scholarly journals Molecular dissection of the male germ cell lineage identifies putative spermatogonial stem cells in rhesus macaques

2009 ◽  
Vol 24 (7) ◽  
pp. 1704-1716 ◽  
Author(s):  
Brian P. Hermann ◽  
Meena Sukhwani ◽  
David R. Simorangkir ◽  
Tianjiao Chu ◽  
Tony M. Plant ◽  
...  
Keyword(s):  
Stem Cells ◽  
Germ Cell ◽  
Rhesus Macaques ◽  
Cell Lineage ◽  
Spermatogonial Stem Cells ◽  
Male Germ Cell ◽  
Molecular Dissection

scholarly journals Male germ cell transplantation in livestock

2006 ◽  
Vol 18 (2) ◽  
pp. 13 ◽  
Author(s):  
J. R. Hill ◽  
I. Dobrinski
Keyword(s):  
Stem Cells ◽  
Germ Cell ◽  
Cell Transplantation ◽  
Large Scale ◽  
Developmental State ◽  
Male Germ Cell ◽  
Seminiferous Tubules ◽  
Cell Transfer ◽  
Donor Sperm ◽  
Germ Cell Transplantation

Male germ cell transplantation is a powerful approach to study the control of spermatogenesis with the ultimate goal to enhance or suppress male fertility. In livestock animals, applications can be expanded to provide an alternative method of transgenesis and an alternative means of artificial insemination (AI). The transplantation technique uses testis stem cells, harvested from the donor animal. These donor stem cells are injected into seminiferous tubules, migrate from the lumen to relocate to the basement membrane and, amazingly, they can retain the capability to produce donor sperm in their new host. Adaptation of the mouse technique for livestock is progressing, with gradual gains in efficiency. Germ cell transfer in goats has produced offspring, but not yet in cattle and pigs. In goats and pigs, the applications of germ cell transplantation are mainly in facilitating transgenic animal production. In cattle, successful male germ cell transfer could create an alternative to AI in areas where it is impractical. Large-scale culture of testis stem cells would enhance the use of elite bulls by providing a renewable source of stem cells for transfer. Although still in a developmental state, germ cell transplantation is an emerging technology with the potential to create new opportunities in livestock production.

217 PROTEIN-INDUCED TRANSDIFFERENTIATION INTO MALE GERM CELL-LIKE LINEAGE FROM CHICKEN FETAL BONE MARROW STEM CELLS

2012 ◽  
Vol 24 (1) ◽  
pp. 220
Author(s):  
J. M. Yoo ◽  
J. J. Park ◽  
K. Gobianand ◽  
J. Y. Ji ◽  
J. S. Kim ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Retinoic Acid ◽  
Germ Cell ◽  
Germ Cells ◽  
Cultured Cells ◽  
Male Germ Cell ◽  
Male Germ Cells ◽  
Germ Cell Differentiation ◽  
Transgenic Chickens

Bone marrow (BM)-derived stem cells are capable of transdifferentiation into multilineage cells like muscle, bone, cartilage, fat and nerve cells. In this study, we investigated the capability of mesenchymal stem cells (MSC) derived from BM into germ cell differentiation in the chicken. Chicken MSCs were isolated from BM of day 20 fertilized fetal chicken with Ficoll-Paque Plus. Isolated cells were cultured in advance-DMEM (ADMEM) supplemented with 10% fetal bovine serum and antibiotics. Once confluent, cells were subcultured until five passages. The cultured cells showed fibroblast-like morphology. The cells had positive expressions of Oct4, Sox2 and Nanog. Two induction methods were conducted to examine the ability of transdifferentation into male germ cells. In group 1, MSC were cultured in ADMEM containing retinoic acid and chicken testicular extracts proteins for 10 to 15 days. In group 2, MSC were permeabilized by streptolysin O and treated with chicken testicular protein extracts. In both treatment groups, MSC were cultured in ADMEM containing retinoic acid for 10 to 15 days. We found that chicken MSC had a positive expression of pluripotent proteins such as Oct4, Sox2, Nanog and a small population of chicken MSC seem to transdifferentiate into male germ cell-like cells. These cells expressed early germ cell markers and male germ-cell-specific markers (Dazl, C-kit, Stra8 and DDX4) as analysed by reverse transcription-PCR and immunohistochemistry. These results demonstrated that chicken MSC may differentiate into male germ cells and the same might be used as a potential source of cells for production of transgenic chickens. This study was carried out with the support of Agenda Program (Project No. PJ0064692011), RDA and Republic of Korea.

428 PRODUCTION OF GERM-LINEAGE CELL AND FUNCTIONAL GAMETES FROM MULTI-POTENT SPERMATOGONIAL STEM CELLS

2010 ◽  
Vol 22 (1) ◽  
pp. 371
Author(s):  
J. E. Lim ◽  
J. H. Eum ◽  
H. J. Kim ◽  
H. S. Lee ◽  
J. H. Kim ◽  
...  
Keyword(s):  
Stem Cells ◽  
Germ Cell ◽  
Germ Cells ◽  
Culture Medium ◽  
Genetically Modified ◽  
Spermatogonial Stem Cells ◽  
Specific Gene ◽  
Specific Marker ◽  
Male Gametes ◽  
Genetically Modified Mice

Multi-potent spermatogonial stem cells (mSSC), derived from uni-potent SSC, are a type of reprogrammed cells with similar characteristics to embryonic stem cells (ESC). Similar to ESC, mSSC are capable of differentiating into 3-germ layers in vitro and teratoma formation in vivo. Additionally, mSSC proliferate rapidly and can be transfected more easily than SSC. In contrast to previous reports, we have found that mSSC also have germ-cell-specific micro (mi)RNA and gene expression profiles. Therefore, the aims of this study were to compare the efficiency of mSSC v. ESC to differentiate into germ lineage and produce male gametes, as well as to develop a novel system for the production of genetically modified mice. Mouse mSSC were transfected with a lentiviral vector expressing green fluorescent protein (GFP) and testis-specific gene and maintained in the ESC-culture medium containing leukemia inhibitory factor (LIF). Embryonic bodies (EB) were formed after the cells were detached from the feeder cells. Bone morphogenetic protein (BMP)-4 (10 ng mL˜1) and retinoic acid (RA, 0.1 μM) were added to the ESC-culture medium for 3 days in order to induce differentiation into germ lineage cells. Then, these cells were changed to germ cell-culture medium (Stem-Pro™ containing GDNF; Invitrogen, Carlsbad, CA, USA) and cultured for 3 days. After 6 days, cultured cells were sorted by magnetic activating cell sorting system using specific marker for germ cells, CD-9. Isolated germ lineage cells were transplanted into a busulfan-treated mouse testis for the production of male germ cells. Three to 6 weeks later, the testis and epididymis were collected, and half of the sample was used to perform histological analysis and the other half for the production of intracytoplasmic sperm injection (ICSI)-derived embryos. The statistical significance of differences between the 2 groups was evaluated by Student’s t-test Immunocytochemical and flow cytometrical analysis performed 6 days after differentiation showed that the ratio of germ cell-specific markers in EB derived from mSSC was higher than those from ESC. Moreover, after 3 to 6 weeks of transplantation the testis produced sperms and germ cells expressing GFP. We have successfully produced embryos by ICSI and offspring by embryo transfer into uteri of poster mothers. These results demonstrate that mSSC can be easily differentiated into germ lineage cells compared with ESC and have the potential to generate functional gametes. Therefore, the differentiation and transgenesis of mSSC may be a useful model for production of genetically modified mice. This work was supported by a grant of the Korea Healthcare Technology R&D Project, Ministry for Health, Welfare & Family Affairs, Republic of Korea (A084923).

286 EXPRESSION OF PLURIPOTENT STEM CELL MARKERS IN DOMESTIC CAT SPERMATOGONIAL CELLS

2013 ◽  
Vol 25 (1) ◽  
pp. 290 ◽  
Author(s):  
R. H. Powell ◽  
M. N. Biancardi ◽  
J. Galiguis ◽  
Q. Qin ◽  
C. E. Pope ◽  
...  
Keyword(s):  
Stem Cells ◽  
Flow Cytometry ◽  
Basement Membrane ◽  
Germ Cell ◽  
Germ Cells ◽  
Spermatogonial Stem Cells ◽  
Seminiferous Tubules ◽  
Cell Populations ◽  
Surface Markers ◽  
Cell Markers

Spermatogonial stem cells (SSC), progenitor cells capable of both self-renewal and producing daughter cells that will differentiate into sperm, can be manipulated for transplantation to propagate genetically important males. This application was demonstrated in felids by the successful xeno-transplantation of ocelot mixed germ cells into the testes of domestic cats, which resulted in the production of ocelot sperm (Silva et al. 2012 J. Androl. 33, 264–276). Spermatogonial stem cells are in low numbers in the testis, but have been identified and isolated in different mammalian species using SSC surface markers; however, their expression varies among species. Until recently, little was known about the expression of SSC surface markers in feline species. We previously demonstrated that many mixed germ cells collected from adult cat testes express the germ cell markers GFRα1, GPR125, and C-Kit, and a smaller population of cells expresses the pluripotent SSC-specific markers SSEA-1 and SSEA-4 (Powell et al. 2011 Reprod. Fertil. Dev. 24, 221–222). In the present study, our goal was to identify germ cell and SSC-specific markers in SSC from cat testes. Immunohistochemical (IHC) localization of germ cell markers GFRα1, GPR125, and C-Kit and pluripotent SSC-specific markers SSEA-1, SSEA-4, TRA-1-60, TRA-1-81, and Oct-4 was detected in testis tissue from both sexually mature and prepubertal males. Testes were fixed with modified Davidson’s fixative for 24 h before processing, embedding, and sectioning. The EXPOSE Mouse and Rabbit Specific HRP/DAB detection IHC kit (Abcam®, Cambridge, MA, USA) was used for antibody detection. Staining for SSEA-1, SSEA-4, TRA-1-60, TRA-1-81, and Oct-4 markers was expressed specifically at the basement membrane of the seminiferous tubules in both adult and prepubertal testes. The GFRα1 and GPR125 markers were detected at the basement membrane of the seminiferous tubules and across the seminiferous tubule section. However, C-Kit was not detected in any cell. Using flow cytometry from a pool of cells from seven adult testes, we detected 45% GFRα1, 50% GPR125, 59% C-Kit, 18% TRA-1-60, 16% TRA-1-81 positive cells, and a very small portion of SSEA-1 (7%) and SSEA-4 (3%) positive cells. Dual staining of germ cells pooled from 3 testes revealed 3 distinct cell populations that were positive for GFRα1 only (23%), positive for both GFRα1 and SSEA-4 (6%), and positive for SSEA-4 only (1%). Our IHC staining of cat testes indicated that cells along the basement membrane of seminiferous tubules were positive for SSC-specific markers, and flow cytometry analysis revealed that there were different cell populations expressing both germ cell and SSC-specific markers. Flow cytometry results show overlapping germ cell populations expressing SSEA-4 and GFRα1, and IHC results reveal that SSEA-4 positive cells are spermatogonia, whereas GFRα1 positive cells include other stages of germ cells, indicating that the small population of cells positive only for SSEA-4 is undifferentiated cat SSC.

182 In Vitro Derivation of Male Germ Cells from Bovine Bone Marrow-Derived Mesenchymal Stem Cells

2018 ◽  
Vol 30 (1) ◽  
pp. 231
Author(s):  
J. Cortez ◽  
J. Bahamonde ◽  
J. Palomino ◽  
M. De los Reyes ◽  
C. Torres ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Germ Cell ◽  
Mrna Expression ◽  
Male Germ Cell ◽  
Bovine Bone ◽  
Mrna Levels ◽  
Germ Cell Differentiation

During the last few years, the in vitro derivation of germ cell lineages from stem cells has emerged as an exciting new strategy for obtaining mature gametes. In vitro gamete derivation technology has potential applications as an alternative method for dissemination of elite animal genetics, production of transgenic animals, and conservation of endangered species. Germ cell differentiation and gametogenesis is a complex process and potential of different stem cell donors (i.e. SSC, ESC, iPSC) for in vitro male germ cell derivation has been inconsistent. Mesenchymal stem cells (MSC) may be suitable candidates for in vitro gamete derivation considering their (1) plasticity that is not limited to mesodermal derivatives, (2) availability of abundant tissues sources for isolation, (3) high proliferative potential, (4) simple and inexpensive isolation, and (5) high potential for cell therapy, including autologous or allogenic transplantation. The present study aimed to induce differentiation of MSC isolated from bone marrow derived from bovine male fetuses (bfMSC) into the germ cell lineage using an in vitro approach based on the exogenous effect of retinoic acid (RA) and bone morphogenetic protein 4 (BMP4). Differentiation media consisted in control media (DMEM with high glucose plus 10% fetal bovine serum, 100 IU mL−1 penicillin, 100 μg mL−1 streptomycin, and 0.25 μg mL−1amphotericin B) supplemented with RA (0.01, 0.1, or 1 µM) or BMP4 (10, 50, or 100 ng mL−1). Cell samples were obtained from differentiating and control bfMSC cultures and analysed for expression of housekeeping genes β-ACTIN and GAPDH, pluripotent genes OCT4 and NANOG, germ cell genes FRAGILLIS, STELLA, and VASA, male germ cell genes DAZL, PIWIl2, and STRA8, and meiotic biomarker SCP3 by quantitative-PCR (Q-PCR). OCT4, NANOG, and DAZL were immunodetected in undifferentiated and differentiated bfMSC using flow-cytometry analysis. The mRNA expression of DAZL was activated by RA or BMP4 supplementation, although no differences (P > 0.05) were detected among different concentrations. DAZL and NANOG mRNA levels increased (P < 0.05) from Day 7 to Day 21 during supplementation of RA (0.1 μM). In comparison, DAZL mRNA levels increased (P < 0.05) at Day 14 during supplementation of BMP4 (100 ng). OCT4 and SCP3 mRNA levels were not affected by RA or BMP4 treatments. Transcripts of FRAGILLIS, STELLA, VASA, PIWIl2, and STRA8 were not detected in control or differentiated bfMSC. Higher (P < 0.05) percentages of undifferentiated bfMSC were positive for NANOG (80.6%) and OCT4 (83.4%). DAZL- and NANOG-positive cells were 2.1% and 2.9%, and 95.9% and 97.8% at Days 0 and 21 of RA treatment, respectively. Data indicated that expression of germ cell biomarker DAZL in bfMSC is activated and increased after in vitro supplementation of RA and BMP4. Moreover, NANOG mRNA levels were regulated by RA treatment. Similar levels of SCP3 mRNA expression suggest that differentiated bfMSC were not induced into meiosis. Thus, exposure of bfMSC to RA or BMP4 under in vitro conditions might induce an early stage of premeiotic germinal differentiation.

Sertoli cell-mediated differentiation of bovine fetal mesenchymal stem cells into germ cell lineage using an in vitro co-culture system

2019 ◽  
Vol 130 ◽  
pp. 8-18
Author(s):  
M.N. Segunda ◽  
J. Bahamonde ◽  
I. Muñoz ◽  
S. Sepulveda ◽  
J. Cortez ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Germ Cell ◽  
Sertoli Cell ◽  
Culture System ◽  
Cell Lineage
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