3H-Thymidine incorporation (DNA synthesis) and radiotoxicity in the ovary of the Japanese quail before and during follicle formation

Summary No evidence was found for ribosomal DNA amplification in the oocytes of the Japanese quail, before or during folliculogenesis. DNA synthesis in the somatic cells, involved in follicle formation, starts at the medullar side of the basement membrane. The localized sterilization of the quail ovary after administration of³H-thymidine (³H-TdR) seems to be due to radiation-induced lesions in the follicle forming somatic cells, rather than to direct radiation damage of the oocyte.The author is very grateful to Prof. Dr L. Vakaet, R.U.C.A.—Antwerpen, for his valuable suggestions and to Mrs D. De Wolf-Van Rompaey for her excellent technical assistance.     

Origin of ovarian follicle cells in birds

Summary In the embryonic Japanese quail ovary, transplanted on chicken chorioallantoic membrane (CAM), follicle cells are derived from somatic cells of the ovarian surface epithelium. No evidence was found for a contribution of other cell groups of the quail ovary in the formation of follicle cells. This may be demonstrated on PAS stained sections, by following the transfer of carbon particles, initially applied on the surface epithelium.The author is very grateful to Prof.L. Vakaet, R.U.C.A. Antwerp, for his valuables suggestions, and to MissC. van Hoecke for her technical assistance.     

La population germinale des gonades chez des embryons chimères obtenus par l'association de fragments de blastodermes de Caille japonaise et de Poulet domestique

Summary The experimental realization of chimerical embryos (Martin's ¹³ technic) permits a quantitative appreciation of the modalities of the colonization of quail gonads by chick germ cells. Results clearly show that nature and origin of the somatic part of the gonad areas settle the characters of the genital ridges populating, and specially the specific index of asymmetry expressed by the percentage of PGC colonizing the right gonad.     

DNA repair mechanisms in embryonic stem cells

Embryonic stem cells (ESCs) can undergo unlimited self-renewal and retain the pluripotency to differentiate into all cell types in the body. Therefore, as a renewable source of various functional cells in the human body, ESCs hold great promise for human cell therapy. During the rapid proliferation of ESCs in culture, DNA damage, such as DNA double-stranded breaks, will occur in ESCs. Therefore, to realize the potential of ESCs in human cell therapy, it is critical to understand the mechanisms how ESCs activate DNA damage response and DNA repair to maintain genomic stability, which is a prerequisite for their use in human therapy. In this context, it has been shown that ESCs harbor much fewer spontaneous mutations than somatic cells. Consistent with the finding that ESCs are genetically more stable than somatic cells, recent studies have indicated that ESCs can mount more robust DNA damage responses and DNA repair than somatic cells to ensure their genomic integrity.     

Reestablishment of the inactive X chromosome to the ground state through cell fusion-induced reprogramming

The restricted gene expression pattern of a differentiated cell can be reversed by fusion of the somatic cell with a more developmentally potent cell type, such as an embryonic stem (ES) cell. During this reprogramming process, somatic cells obtain most of the characteristics of pluripotent cells. Reactivation of an inactive X chromosome (Xi) is an important epigenetic marker confirming the pluripotent reprogramming of somatic cells. Female somatic cells contain one active X chromosome (Xa) and one Xi, and following the fusion of these cells with male ES cells, the Xi becomes activated, resulting in XaXaXaY fusion hybrid cells. To monitor Xi reactivation, transgenic female neural stem cells (fNSCs) carrying a green fluorescent protein (GFP) reporter gene expressed on the Xa (X-GFP), but not on the Xi, were used for reprogramming. XaXi^GFP NSCs, whose GFP reporter was silenced, were fused with HM1 ES cells (XY) to induce pluripotent reprogramming. The Xi^GFP of NSCs were found to be activated on day 4 post-fusion, indicating reactivation of the Xi. Hybrid cells showed pluripotent cell-specific characteristics cells including inactivation of the NSC marker Nestin, DNA demethylation of Oct4, DNA methylation of Nestin, and reactivation of the Xi. Following differentiation of the (GFP-positive) hybrid cells through embryoid body formation, the proportion of GFP-negative cells was found to be approximately 26?%, indicating that there was random inactivation of one of the three Xas. Here, we showed that the Xi of somatic cells is reprogrammed to the Xa state and that cellular differentiation occurs randomly, i.e., regardless of the Xa or Xi state, indicating that the memory of the Xi of somatic cells has been erased and reset to the ground state (i.e., inner cell mass-like state), indicating that random X-chromosome inactivation occurs upon differentiation.     

Apoptosis occurs in granulosa cells but not cumulus cells in the atretic antral follicles in pig ovaries

The porcine antral follicles, 3–6 mm in diameter, were dissected from the ovaries of mature pigs, and then granulosa and cumulus cells were isolated from each follicle. In atretic follicles, high activity of neutral Ca²⁺/Mg²⁺-dependent endonuclease and DNA ladder formation, estimated by electrophoresis, were noted in granulosa cells but not in cumulus cells. Extremely low activity of the endonuclease and no DNA ladder formation were observed in both types of cells obtained from healthy follicles. Moreover, apoptotic cells were observed histochemically among granulosa cells only. A good correlation (r=0.987) between the endonuclease activity of granulosa cells and the progesterone/estradiol ratio of follicular fluid in each follicle was found. These results suggest that apoptosis occurs in granulosa cells but not cumulus cells in the atretic antral follicles in pigs.     

Production of transgenic birds

The avian embryo presents a tremendous challenge for those interested in accessing and manipulating the avian germ line. By far the most successful method of gene transfer is by retrovirus vector. The efficacy of retrovirus vectors has been demonstrated by germ line insertion of replication-competent retroviruses as well as the insertion of replication-defective retrovirus vectors carrying bacterial marker genes. Retroviral vectors have also been shown to be useful for the transfer and expression of genes in somatic cells. Further, germ line transgenesis has been reported in both the chicken and the Japanese quail. In addition, several alternative gene transfer methods are under development. These include transfection of avian sperm, development of germ line chimeras using primordial germ cells and blastodermal cells, and the development of embryonic stem cell lines. Potentially, basic research and the poultry industry will derive substantial benefit from this revolutionary technology.     

Production of transgenic birds

The avian embryo presents a tremendous challenge for those interested in accessing and manipulating the avian germ line. By far the most successful method of gene transfer is by retrovirus vector. The efficacy of retrovirus vectors has been demonstrated by germ line insertion of replication-competent retroviruses as well as the insertion of replication-defective retrovirus vectors carrying bacterial marker genes. Retroviral vectors have also been shown to be useful for the transfer and expression of genes in somatic cells. Further, germ line transgenesis has been reported in both the chicken and the Japanese quail. In addition, several alternative gene transfer methods are under development. These include transfection of avian sperm, development of germ line chimeras using primordial germ cells and blastodermal cells, and the development of embryonic stem cell lines. Potentially, basic research and the poultry industry will derive substantial benefit from this revolutionary technology.     

Development and use of shell-less quail chorio-allantoic-membrane cultures to study developing skeletal tissues; a qualitative study

Summary A technique is described for in vitro culture of the quail embryo from the 1st to the 18th day of development. The embryos are cultured in Teflon hammocks, suspended in glass supports and kept in a humidified atmosphere at 36.5°C. The quail CAM is used as support and cell source for developing non-quail cartilage and bone. The quail cells can be identified histologically and easily recognized by Feulgen-staining which is demonstrated in the presence of quail chondro- or osteoclasts in a mouse long bone rudiment cultured on the CAM.     

Development and use of shell-less quail chorio-allantoic-membrane cultures to study developing skeletal tissues; a qualitative study

A technique is described for in vitro culture of the quail embryo from the 1st to the 18th day of development. The embryos are cultured in Teflon hammocks, suspended in glass supports and kept in a humidified atmosphere at 36.5 degrees C. The quail CAM is used as support and cell source for developing non-quail cartilage and bone. The quail cells can be identified histologically and easily recognized by Feulgen-staining which is demonstrated in the presence of quail chondro- or osteoclasts in a mouse long bone rudiment cultured on the CAM.     

La synthèse de l'ADN prémitotique et préméiotique dans les cellules germinales de l'embryon femelle de la caille japonaise (Coturnix coturnix japonica)

Summary The premeiotic DNA synthesis of the female Japanese quail embryo begins after 9 days incubation. The germ cells in premeiotic S phase present a particular structure different from the structure found in germ cells during premitotic S phase. They are the precursors of the meiocytes in leptotene stage.

---

---

L'auteur remercie vivement le ProfesseurL. Vakaet (R.U.C.A., Anvers) pour ses suggestions très valables.     

Invasive properties of the ovarian cortex in birds

Summary In quail/chicken ovarian cortex associations, grown on chicken chorioallantoic membrane (CAM), epithelial and/or germ cells from the quail ovary may invade the cortical rim of the chicken ovary. In certain experimental conditions, ovarian cortical cells may leave a transplanted ovary and grow over or invade the mesenchyme of the surrounding CAM. So a germinal epithelium completely free from other ovarian cell groups can be obtained.The author is very grateful to Prof. Dr L. Vakaet, R.U.C.A., Antwerp, for his valuable suggestions and to Mrs S. De Wolf-Van Rompaey, for her excellent technical assistance.     

AID and Apobec3G haphazard deamination and mutational diversity

Activation-induced deoxycytidine deaminase (AID) and Apobec 3G (Apo3G) cause mutational diversity by initiating mutations on regions of single-stranded (ss) DNA. Expressed in B cells, AID deaminates C → U in actively transcribed immunoglobulin (Ig) variable and switch regions to initiate the somatic hypermutation (SHM) and class switch recombination (CSR) that are essential for antibody diversity. Apo3G expressed in T cells catalyzes C deaminations on reverse transcribed cDNA causing HIV-1 retroviral inactivation. When operating properly, AID- and Apo3G-initiated mutations boost human fitness. Yet, both enzymes are potentially powerful somatic cell “mutators”. Loss of regulated expression and proper genome targeting can cause human cancer. Here, we review well-established biological roles of AID and Apo3G. We provide a synopsis of AID partnering proteins during SHM and CSR, and describe how an Apo2 crystal structure provides “surrogate” insight for AID and Apo3G biochemical behavior. However, large gaps remain in our understanding of how dC deaminases search ssDNA to identify trinucleotide motifs to deaminate. We discuss two recent methods to analyze ssDNA scanning and deamination. Apo3G scanning and deamination is visualized in real-time using single-molecule FRET, and AID deamination efficiencies are determined with a random walk analysis. AID and Apo3G encounter many candidate deamination sites while scanning ssDNA. Generating mutational diversity is a principal aim of AID and an important ancillary property of Apo3G. Success seems likely to involve hit and miss deamination motif targeting, biased strongly toward miss.     

Genetics of early mammalian folliculogenesis

Early ovarian folliculogenesis begins with the breakdown of germ cell clusters and formation of primordial follicles. Primordial follicles are the smallest ovarian follicle units continuously recruited to grow into primary and more advanced ovarian follicles. Genes expressed in the germ cells such as Figla, Nobox, Kit and Ntrk2, as well as genes expressed in the surrounding somatic cells such as Foxl2, Kitl and Ngf, play critical functions during early folliculogenesis. Transgenic mice continue to provide important insights into the genetic pathways that regulate early mammalian folliculogenesis. Genes critical in early folliculogenesis are important determinants of reproductive life span and represent candidate genes for human ovarian failure. Received 25 August 2005; received after revision 18 October 2005; accepted 21 November 2005     

Immunocytochemical demonstration of contractile cells in the human ovarian follicle

Actin- and myosin-like immunoreactivity is found in cells located in the theca externa of the follicle wall of the human ovary, and corresponding to previously observed myoid cells. The immunocytochemical observation provides direct structural evidence that non-vascular contractile cells are also present in the follicle wall in humans. As expected, perifollicular blood vessels showed a positive immunoreaction for actin and myosin in their smooth muscle walls.     

Localization of amplified DNA in nuclei of the orchid Cymbidium by in situ hybridization

Summary In situ hybridization of³H-polyuridylic acid of low specific activity to somatic nuclei of Cymbidium protocorms is suggested to indicate the location of the highly amplified AT-rich DNA fraction.     

Étude comparée de la multiplication des cellules germinales à la fin de la première semaine de la vie embryonnaire chez trois espèces de Gallinacés (Gallus domesticus,Meleagris gallopavo,Coturnix coturnix japonica)

Summary Counts were made of the numbers of germ cells in quail embryos aged 4–7 days' incubation. The rate of growth in the population of gonocytes was compared to these of chick and turkey embryos during the same period. The increase is exponential in the three species and the rates are more similar between quail and turkey than between each of them and the chick.     

Initiation of genetic instability and tumour formation: a review and hypothesis of a nongenotoxic mechanism

Genetic instability in tumours results in cell-to-cell variability of genome which parallels the cell-to-cell variability of microscopic morphology and of behaviour (tumour cell heterogeneity) of these lesions. Genetic instability is therefore strongly supported as the fundamental process by which normal tissue cells become neoplastic. The commonest current suggestion for the mechanism of initiation of carcinogenesis is a 'direct hit' mutation of a 'cancer critical' gene in a somatic cell by carcinogenic agents. However, this mechanism does not account for the activity of carcinogens which are not mutagens, and does not explain why many mutagens are not carcinogens. This paper proposes a nonmutational (nongenotoxic) mechanism of initiation of genetic instability in previously normal cells as follows: 1) During S phase of local tissue stem cells, carcinogen binds to and disables the proofreading enzyme for a new DNA strand. 2) While it is disabled, the proofreading enzyme fails to correct illicit changes in the nucleotide sequence(s) for one or more genes for proofreading fidelity or repair of DNA in the new strand of DNA, which passes to one daughter cell. 3) When this daughter cell is a continuing stem cell, the resulting cell line remains immortal, and retains its prior differentiation commitment to produce daughter cells of a particular type. However, the acquired genetic instability in this cell line causes secondary mutations which lead to uncontrolled growth, and the heterogeneous morphologic and behavioural features of a tumour resembling the parent cell type.     

Differentiating abilities of avian somatopleural mesoderm

Summary Quail-to-chick grafting experiments were performed on 2-day embryos in order to test the differentiating abilities of the somatopleure. After orthotopic and heterotopic transplantations of different parts of quail somatopleural mesoderm into chick embryos it is demonstrated that avian somatopleural cells differentiate into skeletal elements, smooth muscles, tendons and connective tissues. However, skeletal muscle fibres do not originate from somatopleural cells.Supported by a grant from the Deutsche Forschungsgemeinschaft (Ch 44/3).     

Role of the infundibular complex and its neural afferents in the photosexual reflex in the quail]

The photosexual reflex was suppressed in quail after bilateral preoptic lesions. However an anterior-lateral de-afferentation of the hypothalamus of previously preoptic-lesioned quail resulted in partial photo-induced testis growth. As a working hypothesis, it is suggested that the preoptic area may control some unknown extrahypothalamic influences in order to modulate the gonadotropin controlling function of the infundibular area.