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Göttingen, Germany

Albert S.,University of Munster | Wistuba J.,University of Munster | Eildermann K.,Stem Cell Biology Unit | Ehmcke J.,University of Munster | And 3 more authors.
Cells Tissues Organs | Year: 2012

The marmoset monkey is a valuable model in reproductive medicine. While previous studies have evaluated germ cell dynamics in the postnatal marmoset, the features of testicular somatic cells remain largely unknown. Therefore, the aim of this study was to establish marmoset-specific markers for Sertoli and peritubular cells (PTCs) and to compare protocols for the enrichment and culture of testicular cell types. Immunohistochemistry of Sertoli and PTC-specific markers - anti-müllerian hormone (AMH), vimentin (VIM), α-smooth muscle actin (SMA) - was performed and corresponding RNA expression profiles were established by quantitative PCR analysis (SOX9,AMH, FSHR,VIM, and SMA). For these analyses, testicular tissue from newborn (n = 4), 8-week-old (n = 4) and adult (n = 3) marmoset monkeys was used. Protocols for the enrichment and culture of testicular cell fractions from the 8-week-old marmoset monkeys (n = 3) were evaluated and cells were analyzed using germ cell- and somatic cell-specific markers. The expression of AMH, VIM and SMA reflects the proportion and differentiation status of Sertoli and PTCs at the RNA and the protein levels. While applied protocols did not support the propagation of germ cells in vitro, our analyses revealed that PTCs maintain their proliferative potential and constitute the dominant cell type after short- and long-term culture. Expression of functionally meaningful testicular somatic markers is similar in the human and the marmoset monkey, indicating that this primate can indeed be used as model for human testicular development. The PTC culture system established in this study will facilitate the identification of factors influencing male sex differentiation and spermatogenesis. Copyright © 2012 S. Karger AG, Basel.

Langenstroth D.,Institute of Reproduction and Regenerative Biology | Kossack N.,Institute of Reproduction and Regenerative Biology | Westernstroer B.,Institute of Reproduction and Regenerative Biology | Wistuba J.,Institute of Reproduction and Regenerative Biology | And 3 more authors.
Human Reproduction | Year: 2014

STUDY QUESTION Can primate spermatogonial cultures be optimized by application of separation steps and well defined culture conditions? SUMMARY ANSWER We identified the cell fraction which provides the best source for primate spermatogonia when prolonged culture is desired. WHAT IS KNOWN ALREADY Man and marmoset show similar characteristics in regard to germ cell development and function. Several protocols for isolation and culture of human testis-derived germline stem cells have been described. Subsequent analysis revealed doubts on the germline origin of these cells and characterized them as mesenchymal stem cells or fibroblasts. Studies using marmosets as preclinical model confirmed that the published isolation protocols did not lead to propagation of germline cells. STUDY DESIGN, SIZE, DURATION Testicular cells derived from nine adult marmoset monkeys (Callithrix jacchus) were cultured for 1, 3, 6 and 11 days and consecutively analyzed for the presence of spermatogonia, differentiating germ cells and testicular somatic cells. PARTICIPANTS/MATERIALS, SETTING, METHODS Testicular tissue of nine adult marmoset monkeys was enzymatically dissociated and subjected to two different cell culture approaches. In the first approach all cells were kept in the same dish (non-separate culture, n = 5). In the second approach the supernatant cells were transferred into a new dish 24 h after seeding and subsequently supernatant and attached cells were cultured separately (separate culture, n = 4). Real-time quantitative PCR and immunofluorescence were used to analyze the expression of reliable germ cell and somatic markers throughout the culture period. Germ cell transplantation assays and subsequent wholemount analyses were performed to functionally evaluate the colonization of spermatogonial cells. MAIN RESULTS AND THE ROLE OF CHANCE This is the first report revealing an efficient isolation and culture of putative marmoset spermatogonial stem cells with colonization ability. Our results indicate that a separation of spermatogonia from testicular somatic cells is a crucial step during cell preparation. We identified the overgrowth of more rapidly expanding somatic cells to be a major problem when establishing spermatogonial cultures. Initiating germ cell cultures from the supernatant and maintaining germ cells in suspension cultures minimized the somatic cell contamination and provided enriched germ cell fractions which displayed after 11 days of culture a significantly higher expression of germ cell markers genes (DDX-4, MAGE A-4; P < 0.05) compared with separately cultured attached cells. Additionally, germ cell transplantation experiments demonstrated a significantly higher absolute number of cells with colonization ability (P < 0.001) in supernatant cells after 11 days of separate culture. LIMITATIONS, REASONS FOR CAUTION This study presents a relevant aspect for the successful setup of spermatogonial cultures but provides limited data regarding the question of whether the long-term maintenance of spermatogonia can be achieved. Transfer of these preclinical data to man may require modifications of the protocol. WIDER IMPLICATIONS OF THE FINDINGS Spermatogonial cultures from rodents have become important and innovative tools for basic and applied research in reproductive biology and veterinary medicine. It is expected that spermatogonia-based strategies will be transformed into clinical applications for the treatment of male infertility. Our data in the marmoset monkey may be highly relevant to establish spermatogonial cultures of human testes. STUDY FUNDING/COMPETING INTEREST(S) Funding was provided by the DFG-Research Unit FOR 1041 Germ Cell Potential (SCHL394/11-2) and by the Graduate Program Cell Dynamics and Disease (CEDAD) together with the International Max Planck Research School - Molecular Biomedicine (IMPRS-MBM). The authors declare that there is no conflict of interest. TRIAL REGISTRATION NUMBER Not applicable. © 2014 The Author.

Roos C.,German Primate Center | Zinner D.,German Primate Center | Kubatko L.S.,Ohio State University | Schwarz C.,German Primate Center | And 13 more authors.
BMC Evolutionary Biology | Year: 2011

Background: Colobine monkeys constitute a diverse group of primates with major radiations in Africa and Asia. However, phylogenetic relationships among genera are under debate, and recent molecular studies with incomplete taxon-sampling revealed discordant gene trees. To solve the evolutionary history of colobine genera and to determine causes for possible gene tree incongruences, we combined presence/absence analysis of mobile elements with autosomal, X chromosomal, Y chromosomal and mitochondrial sequence data from all recognized colobine genera. Results: Gene tree topologies and divergence age estimates derived from different markers were similar, but differed in placing Piliocolobus/Procolobus and langur genera among colobines. Although insufficient data, homoplasy and incomplete lineage sorting might all have contributed to the discordance among gene trees, hybridization is favored as the main cause of the observed discordance. We propose that African colobines are paraphyletic, but might later have experienced female introgression from Piliocolobus/ Procolobus into Colobus. In the late Miocene, colobines invaded Eurasia and diversified into several lineages. Among Asian colobines, Semnopithecus diverged first, indicating langur paraphyly. However, unidirectional gene flow from Semnopithecus into Trachypithecus via male introgression followed by nuclear swamping might have occurred until the earliest Pleistocene. Conclusions: Overall, our study provides the most comprehensive view on colobine evolution to date and emphasizes that analyses of various molecular markers, such as mobile elements and sequence data from multiple loci, are crucial to better understand evolutionary relationships and to trace hybridization events. Our results also suggest that sex-specific dispersal patterns, promoted by a respective social organization of the species involved, can result in different hybridization scenarios. © 2011 Roos et al; licensee BioMed Central Ltd.

Muller T.,Stem Cell Biology Unit | Muller T.,Hannover Medical School | Hupfeld T.,Stem Cell Biology Unit | Roessler J.,Hannover Medical School | And 3 more authors.
Journal of Medical Primatology | Year: 2011

Background Common marmoset monkeys (Callithrix jacchus) are readily used in biomedical research. However, superovulation for embryonic stem cell production and developmental research still remain difficult. Inexplicably, follicle-stimulating hormone (FSH) as key player in superovulation has to be administered in extremely high dosages in this non-human primate compared to human. Methods To evaluate whether marmoset FSH (cjFSH) is functionally more competent than its human homologue on the marmoset FSH receptor (cjFSHR), we established in vitro a homologous system characterizing homologous and recombinantly produced cjFSH. Results Upon stimulation of two cell lines stably expressing either the marmoset or the human FSH receptor (cj/hFSHR), respectively, the second messenger signaling of the activated receptor displayed no significant difference in ED50 values. Thermostability of cjFSH was significantly prolonged by roughly 20% on both FSHRs. Conclusion High FSH dosage in marmoset superovulation cannot be explained by enhanced biopotency of the natural animal's gonadotropin. © 2010 John Wiley & Sons A/S.

Aeckerle N.,Stem Cell Biology Unit | Dressel R.,University of Gottingen | Behr R.,Stem Cell Biology Unit
Cells Tissues Organs | Year: 2013

Single-cell suspensions derived from immature rodent and ungulate testes can reconstitute testicular cords upon grafting into immunodeficient mice. In the present study, neonatal common marmoset monkey (Callithrix jacchus) testes were digested to a single-cell suspension, which was transplanted subcutaneously into immunodeficient mice. After 9 or 18 weeks of incubation, the derivatives of the grafted single-cell suspensions were retrieved and analyzed histologically and immunohistochemically. Three of 4 (75%) neonatal grafts exhibited reconstituted seminiferous cords strongly resembling seminiferous cords of the intact neonatal testis. The cords consisted of Sertoli cells, germ cells and peritubular myoid cells, which was confirmed by immunohistochemical marker analysis. Three-dimensional reconstruction models of the grafts revealed elongated tubules. Some of the tubules were branched, which occurs also in vivo, as we show here for the marmoset monkey. Importantly, no teratoma formation by immature pluripotency factor-expressing germ cells was observed. In summary, the reconstituted testicular cords were almost indistinguishable from the cords formed in situ, thereby impressively demonstrating a very high reconstructive potential of a single-cell suspension obtained from the neonatal marmoset monkey testis. To our knowledge, this is the first study demonstrating testicular cord neomorphogenesis for a primate species ex situ. © 2013 S. Karger AG, Basel.

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