Propagation of bovine spermatogonial stem cells in vitro

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.

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

Development ◽

10.1242/dev.127.10.2125 ◽

2000 ◽

Vol 127 (10) ◽

pp. 2125-2131 ◽

Cited By ~ 1

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.

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Direct Stimulation of Adult Neural Stem Cells In Vitro and Neurogenesis In Vivo by Vascular Endothelial Growth Factor

Brain Pathology ◽

10.1111/j.1750-3639.2004.tb00060.x ◽

2006 ◽

Vol 14 (3) ◽

pp. 237-248 ◽

Cited By ~ 227

Author(s):

Anne Schänzer ◽

Frank-Peter Wachs ◽

Daniel Wilhelm ◽

Till Acker ◽

Christiana Cooper-Kuhn ◽

...

Keyword(s):

Stem Cells ◽

Vascular Endothelial Growth Factor ◽

Growth Factor ◽

Neural Stem Cells ◽

Vascular Endothelial ◽

Direct Stimulation ◽

Endothelial Growth Factor ◽

Stimulation Of

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Ex-Vivo Stimulation of Adipose Stem Cells by Growth Factors and Fibrin-Hydrogel Assisted Delivery Strategies for Treating Nerve Gap-Injuries

Bioengineering ◽

10.3390/bioengineering7020042 ◽

2020 ◽

Vol 7 (2) ◽

pp. 42

Author(s):

Katharina M. Prautsch ◽

Lucas Degrugillier ◽

Dirk J. Schaefer ◽

Raphael Guzman ◽

Daniel F. Kalbermatten ◽

...

Keyword(s):

Stem Cells ◽

Growth Factors ◽

Growth Factor ◽

Nerve Regeneration ◽

Ex Vivo ◽

Axonal Outgrowth ◽

Assisted Delivery ◽

Delivery Strategies ◽

Stimulation Of

Peripheral nerve injuries often result in lifelong disabilities despite advanced surgical interventions, indicating the urgent clinical need for effective therapies. In order to improve the potency of adipose-derived stem cells (ASC) for nerve regeneration, the present study focused primarily on ex-vivo stimulation of ASC by using growth factors, i.e., nerve growth factor (NGF) or vascular endothelial growth factor (VEGF) and secondly on fibrin-hydrogel nerve conduits (FNC) assisted ASC delivery strategies, i.e., intramural vs. intraluminal loading. ASC were stimulated by NGF or VEGF for 3 days and the resulting secretome was subsequently evaluated in an in vitro axonal outgrowth assay. For the animal study, a 10 mm sciatic nerve gap-injury was created in rats and reconstructed using FNC loaded with ASC. Secretome derived from NGF-stimulated ASC promoted significant axonal outgrowth from the DRG-explants in comparison to all other conditions. Thus, NGF-stimulated ASC were further investigated in animals and found to enhance early nerve regeneration as evidenced by the increased number of β-Tubulin III+ axons. Notably, FNC assisted intramural delivery enabled the improvement of ASC’s therapeutic efficacy in comparison to the intraluminal delivery system. Thus, ex-vivo stimulation of ASC by NGF and FNC assisted intramural delivery may offer new options for developing effective therapies.

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Loss of Ubiquitin Carboxy-Terminal Hydrolase L1 Impairs Long-Term Differentiation Competence and Metabolic Regulation in Murine Spermatogonial Stem Cells

Cells ◽

10.3390/cells10092265 ◽

2021 ◽

Vol 10 (9) ◽

pp. 2265

Author(s):

Whitney F. Alpaugh ◽

Anna L. Voigt ◽

Rkia Dardari ◽

Lin Su ◽

Iman Al Khatib ◽

...

Keyword(s):

Stem Cells ◽

Metabolic Regulation ◽

Spermatogonial Stem Cells ◽

Seminiferous Tubules ◽

Testis Weight ◽

Stem And Progenitor Cells ◽

Cell Transcriptome ◽

Carboxy Terminal

Spermatogonia are stem and progenitor cells responsible for maintaining mammalian spermatogenesis. Preserving the balance between self-renewal of spermatogonial stem cells (SSCs) and differentiation is critical for spermatogenesis and fertility. Ubiquitin carboxy-terminal hydrolase-L1 (UCH-L1) is highly expressed in spermatogonia of many species; however, its functional role has not been identified. Here, we aimed to understand the role of UCH-L1 in murine spermatogonia using a Uch-l1−/− mouse model. We confirmed that UCH-L1 is expressed in undifferentiated and early-differentiating spermatogonia in the post-natal mammalian testis. The Uch-l1−/− mice showed reduced testis weight and progressive degeneration of seminiferous tubules. Single-cell transcriptome analysis detected a dysregulated metabolic profile in spermatogonia of Uch-l1−/− compared to wild-type mice. Furthermore, cultured Uch-l1−/− SSCs had decreased capacity in regenerating full spermatogenesis after transplantation in vivo and accelerated oxidative phosphorylation (OXPHOS) during maintenance in vitro. Together, these results indicate that the absence of UCH-L1 impacts the maintenance of SSC homeostasis and metabolism and impacts the differentiation competence. Metabolic perturbations associated with loss of UCH-L1 appear to underlie a reduced capacity for supporting spermatogenesis and fertility with age. This work is one step further in understanding the complex regulatory circuits underlying SSC function.

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Cryopreserved fragments of testicular seminiferous tubules of rats as a source of spermatogonial stem cells

Cell and Organ Transplantology ◽

10.22494/cot.v9i1.120 ◽

2021 ◽

Vol 9 (1) ◽

Author(s):

N. Volkova ◽

◽

M. Yukhta ◽

L. Sokil ◽

L. Chernyschenko ◽

...

Keyword(s):

Stem Cells ◽

Cell Culture ◽

Morphological Characteristics ◽

Relative Number ◽

Spermatogonial Stem Cells ◽

Blue Staining ◽

Seminiferous Tubules ◽

Proliferative Potential ◽

Tissue Samples

The use of modern technologies of cryopreservation of testicular tissue samples in prepubertal patients is one of the ways to maintain their fertility in the future. The purpose of the study was to investigate the proliferative potential, morphological characteristics and expression of specific markers of cell culture obtained from cryopreserved and vitrified fragments of seminiferous tubules (FSTs) of rats' testis. Materials and methods. The isolation of cells from native, cryopreserved and vitrified FSTs of immature rats was performed by incubation in a solution of collagenase type IV (1 mg/mL) + DNase (500 μg/mL). Cell viability was determined by Trypan blue staining. Monoclonal antibodies CD9-FITC, CD24-PE, CD45-FITC, CD90-FITC were used for immunophenotype analysis. Morphological characteristics, proliferative activity (MTT assay), relative number of cells positive for MAGE-B1 and vimentin were assessed in the obtained cultures. Results. The analysis of phenotypic characteristics showed that cells from native, cryopreserved and vitrified FSTs were characterized by high expression level of CD9 (≥ 40 %), CD24 (≥ 70 %), CD90 (≥ 70 %) and low expression of the CD45 (≤ 1 %). In cell culture in vitro, the studied cells from cryopreserved and vitrified rat's FSTs had the ability to adhere and proliferate while maintaining a cells population positive for MAGE-B1 and vimentin. Conclusions. The results can be the basis for the development of effective protocols for the cultivation and cryopreservation of testicular spermatogonial stem cells in order to restore fertility in men.

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288 THE EXPRESSION PATTERN OF ACETYLATED ALPHA-TUBULIN IS CONSERVED IN PORCINE AND MURINE SPERMATOGONIAL STEM CELLS

Reproduction Fertility and Development ◽

10.1071/rdv20n1ab288 ◽

2008 ◽

Vol 20 (1) ◽

pp. 223

Author(s):

J. Luo ◽

S. Megee ◽

I. Dobrinski

Keyword(s):

Stem Cells ◽

Basement Membrane ◽

Expression Pattern ◽

Germ Cells ◽

Sertoli Cells ◽

Spermatogonial Stem Cells ◽

Seminiferous Tubules ◽

Histone Deacetylase 6 ◽

Pgp 9.5

During mammalian spermatogenesis, spermatogonial stem cells (SSCs) reside in the stem cell niche on the basement membrane where they undergo self-renewing divisions. Differentiating daughter cells are located progressively more toward the tubular lumen where they ultimately form spermatozoa. The mechanisms responsible for maintenance of SSCs at the basement membrane are unclear. Microtubules consisting of α/β-tubulin heterodimers are associated with many cellular functions. Reversible acetylation of α-tubulin at Lys40 has been implicated in regulating microtubule stability and function. Acetylation of α-tubulin is abundant in stable microtubules but absent from dynamic cellular structures. Deacetylation of α-tubulin is controlled by histone deacetylase 6 which is predominantly expressed in mouse testis. Here, we tested the hypothesis that differential acetylation of α-tubulin might be involved in maintenance of SSCs. Immunohistochemistry for acetylated α-tubulin (Ac-α-Tu) and the spermatogonia specific proteins PGP 9.5, DAZL, and PLZF were used to characterize the expression pattern of Ac-α-Tu in porcine and murine germ cells at different stages of testis development. In immature boar testes, Ac-α-Tu was present exclusively in gonocytes but not in other testicular cells at 1 week of age, and in a subset of spermatogonia at 10 weeks of age. At this age, spermatogonia are migrating to the basement membrane of the seminiferous tubules, and Ac-α-Tu appeared to be polarized toward the basement membrane. In immature mouse testes, Ac-α-Tu was present in germ cells and Sertoli cells at 6 days of age, whereas at 2 weeks of age, Ac-α-Tu expression was stronger in spermatogonia co-expressing PGP 9.5 and in spermatocytes than in Sertoli cells or PGP 9.5-negative spermatogonia. In adult boar and mouse testes, Ac-α-Tu was detected in a few single or paired spermatogonia expressing PGP 9.5 localized on the basement membrane as well as in spermatocytes, spermatids, and spermatozoa. Spermatogonia with high levels of Ac-α-Tu expressed PLZF but did not express DAZL, suggesting that only undifferentiated spermatogonia maintain a high level of Ac-α-Tu. When seminiferous tubules from 1-week-old and adult boar testes were maintained in vitro for 1–2 days, high levels of Ac-α-Tu were detected in single or paired round spermatogonia with a large nucleus, compared to low levels in elongated paired and aligned spermatogonia. The unique expression pattern of Ac-α-Tu in undifferentiated germ cells during postnatal development appears to be conserved in mammalian testes. Since Ac-α-Tu is a component of long-lived stable microtubules and reducing acetylation of α-tubulin enhances cell motility, these results suggest that stabilization of microtubules might contribute to the maintenance of spermatogonial stem cells. This work was supported by 1R01 RR 17359-05.

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Autologous and homologous transplantation of bovine spermatogonial stem cells

Reproduction ◽

10.1530/rep.0.1260765 ◽

2003 ◽

pp. 765-774 ◽

Cited By ~ 119

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.

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Maintenance of adult mouse type A spermatogonia in vitro: influence of serum and growth factors and comparison with prepubertal spermatogonial cell culture

Reproduction ◽

10.1530/rep.0.1240791 ◽

2002 ◽

pp. 791-799 ◽

Cited By ~ 50

Author(s):

LB Creemers ◽

K den Ouden ◽

AM van Pelt ◽

DG de Rooij

Keyword(s):

Germ Cells ◽

Culture Medium ◽

Somatic Cells ◽

Calf Serum ◽

Type A ◽

Dividing Cells ◽

Deficient Mice ◽

Type A Spermatogonia

The culture of spermatogonial cells under well-defined conditions would be an important method for elucidating the mechanisms involved in spermatogenesis and in establishing tissue regeneration in vivo. In this study, a serum-free culture system was established, with type A spermatogonia isolated from adult vitamin A-deficient mice. At days 1, 3 and 7 of culture, the viability and proliferation of cells were monitored. The viability of the cells decreased by day 7 to 10% of the cells present. Proliferation occurred mainly during day 1, when 1% of the germ cells was proliferating. Co-labelling for a germ cell marker (heat shock protein-90alpha, Hsp90alpha), and a marker used to detect dividing cells (bromodeoxyuridine, BrdU), showed that this proliferation was restricted to germ cells. In an attempt to improve these parameters, medium containing fetal calf serum (FCS) was used. Viability was not influenced by serum, but proliferation was markedly enhanced. However, after day 7 of incubation with FCS, co-immunolocalization for Hsp90alpha and BrdU showed a preferential proliferation of somatic cells. Comparison of cultures of adult cells with cultures of prepubertal germ cells, commonly used in studies of spermatogenesis, showed that prepubertal germ cells are twice as viable. In addition, a different proliferation profile was observed, with a peak at day 3. Here, a distinct proliferation of somatic cells was also noted. The results from the present study indicate that the origin of isolated germ cells partly determines culture outcome and that cultures of prepubertal germ cells may not be representative for adult spermatogenesis. Moreover, adding FCS to the culture medium invokes the risk of profound and undesirable effects on cell composition, also underlining the need for identification of germ cells during culture.

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Preservation of fertility in nature and ART

Reproduction ◽

10.1530/rep.0.1230003 ◽

2002 ◽

pp. 3-11 ◽

Cited By ~ 21

Author(s):

R Gosden ◽

M Nagano

Keyword(s):

Stem Cells ◽

Germ Cell ◽

Germ Cells ◽

Reproductive Technologies ◽

Spermatogonial Stem Cells ◽

Seminiferous Tubules ◽

Natural Fertility ◽

Fertilization In Vitro ◽

Gonadal Failure

Individuals may regard reproduction as optional but sufficient number of them must be productive to perpetuate the species. The reproductive system is surprisingly vulnerable and depends, among other things, on a limited endowment of oocytes, controlled proliferation of spermatogonial stem cells and the genetic integrity of both. The developmental competence of oocytes and spermatogonial stem cells is maintained by evolved mechanisms for cellular detoxification and genomic stability, and excess or damaged cells are eliminated by apoptosis. Gonadal failure as a result of germ cell depletion can occur at any age, and from the effects of chemical cytotoxicity, disease and infection as well as genetic predisposition. Among extrinsic factors, alkylating agents and ionizing radiation are important causes of iatrogenic gonadal failure in young women and men. In animal models, there is evidence that hormonal manipulation, deletion of genes involved in apoptotic pathways and dietary manipulation can protect against natural and induced germ cell loss, but evidence in humans is absent or unclear. Assisted reproductive technologies (ARTs) provide an ensemble of strategies for preserving fertility in patients and commercially valuable or endangered species. Semen cryopreservation was the first technology for preserving male fertility, but this cannot serve prepubertal boys, for whom banking of testicular biopsies may provide a future option. In sterilized rodents, cryopreserved spermatogonial stem cells can recolonize seminiferous tubules and reinitiate spermatogenesis, and subcutaneous implantation of intact tubules can generate spermatozoa for fertilization in vitro by intracytoplasmic sperm injection. Transplantation of frozen-banked ovarian tissue is well-established for restoring cyclicity and fertility and is currently undergoing clinical evaluation for cancer patients. When restoration of natural fertility is unnecessary or reimplantation is unsafe, it is desirable to culture the germ cells from thawed tissue in vitro until they reach the stage at which they can be fertilized. Low temperature banking of immature germ cells is potentially very versatile, but storage of embryos and, to a lesser extent, mature oocytes is already practised in a number of species, including humans, and is likely to remain a mainstay for fertility preservation.

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Mouse thy1-positive spermatogonia suppress the proliferation of spermatogonial stem cells by Extracellular vesicles in vitro

10.1101/2020.06.15.153668 ◽

2020 ◽

Author(s):

Yu Lin ◽

Qian Fang ◽

Yue He ◽

Xiaowen Gong ◽

Yinjuan Wang ◽

...

Keyword(s):

Stem Cells ◽

Extracellular Vesicles ◽

Magnetic Beads ◽

Spermatogonial Stem Cells ◽

The Self ◽

Seminiferous Tubules ◽

Self Renewal

ABSTRACTThe self-renewal of mammalian spermatogonial stem cells (SSCs) supports spermatogenesis to produce spermatozoa, and this is precisely controlled in a stem niche microenvironment in the seminiferous tubules. Although studies have revealed the role of the surrounding factors in SSCs, little is known about whether the division of SSCs is controlled by extracellular vesicles. Here, extracellular vesicles were found in the basal compartment of seminiferous tubules in mouse, rat, rabbit and human testes. In the mice, the testicular extracellular vesicles are secreted by spermatogonia and are taken up by SSCs. Further, the extracellular vesicles from thy1-positive spermatogonia were purified by anti-Thy1-coupled magnetic beads, and which suppress their proliferation of SSCs but not lead to the apoptosis in vitro.

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