Regulation of vascular development by fibroblast growth factors

Fibroblast growth factors (FGFs) are potent stimulators of angiogenesis in vitro and in vivo. However, the precise role of FGFs and vascular development in normal and pathological tissue has long remained ill defined. Recently, substantial progress has been made toward a better understanding of their role. Genetic studies in mice or in culture systems indicate a role for FGFs in vessel assembly and sprouting. FGFs also stimulate blood vessel branching and lymphangiogenesis. The molecular mechanisms by which FGFs mediate angiogenesis are also better understood. Finally, the FGF/FGF-receptor system has become a focus for the development of novel therapeutic strategies for the treatment of angiogenesis-related diseases such as tissue ischemia.Work described herein from our laboratory was supported by grants from the Ligue Nationale contre le Cancer, the Association de la Recherche sur le Cancer, Rétina France, the Institut National de la Santé et de la Recherche Médicale (INSERM), and the Ministère de la Recherche     

A new method for toxicity assays on human and mouse fetal testis

Exposure to environmental pollutants (EP) is associated with a wide range of toxic effects, in particular in testis development. Uranium is a potential pollutant of nuclear industry and over the last few years, its environmental concentrations have increased. In animals, the current procedures for evaluating the potential developmental toxicity of uranium are based on in vivo studies. These methods do not allow to know the direct effects on testicular cells and are obviously excluded for human experiments. Consequently, we have developed an in vitro culture system of the whole testis. In the present study we characterized and validated this organ culture system in both mouse fetal testes and human fetal testes recovered during the first trimester (6-12 weeks) of gestation. We compared the histological aspect, the number of germ cells and the testosterone production, before and after culture. Testicular architecture and intercellular communications were preserved, and organ culture appears as a powerful method for studying the early development of testicular gametogenesis and steroidogenesis in both species. Thus by using this method we will be able to investigate the effects of uranium on mouse and human developing testis. The mouse model will allow us to determine the dose range of interest without restriction of material.     

Influence of adenosine 3':5'-cyclic monophosphate analogues on testicular organization of fetal mouse gonads in vitro

Gonadal primordia, isolated from fetal mice on the 11th or 12th day of gestation, differentiated in vitro into morphologically distinct testes or ovaries after 7 days in culture. The addition of cAMP analogues into culture media prevented the differentiation of testis cords. Histological examination indicated that the basement membranes of testis cords disintegrated after treatment with cAMP analogues, while development of germ cells and Leydig cells appeared to be unaffected. Fetal testes in culture secreted testosterone which increased following addition of dibutyryl-cAMP (Bt2 c-AMP). Primordial germ cells reached prespermatogonial stage in the presence or absence of Bt2 cAMP, suggesting that progressive differentiation of primordial germ cells is independent of testis cord organization. The Bt2 cAMP-treated explants resumed testicular development after transplantation into a site beneath the kidney capsules of adult mice, although the inhibitory effect appeared irreversible in vitro. The testicular organization-preventing effect of cAMP analogues was mimicked by prostaglandins or forskolin, which are known to stimulate adenylate cyclase. The inhibitory effect of either cAMP analogues or prostaglandins was potentiated when added in combination with phosphodiesterase inhibitors. The present results suggest that increase of intracellular cAMP prevents the development of basement membrane and the assembly of cells to form testicular structures.     

Zytophotometrische Bestimmung des Histon- und Gesamtproteingehalts von Zellen des lymphatischen Gewebes

Résumé L'étude au microscope électronique a révélé la présence de particules B dans le thymus de la souris NZB, adulte et saine. Les particules se forment dans un type de cellule thymique bien défini, la cellule réticulo-épithéliale à vacuoles et naissent par bourgeonnement des membranes plasmatiques limitant les vacuoles. L'élaboration de particules B dans lethymus d'animaux apparemment sains est un fait nouveau dont la signification n'est pas encore connue.

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Summary An electron microscopical study revealed the presence of virus-like particles in the thymus of adult healthy NZB-mice. The particles are found in the vacuoles of reticuloepithelial cells and are elaborated by a budding process from the membranes surrounding the vacuoles. The formation of B-particles in the thymus of apparently healthy animals is a so far unknown fact the significance of which remains to be determined.

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Avec l'aide du Centre National de la Recherche Scientifique et de l'Institut National de la Santé et de la Recherche Médicale, et la collaboration technique dévouée de M^lle J. Patry et de Mr. B. Fontaine.     

The role of p63 in germ cell apoptosis in the developing testis

The fetal and neonatal development of male germ cells (gonocytes) is a poorly understood but crucial process for establishing fertility. In rodents, gonocytes go through two phases of proliferation accompanied by apoptosis and separated by a quiescent period during the end of fetal development. P63 is a member of the P53 gene family that yields six isoforms. We detected only the p63 protein and no p53 and p73 in the nucleus of the gonocytes of mouse testes. We report for the first time the ontogeny of each p63 mRNA isoform during testis development. We observed a strong expression of p63gamma mRNA and protein when gonocytes are in the quiescent period. In vitro treatment with retinoic acid prevented gonocytes from entering the quiescent period and was correlated with a reduced production of p63gamma isoform mRNA. We investigated the function of p63 by studying the testicular phenotype of P63-null mice. P63 invalidation slightly, but significantly increased the number of gonocytes counted during the quiescent period. As P63-null animals die at birth we used an original organ culture that mimicked neonatal in vivo development to study further the testicular development. P63 invalidation resulted in a sharply increased number of gonocytes during the culture period due to a decrease in spontaneous apoptosis with no change in proliferation. P63 invalidation also caused abnormal morphologies in the germ cells that were also found in P63(+/-) adult male mice. Thus, p63 appears as an important regulator of germ cell development.     

The matricellular protein SPARC is internalized in Sertoli, Leydig, and germ cells during testis differentiation

The gene encoding the matricellular protein secreted protein, acidic and rich in cysteine (SPARC) was identified in a screen for genes expressed sex-specifically during mouse gonad development, as being strongly upregulated in the male gonad from very early in testis development. We present here a detailed analysis of SPARC gene and protein expression during testis development, from 11.5 to 15.5 days post coitum (dpc). Section in situ hybridization analysis revealed that SPARC mRNA is expressed by the Sertoli cells in the testis cords and the fetal Leydig cells, found within the interstitial space between the testis cords. Immunodetection with anti-SPARC antibody showed that the protein was located inside the testis cords, within the cytoplasm of Sertoli and germ cells. In the interstitium, SPARC was present intracellularly within the Leydig cells. The internalization of SPARC in Sertoli, Leydig, and germ cells suggests that it plays an intracellular regulatory role in these cell types during fetal testis development.     

Ultrastructure des cellules de Leydig et des cellules de Sertoli au cours du cycle testiculaire du Canard Pékin

Résumé L'ultrastructure des cellules de Leydig et des cellules de Sertoli du testicule du Canard Pékin a été étudiée au cours de la phase printanière du cycle sexuel, soit de janvier à juillet. Parallèlement on a effectué chez les mêemes animaux la recherche histochimique de la ⁵-3 -hydroxystéroïdedeshydrogénase (⁵-3 -HSDH) ainsi que le dosage, par chromatographie en phase gazeuse des stéroïdes androgènes dans le plasma veineux périphérique et dans le testicule.Les cellules de Leydig du Canard possèdent les organites cytoplasmiques spécifiques des cellules stéroïdogènes (reticulum lisse, mitochondries à crêtes tubulaires) ainsi que d'autres structures souvent rencontrées dans ce type cellulaire (microfilaments, vacuoles, granules denses). Les cellules de Sertoli contiennent un reticulum agranulaire moins développé que celui des cellules de Leydig et, très rarement, des mitochondries à crêtes tubulaires. Ces divers organites cytoplasmiques subissent un cycle saisonnier. La différenciation du reticulum lisse et des crêtes mitochondriales tubulaires commence en janvier et atteint son optimum en mars. Leur régression s'amorce en avril; d'abord accompagnée de structures dégénératives transitoires; elle conduit à la dispartion totale de ces organites en mait. Aucun indice de nécrose n'est observé dans ces cellules. Histochimiquement, une activité ⁵-3 -HSDH est présente dans les cellules de Leydig et, à un degré moindre, dans les tubes séminifères. Son intensité varie au cours du cycle.La confrontation de l'étude morphologique avec les résultats des dosages hormonaux montre qu'il existe une bonne corrélation entre le développement puis la régression du reticulum lisse et des crêtes tubulaires des mitochondries ainsi que des critères histochimiques de la ⁵-3 -HSDH d'une part et l'évolution de la testostérone plasmatique et testiculaire d'autre part. De plus on observe une augmentation du rapport testostérone/⁴-androstènedione testiculaire parallèlement au développement des organites cytoplasmiques. Ces organites semblent donc bien impliqués dans la synthèse et la sécrétion de la testostérone chez le Canard.

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Ultrastructure of Leydig and Sertoli cells in the testicular cycle of the Pekin duckBiochemical and cytoenzymological correlations

Summary Leydig and Sertoli cells of the testis of the Pekin duck were studied ultrastructurally during the spring phase of the sexual cycle, from January to July. Simultaneously, in the same animals, ⁵-3 -hydroxysteroiddehydrogenase (⁵-3 -HSDH) activity was ascertained histochemically and androgenic steroids of the plasma and testes were assayed by gas-liquid chromatography.The Leydig cells of the duck possess cytoplasmic organelles specific to steroidogenic cells (smooth reticulum, tubular mitochondria) as well as other structures often found in this cell type (microfilaments, vacuoles, denses bodies). The Sertoli cells contain an agranular reticulum that is less developed than that of the Leydig cells, and rarely show mitochondria with tubular cristae. These various cytoplasmic organelles undergo a seasonal cycle. The differentiation of the smooth reticulum and the mitochondrial tubular cristae begins in January and reaches a maximum in March. They begin to regress in April, at first with transitory degenerative structures, and then by total disappearance of these organelles by May. No indication of necrosis is observed in the cells. Histochemically ⁵-3 -HSDH activity is present in the Leydig cells, and to a slightly lesser degree in the seminiferous tubules. The intensity varies during the cycle.The comparison of the results of the morphological study with the hormone assays shows that a good correlation exists with the development and regression of the smooth endoplasmic reticulum and tubular cristae in the mitochondria, as well as the histochemical criteria of the ⁵-3 -HSDH on one hand, and the levels of plasma and testicular testosterone on the other hand. In addition there is an increase in the ratio of testicular testosterone to ⁴-androstenedione which parallels the development of the cytoplasmic organelles. These organelles thus seem to be implicated in the synthesis and secretion of testosterone in the duck.

Nous tenons à remercier très vivement Mme G. Collenot qui nous a initiées aux techniques d'histoenzymologie et nous a très gentiment permis de faire cette partie de nos recherches dans son laboratoire. Nous remerçions également M. Claude Pennarun, photographe, pour son excellente collaboration.     

Development of Fetal Testicular Cells in Androgen Receptor Deficient Mice

During mammalian development, androgens produced by the fetal testis are the most important hormones controlling the masculinization of the reproductive tract and the genitalia. New findings show that the male germ line is the most sensitive to anti-androgenic endocrine disruptors during the embryonic period. In a recent study, we reported that endogenous androgens physiologically control germ cell growth in the male mouse fetus during early fetal life. In the present study, we extended this result by showing the presence of a functional androgen receptor in the gonocytes in the latter part of the fetal life. We also studied the effect of androgens on the development of the somatic testicular cells using the Tfm mice which carry a naturally inactivating mutation of the androgen receptor. Fetal Leydig cell are largely independent of endogenous androgens during fetal development whereas fetal Sertoli cell number is decreased following a default of peritubular myoid cells differenciation. They also point to the gonocyte as a special target for androgens during the embryonic period and indicate a novel mechanism of androgen action on gonocytes. Elucidation of this new pathway in the fetal testis will clarify not only fetal testis physiology but also the effects of environmental anti-androgens that act during fetal life and open new perspectives for future investigations into the sensitivity of fetal germ cell to androgens.     

Phosphofructokinase (PFK) isozymes in man

Summary Isozymic heterogeneity of human phosphofructokinase was investigated by means of ATP inhibition, immunoneutralization by antihuman muscle-type and antiliver-type phosphofructokinase antisera, solubility in (NH₄)₂SO₄ solutions, and starch gel and polyacrylamide slab gel electrophoresis. The enzymes studied by these methods were purified from various normal and malignant human adult tissues by chromatography on blue Dextran Sepharose 4 B columns. From the results of these studies we suggest that three basic phosphofructokinase isozymes could exist: muscle-type, fibroblast-type, and liver-type isozymes.Muscle-type isozyme is the single form found in adult muscle, and is involved in the enzymes from heart, brain, red cell, and testis.Fibroblast-type isozyme is found mainly in the placenta, fibroblasts, kidney, and some malignant tissues.Liver-type phosphofructokinase seems to be very definitely the predominant form in mature polymorphonuclear cells, platelets, and liver.Testis and red cell phosphofructokinase enzymes definitely include muscle-type and liver-type subunits, associated in various hybrid forms.With the technical assistance of R. KernempUnité 129 de l'Institut National de la Santé et de la Recherche Médicale, laboratoire associé 85 au Centre National de la Recherche Scientifique     

Individualisation au microscope optique des grains de proteine secretes par la glande mammaire

Resumé Les grains de protéine sécrétés par la glande mammaire, révélés par le microscope électronique, peuvent s'individualiser au microscope optique à condition d'employer une technique assez fine.

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Summary The protein granules secreted by the mammary gland and first revealed by electron microscopy can be seen in the light microscope, if appropriate techniques are emploied.

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Avec l'aide du Centre National de la Recherche Scientifique et de l'Institut National de la Santé et de la Recherche Médicale.     

Différenciation ultrastructurale des jonctions intercellulaires de l'endothélium des capillaires télencéphaliques chez l'embryon de poulet

Résumé Au cours du développement embryonnaire du Poulet, les cellules endothéliales des bourgeons vasculaires et des capillaires néo-formés du télencéphale sont unies par des jonctions transitoires au niveau desquelles les feuillets externes des membranes plasmiques n'ont pas fusionné et sont séparés par un espace intercellulaire mesurable. Des points de fusion de ces feuillets externes apparaissent peu à peu et au hasard dans la zone du contact intercellulaire. Entre ces points persistent des espaces intercellulaires qui assurent encore la communication entre la lumière du capillaire et l'espace extracellulaire du tissu nerveux. L'adjonction de nouveaux points de fusion, entre ceux existant déjà, conduit à la disparition progressive de l'espace intercellulaire et à la formation de véritables zonulae occludentes.

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Ultrastructural differentiation of intercellular junctions in the endothelium of capillaries in the embryonic telencephalon of the chicken

Summary During the embryonic development of the chick, the endothelial cells of the vascular sprouts and of the newly-formed capillaries in the telencephalon are linked by transitory junctions at the level of which the external leaflets of the plasma membrane are not fused and are separated by a measurable intercellular space. Some points of fusion of these external leaflets gradually appear at random in the area of the intercellular contact. Between these points of fusion, some intercellular spaces persist which still allow the communication between the capillary lumen and the extracellular space in the nervous tissue. The adjunction of new points of fusion between the preexisting ones, leads progressively to the disappearance of these intercellular spaces and to the formation of true zonulae occludentes.

L'auteur remercie les Professeurs J. Gayet et G. Grignon de leurs conseils lors de la préparation du manuscrit. Ces recherches ont bénéficié de subventions du Centre National de la Recherche Scientifique (E.R.A. n° 331) et de la Fondation pour la Recherche Médicale Française.     

Separation of germ cells from somatic cells in mouse testis by affinity for a lectin, peanut agglutinin

A new method for the separation of germ cells from somatic cells in the mouse testis was accomplished by making use of the differences in cell surface affinity for a lectin, peanut agglutinin (PNA). The separation procedure was based on the specific presence of PNA receptor on testicular germ cells and its absence on somatic cells, such as Leydig, Sertoli and peritubular cells. As a result, more than 99% of cells in PNA receptor-positive (PNA+) fractions were identified as germ cells by immunoperoxidase reaction with specific antiserum to mouse testicular germ cells. In contrast, Leydig cells were enriched in PNA receptor-negative (PNA-) fractions, i.e., 65% of cells in these fractions were cytochemically stained for 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) activity.     

Erythroid precursors cultured from adult mice are sensitive to H2O2 toxicity

Summary The growth of late erythroid precursors (CFU-Es) from adult bone marrow is inhibited when Iscove's modified Dulbecco's medium supplied in liquid form is used. Catalase and other H₂O₂ destroying compounds restore the capacity of culture medium to support colony development. However early precursors from adult bone marrow and fetal liver CFU-Es were resistant to H₂O₂. This work was supported by grants from the Centre National de la Recherche Scientifique, the Institut National de la Santé et de la Recherche Médicale and the Fédération Nationale des Centres de Lutte contre le Cancer.     

Germ cell deficiency causes testis cord differentiation in reconstituted mouse fetal ovaries

Sex-reversal in fetal ovaries was studied by using a dissociation-reconstitution technique. Gonads of 12.5 gestation-day male and female mouse fetuses were dissociated into single cells. To eliminate germ cells, the dissociated cells were cultured for 14 h, and then somatic cells attached to culture dishes were harvested and aggregated by gyratory culture for 24 h. The aggregates were then transplanted into ovarian bursa in ovary-ectomized nude mice. The recovered explants were examined histologically. Male somatic cells developed into testes containing Sertoli cells, Leidig cells, and tunica albuginea. Female somatic cells formed testis cords and differentiated into Sertoli cells, but they did not differentiate into other testis components or ovarian tissues. However, aggregates consisting of both female and male somatic cells differentiated into well-developed testes containing Leidig cells and tunica albuginea as well as Sertoli cells. Enzyme marker analysis showed significant contributions of female cells in these organized testes. In contrast, aggregates containing both female germ cells and somatic cells developed into ovaries and did not differentiate into any testicular tissues. The results indicate that female somatic cells in fetal gonads at 12.5 gestation day have the potency to form testis cords and differentiate into Sertoli cells. The subsequent steps in testis development require the contributions of male cells. The present study also suggests that testicular differentiation is independent of germ cells but ovarian development involves the interaction between germ cells and somatic cells.     

Effets de O-(β-Hydroxyaethyl)-rutosidea (HR) sur les fibroblastes d'embryon de Poulet cultivés in vitro en différentes concentrations d'oxygène

Résumé Les auteurs ont étudié, au moyen de méthodes histochimiques en association avec la réaction topooptique de Romhànyi, la couche périphérique de fibroblastes cultivés in vitro. On a démontré qu'elle est formée par des macromolécules protéiques (collagène, vraisemblablement), par de l'acide hyaluronique et par du chondroïtinesulfate A et C. En conditions de fortes hyperoxie et hypoxie, la couche périphérique, qui est métachromatique et anisotrope, perd, en degré variable, sa stabilité et son orientation. L'adjonction au milieu de culture d'un flavonoïde (HR) améliore sensiblement la stabilité de la dite couche.

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Effects of O-(-Hydroxyaethyl)-Rutosidea (HR) on chick embryo fibroblasts cultivated in vitro in different oxygen concentrationsIII. Histochemical topooptic studies with polarized light microscopy

Summary The authors have studied, by means of histochemical methods associated with Romhànyi topooptic reaction, the peripheral layer of fibroblasts cultivated in vitro. They demonstrated that this layer is formed by proteic macromolecules (problably collagen), by hyaluronic acid and chondroitin sulfate A and C. In high hyperoxic and hypoxic conditions the peripheral layer, which is metachromatic and anisotropic, loses, in various degree, its stability and its orientation. A flavonoid added to culture medium noticeably improves the stability of this layer.

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Ce travail a bénéficié de l'aide financière du Fonds National Suisse de la Recherche scientifique.     

Established cell lines of mouse marrow adherent cells producing differentiation factor(s) for the granulocyte-macrophage lineage

Summary Two continuous cell lines derived from long-term cultures of AKR mouse bone marrow adherent cells were isolated. These cell lines release colony stimulating activity (CSA), a factor that induces in vitro differentiation of granulocyte-macrophage progenitor cells. The colony forming cells and cluster forming cells in mouse marrow responsive to CSA from cell line conditioned medium were compared with those responsive to CSA from mouse lung conditioned medium (MLCM). Colony forming cells were characterized by analysis of their density distribution after equilibrium centrifugation in density gradient. Cluster forming cells were characterized by analyzing the progeny of individual clusters after transfer to fresh semisolid culture medium containing MLCM. The results obtained indicate that the CSA from cell line conditioned medium closely compares with the CSA from MLCM in terms of the populations of colony and cluster forming cells stimulated. This work was supported by a research grant from the Institut National de la Santé et de la Recherche Médicale (CRL 802620), Paris, France.     

Synthese et reabsorption des proteines dans la glande mammaire en stase,etude autoradiographique au microscope electronique

Resumé L'étude autoradiographique au microscope électronique de la glande mammaire en stase démontre la présence de nombreux grains de protéine marqués trente minutes après l'injection de leucine tritiée. Les grains marqués sont situés dans l'appareil de Golgi des cellules glandulaires, dans les lumières acineuses et dans les espaces conjonctifs inter-acineux. Le mécanisme de passage des grains de protéine dans le tissu conjonctif reste à élucider mais ces protéines sont fraichement synthétisées. Ces constatations indiquent que, dans la stase, la cellule glandulaire conserve une activité sécrétoire normale et n'a aucun caractère involutif.

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Summary Electron microscopic autoradiography has been applied to the study of protein synthesis in the mammary gland during milk stasis. Thirty minutes after injection of tritiated leucine silver grains are localized over the protein granules in the Golgi vacuoles, in the acinar lumina and in the inter-acinar spaces. They indicate that these granules are newly synthetized proteins and that their passage in the inter-acinar spaces is as immediate as it is in the glandular lumina. The mechanism by which the protein granules reach the inter-acinar spaces has still to be elucidated. The observations indicate that during milk stasis the glandular cell preserves its normal secretory activity.

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Avec l'aide du Centre National de la Recherche Scientifique et de l'Institut National de la Santé et de la Recherche Médicale.     

Interleukin-6 expression during normal maturation of the mouse testis

In this study, we examined the cellular origin and the expression levels of interleukin-6 (IL-6) during normal maturation of mouse testis. The levels of IL-6 (protein and mRNA) were higher in testicular homogenates of sexually immature than mature mice. Immunohistochemical staining of testicular tissues of sexually immature and adult mice show that testicular germ cells, at different stages of differentiation, Leydig cells/interstitial cells and peritubular cells express IL-6. Our results demonstrate, for the first time, overexpression of IL-6 in testicular tissues of immature mice, as compared to mature mice, as well as the expression of IL-6 in germ cells of testicular tissues of adult and sexually immature mice. Thus, our results may indicate the involvement of the endocrine system (gonadotropins and testosterone) in the regulation of IL-6, which is involved in the regulation of testicular development, functions and spermatogenesis.     

Changes in the testis interstitium of Sprague Dawley rats from birth to sexual maturity

Changes in the rat testis interstitium from birth to adulthood were studied using Sprague Dawley rats of 1, 7, 14, 21, 28, 40, 60, and 90 days of age. Our objectives were 1) to understand the fate of the fetal Leydig cells (FLC) in the postnatal rat testis, 2) to determine the volume changes in testicular interstitial components and testicular steroidogenic capacity in vitro with age, 3) to differentially quantify FLC, adult Leydig cells (ALC), and different connective tissue cell types by number and average volume, and 4) to investigate the relationship between mesenchymal and ALC numbers during testicular development. FLC were present in rat testes from birth to 90 days, and they were the only steroidogenic cells in the testis interstitium at Days 1 and 7. Except for FLC, all other interstitial cell numbers and volumes increased from birth to 90 days. The average volume of an FLC and the absolute volume of FLC per testis were similar at all ages except at Day 21, when lower values were observed for both parameters. FLC number per testis remained constant from birth through 90 days. The observations suggested that the significance of FLC in the neonatal-prepubertal rat testis is to produce testosterone to activate the hypothalamo-hypophyseal-testicular axis for the continued development of the male reproductive system. ALC were the abundant Leydig cell type by number and absolute volume per testis from Day 14 onwards. The absolute numbers of ALC and mesenchymal cells per testis increased linearly from birth to 90 days, with a slope ratio of 2:1, respectively, indicating that the rate of production of Leydig cells is 2-fold greater than that of mesenchymal cells in the postnatal rat testis through 90 days. In addition, this study showed that the mesenchymal cells are an active cell population during testis development and that their numbers do not decrease but increase with Leydig cell differentiation and testicular growth up to sexual maturity (90 days).