Expression Of Germ Cell Markers Research Articles

247. BMP signalling in the induction of germline precursors from mouse embryonic stem cells in vitro

BMP signalling is critical for germline lineage establishment during mouse embryogenesis. To assess its importance in the induction of germline precursors in vitro, a mouse embryonic stem (ES) cell line harbouring an Oct4 promoter-EGFP reporter construct was used to derive embryoid body (EB) aggregates cultured in the absence or presence of combinations of BMP2, BMP4 and BMP8b for 3 to 10 days. At both day 5 and 10 of culture, clearly defined clusters of Oct4-EGFP were observed in EBs cultured with BMPs, while these clusters were minimal to absent in untreated EBs. Quantitative mRNA analysis of day 3 to day 10 EBs revealed a significant elevation in the expression of genes associated with primordial germ cell specification in EBs grown in the presence of BMPs. Moreover, a transient elevation of early germ cell markers Blimp1, Fragilis and Stella was detected in day 3–4 EBs cultured with BMPs, followed their decline by day 5. In contrast, levels of the pluripotency markers, Oct3/4 and Nanog, and the later germ cell markers, Dazl and Vasa, progressively increased from day 3 to day 5. Levels of TGFβ superfamily signalling components ALK2, Smad1 and Smad5 remained relatively constant during this period. Wholemount immunofluorescence of day 5 Oct4-EGFP EBs exposed to BMP4 demonstrated co-localisation of primordial germ cell markers Oct3/4, Stella, and the cell surface antigen SSEA-1 with EGFP+ clusters. These results demonstrate that signalling by BMP2 and 4 enhances germ cell marker expression within EBs and identifies the day 3 to 5 timeframe in EB differentiation as a critical window when putative germ cells are first specified in vitro.

Expression of Pluripotent Stem Cell Markers in the Human Fetal Testis

Human primordial germ cells (PGCs) have proven to be a source of pluripotent stem cells called embryonic germ cells (EGCs). However, the developmental potency of these cells in the fetal gonad still remains elusive. Thus, this study provides a comprehensive analysis of pluripotent and germ cell marker expression in human fetal testis 7-15 weeks postfertilization (pF) and compares this expression to their ability to derive EGCs. Although the majority of germ cells expressed stem cell markers stage-specific embryonic antigen (SSEA) 1, SSEA4, EMA-1, and alkaline phosphatase, only a small percentage of those (<1%) expressed OCT4, CKIT, and NANOG. Specifically, the number of OCT4(+)/CKIT(+)/NANOG(+) cells significantly increased in the developing cords during weeks 7-9, followed by a gradual decline into week 15 pF. By week 15 pF, the remaining OCT4(+)/CKIT(+)/NANOG(+) cells were found in the cords surrounding the periphery of the testis, and the predominant germ cells, CKIT(+) cells, no longer expressed OCT4 or NANOG. Based on morphology and early germ cell marker expression, including VASA, PUM2, and DAZL, we suggest these cells are mitotically active gonocytes or prespermatogonia. Importantly, the number of OCT4(+) cells correlated with an increase in the number of EGC colonies derived in culture. Interestingly, two pluripotent markers, Tra-1-60 and Tra-1-81, although highly expressed in EGCs, were not expressed by PGCs in the gonad. Together, these results suggest that PGCs maintain expression of pluripotent stem cell markers during and after sexual differentiation of the gonad, albeit in very low numbers.

Germ cell development in the Honeybee (Apis mellifera); Vasa and Nanosexpression

BackgroundStudies of specification of germ-cells in insect embryos has indicated that in many taxa the germ cells form early in development, and their formation is associated with pole plasm, germ plasm or an organelle called the oosome. None of these morphological features associated with germ cell formation have been identified in the Honeybee Apis mellifera. In this study I report the cloning and expression analysis of Honeybee homologues of vasa and nanos, germ cell markers in insects and other animals.ResultsApis vasa and nanos RNAs are present in early honeybee embryos, but the RNAs clear rapidly, without any cells expressing these germ cell markers past stage 2. These genes are then only expressed in a line of cells in the abdomen from stage 9 onwards. These cells are the developing germ cells that are moved dorsally by dorsal closure and are placed in the genital ridge.ConclusionThis study of the expression of germ cell markers in the honeybee implies that in this species either germ cells are formed by an inductive event, late in embryogenesis, or they are formed early in development in the absence of vasa and nanos expression. This contrasts with germ cell development in other members of the Hymenoptera, Diptera and Lepidoptera.

Dazl deficiency leads to embryonic arrest of germ cell development in XY C57BL/6 mice

Genes of the DAZ family play critical roles in germ cell development in mammals and other animals. In mice, Dazl mRNA is first observed at embryonic day 11.5 (E11.5), but previous studies using Dazl-deficient mice of mixed genetic background have largely emphasized postnatal spermatogenic defects. Using an inbred C57BL/6 background, we show that Dazl is required for embryonic development and survival of XY germ cells. By E14.5, expression of germ cell markers (Mvh, Oct4, Dppa3/Stella, GCNA and MVH protein) was reduced in XY Dazl−/− gonads. By E15.5, most remaining germ cells in XY Dazl−/− embryos exhibited apoptotic morphology, and XY Dazl−/− gonads contained increased numbers of TUNEL-positive cells. The rare XY Dazl−/− germ cells that persisted until birth maintained a nuclear morphology that resembled that of wildtype germ cells at E12.5–E13.5, a critical developmental period when XY germ cells lose pluripotency and commit to a spermatogonial fate. We propose that Dazl is required as early as E12.5–E13.5, shortly after its expression is first detected, and that inbred Dazl−/− mice of C57BL/6 background provide a reproducible standard for exploring Dazl's roles in embryonic germ cell development.

Spatial expression of germ cell markers during maturation of human fetal male gonads: an immunohistochemical study

The aim of the present study was to examine fetal male germ cells for expression of proteins associated with differentiation and maturation and to compare them with morphologically defined subpopulations. Testes of 61 fetuses from week 12 of gestation to the newborn period were selected. Immunohistochemistry was performed using antibodies to proteins associated with differentiation of germ cells (c-KIT, AP-2gamma) or pluripotency (OCT3/4), oncofetal protein M2A and spermatogonial marker MAGE-A4. Two subtypes of fetal germ cells were detected by quantification and immunohistochemistry. Nearly all germ cells with morphological criteria of gonocytes and intermediate cells co-expressed OCT3/4, c-KIT, M2A and AP-2gamma. Starting from week 12, their number increased up to week 18/19 and then declined continuously during further development. After week 25, pre-spermatogonia were predominant and expressed MAGE-A4 selectively. Fetal male germ cells are comprised of two major groups with distinct immunohistochemical phenotypes. Germ cells that are predominantly found before week 25 of gestation co-express oncofetal proteins OCT3/4, c-KIT, M2A and AP-2gamma. After week 25, most germ cells have lost their pluripotent potential and acquire a spermatogonial phenotype defined by expression of MAGE-A4.