Artificial milk preference of newborn lambs is prenatally influenced by transfer of the flavor from the maternal diet to the amniotic fluid.

The present study examined in lambs whether exposure to flavors derived from pregnant mother's diet and transferred to amniotic fluid (AF) could induce a preference for artificial milk containing one of these flavors. To test this hypothesis, cumin was added to the maternal diet in the last month of gestation. Preference for artificial milk containing p-cymene, one of the chemosensory compounds of cumin, was tested within the first two days after birth in maternally deprived lambs born from mothers fed a cumin-flavored diet (Cumin group), or an unflavored diet (Control group). Aromatic profile of AF from cumin-fed mothers was analyzed by GC-MS/MS to determine whether p-cymene could be detected. While the control group avoided the flavored artificial milk on day 1, the Cumin group did not and showed a preference for the cumin-scented formula on day 2. GC-MS/MS profile of AF revealed that four of the main volatile cumin compounds, p-cymene, p-cymenene, ß-pinene and γ-terpinene were present in variable amounts in all samples, p-cymene being the most frequently detected. These findings indicate that newborn lambs can memorize flavors from the mother's diet present in AF and that prenatal experience influences their preference for an artificial milk containing one specific flavor.

Endothelin evokes distinct calcium transients in neuronal and non-neuronal cells of rat olfactory mucosa primary cultures.

The olfactory system is regulated by several nervous and hormonal factors, and there is a growing body of evidence that some of these modulations already take place in the olfactory mucosa (OM). We recently suggested that, among others, vasoactive peptides might play multifaceted roles in different OM cells. Here we studied the effect of the vasoconstrictive peptide endothelin (ET) in the rat OM. We identified different components of the ET system both in the olfactory mucosa and in long-term primary culture of OM cells, composed of olfactory sensory neurons (OSNs) lying on a blend of non-neuronal OM cells (nNCs). We demonstrated that ET receptors are differentially expressed on OM cells, and that ET might be locally matured by the endothelin-converting enzyme ECE-1 located in OSNs. Using calcium imaging, we showed that ET triggers robust dose-dependent Ca(2+) responses in most OM cells, which consist of a transient phase, followed, in nNCs, by a sustained plateau phase. All transient responses depended on intracellular calcium release, while the sustained plateau phase also depended on subsequent external calcium entry. Using both pharmacology and spotting lethal (sl/sl) mutant rats, lacking functional ET(B) receptors, we finally demonstrated that these effects of ET are mediated through ET(B) receptors in OSNs and ET(A) receptors in nNCs.The present study therefore identifies endothelin as a potent endogenous modulator of the olfactory mucosa; specific endothelin-mediated Ca(2+) signals may serve distinct signaling functions, and thereby suggest differential functional roles of endothelin in both neuronal and non-neuronal OM cells.

Nutritional status modulates behavioural and olfactory bulb Fos responses to isoamyl acetate or food odour in rats: roles of orexins and leptin.

Food odours are major determinants for food choice, and their detection depends on nutritional status. The effects of different odour stimuli on both behavioural responses (locomotor activity and sniffing) and Fos induction in olfactory bulbs (OB) were studied in satiated or 48-h fasted rats. We focused on two odour stimuli: isoamyl acetate (ISO), as a neutral stimulus either unknown or familiar, and food pellet odour, that were presented to quiet rats during the light phase of the day. We found significant effects of nutritional status and odour stimulus on both behavioural and OB responses. The locomotor activity induced by odour stimuli was always more marked in fasted than in satiated rats, and food odour induced increased sniffing activity only in fasted rats. Fos expression was quantified in periglomerular, mitral and granular OB cell layers. As a new odour, ISO induced a significant increase in Fos expression in all OB layers, similar in fasted and satiated rats. Significant OB responses to familiar odours were only observed in fasted rats. Among the numerous peptides shown to vary after 48 h of fasting, we focused on orexins (for which immunoreactive fibres are present in the OB) and leptin, as a peripheral hormone linked to adiposity, and tested their effects of food odour. The administration of orexin A in satiated animals partially mimicked fasting, since food odour increased OB Fos responses, but did not induce sniffing. The treatment of fasted animals with either an orexin receptors antagonist (ACT-078573) or leptin significantly decreased both locomotor activity, time spent sniffing food odour and OB Fos induction in all cell layers, thus mimicking a satiated status. We conclude that orexins and leptin are some of the factors that can modify behavioural and OB Fos responses to a familiar food odour.

Expression of insulin system in the olfactory epithelium: first approaches to its role and regulation.

Food odours are major determinants for food choice; their detection is influenced by nutritional status. Among different metabolic signals, insulin plays a major role in food intake regulation. The aim of the present study was to investigate a potential role of insulin in the olfactory mucosa (OM), using ex vivo tissues and in vitro primary cultures. We first established the expression of insulin receptor (IR) in rat olfactory mucosa. Transcripts of IR-A and IR-B isoforms, as well as IRS-1 and IRS-2, were detected in OM extracts. Using immunocytochemistry, IR protein was located in olfactory receptor neurones, sustentacular and basal cells and in endothelium of the lamina propria vessels. Moreover, the insulin binding capacity of OM was quite high compared to that of olfactory bulb or liver. Besides the main pancreatic insulin source, we demonstrated insulin synthesis at a low level in the OM. Interestingly 48 h of fasting, leading to a decreased plasmatic insulin, increased the number of IR in the OM. Local insulin concentration was also enhanced. These data suggest a control of OM insulin system by nutritional status. Finally, an application of insulin on OM, aiming to mimic postprandial insulin increase, reversibly decreased the amplitude of electro-olfactogramme responses to odorants by approximately 30%. These data provide the first evidence that insulin modulates the most peripheral step of odour detection at the olfactory mucosa level.

Transcription profile analysis reveals that OBP-1F mRNA is downregulated in the olfactory mucosa following food deprivation.

Neuroanatomical data show that olfactory mucosa (OM) is a possible place for interactions between nutrition and smell. A combination of differential display mRNA analysis together with a macroarray screening was developed to identify transcripts that are differentially expressed in rat OM following food deprivation. Using this method, backed on a stringent statistical analysis, we identified molecules that fell into several Gene Ontology terms including cellular and physiological process, signal transduction, and binding. Among the 15 most differentially expressed molecules, only one was upregulated, but 14 were downregulated in the fasted state among which was, unexpectedly, odorant-binding protein 1F (OBP-1F). Because of its potential relevance to olfactory physiology, we focused our further analysis on OBP-1F using in situ hybridization, quantitative polymerase chain reaction, and western blot analysis. OBP-1F was highlighted in the lateral nasal glands, but its expression (mRNA and protein) did not change following food deprivation. Only the minor fraction of OBP-1F mRNA expressed by the OM itself was downregulated following 48 h fasting. Altogether, our results suggest that the fine transcriptional control of OBP-1F in the OM following food deprivation could be efficient only at the local level, close to its site of secretion to participate in the perireceptor events of the olfactory signal reception.

Functional and molecular reorganization of the nucleolar apparatus in maturing mouse oocytes.

In mammalian preovulatory oocytes, rRNA synthesis is down-regulated until egg fertilization and zygotic genome reactivation, but the underlying regulatory mechanisms of this phenomenon are poorly characterized. We examined the molecular organization of the rRNA synthesis and processing machineries in fully grown mouse oocytes in relation to ongoing rDNA transcription and oocyte progression throughout meiosis. We show that, at the germinal vesicle stage, the two RNA polymerase I (RNA pol I) subunits, RPA116 and PAF53/RPA53, and the nucleolar upstream binding factor (UBF) remain present irrespective of ongoing rDNA transcription and colocalize in stoichiometric amounts within discrete foci at the periphery of the nucleolus-like bodies. These foci are spatially associated with the early pre-rRNA processing protein fibrillarin and in part with the pre-ribosome assembly factor B23/nucleophosmin. After germinal vesicle breakdown, the RNA pol I complex disassembles in a step-wise manner from chromosomes, while UBF remains associated with chromosomes until late prometaphase I. Dislodging of UBF, but not of RNA pol I, is impaired by the phosphatase inhibitor okadaic acid, thus strengthening the idea of a relationship between UBF dynamics and protein phosphorylation. Since neither RNA pol I, UBF, fibrillarin, nor B23 is detected at metaphase II, i.e., the normal stage of fertilization, we conclude that these nucleolar proteins are not transported to fertilized eggs by maternal chromosomes. Together, these data demonstrate an essential difference in the dynamics of the major nucleolar proteins during mitosis and meiosis.

Induction of early transcription in one-cell mouse embryos by microinjection of the nonhistone chromosomal protein HMG-I.

In the mouse embryo, the onset of zygotic transcription occurs at the end of the first cell cycle, upon completion of DNA replication. We show that the nonhistone chromosomal protein HMG-I, whose translocation into the pronuclei of one-cell embryos is linked to this first round of DNA synthesis, plays a critical role in the activation of zygotic transcription. Indeed, microinjection of purified HMG-I results in a higher nuclear accumulation of the protein and triggers an earlier activation of zygotic transcription, an effect which is abolished by the preincubation of the protein with a specific antibody directed against its AT-hook DNA-binding motifs. Significantly, microinjection of this antibody also prevents the normal onset of transcription in the embryo, suggesting that endogenous HMG-I is similarly involved in this process. Finally, microinjection of the exogenous protein modifies chromatin structure as measured by in situ accessibility to DNase I. We propose that general chromosomal architectural factors such as HMG-I can modulate the accessibility of chromatin to specialized regulatory factors, thereby promoting a transcriptionally competent state.

Differential transcriptional activity associated with chromatin configuration in fully grown mouse germinal vesicle oocytes.

It was previously shown that fully grown ovarian germinal vesicle (GV) oocytes of adult mice exhibit several nuclear configurations that differ essentially by the presence or absence of a ring of condensed chromatin around the nucleolus. These configurations have been termed, respectively, SN (surrounded nucleolus) and NSN (nonsurrounded nucleolus). Work from our and other laboratories has revealed ultrastructural and functional differences between these two configurations. The aims of the present study were 1) to analyze the equilibrium between the SN and the NSN population as a function of the age of the mice and the time after hCG-induced ovulation and 2) to study the polymerase I (pol I)- and polymerase II (pol II)-dependent transcription in both types of oocytes through the detection of bromouridine incorporated into nascent RNA. We show 1) that ovarian GV oocytes exhibiting the SN-type configuration can be found as soon as 17 days after birth in the C57/CBA mouse strain and 2) that the SN:NSN ratio of ovarian GV oocytes is very low just after hCG-induced ovulation and then increases progressively with the time after ovulation. Furthermore, we demonstrate that the SN configuration correlates strictly with the arrest of both pol I- and pol II-dependent transcription in mice at any age. Finally, we show that ribosomal genes are located at the outer periphery of the nucleolus in the NSN configuration and that pol I-dependent perinucleolar transcription sites correspond to specific ultrastructural features of the nucleolus. Altogether, these results provide clear-cut criteria delineating transcriptionally active GV oocytes from those that are inactive, and confirm that the SN-type configuration is mostly present in preovulatory oocytes.

Dynamic organization of DNA replication in one-cell mouse embryos: relationship to transcriptional activation.

We have analyzed the spatial and temporal relationship between transcription and replication sites during the first cell cycle in mouse embryos. Embryos were microinjected with both 5-bromouridine-5'-triphosphate and digoxygenin-11-deoxyuridine-5'-triphosphate to visualize transcription and replication sites respectively. We detected six different phases of replication during S phase and dated the onset of zygotic transcription at the end of the S phase. Using confocal microscopy, we showed that there is essentially no colocalization of replication and transcription sites at this stage of development. Moreover, studies on aphidicolin-treated embryos demonstrated that inhibition of DNA replication does not hinder transcriptional activation at the 1-cell stage.