Lorraine Michelet
email: lorraine.michelet@anses.fr

Céline Richomme
email: celine.richomme@anses.fr

Edouard Réveillaud
email: edouard.reveillaud@agriculture.gouv.fr

Krystel De Cruz
email: krystel.decruz@anses.fr

Jean-Louis Moyen
email: jl.moyen@dordogne.fr

Maria Laura Boschiroli

Céline Richom

Maria Laura Boschiroli
email: maria-laura.boschiroli@anses.fr

Mycobacterium microti Infection in Red Foxes in France

Keywords: red fox, bovine tuberculosis, Mycobacterium microti, cross-sectional study

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

Introduction

Mycobacterium microti, member of the Mycobacterium tuberculosis complex (MTBC), was originally described as the agent of tuberculosis (TB) in wild rodents [START_REF] Cavanagh | Mycobacterium microti infection (vole tuberculosis) in wild rodent populations[END_REF]. However, in the last years, an increasing number of cases were described in pets (cat and dog), zoo animals, camelids and, also, in humans [START_REF] Michelet | Infection with Mycobacterium microti in animals in France[END_REF][START_REF] Panteix | Pulmonary tuberculosis due to Mycobacterium microti: A study of six recent cases in France[END_REF][START_REF] Smith | Mycobacterium microti: More diverse than previously thought[END_REF][START_REF] Peterhans | Mycobacterium microti: Not Just a Coincidental Pathogen for Cats[END_REF]. In France, Mycobacterium microti interferes with the diagnosis of bovine TB (bTB) and has been identified through the surveillance system in livestock (cattle and goats) [START_REF] Michelet | Mycobacterium microti infection in a cow in France[END_REF][START_REF] Michelet | Mycobacterium microti Infection in Dairy Goats, France[END_REF][START_REF] Michelet | Mycobacterium microti Interferes with Bovine Tuberculosis Surveillance[END_REF] and in wild animals (wild boars and badgers) [START_REF] Michelet | Infection with Mycobacterium microti in animals in France[END_REF][START_REF] Michelet | Mycobacterium microti detection in French wildlife[END_REF]. The presence of this pathogen was also described in wildlife in other European countries [START_REF] Boniotti | Detection and molecular characterization of Mycobacterium microti in wild boar from northern Italy[END_REF][START_REF] Dondo | Mycobacteriosis in wild boar: Results of 2000-2006 activity in north-western Italy[END_REF][START_REF] Perez De Val | Mycobacterium microti Infection in Free-Ranging Wild Boar, Spain, 2017-2019[END_REF][START_REF] Schoning | Surveillance of bovine tuberculosis and risk estimation of a future reservoir formation in wildlife in Switzerland and Liechtenstein[END_REF]. Some authors hypothesize that M. microti infection might provide some protection to cattle against M. bovis infection (M. bovis belonging to the same MTBC), especially in regions where M. microti is endemic in badgers and cats [START_REF] Smith | Mycobacterium microti: More diverse than previously thought[END_REF][START_REF] Gunn-Moore | Mycobacterial disease in cats in Great Britain: I. Culture results, geographical distribution and clinical presentation of 339 cases[END_REF] and could therefore explain the low incidence of bTB in these areas. However, at least at a population level, this protective immunity does not seem to occur in regions where M. microti and M. bovis infection overlap in some areas in France [START_REF] Michelet | Mycobacterium microti Interferes with Bovine Tuberculosis Surveillance[END_REF]. Increasing data generated in the recent years-mostly by molecular diagnosis-suggest that the presence of this hard-cultivable pathogen has been underestimated and that the epidemiology of this pathogen, which role as zoonotic agent remains an open question, deserves to be explored.

Bovine TB, mainly due to M. bovis, is now considered as a multi-host disease that in Europe affects not only cattle but also wildlife species, such as wild boars (Sus scrofa), badgers (Meles meles) or wild deer (Cervus elaphus, Capreolus capreolus). Infection by M. bovis has been reported in red foxes (Vulpes vulpes) and has been recently identified in four French bTB endemic areas showing prevalence rates similar to those found in badgers in the same areas, raising the question of the role of foxes in the bTB multihost system [START_REF] Richomme | Mycobacterium bovis infection in Red foxes in four animal tuberculosis endemic areas in France[END_REF]. During this same study, M. microti was also identified in foxes. We present here prevalence values and M. microti infection description in this animal species and compare it to that caused by M. bovis in these four areas. This is the first report of M. microti infection in red foxes.

Materials and Methods

Ethics Statement

All fox carcasses used in the present study were provided by hunters who held the appropriate permits for hunting foxes under the supervision of local hunting federations, or trappers duly trained and authorized, supervised by pest control officers (historically called wolf-hunter, nowadays being state but volunteer officers in charge of pest control and who supervised trapping) in agreement with national regulations. As the study did not involve invasive procedures on live animals, no ethical approval was necessary.

Study Areas, Fox Sampling and Sample Collection

The study concerns four areas of about 500 km 2 each in the core of bTB infected areas of Dordogne, Charente and Landes departments, in the South West of France (Nouvelle-Aquitaine region) and of Côte-d'Or department in Eastern France (Bourgogne-Franche-Comté region) [START_REF] Richomme | Mycobacterium bovis infection in Red foxes in four animal tuberculosis endemic areas in France[END_REF]. The studied population was the same than in our previous work [START_REF] Richomme | Mycobacterium bovis infection in Red foxes in four animal tuberculosis endemic areas in France[END_REF]. Sampling took place from March 2017 to January 2020. In Dordogne, foxes were collected in the whole study area until August 2018 (season #1) and afterwards (season #2) animals from only two bTB hotspots-communes (administrative units) were collected [START_REF] Richomme | Mycobacterium bovis infection in Red foxes in four animal tuberculosis endemic areas in France[END_REF]. The expected sample size was determined to enable detection of infection in foxes in each of the four areas, assuming an apparent prevalence of at least 3%, with a 95% confidence interval [START_REF] Richomme | Mycobacterium bovis infection in Red foxes in four animal tuberculosis endemic areas in France[END_REF]. Foxes were sent in double packaging to collect points and then to the local laboratories in charge of the necropsies. The following samples were collected at the lab: retropharyngeal (RP) lymph nodes (LN), respiratory LN (composed of tracheobronchial and mediastinal LN), mesenteric LN and any lesion suggestive of tuberculosis.

Detection of M. microti Infection

TB infection was determined by molecular diagnosis performed in (1) a pool of RP LN and respiratory LN, and (2) in the mesenteric LN on animals from the four regions. DNA extraction was performed after mechanical lysis using an LSI MagVetTM Universal Isolation Kit (Life Technologies, Villebon-sur-Yvette, France) with a KingFisherTM Flex automate (Thermo Electron LED S.A.S., Saint-Herblain, France), following the manufacturer's instructions. For confirming Mycobacterium tuberculosis complex (MTBC) the LSI VetMAXTM MTBC Real-Time PCR kit (Life Technologies, Villebon-sur-Yvette, France), which targets IS6110 and a PCR based on IS1081 were employed [START_REF] Lesellier | Detection of live M. bovis BCG in tissues and IFN-gamma responses in European badgers (Meles meles) vaccinated by oropharyngeal instillation or directly in the ileum[END_REF]. IS1561 and Rv1510 (RD4) based PCRs were used to differentiate M. microti from M. bovis infections [START_REF] Lesellier | Detection of live M. bovis BCG in tissues and IFN-gamma responses in European badgers (Meles meles) vaccinated by oropharyngeal instillation or directly in the ileum[END_REF]. Spoligotyping by Luminex, as described by Zhang et al. [START_REF] Zhang | Mycobacterium tuberculosis complex CRISPR genotyping: Improving efficiency, throughput and discriminative power of 'spoligotyping' with new spacers and a microbead-based hybridization assay[END_REF], using TB-SPOL kits purchased from Beamedex ® (Beamedex SAS, Orsay, France) on Bio-PLex 200/Luminex 200 ® was also employed on PCR positive samples for intra-MTBC differentiation and genotyping. The presence or absence of the 43 spacer sequences contained in the DR locus is represented in a binary code of 43 entries. Spoligotypes are named according to an agreed international convention (www.mbovis.org, accessed on 22 October 2020) [START_REF] Smith | Naming spoligotype patterns for the RD9-deleted lineage of the Mycobacterium tuberculosis complex; www[END_REF]. Results were interpreted following the manufacturer's recommendations and by comparison with negative and positive controls.

M. microti infection was confirmed when a positive PCR with Rv1510 target and/or a specific M. microti spoligotype profile was obtained. With this strategy, and if present, M. microti and M. bovis coinfection can be identified by IS1561 and Rv1510 concomitant positive PCRs.

Bacterial culture was also performed on the tissues as described previously [START_REF] Richomme | Mycobacterium bovis infection in Red foxes in four animal tuberculosis endemic areas in France[END_REF], however they were all negative for M. microti.

Data Analysis

For the present study, an infected fox was defined as an animal with an analytical result demonstrating M. microti infection by molecular diagnosis. Apparent prevalence in each of the 4 areas and 95% confidence intervals (CI 95% ) were calculated using exact binomial tests. For foxes from Nouvelle-Aquitaine region, the exact Fisher test was used to attest or not of a statistically significant difference of the prevalence in each of the three areas. In each area, M. microti prevalence of the present study were compared to M. bovis prevalence found in the same samples [START_REF] Richomme | Mycobacterium bovis infection in Red foxes in four animal tuberculosis endemic areas in France[END_REF] using the exact Fisher test.

Results

In Côte-d'Or, the apparent prevalence is 0.7% (CI 95% : 0.1-3.8%), with one fox found infected by M. microti (n = 1/146) (Table 1). No infected foxes were identified in Charente (n = 0/98), indicating a prevalence lower than 3.7% (upper limit of the 95% confidence interval). In Dordogne (season #1) and Landes, n = 12/184 and n = 3/140 foxes were found infected, with prevalence values of 6.5% (3.4-11.1%) and 2.1% (0.4-6.11%), respectively. Within Nouvelle-Aquitaine, prevalence in Dordogne is significantly higher than in Charente (p-value = 0.0101) and higher than in Landes (with no significant difference, p-value = 0.1073). The rate of infection by M. microti was statistically lower than the rate of M. bovis infection in Charente (p = 0.0035) but not in Dordogne, Landes or Côte-d'Or (Figure 1). During season #2 in Dordogne, all M. microti infected foxes came from hotspot 1 (Table 2). The rate of infection of foxes infected by M. microti was statistically lower than the rate of infected fox by M. bovis in hot spot 2 (Fisher exact test, p-value < 0.05) whereas no statistically significant difference was observed in hotspot 1. During season #1, four M. microti infected foxes came from hotspot 1 and one from hotspot 2.

In Dordogne still, M. microti cases were located in five communes in the northeast of the studied area (Figure 2). Concomitant infection by M. microti and by M. bovis was observed in two communes. One of these two communes is the bTB hotspot 1, which includes 4 and 12 M. microti cases identified in season #1 and season #2, respectively. In Landes, M. microti cases were located in three distant communes (Figure 2). In one of them, one M. bovis infected fox was also identified.

None of the sampled foxes were identified as co-infected by M. microti and M. bovis.

In each area, the genotype of M. microti that infected foxes was the same as that identified in wildlife species of the same region: SB0118 in Côte-d'Or and Landes, and SB2273 in Dordogne [START_REF] Michelet | Infection with Mycobacterium microti in animals in France[END_REF].

None of the infected foxes presented gross TB-like lesions. Among the 28 M. microti infected foxes during the whole study, 27 (96%) exhibited infection in the mesenteric LN (Table S1), suggesting that the infection was acquired by ingestion of infected food. Fox 180814038269 was positive for the MTBC (IS6110 and IS1081 positive PCRs), but intra-MTBC differentiation by PCR (IS1561 and RD4) or spoligotyping was impossible due to a weak DNA concentration. For eight foxes, M. microti infection was disclosed in the pool of RP and respiratory LN. Seven of these foxes exhibited detectable M. microti infection in the mesenteric LN although for one of them (fox 180814038269) only MTBC was confirmed in this type of LN. A ninth fox (fox 170831039977) was MTBC positive in the pool of RP and respiratory LN and was positive for M. microti in the mesenteric LN.

With regards excretion, appropriate samples (swab, urine or kidney and feces) were collected during season #2 in Dordogne for 11 M. microti infected foxes. None of these samples was positive for M. microti or any other MTBC member.

Discussion

With the present study, we demonstrated that infection by M. microti is prevalent in foxes in areas where bTB is endemic in cattle and where foxes are also found infected by M. bovis even at high prevalence [START_REF] Richomme | Mycobacterium bovis infection in Red foxes in four animal tuberculosis endemic areas in France[END_REF]. To our knowledge, this is the first work that describes M. microti infection in red foxes. However, the absence of previous descriptions does not seem surprising. From the one hand, the recent use of molecular diagnostic tools was able to disclose the wide presence and animal host spectrum of M. microti. Indeed, many historical articles describing M. microti only used bacteriology, which in the case of this difficult grower has a particularly low sensitivity. On the other hand, and this also stands for M. bovis, infection by MTBC does not seem to provoke visible lesions in foxes thus considered negligible actors in bTB epidemiology, and then understudied. The important number of M. microti infected foxes found in this study strengthens previous findings showing that M. microti is widely spread in the nature [START_REF] Kipar | Mycobacterium microti Tuberculosis in Its Maintenance Host, the Field Vole (Microtus agrestis): Characterization of the Disease and Possible Routes of Transmission[END_REF][START_REF] Wells | The murine type of tubercle bacillus (the vole acid-fast bacillus)[END_REF].

Differences in prevalence rates exist between the four studied areas. None or very few M. microti cases were identified in Charente, Côte d'Or and Landes, while most of the infected animals were found in Dordogne. For Charente, the absence of cases could be explained by a sampling bias, as the number of sampled animals was lower than in the rest of the studied areas. These results in foxes correlate with those previously found in other wildlife species. In the framework of the TB surveillance program in wildlife in France (Sylvatub) [START_REF] Reveillaud | Infection of Wildlife by Mycobacterium bovis in France Assessment through a National Surveillance System[END_REF], M. microti cases were identified in other species in the four areas of this study between 2011 and 2020. In wild boars, one case was found in Charente, another one in Landes and two cases were found in Côte-d'Or. In Dordogne, a larger number of M. microti cases were identified: 11 in wild boars and 4 in badgers (Bovine Tuberculosis National Reference Laboratory data).

Foxes have a very large feeding tropism for small rodents, and they have proved to be susceptible to M. bovis-thus to MTBC-infection-, therefore, it is not surprising to find them harboring M. microti as well. Another means that foxes become infected could be via ingestion of M. microti-bearing earthworms (Lumbricus terrestris). Indeed, as has been suggested for M. bovis, earthworms could act as vectors of mycobacterium present in contaminated environments [START_REF] Barbier | Rapid dissemination of Mycobacterium bovis from cattle dung to soil by the earthworm Lumbricus terrestris[END_REF], red foxes being great consumers of these annelids [START_REF] Macdonald | The Red fox, Vulpes vulpes, as a predator upon earthworms. Lumbricus terrestris[END_REF][START_REF] Macdonald | Encounters btween two sympatric carnivores: Red foxes (Vulpes vulpes) and European badgers (Meles meles)[END_REF]-as are badgers or wild boars. These types of environments could exist if they were contaminated by highly infected voles. Besides, ingestion of wild boar M. microtiinfected carcasses cannot be also excluded as a means of foxes' infection.

The highest prevalence of M. microti infection in foxes was observed in Dordogne. The landscape in the four studied areas is roughly similar, composed of a mixture of pastures and woods. However, we cannot exclude that one reason for explaining the variation in prevalence rates could be the density of rodents, or even earthworms, as mentioned above, which could be associated to local characteristics of the landscape or the period of sampling (sampling occurred in 2017-2018 for Dordogne and in 2018-2019 for the three other areas).

Contrary to what we observed in M. bovis infected foxes, excretion does not seem to be occurring in M. microti infection, even when mesenteric lymph nodes, as in M. bovis infection, are affected. None of the sampled foxes showed the presence of M. microti in excreta, while for five M. bovis infected foxes-four of which were infected in the mesenteric lymph nodes-this latter bacillus was detected, suggesting active excretion for this pathogen [START_REF] Richomme | Mycobacterium bovis infection in Red foxes in four animal tuberculosis endemic areas in France[END_REF]. According to our data, foxes do not seem to play any role in the active transmission of M. microti, and would likely be playing that of dead end hosts. However, the number infected foxes found in our study is relatively small and this hypothesis deserves to be confirmed with a larger sample.

Almost all red foxes disclosed M. microti infection in mesenteric lymph nodes, suggesting infection by ingestion of contaminated food. European badgers and red foxes can occupy similar ecological niches and can compete over food, including small vertebrates [START_REF] Macdonald | Encounters btween two sympatric carnivores: Red foxes (Vulpes vulpes) and European badgers (Meles meles)[END_REF]. One of the main preys of red foxes' diet in Western Europe are field voles (Microtus agrestis) [START_REF] O'mahony | Fox predation on cyclic field vole populations in Britain[END_REF][START_REF] Kidawa | The effects of sex, age, season and habitat on diet of the red fox Vulpes vulpes in northeastern Poland[END_REF]. Wild mammalian carnivores are consequently likely to have substantial exposure to M. microti both from ingestion and their contaminated environment [START_REF] Kipar | Mycobacterium microti Tuberculosis in Its Maintenance Host, the Field Vole (Microtus agrestis): Characterization of the Disease and Possible Routes of Transmission[END_REF]. The differences in prevalence rates by area observed in our study are probably related to the infection and/or presence of the main reservoir, although no data about infection in voles to confirm or refute this hypothesis are available. Indeed, very few data exist on prevalence of this disease in wild rodents. In 2002, 8% of field voles, bank voles (Clethrionomys glareolus) and wood mice (Apodemus sylvaticus) were found to be infected in England [START_REF] Cavanagh | Mycobacterium microti infection (vole tuberculosis) in wild rodent populations[END_REF]. In another study, prevalence varied between 0% and 50% of infected voles, with an apparent seasonality at the English and Scottish border [START_REF] Burthe | Tuberculosis (Mycobacterium microti) in wild field vole populations[END_REF].

Although we found that in some localizations M. microti or M. bovis presence was exclusive, cohabitation of the two MTBC species could be observed at least at a population level in bTB high burden areas in Dordogne (bTB hotspot 1). Until present, co-infection in a same animal was not demonstrated here or in any other French animal [START_REF] Michelet | Infection with Mycobacterium microti in animals in France[END_REF][START_REF] Michelet | Mycobacterium microti Interferes with Bovine Tuberculosis Surveillance[END_REF][START_REF] Michelet | Mycobacterium microti detection in French wildlife[END_REF]. For two foxes, 170831039977 and 180814038269, we could not confirm the mycobacterium identity due to a low DNA concentration. However, given that M. microti was confirmed in another lymph node of the same animal, it is very probable that the same mycobacterium gave rise to PCR positive results. These observations may be in favor of the hypothesis that M. microti infection might provide some protection against M. bovis infection at individual level and vice versa.

This study confirms the need of an accurate surveillance of TB using specific molecular tools that overcome the particular difficulty of confirming M. microti by bacteriology. Further studies are needed to better characterize the epidemiology of this mycobacteria, to identify the maintenance species and the infection prevalence within their populations as well as climatic, ecological and landscape factors explaining its occurrence and the possible consequences for animal and human public health need to be further explored.

Figure 1 .

 1 Figure 1. Comparison of M. microti (in red) and M. bovis (in blue) prevalence in three TB areas of Nouvelle-Aquitaine-Charente, Dordogne (season #1) and Landes-and in the core TB infected area of Côte-d'Or, France. The lines are the CI 95% of the prevalence.

Figure 2 .

 2 Figure 2. Location of sampled (black dots), M. microti infected (yellow (season #1) and blue (season #2) stars) and M. bovis infected (red stars) foxes in the Dordogne (Do) and Landes (L) study areas (in green). In Dordogne, red circles represent season #2 sampling in hotspot 1 and 2. The grey lines delimit communes (administrative units). The communes in pink belong to the infected areas where surveillance and management are implemented in cattle and in wildlife species (badgers, wild boars and red deer). Within them, the communes in green are those of included in this study. Black dots and stars are positioned at the center of the communes and their size is proportional to the number of foxes (1 to 27 foxes for dots and 1 to 16 foxes for stars).

Table 1 .

 1 M. microti infection of foxes in three TB areas of Nouvelle-Aquitaine and in the core TB infected area of Côte-d'Or, France.

		Analyzed	Number of Infected Foxes	Prevalence in % (IC 95% )
	Charente	98	0	<3.7 *
	Dordogne season #1	184	12	6.

5 (3.4; 11.1) Landes 140 3 2.1 (0.4; 6.11) Côte-d'Or 146 1 0.7 (0.1; 3.8) * Upper limit of the 95% confidence interval.

Table 2 .

 2 Results of M. microti and M. bovis detection in foxes in Dordogne (season #2) and in the two bTB hot spots of this area.

			Number of Infected Foxes
			(% [CI 95% ])
		Analyzed	M. microti *	M. bovis **	p-Value of the Fisher Exact Test
	bTB Hot spot 1	45	12 (26.6 (14.6-41.9))	6 (13.3 (5.0-26.8))	0.3
	bTB Hot spot 2	50	0 (0 (0-7.1))	6 (12 (4.5-24.3))	0.0283
	Total Dordogne #2	95	12 (6.5)	12 (6.5)	1

* Number of M. microti infected foxes in the two hot spots is statistically different (Fisher exact test, p-value = 0.0003); ** data from

[START_REF] Richomme | Mycobacterium bovis infection in Red foxes in four animal tuberculosis endemic areas in France[END_REF]

.

Microorganisms 2021, 9, 1257. https://doi.org/10.3390/microorganisms9061257 https://www.mdpi.com/journal/microorganisms

Acknowledgments:

The authors thank local state veterinary services, local hunting federations, trappers and pest control officers, veterinary laboratories of Côte-d'Or, Dordogne and Charente and the laboratory of Pyrénées and Landes, local Animal Health Defense Association of Dordogne.

L'archive ouverte pluridisciplinaire HAL, est destinée au dépôt et

Data Availability Statement:

The data presented in this study are available in supplementary material Table S1.

Supplementary Materials:

The following are available online at https://www.mdpi.com/article/10 .3390/microorganisms9061257/s1, Table S1: Results of M. microti detection by molecular diagnosis in tissues and feces, when available, for each of the 28 foxes found infected. Funding: The French ministry of Agriculture mainly financed the sampling and the analyses in the framework of the RFSA call on TB projects (Anses-DGAl credit agreement no 2017-326).

Institutional Review Board Statement: Not applicable.

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.


Ibtissam Chakir

Benjamin B Tournier

Hanane Touati

Vincent-Joseph Poirel

Etienne Challet

Paul Pevet

Ali Ouarour

Patrick Vuillez

Pinealectomy and gonadectomy modulate amplitude, but not photoperiodic modulation of Clock gene expression in the Syrian hamster suprachiasmatic nuclei

Keywords: biological rhythms, refractory state, clock genes, pineal gland, testis

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

Introduction

Seasonal mammals show deep changes in physiology, including reproduction and energy metabolism, according to the photoperiod (i.e., duration of daytime light phase). The nocturnal secretion of melatonin from the pineal gland, which is proportional to the duration of nocturnal phase, plays a critical role in the integration of photoperiodic changes [START_REF] Pevet | Melatonin: from seasonal to circadian signal[END_REF]. Among seasonal mammals, Syrian hamsters are called long-day breeders because they are sexually active when exposed to long (summer-like) photoperiod, while regression of the reproductive system, including gonadal atrophy and highly reduced synthesis of sexual hormones, is induced by transfer to short (winter-like) photoperiod. The photorefractory period, or photorefractoriness, is a particular physiological state occurring after a long-term (i.e. over 20 weeks) exposure to a short photoperiod and characterized by a spontaneous recrudescence of gonadal activity, despite the winter-like long-lasting secretion of nocturnal melatonin [START_REF] Reiter | The pineal and its hormones in the control of reproduction in mammals[END_REF].

Clock (Circadian locomotor output cycles kaput) is one of the core clock genes of the master circadian clock localized in the suprachiasmatic nuclei (SCN) [START_REF] Reppert | Coordination of circadian timing in mammals[END_REF]. The dimer of CLOCK protein, in association with BMAL1, forms the key element of a positive control loop that drives the expression of core clock genes of the negative part of the loops (such as Period (Per)1-3 and Cryptochrome (Cry)1-2) and clock-controlled-genes (such as Avp) [START_REF] Jin | A molecular mechanism regulating rhythmic output from the suprachiasmatic circadian clock[END_REF][START_REF] Reppert | Coordination of circadian timing in mammals[END_REF]. Expression of Clock in the SCN of nocturnal rodents such as mice and hamsters is most often described as constitutive across 24 h (i.e., with no daily variations). In the case of defective function of Clock, the expression of other clock genes is blunted. The expression rhythm of clock-controlled-genes is abolished, and the daily rhythm of locomotor behavior, a well characterized circadian output of the SCN, is highly disturbed [START_REF] Jin | A molecular mechanism regulating rhythmic output from the suprachiasmatic circadian clock[END_REF][START_REF] Kume | mCRY1 and mCRY2 are essential components of the negative limb of the circadian clock feedback loop[END_REF][START_REF] Oishi | Rhythmic expression of BMAL1 mRNA is altered in Clock mutant mice: differential regulation in the suprachiasmatic nucleus and peripheral tissues[END_REF][START_REF] Shearman | Interacting molecular loops in the mammalian circadian clock[END_REF][START_REF] Nakamura | Clock mutation lengthens the circadian period without damping rhythms in individual SCN neurons[END_REF][START_REF] Redlin | The circadian Clock mutant mouse: impaired masking response to light[END_REF]. The endogenous rhythmicity within the SCN is entrained at 24h every day by the light/dark cycle (LD). The photic information, carried from the retina to the SCN by the retinohypothalamic tract, is known to regulate the expression of some clock genes, especially from the negative control loop. However, some studies suggested that the CLOCK protein also contributes to the animals' sensitivity to light [START_REF] Redlin | The circadian Clock mutant mouse: impaired masking response to light[END_REF][START_REF] Ramkisoensing | Synchronization of Biological Clock Neurons by Light and Peripheral Feedback Systems Promotes Circadian Rhythms and Health[END_REF]. From the SCN, the circadian or lightsynchronized signals are then distributed to the whole organism by the autonomous nervous system and by the rhythmic control of hormonal secretion [START_REF] Challet | Keeping circadian time with hormones[END_REF]. One of the most studied hormones is the pineal melatonin which may participate to the daily synchronization of peripheral oscillators [START_REF] Pevet | Melatonin: both master clock output and internal time-giver in the circadian clocks network[END_REF]Chakir et al., 2015b).

In addition to the sensitivity to the day-night transitions, expression of clock genes within the SCN and physiology of seasonal species are markedly sensitive to photoperiod changes [START_REF] Johnston | Measuring seasonal time within the circadian system: regulation of the suprachiasmatic nuclei by photoperiod[END_REF]. Notably, the transfer of Syrian hamsters from long-(LP) to shortphotoperiod (SP) causes a daily rhythmicity of the expression of the Clock gene due to diminished transcription at dawn, and transcriptional modifications of other feedback loops [START_REF] Tournier | Photoperiod differentially regulates clock genes' expression in the suprachiasmatic nucleus of Syrian hamster[END_REF][START_REF] Tournier | Gene expression in the suprachiasmatic nuclei and the photoperiodic time integration[END_REF]. Of note, the photoperiodic changes of Clock expression in the SCN are not immediate. Although these changes are already started at 4 days after transfer to SP, the complete SP profile of expression is not yet obtained after 21 days of transfer to SP [START_REF] Tournier | Gene expression in the suprachiasmatic nuclei and the photoperiodic time integration[END_REF]. The photoperiodic signals of SCN are then translated by an increased duration of the nocturnal peak of melatonin, which ultimately leads to a winter-like physiology. One of the simplest photoperiodic indicators to observe is the testicular regression in male hamsters that highlights the period of sexually quiescent of the seasonal reproductive cycle. As mentioned above, a prolonged exposure to SP (over 20 weeks) induces photorefractoriness (i.e. animals become insensitive to the photoperiod, SP REF ). During such SP REF , a spontaneous reversion to a sexually active phenotype of spring/summer is observed, which includes testis growth and elevation of testosterone levels [START_REF] Reiter | The pineal and its hormones in the control of reproduction in mammals[END_REF]. In the SCN, the Clock gene is the only core clock gene to show an altered profile of expression in the photorefractory phase: its daily expression becomes constitutive again, but with lower values as compared to LP [START_REF] Tournier | Gene expression in the suprachiasmatic nuclei and the photoperiodic time integration[END_REF].

Thus, the Clock gene may be at the heart of the photoperiodic time measurement because it is the only circadian gene in Syrian hamster whose expression is different between LP, SP and SP REF conditions. However, the knowledge of the mechanisms underlying its photoperiodic regulation is missing. Because melatonin exerts an action on SCN-driven rhythmicity [START_REF] Pevet | Melatonin: both master clock output and internal time-giver in the circadian clocks network[END_REF], on the expression of some clock genes in the SCN [START_REF] Poirel | Contrary to other non-photic cues, acute melatonin injection does not induce immediate changes of clock gene mRNA expression in the rat suprachiasmatic nuclei[END_REF][START_REF] Johnston | Multiple effects of melatonin on rhythmic clock gene expression in the mammalian pars tuberalis[END_REF][START_REF] Agez | Endogenous melatonin provides an effective circadian message to both the suprachiasmatic nuclei and the pars tuberalis of the rat[END_REF][START_REF] Nagy | Melatonin adjusts the expression pattern of clock genes in the suprachiasmatic nucleus and induces antidepressant-like effect in a mouse model of seasonal affective disorder[END_REF][START_REF] Vriend | Melatonin feedback on clock genes: a theory involving the proteasome[END_REF] and it may interact with CLOCK [START_REF] Shimomura | Genetic suppression of the circadian Clock mutation by the melatonin biosynthesis pathway[END_REF], it could also participate in the photoperiodic reading associated with seasonal profiles of Clock expression within the SCN. Testosterone, another hormone known to act on clock genes in the SCN [START_REF] Iwahana | Gonadectomy reveals sex differences in circadian rhythms and suprachiasmatic nucleus androgen receptors in mice[END_REF][START_REF] Karatsoreos | Androgens modulate structure and function of the suprachiasmatic nucleus brain clock[END_REF][START_REF] Model | Suprachiasmatic nucleus as the site of androgen action on circadian rhythms[END_REF] is up-regulated during SP REF , as aforementioned [START_REF] Matt | Hypothalamic-pituitary-gonadal interactions during spontaneous testicular recrudescence in golden hamsters[END_REF][START_REF] Tate-Ostroff | Correlative changes in the response to castration and the onset of refractoriness in male golden hamsters[END_REF][START_REF] Stetson | Role of the pineal and its hormone melatonin in the termination of photorefractoriness in golden hamsters[END_REF][START_REF] Turek | Pinealectomy and lesions of the suprachiasmatic nucleus affect the castration response in hamsters exposed to short photoperiods[END_REF]. Thus, the distinct profile of Clock expression in SP REF could result from the resumption of gonadal activity. Therefore, our study aims to test if changes of the daily expression of Clock in response to SP (winter-like state of gonadal regression) and to prolonged SP (spring-like state of gonadal reactivation) are sensitive to melatonin and testosterone, respectively.

Material and Methods

Experimental designs

Animals were exposed to different artificial photoperiods defined by the lights turning on at ZT0 (zeitgeber time 0) and turning off at different times (ZT16 or ZT8) to create long and short photoperiods.

In the first experiment, animals were either kept in LP (LD16:8, with lights on at 05:00 AM, defining, ZT0) or transferred in a SP (LD8:16, with lights on at 09:00 AM, defining ZT0) for 10-(SP groups) or 30-weeks (photorefractory phase, SP REF groups). To test the role of melatonin, animals bred in LP were either pinealectomised (PinX) or sham-operated before the photoperiodic change, thus leading to six groups of twenty-five animals (Fig. 1A).

In the second experiment, animals were kept in SP (LD8:16) for 30-weeks (SP REF ). To test the role of testosterone, at the week 17, before spontaneous restoration (Bittman, 1978), animals were gonadectomised and implanted with either empty capsule (GDX) or testosterone-filled capsule (GDX-T), thus, leading to two groups of twenty-five animals per group (Fig. 1B).

The age of individuals at the time of surgery and/or transfer to SP was considered in order that all the animals got the same age (9-10 months) when euthanized at the end of experiments. Whatever the experimental group, animals were weighed and killed by decapitation every 4h throughout the 24h, from ZT 3, 7, 11, 15, 19 to 23. Brains were quickly removed, frozen in cold (-35°C) isopentane and stored at -80°C until use. Testeis were dissected and weighed. Plasma was isolated from trunk blood, aliquoted and stored at -20°C until used for testosterone analysis.

Animals

Adult male Syrian hamsters (Mesocricetus auratus) were obtained from the colony inbred at the Chronobiotron (animal facility, UMS 3415 CNRS/University of Strasbourg, Strasbourg). Animals were group housed in an environmentally controlled room with a light/dark (LD) 16:8 cycle with a light intensity of approximately 150 lux during daytime and a constant dim red light (< 1 lux). Temperature was maintained at 20 ± 1°C. Animals had free access to food and water. Experimental procedures involving Hamsters complied with the French National law (License 67-88) implementing the European Union Directive 2010/63/EU. All efforts were made to minimize the number of animals used and their suffering, and the study met the ethical standards.

Surgery procedures: pinealectomy, gonadectomy, steroid implants

In order to perform the pinealectomy, hamsters were anesthetized during the light phase with i.p. injections of a mixture of zoletil 20 (Virbac, Carros, France; 40 mg/kg i.p.) and rompun 2% (Bayer Pharma, Puteaux, France; 10 mg/kg i.p.). After being placed into a stereotaxic instrument (Kopf), a midline circular incision of the skull was gently performed to expose the pineal gland, trying to avoid bleeding of the median venous sinus. The pineal was removed with a pair of fine forceps, the skull cap replaced and the incision closed. After surgery, hamsters were housed individually until complete recovery. For controls, the same steps were made but only half of the midline circular incision of the skull has been done. Surgical procedure of the gonadectomy was performed under anaesthesia (2% isoflurane in oxygen) and with a mixture of ketamine (100 mg/kg, i.p.) and rompun 2% (10 mg/kg, i.p.).

GDX was realised by abdominal incision and removal of both testes. Muscles and fascia were closed using surgical silk, and the overlying skin was closed.

For the steroid implants, silastic capsules (I.D. 1.47 mm, O.D. 1.95 mm, and 13 mm long; Dow Corning, Belgium), full of testosterone (4-androsten-17β-ol-3-one, Sigma, Saint-Louis, MO, USA), or empty capsules for controls (Recio et al., 1998). Both were sealed with Silastic glue and allowed to dry overnight. All capsules were primed in a 37°C 0.9% saline bath for 36h before implantation.

In Situ Hybridization.

Coronal sections (14-μm thick) covering the rostro-caudal extent of the SCN were cut using a cryostat and mounted onto Superfrost+ slides (VWR International, Leuven, Belgium).

Slides were then air-dried and stored at -20°C until use. The mouse Clock (3441 bp) clone (generous gift of Prof. J.S. Takahashi, HHMI, University of Texas Southwestern Medical Center, Dallas, USA) was used to synthesize antisense mRNA probes. The synthesis of Clock mRNA probes and the hybridization procedure in Syrian and European hamsters were previously described [START_REF] Tournier | Photoperiod differentially regulates clock genes' expression in the suprachiasmatic nucleus of Syrian hamster[END_REF][START_REF] Tournier | Seasonal variations of clock gene expression in the suprachiasmatic nuclei and pars tuberalis of the European hamster (Cricetus cricetus)[END_REF][START_REF] Tournier | Gene expression in the suprachiasmatic nuclei and the photoperiodic time integration[END_REF].

Briefly, slides were exposed to antisense Clock probes (54°C, overnight), then subjected to RNA digestion and treated with stringency washes to remove non-specific probe hybridization. Dehydrated and air-dried slides were exposed to 35 S sensitive films (BioMax MR, Kodak) with 14 C standards. All the samples from the first experiment (role of melatonin)

were treated simultaneously and blind to the treatment. The same methodology was used for the second experiment (role of testosterone). Relative optical densities (ROD) in bilateral SCN (3 sections per animal) and in the anterior periventricular zone (background signal) were measured using NIH-Image J after calibration with the standard [START_REF] Lincoln | Photorefractoriness in mammals: dissociating a seasonal timer from the circadianbased photoperiod response[END_REF]. The specific signal intensity was defined as follows: ROD SCN -ROD background . For each animal, values in bilateral SCN were averaged. Analyses were performed by experimenter blind to the treatment group.

Statistics

Data are presented as individual values and mean ± SEM. Data were analysed with one-, two-and three-way analyses of variance (ANOVA) followed by Fisher's least significant difference (LSD) post hoc test using the Statistica software (Statistica 8.0; StatSoft).

Results

The experimental designs of experiment 1 and 2 is presented in Figure 1.

Physiological parameters

The photoperiodic responsiveness was determined from changes in testicular mass, as shown in Figure 2A according to experimental groups. Two-way ANOVA indicated that the testicular mass is under the control of the photoperiod x treatment interaction (p<0.05). Post hoc analysis showed that 10 weeks in SP induced a testicular regression as compared to LP (LP-sham vs SP-sham, p<0.001). Following 30 weeks in SP, the testicular mass became again very close to the LP group (SP REF -sham vs LP, p>0.05). Pinealectomy did not modify the testicular mass in LP (LP-sham vs LP-PinX, p>0.05). Importantly, pinealectomy completely abolished the regression induced by the short photoperiod (SP-sham vs SP-PinX, p<0.001).

Therefore, no difference in testicular mass was observed between sham-and pinealectomizedanimals placed during 30 weeks in SP (SP REF -sham vs SP REF -PinX, p>0.05), as SP REF -PinX did not have testicular regression.

In addition, the seminal vesicle mass was measured as an indicator of testosterone presence (Fig. 2B). The one-way ANOVA indicated the main effect of the group on seminal vesicle mass (p<0.001). Post hoc test showed significant reductions in seminal vesicle mass in the SP-sham and GDX as compared to LP and GDX-T groups (p<0.001), validating low testosterone level in SP-sham and GDX and its restoration in GDX-T as compared to LP.

Effects of melatonin and photoperiod on Clock expression (experiment 1)

The effects of photoperiod, pinealectomy and ZT on Clock mRNA levels are presented in Figure 3. Three-way ANOVA indicated significant main effects of photoperiod (p<0.001), pinealectomy (p<0.001) and ZT (p<0.01). Although the mean Clock mRNA levels were influenced by the pinealectomy (-10.07  2.25%, p<0.001; ROD values, Sham-treated: 4.5 2.25, PinX: 3.4 2), this effect was independent of both photoperiod and ZT (pinealectomy x ZT x photoperiod interaction: p>0.05; pinealectomy x ZT interaction: p>0.05; pinealectomy x photoperiod interaction: p>0.05). Thus, pinealectomy did not prevent the appearance of a ZT effect in specific profile of Clock mRNA expression in SP and SP REF (Figure 3A-B).

Independently of the pinealectomy, the expression of Clock mRNA was constitutive in LP (ZT effect: p>0.05, Figure 3C). In marked contrast, exposition of animals to 10 weeks of SP (SP-sham and SP-PinX) induced a significant decrease in Clock mRNA levels at ZT15, ZT19 and ZT23, as compared to LP (p<0.05, Figure 3C). Finally, the expression of Clock mRNA was constitutive in SP REF (SP REF -sham and SP REF -PinX, ZT effect: p>0.05) as an additional decrease in Clock mRNA levels was observed at ZT3, ZT7 and ZT11, as compared to SP (SPsham and SP-PinX, Figure 3C). Thus, mean levels of Clock mRNA in SP REF were significantly decreased as compared to LP (-24.08  2.29%; p<0.001, Figure 3D

Effects of testosterone on Clock gene in SP REF (experiment 2)

Two-way ANOVA revealed the absence of ZT effect (p>0.05) and of the ZT x gonadectomy interaction (p>0.05), indicating that gonadectomy did not prevent the occurrence of a constitutive expression profile of Clock mRNA in SP REF (Figure 4A). However, a significant main effect of gonadectomy (p<0.01) was observed. Gonadectomy induced a decrease in Clock mRNA levels as compared to SP REF (-26.11

Discussion

The present study shows that the daily profile of Clock expression in the SCN is not only a marker of the photoperiod, but also of the duration spent in the winter day-length condition.

From constitutively high levels in LP, Clock mRNA expression becomes rhythmic in SP and shows constitutively low levels when reproductive physiology escapes the inhibitory action of the SP (i.e. SP REF ). In addition, the pinealectomy, known to inhibit physiological integration of SP (such a regression of the gonads), did not prevent neither the appearance of the the duration spent in SP seems to be independent of the presence of two main hormones involved in the seasonal physiology, namely melatonin and testosterone. Our data suggest that photoperiodic modulation of Clock levels in the master clock occurs upstream of the variations of activity of the pineal and gonads, and not as a consequence of feedback effects of their respective hormones.

We showed that melatonin, a key hormone in seasonal physiology, does not participate in the photoperiodic adaptation of Clock gene expression. This observation, which corroborates the absence of effect of pinealectomy on the increased light sensitivity phase in terms of c-fos after transfer to SP [START_REF] Vuillez | In Syrian an European hamsters, the duration of sensitive phase to ligtht of the suprachiasmatic nuclei depends on the photoperiod[END_REF], suggests that the photoperiodic integration by the SCN is insensitive to melatonin. Indeed, although the pinealectomized animals have an inhibited physiological integration of SP (i.e. testicular regression cannot be longer induced by the long-lasting melatonin signal), they show a daily expression profile of Clock mRNA level that normally corresponds to winter-like SP conditions. Thus, the adaptation of Clock mRNA levels from LP to SP seems to be a direct response to the changes of photic signals and could therefore participate in the development of a daily synchronization message depending on the photoperiodic information. In agreement with this hypothesis, the expression of clock-controlled genes within SCN is also dependent on the photoperiod, but with a delay of adaptation greater than 21 days [START_REF] Tournier | Gene expression in the suprachiasmatic nuclei and the photoperiodic time integration[END_REF]. In contrast, we showed that melatonin, whatever the photoperiod, may induce an increase in Clock mRNA levels, which could contribute to a strengthened functioning of the master clock. Therefore, our observation corroborates and extends previous data on the effects of melatonin on gene expression within SCN.

During the photorefractory period, reproductive physiology escapes the inhibitory action of prolonged secretion of nocturnal melatonin associated to short days. Pinealectomy performed before the transfer from LP to SP, however, had no effect on the reduction of Clock mRNA levels observed in SP REF as compared to LP. However, as the desensitization of gonadal axis to melatonin in SP REF induces a re-increase in testosterone levels, and as testosterone can act on the SCN (Karatsoreos et al, 2007;Model et al, 2017), we tested if the acquisition of the Clock mRNA expression profile in SP REF depends on the presence of gonadal activity. We showed that testosterone does not modify photoperiodic time integration. However, testosterone showed a positive effect on Clock mRNA levels. Thus, although melatonin and testosterone act increase on Clock mRNA levels, they do not participate in photoperiodic control of its rhythmic or constitutive expression in the SCN.

The modifications of the expression of Clock according to the photoperiodic conditions we showed confirm previous studies. SP induces a decrease in the amount of Clock mRNA at the end of the night in Syrian hamsters housed in LD10:14 or LD8:16 [START_REF] Tournier | Photoperiod differentially regulates clock genes' expression in the suprachiasmatic nucleus of Syrian hamster[END_REF][START_REF] Tournier | Gene expression in the suprachiasmatic nuclei and the photoperiodic time integration[END_REF]. It is interesting to note that the duration during which Clock mRNA levels are low appears to be greater when the animals are placed in LD8:16 (from ZT15 to ZT23 in the present study and from ZT14 to ZT2 in the study published in 2009) than in LD10:14 (from ZT23 to ZT5). This observation must remain a hypothesis because the number of samples taken over 24 hours is different between studies (every 4 hours in the studies in LD8:16 and every 2 hours in the study in LD10:14) and therefore does not allow a direct comparison. Thus, our study confirms the appearance of a rhythm in the daily expression of Clock in SP and its abolition in SP REF and extended the previous observations by showing that these adaptations are independent of the presence of melatonin (appearance of the rhythmic Clock mRNA expression in SP) and testosterone (disappearance of the rhythmic Clock mRNA expression after a prolonged time in SP). Overall these adaptations go in the direction of reducing Clock mRNA levels in SP and SP REF . In the European hamster, a reduction in Clock mRNA levels were also observed but without the appearance of a rhythmic expression.

However, the physiology of the European hamster is sensitive to the photoperiod but also to another endogenous timer, the so-called circannual clock [START_REF] Canguilhem | Photoperiodic regulation of body mass, food intake, hibernation, and reproduction in intact and castrated male European hamsters, Cricetus cricetus[END_REF][START_REF] Masson-Pevet | An attempt to correlate brain areas containing melatonin-binding sites with rhythmic functions: a study in five hibernator species[END_REF]. In addition, the study showing SP-induced reduction in Clock mRNA levels in the European hamster without the appearance of a rhythm was performed in natural photoperiod. These two fundamental differences make it difficult to directly compare the conclusions of these studies, but it is important to note that in both Syrian and European hamsters, an overall decrease in Clock levels is observed. Conversely, these changes are less pronounced in non-photoperiodic animals, whose reproductive physiology is insensitive to photoperiod changes [START_REF] Sumova | Clock gene daily profiles and their phase relationship in the rat suprachiasmatic nucleus are affected by photoperiod[END_REF].

Thus, in all of these studies, the SP induced a decrease in the quantities of Clock mRNA.

However, the effects of SP seem to be different over the daily time: (i) after a short time exposure in SP (less than 1 week), levels are only decreased at the night / day transition; (ii) when the winter-like physiology is in place (8 weeks), the decrease extends to the dark phase of the day; (iv) and finally, when the spontaneous return to a summer-like physiology takes place, the levels are low even at the day / night transition. The consequence of the appearance of Clock level alterations as a function of the photoperiod and of the time spent in SP seems to indicate that Clock represents a seasonal timer, independent of the feedback of two hormones closely related to seasonal physiology (i.e. melatonin and testosterone). The rapid or otherwise slower impact of SP on Clock levels at the day/night and night/day transition, respectively, could contribute to the day-to-day elaboration of a photoperiod-dependent circadian message which would also depend on the duration spent in the short photoperiod.

Thus, expression patterns of Clock mRNA could, to some extent, be markers of the photoperiod, in response to photic stimuli and independent of the hormonal feedback.

Another important issue to address is the species differences of Clock expression in the SCN. Clock seems to be of constitutive expression in nocturnal rodents such as rats [START_REF] Sumova | Clock gene daily profiles and their phase relationship in the rat suprachiasmatic nucleus are affected by photoperiod[END_REF], Syrian [START_REF] Tournier | Gene expression in the suprachiasmatic nuclei and the photoperiodic time integration[END_REF] and European hamsters [START_REF] Tournier | Seasonal variations of clock gene expression in the suprachiasmatic nuclei and pars tuberalis of the European hamster (Cricetus cricetus)[END_REF], when placed in LP. On the contrary, its levels vary throughout the day in the SCN of diurnal species (rodent: (Chakir et al., 2015a); and sheep: [START_REF] Lincoln | Temporal expression of seven clock genes in the suprachiasmatic nucleus and the pars tuberalis of the sheep: evidence for an internal coincidence timer[END_REF]). In addition, adaptations of its expression to a change in photoperiod are also described as speciesdependent with the maintenance of a rhythm of expression [START_REF] Lincoln | Temporal expression of seven clock genes in the suprachiasmatic nucleus and the pars tuberalis of the sheep: evidence for an internal coincidence timer[END_REF], its appearance [START_REF] Tournier | Gene expression in the suprachiasmatic nuclei and the photoperiodic time integration[END_REF] or a simple decrease in its average levels [START_REF] Tournier | Seasonal variations of clock gene expression in the suprachiasmatic nuclei and pars tuberalis of the European hamster (Cricetus cricetus)[END_REF]. These evidences for species-specific clock gene expression patterns in LP and in response to a transfer to SP sustain the hypothesis that Clock gene could participate in the physiological differences observed between mammals at both the daily and annual levels.

As a conclusion, our data clearly demonstrate that pinealectomy did not affect the SP-

Figures and

Graphical abstract (73/75)

Within the hamster master clock, expression rhythm of Clock gene in response to transfer from long-(LP) to short-photoperiod (SP) is melatonin independent. Alterations in Clock mRNA levels during the photorefractory period (SP REF ) are also testosterone independent. However, the average Clock mRNA amount is lower in the absence of both hormones. Thus, the photoperiodic control of daily variations of Clock gene is independent of the positive melatonin/testosterone feedback signals. GDX: gonadectomy; PinX: Pinealectomy

  ; ROD values, LP: 4.5 2.25, SP REF : 3.4 2). Representative examples of the expression of Clock at high, intermediate and low levels are given in the Figure 3E.

   3.70%; p<0.001; ROD values, SP REF : 4.5 2.25, GDX: 3.4 2) that was reversed by testosterone implants (-5.77  4.53%; p>0.05, Figure 4B; ROD values, SP REF : 4.5 2.25, GDX-T: 3.4 2).

Figure 1 .

 1 Figure 1. Experimental design.The solid line curve illustrates the time course of the regression and the spontaneous restoration of sexual activity observed in hamsters transferred from long photoperiod (LP) to short photoperiod (SP). SP induces sexual inhibition within 10 weeks. Nevertheless, hamsters kept in SP for longer time (30 weeks) are refractory to SP (SP REF ) and restore a spring physiology. Name of each group is written in italic, close to a circle whose position show the time of euthanasia as well as sexual physiological state. A. in experiment 1, animals were either pinealectomized (Pin-X) or Sham operated few days before transfer into SP and were euthanized after 10 weeks or 30 weeks passed in SP. Two other groups (Pin-X and sham hamsters) were maintained in LP 10 weeks before euthanasia. B. In experiment 2, animals were transferred into SP for testis regression. After 17 weeks, they were gonadectomised before spontaneous restoration and i.p. implanted with either empty capsule (GDX) or testosterone filled capsule (GDX-T).

Figure 2 .

 2 Figure 2. Physiological validation of the experimental procedures.A. Animals were pinealectomized (PinX) or sham-operated (sham) and bred in LP or transferred in SP for 10 weeks (SP) or 30 weeks (SP REF ). At the end, the testicular mass was measured. Significantly different from the LP group at p<0.001 using a two-way ANOVA followed by a LSD post hoc test. B. The seminal vesicle mass was measured in all experimental groups. Significantly different from the LP group at p<0.001 and from the GDX-T group at ∞∞∞ p<0.001 using a one-way ANOVA followed by a LSD post hoc test.

Figure 3 .

 3 Figure 3. Effects of melatonin and photoperiod on Clock gene expression in the master clock. Clock mRNA levels in the SCN of animals bred in long photoperiod LD16:8 (LP, pink circles) or short photoperiod LD8:16 for 10-(SP, green squares) or 30-weeks (SP REF , grey triangles). A-C. Data present shamoperated (sham, A) pinealectomized (PinX, B) or both groups (C). Data are presented in reference to ZT0 (i.e., lights on) in each condition and expressed in relative optical density (ROD). Significant differences between LP and SP groups at p<0.05 and between SP and SP REF groups at ∞p<0.05 using a two-way ANOVA followed by a LSD post hoc test. D. Values are also presented independently of the ZT. Significant differences between LP groups (Sham and PinX) and SP REF groups (Sham and PinX) at p<0.05 using a two-way ANOVA followed by a LSD post hoc test. E. Representative examples of maximal, intermediate and minimal expression of Clock

Figure 4 .

 4 Figure 4. Regulation of Clock gene expression within the master clock under SP REF by testosterone. Clock mRNA levels in the SCN of animals bred in short photoperiod LD8:16 for 30-weeks (SP REF , grey triangles). After 17 weeks in SP, one part of the animals was gonadectomized and implanted with either empty capsule (GDX, orange squares) or testosterone-filled capsule (GDX-T, black circles). A. Data are presented in reference to ZT0 (i.e., lights on) in each condition and expressed in relative optical density (ROD). B. Values are presented independently of the ZT. Significantly different from the SP REF group at p<0.01 using a two-way ANOVA followed by a LSD post hoc test.

  rhythmicity of expression of the Clock gene in SP nor the presence of constant and low levels of Clock mRNA in SP REF . In fact, pinealectomized animals showed daily expression profiles of Clock comparable to sham-treated animals in both SP and in SP REF . In animals placed in SP with testicular regression, the gonadectomy did not inhibit the Clock mRNA changes in SP

REF , as compared to SP. Thus, changes in Clock mRNA expression induced by SP and by

  induced appearance of rhythmic Clock expression and the abolishment of such rhythm in SP REF . In addition, the gonadectomy and testosterone supply did not affect the specific Clock expression pattern in SP REF . Our data support the hypothesis that expression of Clock gene in the SCN is a time-counter sensitive to photoperiod changes. Further experiments will be necessary to identify if this differential regulation can be transferred to circadian oscillations outside of the SCN and how it can participate in physiological changes controlled by photoperiodism.

211x59mm (300 x 300 DPI)

128x59mm (300 x 300 DPI)

168x114mm (300 x 300 DPI)

Acknowledgements

We thank Pr. J.S. Takahashi (HHMI, University of Texas Southwestern Medical Center, Dallas, USA) for the gift of mClock clone.

Data accessibility

Raw data are available from the corresponding author on request.

Competing Interests

Authors have nothing to disclose.

Author Contributions

IC, BBT, EC, PP, PV designed the experiment. IC, PV, HT, V-JP performed the experiment. IC, BBT, EC, PP, PV analyzed and interpreted the data. IC, BBT, EC, PP, PV wrote the article and AO revised it.

Abbreviations

GDX: gonadectomy; GDX-T: gonadectomy plus testosterone-filled capsule; LD: light/dark cycle; LP: long photoperiod; PinX: pinealectomy; SCN: suprachiasmatic nuclei; SP: short photoperiod; SP REF : photorefractoriness due to prolonged exposure to SP.


Mickaël Riou

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Sandrine Melo

Marie Pierre Foulc

Proof of concept: efficacy of surgical titanium implants coated with linear gentamicin against osteomyelitis in pigs

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

The project was managed and supported by Rescoll, and funded by FUI, INRA and Rescoll. coated against acute postoperative osteomyelitis for human use.

Conclusions: This porcine model therefore validates the proof of concept on the therapeutic efficacy of biomedical implants the clinical condition of the pigs is better and inflammation is less spread in the presence of gentamicin coated implants. and medical imaging (MRI) follow-up and confirmed by the increase in cytokines. As with bacteraemia, the results show that presence of gentamicin-coated implants. In both cases, inflammation of the implanted bone area was observed through clinical concentrations tested, as early as 7 days post-infection. However, a significant reduction in bacterial load is observed in the Results: Clinical and bacteriological results from implants with or without gentamicin confirmed local bone infection at both were deposited.

the bacteria have been deposited in the bone hole, the 4 mm and/or 8 mm titanium implant with or without coated gentamicin bone infection with S. aureus SAHOS (human bone clinical isolate) at two different concentrations (10 2 CFU and 10 6 CFU). Once length by 4 mm diameter). Double and bilateral partial osteotomy surgery of the porcine tibia was performed, followed by local surgical implants against S. aureus osteomyelitis in pigs. We have defined two sizes of titanium cylinder implants (4 and 8 mm Materials/methods: The objective of this study is therefore to demonstrate the concept of the efficacy of gentamicin-coated surgical placement of titanium implants with bone infection and to measure the antibiotic efficacy of coated implants. antibiotics locally only in case of infection. To meet this objective, it is therefore essential to develop an animal model for the significant cost to society. It is necessary to find alternative solutions, such as using a surface treatment on titanium to deliver this type of infection and the surgical procedure is often complex. These practices represent a high risk to the patient and a surgical treatment, as well as implant replacement. However, standard antibiotic treatment protocols have a low efficacy for ment of these infections almost systematically requires the combination of long-term antibiotic treatment and concomitant Background: Implant infections in human surgery are serious clinical conditions that result in high mortality rates. The treat- Mickael Riou 1 , Aurélien Voisin 2 , Nathalie Kasal-Hoc 1 , Céline Barc 1 , Christelle Rossignol 3 , Hans Adriaensen 4 , José Delaval 5 , Anne-Proof of concept: efficacy of surgical titanium implants coated with linear gentamicin against osteomyelitis in pigs

Nouzilly, France

  de la Reproduction et des Comportements, INRA -CNRS -HARAS NATIONAUX -IFCE -Université de Tours, 5 Laboratory Touraine, Parçay-Meslay, France, 4 Plateforme Chirurgie et Imagerie pour la Recherche et l'En seignement, INRA, Nouzilly, France, 3 UE-1282 Infectiol ogie et Santé publique, INRA -Université de Tours, Nouzilly, France, Plateforme d'Infectiologie Expérimentale (PFIE), INRA, Nouzilly, France, Lyse Lainé 6 , Sandrine Mélo 3 , Marie-Pierre Foulc 2