F Amiot

F Kanoufi

F Hild
email: hild@lmt.ens-cachan.fr

J P Roger

Charge redistribution in electrochemically-actuated mechanical sensors

Keywords: Cantilever-based sensors, Full-field measurements, Identification problem, Microscopy of surfaces, Surface strains

Many proofs of concept studies have established the mechanical sensitivity of functionalized microcantilevers to a large spectrum of target molecules. However, moving to real-life applications also requires the monitored mechanical effect to be highly specific. Moving towards more specificity in cantilever-based sensing, monitoring the mechanical response of electrochemically actuated microcantilevers is then thought to provide a fast, reliable and complementary experimental information to the longtime cantilever bending measurement for the detection of target molecules. Full-field measurements are therefore used to investigate the way the electro-elastic coupling is altered when a microcantilever undergoes decane-thiol adsorption. The proposed technique reveals that the latter results in a charge density redistribution along the cantilever in addition to the local surface passivation. Focusing on the cantilever tip displacement under electrochemical actuation, this redistribution partially compensates the electro-elastic coupling alteration due to the surface passivation, therefore possibly yielding an ambiguous detection result. This effect should be taken into account for the optimal design of specific electrochemically actuated mechanical sensors.

Introduction

Decreasing the size of mechanical objects induces an increase of their surface / volume ratio, and thus their ability to interact with their environment.

That is the main reason for the development of MEMS-based bio-chemical sensors. One of the object surfaces is functionalized to allow for chemical recognition of a target molecule. When bonding occurs (regardless of its type), two types of detection schemes are possible [START_REF] Lavrik | Cantilever transducers as a platform for chemical and biological sensors[END_REF], namely, one monitors the resonance frequency shift induced by mass changes, or one measures the quasi-static induced bending arising from surface free-strains.

The measurement of the resonance frequency shift is used to detect mass variations [START_REF] Ilic | Mechanical resonant immunospecific biological detector[END_REF][START_REF] Ilic | Attogram detection using nanoelectromechanical oscillators[END_REF][START_REF] Lavrik | Femtogram mass detection using photothermally actuated nanomechanical resonators[END_REF] and then to resolve small amounts of biological material [START_REF] Gupta | Single virus particle mass detection using microresonators with nanoscale thickness[END_REF].

This resonant frequency shift appears in conjunction with a surface free-strain, which is preferably measured in liquids to detect DNA hybridization [START_REF] Fritz | Translating biomolecular recognition into nanomechanics[END_REF] for instance. In low viscosity media, this quasi-static bending measurement is carried out together with the resonance frequency shift to provide a more robust detection procedure [START_REF] Pinnaduwage | Sensitive detection of plastic explosives with self-assembled monolayer-coated microcantilevers[END_REF]. On the one hand, the detection specificity may be improved by tailoring the used coatings. On the other hand, a fast and user-controlled query mechanism of the cantilever mechanical state is highly desirable to move towards real-life applications.

The coupling between electrochemical and mechanical effects involves small elastic strains, thus requiring the measurement of very small strains [START_REF] Marichev | Partial charge transfer during anion adsorption Methodological aspects Surf[END_REF], so that electrochemical effects have been investigated through the mechanical loading they induce on microcantilevers. An investigation of the double-layer region [START_REF] Fredlein | An electrocapillary study of the goldperchloric acid solution interface Surf[END_REF][START_REF] Raiteri | Measuring electrochemically induced surface stress with an atomic force microscope[END_REF][START_REF] Tian | Observation of the surface stress induced in microcantilevers by electrochemical redox processes Ultramicroscopy 100[END_REF][START_REF] Lee | Electromechanical identification of molecules adsorbed on microcantilevers[END_REF] (i.e., when no electrochemical reaction occurred) as well as adsorption reactions [START_REF] Tabard-Cossa | A differential microcantilever-based system for measuring surface stress changes induced by electrochemical reactions Sens[END_REF][START_REF] Ibach | Potential-induced stress in the solid-liquid interface: Au(111) and Au(100) in an HCl04 electrolyte[END_REF][START_REF] Haiss | Surface stress of clean and adsorbate-covered solids Rep[END_REF][START_REF] Tabard-Cossa | Microcantilever-based sensors: effect of morphology, adhesion, and cleanliness of the sensing surface on surface stress[END_REF] and electrodeposition [START_REF] Brunt | Measuring the surface stresses in an electrochemically deposited metal monolayer: Pb on Au (111)[END_REF] have been carried out using the standard optical lever technique [START_REF] Lavrik | Cantilever transducers as a platform for chemical and biological sensors[END_REF].

In order to further describe electrochemical phenomena at solid electrodes, several interferometric techniques have been used to provide descriptions of the electrochemically driven topographic or chemical changes occurring in the layer adjacent to an electrode [START_REF] White | In situ topographical imaging of electrode surfaces using high-resolution phase measurement interferometric microscopy[END_REF][START_REF] Smith | Phasemeasurement interferometric microscopy of microlithographically fabricated platinum electrodes[END_REF][START_REF] Li | Interferometric measurement of a depletion layer structure and volumetric data in concentrated organic redox solutions[END_REF]. In the special case of microcantilever inspection, it has been proven that using full-field interferometric measurements [START_REF] Amiot | Nomarski imaging interferometry to measure the displacement field of micro-electro-mechanical systems[END_REF] to investigate the electro-elastic coupling provides redundant enough experimental information to build a model of the coupled interfacial phenomena [START_REF] Amiot | Identification of the electroelastic coupling from multi-physical fields measured at the micrometre scale[END_REF]. Using such a spatially resolved information is then necessary, since a micro-cantilever is expected to behave as an "antenna", thus yielding a charge localization near geometric singularities.

It is therefore proposed to adapt such a tool to describe at the local level as well as at the cantilever scale the way the electro-elastic coupling is altered by molecular adsorption. As the mechanical reaction to an electrochemical stimulus is found to be highly adsorption rate dependent, one considers the concept of electrochemically-actuated micromechanical sensors as a potential candidate for various real-life target molecules recognition problems. After a description of the experimental set-up and procedures, the adsorption process is described from the electrochemical and mechanical point of view. A dedicated framework is then proposed to analyze phase maps acquired when the cantilever undergoes an electrochemical actuation at different adsorption rates. This analysis shows that both the coupling intensity and the charge distribution are significantly altered by adsorption, so that using a device as simple as a cantilever is not optimal to provide a reliable detection scheme when the global mechanical response of the sensor is monitored.

2 Experimental procedure

Experimental set-up

A home-made fluid-cell is used to host an array of gold coated microcantilevers. This sample is fastened to a copper substrate with a conducting epoxy glue.

The temperature of this copper substrate is controlled through Peltier effect modules and a temperature sensor. Another copper part, whose temperature is also controlled through Peltier modules, encapsulates this fluid-cell, whose content is modified with a fluid inlet and outlet.

The objects under consideration are micro-cantilevers (70 × 20 × 0.84 µm 3 ) that are made of 770-nm thick silica obtained by thermal oxidation of a silicon wafer. Titanium (20-nm thick) and 50-nm thick gold layers are then deposited onto the sample. The gold layer on top of the cantilevers is utilized as a working electrode in a KCl solution. All the connections are insulated with a thin PDMS layer to ensure that only the gold surface of the working electrode is in contact with the solution. It has a total area of almost 1 cm 2 in constant contact with the electrolytic solution.

Surface activation

After bubbling nitrogen, the fluid cell is then filled with a KCl solution (10 -2 M), prepared from milliQ water. The gold surface is then cleaned electrochemically by performing successive cyclic voltammograms between 0 and 0.8 V at a rate of 10 mV/s. Gold is then successively oxidized and reduced, until a stable voltammogram (i.e., after a few cycles) is obtained, thus ensuring a clean and reproducible metallic surface. The fluid cell is then purged and filled again with a 10 -2 M KCl deoxygenated solution.

Cyclic voltammetry

The potential window was chosen so that the electrode processes are not perturbed by any substance or electrochemical transformation of the electrode material. For potentials less than -0.1 V vs. Ag/AgCl, oxygen reduction may occur while a peak, presumably related to Au oxidation, is observed for potentials greater than 0.5 V. During three potential cycles, an optical phase map [START_REF] Amiot | Nomarski imaging interferometry to measure the displacement field of micro-electro-mechanical systems[END_REF] is recorded every 0.05-V step , thus providing a set of 61 phase maps, that is a 15 × 111 × 61 phase measurements stack covering the cantilever through the three potential cycles. The choice of the potential scan rate (2 mV/s) ensures that the electrode equilibrium is established in times much shorter than the time interval between two snapshots (25 s). The phase map acquisition is carried out during almost 1 s. Therefore the potential of the electrode is considered constant during this observation. Such cyclic voltammetry cycles have been performed before (CV0), during (CV1) and after (CV2) decane-thiol adsorption.

Displacement field measurement

The cantilever bending is monitored using a phase-modulated Nomarski imaging shear-interferometer [START_REF] Amiot | Nomarski imaging interferometry to measure the displacement field of micro-electro-mechanical systems[END_REF]. The interference pattern is obtained as the difference of two topographies of the surface, shifted by a Wollaston prism by a distance d. The Wollaston shear-direction is chosen to be parallel to the cantilever axis, denoted by x, so that the topographies involved in the interference pattern are views of the cantilever shifted along the direction of its larger dimension (Fig. 1). The distance d is chosen to be similar in magnitude with the cantilever length.

Monitoring the induced quasi-static cantilever bending, the recorded phase map changes φ mes are purely mechanical as long as there is no heterogeneous chemical surface modification [START_REF] Amiot | Nomarski imaging interferometry to measure the displacement field of micro-electro-mechanical systems[END_REF], and is thus described solely by φ mec , which arise from both surface out-of-plane displacement and rotation

φ mec (x, y) = 4πn l ιλ (v(x, y) -v(x -d, y)) + ∂φ W ∂γ dv dx (x, y) - dv dx (x -d, y) (1)
v(x, y) is the out-of-plane displacement field and ∂φ W ∂γ is an experimentally identified coefficient, n l the average refractive index of the ambient medium, λ the used wavelength, and ι a scaling coefficient depending on the numerical aperture of the objective lens. Section 3.3 deals with the case where heterogeneous surface reaction is expected. Throughout the reported experiments, the displacement field was observed to be homogeneous across the cantilever width, so that it is expanded onto a user-defined functions basis of the longi-

tudinal coordinate x v(x, n) = R r=1 ζ r (n)f r (x) (2)
where {ζ r (n)} for r ∈ {1 . . . R} is the projection of the displacement field v onto the basis {f r (x)} for the time step n. The displacement is then obtained from the measured phase stack by minimizing

η 2 = 1 N p x,y,n χ(x, y, n) 2 (3) = 1 N p x,y,n 1 µ(x, y, n) 2 (φ test (x, y, n) -φ test (x, y, n -1) -φ mes (x, y, n) + φ mes (x, y, n -1)) 2 (4)
where φ mes (x, y, n) -φ mes (x, y, n -1) is the measured phase change between two consecutive loading steps, φ test (x, y, n) -φ test (x, y, n-1) the phase change estimated from the test parameters ζ r , N p the number of measurement points and µ(x, y, n) 2 the estimated phase change variance at the considered point.

The optimal parameters set then provides the cantilever displacement field.

Decane-thiol adsorption

Decane-thiol from Sigma-Aldrich was diluted in ethanol to get a 7 mM decanethiol solution. The latter was then diluted (1 % in volume) in the supporting electrolyte, namely 10 -2 M KCl, before injection in the above-described fluid-cell. The refractive index change induced when mixing the decane-thiol solution with 10 -2 M KCl is then negligible and does not perturb the imaging properties of the microscope. The cantilever quasi-static bending is then monitored (step 1: 1 image every 2 minutes during 5 hours; step 2: 1 image each 3 minutes during 7h30). In order to monitor the adsorption process, AC voltammetry scans have been carried before (DC0), during (DC1) and after (DC2) the decane-thiol adsorption. The electrode impedance was measured between -0.1 and 0.4 V vs. Ag/AgCl, at 20 Hz with a modulation amplitude of 25 mV. These AC voltammetry scans are performed prior to the three above-described voltammetry cycles. The experiment took place following the sequence DC0, CV0, adsorption step 1, DC1, CV1, adsorption step 2, DC2, CV2, so that the consequence of decane-thiol adsorption on the electro-elastic coupling is assessed (Fig. 2).

3 Results and discussion for Au(111)/KCl 10 -2 M systems [START_REF] Clavilier | Etude de la structure de la couche double sur les electrodes d'or[END_REF]. In addition, the coverage ratio may be roughly estimated from the minimum capacitance values, assuming that the coverage ratio is 0 for DC0, and 1 at the end of the experiment (DC2).

Electrochemical response of the microcantilevers array

Considering that the electrode behavior is described by a capacitor [START_REF] Damaskin | Adsorption of Organic Compounds on Electrodes[END_REF][START_REF] Subramanian | Study of kinetics of adsorption of alkanethiols on gold using electrochemical impedance spectroscopy Electrochim[END_REF] (representing the clean part of the electrode) in a branch parallel to a serial capacitors assembly [START_REF] Becka | Electrochemistry at ω-hydroxy thiol coated electrodes. 4. Comparison of the double-layer at ω-hydroxy thiol and alkanethiol monolayer coated Au electrodes[END_REF] (representing the covered part of the electrode), the coverage ratio is estimated to be 0.71 for DC1, and the decane-thiol layer capacitance is estimated to be about 0.8 µF/cm 2 , in agreement with reported values [START_REF] Porter | Spontaneously organized molecular assemblies. 4. Structural characterization of n-alkyl thiol monolayers on gold by optical ellipsometry, infrared spectroscopy, and electrochemistry[END_REF]. The current flowing through the electrode is recorded during cyclic voltammetry scans.

Adsorption-induced cantilever bending

The adsorption process (i.e., moving the molecules closer to the surface and considering their interaction) is highly favorable from a thermodynamics point of view, so that the interfacial system provides relatively large surface strains to reach mechanical equilibrium. This corresponds to the minimum overall enthalpy, leading to a decreased chemical enthalpy (because of decane-thiol adsorption process) and an increased elastic energy (because of cantilever bending) [START_REF] Amiot | A model for chemically-induced mechanical loading on[END_REF]. Adsorption is assumed to occur uniformly along the cantilever, so that it does not induce any additional phase contribution. The procedure detailed in Section 2.4 then holds. The identified quasi-static displacement fields along the median line of the cantilever using a polynomial basis f r (x) = x r for r ranging from 1 to R = 3 are shown as functions of time in Fig. 4 for both adsorption steps. The minimum value of η 2 min is found to be around 10 -10

for both adsorption steps, thereby proving that the displacement field is well described by a cubic polynomial. As already reported [START_REF] Berger | Surface stress in the self-assembly of alkanethiols on gold probed by a force microscopy technique[END_REF][START_REF] Berger | Güntherodt Surface stress in the self-assembly of alkanethiols on gold[END_REF], upon adsorption of thiol the cantilever experiences a downward bending, suggesting that this adsorption is accompanied by a gold surface expansion. The maximum displacement is about 80 nm after 5 h (end of step 1) and 160 nm after step 2 (the initial states correspond to the beginning of adsorption steps 1 and 2, respectively). Let us underline the different time scales the displacement follows for the two adsorption steps:

• on the one hand, adsorption on a clean surface (step 1) yields a unique characteristic time scale;

• on the other hand, adsorption on a pre-adsorbed surface (step 2) exhibits a mechanical effect following two very different time scales, namely, a first mechanical contribution takes place during a few minutes, whereas the full mechanical effect develops during several hours.

According to the literature, the formation kinetics of alkane-thiol self-assembled monolayer were investigated using various physico-chemical techniques including electrochemical [START_REF] Subramanian | Study of kinetics of adsorption of alkanethiols on gold using electrochemical impedance spectroscopy Electrochim[END_REF], AFM [START_REF] Hu | In situ monitoring of kinetics of charged thiol adsorption on gold using an atomic force microscope[END_REF], surface plasmon resonance (SPR) [START_REF] Debono | Self-assembly of short and long-chain n-alkyl thiols onto gold surfaces: A real-time study using surface plasmon resonance techniques Can[END_REF] and ellipsometry [START_REF] Bain | Formation of monolayer films by the spontaneous assembly of organic thiols from solution onto gold[END_REF][START_REF] Damos | Determination of thickness, dielectric constant of thiol films, and kinetics of adsorption using surface plasmon resonance[END_REF]. Even though there are some contradictions concerning the rate of formation of the layer, most of the studies deal with a two-stage adsorption process in agreement with what is observed herein. In principle, the present results may be compared with the two time scales identified by ellipsometric monitoring of adsorption process [START_REF] Damos | Determination of thickness, dielectric constant of thiol films, and kinetics of adsorption using surface plasmon resonance[END_REF], even though it has been reported that the mechanical effect and the optical thickness of the adsorbed layer growth kinetics are not simply related [START_REF] Godin | Combined in-situ micromechanical cantilever-based sensing and ellipsometry[END_REF]. For this reason and also because the adsorption process homogeneity along the micro-cantilever is difficult to assess, a deeper description of the kinetics of formation of the monolayer lies beyond the scope of the present paper. It is worth noting that this long-term monitoring of the mechanical effect induced by adsorption is achieved using differential interferometric measurements and all possible precautions to avoid any significant drift, which are not easily implemented in real-life applications.

Electrochemically-induced cantilever bending

In order to bypass the difficulty of performing long time measurements, which are likely to be subjected to significant drifts (e.g., thermal), the displacement of the cantilever is modulated at a user-defined frequency. This is achieved by using the electro-elastic coupling that is stimulating electrochemically, during a cyclic voltammetry experiment, the deformation of the cantilever (Subsec-tion 2.3). In that case, the measured phase map arises from both mechanical deformation of the cantilever and change in the interface composition owing to changes in the chemical composition of the double layer adjacent to the electrode, so that the procedure presented in Section 2.4 has to be modified.

Because of the presence of a (partially) adsorbed layer, the method proposed in Ref. [START_REF] Amiot | Identification of the electroelastic coupling from multi-physical fields measured at the micrometre scale[END_REF] has to be enriched. The thin electrolyte layer close to the surface, whose thickness scales as the Debye length λ D , is significantly modified when charging the electrode. The concentration shift induced in this layer modifies locally the refractive index of the solution. In addition, charging the interface modifies the free electrons density in the metal, thus changing the complex refractive index of the metal [START_REF] Paik | The adsorption of anions at the solid-solution interface[END_REF]. Both contributions (as well as decane-thiol adsorption) modify the complex reflection coefficient of the interface, and therefore generate a non-mechanically induced optical phase change

φ mes = φ mec + φ ec (5)
The measured phase φ mes results from the mechanical contribution φ mec and an additional electrochemical term φ ec that is obtained by assuming that reflection occurs at the interface between two homogeneous layers:

• on the metallic side, the thickness is arbitrarily chosen to be 1 nm [START_REF] Paik | The adsorption of anions at the solid-solution interface[END_REF], and the refractive index is obtained according to Drude's model;

• the thickness of the liquid phase layer is chosen to be equal to 2λ D + ϑλ ads , where λ ads is the adsorbed decane-thiol film thickness, and ϑ is a mixing ratio. The parameter ϑ is not a coverage ratio but describes how much the ions penetrate the decane-thiol adsorbed layer during the cyclic voltammetry. The case when ϑ = 0 may thus result either from a clean electrode (i.e., no adsorption) or from an impenetrable adsorbed layer. The composition of this layer is determined to ensure the same surface excess than predicted by the Gouy-Chapman theory, and the refractive index of this homogeneous layer is obtained from the Lorentz-Lorenz formula [START_REF] Stedman | Reflectance and ellipsometry of Metal/Electrolyte Interfaces Trans[END_REF]. This significant enrichment, compared with the modeling we proposed in Ref. [START_REF] Amiot | Identification of the electroelastic coupling from multi-physical fields measured at the micrometre scale[END_REF], allows one to deal with partially covered surfaces.

For ions, the molar volumes and refractivities are taken from the literature [START_REF] Waddington | Ionic radii and the method of the undetermined parameter Trans[END_REF][START_REF] Zana | Ultrasonic vibration potentials and their use in the determination of ionic partial molal volumes[END_REF],

namely, λ ads = 1.5 nm is obtained from published estimates [START_REF] Porter | Spontaneously organized molecular assemblies. 4. Structural characterization of n-alkyl thiol monolayers on gold by optical ellipsometry, infrared spectroscopy, and electrochemistry[END_REF], while the molar refractivity for the decane-thiol is obtained from Ref. [START_REF] Kupchik | General treatment of the heteroatoms with the randic molecular connectivity index Quant[END_REF]. The above assumptions allow one to compute the electrochemical contribution as a function of the surface charge density field and the mixing ratio, using Fresnel's equations for a given equivalent incidence angle θ e and both TE and TM polarizations [START_REF] Born | Principles of optics[END_REF] φ ec (x, y, ϑ, θ e ) = φ T E,T M (σ m (x, y), ϑ, θ e ) -φ T E,T M (σ m (x -d, y), ϑ, θ e ) (

The influence of θ e is found to be negligible when compared with ϑ. In previous investigations of the electro-elastic coupling on clean cantilevers [START_REF] Amiot | Identification of the electroelastic coupling from multi-physical fields measured at the micrometre scale[END_REF] the microcantilever bending was observed to result from a non-uniform mechanical loading. It was interpreted as a singular charging of the microcantilever edge in agreement with non-uniform current distribution at microelectrodes [START_REF] Maus | Imaging of nonuniform current density at microelectrodes by electrogenerated chemiluminescence[END_REF][START_REF] Combellas | Surface modification of halogenated polymers. 10. Redox catalysis induction of the polymerization of vinylic monomers. Application to the localized graft copolymerization of poly(tetrafluoroethylene) surfaces by vinylic monomers[END_REF].

The integration of the current with respect to time then provides the overall charge carried by the working electrode Q as a function of the potential. A good correlation between the mechanical behavior of the cantilever and the surface electrochemical response was obtained when considering that the local charge density σ m at a distance x from the field border on the substrate is distributed according to

σ m (x, Q) = D(x) × Q (7)
where Q is the overall surface charge of the entire electrode, and the localization function is written as

D(x) = c q (x 0 ) (x 0 -x) p (8)
where the constant c q (x 0 ) is defined so that D(x) = 1 when x tends to the field border on the substrate, x 0 and p are used to describe the position and order of the singular charge density field, respectively. A similar framework is used herein.

In addition, the mechanical contribution is obtained by assuming that the cantilever is subjected to a shear-stress field τ (x), which is a continuous function of the local charge density σ m . This local relationship is then expanded onto a polynomial basis

τ (x) = T t=1 δ t σ t m (x) (9)
where {δ t } are the coefficients describing the local coupling relationship to be found. For a given charge density field, the displacement field induced by each component of Eq. ( 9) is derived by using the Euler-Bernoulli beam theory [START_REF] Timoshenko | Theory of Elasticity[END_REF],

thus yielding the functions basis f t (x) defined in Eq.2. To summarize, each cross-section is assumed to remain planar during deformation, so that the local beam curvature is obtained from the bending moment M b generated by the shear-stress τ applied to the cantilever

M b = EI d 2 v(x) dx 2 = - L x
beτ (ξ)dξ [START_REF] Fredlein | An electrocapillary study of the goldperchloric acid solution interface Surf[END_REF] where b denotes the cantilever width, EI its flexural stiffness, and e the distance from the neutral axis to the gold surface. Setting arbitrarily the equivalent incidence angle to θ e = 0.3 rad (this does not significantly affect the following results), one is then able to compute a trial phase field, and thus to find the optimal set of parameters {p, x 0 , ϑ, δ t } describing the electro-elastic test under scrutiny by minimizing the function defined by Eq. ( 3). Linearizing this latter function with respect to its parameters close to the solution, an estimate for the standard deviation of the identified parameters is obtained from the measurement reproducibility.

Table 1 shows the identified parameters for the three electro-elastic tests, namely, CV0, CV1 and CV2. For all the considered tests, T = 3 is found to provide the best compromise between the coupling description and the minimization robustness. Changing the arbitrarily chosen θ e value has no significant effect on the following results. The residual η 2 min is small compared to 1, meaning that the phase change fields all over the 3 potential cycles are well described by the proposed modeling. In addition, an upper bound Γ ϑ for the standard deviation on ϑ resulting from the measurement uncertainties is provided to assess its identifiability.

Figure 5 shows the identified displacement field along the cantilever as a function of the loading step during the potential cycles for CV0 and CV2. Contrary to decane-thiol adsorption, the cantilever subjected to the electro-elastic coupling experiences an upward bending, as already reported [START_REF] Fredlein | An electrocapillary study of the goldperchloric acid solution interface Surf[END_REF][START_REF] Raiteri | Measuring electrochemically induced surface stress with an atomic force microscope[END_REF][START_REF] Amiot | Identification of the electroelastic coupling from multi-physical fields measured at the micrometre scale[END_REF]. The displacement amplitude is significantly decreased when moving from the clean electrode (CV0: above 100 nm maximum displacement) to the blocked electrode (CV2: 45 nm maximum displacement). In addition, the displacement field and its change during the test are altered, and a mechanical description of the measured effect is obtained through the identification procedure.

The identified localization functions D(x), describing the surface charge singularity on the cantilever edge and modeled by the p and x 0 parameters, are plotted in Fig. 6. Similarly to the clean electrode case [START_REF] Amiot | Identification of the electroelastic coupling from multi-physical fields measured at the micrometre scale[END_REF], the surface charge density is highly localized close to the cantilever edge, thereby proving that one should use local shear-stress fields instead of a global "surface stress" concept to describe mechanical effects at the micro-cantilever scale. Similarly, it is preferable to deal with charge densities measured at the level of a single cantilever instead of the global charge density measured on a large electrode. This is not possible with the current experimental set-up, it is currently attempted to improve this issue. Alkane-thiol adsorption induces a significant change in the surface charge distribution, namely, even though the overall electrode charge range decreases when moving to blocked surfaces, the charge distribution tends to be more and more uniform. One should underline that this tendency is not satisfied for CV1, where the "antenna" effect seems to be first enhanced by the adsorption process, probably because of an easier electrode blockage far from the edges.

The significant change in the electrochemical behavior of the electrode is also obtained in Fig. 7, where the identified electro-elastic coupling relationships (defined in Eq. ( 9) and the identified δ t coefficients reported in Table 1) are shown in their identification range, that is in the identified charge density range. The decrease in the overall charge together with the decrease in sin-gularity of the localization function yield a dramatically reduced local charge density range for fully adsorbed state (CV2) compared to the early steps (CV0 and CV1). It is also worth noting that the deformation accumulation observed for CV2 is well described by the charge accumulation on the electrode.

The identified ϑ value for CV0 is close to zero, which is consistent with the fact that the electrode is initially free of thiols. The residual is not significantly affected by enforcing ϑ = 0 during minimization. On the other hand, the identified mixing parameter ϑ remains fairly low for the intermediate step, even though the electrode is partially covered (Subsection 3.1), thereby proving that the ions do not penetrate the added layer, which is thus hydrophobic. This is known as a characteristic feature of alkane-thiol films [START_REF] Finklea | Electroanalytical chemistry[END_REF]. Again, the identification results are not significantly modified by setting ϑ = 0. For the last step (CV2), because the charge density field becomes uniform, the optical properties of the surface no longer influence the measured (i.e., differential) phase field, so that the mixing parameter is no longer identifiable with the proposed technique, as shown by the large value obtained for Γ ϑ (the ϑ value is therefore not displayed in Table 1). There is therefore room for experimental improvements.

If the coupling relationship is not significantly modified by the first adsorption step (i.e., the identified relationship for CV0 lies within the confidence range around the identified relationship for CV1), the increase in the initial slope of the coupling relationship is significant after the second adsorption step, thereby providing an information complementary to the quasi-static bending of the cantilever (Fig. 7). Qualitatively, the alkane-thiol film increases the closest approach distance of ions from the electrode, thereby decreasing the charge carried by the electrode at a given potential. On the other hand, the dielectric constant of the surface is increased by the adsorption process, so that the electrostatic free energy of the surface is significantly higher, for a given charge density, when adsorption occurs. The stored electrochemical energy is then higher for a given surface charge density, so that the electro-elastic coupling (at a given charge) is enhanced [START_REF] Amiot | A model for chemically-induced mechanical loading on[END_REF]. The quantitative evaluation of the electro-elastic coupling modification as a function of the adsorbed layer would require a more detailed characterization of the interface [START_REF] Tabard-Cossa | Microcantilever-based sensors: effect of morphology, adhesion, and cleanliness of the sensing surface on surface stress[END_REF].

Using full-field measurements to investigate the effects of decane-thiol adsorption, it is shown that the significant decrease in the maximum shear-stress (by a factor of 10 as seen from Fig. 7) is partially compensated by the surface charge redistribution induced by adsorption, thus resulting in a limited cantilever tip displacement ratio before and after adsorption (almost equal to 2 as seen from Fig. 5). It is therefore necessary to take into account these global and local effects when designing electrochemically-actuated sensors in order to save their tremendous potential compared with the long-term, drift sensitive, and purely mechanical measurements developed for micro-cantilever sensors.

Conclusion

A metal coated micro-cantilever was utilized simultaneously as a micro-electrode and a micromechanical sensor. Electrochemical and full-field interferometric measurements were carried out to monitor both surface composition changes and cantilever displacements during cyclic voltammetry, before, during and after decane-thiol adsorption. The use of full-field measurements and a dedicated identification technique allows one to monitor the effects of decane-thiol adsorption on the electro-elastic coupling, namely, the charge density quasi-singularity on the cantilever edge vanishes, and the electro-elastic coupling relationship is significantly modified. This result shows that the mechanisms involved in the adsorption-induced electro-elastic coupling alteration on a microcantilever need to be described at the local as well as at the cantilever scale, thus revealing the local and global nature of the alteration. The results reported herein show that even though the local passivation decreases by a factor 10 the maximum stress acting on the cantilever, the tip displacement is reduced by only a factor 2 because of the simultaneous charge redistribution.

These effects should therefore be considered when designing optimized electrochemically actuated sensors in order to make the most of their potential. and after (CV2) decane-thiol adsorption, with estimated confidence range from the standard deviation on the identified parameters for step CV1.

Figure 3

 3 Figure 3 shows the change of the measured overall capacitance during the adsorption process. The capacitance value decreases by a factor of 2 between the experiment start (DC0) and the first stop (DC1). The capacitance then does not evolve as fast, reaching a minimum value at the end of the adsorption process, thereby proving the electrode passivation (DC2). The potential of zero charge is obtained from the minimum of the differential capacity (DC0), and is about -0.05 V vs. Ag/AgCl, in agreement with values already reported

δ1 (C - 1 ) - 3 . 13 × 5 residual η 2 min 6 . 3 × 2 7

 131352632 10 3 -3.18 × 10 3 -5.25 × 10 3 -5.76 × 10 3 -2.18 × 10 5 δ2 (m 2 .C -2 ) -2.04 × 10 2 -2.05 × 10 2 -1.18 × 10 2 -1.31 × 10 2 1.66 × 10 5 δ3 (m 4 .C -3 ) 1.07 × 10 -1 1.09 × 10 -1 8.40 × 10 -2 9.40 × 10 -2 -1.48 × 10 10 -2 6.3 × 10 -2 5.8 × 10 -2 5.8 × 10 -2 8.5 × 10 -Identified electro-elastic coupling relationships before (CV0), during (CV1) and after (CV2) decane-thiol adsorption, with estimated confidence range from the standard deviation on the identified parameters for step CV1. 35

Fig. 1 .

 1 Fig. 1. Schematic view of the imaging set-up and typical interference pattern. The shear direction is parallel to the cantilever direction, and the shear distance d is almost equal to the cantilever length. The axis system is shifted for the sake of clarity and the origin is set at point O.

Fig. 2 .

 2 Fig. 2. Schematic view of the system considered to assess the effect of decane-thiol adsorption on the electro-elastic coupling at both the local and cantilever scales.

Fig. 3 .

 3 Fig. 3. Measured differential capacity of the microcantilevers array in a 10 -2 M KCl solution before (DC0), during (DC1) and after (DC2) decane-thiol adsorption; starting potential -0.1 V vs. Ag/AgCl.

Fig. 4 .

 4 Fig. 4. Identified out-of-plane quasi-static displacement field along the median line of the cantilever as a function of adsorption time: a) step 1, b) step 2.

Fig. 5 .

 5 Fig. 5. Identified out-of-plane displacement field along the median line of the cantilever during cyclic voltammetry cycles: a) CV0, b) CV2.

Fig. 6 .

 6 Fig. 6. Identified localization functions D(x) for the three electro-elastic tests before (CV0), during (CV1) and after (CV2) decane-thiol adsorption.

Fig. 7 .

 7 Fig. 7. Identified electro-elastic coupling relationships before (CV0), during (CV1)

Table 1

 1 Identified parameters for the different cyclic voltammetry experiments, describing the surface charge density, the interface composition, the coupling relationship and the identification quality. The coupling relationship coefficients are normalized with respect to the cantilever flexural stiffness: δi = beδ i

	EI

List of Tables


Aurélie Dewaele

Emilie Dujardin

Marjolaine André

Audrey Albina

Hélène Jammes

Frank Giton

Eli Sellem

Geneviève Jolivet

Eric Pailhoux

Maëlle Pannetier
email: maelle.pannetier@inrae.fr

Aurélie Dewael

Emilie Dujard

Absence of Testicular Estrogen Leads to Defects in Spermatogenesis and Increased Semen Abnormalities in Male Rabbits

Keywords: CYP19A1 knock-out, male fertility, testicular estrogens, epididymis, spermatogenesis, sperm maturation, DNA methylation

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Introduction

Sex steroids are key reproductive system hormones in both sexes. Estrogens have always been considered the female sex steroid hormones and androgens as their male counterparts. This simplistic assessment remains accurate in different species of vertebrate and for several developmental pathways such as ovarian development in non-mammalian species in which estrogen plays a key role [START_REF] Guiguen | Involvement of Estrogens in the Process of Sex Differentiation in Two Fish Species: The Rainbow Trout (Oncorhynchus Mykiss) and a Tilapia (Oreochromis Niloticus)[END_REF][START_REF] Pieau | Temperature-Dependent Sex Determination and Gonadal Differentiation in Reptiles[END_REF][START_REF] Elbrecht | Aromatase Enzyme Activity and Sex Determination in Chickens[END_REF][START_REF] Wade | Functional Testicular Tissue Does Not Masculinize Development of the Zebra Finch Song System[END_REF][START_REF] Vaillant | Sex Reversal and Aromatase in Chicken[END_REF] or in the differentiation of internal and external male genitalia of mammals for androgens [START_REF] Jost | A New Look at the Mechanisms Controlling Sex Differentiation in Mammals[END_REF]. However, it has been clear for many decades that the situation is more complex; on the one hand because the synthesis of estrogens is made from androgens, thus implying the presence, at least transitory, of androgens in females, and on the other hand because estrogens are also produced by the testes of mammals where their roles remain to be elucidated (for review see [START_REF] Hess | Estrogens and Development of the Rete Testis, Efferent Ductules, Epididymis and Vas Deferens[END_REF]).

Cytochrome P450 aromatase, encoded by the CYP19A1 gene, is responsible for the irreversible conversion of androgens to estrogens. This enzyme is expressed in the adult testes in mammals, but its cellular localization is highly variable depending on the species and the laboratory of analyses (for review see [START_REF] Lambard | Aromatase in Testis: Expression and Role in Male Reproduction[END_REF]). Initially, the aromatase expression was Genes 2022, 13, 2070 2 of 15 described in Leydig cells in rats [START_REF] Levallet | Expression and Immunolocalization of Functional Cytochrome P450 Aromatase in Mature Rat Testicular Cells1[END_REF], pigs [START_REF] Fraczek | Cytochrome P450 Aromatase in the Testis of Immature and Mature Pigs[END_REF], stallions [START_REF] Sipahutar | Immunolocalization of Aromatase in Stallion Leydig Cells and Seminiferous Tubules[END_REF] or humans [START_REF] Payne | Intratesticular Site of Aromatization in the Human[END_REF]. Its expression in Sertoli cells was also observed in immature rat testes [START_REF] Papadopoulos | Rat Testis 17β-Estradiol: Identification by Gas Chromatography-Mass Spectrometry and Age Related Cellular Distribution[END_REF] and aromatase was finally described in meiotic and post-meiotic germ cells of mice [START_REF] Nitta | Germ Cells of the Mouse Testis Express P450 Aromatase[END_REF], rats [START_REF] Levallet | Expression and Immunolocalization of Functional Cytochrome P450 Aromatase in Mature Rat Testicular Cells1[END_REF] and humans [START_REF] Lambard | Human Immature Germ Cells and Ejaculated Spermatozoa Contain Aromatase and Oestrogen Receptors[END_REF]. Some studies even detected its expression in spermatozoa in pigs [START_REF] Rago | Cytochrome P450arom, Androgen and Estrogen Receptors in Pig Sperm[END_REF] and humans [START_REF] Lambard | Human Immature Germ Cells and Ejaculated Spermatozoa Contain Aromatase and Oestrogen Receptors[END_REF].

To promote their actions, estrogens are known to use two nuclear receptors ERα/ESR1 and ERβ/ESR2, resulting in genomic effects; and a G-protein-coupled seven-transmembrane receptor (GPER, G-Protein Estrogen coupled Receptor) causing rapid non-genomic effects. On the base of the literature, ERs expression can be detected in all testicular cell types although the results often differ between species and studies (reviewed in details by [START_REF] Dostalova | Of Oestrogens and Sperm: A Review of the Roles of Oestrogens and Oestrogen Receptors in Male Reproduction[END_REF]). For example, in the human testis, ESR1 expression has been described in Leydig cells [START_REF] Pelletier | Immunocytochemical Localization of Estrogen Receptors Alpha and Beta in the Human Reproductive Organs[END_REF], or in spermatogonia, spermatocytes and round spermatids [START_REF] Fietz | Expression Pattern of Estrogen Receptors α and β and G-Protein-Coupled Estrogen Receptor 1 in the Human Testis[END_REF], or in Leydig, Sertoli and germ cells [START_REF] Cavaco | Estrogen Receptors Alpha and Beta in Human Testis: Both Isoforms Are Expressed[END_REF]. This variability of results can be explained by the existence of ER variants (spliced isoforms [START_REF] Hirata | Isoform/Variant MRNAs for Sex Steroid Hormone Receptors in Humans[END_REF]), other proteins that share homology with classical ER, or by the methodologies and antibodies used.

The importance of estrogen signaling in the male fertility has been indicated by the adverse effects of estrogen-like compounds and their interaction with estrogen receptors, which have been shown to cause pathologies. In rats, nuclear receptor overstimulation experiments revealed the presumed role of estrogens in spermatogenesis. Treatment with an ESR1 agonist impaired the formation of elongated spermatids, while administration of an ESR2 agonist induced spermatocyte apoptosis and spermiation failure both leading to reduced sperm count [START_REF] Dumasia | Differential Roles of Estrogen Receptors, ESR1 and ESR2, in Adult Rat Spermatogenesis[END_REF]. In addition, overexposure to estrogen during spermatogenesis resulted in epigenetic defects in sperm, such as increased histone retention (ESR1 agonist) and decreased DNA methylation (ESR2 agonist) [START_REF] Dumasia | Estrogen, through Estrogen Receptor 1, Regulates Histone Modifications and Chromatin Remodeling during Spermatogenesis in Adult Rats[END_REF][START_REF] Dumasia | Estrogen Signaling, through Estrogen Receptor β, Regulates DNA Methylation and Its Machinery in Male Germ Line in Adult Rats[END_REF].

On the other hand, gene modification experiments carried out in mice tend to show that estrogens, in the testes and male genital tract, act mainly via the ESR1 receptor. Indeed, data on ESR2 functions in the male tract are still controversial in mice, since some showed normal Esr2 -/-male fertility [START_REF] Krege | Generation and Reproductive Phenotypes of Mice Lacking Estrogen Receptor Beta[END_REF], while others described infertility of unknown origin [START_REF] Antal | Sterility and Absence of Histopathological Defects in Nonreproductive Organs of a Mouse ERbeta-Null Mutant[END_REF]. In addition, mice deficient for the membrane receptor (Gper -/-) are fertile and show no particular phenotype [START_REF] Otto | GPR30 Does Not Mediate Estrogenic Responses in Reproductive Organs in Mice[END_REF]. On the contrary, a complete infertility was described in male Esr1 -/- [START_REF] Eddy | Targeted Disruption of the Estrogen Receptor Gene in Male Mice Causes Alteration of Spermatogenesis and Infertility[END_REF]. Early in their reproductive life, Esr1 -/-males showed testes with disorganized seminiferous epithelium and dilated lumen. While sperm counts were normal in Esr1 -/-males, spermatozoa presented reduced motility (flagellar defects) [START_REF] Eddy | Targeted Disruption of the Estrogen Receptor Gene in Male Mice Causes Alteration of Spermatogenesis and Infertility[END_REF][START_REF] Joseph | Absence of Estrogen Receptor Alpha Leads to Physiological Alterations in the Mouse Epididymis and Consequent Defects in Sperm Function[END_REF][START_REF] Joseph | Epididymal Hypo-Osmolality Induces Abnormal Sperm Morphology and Function in the Estrogen Receptor Alpha Knockout Mouse[END_REF] and were ineffective in in vitro fertilization (premature acrosomal reaction in mutants) [START_REF] Eddy | Targeted Disruption of the Estrogen Receptor Gene in Male Mice Causes Alteration of Spermatogenesis and Infertility[END_REF][START_REF] Joseph | Epididymal Hypo-Osmolality Induces Abnormal Sperm Morphology and Function in the Estrogen Receptor Alpha Knockout Mouse[END_REF]. The latter phenotypes appeared to be related to epididymal dysfunctions, and alterations of the epididymal fluid milieu were observed in Esr1 -/-mice [START_REF] Joseph | Absence of Estrogen Receptor Alpha Leads to Physiological Alterations in the Mouse Epididymis and Consequent Defects in Sperm Function[END_REF].

Genetic modifications or mutations affecting estrogen production have also been reported. In mice, in the absence of aromatase in males (Cyp19a1 -/-or ArKO), normal testicular morphology was observed up to 14 weeks, with no signs of infertility. Then, a progressive alteration of spermiogenesis was reported, leading to an increase in apoptosis of round spermatids and degeneration of the seminiferous epithelium: the ArKO mice became infertile with advancing age [START_REF] Robertson | Impairment of Spermatogenesis in Mice Lacking a Functional Aromatase (Cyp 19) Gene[END_REF][START_REF] Robertson | Characterization of the Fertility of Male Aromatase Knockout Mice[END_REF]. In humans, aromatase mutations are extremely rare conditions. In these patients, there are no consistent findings regarding the testicular phenotype (review in [START_REF] Haverfield | Teasing out the Role of Aromatase in the Healthy and Diseased Testis[END_REF]). Nevertheless, when semen collection could be done, oligoazoospermia and reduced sperm motility were observed [START_REF] Herrmann | Impact of Estrogen Replacement Therapy in a Male with Congenital Aromatase Deficiency Caused by a Novel Mutation in the CYP19 Gene[END_REF][START_REF] Carani | Effect of Testosterone and Estradiol in a Man with Aromatase Deficiency[END_REF].

The rabbit is an important biomedical model that could help to better understand the function of this testicular estrogen production for spermatogenesis. Thus, we first described CYP19A1, ESR1 and ESR2 expression in the testis and epididymis of adult rabbits. We showed that estrogens are exclusively produced by germ cells, mainly pachytene spermatocytes. Both ESR1 and ESR2 were expressed by round spermatids. Additionally, these receptors were detected in the epididymis, mainly the cauda, where estrogen could be measured. Then, taking advantage of the CYP19A1 mutant rabbit model created by our laboratory [START_REF] Jolivet | Fetal Estrogens Are Not Involved in Sex Determination But Critical for Early Ovarian Differentiation in Rabbits[END_REF], we investigated the effects of estrogen deprivation on testes and sperm production in this species. First, a slight decrease in fertility was observed in homozygous mutant males. Then, abnormalities of the seminiferous epithelium were observed, which were related to impaired spermatogenesis and led to a lower sperm count. Finally, sperm motility was affected and sperm morphological abnormalities were increased in mutant males suffering from estrogen deprivation.

Materials and Methods

Animals

New Zealand rabbits (NZ1777, Hypharm, Rousssay, France) were bred at the SAAJ rabbit facility (Jouy-en-Josas, France). All experiments were performed with the approval of the French Ministry MENESR (accreditation number APAFIS#6775-2016091911407980 vI) following the recommendation given by the local committee for ethic in animal experimentation (COMETHEA, Jouy-en-Josas). All researchers working directly with the animals possessed an animal experimentation license delivered by the French veterinary services. Three independent lines of CYP19A1 mutant rabbits have been generated [START_REF] Jolivet | Fetal Estrogens Are Not Involved in Sex Determination But Critical for Early Ovarian Differentiation in Rabbits[END_REF], and two of them were used in this study, since no phenotypical differences were observed.

From sexual maturity (6 months), heterozygous CYP19A1 +/-and homozygous CYP19A1 -/-males were mated with heterozygous CYP19A1 +/-females, while control males and females were mated together. The number of mating with or without birth per male, as well as the number of pups per litter was monitored.

Histological and Immunohistological Analyses

Immediately after sampling, pieces of adult testes were immersed in Bouin's fixative or paraformaldehyde (4% PFA in PBS 1×), fixed for 72 h then paraffin embedded. Microtome sections of 5 µm thickness were processed. Periodic Acid Schiff (PAS) colorations were performed by the @Bridge platform (INRAE, Jouy-en-Josas, France) using an automatic Varistain Slide Stainer (Thermo Fisher Scientific, France).

In situ hybridization (ISH) was performed using the RNAscope ISH methodology (ACD, Bio-Techne SAS, Rennes, France) as previously described [START_REF] Jolivet | Fetal Estrogens Are Not Involved in Sex Determination But Critical for Early Ovarian Differentiation in Rabbits[END_REF]. The CYP19A1, ESR1 and ESR2 probes that were used were those published previously [START_REF] Jolivet | Fetal Estrogens Are Not Involved in Sex Determination But Critical for Early Ovarian Differentiation in Rabbits[END_REF]. Hybridization was performed on 5µm sections from PFA fixed tissue using labelling kits (RNAscope 2.5HD assay-brown (conjugated to horse radish peroxidase)) as recommended by the manufacturer. Hybridization was considered as positive when at least one dot was observed in one cell. All colored sections (visible) were scanned using a 3DHISTECH panoramic scanner at the @Bridge platform (INRAE, Jouy-en-Josas, France).

Immunofluorescence was performed on rehydrated sections, where epitope retrieval was performed with citrate-based unmasking solution in a pressure cooker. DNA was then denatured 15 min with HCl 2N, and sections were permeabilized by incubation with 0.5% Triton, 1% BSA for 1h30. After an overnight incubation at 4 • C with the primary antibodies (anti-5mC, Eurogentec, ref BI-MECY-0100, 1/500; anti-5hmC, Active Motif, ref 39569, 1/500), and a 1-h incubation at room temperature with secondary antibodies (Dylight 488 anti-mouse, KPL, ref 072-03-18-06, 1/200; anti-rabbit AlexaFluor 488, Life Technologies, ref A21441, 1/200), slides were mounted in Vectashield mounting medium (Vector, Eurobio Scientific, France) containing DAPI and images were acquired with a DP50 CCD camera (Olympus, France).

RNA Extraction and RT-qPCR Analyses

The testis and epididymis (head and cauda) from adult rabbits were collected and immediately frozen at -80 • C. Total RNA from each sample was extracted using the RNeasy ® MicroKit (Qiagen, France). Quantitative PCR was performed on reverse transcribed RNAs (High-Capacity Reverse cDNA Transcription kit with the included set of random primers, Applied Biosystems, ThermoFisher Scientific, France). Based on the output of the GeNorm program, we used H2AFX, YWHAZ and SF1 (Splicing Factor 1) as the reference genes for this study (Table 1). The results were analyzed with qBase Software (qbase+, Biogazelle, France) [START_REF] Hellemans | QBase Relative Quantification Framework and Software for Management and Automated Analysis of Real-Time Quantitative PCR Data[END_REF]. Estradiol, testosterone and DHEA were assayed by GC/MS according to the protocol described by [START_REF] Giton | Evidence of Estrone-Sulfate Uptake Modification in Young and Middle-Aged Rat Prostate[END_REF] with modifications [START_REF] Devillers | FSH Inhibits AMH to Support Ovarian Estradiol Synthesis in Infantile Mice[END_REF]. Sample extraction and purification, derivatization and determination of steroid levels in testes and epididymides from adult rabbits are described in [START_REF] Jolivet | Fetal Estrogens Are Not Involved in Sex Determination But Critical for Early Ovarian Differentiation in Rabbits[END_REF] or can be provided upon request.

Semen Collection and Sperm Parameter Analyses (CASA)

Semen of rabbits from the different genotypes were collected using a specially designed artificial vagina. Two successive samplings were performed when possible for each animal. The ejaculate volume was estimated by pipetting and sperm was then immediately diluted in GALAP media (IMV Technologies, France) which was specifically designed for the conservation of rabbit semen. Each diluted sample was incubated 10 min at 37 • C before analyzing sperm parameters using a CASA Hamilton Thorne IVOS II (Hamilton Thorne, France) device with the ×10 objective.

Luminometric Methylation Assay (LUMA)

DNA extraction from sperm samples was performed as described in [START_REF] Perrier | A Multi-Scale Analysis of Bull Sperm Methylome Revealed Both Species Peculiarities and Conserved Tissue-Specific Features[END_REF]. Global DNA methylation levels were quantified using LUMA [START_REF] Karimi | LUminometric Methylation Assay)-A High Throughput Method to the Analysis of Genomic DNA Methylation[END_REF]. Briefly, 1 µg of genomic DNA was cleaved using the isoschizomers HpaII (methylation sensitive) and MspI (non-methylationsensitive) in two separate reactions and in the presence of EcoRI to standardize for DNA amounts (New England Biolabs, France). The protruding ends were then used as templates for pyrosequencing with the Pyromark Q24 device and Pyromark Gold Q96 reagents (Qiagen). The luminometric signals produced by either the sequential incorporation of C and G nucleotides (reflecting the number of CCGG sites digested by HpaII or MspI) or the sequential incorporation of A and T nucleotides (reflecting the number of AATT sites digested by EcoRI), were then quantified using Pyromark Q24 software (Qiagen, France). Each sample was assayed in duplicate.

The global methylation percentage per sample was then calculated as follows: Methylation% = [100 -(Average signal obtained with HpaII after EcoRI normalization/Average signal obtained with MspI after EcoRI normalization)] × 100

Statistics

The statistical analyses were performed using the GraphPad Prism 7 Software (Graph-Pad Software Inc., La Jolla, CA, USA). Because of the small number of samples in groups, comparisons between values were made by the Mann-Whitney test for non-parametric values. A probability lower than 0.05 was required for significance.

Results

Localization of CYP19A1/Aromatase and ESRs Expression in the Rabbit Testis and Epididymis

To decipher which testicular cell type expressed the CYP19A1 gene and thus, were able to produce estrogens, we carried on in situ hybridization by using the RNAscope technology, giving reproducible results on rabbit gonads [START_REF] Jolivet | Fetal Estrogens Are Not Involved in Sex Determination But Critical for Early Ovarian Differentiation in Rabbits[END_REF] (Figure 1). Aromatase expression was detected inside the seminiferous tubules, mainly in germ cells with a large nucleus and which seemed to correspond to pachytene spermatocytes (Figure 1A). In addition, to determine which cell types could respond to estrogens in the seminiferous compartment, probes corresponding to estrogen receptors ESR1 and ESR2 were used. The expression of both estrogen receptor mRNAs was attested into the seminiferous tubules and was restricted to the round spermatids, with a stronger labelling for ESR1 (Figure 1B).

Results

Localization of CYP19A1/Aromatase and ESRs Expression in the Rabbit Testis and Epididymis

To decipher which testicular cell type expressed the CYP19A1 gene and thus, were able to produce estrogens, we carried on in situ hybridization by using the RNAscope technology, giving reproducible results on rabbit gonads [START_REF] Jolivet | Fetal Estrogens Are Not Involved in Sex Determination But Critical for Early Ovarian Differentiation in Rabbits[END_REF] (Figure 1). Aromatase expression was detected inside the seminiferous tubules, mainly in germ cells with a large nucleus and which seemed to correspond to pachytene spermatocytes (Figure 1A). In addition, to determine which cell types could respond to estrogens in the seminiferous compartment, probes corresponding to estrogen receptors ESR1 and ESR2 were used. The expression of both estrogen receptor mRNAs was attested into the seminiferous tubules and was restricted to the round spermatids, with a stronger labelling for ESR1 (Figure 1B). The expression level of both aromatase and estrogen receptors transcripts was studied and compared within testes and epididymides (caput and cauda) by RT-qPCR The expression level of both aromatase and estrogen receptors transcripts was studied and compared within testes and epididymides (caput and cauda) by RT-qPCR (Figure 1C). Although CYP19A1 expression was restricted to the testis, ESR1 expression was strongly detected in the testis and in the tail of the epididymis (cauda). ESR2 expression was also observed in testes, and was faintly detected in both the head and the tail of the epididymis. However, in situ hybridization failed to detect ESR2 (or ESR1) expression in the caput epididymis (Figure 1B). On the contrary, strong staining was obtained in the mesenchyma with ESR1 probe and a faint staining for ESR2 in the epithelial cells of the cauda epididymis.

Genes 2022, 13, 2070 6 of 15

CYP19A1 Gene-Targeting in Rabbits Efficiently Suppresses Testicular Estrogen Secretion

We have previously established three strains of genetically modified rabbits carrying deletions of exon 2 (including the initiator ATG codon) of the CYP19A1 gene. These rabbit strains were initially created to evaluate the role of fetal estrogens produced by early developing ovaries (i.e.,: before meiosis initiation in the germinal lineage) in a nonrodent mammalian species [START_REF] Jolivet | Fetal Estrogens Are Not Involved in Sex Determination But Critical for Early Ovarian Differentiation in Rabbits[END_REF]. To confirm that knocking out CYP19A1 in male really led to testicular estrogen deprivation, we measured estrogen concentrations in CYP19A1 -/-testes in comparison with wild type ones (Figure 2A). As expected, consistent with estrogen assays performed for their female counterparts [START_REF] Jolivet | Fetal Estrogens Are Not Involved in Sex Determination But Critical for Early Ovarian Differentiation in Rabbits[END_REF], no testicular estrogen remained detectable in mutant gonads compared to wild type ones where the median 17β-Estradiol value was about 55 pg/g of tissue. Even if testicular estrogen production was abolished, testosterone and dehydroepiandrosterone (DHEA) concentrations remained similar between mutant and wild type testes with, respectively, around 9 ng/g and 40 ng/g (median values) in each condition (Figure 2A). These results confirm that the deletions engineered on CYP19A1 exon 2 in the three rabbit lines efficiently suppressed aromatase activity and estrogen secretion as previously demonstrated in females [START_REF] Jolivet | Fetal Estrogens Are Not Involved in Sex Determination But Critical for Early Ovarian Differentiation in Rabbits[END_REF].

tail of the epididymis. However, in situ hybridization failed to detect ESR2 (or ESR expression in the caput epididymis (Figure 1B). On the contrary, strong staining w obtained in the mesenchyma with ESR1 probe and a faint staining for ESR2 in t epithelial cells of the cauda epididymis.

CYP19A1 Gene-Targeting in Rabbits Efficiently Suppresses Testicular Estrogen Secretion

We have previously established three strains of genetically modified rabbits carryi deletions of exon 2 (including the initiator ATG codon) of the CYP19A1 gene. These rab strains were initially created to evaluate the role of fetal estrogens produced by early d veloping ovaries (i.e.,: before meiosis initiation in the germinal lineage) in a non-rode mammalian species [START_REF] Jolivet | Fetal Estrogens Are Not Involved in Sex Determination But Critical for Early Ovarian Differentiation in Rabbits[END_REF]. To confirm that knocking out CYP19A1 in male really led to t ticular estrogen deprivation, we measured estrogen concentrations in CYP19A1 -/-testes comparison with wild type ones (Figure 2A). As expected, consistent with estrogen assa performed for their female counterparts [START_REF] Jolivet | Fetal Estrogens Are Not Involved in Sex Determination But Critical for Early Ovarian Differentiation in Rabbits[END_REF], no testicular estrogen remained detectab in mutant gonads compared to wild type ones where the median 17β-Estradiol value w about 55 pg/g of tissue. Even if testicular estrogen production was abolished, testostero and dehydroepiandrosterone (DHEA) concentrations remained similar between muta and wild type testes with, respectively, around 9 ng/g and 40 ng/g (median values) in ea condition (Figure 2A). These results confirm that the deletions engineered on CYP19 exon 2 in the three rabbit lines efficiently suppressed aromatase activity and estrogen cretion as previously demonstrated in females [START_REF] Jolivet | Fetal Estrogens Are Not Involved in Sex Determination But Critical for Early Ovarian Differentiation in Rabbits[END_REF]. We have previously shown that estrogens are produced in the seminiferous tubul Due to the presence of the blood-testis barrier, estrogens were expected to circula We have previously shown that estrogens are produced in the seminiferous tubules. Due to the presence of the blood-testis barrier, estrogens were expected to circulate through the efferent ducts and epididymides. 17β-Estradiol levels were thus measured in the epididymides, showing 25 pg/g and 20 pg/g as median values in the head and the tail of the epididymis, respectively (Figure 2B). No estrogen could be detected in CYP19A1 homozygous mutant epididymides.

Genes 2022, 13, 2070 7 of 15

CYP19A1 Knockout Male Rabbits Show Subfertility Parameters

During our previous study on CYP19A1 -/-fetal ovary [START_REF] Jolivet | Fetal Estrogens Are Not Involved in Sex Determination But Critical for Early Ovarian Differentiation in Rabbits[END_REF], spanning 8 years, CYP19A1 genetically modified male rabbits from three different strains were mated with heterozygous females (CYP19A1 +/-) to expand the lines and produce biological materials. Either heterozygous carrier males (XY CYP19A1 +/-) or homozygous mutant males (XY CYP19A1 -/-) were used. Interestingly, a slight subfertility of homozygous mutant males compared to heterozygous carrier ones was observed on two recorded parameters. First, the percentage of mating without birth was increased when using homozygous mutant males (58.9%) compared to heterozygous carrier males (32.3%) or control rabbits (35.5%) (Figure 3A). Secondly, when mating was successful, the recorded litter size was statistically reduced, as the number of pups per litter dropped from 7.9 (± 3.3) to 6 (± 2.8) by using heterozygous or homozygous mutant males, respectively (Figure 3B,C).

the epididymides, showing 25 pg/g and 20 pg/g as median values in the head an of the epididymis, respectively (Figure 2B). No estrogen could be detected in homozygous mutant epididymides.

CYP19A1 Knockout Male Rabbits Show Subfertility Parameters

During our previous study on CYP19A1 -/-fetal ovary [START_REF] Jolivet | Fetal Estrogens Are Not Involved in Sex Determination But Critical for Early Ovarian Differentiation in Rabbits[END_REF], spanning CYP19A1 genetically modified male rabbits from three different strains were ma heterozygous females (CYP19A1 +/-) to expand the lines and produce biological m Either heterozygous carrier males (XY CYP19A1 +/-) or homozygous mutant m CYP19A1 -/-) were used. Interestingly, a slight subfertility of homozygous muta compared to heterozygous carrier ones was observed on two recorded paramet the percentage of mating without birth was increased when using homozygou males (58.9%) compared to heterozygous carrier males (32.3%) or control rabbit (Figure 3A). Secondly, when mating was successful, the recorded litter size wa cally reduced, as the number of pups per litter dropped from 7.9 (± 3.3) to 6 (± 2.8) heterozygous or homozygous mutant males, respectively (Figure 3B,C).

Absence of Testicular Estrogens Leads to Spermatogenesis Defects

The effects of estrogen deficiency on testicular morphogenesis and function were evaluated in 2-to 3-year-old rabbits. Histological analyses of the testes showed some abnormal seminiferous tubules in the homozygous mutants (Figure 4A-F). These were found clustered in a few testicular lobules or scattered through the testis (Figure 4C andD and Figure 4E and F, respectively). In these abnormal tubules, the thickness of the seminiferous epithelium was reduced, and the lumen appeared larger: a drastic decrease in the spermatid layer was observed.

Genes 2022, 13, 2070 8 o

Absence of Testicular Estrogens Leads to Spermatogenesis Defects

The effects of estrogen deficiency on testicular morphogenesis and function w evaluated in 2-to 3-year-old rabbits. Histological analyses of the testes showed some normal seminiferous tubules in the homozygous mutants (Figure 4A-F). These w found clustered in a few testicular lobules or scattered through the testis (Figure 4C a D and Figure 4E and F, respectively). In these abnormal tubules, the thickness of the se iniferous epithelium was reduced, and the lumen appeared larger: a drastic decrease the spermatid layer was observed. The expression levels of the markers of spermatocytes I (SYCP3) and spermat (PRM1, Protamine 1) were evaluated and indeed showed that the fraction of spermat could be affected in the mutants because the mRNA levels of PRM1 were diminished Value = 0.02) (Figures 5A,B). Consistent with spermatogenesis abnormalities, testis body weight ratio was found to be significantly lower in CYP19A1 -/-males compared control ones (Figure 5C). In addition, total sperm number was estimated using the IV II CASA system (computer assisted sperm analysis), showing that the sperm count w significantly decreased in absence of estrogen synthesis (Figure 5D). The expression levels of the markers of spermatocytes I (SYCP3) and spermatids (PRM1, Protamine 1) were evaluated and indeed showed that the fraction of spermatids could be affected in the mutants because the mRNA levels of PRM1 were diminished (p-Value = 0.02) (Figure 5A,B). Consistent with spermatogenesis abnormalities, testis to body weight ratio was found to be significantly lower in CYP19A1 -/-males compared to control ones (Figure 5C). In addition, total sperm number was estimated using the IVOS II CASA system (computer assisted sperm analysis), showing that the sperm count was significantly decreased in absence of estrogen synthesis (Figure 5D).

Absence of Testicular Estrogens Has No Impact on Germ Cell DNA Methylation

Since estrogen receptor over activation has been linked to epigenetic modifications [START_REF] Dumasia | Estrogen Signaling, through Estrogen Receptor β, Regulates DNA Methylation and Its Machinery in Male Germ Line in Adult Rats[END_REF], we were interested in the DNA methylation of germ cells. Nevertheless, immunofluorescence studies of the deposition of 5-methyl Cytosine (DNA methylation) or the 5-hydroxymethyl Cytosine (DNA hydroxymethylation, i.e., DNA demethylation) showed no difference between control and CYP19A1 -/-testes (Figure 6A). In addition, the DNA methylation rate of ejaculated sperm was determined by luminometric methylation assay (LUMA). The percentage of DNA methylation, around 70%, was found identical between sperm from control and mutant rabbits (Figure 6B), suggesting that if estrogen plays a role in epigenetics of the male gamete, this might not have been detected by global DNA methylation assessment.

Absence of Testicular Estrogens Has No Impact on Germ Cell DNA Methylation

Since estrogen receptor over activation has been linked to epigenetic modifications [START_REF] Dumasia | Estrogen Signaling, through Estrogen Receptor β, Regulates DNA Methylation and Its Machinery in Male Germ Line in Adult Rats[END_REF], we were interested in the DNA methylation of germ cells. Nevertheless, immunofluorescence studies of the deposition of 5-methyl Cytosine (DNA methylation) or the 5hydroxymethyl Cytosine (DNA hydroxymethylation, i.e., DNA demethylation) showed no difference between control and CYP19A1 -/-testes (Figure 6A). In addition, the DNA methylation rate of ejaculated sperm was determined by luminometric methylation assay (LUMA). The percentage of DNA methylation, around 70%, was found identical between sperm from control and mutant rabbits (Figure 6B), suggesting that if estrogen plays a role in epigenetics of the male gamete, this might not have been detected by global DNA methylation assessment.

Absence of Testicular Estrogen Leads to Sperm Defects

In order to better understand the subfertility of CYP19A1 -/-males, the sperm parameters of mutant and control rabbits were evaluated using the IVOS II CASA system on ejaculated sperm. Of the parameters assessed by this procedure, six were found statistically divergent between control and mutant sperm (Figure 7A-F). First, we noticed a decrease in total (from 90% to 55%) and progressive (from 60% to 30%) sperm motility in mutant rabbits (Figure 7A,B). Second, the mutants displayed increased sperm malformations such as bent tails and Distal Midpiece Reflex curvatures (DMR) (Figure 7C,D) Finally, the spermatozoa from mutant animals retained more proximal and distal (Figure 7E,F) cytoplasmic droplets than the control sperm, suggesting an imperfect maturation of the gametes during their transit in the epididymis [START_REF] Cooper | Cytoplasmic Droplets: The Good, the Bad or Just Confusing?[END_REF].

Absence of Testicular Estrogen Leads to Sperm Defects

In order to better understand the subfertility of CYP19A1 -/-males, the sperm parameters of mutant and control rabbits were evaluated using the IVOS II CASA system on ejaculated sperm. Of the parameters assessed by this procedure, six were found statistically divergent between control and mutant sperm (Figure 7A-F). First, we noticed a decrease in total (from 90% to 55%) and progressive (from 60% to 30%) sperm motility in mutant rabbits (Figure 7A,B). Second, the mutants displayed increased sperm malformations such as bent tails and Distal Midpiece Reflex curvatures (DMR) (Figure 7C,D). Finally, the spermatozoa from mutant animals retained more proximal and distal (Figure 7E,F) cytoplasmic droplets than the control sperm, suggesting an imperfect maturation of the gametes during their transit in the epididymis [START_REF] Cooper | Cytoplasmic Droplets: The Good, the Bad or Just Confusing?[END_REF].

Discussion

In rabbits, the production of estrogen by the adult testes is strictly limited to the germ cells inside the seminiferous tubules: mainly to the meiotic germ cells (pachytene spermatocytes). While the location of aromatase expression differs according to the published studies, several of them are concordant on this point in rodents and in humans [START_REF] Levallet | Expression and Immunolocalization of Functional Cytochrome P450 Aromatase in Mature Rat Testicular Cells1[END_REF][START_REF] Nitta | Germ Cells of the Mouse Testis Express P450 Aromatase[END_REF][START_REF] Lambard | Human Immature Germ Cells and Ejaculated Spermatozoa Contain Aromatase and Oestrogen Receptors[END_REF]. Thus, because of the blood-testis barrier established in the tubules, testicular estrogens should not pass through the general circulation in rabbits, but rather have a local function on the germ cells themselves, or on the male genital tract. Accordingly, 17β-Estradiol could be measured in the head and the tail of epididymis, showing that testicular estrogens diffuse into the fluid during the epididymal transit.

Testicular Estrogens Are Involved in Germ Cells Differentiation

In the testis, both ESR1 and ESR2 receptor mRNAs were detected in round spermatids, suggesting that estrogens may have a role on post-meiotic germ cells. Accordingly, hypo-spermatogenesis has been observed in homozygous CYP19A1 -/- mutant males, with some testicular lobules showing thinner seminiferous epithelium, with a lack of round spermatids, as described in the ArKO mouse model [START_REF] Robertson | Impairment of Spermatogenesis in Mice Lacking a Functional Aromatase (Cyp 19) Gene[END_REF][START_REF] Robertson | Characterization of the Fertility of Male Aromatase Knockout Mice[END_REF]. In the rat, overstimulation of ESR1 or ESR2 leads to spermiogenesis defects [START_REF] Dumasia | Differential Roles of Estrogen Receptors, ESR1 and ESR2, in Adult Rat Spermatogenesis[END_REF]. Thus, a lack or

Discussion

In rabbits, the production of estrogen by the adult testes is strictly limited to the germ cells inside the seminiferous tubules: mainly to the meiotic germ cells (pachytene spermatocytes). While the location of aromatase expression differs according to the published studies, several of them are concordant on this point in rodents and in humans [START_REF] Levallet | Expression and Immunolocalization of Functional Cytochrome P450 Aromatase in Mature Rat Testicular Cells1[END_REF][START_REF] Nitta | Germ Cells of the Mouse Testis Express P450 Aromatase[END_REF][START_REF] Lambard | Human Immature Germ Cells and Ejaculated Spermatozoa Contain Aromatase and Oestrogen Receptors[END_REF]. Thus, because of the blood-testis barrier established in the tubules, testicular estrogens should not pass through the general circulation in rabbits, but rather have a local function on the germ cells themselves, or on the male genital tract. Accordingly, 17β-Estradiol could be measured in the head and the tail of epididymis, showing that testicular estrogens diffuse into the fluid during the epididymal transit.

Testicular Estrogens Are Involved in Germ Cells Differentiation

In the testis, both ESR1 and ESR2 receptor mRNAs were detected in round spermatids, suggesting that estrogens may have a role on post-meiotic germ cells. Accordingly, hypospermatogenesis has been observed in homozygous CYP19A1 -/-mutant males, with some testicular lobules showing thinner seminiferous epithelium, with a lack of round spermatids, as described in the ArKO mouse model [START_REF] Robertson | Impairment of Spermatogenesis in Mice Lacking a Functional Aromatase (Cyp 19) Gene[END_REF][START_REF] Robertson | Characterization of the Fertility of Male Aromatase Knockout Mice[END_REF]. In the rat, overstimulation of ESR1 or ESR2 leads to spermiogenesis defects [START_REF] Dumasia | Differential Roles of Estrogen Receptors, ESR1 and ESR2, in Adult Rat Spermatogenesis[END_REF]. Thus, a lack or an excess of estrogens may impact spermatid differentiation. Additionally, epigenetic defects in the spermatids have been reported when estrogen receptors were overstimulated [START_REF] Dumasia | Estrogen, through Estrogen Receptor 1, Regulates Histone Modifications and Chromatin Remodeling during Spermatogenesis in Adult Rats[END_REF][START_REF] Dumasia | Estrogen Signaling, through Estrogen Receptor β, Regulates DNA Methylation and Its Machinery in Male Germ Line in Adult Rats[END_REF]. Nevertheless, in this present study, we could not detect any changes in DNA methylation in the absence of estrogen. This may be related to the use of inappropriate methods, but estrogen might also not be involved in epigenetic reprograming in normal situations.

In humans, loss-of-function mutations affecting CYP19A1/Aromatase gene are very rare and poorly documented. In one reported case, the authors described abnormal skeletal growth and bone maturation in a male patient, which were associated with testicular hypoplasia and infertility [START_REF] Carani | Effect of Testosterone and Estradiol in a Man with Aromatase Deficiency[END_REF]. The initiation of a replacement therapy by daily injection of estrogens restored the bone/skeletal phenotype, but had no effect on the testicles or fertility disorders. This last aspect could be linked to the fact that estrogens must be produced locally in the seminiferous tubules in order to be able to act on the differentiation of germ cells. In addition, a testicular biopsy, performed in this patient, revealed hypospermatogenesis and an arrest of germ differentiation, mainly at the level of primary spermatocytes [START_REF] Carani | Effect of Testosterone and Estradiol in a Man with Aromatase Deficiency[END_REF]. This phenotype is close to that observed in rabbits, where round spermatids were rare in some tubules of the CYP19A1 -/-males, suggesting either that estrogens are necessary for their differentiation or maintenance, or that estrogens may be involved in the completion of meiosis. Interestingly, overexposure to estrogen or BPA has the same impact on spermatogenesis, with meiotic progression being defective and stopping at the pachytene stage [START_REF] Liu | Exposure to Bisphenol A Disrupts Meiotic Progression during Spermatogenesis in Adult Rats through Estrogen-like Activity[END_REF]. In the present study, in rabbits deficient for aromatase, PRM1 mRNAs levels were found to be decreased which could reflect spermiation disorder or could be an additional clue to consider the function of estrogens on meiotic process. Further investigations involving high throughput transcriptome sequencing may highlight the potential implication of estrogens into meiosis in males.

Testicular Estrogens Are Involved in Sperm Production, Maturation and Motility

As a consequence of the described defect in spermatogenesis, mild but significant testicular hypoplasia was observed in CYP19A1 -/-rabbits and the number of ejaculated spermatozoa decreased. These animals presented slight subfertility with conception difficulties (mating without birth), as well as a decrease in the number of offspring. In addition of a decrease in sperm count, an increase in sperm abnormalities was observed. First, like in humans [START_REF] Herrmann | Impact of Estrogen Replacement Therapy in a Male with Congenital Aromatase Deficiency Caused by a Novel Mutation in the CYP19 Gene[END_REF][START_REF] Carani | Effect of Testosterone and Estradiol in a Man with Aromatase Deficiency[END_REF] and mice [START_REF] Joseph | Absence of Estrogen Receptor Alpha Leads to Physiological Alterations in the Mouse Epididymis and Consequent Defects in Sperm Function[END_REF][START_REF] Robertson | Impairment of Spermatogenesis in Mice Lacking a Functional Aromatase (Cyp 19) Gene[END_REF][START_REF] Robertson | Characterization of the Fertility of Male Aromatase Knockout Mice[END_REF], sperm motility was affected, with a 50% reduction in the progressive motility in CYP19A1 -/-compared to control males. Then, related to the motility defects, increased percentages of flagellar abnormalities were noted, including bent tail and Distal Midpiece Reflex (DMR) which were doubled in mutants. In addition, proportion of sperm with cytoplasmic residual droplets was increased. These last phenotypes could be related to sperm maturation trouble in the epididymis, where sperm motility is acquired. In particular, the cytoplasmic droplet is expected to migrate caudally along the sperm during epididymal transit, and this droplet has been implicated in affecting some biochemical aspects of motility [START_REF] Gervasi | Molecular Changes and Signaling Events Occurring in Sperm during Epididymal Maturation[END_REF]. Some signaling pathways have been associated with sperm motility, and evidence suggests that sperm may have functional flagellar machinery that is activated during epididymal transit [START_REF] Gervasi | Molecular Changes and Signaling Events Occurring in Sperm during Epididymal Maturation[END_REF]. Indeed, in the epididymis, sperm undergo protein changes. As sperm are translationally silent, proteins appearing in them are thought to be synthesized by the epididymal epithelium and then incorporated to the sperm cells, thanks to exosomes for instance, called epididymosomes [START_REF] Sullivan | Prostasomes, and Liposomes: Their Roles in Mammalian Male Reproductive Physiology[END_REF]. ESR1 and ESR2 receptor mRNAs were both detected in the epididymis of male rabbits, mainly in the tail, where estrogens seem thus to exert their functions. In particular, ESR2 transcripts were found in the epithelial cells, which are supposed to secrete epididymosomes. Additional analyses on the transcriptomes and proteomes of mutant epididymides could provide clues to better understand how estrogen pathway dysfunctions impact sperm maturation and motility.

Conclusions

In the rabbit, testicular estrogens are produced inside the seminiferous tubules, mainly by meiotic germ cells. They play two main roles in promoting the fertility of the male ga-

Figure 1 .

 1 Figure 1. CYP19A1 and estrogen receptors expression in the testis and epididymis of adult rabbits. (A) Location of CYP19A1 expression by in situ hybridization (RNAscope technology) in the adult testis. (B) Location of ESR1 and ESR2 expression by in situ hybridization in the testis and the cauda epididymis. (C) Relative expression levels of CYP19A1, ESR1 and ESR2 analyzed by RT-qPCR in the adult testis and epididymis (caput and cauda). Testis n = 4; Epididymis n = 4.

Figure 1 .

 1 Figure 1. CYP19A1 and estrogen receptors expression in the testis and epididymis of adult rabbits. (A) Location of CYP19A1 expression by in situ hybridization (RNAscope technology) in the adult testis. (B) Location of ESR1 and ESR2 expression by in situ hybridization in the testis and the cauda epididymis. (C) Relative expression levels of CYP19A1, ESR1 and ESR2 analyzed by RT-qPCR in the adult testis and epididymis (caput and cauda). Testis n = 4; Epididymis n = 4.

Figure 2 .

 2 Figure 2. Steroid levels in the testis and epididymis in adults. (A) Dosage of 17β-estradiol (E2), testosterone and DHEA concentrations in control and CYP19A1 -/-adult testes by GC/MS. (B) Dosage of 17β-estradiol (E2) in the head and the tail of epididymis. Con trol n = 4 to 6; CYP19A1 -/-n = 5. The median is shown in red. ND: not detected. Mann-Whitney test: p-value < 0.05. ns: non-significant.

Figure 2 .

 2 Figure 2. Steroid levels in the testis and epididymis in adults. (A) Dosage of 17β-estradiol (E2), testosterone and DHEA concentrations in control and CYP19A1 -/-adult testes by GC/MS. (B) Dosage of 17β-estradiol (E2) in the head and the tail of epididymis. Control n = 4 to 6; CYP19A1 -/-n = 5. The median is shown in red. ND: not detected. Mann-Whitney test: p-value < 0.05. ns: non-significant.

Figure 3 .

 3 Figure 3. Effect of the CYP19A1 gene knock-out on the male fertility. (A) Numbe cessful and unsuccessful mating, depending on the genotype of the parents. (B) of different males and females used and pups per litter depending on the genoty Number of pups per litter obtained by crossing heterozygous females with hete (CYP19A1 +/-) or homozygous (CYP19A1 -/-) males. The median is shown in red. M Whitney test: ** p < 0.01.

Figure 3 .

 3 Figure 3. Effect of the CYP19A1 gene knock-out on the male fertility. (A) Number of successful and unsuccessful mating, depending on the genotype of the parents. (B) Number of different males and females used and pups per litter depending on the genotype. (C) Number of pups per litter obtained by crossing heterozygous females with heterozygous (CYP19A1 +/-) or homozygous (CYP19A1 -/-) males. The median is shown in red. Mann-Whitney test: ** p < 0.01.

Figure 4 .

 4 Figure 4. Spermatogenesis defects in absence of estrogen production. (A,B) PAS staining on con testis. (C,F) PAS staining on CYP19A1 -/-testis from two different rabbits (C-F). Males are 2 to 3 ye old.

Figure 4 .

 4 Figure 4. Spermatogenesis defects in absence of estrogen production. (A,B) PAS staining on control testis. (C,F) PAS staining on CYP19A1 -/-testis from two different rabbits (C-F). Males are 2 to 3 years old.

Figure 5 .

 5 Figure 5. Hypo-spermatogenesis in CYP19A1 -/-males. (A,B) RT-qPCR analyses of mRNA levels of SYCP3 and PRM1 in control and CYP19A1 -/-adult testis (n = 5 for each genotype). (C) Testis on body weight ratio in control and CYP19A1 -/-rabbits. Control, n = 6; CYP19A1 -/-n = 5. The median is shown in red. (D) Total sperm counts per million per ejaculated sample in control and CYP19A1 -/-rabbits. Control, n = 10; CYP19A1 -/-n = 5. Dots represent the average of two successive semen collections per animal. For CYP19A1 -/-rabbits, three sets of two successive ejaculations were collected over a oneyear interval. The median is shown in red. Mann-Whitney test: * p-value < 0.05. *** p-Value < 0.0005. ns: non-significant.

Figure 5 .

 5 Figure 5. Hypo-spermatogenesis in CYP19A1 -/-males. (A,B) RT-qPCR analyses of mRNA levels of SYCP3 and PRM1 in control and CYP19A1 -/-adult testis (n = 5 for each genotype). (C) Testis on body weight ratio in control and CYP19A1 -/-rabbits. Control, n = 6; CYP19A1 -/-n = 5. The median is shown in red. (D) Total sperm counts per million per ejaculated sample in control and CYP19A1 -/- rabbits. Control, n = 10; CYP19A1 -/-n = 5. Dots represent the average of two successive semen collections per animal. For CYP19A1 -/-rabbits, three sets of two successive ejaculations were collected over a one-year interval. The median is shown in red. Mann-Whitney test: * p-value < 0.05. *** p-Value < 0.0005. ns: non-significant.

Figure 6 .

 6 Figure 6. DNA methylation in the testis and sperm in absence of estrogen synthesis. (A) Immuno detection of 5mC and 5hmC in control and CYP19A1 -/-testis (green). Nuclei were stained in blue (DAPI). (B) Percentage of sperm DNA methylation from control and CYP19A1 -/-male rabbits deter mined by Luminometric Methylation Assay (LUMA). The median is shown in red. Mann-Whitney test: nonsignificant.

Figure 6 .

 6 Figure 6. DNA methylation in the testis and sperm in absence of estrogen synthesis. (A) Immunodetection of 5mC and 5hmC in control and CYP19A1 -/-testis (green). Nuclei were stained in blue (DAPI). (B) Percentage of sperm DNA methylation from control and CYP19A1 -/-male rabbits determined by Luminometric Methylation Assay (LUMA). The median is shown in red. Mann-Whitney test: nonsignificant.

Figure 7 .

 7 Figure 7. Spermatic parameters in control and CYP19A1 -/-rabbits. Motility and morphometric parameters of the sperm from control and CYP19A1 -/-rabbits were obtained by Computer Assisted Sperm Analysis. Percentages of (A) total motility and (B) progressive motility of the sperm were decreased in mutants. Percentages of (C) bent tails and (D) Distal Midpiece Reflex were increased in CYP19A1 -/-semen, together with (E) proximal droplets and (F) distal droplets. Dots represent the average of two successive semen collections per animal. For CYP19A1 -/-rabbits, three sets of two successive ejaculations were collected over a one-year interval. The median is shown in red. Control n = 10; CYP19A1 -/-n = 5. Mann-Whitney test: * p-Value < 0.05; ** p-Value < 0.005; *** p-Value < 0.0005.

Figure 7 .

 7 Figure 7. Spermatic parameters in control and CYP19A1 -/-rabbits. Motility and morphometric parameters of the sperm from control and CYP19A1 -/-rabbits were obtained by Computer Assisted Sperm Analysis. Percentages of (A) total motility and (B) progressive motility of the sperm were decreased in mutants. Percentages of (C) bent tails and (D) Distal Midpiece Reflex were increased in CYP19A1 -/-semen, together with (E) proximal droplets and (F) distal droplets. Dots represent the average of two successive semen collections per animal. For CYP19A1 -/-rabbits, three sets of two successive ejaculations were collected over a one-year interval. The median is shown in red. Control n = 10; CYP19A1 -/-n = 5. Mann-Whitney test: * p-Value < 0.05; ** p-Value < 0.005; *** p-Value < 0.0005.

Table 1 .

 1 Primers used by RT-qPCR.

	Gene Name	Forward Primer	Reverse Primer
	H2AFX	ACCTGACGGCCGAGATCCT	CGCCCAGCAGCTTGTTGAG
	YWHAZ	GGGTCTGGCCCTTAACTTCTCT	AGCAATGGCTTCATCAAAAGC
	SF1	GCTTCCGACTGCAAATTCCA	TCACCCAGTTCAGCCATGAG
	CYP19A1	GGAAGAATGCATCGACTTGAGTT	GGGCCCAAAACCAAATGGT
	ESR1	TCCTCATCCTCTCCCACATC	AGCATCTCCAGCAACAGGTC
	ESR2	CTCACCAAGCTGGCTGACAA	AGAGGCGCACTTGGTCCAA
	SYCP3	AAAAGAAATGGCCATGTTGCA GAGTCATCAAAGTAACACGGATTGAA
	PRM1	CCAGAGGCGAAGAGTCAGGAA	TCTGGTGGGTCTGCTGTTCTGT
	2.4. Measurement of Estradiol, Testosterone and DHEA Hormone Levels in Testis and Epididymis
	of Adult Rabbits

mete: (i) on germ cells and their progression in spermatogenesis; (ii) on the epididymis and indirectly on sperm maturation and motility acquisition. The phenotype of the CYP19A1 -/- rabbits is very similar to the rare cases of aromatase mutations reported in humans, making the rabbit a relevant biomedical model for understanding and preventing male fertility.

Acknowledgments:

The authors would like to thank Patrice Congar, Gwendoline Morin and all the staff of the facility (SAAJ, INRAE, Jouy-en-Josas, France) for the care of the rabbits and semen collection, and Julie Rivière and Marthe Vilotte (UMR GABI, INRAE, Jouy-en-Josas, France) for their assistance on the histological platform (@Bridge platform) and for the access to the virtual slide scanner.

Data Availability Statement: Not applicable.

Author Contributions: Conceptualization, G.J. and M.P.; Methodology, A.D., E.D., M.A., A.A., F.G., H.J., E.S., G.J. and M.P.; Validation, A.D., E.D., A.A., M.A. and H.J.; Formal Analysis, A.D., E.D., E.P. and M.P. Investigation, A.D., G.J. and M.P.; Writing-Original Draft Preparation, M.P., E.P. and A.D.; Writing-Review and Editing, G.J., E.P. and M.P.; Supervision, M.P.; Funding Acquisition, G.J. and E.P. All authors have read and agreed to the published version of the manuscript.

Funding: This research was funded by ANR (Agence Nationale de la Recherche) grants (GENIDOV: ANR-09-GENM-009; ARGONADS: ANR-13-BSV2-0017; ARDIGERM: ANR-2020-CE14-0022), and CELPHEDIA infrastructure (CELPHEDIA France, 2017).

Institutional Review Board Statement:

The study was conducted according to the guidelines of Declaration of Helsinki, and approved by the French Ministry MENESR (accreditation number APAFIS#6775-2016091911407980 vI) following the recommendation given by the local committee for ethic in animal experimentation (COMETHEA, Jouy-en-Josas, France). All researchers working directly with the animals possessed an animal experimentation license delivered by the French veterinary services.

Informed Consent Statement: Not applicable.

Conflicts of Interest:

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.