Germ Cell Markers Research Articles

Xenotransplantation of canine spermatogonial stem cells (cSSCs) regulated by FSH promotes spermatogenesis in infertile mice

BackgroundXenotransplantation of spermatogonial stem cells (SSCs) has become a popular topic in various research fields because manipulating these cells can provide insights into the mechanisms associated with germ cell lines and the entire spermatogenesis process; moreover, these cells can be used in several biotechnology applications. To achieve successful xenotransplantation, the in vitro microenvironment in which SSCs are cultured should be an ideal microenvironment for self-renewal and similar to the in vivo testicular microenvironment. The age of the donor, the correct spermatogenesis cycle, and the quality of the donor tissue are also important. Although cell culture-related factors, such as the in vitro supplementation of hormonal factors, are known to promote successful xenotransplantation in mice, little is known about the influence of these factors on SSCs in vitro or in vivo in other mammalian species, such as dogs (Canis lupus familiaris). In this context, the goals of this study were to test the effect of follicle-stimulating hormone (FSH) on canine spermatogonial stem cell (cSSC) cultures since this hormone is related to the glial cell-derived neurotrophic factor (GDNF) signaling pathway, which is responsible for the self-renewal and maintenance of these cells in vivo, and to investigate the microenvironment of the SSC culture after FSH supplementation. Additionally, in vivo analyses of transplanted FSH-supplemented cSSCs in the testes of infertile mice were performed to assess the capacity of cSSCs to develop, maintain, and restore spermatogenesis.MethodsSSCs from canine prepubertal testes (aged 3 months) were cultured in vitro in the presence of FSH (10 IU L−1). GFRA1 transcript expression was detected to confirm the spermatogonia population in culture and the effect of FSH on these cells. The protein and transcript levels of late germ cell markers (GFRA1, DAZL, STRA8, PLZF, and CD49f) and a pluripotency marker (OCT4) were detected at 72 and 120 h to confirm the cSSC phenotype. In vivo experiments were performed by transplanting GFP+ cSSCs into infertile mice, and a 10-week follow-up was performed. Histological and immunofluorescence analyses were performed to confirm the repopulation capacity after cSSC xenotransplantation in the testis.ResultsSupplementation with FSH in cell culture increased the number of cSSCs positive for GFRA1. The cSSCs were also positive for the pluripotency and early germline marker OCT4 and the late germline markers PLZF, DAZL, C-kit, and GFRA-1. The in vivo experiments showed that the cSSCs xenotransplanted into infertile mouse testes were able to repopulate germline cells in the seminiferous tubules of mice.ConclusionsIn conclusion, our results showed for the first time that the treatment of cSSC cultures with FSH can promote in vitro self-renewal, increase the population of germline cells, and possibly influence the success of spermatogenesis in infertile mice in vivo.

GDNF family receptor alpha 1 is a reliable marker of undifferentiated germ cells in bulls

Undifferentiated germ cells, including spermatogonial stem cells (SSCs), make up only a very small proportion of germ cells within the testis; for example, 0.03% of germ cells in the mouse testis are SSCs. In this study, we investigated the characteristics of bovine undifferentiated germ cells and developed an enrichment procedure for these cells on the basis of fluorescence-activated cell sorting (FACS), using the specific cell surface marker glial cell line-derived neurotrophic factor family receptor alpha 1 (GFRα1). FACS analysis showed that only 0.6% of the total testicular cells were GFRα1-positive. These GFRα1-positive cells had a significantly higher expression of UCHL1, ZBTB16, and DDX4 (all markers of undifferentiated spermatogonial and germ cells) than that of fresh testicular cells. Quantitative reverse-transcription PCR analyses also indicated that the gene expression of BCL6B and NANOS2 was significantly higher in GFRα1-positive cells. Furthermore, xenogeneic transplantation of bovine testicular cells into immunodeficient mice resulted in 4.4-fold more colonies of GFRα1-positive cells than those of fresh testicular cells, indicating that FACS with antibodies to GFRα1 had efficiently enriched putative SSCs from total testicular cells. Collectively, these results demonstrate that GFRα1 could be used as a marker of bovine undifferentiated germ cells, including putative SSCs, and that its expression on SSCs has important implications for the further development of techniques for enriching stem cells from other species.

Induction of Mouse Peritoneum Mesenchymal Stem Cells into Germ Cell-Like Cells Using Follicular Fluid and Cumulus Cells-Conditioned Media.

The peritoneum mesothelium lines body cavities and has the same origin as ovarian surface epithelium with probable existence of peritoneum mesenchymal stem cells (PMSCs). In the present research, PMSCs were isolated from peritoneum and differentiated into ovarian cell-like cells using human follicular fluid (HFF) and human cumulus-conditioned medium (HCCM). Anterior abdominal wall and intestinal peritoneum explants were used for cells isolation and cultured in Dulbecco's modified Eagle's medium. After passage 3, purified PMSCs were assessed for morphology, proliferation rate, and cell viability. Then, isolated PMSCs underwent two characterization procedures: (1) differentiation to mesodermal lineage and (2) expression of mesenchymal (CD90 and CD44) and epithelial cell (CK19) markers. The characterized PMSCs were differentiated into ovarian cell-like cells using 10% HFF and 50% HCCM for 21 days, and the expressions of oocyte (Zp3, Gdf9), germ cell (Ddx4, Dazl), granulosa cell (Amh), and theca cell (Lhr) markers were assessed using real-time polymerase chain reaction and immunocytofluorescence assay. Both anterior abdominal wall and intestinal peritoneum mesenchymal stem cells (AP-MSCs and IP-MSCs) showed mesenchymal characters and differentiated to adipocyte and osteocyte. AP-MSCs expressed mesenchymal- and epithelial cell-specific markers more than IP-MSCs and showed an analytically better proliferation rate. The induced AP-MSCs and IP-MSCs were expressed as germ and oocyte cell-specific markers, and this expression increased in the third week of culture. In both groups of AP-MSCs and IP-MSCs, the expressions of Gdf9, Zp3, Ddx4, Dazl, and Amh genes under just HCCM induction showed upregulation significantly on the 21st day of culture compared with day 0. But in protein synthesis of all mentioned genes, both HFF and HCCM had equal induction effect on the 21st day of culture against the 0th day. In addition, LHR was not expressed in any groups. Finally, in both characterization and differentiation procedures, the AP-MSCs respond to inducers better than IP-MSCs.

Dmrt1 (doublesex and mab-3-related transcription factor 1) expression during gonadal development and spermatogenesis in the Japanese eel

Dmrt1, doublesex- and mab-3-related transcription factor-1, has been suggested to play critical roles in male gonadogenesis, testicular differentiation and development, including spermatogenesis, among different vertebrates. Vasa is a putative molecular marker of germ cells in vertebrates. In this study, we cloned the full-length dmrt1 cDNA from Japanese eel, and the protein comprised 290 amino acids and presented an extremely conserved Doublesex and Mab-3 (DM) domain. Vasa proteins were expressed in gonadal germ cells in a stage-specific manner, and were expressed at high levels in PGC and spermatogonia, low levels in spermatocytes, and were absent in spermatids and spermatozoa of Japanese eels. Dmrt1 proteins were abundantly expressed in spermatogonia B cells, spermatocytes, spermatids, but not in spermatozoa, spermatogonia A and Sertoli cells. To our knowledge, this study is the first to show a restricted expression pattern for the Dmrt1 protein in spermatogonia B cells, but not spermatogonia A cells, of teleosts. Therefore, Dmrt1 might play vital roles at the specific stages during spermatogenesis from spermatogonia B cells to spermatids in the Japanese eel. Moreover, the Dmrt1 protein exhibited a restricted localization in differentiating oogonia in the early differentiating gonad (ovary-like structure) of male Japanese eels and in E2-induced feminized Japanese eels. We proposed that dmrt1 may be not only required for spermatogenesis but might also play a role in oogenesis in the Japanese eel.

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

In vitro gamete derivation based on differentiation of germ cells (GC) from stem cells has emerged as a potential new strategy for the treatment of male infertility. This technology also has potential applications in animal reproduction as an alternative method for dissemination of elite animal genetics, production of transgenic animals, and conservation of endangered species. Mesenchymal stem cells (MSC) are multipotent progenitor cells defined by their ability to differentiate into mesodermal lineages. Under the effect of selected bioactive factors, MSC upregulate expression of pluripotent and GC specific-markers revealing their potential for GC differentiation. In addition to the effect of trophic factors, cell-to-cell interaction with Sertoli cells (SC) may be required to guide the sequential differentiation of MSC into GC. Thus, the aim of the present study was to investigate the effect of coculture with SC on the potential for in vitro GC differentiation of bovine fetal MSC (bfMSC) derived from bone marrow (BM-MSC) and adipose tissue (AT-MSC). bfMSC were isolated from male bovine fetuses and SC were collected from adult bull testes. The effect of SC interaction with BM-MSC or AT-MSC was analyzed on the expression of pluripotent factors OCT4 and NANOG, GC genes FRAGILLIS, STELLA and VASA and male GC markers DAZL, PIWIL2, STRA8 and SCP3 at Day 14 of coculture. Flow cytometry analyses detected that the majority (95,5% ± 2.5; P < 0.05) of the isolated population of SC cultures were positive for SC-specific marker WT1. Levels of mRNA of WT1 in BM-MSC and AT-MSC were lower (P < 0.05) compared to SC; whereas, WT1 expression was not detected in bovine fetal fibroblasts (FB). Cocultures of BM-MSC and AT-MSC with SC had higher (P < 0.05) OCT4 mRNA levels compared to monocultures of BM-MSC, AT-MSC and SC. Moreover, cocultures of BM-MSC with SC had higher (P < 0.05) proportion of cells positive for Oct4 and Nanog compared to monocultures of BM-MSC and SC. Levels of mRNA of DAZL, PIWIL2 and SCP3 were upregulated in cocultures of AT-MSC with SC compared to monocultures of AT-MSC and SC. Accordingly, the proportion of cells positive for Dazl were higher (P < 0.05) in cocultures of AT-MSC with SC compared to monocultures of AT-MSC and SC. Changes in gene expression profiles during coculture of SC with AT-MSC suggest that cell-to-cell interaction or bioactive factors provided by SC may induce progression of AT-MSC into early stages of GC differentiation.

The germ cell marker dead end reveals alternatively spliced transcripts with dissimilar expression

Since the late 19th century, the Amazon species Colossoma macropomum (tambaqui) has been exploited commercially and the climate change has contributed to decline in tambaqui numbers. Although germ cell cryopreservation and transplantation can help preserve the species’ genetic resources semipermanently, its germ cell behavior has not been analyzed to date. In this study, we isolated the tambaqui’s dead end gene (dnd) homolog (tdnd) and used it as a molecular marker for germ cells to obtain basic information essential for transplantation. The amino acid sequence showed 98% similarity and 53% identity with the zebrafish dnd. Phylogenetic analysis and the presence of consensus motifs known for dnd revealed that tdnd encodes the dnd ortholog and its transcript is detectable only in the testes and ovaries, showing a strong positive signal in oocytes and spermatogonia. The tambaqui possesses, at least, three different transcripts of tdnd which show dissimilar expression profile in undifferentiated and sexually mature animals, suggesting that they play distinct roles in germline development and they may influence the choice of donors for the cell transplantation study.

Hepatoid Carcinoma of the Ovary: Clinical, Histopathologic, and Immunophenotypic Features.

Hepatoid carcinoma of the ovary (HCO) is a rare malignant tumor of uncertain histogenesis that was first described by Ishikura and Scully in the late 1980s. Unlike hepatoid yolk sac tumor (HYST), one of its main differential diagnoses, HCO usually presents in perimenopausal and postmenopausal women without gonadal dysgenesis. Most cases show advanced local disease at initial presentation, with diffuse intraperitoneal dissemination. Despite aggressive treatment, including surgery and adjuvant chemotherapy, 61.5% of patients either die of the disease (11 of 26; 42.3%) or are alive with recurrent or residual disease (5 of 26; 19.2%) after a median follow-up of 11.5 months (range, 1-60 months). Most HCOs are solid, with high-grade histology, significant nuclear pleomorphism, scattered giant cells, and a high mitotic index. Their immunophenotype is defined by the expression of broad-spectrum cytokeratins, α-fetoprotein, and hepatocellular antigens with absence of sex cord and germ cell markers. Although immunohistochemistry can be very helpful to distinguish between sex cord-stromal tumors and HCO, differentiation of the latter from HYST, metastatic hepatocellular carcinoma, and metastatic gastrointestinal tumors with hepatoid phenotype requires integration of clinical, radiologic, and pathologic information.

Efficient generation of human primordial germ cell-like cells from pluripotent stem cells in a methylcellulose-based 3D system at large scale.

BackgroundThe mechanisms underlying human germ cell development and infertility remain largely unknown due to bioethical issues and the shortage of experimental materials. Therefore, an effective in vitro induction system of human primordial germ-like cells (hPGCLCs) from human pluripotent stem cells (hPSC) is in high demand. The current strategies used for the generation of hPGCLCs are not only costly but also difficult to perform at a large scale, thereby posing barriers to further research. In this study, we attempted to solve these problems by providing a new 3D culture system for hPGCLC differentiation.MethodsThe efficiency and relative yield of a methylcellulose (MC)-based 3D hPGCLC induction system were first compared with that of a conventional U96 system. Then, we examined the gene expression of germ cell marker genes and the key epigenetic modifications of the EpCAM-/INTEGRINα6-high cells from the 3D MC induction system and the U96 system via quantitative PCR and immunofluorescence. Finally, the reliability of the MC-based 3D hPGCLC induction system was evaluated via the generation of induced pluripotent stem cells (iPSCs) from the testicular cells of one patient with obstructive azoospermia (OA) and followed by the subsequent differentiation of iPSCs into the germ cell lineage.ResultsIn the present study, we demonstrated that the 3D MC induction system combined with low-cell attachment plates facilitated the generation of hPGCLCs at a large scale. We found that the hPGCLCs generated via the MC system shared similar characteristics to that via the U96 system in terms of the gene expression profiles, germ cell-specific markers, epigenetic modification states and cellular states. In addition, hPGCLCs from iPSCs derived from one OA patient were generated with high efficiency via the present 3D MC induction system.DiscussionThe in vitro induction of hPGCLCs from human embryonic stem cells (hESCs)/human induced pluripotent stem cells (hiPSCs) has significant implications in exploring the underlying mechanisms of the origin and specification of hPGCs and the epigenetic programming of the human germ line as well as treating male infertility. Here, we developed a simple and efficient 3D induction system to generate hPGCLCs from hESCs/hiPSCs at a large scale, which facilitated the study of human germ cell development and stem cell-based reproductive medicine.

Pluripotency Potential of Embryonic Stem Cell-Like Cells Derived from Mouse Testis.

Objective During the cultivation of spermatogonial stem cells (SSCs) and their conversion into embryonic stem-like (ES-like) cells, transitional ES-like colonies and epiblast-like cells were observable. In the present experimental study, we aimed to analyze the efficiency of the multipotency or pluripotency potential of ES-like cells, transitional colonies and epiblast-like cells. Materials and Methods In this experimental study, SSCs were isolated from transgenic octamer-binding transcription factor 4 (Oct4)-green fluorescent protein (GFP)-reporter mice. During cell culture ES-like, transitional and epiblast- like colonies developed spontaneously. The mRNA and protein expression of pluripotency markers were analyzed by Fluidigm real-time polymerase chain reaction (RT-PCR) and immunocytochemistry, respectively. Efficiency to produce chimera mice was evaluated after injection of ES and ES-like cells into blastocysts. Results Microscopic analyses demonstrated that the expression of Oct4-GFP in ES-like cells was very strong, in epiblast-like cells was not detectable, and was only partial in transitional colonies. Fluidigm RT-PCR showed a higher expression of the germ cell markers Stra-8 and Gpr-125 in ES-like cells and the pluripotency genes Dppa5, Lin28, Klf4, Gdf3 and Tdgf1 in ES-like colonies and embryonic stem cells (ESCs) compared to the epiblast-like and transitional colonies. No significant expression of Oct-4, Nanog, Sox2 and c-Myc was observed in the different groups. We showed a high expression level of Nanog and Klf4 in ES-like, while only a partial expression was observed in transitional colonies. We generated chimeric mice after blastocystic injection from ES and ES-like cells, but not from transitional colonies. We observed that the efficiency to produce chimeric mice in ES cells was more efficient (59%) in comparison to ES-like cells (22%). ConclusionThis new data provides more information on the pluripotency or multipotency potentials of testis-derived ES-like cells in comparison to transitional colonies and epiblast-like cells.

Molecular characterization of vasa homologue in marbled goby, Oxyeleotris marmorata: Transcription and localization analysis during gametogenesis and embryogenesis.

Identification of germ cell markers is important for investigating reproduction biology in fish. Vasa is one of the most studied germ cell markers in mammals and lower vertebrates including fish. Here, we characterized a vasa homologue from the fish marbled goby (Oxyeleotris marmorata), termed omvasa. The full length of omvasa cDNA is 2344 bp and encodes 658 amino acids, sharing high identities with Vasa homologues of other vertebrates. OmVasa protein contains 15 RG/RGG repeats at N-terminus, 2 ATPase motifs, as well as RNA unwinding and RNA binding domains at C-terminus. Phylogenetic tree showed that omVasa had the closest relationship with the Vasa homologue from the fish Boleophthalmus pectinirostris, the great blue-spotted mudskipper. qRT-PCR analysis indicated that omvasa was specifically transcribed in gonads, and the transcription level was gradually increased during oocyte development. The germ cell-specific distribution of omvasa mRNA was revealed by fluorescent in situ hybridization. In ovary, the signal of omvasa RNA displayed strong-weak-strong dynamics from oogonia over pre-vitellogenic oocytes to vitellogenic oocytes. In testis, omvasa signal was strong in spermatogonia, modest in spermatocytes but undetectable in spermatids and somatic cells. During embryogenesis, the transcription of omvasa mRNA was high at blastula stage, gradually decreased from gastrula stage and maintained at a low level in later developmental stages. Whole mount in situ hybridization indicated that omvasa mRNA was specific to primordial germ cells (PGCs). In summary, marbled goby vasa is a germ cell-specific transcript during gametogenesis, and can be used as an ideal marker for tracing PGC formation and migration, which is pivotal to germ cell manipulation in this species.

189 Isolation and purification of rhinoceros and horse spermatogonial stem cells

The northern white rhinoceros (NWR; Ceratotherium simum cottoni) is critically endangered. Testicular tissue (TT) of an NWR that died at 46 years was cryopreserved and stored. Spermatogonial stem cells (SSC) have been isolated from TT and, following transplantation or in vitro culture, differentiated into mature spermatozoa. These SSC have been phenotypically characterised by the expression of markers specific for SSC, and in vitro culture methods optimized to isolate, purify, and enrich populations of SSC from mixed germ cells. The reproductive age may affect the percent of SSC in TT, affecting recovery from older animals. We previously identified in TT of NWR the surface markers GFRa1 and GPR125, both of which were expressed at various stages of spermatogenesis. However, characterisation, isolation, and purification of rhinoceros SSC have not been studied. Because of the limited availability of rhinoceros TT, we used the horse as a model for rhinoceros (both species are in the same order: Perissodactyla). In this study, we (1) identified and compared the expression of markers specific for SSC (GFRa1, GPR125, PLZF) and pluripotent markers (SSEA-1, SSEA-4, OCT-4) in rhinoceros and horse mixed germ cells; (2) evaluated whether rhinoceros and horse SSC could be purified and enriched by sequential culture with collagen (2 days) and laminin (3 weeks); and (3) determined whether age may affect the percentages of SSC by comparing the expression of SSC and pluripotent markers in horse mixed germ cells at different reproductive stages: pubertal (PU=1-1.5 years) v. post-pubertal (PP=2-3 years) v. adult (AD=&amp;gt;5 years). Mixed germ cells were isolated from testes of one male of each of 3 rhinoceros species: NWR, southern white rhinoceros (SWR; Ceratotherium simum simum, 49 years), and greater one-horned rhinoceros (GOHR; Rhinoceros unicornis, 5 years), and horses at different reproductive stages (PU=2, PP=2, AD=7). The SWR and GOHR testes were refrigerated overnight, whereas NWR mixed germ cells were isolated from frozen-thawed TT. Flow cytometry analysis showed expression of SSC and pluripotent markers but not for transcription factor PLZF. The marker expression was similarly distributed between the 3 species of rhinoceros, with a larger portion of cells positive for SSEA-4 (mean%±s.e.m.: 6.3±1.1%) and smaller proportion for GDNFa1 (0.4±0.2%), GRP125 (0.1±0.1%), OCT-4 (0.4±0.2%), and SSEA-1 (0.4±0.2%; P&amp;lt;0.05). Similarly, horse expressed the markers GDNFa1 (0.9±0.3%), GRP125 (0.9±0.4%), OCT-4 (1.7±0.5%), and SSEA-1 (0.12±0.1%), but the abundance of SSEA-4 (1.8±0.8%) was less than that of rhinoceros (P&amp;lt;0.05). In rhinoceros, sequential culture enhanced the numbers of cells expressing all markers compared with that before culture, whereas in horses, the increase was observed only for cells expressing GDNFa1, GPR125, and SSEA-1. Reproductive stage did not affect the percentages of horse cells expressing germ cell and pluripotent markers. Overall, these results showed that rhinoceros SSC can be isolated from TT and expressed the same SSC markers as horses, and that differential culture enriched a population of SSC.

All-trans Retinoic Acid Disrupts Development in Ex Vivo Cultured Fetal Rat Testes. II: Modulation of Mono-(2-ethylhexyl) Phthalate Toxicity.

Humans are universally exposed to low levels of phthalate esters (phthalates), which are used to plasticize polyvinyl chloride. Phthalates exert adverse effects on the development of seminiferous cords in the fetal testis through unknown toxicity pathways. To investigate the hypothesis that phthalates alter seminiferous cord development by disrupting retinoic acid (RA) signaling in the fetal testis, gestational day 15 fetal rat testes were exposed for 1-3 days to 10-6 M all-trans retinoic acid (ATRA) alone or in combination with 10-6-10-4 M mono-(2-ethylhexyl) phthalate (MEHP) in ex vivo culture. As previously reported, exogenous ATRA reduced seminiferous cord number. This effect was attenuated in a concentration-dependent fashion by MEHP co-exposure. ATRA and MEHP-exposed testes were depleted of DDX4-positive germ cells but not Sertoli cells. MEHP alone enhanced the expression of the RA receptor target Rbp1 and the ovary development-associated genes Wnt4 and Nr0b1, and suppressed expression of the Leydig cell marker, Star, and the germ cell markers, Ddx4 and Pou5f1. In co-exposures, MEHP predominantly enhanced the gene expression effects of ATRA, but the Wnt4 and Nr0b1 concentration-responses were nonlinear. Similarly, ATRA increased the number of cells expressing the granulosa cell marker FOXL2 in testis cultures, but this induction was attenuated by addition of MEHP. These results indicate that MEHP can both enhance and inhibit actions of ATRA during fetal testis development and provide evidence that RA signaling is a target for phthalate toxicity in the fetal testis.

Isolation and characterization of a germ cell marker in teleost fish Colossoma macropomum

The native Amazonian fish tambaqui (Colossoma macropomum) is the second-largest scaled fish in South America and the most common native species in Brazil. To preserve genetic resources with sufficient genetic diversity through germ cell cryopreservation and transplantation techniques, a molecular marker for identifying the cells is required to trace them during the manipulation processes. The vasa gene is a promising candidate, as its specific expression in germ cell lineage has been well-conserved throughout animal evolution. In this study, the full sequence of the vasa cDNA homolog from tambaqui was isolated and characterized, showing an open reading frame of 2010 bp encoding 669 amino acids. The putative protein was shown to contain eight conserved motifs of the DEAD-box protein family and high similarity to vasa homologs of other species. Tambaqui vasa (tvasa) mRNA expression was specific to the gonads, and in situ hybridization showed signals only in oocytes and spermatogonia. The results suggested that tvasa could be a useful germ cell marker in this species.

The combination of retinoic acid and estrogen can increase germ cells genes expression in mouse embryonic stem cells derived primordial germ cells

Generation of germ cells from embryonic stem cells in vitro could have great application for treating infertility. The temporal expression profile of several genes was expressed at different stages of germ cell development and examined in differentiation the mouse embryonic stem cells. Cells were treated in three groups of control, with 10−8 M of all-trans retinoic acid and the combination of 10−9 M of 17β-Estradiol and retinoic acid for 7, 12, 17 or 22 days. Quantitative RT-PCR and Immunofluorescent were used to investigate the possible inductive effects of estrogen on mouse embryonic stem cell-derived primordial germ cells. mRNA expression of Oct4 and Dazl were downregulated in embryonic stem cells by the retinoic acid group, whereas Mvh transcription was reduced by retinoic acid and estrogen group in these cells compared to the control group. But, retinoic acid with estrogen group-treated cells exhibited increased mRNA expression of Stra8, Fragilis, Sycp3, GDF9, and Stella compared to untreated controls. The expression of Stella and Mvh proteins were remarkably increased in cell colonies. This study shows that estrogen affects the expression of specific markers of primordial germ cells. Also, estrogen and retinoic acid speed up and increase the level of expression of specific markers.

Effect of Fe and Cd Co-Exposure on Testicular Steroid Metabolism, Morphometry, and Spermatogenesis in Mice.

The mechanism of testicular toxicity of simultaneous multiple exposures to metals is poorly understood. Previous studies reported that the toxic effect of cadmium (Cd) is modified by tissue concentration of iron (Fe). Using the mice (Mus musculus) model in the present study, we demonstrated that combined Cd (25mgkg-1 bw) and Fe (100mgkg-1 bw) treatment increased both Cd and Fe testicular concentrations much more than separate exposures to either of the metals. Intratesticular Cd and Fe concentrations were inversely correlated (r = - 0.731, p < 0.05) on administration of Fe but not on combined exposure to both metals when they were positively correlated (versus Cd; r = 0.793, versus Fe; r = 0.779, p < 0.05). Additionally, Cd + Fe treatment increased testicular lipid peroxidation and depleted intratestesticular testosterone, cholesterol and glutathione concentrations much more than their separate treatment. This was also associated with decreased activity of the germ cell marker, testicular lactate dehydrogenase, and increased testicular myeloperoxidase activity. These changes resulted in decreased seminiferous epithelial height, tubular diameter, germ cell (spermatogonia, spermatocytes, and spermatids) numbers, and severe tissue damage. In conclusion, Cd + Fe intake have synergistic toxic effects on testicular steroid formation and spermatogenesis due to the high testicular concentrations of both metals.

Transcriptional landscape changes during human embryonic stem cell derivation.

What are the transcriptional changes occurring during the human embryonic stem cell (hESC) derivation process, from the inner cell mass (ICM) to post-ICM intermediate stage (PICMI) to hESC stage, that have downstream effects on pluripotency states and differentiation? We reveal that although the PICMI is transcriptionally similar to the hESC profile and distinct from ICM, it exhibits upregulation of primordial germ cell (PGC) markers, dependence on leukemia inhibitory factor (LIF) signaling, upregulation of naïve pluripotency-specific signaling networks and appears to be an intermediate switching point from naïve to primed pluripotency. It is currently known that the PICMI exhibits markers of early and late-epiblast stage. It is suggested that hESCs acquire primed pluripotency features due to the upregulation of post-implantation genes in the PICMI which renders them predisposed towards differentiation cues. Despite this current knowledge, the transcriptional landscape changes during hESC derivation from ICM to hESC and the effect of PICMI on pluripotent state is still not well defined. To gain insight into the signaling mechanisms that may govern the ICM to PICMI to hESC transition, comparative RNA sequencing (RNA-seq) analysis was performed on preimplantation ICMs, PICMIs and hESCs in biological and technical triplicates (n = 3). Primed hESCs (XX) were maintained in feeder-free culture conditions on Matrigel for two passages and approximately 50 cells were collected in biological and technical triplicates (n = 3). For ICM sample collection, Day 3, frozen-thawed human embryos were cultured up to day five blastocyst stage and only good quality blastocysts were subjected to laser-assisted micromanipulation for ICM collection (n = 3). Next, day six expanded blastocysts were cultured on mouse embryonic fibroblasts and manual dissection was performed on the PICMI outgrowths between post-plating Day 6 and Day 10 (n = 3). Sequencing of these samples was performed on NextSeq500 and statistical analysis was performed using edgeR (false discovery rate (FDR) < 0.05). Comparative RNA-seq data analysis revealed that 634 and 560 protein-coding genes were significantly up and downregulated in hESCs compared to ICM (FDR < 0.05), respectively. Upon ICM to PICMI transition, 471 genes were expressed significantly higher in the PICMI compared to ICM, while 296 genes were elevated in the ICM alone (FDR < 0.05). Principle component analysis showed that the ICM was completely distinct from the PICMI and hESCs while the latter two clustered in close proximity to each other. Increased expression of E-CADHERIN1 (CDH1) in ICM and intermediate levels in the PICMI was observed, while CDH2 was higher in hESCs, suggesting a role of extracellular matrix components in facilitating pluripotency transition during hESC derivation. The PICMI also showed regulation of naïve-specific LIF and bone morphogenetic protein signaling, differential regulation of primed pluripotency-specific fibroblast growth factor and NODAL signaling pathway components, upregulation of phosphatidylinositol 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) pathway (PI3K/AKT/mTORC), as well as predisposition towards the germ cell lineage, further confirmed by gene ontology analysis. Hence, the data suggest that the PICMI may serve as an intermediate pluripotency stage which, when subjected to an appropriate culture niche, could aid in enhancing naïve hESC derivation and germ cell differentiation efficiency. Gene Expression Omnibus (GEO) Accession number GSE119378. Owing to the limitation in sample availability, the sex of ICM and PICMI have not been taken into consideration. Obtaining cells from the ICM and maintaining them in culture is not feasible as it will hamper the formation of PICMI and hESC derivation. Single-cell quantitative real-time PCR on low ICM and PICMI cell numbers, although challenging due to limited availability of human embryos, will be advantageous to further corroborate the RNA-seq data on transcriptional changes during hESC derivation process. We elucidate the dynamics of transcriptional network changes from the naïve ICM to the intermediate PICMI stage and finally the primed hESC lines. We provide an in-depth understanding of the PICMI and its role in conferring the type of pluripotent state which may have important downstream effects on differentiation, specifically towards the PGC lineage. This knowledge contributes to our limited understanding of the true nature of the human pluripotent state in vitro. This research is supported by the Concerted Research Actions funding from Bijzonder Onderzoeksfonds University Ghent (BOF GOA 01G01112).The authors declare no conflict of interest.

Differentiation of human male germ cells from Wharton's jelly-derived mesenchymal stem cells.

ObjectiveRecapitulation of the spermatogenesis process in vitro is a tool for studying the biology of germ cells, and may lead to promising therapeutic strategies in the future. In this study, we attempted to transdifferentiate Wharton's jelly-derived mesenchymal stem cells (WJ-MSCs) into male germ cells using all-trans retinoic acid and Sertoli cell-conditioned medium.MethodsHuman WJ-MSCs were propagated by the explant culture method, and cells at the second passage were induced with differentiation medium containing all-trans retinoic acid for 2 weeks. Putative germ cells were cultured with Sertoli cell-conditioned medium at 36℃ for 3 more weeks.ResultsThe gene expression profile was consistent with the stage-specific development of germ cells. The expression of Oct4 and Plzf (early germ cell markers) was diminished, while Stra8 (a premeiotic marker), Scp3 (a meiotic marker), and Acr and Prm1 (postmeiotic markers) were upregulated during the induction period. In morphological studies, approximately 5% of the cells were secondary spermatocytes that had completed two stages of acrosome formation (the Golgi phase and the cap phase). A few spermatid-like cells that had undergone the initial stage of tail formation were also noted.ConclusionHuman WJ-MSCs can be transdifferentiated into more advanced stages of germ cells by a simple two-step induction protocol using retinoic acid and Sertoli cell-conditioned medium.

Evolutionary conservation and divergence of Vasa, Dazl and Nanos1 during embryogenesis and gametogenesis in dark sleeper (Odontobutis potamophila)

Germline-specific genes, Vasa, Dazl and Nanos1, have highly conserved functions in germline development and fertility across animal phyla. In this study, the full-length sequences of Opvasa, Opdazl and Opnanos1 were cloned and characterized from the dark sleeper (Odontobutis potamophila). Gonad-specific expression patterns of Opvasa and Opdazl were confirmed in adult tissues by quantitative real-time PCR (qRT-PCR). Different from Opvasa and Opdazl, the expression of Opnanos1 was ubiquitously detected in all examined tissues except for the liver and spleen. Time-course dynamic expressions during embryogenesis were assessed, and all three genes (Opvasa, Opdazl and Opnanos1) persisted at a high level until gastrulation. qRT-PCR and Western blotting analyses revealed that all three genes were highly expressed throughout gametogenesis. In testis, the expressions of all three genes at the mRNA and protein levels were down-regulated during spermatogenesis. In ovary, different expression patterns were found, and all three genes had a differential role in translational regulation during oogenesis. The expressions of Opvasa, Opdazl and Opnanos1 at the mRNA but not the protein level were high in stage IV. Different expression patterns were found in premeiotic gonads treated by HPG axis hormones (HCG and LHRH-A). Immunolocalization analysis demonstrated that in testis, Opvasa, Opdazl and Opnanos1 were detected in spermatogonia and spermatocytes but absent in the meiotic products, such as spermatids and spermatozoa. In ovary, Opvasa, Opdazl and Opnanos1 persisted at a high level throughout oogenesis. These findings indicated that Opvasa, Opdazl and Opnanos1 played an important role in mitotic and early meiotic phases of oogenesis and spermatogenesis, and they functioned as maternal factors in early embryogenesis. Their proteins could be used as three new markers for germ cells during gametogenesis in O. potamophila gonad. Our data laid a good foundation for improving the breeding efficiency of O. potamophila.

Repressible Transgenic Sterilization in Channel Catfish, Ictalurus punctatus, by Knockdown of Primordial Germ Cell Genes with Copper-Sensitive Constructs.

Repressible knockdown approaches were investigated to manipulate for transgenic sterilization in channel catfish, Ictalurus punctatus. Two primordial germ cell (PGC) marker genes, nanos and dead end, were targeted for knockdown and an off-target gene, vasa, was monitored. Two potentially copper-sensitive repressible promoters, yeast ctr3 (M) and ctr3-reduced (Mctr), were coupled with four knockdown strategies separately including: ds-sh RNA targeting the 5' end (N1) or 3' end (N2) of channel catfish nanos, full-length cDNA sequence of channel catfish nanos for overexpression (cDNA), and ds-sh RNA-targeting channel catfish dead end (DND). Each construct had an untreated group and treated group with copper sulfate as the repressor compound. Spawning rates of full-sibling P1 fish exposed or not exposed to the constructs as treated and untreated embryos were 85 and 54%, respectively, indicating potential sterilization of fish and repression of the constructs. In F1 fish, mRNA expressions of PGC marker genes for most constructs were downregulated in the untreated group and the knockdown was repressed in the treated group. Gonad development in transgenic, untreated F1 channel catfish was reduced compared to non-transgenic fish for MctrN2, MN1, MN2, and MDND. For 3-year-old adults, gonad size in the transgenic untreated group was 93.4% smaller than the non-transgenic group for females and 92.3% for males. However, mean body weight of transgenic females (781.8g) and males (883.8g) was smaller than of non-transgenic counterparts (984.2 and 1254.3g) at 3years of age, a 25.8 and 41.9% difference for females and males, respectively. The results indicate that repressible transgenic sterilization is feasible for reproductive control of fish, but negative pleiotropic effects can result.

A potential germ cell-specific marker in Japanese flounder, Paralichthys olivaceus: identification and characterization of lymphocyte antigen 75 (Ly75/CD205)

Some germ cell marker genes, such as vasa, nanos, and dead end (dnd), have been identified in fish. Recently, lymphocyte antigen 75 (Ly75/CD205) has been identified as a mitotic germ cell-specific cell-surface marker in several fish species. In this study, the Japanese flounder Paralichthys olivaceus ly75 homolog (ly75) was cloned and its expression pattern in gonads was analyzed. The full-length cDNA of ly75 was 7 346 bp, with an open reading frame (ORF) of 5 229 bp. The ORF encoded a protein containing 1 742 amino acids with a predicted molecular mass of 196.89 kDa. In adult tissues, ly75 transcripts were detected in all analyzed tissues but abundantly in the testis. In in-situ hybridization analyses, ly75 mRNA was predominantly localized in oocytes in the ovary and spermatogonia in the testis, but ly75 mRNA was not detected in oogonia, spermatocytes, spermatids, or spermatozoa. These results indicated that ly75 could be a potential germ cell-specific marker in P. olivaceus, as in other fishes.