Testis Tissue Explant Culture Supports Survival and Proliferation of Bovine Spermatogonial Stem Cells1

Oatley, Jon M.; Avila, David M. de; Reeves, Jerry J.; McLean, Derek J.

doi:10.1095/biolreprod.103.022483

Cited by 42 publications

(40 citation statements)

References 28 publications

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“…Unfortunately, efficient and reproducible SSC transplantation between bulls is not available. However, a xenogeneic transplantation assay in which bovine germ cells are microinjected into seminiferous tubules of recipient immunodeficient mice provides an assessment of possible stem cell content based on the formation of colonies of bovine cells (Dobrinski et al 2000, Oatley et al 2004a, 2004b. To assay for possible SSC enrichment, MACS-isolated THY1C cells from prepubertal bull testes and corresponding unselected total testis cell populations were labeled with a fluorescent membrane linker dye and microinjected into seminiferous tubules of immunodeficient recipient mice.…”

Section: Resultsmentioning

confidence: 99%

“…Xenogeneic transplantation assays were conducted to compare SSC content of MACS-isolated THY1C cells to unselected total testis cell populations of pre-pubertal bull testes as described previously (Oatley et al 2004a(Oatley et al , 2004b. Briefly, recipient NCr Swiss nude mice (Taconic, Germantown, NY, USA) were treated with busulfan (33 mg/kg of body weight) 6 weeks prior to transplantation to deplete endogenous spermatogenesis.…”

Section: Xenogeneic Transplantation Of Bull Testis Cells and Analysismentioning

confidence: 99%

“…Currently, methods for efficiently culturing and transplanting SSCs are only available for rodents (Brinster & Avarback 1994, Brinster & Zimmermann 1994 and have yet to be fully established for livestock species, though some initial progress has been made (Dobrinski et al 2000, Izadyar et al 2003, Oatley et al 2002, 2004b, Aponte et al 2008. Development of these procedures in rodents has been aided greatly by identification of surface markers expressed by undifferentiated spermatogonia, allowing for isolation of cell fractions enriched for these cells compared to the total testis cell population.…”

Section: Introductionmentioning

confidence: 99%

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THY1 is a conserved marker of undifferentiated spermatogonia in the pre-pubertal bull testis

Reding¹,

Stepnoski²,

Cloninger³

et al. 2010

Reproduction

105

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The undifferentiated spermatogonial population consists of stem and progenitor germ cells which function to provide the foundation for spermatogenesis. The stem cell component, termed spermatogonial stem cells (SSCs), is capable of self-renewal and differentiation. These unique attributes have made them a target for novel technologies to enhance reproductive function in males. With bulls, culture and transplantation of SSCs have the potential to enhance efficiency of cattle production and provide a novel avenue to generate transgenic animals. Isolation of SSCs is an essential component for the development of these techniques. In rodents and non-human primates, undifferentiated spermatogonia and SSCs express the surface marker THY1. The hypothesis tested in this study was that THY1 is a conserved marker of the undifferentiated spermatogonial population in bulls. Flow cytometric analyses showed that the THY1C cell fraction comprises a rare sub-population in testes of pre-pubertal bulls. Immunocytochemical analyses of the isolated THY1C fraction for expression of VASA showed that this cell population is comprised mostly of germ cells. Additionally, expression of the undifferentiated spermatogonial specific transcription factor promyelocytic leukemia zinc finger (PLZF, ZBTB16) protein was found to be enriched in the isolated THY1C testis cell fraction. Lastly, xenogeneic transplantation of bull testis cells into seminiferous tubules of immunodeficient mice resulted in greater than sixfold more colonies from isolated THY1C cells compared to the unselected total testis cell population indicating SSC enrichment. Collectively, these results demonstrate that THY1 is a marker of undifferentiated spermatogonia in testes of pre-pubertal bulls, and isolation of THY1C cells results in their enrichment from the total testis cell population.Reproduction (2010) 139 893-903

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Section: Resultsmentioning

confidence: 99%

Section: Xenogeneic Transplantation Of Bull Testis Cells and Analysismentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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THY1 is a conserved marker of undifferentiated spermatogonia in the pre-pubertal bull testis

Reding¹,

Stepnoski²,

Cloninger³

et al. 2010

Reproduction

105

View full text Add to dashboard Cite

show abstract

“…Bovine spermatogonia were incapable of resuming differentiation when seminiferous tubule fragments containing Sertoli and stem germ cells were cultured for 2 wk [6]. If rat seminiferous tubules also contained spermatocytes, then new round spermatids (6%-10%) formed after 21 days of culture, confirming successful meiosis resumption in vitro [7].…”

Section: Introductionmentioning

confidence: 92%

Cytological and Expression Studies and Quantitative Analysis of the Temporal and Stage-Specific Effects of Follicle-Stimulating Hormone and Testosterone During Cocultures of the Normal Human Seminiferous Epithelium1

Sá

Neves

Fernandes

et al. 2008

Biology of Reproduction

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In vitro culturing of normal human seminiferous epithelium remains largely unexplored. To study normal human spermatogenesis in vitro, we used a micromethod for the purification and culture of Sertoli cells, spermatogonia A, spermatocytes, and early round spermatids. Cytological quantitative data for Sertoli and premeiotic germ cell cocultures isolated from normal testicular biopsies demonstrated that cells were able to proliferate (4%), complete meiosis (6.7%), and differentiate into late round (54%), elongating (49%), and elongated (17%) spermatids at similar in vivo time delays (up to 16 days) in response to FSH + testosterone stimulation. Cells maintained normal meiotic segregation, chromosome complements, and specific gene expression profiles. Follicle-stimulating hormone + testosterone stimulated spermatogonia proliferation and Sertoli cell survival. Follicle-stimulating hormone and especially FSH + testosterone increased diploid germ cell survival during the first week, whereas only FSH + testosterone was able to inhibit cell death during the second week of culture. Follicle-stimulating hormone and especially FSH + testosterone also stimulated meiosis resumption, although this was restricted to late pachytene and secondary spermatocytes. In contrast, spermiogenesis was only stimulated by FSH + testosterone. Expression studies showed that apoptosis was induced in the nucleus of diploid cells, and in nuclear and cytoplasmic compartments of spermatids, mainly triggered by the Fas pathway. Although junctional complexes between Sertoli and premeiotic germ cells were partially reacquired, the same did not apply to spermatids, suggesting that FSH potentiated by testosterone was unable to render Sertoli cells competent to bind round spermatids.

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“…The rationale of this suggestion is based on the morphological and functional differences among areas or zones along the reproductive tract. For example, the six zones of the epididymis each possess cellular differences (Clermont and Flannery, 1970) and within each zone a mixture of cell types are found, including basal, secretory epithelial, endocrine, stem cells, and leucocytes (Clermont and Flannery, 1970;Dym and Romrell, 1975;Oatley et al, 2004). Phillips et al (1978) used human semen as a source of epithelial cells for in vitro culture, although the cell origin was not indicated.…”

Section: Culture Of Semen Epithelial Cells 153mentioning

confidence: 99%

Cloned Embryos from Semen. Part 1:In VitroProliferation of Epithelial Cells on Embryonic Fibroblasts after Isolation from Semen by Gradient Centrifugation

Nel-Themaat

Gómez

Pope

et al. 2008

Cloning and Stem Cells

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Although epithelial-like somatic cells have been previously isolated from semen, cell proliferation rates were low. Culture of whole semen samples resulted in loss of potentially valuable spermatozoa. The aims of the present study were to: (1) isolate somatic cells from semen, while preserving sperm viability, and (2) optimize in vitro culture conditions for semen-derived epithelial cells. Density gradient centrifugation of washed ejaculates of two rams (Ovis aries) (n = 24) and one eland bull (Taurotragus oryx) (n = 4) was performed using a three-layer discontinuous Percoll column consisting of 90% (P-90), 50% (P-50), and 20% (P-20) Percoll. In vitro culture and Trypan Blue staining indicated that live somatic cells settled in the P-20 layer. Nonmotile spermatozoa were recovered at the P-50 and P-90 interfaces, whereas motile spermatozoa were collected in the pellet from the P-90 layer. Subsequently, somatic cells isolated from the P-20 layer were plated either on inactivated 3T3 mouse embryonic fibroblast feeder layers, collagen-coated plates with 3T3 feeder cell inserts, or on collagen-coated plates. Initial somatic cell plating was similar among treatments, but proliferation significantly increased when cocultured with 3T3 cells (feeder or insert). Furthermore, two different types of epithelial cells were obtained. The exact origin of the cells in the male reproduction system is uncertain and probably variable. The present method of cell isolation and in vitro culture may be of value for preserving endangered species. Specifically, cells isolated and cultured from cryopreserved semen of nonliving males could be used for producing embryos by somatic cell nuclear transfer.

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Testis Tissue Explant Culture Supports Survival and Proliferation of Bovine Spermatogonial Stem Cells1

Cited by 42 publications

References 28 publications

THY1 is a conserved marker of undifferentiated spermatogonia in the pre-pubertal bull testis

THY1 is a conserved marker of undifferentiated spermatogonia in the pre-pubertal bull testis

Cytological and Expression Studies and Quantitative Analysis of the Temporal and Stage-Specific Effects of Follicle-Stimulating Hormone and Testosterone During Cocultures of the Normal Human Seminiferous Epithelium1

Cloned Embryos from Semen. Part 1:In VitroProliferation of Epithelial Cells on Embryonic Fibroblasts after Isolation from Semen by Gradient Centrifugation

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