M Séger

R Guérin

A Frison

H Bourennane

G Richard

I Cousin

A 3D electrical resistivity tomography survey to characterise the structure of a albeluvic tonguing horizon composed of distinct elementary pedological volumes

Keywords: soil electrical resistivity, heterogeneous structure, Elementary Pedological Volumes, electrical resistivity tomography, degraded horizon, hydraulic modelling, geostatistical filtering, kriging component

Water and gas transfer in porous media like soils are determined by their porous network, described by their structure. In soil, the horizon is usually considered to be elementary and homogeneous functioning system in the description of gas and water functioning. However, in some cases, a horizon is heterogeneous, and its structure is defined by the 3D arrangement of Elementary Pedological Volumes (EPVs). The horizon needs to be described in three dimensions to improve the characterisation of the structure and, consequently, the prediction of its hydraulic functioning. The aim of this study was to determine the feasibility of describing the 3D structure of a heterogeneous albeluvic tonguing soil horizon composed of a juxtaposition of silty white and clayey ochre EPVs, using 3D electrical resistivity tomography (ERT).

Electrical measurements were compared with geostatistical analyses from soil photographs. We demonstrated that the resistivity of the white EPVs was greater than that of the ochre EPVs. In addition, the general soil structure and organisation of the soil horizon could be derived from the electrical resistivity data. We proposed a method to discretise the soil electrical resistivity into a binary system that corresponded to white and ochre volumes. Finally, a 3D representation of the soil structure was created that could be used to improve soil hydraulic models.

Introduction

Soil naturally filters water as it is transported to groundwater reservoirs. Thus, soil structure influences water storage and transfer and gas diffusivity, which have major environmental and agronomic consequences. Therefore, it is important to know the soil structure to predict soil hydraulic functions.

Typically, soil horizons are considered to be elementary and homogeneous systems for the pedological description of soils [START_REF] Jamagne | The current French approach to soilscape typology[END_REF][START_REF] Bockheim | Historical development of key concepts in pedology[END_REF]. Furthermore, soil horizons are used for modelling water transfer properties and consist in the basic information of soil databases, such as the UNSODA 1 [START_REF] Nemes | Description of the unsaturated soil hydraulic database UNSODA version 2.0[END_REF] or HYPRES 2 [START_REF] Wösten | Development and use of a database of hydraulic properties of European soils[END_REF] databases, which are populated with soil data at the horizon scale. Nevertheless, in some cases, the horizon is heterogeneous and its structure, at the so-called n scale, is defined by the arrangement of pedological volumes at the (n-1) scale. Heterogeneous horizons are very common and include ploughed horizons [START_REF] Richard | Effect of compaction on the porosity of a silty soil: influence on unsaturated hydraulic properties[END_REF], stony horizons [START_REF] Cousin | Influence of rock fragments on the water retention and water percolation in a calcareous soil[END_REF], or horizons with vertical leaching tongues [START_REF] Diab | Water movement in a glossaqualf as measured by two tracers[END_REF][START_REF] Frison | Soil hydraulic properties in relation to local rapid soil changes induced by field drainage: a case study[END_REF].

To improve the characterisation of the soil structure and, consequently, its hydraulic function, the soil horizon needs to be described in three dimensions (3D).

Several methods already exist to characterise heterogeneous soil horizons. These methods are based on (i) the visual description of the soil profile in a ploughed horizon [START_REF] Roger-Estrade | Morphological characterisation of soil structure in tilled fields: from a diagnosis method to the modelling of structural changes over time[END_REF], (ii) the characterisation of water infiltration or (iii) soil property measurements, such as bulk density, porosity, and penetrability.

However, these methods do not describe the horizon structure in 3D. Serial sections, X-ray tomography (XRT) and electrical resistivity tomography (ERT) are three techniques that can be used to obtain a 3D description of soil structure. The serial sections method is conducted by taking photographs of successive thin twodimensional (2D) slices of an impregnated (e.g., with dye tracer - [START_REF] Trojan | Macroporosity and hydraulic properties of earthworm-affected soils as influenced by tillage and residue management[END_REF][START_REF] Forrer | Quantifying dye tracers in soil profiles by image processing[END_REF] or non-impregnated [START_REF] Pereira | Three-dimensional representation of tubular horizons in sandy soils[END_REF]) volume of soil. These images are superimposed to reproduce the 3D soil volume (e.g. [START_REF] Cousin | Three-dimensional analysis of a loamy-clay soil using pore and solid chord distributions[END_REF]. This method can be applied to obtain a resolution from a few hundred micrometres [START_REF] Cousin | Three-dimensional analysis of a loamy-clay soil using pore and solid chord distributions[END_REF] to approximately one centimetre in the 1 UNsaturated SOil hydraulic DAtabase 2 HYdraulic PRoperties of European Soils Comment citer ce document : Seger, M. (Auteur de correspondance), Guérin, R., Frison, A., Bourennane, H., Richard, G., Cousin, I. (2014). A 3D electrical resistivity tomography survey to characterise the structure of a albeluvic tonguing horizon composed of distinct elementatry pedological volumes. [START_REF] Vogel | Quantification of pore structure and gas diffusion as a function of scale[END_REF]. Since the work reported by [START_REF] Petrovic | Soil bulk density analysis in three dimensions by computed tomographic scanning[END_REF], XRT had enabled researchers to characterise soil structure on large undisturbed samples of a few cubic decimetres. Similar to the serial sections method, the XRT method is often used to obtain topological information and porous network descriptions of the studied sample. Unfortunately, the serial sections and XRT methods require destructive sampling, which does not allow monitoring of the soil structure evolution with time.

Therefore, the use of non-destructive techniques should be encouraged. Among the non-destructive techniques, ERT is of primary interest for obtaining a 3D representation of the subsurface [START_REF] Dahlin | The development of DC resistivity imaging techniques[END_REF]. ERT has been used to describe changes in bulk density (e.g. [START_REF] Abu-Hassanein | Electrical resistivity of compacted clays[END_REF][START_REF] Besson | Structural heterogeneity of the soil tilled layer as characterized by 2D electrical resistivity surveying[END_REF], stony inclusions (e.g. [START_REF] Rey | Characterisation of soils with stony inclusions using geoelectrical measurements[END_REF], and soil morphological structures [START_REF] Séger | Characterisation of the structural heterogeneity of the soil tilled layer by using in situ 2D and 3D electrical resistivity measurements[END_REF].

The aim of this work was to determine the 3D structure of a heterogeneous albeluvic tonguinghorizon for an area of approximately one square metre using 3D

ERT. At this site, the heterogeneity is a juxtaposition of ochre clay and white silt pedological volumes of a few cubic centimetres, called 'Elementary Pedological

Volumes' (EPVs) as described in [START_REF] Frison | Soil hydraulic properties in relation to local rapid soil changes induced by field drainage: a case study[END_REF]. These types of horizons are very common in soils, but their three-dimensional heterogeneity is usually difficult to describe and to be considered in soil hydric functioning models. However, the hydraulic properties of the two types of EPV in this soil horizon are strongly different and lead to complex hydric functioning [START_REF] Frison | Soil hydraulic properties in relation to local rapid soil changes induced by field drainage: a case study[END_REF]. Data were analysed and compared with soil photographs to assess the potential of electrical resistivity measurements for describing the clay and silt volume arrangements.

Materials and methods

Study site

Soil description

The studied soil is located on the crest of the Yonne plateau, France, in a cultivated field. This soil is an Albeluvisol (WRB, 1998) that was developed on aeolian Quaternary loam that was deposited over an Eocene clay layer. This soil is characterised by the three following horizons (Figure 1). -From 0.35 to 0.60 m, a transitional albeluvic tonguing E&Bt horizon with a polyhedral structure that is characterised by the juxtaposition of different sub-units [START_REF] Frison | Soil hydraulic properties in relation to local rapid soil changes induced by field drainage: a case study[END_REF] from the upper and the lower horizons. These subunits include white-grey (10YR8/2 to 10YR7/1) to pale-brown (10YR7/4) albeluvic tongues that are called "white Elementary Pedological Volumes (EPVs)" and ochre (10YR5/6 with 10YR5/8) volumes that are called "ochre EPVs". Black concretions and inclusions were observed in the ochre EPVs. This E&Bt horizon, that exhibited the maximum structural heterogeneity [START_REF] Diab | Water movement in a glossaqualf as measured by two tracers[END_REF][START_REF] Montagne | Quantification of soil volumes in the Eg&Bt-horizon of an Albeluvisol using image analysis[END_REF][START_REF] Montagne | Impact of drainage on soil-forming mechanisms in a French Albeluvisol: input of mineralogical data in mass-balance modelling[END_REF][START_REF] Frison | Soil hydraulic properties in relation to local rapid soil changes induced by field drainage: a case study[END_REF] was the object of this study.

-From 0.60 to 1.00 m, a Bt horizon that exhibits white albeluvic tongues (equivalent to those of the E&Bt horizon) in the ochre matrix. The structure of this horizon is more prismatic than that of the E&Bt horizon.

EPV description

Several EPVs (15 -30 cm 3 ) were collected from the E&Bt horizon in the field by gently separating this soil by hand according to colour. These samples were collected at two depths, from 0.35 to 0.45 and from 0.45 to 0.55 m. The samples were stored at their field moisture contents at 4°C in hermetic boxes prior to conducting any measurements. The water content was measured on a mass basis for each EPV using approximately six replicates. The bulk density was determined using the kerosene method [START_REF] Blake | Bulk density[END_REF] on approximately ten replicates. The clay content of each EPV was determined by blending ten EPVs and using Robison's pipette method following ultrasonic dispersion in water and the addition of NaPO 3 and Na 2 CO 3 (according to the French normalised protocol, X 31-107).

The bulk density of the white EPVs was slightly lower than the bulk density of the ochre EPVs (Table 1). The water content in the white EPVs was slightly higher than that in the ochre EPVs. However, in both cases, the water content did not depend on the position of the EPVs in the profile (Table 1). The clay content was higher in the ochre EPVs and increased with depth (Table 1). The measurements were realised at the depth 0.35 to 0.55 in the highly heterogeneous E&Bt horizon. A trench 0.5 m x 1.0 m, with a depth of 1 m was dug for the people to stay. Before the electrical measurements, the ploughed horizon was removed from the upper surface of the E&Bt horizon to obtain a regular and planar xy surface of approximately one square metre. This x-y plane was called "A level" and was located at a depth of z = 0.35 m (Figure 1). Colour photographs were taken of the A level before conducting electrical measurements. A 0.06 m layer of the soil was removed to obtain a second x-y surface. This second x-y surface is referred to as "B level" and was at a depth of z = 0.41 m. A second sequence of electrical measurements and colour photographs was collected from the B level. The same procedure was repeated to obtain a third x-y surface at a depth of z = 0.47 m, which is referred to as the "C level". All colour photographs were taken with a digital camera (Panasonic, DMC-FZ5, focal distance 2.8, speed 1/60 s, ISO-150) and with natural sunlight.

Electrical measurements

Electrical resistivity measurements were performed with a programmable multi-electrode system that was connected to a resistivity meter (Syscal R1 Plus, IRIS Instruments). Brass electrodes of 2 mm diameter were positioned on supports and inserted approximately 2 mm into the soil. To obtain high-quality electrical resistivity measurements, the resistance between the soil and electrode was measured to check if the contacts were reliable and steady. Indeed, it then ensure that the current intensity and resulting potential are high enough to be measured with a correct significativity. Electrodes were positioned far enough from the excavation (60 cm) to avoid border effects.

Three types of electrode configurations (Figure 2) were employed for the electrical resistivity measurements, including (i) a line of 32 electrodes spaced at 0.03 m apart (Wen32), (ii) a line of 16 electrodes spaced at 0.03 m apart (Wen16) and (iii) a regular square grid of 32 electrodes spaced at 0.09 m apart (Sqr32). The

Comment citer ce document : Seger, M. (Auteur de correspondance), Guérin, R., Frison, A., Bourennane, H., Richard, G., Cousin, I. (2014). A 3D electrical resistivity tomography survey to characterise the structure of a albeluvic tonguing horizon composed of distinct elementatry pedological volumes. Wenner-α array was used for the line configurations (Wen32 and Wen16) because it is known to be relatively sensitive to vertical changes and has the best signal-tonoise ratio. In addition, this array type can be used in areas with high background noise [START_REF] Loke | Tutorial : 2D and 3D electrical imaging surveys[END_REF][START_REF] Dahlin | A numerical comparison of 2D resistivity imaging with 10 electrode arrays[END_REF][START_REF] Samouëlian | Electrical resistivity survey in soil science: a review[END_REF], which occurs in the studied E&Bt horizon. The square array (Sqr32) is important for investigating anisotropic soil resistivity measurements [START_REF] Samouëlian | Three-dimensional crack monitoring by electrical resistivity measurement[END_REF] and is considered relevant for recognising 3D objects [START_REF] Matias | Square array anisotropy measurements and resistivity sounding interpretation[END_REF].

For the A level, seven Wen32 profiles were realised in the x direction. The distance between two successive lines was 0.09 m. The recorded apparent electrical resistivity measurements for one Wen32 profile resulted in 129 measurements at six pseudo-depths. The same measurement procedure with Wen32 was repeated for the B and C levels. The x and y coordinates of each Wen32 array did not change.

For the A level, four Wen16 profiles were realised in the y direction; these profiles were perpendicular to the Wen32 profiles. Seven Wen16 profiles were realised for the C level. For one Wen16 profile, each experiment resulted in 34 measurements that were distributed across four pseudo-depths.

At the A and C levels, the square array (Sqr32) was used. This sequence resulted in thirty measurements that were distributed across two pseudo-depths.

The temperature was recorded in the E&Bt horizon during the experiments with PT100 probes. The temperature was constant during the electrical resistivity measurements and was approximately 9°C. Thus, there is no need to consider temperature effects on the electrical measurements.

Data processing

3D inversion of the electrical resistivity data

For each level, a complete dataset of georeferenced apparent resistivity measurements was built. This dataset included measurements from the Wen32, Wen16 and Sqr32 configurations. To obtain quantitative information regarding the resistivity distribution at the actual investigated depths, the apparent resistivity measurements were interpreted with the RES3DINV software (Geotomo Software, Malaysia) based on the [START_REF] Loke | Rapid least-squares inversion of apparent resistivity pseudosections using a quasi-Newton method[END_REF] inversion procedure. To help detect the E&Bt horizon vertical structures, we chose a damping factor so that the vertical All of the photographs were ortho-rectified to the same (0, x, y, z) coordinates of the electrical resistivity data so that they could be compared. On each photograph, the ochre and white EPVs were separated by hand with a visual analysis. This analysis created binary images that could be used to count the proportion of each EPV and to evaluate the ability of the electrical resistivity measurements for identifying ochre and white EPVs. As included in the ochres ones, the black concretions were labelled as ochre ones in the binary images.

Comparison between the electrical resistivity data and the soil photograph pictures

To compare electrical resistivity data, which were in 3D, and soil photographs, which were in 2D, we extracted 2D planes from the 3D bloc diagram of inverted electrical resistivity at a depth of 0.35, 0.41 and 0.47 m (i.e., at the A, B and C levels).

A variographic analysis was conducted on the three colour channels of the photographs that were taken at the surface of each level and on the 2D resistivity planes at depths of 0.35, 0.41 and 0.47 m. For each variogram, the local and regional components were identified and used to create new datasets that corresponded to each spatial component, except for nugget (see Appendix for the complete description of the method).

Whatever the type of dataset (original dataset, local component dataset or regional component dataset), 2D resistivity data were compared to each channel of the photographs.

Results

Comment citer ce document : Seger, M. (Auteur de correspondance), Guérin, R., Frison

Proportion of the EPVs at each level

The corresponding binary images of the photographs of the three levels were used to determine the proportion of ochre and white EPVs (Figure 3). The proportions of the ochre EPVs were 46, 45 and 55% for the A, B and C levels, respectively. A concentric zone (see the arrow symbol on the C level) with a higher proportion of white EPVs between 0.18 and 0.45 m along the Ox axis was observed, especially for the C level.

3D electrical resistivity

The graphic representations of the planes that corresponded to the A, B and C level depths were obtained from the first layer of each inverted dataset for each A or B or C levels (Figure 4). They exhibited resistivity values of between 15 Ω.m (i.e. 66 mS.m -1 ) and 80 Ω.m (i.e. 12 mS.m -1 ). Generally, the resistivity values decreased with depth. For example, the resistivity values were higher for the A level (40 Ω.m on average i.e. 25 mS.m -1 ) than for the B and C levels (30 Ω.m on average i.e. 33 mS.m-1). At each level, the resistivity values varied spatially.

On the C level, a more resistive zone of approximately 45 Ω.m (i.e. 22 mS.m -1 )

was observed between 0.20 and 0.40 m along the Ox axis (see the arrow symbol), which corresponded to the location of the concentric zone that was previously described in section 2.2.

Comparison between the electrical resistivity data and the photographs

Variographic analysis

To compare the soil photographs at the three levels to the 2D planes from the 3D electrical resistivity inversion, the original, local component and regional component datasets were analysed. Figure 5 provides an example for level C.

We then analyse the coefficient correlation between the soil photograph pixel values for each colour channel and the 2D electrical resistivity values of the 2D planes that were extracted by 3D inversion for the A, B and C levels (Table 2). These correlation coefficients were calculated for the original, local component and regional In contrast, for levels B and C, the correlation coefficients between the regional components of the photographs and the spatial components of resistivity maps were considered significant for all colour channels.

Creation of a 3D soil structure image

The 3D inverted resistivity values were interpolated using a 3D kriging routine.

In addition, we proposed the following methodology to transform the electrical resistivity values into ochre and white EPVs. At each level, we separated the electrical resistivities into two classes with different threshold values and calculated the resulting proportions of ochre and white. The best threshold value should provide the ochre and white proportion that is closest to the one calculated from the binary photographs. For each level, the best agreement was observed when the threshold value corresponded to the mean resistivity value. Next, we used the mean value of the entire 3D resistivity dataset to discretise the 3D data into white and ochre EPVs. This 3D discretisation (Figure 7) was composed of 43.8 and 56.2 % of white and ochre electrical EPVs, respectively, which is consistent with the values obtained from the soil photographs at the three levels. In addition, these results were consistent with those were obtained by [START_REF] Montagne | Quantification of soil volumes in the Eg&Bt-horizon of an Albeluvisol using image analysis[END_REF], who used image analysis on large impregnated blocks.

Discussion

The structure of the heterogeneous horizon: what do we learn from electrical measurements?

Comment citer ce document : Seger, M. (Auteur de correspondance), Guérin, R., Frison, A., Bourennane, H., Richard, G., Cousin, I. (2014). A 3D electrical resistivity tomography survey to characterise the structure of a albeluvic tonguing horizon composed of distinct elementatry pedological volumes. For our experiment, several soil characteristics, including the bulk density, the clay content and the water content, were determined. The clay contents were significantly higher in the ochre than in the white EPVs, which should result in lower resistivity values in the ochre EPVs. For the water and the bulk density, the maximum differences between the white and ochre EPVs were 0.005 g.g -1 and 0.06 g.cm -3 , respectively (Table 1). Such small variations cannot be detected by the electrical resistivity measurements (Seladji et al., 2010). Thus, we can assume that only the soil texture (due to its clay content) would induce large soil electrical variations.

Incidentally, soil texture measurements throughout the soil profile indicated that the clay content increased with depth due to the decreasing white volumes. This finding is consistent with the general decrease in resistivity with depth.

The comparisons between the photographs of each level and the interpreted electrical resistivity measurements did not lead to significant results when the raw values or the local components of the two datasets were considered. Indeed, the precise geometrical limits of the EPVs were not detected by the electrical resistivity because of either the inter-electrode spacing or the loss of information with depth. In contrast, because the ERT method is integrative, these comparisons were more relevant for the regional components of the two datasets. The current lines are threedimensional and represent a certain soil volume whose size increases and resolution decreases as the inter-electrode spacing increases. As a result, this method is efficient for locating large areas (several centimetres) of white or ochre soil volumes without determining their exact local geometry. Regarding geometry, the electrical structure exhibits large electrical resistivity values that correspond to white volumes near the surface and along the Ox axis (i.e., parallel to the surface plane). Deeper, the large resistivity values are organised by geometrical volumes and are oriented more along the Oz axis (i.e., along a vertical axis). This orientation is consistent with the general organisation of the E&Bt horizon in an Albeluvisol. Near the soil surface, the amount of white EPVs (i.e. albeluvic tongues) is greater. These white volumes are less numerous and more vertical. In addition, they occur in tongues that range from a few centimetres to several tens of centimetres in size [START_REF] Wrb | A world reference base for soil resources[END_REF]. These

Comment citer ce document : Seger, M. (Auteur de correspondance), Guérin, R., Frison, A., Bourennane, H., Richard, G., Cousin, I. (2014). A 3D electrical resistivity tomography survey to characterise the structure of a albeluvic tonguing horizon composed of distinct elementatry pedological volumes. tongues have a higher hydraulic conductivity than the ochre volumes and constitute preferential pathways for water transfer [START_REF] Frison | Soil hydraulic properties in relation to local rapid soil changes induced by field drainage: a case study[END_REF]. According to the field observations of several authors, the functionality of some of these tongues depends of their texture [START_REF] Diab | Water movement in a glossaqualf as measured by two tracers[END_REF]. However, these differences are difficult to identify from soil profile photographs. Based on the electrical structure analyses of the tongues, lower electrical resistivities corresponded to higher hydraulic conductivities.

To interpret the electrical resistivity in terms of hydraulic conductivity, some complementary measurements are necessary, such as the water content and the resistivity of several soil volumes. Nevertheless, the 3D structure of a heterogeneous horizon, which is derived from electrical measurements, contains a relevant 3D structure of the hydraulic properties. Thus, this 3D structure could be used to model the water transfer at this scale, as demonstrated by [START_REF] Samouëlian | Determining the effective properties of a highly heterogeneous horizon[END_REF].

Determination of the structure's Representative Elementary Volume (REV)

Modelling the hydric function of the horizon with albeluvic tonguing is only feasible if (i) the structure is studied in 3D and, (ii) the volume of studied soil is greater than the Representative Elementary Volume (REV). It is possible that the REV of some hydric properties is different than the REV of some structural characteristics. However, we can determine if this difference occurs by determining if the volume analysed by electrical resistivity is greater than the REV that is necessary to describe the horizon morphology (i.e., the 3D geometrical arrangement of the ochre and the white EPVs).

From the 3D resistivity values, a variogram was calculated for each direction (Ox, Oy, Oz) (Figure 8) to represent the distribution of the ochre and white EPVs based on distance. A limit value was observed at a distance of 0.25 m for both variograms in the Ox and Oz directions. This result indicated that the REV for the horizon structure was reached. In addition, this value was consistent with the value obtained by [START_REF] Montagne | Quantification of soil volumes in the Eg&Bt-horizon of an Albeluvisol using image analysis[END_REF] from 2D images of the same horizon. In contrast, the REV was not reached in the Oy direction. Thus, further investigations are needed to determine the exact structure in the Oy direction.

Conclusions

Comment citer ce document : Seger, M. (Auteur de correspondance), Guérin, R., Frison, A., Bourennane, H., Richard, G., Cousin, I. (2014). A 3D electrical resistivity tomography survey to characterise the structure of a albeluvic tonguing horizon composed of distinct elementatry pedological volumes. The studied horizon had a complex structure composed of a juxtaposition of silty white and clayey ochre EPVs. We demonstrated that the resistivity of the white EPV was larger than the resistivity of the ochre EPV. In addition, the structure of the soil horizon can be derived from electrical resistivity data. Nevertheless, to obtain accurate electrical measurements that could describe the horizon structure, our work required the destruction of the studied soil. Other destructive methods, such as serial sectioning with numerical photograph analysis, could also be used to produce the 3D structure of the studied horizon. However, more electrodes could be used in the future to improve the accuracy of the ERT measurements without disturbing the soil horizon.

The resistivity was higher in the upper portion of the studied horizon. In this layer, zones were identified that were parallel to the surface and corresponded to the white EPVs. The resistivity was lower at depth due to the higher amount of ochre EPVs. However, the white EPVs occurred in structures that were predominantly orientated perpendicular to the soil surface, similarly to tongues. Depending on their actual texture, these tongues may or may not transfer water. Electrical resistivity measurements could enable the identification of functional tongues if they are collected several times during infiltration or evaporation periods. In the future, the 3D structure of such a complex horizon obtained by electrical resistivity measurements will improve water and solute transport models.  must sum to zero, and not to one as in ordinary kriging [START_REF] Wackernagel | Multivariate geostatistics[END_REF]. This leads to the following kriging system for lth component:

0 ..., , 1 = for ) ( ) ( 1 , 0 1 ,                 n l l l l n l w n b w            x x x x (4)
where l  is the Lagrange parameter for the lth component. This system of n+ 1 equations must be solved to find the weights l w ,  that are then inserted into equation ( 4) to estimate the lth spatial component of variable Z at location x0. The operation is repeated for all locations where such decomposition is required. and on the local (-b-and -e-) and regional components (-c-and -f-) that were obtained from variographic analysis. For conductivity equivalent values, see Comment citer ce document : Seger, M. (Auteur de correspondance), Guérin, R., Frison, A., Bourennane, H., Richard, G., Cousin, I. (2014). A 3D electrical resistivity tomography survey to characterise the structure of a albeluvic tonguing horizon composed of distinct elementatry pedological volumes.

Figure 6

 6 Figure 6 contains the maps of agreement between the binary electrical resistivity data and the binary soil photographs. A large zone of agreement between 0.20 and 0.40 m along the Ox axis was observed that corresponded to the concentric zone position of the white EPVs.

  to l = 0 is called nugget and represents the spatial unstructured part of the total variance. Based on the linear model of regionalization (1), the random function ) (x Z can be decomposed into a sum of (L+1) independent random functions, called spatial component, and its local mean m(x): component can thus be individually mapped by filtering out the other components. The estimator of the lth spatial component of variable Z at location x0 is: of all L + 1 components which are considered mutually independent, only the contribution of the lth component must be taken into account in the right hand side term of the kriging system. Moreover to ensure unbiased estimations and provided the variogram model ) (h l  is bounded (stationary case), the

Figure 1 :

 1 Figures captions:Figure1: 3D representation of the soil profile. The underlined white zones correspond to some white EPVs. The ochre EPVs were juxtaposed to the white ones.

Figure 2 :

 2 Figure 2: Experimental design. Location of the Wen32, Wen16 and Sqr32 electrodes at each studied level.

Figure 3 :

 3 Figure 3: Photographs of the three surface levels (-a-, -c-and -e-) and their corresponding binary representations (-b-, -d-and -f-) for white and ochre EPVs after the visual analysis. The surface proportions of the white and ochre EPVs are mentioned.

Figure 4 :

 4 Figure 4: Interpreted electrical resistivity for the three levels extracted from the inverted electrical resistivity 3D data.

Figure 5 :

 5 Figure5: Comparisons of the electrical resistivity planes and the real surface level photographs. For example, the C level between the resistivity plane that was extracted from the 3D data and the blue channel of the real photograph were compared. The comparisons are made on the original images(-a-and -d-)

  Figure 4.

Figure 6 :

 6 Figure 6: Map of agreement between the binary classifications that were determined from visual analysis and from electrical resistivity maps separated by the threshold value.

Figure 7 :

 7 Figure 7: A 3D representation of the electrical structure discretisation in two EPVs.

Figure 8 :

 8 Figure 8: Variograms of the 3D block of the electrical resistivity discretised for the three Ox, Oy and Oz directions.

  Version définitive du manuscrit publié dans / Final version of the manuscript published in : Geoderma, 2014, 219-220, 168-176 http://dx.doi.org/10.1016/j.geoderma.2013.12.018

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Geoderma, 219-220, 168-176. DOI : 10.1016/j.geoderma.2013.12.018

  Version définitive du manuscrit publié dans / Final version of the manuscript published in : flatness filter was twice the horizontal flatness filter. We used a model with narrower cells, which is recommended when the resistivity varies greatly near the surface.The 3D inversion of the electrical resistivity data provided a bloc diagram of the interpreted electrical resistivity values for the three levels A, B and C.

		Geoderma, 2014, 219-220, 168-176	http://dx.doi.org/10.1016/j.geoderma.2013.12.018
	Manuscrit d'auteur / Author Manuscript	2.3.2. Processing of the soil photographs
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Comment citer ce document : Seger, M. (Auteur de correspondance), Guérin, R., Frison, A., Bourennane, H., Richard, G.,

Cousin, I. (2014)

. A 3D electrical resistivity tomography survey to characterise the structure of a albeluvic tonguing horizon composed of distinct elementatry pedological volumes. Geoderma, 219-220, 168-176. DOI : 10.1016/j.geoderma.2013.12.018

  Version définitive du manuscrit publié dans / Final version of the manuscript published in : Geoderma, 2014, 219-220, 168-176 http://dx.doi.org/10.1016/j.geoderma.2013.12.018

	Manuscrit d'auteur / Author Manuscript
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, A., Bourennane, H., Richard, G.,

Cousin, I. (2014)

. A 3D electrical resistivity tomography survey to characterise the structure of a albeluvic tonguing horizon composed of distinct elementatry pedological volumes. Geoderma, 219-220, 168-176. DOI : 10.1016/j.geoderma.2013.12.018

  Version définitive du manuscrit publié dans / Final version of the manuscript published in :The use of electrical resistivity in soil science studies has one considerable advantage over the use of geological studies because non-destructive and rapid observations of the structure are feasible. The E&Bt horizon appears to be a good case study because the electrical resistivity values are highly heterogeneous.

	Geoderma, 2014, 219-220, 168-176	http://dx.doi.org/10.1016/j.geoderma.2013.12.018
	Manuscrit d'auteur / Author Manuscript
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Geoderma, 219-220, 168-176. DOI : 10.1016/j.geoderma.2013.12.018

  Version définitive du manuscrit publié dans / Final version of the manuscript published in :

	Geoderma, 2014, 219-220, 168-176	http://dx.doi.org/10.1016/j.geoderma.2013.12.018
	Manuscrit d'auteur / Author Manuscript
	Manuscrit d'auteur / Author Manuscript
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Geoderma, 219-220, 168-176. DOI : 10.1016/j.geoderma.2013.12.018

  Geoderma, 219-220, 168-176. DOI : 10.1016/j.geoderma.2013.12.018 Version définitive du manuscrit publié dans / Final version of the manuscript published in :ERT surveys were conducted on a highly heterogeneous horizon over a surface area of approximately one square metre. The E&Bt horizon of an Albeluvisol was studied with 3D electrical resistivity prospecting.

	Geoderma, 2014, 219-220, 168-176	http://dx.doi.org/10.1016/j.geoderma.2013.12.018
	Manuscrit d'auteur / Author Manuscript
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  Version définitive du manuscrit publié dans / Final version of the manuscript published in :

		Geoderma, 2014, 219-220, 168-176	http://dx.doi.org/10.1016/j.geoderma.2013.12.018
	Manuscrit d'auteur / Author Manuscript	kriging weights	w ,	l
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Comment citer ce document : Seger, M. (Auteur de correspondance), Guérin, R., Frison, A., Bourennane, H., Richard, G.,

Cousin, I. (2014)

. A 3D electrical resistivity tomography survey to characterise the structure of a albeluvic tonguing horizon composed of distinct elementatry pedological volumes. Geoderma, 219-220, 168-176. DOI : 10.1016/j.geoderma.2013.12.018

Table 1 :

 1 Version définitive du manuscrit publié dans / Final version of the manuscript published in : Geoderma, 2014, 219-220, 168-176 http://dx.doi.org/10.1016/j.geoderma.2013.12.018 Soil characteristics for the white and ochre EPVs. The standard errors of measurements are presented in brackets.

	Manuscrit d'auteur / Author Manuscript	Bulk density (g cm -3 ) Clay content (g kg -1 ) Field water content (g g -1 ) White EPV Ochre EPV White EPV Ochre EPV White EPV Ochre EPV 0.35-0.45 1.61 (0.06) 1.63 (0.02) Depth (m) 182 248 0.211 (0.01) 0.206 (0.01) 0.45-0.55 1.59 (0.02) 1.65 (0.03) 213 322 0.209 (0.01) 0.205 (0.01)
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Geoderma, 219-220, 168-176. DOI : 10.1016/j.geoderma.2013.12.018

Table 2 :

 2 Correlation coefficients between the colour channel pixels of the numerical photographs and the interpreted resistivity of the x-y maps that were extracted from the 3D data in the A, B and C levels (at z coordinates of 0.35, 0.41 and 0.47 m respectively). The comparisons were made for the raw data, the local and regional components of each level.

		Level A (0.35 m)
	Correlation	Red channel	Green channel	Blue channel
	Raw data	0.05	0.09	0.08
	Local component	0.01	0.02	0.02
	Regional component	0.14	0.21	0.20
		Level B (0.41 m)
	Correlation	Red channel	Green channel	Blue channel
	Raw data	0.23	0.27	0.27
	Local component	0.08	0.15	0.18
	Regional component	0.55 *	0.64 *	0.64 *
		Level C (0.47 m)
	Correlation	Red channel	Green channel	Blue channel
	Raw data	0.32	0.40	0.39
	Local component	0.21	0.33	0.35
	Regional component	0.71 *	0.75 *	0.76 *
	* correlation coefficient considered as significant

Acknowledgments

Guillaume Giot, Christian Lelay, Sophie Cornu and Anatja Samouëlian are gratefully acknowledged for their help with field experiments and laboratory measurements.

Version postprint

Comment citer ce document : Seger, M. (Auteur de correspondance), Guérin, R., Frison, A., Bourennane, H., Richard, G., Cousin, I. (2014). A 3D electrical resistivity tomography survey to characterise the structure of a albeluvic tonguing horizon composed of distinct elementatry pedological volumes. Geoderma, 219-220, 168-176. DOI : 10.1016/j.geoderma.2013.12.018 Version définitive du manuscrit publié dans / Final version of the manuscript published in : Geoderma, 2014, 219-220, 168-176 http://dx.doi.org/10.1016/j.geoderma.2013.12.018 Manuscrit d'auteur / Author Manuscript Manuscrit d'auteur / Author Manuscript Manuscrit d'auteur / Author Manuscript -14 -

The authors wish to thank the Region Centre for the funding of the PhD thesis of Anthony Frison and the program SPATIOFLUX.

Appendix

Indeed, a spatial random process can be a combination of several constituent random processes one nested within another and acting different charachteristic spatial scales. In these circumstances the variogram of a random function

is a nested combination of two or more individual variograms leading to so-called nested variogram that displays different structures (e.g. different ranges of spatial dependence). The variogram can thus be modelled as the sum of L + 1 basic variograms (e;g. [START_REF] Goovaerts | Geostatistics for Natural resources Evaluation[END_REF][START_REF] Bourennane | Geostatistical filtering for improved soil water content setimation from electrical resistivity data[END_REF], each corresponding to a disctinct spatial structure:


domain: sdv info

text:
Elie Aur

Allais-Bonnet

Johan Castille

Ma€ Elle Pannetier

Bruno Passet

Ma€ Eva Elza€ Iat

Marjolaine Andr

Fatemeh Montazer-Torbati

Katayoun Moazami-Goudarzi

Jean-Luc Vilotte

Eric Pailhoux
email: eric.pailhoux@jouy.inra.fr

A specific role for PRND in goat foetal Leydig cells is suggested by prion family gene expression during gonad development in goats and mice

Keywords: Doppel, foetal Leydig, goat, mouse, prion, Shadoo CNS, central nervous system, dpc, days post co€ ıtum, Dpl, Doppel, dpp, days post partum, ECL, enhanced chemiluminescence, GPI, glycophosphatidylinositol, IHC, immuno-histo-chemistry, mpp, months post partum, PBS, phosphate saline buffer, qRT-PCR, quantitative RT-PCR, Sho, Shadoo, TSE, transmissible spongiform encephalopathy

Three genes of the prion protein gene family are expressed in gonads. Comparative analyses of their expression patterns in mice and goats revealed constant expression of PRNP and SPRN in both species and in both male and female gonads, but with a weaker expression of SPRN. By contrast, expression of PRND was found to be sex-dimorphic, in agreement with its role in spermatogenesis. More importantly, our study revealed that PRND seems to be a key marker of foetal Leydig cells specifically in goats, suggesting a yet unknown role for its encoded protein Doppel during gonadal differentiation in nonrodent mammals.

.These observations suggested a biological redundancy between PrP and another PrP-like protein in mammals.

Sho is expressed in the CNS and both Sho and PrP share neuro-protective properties [START_REF] Watts | The CNS glycoprotein Shadoo has PrP(C)-like protective properties and displays reduced levels in prion infections[END_REF]. Using Sprn reporter mice, a recent study describes expression of Sprn in the male and female gonads suggesting an involvement of Sho in reproduction [START_REF] Young | Generation of Sprn-regulated reporter mice reveals gonadic spatial expression of the prion-like protein Shadoo in mice[END_REF]. The Sprn mRNA knockdown in Prnp 0/0 embryos produces early embryonic lethality, suggesting that Sho and PrP could play a role in early developmental stages [START_REF] Young | The prion or the related Shadoo protein is required for early mouse embryogenesis[END_REF][START_REF] Passet | Prion protein and Shadoo are involved in overlapping embryonic pathways and trophoblastic development[END_REF]. However, the Sprn ablation (Sprn 0/0 ) or the doubleknockout of Sprn and Prnp (Sprn 0/0 /Prnp 0/0 ) in mice resulted in no drastic developmental phenotype [START_REF] Daude | Knockout of the prion protein (PrP)-like Sprn gene does not produce embryonic lethality in combination with PrP(C)-deficiency[END_REF].

Dpl is mainly expressed in the testis of adult mammals. Its ectopic expression induces some neurodegeneration in the CNS only in the absence of PrP [START_REF] Rossi | Onset of ataxia and Purkinje cell loss in PrP null mice inversely correlated with Dpl level in brain[END_REF][START_REF] Moore | Ataxia in prion protein (PrP)-deficient mice is associated with upregulation of the novel PrP-like protein doppel[END_REF][START_REF] Anderson | Transgene-driven expression of the Doppel protein in Purkinje cells causes Purkinje cell degeneration and motor impairment[END_REF][START_REF] Bersaoui | Purkinje-cell degeneration in prion protein-deficient mice is associated with a cerebellumspecific Doppel protein species signature[END_REF], suggesting a biological link between Dpl and PrP. Prnd ablation in mice (Prnd 0/0 ) resulted in male infertility characterized by the sperm's inability to perform the acrosome reaction and by an elevated level of oxidative DNA damage [START_REF] Behrens | Absence of the prion protein homologue Doppel causes male sterility[END_REF][START_REF] Paisley | Male infertility and DNA damage in Doppel knockout and prion protein/Doppel double-knockout mice[END_REF]. The double-knockout of Prnd and Prnp (Prnd 0/0 /Prnp 0/0 ) only mimicked the effect of the Prnd single inactivation [START_REF] Paisley | Male infertility and DNA damage in Doppel knockout and prion protein/Doppel double-knockout mice[END_REF]. Immunohistochemical studies of Dpl were performed in gonads of various species, such as humans, rodents, boars and bovidae. The cellular localization of Dpl depends on the maturation stage of the gonads, on the studied species and the antibodies [START_REF] Pailhoux | Role of the prion protein family in the gonads[END_REF]. For instance, in rodents and sheep, Dpl was only detected in germinal and somatic cells in mature testis, whereas in humans, boars and bovine, DPL seems to be present during most of the developing stages of the germ cells and in the Sertoli cells of foetal and mature gonads [START_REF] Peoc'h K | The human "prion-like" protein Doppel is expressed in both Sertoli cells and spermatozoa[END_REF][START_REF] Espenes | Dynamic expression of the prion-like protein Doppel in ovine testicular tissue[END_REF][START_REF] Rondena | Identification of bovine doppel protein in testis, ovary and ejaculated spermatozoa[END_REF][START_REF] Serres | Spatio-developmental distribution of the prion-like protein doppel in Mammalian testis: a comparative analysis focusing on its presence in the acrosome of spermatids[END_REF]. In goats and bovine, DPL was detected both in immature testis and in young female follicles [START_REF] Rondena | Identification of bovine doppel protein in testis, ovary and ejaculated spermatozoa[END_REF][START_REF] Kocer | Goat PRND expression pattern suggests its involvement in early sex differentiation[END_REF]. Nevertheless, these different observations suggested a role of Dpl in early and/or mature sex differentiation [START_REF] Pailhoux | Role of the prion protein family in the gonads[END_REF].

To get deeper into the potential role of the prion protein gene family during gonad development, we report the comparative expression profiles of the three members of the prion protein gene family and the comparative localizations of their encoded proteins during ovary and testis development in two different species: goats and mice. These data suggest that Prnd may exert a yet unknown specific role in goat foetal Leydig cells.

Materials and methods

Animals and tissue samples

Procedures for handling goats were conducted in compliance with the guidelines for Care and Use of Agricultural Animals in Agricultural Research and Teaching (authorization no. 78-34). All goat foetuses and young goats were obtained from pregnant females, following hormonal treatment as previously described [START_REF] Pailhoux | Ontogenesis of femaleto-male sex-reversal in XX polled goats[END_REF].

For mice, animal experiments were carried out in strict accordance with the recommendations in the guidelines of the Code for methods and Welfare Considerations in Behavioral Research with Animals (Directive 86/609EC). Experiments were approved by the Local ethics committee of Jouy-en-Josas on the Ethics of Animals Experiments of the author's institution, INRA (Permit Number RTA06-091). All transgenic animal manipulations were performed according to the recommendations of the Haut Conseil des Biotechnologies (Permit number 6461). All mouse foetuses and pups were obtained from pregnant FVB/N, FVB/N Prnp 0/0 [quoted in 17] and Prnd 0/0 females [START_REF] Behrens | Absence of the prion protein homologue Doppel causes male sterility[END_REF].

Day 0 post co€ ıtum corresponds to the day of mating. The genetic sex of all foetuses was determined by PCR amplification of SRY and ZFY/ZFX genes, on liver genomic DNA [START_REF] Pailhoux | Ontogenesis of femaleto-male sex-reversal in XX polled goats[END_REF]. For each goat sample, one gonad was frozen in liquid nitrogen for molecular analysis; the other one was fixed for immuno-histological studies. For mice, samples of a same sex were pooled before molecular analysis. Two or 3 gonads were fixed for immuno-histological studies at each developmental stage. Table 1 summarized the number of individuals used at each developmental stage in mice and goats.

PCR primers

PCR primers were designed using PRIMER EXPRESS Software for Real-Time PCR 3.0 (Applied Biosystems, ThermoFisher Scientific, Courtaboeuf, France) analysis of Prnp, Prnd and Sprn expression in mice and goats (Table 2). Mice and goats gene sequences were obtained from GenBank. Primer efficiencies and specificities were evaluated on genomic DNA. The chosen sets of primers share similar efficiencies (not below 90%).

Quantitative RT-PCR

RNAs were extracted using the RNeasy Mini kit (Qiagen, Courtaboeuf, France). Super-Script II (Invitrogen, Thermo-Fisher Scientific) was used to synthesize cDNA for qRT-PCR from 1lg (mice) or 2 lg (goats) of gonad RNA (Table 1). To identify appropriate qRT-PCR normalizing genes for foetal and postnatal gonads in mice, expression stability of seven genes (Gapdh, Actb, B2 m, Mapk1, H2afz, Ywhaz and Hprt1) was tested at each time point and the GeNorm program [START_REF] Vandesompele | Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes[END_REF] used to select a combination of the most stable genes. The three retained genes were Ywhaz, H2afz and Hprt1 (Table 2). For goats, the previously described ACTB, YWhAZ and H2AFZ genes were used [START_REF] Montazer-Torbati | A study of goat SRY protein expression suggests putative new roles for this gene in the developing testis of a species with long-lasting SRY expression[END_REF] (Table 2). qRT-PCR was performed on all genes at all time points, in triplicates, using the Absolute Blue SYBR Green ROX mix (ThermoFisher Scientific) and the StepOnePlus Real-Time PCR System (Applied Biosystems). The results were analysed a Three different pieces of the same testis was used to realize three independent RT.

by the relative standard curve method with the QBASE Software [START_REF] Hellemans | qBase relative quantification framework and software for management and automated analysis of real-time quantitative PCR data[END_REF]. Data points were plotted using Excel. Statistical analyses were performed using the INVIVOSTAT software [START_REF] Clark | A comparison of InVivoStat with other statistical software packages for analysis of data generated from animal experiments[END_REF] that combines an ANOVA approach followed by a Fisher's Least Significant Difference (LSD)-test.

Immunostaining

Freshly dissected gonads were fixed in 4% paraformaldehyde in phosphate saline buffer (PBS) at 4 °C for 1 h or overnight (according to the size of the gonad). After washes in PBS with increasing concentrations of sucrose (0, 12%, 15% and 18%), tissue specimens were embedded in Jung Tissue Freezing Medium (Leica Biosystems, Nanterre, France) and frozen at À80 °C. Cryo-sections (7 lm thick) were obtained and stored at À80 °C until used. The sections were air-dried, rehydrated in PBS and permeabilized during 30 min in PBS with 0.5% triton and 1% BSA. The primary antibodies were then applied overnight at 4 °C. Table 3 describes the antibody references and concentrations [START_REF] Watts | The CNS glycoprotein Shadoo has PrP(C)-like protective properties and displays reduced levels in prion infections[END_REF][START_REF] Rondena | Identification of bovine doppel protein in testis, ovary and ejaculated spermatozoa[END_REF][START_REF] Morel | Screening of 145 anti-PrP monoclonal antibodies for their capacity to inhibit PrPSc replication in infected cells[END_REF][START_REF] Lampo | Distribution of the Shadoo protein in the ovine brain assessed by immunohistochemistry[END_REF][START_REF] Hales | Testosterone inhibits cAMP-induced de Novo synthesis of Leydig cell cytochrome P-450(17 alpha) by an androgen receptor-mediated mechanism[END_REF]. After several washes, the sections were incubated with secondary antibodies for 1 h at room temperature. The slides were then rinsed in PBS, mounted in Vectashield mounting medium with DAPI (Vector) and observed as above.

Western blot analysis

Frozen tissues of adult mice and goats were homogenized in 50 nM Tris HCl, 150 mM NaCl, 0.5% sodium deoxycholate (w/v), 0.1% sodium dodecyl sulphate (w/v), 1% of a nonionic nondenaturing detergent (NP-40), one complete EDTA free mini-protease inhibitor tablet per 10 mL (Roche Diagnostic, Saint-Egr eve, France). Whole extracts (20 lg of total protein) were subject to 4-15% gradient SDS/PAGE and transferred to a poly(vinylidene difluoride) membrane (GE healthcare Life Sciences, V elizy-Villacoublay, France). The membrane was probed with anti-biotinylated-PrP antibody (bSha 31; Table 3). The secondary antibody used was horseradish peroxydase/ streptavidin conjugated antirabbit (Table 3). Immunodetection using the enhanced chemiluminescence (ECL) method (PIERCE) was performed according to the manufacturer's instruction and the images were recorded on an image analysis station (Luminescent Image analyse Las-1000plus Fujifilm).

Results and discussion

By contrast with mice, PRND is highly expressed in goat foetal testis

We have previously carried out an expressional study of the Prion gene family in the goat species [START_REF] Kocer | Goat PRND expression pattern suggests its involvement in early sex differentiation[END_REF] that suggested an involvement of PRND in early gonadal differentiation. The aim of the present study was to complete this observation by (i) including the recently discovered SPRN gene and (ii) establishing a comparative view of the expression of the three members of the Prion gene family throughout all gonad developmental stages, from differentiation to adulthood (Fig. 1A), in (iii) two mammalian species, mice and goats. Gonadal expression profiles of PRNP, SPRN and PRND have been precisely determined using quantitative RT-PCR instead of classical RT-PCR as previously used [START_REF] Kocer | Goat PRND expression pattern suggests its involvement in early sex differentiation[END_REF], at 7 and 15 developmental stages in mice and goats respectively (Fig. 1B-G), and during ageing in mice where four additional stages were studied. A stable gonadal expression of these three genes in the mouse species was observed during ageing since 50 dpp until 10-12 months of age (Fig. 2). In both species, Prnp/ PRNP gene expression slightly increases during development and appears to be more intense around birth (Fig. 1B,C). During gonadal development, SPRN was found to be expressed in male and female gonads of mice and goats at all tested stages (Fig. 1B-E) but only faintly when compared to PRNP and PRND (as for example in goat testis samples, the cycle thresholds, CT, values are for PRNP and PRND between 21 and 30, but only between 31 and 36 for SPRN). Sprn/ SPRN appears to be more expressed in ovaries of both species during early follicles formation (i.e. from 18.5 dpc to 5 dpp in mice and 70-90 dpc in goats); and specifically in goat ovaries before the beginning of germ cell meiosis (Fig. 1D,E). Indeed, the highest SPRN expression level is found at this premeiotic stage (i.e. 41-50 dpc) only in the goat species. The duration of this premeiotic stage, which starts after gonad commitment in one sex or the other (i.e. 12.5 dpc in mice, 36 dpc in goats), appears quite different in mice and goats, 24 h in mice instead of 2 weeks in goats. Although no profound change could be noticed in mouse ovaries during this period, goat ovaries were organized into cortical and medullar compartments where germ cells were concentrated in the cortex and estrogens were produced by the medulla part under the control of the FOXL2 gene that has been shown to be a major ovarian-determining gene in goats, by contrast with mice [START_REF] Boulanger | FOXL2 is a female sex-determining gene in the goat[END_REF][START_REF] Pannetier | Telling the story of XX sex reversal in the goat: highlighting the sex-crossroad in domestic mammals[END_REF][START_REF] Pannetier | FOXL2 activates P450 aromatase gene transcription: towards a better characterization of the early steps of mammalian ovarian development[END_REF].

Finally and as previously noticed [START_REF] Kocer | Goat PRND expression pattern suggests its involvement in early sex differentiation[END_REF], Prnd/PRND expression is found higher in testes than in ovaries at adulthood for both species, but this sex-dimorphism appears since early testicular differentiation specifically in the goat species (Fig. 1F,G). The expression of Prnd/PRND in adult testes is welldocumented and Prnd has been shown to be required for spermiogenesis in mice [START_REF] Behrens | Absence of the prion protein homologue Doppel causes male sterility[END_REF][START_REF] Paisley | Male infertility and DNA damage in Doppel knockout and prion protein/Doppel double-knockout mice[END_REF]. However, the concomitant increased level of PRND expression with the start of testicular differentiation appears specific to the goat species as it was not observed in mice and it has not been described to date in any other species (Fig. 1G). (two-way ANOVA approach, followed by a Fisher's LSD-test). For each stage, significant differences between the two sexes are showed by stars (*P-value ≤ 0.05; **P-value ≤ 0.01; ***P-value < 0.001).

Sho and PrP cellular localizations remain difficult to ascertain

In order to gain more information on the 3 proteins encoded by the 3 Prion gene family members, we carried out IHC (Immuno-Histo-Chemistry) and western blot experiments using available antibodies (Table 3).

Two antibodies have been tested against Sho. Each gives a specific staining by IHC that remains different (i) from each other, (ii) from one species to another and also (iii) from Sho gonadal expression already described by additive transgenesis of a Sprn-LacZ mini-gene in mice [START_REF] Young | Generation of Sprn-regulated reporter mice reveals gonadic spatial expression of the prion-like protein Shadoo in mice[END_REF]. Thus, we are unable to ascer- tain in what gonadal cell type Sho could be detected.

The recent derivation of Sho-knockout mice [START_REF] Daude | Knockout of the prion protein (PrP)-like Sprn gene does not produce embryonic lethality in combination with PrP(C)-deficiency[END_REF] might help to decipher the real expression pattern of this protein by comparative IHC analysis, but these mice were not available to us at the time of this experiment.

In the same way, the PrP protein was detected by IHC and western blot at adulthood in both species and at 130 dpc in goats (Fig. 3). By discarding the strong nonspecific staining in the interstitial area of adult mouse testes, identified by using Prnp 0/0 testicular samples (Fig. 3A), the PrP protein appears to be present at all stages mainly inside the seminiferous tubules, most likely in the Sertoli cells (clearly visible at 130 dpc in goats) and in the germ cells at the end of spermiogenesis (see the staining of elongated spermatids on Fig. 3B,C). Presence of PrP in the testis was confirmed by western blot performed on adult mice and goats testes and spermatozoa (Fig. 3F). The presence of PrP on ejaculated sperm cells has already been reported in humans, cattle and mice [START_REF] Shaked | A C-terminal-truncated PrP isoform is present in mature sperm[END_REF], but its testicular or epididymal origin remains debated, as recently discussed [START_REF] Pailhoux | Role of the prion protein family in the gonads[END_REF]. To our knowledge, this is the first time that PrP is clearly detected in the Sertoli cells of imma-ture (130 dpc in goats) and mature (in adult mice) testes.

By contrast with mice, DPL is detected in goat Leydig cells

We have previously shown that the DPL protein could be detected in germ cells of both sexes and in foetal Leydig cells of early goats developing testes at 44 and 62 dpc [START_REF] Kocer | Goat PRND expression pattern suggests its involvement in early sex differentiation[END_REF]. According to qRT-PCR results (Fig. 1F,G) showing a high expression of PRND in goat developing testes, we checked its cellular localization throughout testis development in immature and mature testes and on spermatozoa of both species (Fig. 4). First, the specificity of anti-DPL antibody boDpl67-81 [START_REF] Rondena | Identification of bovine doppel protein in testis, ovary and ejaculated spermatozoa[END_REF] was confirmed as no signal was obtained on adult Prnd 0/0 mouse testes (Fig. 4A,B). Using this antibody, DPL is detected in the cytoplasm of some germ cells in both species; germ cells that have passed the zygotene stage of meiosis (i.e. pachytene and spermatides)(Fig. 4C,F and K,N) and its presence persists on spermatozoa with a high staining of the acrosomal vesicle (Fig. 4G,H and O, P). These results are in complete agreement with the role of DPL in spermiogenesis [START_REF] Behrens | Absence of the prion protein homologue Doppel causes male sterility[END_REF][START_REF] Paisley | Male infertility and DNA damage in Doppel knockout and prion protein/Doppel double-knockout mice[END_REF]. More interestingly, a strong staining is detected in some cells of the interstitial testicular space, specifically in the goat species and at immature stages, as the staining disappears in adult testes (compare Fig. 4I,L with M,N). In order to precisely define this DPL staining in the interstitial testicular compartment, we carried out double IHC with an anti-CYP17 antibody detecting a Leydig cell-specific marker corresponding to a key enzyme of steroid synthesis, the cytochrome P 450 17alpha-hydroxylase/17,20-lyase [START_REF] Hales | Testosterone inhibits cAMP-induced de Novo synthesis of Leydig cell cytochrome P-450(17 alpha) by an androgen receptor-mediated mechanism[END_REF]. DPL and CYP17 are found colocalized in the same interstitial cells (Fig. 5A-F). DPL staining in goat testes disap- pears between the prepubertal 3-month and the pubertal 7-month stages (Fig. 4K-N), suggesting that PRND is specifically expressed in the foetal Leydig cell population. These cells disappear after birth and are replaced by adult Leydig cells at puberty [START_REF] Dong | Gene expression during development of fetal and adult Leydig cells[END_REF]. Finally, the PRND expression profile appear hugely similar to that of 3bHSD, another Leydig cell-specific marker, until 1 month after birth indicating that during testis development the major part of PRND expression is Leydig specific (Fig. 5G). This observation could explain the high testicular levels of PRND transcripts detected from 41 dpc to 1 mpp specifically in goat testes (Fig. 1G) and not in the mouse testes of the corresponding stages, from 12.5 dpc to 5 dpp (Fig. 1F).

During the prepubertal period, spermatogenesis starts (25 dpp in mice, 3 mpp in goats), the number of germ cells increases and more importantly differentiated meiotic germ cells expressing PRND/Prnd appear. From these prepubertal stages, the PRND/Prnd expression profiles are similar between mice and goats (Fig. 1F,G), but diverge from that of 3bHSD in goats (Fig. 5G). It indicates that around puberty PRND expression increases in postmeiotic germ cells and disappears from foetal Leydig cells because of their own disappearance.

In conclusion, we report the differential expression of the three members of the prion protein gene family in the developing gonads of mice and goats. Only relatively low levels of expression were detected for Sho, an observation that might relate with the lack of reproductive-associated phenotype in mouse Sprnknockout mice [START_REF] B€ Ueler | Normal development and behaviour of mice lacking the neuronal cell-surface PrP protein[END_REF][START_REF] Daude | Knockout of the prion protein (PrP)-like Sprn gene does not produce embryonic lethality in combination with PrP(C)-deficiency[END_REF]. By contrast and in addition to its conserved role in spermiogenesis, PRND seems to be a key marker of foetal Leydig cells in goats. This observation adds PRND to the list of genes having potentially different biological roles in mice and humans or ruminants (see [START_REF] Boulanger | FOXL2 is a female sex-determining gene in the goat[END_REF][START_REF] Elzaiat | High-throughput sequencing analyses of XX genital ridges lacking FOXL2 reveal DMRT1 up-regulation before SOX9 expression during the sex-reversal process in goats[END_REF] for recent examples). The present results highlight that PRND is a key candidate gene for functional studies in goats because its involvement in foetal Leydig cells cannot be studied in the widely used mouse mammalian model. Deciphering this role may have important implications for human reproduction. In order to determine if PRND could be a crucial actor of foetal Leydig cell develop- ment, its targeting is currently under way in the goat species by using genome editing technologies, recently proven successful by us in this species [START_REF] Boulanger | FOXL2 is a female sex-determining gene in the goat[END_REF].

Fig. 1 .

 1 Fig. 1. Expression of Prion family genes during gonad development. (A) Chronology of gonad differentiation in mice and goats. Expression of the Prnp/PRNP (B, C), Sprn/SPRN (D, E) and PrndPRND (F, G) genes was quantified using real-time RT-PCR in ovaries (pink histograms) and testes (blue histograms) in mice (B, D, F) and goats (C, E, G). From 12.5 dpc to 13.5 dpc (mice) and 30 dpc to 36 dpc (goats): gonad + mesonephros, others stages: gonad only. Prnp/PRNP, PrP protein gene; Sprn/SPRN, Sho gene; Prnd/PRND, Dpl gene; dpc, days post co€ ıtum; dpp, days post partum; mpp, months post partum. Values are expressed in percentage according to the highest one noted 100%. Means AE SD were plotted. Planned comparisons were made on the predicted means with 'sex' and 'stage' as treatment factors (two-way ANOVA approach, followed by a Fisher's LSD-test). For each stage, significant differences between the two sexes are showed by stars (*P-value ≤ 0.05; **P-value ≤ 0.01; ***P-value < 0.001).

Fig. 2 .

 2 Fig. 2. Expression of Prion family genes during postnatal gonad development in mice. Sprn (A), Prnp (B) and Prnd (C) gene expressions were quantified using realtime RT-PCR in postnatal ovaries (pink histograms) and testes (blue histograms) in mice. Sprn, Sho gene; Prnp, PrP protein gene; Prnd, Dpl gene; dpp, days post partum; mpp, months post partum.

Fig. 3 .

 3 Fig. 3. PrP immunodetection in mice and goat testes. (A): The specificity of PrP antibody (sha31) was tested on adult Prnp 0/0 testis in mice. Testicular interstitial cells showed a nonspecific fluorescence. PrP immunodetection was performed on adult testis in mice (B, C) and on 130 dpc testis in goats (D, E). The fluorescent staining is presented with a 4, 6-diamine-2-phenylidole-dihydrochloride (DAPI) blue nuclear-specific counterstaining. (C, E) photographs correspond to an enlargement of the white rectangles depicted on (B, D) photographs. Some cells are marked as followed: Sertoli cells (white arrow, pointed on a Sertoli cell nucleus), Leydig cells (yellow star) and germinal cells (red arrowhead). dpc, days post co€ ıtum. (F): Western blot assays of endogenous PrP (Sha31b) of adult homogenates tissues (brain, testis and lung) and sperm cells in mice and goats. Bands denoted the presence of various PrP isoforms in these tissues. No protein was detected in Prnp 0/0 brain mice used as negative control.

Fig. 4 .

 4 Fig. 4. Dpl immunodetection during testis development in mice and goats. In mice, the specificity of the Dpl antibody (boDpl67-81) was tested on adult Prnd 0/0 testis (A, B). Dpl immunodetection was performed on 25 dpp (C, D), adult (E, F) testes and on spermatozoa (G, H). In goats, Dpl immunostaining was carried out 130 dpc (I, J), 3 mpp (K, L), adult (M, N) testes and on spermatozoa (O, P). The fluorescent staining is presented with a 4, 6-diamine-2-phenylidole-dihydrochloride (DAPI) blue nuclear-specific counterstaining. Thesecond (B, D, F, H) and fourth (J, L, N, P) columns correspond to an enlargement of the white rectangles depicted on the first (A, C, E, G) and third (I, K, M, O) columns respectively. Cells are marked as followed: Sertoli (white arrow), Leydig (yellow star) and germinal (red arrowhead). dpc, days post co€ ıtum; dpp, days post partum; mpp, months post partum; SPZ, spermatozoa.

Fig. 5 .

 5 Fig. 5. Expression of Leydig cell markers in goat testis. Dpl (A-F), VASA (B, E) and CYP17 (C, F) immunodetections were performed in a goat testis at 130 dpc. (D-F) photographs correspond to an enlargement of the white rectangles depicted on (A-C) photographs. (G): 3bHSD (black curve) and PRND (blue curve) expression was quantified using real-time RT-PCR during testis development in goat and represented on the same graph. At 30 and 36 dpc gonads are not dissected from mesonephros. dpc, days post co€ ıtum; dpp, days post partum; mpp, months post partum.

Table 1 .

 1 Number of animals used at each developmental stage for gene expression studies with real-time PCR.

					Number of	Number of
	Species	Stages	Sex	Tissues	individual	independent RT
	Mouse	12.5 dpc	Female	2 Gonads + Mesonephros	9
			Male	2 Gonads + Mesonephros	7
		13.5 dpc	Female	2 Gonads + Mesonephros	6
			Male	2 Gonads + Mesonephros	12
		14.5 dpc	Female	2 Gonads	4
			Male	2 Gonads	6
		18.5 dpc	Female	2 Gonads	6
			Male	2 Gonads	9
		5 dpp	Female	2 Gonads	11
			Male	1 Gonad	4
		25 dpp	Female	1 Gonad	5
			Male	1/2 Gonad	4
		50 dpp	Female	1 Gonad	3
			Male	1/2 Gonad	3
		4 mpp	Female	1 Gonad	3
			Male	1/2 Gonad	3
		6 mpp	Female	1 Gonad	2
			Male	1/2 Gonad	3
		6-8 mpp	Female	1 Gonad	3
			Male	1/2 Gonad	3
		10-12 mpp	Female	1 Gonad	3
			Male	1/2 Gonad	3
	Goat	30 dpc	Female	1 Gonads + Mesonephros	1
			Male	1 Gonads + Mesonephros	2
		32 dpc	Female	1 Gonads + Mesonephros	1
			Male	1 Gonads + Mesonephros	2
		34 dpc	Female	1 Gonads + Mesonephros	2
			Male	1 Gonads + Mesonephros	2
		36 dpc	Female	1 Gonads + Mesonephros	3
			Male	1 Gonads + Mesonephros	2
		41 dpc	Female	1 Gonad	4
			Male	1 Gonad	4
		45 dpc	Female	1 Gonad	9
			Male	1 Gonad	4
		50 dpc	Female	1 Gonad	4
			Male	1 Gonad	3
		60 dpc	Female	1 Gonad	2
			Male	1 Gonad	2
		70 dpc	Female	1 Gonad	2
			Male	1 Gonad	2
		90 dpc	Female	1 Gonad	2
			Male	Piece of gonad	2
		130 dpc	Female	1 Gonad	1
			Male	Piece of gonad	1
		5 dpp	Female	Piece of gonad	1
			Male	Piece of gonad	1
		1 mpp	Female	Piece of gonad	1
			Male	Piece of gonad	1
		3 mpp	Female	Piece of gonad	1
			Male	Piece of gonad	1
		7 mpp	Female	Piece of gonad	1
			Male	Piece of gonad	1
		4 ypp	Male	Piece of gonad	1 a

Table 2 .

 2 Primers used in the present study.

	Amplicon size (bp)
	E-efficiency
	Reverse primer
	Forward primer
	Ref seq
	Gene ID
	Gene	Mice primers	Prnp

Table 3 .

 3 Antibodies used in the present study.

	Primary antibody	Reference		Source	IHC dilution	WB dilution
	DPL -purified boDpl67-81	Gift of Dr Paltrinieri (Rondena et al. [28])	Rabbit	1 : 150
	PRP -Sha31	(Feraudet et al. [36])		Mouse	1 : 1000
	Biotinylated PRP -b Sha31	(Feraudet et al. [36])		Mouse		1 : 50 000
	Murine SHO	Gift of Dr Westaway (Watts et al. [15])	Rabbit	1 : 500
	Ovine SHO	Gift of Dr Peelman (Lampo et al. [37])	Rabbit	1 : 50
	Cyp17A	Gift of Dr Hales (Hales et al.[38])	Rabbit	1 : 200
	Secondary antibody	Reference	Conjugate		IHC dilution	WB dilution
	Anti-rabbit IgG-Dylight 488	072-03-15-06, KPL	Fluorescein		1 : 200
	Anti-rabbit IgG-Dylight 594	072-09-15-06, KPL	Fluorescein		1 : 200
	Anti-mouse IgG-Cy3	AP160C, Millipore	Cyanine 3		1 : 200
	Anti-rabbit IgG-B	BA-1000, Vector	Biotin		1 : 200
	Anti-mouse-IgG-HRP strep		Horseradish peroxydase		1/20 000
			Streptavidin
	Fluorescein Streptavidin	SA-5001, Vector			1 : 200
	Texas Red â Streptavidin	SA-5006, Vector			1 : 200
	Mounting medium	Reference
	Vectashield for fluorescence	H-1200, Vector
	with DAPI

Prion family gene expression during gonad developmentA. Allais-Bonnet et al.

FEBS Open Bio 6 (2016) 4-15 ª 2015 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.

FEBS Open Bio 6 (2016) 4-15 ª 2015 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.Prion family gene expression during gonad developmentA. Allais-Bonnet et al.

Acknowledgements

We thank all the staff of UCEA (INRA's Joint Animal Experimental Unit) for management of the goats, and Mrs Elaine R emy for English corrections. We also thank the ANR (National Research Agency) for its financial support via the following grant, ANR-09-BLAN-0015-01, PRIFAGENE.

Author contributions

AAB, MP, KMG, JLV and EP participated in the conception of the study, interpretation of data and in the drafting of the article. BP and JC participated in the design and realization of animal experiments. AAB, ME, MA and FMT performed experiments.