Salomé Cojean
email: salome.cojean@univ-grenoble-alpes.fr

Nicolas Martin
email: nicolas.martin6@univ-grenoble-alpes.fr

Effets des sous-titres et des mots-clés en apprentissage vidéo

Keywords: Video, subtitles, keywords, split-attention, learning Vidéo, sous-titres, mots-clés, attention divisée, apprentissage

Reducing the split-attention effect of subtitles during video learning: might the use of occasional keywords be an effective solution? Réduire l'effet d'attention divisée des sous-titres pendant l'apprentissage en vidéo : les mots-clés ponctuels sont-ils une solution efficace ?

Introduction

The use of pedagogical videos has increased over the last 20 years, a trend marked by three main factors: 1) Massive Online Open Courses (MOOC), generally in the form of videorecorded lectures [START_REF] Giannakos | Exploring the video-based learning research: A review of the literature[END_REF]Schacter & Szpunar, 2015); 2) flipped classrooms involving videos for the lesson to be studied before the class [START_REF] Haagsman | Pop-up questions within educational videos: Effects on students' learning[END_REF][START_REF] Van Der Meij | Effects of embedded questions in recorded lectures[END_REF]; and 3) distance learning in schools and universities due to the COVID-19 pandemic [START_REF] Dietrich | Attempts, successes, and failures of distance learning in the time of COVID-19[END_REF][START_REF] Kim | Learning and teaching online during Covid-19: Experiences of student teachers in an early childhood education practicum[END_REF]. Presenting the pedagogical content via video may have some benefits, but it also has some constraints compared to traditional textbased content. Research on video learning has grown since 2007 [START_REF] Giannakos | Looking at MOOCs rapid growth through the lens of video-based learning research[END_REF], but questions remain on how to efficiently design the videos, taking into account new technological advances.

Indeed, technical progress in artificial intelligence [START_REF] Lecun | Deep learning[END_REF] has allowed new possibilities to improve education. Relying on deep learning methods [START_REF] Goksel | Artificial intelligence in education: Current insights and future perspectives[END_REF], it is possible to automatically computerize some tasks, such as the classification, segmentation, or generation of various types of documents (e.g., text, images). In the context of pedagogical videos, some tasks previously requiring human intervention such as subtitles (e.g., Qiu, 2020;Shi et al., 2019) or summaries (e.g., [START_REF] Yousefi-Azar | Text summarization using unsupervised deep learning[END_REF] can now be automatically generated. Even if such operations are now technically feasible, their contributions to learning must still be evaluated.

Although many studies have been conducted on the effect of subtitles in video in foreign language situations (e.g., d'Ydewalle & De Bruycker, 2007;[START_REF] Guichon | The effects of multimodality on L2 learners: Implications for CALL resource design[END_REF][START_REF] Guillory | The effects of keyword captions to authentic French video on learner comprehension[END_REF][START_REF] Kanellopoulou | Film Subtitles as a Successful Vocabulary Learning Tool[END_REF][START_REF] Lavaur | Languages on the screen: Is film comprehension related to the viewers' fluency level and to the language in the subtitles[END_REF][START_REF] Lee | The Impact of Subtitles on Comprehension of Narrative Film[END_REF]Liao, Kruger, et al., 2020;[START_REF] Szarkowska | Six-second rule revisited: An eye-tracking study on the impact of speech rate and language proficiency on subtitle reading[END_REF][START_REF] Van Der Zee | Effects of subtitles, complexity, and language proficiency on learning from online education videos[END_REF][START_REF] Winke | Factors influencing the use of captions by foreign language learners: An eye-tracking study[END_REF][START_REF] Zanón | Using subtitles to enhance foreign language learning[END_REF], the processes involved may differ [START_REF] Kanellopoulou | The Dual-Coding and Multimedia Learning Theories: Film Subtitles as a Vocabulary Teaching Tool[END_REF][START_REF] Lin | Reading subtitles and taking enotes while learning scientific materials in a multimedia environment: Cognitive load perspectives on EFL students[END_REF] from learning with same-language subtitles (or video captions; see [START_REF] Gernsbacher | Video Captions Benefit Everyone[END_REF]. In the current study, the impact of same-language subtitles and keywords on learning will be investigated. This topic seems particularly relevant, as designers might be tempted to insert them mechanically into video-based environments. In the light of recent studies about signaling information in videos, keywords will be associated with markers in the timeline in the present study. This implies less frequent keywords than those commonly studied in the literature about partial redundancy but may maximize their impact on learning.

1.1.Video-based learning

Multimedia learning

Multimedia learning implies that information is delivered by texts (written or heard) and pictures (Mayer, 2014b). According to Paivio's dual coding theory (1990), two separated channels are devoted to information processing: one is a verbal system devoted to language processing (e.g., written or oral words) and the other is an imagery system devoted to nonverbal information (e.g., visual objects, sounds, tactile sensations) in which information can be processed in parallel (see Figure 1). These channels are also considered as capacity-limited.

The theory relies on the general assumption of working memory as capacity-limited, and the possibility of overloading it with too much incoming information for it to process. This cognitive overload may be detrimental to learning [START_REF] Paas | Implications of cognitive load theory for multimedia learning[END_REF][START_REF] Sweller | Cognitive architecture and instructional design: 20 years later[END_REF].

In the cognitive load theory, three types of cognitive load are usually distinguished: the intrinsic cognitive load that refers to the complexity of information; the extraneous cognitive load related to information presentation; and the germane cognitive load, defined as the resources left to process information [START_REF] Paas | Implications of cognitive load theory for multimedia learning[END_REF]Sweller et al., 2019, p. 201). The aim of pedagogical design is thus to: 1) reduce the extraneous cognitive load to 2) increase the germane cognitive load to 3) process the intrinsic cognitive load [START_REF] Paas | Implications of cognitive load theory for multimedia learning[END_REF]. (2014a), and in line with Paivio's work as outlined above, incoming information is processed through two capacity-limited channels. But more precisely, this process implies the active participation of learners, who select the relevant information from the document, organize it into mental representations, and then integrate these representations with their previous knowledge. This succession of steps is common to various other models, and may be designated as the Selection-Organization-Integration model [START_REF] Fiorella | Eight ways to promote generative learning[END_REF]. However, theories about multimedia learning have not been specifically designed for video-based learning.

Therefore, some particularities related to the video format need to be considered.

Benefits of the video format to learning

The video format combines two major benefits over traditional written pedagogical documents (van der Meij & van der Meij, 2014) that are not exclusive to videos, but are combined in this format. The first is that videos make it possible to show animated illustrations, which are beneficial compared to static pictures according to the literature (for a meta-analysis, see [START_REF] Höffler | Instructional animation versus static pictures: A metaanalysis[END_REF]. This makes it possible to represent dynamic content, and gives the learner access to the perception of changes in time and/or space [START_REF] Lowe | Animation principles in multimedia learning[END_REF][START_REF] Ploetzner | A review of learning demands in instructional animations: The educational effectiveness of animations unfolds if the features of change need to be learned[END_REF][START_REF] Van Der Meij | A comparison of paper-based and video tutorials for software learning[END_REF].

The second benefit of the video format is that visual and auditory information can be delivered simultaneously [START_REF] Van Der Meij | A comparison of paper-based and video tutorials for software learning[END_REF]. This benefit is related to the way the information is processed. On the basis of Paivio's work, learning models such as the Cognitive Theory of Multimedia Learning (Mayer, 2014a) have emerged, alongside the Integrated Model of Text and Picture Comprehension (Schnotz, 2014). According to these models, information is still processed in two separated capacity-limited channels, but a preliminary sensory level is added in which the two channels distinguish between auditory and visual modalities. After this, information is processed in verbal and pictorial models in Mayer's model, and in depictive and descriptive subsystems according to Schnotz's model. This distinction at a cognitive level between verbal and non-verbal channels appears very similar to that described by [START_REF] Paivio | Mental representations: A dual coding approach[END_REF] for verbal and imagery systems (see Figure 2). [START_REF] Low | The modality principle in multimedia learning[END_REF][START_REF] Sweller | Cognitive architecture and instructional design: 20 years later[END_REF]. In the multimedia learning literature, this beneficial effect is called the modality effect (for a meta-analysis, see [START_REF] Ginns | Meta-analysis of the modality effect[END_REF]. Furthermore, in videos, auditory information is usually in the form of verbal narration, and visual information is usually provided as animated pictures. So, irrespective of the theoretical distinction considered to exist between the two channels (auditory/visual or verbal/non-verbal), the video format should be particularly adapted to solicit both of them. In summary, the video format delivers information in a particular way that can lead to better information processing. Liao et al. (2020) proposed a model specifically including a subtitles process. This model also presents benefits from the parallel use of visual and auditory channels. However, the video format also brings some constraints, that must now be considered.

Negative aspects of the video format to learning

Along with the positive aspects of video over written documents, some negative impacts of videos must also be noted. The main concern related to information delivered transiently [START_REF] Höffler | Instructional animation versus static pictures: A metaanalysis[END_REF][START_REF] Leahy | Cognitive load theory, modality of presentation and the transient information effect[END_REF][START_REF] Merkt | Learning with videos vs. learning with print: The role of interactive features[END_REF]. Contrary to static information (e.g., written text), when individuals watch some animated content, they need to integrate the delivered information with previous information that is no longer available [START_REF] Hasler | Learner control, cognitive load and instructional animation[END_REF]Schwan & Riempp, 2004;[START_REF] Singh | The Transient Information Effect: Investigating the Impact of Segmentation on Spoken and Written text: The transient information effect[END_REF]. This kind of information processing is cognitively costly (i.e., it increases the extraneous cognitive load; see [START_REF] Wong | The effects of transient information and element interactivity on learning from instructional animations[END_REF], and could exceed individuals' resources [START_REF] Bétrancourt | Computer technologies in powerful learning environments: The case of using animated and interactive graphics for teaching financial concepts[END_REF][START_REF] Hasler | Learner control, cognitive load and instructional animation[END_REF][START_REF] Spanjers | Explaining the segmentation effect in learning from animations: The role of pausing and temporal cueing[END_REF][START_REF] Sweller | Cognitive architecture and instructional design: 20 years later[END_REF]. Indeed, the process of maintaining access to representations and integrating new information into these representations mainly relies on the working memory capacity, which is limited [START_REF] Wiley | The Individual Differences in Working Memory Capacity Principle in Multimedia Learning[END_REF].

Another constraint of the video format is related to the difficulty of retrieving specific information. Rather than looking for information in a written document by visually browsing it, individuals need to navigate through the timeline of the video until they find the information they seek [START_REF] Zhang | Instructional video in elearning: Assessing the impact of interactive video on learning effectiveness[END_REF]. However, it appears that the ability to identify content's organization in a document is a key component in information processing (Sanchez et al., 2001).

This ability may also rely on working memory capacity: indeed, individuals with higher working memory capacity may better maintain and integrate information during retrieval activity [START_REF] Banas | Working Memory Capacity and Learning Underlying Conceptual Relationships Across Multiple Documents: Working Memory and Underlying Relationships[END_REF].

Overall, the aims when designing a video-based learning environment remain the same as those of multimedia learning to 1) avoid cognitive overload; and 2) facilitate the processes of selection, organization (both promoting information seeking process), and integration (constrained by transience and limited working memory capacity) of relevant information. For this purpose, the presence of subtitles needs to be analyzed in view of these aims. It appears that, in line with the previously presented models, subtitles may induce cognitive overload. The next section will explain how full subtitles may be more detrimental than beneficial to the learner, but partial subtitles such as keywords may resolve some of the negative aspects.

Presence of subtitles in a video-based environment

Subtitles and split-attention

Contrary to expectations based on the modality effect, in a study conducted by [START_REF] Leahy | Cognitive load theory, modality of presentation and the transient information effect[END_REF], participants had a lower level of learning performance when visual illustration was associated with oral narration rather than written text. According to the authors, this result can be explained by the transitory nature of oral narration, that makes it too difficult to process in working memory when the speech is too long. In this specific situation, the negative transience effect would hinder the beneficial modality effect. A shorter, less complex oral narration or written text would be easier to process. Thus, it might appear that adding written text (i.e., subtitles) to oral narration could limit the negative transitory aspect of information, but the combination of written text and oral narration (with an illustration) could actually be more detrimental than beneficial. Indeed, a negative split-attention effect could then occur when learners have to simultaneously mentally integrate different sources of information [START_REF] Hasler | Learner control, cognitive load and instructional animation[END_REF]. A subtitled video would provide three concurrent sources of information at the same time: oral narration, written text, and visual illustration [START_REF] Zanón | Using subtitles to enhance foreign language learning[END_REF]. According to Paivio's model (1990), this implies a risk of cognitive overload in the verbal system, while in Mayer's (2014a) and Schnotz's (2014) models the risk of overload is situated in the visual channel [START_REF] Van Der Zee | Effects of subtitles, complexity, and language proficiency on learning from online education videos[END_REF][START_REF] Yue | Reducing verbal redundancy in multimedia learning: An undesired desirable difficulty[END_REF]. This situation may lead to losing the benefit of the modality effect (i.e., the use of two channels instead of one to avoid overload).

Furthermore, the association of oral narration and written subtitles may lead to redundancy, since these two sources deliver identical information [START_REF] Adesope | Verbal redundancy in multimedia learning environments: A meta-analysis[END_REF][START_REF] Sorden | A cognitive approach to instructional design for multimedia learning[END_REF].

Generally, redundancy is considered as negative, individuals learn better when the pedagogical document is constructed of pictures and spoken words rather than pictures, spoken and written text presented simultaneously [START_REF] Jamet | The effect of redundant text in multimedia instruction[END_REF][START_REF] Kalyuga | When redundant on-screen text in multimedia technical instruction can interfere with learning[END_REF][START_REF] Mayer | Principles for reducing extraneous processing in multimedia learning: Coherence, signaling, redundancy, spatial contiguity, and temporal contiguity principles[END_REF].

Lastly, the presence of subtitles has specific constraints related to written content: learning is mainly driven by the text. In a study led by Hannus and Hyönä (1999, experience 2), individuals were given a document with static illustration and written text. The authors' analysis of eye movements showed that learners spent only 6% of learning time inspecting the illustrations. Overall, when written text is presented, individuals start reading before alternating between text and pictures, and ultimately spend more time on the text (Schmidt-Weigand et al., 2010). However, in video, visual information is transient [START_REF] Ayres | Factors that impact on the effectiveness of instructional animations[END_REF]. Even if it can be considered less transient than oral speech, adding subtitles implies adding partially transient written information, and the reading rhythm is no longer controlled by the learner [START_REF] Kruger | Subtitles on the moving image: An overview of eye tracking studies[END_REF]. Attention being mainly focused on the subtitles may lead to not enough time being devoted to inspecting illustrated information that may quickly disappear.

So, the main constraints of subtitles in videos are that a split-attention effect can occur between the text and pictures, and that the text is considered as the principal source of information. The literature generally agrees on the potential negative effect of redundancy that full subtitles may provide with oral narration, even if some studies contradict these results (e.g., [START_REF] Gernsbacher | Video Captions Benefit Everyone[END_REF][START_REF] Szarkowska | The effects of text editing and subtitle presentation rate on the comprehension and reading patterns of interlingual and intralingual subtitles among deaf, hard of hearing and hearing viewers[END_REF]. A way to limit the amount of time allocated to redundant information could be to present only keywords associated with the oral narration in writing [START_REF] Adesope | Verbal redundancy in multimedia learning environments: A meta-analysis[END_REF].

Full versus partial subtitles

In studies by [START_REF] Mayer | Revising the redundancy principle in multimedia learning[END_REF], learners were exposed to a diagram accompanied by oral narration. Written keywords were proposed or not. The presence of redundant keywords was found to lead to better memorization of the information. In a metaanalysis conducted by [START_REF] Adesope | Verbal redundancy in multimedia learning environments: A meta-analysis[END_REF], the degree of redundancy between oral narration and written text seemed to moderate the redundancy effect. Indeed, partial redundancy (e.g., oral narration associated with written keywords only) may lead to better learning than full redundancy (e.g., oral narration, and identical written text). The positive effect of keywords may be explained by the fact that keywords are supposed to guide learners in selecting relevant information [START_REF] Adesope | Verbal redundancy in multimedia learning environments: A meta-analysis[END_REF][START_REF] Mayer | Revising the redundancy principle in multimedia learning[END_REF][START_REF] Mccrudden | Exploring the Boundary Conditions of the Redundancy Principle[END_REF]. As it corresponds to the first step of the learning process according to the Selection-Organization-Integration model [START_REF] Fiorella | Eight ways to promote generative learning[END_REF]Mayer, 2014a), facilitating information selection may be particularly useful in improving learning [START_REF] De Koning | Towards a framework for attention cueing in instructional animations: Guidelines for research and design[END_REF].

In fact, this highlighting benefit attributed to keywords could be related to the definition of signaling devices in written documents (e.g., titles, typographical variations) according to [START_REF] Lorch | Text-signaling devices and their effects on reading and memory processes[END_REF], aiding the identification of important information. When dealing with transient content (i.e., video), it may be particularly helpful for learners to know which information is important but also when it is presented, which could decrease extraneous cognitive load (de [START_REF] De Koning | Towards a framework for attention cueing in instructional animations: Guidelines for research and design[END_REF]. Further, studies on the effect of titles in videos (when associated with markers in the timeline, e.g., [START_REF] Cojean | Facilitating information-seeking activity in instructional videos: The combined effects of micro-and macroscaffolding[END_REF][START_REF] Cojean | The role of scaffolding in improving information seeking in videos[END_REF] have shown that they may facilitate navigation in the video and the retrieval of specific information (which is one of the major constraints of videos). Thus, it can be supposed that occasional keywords with markers in the timeline could be beneficial in the same way in dealing with transient information and facilitating the retrieval of information during learning, especially during reviewing activity.

The current study

The current study aims to investigate the impact of full subtitles and keywords (i.e., partial subtitles) on learning in video. When focused on the redundant effect, previous studies appeared either to focus on the effect of subtitles (e.g., [START_REF] Jamet | The effect of redundant text in multimedia instruction[END_REF] or on the effect of keywords (e.g., [START_REF] Mayer | Revising the redundancy principle in multimedia learning[END_REF]. In a study conducted by de Koning, van Hooijdonk, and Lagerwerf (2017), the effects of oral speech, written subtitles and on-screen labels on procedural learning were analyzed. However, the animation only lasted 84 seconds.

According to results from [START_REF] Leahy | Cognitive load theory, modality of presentation and the transient information effect[END_REF], difficulties due to transience may depend on the length of the video. Perhaps the video in the study of de Koning, van Hooijdonk, and Lagerwerf (2017) was too short to induce a negative impact of redundancy and split-attention.

It may therefore be interesting to analyze the impact of subtitles and keywords when learning using a longer video that might exceed learners' cognitive resources. The current study aims to evaluate these effects through the use of a 12-minute video. Moreover, in the current study, and contrary to de Koning, van Hooijdonk, and Lagerwerf (2017), keywords were associated with markers in the timeline to optimize their beneficial guiding effect. Following a 2x2 experimental design, learners were shown a video with full subtitles, only some keywords, both (subtitles and keywords), or none. Our main hypotheses are that: 1) subtitles may be totally redundant with oral narration, split attention between too many sources of information, and then have a negative impact on learning performance; and 2) occasional keywords may provide only partial redundancy and guide learners into relevant strategies (i.e., the selection of relevant information, navigation) that have an overall positive effect on learning.

In this context, learning corresponds to the increase in knowledge in the long-term memory [START_REF] Hasler | Learner control, cognitive load and instructional animation[END_REF], and it implies the memorization as well as comprehension of the presented information [START_REF] Eitel | How inspecting a picture affects processing of text in multimedia learning[END_REF]. On the one hand, memorization can be defined as the ability to reproduce or recognize presented material (Mayer, 2014b), and is usually measured through retention tests [START_REF] Bétrancourt | Computer technologies in powerful learning environments: The case of using animated and interactive graphics for teaching financial concepts[END_REF]. On the other hand, comprehension corresponds to the interaction between incoming information and prior knowledge [START_REF] Woodall | From the boob tube to the black box: Television news comprehension from an information processing perspective[END_REF], and is usually measured through transfer tests [START_REF] Bétrancourt | Computer technologies in powerful learning environments: The case of using animated and interactive graphics for teaching financial concepts[END_REF] in which learners can reuse what they have learned in new contexts.

A series of predictions was made in the present study, as expressed in the following hypotheses:

Hypothesis 1. Effect of subtitles on video learning. Due to the split-attention effect between three concurrent sources of information (oral narration, written text, and visual illustration), full redundancy between two sources (oral narration and written text) and attention being focused on the written text at the expense of visual illustration, the presence of subtitles may be detrimental to the learner's comprehension (Hypothesis 1a) and memorization (Hypothesis 1b) of the video content.

Hypothesis 2. Effects of keywords on video learning. Keywords may help learners to select important information and facilitate the retrieval of specific information. So, the presence of keywords may be beneficial to comprehension (Hypothesis 2a) and the memorization of video content. However, previous studies have shown that the memorization of non-highlighted information could be poorer in the presence of keywords, because it would be considered as less important information [START_REF] Lorch | Text-signaling devices and their effects on reading and memory processes[END_REF][START_REF] Lorch | Effects of organizational signals on free recall of expository text[END_REF]. Thus, a distinction is made by the learner between important information (i.e., highlighted by keywords when they are provided) and less important information (i.e., not highlighted by keywords). The presence of keywords may be beneficial to the memorization of important information (Hypothesis 2b), but they may have a negative impact on the memorization of less important information (Hypothesis 2c).

Method

Participants

113 students attending the University of Angers (France) participated in the study. Data from 17 participants were excluded from the analyses due to video issues. Of the 96 remaining students (87 women, 9 men), the average age was 20.07 years (SD = 2.15). All the participants volunteered to take part in the study, and were recruited via an email sent to all students at the university. The experiment was conducted in accordance with the principles of the Declaration of Helsinki (World Medical Association, 2013).

Materials and experimental design

Each participant had access to a computer and headphones. A video-based environment was designed to display a video in French about astronomy (TEDx Talks, 2013). The videobased environment is presented in Figure 3, and consisted of the video (lasting 11 minutes and 51 seconds), a table of contents presenting the five main parts of the video, and the timeline with segmentation corresponding to the five chapters. A timer was designed just below the table of contents to indicate how much time was left for learning (maximum 20 minutes). A button reading "Go to the questionnaire" was also displayed below the timer. A table of contents representing the chapters of the video was available in each experimental condition, as were segments in the timeline corresponding to these chapters. All the participants could navigate through the video using the timeline and click on a chapter in the table of contents to directly access it. Participants were evenly and randomly distributed among the four experimental conditions. In the "Subtitles" condition (n = 24), subtitles transcribing the full oral narration (i.e., verbatim;[START_REF] Szarkowska | Verbatim, standard, or edited? Reading patterns of different captioning styles among deaf, hard of hearing, and hearing viewers[END_REF] were included below the screen (see Figure 3). In the "Keywords" condition (n = 25), seven keywords were available instead of full subtitles. They appeared when they were pronounced by the oral narration and corresponded to the actual written version of the spoken text. No other written information was included except the (i.e., the key ideas) in the delivered content. According to de [START_REF] De Koning | Towards a framework for attention cueing in instructional animations: Guidelines for research and design[END_REF], highlighting information in transient content implies that the learner should guide their attention onto which information is highlighted, and when. Furthermore, previous studies in the literature (e.g., [START_REF] Cojean | Facilitating information-seeking activity in instructional videos: The combined effects of micro-and macroscaffolding[END_REF][START_REF] Cojean | The role of scaffolding in improving information seeking in videos[END_REF] have shown that titles only have a beneficial impact on information retrieval and navigation in a video when they are associated with markers in the timeline. Then, markers were situated in the timeline to represent the localization of keywords (see Figure 3). In the "Subtitles and Keywords" condition (n = 23), full subtitles were displayed, and keywords were highlighted in these subtitles. Corresponding markers in the timeline were also available (see Figure 3). Finally, in the "Control" condition (n = 24), the video was displayed without any subtitles or keywords (see Figure 3).

Measures

Control variables

Interest in the topic and perceived competence. A pre-task questionnaire was proposed to participants before they undertook the learning task. They were asked to indicate from 0 to 10 (on an 11-point Likert scale) their degree of interest in the topic of the video (i.e., galaxies in the universe). They also had to indicate from 0 to 10 (11-point Likert scale) how much they felt competent on this subject. The aim of these two questions was to ensure that participants were equitably distributed among the four experimental conditions.

Learning time

The video lasted 11 minutes and 51 seconds, but learners had 20 minutes to learn it.

Then, time was left for the learners to revise the information. They could navigate through the video as much as they wanted during these 20 minutes. In the current study, this could be considered as a measure of learning strategies. If they considered that their learning was done before the end of the 20 minutes, they could go to the post-task questionnaire whenever they wanted. The learning time was measured from when the learners launched the video (i.e., when the timer started) to when they skipped to the post-task questionnaire (i.e., when they clicked on the "Go to the questionnaire" button or directly accessed the questionnaire at the end of the 20 minutes).

Comprehension

A post-task questionnaire was proposed to the learners after the learning task. Five comprehension questions (i.e., transfer questions) were asked (four open-ended questions and one multiple-choice question). The answers were not explicitly given in the video; rather, learners had to infer responses (e.g., "What process in space can be related to the famous Lavoisier sentence "Nothing is lost, nothing is created, everything is transformed"?"). Each question was worth one point. In total, the comprehension questions were scored as five points, then transformed into a percentage. Questions were scored by one rater.

Memorization

In the post-task questionnaire, 13 memorization questions (12 open-ended questions and one multiple-choice question) were asked, for a total of 15 points. Eleven questions were worth one point each, and the other two questions were worth two points each because the expected responses were composed of two parts. All the information to answer these questions was explicitly contained in the video. Six memorization questions (on six points) were directly about important information that the keywords highlighted when they were available. For example, in the "Keywords" and "Subtitles and Keywords" conditions, one of the keywords (or key phrases) was "13 million light years", because it was considered as important information in the video (see Figure 3). Then, one question was "What is the maximum distance, with a telescope, at which we can see galaxies?". Seven other questions (on nine points) were about information considered as less important and thus not related to potential keywords. For example, one of those questions was "In its beginnings, what was the temperature of the universe?", and this information was not related to any keywords. Since scores on important and less important information were not on the same scale, they were transformed into percentages. As for comprehension questions, memorization questions were scored by one rater.

Procedure

One to eight participants could perform the experiment at the same time. Once they were installed, each one in front of a computer, the experimenter presented the study and the instructions. Each participant started when they wanted. As was mentioned above, first, the participants answered a pre-task questionnaire about their interest and perceived competence in the topic of the video. Then, they had to read the instructions for the learning task. It was stipulated that they would have 20 minutes to learn the maximum information from the video, and that they could navigate as much as they wanted during those 20 minutes. When they wanted to go to the knowledge test, they would have to click on the "Go to the questionnaire" button. If they had not finished in 20 minutes, the video would stop and they would automatically go to the questionnaire. The post-task questionnaire was presented on the computer screen and was composed of 13 memorization and 5 comprehension questions presented in a random order. At the end of the questionnaire, two demographic questions about age and gender were asked.

Results

All statistical analyses were performed using the R statistical software (R Core Team, 2019).

Control variables

An ANalysis Of VAriances (ANOVA) was performed on the prior interest and perceived competence scores (from 0 to 10) to ensure that the participants were equitably distributed among the experimental conditions. The ANOVA showed no differences between conditions (see Table 1 for descriptive statistics), either for interest in the topic, F(3, 92) = 0.78, p = .510, η² = .02, or perceived competence, F(3, 92) = 0.88, p = .454, η² = .03. F(1, 92) = 0.06, p = .806, partial η² = .00, nor of interaction between keywords and subtitles, F(1, 92) = 0.38, p = .538, partial η² = .00, were found.

2) redundancy between oral and written narration; and 3) learners' attention being focused on the written text instead of illustrations. In contrast, using occasional keywords with markers on the timeline was supposed to be positive since it could 1) highlight relevant information and guide learners to be more efficient in their selection process [START_REF] Mayer | Revising the redundancy principle in multimedia learning[END_REF]; and

2) facilitate navigation through the video [START_REF] Cojean | Facilitating information-seeking activity in instructional videos: The combined effects of micro-and macroscaffolding[END_REF].

The results showed no effect of subtitles on learning performance. Hypotheses 1a and 1b are therefore not validated. If a split-attention effect occurred, it had no impact on learning in this study. Two explanative hypotheses could be considered and explored in future works.

The first of these is that since learning seems to be generally driven by the text (e.g., [START_REF] Hannus | Utilization of illustrations during learning of science textbook passages among low-and high-ability children[END_REF]Schmidt-Weigand et al., 2010), learners may have focused on subtitles when they were present and totally inhibited information from the oral speech. However, this is not in line with the theoretical concept of redundancy [START_REF] Mayer | Principles for reducing extraneous processing in multimedia learning: Coherence, signaling, redundancy, spatial contiguity, and temporal contiguity principles[END_REF]. Secondly, a reverse possibility is that the learners ignored the written text (i.e., subtitles). Maybe it is easier to focus only on oral speech and visual illustrations when the video is long and when subtitles are present at a high frequency, contrary to keywords being less frequent and way more salient in drawing learners' attention. Studies could therefore be conducted with slightly different information delivered via written text and oral speech in order to explore which source was favored in this context. Further, the effect of keywords surprisingly seemed to be negative on learning performance. Learners with keywords had lower comprehension scores than learners without keywords. Hypothesis 2a is thus not validated. Moreover, the presence of keywords had a negative impact on the memorization of less important information as well as on the memorization of important information, thus confirming Hypothesis 2c but not validating Hypothesis 2b. Regarding learning time, it appears that learners with keywords spent less time on the video, which indicates less time reviewing the information that could have been relevant.

The current study has some limits. First, all the participants were college students. A positive side of this is that students are daily engaged in learning activities, and are probably more likely to deal with video formats than other people. However, to generalize the observed effects, a larger sample of participants would be needed. Further, only one video was used in the proposed learning activity. Replication of the study using different video-based environments and different types of content would be necessary to generalize the conclusions.

Next, only one rater scored the memorization and comprehension questionnaires. Even if the rater followed a detailed scale, double blind scoring should be considered in future studies. Also in future studies, additional measures could be recorded as verbalizations to gain access to learners' strategies and self-regulation processes [START_REF] Fernandez | Extending the testing effect to self-regulated learning[END_REF][START_REF] Greene | Domain-specificity of self-regulated learning processing in science and history[END_REF][START_REF] Kruger | Subtitles and Eye Tracking: Reading and Performance[END_REF]. Eye-tracking measures could also be used to gain insights into learners' visual strategies (e.g., [START_REF] Kruger | Subtitles and Eye Tracking: Reading and Performance[END_REF]: it may be the case that a split-attention effect between picture, oral narration, and written text doesn't appear because learners strongly rely on one source rather than another. Additional measures could also explore the attention specifically given to oral narration when written text is read. Finally, the linguistic characteristics of keywords (e.g., length, level of abstraction) could be considered, as well as subtitles and keyword editing (i.e., verbatim or edited text; [START_REF] Szarkowska | Verbatim, standard, or edited? Reading patterns of different captioning styles among deaf, hard of hearing, and hearing viewers[END_REF][START_REF] Szarkowska | The effects of text editing and subtitle presentation rate on the comprehension and reading patterns of interlingual and intralingual subtitles among deaf, hard of hearing and hearing viewers[END_REF].

Conclusion

In conclusion, contrary to theoretical redundancy and split-attention effects, the current study observed no impact on learning performance when subtitles are totally redundant with oral narration. However, the presence of some keywords had a negative impact on learning.

Learners' attention was probably even more divided between the oral speech, visual illustration, and keywords, because they were only occasional. There is also the possibility that learners were misled by the keywords and did not pursue efficient learning strategies. Future studies should focus on a deeper comprehension of learning strategies and the processes involved in

Figure 1 .

 1 Figure 1. Paivio's distinction between verbal and imagery systems for information

Figure 2 .

 2 Figure 2. Information processing in two channels at sensory and cognitive levels

  18

Figure 3 .

 3 Figure 3. Screenshots of video-based environment in Control (1), Subtitles (2),

Table 1 .

 1 Descriptive statistics for control variables Tableau 1. Statistiques descriptives pour les variables contrôles

	Condition	M	Prior interest	SD	Perceived competence M SD
	Control	6.29	1.30	2.00	1.22
	Subtitles	5.83	2.39	2.67	1.95
	Keywords	6.00	1.55	2.16	1.34
	Subtitles and Keywords	5.39	2.74	2.43	1.56
	3.2. Learning
	3.2.1. Learning time
	Concerning learning time, the ANOVA showed a significant effect of keywords on
	learning times, F(1, 92) = 5.88, p = .017, partial η² = .06. Participants with keywords seemed
	to spend less time learning the video than participants without keywords (see Table 2 for
	descriptive statistics). Since the video lasted 11 minutes and 51 seconds (i.e., 711 seconds),
	participants with keywords spent an average of 2 minutes and 3 seconds (i.e., 123.41 seconds)
	engaged in reviewing activity, whereas participants without keywords spent 3 minutes and 31
	seconds on average (i.e., 211.07 seconds) reviewing information. No effects of subtitles,

Comprehension

The ANOVA showed a significant effect of keywords on comprehension scores, F(1, 92) = 5.05, p = .027, partial η² = .05. Participants with keywords seemed to have a lower comprehension score than participants without keywords (see Table 3 for descriptive statistics).

No effects of subtitles, F(1, 92) = 1.15, p = .287, partial η² = .01, nor of interaction between keywords and subtitles, F(1, 92) = 0.18, p = .675, partial η² = .00, were found.

Memorization

Concerning the global memorization score, the results showed a significant effect of keywords, F(1, 92) = 6.67, p = .011, partial η² = .07, but no effect of subtitles, F(1, 92) = 2.50, p = .117, partial η² = .03, nor any interaction between keywords and subtitles, F(1, 92) = 0.38, p = .538, partial η² = .00.

Questions about important information (i.e., information highlighted in conditions with keywords) and questions about less important information (i.e., that which was not highlighted by keywords in conditions with keywords) were also distinguished. For questions about important information, the ANOVA showed a significant effect of keywords on memorization scores, F(1, 94) = 6.64, p = .012, η² = .07. For questions about less important information, the results also showed a significant effect of keywords, F(1, 94) = 4.63, p = .034, η² = .05.

Overall, participants with keywords seemed to have lower memorization scores than participants without keywords, both for highlighted information and non-highlighted information (see Table 4 for descriptive statistics).

Discussion

The aim of this study was to test the effects of subtitles and keywords in a video on learning performance. Full subtitles were supposed to have a negative impact on learning due to 1) the split-attention effect between illustration, oral narration, and written text;

An explicative hypothesis could be that learners don't perceive keywords as particularly relevant (de [START_REF] De Koning | Towards a framework for attention cueing in instructional animations: Guidelines for research and design[END_REF]. Also, in the current study, the presence of occasional and salient keywords could have led to the implementation of non-relevant strategies by learners.

Keywords are supposed to help the selection of important information by signaling it, but learners may have considered that instead of supporting the selection process, the keywords replaced it. Then, they may overestimate their learning performance, which is consistent with less time spent watching the video. This difficulty for learners in pursuing a relevant strategy (i.e., using keywords to facilitate the selection process) must be explored, and may be explained in terms of difficulties [START_REF] Fernandez | Extending the testing effect to self-regulated learning[END_REF][START_REF] Winne | nStudy: Tracing and Supporting Self-Regulated Learning in the Internet[END_REF].

Lastly, keywords were supposed to reduce the split-attention effect between the spoken and written text. However, in relation to the results of the current study, it can be hypothesized that keywords caused more split-attention than subtitles due to their low frequency of appearances and disappearances (i.e., only seven instances). In fact, the number and frequency of keywords may be an important factor in the process: a lower number of more salient keywords would be worse than partial subtitles throughout the video. In studies conducted by [START_REF] Mayer | Revising the redundancy principle in multimedia learning[END_REF], the term "keywords" actually referred to partial subtitles present at each slide (i.e., at a high frequency), and were found to have a positive impact on learning. further, in a study led by [START_REF] Angerbauer | Automatic Compression of Subtitles with Neural Networks and its Effect on User Experience[END_REF], full subtitles were compared to compressed subtitles (the latter with about 50% of the words presented in the full subtitles). No differences were found in comprehension between the conditions. The keywords presented in the current study represented far less than 50% of the subtitles. The small number of keywords may have had a negative impact on learners' attention. It may therefore be necessary to find the right ratio between too much redundancy and too little salient information. Further work could also help to establish which information to highlight, as this may guide learners' attention and strategies.

the presence of keywords. It is also necessary to look for a specific design promoting the potentially beneficial effect of keywords on the selection process.

Acknowledgments

The authors would like to thank students and members of the LPPL (Laboratoire de Psychologie des Pays de la Loire) for their material support.

Declaration of Conflicting Interest

The authors declare that there is no conflict of interest. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Information Hiding and Multimedia Signal Processing (Vol. 156,.

Springer Singapore. https://doi.org/10.1007/978-981-13-9714-1_34


Blondet Antonine
email: antonine.blondet@kallistem.com

Martin Guillaume
email: guillaume.martin@kallistem.com

Durand Philippe
email: philippe.durand@kallistem.com

Perrard Marie-Hélène
email: marie-helene.perrard-durand@inserm.fr

Low concentrations of glyphosate alone affect the pubertal male rat meiotic step: an in vitro study

Keywords: STMN1, Transition Protein 1: TP1, Transition Protein 2: TP2, Protamine 3: PRM3, Glyphosate, low concentrations, meiosis, in vitro study

Glyphosate is the most used herbicide in the world. Controversial studies exist on its effect on the male reproductive system. We used the validated BioAlter® model to test the effects of low concentrations of Glyphosate. Pubertal rat seminiferous tubules were treated with Glyphosate 50 nM, 500 nM, 5 µM or 50 µM over a 3-week culture period. The Trans-Epithelial Electrical Resistance was not modified by any of the concentrations. The decrease of Clusterin mRNAs suggested that glyphosate would target the integrity of Sertoli cells. The decrease of the numbers of germ cells from day 14 onward highlighted the chronic effect of glyphosate at 50 nM, 500 nM or 5 µM. No consistent effect of glyphosate was observed on the numbers of spermatogonia or on their specific mRNA levels. However, those low concentrations of glyphosate targeted young spermatocytes and middle to late pachytene spermatocytes resulting in a decrease of the numbers of round spermatids, the direct precursors of spermatozoa. This study underlines that the effect of a toxicant should be also studied at low doses and during the establishment of the blood-testis barrier.

Introduction

Glyphosate dates from the 1970s and has become the most used herbicide in the world [START_REF] Benbrook | Trends in glyphosate herbicide use in the United States and globally[END_REF]. Glyphosate has been mixed with other chemicals to constitute widely used glyphosate-based herbicides. Among them Roundup® has been used in agricultural fields and home gardens [START_REF] Gillezeau | The evidence of human exposure to glyphosate: a review | Environmental Health | Full Text[END_REF]. There is increasing interest in potential chronic effects of formulated glyphosate and its degradation products as they accumulate in the environment [START_REF] Bai | Glyphosate: environmental contamination, toxicity and potential risks to human health via food contamination[END_REF][START_REF] Battaglin | Glyphosate and its degradation product AMPA occur frequently and widely in U.S. soils, surface water, groundwater, and precipitation[END_REF]. Glyphosate concentrations vary from 2 to 430 μg/L (11.8 nM to 2.5 µM) in river water and stream water in the USA [START_REF] Battaglin | The occurrence of glyphosate, atrazine, and other pesticides in vernal pools and adjacent streams in Washington, DC, Maryland, Iowa, and Wyoming, 2005-2006[END_REF][START_REF] Battaglin | Glyphosate, other herbicides, and transformation products in Midwestern streams[END_REF][START_REF] Coupe | Fate and transport of glyphosate and aminomethylphosphonic acid in surface waters of agricultural basins[END_REF][START_REF] Mahler | Similarities and differences in occurrence and temporal fluctuations in glyphosate and atrazine in small Midwestern streams (USA) during the 2013 growing season -News -Elsevier[END_REF]. In Europe, glyphosate has also been detected in various waters (from 0.1 to 2.5 µg/L i.e 0.6 nM to 14.8 nM) (Poiger et al., 2017;[START_REF] Skark | The occurrence of glyphosate in surface water[END_REF]. Higher levels (up to 165 μg/L i.e 976 nM) were found in France [START_REF] Villeneuve | Herbicide Contamination of Freshwater Ecosystems: Impact on Microbial Communities[END_REF] and Denmark (Rosenbom et al., 2015).

Human beings may be exposed to glyphosate through food and drinking water [START_REF] Bai | Glyphosate: environmental contamination, toxicity and potential risks to human health via food contamination[END_REF]. Glyphosate was detected in human urine samples. Its concentrations were higher among subjects in the USA (mean 2-3 μg/L i.e 11.8-17.6 nM) with maximum up to 233 μg/L i.e 1.4 µM) than in Europe (mean: 1 μg/L i.e 5.9 nM and maximum 5 μg/L i.e 29.5 nM) [START_REF] Niemann | A critical review of glyphosate findings in human urine samples and comparison with the exposure of operators and consumers[END_REF].

There is a growing interest in toxicology of male reproduction because human semen quality has declined worldwide over the last decades [START_REF] Carlsen | Evidence for decreasing quality of semen during past 50 years[END_REF]Geoffroy-Siraudin et al., 2012a;[START_REF] Le Moal | Semen quality trends in French regions are consistent with a global change in environmental exposure[END_REF][START_REF] Olesen | Decrease in semen quality and Leydig cell function in infertile men: A longitudinal study[END_REF]. In addition, semen quality may be a fundamental biomarker of overall male health [START_REF] Eisenberg | Semen quality, infertility and mortality in the USA[END_REF][START_REF] Jensen | Good semen quality and life expectancy: A cohort study of 43,277 men[END_REF].

Controversial in vivo studies exist on the effect of glyphosate alone on male reproductive system. Two studies have shown that glyphosate affects spermatogenesis (i) in mice by decreasing the number of spermatozoa and the plasma levels of testosterone [START_REF] Pham | Perinatal exposure to glyphosate and a glyphosate-based herbicide affect spermatogenesis in mice[END_REF], (ii) in rabbits resulting in a decline in ejaculate volume and sperm concentration (Yousef et al., 1995). By contrast, Li's group has shown that glyphosate does not affect male reproduction in rat [START_REF] Dai | Effect of glyphosate on reproductive organs in male rat[END_REF]) and Svingen's team found no significant effect of glyphosate on testes or testosterone synthesis in rats [START_REF] Johansson | Exposure to a glyphosate-based herbicide formulation, but not glyphosate alone, has only minor effects on adult rat testis[END_REF]. There are only very few in vitro studies on the effect of glyphosate alone on primary cultures of testicular cells. Séralini's team tested glyphosate on mature rat fresh testicular cells and showed that within 24-48 hours glyphosate was essentially toxic to Sertoli cells [START_REF] Clair | A glyphosate-based herbicide induces necrosis and apoptosis in mature rat testicular cells in vitro, and testosterone decrease at lower levels[END_REF]. The group of Meroni showed that glyphosate could alter the blood-testis barrier which is essential for spermatogenesis, but only at a high concentration: 100 ppm ie 100 µg/ml (588 µM) [START_REF] Gorga | In vitro effects of glyphosate and Roundup on Sertoli cell physiology[END_REF]. Toxic effects on sperm progressive motility but not on sperm DNA integrity were induced in human by glyphosate alone at 360 µg/L (2.1 µM), a concentration that greatly exceed environmental exposures in Europe [START_REF] Anifandis | The Effect of Glyphosate on Human Sperm Motility and Sperm DNA Fragmentation[END_REF].

The purpose of the present in vitro study is to investigate the effect of low doses of glyphosate alone at 50 nM (8.45 ng/ml), 500nM (84.5 ng/ml), 5µM (845 ng/ml) or 50 µM (8.45 µg/ml), in our 20-22-day-old rat seminiferous tubule culture model, called BioAlter®. These concentrations of glyphosate were selected on the basis of the mean plasma concentrations despite they may be somewhat different from the concentrations present in the seminiferous tubules. These concentrations are partially overlapping the levels found in occupational workers (11.73 ng/ml i.e 69 nM) [START_REF] Pan | The association between coronary artery disease and glyphosate exposure found in pesticide factory workers[END_REF], or in patients with acute selfpoisoning reported as asymptomatic (2.9 µg/ml i.e 17 µM) or suffering from minor to moderate symptoms (40.8 µg/ml i.e 243 µM) (Roberts et al., 2010). BioAlter® mimics the in vivo situation and has been carefully validated from a physiological [START_REF] Geoffroy-Siraudin | Validation of a rat seminiferous tubule culture model as a suitable system for studying toxicant impact on meiosis effect of hexavalent chromium[END_REF][START_REF] Perrard | European regulation REACH and the assessment of testicular toxicity[END_REF][START_REF] Perrard | Development of the meiotic step in testes of pubertal rats: Comparison between the in vivo situation and under in vitro conditions[END_REF] and a toxicological points of view [START_REF] Goldstein | Use of a rat exvivo testis culture method to assess toxicity of select known male reproductive toxicants[END_REF], and is relevant to investigate the mechanisms of action on spermatogenesis of heavy metals [START_REF] Carette | Hexavalent chromium at low concentration alters Sertoli cell barrier and connexin 43 gap junction but not claudin-11 and N-cadherin in the rat seminiferous tubule culture model[END_REF]Geoffroy-Siraudin et al., 2012b, 2010) pesticides or pollutants either separately or in cocktail [START_REF] Ali | Exposure to low-dose bisphenol a impairs meiosis in the rat seminiferous tubule culture model: A physiotoxicogenomic approach[END_REF][START_REF] Carette | Endocrine Disrupting Effects of Noncytotoxic Doses of Carbendazim on the Pubertal Rat Seminiferous Epithelium: An Ex Vivo Study[END_REF][START_REF] Durand | Effects of a mixture of low doses of atrazine and benzo[a]pyrene on the rat seminiferous epithelium either during or after the establishment of the blood-testis barrier in the rat seminiferous tubule culture model[END_REF][START_REF] Durand | Effects of low doses of carbendazim or iprodione either separately or in mixture on the pubertal rat seminiferous epithelium: An ex vivo study[END_REF]Pisani et al., 2016) during a 3-week culture period. In addition, BioAlter® decreases the number of sacrificed rats by 20 to 30 compared to in vivo studies; in agreement with the European REACH directive.

One originality of this work is that the effect of glyphosate could be studied: (i) at the level of the blood-testis barrier; (ii) on the numbers of Sertoli cells and of each germ cell type (spermatogonia, spermatocytes, secondary spermatocytes and round spermatids which are the direct precursors of spermatozoa); (iii) on the levels of transcripts specific of either the bloodtestis barrier proteins, or of Sertoli cells, or of germ cells (spermatogonia, pachytene spermatocytes, round spermatids) and of steroid hormone receptors.

Material and methods

Animals

Experiments were performed with 20-22-day-old male Sprague-Dawley rats (Charles River, Saint-Germain-Nuelles, France) having undergone no treatment. The experimental protocol was designed in compliance with recommendations of the European Economic Community (EEC) (86/609/EEC) for the care and use of laboratory animals. Testes from four 20-22-dayold rats were pooled in every culture experiment.

Preparation and culture of seminiferous tubule segments

Pooled seminiferous tubule segments from 4 to 6 rats were cultured in a serum-free culture medium as previously described [START_REF] Hue | Meiotic differentiation of germinal cells in three-week cultures of whole cell population from rat seminiferous tubules[END_REF][START_REF] Staub | The whole meiotic process can occur in vitro in untransformed rat spermatogenic cells[END_REF]. The culture medium consisted of 15 mM Hepes-buffered F12/DMEM supplemented with antibiotics, 1.2 g/L NaHCO 3 ,10 µg/ml insulin, 10 µg/ml transferrin, 10 -4 M vitamin C, 10 µg/ml vitamin E, 10 -7 M testosterone, 3.3 X 10 -7 M retinoic acid, 3.3 X 10 -7 M retinol, 10 -3 M pyruvate (all from Sigma), and 50 ng/ml NIH (Bethesda, MD) porcine FSH (lot #AFP 5600; obtained through NIDDK, Rockville, MD). Some cultures were performed in the absence of testosterone. A stock solution of glyphosate (Sigma-Aldrich, St. Quentin Fallavier , France) 25 mM (4.23 mg/ml) was prepared in water, then diluted in the culture medium in order to obtain the final concentrations 50 µM (8.45 µg/ml), 5 µM (845 ng/ml), 500 nM (84.5 ng/ml) and 50 nM (8.45 ng/ml) of glyphosate in the basal compartment of the bicameral chambers of culture. In the wells containing the treated cells, glyphosate was added to the culture medium from day 2 onward. Basal media (with or without glyphosate) were renewed every 2 days. At selected days of culture, cells were detached from the culture dishes and isolated with trypsin for determining cell number, and cell viability by trypan blue exclusion or cell flow cytometry analysis.

Trans-epithelial electrical resistance

Trans-epithelial electrical resistance (TEER) measurement was performed with an EVOM2 (World Precision Instruments, Florida, USA) [START_REF] Durand | Effects of low doses of carbendazim or iprodione either separately or in mixture on the pubertal rat seminiferous epithelium: An ex vivo study[END_REF]. The culture was equilibrated at room temperature before the assay. The TEER was calculated according to the following equation: TEER = (R total -R control).A (Ω cm 2 ), where R total is the resistance measured, R control is the resistance of the control insert (insert alone with the culture medium), and A is the surface area of the insert.

Immunolabeling of cultured cells for flow cytometric analysis

Cultured cells from seminiferous tubules were fixed in 70% ethanol. To distinguish germ cells from somatic cells, the latter are mainly Sertoli cells as rat possess only one layer of peri-tubular/myoid cells [START_REF] Maekawa | Peritubular myoid cells in the testis: their structure and function[END_REF], cells were immunolabeled with a monoclonal antibody against vimentin (clone V9; DAKO SA, Trappes, France) that was conjugated with a green fluorochrome, Alexa 488, by using a Zenon Mouse labeling kit (Life Technologies, Cailloux-sur-Fontaines, France) as previously described [START_REF] Durand | Effects of low doses of carbendazim or iprodione either separately or in mixture on the pubertal rat seminiferous epithelium: An ex vivo study[END_REF][START_REF] Perrard | Cytostatic factor proteins are present in male meiotic cells and β-nerve growth factor increases Mos levels in rat late spermatocytes[END_REF]. Then, cells were analyzed using an Attune Acoustic Focusing Cytometer (Applied biosystems) as described [START_REF] Carette | Endocrine Disrupting Effects of Noncytotoxic Doses of Carbendazim on the Pubertal Rat Seminiferous Epithelium: An Ex Vivo Study[END_REF][START_REF] Durand | Effects of low doses of carbendazim or iprodione either separately or in mixture on the pubertal rat seminiferous epithelium: An ex vivo study[END_REF]. Acquisition and analysis were performed using the Attune Cytometric Software. The vimentin-positive somatic/Sertoli cells and the vimentin-negative tetraploid, diploid and haploid germ cells were separated with the bivariate analysis of DNA content/vimentin. Then, the bivariate linear forward light scatter and linear side angle light scatter analyses allowed the identification of the different populations of cells [START_REF] Durand | Effects of low doses of carbendazim or iprodione either separately or in mixture on the pubertal rat seminiferous epithelium: An ex vivo study[END_REF][START_REF] Godet | Developmental changes in cyclin B1 and cyclin-dependent kinase 1 (CDK1) levels in the different populations of spermatogenic cells of the post-natal rat testis[END_REF][START_REF] Perrard | Cytostatic factor proteins are present in male meiotic cells and β-nerve growth factor increases Mos levels in rat late spermatocytes[END_REF].

RT-qPCR

Total RNA was extracted from cultured rat seminiferous tubules cells using TRI-REAGENT (Euromedex, Souffelweyersheim, France) according to the manufacturer's protocol. All samples were treated by DNase I before the reverse transcription; first strand cDNA was synthesized from 2 μg RNA with Super Script III in the presence of oligodT and Random Primer (all from Thermo Fisher Scientific, Villebon sur Yvette, France). Realtime PCR was performed in a Step One Plus instrument (Applied biosystems) by using SYBR-Green (Qiagen, Courtaboeuf, France). Primers sequences are presented in Table 1.

Mitochondrial ribosomal proteins S18 and S16 mRNAs were used as housekeeping gene controls.

Statistical analyses

Statistical analyses were performed using Graphpad and R softwares. Data are presented as the means ± S.E.M. Data were analysed by paired Student's t-test to compare treated cells with their corresponding controls. When required, values were logarithmically transformed to eliminate heterogeneity of variance and achieve a reasonable assumption of normal distribution [START_REF] Bland | Statistics Notes: The use of transformation when comparing two means[END_REF] P<0.05 was considered significant.

Results

Effect of glyphosate on the TEER

Seminiferous tubules segments were cultured for 23 days in the absence or presence of 50 nM, 500 nM, 5 µM or 50 µM of glyphosate. Under control conditions, the TEER increased from 12 to 95 ohm.cm 2 between day 2 and day 12 of culture and slightly decreased down to 59 ohm.cm 2 on day 23 (Fig. 1). This high TEER should be associated with the presence of tight junctions between the Sertoli cells, the main component of the blood-testis barrier [START_REF] Carette | Endocrine Disrupting Effects of Noncytotoxic Doses of Carbendazim on the Pubertal Rat Seminiferous Epithelium: An Ex Vivo Study[END_REF][START_REF] Su | Germ cells contribute to the function of the Sertoli cell barrier[END_REF]. The TEER of seminiferous tubules culture was not modified at any concentration of glyphosate.

For further studies only the lower concentrations (50 nM, 500 nM and 5 µM) of glyphosate were chosen.

Effect of glyphosate on cell viability and on the numbers of Sertoli cells and of germ cells

Figure 2 shows the changes in the numbers of total cells, Sertoli cells, and of germ cells in 4 different control cultures. As expected [START_REF] Staub | The whole meiotic process can occur in vitro in untransformed rat spermatogenic cells[END_REF][START_REF] Perrard | Development of the meiotic step in testes of pubertal rats: Comparison between the in vivo situation and under in vitro conditions[END_REF] , the number of each type of tubular cells varied according to the number of seeded total cells (Fig. 2) as may vary also the yield of each step of the spermatogenic differentiation, as it does in vivo.

Hence, in every experiment, this was the same population of tubular cells which was exposed (treated cells) or not (control cells) to glyphosate (paired samples). Therefore, in the following figures, the results were expressed as a percent of the values obtained with the corresponding control cells [START_REF] Carette | Endocrine Disrupting Effects of Noncytotoxic Doses of Carbendazim on the Pubertal Rat Seminiferous Epithelium: An Ex Vivo Study[END_REF]. The proportion of viable cells ranged between 91% and 99% of total cells during the whole culture period and was not affected by 50 nM, 500 nM or 5 µM of glyphosate, at any day of culture (data not shown). Neither the numbers of total cells nor the numbers of Sertoli cells were modified by glyphosate at any day of culture (Fig. 3A, 3B). The numbers of total germ cells were decreased by glyphosate at 50 nM or 500 nM on day 14 (p ≤ 0.05 to p ≤ 0.01) and at 50 nM or 5 µM on day 21 (p ≤ 0.05). The numbers of spermatogonia were not modified but decreased for the lower concentration at the end of the culture (p ≤ 0.05). The numbers of young spermatocytes were decreased only at the end of the culture by the two higher concentrations (p ≤ 0.05), while the numbers of middle to late pachytene spermatocytes were decreased (or tended to be decreased) by glyphosate at 500 nM (p ≤ 0.05) or 5 µM (p = 0.06) on day 14 and by the 3 concentrations on day 21 (p ≤ 0.05, p = 0.08 and p ≤ 0.01 for 50nM, 500 nM and 5 µM respectively). The numbers of secondary spermatocytes were decreased (or tended to be decreased) by the 3 concentrations on day 14 (p ≤ 0.05, p = 0.1 and p = 0.06 for 50nM, 500 nM and 5 µM respectively and only by the higher concentration at the end of the culture (p ≤ 0.05). The numbers of round spermatids were decreased (or tended to be decreased) by the 3 concentrations all along the cell culture (p = 0.06 to p ≤ 0.01 on day 7, p = 0.1 to p ≤ 0.05 on day 14 and p ≤ 0.05 to p ≤ 0.01 on day 21 (Fig. 3B).

Effect of glyphosate on the relative levels of some "specific" mRNAs

In order to complete the flow cytometric analyses, some mRNA levels were measured in seminiferous tubules cultured for 7, 14, or 21 days in the absence or presence of glyphosate.

For CHK2, VASA, GFRα1 and α6 Integrin which are specific of spermatogonia [START_REF] Bartkova | Chk2 tumour suppressor protein in human spermatogenesis and testicular germ-cell tumours[END_REF][START_REF] Phillips | Pesticide leaching through sandy and loamy fields -Long-term lessons learnt from the Danish pesticide leaching assessment programme[END_REF], for TH2B and STMN1 which are more specific of pachytene spermatocytes and for TP1, TP2 and PRM3 which are specifically expressed by round spermatids [START_REF] Marret | Localization and quantitative expression of mRNAS encoding the testis-specific histone TH2B, the phosphoprotein p19, the transition proteins 1 and 2 during pubertal development and throughout the spermatogenic cycle of the rat[END_REF][START_REF] Schluter | The rat Prm3 gene is an intronless member of the protamine gene cluster and is expressed in haploid male germ cells[END_REF], the numbers of transcripts were expressed by % of spermatogonia, % of 4C germ cells and % of round spermatids respectively [START_REF] Durand | Effects of a mixture of low doses of atrazine and benzo[a]pyrene on the rat seminiferous epithelium either during or after the establishment of the blood-testis barrier in the rat seminiferous tubule culture model[END_REF]. Indeed, the relative cell composition of the cultures changes according to the day of sampling, as it does in vivo during development [START_REF] Almstrup | Analysis of Cell-Type-Specific Gene Expression During Mouse Spermatogenesis1[END_REF]. Hence, the measured levels of a specific mRNA depend on both the amount of mRNA produced by the specific cells and the proportion of these cells in the sample. Taking into account these two parameters provide more significant data (Plotton et al., 2006(Plotton et al., , 2005)).

CHK2, VASA, α6 Integrin and GFRα1 mRNA levels were not modified by glyphosate treatment at any day (data not shown). But, as shown in Fig. 4, STMN1 mRNAs were increased on day 7 and on day 21 by glyphosate at 5 µM (p ≤ 0.01), whereas TH2B mRNAs were not modified by glyphosate treatment at any day. TP1 mRNAs were increased on days 7 and 14 by the higher concentration (p ≤ 0.05 to p ≤ 0.01), and on day 21 at 500 nM p ≤ 0.05).

TP2 mRNAs were increased by glyphosate at 5 µM on days 7 and 14 (p ≤ 0.05) whereas PRM3 mRNAs were increased at this concentration only on day 7 (p ≤ 0.05) (Fig. 5). In order to determine if the effect of glyphosate on the mRNAs levels of TP1, TP2 or PRM3 mRNAs was due to an androgen-like effect, as we found for carbendazim [START_REF] Carette | Endocrine Disrupting Effects of Noncytotoxic Doses of Carbendazim on the Pubertal Rat Seminiferous Epithelium: An Ex Vivo Study[END_REF], seminiferous tubules were cultured without or with glyphosate at 5 µM in the absence of testosterone in the culture medium for 7 days, since the maximum effect of glyphosate 5 μM in the presence of testosterone was observed on Day 7. In the absence of testosterone, the mRNAs levels of TP1, TP2 or PRM3 were not modified by glyphosate (data not shown). To determine if glyphosate alters Sertoli cell function, mRNA levels of Clusterin (Plotton et al., 2005) and Vimentin [START_REF] Franke | Intermediate-sized filaments present in Sertoli cells are of the vimentin type[END_REF][START_REF] Kopecky | Vimentin expression during altered spermatogenesis in rats[END_REF], specific of Sertoli cells, were measured. For these two genes the numbers of transcripts were expressed by % of sertoli cells. As shown in Fig. 6, the vimentin mRNA levels were never modified by glyphosate treatment at any day, while Clusterin mRNA levels were decreased (or tended to be decreased) by the 3 concentrations at the end of the culture (p ≤ 0.01 for 50 nM and 500 nM and p = 0.1 for 5 µM).

In order to complete the study of the blood-testis barrier, mRNAs levels of Connexin 43, Occludin, TJP1, Claudin 11 and Claudin 4 were measured in the presence or absence of glyphosate. The transcripts of these five genes were not modified by glyphosate treatments at any day of culture (data not shown).

Because spermatogenesis is an androgen-and estrogen-regulated process the levels of AR, ERα, ERβ and aromatase mRNAs were further determined (Fig. 7). For AR the numbers of transcripts were expressed by % of Sertoli cells. Because ERα, ERβ and aromatase are expressed by both Sertoli cells and germ cells their mRNA transcripts were not corrected by any cell population. ERα mRNA levels were decreased only by 500 nM glyphosate at the end of the culture (p ≤ 0.01), and aromatase mRNAs were decreased only by 50 nM glyphosate on day 14. AR and ERβ mRNA levels were never modified by glyphosate treatment.

Discussion

The aim of our study was to investigate the possible impact of low concentrations of glyphosate alone on pubertal rat spermatogenesis by using our validated rat seminiferous tubule culture model, BioAlter®. It is important to note that our model decreases the interanimal variations existing between the rats, in in vivo studies, since in every experiment, this is the same population of tubular cells which was exposed (treated cells) or not (control cells)

to glyphosate (paired samples). For the first time, our study has shown that glyphosate alone has a detrimental effect on the meiotic step.

The absence of an effect of glyphosate on the TEER is in agreement with the in vitro study of Gorga et al. which did not find any alteration of the TEER, in cultured 20-day-old rat Sertoli cells, after a short (5 days) treatment by glyphosate at 10 ppm ie 10 µg/ml (58.8 µM), which is very close to our highest tested concentration for TEER, 8.45 µg/ml (50 µM) [START_REF] Gorga | In vitro effects of glyphosate and Roundup on Sertoli cell physiology[END_REF]. But, it is important to underline that our culture system, BioAlter®, is closer to the in vivo situation than that of Gorga et al, since we culture Sertoli cells together with germ cells. In addition, BioAlter® allows studying the chronic effect of a toxicant over a 3 week culture period on Sertoli cells and on the germ cell types (from spermatogonia to round spermatids).

Under control condition, the decrease of the number of germ cells from day 14 onward was mainly due to the decrease of the numbers of young spermatocytes and of middle to late pachytene spermatocytes. This fact would mean that the replenishment of the 4C cell pool became less efficient after several days of culture. Indeed, seminiferous tubules were cultured in a chemically defined medium containing testosterone which is necessary for the meiotic step [START_REF] Smith | The regulation of spermatogenesis by androgens[END_REF], but which leads to the inhibition of spermatogonial differentiation [START_REF] Meistrich | Inhibition of spermatogonial differentiation by testosterone[END_REF] inducing the depletion of the pool of 4C cells. In addition, the decrease of the 4C cell pool could also be due to their apoptosis. Indeed, the proportions of apoptotic 2C and 4C cell in BioAlter® were rather close to those observed during puberty of the rat [START_REF] Perrard | Development of the meiotic step in testes of pubertal rats: Comparison between the in vivo situation and under in vitro conditions[END_REF]. Nevertheless, as explained above, this is the same population of tubular cells which was exposed or not to glyphosate.

The decrease of the numbers of total germ cells by the 3 concentrations of glyphosate from day 14 onward shows its chronic effect, and is due to the disruption of the meiotic step leading to a decrease of the numbers of young spermatocytes, middle to late pachytene spermatocytes and secondary spermatocytes resulting in the decrease of round spermatids. In addition, it might be noted that the numbers of round spermatids were already decreased from day 7 onward (i.e as soon as they appeared) [START_REF] Staub | The whole meiotic process can occur in vitro in untransformed rat spermatogenic cells[END_REF], which could be due to a blockage of the two meiotic division. To our knowledge, no in vitro or in vivo study exists on the effects of glyphosate alone on the meiotic step. Nevertheless, two previous studies have shown that the two herbicides, Roundup and Pondmaster, which contain glyphosate as the active chemical, target spermatocytes: (i) Kale et al have shown that Roundup® and Pondmaster induce a very high frequency of lethals in larval spermatocytes by using the Drosophila sexlinked recessive lethal mutation assay [START_REF] Kale | Mutagenicity testing of nine herbicides and pesticides currently used in agriculture[END_REF]; (ii) the study of Jiang et al. has shown that an excessive apoptosis of Kunming mice male germ cells, mainly spermatocytes and spermatids, occurs after Roundup® exposure [START_REF] Jiang | A commercial Roundup® formulation induced male germ cell apoptosis by promoting the expression of XAF1 in adult mice[END_REF].

The decrease of the round spermatid numbers we observed is in agreement with two previous in vivo studies: (i) mice with glyphosate at 0.5 and 5 mg/kg/day [START_REF] Pham | Perinatal exposure to glyphosate and a glyphosate-based herbicide affect spermatogenesis in mice[END_REF]. By contrast, two other in vivo studies showed either no effect on testes in adult rats exposed to glyphosate at levels up to 25 mg/kg bw/day [START_REF] Johansson | Exposure to a glyphosate-based herbicide formulation, but not glyphosate alone, has only minor effects on adult rat testis[END_REF], or a decrease of the total sperm production only at the dose of 500 mg/kg/day [START_REF] Dai | Effect of glyphosate on reproductive organs in male rat[END_REF]. It is important to note that these last two teams used sexually mature rats while Nardi et al., like us, worked with rats at the critical age of puberty [START_REF] Nardi | Prepubertal subchronic exposure to soy milk and glyphosate leads to endocrine disruption[END_REF].

Previous in vivo studies have shown that glyphosate at 5 mg/kg/day decreases the numbers of undifferentiated spermatogonia [START_REF] Pham | Perinatal exposure to glyphosate and a glyphosate-based herbicide affect spermatogenesis in mice[END_REF], and that Roundup and Pondmaster induce a very high frequency of lethals in larval spermatocytes (see above) and in spermatogonia [START_REF] Kale | Mutagenicity testing of nine herbicides and pesticides currently used in agriculture[END_REF]. In our study, no consistent effect of glyphosate was observed on the number of spermatogonia, or on CHK2, VASA, α6 Integrin and GFRα1 mRNA levels; this might be due to the lower concentrations we used. No correspondence between the diets of the rats and a serum level of glyphosate was indicated in the above cited in vivo studies, which leads to an impossibility in the comparison of our results with those of in vivo studies.

The young spermatocyte population is composed of leptotene, zygotene spermatocytes and pachytene spermatocytes from stages XIV to IV whereas middle to late pachytene spermatocytes are from stages V to XIII of spermatogenesis [START_REF] Godet | Developmental changes in cyclin B1 and cyclin-dependent kinase 1 (CDK1) levels in the different populations of spermatogenic cells of the post-natal rat testis[END_REF][START_REF] Perrard | Development of the meiotic step in testes of pubertal rats: Comparison between the in vivo situation and under in vitro conditions[END_REF]. TH2B mRNAs are expressed by pachytene spermatocytes from stages III-XIV onward with higher levels in stages VIII to X whereas STMN1 mRNA levels are roughly similar between stages III to X [START_REF] Marret | Localization and quantitative expression of mRNAS encoding the testis-specific histone TH2B, the phosphoprotein p19, the transition proteins 1 and 2 during pubertal development and throughout the spermatogenic cycle of the rat[END_REF]. The fact that STMN1 mRNAs were increased on days 7 and 21 by glyphosate at 5 µM while the numbers of young spermatocytes were not modified or decreased on days 7 and 21 respectively, might suggest an effect of glyphosate on the regulation of STMN1 mRNAs. Likewise, glyphosate could regulate TH2B mRNAs which

were not modified at the end of culture despite the numbers of young spermatocytes and of middle to late pachytene spermatocytes were decreased.

Despite the decrease in the numbers of round spermatids by glyphosate, TP1, TP2 and PRM3 mRNA levels were sometimes increased by glyphosate at 5 µM or 500 nM. As, we have previously shown that testosterone increases the amount of TP1 mRNAs transcribed by round spermatids in vitro [START_REF] Vigier | The effects of FSH and of testosterone on the completion of meiosis and the very early steps of spermiogenesis of the rat: An in vitro study[END_REF], the hypothesis of an androgen like effect of glyphosate could have been suggested. But, no effect of glyphosate was found on the mRNA levels of TP1, TP2 or PRM3 in the absence of testosterone in the medium. Nevertheless, the increase of TP1, TP2 or PRM3 mRNA levels by glyphosate 500 nM or 5 µM in the presence of testosterone might suggest an effect of glyphosate on the regulation of those mRNAs.

Moreover, no modification in the levels of AR or Erα or Erβ mRNAs was observed.

Glyphosate at 50 nM or 500 nM induced a decrease of Clusterin mRNAs which is a good marker of the amount of RNA of Sertoli cell origin in rat testis samples at day 20 or later (Plotton et al., 2005). This result might suggest that Glyphosate induced some alteration of the function of Sertoli cells. Two previous in vitro studies reported a harmful effect of glyphosate on Sertoli cells: (i) a deleterious impact of glyphosate at 3.6 µg/ml (21.3 µM) (i.e 42.6 to 426 fold higher than our tested concentrations) was shown on the immature mouse Sertoli cell line (TM4) [START_REF] Vanlaeys | Formulants of glyphosatebased herbicides have more deleterious impact than glyphosate on TM4 Sertoli cells[END_REF]; (ii) Glyphosate at concentrations ranging from some human urine and environment to agricultural levels [1 ppm ie 1 µg/ml (5.92 µM), similar to our 5 µM concentration, to 10000 ppm ie 10000 µg/ml (59.2 mM), 11840 fold higher than our higher tested concentration] was essentially toxic on cultured mature rat Sertoli cells within 24-48 hours [START_REF] Clair | A glyphosate-based herbicide induces necrosis and apoptosis in mature rat testicular cells in vitro, and testosterone decrease at lower levels[END_REF]. Since Sertoli cells are essential for spermatogenesis, some alteration of their function by glyphosate could contribute to the disruption of the meiotic stage that we observed (see above).

The absence of modification of the mRNAs levels of Connexin 43 (the predominant testicular gap junction protein) (Pointis and Segretain, 2005), and Occludin, TJP1, Claudin 11 and Claudin4 (four tight junction proteins) [START_REF] Mruk | The mammalian blood-testis barrier: Its biology and regulation[END_REF] is in line with the absence of effect of glyphosate on the TEER. These last results suggest that the main target of glyphosate would be the function of Sertoli cell and not the Sertoli cell junctions at the bloodtestis barrier level.

Conclusion:

The present study clearly highlights the chronic effects of low concentrations of glyphosate on prepubertal rat seminiferous tubules. These low concentrations of glyphosate target (i) some function of the Sertoli cells, as observed with the decrease in clusterin mRNAs, and (ii) the meiotic step resulting in a decrease of the number of round spermatids, the direct precursors of spermatozoa. This study underlines that the effect of a toxicant should be also studied at low doses and during the establishment of the blood-testis barrier. Results are means ± SEM of 6 wells per day for each condition.

Results are representative of two different experiments. For AR the numbers of transcripts were expressed by % of Sertoli cells. Given that oestrogen receptors ERα and ERβ and Aromatase are expressed by both Sertoli and germ cells, their mRNA transcripts were not corrected by any cell population.

Results are expressed as a percentage of values obtained from cells cultured under control conditions. Each value is the mean ± SEM of 4 cultures. * p<0.05; ** p < 0.01 vs control.

Figure 1 :

 1 Figure 1:

Figure 2 :

 2 Figure 2:

Figure 3 :

 3 Figure 3:

Figure 4 :Figure 4 :

 44 Figure 4:

  Figure 5: Figure 5: Changes in the levels of TP1, TP2 or PRM3 mRNA-related qPCR products in seminiferous tubule segments cultured in the absence (control) or presence of 50 nM or 500 nM or 5 µM glyphosate. The numbers of transcripts were expressed by % of 1C cells in the cultures. Results are expressed as a percentage of values obtained from cells cultured under control conditions. Each value is the mean ± SEM of 4 cultures. * p<0.05; ** p < 0.01 vs control.

Figure 6 :Figure 6 :

 66 Figure 6:

  Figure 7: Figure 7: Changes in the levels of AR, Erα, Erβ or Aromatase mRNA-related qPCR products in seminiferous tubule segments cultured in the absence (control) or presence of 50 nM or 500 nM or 5 µM glyphosate.

Table 1 .

 1 Sequence of the primers used for RTqPCR

	365, 1663-1678.
	Pisani, C., Voisin, S., Arafah, K., Durand, P., Perrard, M.H., Guichaoua, M.R., Bulet, P., Prat,
	O., 2016. Ex vivo assessment of testicular toxicity induced by carbendazim and
	iprodione,	alone	or	in	a	mixture.	Altex	33,	393-413.
	https://doi.org/10.14573/altex.1601253
	Plotton, I., Sanchez, P., Durand, P., Lejeune, H., 2006. Decrease of both stem cell factor and
	clusterin mRNA levels in testicular biopsies of azoospermic patients with constitutive or
	idiopathic but not acquired spermatogenic failure. Human Reproduction 21, 2340-2345.
	Plotton, I., Sanchez, P., Perrard, M.H., Durand, P., Lejeune, H., 2005. Quantification of stem
	cell factor mRNA levels in the rat testis: Usefulness of clusterin mRNA as a marker of
	the amount of mRNA of sertoli cell origin in post pubertal rats. Journal of Endocrinology
	186, 131-143.
	Poiger, T., Buerge, I.J., Bächli, A., Müller, M.D., Balmer, M.E., 2017. Occurrence of the
	herbicide glyphosate and its metabolite AMPA in surface waters in Switzerland
	determined with on-line solid phase extraction LC-MS/MS. Environmental Science and
	Pollution Research 24, 1588-1596. https://doi.org/10.1007/s11356-016-7835-2
	Pointis, G., Segretain, D., 2005. Role of connexin-based gap junction channels in testis.
	Trends	in	Endocrinology	and	Metabolism	16,	300-306.
	https://doi.org/10.1016/j.tem.2005.07.001
	Roberts, D.M., Buckley, N.A., Mohamed, F., Eddleston, M., Goldstein, D.A., Mehrsheikh,
	A., Bleeke, M.S., Dawson, A.H., 2010. A prospective observational study of the clinical
	toxicology of glyphosate-containing herbicides in adults with acute self-poisoning.

Clinical Toxicology 48, 129-136. https://doi.org/10.3109/15563650903476491

Acknowledgements

We thank Clothilde Egraz and Virginie Nakad for her technical help in the TEER experiments. Ovine FSH-20 was obtained through the National Hormone and Peptide Programme, the National Institute of Diabetes and Digestive and Kidney Diseases (Dr AF Parlow).

This work was supported by Kallistem SAS. https://doi.org/10.5772/13515 Yousef, M.I., Ibrahim, H.Z., Helmi, S., Salem, M.H., Seehy, M.A., Bertheussen, K., 1995. Toxic effects of carbofuran and glyphosate on semen characteristics in rabbits. Journal of Environmental Science and Health, Part B 30, 513-534. https://doi.org/10.1080/03601239509372951