Bénédicte Grimard
email: benedicte.grimard@vet-alfort.fr

Alice De Boyer

Des Roches

Maud Coignard

Anne Lehebel

A Chuiton

Luc Mounier

Isabelle Veissier

Raphaël Guatteo

Nathalie Bareille

A De Boyer Des Roches

A Lehébel

Relationships between welfare and reproductive performance in French dairy herds

Keywords: Calving rate, Calving to first service interval, Dairy cow, Fertility, Welfare

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Introduction

There is currently a serious concern about farm animal welfare in western countries: the vegan and anti-species movements call into question rearing animals. Moreover, consumers want to know how their food is produced. Identifying positive relationships between welfare and productivity might reassure consumers and encourage producers to improve animal welfare in their farms. Moreover, relationships between welfare and routinely recorded data (date of birth, calving date, date of insemination, milk production, milk quality, date of culling…) might help in selecting herds for welfare assessment [START_REF] Sandgren | Using a national dairy database to identify herds with poor welfare[END_REF][START_REF] De Vries | Exploring the value of routinely collected herd data for estimating dairy cattle welfare[END_REF]. This has been extensively studied in dairy cows (for a review: [START_REF] De Vries | Invited review: Associations between variables of routine herd data and dairy cattle welfare indicators[END_REF]. For example, in their meta-analysis, [START_REF] Lopez-Gatius | Effects of body condition score and score change on the reproductive performance of dairy cows: a meta-analysis[END_REF] and [START_REF] Bedere | Meta-analysis of the relationships between reproduction, milk yield and body condition score in dairy cows[END_REF] animals with an intermediate or low BCS. A severe loss (>0.5 points on a 5 point scale) in BCS during early lactation and a low BCS (BCS ≤ 2) at nadir was related to an increase in days open and to a reduction of conception rate compared to a slight BCS loss and BCS ≥ 2.5 at nadir [START_REF] Chagas | Invited review: New perspectives on the roles of nutrition and metabolic priorities in the subfertility of high-producing dairy cows[END_REF][START_REF] Bedere | Meta-analysis of the relationships between reproduction, milk yield and body condition score in dairy cows[END_REF]. However, if days open is a good indicator of the effects of BCS or change in score on reproductive performance, the measurement of conception rate at first service produced variable results among studies. Likewise, lameness [START_REF] Hernandez | Effect of lameness on the calving-toconception interval in dairy cows[END_REF][START_REF] Hernandez | Comparison of the calving-to-conception interval in dairy cows with different degrees of lameness during the prebreeding postpartum period[END_REF] and disease [START_REF] Fourichon | Effect of disease on reproduction in the dairy cow: a meta-analysis[END_REF][START_REF] Raboisson | Diseases, reproductive performance, and changes in milk production associated with subclinical ketosis in dairy cows: a metaanalysis and review[END_REF] were clearly related to reproductive performance: affected cows had longer calving to first service interval and required more AIs to become pregnant than healthy cows. The response to the approach of a human was positively associated with conception rate at first service [START_REF] Hemsworth | Relationships between humananimal interactions and productivity of commercial dairy cows[END_REF]. However, most studies have focused on only one specific aspect of welfare rather than considering welfare as a whole.

A protocol allowing the overall assessment of animal welfare at the herd level was developed in Europe in the Welfare Quality project [START_REF] Botreau | Definition of criteria for overall assessment of animal welfare[END_REF]Welfare Quality, 2009;[START_REF] De Vries | Evaluating results of the Welfare Quality multi-criteria evaluation model for classification of dairy cattle welfare at the herd level[END_REF]. This protocol was used in the winter of 2010-2011 in 131 French dairy farms and conducted to identify the main welfare problems and associations between welfare and milk production [START_REF] Coignard | Description and factors of variation of the overall health score in French dairy cattle herds using the Welfare Quality assessment protocol[END_REF][START_REF] Coignard | Does milk yield reflect the level of welfare in dairy herds?[END_REF][START_REF] De Boyer Des Roches | The major welfare problems of dairy cows in French commercial farms: an epidemiological approach[END_REF]. [START_REF] Coignard | Does milk yield reflect the level of welfare in dairy herds?[END_REF] identified positive associations between milk yield and low levels of aggression between cows and good emotional state of the herd. However, milk yield was also associated with low welfare principle scores for Good health assessed by the occurrence of diseases and injuries.

In dairy systems, production performance refers to milk yield and composition but is also related to reproductive performance and longevity. Calving and insemination dates are routinely recorded in most countries. Sangden et al. (2009) and[START_REF] De Vries | Exploring the value of routinely collected herd data for estimating dairy cattle welfare[END_REF] have already explored the relationship between reproductive performance and welfare measured at the herd level. However, they did not consider confounding factors in their models. Therefore, the objective of this study was to investigate the relationship between cow reproductive performance and welfare scores measured at the herd level through the Welfare Quality Protocol, in French commercial dairy herds, taking into account confounding factors for reproductive performance recorded in the national database.

Materials and methods

The study was carried out in 2010 and 2011 using only remote observations; ethical review was not required for this kind of work in 2010 and animal studies were compliant with all applicable provisions established by the European Directive 2010/63/EU. However, approval was sought and granted by the VetAgro Sup Ethics Committee for Experiments on Animals (France, agreement number CEEA18) on 5 March, 2019.

Dairy farms and welfare assessment

This investigation was conducted in the 131 French commercial dairy farms previously studied by de Boyer des [START_REF] De Boyer Des Roches | The major welfare problems of dairy cows in French commercial farms: an epidemiological approach[END_REF]. The farms were selected to be representative of the diversity of French dairy systems. They were visited once during the winter, between December 2010 and March 2011.

Animal welfare was estimated using the Welfare Quality (WQ) assessment protocol for dairy cattle (Welfare Quality, 2009). Briefly, 11 welfare criteria were assessed using one to seven measurements (Table 1). Each criterion was evaluated on a scale of 0 (lowest score) to 100 (highest score). These criterion scores were then grouped into four principle welfare scores giving the highest importance to the worst criterion. The four principle scores (good feeding, good housing, good health and appropriate behavior) were also expressed on a scale of 0 (lowest score) to 100 (highest score). Finally, the farm was assigned to an overall welfare category according to their principles scores. Overall welfare was considered to be excellent (at the highest level), if all the scores were >55 with two principle welfare scores >80, enhanced (good), if the principle welfare scores were all >20 with two principle scores >55, acceptable (above or meets minimal requirements), if the four principle welfare scores were >10 with at least three principle scores > 20. In the other situations, welfare was considered to be very low and the term used to describe the herd was not classified (poor welfare).

Data recorded and reproductive performance of the cows

The following data were extracted from the national performance database (Système National d'Information Génétique): cow breed, dates of calving, parity, milk production during the first 3 months of lactation, insemination dates, breed of the bull used, type of semen (sex sorted or not). Rank 1 to 3 artificial inseminations (AI) performed between the 1 December 2010 to the 31 March 2011 were selected. During this period, cows are housed in France and welfare was considered to be unchanged. In April, most of the cows are put out to grass and this might change their welfare situation. When first service occurred before 20 days postcalving or >200 days post-calving, the cows were excluded from the study. When two AIs were separated by an interval shorter than 5 days only the second AI was used and included in the analysis. The use of these inclusion criteria meant that only the data from 124 herds of the 131 were used. The exclusion of the remaining seven herds was due to the fact that an eligible AI was not observed during the period.

Two parameters were calculated to estimate reproductive performance: the calving to first service interval (CFSI) and the calving rate after AI (CR). A cow was considered to be pregnant if its subsequent calving occurred during an interval which was compatible with the average length of pregnancy for the breed ± 2 standard deviations (SD; as evaluated by [START_REF] Marceau | Can routinely recorded reproductive events be used as indicators of disease emergence in dairy cattle? An evaluation of 5 indicators during the emergence of bluetongue virus in France in 2007 and 2008[END_REF]. Cows culled between AI and the end of pregnancy were considered to be non-pregnant as recommended by the International Committed for Animal Recording1 .

Statistical analysis

The statistical unit was the AI. The overall welfare category, each principle and each criterion scores were taken into account in the analysis. As previously proposed by Coignard et al. (2014), the quantitative evaluations of each principle and criterion welfare scores were split into two categories: the first category corresponded to the lowest scores (i.e. scores lower than or equal to the first quartile) observed at cow level and the second category corresponded to the scores above the first quartile. This methodology was chosen to investigate the effects of high welfare impairment rather than to compare the best half to the worst half of herds since in the herds included in this study welfare variability was low (absence of enhanced and low number of poor welfare scored herds).

The relationships between CFSI and the welfare indicators (i.e. overall welfare category, principles and criteria scores) were evaluated using multivariate Cox proportional hazard models. Models were adjusted to take into account the factors likely to influence CFSI: cow breed (Prim'Holstein vs. Montbéliarde and other breeds), parity (primiparous vs. multiparous), season of calving (spring and summer, autumn, winter), mean milk production during the 3 months after calving, season of insemination (autumn vs. winter) and two interactions: parity*milk production and breed*milk production. These factors were selected because they have already been associated with CFSI in the literature [START_REF] Crowe | Parturition to resumption of ovarian cyclicity: comparative aspects of beef and dairy cows[END_REF].

The following model was used:

ℎ , = ℎ * exp + ) ~ 0,
where ℎ is the hazard function at time t for the probability of AI after a calving for the ! "# cows of the $ "# herd, ℎ is the unspecified hazard function, is a matrix containing all adjustment variables previously cited, is the vector of coefficients associated with these variables and and is a herd random effect. It was assumed to be normally distributed with a mean of 0 and a variance % .

Mixed logistic regression was used to analyze the relationships between CR and welfare indicators. As previously described, the models were adjusted for factors likely to influence CR [START_REF] Walsh | A review of the causes of poor fertility in high milk producing dairy cows[END_REF] : cow breed, parity, season of calving, mean milk production during the 3 months after calving, season of insemination, rank of AI (1, 2 or 3), interval between calving and AI, type of semen (sex-sorted vs. not sorted) and crossbreeding (bull breed different from the cow breed), parity*milk production and breed*milk production. The model used was:

& ~'( ) * ! + ln . + 1 -+ 1 = + * + * ~ 0, 2 ~ 0,
where the outcome & is the binary variable representing the occurrence of a calving for the cow ! in the herd $, is a matrix containing all adjustment variables previously cited, is the vector of coefficients associated with these variables, * is the random nested effect for a cow ! in a herd $ and is a herd random effect for the herd $. The random effect * and were assumed to be normally distributed with a mean of 0 and a variance % 2 and respectively.

The quality of the models was estimated using the Akaike Information Criterion (AIC).

The model containing the most variables with the lowest AIC was retained. Sixteen Cox proportional hazard models were constructed: for each reproductive performance: one for the overall welfare category, four for each welfare principle scores and 11 for each criteria score.

Each welfare score was taken into account alone, and its introduction in the multivariate models aimed to evaluate its effect corrected for the other variation factors susceptible to influence reproductive performance.

All statistical analyses were performed by using R software2 . Cox models were performed using the survival package3 [START_REF] Therneau | Modeling Survival Data: Extending the Cox Model[END_REF] and mixed logistic regression using the lme4 package [START_REF] Bates | Fitting Linear Mixed-Effects Models Using lme4[END_REF]. The effect of each welfare score in the different models was evaluated using drop 1 function of stat package and ANOVA function of car package.

Results

Characteristics of the sample studied

The Briefly, the main welfare problems highlighted by the median of criterion welfare scores were Absence of pain induced by management procedures and Absence of disease. In the first quartile, the Good feeding score was low mainly due to a low Absence of prolonged thirst score which was attributed when there were too few drinkers (>15 cows by drinking point). The absence of prolonged hunger score was also low in the first quartile, which was related to >23% of cows being very thin (<2.5). The good housing score was low when comfort around resting was not adequate. This was related to a long time taken to lie down and to a high percentage of cows which had dirty lower legs. Ease of movement score was high in all the farms because all the cows were loose-housed. The good health score was low in the first quartile mostly related to dehorning practices (disbudding without anesthetic or analgesic), to disease, essentially due to a high percentage of cows with somatic cell counts > 400 000 cells/mL and finally to small integument alterations and lameness. for the whole sample.

Relationship between animal welfare and CFSI

When introduced in the multivariate model the overall welfare category was significantly related to CFSI (P=0.02). CFSI tended to be associated with the criterion C6

(absence of injuries, P=0.06) and C9 (expression of social behavior, P=0.07; Table 4). For the overall welfare category and C6 (absence of injuries), the higher the welfare category/score the shorter was the CFSI (-11 to -7 days). The relationship was the opposite for C9. CFSI was shorter in the herds where high levels of agonistic interactions were observed between cows (-2 days).

Among the covariates, only breed, season of calving and season of AI were significantly related to CFSI. Median CFSI was shorter in Montbéliarde and other breeds compared to Prim'Holstein cows (72.0 vs. 83.0 days; P < 0.001), when calving occurred in autumn compared to spring/summer (72.0 vs. 98.0 days; P < 0.0001) and when insemination occurred in autumn compared to winter/spring (73.0 vs. 77.0 days; P < 0.0001).

Relationship between animal welfare and CR

The variables crossbreeding and type of semen were not retained in the multivariate model because their contribution to model quality was not significant.

The overall welfare category was not related to CR (P=0.54). CR was significantly related with the P2 criteria (good housing, P=0.05; Table 5) and this effect was explained by the relationship between CR and C3 (comfort around resting; P=0.05). Two trends were observed: CR tended to be related to C1 (absence of prolonged hunger; P=0.07) and C6 (absence of injuries; P=0.09). For all these associations the higher the score, the higher was the CR (odds ratio 1.21 to 1.24).

Among the covariates taken into account in the models, breed and milk yield were significantly associated with CR. CR was lower in Prim'Holstein cows than in Montbéliardes and other breeds (OR=0.40; P<0.0001) and was reduced in lower producing (≤26.3 kg/d) vs higher producing cows (>32.8 kg/d; OR=0.66; P=0.002).

Discussion

This study aimed to assess the relationship between welfare measured at the herd level and reproductive performance in a population selected to reflect the diversity of French dairy farms. We considered potential confounding factors of our two indicators of reproductive performance: calving to first service interval (CFSI) and calving rate (CR). In most of our herds, cows go to pasture for the grazing period. This is likely to affect their welfare: access to food and water, comfort around resting, cleanliness, integument alterations, lameness, diseases, somatic cell count, expression of social behavior might be modified when grazing. We therefore limited our observations to the indoor period. This reduced the size of our dataset. However, the number of inseminations selected was high enough to investigate relationships between welfare and reproduction performance considering known confounding factors.

The herds included in our sample were slightly larger in size than the average French herd (51 vs. 41 dairy cows, [START_REF] Agreste | Produits agroalimentaires : Bovins, In: Graphagri[END_REF] only parity from 1 to 3. In our analysis, all the culled cows were considered to be not pregnant and we included all ranks of lactation (range from 1 to 13). This might have reduced our estimation of CR [START_REF] Walsh | A review of the causes of poor fertility in high milk producing dairy cows[END_REF].

In our study, none of the herds were classified as excellent after welfare was evaluated and only five herds were not classified (poor welfare, considered to be unacceptable). The absence of excellent herds has already been observed by de [START_REF] De Vries | Evaluating results of the Welfare Quality multi-criteria evaluation model for classification of dairy cattle welfare at the herd level[END_REF] in a sample of 196 herds in The Netherlands even though they were selected to have a wide range of levels of animal welfare. This could mean that the level of dairy herd welfare is low in Europe or that the system of evaluation is not adapted to current dairy cow rearing practices. The low number of poor welfare farms might be explained by the fact that herd recruitment was voluntary in our study. Consequently, the relatively low variability in the welfare score in our sample might have reduced the statistical power of our investigation.

The calving to first service interval was related to the overall welfare category calculated with the Welfare Quality Protocol: the median CFSI was reduced by 11 days in the herds with the best score (enhanced and acceptable in our study) compared to the worst (poor welfare).

This is in favor of a positive relationship between welfare and productivity.

However, CFSI was not significantly associated with the principle or criterion scores were significantly associated to CFSI. Two welfare criteria tented to be associated with CFSI:

Absence of injuries and expression of social behavior. The higher the score for Absence of injuries, the shorter was the median CFSI. Lameness is taken into account in this criterion and a negative relationship with CFSI has already been observed by [START_REF] Fourichon | Effect of disease on reproduction in the dairy cow: a meta-analysis[END_REF] in their meta-analysis and by [START_REF] Hernandez | Effect of lameness on the calving-toconception interval in dairy cows[END_REF] in 837 Holstein cows. For [START_REF] Hernandez | Comparison of the calving-to-conception interval in dairy cows with different degrees of lameness during the prebreeding postpartum period[END_REF], calving to conception interval was increased in lame cows which may be related to an effect on CFSI but also on conception rate. Moreover, [START_REF] Sandgren | Using a national dairy database to identify herds with poor welfare[END_REF], explored the potential of using information recorded in the national database for use as indicators of animal welfare and they identified a relationship between calving interval with a score for injuries and inflammation and animal cleanliness (the better the score the shorter was the calving interval).

This is in agreement with our observation.

Herds with a high frequency of agonistic interactions (taken into account in C9, expression of social behavior) tended to have a shorter CFSI. We can hypothesise that, the shorter the CFSI, the higher was the probability to observe cows in heat in the herd. Indeed, agonistic interactions between cows are observed when they are in oestrus [START_REF] Orihuela | Some factors affecting the behavioural manifestation of oestrus in cattle: a review[END_REF][START_REF] Kerbrat | A proposition for an updated behavioural characterisation of the oestrus period in dairy cows[END_REF][START_REF] Roelofs | When is a cow in estrus? Clinical and practical aspects[END_REF].

Surprisingly, the criterion absence of prolonged hunger estimated by the proportion of very thin cows, was not related to CFSI even though low body condition score at calving and high BCS loss after calving are known to increase the length of postpartum anoestrus in dairy cows [START_REF] Chagas | Invited review: New perspectives on the roles of nutrition and metabolic priorities in the subfertility of high-producing dairy cows[END_REF][START_REF] Crowe | Parturition to resumption of ovarian cyclicity: comparative aspects of beef and dairy cows[END_REF][START_REF] Bedere | Meta-analysis of the relationships between reproduction, milk yield and body condition score in dairy cows[END_REF]. This might be related to the methodology used to estimate this criterion in the Welfare Quality protocol. BCS was evaluated on a sample of lactating and dry cows and pregnant heifers. A very lean cow corresponding to a BCS < 2. The criterion was noted at a herd level and the score is lower than 20 (as in our first quartile) if between 20% to 100% of cows are very lean. In high producing dairy herds, a physiological loss of BCS is observed after calving. A proportion of 20% of thin cows is perhaps not detrimental enough to affect CFSI if only observed in early lactating cows. It might be of interest to differentiate between two criteria in the Welfare Quality Protocol: very low BCS observed in dry cows, pregnant heifers, late lactating vs. early lactating cows. Therefore, it seems that an improvement in animal welfare is likely to reduce the interval between calving and insemination even though no specific aspect of animal welfare seems to have a prominent effect.

In our study, CR rate was not related to the overall welfare category. However, a significant relationship was observed between the principle good housing, the criterion comfort around resting and CR. In our study, this was not related to the type of housing (cubicles vs. straw bedding, data not shown). To our knowledge, the link between cow comfort and CR has not been previously documented. The score for comfort around resting was the main factor which influence the good housing score in our study. The other parameter taken into account to calculate the P2 principle (good housing) is ease of movement which was high in all our herds because all the cows were reared in loose housing systems. CR also tended to be associated with absence of prolonged hunger and absence of injuries which is in agreement with previous observations: low body condition score, lameness and claw disorders have been shown

to be related to low conception rate [START_REF] Fourichon | Effect of disease on reproduction in the dairy cow: a meta-analysis[END_REF][START_REF] Hernandez | Effect of lameness on the calving-toconception interval in dairy cows[END_REF][START_REF] Lopez-Gatius | Effects of body condition score and score change on the reproductive performance of dairy cows: a meta-analysis[END_REF][START_REF] Hernandez | Comparison of the calving-to-conception interval in dairy cows with different degrees of lameness during the prebreeding postpartum period[END_REF][START_REF] Bedere | Meta-analysis of the relationships between reproduction, milk yield and body condition score in dairy cows[END_REF]. All the association or trends observed were positive: CR was higher when welfare scores were high. However, CR seemed only to be related to certain specific aspect of welfare aspect (comfort around resting) and this needs further investigations to be understand.

Finally, CFSI was more closely related to animal welfare measured at herd level than CR. Therefore, the CFSI seems therefore to be more relevant when selecting herds before conducting visits for on farm welfare assessment.

The other risk factors for poor reproductive performance identified in this work have often been observed in other studies. CFSI was related to cow breed, season of calving and season of AI as observed for French cattle in general (Le [START_REF] Mezec | Fertilité des principales races laitières[END_REF]. CR was related to cow breed and milk yield. The breed effect observed in this study has already been described in the French and in the European cattle populations (European Food Safety Authority, 2009;[START_REF] Mezec | Fertilité des principales races laitières[END_REF]. The positive association between milk yield and CR (increase of CR with the increase in milk yield) is not consistent with the generally accepted idea that there is a negative relationship between milk yield and reproductive performance. However, some authors have highlighted potential bias in this association [START_REF] Leblanc | Assessing the association of the level of milk production with reproductive performance in dairy cattle[END_REF]. In our study, the lower category of milk production during the three first months of lactation was very low (≤26.3 kg/d). Animals which produce less than this might have an alteration in health or a nutritional deficiency if the diet is insufficient/unbalanced.

Conclusions

We identified positive associations between good welfare and reproductive performance. CFSI was shorter for the cows bred in herds with enhanced and acceptable overall welfare category and a trend was observed for the association with absence of injuries. CR was not related to overall welfare category but was significantly higher in the farms with a high comfort around resting score. Our study suggests that CFSI might be reduced by better animal welfare. This result is likely to encourage dairy producers to improve welfare on their farms.

Moreover, a long CFSI which is routinely recorded, could be used as a criterion to help in the identification of herds where it would be appropriate look more closely at animal welfare.

Tables Table 1 The Welfare Quality protocol in dairy cattle: observations used to estimate the four principles (P) and the 12 criteria (C) welfare scores.

  reported significant associations between body condition score (BCS) at calving, BCS change between calving and first service, BCS at nadir and number of days open. Days open were significantly reduced in animals with high BCS at calving showed a significant reduction in days open vs.

  Parity ranged from 1 to 13. Maximum and mean daily milk production during the first 3 months of lactation were respectively 32.8 ± 7.8 and 30.1 ± 7.3 kg/d. The main cow characteristics recorded for the cows are presented in Table3. Crossbreeding was used for 16.4% of the AIs (n=649/3951) and sex-sorted semen was used for only 75 AI of the 3951 performed.The median of CFSI was 76 days (mean 81.5±27.7 days; n=2172) and CR was 34.5%

	enhanced (n=1145/2755; 41.6%) and acceptable (n=1526/2755; 55.4%) welfare. Only 3.0%
	(n=84/2755) were bred in the poor welfare herds. The welfare scores observed for the 2755
	cows are described in Supplementary material S1. The sample was comprised of mainly
	Montbéliarde (50.9%) and Prim'Holstein (45.7%) cows. Reproductive performance was for the
	Prim'Holstein, Montbéliarde and other breeds respectively 83, 72, 73 days for the CFSI median

The low appropriate behavior score in the first quartile was related to a high number of head butts or other aggressive events per cow per hour (low score for expression of social behaviors), to the low percentage of cows which could be touched or approached within less than 50 cm (low score for good human-animal relationship) and finally, to negative terms attributed to describe cow behavior (low score for positive emotional state).

The number of cows per herd inseminated during the observation period ranged from one (in one herd) to 56 (in one herd). Most of the cows were bred in the herds classified as and 27.1, 40.9 and 43.2% for the CR. The result from the 95 cows (3.4%) classed as other breeds were grouped with the Montbéliarde cows since their reproductive performance was similar.

  ). We included mainly Prim'Holstein and Montbéliarde cows in our study which are the main two French dairy breeds (66 and 17% of the French population, (France Génétique Elevage, 2016).

The reproductive performance of the cows enrolled in this study were close to that observed for the French cow population (Le

[START_REF] Mezec | Fertilité des principales races laitières[END_REF]

. Mean CFSI was slightly shorter than that observed in the French cow population (98 days for Prim'Holstein and 80 for Montbéliarde cows, (Le Mezec, 2017)). The high proportion of Montbéliarde cows in our sample reduced the mean CFSI observed. Moreover, we excluded CFSIs over to 200 days from our analysis. CR was slightly lower than that observed in the French population (37.1% in Prim'Holstein cows and 49.2% in Montbéliarde ones,

[START_REF] Mezec | Fertilité des principales races laitières[END_REF]

). However, Le Mezec calculated the calving rate without taking into account culled cows. Moreover, they included

Table 2

 2 Welfare scores a recorded in 124 herds according to the Welfare Quality protocol assessment Scores were estimated on a scale of 0 (poor score) to 100 (best score)

	Variable	Minimum	First	Median	Third	Maximum
			quartile		quartile
	Good feeding (P1)	3.5	14.6	43.5	64.8	100.0
	Absence of prolonged hunger (C1)	2.5	27.9	49.3	100.0	100.0
	Absence of prolonged thirst (C2)	3.0	3.0	60.0	100.0	100.0
	Good housing (P2)	35.2	51.6	59.4	66.2	79.8
	Comfort around resting (C3)	0.0	26.1	38.6	49.2	70.9
	Ease of movement (C5)	95.0	95.0	95.0	95.0	95.0
	Good health (P3)	13.4	25.5	29.5	35.8	52.4
	Absence of injuries (C6)	8.7	39.1	55.7	69.2	100.0
	Absence of disease (C7)	11.9	26.6	33.4	44.9	86.1
	Absence of pain induced by	2.0	20.0	20.0	28.0	100.0
	management procedures (C8)
	Appropriate behavior (P4)	6.5	26.6	37.4	44.7	69.0
	Expression of social behaviors (C9)	0.0	20.5	41.3	61.7	95.8
	Expression of other behaviors (C10)	0.0	77.2	82.3	86.5	100.0
	Good human-animal relationship (C11)	13.7	33.5	41.6	51.0	69.6
	Positive emotional state (C12)	1.3	34.9	49.0	66.5	93.3

a

Table 3 512

 3 Characteristics of the 2,755 dairy cows included in the study.

	513
	514
	Variable

Table 4 517

 4 Associations between overall welfare category, principles and criteria scores (measured at herd 518 level) and calving to first service interval (CFSI) in 124 French dairy herds (n= 2172 AI1). The 519 scores were introduced one by one in a multivariate model taking into account breed, parity, 520 season of calving, season of AI, milk production and interactions between milk 521 production*breed and milk production*parity.

	522
				CFSI
		Score	n		HR	95% CI	P
				median
	Overall welfare category	Acceptable	1202	76	Ref	-
		Enhanced	907	75	0.93	0.78 -1.12	0.02
		Poor welfare	63	86	0.68	0.86 -1.06
	Good feeding (P1)	≤14.6	573	75	Ref.	-
							0.65
		>14.6	1599	76	1.05	0.86 -1.28
	Good housing (P2)	≤52.0	601	79	Ref.	-
							0.26
		>52.0	1571	74	1.12	0.92 -1.35
	Good Health (P3)	≤25.5	561	80	Ref.	-
							0.14
		>25.5	1611	75	1.16	0.95 -1.41
	Appropriate behavior	≤26.7	576	77	Ref.	-
							0.67
	(P4)	>26.7	1596	76	0.96	0.79 -1.17
	Absence of prolonged	≤28.9	552	82	Ref.	-
							0.19
	hunger (C1)	>28.9	1620	74	1.15	0.94 -1.40
	Absence of prolonged	≤3.0	573	75	Ref.	-
							0.65
	thirst (C2)	>3.0	1599	76	1.05	0.86 -1.28
	Comfort around resting	≤26.8	601	79	Ref.	-
							0.26
	(C3)	>26.8	1571	74	1.12	0.92 -1.35

See: International Committee for Animal Recording,

International Recording Guidelines. https://www.icar.org/index.php/icar-recording-guidelines/ (Accessed 17 March 2019).

See: The R Project for Statistical Computing https://www.r-project.org (Accessed 19 March 2019)

See : Therneau T., 2015. A package for survival analysis in R. version 2.38 https://CRAN.Rproject.org/package=survival. (Accessed 21 March 2019)

Acknowledgements

The authors thank François Beaudeau (Oniris) for his useful advice on statistical analysis and Andrew Ponter for checking the English language of the manuscript. This work received financial support from Danone Research and pole ESTIVE. We thank the Groupements de Défense Sanitaire (GDS) for providing lists of eligible farms, France Génétique Elevage for access to data and all the farmers who co-operated in this survey. We are also particularly grateful to Eric Delval (INRA), Christophe Mallet (INRA) and Remi Debauchez (ISARA) for their help in data collection, Jean-Yves Audiart (ONIRIS) and Didier Billon (ONIRIS) for their help in data entry, Anne Lamadon (INRA), Yoan Gaudron (INRA) and Pascal Champciaux (INRA) for calculating the welfare scores.

Conflict of interest statement

This work was supported by Danone Research. None of the authors has any other financial or personal relationships that could inappropriately influence or bias the content of the paper.

Appendix: Supplementary material

Supplementary data associated with this article can be found in the online version at


Marion Gabel

Franck Delavoie

Cathy Royer

Tam Tahouly

Stephane Gasman

Marie-France Bader

Nicolas Vitale

Sylvette Chasserot
email: chasserot@inci-cnrs.unistra.fr

Cathy Royer #

Stéphane Gasman

Sylvette Chasserot-Golaz

Phosphorylation cycling of Annexin A2 Tyr23 is critical for calcium-regulated exocytosis in neuroendocrine cells

Keywords:

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

INTRODUCTION

Molecules such as neurotransmitters and hormones are secreted by calcium-regulated exocytosis [START_REF] Tanguy | Lipids implicated in the journey of a secretory granule: from biogenesis to fusion[END_REF][START_REF] Bombardier | Three steps forward, two steps back: mechanistic insights into the assembly and disassembly of the SNARE complex[END_REF]. In neuroendocrine cells, exocytosis implies the recruitment and subsequent fusion of secretory granules at specific sites of the plasma membrane. Annexin A2 (AnxA2) was the first protein identified at these exocytotic sites in chromaffin cells releasing catecholamine [START_REF] Nakata | Conformational change and localization of calpactin I complex involved in exocytosis as revealed by quick-freeze, deep-etch electron microscopy and immunocytochemistry[END_REF]. Since then, AnxA2 was shown to bind two major actors of exocytosis, actin and phospholipids. Electron tomography of chromaffin cells revealed that actin filaments bundled by AnxA2 contribute to the formation of GM1-enriched lipid microdomains required for the spatial organisation of fusion sites at the plasma membrane [START_REF] Bharadwaj | Annexin A2 heterotetramer: structure and function[END_REF][START_REF] Gabel | Annexin A2, an essential partner of the exocytotic process in chromaffin cells[END_REF].

Yet, the mechanism(s) regulating AnxA2 activity during exocytosis is an important aspect that remains poorly understood.

The N-terminal domain of AnxA2 has many potential regulatory sites. These include phosphorylation sites at Tyr23 and Ser25 that have been proposed to control the activity and functions of AnxA2. In chromaffin cells, AnxA2-Ser25 phosphorylation by protein kinase C occurs in response to nicotine stimulation and has been linked to granule exocytosis [START_REF] Gould | The protein-tyrosine kinase substrate p36 is also a substrate for protein kinase C in vitro and in vivo[END_REF][START_REF] Chasserot-Golaz | Annexin II in exocytosis: catecholamine secretion requires the translocation of p36 to the subplasmalemmal region in chromaffin cells[END_REF][START_REF] Delouche | Phosphorylation by protein kinase C of annexin 2 in chromaffin cells stimulated by nicotine[END_REF][START_REF] Regnouf | In vitro" phosphorylation of annexin 2 heterotetramer by protein kinase C. Comparative properties of the unphosphorylated and phosphorylated annexin 2 on the aggregation and fusion of chromaffin granule membranes[END_REF].

AnxA2 has also been identified as the major substrate for the Src family of tyrosine kinases [START_REF] Erikson | Biochemical characterization of a 34kilodalton normal cellular substrate of pp60v-src and an associated 6-kilodalton protein[END_REF]. Src-dependent phosphorylation of AnxA2-Tyr23 has been reported to modulate actin and membrane binding properties of AnxA2 without affecting the tetramer formation with S100A10 [START_REF] Glenney | Calpactins: two distinct Ca++-regulated phospholipid-and actin-binding proteins isolated from lung and placenta[END_REF][START_REF] Gerke | Annexins: linking Ca2+ signalling to membrane dynamics[END_REF][START_REF] Grindheim | Protein phosphorylation and its role in the regulation of Annexin A2 function[END_REF]. Moreover, AnxA2-Tyr23 phosphorylation induces a conformational change that stabilizes AnxA2 at the membrane and promotes the binding of AnxA2 with phosphatidic acid (PA) [START_REF] Morel | Annexin A2 binding to endosomes and functions in endosomal transport are regulated by tyrosine 23 phosphorylation[END_REF], a lipid described as an essential component for exocytosis [START_REF] Morel | Annexin A2 binding to endosomes and functions in endosomal transport are regulated by tyrosine 23 phosphorylation[END_REF]. Chromaffin cells are enriched in tyrosine kinases such as c-Src [START_REF] Parsons | p60c-src activity detected in the chromaffin granule membrane[END_REF] and Fyn [START_REF] Allen | Activation of Fyn tyrosine kinase upon secretagogue stimulation of bovine chromaffin cells[END_REF]. Additionnally, nicotine-induced stimulation of chromaffin cells and catecholamine secretion is accompanied by the activation of pp60c-src kinase [START_REF] Oddie | Modulation of pp60c-src tyrosine kinase activity during secretion in stimulated bovine adrenal chromaffin cells[END_REF][START_REF] Olivares | Src kinases regulate de novo actin polymerization during exocytosis in neuroendocrine chromaffin cells[END_REF] and rapid tyrosine phosphorylation of multiple cellular proteins [START_REF] Cox | Tyrosine kinases are required for catecholamine secretion and mitogen-activated protein kinase activation in bovine adrenal chromaffin cells[END_REF].

Tyr23 phosphorylation of AnxA2 was first reported to be involved in endocytosis, particularly in the internalisation of the insulin receptor [START_REF] Biener | Annexin II is a novel player in insulin signal transduction. Possible association between annexin II phosphorylation and insulin receptor internalization[END_REF]. Phosphorylated Tyr23 seems to be necessary for the stable association of AnxA2 with endosomes, and for their early-stage transport [START_REF] Morel | Annexin A2 binding to endosomes and functions in endosomal transport are regulated by tyrosine 23 phosphorylation[END_REF]. The role of AnxA2-Tyr23 phosphorylation during regulated secretion has not been studied. Here, using phosphodead and phosphomimetic mutants of AnxA2-Tyr23 generated by site-directed mutagenesis and a combination of immunocytochemical and biochemical approaches, we investigated the functional importance of Tyr23 phosphorylation during exocytosis in chromaffin cells. Our results suggest a model in which phosphorylation/dephosphorylation of AnxA2-Tyr23 regulates both lipid microdomains formation and actin filament bundling at exocytotic sites, thereby contributing to the fine tuning of regulated exocytosis.

MATERIALS & METHODS

Antibodies and reagents

Rabbit polyclonal antibodies directed against Anx-A2 (p36) purified from bovine aorta were diluted 1:200 (generous gift from J.C. Cavadore, Inserm U-249). Rabbit polyclonal antibodies directed against dopamine ß-hydroxylase (EC.1.14.17.1: DBH) were diluted 1:75 to specifically label exocytotic sites and 1:300 to label secretory granules in chromaffin cells [START_REF] Chasserot-Golaz | Annexin II in exocytosis: catecholamine secretion requires the translocation of p36 to the subplasmalemmal region in chromaffin cells[END_REF]. Monoclonal antibodies anti-pTyr23-AnxA2 (85.Tyr24) and anti-phosphotyrosine (clone 4G10) were purchased by Santa Cruz and Merck-Millipore respectively. Polyclonal antibodies against GFP (IhIP grade) were from Abcam. Secondary coupled to Alexa Fluor® conjugates (488 or 647) or gold particles were from Molecular Probes (Invitrogen, Cergy Pontoise, France) and Aurion (Wageningen, NL) respectively. Fluorescent cholera toxin B subunit coupled to Alexa Fluor® 598, was from Molecular Probes (Thermo Fisher Scientific Inc., Waltham, MA, USA).

Chromaffin cells

Chromaffin cells were isolated from fresh bovine adrenal glands by retrograde perfusion with collagenase, purified on self-generating Percoll gradients and maintained in culture as previously described [START_REF] Bader | Characterization of hormone and protein release from alpha-toxin-permeabilized chromaffin cells in primary culture[END_REF]. To induce exocytosis, chromaffin cells were washed twice with Locke's solution (140 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl 2 , 1.2 mM KH 2 PO 4 , 1.2 mM MgSO 4 , 11 mM glucose, 0.56 mM ascorbic acid, 0.01 mM EDTA and 15 mM Hepes, pH 7.5), and then stimulated with Locke's solution containing 20 µM nicotine or depolarising solution, K + 59mM.

Quantification of catecholamine secretion

Chromaffin cells maintained in 96-well plates (Thermo Fisher Scientific, France) were briefly washed twice with Locke and processed as stated in the figure legends. Aliquots of the medium were collected at the end of each experiment and cells were lysed with 1% (v/v) Triton X-100 (Sigma, UK). Both sets of samples were assayed fluorimetrically for catecholamine content as previously described [START_REF] Meunier | Glycerotoxin from Glycera convoluta stimulates neurosecretion by up-regulating N-type Ca2+ channel activity[END_REF]. Briefly, 20 µl of sample were transferred to 96-well black-plates (Thermo Fisher Scientific, France), 150 μl of CH3COONa (1M, pH 6) and 15 μl of K3Fe (CN) 6 (0.25%) were added to each well to oxidize catecholamines to adrenochrome. Next 50 µl of NaOH (5M) containing ascorbic acid (0.3 mg/ml) were added, to convert adrenochrome to adrenolutin [START_REF] Heacock | Reduction of adrenochrome with ascorbic acid[END_REF]. The fluorescence emitted by adrenolutin (λex: 430 nm, λem: 520 nm) is measured with a spectrofluorometer (LB940 Mithras, Berthold). For each experiment a standard curve was determined using known concentration of adrenaline and noradrenaline to demonstrate that the values obtained were in the linear range of detection of the assay. Amounts released were expressed as a percentage of the total amount of catecholamine present in the cells.

DNA constructs and cell transfection

Using Quick Change mutagenesis kit (Agilent Technologies, Massy, France), Anx-A2-Y23A-GFP and Anx-A2-Y23E-GFP were generated in human recombinant Anx-A2 fused with GFP [START_REF] Rescher | Annexin 2 is a phosphatidylinositol (4,5)-bisphosphate binding protein recruited to actin assembly sites at cellular membranes[END_REF]. Recombinant GST-Anx-A2 WT GST-Anx-A2-Y23A and GST-Anx-A2-Y23E were produced in E. coli and purified on glutathione-sepharose (GE Healthcare, Upsala, Sweden) as previously described [START_REF] Vitale | ARD1, a 64-kDa bifunctional protein containing an 18-kDa GTP-binding ADP-ribosylation factor domain and a 46-kDa GTPase-activating domain[END_REF]. The purity was estimated at 98% by Coomassie blue staining of SDS-PAGE gels. Plasmids (3 µg) were transfected into chromaffin cells (5 x 10 6 cells) by electroporation (Amaxa Nucleofactor systems, Lonza, Levallois, France) according to the manufacturer's instructions. Electroporated cells were immediately recovered in warm culture medium and plated onto fibronectin-coated glass coverslips. Experiments were performed 48 h after transfection.

In vitro liposomes binding assays

Liposome preparation was performed as described previously [START_REF] Kassas | Comparative Characterization of Phosphatidic Acid Sensors and Their Localization during Frustrated Phagocytosis[END_REF]. Lipids solubilized in chloroform were purchased from Avanti Polar Lipids. Liposome mixtures were prepared in mass ratios composed of 85% PC, 5% PE-NBD, and 10% PA, PS, or PI(4,5)P2. Lipids were dried in a stream of nitrogen gas and kept under vacuum for at least 2 h. Dried lipids were then suspended at a concentration of 1.65 mg/ml in liposome-binding buffer (LBB: 20 mM HEPES, pH 7.4, 150 mM NaCl, 1 mM MgCl2) by three freeze and thaw cycles and were extruded using a Mini-Extruder (Avanti) through polycarbonate track-etched membrane filters to produce liposomes usually 200 nm in diameter. The size distribution of liposomes was estimated by dynamic light scattering using a Zetasizer NanoS from Malvern Instruments equipped with a 4-milliwatt laser. Samples were diluted 1:100 in LBB and analyzed at 20°C using 10 runs, each composed of 14 measurements. Quantification of liposome binding to 330 pmol of GST-Anx-A2 WT GST-Anx-A2-Y23A and GST-Anx-A2-Y23E linked to GSH-Sepharose beads was estimated after 20 min incubation in the dark at room temperature and under agitation with liposomes containing a 10-fold molar excess of PA, PS, or PI(4,5)P2 relative to the quantity of GST proteins in a final volume of 200 μl of LBB. Beads were washed three times with 1 ml of ice-cold LBB and collected by centrifugation at 3000 rpm for 5 min. Liposome binding to beads was estimated by measuring the fluorescence at 535 nm with a Mithras (Berthold) fluorimeter. Triplicate measurements were performed for each condition. Fluorescence measured with GST linked to GSH-Sepharose beads alone was between 3 and 4 A.U. and was subtracted from sample measurements.

In vitro F-actin binding experiments

The F-actin-bundling activity of recombinant GST-Anx-A2s was tested in vitro using the non-muscle actin binding protein Spin-Down Biochem kit (Cat BK013, Cytoskeleton, Denver, CO, USA). Formation of actin bundles was assessed by electron microscopy [START_REF] Ikebuchi | Calcium-dependent regulation of actin filament bundling by lipocortin-85[END_REF], recombinant AnxA2 proteins were incubated with actin filament during 20 min and an aliquots (5 µl) were spread on electron grids, negatively stained with phosphotungstic acid 1% in water. The samples were observed using a Hitachi 7500 transmission electron microscope.

Immunofluorescence and confocal microscopy

For immunocytochemistry, chromaffin cells, grown on fibronectin-coated glass coverslips, were fixed and labelled as described previously [START_REF] Chasserot-Golaz | Annexin II in exocytosis: catecholamine secretion requires the translocation of p36 to the subplasmalemmal region in chromaffin cells[END_REF]. GM1 was labelled on live chromaffin cells incubated for 10 min with 8 µg/ml fluorescent cholera toxin B subunit (coupled with Alexa Fluor® 598) in Locke's solution with or without 20 µM nicotine. The transient accessibility of DBH to the plasma membrane of chromaffin cells was tested by incubating cells for 10 min in Locke's solution containing 20µM nicotine and anti-DBH antibodies diluted to 1:75. F-actin was stained with TRITC-conjugated phalloidin (0.5 µg/ml) for 15 min in the dark at room temperature. Labelled cells were visualised using a Leica SP5II confocal microscope. The amount of cholera toxin or DBH labelling associated with the plasma membrane was measured with ICY software and expressed as the average fluorescence intensity normalised to the corresponding surface area, and divided by the total perimeter of each cell. This allowed a quantitative cell-to-cell comparison of the fluorescence detected in cells.

Plasma membrane sheet preparation and transmission electron microscopy observation

Cytoplasmic face-up membrane sheets were prepared and processed as previously described [START_REF] Umbrecht-Jenck | Chasserot-Golaz, S100A10-mediated translocation of annexin-A2 to SNARE proteins in adrenergic chromaffin cells undergoing exocytosis[END_REF]. Briefly, carbon-coated Formvar films on nickel electron grids were spilled on unstimulated or nicotine-stimulated chromaffin cells. To prepare membrane sheets, a pressure was applied to the grids for 20 s then grids were lifted so that the fragments of the upper cell surface adhered to the grid. These fragments were fixed in 2% paraformaldehyde for 10 min at 4°C immediately after cell stimulation and sheet preparation, which required less than 30 s.

After blocking in PBS with 1% BSA and 1% acetylated BSA, the immune labelling was performed and revealed with 25 nm gold particles-conjugated secondary antibodies. These membrane sheets were fixed in 2.5% glutaraldehyde in PBS, postfixed with 0.5% OsO4, dehydrated in a graded ethanol series, treated with hexamethyldisilazane (Sigma-Aldrich, St.

Louis, MO, USA), air-dried and observed using a Hitachi 7500 transmission electron microscope.

Electron tomography and image processing

Tilt series were acquired on a JEOL JEM-2100 transmission electron microscope operated at 200 kV. Data were automatically acquired using Digital Micrograph software.

Typically, the tilt ranged between -60 and + 60 with 2° angular increments. Images were recorded at a nominal magnification of 5,000 X or 10,000 X on a Gatan Ultrascan 2K×2K CCD camera with a defocus range of -4 µm to -8 µm. Alignments and weighted backprojection-based reconstructions of raw tilt series, using patch tracking algorithm, were computed with IMOD software package [START_REF] Kremer | Computer visualization of threedimensional image data using IMOD[END_REF]. Three-dimensional reconstructions were visualised with UCSF Chimera [START_REF] Pettersen | UCSF Chimera--a visualization system for exploratory research and analysis[END_REF] using the surface or solid rendering mode.

Statistical analysis

As specified in figure legends, group of data are presented as mean (± SEM) or median and were analysed using a Mann-Whitney test. Asterisks in each box and whisker plot indicate statistical significance (*= P < 0.05; **= P < 0.01; ***= P < 0.001).

RESULTS

AnxA2 is phosphorylated on Tyr23 at an early stage of exocytosis

To determine whether AnxA2 is tyrosine phosphorylated during exocytosis, doublelabeling experiments to visualize tyrosine phosphorylated proteins and AnxA2 were performed. We have previously shown that AnxA2 translocates to the periphery of 80% of chromaffin cells after 3 minutes of stimulation with nicotine [START_REF] Chasserot-Golaz | Annexin 2 promotes the formation of lipid microdomains required for calcium-regulated exocytosis of dense-core vesicles[END_REF]. Thus in this condition, we found that nicotinic stimulation of chromaffin cells triggered the translocation of AnxA2 at the plasma membrane together with a significant increase of tyrosine phosphorylated proteins at the cell periphery (Figure 1A), in agreement with a previous report [START_REF] Cox | Tyrosine kinases are required for catecholamine secretion and mitogen-activated protein kinase activation in bovine adrenal chromaffin cells[END_REF]. The colocalization of AnxA2 with tyrosine-phosphorylated protein labeling in the cell periphery (Figure 1A) suggested that AnxA2 might be phosphorylated in the subplasmalemal area of stimulated cells. To test this hypothesis, western-blot analyses were performed using an antibody specifically directed against pTyr23AnxA2 (Figure 1B). Densitometric scanning revealed that the ratio of pTyr23AnxA2/total AnxA2 increased by 5 in stimulated cells, indicating that AnxA2 is phosphorylated on Tyr23 during exocytosis. As shown in Figures 1C andD, Tyr23 phosphorylation preceded the peak of chromogranin A and catecholamine secretion, being maximal after 30 seconds of stimulation. It is interesting to note that AnxA2 was much less phosphorylated at maximal secretion, suggesting that dephosphorylation already starts during the exocytotic process. Altogether, these data demonstrate that a dynamic tyrosine phosphorylation/dephosphorylation cycle of AnxA2 occurs during exocytosis.

Tyr23 phosphorylation of AnxA2 increases in vitro binding to phosphatidylserine (PS) and phosphatidic acid (PA) but decreases in vitro actin bundling activity.

To study the functional importance of AnxA2 phosphorylation on Tyr23, two AnxA2 mutants were generated by site-directed mutagenesis: a phosphodead mutant in which Tyr23 is replaced by an alanine (AnxA2-Y23A) and a phosphomimetic mutant in which Tyr23 is replaced by glutamic acid (AnxA2-Y23E). AnxA2-Wild Type (AnxA2-WT) and the mutants were expressed as recombinant proteins fused to glutathione S-transferase (GST) for in vitro experiments. Since Tyr23 phosphorylation might affect lipid interaction, we examined the binding of AnxA2-WT and mutated AnxA2 to different phospholipids potentially present at the exocytotic sites [START_REF] Ammar | Lipids in Regulated Exocytosis: What are They Doing?[END_REF][START_REF] Chasserot-Golaz | Lipid dynamics in exocytosis[END_REF]. Binding assays were performed with glutathione S-transferase (GST)-fused recombinant AnxA2 proteins incubated with fluorescent liposomes enriched in PIP2, PS or PA, in presence or absence of calcium [START_REF] Kassas | Comparative Characterization of Phosphatidic Acid Sensors and Their Localization during Frustrated Phagocytosis[END_REF]. In line with a previous report [START_REF] Gerke | Annexins: linking Ca2+ signalling to membrane dynamics[END_REF],

AnxA2-WT was found to bind more efficiently to negative phospholipids in the presence of calcium (Figure 2A). Binding of AnxA2 to PIP2 was not significantly modified by the mutations (Figure 2A). In contrast, binding of the phosphomimetic AnxA2-Y23E mutant to PA or PS was enhanced by 2.5 fold compared to AnxA2-WT or to AnxA2-Y23A. This observation is in agreement with previous results showing that AnxA2-Y23E interacts more efficiently than AnxA2-Y23A with liposomes containing PA [START_REF] Morel | Annexin A2 binding to endosomes and functions in endosomal transport are regulated by tyrosine 23 phosphorylation[END_REF]. This suggests that AnxA2 phosphorylation on Tyr23 does not seem to affect PIP2 binding but increased binding to other negatively charged phospholipids such as PA and PS.

Next, the in vitro actin binding and aggregating activities of AnxA2-WT and AnxA2 mutants were compared using GST-coupled recombinant proteins incubated with F-actin in the absence or presence of calcium [START_REF] Gabel | Annexin A2-dependent actin bundling promotes secretory granule docking to the plasma membrane and exocytosis[END_REF]. GST-AnxA2-WT, GST-AnxA2-Y23A and GST-AnxA2-Y23E were able to bind and aggregate F-actin in a calcium-dependent manner (data not shown). Figure 2B illustrates the ultrastructure of the F-actin/Anx-A2 aggregates examined by electron microscopy. GST-AnxA2-WT and GST-AnxA2-Y23A formed actin bundles with linearly ordered filaments. In contrast, GST-AnxA2-Y23E was unable to bundle polymerized actin, which appeared as disordered filaments. To summarize these in vitro observations, the phosphomimetic mutation Y23E enhances the binding of monomeric AnxA2 to PS and PA but prevent its capacity to form actin bundles.

The phosphorylation of Tyr23 is necessary for the formation of lipid microdomains formed at the plasma membrane of chromaffin cells undergoing exocytosis

To examine whether Tyr23 phosphorylation may interfere with the AnxA2-mediated lipid micro-domains formed at the exocytotic sites in stimulated cells [START_REF] Chasserot-Golaz | Annexin 2 promotes the formation of lipid microdomains required for calcium-regulated exocytosis of dense-core vesicles[END_REF], AnxA2-WT and AnxA2 mutants were expressed in chromaffin cells as C-terminal GFP fusion proteins [START_REF] Zobiack | Complex formation and submembranous localization of annexin 2 and S100A10 in live HepG2 cells[END_REF][START_REF] Rescher | Tyrosine phosphorylation of annexin A2 regulates Rho-mediated actin rearrangement and cell adhesion[END_REF] and cells were stimulated in the presence of fluorescent cholera toxin to visualize the formation of GM1 domains [START_REF] Chasserot-Golaz | Annexin 2 promotes the formation of lipid microdomains required for calcium-regulated exocytosis of dense-core vesicles[END_REF]. As illustrated in Figure 3A, cell stimulation triggered equally well the translocation of the three forms of AnxA2 to the cell periphery. Cholera toxin labeling was similar in cells expressing AnxA2-WT or AnxA2-Y23E mutant than in nontransfected cell (control). In contrast, cholera toxin labeling decreased in cells expressing the phosphodead AnxA2-Y23A mutant. Semi-quantitative analysis confirmed that expression of the mutant Y23A reduced GM1 labeling suggesting a defect in lipid micro-domains formation (Figures 3A andB). Altogether these observations suggest that Tyr23 phosphorylation of AnxA2 is implicated in the formation of lipid micro-domains at the plasma membrane of secretagogue-activated chromaffin cells.

Tyr23 phosphorylation of AnxA2 plays a role in exocytosis

To investigate the impact of AnxA2 Tyr23 phosphorylation on secretion, chromaffin cells expressing AnxA2-WT and AnxA2 mutants were stimulated in the presence in the extracellular medium of an antibody directed against dopamine-beta-hydroxylase (DBH) [START_REF] Chasserot-Golaz | Annexin 2 promotes the formation of lipid microdomains required for calcium-regulated exocytosis of dense-core vesicles[END_REF]. As DBH is a luminal secretory granule marker, it becomes accessible to the extracellular anti-DBH antibodies only after full fusion of secretory granules with the plasma membrane, thereby revealing the sites of exocytosis. Figures 4A andB show that expression of phosphodead AnxA2-Y23A and phosphomimetic AnxA2-Y23E decreased cell surface DBH labeling compared to AnxA2-WT or to non-transfected cells by approximately 60%, indicating a significant reduction of exocytotic activity in stimulated cells expressing AnxA2 mutants. These effects are not likely the consequence of a decrease of DBH level in these cells because DBH labelling in permeabilized chromaffin cells expressing the different AnxA2 constructions were not significantly affected (Figure 4C). These results support the idea that the phosphorylation status of the AnxA2 Tyr23 residue is of functional significance for neuroendocrine secretion. The inhibition of secretion observed in cells expressing the phosphodead AnxA2 mutant is in line with the inhibition of lipid domain formation induced by this mutant (Figure 3). The inhibition of secretion observed in cells expressing the phosphomimetic mutant of AnxA2 could be a consequence of its decreased actin bundling activity (Figure 2B).

Tyr23 phosphorylation of AnxA2 affects the recruitment of secretory granules to the plasma membrane

To determine more precisely which stage of exocytosis is controlled by Tyr23 phosphorylation, we prepared plasma membrane sheets from cells expressing the two AnxA2 mutants and counted the number of docked granules. Membrane sheets of transfected cells were identified using an anti-GFP antibody visualized by 25 nm beads [START_REF] Gabel | Annexin A2-dependent actin bundling promotes secretory granule docking to the plasma membrane and exocytosis[END_REF]. As illustrated Figure 5, the number of docked granules is largely decreased in cells expressing the AnxA2-Y23A mutant compared to non-transfected cells (control) or cells expressing AnxA2-WT.

Expression of the phosphomimetic mutant also decreased the amount of docked secretory granule although to a smaller extent. These results together with the decrease of exocytotic activity revealed by DBH labelling (Figure 4) suggest that the phosphorylation status of AnxA2 Tyr23 plays a role in exocytosis by controlling secretory granule docking at the plasma membrane.

At higher magnification, docked granules appeared somewhat different in cells expressing the two AnxA2 mutants (Figure 5C). In cells expressing AnxA2-Y23A, granules were smooth devoid of actin filaments. In cells expressing AnxA2-Y23E, granules displayed actin filaments connecting them to plasma membrane but filaments were much thinner than in cells expressing AnxA2-WT (Figure 5C), in agreement with the in vitro observations indicating that GST-AnxA2-Y23E was unable to form bundles of actin filaments (Figure 3).

To probe this model, electron tomography was performed on plasma membrane sheets obtained from cells expressing the phosphomimetic mutant AnxA2-Y23E. After 3D reconstruction, defects in the actin bundles connecting secretory granules to the plasma membrane clearly appeared in stimulated cells expressing AnxA2-Y23E (Figure 6B) compared to AnxA2-WT cells (Figure 6A). In the phosphomimetic mutant expressing cells, secretory granules appeared linked to the plasma membrane by a thin structure, most likely a thin actin microfilament (Figure 6D) whereas in the AnxA2-WT cells, F-actin connecting granules to the plasma membrane was organized into a complex network of bundles and branches (Figure 6C). These results are consistent with the hypothesis that the phosporylationdephosphorylation cycle of AnxA2 has a key role in the cortical actin rearrangement occurring at the stage of granule docking during exocytosis.

DISCUSSION

During neuroendocrine secretion, AnxA2 has been shown to participate together with the actin cytoskeleton in the formation of lipid microdomains required for docking and fusion of secretory granules with the plasma membrane. [START_REF] Umbrecht-Jenck | Chasserot-Golaz, S100A10-mediated translocation of annexin-A2 to SNARE proteins in adrenergic chromaffin cells undergoing exocytosis[END_REF][START_REF] Chasserot-Golaz | Annexin 2 promotes the formation of lipid microdomains required for calcium-regulated exocytosis of dense-core vesicles[END_REF][START_REF] Gabel | Annexin A2-dependent actin bundling promotes secretory granule docking to the plasma membrane and exocytosis[END_REF]. The N-terminal domain of AnxA2 contains many phosphorylation sites that modulate the three-dimensional structure of the protein and regulate its affinity for different partners [START_REF] Gerke | Annexins: linking Ca2+ signalling to membrane dynamics[END_REF][START_REF] Grindheim | Protein phosphorylation and its role in the regulation of Annexin A2 function[END_REF][START_REF] Ecsedi | Regulation of the Equilibrium between Closed and Open Conformations of Annexin A2 by N-Terminal Phosphorylation and S100A4-Binding[END_REF]. In particular, phosphorylation of Tyr23 has been described to modify AnxA2 binding to membranes and actin [START_REF] Hubaishy | Modulation of annexin II tetramer by tyrosine phosphorylation[END_REF], two major players of the exocytotic machinery [START_REF] Chasserot-Golaz | Annexin 2 promotes the formation of lipid microdomains required for calcium-regulated exocytosis of dense-core vesicles[END_REF][START_REF] Gabel | Annexin A2-dependent actin bundling promotes secretory granule docking to the plasma membrane and exocytosis[END_REF]. Yet, until recently the importance of AnxA2-Tyr23 phosphorylation in the context of exocytosis has never been studied. The present report describes that AnxA2 is phosphorylated on Tyr23 during an early step of the secretory process and provides further evidence that this phosphorylation step is critical for exocytosis. Expression of AnxA2-Tyr23 phosphomimetic and phosphodead mutants in chromaffin cell indicate that a Tyr23 phosphorylation/dephosphorylation cycle is required for both the formation of lipid domains and the docking of secretory granules to the plasma membrane. We propose that AnxA2-Tyr23 is first phosphorylated to promote lipid domain formation required for secretory granule recruitment and subsequently dephosphorylated to facilitate the formation of actin bundles that stably anchor secretory granules to the plasma membrane. On the other hand, phosphorylation/dephosphorylation cycle of the AnxA2-Ser-25 has also been involved in chromaffin cells secretion [START_REF] Delouche | Phosphorylation by protein kinase C of annexin 2 in chromaffin cells stimulated by nicotine[END_REF] and phosphorylation of AnxA2-Ser11 by PKC induces the dissociation of AnxA2 tetramer [START_REF] Bharadwaj | Annexin A2 heterotetramer: structure and function[END_REF]. In the light of these previous studies and on our results concerning the role of phosphorylation/dephosphorylation cycle of Tyr23 during exocytosis, we propose the following sequence of events depicted in Figure 7. Upon cell stimulation, AnxA2 translocates to the plasma membrane and is phosphorylated on Tyr23 to organize lipid microdomains required for the sites of exocytosis. Tyr23 is then dephosphorylated to activate the actin bundling activity and form an actin network that stably anchors docked granules and Ser25 is phosphorylated by PKC for the late fusion step. Finally, phosphorylation on Ser11 triggers the dissociation of the AnxA2 tetramer, the relocation of AnxA2 monomer into the cytoplasm and ends the exocytotic process. Thus, the fine-tuning of AnxA2 functions in the exocytotic machinery is likely to be mediated by the coordinated modification of its three main phosphorylation sites.

Phosphorylation of AnxA2-Tyr23 is necessary for the formation of exocytotic sites

We show here that phosphorylation of AnxA2-Tyr23 is an early event after stimulation, preceding the bulk of chromogranin A and catecholamine secretion. In line, we previously described in chromaffin cells that nicotine triggers within seconds the rapid incorporation of 32 P into AnxA2 [START_REF] Chasserot-Golaz | Annexin II in exocytosis: catecholamine secretion requires the translocation of p36 to the subplasmalemmal region in chromaffin cells[END_REF]. This incorporation of 32 P was primarily observed in Anx2 associated to a detergent-insoluble fraction, an observation suggesting that the translocation of AnxA2 to the cell periphery may precede the phosphorylation process [START_REF] Chasserot-Golaz | Annexin II in exocytosis: catecholamine secretion requires the translocation of p36 to the subplasmalemmal region in chromaffin cells[END_REF].

Indeed, Tyr23 becomes more exposed and accessible when AnxA2 is associated with the plasma membrane [START_REF] Bellagamba | Tyrosine phosphorylation of annexin II tetramer is stimulated by membrane binding[END_REF] and phosphorylation induces a conformational change that stabilizes AnxA2 at the membrane [START_REF] Morel | Annexin A2 binding to endosomes and functions in endosomal transport are regulated by tyrosine 23 phosphorylation[END_REF], further supporting the idea that AnxA2 is phosphorylated just after translocation to the membrane. The fact that the AnxA2-Y23E mutant has a higher affinity for PA and PS than AnxA2-WT, and conversely that the mutant AnxA2-Y23A inhibits the formation of lipid microdomains, led us to conclude that AnxA2 must be phosphorylated to form lipid domains. It is likely that the increased affinity of pTyr23AnxA2 for PA and PS might recruit and locally increase the concentration of these lipids and thus favor the formation of lipid microdomains.

In cells expressing the phosphodead mutant AnxA2-Y23A, analysis of membrane sheets by electron microscopy revealed that the number of granules docked at the plasma membrane was greatly reduced. Docking inhibition might be a direct consequence of the inhibition of lipid platform formation where specific proteins required for efficient docking are concentrated. It is noteworthy that in cells expressing the AnxA2-Y23A mutant, secretory granules docked at the plasma membrane appeared smoother, presumably reflecting the absence of an actin coat [START_REF] Gabel | Annexin A2-dependent actin bundling promotes secretory granule docking to the plasma membrane and exocytosis[END_REF]. Thus, disorganization of the lipid platform due to the absence of AnxA2-Tyr23 phosphorylation results also in a defect of the actin cytoskeleton surrounding granules. A possible explanation is that inhibition or destabilization of lipid micro-domains in the absence of Tyr23 phosphorylation affects also the recruitment of proteins, such as the Rho GTPases, involved in the synthesis of actin filaments and granule coat formation in the late stages of exocytosis [START_REF] Gasman | Involvement of Rho GTPases in calcium-regulated exocytosis from adrenal chromaffin cells[END_REF].

AnxA2-Tyr23 needs to be dephosphorylated in the final stages of exocytosis

In cells expressing AnxA2-Y23E mutant, lipid microdomain formation was apparently not impaired but yet exocytosis was strongly inhibited and less secretory granules were found docked to the plasma membrane. These findings suggest that an AnxA2-Tyr23 dephosphorylation step might be needed to further proceed for exocytosis. Phosphorylation of AnxA2-Tyr23 and Ser25 are mutually exclusive [START_REF] Ecsedi | Regulation of the Equilibrium between Closed and Open Conformations of Annexin A2 by N-Terminal Phosphorylation and S100A4-Binding[END_REF]. Thus, a first explanation might be that Tyr23 needs to be dephosphorylated to allow the phosphorylation of Ser25 required for the late fusion step of exocytosis [START_REF] Delouche | Phosphorylation by protein kinase C of annexin 2 in chromaffin cells stimulated by nicotine[END_REF]. Alternatively, we show here that the AnxA2-Y23E mutant is unable to bundle actin filaments in vitro. Moreover, in stimulated cells expressing the phosphomimetic AnxA2-Y23E mutant, the actin network wrapping docked secretory granule and possibly connecting them to the plasma membrane appeared reduced in size and organization. Thus, an interesting possibility is that dephosphorylation of Tyr23 might be needed to relieve the actin bundling activity of AnxA2. Thereafter, the AnxA2-mediated actin network might be required to stabilize granules docked to the plasma membrane by a minimal tethering machinery and/or to squeeze granules for effective content release after fusion [START_REF] Gabel | Annexin A2-dependent actin bundling promotes secretory granule docking to the plasma membrane and exocytosis[END_REF][START_REF] Toonen | Dissecting docking and tethering of secretory vesicles at the target membrane[END_REF].

Regulation of the AnxA2-Tyr23 phosphorylation/dephosphorylation cycle in the course of exocytosis

The nature of the kinase and phosphatase involved in this phosphorylation/dephosphorylation cycle of the AnxA2-Tyr23 remains to be identified.

AnxA2 is the major substrate of the pp60Src kinase in bovine intestinal epithelial cells [START_REF] Glenney | Amino-terminal sequence of p36 and associated p10: identification of the site of tyrosine phosphorylation and homology with S-100[END_REF].

Other Src family kinases, such as Lyn tyrosine kinase are associated with lipid domains [START_REF] Matsuda | Involvement of Golgi-associated Lyn tyrosine kinase in the translocation of annexin II to the endoplasmic reticulum under oxidative stress[END_REF] and Fyn tyrosine kinase is calcium sensitive and associated with chromaffin cell membranes [START_REF] Spijkers-Hagelstein | Src kinase-induced phosphorylation of annexin A2 mediates glucocorticoid resistance in MLLrearranged infant acute lymphoblastic leukemia[END_REF]. Thus, at first view, the Src kinase family members represent potential candidates.

However, Tyr23 phosphorylation of AnxA2 is an early event of exocytosis, whereas Src kinases have been involved in late stages of exocytosis such as the fusion pore expansion [START_REF] Olivares | Src kinases regulate de novo actin polymerization during exocytosis in neuroendocrine chromaffin cells[END_REF]. Moreover, the Src family tyrosine kinase inhibitor PP2 did not inhibit phosphorylation of AnxA2-Tyr23 in chromaffin cells (data not shown) questioning the role of the Src family kinases in the Tyr23 phosphorylation of AnxA2 during secretion. Another interesting candidate would be the tyrosine kinase Erythropoietin-producing human hepatocellular receptor (EPH) B6 (EPHB6) recently described to regulate catecholamine release in chromaffin cells [START_REF] Wang | EPHB6 and testosterone in concert regulate epinephrine release by adrenal gland chromaffin cells[END_REF] but this remains to be investigated.

An inportant observation is that AnxA2 must be dephosphorylated after formation of lipid domains. Thus, it would also be interesting to identify the phosphatase involved. In chromaffin cells, molecular complexes displaying both tyrosine kinase and tyrosine phosphatase activity have been described at the plasma membrane [START_REF] Van Hoek | Phosphotyrosine phosphatase activity associated with c-Src in large multimeric complexes isolated from adrenal medullary chromaffin cells[END_REF]. In addition, some phosphatases are preferentially localized in lipid domains [START_REF] Simons | Lipid rafts and signal transduction[END_REF], and thus could dephosphorylate AnxA2 at this stage. The calcium-sensitive phosphatase Src homology 2 (SH2)-containing protein tyrosine phosphatase (Shp2) is an interesting candidate because Shp2 can interact with AnxA2 in the presence of cholesterol [START_REF] Yoo | Annexin II binds to SHP2 and this interaction is regulated by HSP70 levels[END_REF][START_REF] Burkart | Regulation of the SHP-2 tyrosine phosphatase by a novel cholesterol-and cell confluence-dependent mechanism[END_REF], Shp2 is highly specific for phosphorylated AnxA2-Tyr23 [START_REF] Wang | DephosSite: a machine learning approach for discovering phosphotase-specific dephosphorylation sites[END_REF] and Shp2 is expressed in chromaffin cells.

CONCLUSION

The multifunctionality of AnxA2 in chromaffin cells is subjected to a particularly complex regulation via ligand binding such as S100A10, subcellular targeting (cytosol versus plasma membrane) and post-translational modifications i.e. phosphorylation and dephosphorylation on tyrosine and serine residues. We show here that two essential functions of AnxA2 during regulated exocytosis i.e. the regulation of lipid micro-domain formation and cytoskeleton organization may be under the control of the phosphorylation/dephosphorylation cycle of Tyr23. The values were expressed in arbitrary units (± SEM ; n ≥35 cells/condition). The control condition corresponds to non transfected cells. Statistical significance for medians was determined using a Mann-Whitney test, there was not statistically significant difference between the different groups. The experiment has been done on two different cell cultures.

Figure 1 :

 1 Figure 1: AnxA2 is phosphorylated on Tyr23 during exocytosis A. Unstimulated (U) or 20 µM nicotine stimulated (S) chromaffin cells were labelled with anti-AnxA2 and anti-P-Tyrosine antibodies and revealed with Alexa Fluor®488-conjugated antirabbit and Alexa Fluor®555-conjugated anti-mouse antibodies, respectively. Images were recorded in the same optical section by a dual exposure procedure. Masks representing the region of co-localization were generated by selecting the double-labelled pixels. Bars = 5µm. B. Lysates obtained from unstimulated (U) or nicotine (20µM)-stimulated (S) chromaffin cells were analyzed by western blot and revealed with anti-actin, anti-AnxA2 and anti-pTyr23-AnxA2 antibodies. Data correspond to a typical experiment representative of 3 independent experiments. C. Kinetics of AnxA2-Tyr23 phosphorylation in response to cell stimulation. Chromaffin cells were stimulated with 20 µM nicotine for the indicated periods of time. Secreted products were collected and cells lysed. Lysates and secreted proteins were analyzed by western blot on a 12% polyacrylamide gel and revealed with anti-chromogranin A (CGA, secreted proteins) or anti-actin and anti-pTyr23-AnxA2 antibodies (lysates). Data correspond to a typical experiment representative of 3 experiments. D. Time course of AnxA2 phosphorylation, chromogranin A and catecholamine secretion in nicotine-stimulated chromaffin cells. Data correspond to the same experiment presented pannel C, and are representative of 3 independent experiments.

Figure 2 :Figure 3 :Figure 4 :

 234 Figure 2: Characterization of AnxA2 mutants A. Semi-quantitative fluorescent liposome assays performed with GST-AnxA2-beads and liposomes (5% PE-NBD, 85% DOPC, and 10% PIP 2 or PS or PA). Binding of liposomes to GST-AnxA2 beads was estimated by measuring the total fluorescence and subtracting the background (see under "Materials and Methods"). Fluorescence measured when liposomes were incubated with GST alone was around 3 A.U. and was subtracted. Results are presented as means ± S.D. Triplicate measurements pooled from a typical experiment representative of 5 experiments. B. Electron micrographs showing recombinant Anx-A2/F-actin aggregates. Purified recombinant Anx-A2 proteins fused with GST (4µM) were incubated with pre-formed actin filaments (18µM) for 30 min at room temperature. An aliquot (5µl) was spread on electron grids and prepared for electron microscopy. Data are representative of 2 samples analysed in 3 independent experiments. Bars = 50nm.

Figure 5 :Figure 6 :

 56 Figure 5 : Phosphorylation state of AnxA2-Tyr23 affects docking of secretory granules. A. Representative electron micrographs of plasma membrane sheets prepared from stimulated chromaffin cells transfected with AnxA2-WT-GFP (WT), AnxA2-Y23A-GFP (Y23A), or AnxA2-Y23E-GFP (Y23E). Transfected cells were labelled with anti-GFP antibodies and revealed with 25 nm gold particles. Bars = 500nm. B. Number of granules morphologically docked on plasma membrane sheets prepared from stimulated non transfected cells (Cont) or stimulated cells expressing A2-WT, A2-Y23A, or A2-Y23E (± SEM n > 30 images). Asterisks indicate statistical significance for medians (black line) determined using a Mann-Whitney test; the dotted lines represent the means. Images were acquired from three different culture preparations. C. Representative electron micrographs at higher magnification of plasma membrane sheets prepared from stimulated cells transfected with the indicated AnxA2 constructs. Bars = 100 nm.

Figure 7 :

 7 Figure 7 : Multi-functions of AnxA2 during exocytosis : control by Tyr and Ser phosphorylationsUpon cell stimulation, AnxA2 is recruited to the plasma membrane to form a tetramer with the S100A10 protein. At the plasma membrane, AnxA2 phosphorylated on Tyr23 stabilizes lipid microdomains required to recruit/organize the priming/docking machinery. Tyr23 dephosphorylation is then needed to promote the formation of actin bundles that strongly anchor secretory granules to the exocytotic sites and to allow Ser25 phosphorylation necessary to secretory granule fusion. During this step, AnxA2 and actin bundles regulate the fusion pore opening and participate in the expulsion of the granular content. Finally, phosphorylation of AnxA2-Ser 11 leads to the dissociation of the heterotetramer and the release of the AnxA2 monomer in cytoplasm.

ACKNOWLEDGEMENTS

We are grateful to V. Gerke for generous gift of AnxA2-GFP. We thank the municipal slaughterhouse of Haguenau (France) to provide bovine adrenal glands. We acknowledge N.

Grant for helpful comments and critical reading of the manuscript, B Lorber for dynamic light scattering to control liposome size. This work was supported by CNRS, Université de Strasbourg, Inserm and by a grant from Fondation pour la Recherche Médicale to N.V.