Intracellular bacterial symbiosis in the genus Sitophilus: the 'biological individual' concept revisited.

Eukaryotic cells, as genetic entities, most often involve several physically associated genomes that direct the metabolic cell equilibrium. In the coleopteran insects of the genus Sitophilus, in addition to the nucleus and the mitochondrial genomes, two other intracellular bacterial genomes belonging to the alpha and the gamma groups of Proteobacteria are also present. Coexisting with the eukaryotic host cell genomes, they intervene in the physiology and reproduction of the host. They are both transmitted vertically to the progeny and exhibit different levels of symbiont integration in insects. Their coexistence within a eukaryotic cell system illustrates the genetic complexity of animal tissue and questions the concept of the 'biological individual'.

Dynamics of Wolbachia populations in transfected lines of Trichogramma.

Fluorescence in situ hybridization was tested to specifically detect symbionts of the genus Wolbachia in Trichogramma and to allow for semiquantitative estimations of symbiont abundance. Extraction solutions used for horizontal transfers of symbionts contain a high abundance of Wolbachia, but Wolbachia have a low and decreasing abundance in microinjected lines (transfected lines). Moreover, eggs of microinjected lines were shown to be polymorphic for the infection. In naturally infected lines, Wolbachia are localized at the posterior pole of the eggs; they are scattered during the early stages of larval development and then concentrated in the ovaries at the end of the female pupal development. Scattering and concentration are probably not active but rather the result of replications or morphogenesis. Conversely, Wolbachia are not concentrated at the posterior pole of eggs in microinjected lines. Comparison of the within-family and between-family variances of the symbiont abundance in a microinjected line did not lead us to conclude that this character shows a genetic variability.

Four intracellular genomes direct weevil biology: nuclear, mitochondrial, principal endosymbiont, and Wolbachia.

Cell physiology in the weevil Sitophilus oryzae is coordinated by three integrated genomes: nuclear, mitochondrial, and the "S. oryzae principal endosymbiont" (SOPE). SOPE, a cytoplasmic bacterium (2 x 10(3) bacteria per specialized bacteriocyte cell and 3 x 10(6) bacteria per weevil) that belongs to the proteobacteria gamma3-subgroup, is present in all weevils studied. We discovered a fourth prokaryotic genome in somatic and germ tissues of 57% of weevil strains of three species, S. oryzae, Sitophilus zeamais, and Sitophilus granarius, distributed worldwide. We assigned this Gram-negative prokaryote to the Wolbachia group (alpha-proteobacteria), on the basis of 16S rDNA sequence and fluorescence in situ DNA-RNA hybridization (FISH). Both bacteria, SOPE and Wolbachia, were selectively eliminated by combined heat and antibiotic treatments. Study of bacteria involvement in this insect's genetics and physiology revealed that SOPE, which induces the specific differentiation of the bacteriocytes, increases mitochondrial oxidative phosphorylation through the supply of pantothenic acid and riboflavin. Elimination of this gamma3-proteobacterium impairs many physiological traits. By contrast, neither the presence nor the absence of Wolbachia significantly affects the weevil's physiology. Wolbachia, disseminated throughout the body cells, is in particularly high density in the germ cells, where it causes nucleocytoplasmic incompatibility. The coexistence of two distinct types of intracellular proteobacteria at different levels of symbiont integration in insects illustrates the genetic complexity of animal tissue. Furthermore, evolutionary timing can be inferred: first nucleocytoplasm, then mitochondria, then SOPE, and finally Wolbachia. Symbiogenesis, the genetic integration of long-term associated members of different species, in the weevil appears to be a mechanism of speciation (with Wolbachia) and provides a means for animals to acquire new genes that permit better adaptation to the environment (with SOPE).

Molecular characterization of the principal symbiotic bacteria of the weevil Sitophilus oryzae: a peculiar G + C content of an endocytobiotic DNA.

The principal intracellular symbiotic bacteria of the cereal weevil Sitophilus oryzae were characterized using the sequence of the 16S rDNA gene (rrs gene) and G + C content analysis. Polymerase chain reaction amplification with universal eubacterial primers of the rrs gene showed a single expected sequence of 1,501 bp. Comparison of this sequence with the available database sequences placed the intracellular bacteria of S. oryzae as members of the Enterobacteriaceae family, closely related to the free-living bacteria, Erwinia herbicola and Escherichia coli, and the endocytobiotic bacteria of the tsetse fly and aphids. Moreover, by high-performance liquid chromatography, we measured the genomic G + C content of the S. oryzae principal endocytobiotes (SOPE) as 54%, while the known genomic G + C content of most intracellular bacteria is about 39.5%. Furthermore, based on the third codon position G + C content and the rrs gene G + C content, we demonstrated that most intracellular bacteria except SOPE are A + T biased irrespective of their phylogenetic position. Finally, using the hsp60 gene sequence, the codon usage of SOPE was compared with that of two phylogenetically closely related bacteria: E. coli, a free-living bacterium, and Buchnera aphidicola, the intracellular symbiotic bacteria of aphids. Taken together, these results show a peculiar and distinctly different DNA composition of SOPE with respect to the other obligate intracellular bacteria, and, combined with biological and biochemical data, they elucidate the evolution of symbiosis in S. oryzae.

Glycine and methionine transport by bovine embryos.

As glycine is one of the most concentrated amino acids in the female genital tract, we investigated its uptake by bovine in vitro matured/in vitro fertilised blastocysts in the presence of increasing concentrations of radiolabelled glycine. We also determined methionine uptake by in vitro and in vivo produced embryos. In our study, the hypothesis of more than one site of enzyme activity for glycine substrate was not validated. We determined a Vmax of 23.4 fmol/min per embryo and a K(m) value of 13.3 microM. No significant difference was observed either between in vivo and in vitro derived embryos or between grade 1 and grade 2 embryos for methionine uptake. The methionine and glycine uptake of a day 7 bovine was similar to that of a day 4 mouse blastocyst. This is rather low if we consider the relative cell numbers.

[Embryonal metabolism]. / Le métabolisme embryonnaire.

It is very difficult to have a clear and homogeneous idea of the embryo metabolism. In fact it may vary from one species to another and also according to the embryonic stage: i.e. before and after genomic activation. Basic compounds such as glucose may be toxic, but obviously, it is more the problem of the quantity introduced in the culture media and an unsuitable balance between the metabolites which may impair the embryonic development. At low concentration glucose is actively metabolised by embryos. High levels of amino acids are deleterious (due to release of ammonia), but they are necessary at low concentrations. Addition of serum or other biological fluids is generally useless. Further knowledge on embryo metabolism is necessary to avoid culture medium related delay or developmental blocks. Sequential media are at least partly the answer.

Comparative glucose and fructose incorporation and conversion by in vitro produced bovine embryos.

We have investigated the quality of bovine IVM/IVF embryos co-cultured on Vero cells. Blastocyst cell numbers are very similar to those obtained in vivo, and higher than those obtained by co-culture with oviduct cells. The metabolism and conversion of fructose and glucose are not equivalent even though carbon dioxide production is similar and increasing from morula to blastocyst. Formation of free amino acids and incorporation into proteins are higher and faster for glucose than for fructose, but this conversion is rather stable with embryonic growth. Moreover, the by-products formed are not the same. Glucose at physiological concentrations (i.e. 2 mM) seems to be a more appropriate fuel for the burst of embryonic development at the blastocyst stage in preparation for hatching.

Immunolocalization of transforming growth factor-beta 1 and transforming growth factor-beta 2 in the mouse ovary during gonadotrophin-induced follicular maturation.

An immunohistochemical approach was utilized to evaluate the cellular distribution of transforming growth factor-beta 1 (TGF beta 1) and transforming growth factor beta 2 (TGF beta 2) at different stages of follicle development in the prepubertal mouse ovary under the following conditions: (i) after pregnant mare's serum gonadotrophin (PMSG) treatment; (ii) after PMSG and human chorionic gonadotrophin (HCG) treatment; (iii) after PMSG and HCG treatment plus mating. In the immature ovary, TGF beta 1 and TGF beta 2 immunoreactivities are localized in theca and granulosa cells and in oocytes. After PMSG treatment, TGF beta 1 and TGF beta 2 immunoreactivities are localized in granulosa cells; in addition, TGF beta 2 staining is noted in the matrix surrounding antral cells. Staining for both TGR beta 1 and TGF beta 2 drops in the theca but persists in the oocyte. PMSG plus HCG treatment results in a significant increase in TGF beta 1 and TGF beta 2 immunoreactivity in the theca and in the maintenance of TGF beta 1 staining in both basal granulosa cells and cumulus cells whereas TGF beta 2 immunoreactivity is essentially localized in the matrix surrounding cumulus cells. Staining for TGF beta 1 and TGF beta 2 persists in the oocyte. Following PMSG plus HCG treatment and mating, TGF beta 1 immunoreactivity is localized in the luteal cells of corpora lutea and TGF beta 2 shows a similar localization pattern. This study provides evidence that TGF beta 1 and TGF beta 2 peptides are expressed in specific cell types during induced follicular maturation in the mouse ovary.

Interactions in glycine and methionine uptake, conversion and incorporation into proteins in the preimplantation mouse embryo.

Glycine is the most concentrated amino acid in the female genital tract. In this study, we report its conversion and incorporation into proteins in the presence or absence of methionine, in both 1-cell and blastocyst mouse embryos. The uptake, incorporation and conversion of radiolabelled glycine were studied in the presence or absence of unlabelled methionine. For control purposes, the reciprocal experiment was performed with labelled methionine in the presence or absence of unlabelled glycine. At the 1-cell stage neither glycine uptake nor its incorporation into proteins is inhibited by methionine. Glycine is, however, highly used as an oxidisable energy substrate, via glycolate. At the blastocyst stage, glycine conversion into other amino acids is high and mainly utilised in the formation of glutamic acid. Glycine is highly incorporated into proteins, resulting in a poor exchange of glycine from the preloaded embryos. Methionine competes for glycine uptake and consequently reduces its overall incorporation into proteins. For methionine, neither its uptake nor its incorporation into proteins is reduced in the presence of glycine for the two embryonic stages tested here. The embryo has different mechanisms for incorporation and utilisation of methionine and glycine. Glycine, which has an important function in the embryo, has an inefficient transport system compared with methionine. We were unable to demonstrate the presence of methylglycine since SAM-glycine-methyltransferase (EC 2.1.1.20) was not detected. The same results were obtained when exogenous methionine was added. We therefore concluded that glycine does not compete in transmethylation within the embryo.

[Implication of glucose 6 phosphate isomerase (EC 5.3.1.9) activity in blocking segmentation of the mouse ovum at the 2 cell stage in vitro]. / Implication de l'activité glucose 6 phosphate isomérase (EC 5.3.1.9) dans l'arrêt de la segmentation de l'oeuf de souris au stade 2 cellules in vitro.

The mouse embryo, when grown in media containing glucose, synthesizes and accumulates glycogen. In certain strains, glucose could be responsible for the 2 cell block; it can be replaced for a short time by glutamine, but with an increase in embryo degeneration. We have tested the hypothesis according to which the problems of glycogen accumulation and the developmental arrest could be due to a metabolic lock involving glucose 6 phosphate isomerase (EC 5.3.1.9). Fructose is located immediately downstream from this metabolic lock. We have exactly replaced the glucose in Whittingham M16 culture medium by fructose: Medium F.M. With this culture medium, Swiss one cell embryos, which normally block in M16 medium (greater than 90%), do not block any longer at the 2 cell stage (11.6%). They can be cultured for 4 days with high rates of blastocyst formation (53.6%). Embryo degeneration (23.6%) is lower than that observed in CZB (31%), and not different from our control degeneration in vivo. Moreover fructose seems to be adequate all through the culture period as there is no need for further addition of metabolites, to obtain blastocysts, as observed for CZB. This demonstrates that a lock at the Glucose phosphate isomerase level is responsible for glycogen accumulation and the 2 cell block in the mouse embryo in vitro.

Regulation of S-adenosyl methionine synthesis in the mouse embryo.

In early embryos, methylation is involved in "gamete imprinting" and inactivation of artificially introduced foreign genes. We studied the biosynthesis of the universal methylation cofactor: S-Adenosyl methionine (SAM). In the mouse, SAM conversion from methionine is limited by saturation of the methionine endogenous pool. SAM is present at a practically unchanged level from the unfertilized oocyte to early morula. SAM synthesis is increased at the time of compaction. In blastocysts, although methionine uptake is increased, the conversion rate from methionine is lowered. We observed no differences between C57 Black and Swiss albino random bred strains. In few experiments with human unfertilized oocytes and spared embryos, we observed higher methionine incorporation, and higher conversion to SAM. Next, the effect of two methylation inhibitors was tested, on early mouse embryonic development, at the one-cell and the two-cell stage. We found that ethionine is very toxic, even at the lowest tested concentration of 25 microM. Homocysteine is more potent at the one-cell stage than at the 2-cell stage, and it only partially blocks blastocyst formation from the 2-cell stage even at a concentration of 500 microM. It clearly acts as a methylation inhibitor; it lowers the SAM pool and the methylation index, SAH/SAM ratio (SAH: S-Adenosyl Homocysteine). We also found that homocysteine is an unexpected competitor for methionine influx and efflux.

Catecholamines within the rabbit oviduct at fertilization time.

Dopamine, norepinephrine and epinephrine levels of rabbit oviductal fluids were measured in order to establish whether the ampullary segment contained higher concentrations correlating with the sperm hyperactivity observed in the ampulla of different mammals. In this study, we found no statistically significant differences between flushings from the ampulla or the isthmus at oestrus, ovulation or 72 h post-coitum (p.c.). However, after correcting for the volume secreted, the concentrations of catecholamines are always lower in the ampulla than in the isthmus. In all cases the levels of biogenic amines were highest at oestrus then fell at ovulation. Individual variation of these levels at the time of ovulation was observed only in the ampulla, probably due to some contribution from follicular fluid. During the early luteal phase (72 h p.c.) the catecholamine levels again increase slightly, especially in the isthmus. We conclude that whiplash motility cannot be explained by a higher level of catecholamines in the ampulla.

Peptides bound to albumin.

Preparations of albumin (placenta or serum) do bind peptides. Dissociation of these peptides from the albumin core can occur partially in water but is much more efficient in the presence of amino acids and salts, in complex culture media for example. Contrary to common belief, we observed that arginine vasopressin (AVP) can bind to serum albumin, at least in commercial powder form.

Catecholamine levels in relation to number of spermatozoa inseminated "in vitro" in the human being.

We tried to understand and to explain why such a high number of sperm cells is necessary for human in vitro fertilization in relation to the possible role of catecholamines. Sperm cells, even washed several times, bring catecholamines (epinephrine, nor-epinephrine and dopamine). When incubated in a suitable medium, with Taurine and reducing agents, the catecholamine level remains constant for 5 hours. The origin of these amines and the positive vs negative effect of "in vitro" insemination with a high number of spermatozoa are discussed.

Human preovulatory follicular fluid: the lipids. Are they the trigger for capacitation?

The lipid content of human follicles within 6 h of ovulation was determined. Levels for triglycerides, phospholipids, and total cholesterol were 1/4 to 1/3 of those in serum. An uncommonly high level of free cholesterol was observed, possibly resulting from a high synthetic rate by the granulosa. Follicular lipids appear to be of serum origin since the relative proportions of the various fatty acids are the same. Arachidonic acid was not found to be present at the high levels reported for "large" porcine follicles, possibly due to its active utilization in the synthesis of prostaglandins necessary for the ovulation process. Precise timing relative to ovulation is clearly essential for follicular classification in order to distinguish between before and after the triggering of the ovulation process. A possible role for lipids in the induction of capacitation is proposed.

Role of thiol compounds in mammalian melanin pigmentation. II. Glutathione and related enzymatic activities.

Previously, we reported evidence suggesting that, in addition to tyrosinase, glutathione-reductase plays an important role in the regulation and control of the biosynthetic activity of melanocytes. Further investigations were performed on a mammal presenting a well-defined genotype for coat pigmentation, the mutant mouse [subline C57 BL (6J)], namely the nonagouti black (a/a) mutant and the yellow (Ay/a) mutant showing, respectively, pure uniform eumelanin and phaeomelanin pigmentation. Analysis of thiol compounds and glutathione-related enzyme levels in mouse skin gave similar results to those found in tortoise-shell guinea pig skin. The observed differences in the glutathione and glutathione-related enzyme content between black and yellow (or red) skin provide evidence that the increase of glutathione-reductase activity in the environment of the melanocytes may stimulate the pigment cells to produce phaeomelanin instead of eumelanin pigment.

Amelanotic changes in B 16 melanoma after transplantation to 'Yellow' Ay/a mice.

B 16 mouse melanoma maintained on nonagouti a/a mice (C 57 Bl 6j subline) was transplanted to 'Yellow' Ay/a mutants. B 16 melanoma has now been maintained for 1 year on the 'Yellow' strain. A microscopic and ultrastructural study of transplanted tumors is described. Several enzymatic activities including tyrosinases are investigated. A marked depigmentation of the B 16 melanoma is noted after its transplantation to the 'Yellow' strain, and melanogenic characteristics of the tumor are modified.

The preovulatory follicular fluid in the human: influence of hormonal pretreatment (clomiphene-hCG) on some biochemical and biophysical variables.

Some biochemical and biophysical variables of human follicular fluid have been studied both in normal women after the LH peak (preovulatory follicles) and in women treated with clomiphene and hCG (clomiphene treated follicles). The results showed that lactate and glucose levels are different from those found in serum but are not influenced by the clomiphene treatments. Osmolarity and enzymatic contents are neither different from those of serum nor modified by the hormonal treatments. The transfer of water across the follicle wall was found to be closely regulated because only slight variations were observed in the wide range of volumes collected in the samples examined.

Role of thiol compounds in mammalian melanin pigmentation: Part I. Reduced and oxidized glutathione.

Evidence for the postulated role of glutathione reductase in melanin pigmentation has been obtained by determinations of the glutathione concentrations in Tortoiseshell guinea pig skin of different colors (black, yellow, red, and white). As expected, the lowest levels of reduced glutathione (GSH) were found associated with eumelanin type pigmentation, whereas the highest ones were found in the skin with phaeomelanin producing melanocytes. On the other hand, white skin of guinea pig having no active melanocytes showed GSH levels which were intermediate between those of the black and yellow areas. These results are consistent with the view that the activity of the enzyme glutathione reductase, though not primarily related to pigmentation, plays an important role in the regulation and control of the biosynthetic activity of melanocytes leading to various types of melanin pigments.

["In vitro" culture of the oviduct of women, rats and rabbits. Preliminary results (author's transl)]. / Culture "in vitro" de l'oviducte de femme, de rate et de lapine. Résultats préliminaires.

A continuous flow perfusion system which has been improved for the culture of large pieces of tissue, e.g. epididymis and ovarian follicles was tested for the entire oviduct in women, rabbits and rats. The histological properties of the organ were maintained after a day or two of culture; but only in the presence of oestrogens. Progesterone seems to induce a weakness of epithelial cell attachment and these become scattered in the lumen. In women the cultures seemed to be more or less permanently contamined, probably due to commensals, because this problem did not occur with rat or rabbit oviducts.