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Brian P Hermann, Meena Sukhwani, Marc C Hansel and Kyle E Orwig

Spermatogonial stem cells (SSCs) maintain spermatogenesis throughout the reproductive life of mammals. While Asingle spermatogonia comprise the rodent SSC pool, the identity of the stem cell pool in the primate spermatogenic lineage is not well established. The prevailing model is that primate spermatogenesis arises from Adark and Apale spermatogonia, which are considered to represent reserve and active stem cells respectively. However, there is limited information about how the Adark and Apale descriptions of nuclear morphology correlate with the clonal (Asingle, Apaired, and Aaligned), molecular (e.g. GFRα1 (GFRA1) and PLZF), and functional (SSC transplantation) descriptions of rodent SSCs. Thus, there is a need to investigate primate SSCs using criteria, tools, and approaches that have been used to investigate rodent SSCs over the past two decades. SSCs have potential clinical application for treating some cases of male infertility, providing impetus for characterizing and learning to manipulate these adult tissue stem cells in primates (nonhuman and human). This review recounts the development of a xenotransplant assay for functional identification of primate SSCs and progress dissecting the molecular and clonal characteristics of the primate spermatogenic lineage. These observations highlight the similarities and potential differences between rodents and primates regarding the SSC pool and the kinetics of spermatogonial self-renewal and clonal expansion. With new tools and reagents for studying primate spermatogonia, the field is poised to develop and test new hypotheses about the biology and regenerative capacity of primate SSCs.

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Jason G Knott and Soumen Paul

Mammalian reproduction is critically dependent on the trophoblast cell lineage, which assures proper establishment of maternal–fetal interactions during pregnancy. Specification of trophoblast cell lineage begins with the development of the trophectoderm (TE) in preimplantation embryos. Subsequently, other trophoblast cell types arise with the progression of pregnancy. Studies with transgenic animal models as well as trophoblast stem/progenitor cells have implicated distinct transcriptional and epigenetic regulators in trophoblast lineage development. This review focuses on our current understanding of transcriptional and epigenetic mechanisms regulating specification, determination, maintenance and differentiation of trophoblast cells.

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I. A. Polejaeva, W. A. Reed, T. D. Bunch, L. C. Ellis and K. L. White

The mink reproductive cycle includes an obligatory period of embryonic diapause and delayed implantation, which continues in vitro and reduces the efficiency of embryonic stem (ES) cell establishment. Blastocysts recovered on day 7 and on days 13–16 after final mating were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with various concentrations of prolactin to determine optimal conditions for embryo attachment and subsequent establishment of embryonic stem cells. Five treatments were applied to both ages of blastocyst: A, DMEM control (n = 16); B, DMEM ± 5 μg prolactin ml−1 after 10 days initial culture in DMEM alone (n=17); after 1 day of initial culture: C, DMEM ± 10 ng prolactin ml−1 (n= 17); D, DMEM ± 1 μg prolactin ml−1 (n= 19); and E, DMEM ± 5 μg prolactin ml−1 (n=17). Prolactin terminated diapause of day 13–16 blastocysts at all concentrations tested. The maximum attachment of embryos in vitro and subsequent production of ES-like cells occurred in medium supplemented with 5 μg prolactin ml−1. Prolactin did not affect attachment rates for day 7 blastocysts when 5 μg prolactin ml−1 was added, but prolactin at concentrations of 1 μg ml−1 and 5 μg ml−1 when added on day 1 of culture enhanced ES-like cell line establishment. Two principal cell types were observed in the colonies: small stem cells and trophoblast-like cells with large areas of cytoplasm. The morphological evaluation of mink ES-like cell colonies was confirmed by cytochemical staining for alkaline phosphatase. Mink embryonic stem-like cells were found to stain positive for alkaline phosphatase. Alkaline phosphatase activity was lost upon cellular differentiation.

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Nady Golestaneh, Elspeth Beauchamp, Shannon Fallen, Maria Kokkinaki, Aykut Üren and Martin Dym

Spermatogonial stem cells (SSCs) self-renew throughout life to produce progenitor cells that are able to differentiate into spermatozoa. However, the mechanisms underlying the cell fate determination between self-renewal and differentiation have not yet been delineated. Culture conditions and growth factors essential for self-renewal and proliferation of mouse SSCs have been investigated, but no information is available related to growth factors that affect fate determination of human spermatogonia. Wnts form a large family of secreted glycoproteins, the members of which are involved in cell proliferation, differentiation, organogenesis, and cell migration. Here, we show that Wnts and their receptors Fzs are expressed in mouse spermatogonia and in the C18-4 SSC line. We demonstrate that WNT3A induces cell proliferation, morphological changes, and cell migration in C18-4 cells. Furthermore, we show that β-catenin is activated during testis development in 21-day-old mice. In addition, our study demonstrates that WNT3A sustained adult human embryonic stem (ES)-like cells derived from human germ cells in an undifferentiated stage, expressing essential human ES cell transcription factors. These results demonstrate for the first time that Wnt/β-catenin pathways, especially WNT3A, may play an important role in the regulation of mouse and human spermatogonia.

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Jin Gyoung Jung, Young Mok Lee, Jin Nam Kim, Tae Min Kim, Ji Hye Shin, Tae Hyun Kim, Jeong Mook Lim and Jae Yong Han

We recently developed bimodal germline chimera production approaches by transfer of primordial germ cells (PGCs) or embryonic germ cells (EGCs) into embryos and by transplantation of spermatogonial stem cells (SSCs) or germline stem cells (GSCs) into adult testes. This study was undertaken to investigate the reversible developmental unipotency of chicken germ cells using our established germline chimera production systems. First, we transferred freshly isolated SSCs from adult testis or in vitro cultured GSCs into stage X and stage 14–16 embryos, and we found that these transferred SSCs/GSCs could migrate to the recipient embryonic gonads. Of the 527 embryos that received SSCs or GSCs, 135 yielded hatchlings. Of 17 sexually mature males (35.3%), six were confirmed as germline chimeras through testcross analysis resulting in an average germline transmission efficiency of 1.3%. Second, PGCs/EGCs, germ cells isolated from embryonic gonads were transplanted into adult testes. The EGC transplantation induced germline transmission, whereas the PGC transplantation did not. The germline transmission efficiency was 12.5 fold higher (16.3 vs 1.3%) in EGC transplantation into testis (EGCs to adult testis) than that in SSC/GSC transfer into embryos (testicular germ cells to embryo stage). In conclusion, chicken germ cells from different developmental stages can (de)differentiate into gametes even after the germ cell developmental clock is set back or ahead. Use of germ cell reversible unipotency might improve the efficiency of germ cell-mediated germline transmission.

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F Izadyar, GT Spierenberg, LB Creemers, K den Ouden and DG de Rooij

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.

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Suzanne C Reding, Aaron L Stepnoski, Elizabeth W Cloninger and Jon M Oatley

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 THY1+ cell fraction comprises a rare sub-population in testes of pre-pubertal bulls. Immunocytochemical analyses of the isolated THY1+ 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 THY1+ 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 THY1+ 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 THY1+ cells results in their enrichment from the total testis cell population.

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Eleonora Iacono, Lara Brunori, Alessandro Pirrone, Pasquale Paolo Pagliaro, Francesca Ricci, Pier Luigi Tazzari and Barbara Merlo

Mesenchymal stem cells (MSCs) have been derived from multiple sources of the horse including umbilical cord blood (UCB) and amnion. This work aimed to identify and characterize stem cells from equine amniotic fluid (AF), CB and Wharton's Jelly (WJ). Samples were obtained from 13 mares at labour. AF and CB cells were isolated by centrifugation, while WJ was prepared by incubating with an enzymatic solution for 2 h. All cell lines were cultured in DMEM/TCM199 plus fetal bovine serum. Fibroblast-like cells were observed in 7/10 (70%) AF, 6/8 (75%) CB and 8/12 (66.7%) WJ samples. Statistically significant differences were found between cell-doubling times (DTs): cells isolated from WJ expanded more rapidly (2.0±0.6 days) than those isolated from CB (2.6±1.3 days) and AF (2.3±1.0 days) (P<0.05). Positive von Kossa and Alizarin Red S staining confirmed osteogenesis. Alcian Blue staining of matrix glycosaminoglycans illustrated chondrogenesis and positive Oil Red O lipid droplets staining suggested adipogenesis. All cell lines isolated were positive for CD90, CD44, CD105; and negative for CD34, CD14 and CD45. These findings suggest that equine MSCs from AF, UCB and WJ appeared to be a readily obtainable and highly proliferative cell lines from a uninvasive source that may represent a good model system for stem cell biology and cellular therapy applications in horses. However, to assess their use as an allogenic cell source, further studies are needed for evaluating the expression of markers related to cell immunogenicity.

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Eight stages of spermatogenesis, each with a characteristic frequency and germ cell association could be recognized in the vole. There was no difference between laboratory bred and field animals in the frequency of the stages. Counts of the different types of germ cell showed that there was considerable cell loss during spermatogonial mitotic and spermatocytic meiotic divisions. Only 60% of germ cells became spermatozoa in sexually mature animals, and 19% in the regressing testes of voles exposed to short photoperiods. Animals with regressed testes probably have lowered circulating levels of gonadotrophins and testicular hormones, so that the greater germ loss suggests the importance of these hormones in the regulation of germ cell wastage. From the cell counts in mature animals, a scheme of cell divisions has been suggested by which spermatogonia produce progressively more highly differentiated germ cells while continuing to perpetuate stem cells.

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S. Hasthorpe, S. Barbic, P. J. Farmer and J. M. Hutson

Survival and proliferation of mouse gonocytes was studied using a single cell clonogenic assay in vitro. The effect of growth factors and extracellular matrix on clonogenic development was quantitated. Fundamental requirements for growth of neonatal gonocytes included addition of fetal calf serum and coating culture wells with collagen IV alone or with added fibronectin. After 4–5 days, colonies ranged in size from four to > 128 cells, and some contained very elongated cells indicating migratory behaviour. Soluble stem cell factor did not have any effect on clonogenicity, although STO (subline of SIM mouse fibroblasts) cells, which produce membrane-bound stem cell factor, reduced colony formation from 79 ± 5.9% to 20 ± 3.3% without added growth factor. The majority of gonocytes and type A spermatogonia express the c-kit receptor according to in situ hybridization studies. However, the results indicate that the receptor may not be functional in neonatal gonocytes and their immediate progeny. The current assay for gonocytes can be extended to test new growth factors or proliferation-inducing agents directly, as well as to study cell–cell interactions. This assay and long-term propagation of neonatal germ cells will provide the much needed resources to enable greater understanding of the early development of germ cells.