scholarly journals Autologous and homologous transplantation of bovine spermatogonial stem cells

Reproduction ◽  
2003 ◽  
pp. 765-774 ◽  
Author(s):  
F Izadyar ◽  
K Den Ouden ◽  
TA Stout ◽  
J Stout ◽  
J Coret ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cross Sections ◽  
Autologous Transplantation ◽  
Type A ◽  
Spermatogonial Stem Cells ◽  
Rete Testis ◽  
X Rays ◽  
Spermatogonial Transplantation ◽  
Homologous Transplantation ◽  
Type A Spermatogonia

The aim of this study was to develop a method for spermatogonial stem cell transplantation into the bovine testis. Five-month-old Holstein-Friesian calves were used and half of the calves were hemicastrated to allow autologous transplantation and the other half were used for homologous transplantation. Approximately 20 g of each testis was used for cell isolation. On average 106 cells per gram of testis containing about 70% type A spermatogonia were isolated. The cells were frozen in liquid nitrogen until transplantation. Testes were irradiated locally with 10-14 Gy of X-rays to deplete endogenous spermatogenesis. At 2 months after irradiation, cells (approximately 10 x 10(6) were injected into the rete testis through a long injection needle (18 gauge), using ultrasonography and an ultrasound contrast solution. At 2.5 months after transplantation, calves were castrated and samples of testes were taken for histological examination. After 2.5 months in the irradiated non-transplanted control testes, only 45% of the tubules contained type A spermatogonia. However, after autologous spermatogonial transplantation, >80% of the tubule cross-sections contained type A spermatogonia. In addition, only 20% of the tubules of the control testes contained spermatocytes and, except for a few tubules (5%) with round spermatids, no more advanced germ cells were found. After autologous spermatogonial transplantation, about 60% of the tubules contained spermatocytes; 30% contained spermatids and in about 15% of tubules spermatozoa were found. No improvement in spermatogonial repopulation was found after homologous transplantation. The results of this study demonstrate, for the first time, successful autologous transplantation of bovine spermatogonial stem cells resulting in a complete regeneration of spermatogenesis.

scholarly journals Propagation of bovine spermatogonial stem cells in vitro

Reproduction ◽  
2008 ◽  
Vol 136 (5) ◽  
pp. 543-557 ◽  
Author(s):  
Pedro M Aponte ◽  
Takeshi Soda ◽  
Katja J Teerds ◽  
S Canan Mizrak ◽  
Henk J G van de Kant ◽  
...  
Keyword(s):  
Stem Cells ◽  
Growth Factor ◽  
Cultured Cells ◽  
Somatic Cells ◽  
Type A ◽  
Spermatogonial Stem Cells ◽  
Seminiferous Tubules ◽  
Type A Spermatogonia ◽  
Stimulation Of

The access to sufficient numbers of spermatogonial stem cells (SSCs) is a prerequisite for the study of their regulation and further biomanipulation. A specialized medium and several growth factors were tested to study thein vitrobehavior of bovine type A spermatogonia, a cell population that includes the SSCs and can be specifically stained for the lectin Dolichos biflorus agglutinin. During short-term culture (2 weeks), colonies appeared, the morphology of which varied with the specific growth factor(s) added. Whenever the stem cell medium was used, round structures reminiscent of sectioned seminiferous tubules appeared in the core of the colonies. Remarkably, these round structures always contained type A spermatogonia. When leukemia inhibitory factor (LIF), epidermal growth factor (EGF), or fibroblast growth factor 2 (FGF2) were added, specific effects on the numbers and arrangement of somatic cells were observed. However, the number of type A spermatogonia was significantly higher in cultures to which glial cell line-derived neurotrophic factor (GDNF) was added and highest when GDNF, LIF, EGF, and FGF2 were all present. The latter suggests that a proper stimulation of the somatic cells is necessary for optimal stimulation of the germ cells in culture. Somatic cells present in the colonies included Sertoli cells, peritubular myoid cells, and a few Leydig cells. A transplantation experiment, using nude mice, showed the presence of SSCs among the cultured cells and in addition strongly suggested a more than 10 000-fold increase in the number of SSCs after 30 days of culture. These results demonstrate that bovine SSC self-renew in our specialized bovine culture system and that this system can be used for the propagation of these cells.

scholarly journals Expression and functional analyses of ephrin type-A receptor 2 in mouse spermatogonial stem cells†

2019 ◽  
Vol 102 (1) ◽  
pp. 220-232 ◽  
Author(s):  
Hiroko Morimoto ◽  
Mito Kanatsu-Shinohara ◽  
Kyle E Orwig ◽  
Takashi Shinohara
Keyword(s):  
Stem Cells ◽  
Cell Sorting ◽  
Type A ◽  
Spermatogonial Stem Cells ◽  
Fibroblast Growth Factor 2 ◽  
Self Renewal ◽  
Spermatogonial Transplantation ◽  
Short Hairpin ◽  
Functional Analyses ◽  
Magnetic Activated Cell Sorting

Abstract Spermatogonial stem cells (SSCs) undergo continuous self-renewal division in response to self-renewal factors. The present study identified ephrin type-A receptor 2 (EPHA2) on mouse SSCs and showed that supplementation of glial cell-derived neurotrophic factor (GDNF) and fibroblast growth factor 2 (FGF2), which are both SSC self-renewal factors, induced EPHA2 expression in cultured SSCs. Spermatogonial transplantation combined with magnetic-activated cell sorting or fluorescence-activated cell sorting also revealed that EPHA2 was expressed in SSCs. Additionally, ret proto-oncogene (RET) phosphorylation levels decreased following the knockdown (KD) of Epha2 expression via short hairpin ribonucleic acid (RNA). Although the present immunoprecipitation experiments did not reveal an association between RET with EPHA2, RET interacted with FGFR2. The Epha2 KD decreased the proliferation of cultured SSCs and inhibited the binding of cultured SSCs to laminin-coated plates. The Epha2 KD also significantly reduced the colonization of testis cells by spermatogonial transplantation. EPHA2 was also expressed in human GDNF family receptor alpha 1-positive spermatogonia. The present results indicate that SSCs express EPHA2 and suggest that it is a critical modifier of self-renewal signals in SSCs.

scholarly journals Radioprotective effect of misoprostol on mouse spermatogonial stem cells

1998 ◽  
Vol 72 (3) ◽  
pp. 185-189 ◽  
Author(s):  
DIRK G. DE ROOIJ ◽  
MARIA E. A. B. VAN BEEK ◽  
DERK H. RUTGERS ◽  
ANNEMARIE VAN DUYN-GOEDHART ◽  
PAUL P. W. VAN BUUL
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Cross Sections ◽  
Dose Response ◽  
Spermatogonial Stem Cells ◽  
Cell Killing ◽  
Prostaglandin E ◽  
X Rays ◽  
Response Curves ◽  
F1 Hybrid

The radioprotective effects of misoprostol, a synthetic stable analogue of prostaglandin E1, on spermatogonial stem cells of C3H/HeH×101/F1 hybrid mice (3H1) were analysed by establishing dose–response relationships for stem cell killing by X-rays in mice that were pretreated with misoprostol. Spermatogonial stem cell killing was studied through determination of the percentage of tubular cross-sections showing repopulation at 10 days after irradiation. In control mice, the D0 values ranged between 1·7 and 3·6 Gy, dependent on the stage of the cycle of the seminiferous epithelium the cells were in. As found previously, proliferating spermatogonial stem cells were much more radioresistant than quiescent stem cells. In the misoprostol-pretreated animals the spermatogonial stem cells were more radioresistant, the D0 values ranging from 3·6 to 5·0 Gy. Both proliferating and quiescent spermatogonial stem cells were protected by misoprostol. As the dose–response curves in control and misoprostol-pretreated mice showed about the same extrapolation number to the y-axis it was concluded that the misoprostol pretreatment did not alter the kinetics of the repopulation process.

scholarly journals Isolation and purification of type A spermatogonia from the bovine testis

Reproduction ◽  
2002 ◽  
pp. 85-94 ◽  
Author(s):  
F Izadyar ◽  
GT Spierenberg ◽  
LB Creemers ◽  
K den Ouden ◽  
DG de Rooij
Keyword(s):  
Stem Cells ◽  
Cell Suspension ◽  
Light Microscope ◽  
Type A ◽  
Spermatogonial Stem Cells ◽  
Recipient Mouse ◽  
Isolated Cells ◽  
Isolation And Purification ◽  
Bovine Type ◽  
Type A Spermatogonia

The aim of this study was to isolate and purify bovine type A spermatogonia. Testes from 5-7-month-old calves were used to isolate germ cells using a two-step enzymatic digestion. During the isolation and purification steps, the viability of cells was determined using live/dead staining. The identity of type A spermatogonia during isolation and purification was determined under a light microscope equipped with a Nomarski lens. Isolated cells were characterized further by using specific markers for type A spermatogonia, including Dolichos biflorus agglutinin (DBA) and c-kit. The cell suspension was transplanted into immunodeficient recipient mouse testes and the colonization was assessed 1-3 months after transplantation, to assess the stem cell population among the isolated cells. After isolation, a cell suspension was obtained containing about 25% type A spermatogonia, which was enriched further by differential plating and separation on a discontinuous Percoll gradient. Finally, fractions containing 65-87% pure type A spermatogonia were obtained. Large and small type A spermatogonia with different numbers and sizes of nucleoli were found. DBA stained both large and small type A spermatogonia and its application in fluorescence-activated cell sorting (FACS) resulted in comparable percentages of type A spermatogonia to those determined by morphological examination under a light microscope equipped with a Nomarski lens. Nearly all of the large type A spermatogonia showed strong c-kit immunoreactivity, indicating that these cells had undergone at least an initial differentiation step. In contrast, approximately half of the small type A spermatogonia were negative for c-kit, indicating the presence of the spermatogonial stem cells in this population. At 3 months after transplantation, groups of bovine type A spermatogonia were found in most tubule cross-sections of the recipient mouse testes, showing the presence of spermatogonial stem cells among the isolated cells.

Regulation of proliferation and differentiation in spermatogonial stem cells: the role of c-kit and its ligand SCF

Development ◽  
2000 ◽  
Vol 127 (10) ◽  
pp. 2125-2131 ◽  
Author(s):  
H. Ohta ◽  
K. Yomogida ◽  
K. Dohmae ◽  
Y. Nishimune
Keyword(s):  
Stem Cells ◽  
Germ Cells ◽  
Type A ◽  
Spermatogonial Stem Cells ◽  
Mutant Mice ◽  
Seminiferous Tubules ◽  
Kit Receptor ◽  
Proliferation And Differentiation ◽  
Cell Niche ◽  
Type A Spermatogonia

To study self-renewal and differentiation of spermatogonial stem cells, we have transplanted undifferentiated testicular germ cells of the GFP transgenic mice into seminiferous tubules of mutant mice with male sterility, such as those dysfunctioned at Steel (Sl) locus encoding the c-kit ligand or Dominant white spotting (W) locus encoding the receptor c-kit. In the seminiferous tubules of Sl/Sl(d) or Sl(17H)/Sl(17H) mice, transplanted donor germ cells proliferated and formed colonies of undifferentiated c-kit (−) spermatogonia, but were unable to differentiate further. However, these undifferentiated but proliferating spermatogonia, retransplanted into Sl (+) seminiferous tubules of W mutant, resumed differentiation, indicating that the transplanted donor germ cells contained spermatogonial stem cells and that stimulation of c-kit receptor by its ligand was necessary for maintenance of differentiated type A spermatogonia but not for proliferation of undifferentiated type A spermatogonia. Furthermore, we have demonstrated that their transplantation efficiency in the seminiferous tubules of Sl(17H)/Sl(17H) mice depended upon the stem cell niche on the basement membrane of the recipient seminiferous tubules and was increased by elimination of the endogenous spermatogonia of mutant mice from the niche by treating them with busulfan.

Studies of Sl/Sld in equilibrium with +/+ mouse aggregation chimaeras. II. Effect of the steel locus on spermatogenesis

Development ◽  
1988 ◽  
Vol 102 (1) ◽  
pp. 117-126 ◽  
Author(s):  
H. Nakayama ◽  
H. Kuroda ◽  
H. Onoue ◽  
J. Fujita ◽  
Y. Nishimune ◽  
...  
Keyword(s):  
Mast Cells ◽  
Germ Cell ◽  
Germ Cells ◽  
Cross Sections ◽  
Sertoli Cells ◽  
Type A ◽  
Precursor Cells ◽  
Intrinsic Defect ◽  
Serial Sections ◽  
Type A Spermatogonia

Mutant mice of Sl/Sld genotype are deficient in melanocytes, erythrocytes, mast cells and germ cells. Deficiency of melanocytes, erythrocytes and mast cells is not attributable to an intrinsic defect in their precursor cells but to a defect in the tissue environment that is necessary for migration, proliferation and/or differentiation. We investigated the mechanism of germ cell deficiency in male Sl/Sld mice by producing aggregation chimaeras from Sl/Sld and +/+ embryos. Chimaeric mice with apparent white stripes were obtained. Two of four such chimaeras were fertile and the phenotypes of resulting progenies showed that some Sl/Sld germ cells had differentiated into functioning sperms in the testis of the chimaeras. In cross sections of the testes of chimaeras, both differentiated and nondifferentiated tubules were observed. However, the proportions of type A spermatogonia to Sertoli cells in both types of tubules were comparable to the values observed in differentiated tubules of normal +/+ mice. We reconstructed the whole length of four tubules from serial sections. Differentiated and nondifferentiated segments alternated in a single tubule. The shortest differentiated segment contained about 180 Sertoli cells and the shortest nondifferentiated segment about 150 Sertoli cells. These results suggest that Sertoli cells of either Sl/Sld or +/+ genotype make discrete patches and that differentiation of type A spermatogonia does not occur in patches of Sl/Sld Sertoli cells.

Expression and intracellular localization of Nanos2-homologue protein in primordial germ cells and spermatogonial stem cells

Zygote ◽  
2019 ◽  
Vol 27 (02) ◽  
pp. 82-88 ◽  
Author(s):  
Vivek Pandey ◽  
Anima Tripathi ◽  
Pawan K. Dubey
Keyword(s):  
Stem Cells ◽  
Flow Cytometry ◽  
Germ Cells ◽  
Expression Patterns ◽  
Intracellular Localization ◽  
Primordial Germ Cells ◽  
Type A ◽  
Spermatogonial Stem Cells ◽  
Single Type ◽  
Rt Pcr

SummaryThe decision by germ cells to differentiate and undergo either oogenesis or spermatogenesis takes place during embryonic development and Nanos plays an important role in this process. The present study was designed to investigate the expression patterns in rat of Nanos2-homologue protein in primordial germ cells (PGCs) over different embryonic developmental days as well as in spermatogonial stem cells (SSCs). Embryos from three different embryonic days (E8.5, E10.5, E11.5) and SSCs were isolated and used to detect Nanos2-homologue protein using immunocytochemistry, western blotting, reverse transcription polymerase chain reaction (RT-PCR) and flow cytometry. Interestingly, Nanos2 expression was detected in PGCs at day E11.5 onwards and up to colonization of PGCs in the genital ridge of fetal gonads. No Nanos2 expression was found in PGCs during early embryonic days (E8.5 and 10.5). Furthermore, immunohistochemical and immunofluorescence data revealed that Nanos2 expression was restricted within a subpopulation of undifferentiated spermatogonia (As, single type A SSCs and Apr, paired type A SSCs). The same results were confirmed by our western blot and RT-PCR data, as Nanos2 protein and transcripts were detected only in PGCs from day E11.5 and in undifferentiated spermatogonia (As and Apr). Furthermore, Nanos2-positive cells were also immunodetected and sorted using flow cytometry from the THY1-positive SSCs population, and this strengthened the idea that these cells are stem cells. Our findings suggested that stage-specific expression of Nanos2 occurred on different embryonic developmental days, while during the postnatal period Nanos2 expression is restricted to As and Apr SSCs.

scholarly journals Circulating stem cells in mice treated with cyclophosphamide

Blood ◽  
1992 ◽  
Vol 80 (1) ◽  
pp. 264-269 ◽  
Author(s):  
CF Craddock ◽  
JF Apperley ◽  
EG Wright ◽  
LE Healy ◽  
CA Bennett ◽  
...  
Keyword(s):  
Stem Cells ◽  
Peripheral Blood ◽  
Blood Cells ◽  
Cultured Cells ◽  
Autologous Transplantation ◽  
White Blood Cells ◽  
X Rays ◽  
Donor Cells ◽  
Macrophage Colony

Abstract Chemotherapy has been used clinically to mobilize hematopoietic progenitor cells into the peripheral blood so that they can be harvested for autologous transplantation. In humans, this is demonstrated by the presence of circulating granulocyte-macrophage colony-forming cells (CFU-GM) and CD34-positive cells, but it has not been possible to confirm the presence of marrow-repopulating stem cells. In this study, we treated mice with 200 mg/kg cyclophosphamide (CY) and measured the numbers of white blood cells, day 12 CFU-S (CFU- S12), and CFU-GM in the peripheral blood. There was a peak in the numbers of CFU-S12 and CFU-GM 8 days after treatment with cyclophosphamide. Peripheral blood cells taken at this time rescued lethally irradiated mice and engraftment of donor cells was confirmed after 140 days in sex mismatched recipients using a Y chromosome- specific probe. In vitro culture of the blood cells harvested after cyclophosphamide showed that they proliferated in suspension cultures for at least a year in the presence of interleukin-3. The cultured cells rapidly lost their abilities to rescue irradiated mice and to form colonies in vitro, but they did not become leukemic. Also, CY- treated mice were irradiated with a leukemogenic dose of x-rays to coincide with peak circulating cell numbers but these animals did not develop an excess of leukemias over mice given irradiation alone.

scholarly journals Autologous transplantation of spermatogonial stem cells restores fertility in congenitally infertile mice

2020 ◽  
Vol 117 (14) ◽  
pp. 7837-7844
Author(s):  
Mito Kanatsu-Shinohara ◽  
Narumi Ogonuki ◽  
Shogo Matoba ◽  
Atsuo Ogura ◽  
Takashi Shinohara
Keyword(s):  
Stem Cells ◽  
Sertoli Cells ◽  
Expression Patterns ◽  
Autologous Transplantation ◽  
Spermatogonial Stem Cells ◽  
Congenital Defects ◽  
Seminiferous Tubules ◽  
Special Environment ◽  
Deficient Mice

The blood–testis barrier (BTB) is thought to be indispensable for spermatogenesis because it creates a special environment for meiosis and protects haploid cells from the immune system. The BTB divides the seminiferous tubules into the adluminal and basal compartments. Spermatogonial stem cells (SSCs) have a unique ability to transmigrate from the adluminal compartment to the basal compartment through the BTB upon transplantation into the seminiferous tubule. Here, we analyzed the role ofCldn11, a major component of the BTB, in spermatogenesis using spermatogonial transplantation.Cldn11-deficient mice are infertile due to the cessation of spermatogenesis at the spermatocyte stage.Cldn11-deficient SSCs failed to colonize wild-type testes efficiently, andCldn11-deficient SSCs that underwent double depletion ofCldn3andCldn5showed minimal colonization, suggesting that claudins on SSCs are necessary for transmigration. However,Cldn11-deficient Sertoli cells increased SSC homing efficiency by >3-fold, suggesting that CLDN11 in Sertoli cells inhibits transmigration of SSCs through the BTB. In contrast to endogenous SSCs in intactCldn11-deficient testes, those from WT orCldn11-deficient testes regenerated sperm inCldn11-deficient testes. The success of this autologous transplantation appears to depend on removal of endogenous germ cells for recipient preparation, which reprogrammed claudin expression patterns in Sertoli cells. Consistent with this idea, in vivo depletion ofCldn3/5regenerated endogenous spermatogenesis inCldn11-deficient mice. Thus, coordinated claudin expression in both SSCs and Sertoli cells expression is necessary for SSC homing and regeneration of spermatogenesis, and autologous stem cell transplantation can rescue congenital defects of a self-renewing tissue.

Sign in / Sign up

Export Citation Format

Share Document