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OPEN ACCESS ARTICLE
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TISSUE-SPECIFIC STEM CELLS |
Departments of aOb/Gyn & Reproductive Sciences,
bMolecular Genetics and Biochemistry, and
cCell Biology and Physiology,
dCenter for Research in Reproductive Physiology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA;
ePittsburgh Development Center, Magee-Womens Research Institute & Foundation, Pittsburgh, Pennsylvania, USA;
fCancer Biology and Genetics Program and Department of Pathology, Sloan-Kettering Institute, Memorial Sloan-Kettering Cancer Center, New York, New York, USA
Key Words. Busulfan • Chemotherapy • Infertility • Spermatogenesis • Spermatogonial stem cells • Xenotransplantation
Correspondence: Kyle E. Orwig, Ph.D., University of Pittsburgh School of Medicine, Magee-Womens Research Institute & Foundation, 204 Craft Avenue, Pittsburgh, Pennsylvania 15213, USA. Telephone: 412-641-2460; Fax: 412-641-3899; e-mail: korwig{at}pdc.magee.edu
Received on February 25, 2007;
accepted for publication on June 8, 2007.
First published online in STEM CELLS EXPRESS June 21, 2007.
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ABSTRACT |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionConclusionDisclosure of Potential...AcknowledgmentsReferences |
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1), and cells expressing these markers were depleted following high-dose busulfan treatment. Furthermore, fresh or cryopreserved germ cells from normal rhesus testes produced colonies of spermatogonia, which persisted as chains on the basement membrane of mouse seminiferous tubules in the primate to nude mouse xenotransplant assay. In contrast, testis cells from animals that received high-dose busulfan produced no colonies. These studies provide basic information about rhesus SSC activity and the impact of busulfan on the stem cell pool. In addition, the germ cell-depleted testis model will enable autologous/homologous transplantation to study stem cell/niche interactions in nonhuman primate testes. Disclosure of potential conflicts of interest is found at the end of this article.
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INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionConclusionDisclosure of Potential...AcknowledgmentsReferences |
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In primates, human and nonhuman alike, classic histological studies of nuclear morphology indicate that two types of undifferentiated spermatogonia are present on the basement membrane of testicular seminiferous tubules, designated Adark and Apale [5, 6]. The prevailing model of spermatogonial proliferation and differentiation is that Adark and Apale represent reserve and active stem cells, respectively. According to this model, Adark spermatogonia rarely divide and are activated by cytotoxic insult, whereas Apale spermatogonia undergo regular self-renewing divisions to maintain a pool of undifferentiated germ cells, which support spermatogenesis under normal circumstances [7–12]. However, these stem cell designations in primates are subject to debate and are clearly different from rodents, in which the entire spermatogenic lineage is derived from Asingle spermatogonia, the rodent SSC [13, 14]. Thus, there is justification for studying the biology of SSCs in a nonhuman primate model that exhibits germ cell organization similar to humans.
Although tools and reagents for studying SSCs in rodents are well-established (e.g., SSC transplantation), the resources for studying these cells in primates are limited. Establishment of a germ cell depleted model of male infertility in nonhuman primates will enhance investigation of SSCs by facilitating experiments that evaluate their regenerative potential and stem cell/niche interactions. Furthermore, depletion of spermatogenesis and infertility is a common side effect experienced by cancer survivors who have undergone chemotherapy and radiation treatments [15, 16]. Therefore, a nonhuman primate model of chemotherapy-induced infertility constitutes a valuable tool for both fundamental and applied investigations (reviewed in [17]).
In the current study, we validated antibodies for germ cell and stem/progenitor markers in the rhesus testis and optimized rhesus-to-nude mouse xenotransplantation as a routine biological assay to study rhesus SSCs. We used these tools to obtain baseline information about stem cell activity in normal rhesus testes, evaluate the effect of cryopreservation on SSC biological activity, and test the effect of busulfan treatment on spermatogenesis and the stem cell pool to identify a treatment regimen that causes long-term infertility.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionConclusionDisclosure of Potential...AcknowledgmentsReferences |
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Experimental Design
Six adult rhesus macaques were assigned to three treatment groups (two animals per group; 4, 8, and 12 mg/kg busulfan). Two additional adult males were used as unmanipulated controls (i.e., 0 mg/kg treatment group) and provided a baseline for weekly and seasonal fluctuations in sperm counts, testis volume measurements, and hematological parameters. For the purpose of histological evaluation and isolation of testicular cells, testis tissue was recovered by biopsy or hemicastration, before and after busulfan treatment (Fig. 1A, 1E). A small piece of each hemicastrated testis was used for morphological analyses, and the remainder was used to generate a single cell suspension by two-step enzymatic digestion (see below; Fig. 1A).
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Semen Collection and Analysis
Semen samples were collected from experimental and control animals at weekly intervals (Fig. 1D) as described [18]. Total sperm count per ejaculate was determined by hemocytometer.
Testis Volume Measurements
At weekly intervals, calipers were used to measure the longest diameter (height) and shortest diameter (width) of each testis through the scrotum of each experimental and control animal (Fig. 1D). Testis volume was estimated using the equation for the volume of a prolate spheroid (volume = 4/3 
x width2 x height). For animals with both testes, testis volumes were averaged.
Blood Collection and Analysis
Blood was collected at weekly intervals and processed for complete blood count (Antech Diagnostics, Lake Success, NY, http://www.antechdiagnostics.com) to measure hematopoietic parameters and determine the general health status of experimental animals (Fig. 1D).
Histology
Portions of testicular tissue collected above were fixed with Bouin's solution (Accustain; Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com), paraffin embedded, sectioned (5 µm), and stained with hematoxylin and eosin (Fig. 1A, 1E). Stained seminiferous tubule cross-sections were scored for the extent of spermatogenesis as empty (few or no germ cells, Sertoli cell only), multiple germ cell layers (at least two layers of germ cells but no elongating/elongated spermatids), or complete spermatogenesis (presence of elongating or elongated spermatids). A minimum of 100 seminiferous tubule cross-sections were evaluated per animal, except where noted.
Immunofluorescent Detection of Germ Cell and SSC Markers
Biopsies of rhesus testes collected above were fixed in 4% paraformaldehyde, paraffin embedded, and sectioned (5 µm) (Fig. 1A, 1E). Following high-temperature citrate buffer antigen retrieval, sections were stained with rabbit anti-VASA (DEAD box polypeptide 4, 1:200 dilution; Abcam, Cambridge, MA, http://www.abcam.com), goat anti-human DAZL (deleted in a zoospermia-like; 1:200; Novus Biologicals, Littleton, CO, http://www.novusbio.com), mouse anti-human GFR1 IgG (1:250; R&D Systems Inc., Minneapolis, http://www.rndsystems.com), or Armenian hamster anti-PLZF IgG (1:200 dilution; R.M. Hobbs, P.P. Pandolfi). Primary antibodies were detected with goat anti-rabbit IgG AlexaFluor 488 (1:200; Invitrogen, Carlsbad, CA, http://www.invitrogen.com), donkey anti-goat IgG AlexaFluor 568 (1:200; Invitrogen), goat anti-mouse IgG AlexaFluor 488 (1:200; Invitrogen), or goat anti-Armenian hamster IgG fluorescein isothiocyanate (FITC) (1:50; Jackson Immunoresearch Laboratories, West Grove, PA, http://www.jacksonimmuno.com). Samples were mounted with Vectashield mounting medium containing 4,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, Burlingame, CA, http://www.vectorlabs.com). For quantification of rhesus spermatogonia expressing PLZF, positive nuclei and seminiferous tubule cross-sections were counted from four normal adult rhesus macaques.
Preparation of Donor Rhesus Macaque Testis Cell Suspensions
Donor cells were recovered from rhesus macaque testes by modification of a previously published two-step enzymatic digestion procedure ([19, 20]; Fig. 1A, 1E). Upon castration, seminiferous tubules were removed from the tunica albuginea and digested with collagenase type IV (1 mg/ml; Sigma) in Hanks balanced salt solution (HBSS; Invitrogen) for 5–10 minutes at 37°C with vigorous shaking. Dispersed seminiferous tubules were sedimented by gentle centrifugation at 100g and washed three times in HBSS to remove interstitial cells. Isolated seminiferous tubules were further digested with trypsin (2.0 mg/ml trypsin plus 1.04 mM EDTA; Invitrogen) and DNase I (1.4 mg/ml; Sigma) in HBSS for 10–15 minutes at 37°C with mild trituration every five minutes. Digestions were quenched with 10% fetal bovine serum (FBS; HyClone, Logan, UT, http://www.hyclone.com) and filtered through a nylon mesh (70-µm Cell Strainer; Becton, Dickinson and Company, Franklin Lakes, NJ, http://www.bd.com), producing a single cell suspension. Cells were pelleted by centrifugation at 600g and resuspended in minimum essential medium alpha (MEM; Invitrogen) containing 10% FBS. In some experiments, testis cells were xenotransplanted immediately (Fig. 1F), whereas the remaining cells were cryopreserved for future studies (Fig. 1B). For cryopreservation, cells were resuspended at 40 x 106 per milliliter in medium (MEM + 10% FBS) and aliquoted in cryovials, and an equal volume of freezing medium (MEM + 20% FBS + 20% dimethyl sulfoxide [DMSO]) was added drop-wise. Vials were frozen in –1°C/minute controlled-rate freezing containers (Nunc, Rochester, NY, http://www.nuncbrand.com) to –80°C and stored in liquid nitrogen (final cryopreserved cell concentration was 20 x 106 per milliliter in MEM + 15% FBS + 10% DMSO). For experiments using cryopreserved cells, vials were thawed rapidly at 37°C, excess medium (MEM + 10% FBS) was added to the cell mixture drop-wise, and cells were washed three times in medium and were used for xenotransplantation (Fig. 1F).
Recipient Mouse Preparation and Xenotransplantation Procedure
Nude mice (NCr nu/nu; Taconic, Germantown, NY, http://www.taconic.com) were treated with busulfan (40 mg/kg; Sigma) at 6 weeks of age to eliminate endogenous spermatogenesis as described [1, 21]. Six weeks after busulfan treatment, approximately 7 µl of fresh or cryopreserved donor testis cell suspension containing 10% trypan blue (Invitrogen) at 100–200 x 106 cells per milliliter were injected into the seminiferous tubules of recipient testes via the efferent ducts (Fig. 1F; [22]).
Antibody Generation
For generation of the rhesus testis cell polyclonal antiserum, testis cell suspensions from two juvenile rhesus macaques (ages 1.5 and 1.33 years) were prepared as above and used to inoculate immune-naïve rabbits (Covance Research Products, Denver, PA, http://www.crpinc.com). The IgG fraction of the resulting rabbit antiserum was purified by affinity chromatography (MAbTrap kit; GE Healthcare, Piscataway, NJ, http://www6.gelifesciences.com) and preabsorbed against acetone-precipitated mouse testis proteins (1:10; IgG:mouse testis acetone powder, wt/wt) to minimize cross-reactivity to mouse antigens.
The PLZF antibody was raised in Armenian hamsters to a peptide corresponding to sequence within the hinge domain of mouse Plzf. Details of PLZF antibody derivation will be described elsewhere (R.M. Hobbs, P.P. Pandolfi).
Whole-Mount Immunofluorescent Staining of Xenotransplanted Testes
For quantitative analysis of donor rhesus testis cell colonization, intact seminiferous tubules were prepared from nude mouse recipient testes, collected 2 months after transplantation as described [19, 20, 23]. Several improvements to previously reported staining procedures were employed. Donor-derived colonies of spermatogonia were detected in intact seminiferous tubules by whole-mount immunofluorescent staining with the rhesus testis cell antibody, as described with modifications [24]. Samples were dehydrated in a graded methanol dilution series prior to incubation in MeOH:DMSO:H2O2 (4:1:1), which was limited to 2–3 hours. All dehydration, rehydration, blocking, and washing steps were performed in 12-mm Transwell baskets (12-µm pore size; Corning Life Sciences, Acton, MA, http://www.corning.com/lifesciences) to facilitate washing and prevent loss of seminiferous tubules and data. The rhesus testis cell antibody was used at a 1:200 dilution and detected with goat anti-rabbit IgG conjugated to AlexaFluor 488 (1:200 dilution; Invitrogen). Following staining, samples were mounted with Vectashield mounting medium containing DAPI (Vector Laboratories) on glass slides with raised coverslips (to preserve seminiferous tubule dimensions). The DAPI staining was used to locate donor spermatogonia nuclei in relation to recipient Sertoli cell and peritubular myoid cell nuclei and, thus, to determine whether they were positioned on the basement membrane. Samples were visualized by fluorescent microscopy using a FITC/TRITC B isothiocyanate dual-emission filter (Nikon Instruments, Melville, KY, http://www.nikoninstruments.com) to distinguish specific signal and tissue autofluorescence. In costaining experiments, the rhesus testis cell antibody was detected with donkey anti-rabbit IgG AlexaFluor 488 (Invitrogen), and VASA was detected with a goat anti-human VASA IgG (1:200; R&D Systems) together with donkey anti-goat IgG AlexaFluor 568 (Invitrogen).
For histological analysis of rhesus spermatogonia in xenotransplant colonies, a recipient nude mouse testis was fixed, embedded, and serial sectioned 5 months after transplantation with a rhesus testis cell suspension. Recipient nude mouse testis sections were stained with the rhesus testis cell antibody (1:200 dilution), mounted, and analyzed as described above.
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RESULTS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionConclusionDisclosure of Potential...AcknowledgmentsReferences |
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Compared with two untreated control males, ejaculated sperm counts in experimental animals sharply declined with all three doses of busulfan and reached 0 by 10 weeks after treatment, indicating that busulfan treatment acutely disrupted spermatogenesis (Fig. 2A). Although sperm counts returned to the normal range by 24 weeks in animals treated with 4 mg/kg busulfan, sperm counts remained undetectable 1 year following high-dose treatment with 8 and 12 mg/kg busulfan, suggesting long-term infertility (Fig. 2A). Testis volume measurements closely mirrored the sperm counts, indicating changes in sperm production were due to spermatogenic defects, not ductile obstruction (supplemental online Fig. 2). Importantly, red blood cell, white blood cell, and platelet counts declined acutely with 8 and 12 mg/kg busulfan but returned to baseline in surviving animals within 3 months, indicating that these doses can be compatible with long-term hematopoietic recovery (supplemental online Fig. 1).
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Immunohistochemical studies demonstrated that adult rhesus macaque germ cells expressed two proteins known to be present in germ cells from a variety of species, VASA [25] and DAZL [26], and both proteins were absent following 12 mg/kg busulfan treatment (Fig. 3A–3D). Furthermore, GFR1 [27–29] and PLZF [30, 31] are consensus markers of stem and progenitor spermatogonia, which comprise the undifferentiated pool (defined as As, Apr, and Aaligned4–16 [13]) in rodents and exhibit a staining pattern consistent with this designation in rhesus testes (Fig. 3E, 3G). There were 1.84 ± 0.59 PLZF expressing spermatogonia per seminiferous tubule cross-section (Fig. 3G; supplemental online Table 2), which is slightly less than the reported combined frequency of Adark and Apale spermatogonia (
2.2 per seminiferous tubule [32]). Notably, expression of GFR1 and PLZF was absent following 12 mg/kg busulfan treatment, suggesting that high-dose busulfan depleted the SSC pool (Fig. 3F, 3H). To gain insight into the progression of spermatogenic depletion caused by busulfan treatment, we analyzed the testis of animal 204, which was euthanized 10 weeks after 8 mg/kg busulfan treatment. At this time point, 99.6% of seminiferous tubules were empty (supplemental online Table 1). In the 0.4% of seminiferous tubules that contained two or more layers of germ cells (supplemental online Table 1), no GFR1 expression was observed (supplemental online Fig. 3D, 3F), suggesting that those tubules also would eventually become empty. However, a few empty seminiferous tubules had GFR1+ cells (supplemental online Fig. 3G–3I). It is tempting to speculate that these tubules would eventually exhibit spermatogenic recovery, as observed for animal 72, 60 weeks after busulfan treatment (Fig. 2D, 2H). Although these staining results are compelling, a functional assay that measures the biological potential of SSCs to colonize and persist in recipient seminiferous tubules provides a more definitive assessment of stem cell activity.
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100 µm between cells). In addition, we confirmed that colonies of rhesus cells expressed VASA, a consensus germ cell marker (Fig. 4G–4I).
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionConclusionDisclosure of Potential...AcknowledgmentsReferences |
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In order for rhesus testis cell xenotransplantation to serve as a quantitative assay, we developed strict criteria to identify putative donor stem cell-derived colonies (Results). These criteria provide a framework for interpreting the results of the current study in the context of previous and future investigations. The xenotransplantation assay detected 4.64 ± 0.95 colonies of rhesus spermatogonia per 106 viable cells transplanted to recipient mouse testes. Colonization foci in recipient nude mouse testes arose from transplanted rhesus testis cells that migrated to the basement membrane of recipient seminiferous tubules and produced chains of spermatogonia (demonstrated by VASA staining) that persisted long-term. These attributes are consistent with spermatogonial stem cell behavior, although evolutionary distance between rhesus and mouse (65–85 million years [38, 39]) precluded the establishment of complete rhesus spermatogenesis. While colonizing activity correlated with the presence or absence of consensus stem/progenitor markers (i.e., GFR1 and PLZF), we cannot exclude the possibility that differentiating spermatogonia (e.g., type B) produce some colonization foci. Ultimate validation of the xenotransplant assay will depend on future rhesus-to-rhesus testis cell transplantations, where donor cells are expected to produce complete spermatogenesis.
Based on the assumption that xenotransplant colonization data reflect the activity of SSCs, the concentration of putative SSCs identified in this study (4.64 colonies per 106 cells transplanted) is comparable to those detected from adult mice (9 colonies per 106 cells [34]) and baboons (3.9 colonies per 106 cells [20]) but lower than adult rat (169 colonies per 106 cells [40]). Based on the average number of cells obtained per gram of adult rhesus testis (82.1 ± 28.1 x 106 cells per gram testis tissue), there are approximately 381 SSCs per gram rhesus testis tissue ([4.64 SSCs per 106 viable cells transplanted] x [82.1 x 106 cells per gram donor testis tissue]) or 8,517 SSCs per testis (for the average 22.36 g testis in this study). Since the number of functional rodent stem cells observed by SSC transplantation is thought to represent 5%–12% of actual stem cells (i.e., engraftment efficiency), our estimates likely under-represent the total rhesus SSC pool [40–43]. Previous morphological analyses estimated that there are approximately 300 x 106 Adark and 300 x 106 Apale spermatogonia per adult rhesus testis [32]. This suggests that either the efficiency of rhesus SSC engraftment in mouse seminiferous tubules is extraordinarily low (e.g., 0.0015%–0.003%, depending on whether stem cell activity resides in one or both subpopulation[s] of rhesus spermatogonia) and/or that rhesus stem cells are a subpopulation of morphologically defined undifferentiated Adark and/or Apale spermatogonia.
The PLZF staining data suggest that rhesus SSCs represent a subpopulation of Adark and/or Apale spermatogonia. In rodents, Plzf marks undifferentiated spermatogonia (As, Apr, Aal4–16 [30, 31]), and SSCs (As) comprise 10% of the undifferentiated pool [44]. The frequency of PLZF+ cells in adult rhesus testes (1.86 per tubule cross section; supplemental online Table 2) is similar to the frequency of undifferentiated spermatogonia in rhesus testes ([1.1 Apale per tubule cross-section] + [1.1 Adark per tubule cross-section] = 2.2) [32]. If PLZF marks stem and progenitor (undifferentiated) spermatogonia in rhesus, and stem cells are a small fraction of the stem/progenitor pool (i.e., 10% as for rodents), then the SSC pool is a smaller subpopulation of Adark and/or Apale spermatogonia that remains to be defined.
Cytotoxic cancer treatments (i.e., chemotherapy and radiation) can cause temporary or permanent infertility [15] because these treatments fail to discriminate between cancerous and naturally proliferative tissues in the body (e.g., spermatogenesis). Cryopreservation of SSCs prior to cancer treatment and autologous transplantation of these cells back into the testis after cure may restore fertility and allow male cancer survivors to father their own genetic children [17, 45]. Our data demonstrate that rhesus SSCs retain their engraftment potential after cryopreservation. Although other fertility preserving options exist for adult male cancer survivors (semen cryopreservation or testicular sperm extraction followed by in vitro fertilization or intracytoplasmic sperm injection), some adult survivors remain unable to father children because of the advanced stage of their disease at the time of diagnosis [46]. In addition, there are currently no fertility-preserving options for preadolescent cancer survivors who are not yet producing sperm at the time of treatment [47]. Responsible development of SSC transplantation to treat male infertility requires investigation of the fundamental characteristics of the SSC pool in primates and the consequences of cytotoxic therapies.
The current study investigated the long-term effects of busulfan treatment on spermatogenesis and SSCs in rhesus macaques. Several studies have documented the hematological effects of busulfan in this species [48–52], but its gonadotoxicity has received little attention [53]. Although intraperitoneal busulfan treatment (10 mg/kg) caused transient disruption of rhesus spermatogenesis in a previous study [53], we observed long-term infertility following i.v. treatment with 8 and 12 mg/kg Busulfex IV. This apparent discrepancy may result from pharmacokinetic differences arising from the route of busulfan administration. In addition, our analysis suggests that busulfan-induced loss of germ cells correlated with SSC depletion because colonizing activity was not detectable in the xenotransplant assay, and markers of stem/progenitor spermatogonia (PLZF and GFR1) were absent. Thus, we have established a nonhuman primate model of chemotherapy-induced infertility.
To maximize the potential for using this rhesus model of infertility to study the fundamental characteristics and regenerative potential of rhesus SSCs, some refinement of the experimental approach is required. Future studies will employ the lowest dose of busulfan that causes long-term infertility (probably between 8 and 10 mg/kg) and use prophylactic peripheral blood stem cell transplant to guard against the hematopoietic deficits caused by busulfan treatment in some animals. High-dose busulfan treatment produces an ablated testis model that will facilitate rhesus-to-rhesus transplantation in future studies to evaluate stem cell/niche interactions and optimize SSC transplantation in a primate model.
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CONCLUSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionConclusionDisclosure of Potential...AcknowledgmentsReferences |
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1) between mice and nonhuman primates. Although the xenotransplantation assay is limited because of the evolutionary distance between rhesus and mouse, it serves as a practical tool that lays the foundation to test future hypotheses. The ablated rhesus testis model established in this study will facilitate future rhesus-to-rhesus testis cell transplantations to validate the xenotransplantation results, investigate the full regenerative potential of nonhuman primate SSCs, and study stem cell/niche interactions. |
DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST |
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ACKNOWLEDGMENTS |
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The authors thank Kevin Grund, Cynthia Oberley, Kari Panza, and Felicity Winkler for assistance with semen collection and analysis. Tony Battelli, Michelle Walsh, and Michael Bodenheimier provided rodent colony maintenance. The rhesus testis cell antibody was produced in collaboration with Dr. Tony Plant (supported by a Specialized Cooperative Centers Program in Reproduction and Infertility Research grant to T. Plant and K.E.O. [U54 HD008610]), who also assisted with hemicastrations. Dr. Stefan Schlatt assisted with testis biopsy collection.
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REFERENCES |
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4 rhesus cells with spermatogonial morphology on the basement membrane of recipient mouse seminiferous tubules) per 106 viable transplanted cells. Cryopreserved cells in four replicate experiments from different animals were maintained in liquid nitrogen for 336, 289, 216, or 214 days prior to thawing and xenotransplantation. The number of individual rhesus donors (animals) and nude mouse recipient testes is indicated below the bars. Statistical significance is noted above each set of bars and was determined by a two-tailed heteroscedastic Student's t test. The histogram in (C) displays the distribution of colony sizes from fresh (white bars) and cryopreserved (black bars) rhesus testis cells reported in (B). Inset images in (C) show representative xenotransplant colonies 2 months after transplantation from fresh and cryopreserved rhesus testis cells. Colonies were detected by whole-mount staining with the rhesus testis cell antibody (green) and counterstained with 4,6-diamidino-2-phenylindole (blue). Dashed white lines mark seminiferous tubule margins. Scale bars = 25 µm.