Gilles Bruneau

Short-Term Effect of Oestradiol on Neurokinin B mRNA Expression in the Infundibular Nucleus of Ewes

In sheep, essentially all the neurokinin B (NKB) neurones of the infundibular nucleus express oestradiol receptor α, and analysis of female and male brains has revealed an exceptionally marked female‐dominant sex difference in the numbers of NKB neurones in the infundibular nucleus. This neuronal population is located in an oestradiol‐sensitive brain area involved in the control of gonadotropin‐releasing hormone (GnRH) secretion and oestrous behaviour, but its physiological role is poorly documented. The aim of the present study was to analyse NKB mRNA expression at a crucial time when the steroid has stimulated the pathways leading to the induction of these two events. After cloning a specific ovine NKB antisense riboprobe, we examined the effects of a short oestradiol treatment (4 h subcutaneously) on the expression of NKB mRNA in the caudal part of the infundibular nucleus of progesterone‐primed ovariectomized ewes. We demonstrated that oestradiol decreased both the level of NKB mRNA expression (34%) and the number of cells containing NKB mRNA (43%). Oestradiol acts strongly on these NKB cells in the short term. We suggest that this early change in NKB mRNA expression during the preovulatory period might be involved in the control of the induction of GnRH secretion or oestrous behaviour.

Is there a leptin gene in the chicken genome? Lessons from phylogenetics, bioinformatics and genomics

The first publication describing the cloning of Gallus gallus leptin cDNA by Taouis et al. (1998) led to a controversy between some who succeeded in confirming these results (Ashwell et al., 1999) and others who failed to reproduce them (Friedman-Einat et al., 1999), controversy that has been recently debated in this journal (Sharp et al., 2008; Simon et al., 2009). Now, more than 10 years after, an increasing number of papers have been published describing the effects of exogenous (human as well as ‘‘chicken/mouse like”) recombinant leptin on chicken cell culture in vitro and on poultry in vivo. If the leptin gene really does not exist in these species, all these works would not have any relevant physiological significance, except to confirm the existence of a leptin receptor that binds human ligand. In the present paper, we will summarize arguments (mainly previously debated) that do not favor the presence of a leptin gene, and presents new genomic and bioinformatic data to enlighten the discussion.

Early decrease of proopiomelanocortin but not neuropeptide Y mRNA expression in the mediobasal hypothalamus of the ewe, during the estradiol-induced preovulatory LH surge.

In sheep, the mediobasal hypothalamus (MBH) has been shown to be the primary central site of estradiol (E2) action that induces both the preovulatory surge and sexual behaviour. However, the nature of the neurotransmitters or neuromodulators synthesized in the MBH during E2 stimulation remains to be clearly defined. After the cloning of the ovine cDNA sequences and using in situ hybridization, hypothalamic proopiomelanocortin (POMC), and preproneuropeptide Y (preproNPY) mRNA expression was studied in ovariectomized ewes that received a sequential treatment of progesterone and E2. As we showed that an exposition to E2 only for 4h well in advance on the LH surge onset is sufficient to induce the preovulatory surge and estrous behaviour, mRNA expression was evaluated in ewes treated with 6x30-mm E2 implants (experimental group) or with empty implants (control group) and slaughtered 4h after the start of the E2 treatment. Our results demonstrate that this short E2 treatment significantly decreased both the mean number of silver grains per POMC-containing cell (35%) and the mean number of POMC-cells (38%) in the ovine infundibular nucleus, whereas the treatment had no effect on preproNPY mRNA expression. These observations suggest that a reduction of POMC gene transcription could participate to the early neural mechanism of E2 feedback.

A one-step exclusion-binding procedure for the purification of functional heavy-chain and mammalian-type γ-globulins from camelid sera

A new approach has recently been proposed for the purification of ‘mammalian‐type’ IgG, consisting of exclusion binding. The technique uses a gel (‘Melon gel’; Pierce) that binds to all plasma proteins, but not to IgGs, thus allowing IgGs to be recovered in the FT (flow‐through) fraction. Here, the technique was applied to camelid IgGs, which are known to be composed of not only classic mammalian‐type IgGs (IgG1) but also HC‐IgGs (heavy chain IgGs). Both mammalian type and HC‐IgGs can be purified in the FT fraction of dromedary (Camelus dromedarius) plasma samples with less than 8.5% contamination, by making minor improvements to the conditions recommended by the manufacturer. The contaminant proteins, as determined by LC‐MS/MS (liquid chromatography–tandem MS), are mainly transferrin and albumin. The recovery rate is elevated for both types of IgGs (95±14% and 88±25% for IgG1 and HC‐IgGs respectively). IgGs thus purified maintain their ability to bind to their antigen, as measured by surface plasmon resonance and Western ligand blotting. Furthermore, IgGs can be purified from plasma samples of all camelid species in a similar manner, although the ratio of HC‐IgGs to total IgGs was lower for Lama (llama) and Vicugna (vicuña) than for Camelus species. The ‘Melon gel’ technique can thus be used to satisfactorily purify IgG1 and HC‐IgGs from all camelid species.

A new method to discriminate immunogen-specific heavy-chain homodimer from heterotetramer immunoglobulin G directly in immunized dromedary whole plasma proteins: western ligand blotting.

The variable domain of heavy-chain camelid antibodies (VHH), exclusively present in the homodimer IgGs (HC IgG), provides valuable ligands for diagnosis, imaging and therapy. These VHHs are efficiently produced from lymphocytes of immunized animals through phage display and recombination biotechnology. For VHH development it is desirable to identify animals with high affinity HC IgG response by monitoring antigen-binding in the course of immunization. The aim of this study was to propose a direct approach on whole plasma samples to distinguish between homodimer IgG and heterotetramer (IgG(1)) responses, and quantify them, using western ligand blotting (WLB). WLB consists here in electrophoretic separation of the target IgG subclasses on the basis of molecular size and binding of (125)I labeled antigen. First, we established the WLB parameters for titration of antigen-binding homodimers in relation to antigen-binding total IgGs in ovalbumin-immunized dromedary plasma samples and demonstrated that the WLB is an alternative to ELISA for IgG subclass titration. As purification of IgG subclasses or availability of IgG subclass-specific antibodies is not necessary, WLB is more direct and practical for screening a large number of samples. Second, WLB was applied to study the pattern of homodimer and heterotetramer responses during the time-course of immunizations against three different types of immunogens. As these patterns can differ between animals and immunogens, the method may be useful for identifying animals displaying the desired antigen-specific homodimer IgG response. Lastly, WLB was also described in its variant form of dot ligand blotting for identifying antigen-binding phages at the final step of a phage display experiment.

Resistant ribonuclease activity in preparations of total RNA extracted from artiodactyl brain with GITC

The protocol of Chomczynski and Sacchi (1) constitutes an established method widely used for extracting total RNA from many mammalian species. In general, this method produces goodquality total RNA suitable for various techniques such as Northern blot, RTPCR, or RNase mapping. The combined inhibitory effect of both phenol and guanidium isothiocyanate (GITC) allows inactivation of most ribonuclease activity during RNA isolation. RNA quality is usually tested in the following three ways: visualization of 18S and 28S ribosomal RNA with agarose gel electrophoresis, detection of a particular mRNA expression by RT-PCR, and the RNA A260/280 ratio, which gives information about protein contamination. However, in our experiments, a residual degrading activity in ovine RNA prepared with the Chomczynski and Sacchi method has been detected. This degradation was not observed when RNA was dissolved in water, and the controls described above gave correct results. The degradation occurred at a later stage when RNA was incubated in an enzyme buffer such as DNase I buffer, reverse transcriptase buffer, or in vitro transcription buffer. Brain (hypothalamus and cortex) and liver total RNA were extracted from frozen artiodactyl and rodent tissues (-70°C) using the Chomzynski and Sacchi method (1). Briefly, tissue was homogenized at ambient temperature or at 4°C in a denaturing solution (Solution D) composed of 4 M GITC; 25 mM sodium citrate, pH 7.0; 0.5% sarcosyl; 0.1 M β-mercaptoethanol. Onetenth volume 2 M sodium acetate, pH 4.0, 1 volume phenol, and 0.2 volume choroform:isoamyl alcohol mixture (49:1) were successively added. The mixture was vigorously shaken and left for 15 min on ice. After centrifuging at 11 000× g for 20 min, the aqueous phase was mixed with one volume of isopropanol. The precipitated RNA was pelleted by centrifugation at 11 000× g for 30 min and resuspended in Solution D. After a second centrifugation, the pellet was rinsed with 75% ethanol and dissolved in Milli-Q® water (Millipore S.A., St. Quentin Yvelines, France) (2). Ten micrograms of RNA were treated with proteinase K (50 μg/mL final in 10 mM Tris-HCl, pH 7.5; 5 mM EDTA; 0.5% SDS) for 30 min at 37°C and then subjected to one phenol:chloroform extraction, followed by two chloroform extractions and ethanol precipitation with sodium acetate. RNA was dissolved in Milli-Q water. Ten micrograms of untreated RNA and proteinase K-treated RNA were incubated in either Milli-Q water or enzyme buffer for 30 min at 37°C and recovered by ethanol precipitation. After centrifugation, the pellet was resuspended in a denaturing loading buffer, and RNA was separated on 1% agarose gel containing formaldehyde and visualized with ethidium bromide staining. The electrophoresis on the denaturing gel in Figure 1A showed that the method of Chomczynski and Sacchi leads to the preparation of intact ovine hypothalamus RNA, when in Milli-Q water solution, even after 30 min incubation at 37°C (lanes 1 and 2). The A260/280 ratio was generally within an acceptable range (1.6–1.8), considering the slightly acidic pH of the solution after RNA dissolution in water (3). When incubated in DNase I buffer (33 mM Tris-acetate, pH 7.8, 66 mM potassium acetate, 10 mM magnesium acetate, 0.5 mM DTT; BD Biosciences Clontech, Palo Alto, CA, USA) for 10, 20, and 30 min at 37°C (lanes 3–5), RNA was detected as smearing with total disappearance of the 18S and 28S RNA (lanes 4 and 5). This degradation did not occur when the RNA preparation was first subjected to treatment with proteinase K. Proteinase K-treated RNA retained its integrity when incubated for 30 min at 37°C in DNase I buffer (lane 10). Moreover, this degrading activity disappeared when proteinase K-untreated RNA was incubated in DNase I buffer in the presence of RNase inhibitor (Rnasin®; Promega France, Charbonniéres, France) (lane 6). This may mean that the degradation is due to the action of a ribonuclease co-purified with RNA. The same pattern was observed in preparations of bovine and porcine brain RNA (Table 1), from which it could be concluded that this degrading activity may be found in artiodactyl species. This activity is not restricted to the hypothalamus, since we have also found it in cortex RNA preparations (Table 1). In contrast, we did not observe any degradation in rodent brain RNA, even when incubated in DNase I buffer at 37°C. The migration pattern of rat RNA on denaturing agarose gel showed profiles of intact RNA from the hypothalamus or cortex, incubated in either Milli-Q water or DNase I buffer (Figure 1B, lanes 1–4). The same result was obtained with mouse RNA (data not shown). This showed that the residual activity depicted above is species-specific in the brain. While it is found co-purified with RNA from artiodactyl species, no degradation can be detected in rodent RNA preparation when incubated with DNase I buffer at 37°C (results are summarized in Table 1). In sheep and rodents, we observed no difference between males and females (Table 1). To eliminate the latent degrading activity, two additional phenol:chloroform extractions were performed on the aqueous phase before isopropanol precipitation. This modification, intended to remove some residual proteins from preparations, did not alter RNA integrity when incubated in Milli-Q water (Figure 2A, lane 2). Surprisingly, the degrading activity was resistant to the three successive phenol:chloroform extractions (Figure 2A, lane 3). The degradation was also observed when sheep RNA was incubated in a reverse transcriptase buffer (50 mM Tris-HCl, pH 8.3, 75 mM KCl, 3 mM MgCl2; SuperScript II®; Invitrogen S.A.R.L., Cergy Pontoise, France) and in an in vitro transcription buffer (40 mM Tris-HCl, pH 8.0, 8 mM MgCl2, 2 mM spermidine, 50 mM NaCl, Stratagene, La Jolla, CA, USA) (Figure 2B, lanes 3 and 5). The integrity of proteinase K-treated RNA after incubation in these two buffers demonstrated that there was no exogenous RNase contamination during the experiment (lanes Benchmarks

Follicle-Stimulating Hormone Receptor: Advances and Remaining Challenges

Follicle-stimulating hormone (FSH) is produced in the pituitary and is essential for reproduction. It specifically binds to a membrane receptor (FSHR) expressed in somatic cells of the gonads. The FSH/FSHR system presents many peculiarities compared to classical G protein-coupled receptors (GPCRs). FSH is a large naturally heterogeneous heterodimeric glycoprotein. The FSHR is characterized by a very large NH2-terminal extracellular domain, which binds FSH and participates to the activation/inactivation switch of the receptor. Once activated, the FSHR couples to Gαs and, in some instances, to other Gα-subunits. GPCR kinases and β-arrestins are also recruited to the FSHR and account for its desensitization, the control of its trafficking and its intracellular signaling. Of note, the FSHR internalization and recycling are very fast and involve very early endosomes (EE) instead of EE. All the transduction mechanisms triggered upon FSH stimulation lead to the activation of a complex signaling network that controls gene expression by acting at multiple levels. The integration of these mechanisms not only leads to context-adapted responses from the target gonadal cells but also indirectly affects the fate of germ cells. Depending on the physiological/developmental stage, FSH elicits proliferation, differentiation, or apoptosis in order to maintain the homeostasis of the reproductive system. Pharmacological tools targeting FSHR recently came to the fore and open promising prospects both for basic research and therapeutic applications. This chapter provides an updated review of the most salient aspects and peculiarities of FSHR biology and pharmacology.

Evidence that histaminergic neurons are devoid of estrogen receptor alpha in the ewe diencephalon during the breeding season.

In sheep as in rat, it has been highly suggested that neuronal histamine (HA) participates to the estradiol (E2)-induced GnRH and LH surges, through H1 receptor. With the aim of determining if E2 could act directly on HA neurons, we examined here whether HA neurons express estrogen receptor alpha (ERα) in the ewe diencephalon during the breeding season. We first produced a specific polyclonal antibody directed against recombinant ovine histidine decarboxylase (oHDC), the HA synthesizing enzyme. Using both this anti-oHDC antibody and an anti-ERα monoclonal antibody in double label immunohistochemistry, we showed that HA neurons do not express ERα in diencephalon of ewes with different hormonal status. This result diverges from those obtained in rat, in which around three quarters of HA neurons express ERα in their nucleus. This discrepancy between these two mammal species may reflect difference in their neuronal network.

Selection and characterization of recombinant dromedary antiovalbumin antibody fragments that do not cross-react with ovalbumin-related protein x: use for immunoaffinity

Ovalbumin-related protein X (OVAX) and ovalbumin are two very close ovalbumin-related serpins. As primary data on OVAX remain recent, information about possible cross-reaction of available antiovalbumin antibodies with OVAX is still missing. Using labeled purified OVAX and dot ligand blotting, we identified 49 recombinant dromedary antiovalbumin single domain antibody (sdAb) fragments that were unable to bind OVAX. Discrimination between OVAX and ovalbumin was confirmed for two of the corresponding sdAb fragments by surface plasmon resonance and Western ligand blotting (WLB) characterizations. Furthermore, they were covalently linked to Sepharose and used as an affinity matrix for ovalbumin depletion. At least 90% of the original ovalbumin was eliminated from the allantoic fluid of 14 day old chicken embryo in one step. These sdAb fragments, which bind ovalbumin with nanomolar affinity, should also contribute to a better characterization of ovalbumin preparations.