FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W1584801101> ?p ?o ?g. }

• W1584801101 endingPage "908" @default.
• W1584801101 startingPage "899" @default.
• W1584801101 abstract "To assess adult stem cell differentiation in the testis, we injected bone marrow cells from adult green fluorescent protein (GFP) transgenic mice into the seminiferous tubules and the testicular interstitium of busulfan-treated wild-type or c-kit mutant (W/Wv) mice. Ten to 12 weeks after transplantation, we examined the fate of the transplanted bone marrow cells and found that they survived in recipient testes. In both the busulfan-treated and W/Wv mice, some of the GFP-positive donor cells had a Sertoli cell appearance and expressed follicle-stimulating hormone receptor within the seminiferous tubules. In addition, GFP-positive donor cells were found in the interstitium of recipient testes, and they expressed the cytochrome P450 side chain cleavage enzyme (P450scc). In the seminiferous tubules of busulfan-treated mice, GFP-positive donor cells had the appearance of spermatogonia or spermatocytes and expressed VASA. However, this was not found in the seminiferous tubules of W/Wv mice. We conclude that adult bone marrow cells, in a favorable testicular environment, differentiate into somatic and germ cell lineages. The resident neighboring cells in the recipient testis may control site-appropriate stem cell differentiation. This clinically relevant finding raises the possibility for treatment of male infertility and testosterone deficiency through the therapeutic use of stem cells. To assess adult stem cell differentiation in the testis, we injected bone marrow cells from adult green fluorescent protein (GFP) transgenic mice into the seminiferous tubules and the testicular interstitium of busulfan-treated wild-type or c-kit mutant (W/Wv) mice. Ten to 12 weeks after transplantation, we examined the fate of the transplanted bone marrow cells and found that they survived in recipient testes. In both the busulfan-treated and W/Wv mice, some of the GFP-positive donor cells had a Sertoli cell appearance and expressed follicle-stimulating hormone receptor within the seminiferous tubules. In addition, GFP-positive donor cells were found in the interstitium of recipient testes, and they expressed the cytochrome P450 side chain cleavage enzyme (P450scc). In the seminiferous tubules of busulfan-treated mice, GFP-positive donor cells had the appearance of spermatogonia or spermatocytes and expressed VASA. However, this was not found in the seminiferous tubules of W/Wv mice. We conclude that adult bone marrow cells, in a favorable testicular environment, differentiate into somatic and germ cell lineages. The resident neighboring cells in the recipient testis may control site-appropriate stem cell differentiation. This clinically relevant finding raises the possibility for treatment of male infertility and testosterone deficiency through the therapeutic use of stem cells. Testes have two main functions in mammals: they produce androgens and gametes. Spermatogenesis occurs in the seminiferous tubules, an immunoprivileged site, and requires functionally competent Sertoli and Leydig cells. Spermatogonial stem cells are undifferentiated cells defined by their ability to both self-renew and differentiate into mature spermatozoa. When spermatogonial stem cells are harvested from donor testes and transplanted into a sterilized recipient testis, morphologically and functionally normal spermatogenesis is re-established.1Brinster RL Zimmermann JW Spermatogenesis following male germ-cell transplantation.Proc Natl Acad Sci USA. 1994; 91: 11298-11302Crossref PubMed Scopus (1283) Google Scholar, 2Brinster RL Avarbock MR Germline transmission of donor haplotype following spermatogonial transplantation.Proc Natl Acad Sci USA. 1994; 91: 11303-11307Crossref PubMed Scopus (927) Google Scholar Transplantation of Leydig stem cells isolated from mice can restore the serum testosterone concentration in luteinizing hormone receptor knockout mice.3Lo KC Lei Z Rao Ch V Beck J Lamb DJ De novo testosterone production in luteinizing hormone receptor knockout mice after transplantation of Leydig stem cells.Endocrinology. 2004; 145: 4011-4015Crossref PubMed Scopus (67) Google Scholar Transplantation of wild-type Sertoli cells into infertile Steel (SI) testes creates a permissive testicular microenvironment for restoration of spermatogenesis.4Shinohara T Orwig KE Avarbock MR Brinster RL Restoration of spermatogenesis in infertile mice by Sertoli cell transplantation.Biol Reprod. 2003; 68: 1064-1071Crossref PubMed Scopus (125) Google Scholar, 5Kanatsu-Shinohara M Miki H Inoue K Ogonuki N Toyokuni S Ogura A Shinohara T Germline niche transplantation restores fertility in infertile mice.Hum Reprod. 2005; 20: 2376-2382Crossref PubMed Scopus (42) Google Scholar In general, homeostatic cell replacement and tissue regeneration in the adult is dependent on tissue-resident stem cells. The resident stem cell in adult proliferates and differentiates into mature cell types that correspond to their tissue of origin and do not cross tissue boundaries to generate cell types of different lineages.6Weissman IL Stem cells: units of development, units of regeneration, and units in evolution.Cell. 2000; 100: 157-158Abstract Full Text Full Text PDF PubMed Scopus (1486) Google Scholar However, a growing body of literature has challenged this notion and demonstrated stem cells crossing tissue boundaries in response to a variety of microenvironmental regenerative cues.7Wagers AJ Weissman IL Plasticity of adult stem cells.Cell. 2004; 116: 639-648Abstract Full Text Full Text PDF PubMed Scopus (990) Google Scholar, 8Kucia M Reca R Jala VR Bawn B Ratajczak J Ratajczak MZ Bone marrow as a home of heterogenous populations of nonhematopoietic stem cells.Leukemia. 2005; 19: 1118-1127Crossref PubMed Scopus (170) Google Scholar Recent studies have demonstrated that spermatogonial stem cells from either neonatal mouse testes9Kanatsu-Shinohara M Inoue K Lee J Yoshimoto M Ogonuki N Miki H Baba S Kato T Kazuki Y Toyokuni S Toyoshima M Niwa O Oshimura M Heike T Nakahata T Ishino F Ogura A Shinohara T Generation of pluripotent stem cells from neonatal mouse testis.Cell. 2004; 119: 1001-1012Abstract Full Text Full Text PDF PubMed Scopus (674) Google Scholar or adult mouse testes10Guan K Nayernia K Maier LS Wagner S Dressel R Lee JH Nolte J Wolf F Li M Engel W Hasenfuss G Pluripotency of spermatogonial stem cells form adult mouse testis.Nature. 2006; 440: 1199-1203Crossref PubMed Scopus (742) Google Scholar possess pluripotency and have the potential to differentiate into embryonic stem cells. Adult stem cell plasticity and its capacity to transdifferentiate create a huge promise for understanding and treating diseases. Studies of bone marrow stem cell transdifferentiation in adopted tissues have been controversial,7Wagers AJ Weissman IL Plasticity of adult stem cells.Cell. 2004; 116: 639-648Abstract Full Text Full Text PDF PubMed Scopus (990) Google Scholar, 11Vogel G Controversial study finds an unexpected source of oocytes.Science. 2005; 309: 678-679Crossref PubMed Scopus (11) Google Scholar Recent literature suggests that bone marrow stem cells transferred into recipient mice can contribute to multiple nonhematopoietic tissues including myocytes, hepatocytes, lung epithelial cells, and neurons.12Herzog EL Chai L Krause DS Plasticity of marrow-derived stem cells.Blood. 2003; 102: 3483-3493Crossref PubMed Scopus (682) Google Scholar, 13Jiang Y Jahagirdar BN Reinhardt RL Schwartz RE Keene CD Ortiz-Gonzalez XR Reyes M Lenvik T Lund T Blackstad M Du J Aldrich S Lisberg A Low WC Largaespada DA Verfaillie CM Pluripotency of mesenchymal stem cells derived from adult marrow.Nature. 2002; 418: 41-49Crossref PubMed Scopus (5195) Google Scholar, 14Kørbling M Estrov Z Adult stem cells for tissue repair—a new therapeutic concept?.N Engl J Med. 2003; 349: 570-582Crossref PubMed Scopus (667) Google Scholar Research on bone marrow cells delivered to the ovaries via the blood stream giving rise to bona fide oocytes in mice15Johnson J Bagley J Skaznik-Wikiel M Lee HJ Adams GB Niikura Y Tschudy KS Tilly JC Cortes ML Forkert R Oocyte generation in adult mammalian ovaries by putative germ cells in bone marrow and peripheral blood.Cell. 2005; 122: 303-315Abstract Full Text Full Text PDF PubMed Scopus (562) Google Scholar has been challenged by a new study.16Eggan K Jurga S Gosden R Min IM Wagers AJ Ovulated oocytes in adult mice derive from non-circulating germ cells.Nature. 2006; 441: 1109-1114Crossref PubMed Scopus (213) Google Scholar In male mice, a recent study has demonstrated that bone marrow stem cells are able to differentiate into primordial germ cells and spermatogonia both in vitro and in vivo.17Nayernia K Lee JH Drusenheimer N Nolte J Wulf G Dressel R Gromoll J Engel W Derivation of male germ cells from bone marrow stem cells.Lab Invest. 2006; 86: 654-663Crossref PubMed Scopus (276) Google Scholar In addition, another study has demonstrated that adult stem cells derived from bone marrow stroma can differentiate into Leydig cells in rat testes.18Yazawa T Mizutani T Yamada K Kawata H Sekiguchi T Yoshino M Kajitani T Shou Z Umezawa A Miyamoto K Differentiation of adult stem cells derived from bone marrow stroma into Leydig or adrenocortical cells.Endocrinology. 2006; 147: 4104-4111Crossref PubMed Scopus (117) Google Scholar In this study, we provide evidence showing that adult bone marrow cells injected into seminiferous tubules or interstitial spaces are not only able to differentiate into germ cells (spermatogonia and spermatocytes) as well as Sertoli and Leydig cells. This finding may be of clinical relevance to the understanding of testicular pathology and may lead to unique treatment of male infertility and testosterone deficiency. Male wild-type (C57BL/6), green fluorescent protein (GFP) transgenic breeder mice (C57BL/6-Tg-UBC-GFP) and c-kit mutant homozygous (W/Wv) mice were purchased from the Jackson Laboratory (Bar Harbor, ME). Adult GFP transgenic male mice were generated from our colony and used as bone marrow cell donors. These GFP transgenic mice express GFP under the direction of the human ubiquitin C promoter. These mice express GFP in all tissues examined (Figure 8). We used two kinds of recipient mice: busulfan-treated mice and W/Wv mice (homozygous), which both have been used as standardized recipients for germ cell transplantation.19Ogawa T Arechaga JM Avarbock MR Brinster RL Transplantation of testis germinal cells into mouse seminiferous tubules.Int J Dev Biol. 1997; 41: 111-122PubMed Google Scholar, 20Johnston DS Russell LD Friel PJ Griswold MD Murine germ cells do not require functional androgen receptors to complete spermatogenesis following spermatogonial stem cell transplantation.Endocrinology. 2001; 142: 2405-2408Crossref PubMed Google Scholar Busulfan is a chemotherapeutic agent that can eliminate spermatogenesis and induce male infertility. For our study, at 4 weeks of age, recipient wild-type mice were given a single dose of busulfan (50 mg/kg body weight) by intraperitoneal injection to destroy endogenous spermatogenesis. Recipients were then used for transplantation 4 weeks after the busulfan injection.21Nagano M Ryu BY Brinster CJ Avarbock MR Brinster RL Maintenance of mouse male germ line stem cells in vitro.Biol Reprod. 2003; 68: 2207-2214Crossref PubMed Scopus (262) Google Scholar We also used recipient W/Wv mice that have no germ cells as a result of mutations in the c-kit receptor. Animal handling, experimentation, and the bone marrow and testicular tissue harvesting protocol were in accordance with the recommendations of the American Veterinary Medical Association and approved by the Institutional Animal Care and Use Review Committee of Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center. To determine whether bone marrow stem cells can differentiate into somatic or germinal cells, we inserted bone marrow cells isolated from GFP transgenic mice directly into the seminiferous tubules and interstitial space of recipient testes in groups of eight busulfan-treated wild-type and W/Wv mice. Recipients were sacrificed at 10 and 12 weeks after transplantation, and the results were evaluated. One side of the testes was dissected out and decapsulated. Seminiferous tubules were dispersed gently in 2 ml of Hanks’ balanced salt solution held in a Petri dish and the live tissue visualized under Zeiss Axioskop 40 fluorescence microscope (Zeiss, Thornwood, NY). The contralateral testis from each animal was fixed in Bouin's solution, embedded in paraffin, and sectioned for immunohistochemistry to detect cells with GFP alone or GFP co-localized with Sertoli, Leydig, and germ cell markers. The Sertoli cell marker used was follicle-stimulating hormone receptor (FSH-R); the Leydig cell marker used was P450scc, and the germ cell marker used was VASA. Co-staining of GFP with various cell markers was detected by double-immunofluorescence technique in combination with confocal laser-scanning microscopy. Donor bone marrow cells were isolated from 6- to 8-week-old GFP transgenic mice by flushing dissected femurs and tibias with phosphate-buffered saline (PBS) (pH 7.4). The cells were pelleted by centrifugation at 600 × g for 5 minutes, after which a single cell suspension was obtained at 34°C by gentle digestion in calcium- and magnesium-free Hanks’ balanced salt solution, which contained 0.05 μg of collagenase/ml (Life Technologies, Inc., Grand Island, NY), 0.05 mg/ml DNase (Sigma, St. Louis, MO), and 0.025% trypsin (Life Technologies, Inc.). After adding trypsin inhibitor (Sigma), centrifuging, and washing with Dulbecco's modified Eagle medium (Invitrogen Corp. Carlsbad, CA), the cells were then counted, pelleted by centrifugation at 600 × g for 5 minutes, and resuspended in injection media with 0.04% trypan blue stain (Invitrogen Corp.)1Brinster RL Zimmermann JW Spermatogenesis following male germ-cell transplantation.Proc Natl Acad Sci USA. 1994; 91: 11298-11302Crossref PubMed Scopus (1283) Google Scholar, 22Boettger-Tong HL Johnston DS Russell LD Griswold MD Bishop CE Juvenile spermatogonial depletion (jsd) mutant seminiferous tubules are capable of supporting transplanted spermatogenesis.Biol Reprod. 2000; 63: 1185-1191Crossref PubMed Scopus (62) Google Scholar at a concentration of 5 to 15 million cells/ml. Microinjection needles were constructed from 20-μl glass micropipettes (catalog 53432-740; VWR, West Chester, PA) drawn on a pipette puller (model P-97; Sutter Instruments, Novato, CA). The tip of each pipette was grounded to a sharp beveled point on a microbeveler (model 48000-F; World Precision Instruments, Sarasota, FL). The injection procedure was a modification of the efferent duct injection procedure previously described.19Ogawa T Arechaga JM Avarbock MR Brinster RL Transplantation of testis germinal cells into mouse seminiferous tubules.Int J Dev Biol. 1997; 41: 111-122PubMed Google Scholar, 20Johnston DS Russell LD Friel PJ Griswold MD Murine germ cells do not require functional androgen receptors to complete spermatogenesis following spermatogonial stem cell transplantation.Endocrinology. 2001; 142: 2405-2408Crossref PubMed Google Scholar A small incision was made with a sterile 30-gauge needle ∼3 mm from the efferent bundles’ junction with the testis. The tip of the injection pipette was inserted into the bundle and then gently pushed toward the rete testis. As the tip entered the area of the rete, 10 μl of the cell suspension was injected under constant pressure. In addition to transplanting the cells into the seminiferous tubules, they were directly injected into the interstitium via rete testis puncture. Immunohistochemistry was performed on Bouin's fixed and paraffin-embedded testicular sections from recipient mice as previously described.23Lue YH Sinha Hikim AP Wang C Leung A Swerdloff RS Functional role of inducible nitric oxide synthase in the induction of male germ cell apoptosis, regulation of sperm number, and determination of testes size: evidence from null mutant mice.Endocrinology. 2003; 144 (1000): 3092Crossref PubMed Scopus (96) Google Scholar Testicular sections were briefly deparaffinized, hydrated by successive series of ethanol, rinsed in distilled water, and then incubated in 2% H2O2 to quench endogenous peroxidases. Sections were blocked with 5% normal horse serum for 20 minutes to prevent nonspecific binding of IgG and subsequently incubated with a 1:500 dilution of a monoclonal anti-GFP antibody (sc-9996; Santa Cruz Biotechnology, Santa Cruz, CA).15Johnson J Bagley J Skaznik-Wikiel M Lee HJ Adams GB Niikura Y Tschudy KS Tilly JC Cortes ML Forkert R Oocyte generation in adult mammalian ovaries by putative germ cells in bone marrow and peripheral blood.Cell. 2005; 122: 303-315Abstract Full Text Full Text PDF PubMed Scopus (562) Google Scholar Immunoreactivity was detected using biotinylated anti-mouse IgG secondary antibody followed by avidin-biotinylated horseradish peroxidase complex visualized with diaminobenzidine tetrahydrochloride (DAB) as per the manufacturer's instructions (Mouse UniTect ABC immunohistochemistry detection system; Calbiochem, La Jolla, CA). Slides were counterstained with hematoxylin and reviewed with a Zeiss Axioskop 40 microscope. Busulfan-treated testes without transplantations and testes from either wild-type or W/Wv mice were processed identically as negative controls. Testes from GFP mice were used analogously as a positive control. Bouin's fixed testicular sections were used for immunohistochemistry to detect cells with co-localized expression of GFP (green, 1:500) and Sertoli, Leydig, or germ cell markers. The Sertoli cell marker used was FSH receptor (FSH-R), the Leydig cell marker used was P450scc, and the germ cell marker used was VASA. We did not find any immunostaining of FSH-R, P450scc, and VASA in isolated bone marrow cells before transplantation. The specificity of the primary antibodies has been previously described.3Lo KC Lei Z Rao Ch V Beck J Lamb DJ De novo testosterone production in luteinizing hormone receptor knockout mice after transplantation of Leydig stem cells.Endocrinology. 2004; 145: 4011-4015Crossref PubMed Scopus (67) Google Scholar, 24Castrillon DH Quade BJ Wang TY Quigley C Crum CP The human VASA gene is specifically expressed in the germ cell lineage.Proc Natl Acad Sci USA. 2000; 97: 9585-9590Crossref PubMed Scopus (469) Google Scholar, 25Baccetti B Collodel G Costantino-Ceccarini E Eshkol A Gambera L Moretti E Strazza M Piomboni P Localization of human follicle-stimulating hormone in the testis.FASEB J. 1998; 12: 1045-1054PubMed Google Scholar After deparaffinization and rehydration, tissue sections were treated with 2% H2O2 in PBS for 10 minutes followed by 20 minutes of incubation with blocking serum (5% normal horse serum) at room temperature. After washing the slides three times in PBS (pH 7.4), sections were incubated with a 1:500 dilution of a monoclonal anti-GFP antibody (Santa Cruz Biotechnology) for 1 hour and then incubated with goat anti-mouse Alexa Fluor 488 (green)-labeled secondary antibody (Molecular Probes, Eugene, OR) for 30 minutes. Then the sections were incubated with one of the following antibodies for 1 hour: FSH-R goat polyclonal antibody (1:100; Santa Cruz Biotechnology, Inc.), P450scc rabbit polyclonal antibody (1:100; Chemicon Inc., Temecula, CA), or VASA (DDX4/MVH) rabbit polyclonal antibody (1:100; Abcam Inc., Cambridge, MA). The slides were then treated with another fluorescent secondary antibody for 30 minutes at room temperature. Goat anti-rabbit Alexa Fluor 594 (red)-labeled secondary antibody (Molecular Probes) was used for P450scc and VASA; donkey anti-goat Alexa Fluor 594 (red)-labeled secondary antibody (Molecular Probes) was used for FSH-R. Slides were washed and then mounted in Vectashield mounting medium (Vector Laboratories, Inc., Burlingame, CA). For negative controls, sections were processed without the primary antibody, and no signals were detected. Confocal imaging was performed using a TCS-SP-MP confocal microscope (Leica Corp., Deerfield, IL). The method used for germ cell quantitation was similar to that described previously.26Lue YH Jentsch JD Wang C Rao PN Hikim AP Salameh W Swerdloff RS XXY mice exhibit gonadal and behavioral phenotypes similar to Klinefelter syndrome.Endocrinology. 2005; 146: 4148-4154Crossref PubMed Scopus (58) Google Scholar, 27Lue YH Rao PN SinhaHikim AP Im M Salameh WA Yen PH Wang C Swerdloff RS XXY male mice: an experimental model for Klinefelter syndrome.Endocrinology. 2001; 142: 1461-1470Crossref PubMed Scopus (54) Google Scholar In brief, testicular sections were examined with an American Optical Microscope (Buffalo, NY) with a ×40 objective and a ×10 eyepiece. A square grid fitted within the eyepiece provided a reference area of 62,500 μm2. GFP-positive Leydig cells, Sertoli cells, and germ cells within 40 grids of testicular sections from each animal were counted. Statistical analyses were performed using the SigmaStat 2.0 program (Jandel, San Rafael, CA). Results were tested for statistical significance using a t-test. Differences were considered significant if P < 0.05. We found that GFP-positive bone marrow-derived cells survived in both busulfan-treated (Figure 1) and W/Wv testes (Figure 2) for at least 12 weeks after transplantation. GFP-positive cells were observed within seminiferous tubules and in the interstitium in both busulfan-treated (Figure 1A1) and W/Wv (Figure 2A1) recipient testes. In some of the seminiferous tubules, the green florescent cells extended from the basal lamina toward the luminal compartment and demonstrated a spatial and morphological pattern characteristic of typical Sertoli cells (Figure 1, Figure 2).Figure 2Seminiferous tubules from W/Wv recipient testis show adult bone marrow-derived cells as GFP-positive (A1, fluorescence microscopy; A2, transillumination). GFP-positive cells in seminiferous tubules exhibit a spatial and morphological pattern typical for Sertoli cells in recipient testis (B1, fluorescence microscopy; B2, transillumination). GFP-positive cells in the interstitium are not readily visible. Scale bar = 0.2 mm.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Bone marrow-derived GFP-positive donor cells were further examined by immunohistochemistry and were present in recipient testicular sections from busulfan-treated (Figure 3A) and W/Wv (Figure 3B) mice. Further morphological examination showed that some of these GFP-positive cells in the busulfan-treated recipient testis had a Sertoli cell appearance (Figure 4, A–C) characterized by an irregular nucleus containing a tripartite nucleolus located near the basal lamina as well as cytoplasm extending from the basal lamina toward luminal compartment. GFP staining was found in both nuclear and cytoplasm of bone marrow cell-derived Sertoli cells. Some of these GFP-positive cells in seminiferous tubules exhibited as a clone consisting of interconnected preleptotene and/or pachytene spermatocytes (Figure 4, E and F). In busulfan-treated mice, the donor-derived germ cells were surrounded and embedded in recovered and endogenous spermatogenesis. No GFP-positive round spermatids were found in the seminiferous tubules of testicular sections examined. In the interstitium, the GFP-positive Leydig cells were readily found embedded in the native Leydig cells in the interstitium (Figure 4D) of busulfan-treated and W/Wv mice. Quantitative data (Table 1) of GFP-positive Leydig, Sertoli, and germ cells show significantly lower differentiation rates of bone marrow-derived cells in W/Wv mice when compared with busulfan-treated mice. Testicular serial sections under confocal microscopy showed that GFP-positive donor-derived Sertoli, Leydig, and germ cells have a single nucleus.Figure 4Immunohistochemistry of GFP in Bouin's fixed, paraffin-embedded testicular sections. GFP-positive Sertoli cell (arrow) in recipient testis was shown in A–C. Note of typical morphology of Sertoli cell with both nuclear and cytoplasm staining. A: GFP-positive Sertoli cell was detected by using fluorescent secondary antibody. E: GFP-positive preleptotene spermatocytes are shown. F: A clone of germ cells including spermatogonia and spermatocytes was embedded in endogenous spermatogenesis. D: GFP-positive Leydig cells (arrow) were found in the testicular interstitium. Scale bars: 20 μm (A, C, F); 50 μm (B, D, E).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table 1GFP-Positive Bone Marrow-Derived Cells in Recipient TestesBusulfan-treated miceW/Wv miceLeydig cells/106 μm40.2 ± 17.8*Significant at P < 0.05.11.4 ± 1.98Sertoli cells/106 μm10.2 ± 1.4*Significant at P < 0.05.4.2 ± 1.0Germ cells/106 μm6.6 ± 1.8NoneValues are the mean ± SEM.Note: Germ cells, Sertoli cells, and Leydig cells were characterized based on their morphologic criteria.37Russell LD Ettlin RA SinhaHikim AP Clegg ED Histological, Histopathological Evaluation of the Testis. Cache River Press, Clearwater1990Google Scholar Germ cells included spermatogonia and spermatocytes.* Significant at P < 0.05. Open table in a new tab Values are the mean ± SEM. Note: Germ cells, Sertoli cells, and Leydig cells were characterized based on their morphologic criteria.37Russell LD Ettlin RA SinhaHikim AP Clegg ED Histological, Histopathological Evaluation of the Testis. Cache River Press, Clearwater1990Google Scholar Germ cells included spermatogonia and spermatocytes. Confocal microscopy demonstrated co-localization of GFP-positive donor-derived germ cells with VASA, a germ cell-specific marker in the testis (Figure 5). In bone marrow cell-derived germ cells, GFP was expressed in both the cytoplasm and nucleus. VASA protein was detected in both endogenous and donor-derived germ cells in the busulfan-treated recipient testis. Bone marrow cell-derived GFP-positive Sertoli cells expressed FSH-R (Figure 6). In the interstitium, GFP-positive donor-derived Leydig cells expressed P450scc (Figure 7), which is a Leydig cell marker in the testis. Twelve weeks after engraftment in W/Wv recipient testes, GFP-positive donor cells expressed FSH-R in the seminiferous tubules and P450scc in the interstitium (Figure 8). However, donor-derived germ cells were not observed in the seminiferous tubules in W/Wv recipient testes. A few GFP-positive donor cells with macrophage appearance were occasionally found in the center of the seminiferous tubules in W/Wv recipient testes.Figure 6Confocal images show GFP-positive donor-derived Sertoli cells (green; A and D), FSH-R expression (red; B and E), and co-localization of GFP with FSH-R (yellow; C and F). Scale bars = 10 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 7Confocal images show GFP-positive donor-derived Leydig cells (green, A), P450scc expression (red, B), and co-localization of GFP with P450scc (yellow, C). Scale bar = 50 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT) We demonstrated that donor-derived GFP-positive cells were present in seminiferous tubules and in the interstitium, indicating that bone marrow-derived cells survive in recipient testes for at least 12 weeks after transplantation. The donor cells used in this study were unfractionated bone marrow cells containing hematopoietic stem cells, endothelial stem/progenitor cells, mesenchymal stem cells, and multipotent adult progenitor cells. Thus, we do not know which type of bone marrow stem cells generated the testicular cells. Previous reports demonstrated that bone marrow-derived mesenchymal stem cells are capable of differentiating into germ cells and Leydig cells in the testis.17Nayernia K Lee JH Drusenheimer N Nolte J Wulf G Dressel R Gromoll J Engel W Derivation of male germ cells from bone marrow stem cells.Lab Invest. 2006; 86: 654-663Crossref PubMed Scopus (276) Google Scholar, 18Yazawa T Mizutani T Yamada K Kawata H Sekiguchi T Yoshino M Kajitani T Shou Z Umezawa A Miyamoto K Differentiation of adult stem cells derived from bone marrow stroma into Leydig or adrenocortical cells.Endocrinology. 2006; 147: 4104-4111Crossref PubMed Scopus (117) Google Scholar We intended to use busulfan-treated and W/Wv mice as our recipients. Busulfan treatment induces chemical injury of spermatogenesis, leading to infertility in male mice. In busulfan-treated recipient testes, we found GFP-positive Sertoli cells, spermatogonia, and early spermatocytes in the seminiferous tubules and Leydig cells in the interstitium. The GFP-positive germ cells were halted at the early spermatocyte stage without further differentiation into spermatids. The mechanisms of donor-derived germ cells that failed to go through meiosis remain unknown. We speculate that donor-derived germ cells arrest at the spermatocyte stage because of their inert genetic imprinting or they are incompatible with the support by Sertoli cells. In the W/Wv recipient testes, which are devoid of endogenous germ cells as a result of mutations in the c-kit receptor, we did not find germ cells. A major difference in the two recipient mice is the complete absence of endogenous germ cells in W/Wv mice and the presence of spontaneously recovered endogenous germ cells along with donor-derived germ cells in busulfan-treated mice. We found donor-derived germ cells embedded in or surrounded by the spontaneously recovered endogenous germ cells. Donor-derived Sertoli and Leydig cells were also observed among endogenous Sertoli and Leydig cells, respectively. Our observation suggests an essential role of recovering endogenous germ cells in inducing transdifferentiation of donor bone marrow cells into germ cells in the microenvironment of the seminiferous tubules. Endogenous Sertoli and Leydig cells may also play a role in inducing the differentiation of GFP-positive donor-derived Sertoli and Leydig cells because the numbers of GFP-positive cells were significantly higher in busulfan-treated recipient than W/Wv mice. Based on this observation, we conclude that on a proper migration of donor stem cells, the resident neighboring cells in the recipient testis may control site-appropriate stem cell differentiation. We found GFP-positive donor-derived cells had a single nucleus in each cell, but we cannot completely exclude the possibility of donor cell fusion with native germ, Sertoli, or Leydig cells. The percentage of GFP-positive germ, Sertoli, and Leydig cells were low in the recipient testes. The functional status of donor-derived germ, Sertoli, and Leydig cells remains to be determined. In future experiments we will use flow cytometric analysis for quantitative evaluation of donor-derived cells from both interstitial space and seminiferous tubules of recipient testes with cell-specific markers.28Kubota H Avarbock MR Brinster RL Spermatogonial stem cells share some, but not all, phenotypic and functional characteristics with other stem cells.Proc Natl Acad Sci USA. 2003; 100: 6487-6492Crossref PubMed Scopus (404) Google Scholar, 29Nayernia K Drabent B Meinhardt A Adham IM Schwandt I Muller C Sancken U Kleene KC Engel W Triple knockouts reveal gene interactions affecting fertility of male mice.Mol Reprod Dev. 2005; 70: 406-416Crossref PubMed Scopus (25) Google Scholar To increase the uptake and transdifferentiation of bone marrow cells, we plan to inject cultured and/or isolated and enriched adult stem cells alone with growth factors such as glial cell line-derived neurotrophic factor, stem cell factor, and insulin-like growth factors. Isolation of hematopoietic stem cells has been achieved.30Spangrude GJ Heimfeld S Weissman IL Purification and characterization of mouse hematopoietic stem cells.Science. 1988; 241: 58-62Crossref PubMed Scopus (2235) Google Scholar Clonogenic in vivo and in vitro assays suggest a high level of purity (∼85 to 95%) is attainable for these cells.31Wagers AJ Sherwood RI Christensen JL Weissman IL Little evidence for developmental plasticity of adult hematopoietic stem cells.Science. 2002; 297: 2256-2259Crossref PubMed Scopus (1311) Google Scholar Multipotent marrow stromal cells, which give rise to multiple mesenchymal lineages, can also be isolated from bone marrow.32Prockop DJ Gregory CA Spees JL One strategy for cell and gene therapy: harnessing the power of adult stem cells to repair tissues.Proc Natl Acad Sci USA. 2003; 100: 11917-11923Crossref PubMed Scopus (371) Google Scholar, 33Gronthos S Zannettino AC Hay SJ Shi S Graves SE Kortesidis A Simmons PJ Molecular and cellular characterization of highly purified stromal stem cells derived from human bone marrow.J Cell Sci. 2003; 116: 1827-1835Crossref PubMed Scopus (917) Google Scholar The molecular mechanism of adult stem cell plasticity is not completely understood. The testis creates a unique microenvironment for donor stem cell migration, proliferation, differentiation, and apoptosis. The testis is protected from immunological influences by the blood-testis barrier allowing the recipient to host donor cells without rejection. By transplanting adult stem cells isolated from gene knockout or transgenic mice into wild-type mice, or vice versa, we will be able to study the effect of gene mutations on stem cell biology. The pathogenesis of male infertility is attributable either to the failure in germ cell proliferation and differentiation or to somatic cell dysfunction. In many cases, germ cells are present. The presence of donor-derived somatic cells is critical because both Leydig and Sertoli cells support spermatogenesis. Defects in these cells have been believed to contribute to abnormal spermatogenesis.34Salameh WA Swerdloff RS Conditions affecting Sertoli cells.in: Skinner MK Griswold MD Sertoli Cell Biology. Elsevier Academic Press, San Diego2005: 383-416Crossref Scopus (3) Google Scholar The possibility of beneficial hormonal effects of Leydig cell transplantation independent of their support of spermatogenesis also exits. Because Leydig cells are responsible for testosterone production, stem cell transplantations may replace the need of life-long testosterone supplementation in male hypogonadism or aging.35Swerdloff RS Wang C Androgen and the aging male.Best Proc Res Clin Endocrinol Metab. 2004; 18: 349-362Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 36Liu PY Swerdloff RS Veldhuis JD Clinical review 171: the rationale, efficacy and safety of androgen therapy in older men: future research and current practice recommendations.J Clin Endocrinol Metab. 2004; 89: 4789-4796Crossref PubMed Scopus (136) Google Scholar Additional studies are required to demonstrate such hormone benefits. Thus, the present finding may have a major impact in understanding reproductive physiology and recovery from testicular pathology and also may have the potential for novel future therapies in patients with testicular failure. We thank Michael D. Griswold for training Y.L. in the germ cell transplantation technique, and Vince Atienza for technical assistance." @default.
• W1584801101 created "2016-06-24" @default.
• W1584801101 creator A5007389731 @default.
• W1584801101 creator A5008434600 @default.
• W1584801101 creator A5040182243 @default.
• W1584801101 creator A5048381041 @default.
• W1584801101 creator A5053829971 @default.
• W1584801101 creator A5075943191 @default.
• W1584801101 creator A5090807122 @default.
• W1584801101 date "2007-03-01" @default.
• W1584801101 modified "2023-09-30" @default.
• W1584801101 title "Fate of Bone Marrow Stem Cells Transplanted into the Testis" @default.
• W1584801101 cites W126539919 @default.
• W1584801101 cites W1770672913 @default.
• W1584801101 cites W1968442155 @default.
• W1584801101 cites W1970159856 @default.
• W1584801101 cites W1973035870 @default.
• W1584801101 cites W1993491850 @default.
• W1584801101 cites W1997377831 @default.
• W1584801101 cites W2006368622 @default.
• W1584801101 cites W2008472259 @default.
• W1584801101 cites W2016789158 @default.
• W1584801101 cites W2021971921 @default.
• W1584801101 cites W2032847865 @default.
• W1584801101 cites W2033042516 @default.
• W1584801101 cites W2042686954 @default.
• W1584801101 cites W2047443703 @default.
• W1584801101 cites W2048258494 @default.
• W1584801101 cites W2048329493 @default.
• W1584801101 cites W2055988619 @default.
• W1584801101 cites W2060702385 @default.
• W1584801101 cites W2062601134 @default.
• W1584801101 cites W2076069593 @default.
• W1584801101 cites W2082617977 @default.
• W1584801101 cites W2104592236 @default.
• W1584801101 cites W2105159857 @default.
• W1584801101 cites W2108030133 @default.
• W1584801101 cites W2115183068 @default.
• W1584801101 cites W2115929799 @default.
• W1584801101 cites W2128786355 @default.
• W1584801101 cites W2133685760 @default.
• W1584801101 cites W2142207476 @default.
• W1584801101 cites W2145199573 @default.
• W1584801101 cites W2158048826 @default.
• W1584801101 cites W2166741387 @default.
• W1584801101 cites W2170748214 @default.
• W1584801101 cites W30145574 @default.
• W1584801101 doi "https://doi.org/10.2353/ajpath.2007.060543" @default.
• W1584801101 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/1864883" @default.
• W1584801101 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/17322375" @default.
• W1584801101 hasPublicationYear "2007" @default.
• W1584801101 type Work @default.
• W1584801101 sameAs 1584801101 @default.
• W1584801101 citedByCount "104" @default.
• W1584801101 countsByYear W15848011012012 @default.
• W1584801101 countsByYear W15848011012013 @default.
• W1584801101 countsByYear W15848011012014 @default.
• W1584801101 countsByYear W15848011012015 @default.
• W1584801101 countsByYear W15848011012016 @default.
• W1584801101 countsByYear W15848011012017 @default.
• W1584801101 countsByYear W15848011012018 @default.
• W1584801101 countsByYear W15848011012019 @default.
• W1584801101 countsByYear W15848011012020 @default.
• W1584801101 countsByYear W15848011012021 @default.
• W1584801101 countsByYear W15848011012022 @default.
• W1584801101 countsByYear W15848011012023 @default.
• W1584801101 crossrefType "journal-article" @default.
• W1584801101 hasAuthorship W1584801101A5007389731 @default.
• W1584801101 hasAuthorship W1584801101A5008434600 @default.
• W1584801101 hasAuthorship W1584801101A5040182243 @default.
• W1584801101 hasAuthorship W1584801101A5048381041 @default.
• W1584801101 hasAuthorship W1584801101A5053829971 @default.
• W1584801101 hasAuthorship W1584801101A5075943191 @default.
• W1584801101 hasAuthorship W1584801101A5090807122 @default.
• W1584801101 hasBestOaLocation W15848011011 @default.
• W1584801101 hasConcept C142724271 @default.
• W1584801101 hasConcept C203014093 @default.
• W1584801101 hasConcept C2780007613 @default.
• W1584801101 hasConcept C28328180 @default.
• W1584801101 hasConcept C71924100 @default.
• W1584801101 hasConcept C86803240 @default.
• W1584801101 hasConcept C95444343 @default.
• W1584801101 hasConceptScore W1584801101C142724271 @default.
• W1584801101 hasConceptScore W1584801101C203014093 @default.
• W1584801101 hasConceptScore W1584801101C2780007613 @default.
• W1584801101 hasConceptScore W1584801101C28328180 @default.
• W1584801101 hasConceptScore W1584801101C71924100 @default.
• W1584801101 hasConceptScore W1584801101C86803240 @default.
• W1584801101 hasConceptScore W1584801101C95444343 @default.
• W1584801101 hasIssue "3" @default.
• W1584801101 hasLocation W15848011011 @default.
• W1584801101 hasLocation W15848011012 @default.
• W1584801101 hasLocation W15848011013 @default.
• W1584801101 hasLocation W15848011014 @default.
• W1584801101 hasOpenAccess W1584801101 @default.
• W1584801101 hasPrimaryLocation W15848011011 @default.
• W1584801101 hasRelatedWork W1970707390 @default.
• W1584801101 hasRelatedWork W2027717767 @default.

• next