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The Effects of Dexamethasone on the Differentiation and the Fertilisation of the Germinal Primordium in the Chick Embryo Danièle Cuminge, Julian Smith, Régis Dubois

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The Effects of Dexamethasone on the Differentiation and the Fertilisation of the Germinal Primordium in the Chick Embryo Danièle Cuminge, Julian Smith, Régis Dubois The effects of dexamethasone on the differentiation and the fertilisation of the germinal primordium in the chick embryo Danièle Cuminge, Julian Smith, Régis Dubois To cite this version: Danièle Cuminge, Julian Smith, Régis Dubois. The effects of dexamethasone on the differentiation and the fertilisation of the germinal primordium in the chick embryo. Reproduction Nutrition Devel- opment, EDP Sciences, 2000, 40 (2), pp.127-148. 10.1051/rnd:2000125. hal-00900392 HAL Id: hal-00900392 https://hal.archives-ouvertes.fr/hal-00900392 Submitted on 1 Jan 2000 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Reprod. Nutr. Dev. 40 (2000) 127–148 127 © INRA, EDP Sciences Original article The effects of dexamethasone on the differentiation and the fertilisation of the germinal primordium in the chick embryo Danièle CUMINGEa, Julian SMITHb, Régis DUBOISa* a Institut d’Embryologie Cellulaire et Moléculaire, 49 bis avenue de la Belle Gabrielle, Collège de France et CNRS, 94736 Nogent-sur-Marne Cedex, France b Centre de Biologie du Développement, Université Paul Sabatier, 31062 Toulouse, France (Received 15 December 1999; accepted 24 February 2000) Abstract — We showed that, in the chick embryo, the fertilisation of the attractive germinal epithe- lium by primary germ cells can be represented by a three-dimensional diagram in which the space and time co-ordinates are graduated in terms of the segmentation of the axial and paraxial mesoderm. We thus established that the differentiation of the coelomic epithelium into an attractive germinal epithe- lium and the fertilisation of the gonadal primordium both occur by mechanisms that are tightly linked to somitogenesis. In the continuous presence of a constant concentration of Dexamethasone, a marked inhibition of the rate of fertilisation of the gonadal primordium was observed. A mathematical analysis of the mode of action of the inhibitor revealed the progressive establishment of a competi- tion between Dexamethasone and the molecule(s) responsible for the process of attraction. Given the chemical nature of the inhibitor, we propose that the endogenous factor that triggers the first step of the differentiation of the germinal primordium is a steroid-containing complex. avian germ cells / chemotactism / differentiation / fertilisation / attractive field / steroid / dexamethasone Résumé — Les effets de la dexaméthasone sur la différenciation et la fertilisation de l’ébauche gonadique chez l’embryon de poulet. La fertilisation du primordium gonadique par les cellules germinales primordiales (CGP) en transit dans la circulation embryonnaire est représentée par un dia- gramme à 3 dimensions dans lequel l’espace et le temps sont inscrits sur une graduation obtenue par la projection de la segmentation du mésoderme axial et paraxial. C’est la capture des CGP par chi- miotactisme qui révèle l’existence et la localisation d’un champ attractif. Les gonocytes fixés dans l’ébauche gonadique sont les marqueurs de l’extension de celle-ci dans le sens céphalo-caudal. Il a ainsi été démontré que la différenciation de l’épithélium cœlomique en épithélium germinatif attrac- tif (E.G) est étroitement couplée à la somitogenèse dans la région bornée par les somites 22 à 30. Ces observations posent le problème de savoir si la différenciation de l’E.G est progressivement déclenchée par les somites nouvellement formés, ou si elle est due à une propriété intrinsèque en * Correspondence and reprints 128 D. Cuminge et al. corrélation avec le stade de développement de l’embryon. La discussion conclut en faveur de cette seconde hypothèse qui suggère le couplage de mécanismes génétiques homéotiques. En présence continue et à concentration constante de Dexaméthasone, une nette inhibition de la fertilisation de l’épi- thélium germinatif est observée. Le taux d’inhibition est proportionnel au logarithme de la concen- tration en corticoïde dans la gamme [10–9 M–10–5 M]. L’analyse mathématique du mode d’action de l’inhibiteur révèle l’établissement progressif d’une compétition entre la DEXA et le facteur chi- mique naturel qui déclenche le processus de capture des CGP dans les conditions normales du déve- loppement. Le mode d’action de la DEXA est discuté dans le cadre des acquisitions récentes sur la description des récepteurs des stéroïdes. La nature chimique de l’inhibiteur suggère que le facteur endo- gène qui active l’apparition des forces attractives du chimiotactisme est un complexe stéroïdien. Selon ces conceptions, c’est la mise en œuvre d’une machinerie moléculaire d’une extrême complexité, intégrant notamment un stéroïde et son récepteur cytosolique, qui déclenche la différenciation d’une région particulière de l’épithélium cœlomique en épithélium germinatif attractif. cellules germinales des oiseaux / chimiotactisme / différenciation / fertilisation / champ attractif / stéroïde / dexaméthasone 1. INTRODUCTION notions of “attractive forces” and “attrac- tive field” are extensively used in the text. A previous study [7] has shown that the overall kinetics of fertilisation of the ger- 2. MATERIALS AND METHODS minal epithelia (GE) of the chick embryo by primordial germ cells (PGC) conforms The development of the chick embryo is to the theoretical predictions of the logistic classically described in terms of a “biolog- law (P.F. Verhulst, 1838; R. Pearl, 1924; ical” progression in which morphological see [21, 22]). In earlier work, the colonisa- aspects play a key role. This option for a tion of the gonadal primordium was time axis (t) graduated by a succession of described in terms of a simple function of somitic stages (s) is explained by the fact time. The present study completes previous that fertilised eggs are laid at different stages preliminary investigations by considering and attain given degrees of development an additional parameter, space {Sp}. Explic- after periods of incubation that can be very itly, we studied the variation of the num- variable. In the present work, the somitic bers of gonocytes (Go) that become fixed stage was used as a co-ordinate either of at different levels of the gonadal primordium time (t) according to Hamburger and Hamil- during development. The evolution of fer- ton [14] or of space {Sp} along the antero- tilisation of the GE by PGC transiting in the posterior axis. We designate the time co- embryonic circulation is represented in a ordinate by (s) and the space co-ordinate by three-dimensional grill in which the num- {s}. ber of gonocytes captured (N) is a point function of the form: N = f [(t), {Sp}]. Observations under normal conditions were made on Hubbard chick embryos cul- In the second part of this study, we devel- tured on a gelose-containing medium [32]. oped the analysis and interpreted the exper- Blastoderms, explanted at the 20-somite imental results obtained using Dexametha- stage, were cultured at random for differ- sone (DEXA) in this system. For reasons ent times up to the 28-somite stage. Beyond that are presented in detail in our earlier this point the fertilisation process is dis- work [8], our observations are described turbed by the proliferation of the gonocytes within the framework of the chemotactic installed in the germinal epithelia. At the theory of migration of chick PGC: the end of its imparted culture period, each Dexamethasone and attractive epithelium differentiation 129 blastoderm was fixed in toto in Halmi solu- blastoderms were removed and fixed in tion. The gonadic regions were dissected at Halmi at a time (t) within the interval [(24s) the level of the 19th pair of somites and to (28s)]. For a concentration C = 10–5 M, reoriented as accurately as possible during the interval extended from stage (26s) to their subsequent inclusion in paraffin, with stage (28s). Histological preparations were the aim of facilitating the identification of prepared and analysed as described for the homologous pairs of somites on cross sec- control embryos. tions. Whenever the homologous somites were not rigorously parallel to the plane of the section, the observer made corrections, 3. RESULTS which have a potential resolving power equal to the thickness of a section, that is 3.1. Properties of the control 6.6 µm. Gonocyte counts in the GE were population: three-dimensional analysis carried out on sections stained with gly- of the fertilisation of the germinal chaemalun-eosin. epithelium The spatial co-ordinate {Sp} of the The results were obtained from a series of gonadal primordium was defined as follows: 191 blastoderms selected from a population the segmentation of the paraxial mesoderm of 271 explants. Embryos exhibiting abnor- into somites is projected on the coelomic mal development or cardiovascular defects epithelium. One thus obtains an imaginary, (haemorrhage, venous stasis or ischaemic anatomically continuous subdivision of the zones) were excluded from the statistics (it gonadal primordium into a series of {s} and has been shown that haemodynamic plays an {i-s} regions that follow each other in phase important role as an external cause of with the sequence of somitic and inter- chemotactism in the fertilisation of the somitic spaces of the segmented paraxial gonadal primordium [8, 16]. mesoderm. This virtual regionalisation of the GE allowed an interesting sectorial anal- 3.1.1. Colonisation of the GE ysis to be made of the evolution of the as a function of time(s) colonisation processes, as will be apparent later (see Results). Finally, the numbers of The results are displayed in Table I and histological sections of the total gonadal are expressed graphically as a function of region, as well as of the {s} and {i-s} stage (time) by curve 1 in Figure 1 (d).
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