Maria Greco

Adriana Chiappetta

Leonardo Bruno

Maria Beatrice Bitonti
email: b.bitonti@unical.it

Sébastien Bellow

Gwendal Latouche

Spencer C Brown

Anne Poutaraud

Zoran G Cerovic
email: zoran.cerovic@u-psud.fr

In Posidonia oceanica cadmium induces changes in DNA methylation and chromatin patterning

Keywords: 5-Methylcytosine-antibody, cadmium-stress condition, chromatin reconfiguration, CHROMOMETHYLASE, DNA-methylation, Methylation-Sensitive Amplification Polymorphism (MSAP), Posidonia oceanica (L.) Delile Disease diagnostics, optical proximal sensors, phenolic compounds, phytoalexin fluorescence, Plasmopara viticola, Vitis vinifera

In mammals, cadmium is widely considered as a non-genotoxic carcinogen acting through a methylation-dependent epigenetic mechanism. Here, the effects of Cd treatment on the DNA methylation patten are examined together with its effect on chromatin reconfiguration in Posidonia oceanica. DNA methylation level and pattern were analysed in actively growing organs, under short-(6 h) and long-(2 d or 4 d) term and low (10 mM) and high (50 mM) doses of Cd, through a Methylation-Sensitive Amplification Polymorphism technique and an immunocytological approach, respectively. The expression of one member of the CHROMOMETHYLASE (CMT) family, a DNA methyltransferase, was also assessed by qRT-PCR. Nuclear chromatin ultrastructure was investigated by transmission electron microscopy. Cd treatment induced a DNA hypermethylation, as well as an up-regulation of CMT, indicating that de novo methylation did indeed occur. Moreover, a high dose of Cd led to a progressive heterochromatinization of interphase nuclei and apoptotic figures were also observed after long-term treatment. The data demonstrate that Cd perturbs the DNA methylation status through the involvement of a specific methyltransferase. Such changes are linked to nuclear chromatin reconfiguration likely to establish a new balance of expressed/repressed chromatin. Overall, the data show an epigenetic basis to the mechanism underlying Cd toxicity in plants.

Introduction

In the Mediterranean coastal ecosystem, the endemic seagrass Posidonia oceanica (L.) Delile plays a relevant role by ensuring primary production, water oxygenation and provides niches for some animals, besides counteracting coastal erosion through its widespread meadows (Ott, 1980;Piazzi et al., 1999;Alcoverro et al., 2001). There is also considerable evidence that P. oceanica plants are able to absorb and accumulate metals from sediments (Sanchiz et al., 1990;Pergent-Martini, 1998;Maserti et al., 2005) thus influencing metal bioavailability in the marine ecosystem. For this reason, this seagrass is widely considered to be a metal bioindicator species (Maserti et al., 1988;Pergent et al., 1995;Lafabrie et al., 2007). Cd is one of most widespread heavy metals in both terrestrial and marine environments.

Although not essential for plant growth, in terrestrial plants, Cd is readily absorbed by roots and translocated into aerial organs while, in acquatic plants, it is directly taken up by leaves. In plants, Cd absorption induces complex changes at the genetic, biochemical and physiological levels which ultimately account for its toxicity (Valle and Ulmer, 1972;Sanitz di Toppi and Gabrielli, 1999;Benavides et al., 2005;Weber et al., 2006;Liu et al., 2008). The most obvious symptom of Cd toxicity is a reduction in plant growth due to an inhibition of photosynthesis, respiration, and nitrogen metabolism, as well as a reduction in water and mineral uptake (Ouzonidou et al., 1997;Perfus-Barbeoch et al., 2000;Shukla et al., 2003;Sobkowiak and Deckert, 2003).

At the genetic level, in both animals and plants, Cd can induce chromosomal aberrations, abnormalities in ª 2011 The Author(s). This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/bync/3.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Introduction

Early detection of biotic and abiotic stresses is a major issue in precision agriculture. Accordingly, the development of tools for proximal sensing to monitor plant health has gained in importance [START_REF] Sankaran | A review of advanced techniques for detecting plant diseases[END_REF]. Plant health survey and disease detection can be classed in two categories: indirect ground-based methods (reflectance and fluorescence spectroscopy and imaging, and gaseous metabolite profiling techniques) and direct laboratory-based detection methods (serological or molecular). The latter allows highly reliable assessment, but has a delayed response. Laboratory methods are time-consuming, labour intensive, and very dependent on sampling. Indirect methods based on optical properties are rapid, non-invasive, with immediate response, and above all they can be implemented on vehicles for mapping. Unfortunately optical methods are often less specific. In viticulture, downy mildew, caused by the oomycete Plasmopara viticola (Berk. & M.A. Curtis) Berl. & de Toni, is one of the main diseases, requiring numerous fungicide treatments in order to avoid damage and significant economic losses.

As a protection mechanism, grapevine produces stilbene phytoalexins. The role of stilbenes in the resistance of grapevine to P. viticola is well documented [START_REF] Jeandet | Phytoalexins from the Vitaceae: biosynthesis, phytoalexin gene expression in transgenic plants, antifungal activity, and metabolism[END_REF][START_REF] Chong | Metabolism and roles of stilbenes in plants[END_REF][START_REF] Jeandet | Biosynthesis, metabolism, molecular engineering and biological functions of stilbene phytoalexins in plants[END_REF], but it is still not fully elucidated. Stilbenes show a bright UV-induced violet-blue fluorescence (VBF) [START_REF] Hillis | Chromatographic and spectral properties of stilbene derivatives[END_REF]) that can be measured in vivo on grapevine leaves [START_REF] Langcake | Relationship of resveratrol production to infection of grapevine leaves by Botrytis cinerea[END_REF]Dai et al., 1995a, b). Indeed, under UV illumination, grapevine leaves are known to emit three types of autofluorescence: (i) the red fluorescence of chlorophyll (ChlF); (ii) the blue-green fluorescence (BGF) assigned to hydroxycinnamic acids [START_REF] Cerovic | Ultraviolet-induced fluorescence for plant monitoring: present state and prospects[END_REF][START_REF] Pfündel | Optical properties of plant surfaces[END_REF]; and (iii) a VBF of induced stilbenes in inoculated grapevine leaves [START_REF] Poutaraud | Fast and local assessment of stilbene content in grapevine leaf by in vivo fluorometry[END_REF][START_REF] Bellow | In vivo localization at the cellular level of stilbene fluorescence induced by Plasmopara viticola in grapevine leaves[END_REF]. In a previous study [START_REF] Bellow | In vivo localization at the cellular level of stilbene fluorescence induced by Plasmopara viticola in grapevine leaves[END_REF], confocal spectral microscopy was used for in vivo localization at the cellular level of stilbene fluorescence induced by P. viticola in grapevine leaves. Compartmentation differed between cultivars (resistant versus susceptible). Moreover, it was shown that due to microenvironment effects, compartmentation should strongly affect the fluorescent yield of stilbenes.

Autofluorescence can therefore be used as a non-destructive indicator of the presence of infection. On the one hand, fluorescence imaging is a particularly useful technique to assess autofluorescence changes due to stilbene accumulation in grapevine leaves at both macroscopic [START_REF] Poutaraud | Fast and local assessment of stilbene content in grapevine leaf by in vivo fluorometry[END_REF] and microscopic scales [START_REF] Poutaraud | Fast and local assessment of stilbene content in grapevine leaf by in vivo fluorometry[END_REF][START_REF] Bellow | In vivo localization at the cellular level of stilbene fluorescence induced by Plasmopara viticola in grapevine leaves[END_REF]. On the other hand, laboratory spectrofluorimetry allows an accurate and quantitative assessment of the intrinsic spectral signature of stilbenes that has been correlated to the stilbene content of leaves analysed by HPLC [START_REF] Poutaraud | Fast and local assessment of stilbene content in grapevine leaf by in vivo fluorometry[END_REF]. However, there is no report yet on the behaviour of stilbene VBF during the full course of P. viticola infections until the late visible symptoms (oily spots).

The aims of this study were 2-fold: (i) to characterize on a daily basis the kinetics of autofluorescence in plant-attached grapevine leaves responding to P. viticola infection during at least 2 weeks following their inoculation until characteristic visible symptoms are established; and (ii) to analyse different fluorescence indices as the basis for downy mildew diagnosis in the field. To reach these goals, the monitoring of BGF, VBF, ChlF, and blue-excited green fluorescence (GF) at three spatial scales was performed simultaneously: imaging by fluorescence macroscopy (submillimetric), spectrofluorimetry (millimetric), and proximal sensing (whole-leaf scale) with two sensors, the Multiplex 3 sensor [START_REF] Ben Ghozlen | Non-destructive optical monitoring of grape maturation by proximal sensing[END_REF] and a proximal fluorescence sensor prototype with short-wave UV sources.

Materials and methods

Plants of Vitis vinifera cv. Cabernet Sauvignon (a genotype susceptible to P. viticola) were grown from cuttings in Colmar (France) at 22 ± 3 °C, 13/11 h light/dark in the greenhouse. The study was started when plants attained the stage of ~15 leaves. The plants were settled outdoors next to the greenhouse in a place that was never in the shade in Colmar (latitude 48°05N, longitude 07°20E) in August 2010 for 15 d. This outdoor regimen guaranteed that the plants which were initially grown in the greenhouse protected from biotic or abiotic stresses had a flavonol content equivalent to vineyard leaves at the moment of inoculation [START_REF] Kolb | Effects of natural intensities of visible and ultraviolet radiation on epidermal ultraviolet screening and photosynthesis in grape leaves[END_REF]. As the constitutive flavonol have been shown to participate in the resistance of grapevine leaves to P. viticola [START_REF] Agati | Optically-assessed preformed flavonoids and susceptibility of grapevine to Plasmopara viticola under different light regimes[END_REF], the outdoor treatment was fundamental for the results to be applicable to vineyard-grown leaves. Plasmopara viticola was obtained from naturally infected plants in Colmar. Sporangia were periodically grown in order to prepare inoculum. Sporangia were diluted in distilled water, counted, and then adjusted to a concentration of ~5 × 10 4 sporangia ml -1 . The fifth fully expanded leaf counted from the apex, still attached to the plant, was inoculated. Three protocols of inoculation were used depending on the experiment. For protocols 1 and 2, the shoot was positioned on a horizontal surface and the leaf laid flat with the abaxial side upwards. For fluorescence macroscopy, protocol 1, five drops of 12 µl of inoculum suspension and five drops of distilled water (control) were applied on the abaxial side of each leaf. For spectrofluorimetry, protocol 2, six drops of 200 µl of inoculum suspension and three drops of distilled water were applied on the abaxial side of each leaf. For proximal sensing, protocol 3, nine fifth leaves of different plants were each totally immersed in a test tube containing 60 ml of inoculum suspension. After 5 h of incubation, leaves were cleaned with distilled water and superficially dried. Plants were then transported (by land transport, in humidified portable mini-greenhouses) from Colmar to Gif-sur-Yvette (France) for fluorescence macroscopy, and to Orsay (France) for spectrofluorimetry and proximal sensing. During the 15 d of experiments, except when being measured, plants were maintained either in their humidified portable mini-greenhouses inside a growth chamber (22 °C, 13/11 h light/ dark) or directly in the greenhouse for proximal sensing. The success of infection was attested to by the presence of sporulation on leaves kept in a humid environment, or by oily spots (without sporulation) on other leaves. All measurements were done with leaves attached to the plant.

Fluorescence imaging

Images were acquired using a macroscope (AZ100 multizoom, Nikon, Champigny-sur-Marne, France) equipped with a 130 W metal halide lamp white source (Intensilight, Nikon) and a high-resolution colour camera (Ds-Ri, Nikon). The UV-suppression filter of this source was removed. The images of UV-excited visible autofluorescence were recorded using a custom-made filter block from AHF (Tübingen, Germany) with an excitation bandpass filter 340/26 (FF01 Brightline, Semrock, Rochester, NY, USA), a dichroic filter Q380LP (Chroma Technology Corp., Bellows Falls, VT, USA), and a long-pass 371 nm emission filter (LP02-364RS, Semrock). The images of blue-excited green autofluorescence were recorded using a GFP-B filter set (excitation band pass filter 472/30, dichroic filter 495 nm, and emission bandpass filter 520/35, Nikon). A ×2 objective (NA 0.2, working distanced 45 mm, AZ-Plan Fluor, Nikon) was used, and 24-bit RGB colour images were acquired with a 1284 × 1024 pixel resolution. Imaged leaves, still attached to the plant, were carefully flattened (abaxial side facing the objective) on the glass sample holder (adaxial side lightly moistened for adhesion). The flatness of the imaged area was necessary for a good-quality acquisition. Sporangiophores were washed from the sporulating leaves to avoid their contribution to VBF. Image acquisition was performed using the NIS-Elements software (Nikon). Image analysis, including composition, was performed using the software ImageJ (http://rsbweb.nih.gov/ij/). The presented data are representative results of several experiments.

Spectrofluorimetry

Excitation and emission fluorescence spectra were acquired with a spectrofluorimeter (Cary Eclipse, Varian, Les Ulis, France) using a configuration adapted to attached leaves based on a double-arm optical fibre bundle (C Technologies, Cedar Knolls, NJ, USA) made of 147 randomized fibres. The two arms of the bundle were coupled to the excitation and emission part of the spectrofluorimeter via a fibre-optic coupler accessory provided by Varian (part no. FA-VAR00-AP15). The common part of the fibre bundle was maintained at a fixed distance (5 mm) from the samples by a proprietary clip. Under these conditions, every day from 1 DPI to 15 DPI at about the same time, the spectra of the same marked circular regions (diameter 5.5 mm) of the abaxial side of each leaf were recorded. Excitation spectra were corrected with a calibrated photodiode (S1337-1010BQ, Hamamatsu, Massy, France), and emission spectra were corrected using a standard lamp with a known spectrum (LI-COR 1800-02, LI-COR, Lincoln, NE, USA) as described in detail previously [START_REF] Louis | Quantitative study of fluorescence excitation and emission spectra of bean leaves[END_REF]. In addition, fluorescence was expressed in quinine sulphate equivalent units (QSEU) [START_REF] Cerovic | Ultraviolet-induced fluorescence for plant monitoring: present state and prospects[END_REF]: 1000 QSEU correspond to the fluorescence of 1 µM quinine sulphate dihydrate in 0.105 M perchloric acid for 1 cm light path square cells or, in general, the fluorescence of 1 nmol cm -2 of this standard excited at 347.5 nm and emitting at 450 nm under the identical conditions used to acquire the sample fluorescence spectrum.

Proximal sensing

Multiplex ® 3 (FORCE-A, Orsay, France) is a hand-held, multiparametric fluorescence sensor based on light-emitting diode excitation and filtered photodiode detection that is designed to work in the field under daylight conditions (for detailed description and specifications, see Ben [START_REF] Ben Ghozlen | Non-destructive optical monitoring of grape maturation by proximal sensing[END_REF]. In the present investigation, the Multiplex 3 (with a 6 cm diameter measuring area) was used for daily measurements of the abaxial and adaxial sides of infected (protocol 3) leaves (fifth from the apex) and control leaves (sixth from the apex).

Several Multiplex 3 indices were followed: (i) SFR_R, a chlorophyll fluorescence emission ratio linked to the leaf chlorophyll content; (ii) R-590, the leaf reflectance at 590 nm, named YF_G in the Multiplex 3, that also reflects changes in leaf chlorophyll content, increasing with chlorophyll content decrease; (iii) FER_RG, a chlorophyll fluorescence red-to-green (635/516 nm) excitation ratio originally designed for fruit anthocyanin content, but which is here inversely correlated to the chlorophyll content of leaves devoid of anthocyanins; (iv) FLAV, a chlorophyll fluorescence red-to-UV (635/375 nm) excitation ratio that is a measure of epidermal flavonols; and (v) YF_UV, the 375 nm excited yellow autofluorescence (590 nm, FWHM 10 nm) of the leaves.

The prototype of a new proximal sensor, the Mx-330 (FORCE-A, Orsay, France), was used to measure daily the in vivo VBF of the abaxial and adaxial sides of infected (protocol 3) leaves (fifth from the apex) and control leaves (sixth from the apex), in parallel with the Multiplex 3 measurements (the same region of the leaf). The Mx-330 sensor was based on the Multiplex 3 design (mechanical structure and electronics) but specifically adapted to measure in vivo the stilbene VBF on grapevine leaves (335 nm excitation-400 nm emission). The sensor illuminates a 6 cm diameter surface at a 4 cm distance from the source and detectors. The leaves were flattened as much as possible during the measurements.

Statistical analyses

Statistical analyses were performed using the software Statistica 6.1 (StatSoft Inc., Maison-Alfort, France). As normality (Shapiro-Wilk test) or homoscedasticity (Levene test and Brown-Forsythe test) were often violated, the analysis of variance (ANOVA) could not be used. Instead, the significance of the difference between means was assessed by three non-parametric tests: a Mann-Whitney U-test, a Wald-Wolfowitz runs test, and a Kolmogorov-Smirnov test. The three non-parametric tests usually produced the same results. In the event of discordance, the result of the most stringent test was retained and presented.

Results

Daily imaging of autofluorescence of a grapevine leaf infected by P. viticola

For each treatment (inoculated or control), similar results were obtained in five regions. Images presented in Fig. 1D-F are representative of an inoculated region occupied by one inoculum drop of 12 µl followed during 15 d. The first tiny necrotic spots could be seen on transmission images in the middle of infected areoles at 3 DPI (Fig. 1D). A thickening and an extension of the necrotic spots were observed on the following days. At 7 DPI, a yellowing of the abaxial surface was observed due to a local decrease of the chlorophyll content in leaf tissues (Fig. 1D). This chlorosis of infected areoles increased daily during the remaining course of the survey. At 15 DPI, which corresponds to the end of the survey, the chlorosis appeared clearly limited by veins of infected areoles. The UV-induced autofluorescence images revealed a few spots of VBF at 1 DPI (Fig. 1E). At 2 DPI, these early spots of VBF were no longer visible. At 3 DPI, infected areoles displayed an intense VBF. This signal reached a maximum intensity at 5 DPI and then decreased at 7 DPI and remained at a low but noticeable level for the rest of the survey. From 5 to 15 DPI, the VBF appeared clearly limited to the infected areoles. The chlorosis and the stilbene VBF appeared co-localized. Bright GF spots also appeared at 3 DPI (Fig. 1F). A larger and more diffuse GF was present at 7 DPI in infected tissue, but also restricted to infected areoles. Thereafter, GF covered a region (Fig. 1F) seemingly superimposed upon the chlorotic region (Fig. 1D). From 7 DPI, GF progressively increased until the end of the survey. Images of control regions displayed no changes during the whole survey. Transmission images remained green due to the presence of chlorophyll, with the exception of white veins (Fig. 1A), and no variation was observed on fluorescence images (Fig. 1B,C). The UV-induced visible fluorescence of the abaxial side of control regions appeared purple due to the mix between the red ChlF abundant in the mesophyll and the BGF of constitutive phenolic compounds present in epidermal cell walls (Fig. 1B). The GF displayed no variation in the control region (Fig. 1C).

Daily kinetics of autofluorescence measured by spectrofluorimetry at the abaxial side of grapevine leaf infected by P. viticola

Changes in both BGF excitation and emission spectra associated with the P. viticola infection have been investigated at the abaxial side of a leaf from 1 to 15 DPI (Fig. 2). The spectra were obtained from samples independent of but comparable with those used for images presented in Fig. 1. The results shown are means of measurements on six infected regions and three control regions. From 1 to 12 DPI on control regions (Fig. 2B) and at 1 and 2 DPI on the inoculated regions (Fig. 2A), fluorescence excitation and emission spectra had the usual shape of control grapevine leaves [START_REF] Cerovic | Ultraviolet-induced fluorescence for plant monitoring: present state and prospects[END_REF][START_REF] Poutaraud | Fast and local assessment of stilbene content in grapevine leaf by in vivo fluorometry[END_REF]. From 4 DPI onwards, a second peak emerged at 330 nm on fluorescence excitation spectra of inoculated regions due to the accumulation of stilbenes (Fig. 2A). In fluorescence emission spectra excited at 330 nm, this resulted in a hypsochromic shift of the maximum emission from 440 nm to 400 nm (Fig. 2A). These spectral signatures that reflect the accumulation of stilbenes [START_REF] Poutaraud | Fast and local assessment of stilbene content in grapevine leaf by in vivo fluorometry[END_REF][START_REF] Bellow | In vivo localization at the cellular level of stilbene fluorescence induced by Plasmopara viticola in grapevine leaves[END_REF] were observed from 4 to 15 DPI on the inoculated region and the two last DPI, 14 and 15, on control regions (Fig. 2B). It should be noted here that the control regions were situated on the same leaf, close to infected regions. The daily dynamics of the VBF (330 nm excitation and 400 nm emission) are represented in the inset in Fig. 2C. The increase in VBF on inoculated regions is clear at 4 DPI. The maximum was reached at 6 DPI and then it decreased to reach a plateau from 8 DPI onwards. On control regions, as expected, the VBF value remained low from 1 to 12 DPI. However, at 12 DPI, the autofluorescence started to increase to reach a value close to the plateau of VBF in inoculated regions at 15 DPI.

Fluorescence excitation and emission spectra of Fig. 3 were acquired in order to analyse the GF. In inoculated regions (Fig. 3A), on the excitation spectra (emission at 525 nm), a small peak appeared at 400 nm in addition to the increase at 330 nm due to stilbenes. On control regions, fluorescence spectra changed neither in shape nor in amplitude (Fig. 3B) until 14 DPI. Indeed, the GF emission spectra did not change in shape during the whole survey, both in inoculated and in control regions. The daily dynamics of the fluorescence emission at 525 nm excited at 430 nm are represented in the inset (Fig. 3C). On inoculated regions, the GF, which significantly increased from 4 to 6 DPI, remained higher for infected regions than for control regions until 15 DPI, with a slight increase from 10 to 15 DPI. On control regions, a very small but continuous increase in the GF was observed from 4 to 15 DPI.

Proximal sensing with the Mx-330 of the daily kinetics of violet-blue fluorescence in leaves infected by P. viticola

Mx-330 is a new prototype sensor optimized for the detection of stilbene VBF conceived for outdoor field studies. In order to profit from the large surface sensed by the Mx-330, the entire leaves were inoculated. A significant difference in VBF was measured already at 1 DPI at the abaxial side of infected leaves (Fig. 4B). At the adaxial side, the difference in stilbene VBF between inoculated and control leaves started to be significant at 3 DPI (Fig. 4A). The signal increased on both sides to reach a maximum intensity at ~6-7 DPI. A decrease of the signal was observed from 7 to 8 DPI and then it tended to stabilize until the end of the survey at a significantly larger value than the control. The signal measured at the abaxial side of infected leaves was about double that of the adaxial side. Apart from this difference of fluorescence level, the same dynamics of stilbene VBF were measured on both sides of the leaf.

Proximal sensing with the Multiplex 3 of the daily kinetics of optical parameters in leaves infected by P. viticola

The Multiplex 3 is a field-portable proximal sensor that can gather information on the changes in leaf chlorophyll content, flavonol content, and yellow fluorescence. The sensing of whole leaves was done on the same leaves analysed with the Mx-330. Globally, at the adaxial leaf side, the SFR_R index, reflecting the leaf chlorophyll content [START_REF] Tremblay | Sensing crop nitrogen status with fluorescence indicators. A review[END_REF], had values about double those of the abaxial side (Fig. 5A,B). A slight increase in SFR_R, corresponding to an accumulation of chlorophyll by the still developing leaves, was observed from 1 to 5 DPI, both in inoculated and in control leaves, especially at the adaxial side. As expected, the SFR_R index remained constant in control leaves throughout the rest of the survey, while a significant decrease was observed on both abaxial and adaxial sides of inoculated leaves (Fig. 5A,B). Two other Multiplex 3 indices, the reflectance at 590 nm, R-590 (Fig. 5C,D), and the red-to-green fluorescence excitation ratio, FER_RG (Fig. 5E,F), reflected the same changes in chlorophyll. The latter index is sensitive to chlorophyll changes in the absence of anthocyanins. The observed loss of chlorophyll confirmed by three Multiplex 3 indices corroborated the visual observations (data not shown) and the chlorosis reported in Fig. 1D.

The FLAV index was ~5 times larger on the adaxial side than on the abaxial leaf side (Fig. 5G,H). This reflects the difference in epidermal flavonol contents between adaxial and abaxial side of leaves, a well-known characteristic of dicot plants [START_REF] Cerovic | The use of chlorophyll fluorescence excitation spectra for the non-destructive in situ assessment of UV-absorbing compounds in leaves[END_REF]. While the FLAV index remained constant through the entire study on the adaxial side, an increase could be seen on the abaxial side of inoculated leaves. An increase of the YF_UV index was also recorded for Fig. 2. Changes in UV-excited visible autofluorescence spectra of grapevine leaves during P. viticola infection. Daily fluorescence excitation spectra with an emission wavelength at 400 nm and emission spectra with the UV-excitation wavelength set at 330 nm were acquired from the abaxial side of a Cabernet Sauvignon leaf on circular spots (5.5 mm diameter). The presented results are means of spectra acquired on six inoculated spots (A) and on three control spots (B). Note that the y-axis scale in A is double that in B. All spots were exactly the same every day. Standard deviations of the spectra can be visualized from the inset C that displays the daily means and standard deviation of VBF (330 nm excitation and 400 nm emission). inoculated leaves, especially at the abaxial leaf side. Although not optimized for the present study, this narrow-band index of yellow fluorescence excited by a 375 nm UV light is related to the GF presented in Figs 1C, F, and3.

Discussion

Fluorescence measured by spectroscopy or imaging has two advantages: it is very sensitive and can be applied at different scales, from microscopy to remote sensing. Imaging allows assessment of the spatial distribution of signals, while spectroscopy can assess the variation of signals with larger sensitivity and from a distance. Works of [START_REF] Poutaraud | Fast and local assessment of stilbene content in grapevine leaf by in vivo fluorometry[END_REF] and [START_REF] Bellow | In vivo localization at the cellular level of stilbene fluorescence induced by Plasmopara viticola in grapevine leaves[END_REF] have shown that in vivo VBF is a good indicator of the presence of stilbene phytoalexins in grapevine leaves induced upon infection by P. viticola. In addition, changes in variable ChlF were shown to be a good indicator of downy mildew due to its impairment of photosynthesis [START_REF] Csefalvay | Pre-symptomatic detection of Plasmopara viticola infection in grapevine leaves using chlorophyll fluorescence imaging[END_REF].

Spatiotemporal characteristics of the autofluorescence of grapevine leaves during infection by P. viticola

Images acquired in this study showed that the stilbene VBF was strictly limited to the vein-delimited areoles inoculated by P. viticola. Outside this region, no extensions of visible symptoms of chlorosis or necrosis were observed. Apparently, the development of P. viticola hyphae is generally stopped by veins in leaf tissues [START_REF] Unger | The course of colonization of two different Vitis genotypes by Plasmopara viticola indicates compatible and incompatible host-pathogen interactions[END_REF]. Therefore, the results confirm that visible symptoms and fluorescence induced by the P. viticola infection are both limited to the primary infection site areoles. Sporulation appeared at 4 DPI for the leaves kept in a water-saturated atmosphere in portable minigreenhouses; this confirmed the success of the infection (not shown). Sporangiophores were washed away daily so they appear neither on transmission (Fig. 1D) nor on fluorescence images (Fig. 1E,F), nor did they influence the fluorescence spectra (Figs 2, 3) (cf. Supplementary Fig. S1 available at JXB online). For proximal sensing, plants were kept in the dry atmosphere of a greenhouse and there was no sporulation, hence also no influence of the sporangiophores on the measurements.

The results of this study set the detection threshold of stilbene VBF at 3 DPI at the adaxial leaf side of a susceptible grapevine leaf. On the abaxial side, inoculated leaves had significantly larger VBF than control leaves from day 1 after inoculation. Therefore, the stilbene VBF seems a promising asymptomatic signal to detect infections. These 15 d of the survey revealed the transient nature of the stilbene VBF summarized in Fig. 6, with three characteristic phases: a lag phase, a transient phase characterized by an increase in VBF to a maximum around 6 DPI, followed by a decrease and a stabilization of VBF at a level significantly above the control in the third phase. This last phase was concomitant with the appearance of visible symptoms of chlorosis. The same phases were observed with fluorescence imaging on 0.2 mm 2 , measured with the spectrofluorimeter on 20 mm 2 , and assessed with the new proximal sensor Mx-330 on 2000 mm 2 on both the abaxial and the adaxial side of the whole infected leaves. Blueexcited GF increased continuously during the third phase. Sporulation appeared at the beginning of the transient phase (4 DPI). The transitory nature of VBF raises the question of the fate of stilbenes during the infection. The dynamics of stilbene VBF suggest that a large proportion of phytoalexins detected in infected grapevine leaves are either degraded or metabolized into molecules with a significantly lower fluorescence yield soon after the transient phase. Laccases are fungal enzymes that have the capability to metabolize stilbene phytoalexins produced by the hosts, as seen in the case of Botrytis cinerea infections [START_REF] Pezet | Evidence for oxidative detoxication of pterostilbene and resveratrol by a laccase-like stilbene oxidase produced by Botrytis cinerea[END_REF][START_REF] Sbaghi | Degradation of stilbene-type phytoalexins in relation to the pathogenicity of Botrytis cinerea to grapevines[END_REF]. Laccase activity is assumed to detoxify trans-resveratrol by oxidative polymerization and subsequently to facilitate the infection [START_REF] Jeandet | Phytoalexins from the Vitaceae: biosynthesis, phytoalexin gene expression in transgenic plants, antifungal activity, and metabolism[END_REF]. Although laccases have not been found in P. viticola, a multicopper oxidase was identified recently in this oomycete that could have the same role [START_REF] Mestre | Identification of effector genes from the phytopathogenic Oomycete Plasmopara viticola through the analysis of gene expression in germinated zoospores[END_REF].

The origin of the GF has yet to be determined. However, GF seems both temporally and spatially correlated with the loss of chlorophyll, and therefore the visual symptoms of chlorosis. The decrease in chlorophyll content in infected leaves was sensed by Multiplex 3 indices of chlorophyll content (SFR_R, R-590 and FER_RG) on both the adaxial and the abaxial side of leaves. Dicot leaves, such as in V. vinifera, have palisade (on the adaxial side) and spongy (on the abaxial side) parenchyma with very different histological and optical properties and chlorophyll content. Palisade parenchyma had higher chlorophyll content than spongy parenchyma, shown by the SFR difference in Fig. 5A versus B, and the reflectance index (R-590) in Fig. 5C versus D (opposite response to SFR) for both inoculated and control leaves. The chlorophyll content of a leaf will be the sum of the index measured on each of the two sides. The highly diffusive properties of the abaxial leaf side precluded the detection of significant differences between inoculated and control leaves for R-590. The green-tored fluorescence excitation ratio (FER_RG) has only recently been linked to leaf chlorophyll content when anthocyanins are absent (Z.G. Cerovic, unpublished). It will have higher values for lower chlorophyll contents (opposite response to SFR) due to lower green light absorption. The local loss of chlorophyll, seen by fluorescence indices in the third phase of the infection, will decrease the reabsorption of pre-existing green-fluorescing compounds and lead to an increase in GF [START_REF] Cerovic | Ultraviolet-induced fluorescence for plant monitoring: present state and prospects[END_REF]. This is supported by the absence of changes in the shape of GF spectra during the infection.

Towards new non-destructive indices to detect downy mildew

The similar results obtained by the three approaches used in this study, albeit a four order difference in sensed area size, prove the universality and robustness of the stilbene VBF and GF as indicators of P. viticola infection. In addition to the use of VBF in microscopy to study P. viticola-grapevine interactions [START_REF] Bellow | In vivo localization at the cellular level of stilbene fluorescence induced by Plasmopara viticola in grapevine leaves[END_REF], stilbene fluorescence seems suited for the development of an accurate tool for field detection. The spectra obtained allow the optimal wavelengths (330 nm excitation, 400 nm emission) most specific for stilbene fluorescence in grapevine leaves to be defined. Although the stilbene signal measured at the adaxial side of the leaves was half that measured at the abaxial side, the Mx-330 index allowed a promising discrimination as early as 3 DPI. However, the inoculation performed on entire leaves reached all parts of the leaves. Natural P. viticola droplet infections rarely cover the whole leaf surface. So a lower stilbene-dependent signal can be expected in the field. Still, the specificity and advantage of fluorescence over reflectance is its very high sensitivity. Up to a limit (concentration quenching), fluorescence will be the same from a given amount of fluorophore distributed uniformly over an area or aggregated on small spots. Further experiments in the field on spontaneous infections are required, and these are now feasible thanks to the availability of the Mx-330.

VBF appears as an early indicator of downy mildew, while GF is specific to the late stage of the infection, temporally correlated with visible symptoms of chlorosis. Stilbenes are phytoalexins of grapevine, induced by either biotic or abiotic stresses [START_REF] Jeandet | Phytoalexins from the Vitaceae: biosynthesis, phytoalexin gene expression in transgenic plants, antifungal activity, and metabolism[END_REF] and not specific to a particular disease. Using stilbene VBF to probe downy mildew in the field might not be specific enough because other factors could lead to the production of stilbenes. Therefore, additional signatures of the infection could be advantageous to discriminate specifically P. viticola infections. In the present study, it was found that GF of grapevine leaves was both spatially and temporally correlated with downy mildew late symptoms of chlorosis. Even if this signal does not provide an early diagnosis, it provides evidence of the presence of downy mildew. The comparison of four independent sensing techniques-imaging, specrofluorimetry, UV-excited blue fluorescence sensing (Mx-330), and multiwavelength proximal sensing (Multiplex 3)-has allowed a robust description of the three characteristic phases of autofluorescence of grapevine leaves during the infection by P. viticola (Fig. 6). Variable ChlF [START_REF] Csefalvay | Pre-symptomatic detection of Plasmopara viticola infection in grapevine leaves using chlorophyll fluorescence imaging[END_REF] needs imaging and is not available for measurement with mobile platforms. This seems not to be the case with simple autofluorescence signals or ratios such as VBF, VBF/GF, SFR, and FER_RG. Furthermore, the availability of several signals could help to discriminate downy mildew among the biotic and abiotic stresses.

This study was performed on attached leaves in order to maintain them under physiological conditions. However, plants were grown in a controlled environment compared with field-grown plants that are potentially facing multiple stresses. So the next step would be to perform a similar kinetic investigation involving Multiplex 3 and Mx-330 in the field during spontaneous infections. Although demonstrated here only for the V. vinifera-P. viticola pathosystem, the approach presented herein can be extended to other pathosystems involving fluorescent phytoalexins found in other species, such as coumarins in sunflower or isoflavonoids in soybean [START_REF] Grayer | A survey of antifungal compounds from higher-plants, 1982-1993[END_REF].

Fig. 1 .

 1 Fig. 1. Images of the abaxial side of a V. vinifera cv. Cabernet Sauvignon leaf during P. viticola infection. Numbers of days postinoculation (DPI) by P. viticola are increasing from left to right. (A-C) Control region (2.28 × 1.71 mm). (D-F) Inoculated region (4.07 × 3.04 mm). A and D are white-light transmission images. B and E are images of UV-excited visible autofluorescence. C and F are images of blue-excited green autofluorescence. Images were taken daily, but not all are shown. Bars, 500 µm. For details, see the Materials and methods.

Fig. 3 .

 3 Fig. 3. Green autofluorescence (GF) emission spectra and associated excitation spectra of grapevine leaves during P. viticola infection. Daily fluorescence excitation spectra with an emission at 525 nm and emission spectra with the blue-excitation wavelength set at 430 nm were obtained as described in Fig. 2. (A) Mean spectra of inoculated spots, (B) mean spectra of control spots, (C) daily means and standard deviation of GF (430 nm excitation and 525 nm emission).

Fig. 4 .

 4 Fig. 4. Changes in violet-blue autofluorescence of grapevine leaves during P. viticola infection followed by the Mx-330 sensor.A large surface (6 cm diameter) of both the adaxial (A) and the abaxial (B) side of Cabernet Sauvignon leaves was sensed from the first to the 15th day after inoculation. Note that the y-axis scale in B is double that in A. Grey lines are daily kinetics of individual leaves and bold lines are means with standard deviations of eight inoculated leaves and eight control leaves. The significance of the difference between control and inoculated leaves for each day is indicated on the top of the graphs: NS, not significant; *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 5 .

 5 Fig. 5. Changes in Multiplex 3 indices during P. viticola infection of grapevine leaves. Both adaxial (A, C, E, G, I) and abaxial (B, D, F, H, J) side of leaves were measured on the same leaves shown in Fig. 4. Grey lines are individual leaves and bold lines are means with standard deviations of nine inoculated leaves and nine control leaves. For the description of different Multiplex 3 indices, see the Materials and methods.

at INRA Avignon on December 21, 2012 http://jxb.oxfordjournals.org/ Downloaded from

Acknowledgements

This work was supported by FORCE-A (Orsay) in a joint project with Centre National de la Recherche Scientifique (CNRS), Institut National de la Recherche Agronomique (INRA), and Université Paris-Sud 11 (UPS) (grant nos CNRS 065103, INRA 28000039, and UPS N73800) and by IFR87 'La plante et son environnement' local grant. Fluorescence macroscopy was done on the Imagif Platform with the support of Marie-Noëlle Soler (IFR87). We are grateful to Marie Annick Dorne and Pascale Coste (INRA, Colmar) for growing the grapevine plants and for the inoculation, and to Sabine Merdinoglu and Didier Merdinoglu (INRA, Colmar) for their support.

Supplementary data

Supplementary data are available at JXB online.

Figure S1. Images of sporulating leaves before and after removal of sporangiophores.


Laura L Torres-Rovira

Anne Couturier-Tarrade

Susana S Astiz

Eve Mourier Mourier

Mariluz M Perez-Solana

Paloma P De

La Cruz

Ernesto E Gomez-Fidalgo

Raul R Sanchez-Sanchez

Pascale Chavatte-Palmer

Antonio A Gonzalez-Bulnes

Anne Tarrade

Paloma De La Cruz

Antonio Gonzalez- Bulnes

Sex and Breed-Dependent Organ Development and Metabolic Responses in Foetuses from Lean and Obese/ Leptin Resistant Swine

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Obesity and obesity-associated hyperleptinaemia caused by leptin resistance have been linked to reproductive disorders in human beings [START_REF] Metwally | Reproductive endocrinology and clinical aspects of obesity in women[END_REF][START_REF] Brewer | The adverse effects of obesity on conception and implantation[END_REF] and some animal species like the pig [START_REF] Lo ´pez-Bote | Sustained utilization of the Iberian pig breed[END_REF][START_REF] Ra ´tky | Comparison of follicular and oocyte development and reproductive hormone secretion during the ovulatory period in Hungarian native breed, Mangalica, and Landrace gilts[END_REF]. In both obese human and pigs, poor reproductive outcomes have been related to impairments of ovarian function and, mainly, to deficiencies in endometrial receptivity, implantation and early embryo development [START_REF] Alfer | The endometrium as a novel target for leptin: differences in fertility and subfertility[END_REF][START_REF] Gonzalez | Leptin and leptin receptor are expressed in the human endometrium and endometrial leptin secretion is regulated by the human blastocyst[END_REF][START_REF] Pasquali | Metabolic effects of obesity on reproduction[END_REF][START_REF] Bellver | Obesity and poor reproductive outcome: the potential role of the endometrium[END_REF][START_REF] Pasquali | Obesity and infertility[END_REF][START_REF] Gonzalez-An ˜over | Ovulation rate, embryo mortality and intrauterine growth retardation in obese swine with gene polymorphisms for leptin and melanocortin receptors[END_REF][START_REF] Gonzalez-Bulnes | Reproductive, endocrine and metabolic feto-maternal features and placental gene expression in a swine breed with obesity/leptin resistance[END_REF][START_REF] Torres-Rovira | The interaction between ovulation rate and embryo survival in determining prolificacy of different strains of obese swine with gene polymorphisms for leptin receptors[END_REF].

In human pregnancies, foetal development may be afterwards hampered by either restricted-or excess-growth. Offspring will be born small-or large-for-gestational-age at birth, respectively, as early described in humans by Prof. Smith in the late 1940's [START_REF] Smith | Effects of maternal malnutrition on fetal development[END_REF][START_REF] Cochrane | Overnutrition in prenatal and neonatal life: a problem?[END_REF]. The most troubling fact is that, in both cases, life-long health and fitness of offspring are affected. Evidences of the determinant role of the prenatal environment on the adult phenotype, strongly reinforced by birth-records of the Dutch Hunger Winter [START_REF] Stein | The Dutch famine, 1944-1945, and the reproductive process. I. Effects on six indices at birth[END_REF][START_REF] Stein | The Dutch famine, 1944-1945, and the reproductive process. II. Interrelations of caloric rations and six indices at birth[END_REF][START_REF] Ravelli | Obesity in young men after famine exposure in utero and early infancy[END_REF] and the studies of Prof. Barker [START_REF] Barker | Fetal origins of coronary heart disease[END_REF], gave way to the concept of the Developmental Origin of Health and Disease (DOHaD; [START_REF] Gluckman | Living with the past: evolution, development, and patterns of disease[END_REF]). Currently, the hypothesis of DOHaD is well-supported by abundance of data from different mammalian species [START_REF] Gonzalez-Bulnes | Genetic basis, nutritional challenges and adaptive responses in the prenatal origin of obesity and type-2 diabetes[END_REF].

In pigs, it is important to study the effects of prenatal nutrition on the adult phenotype both from the viewpoint of their use as biomedical models but also from the viewpoint of animal production, health and welfare [START_REF] Wu | Intrauterine growth retardation: Implications for the animal sciences[END_REF]. The failure of some foetuses in the litter for achieving their genetically determined potential size and, thus, the birth of some piglets smaller than normal size (intrauterine growth retardation; IUGR) are common and widely described for lean swine. IUGR in lean pigs is mainly due to intrauterine crowding and subsequent placental insufficiency in high-prolific lines [START_REF] Wu | Intrauterine growth retardation: Implications for the animal sciences[END_REF][START_REF] Ashworth | Causes and consequences of fetal growth retardation in pigs[END_REF][START_REF] Foxcroft | The biological basis for prenatal programming of postnatal performance in pigs[END_REF]. Information on the occurrence of IUGR in obese breeds is scarcer than in lean swine, but previous studies in obese Iberian pigs provide strong evidence of a high incidence of IUGR occurring from very early-pregnancy stages [START_REF] Gonzalez-An ˜over | Ovulation rate, embryo mortality and intrauterine growth retardation in obese swine with gene polymorphisms for leptin and melanocortin receptors[END_REF][START_REF] Gonzalez-Bulnes | Gender-specific early postnatal catch-up growth after intrauterine growth retardation by food restriction in swine with obesity/leptin resistance[END_REF], in spite of a low prolificacy. Piglets affected by IUGR have high rates of mortality and morbidity at early postnatal stages. The health and welfare of the surviving IUGR offspring are compromised by gastrointestinal, metabolic, respiratory and immune dysfunctions, and they have reduced growth potential and a high predisposition for adiposity [START_REF] Gonzalez-Bulnes | Gender-specific early postnatal catch-up growth after intrauterine growth retardation by food restriction in swine with obesity/leptin resistance[END_REF][START_REF] Quiniou | Variation of piglets' birth weight and consequences on subsequent performance[END_REF][START_REF] Kuhn | Heavy newborn piglets develop a high carcass quality[END_REF][START_REF] Bee | Effect of early gestation feeding, birth weight, and gender of progeny on muscle fibre characteristics of pigs at slaughter[END_REF][START_REF] Rehfeldt | Consequences of birth weight for postnatal growth performance and carcass quality in pigs as related to myogenesis[END_REF], which bears a resemblance with findings in human medicine.

The concept of DOHaD states that the adult phenotype is the result of the interaction between prenatal nutrition and early postnatal environments, but there is also a strong influence of genetic background [START_REF] Hanson | Developmental plasticity and developmental origins of non-communicable disease: theoretical considerations and epigenetic mechanisms[END_REF]. In this context, the availability of obese and lean swine breeds displaying IUGR provides the possibility of an interesting model for the study of the role of such genetic predisposition. The most representative obese breed is the Iberian pig (Sus scrofa meridionalis), which is genetically different from the modern commercial breeds (Sus scrofa ferus, [START_REF] Sancristobal | Genetic diversity within and between European pig breeds using microsatellite markers[END_REF][START_REF] Ollivier | European pig genetic diversity: a minireview[END_REF]). These animals have been reared in semi-feral conditions for centuries and have coped with seasonal cycles of feasting and famine by storing excess fat during food abundance [START_REF] Silio | Developing breeding strategies for lower input animal production environments[END_REF][START_REF] Lo ´pez-Bote | Sustained utilization of Iberian pig breed[END_REF]. In fact, Iberian pigs have a higher voluntary food intake and a higher trend towards adiposity than lean swine breeds [START_REF] Nieto | The effect of dietary protein content and feeding level on the rate of protein deposition and energy utilization in growing Iberian pigs from 15 to 50 kg body weight[END_REF][START_REF] Ovilo | Fine mapping of porcine chromosome 6 QTL and LEPR effects on body composition in multiple generations of an Iberian by Landrace intercross[END_REF][START_REF] Mun ˜oz | Single and joint population analyses of two experimental pig crosses to confirm QTL on SSC6 and LEPR effects on fatness and growth traits[END_REF]. The abundance of fat increases secretion of leptin [START_REF] Fernandez-Figares | Serum profile of metabolites and hormones in obese (Iberian) and lean (Landrace) growing gilts fed balanced or lysine deficient diets[END_REF], which should diminish food intake. However, the Iberian pig has a gene polymorphism for the leptin receptor (LEPR), which has not been found in lean swine breeds, similar to the syndrome of leptin resistance described in human medicine [START_REF] Myers | Mechanisms of leptin action and leptin resistance[END_REF][START_REF] Lubis | The role of SOCS-3 protein in leptin resistance and obesity[END_REF][START_REF] Mizuta | Leptin gene and leptin receptor gene polymorphisms are associated with sweet preference and obesity[END_REF]. The LEPR gene polymorphism of the Iberian breed has effects on food intake and fat deposition [START_REF] Ovilo | Fine mapping of porcine chromosome 6 QTL and LEPR effects on body composition in multiple generations of an Iberian by Landrace intercross[END_REF][START_REF] Mun ˜oz | Single and joint population analyses of two experimental pig crosses to confirm QTL on SSC6 and LEPR effects on fatness and growth traits[END_REF] and, thus, Iberian pigs are prone to obesity. Hence, the Iberian swine, having in mind is unique characteristic of leptin resistance, is specially useful as a robust, amenable and reliable translational model for studies on obesity, metabolic syndrome and nutritionassociated diseases in humans [START_REF] Torres-Rovira | Dietinduced swine model with obesity/leptin resistance for the study of metabolic syndrome and type 2 diabetes[END_REF][START_REF] Torres-Rovira | Effect of an obesogenic diet during the juvenile period on growth pattern, fatness and metabolic, cardiovascular and reproductive features of swine with obesity/leptin resistance[END_REF].

Thus, the aim of the present study was to set a basis for such studies by determining possible breed-related differences in growth patterns and metabolic features of the feto-placental unit at advanced pregnancy stages through the comparison of obese Iberian and lean commercial crossbred sows. Foetal development and viability are largely known to be affected by the litter size and, thus, availability of uterine space [START_REF] Webel | Effect of stage of gestation and uterine space on prenatal survival in the pig[END_REF]. Hence, we used a commercial lean crossbred strain (25% Large White 625% Landrace 650% Pietrain) selected for meat efficiency but not for prolificacy to match the low prolificacy of the Iberian breed. At the same time, to avoid any influence from maternal nutrition, both breeds were fed with the same diet, adjusted to fulfil pregnancy requirements according to individual weights.

Materials and Methods

Ethics statement

The experiment was carried out under Project License from the INIA Scientific Ethic Committee and animal manipulations were performed according to the Spanish Policy for Animal Protection RD1201/05, which meets the European Union Directive 86/609 about the protection of animals used in research. We hereby confirm that the INIA Scientific Ethic Committee, which is the named IACUC for the INIA, specifically approved this study (report CEEA 2010/003).

Animals and management

The study involved seven Iberian (group IB) and six lean crossbred sows (25% Large White 625% Landrace 650% Pietrain; group L) that became pregnant after cycle synchronization with altrenogest (RegumateH, MSD, AH, Boxmeer, The Netherlands) and insemination to purebred litters. All the females were nulliparous, were treated at the same time, and have twelve months of age when they were inseminated. The IB sows had been genotyped for polymorphism on LEPR gene with protocols previously described [START_REF] Ovilo | Fine mapping of porcine chromosome 6 QTL and LEPR effects on body composition in multiple generations of an Iberian by Landrace intercross[END_REF] and found to be homozygous for the allele LEPRc.1987T. All the females from both groups were housed together in collective pens at the facilities of the INIA Animal Laboratory Unit (Madrid, Spain), which meets the requirements of the European Union for Scientific Procedure Establishments. The sows were individually fed with a standard grain-based diet (89.8% of dry matter, 15.1% of crude protein, 2.8% of polyunsaturated fat and 3.00 Mcal/kg of metabolizable energy) with ad libitum access to water. The amount of food offered to each sows was adjusted to body-weight and calculated for fulfilling the requirements of pregnancy development, based on data from the British Society of Animal Science [START_REF]Nutrient requirement standards for pigs[END_REF].

The experimental sampling was performed in all the sows at Day 62 of pregnancy.

Morphometric evaluation and sampling of sows, genital tracts and foetuses

At Day 62 of pregnancy (around mid-pregnancy), all sows were weighed and measured for back-fat depth. Fat depth was determined by using a SonoSite S-Series ultrasound machine equipped with a multifrequency (5-8 MHz) lineal array probe (SonoSite Inc., Bothell, WA). The probe was placed against the skin, in a point at the right side of the animal located at 4 cm from the midline and transversal to the head of the last rib as determined by palpation. Concurrently, blood samples were drawn by puncture of the orbital sinus [START_REF] Huhn | Application of the orbital sinus bleeding technique to swine[END_REF], into 5 ml sterile heparinized vacuum tubes (Vacutainer TM Systems Europe). Immediately after recovery, the blood was centrifuged at 1500 g for 15 min and the plasma was separated and stored into polypropylene vials at 220uC until assay for determination of leptin and parameters of carbohydrate, protein and lipids metabolism. Subsequently, the sows were euthanized by i.v. injection with a euthanasia solution (T-61, MSD AH, Boxmeer, The Netherlands). The entire genital tracts were immediately collected for morphometric examination, weighing and sampling.

Ovulatory sites in the ovaries were assessed for the determination of ovulation rate and for the evaluation of morphologically normal and regressing corpora lutea. Regressing corpora lutea were white opaque, smaller in size than normal corpora lutea and scarcely vascularised, as previously described [START_REF] Gonzalez-An ˜over | Ovulation rate, embryo mortality and intrauterine growth retardation in obese swine with gene polymorphisms for leptin and melanocortin receptors[END_REF]. Samples of two normal corpora lutea from each ovary were used for in vitro culture and determination of progesterone secretion.

Each uterus was measured in length and weighted. Afterwards, its content was exposed and the conceptuses were recorded according to their position. Immediately, for each conceptus, blood samples were drawn from heart and/or umbilical cord with heparinised syringes, samples of allantoic and amniotic fluids were obtained by aspiration through the chorioallantoic and amniochorion membranes, respectively, and one sample of around 1 cm 2 of placental tissue (including trophoblast and uterine endometrium) was collected for histological evaluation. Allantoic and amniotic fluids were centrifuged at 1500 g for 15 min and supernatants were stored into polypropylene vials at 220uC until assay. Samples of placental tissue were immediately fixed with 4% paraformaldehyde. Finally, all the conceptuses were recovered to be sexed, measured and weighted and the empty uterus was weighted.

Culture of luteal tissue and assessment of progesterone secretion

The technique for in vitro culture of luteal tissue was adapted from procedures previously described [START_REF] Astiz | In vitro release of ovarian progesterone is decreased during the oestrous cycle and pregnancy of swine with obesity/leptin resistance[END_REF]. Briefly, each corpus luteum was sliced into small strips (each one weighing around 40 mg) and washed several times in sterile saline solution. Two individual strips from each corpus luteum were placed in culture glass tubes (one strip/tube) with culture medium (Dulbecco's Modified Eagle's Medium and Ham's F-12 medium 1:1 [v.v]; Sigma-Aldrich Chemie, Steinheim, Germany) and one of the tubes was supplemented with 100 ng/ml of porcine LH (bio-potency 428 UI/mg; Laboratorios Calier, S.A, Barcelona, Spain). The strips were then incubated, on a shaking platform, for 24 hours at 38.5uC and 5% CO 2 atmosphere. After incubation, the media was recovered and frozen at 220uC until measurement of progesterone. The strips were blotted and weighed, in order to determine the hormone secretion in terms of ng per mg of luteal tissue.

Progesterone concentrations in plasma and culture media were measured in a single analysis using enzymeimmunoassay kits (Demeditec Diagnostics GmbH, Kiel-Wellsee, Germany). The assay sensitivity was 0.04 ng/ml and the intra-assay variation coefficient was 5.4%. Subsequently, the percentage of LHstimulated strips for which progesterone secretion was more than 5% of the basal production of their untreated counterparts (positive reactivity) was also assessed.

Evaluation of absolute and relative measures and weights of the foetuses

Immediately after recovery, crown-rump length, thoracic diameter, occipito-nasal length and biparietal diameter were measured in all the foetuses. The total weight of the foetus and the weights of the head, trunk and main organs (brain, heart, liver, intestine and kidneys) were also assessed. Afterwards, the following weight ratios were considered: weight of the uterus relative to the individual and total foetal weight, weight of the foetal head relative to total weight and trunk weight and brain weight relative to the weight of total viscera, heart, liver, kidneys and intestine.

Evaluation of maternal and foetal metabolic status

Concentrations of leptin in maternal plasma were determined in a single analysis using the Multi-species Leptin RIA kit (Demeditec Diagnostics GmbH, Kiel-Wellsee, Germany). The assay sensitivity was 1.0 ng/ml; the intra-assay variation coefficient was 3.1%.

Parameters related to the metabolism of glucose and lipids (triglycerides, total cholesterol, high-density lipoproteins cholesterol [HDL-c] and low-density lipoproteins cholesterol [LDL-c]) were measured in maternal and foetal plasma and allantoic and amniotic fluids with a clinical chemistry analyzer (Screen Point, Hospitex Diagnostics, Sesto Fiorentino, Italy), according to the manufacturer's instructions.

Evaluation of foetal endocrine and inflammatory status

Concentrations of oestradiol and IL-6 in foetal plasma and allantoic and amniotic fluids were measured by using enzymeimmunoassay kits (Demeditec Diagnostics GmbH, Kiel-Wellsee, Germany for estradiol and DuoSet ELISA Development kit from R&D Systems Europe, Ltd. Abingdon, UK for IL-6). The assay sensitivities were 1.4 pg/ml and 100 pg/ml for oestradiol and IL-6, respectively; intra-assay variation coefficients were 5.7% for estradiol and 6.8% for IL-6, respectively.

Evaluation of placental histology

Samples of placental tissue were maintained in 4% paraformaldehyde for three hours at room temperature and overnight at 4uC. They were transferred to PBS the next day and maintained at 4uC until processed for histology and paraffin embedded. Sections of 7 mm were cut and subsequently stained with haematoxylin-eosin, mounted and scanned (Nanozoomer Digital Pathology 2.3, Hamamatsu Photonics, Hamamatsu, Japan). Stereology was performed on each section using the One Stop Stereology technique of Mercator software (Explora Nova, La Rochelle, France) for determining volume fractions of foetal vessels, connective tissue, trophoblast, uterine glands and maternal vessels, as described [START_REF] Reed | One-stop stereology: the estimation of 3D parameters using isotropic rulers[END_REF].

Statistical analyses

Effects of breed on metabolic and reproductive features of the sows and effects of the breed, sex and normal/IUGR status of the foetus on morphological, endocrine, metabolic and inflammatory characteristics of the conceptuses were assessed by analysis of variance (one-way ANOVA) or by a Kruskall-Wallis test when a Levene's test showed non-homogeneous variables. IUGR was defined as the observation of an individual weight under one standard deviation of the mean litter value, as described elsewhere [START_REF] Stein | The Dutch famine, 1944-1945, and the reproductive process. I. Effects on six indices at birth[END_REF]. All the results were expressed as mean 6 SEM and statistical significance was accepted from P,0.05.

Results

Effect of breed on maternal fatness, metabolism and reproductive features

Weight, subcutaneous fat depth and plasma concentrations of lipid metabolites were significantly different between Iberian (group IB) and lean crossbred sows (group L). The IB females were lighter (131.162.6 vs. 164.769.5 kg, P = 0.01), but had more back-fat depth (36.960.7 vs. 16.761.5 mm, P = 0.0005). Plasma concentrations of leptin (2.660.4 vs. 1.860.1 ng/ml, P = 0.08), triglycerides (56.668.1 vs. 27.767.9 mg/dl, P = 0.02) and cholesterol (94.365.3 vs. 72.864.3 mg/dl, P = 0.01; mainly, LDL-c: 57.964.7 vs 44.965.4 mg/dl, P = 0.09) were significantly higher in IB sows.

The mean number of ovulatory sites on the ovaries was significantly lower in the IB compared to the L group (14.460.7 vs. 22.561.4, P = 0.0005). The same was found when comparing only corpora lutea with adequate development (10.760.4 vs. 15.561.5, P = 0.007). The number of viable foetuses, however, was not different between the two breeds (7.161.2 in the IB vs. 8.261.6 in the L group, P = 0.623).

Plasma progesterone concentrations were significantly lower in IB compared to lean sows (37.962.6 vs. 64.563.6 ng/ml, P,0.005). Similarly, both basal and LH-induced in vitro secretion of progesterone were significantly lower in IB than in L females (39.963.3 vs. 70.765.3 and 52.565.7 vs. 76.766.4 ng/mg of luteal tissue, P,0.0001 for both).

Effect of breed and sex on foetal development

General observations. The data obtained from the evaluation of a total of 50 IB and 49 L foetuses (around 50% of males and females in both groups) are detailed in Table 1. Foetuses from the L group were 11% larger and 20% heavier than IB foetuses (P = 0.0001 for both variables). In contrast, the total foetal weight to total uterine weight ratio was higher in IB foetuses (21.261.7 vs 15.561.2; P = 0.005).

Organ weights in all foetuses. Individual organs were also significantly heavier in L foetuses (Table 1). However, the relative weight of the head and brain compared to total body weight (Figure 1, P = 0.0005 for both) and to other organs (Figure 1, P,0.005 for all) was significantly higher in IB foetuses. The relative weight of intestine/body also tended to be higher in IB foetuses (P = 0.05), whilst the ratios of heart and kidneys weights to body weight were higher in the L foetuses (P = 0.0005 for both).

IUGR foetuses. IUGR was found in 16.7% of the L and 21.9% of the IB foetuses. Relative brain to body weight, weight of total thoracic viscerae, of total abdominal viscerae, of liver (P,0.005 for all) and weight of intestine (P = 0.04) was significantly higher in the IB foetuses affected by IUGR when compared to littermates with adequate development, but this was not the case in the L conceptuses with IUGR.

Sex differences. In the L group, male and female foetuses had similar sizes but male foetuses were 9% heavier (Table 1, P = 0.006). There were no significant sex-related differences in the IB group. There were no sex-related effects on the head and brain to entire body and organs ratios in any breed. In the IB foetuses, however, the liver to body weight ratio was higher in females compared to males (P = 0.04). There was also a trend (P = 0.06) for a higher ratio of intestine to body weight in females than males.

Effect of breed, sex and IUGR on foetal endocrine, metabolic and inflammatory features

Oestradiol concentrations were significantly higher in the amniotic fluids of IB conceptuses (266.3631.0 vs. 142.6612.9 pg/ml in the L group, P = 0.002); without differences related to IUGR or sex.

Parameters related to glucose and lipid metabolism in foetal plasma are described in Tables 2 and3. Plasma glucose and cholesterol concentrations, mainly LDL-c, were significantly higher in IB compared to L conceptuses (P,0.01 for all), due to significantly higher values in IB males than in L males for total cholesterol and LDL cholesterol (P,0.005) and for triglycerides (P = 0.06). On the other hand, triglyceride concentrations were lower in the IB foetuses (P = 0.009), without differences between sex. Amniotic fluid concentrations were consistently lower in IB compared to L foetuses (P,0.05 for all) whereas only total and LDL cholesterol were found to be lower in IB compared to L groups for allantoic fluid (P = 0.06 and P = 0.006, respectively).

The comparison between foetuses with adequate development and foetuses affected by IUGR showed no significant differences in the IB breed, but IUGR conceptuses from the L group showed a trend for a higher plasma cholesterol concentrations (66.7611.0 vs. 54.7 2.2, P = 0.09), due to a significant higher level of HDL-c (14.462.2 vs. 5.060.7, P,0.0005).

IL-6 concentrations in the foetal plasma and allantoic and amniotic fluids were significantly lower in IB than in L conceptuses (3046.96489.7 vs. 5531.66560.4 pg/ml, P = 0.001). Moreover, IL-6 concentration was significantly lower in IB than in L males (2746.36488.3 vs. 6569.86694.5 pg/ml, P,0.0005) but not in females. IL-6 concentrations were also significantly lower in IUGR compared to normal foetuses of both breeds (P,0.05).

Effect of breed, sex and IUGR on placental features

There were no significant differences in the stereological analysis of the placental components among breeds, sexes and normal/IUGR foetuses, except for a higher volume fraction of trophoblastic vessels in the IB IUGR foetuses when compared to normal littermates (10.961.3 vs. 7.660.6, P = 0.02), with no difference between males and females.

Discussion

The present study evidences the existence of differential developmental patterns in Iberian foetuses, which would be protective for the adequate growth and postnatal survival of the individuals. Such differential patterns were more obvious in female compared to male foetuses which, in addition to sex-related differences in the hypothalamic expression of anorexigenic and orexigenic peptides at birth (Ovilo, unpublished data), would explain the early-postnatal catch-up growth previously reported in female, rather than male, piglets of the Iberian breed [START_REF] Gonzalez-Bulnes | Gender-specific early postnatal catch-up growth after intrauterine growth retardation by food restriction in swine with obesity/leptin resistance[END_REF].

The analysis of maternal phenotypes in the present study clearly confirmed the trend for the Iberian pig for a higher adiposity than modern commercial lean crosses, despite the fact that they ate the same maintenance diet. The Iberian sows of the current trial were fatter and had higher plasma concentrations of leptin, triglycerides and cholesterol than their lean crossbred counterparts. The results of the present study also indicate a lower ovulatory quota for the Iberian breed when compared to the lean females, in agreement with some previous studies [START_REF] Gonzalez-An ˜over | Ovulation rate, embryo mortality and intrauterine growth retardation in obese swine with gene polymorphisms for leptin and melanocortin receptors[END_REF] but opposite to other results obtained by our group [START_REF] Gonzalez-An ˜over | Preovulatory follicle dynamics and ovulatory efficiency in sows with thrifty genotype and leptin resistance due to leptin receptor gene polymorphisms (Iberian pig)[END_REF][START_REF] Gonzalez-An ˜over | Patterns of corpora lutea growth and progesterone secretion in sows with thrifty genotype and leptin resistance due to leptin receptor gene polymorphisms (Iberian pig)[END_REF]. The inconsistencies among these studies may be explained by the results of a subsequent trial which highlighted differences in ovulatory patterns between different Iberian dam-lines and their critical importance when studying prolificacy in the Iberian breed [START_REF] Torres-Rovira | The interaction between ovulation rate and embryo survival in determining prolificacy of different strains of obese swine with gene polymorphisms for leptin receptors[END_REF]. The data of the present trial also evidence a lower progesterone secretion in the Iberian than in the lean sows, supporting previous findings obtained when comparing Iberian and lean crosses [START_REF] Astiz | In vitro release of ovarian progesterone is decreased during the oestrous cycle and pregnancy of swine with obesity/leptin resistance[END_REF]. Overall, the results of the current study emphasize previous information suggesting that the main factor determining prolificacy in both Iberian and lean crossbred swine is embryo/foetal viability [START_REF] Gonzalez-An ˜over | Ovulation rate, embryo mortality and intrauterine growth retardation in obese swine with gene polymorphisms for leptin and melanocortin receptors[END_REF][START_REF] Torres-Rovira | The interaction between ovulation rate and embryo survival in determining prolificacy of different strains of obese swine with gene polymorphisms for leptin receptors[END_REF][START_REF] Foxcroft | The biological basis for prenatal programming of postnatal performance in pigs[END_REF][START_REF] Freking | Number of fetuses and conceptus growth throughout gestation in lines of pigs selected for ovulation rate or uterine capacity[END_REF]. The analysis of foetal phenotypes in the present study clearly indicates breed-related differences in weight and size between foetuses of Iberian and lean-crossbred sows. Such differences may be explained by intrinsic differences in body size and weight between both groups rather than by a reduced developmental rate of Iberian foetuses. Indeed, Iberian pigs are significantly smaller than commercial lean pigs, even in neonatal stages [START_REF] Charneca | Body composition and blood parameters of newborn piglets from Alentejano and conventional (Large White6Landrace) genotype[END_REF].

Apart from breed-related differences in body weight and size, a crucial result of the present study is the existence of a very clearly different developmental pattern in Iberian and lean-crossbred conceptuses. Organ analysis indicates that the relative weight of the brain was higher in the Iberian foetuses, whilst the relative weights of other organs like heart and kidneys were lower, when compared to lean counterparts. These data suggest that brain growth is prioritized in the Iberian foetuses and resemble the ''brain-sparing effect'', first described in humans [START_REF] Rudolph | The fetal circulation and its response to stress[END_REF]. The brainsparing effect has been associated in many mammals to intrauterine growth retardation (IUGR) by nutritional and metabolic deficiencies [START_REF] Wu | Intrauterine growth retardation: Implications for the animal sciences[END_REF][START_REF] Vuguin | Animal models for small for gestational age and fetal programing of adult disease[END_REF][START_REF] Metges | Intrauterine growth retarded progeny of pregnant sows fed high protein:low carbohydrate diet is related to metabolic energy deficit[END_REF]. In the current study, there were no significant differences in the occurrence of IUGR between groups. However, similarly to the normal foetuses, the brainsparing effect was more evident in the Iberian IUGR foetuses. The objective of brain-sparing is to assure that brain growth is protected, since a failure in the supply of oxygen and/or nutrients to the developing brain causes a broad spectrum of adverse neurological outcomes [START_REF] Peeling | Review of literature showing that undernutrition affects the growth rate of all processes in the brain to the same extent[END_REF][START_REF] Salihagic | Fetal cerebrovascular response to chronic hypoxia-implications for the prevention of brain damage[END_REF]. Such deficiencies can compromise the critical functions such as breathing, suckling and/or any of the so called autonomic functions [START_REF] Perry | Childhood experience and the expression of genetic potential: What childhood neglect tells us about nature and nurture?[END_REF]. Hence, brain sparing assures vitality and survival of the neonate.

The relatively more advanced growth of the brain in the Iberian foetuses suggests the existence of a strong protective mechanism to improve postnatal survival of Iberian piglets. The main factors determining optimal brain development are genetics and environment [START_REF] Shonkoff | From neurons to neighborhoods: The science of early childhood development[END_REF][START_REF] Welberg | Prenatal stress, glucocorticoids and the programming of the brain[END_REF]. The Iberian swine has an ancient origin, being traced back approximately to year 1000 BC. It has been scarcely selected and has been reared in extensive semi-feral conditions for centuries. In such conditions, the survival of the piglets is largely dependent on their vitality, vivacity and ability for suckling, moving and interacting with their mother. We can conjecture that the brain-sparing characteristics observed in Iberian piglets may support these features and, hence, may favour a better postnatal survival.

The occurrence of breed-related protective effects is also supported in our study by the finding of a significant higher volume fraction of foetal vessels in the Iberian IUGR foetuses when compared to normal littermates; such difference was not found in the lean foetuses. This increase in foetal vessels may be a compensatory response to improve the blood flow and the efficiency of nutrients transport. These data are consistent with those of Blomberg [60] which demonstrated an increase of the expression of endothelial-constitutive nitric oxide synthases (eNOS or NOS3), implicated in blood vessel dilatation or growth, in the IUGR placenta at the same period. The nitric oxide (NO) pathway for increasing angiogenesis and adequate development of embryo blood flow is associated with the secretion of estrogens by the conceptus [START_REF] Andronowska | Influence of estradiol-17 and progesterone on nitric oxide (NO) production in the porcine endometrium during first half of pregnancy[END_REF], which, concurrently, was found to be higher in the Iberian foetuses of the current trial.

The other main result of the present study is the existence of sex-related effects in developmental patterns of the Iberian foetuses, which were not found in the lean crossbred conceptuses. The males were significantly heavier than the female foetuses in the lean group whilst the weight of male and female Iberian foetuses was similar, which indicates an enhanced development of the females in this breed. As a matter of fact, male and female Iberian offspring have similar weights and body measurements at birth. In case of critical maternal malnutrition, however, newborn females are lighter but develop early-postnatal catch-up growth to reach higher body weights than their brothers [START_REF] Gonzalez-Bulnes | Gender-specific early postnatal catch-up growth after intrauterine growth retardation by food restriction in swine with obesity/leptin resistance[END_REF]. Such sexrelated catch-up growth would be favoured by a lower hypothalamic expression of anorexigenic peptides and a higher expression of orexigenic ones (Ovilo, unpublished results) and possibly by faster intestinal development and improved food absorption, since the Iberian female foetuses showed a trend for higher intestine to body weight ratios than their male counterparts. Advanced development of the intestine improves neonatal absorption and utilization of nutrients and other substances like immunoglobulins and, thus, would increase the survival possibilities and the performance of the piglet [START_REF] D'inca | Intrauterine growth restriction modifies the developmental pattern of intestinal structure, transcriptomic profile, and bacterial colonization in neonatal pigs[END_REF].

Furthermore, the relative liver to body weight ratio was higher in Iberian female foetuses compared to males. Faster liver development may favour postnatal growth [START_REF] Antipatis | Moderate maternal vitamin A deficiency affects perinatal organ growth and development in rats[END_REF], through a ''liver-sparing effect'', which appears in addition to the ''brainsparing effect'' in foetuses undergoing adverse conditions [START_REF] Haugen | Fetal liversparing cardiovascular adaptations linked to mother's slimness and diet[END_REF]. The equilibrium or the predominance of one or another effect is driven by the availability of nutrients and even by the foetal sex [START_REF] Godfrey | Fetal liver blood flow distribution: role in human developmental strategy to prioritize fat deposition versus brain development[END_REF].

Adequate liver development is associated with an increase in hepatic glycogen deposition and activity of key gluconeogenic enzymes, thus enhancing hepatic glucogenic capacity in preparation for the nutritional transition from continuous maternal supply of nutrients via the placenta to a more intermittent supply from the milk via the intestine after birth [START_REF] Shelley | Glycogen reserves and their changes at birth and in anoxia[END_REF][START_REF] Dawkins | Biochemical aspects of developing function in newborn mammalian liver[END_REF][START_REF] Trahair | Systemic and luminal influences on the perinatal development of the gut[END_REF]. Differences in foetal liver developmental patterns may interfere in the pre-and postnatal metabolism of nutrients other than glucose, such as amino-acids, proteins, lipids, vitamins and minerals [START_REF] Liu | Intrauterine growth restriction alters the hepatic proteome in fetal pigs[END_REF]. The foetal liver is also actively participating in the synthesis of complex lipids (mainly cholesterol, [START_REF] Seccombe | Fetal utilization of maternally derived ketone bodies for lipogenesis in the rat[END_REF]) that are essential for pre-and postnatal development [START_REF] Mcneil | The effect of fetal pig size and stage of gestation on tissue fatty acid metabolism and profile[END_REF]. In this way, foetal development is highly dependent on the adequacy of the maternal supply of cholesterol and triglycerides and foetal growth may be disturbed either by deficiency [START_REF] Metges | Intrauterine growth retarded progeny of pregnant sows fed high protein:low carbohydrate diet is related to metabolic energy deficit[END_REF][START_REF] Ruwe | Effect of maternal fasting on fetal and placental lipid metabolism in swine[END_REF][START_REF] Sattar | Lipid and lipoprotein concentrations in pregnancies complicated by intrauterine growth restriction[END_REF][START_REF] Cetin | Intrauterine growth restriction is associated with changes in polyunsaturated fatty acid fetalmaternal relationships[END_REF] or excess [START_REF] Woollett | Maternal cholesterol in fetal development: transport of cholesterol from the maternal to the fetal circulation[END_REF][START_REF] Woollett | Review: transport of maternal cholesterol to the fetal circulation[END_REF]. In the current study, the availability of triglycerides and cholesterol in female Iberian foetuses was similar to that of lean crossbred foetuses. Moreover, the availability of IL-6 was lower in Iberian males but similar in female Iberian and lean crossbred foetuses. Since IL-6 is a pleiotropic cytokine that it is also involved in glucose and lipid metabolism [START_REF] Pittas | Adipocytokines and insulin resistance[END_REF][START_REF] Street | Impairment of insulin receptor signal transduction in placentas of intra-uterine growth restricted newborns and its relationship with foetal growth[END_REF][START_REF] Street | Markers of insulin sensitivity in placentas and cord serum of intrauterine growth restricted newborns[END_REF], these data strengthen the evidences of sexdriven differences in metabolism. More work is needed to understand the physiological implications of such differences.

Overall, all the features described above indicate a better postnatal survival and development of females, which are more critical for the survival of the species. Thus, these results support previous evidences in obese humans and murine models exposed to malnutrition indicating that female survival is higher compared to males [START_REF] Ozaki | Dietary restriction in pregnant rats causes gender-related hypertension and vascular dysfunction in offspring[END_REF][START_REF] Woods | Programming of adult blood pressure by maternal protein restriction: Role of nephrogenesis[END_REF][START_REF] Grigore | Sex differences in the fetal programming of hypertension[END_REF][START_REF] Mingrone | Influence of maternal obesity on insulin sensitivity and secretion in offspring[END_REF][START_REF] Mao | Contrasting effects of different maternal diets on sexually dimorphic gene expression in the murine placenta[END_REF].

In conclusion, the current study is supporting the existence of sex-and breed-related differences in morphometric, metabolic and inflammatory patterns in swine foetuses of obese and lean phenotypes that may be extended to postnatal stages. These findings set the basis for future translational studies aimed at increasing the knowledge on the interaction between genetic and environmental factors in the determination of adult phenotype [START_REF] Zimmet | The pathogenesis and prevention of diabetes in adults. Genes, autoimmunity and demography[END_REF]. Such studies are of paramount importance for understanding and preventing the rise of obesity and associated diabetes and cardiovascular diseases in populations adapted for surviving in harsh environments but currently exposed to nutrient excess, like China, India and Middle East countries [START_REF] Scully | Diabetes in numbers[END_REF].

Figure 1 .

 1 Figure 1. Effects of breed on the relative development of foetuses and their organs. Mean 6 S.E.M. ratios of head weight to total body weight (A), of individual weight of selected organs to total body weight (B) and of brain weight to the weights of total viscera and selected organs (C) in Iberian purebred (black bars) and lean crossbreed foetuses (white bars). Asterisk indicate significant differences between breeds (* = p,0.05, ** = p,0.01, *** = p,0.005, **** = p,0.001, ***** = p,0.0005). doi:10.1371/journal.pone.0066728.g001

Table 1 .

 1 Effects of breed and sex on foetal development.

		Iberian breed		Lean crossbred
		Female	Male	Female	Male
	Body length (cm)	10.360.1 i		11.560.1 j
		10.260.1 i	10.360.2 i	11.460.2 j	11.660.1 j
	Occipito-nasal length (cm)	4.960.1 e		5.160.1 f
		4.960.1 a	4.960.1 a	5.160.1 b	5.160.1 b
	Biparietal diameter (cm)	2.560.1 i		2.760.1 j
		2.560.1 e	2.660.1 c	2.860.1 f	2.760.1 d
	Trunk diameter (cm)	4.060.1 i		4.360.1 j
		4.060.1 a	4.060.1 e	4.260.1 b	4.360.1 f
	Body weight (g)	122.862.2 i		152.862.5 j
		119.062.7 i	126.063.3 i	146.063.2 j,3	159.463.4 j,4
	Head weight (g)	30.460.5 i		34.960.5 j
		30.460.6 i	30.660.8 i	33.860.6 j ,1	36.060.8 j,2
	Trunk weight (g)	90.661.8 i		116.262.1 j
		87.962.2 i	93.562.7 i	110.462.7 j,3	121.762.9 j,4
	Brain weight (g)	4.260.1 a		4.060.1 b
		4.260.1 c	4.360.1	3.860.1 d,1	4.160.1 2
	Heart weight (g)	0.860.1 i		1.160.1 j
		0.760.1 i	0.760.1 i	1.160.1 j	1.260.1 j
	Liver weight (g)	6.160.2 i		7.460.2 j
		6.260.2 a	6.060.2 i	6.960.2 b,1	7.760.2 j, 2
	Intestine weight (g)	3.060.1 e		3.460.1 f
		3.160.1	2.960.2 e	3.360.1	3.660.2 f
	Kidneys weight (g)	1.460.1 i		2.360.1 j
		1.460.1 i	1.460.1 i	2.260.1 j	2.460.1

j Mean 6 S.E.M. measurements and weights of Iberian purebred and lean crossbreed foetuses and selected organs. Different letter in superscripts indicate significant differences between breeds (a?b, p,0.05; c?d, p,0.01; e?f, p,0.005; g?h, p,0.001; i?j, p,0.0005) whilst different number denotes significant differences between sexes within the same breed (1?2, p,0.05; 3?4, p,0.01; 5?6, p,0.005). doi:10.1371/journal.pone.0066728.t001

Table 2 .

 2 Effects of breed and sex on foetal glucose metabolism.

		Iberian breed		Lean crossbred
		Female	Male	Female	Male
	Glucose in	52.664.1 i		24.563.7 j
	foetal plasma
	(mg/dl)
		55.064.6 i	50.966.4 e 25.663.7 j	23.162.7 f
	Glucose in	32.861.7 a		39.162.4 b
	amniotic fluid
	(mg/dl)
		34.262.8	31.161.8	40.163.6	38.263.2
	Glucose in	14.661.9		13.361.4
	allantoic fluid
	(mg/dl)
		17.162.8	11.762.3	14.161.9	12.661.9

Mean 6 S.E.M. glucose concentrations in plasma and allantoic and amniotic fluids of Iberian purebred and lean crossbreed foetuses. Different letter in superscripts indicate significant differences between breeds (a?b, p,0.05; c?d, p,0.01; e?f, p,0.005; g?h, p,0.001; i?j, p,0.0005). doi:10.1371/journal.pone.0066728.t002

Table 3 .

 3 Effect of breed and sex on parameters related to lipids metabolism.

	Iberian breed		Lean crossbred
	Female	Male	Female	Male

PLOS ONE | www.plosone.org

July 2013 | Volume 8 | Issue 7 | e66728

Acknowledgments

The authors thank the staff of the INIA for skilled technical assistance, Dr. J.M. Navas for technical support and P. Cuesta and I. Cano (Department of Research Support, Universidad Complutense de Madrid) for statistical analyses. The generous donation of LH from A. Ferro and R. Buch (Laboratorios Calier) is gratefully acknowledged.

The collaborative work between INIA and INRA groups was supported by GEMINI COST ACTION FA0702 (STSM). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Author Contributions