool as was proposed by van Alphen et al. (1988b)
. In conclusion, which cell acts as the primate male germline stem cell can not be unequivocally determined from the previous radiation study as well as from our study. The questions which need to be answered are (i) are label-retaining cells exclusively found among the population of one A-spermatogonial subtype, (ii) to what extent are the pools of Apale and Adark spermatogonia separate populations and how intense is the transition between both subtypes under normal conditions as well as during testis growth or recolonization after germ cell depletion. We can, however, confirm that Apale spermatogonia are the only cells which cycle regularly, replenish their own numbers and give rise to B-spermatogonia. Due to their effective self-renewal, only very few Apale spermatogonia would need to be generated by stem cells.
In the present study we also detect a very weak and granular BrdU label in Adark spermatogonia and in round-to-elongating spermatids at stages VII and VIII of the cycle of the seminiferous epithelium. This label is BrdU-specific because it is absent in tissue derived from animals which have not received a BrdU injection, and in immunohistochemical controls after omission of the primary antibody. This finding was surprising as we did not expect significant numbers of Adark spermatogonia entering mitotic S-phase, and as round spermatids (haploid germ cells) by definition can not enter the S-phase of the cell cycle. Therefore, the weak BrdU label must be the result of a different BrdU incorporation process.
It is well known that during the condensation of DNA in elongating spermatids, histones are removed from the supercoiled DNA. As part of this process, nuclease activity induces multiple single-strand breaks to release tension of the coiled DNA in order to enable stretching (McPherson and Longo, 1993). Nuclear transient proteins attach to the single-strand breaks and help stabilize the DNA until the breaks are repaired by an as yet unidentified ligase (Caron et al., 2001
; Brewer et al., 2002
; Zhao et al., 2004
). Extensive chromatin remodeling is not an exception, but the rule during spermiogenesis (Kierszenbaum, 2001
; Boissonneault, 2002
; Marcon and Boissonneault, 2004
). During chromatin remodeling, BrdU incorporation into the DNA of the elongating spermatids occurs, however, at a much lower level than during mitotic S-phase. A highly sensitive BrdU-detection system as we used here is able to detect such small amounts of incorporated BrdU as was shown previously for repair processes of the lesioned mouse retina (Menu Dit Huart et al., 2004
). We therefore conclude that our weak granular label in elongating spermatids represents BrdU-incorporation processes occuring during chromatin remodeling in spermiogenesis.
About 18% of the Adark spermatogonia showed the same pattern of weak granular BrdU label. We certainly were surprised by the high number of weakly positive cells which were highly specific for Adark spermatogonia but were never seen in Apale spermatogonia. At this time, we cannot present a decisive explanation for the weak granular BrdU label. As these cells seem to proliferate only very occasionally, the labeling may be caused by DNA-repair processes enabling Adark spermatogonia to preserve their DNA integrity over extended periods of quiescence.
In conclusion, we have presented further evidence supporting our earlier conclusion (Ehmcke et al., 2005) that rhesus monkey spermatogenesis is initiated by a first mitotic division of Apale spermatogonia at stage VII of the seminiferous epithelial cycle. Under in vivo and in vitro conditions the Apale spermatogonial population apparently generates both Apale and B1-spermatogonia after two mitotic divisions at stages VII and IX of the cycle of the seminiferous epithelium. Single proliferating spermatogonia are very rare and proliferate independently of the seminiferous epithelial cycle.
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Acknowledgements |
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References |
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Submitted on October 7, 2004; resubmitted on November 22, 2004; accepted on January 4, 2005.