Expression patterns and miRNA regulation of DNA methyltransferases in chicken primordial germ cells

DNA methylation is widespread in most species, from bacteria to mammals, and is crucial for genomic imprinting, gene expression, and embryogenesis. DNA methylation occurs via two major classes of enzymatic reactions: maintenance-type methylation catalyzed by DNA (cytosine-5-)-methyltransferase (DNMT) 1, and de novo methylation catalyzed by DNMT 3 alpha (DNMT3A) and -beta (DNMT3B). The expression pattern and regulation of DNMT genes in primordial germ cells (PGCs) and germ line cells has not been sufficiently established in birds. Therefore, we employed bioinformatics, RT-PCR, real-time PCR, and in situ hybridization analyses to examine the structural conservation and conserved expression patterns of chicken DNMT family genes. We further examined the regulation of a candidate de novo DNA methyltransferase gene, cDNMT3B by cotransfection of cDNMT3B 3'UTR- and cDNMT3B 3'UTR-specific miRNAs through a dual fluorescence reporter assay. All cDNMT family members were differentially detected during early embryonic development. Of interest, cDNMT3B expression was highly detected in early embryos and in PGCs. During germ line development and sexual maturation, cDNMT3B expression was reestablished in a female germ cell-specific manner. In the dual fluorescence reporter assay, cDNMT3B expression was significantly downregulated by four miRNAs: gga-miR-15c (25.82%), gga-miR-29b (30.01%), gga-miR-383 (30.0%), and gga-miR-222 (31.28%). Our data highlight the structural conservation and conserved expression patterns of chicken DNMTs. The miRNAs investigated in this study may induce downregulation of gene expression in chicken PGCs and germ cells.     

鸡胚胎原始生殖细胞的培养和传代

本文旨在探索鸡胚胎原始生殖细胞(Primordialgermcells,PGCs)培养、传代以及各种因素对PGCs培养的影响。实验结果表明,在添加10%的胎牛血清、2%的鸡血清、2mmol/LL谷氨酰胺、1mmol/L丙酮酸钠、5.5×10-5mol/Lβ巯基乙醇、10μl/ml非必需氨基酸、以及5ng/ml人干细胞生长因子(Humanstemcellfactor,hSCF)、10U/ml鼠白血病抑制因子(Mouseleukemiainhibitoryfactor,mLIF)、10ng/ml碱性成纤维生长因子(Fibroblastgrowthfactorbasic,bFGF)、0.04ng/ml人白细胞介素11(Humaninterleukin11,hIL11),10ng/ml胰岛素样生长因子(Humaninsulinlikegrowthfactor,hIGF)的高糖DMEM培养体系中,以多次离散法进行传代获得5-6代鸡胚胎PGCs,有效地维持了PGCs的未分化状态和正常二倍体核型,同时能够定向地诱导分化为神经细胞,具有作为多能性胚胎干细胞的特征     

影响鸡原始生殖细胞分离克隆因素的研究（简报）

具有多向分化潜能的胚胎干细胞有两种来源：一是来自于早期胚胎内细胞团的胚胎干细胞(Em．bryonic Stem Cells,ESCs),另一种是来自于胚胎生殖腺原始生殖细胞(Primordial Germ Cells,PGCs)的胚胎生殖细胞(Embryonic Germ Cells,EGCs)。     

第二十八期鸡胚原始生殖细胞的冷冻保存

采用Ficoll密度梯度离心 ,提取第 2 8期 (孵化 13 2h)性腺中的PGCs ,对其应用不同的冷冻保护剂和不同的平衡方法进行冷冻保存 ,并于复苏后进行体外培养。复苏后的PGCs用台盼蓝染色检测其存活率 ,结果发现 :从第 2 8期性腺中获取的PGCs在同一种冷冻保护液下 ,采用不同的平衡方法进行冷冻 ,对PGCs的存活率有显著影响 (P <0 .0 5 ) ;平衡方法相同 ,在不同冷冻保护液条件下 ,冷冻保护液III的冻存效果最好 ,与其他保护液之间存在显著 (P <0 .0 5 )或极显著 (P <0 .0 1)差异。冷冻保护液V和冷冻保护液VI二者对PGCs的冻存效果次之。冷冻保护液I、冷冻保护液II和冷冻保护液IV三者对PGCs的冻存效果最差。PGCs经体外培养 2 4小时后再进行冷冻保存 ,复苏后其存活率、体外培养存活时间均极 (P <0 .0 1)显著短于分离后直接冷冻的PGCs。     

Differentiation of female chicken primordial germ cells into spermatozoa in male gonads

In avian species, the developmental fate of different-sex germ cells in the gonads is unclear. The present study attempted to confirm whether genetically female germ cells can differentiate into spermatozoa in male gonads using male germline chimeric chickens produced by the transfer of primordial germ cells (PGC), and employing molecular biological methods. As a result of Southern hybridization, specific sequences of the W chromosome (the female specific sex chromosome in birds) were detected in the genomic DNA extracted from one out of four male germline chimeric chickens. When two-color in situ hybridization was conducted on the spermatozoa of this germline chimera, 0.33% (average) of the nuclei of each semen sample showed the fluorescent signal indicating the presence of the W chromosome. The present study shows that female PGC can differentiate into spermatozoa in male gonads in the chicken. However, the ratio of produced W chromosome-bearing (W-bearing) spermatozoa fell substantially below expectations. It is therefore concluded that most of the W-bearing PGC could not differentiate into spermatozoa because of restricted spermatogenesis.     

Ontogenesis of primordial germ cells

In sexually reproducing animals all gametes of either sex arise from primordial germ cells (PGC). PGC represent a small cell population, appearing early during embryo development. They represent a key cell population responsible for the survival and the evolution of a species. Indeed, the production of gametes will assure fertilisation and therefore the establishment of the next generation. Until recently only few laboratories were working on PGC biology. A new interest emerged since these cells have the ability to function as pluripotent stem cells when established as cell lines. Indeed, like embryonic stem cells (ESC), embryonic germ cells (EGC) are able to differentiate in a wide variety of tissues. In vivo, EGC are able, after injection into a host blastocyst cavity to colonise the inner cell mass and to participate in embryonic development. In vitro studies in human and mouse have also shown their capacity to differentiate into a large variety of cell types allowing the study of processes involved in cardiomyocyte, haematopoietic, neuronal and myogenic differentiation pathways. We present here the last updates of PGC ontogeny focusing mainly on the murine model.     

Activation of protein kinases A and C promoted proliferation of chicken primordial germ cells

Many growth factors or cytokines regulate cell proliferation via different intracellular signaling pathways. The mechanisms remained quite unclear in avian primordial germ cells (PGCs). In the present study, two major protein kinases, PKA and PKC, were investigated to be involved in signal transduction of PGC proliferation. PGCs were isolated from genital ridge of 3.5-day chicken embryos and primary culture was performed with 5% fetal calf serum (FCS)-supplemented medium 199. After culture for 24 h, PGCs were subcultured on chicken embryonic fibroblast feeder (CEF) and the cells were characterized by histochemical stainings of alkaline phosphatase (ALP) and periodic acid-Schiff (PAS) reagent as well as immunocytochemical stainings of c-kit and stage-specific embryonic antigen-1 (SSEA-I). In addition, cells were challenged with adenylate cyclase activator forskolin (FRSK) and PKC activator phorbol-12-myristate-13-acetate (PMA) alone or in combinations with PKA inhibitor H(89) and PKC inhibitor H(7), respectively. Results showed that subcultured PGCs on CEF displayed positive histochemical and immunocytochemical stainings for ALP, PAS, c-kit and SSEA-I and manifested intensive proliferating activity by colony formation. Downstream activation of PKA by FRSK (10(-7) to 10(-5)M) significantly promoted the proliferation of PGCs by increasing colony number (ALP-stained) in a dose-dependant manner. PMA (10(-8)M) also increased PGC colony number (P<0.05). However, the proliferating effects elicited by FRSK or PMA could be inhibited by the respective protein kinase inhibitor H(89) or H(7). Therefore, the above results suggest that activation of intracellular protein kinases A and C by external factors may promote proliferation of cultured PGCs and PKA represents the most likely mediator of PGC proliferation in embryonic chickens.     

Isolation of chicken vasa homolog gene and tracing the origin of primordial germ cells

To obtain a reliable molecular probe to trace the origin of germ cell lineages in birds, we isolated a chicken homolog (Cvh) to vasa gene (vas), which plays an essential role in germline formation in Drosophila. We demonstrate the germline-specific expression of CVH protein throughout all stages of development. Immunohistochemical analyses using specific antibody raised against CVH protein indicated that CVH protein was localized in cytoplasm of germ cells ranging from presumptive primordial germ cells (PGCs) in uterine-stage embryos to spermatids and oocytes in adult gonads. During the early cleavages, CVH protein was restrictively localized in the basal portion of the cleavage furrow. About 30 CVH-expressing cells were scattered in the central zone of the area pellucida at stage X, later 45-60 cells were found in the hypoblast layer and subsequently 200-250 positive cells were found anteriorly in the germinal crescent due to morphogenetic movement. Furthermore, in the oocytes, CVH protein was predominantly localized in granulofibrillar structures surrounding the mitochondrial cloud and spectrin protein-enriched structure, indicating that the CVH-containing cytoplasmic structure is the precursory germ plasm in the chicken. These results strongly suggest that the chicken germline is determined by maternally inherited factors in the germ plasm.     

Serum-free culture of murine primordial germ cells and embryonic germ cells

Fetal calf serum (FCS) has usually been used for culture of embryonic stem (ES) cell as a component of the culture medium. However, FCS contains undefined factors, which promote cell proliferation and occasionally stimulate differentiation of ES cells. Recently, a chemically-defined serum replacement, Knockout Serum Replacement (KSR), was developed to maintain ES cells in an undifferentiated state. In this experiment, we examined the effects of KSR on the growth and differentiation of primordial germ cells (PGCs) and embryonic germ (EG) cells. PGCs were collected 8.5 days postcoitum (dpc) from B6D2F1 (C57BL/6JxDBA/2J) female mice mated with B6D2F1 males. Most of the PGCs that were cultured in FCS-supplemented medium (FCS medium) had alkaline phosphatase (AP) activity and acquired a fibroblast cell shape. In contrast, PGCs in KSR-supplemented medium (KSR medium) proliferated, maintaining round and stem cell-like morphology. In addition, EG cells were established more easily from PGCs cultured in KSR medium than from PGCs cultured in FCS medium. The percentage of undifferentiated colonies of EG cells was significantly higher in KSR medium than in FCS medium. The germ line chimera was also produced from EG cells established in KSR medium. These results suggest that KSR can be used for sustaining an undifferentiated state of PGCs and EG cells in vitro.     

In vitro differentiation of germ cells from stem cells: a comparison between primordial germ cells and in vitro derived primordial germ cell-like cells

Stem cells are unique cell types capable to proliferate, some of them indefinitely, while maintaining the ability to differentiate into a few or any cell lineages. In 2003, a group headed by Hans R. Schöler reported that oocyte-like cells could be produced from mouse embryonic stem (ES) cells in vitro. After more than 10 years, where have these researches reached? Which are the major successes achieved and the problems still remaining to be solved? Although during the last years, many reviews have been published about these topics, in the present work, we will focus on an aspect that has been little considered so far, namely a strict comparison between the in vitro and in vivo developmental capabilities of the primordial germ cells (PGCs) isolated from the embryo and the PGC-like cells (PGC-LCs) produced in vitro from different types of stem cells in the mouse, the species in which most investigation has been carried out. Actually, the formation and differentiation of PGCs are crucial for both male and female gametogenesis, and the faithful production of PGCs in vitro represents the basis for obtaining functional germ cells.     

Expression of EGFL7 in primordial germ cells and in adult ovaries and testes

We have previously reported the isolation and characterization of a novel endothelial-restricted gene, Egfl7, that encodes a secreted protein of about 30-kDa. We and others demonstrated that Egfl7 is highly expressed by endothelial cells during embryonic development and becomes down-regulated in the adult vasculature. In the present paper, we show that during mouse embryonic development, Egfl7 is also expressed by primordial germ cells (PGC). Expression is down-regulated when PGCs differentiate into pro-spermatogonia and oogonia, and by 15.5 dpc Egfl7 can no longer be detected in the germ line of both sexes. Notably, Egfl7 is again transiently up-regulated in germ cells of the adult testis. In contrast, expression in the ovary remains limited to the vascular endothelium. Our results provide the first evidence of a non-endothelial expression of EGFL7 and suggest distinctive roles for Egfl7 in vascular development and germ cell differentiation.     

鸡胚原始生殖细胞的分离和鸡鸭嵌和体的制备

探索了鸡胚12～17期血液中的原始生殖细胞(PGCs)迁移数量变化规律,将其在液氮中冷冻保存.并以Ficoll密度梯度离心、MiniMACS磁气分离、滤膜三种方法对PGCs进行分离,结果发现12～17期血液中均有PGCs存在,13期达到高峰约47.1±10.5个/μl,在血液中比例为0.0126%.冷冻保存3个月后解冻成活率达80%以上.三种分离方法所得的分离效果分别为95.7%、39.2%、63.0%,纯度为27.5%、8.4%、3.1%.将分离的原始生殖细胞以微注射法转移至14～15期麻鸭胚胎中制备了鸡鸭种间嵌和体,获得8只雏鸭(8/110).以鸡W染色体探针原位杂交法在早期鸭胚性腺中检测到鸡原始生殖细胞,嵌和率达84.2%(16/19).表明鸡原始生殖细胞能够迁移定居到鸭胚性腺中,并有可能增殖分化成有功能的配子.     

Induction of pluripotency in primordial germ cells

Primordial germ cells (PGCs) are the founder cells of all gametes. PGCs differentiate from pluripotent epiblasts cells by mesodermal induction signals during gastrulation. Although PGCs are unipotent cells that eventually differentiate into only sperm or oocytes, they dedifferentitate to pluripotent stem cells known as embryonic germ cells (EGCs) in vitro and give rise to testicular teratomas in vivo, which indicates a "metastable" differentiation state of PGCs. We have shown that an appropriate level of phosphoinositide-3 kinase (PI3K)/Akt signaling, balanced by positive and negative regulators, ensures the establishment of the male germ lineage by preventing its dedifferentiation. Specifically, hyper-activation of the signal leads to testicular teratomas and enhances EGC derivation efficiency. In addition, PI3K/Akt signaling promotes PGC dedifferentiation via inhibition of the tumor suppressor p53, a downstream molecule of the PI3K/Akt signal. On the other hand, Akt activation during mesodermal differentiation of embryonic stem cells (ESCs) generates PGC-like pluripotent cells, a process presumably induced through equilibrium between mesodermal differentiation signals and dedifferentiation-inducing activity of Akt. The transfer of these cells to ESC culture conditions results in reversion to an ESC-like state. The interconversion between ESC and PGC-like cells helps us to understand the metastability of PGCs. The regulatory mechanisms of PGC dedifferentiation are discussed in comparison with those involved in the dedifferentiation of testicular stem cells, ESC pluripotency, and somatic nuclear reprogramming.