- Email: [email protected]
Intrastriatal transplantation of Sertoli cells may improve amphetamine-induced rotation and tyrosine hydroxylase immunoreactivity of the striatum in hemiparkinsonian rats
Brain Research 838 Ž1999. 227–233 www.elsevier.comrlocaterbres
Short communication
Intrastriatal transplantation of Sertoli cells may improve amphetamine-induced rotation and tyrosine hydroxylase immunoreactivity of the striatum in hemiparkinsonian rats Hwan-Wun Liu
a, )
a b
, Ya-Jane Kuang a , Jiahn-Chun Wu b , Kuo-Hsing Ma a , Shwun-De Wang a , Jiang-Chuan Liu a
Department of Biology and Anatomy, National Defense Medical Center, Taipei 100, Taiwan Department of Anatomy, College of Medicine, National Taiwan UniÕersity, Taipei, Taiwan Accepted 18 May 1999
Abstract This study investigated survival and neurotrophic effects of Sertoli cells transplanted in the striatum of 6-hydroxydopamine Ž6-OHDA.-induced hemiparkinsonian rats. Primary cultures of Sertoli cells were established from 3-week old rats and characterized by associated marker, placental cadherin ŽP-cadherin.. Two months after transplantation, amphetamine-induced rotations of rats transplanted with Sertoli cells were significantly lower than those of control rats. However, restoration of tyrosine hydroxylase ŽTH. immunoreactivity and Sertoli cells that expressed P-cadherin were only found in the striatum of the rat that showed full recovery from amphetamine-induced rotation 3 months after transplantation without immunosuppression. These results suggest that Sertoli cells transplanted in striatum of hemiparkinsonian rats may survive for at least 3 months, and improve amphetamine-induced rotation and restore TH immunoreactivity. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Parkinson’s disease; Sertoli cell; Transplantation; Striatum; Substantia nigra
Fetal substantia nigra tissue transplanted into the striatum of patients with Parkinson’s disease survives for a long period, restores dopaminergic innervation and improves the clinical symptoms w11,12x. Although other trials failed to obtain these favorable results w9,16x, neurotransplantation has been recognized as a potential treatment w15x. The major challenges to transplantation of nigral tissue as a therapy are obtaining adequate amount of donor tissue and immunological rejection of the transplanted tissue. Sertoli cells are the major epithelial cells of seminiferous tubules that support spermatogenesis in testis w5x. Sertoli cells secrete basic fibroblast growth factor ŽbFGF.w8x, insulin-like growth factor I w6x, transforming growth factor-a ŽTGF a . w23x and b w14x to regulate proliferation and differentiation of germ cells w5,24x. Fas ligand ŽFasL. secreted by Sertoli cells may play an impor-
) Corresponding author. Fax: [email protected]
q 886-2-23681444;
E-mail:
tant role in immunoprivileged status of testis w2x and local immunosuppression when co-cultured with other cells w22x. Sanberg et al. w17–19x reported that porcine Sertoli cells xenografted into rat brain suppress microglial response and promote the survival of co-transplanted bovine adrenal chromaffin cells. In their work, however, Sertoli cells were not well-characterized before transplantation, and survival of Sertoli cells was evaluated by non-specific markers such as latex bead or chloromethyl-benzamido-indocarbocyanine ŽDiI. w4,18x. Since both Northern blot and immunoblot analysis revealed placental cadherin ŽP-cadherin., a Caq2 -dependent cell adhesion molecule, in Sertoli cells cultured from 3-week old rat testes w26x. This study investigated amphetamine-induced rotation, tyrosine hydroxylase ŽTH. immunoreactivity and survival of Sertoli cells in the striatum of hemiparkinsonian rats that received intrastriatal transplantation of Sertoli cells, with special efforts to characterize Sertoli cells with P-cadherin. Male Sprague–Dawley ŽSD. rats were housed in a controlled environment with a 12-h lightrdark cycle at animal facility of National Defense Medical Center, Taipei,
0006-8993r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 9 9 . 0 1 6 5 6 - X
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Taiwan. Rats received Purina Lab Chow and water ad libitum. To induce hemiparkinsonism, male SD rats of 200–250 g were anesthetized with choral hydrate Ž400 mgrkg, i.p... Ten micrograms of 6-hydroxydopamine Ž6-OHDA. and 0.2 mgrml of ascorbic acid in 4 ml of 0.9% saline ŽResearch Biochemicals International, Natick, MA, USA. was injected in medial forebrain bundle Žy4.4 mm AP and 1.2 mm ML relative to bregma and 7.8 mm below dural surface. over 4 min. Depletion of dopamine in the striatum was evaluated by rotation behavior induced by amphetamine Ž0.15 mgr100 g, s.c.. and recorded by an automated rotometer in hemispherical bowls, 30 cm in diameter, every 2 weeks after injection w25x. Since rotation over 250 turnsrh indicated more than 90% depletion of dopamine in striatum w10x, rats that exhibited rotation over 300 turnsrh were selected for experiments. Primary cultures of Sertoli cells were prepared by the method modified from Wu et al. w26x. Testes were recovered from 3-week old male SD rats under aseptic techniques. After the tunica albuginea was removed, the testes were cut into pieces in Hank’s balanced salt solution ŽHBSS, Gibco, Grand Island, NY, USA. containing 0.25% trypsin for 10 min, then 5 mgrml Dnase I was added. The tissue pieces were incubated in a water bath, and shaken 60 cyclermin, 338C for 40 min to remove interstitial cells. Sedimentary tissue pieces were treated with 0.25% soybean trypsin inhibitor ŽSigma, St. Louis, MO, USA.. After two washes in HBSS, tissue pieces were treated with HBSS containing 0.1% hyaluronidase and 0.1% collagenase, shaken 60 cyclermin, 338C for 60 min to remove peritubular basement membrane. Sedimentary tissue pieces were washed in HBSS twice, then treated with 0.1% collagenase in HBSS, shaken 60 cyclermin, 338C for 30 min to remove peritubular cells and germ cells. Sedimentary tissue pieces were washed in Ca2qrMg 2q-free HBSS twice, and treated with Ca2qrMg 2q-free HBSS containing 0.1 mM EGTA for 10 min. The tissue pieces were brought into cell aggregates by pippetting for 1 min. Cell aggregates were collected and washed in serum-free hormonesupplied Dulbecco’s minimum essential medium ŽSF-HS DMEM. by centrifugation at 600 = g for 5 min twice. SF-HS DMEM was DMEM ŽGibco. supplemented with 0.1 mM non-essential amino acid, 4 mM glutamine, 1 mM sodium pyruvate, 5 mgrml insulin, 5 mgrml transferrin, 0.1 mM testosterone, 10 mM retinol and 50 mgrml gentamycin. Some of the aggregates were further digested into free cells by 0.25% trypsin and the cell density was measured. Aggregates of Sertoli cells were resuspended into 1 = 10 7 cellsrml and cultured in SF-HS DMEM, 5% CO 2 , 338C. Sertoli cells were transplanted within 7–10 days in vitro. Before transplantation, primary cultures of Sertoli cells were treated with 20 mM Tris buffer, pH 7.4 to remove germ cells. Homogeneity of Sertoli cells was evaluated by immunofluorescent staining. Sertoli cells were fixed with
10% formalin in PBS and non-specific bindings were blocked by 5% skim milk in PBS. The cells were incubated with 1:200 P-cadherin specific mouse monoclonal antibody Žfrom 72–259 aa of P-cadherin; Transduction Lab., Lexington, KY, USA. for 1 h at room temperature. After three washes in PBS, the cells were incubated with FITC-conjugated goat anti mouse IgG ŽTransduction Lab.. for 1 h at room temperature. After three washes in PBS, the cells were further stained with 1 mgrml of 4,6-diamidino-2-phenyl indole ŽDAPI, Sigma.. The cells were mounted with Vectashield mounting medium ŽVector Laboratories, Burlingame, CA, USA. and viewed with a Nikon Microphot microscope equipped with a vertical illuminator for epifluorescence. Sertoli cells, 6 ml of 1 = 10 7 cellsrml Žtotal of 60 000 cells. were transplanted into striatum Ž1.0 mm AP and 2.4 mm ML relative to bregma and 5.0 mm below dural surface. of selected rats over 4 min. After transplantation, amphetamine-induced rotation behavior w25x was evaluated every month. Three months after transplantation, rats were anesthetized with i.p. injected chloral hydrate Ž400 mgrkg. and perfused with 0.9% normal saline, followed by 4% formaldehyde in 0.1 M PB, pH 7.4, via the left ventricle. The brains were removed and further fixed by 30% sucrose, 4% formaldehyde in 0.1 M PB. Frozen sections of 30 mm were cut on a Reichert-Jung, 280 Frigocut N microtome, and stored in 0.1 M PBS until use. Frozen sections were washed in PBS and non-specific bindings were blocked by 30% Žvrv. normal goat serum, 0.4 Žvrv. Triton X-100 in PBS for 30 min. The sections were incubated with 1:1000 rabbit anti-TH antiserum ŽEugene Tech International, Ridgefield, NJ, USA. overnight at room temperature. After three washes in PBS, the sections were reacted with 1:100 biotinylated goat anti-rabbit IgG ŽVector. for 1 h at room temperature. After three washes in PBS, the sections were further reacted with 1:100 avidin–biotin–peroxidase ŽVector. for 1 h at room temperature. After one wash in PBS and one wash in 0.05 M Tris buffer, pH 7.6, the immunoreaction was visualized by 0.05% diaminobenzidine 4 HCl ŽDAB, Sigma., 0.03% H 2 O 2 in 0.05 M Tris, pH 7.6 for 7–10 min at room temperature. The sections were transferred onto pre-cleaned slides and counter-stained with 0.5% Žwrv. methylene blue. After dehydrated in ethanol and hyalinized in xylene, the sections were mounted with Permount and observed using a Nikon Microphot microscope. For P-cadherin immunoreactivity, frozen sections of brain were incubated with 1:200 P-cadherin specific mouse monoclonal antibody Žfrom 72–259 aa of P-cadherin; Transduction Lab.. overnight, and 1:100 peroxidase conjugated goat anti-mouse IgG ŽTransduction Lab.. for 1 h at room temperature subsequently. Immunoreaction was visualized by 0.05% DAB, 0.03% H 2 O 2 in 0.05 M Tris, pH 7.6. The sections were transferred to pre-cleaned slides, counter-stained with 0.5% Žwrv. methylene blue, dehy-
H.-W. Liu et al.r Brain Research 838 (1999) 227–233 229
Fig. 1. Sertoli cells recovered from 3-week old rats grew into confluent single layer after 5–6 days in culture ŽA.. In addition to Sertoli cells, the primary culture might contain spermatogenic cells Ž10%, arrowhead., myoepithelial cells Ž1–2%. and interstitial cells Ž -1%.. Non-Sertoli cells were removed by 20 mM Tris buffer, pH 7.4 ŽB., and double fluorescent staining revealed almost all the cells Ž )95%. identified by general nuclear stain, DAPI ŽD. expressed specific marker, P-cadherin Žarrow, C.. DAPI stain did not reveal any apparent apoptosis in Sertoli cells ŽD..
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drated in ethanol, hyalinized in xylene and mounted with permount and observed by a Nikon Microphot microscope. Aggregates of 10–50 Sertoli cells were recovered from 3-week old rats as described. The cells attached to culture plate in 24 h and grew into confluent single layer of cells with polygonal shape ŽFig. 1.. After 5–6 days in culture, morphological observation and immunofluorescent stain with Sertoli cells-associated marker, P-cadherin w26x indicated that primary culture of Sertoli cells contained 5% spermatogenic cells, 1–2% myoepithelial cells and - 1% interstitial cells. Those non-Sertoli cells were removed by 20 mM Tris buffer, pH 7.4 before transplantation. Immunofluorescent stain for P-cadherin w26x and general nuclear stain by DAPI revealed that almost all the cells identified by DAPI stain revealed P-cadherin ŽFig. 1.. In addition, DAPI stain did not reveal any apparent apoptosis in Sertoli cells. After injection of 6-OHDA in right forebrain bundle, amphetamine-induced rotation of the rats was evaluated every 2 weeks. Rats that exhibited ipsilateral rotation for more than 300 turnsrh in two consecutive tests were selected. Among 10 rats selected, six received intrastriatal transplantation of Sertoli cells, and four received intrastriatal injection of culture medium served as the controls. Amphetamine-induced ipsilateral rotations of control rats remained as more than 544 turnsrh 3 month after injection. However, amphetamine-induced rotations were improved in those six rats transplanted with Sertoli cells, and the improvement was related to the rotation behavior before transplantation. For example, the improvement was very limited in the rat Ža45. whose rotation was 630 turnsrh before transplantation. Whereas, the rat Ža97. with rotation of 349 turnsrh revealed excellent recovery, its rotation behavior recovered to normal level 2 M after transplantation. The rats with rotation of 436–482 turnsrh showed moderate improvement. Since rotation of control rats were between 410 and 493 turnsrh before transplantation, transplanted rats with rotation of 410–493 turnsrh before transplantation were selected to match control rats in evaluating effects of Sertoli cells transplantation on amphetamine-induced rotation statistically ŽFig. 2E.. Simple t-test was used because both control and transplanted rats were selected from 6OHDA-treated rats and match in rotation behaviors. Amphetamine-induced rotations were not significantly different between transplanted and control rats 1 month after transplantation. However, Sertoli cells transplantation revealed a significant improvement on amphetamine-induced rotation 2 months later. In control rats, TH immunoreactivity was not found in striatum or substantia nigra of which medial forebrain bundle was injected with 6-OHA ŽFig. 2.. In five of six rats transplanted with Sertoli cells, no obvious TH immunoreactivity was found in striatum or substantia nigra. However, remarkable amount of TH immunoreactivity was found in striatum and substantia nigra of the rat Ža97.
which showed excellent recovery in rotation behavior ŽFig. 2.. In the striatum of rat that received transplantation, especially the rat Ža97. that revealed excellent recovery, Sertoli cells expressed P-cadherin were found along the path of injection ŽFig. 2F.. In this study, amphetamine-induced rotations were significantly lower in hemiparkinsonian rats that received intrastriatal transplantation of Sertoli cells than in the control rats ŽFig. 2E., and one rat Ža97. recovered to normal level by 2 months. TH immunoreactivities were found in striatum and substantia nigra of the animal with best recovery ŽFig. 2.. These results suggest that intrastriatal transplantation of Sertoli cells may provide neurotrophic effects to dopaminergic neurons remained in substantia nigra after lesion ŽFig. 2D., and stimulate secretion of dopamine in this hemiparkinsonian rat ŽFig. 2A, B.. Although the mechanism by which Sertoli cells exert these effects is not clear yet. bFGF, one of the trophic factors secreted by Sertoli cells w13x, is found in substantia nigra w1x and may promote survival of mesencephalic neurons in vitro w7x. Therefore, the effects may be attributed to trophic factors secreted by Sertoli cells. In rats transplanted with Sertoli cells, improvement of amphetamine-induced rotation was closely related to the rotation behavior before transplantation. The rat Ža45. with rotation of 630 turnsrh revealed limit improvement after transplantation. However, the rat Ža97. with 349 turnsrh recovered to normal level and rats with 436–482 turnsrh showed moderate improvement. Since amphetamine-induced rotation in hemiparkinsonian rat is linearly relative to depletion of striatal dopamine when depletion is under 90%, and over 250 turnsrh when depletion is over 90% w10x. These findings indicate that effects of Sertoli cells on rotation behavior depend on the extent of dopaminergic neurons are damaged by 6-OHDA. In the rat with 349 turnsrh, there might be some neurons left, and their secretions of dopamine were restored by Sertoli cells. Whereas a little neurons were left for Sertoli cells to stimulate in the rat with rotation of 630 turnsrh, and the rotation behavior was barely improved. This may also explain why in six rats transplanted with Sertoli cells, only the rat with 349 turnsrh revealed full recovery in rotation behavior and restoration of TH immunoreactivity in the striatum. Both rat and porcine Sertoli cells transplanted to rat brain survived for more than 2 months without immunosuppression w4,21x. In this study, Sertoli cells expressing P-cadherin were found along the path of injection in striatum of rats which were not treated with cyclosporine-A 3 months after transplantation. The expression of Pcadherin indicates that Sertoli cells not only survive for 3 months without immunosuppression, but also continue to express this marker. This finding supports the idea that Sertoli cells exhibit immunosuppressive effects. FasL was found in primary culture of Sertoli cells by immunocytochemical stain and suggested to be responsible for
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Fig. 2. In control hemiparkinsonian rats, immunohistochemical localization revealed presence of TH in striatum ŽST, C. and substantia nigra ŽSN, D. of intact side, whereas TH immunoreactivity was not found in 6-OHA injected side. In hemiparkinsonian rat, a97 which revealed excellent improvement in rotation behavior after intrastriatal transplantation of Sertoli cells, recovering TH immunoreactivity was found in striatum Žarrowhead, A. and substantia nigra Žarrow, B.. In addition, P-cadherin positive Sertoli cells Žarrowhead, F. were found in striatum of a97 along the path of injection 3 months after transplantation. Amphetamine-induced rotation Žmean " S.E.M.. was significantly lower in rats Ž n s 4. that transplanted with Sertoli cells than in control rats Ž n s 4. 2 months after transplantation Ž t-test, p - 0.05 at 2 months, p - 0.01 at 3 months..
immunosuppressive effects of Sertoli cells w3,20x, but a variety of trophic factors including bFGF w8x, insulin-like
growth factor I w6x, TGFa w23x and TGFb w14x are secreted by Sertoli cells, the specific role of FasL in survival and
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immunosuppression of Sertoli cell requires further investigation. In a series of studies, Sanberg et al. w18x and Saporta et al. w21x showed that Sertoli cells transplanted into rat brain survived and Borlongan et al. w4x showed improved functional recovery. Since neither percentage of Sertoli cells nor nature of non-Sertoli cells to be transplanted were identified by specific markers, it was difficult to attribute neurotrophic effects after transplantation to Sertoli cells. In this study, primary culture of Sertoli cells were found to contain 5% spermatogenic cells, 1–2% myoepithelial cells and - 1% interstitial cells by immunofluorescent localization ŽFig. 1.. Non-Sertoli cells were removed by 20 mM Tris buffer before transplantation, and Sertoli cells were shown more than 95% in cells to be transplanted by immunofluorescent stain ŽFig. 1.. After transplantation, Sertoli cells survived in striatum of the rat which revealed recovery in rotation behavior, and TH immunoreactivity in stratum and substantia nigra ŽFig. 2. were confirmed by P-cadherin ŽFig. 2F.. Since Sertoli cells were evaluated with immunofluorescent stain before and after transplantation, these results may provide further support to immunosuppressive and neurotrophic effects of Sertoli cells. In this study, TH immunoreactivity was not found in striatum or substantia nigra of control rats whose amphetamine-induced rotation were more than 300 turnsrh. Since depletion of dopamine in striatum was more than 90% when amphetamine-induced rotation was more than 300 turnsrh w10x. These findings indicated that hemiparkinsonian was successfully induced in the rats used for experiments. However, among six rats received Sertoli cells transplantation, only one Ža97. showed obvious recovering TH immunoreactivity in striatum and substantia nigra ŽFig. 2.. These results are consistent to those results of amphetamine-induced rotation, i.e., recovery after Sertoli cells transplantation is closely related to extent of lesion before transplantation. The present study replicated the behavioral effects of Sertoli cells in hemiparkinsonian rats, and characterized Sertoli cells with associated marker, the P-cadherin. The results indicate that Sertoli cells may be used as alternative graft source for treatment of Parkinson’s disease and other neurological disorders.
Acknowledgements Authors are grateful to Professor Marion Murray of Department of Neurobiology and Anatomy, Allegheny University of the Health Sciences, Philadelphia, PA, and Dr. Cesario V. Borlongan of Cellular Neurophysiology Section, National Institute on Drug Abuse, Baltimore, MD, USA, for their generous help in preparing this paper. This study is supported by grants ŽNSC 86-2314-B016-066-M20 and NSC 87-2314-B016-066-M20. from National Science Council of Republic of China.
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H.-W. Liu et al.r Brain Research 838 (1999) 227–233 w17x P.R. Sanberg, C.V. Borlongan, S. Saporta, D.F. Cameron, Sertoli cells: an alternative cell source for neural transplantation in Parkinson’s disease, Exp. Neurol. 135 Ž1995. 169. w18x P.R. Sanberg, C.V. Borlongan, S. Saporta, D.F. Cameron, Testis-derived Sertoli cells survive and provide localized immunoprotection for xenografts in rat brain, Nat. Biotechnol. 14 Ž1996. 1692–1695. w19x P.R. Sanberg, C.V. Borlongan, A.I. Othberg, S. Saporta, T.B. Freeman, D.F. Cameron, Testis-derived Sertoli cells have a trophic effect on dopamine neurons and alleviate hemiparkinsonism in rats, Nat. Med. 3 Ž1997. 1129–1132. w20x P.R. Sanberg, S. Saporta, C.V. Borlongan, A.I. Othberg, R.C. Allen, D.F. Cameron, The testis-derived cultured Sertoli cell as a natural Fas-L secreting cell for immunosuppressive cellular therapy, Cell Transplant. 6 Ž1997. 191–193. w21x S. Saporta, D.F. Cameron, C.V. Borlongan, P.R. Sanberg, Survival of rat and porcine Sertoli cell transplants in the rat striatum without cyclosporine-A immunosuppression, Exp. Neurol. 146 Ž1997. 299– 304.
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w22x H. Selawry, D.F. Cameron, Sertoli cell-enriched fractions in a successful islet cell transplantation, Cell Transplant. 2 Ž1993. 123– 129. w23x M.K. Skinner, K. Takacs, R.J. Coffey, Transforming growth factor-a gene expression action in the seminiferous tubule: peritubular cell– Sertoli cell interaction, Endocrinology 124 Ž1989. 845–854. w24x M.K. Skinner, Secretion of growth factors and other regulatory factors, in: L.D. Russell, M.D. Griswold ŽEds.., The Sertoli Cell, Cache River Press, FL, 1993, pp. 237–247. w25x U. Ungerstedt, G.W. Arbuthnott, Quantitative recording of rotational behavior rats after 6-hydroxy-dopamine lesion of the nigrostriatal dopamine system, Brain Res. 24 Ž1970. 485–493. w26x J.C. Wu, C.E. Gregory, R.M. Dephilip, Expression of E-cadherin in immature rat and mouse testis and in rat Sertoli cell cultures, Biol. Reprod. 49 Ž1993. 1353–1361.
Short communication
Intrastriatal transplantation of Sertoli cells may improve amphetamine-induced rotation and tyrosine hydroxylase immunoreactivity of the striatum in hemiparkinsonian rats Hwan-Wun Liu
a, )
a b
, Ya-Jane Kuang a , Jiahn-Chun Wu b , Kuo-Hsing Ma a , Shwun-De Wang a , Jiang-Chuan Liu a
Department of Biology and Anatomy, National Defense Medical Center, Taipei 100, Taiwan Department of Anatomy, College of Medicine, National Taiwan UniÕersity, Taipei, Taiwan Accepted 18 May 1999
Abstract This study investigated survival and neurotrophic effects of Sertoli cells transplanted in the striatum of 6-hydroxydopamine Ž6-OHDA.-induced hemiparkinsonian rats. Primary cultures of Sertoli cells were established from 3-week old rats and characterized by associated marker, placental cadherin ŽP-cadherin.. Two months after transplantation, amphetamine-induced rotations of rats transplanted with Sertoli cells were significantly lower than those of control rats. However, restoration of tyrosine hydroxylase ŽTH. immunoreactivity and Sertoli cells that expressed P-cadherin were only found in the striatum of the rat that showed full recovery from amphetamine-induced rotation 3 months after transplantation without immunosuppression. These results suggest that Sertoli cells transplanted in striatum of hemiparkinsonian rats may survive for at least 3 months, and improve amphetamine-induced rotation and restore TH immunoreactivity. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Parkinson’s disease; Sertoli cell; Transplantation; Striatum; Substantia nigra
Fetal substantia nigra tissue transplanted into the striatum of patients with Parkinson’s disease survives for a long period, restores dopaminergic innervation and improves the clinical symptoms w11,12x. Although other trials failed to obtain these favorable results w9,16x, neurotransplantation has been recognized as a potential treatment w15x. The major challenges to transplantation of nigral tissue as a therapy are obtaining adequate amount of donor tissue and immunological rejection of the transplanted tissue. Sertoli cells are the major epithelial cells of seminiferous tubules that support spermatogenesis in testis w5x. Sertoli cells secrete basic fibroblast growth factor ŽbFGF.w8x, insulin-like growth factor I w6x, transforming growth factor-a ŽTGF a . w23x and b w14x to regulate proliferation and differentiation of germ cells w5,24x. Fas ligand ŽFasL. secreted by Sertoli cells may play an impor-
) Corresponding author. Fax: [email protected]
q 886-2-23681444;
E-mail:
tant role in immunoprivileged status of testis w2x and local immunosuppression when co-cultured with other cells w22x. Sanberg et al. w17–19x reported that porcine Sertoli cells xenografted into rat brain suppress microglial response and promote the survival of co-transplanted bovine adrenal chromaffin cells. In their work, however, Sertoli cells were not well-characterized before transplantation, and survival of Sertoli cells was evaluated by non-specific markers such as latex bead or chloromethyl-benzamido-indocarbocyanine ŽDiI. w4,18x. Since both Northern blot and immunoblot analysis revealed placental cadherin ŽP-cadherin., a Caq2 -dependent cell adhesion molecule, in Sertoli cells cultured from 3-week old rat testes w26x. This study investigated amphetamine-induced rotation, tyrosine hydroxylase ŽTH. immunoreactivity and survival of Sertoli cells in the striatum of hemiparkinsonian rats that received intrastriatal transplantation of Sertoli cells, with special efforts to characterize Sertoli cells with P-cadherin. Male Sprague–Dawley ŽSD. rats were housed in a controlled environment with a 12-h lightrdark cycle at animal facility of National Defense Medical Center, Taipei,
0006-8993r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 9 9 . 0 1 6 5 6 - X
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Taiwan. Rats received Purina Lab Chow and water ad libitum. To induce hemiparkinsonism, male SD rats of 200–250 g were anesthetized with choral hydrate Ž400 mgrkg, i.p... Ten micrograms of 6-hydroxydopamine Ž6-OHDA. and 0.2 mgrml of ascorbic acid in 4 ml of 0.9% saline ŽResearch Biochemicals International, Natick, MA, USA. was injected in medial forebrain bundle Žy4.4 mm AP and 1.2 mm ML relative to bregma and 7.8 mm below dural surface. over 4 min. Depletion of dopamine in the striatum was evaluated by rotation behavior induced by amphetamine Ž0.15 mgr100 g, s.c.. and recorded by an automated rotometer in hemispherical bowls, 30 cm in diameter, every 2 weeks after injection w25x. Since rotation over 250 turnsrh indicated more than 90% depletion of dopamine in striatum w10x, rats that exhibited rotation over 300 turnsrh were selected for experiments. Primary cultures of Sertoli cells were prepared by the method modified from Wu et al. w26x. Testes were recovered from 3-week old male SD rats under aseptic techniques. After the tunica albuginea was removed, the testes were cut into pieces in Hank’s balanced salt solution ŽHBSS, Gibco, Grand Island, NY, USA. containing 0.25% trypsin for 10 min, then 5 mgrml Dnase I was added. The tissue pieces were incubated in a water bath, and shaken 60 cyclermin, 338C for 40 min to remove interstitial cells. Sedimentary tissue pieces were treated with 0.25% soybean trypsin inhibitor ŽSigma, St. Louis, MO, USA.. After two washes in HBSS, tissue pieces were treated with HBSS containing 0.1% hyaluronidase and 0.1% collagenase, shaken 60 cyclermin, 338C for 60 min to remove peritubular basement membrane. Sedimentary tissue pieces were washed in HBSS twice, then treated with 0.1% collagenase in HBSS, shaken 60 cyclermin, 338C for 30 min to remove peritubular cells and germ cells. Sedimentary tissue pieces were washed in Ca2qrMg 2q-free HBSS twice, and treated with Ca2qrMg 2q-free HBSS containing 0.1 mM EGTA for 10 min. The tissue pieces were brought into cell aggregates by pippetting for 1 min. Cell aggregates were collected and washed in serum-free hormonesupplied Dulbecco’s minimum essential medium ŽSF-HS DMEM. by centrifugation at 600 = g for 5 min twice. SF-HS DMEM was DMEM ŽGibco. supplemented with 0.1 mM non-essential amino acid, 4 mM glutamine, 1 mM sodium pyruvate, 5 mgrml insulin, 5 mgrml transferrin, 0.1 mM testosterone, 10 mM retinol and 50 mgrml gentamycin. Some of the aggregates were further digested into free cells by 0.25% trypsin and the cell density was measured. Aggregates of Sertoli cells were resuspended into 1 = 10 7 cellsrml and cultured in SF-HS DMEM, 5% CO 2 , 338C. Sertoli cells were transplanted within 7–10 days in vitro. Before transplantation, primary cultures of Sertoli cells were treated with 20 mM Tris buffer, pH 7.4 to remove germ cells. Homogeneity of Sertoli cells was evaluated by immunofluorescent staining. Sertoli cells were fixed with
10% formalin in PBS and non-specific bindings were blocked by 5% skim milk in PBS. The cells were incubated with 1:200 P-cadherin specific mouse monoclonal antibody Žfrom 72–259 aa of P-cadherin; Transduction Lab., Lexington, KY, USA. for 1 h at room temperature. After three washes in PBS, the cells were incubated with FITC-conjugated goat anti mouse IgG ŽTransduction Lab.. for 1 h at room temperature. After three washes in PBS, the cells were further stained with 1 mgrml of 4,6-diamidino-2-phenyl indole ŽDAPI, Sigma.. The cells were mounted with Vectashield mounting medium ŽVector Laboratories, Burlingame, CA, USA. and viewed with a Nikon Microphot microscope equipped with a vertical illuminator for epifluorescence. Sertoli cells, 6 ml of 1 = 10 7 cellsrml Žtotal of 60 000 cells. were transplanted into striatum Ž1.0 mm AP and 2.4 mm ML relative to bregma and 5.0 mm below dural surface. of selected rats over 4 min. After transplantation, amphetamine-induced rotation behavior w25x was evaluated every month. Three months after transplantation, rats were anesthetized with i.p. injected chloral hydrate Ž400 mgrkg. and perfused with 0.9% normal saline, followed by 4% formaldehyde in 0.1 M PB, pH 7.4, via the left ventricle. The brains were removed and further fixed by 30% sucrose, 4% formaldehyde in 0.1 M PB. Frozen sections of 30 mm were cut on a Reichert-Jung, 280 Frigocut N microtome, and stored in 0.1 M PBS until use. Frozen sections were washed in PBS and non-specific bindings were blocked by 30% Žvrv. normal goat serum, 0.4 Žvrv. Triton X-100 in PBS for 30 min. The sections were incubated with 1:1000 rabbit anti-TH antiserum ŽEugene Tech International, Ridgefield, NJ, USA. overnight at room temperature. After three washes in PBS, the sections were reacted with 1:100 biotinylated goat anti-rabbit IgG ŽVector. for 1 h at room temperature. After three washes in PBS, the sections were further reacted with 1:100 avidin–biotin–peroxidase ŽVector. for 1 h at room temperature. After one wash in PBS and one wash in 0.05 M Tris buffer, pH 7.6, the immunoreaction was visualized by 0.05% diaminobenzidine 4 HCl ŽDAB, Sigma., 0.03% H 2 O 2 in 0.05 M Tris, pH 7.6 for 7–10 min at room temperature. The sections were transferred onto pre-cleaned slides and counter-stained with 0.5% Žwrv. methylene blue. After dehydrated in ethanol and hyalinized in xylene, the sections were mounted with Permount and observed using a Nikon Microphot microscope. For P-cadherin immunoreactivity, frozen sections of brain were incubated with 1:200 P-cadherin specific mouse monoclonal antibody Žfrom 72–259 aa of P-cadherin; Transduction Lab.. overnight, and 1:100 peroxidase conjugated goat anti-mouse IgG ŽTransduction Lab.. for 1 h at room temperature subsequently. Immunoreaction was visualized by 0.05% DAB, 0.03% H 2 O 2 in 0.05 M Tris, pH 7.6. The sections were transferred to pre-cleaned slides, counter-stained with 0.5% Žwrv. methylene blue, dehy-
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Fig. 1. Sertoli cells recovered from 3-week old rats grew into confluent single layer after 5–6 days in culture ŽA.. In addition to Sertoli cells, the primary culture might contain spermatogenic cells Ž10%, arrowhead., myoepithelial cells Ž1–2%. and interstitial cells Ž -1%.. Non-Sertoli cells were removed by 20 mM Tris buffer, pH 7.4 ŽB., and double fluorescent staining revealed almost all the cells Ž )95%. identified by general nuclear stain, DAPI ŽD. expressed specific marker, P-cadherin Žarrow, C.. DAPI stain did not reveal any apparent apoptosis in Sertoli cells ŽD..
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drated in ethanol, hyalinized in xylene and mounted with permount and observed by a Nikon Microphot microscope. Aggregates of 10–50 Sertoli cells were recovered from 3-week old rats as described. The cells attached to culture plate in 24 h and grew into confluent single layer of cells with polygonal shape ŽFig. 1.. After 5–6 days in culture, morphological observation and immunofluorescent stain with Sertoli cells-associated marker, P-cadherin w26x indicated that primary culture of Sertoli cells contained 5% spermatogenic cells, 1–2% myoepithelial cells and - 1% interstitial cells. Those non-Sertoli cells were removed by 20 mM Tris buffer, pH 7.4 before transplantation. Immunofluorescent stain for P-cadherin w26x and general nuclear stain by DAPI revealed that almost all the cells identified by DAPI stain revealed P-cadherin ŽFig. 1.. In addition, DAPI stain did not reveal any apparent apoptosis in Sertoli cells. After injection of 6-OHDA in right forebrain bundle, amphetamine-induced rotation of the rats was evaluated every 2 weeks. Rats that exhibited ipsilateral rotation for more than 300 turnsrh in two consecutive tests were selected. Among 10 rats selected, six received intrastriatal transplantation of Sertoli cells, and four received intrastriatal injection of culture medium served as the controls. Amphetamine-induced ipsilateral rotations of control rats remained as more than 544 turnsrh 3 month after injection. However, amphetamine-induced rotations were improved in those six rats transplanted with Sertoli cells, and the improvement was related to the rotation behavior before transplantation. For example, the improvement was very limited in the rat Ža45. whose rotation was 630 turnsrh before transplantation. Whereas, the rat Ža97. with rotation of 349 turnsrh revealed excellent recovery, its rotation behavior recovered to normal level 2 M after transplantation. The rats with rotation of 436–482 turnsrh showed moderate improvement. Since rotation of control rats were between 410 and 493 turnsrh before transplantation, transplanted rats with rotation of 410–493 turnsrh before transplantation were selected to match control rats in evaluating effects of Sertoli cells transplantation on amphetamine-induced rotation statistically ŽFig. 2E.. Simple t-test was used because both control and transplanted rats were selected from 6OHDA-treated rats and match in rotation behaviors. Amphetamine-induced rotations were not significantly different between transplanted and control rats 1 month after transplantation. However, Sertoli cells transplantation revealed a significant improvement on amphetamine-induced rotation 2 months later. In control rats, TH immunoreactivity was not found in striatum or substantia nigra of which medial forebrain bundle was injected with 6-OHA ŽFig. 2.. In five of six rats transplanted with Sertoli cells, no obvious TH immunoreactivity was found in striatum or substantia nigra. However, remarkable amount of TH immunoreactivity was found in striatum and substantia nigra of the rat Ža97.
which showed excellent recovery in rotation behavior ŽFig. 2.. In the striatum of rat that received transplantation, especially the rat Ža97. that revealed excellent recovery, Sertoli cells expressed P-cadherin were found along the path of injection ŽFig. 2F.. In this study, amphetamine-induced rotations were significantly lower in hemiparkinsonian rats that received intrastriatal transplantation of Sertoli cells than in the control rats ŽFig. 2E., and one rat Ža97. recovered to normal level by 2 months. TH immunoreactivities were found in striatum and substantia nigra of the animal with best recovery ŽFig. 2.. These results suggest that intrastriatal transplantation of Sertoli cells may provide neurotrophic effects to dopaminergic neurons remained in substantia nigra after lesion ŽFig. 2D., and stimulate secretion of dopamine in this hemiparkinsonian rat ŽFig. 2A, B.. Although the mechanism by which Sertoli cells exert these effects is not clear yet. bFGF, one of the trophic factors secreted by Sertoli cells w13x, is found in substantia nigra w1x and may promote survival of mesencephalic neurons in vitro w7x. Therefore, the effects may be attributed to trophic factors secreted by Sertoli cells. In rats transplanted with Sertoli cells, improvement of amphetamine-induced rotation was closely related to the rotation behavior before transplantation. The rat Ža45. with rotation of 630 turnsrh revealed limit improvement after transplantation. However, the rat Ža97. with 349 turnsrh recovered to normal level and rats with 436–482 turnsrh showed moderate improvement. Since amphetamine-induced rotation in hemiparkinsonian rat is linearly relative to depletion of striatal dopamine when depletion is under 90%, and over 250 turnsrh when depletion is over 90% w10x. These findings indicate that effects of Sertoli cells on rotation behavior depend on the extent of dopaminergic neurons are damaged by 6-OHDA. In the rat with 349 turnsrh, there might be some neurons left, and their secretions of dopamine were restored by Sertoli cells. Whereas a little neurons were left for Sertoli cells to stimulate in the rat with rotation of 630 turnsrh, and the rotation behavior was barely improved. This may also explain why in six rats transplanted with Sertoli cells, only the rat with 349 turnsrh revealed full recovery in rotation behavior and restoration of TH immunoreactivity in the striatum. Both rat and porcine Sertoli cells transplanted to rat brain survived for more than 2 months without immunosuppression w4,21x. In this study, Sertoli cells expressing P-cadherin were found along the path of injection in striatum of rats which were not treated with cyclosporine-A 3 months after transplantation. The expression of Pcadherin indicates that Sertoli cells not only survive for 3 months without immunosuppression, but also continue to express this marker. This finding supports the idea that Sertoli cells exhibit immunosuppressive effects. FasL was found in primary culture of Sertoli cells by immunocytochemical stain and suggested to be responsible for
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Fig. 2. In control hemiparkinsonian rats, immunohistochemical localization revealed presence of TH in striatum ŽST, C. and substantia nigra ŽSN, D. of intact side, whereas TH immunoreactivity was not found in 6-OHA injected side. In hemiparkinsonian rat, a97 which revealed excellent improvement in rotation behavior after intrastriatal transplantation of Sertoli cells, recovering TH immunoreactivity was found in striatum Žarrowhead, A. and substantia nigra Žarrow, B.. In addition, P-cadherin positive Sertoli cells Žarrowhead, F. were found in striatum of a97 along the path of injection 3 months after transplantation. Amphetamine-induced rotation Žmean " S.E.M.. was significantly lower in rats Ž n s 4. that transplanted with Sertoli cells than in control rats Ž n s 4. 2 months after transplantation Ž t-test, p - 0.05 at 2 months, p - 0.01 at 3 months..
immunosuppressive effects of Sertoli cells w3,20x, but a variety of trophic factors including bFGF w8x, insulin-like
growth factor I w6x, TGFa w23x and TGFb w14x are secreted by Sertoli cells, the specific role of FasL in survival and
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immunosuppression of Sertoli cell requires further investigation. In a series of studies, Sanberg et al. w18x and Saporta et al. w21x showed that Sertoli cells transplanted into rat brain survived and Borlongan et al. w4x showed improved functional recovery. Since neither percentage of Sertoli cells nor nature of non-Sertoli cells to be transplanted were identified by specific markers, it was difficult to attribute neurotrophic effects after transplantation to Sertoli cells. In this study, primary culture of Sertoli cells were found to contain 5% spermatogenic cells, 1–2% myoepithelial cells and - 1% interstitial cells by immunofluorescent localization ŽFig. 1.. Non-Sertoli cells were removed by 20 mM Tris buffer before transplantation, and Sertoli cells were shown more than 95% in cells to be transplanted by immunofluorescent stain ŽFig. 1.. After transplantation, Sertoli cells survived in striatum of the rat which revealed recovery in rotation behavior, and TH immunoreactivity in stratum and substantia nigra ŽFig. 2. were confirmed by P-cadherin ŽFig. 2F.. Since Sertoli cells were evaluated with immunofluorescent stain before and after transplantation, these results may provide further support to immunosuppressive and neurotrophic effects of Sertoli cells. In this study, TH immunoreactivity was not found in striatum or substantia nigra of control rats whose amphetamine-induced rotation were more than 300 turnsrh. Since depletion of dopamine in striatum was more than 90% when amphetamine-induced rotation was more than 300 turnsrh w10x. These findings indicated that hemiparkinsonian was successfully induced in the rats used for experiments. However, among six rats received Sertoli cells transplantation, only one Ža97. showed obvious recovering TH immunoreactivity in striatum and substantia nigra ŽFig. 2.. These results are consistent to those results of amphetamine-induced rotation, i.e., recovery after Sertoli cells transplantation is closely related to extent of lesion before transplantation. The present study replicated the behavioral effects of Sertoli cells in hemiparkinsonian rats, and characterized Sertoli cells with associated marker, the P-cadherin. The results indicate that Sertoli cells may be used as alternative graft source for treatment of Parkinson’s disease and other neurological disorders.
Acknowledgements Authors are grateful to Professor Marion Murray of Department of Neurobiology and Anatomy, Allegheny University of the Health Sciences, Philadelphia, PA, and Dr. Cesario V. Borlongan of Cellular Neurophysiology Section, National Institute on Drug Abuse, Baltimore, MD, USA, for their generous help in preparing this paper. This study is supported by grants ŽNSC 86-2314-B016-066-M20 and NSC 87-2314-B016-066-M20. from National Science Council of Republic of China.
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