Anthony Estienne

Belén Lahoz

Peggy Jarrier-Gaillard

Loys Bodin

José Folch

José-Luis Luis Alabart

Stéphane Fabre

Danielle Monniaux
email: danielle.monniaux@inra.fr

Peggy Jarrier

BMP15 regulates the inhibin/activin system independently of ovulation rate control in sheep

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

elements, and the differential regulation of these elements by BMP15 and activin can explain that the effects of BMP15 mutations differ when present in different genetic backgrounds. In conclusion, the ovarian inhibin/activin system is unlikely to participate in the increase of ovulation rate associated with BMP15 mutations in sheep.

Introduction

Increasing evidence support the involvement of cytokines belonging to the transforming growth factor beta (TGFß) family in the control of ovulation number at each sexual cycle (ovulation rate) in adult females. In particular, the bone morphogenetic proteins BMP15 (bone morphogenetic protein 15) and GDF9 (growth differentiation factor 9), known to be specifically expressed in the oocyte [START_REF] Mcgrath | Oocyte-specific expression of growth/differentiation factor-9[END_REF]Dube et al. 1998), could play a key role in this process. In sheep, heterozygous carriers of various missense mutations affecting BMP15 or GDF9, or carriers of a partial loss-of-function mutation in their receptor BMPR1B, exhibit systematically increased ovulation rates and litter sizes (reviews: (Montgomery et al. 2001;Galloway et al. 2002;McNatty et al. 2003;Fabre et al. 2006;Persani et al. 2014). Polymorphisms in GDF9, BMPR1B and SMAD3 (encoding a BMP15/GDF9 signaling factor) have also been shown to occur among human cohorts with a propensity for dizygotic twins (Palmer et al. 2006;Hoekstra et al. 2008;[START_REF] Luong | Variation in BMPR1B, TGFRB1 and BMPR2 and control of dizygotic twinning[END_REF][START_REF] Mbarek | Identification of Common Genetic Variants Influencing Spontaneous Dizygotic Twinning and Female Fertility[END_REF] and the presence of non-synonymous substitutions in BMP15 or GDF9 genes has been recently associated with twinning in callitrichine primates (Harris et al. 2014).

From a phylogenetic analysis comparing mono-and poly-ovulating mammalian species, high variations have been observed in specific areas of BMP15 and GDF9, able to modify the biological activity of the proteins in poly-ovulating mammals (Monestier et al. 2014).

The mechanism by which BMP15 and GDF9 control the ovulation rate is not fully understood. BMP15, acting alone or in synergy with GDF9, can modulate follicle growth and maturation by controlling granulosa cell proliferation and their responsiveness to FSH (Otsuka et al. 2000;Otsuka et al. 2001;[START_REF] Liao | Effect of intracellular interactions on the processing and secretion of bone morphogenetic protein-15 (BMP-15) and growth and differentiation factor-9. Implication of the aberrant ovarian phenotype of BMP-15 mutant sheep[END_REF]McNatty et al. 2005b;Moore & Shimasaki 2005). Recently, the anti-Müllerian hormone (AMH), another member of the TGFß family known to importantly modulate FSH sensitivity (Durlinger et al. 2001;Visser & Themmen 2005;Knight & Glister 2006;Visser & Themmen 2014), has also been proposed to Comment citer ce document : Estienne, A., Lahoz, B., Jarrier, P., Bodin, L., Folch, J., Alabart, J. L., Fabre, S., [START_REF] Estienne | BMP15 regulates the inhibin/activin system independently of ovulation rate control in sheep[END_REF]. BMP15 regulates the inhibin/activin system independently of ovulation rate control in sheep. Reproduction, 153 (4), 395-404. DOI : 10.1530/REP-16-0507 4 participate in this mechanism. Indeed, BMP15 enhances the expression of AMH and its specific receptor AMHR2 in the granulosa cells of sheep antral follicles, and hyperprolific ewes carrying a loss-of-function mutation in BMP15 or its receptor BMPR1B show respectively impaired expression of AMHR2 or AMH (Estienne et al. 2015;Pierre et al. 2016). It is suggested that low BMP15 and low AMH signaling in follicles of these mutant ewes contribute to sensitizing granulosa cells to FSH, thereby promoting the selection and maintenance of follicles during the follicular phase, and thus enhancing the ovulation rate.

Inhibins and activins, other members of the TGFß family, could also be involved in the mechanisms leading to multiple ovulations. Inhibins and activins are produced by the granulosa cells of growing follicles and known to act in an opposite way on their target cells.

Activins are importantly implicated in follicular growth, acting in an intra-ovarian way by promoting granulosa cell proliferation, enhancing FSHR (follicle stimulating hormone receptor) and CYP19A1 (cytochrome P450 family 19 subfamily A member 1, also known as aromatase) expression, and inhibiting follicle atresia and luteinization, whereas inhibins oppose the activin signaling, thus modulating these processes (Knight et al. 2012). Inhibins are secreted by the largest follicles during terminal follicular development and are important inhibitors of FSH secretion by pituitary gonadotrophs. In various animal species, immunization against inhibin induces multiple ovulations and an increase in pituitary secretion of FSH appears to be the main mechanism by which the growth of additional follicles is stimulated (Henderson et al. 1984;Forage et al. 1987;Hillard et al. 1995;[START_REF] Drummond | Animal models of inhibin action[END_REF]Yan et al. 2015). However, immunization against inhibin is not always accompanied by the expected rise in plasma FSH, and intra-ovarian effects of neutralizing endogenous inhibin bioactivity might also be involved in the stimulation of follicular development (Tannetta et al. 1998). Interestingly, the specific intra-ovarian glycosylation of the inhibin alpha and beta A subunits is associated with multiple ovulations

Comment citer ce document : Estienne, A., Lahoz, B., Jarrier, P., Bodin, L., Folch, J., Alabart, J. L., Fabre, S., [START_REF] Estienne | BMP15 regulates the inhibin/activin system independently of ovulation rate control in sheep[END_REF]. BMP15 regulates the inhibin/activin system independently of ovulation rate control in sheep. Reproduction, 153 (4), 395-404. DOI : 10.1530/REP-16-0507 5 and high prolificacy in ewes carrying the FecL L mutation which induces an ectopic expression of the B4GALNT2 (beta-1,4-N-acetyl-galactosaminyl transferase 2) gene within the ovary [START_REF] Drouilhet | The highly prolific phenotype of Lacaune sheep is associated with an ectopic expression of the B4GALNT2 gene within the ovary[END_REF].

We assumed that the ovarian inhibin/activin system could mediate part of the effect of BMP15 mutations in the regulation of ovulation rate in sheep. This assumption is supported by the findings that BMP15 can activate in vitro the transcription of genes encoding inhibin subunits and follistatin (the specific activin binding protein) in mouse granulosa cells [START_REF] Li | Stable expression and characterization of N-terminal tagged recombinant human bone morphogenetic protein 15[END_REF]) and cooperates with GDF9 in enhancing immunoreactive inhibin production in rat granulosa cells (McNatty et al. 2005a). To answer this question, we have analyzed the in vivo effects of two natural loss-of-function mutations of BMP15 in two ovine breeds on the expression of components of the inhibin/activin system in relationship with the hyperprolific phenotype of mutant ewes. We took into consideration various components of this system, including the INHA and INHBA subunits, the activin signaling receptors ACVR1B and ACVR2A and the known antagonists of activin, which are follistatin (FST) and the TGFß type 3 receptor (TGFBR3, also known as betaglycan, a co-receptor of inhibin, able to block activin signaling) (Walton et al. 2012). In addition, we studied the acute in vitro effects of BMP15 on the mRNA expression of these components in ovine granulosa cells, in comparison with those of other factors of the TGFß family such as BMP4, GDF9 and Activin A.

Materials and Methods

Animals

For in vivo expression studies, granulosa cells were recovered from hyperprolific ewes carrying mutations in the BMP15 gene and their corresponding non-carrier ewes of the same breed. The BMP15 W154NFsX55 /FecX R mutation is a 17 bp deletion creating a premature stop

Comment citer ce document : Estienne, A., Lahoz, B., Jarrier, P., Bodin, L., Folch, J., Alabart, J. L., Fabre, S., Monniaux, D. (2017). BMP15 regulates the inhibin/activin system independently of ovulation rate control in sheep. Reproduction, 153 (4), 395-404. DOI : 10.1530/REP-16-0507 6 codon in the BMP15 pro-region; it causes hyperprolificacy in the Spanish Rasa Aragonesa breed when the mutation is present in ewes at the heterozygous state and sterility when present at the homozygous state [START_REF] Martinez-Royo | A deletion in the bone morphogenetic protein 15 Comment citer ce document[END_REF]. The BMP15 T317I /FecX Gr mutation is a substitution of a threonine for an isoleucine which affects the hydrophobicity of the protein and impairs its signaling activity; it causes hyperprolificacy in the French Grivette breed when the mutation is present at the heterozygous state and even more at the homozygous state [START_REF] Demars | Genome-wide association studies identify two novel BMP15 mutations responsible for an atypical hyperprolificacy phenotype in sheep[END_REF]

Collection and culture of granulosa cells

Collected ovaries were finely dissected to isolate all the antral follicles larger than 1 mm in diameter. For each animal, the follicles were then classified according to their size and genotype. For the evaluation of in vivo gene expression in Rasa Aragonesa and Grivette ewes,

Comment citer ce document : Estienne, A., Lahoz, B., Jarrier, P., Bodin, L., Folch, J., Alabart, J. L., Fabre, S., Monniaux, D. (2017). BMP15 regulates the inhibin/activin system independently of ovulation rate control in sheep. Reproduction, 153 (4), 395-404. DOI : 10.1530/REP-16-0507 7 two follicular classes were defined, small (1-3 mm) and large (> or equal to 5 mm) follicles, corresponding to follicle growth and dominance stages, respectively. Follicular fluid and granulosa cells were recovered from small and large follicles as described (Estienne et al. 2015). For small follicles, follicular fluid and granulosa cells were pooled within animal. The volumes of follicular fluid recovered by follicular puncture from each animal were at least 50 µl for individual large follicles and 5 µl for pooled small follicles. Follicular fluids and granulosa cells were stored at -80°C for further analysis.

For culture experiments, ovaries were recovered from ewes of various breeds wildtype at the two BMP15 loci after slaughtering in a local slaughterhouse at Nouzilly (France).

Granulosa cells from 1-3 mm follicles were cultured in serum-free conditions in McCoy's 5a medium (Sigma, L'Isle d'Abeau Chesnes, France) containing insulin (100 ng/ml) according to a previously described method (Campbell 1996). Cells were seeded at 10 5 viable cells/well in 96-well plates with and without recombinant human BMP4, human BMP15, mouse GDF9 or human/mouse/rat Activin A (50 ng/ml each; R&D System Europe, Lille, France), each treatment being tested in 12 replicate wells. After 48 h of culture, cells were recovered using accutase (Sigma), then pooled according to treatment, centrifuged, and cell pellets were stored at -80°C for further RNA extraction. In total, 15 independent culture experiments were performed.

RNA extraction and quantitative real-time PCR

Samples of granulosa cell recovered in vivo and after culture were analyzed for mRNA content of genes encoding components of the inhibin/activin system (INHA, INHBA, ACVR1B, ACVR2A, FST, TGFBR3). For in vivo gene expression studies, the stage of follicular maturation and health was assessed using classic markers of differentiation (CYP19A1) and atresia (IGFBP5) in ovine granulosa cells [START_REF] Besnard | Expression of messenger ribonucleic acids of insulin-like growth factor binding protein-2, -4, and -5 in the ovine ovary: localization and changes during growth and atresia of antral follicles[END_REF][START_REF] Logan | Onset of steroidogenic enzyme gene expression during ovarian follicular development in sheep[END_REF]. Total RNA was extracted with a Nucleospin RNA II kit (Macherey-Nagel, Hoerdt, ]. RPL19 is known to be easily detectable and not regulated during ovine follicular growth [START_REF] Bonnet | Transcriptome profiling of sheep granulosa cells and oocytes during early follicular development obtained by laser capture microdissection[END_REF] and it has been used and validated as a reference gene in the granulosa cells of sheep follicles in previous studies [START_REF] Drouilhet | The highly prolific phenotype of Lacaune sheep is associated with an ectopic expression of the B4GALNT2 gene within the ovary[END_REF]Estienne et al. 2015;Pierre et al. 2016).

Inhibin and activin assays

Inhibin A concentration in follicular fluid was measured using the Active Inhibin A ELISA kit (Beckman Coulter France) according to the manufacturer's protocol. The use of the Active Inhibin A ELISA kit in sheep has been validated by studying serial dilutions of different ovine plasma and follicular fluid samples in steer plasma. Results showed that the dilution curves were linear and parallel to the standard curve (Supplementary Figure 1A). In the present study, Inhibin A concentrations were determined in 30 µl aliquots of follicular fluid diluted at 1/100 and 1/1000 for small and large follicles, respectively. In our working conditions, the limit of detection of the assay was found to be 0.5 pg/well and the intra-and inter-assays coefficients of variation were lower than 5%.

Activin A concentration in follicular fluid was measured using the Human/Mouse/Rat Activin A Quantikine ELISA kit (R&D Systems Europe LTD) according to the manufacturer's protocol. The use of the Human/Mouse/Rat Activin A Quantikine ELISA kit in sheep has been

Comment citer ce document : Estienne, A., Lahoz, B., Jarrier, P., Bodin, L., Folch, J., Alabart, J. L., Fabre, S., Monniaux, D. (2017). BMP15 regulates the inhibin/activin system independently of ovulation rate control in sheep. Reproduction, 153 (4), 395-404. DOI : 10.1530/REP-16-0507 9 validated by studying serial dilutions of different ovine plasma and follicular fluid samples in steer plasma. Results showed that the dilution curves were linear and parallel to the standard curve (Supplementary Figure 1B). In the present study, Activin A concentrations were determined in 100 µl aliquots of follicular fluid diluted at 1/1000 and 1/10000 for small and large follicles, respectively. In our working conditions, the limit of detection of the assay was found to be 1.5 pg/well and the intra-and inter-assays coefficients of variation were lower than 5% and 7%, respectively.

Statistics

All data are presented as mean ± SEM. Data were analyzed using the GraphPad Prism 6 Software (GraphPad Software Inc., La Jolla, CA, USA). In the wild-type (+/+) ewes, comparisons of follicle numbers and sizes between the Rasa Aragonesa and the Grivette breeds were made using Mann Whitney test, and comparisons of gene expression and hormonal concentrations in follicular fluid between breeds and follicular size classes were made using two-way ANOVA. Within each ovine breed, gene expression and hormonal concentrations were compared between follicular classes and genotypes using two-way ANOVA. The ANOVA analyses were followed by Fisher's protected Least Significant Difference (LSD) test for multiple comparisons. For granulosa cell culture experiments, data were analyzed using repeated measures one-way ANOVA with the Greenhouse-Geisser correction of the degrees of freedom. For all analyses, a probability of at least 0.05 was required for significance.

Results

Follicular development in the Rasa Aragonesa and Grivette breeds and effects of BMP15 mutations

Comment citer ce document : Estienne, A., Lahoz, B., Jarrier, P., Bodin, L., Folch, J., Alabart, J. L., Fabre, S., Monniaux, D. ( 2017). BMP15 regulates the inhibin/activin system independently of ovulation rate control in sheep. Reproduction, 153 (4), 395-404. DOI : 10.1530/REP-16-0507 10

There was no difference between the Rasa Aragonesa and Grivette breeds for the numbers and sizes of follicles present in the ovaries of the wild-type (+/+) ewes, but follicular development was affected by the presence of BMP15 mutations in both breeds. In the Rasa Aragonesa breed, the number of large dominant follicles (> or equal to 5 mm in the follicular phase of the estrous cycle) was higher in the prolific R/+ than in the non-prolific +/+ ewes (p<0.05) and the mean size of these large follicles was smaller (p<0.01, Table 2). In the Grivette breed, the number of large follicles was not statistically different between genotypes, although the mean size of the large follicles was smaller in the prolific Gr/Gr ewes than in the +/+ ewes (p<0.001, Table 2). In both breeds, the number of small (1-3 mm) follicles was similar between genotypes.

In +/+ ewes, there was no difference between the Rasa Aragonesa and Grivette breeds for CYP19A1 (a marker of follicle maturation) and IGFBP5 (a marker of follicle atresia) expression in granulosa cells. As expected, in both breeds and all genotypes, CYP19A1 mRNA levels were strongly increased in the granulosa cells of the large follicles, compared to the small ones (p<0.001 for all comparisons, Figure 1). This increase in granulosa cell differentiation was associated with a lower expression of the atresia marker IGFBP5 in the large follicles (Rasa Aragonesa breed: p<0.001 for both +/+ and R/+ follicles; Grivette breed: p<0.05 for +/+ follicles and p<0.01 for Gr/Gr follicles). There was no difference between genotypes, except a 1.9-fold lower CYP19A1 expression in R/+, compared to +/+ large follicles, in the Rasa Aragonesa breed (p<0.05, Figure 1).

Ovarian expression of Inhibin A and Activin A in the Rasa Aragonesa and Grivette breeds and effects of BMP15 mutations

The mRNA levels of INHA and INHBA were higher (p<0.001 for both genes) in +/+ granulosa cells from large follicles of the Grivette, compared to the Rasa Aragonesa breed. In both breeds and all genotypes, INHA and INHBA mRNA levels were strongly increased in the

Comment citer ce document : Estienne, A., Lahoz, B., Jarrier, P., Bodin, L., Folch, J., Alabart, J. L., Fabre, S., Monniaux, D. ( 2017). BMP15 regulates the inhibin/activin system independently of ovulation rate control in sheep. Reproduction, 153 (4), 395-404. DOI : 10.1530/REP-16-0507 11 granulosa cells of the large follicles, compared to the small ones (p<0.001 for both genes and all comparisons, Figure 2). Within each breed, there was no difference between genotypes for INHA and INHBA mRNA levels.

The intra-follicular concentrations of Inhibin A and Activin A were both higher (p<0.001 and p<0.01, respectively) in +/+ large follicles of the Grivette, compared to the Rasa Aragonesa breed, but the Activin A / Inhibin A concentration ratio was similar between breeds. In both breeds and all genotypes, the intra-follicular concentrations of Inhibin A were higher in the large follicles, compared to the small ones (p<0.001 for all comparisons) and, except for the Rasa Aragonesa +/+ ewes, the intra-follicular concentrations of Activin A were also significantly higher in the large follicles (p<0.05 for all comparisons, Figure 3). In both breeds and all genotypes, the Activin A / Inhibin A concentration ratio was lower in the large follicles, compared to the small ones (p<0.001 for all comparisons). No significant differences were found between genotypes, except a 2.3-fold higher concentration of Inhibin A in R/+, compared to +/+ large follicles, in the Rasa Aragonesa breed (p<0.05, Figure 3).

Ovarian expression of activin receptors and activin/inhibin binding proteins in the Rasa Aragonesa and Grivette breeds and effects of BMP15 mutations

The mRNA levels of ACVR1B and ACVR2A were higher in +/+ granulosa cells from large follicles (p<0.001 for both genes) and small follicles (p<0.05 and p<0.001 for ACVR1B

and ACVR2A, respectively) of the Grivette, compared to the Rasa Aragonesa breed. In the Grivette breed, ACVR1B and ACVR2A mRNA levels were higher in the granulosa cells of the large follicles, compared to the small ones for both genotypes (ACVR1B and ACVR2A: p<0.001 for +/+ follicles and p<0.05 for Gr/Gr follicles, Figure 4), but there was no significant difference between follicular sizes in the Rasa Aragonesa breed, except higher ACVR2A mRNA levels in the large R/+ follicles, compared to the small ones of the same genotype (p<0.01). In the Grivette breed, ACVR1B mRNA levels were 1.9-fold lower

Comment citer ce document : Estienne, A., Lahoz, B., Jarrier, P., Bodin, L., Folch, J., Alabart, J. L., Fabre, S., Monniaux, D. ( 2017). BMP15 regulates the inhibin/activin system independently of ovulation rate control in sheep. Reproduction, 153 (4), 395-404. DOI : 10.1530/REP-16-0507 12 (p<0.01) and ACVR2A mRNA levels were 1.6-fold lower (p<0.01) in Gr/Gr, compared to +/+ large follicles (Figure 4), but no difference between genotypes was observed in the Rasa Aragonesa breed.

The mRNA levels of FST and TGFBR3 were lower (p<0.05 and p<0.01, respectively) in +/+ granulosa cells from large follicles of the Grivette, compared to the Rasa Aragonesa breed. In both breeds and all genotypes, FST and TGFBR3 mRNA levels were increased in the granulosa cells of the large follicles, compared to the small ones (FST: p<0.001 for both breeds and all genotypes, TGFBR3: p<0.001 for both genotypes in Rasa Aragonesa breed, p<0.001 for +/+ and p<0.01 for Gr/Gr genotypes in Grivette breed, Figure 5). FST mRNA levels were 1.6-fold lower (p<0.05) in R/+, compared to +/+ small follicles in the Rasa Aragonesa breed (Figure 5), but no difference in FST expression was observed between genotypes in the Grivette breed. TGFBR3 mRNA levels were 1.4-fold lower (p<0.05) in

Gr/Gr, compared to +/+ large follicles in the Grivette breed (Figure 5), but no difference between genotypes was observed in the Rasa Aragonesa breed.

Regulation of the components of the inhibin/activin system by BMP15 and Activin A in ovine granulosa cells in vitro

The small (< 3-fold changes), or non-existent differences observed between genotypes and the inconsistency of the changes found associated with the presence of BMP15 mutations between breeds led us to investigate whether the components of the inhibin/activin system could be direct target genes of BMP15. As BMP15 is known to act alone or in interaction with GDF9, and thus activate different SMAD signaling pathways involved in BMP or/and activin signaling (Mottershead et al. 2015), the effects of BMP15 and GDF9, acting alone or in combination, were compared with those of BMP4 (a canonical inducer of the BMP signaling pathway) and Activin A (Figure 6). In 48h

Discussion

To our knowledge, this is the first study dedicated to the regulation by BMP15 of the ovarian expression of the whole inhibin/activin system, i.e. inhibin and activin subunits, activin receptors and antagonists, based on complementary investigations in vitro and in vivo.

We have identified the direct target genes of BMP15 among these components and evaluated the consequence of the presence of BMP15 loss-of-function mutations on their ovarian expression in two different genetic models of hyperprolific sheep.

In vitro, the effects of BMP15, BMP4 and Activin A itself were compared on the expression of the different components of the inhibin/activin system. In ovine granulosa cells recovered from small antral follicles, BMP15 (acting alone or in combination with GDF9), In vivo, the FecX R mutation, when present at the heterozygous state in the Spanish Rasa Aragonesa breed, was associated with a 1.9-fold increase in ovulation rate, compared to the ovulation rate of non-carrier ewes of the same breed, whereas the recently discovered 1985). However, there was no clear effect of the FecX R and the FecX Gr mutations on CYP19A1 mRNA levels in the granulosa cells recovered from the two studied follicular size classes, i.e. small (1-3 mm) and large (> or equal to 5 mm) follicles. It is possible that the choice of these follicular classes did not allow detecting any fine difference in the maturation stage of the granulosa cells between genotypes. However, the intermediary class of 3-5 mm follicles was discarded from analysis, due to the high expression of IGFBP5 and other

Comment citer ce document : Estienne, A., Lahoz, B., Jarrier, P., Bodin, L., Folch, J., Alabart, J. L., Fabre, S., Monniaux, D. ( 2017 The components of the inhibin/activin system were affected by the presence of a lossof-function BMP15 mutation differently in the Rasa Aragonesa and the Grivette breed. For example, in the granulosa cells of the large follicles, ACVR1B, ACVR2A and TGFBR3 mRNA levels were lower in the homozygous FecX Gr , compared to non-carrier Grivette ewes, but such differences were not observed between the heterozygous FecX R and non-carrier Rasa Aragonesa ewes. Other examples of discrepancies could be cited, concerning Inhibin A concentrations in large follicles and FST expression in the granulosa cells of small follicles.

These discrepancies might be related to the mutation itself or its homozygous or heterozygous state. The FecX R mutation is known to preclude BMP15 production (Martinez-Royo et al.

2008

) and the FecX Gr mutation totally impairs BMP15 signaling in target cells [START_REF] Demars | Genome-wide association studies identify two novel BMP15 mutations responsible for an atypical hyperprolificacy phenotype in sheep[END_REF]. In both models, a lowering of the biological activity of BMP15 is expected, due to lower amounts of the mature protein (as hypothesized in R/+ follicles) or lower functional interactions between BMP15 and GDF9 (as hypothesized in Gr/Gr follicles), but the TGFBR3 mRNA than the granulosa cells of non-mutant Rasa Aragonesa ewes. Consistently, the concentrations of Activin A and Inhibin A proteins were higher in the follicles of Grivette ewes, also illustrating pre-existing differences in the inhibin/activin system between breeds, in the absence of BMP15 mutation.

The complexity of the inhibin/activin system may explain that (1) the effects of BMP15 on granulosa cells in vitro are not consistent with the expressional changes observed when a loss-of-function BMP15 mutation is present in vivo (2) the consequences of a BMP15 mutation differ between breeds. Activin A and its receptors are positive elements of activin signaling, whereas inhibin, follistatin and the inhibin co-receptor called TGFBR3 (or betaglycan) are antagonistic elements. From our in vitro results, BMP15 and Activin A itself can acutely regulate the expression of both positive and negative components of the inhibin/activin system. We propose that a re-balancing of the components of this system in vivo can explain the small effects of BMP15 loss-of-function mutations and the inconsistency of the changes found associated with the presence of BMP15 mutations in different genetic backgrounds. Therefore, the inhibin/activin system would not be implicated in the mechanisms leading to multiple ovulations in ewes carrying BMP15 mutations, in contrast to the recently evidenced AMH system (Pierre et al. 2016).

In conclusion, the presence of a loss-of-function BMP15 mutation, such as the FecX R mutation when present at the heterozygous state, or the FecX Gr mutation when present at the homozygous state, is associated with a clear increase in ovulation rate leading to a higher

Comment citer ce document : Estienne, A., Lahoz, B., Jarrier, P., Bodin, L., Folch, J., Alabart, J. L., Fabre, S., Monniaux, D. ( 2017). BMP15 regulates the inhibin/activin system independently of ovulation rate control in sheep. Reproduction, 153 (4), 395-404. DOI : 10.1530/REP-16-0507

prolificacy. In this study, we have challenged a potential role of the ovarian inhibin/activin system in participating in this phenotype. From our in vitro results, the genes encoding different components of the inhibin/activin system such as INHA, INHBA, FST and TGFBR3 are potential targets of BMP15 in ovine granulosa cells. However, in vivo, the presence of a loss-of-function BMP15 mutation in ewes was not found associated with strong changes in the expression of these components in granulosa cells. Moreover, the observed expressional changes were dependent on either the BMP15 mutation itself or the ovine breed in which the mutation was present. Altogether, these results suggest that the ovarian inhibin/activin system is not a key component of the mechanisms regulating the ovulation rate in BMP15 mutant ewes.

Comment citer ce document : Estienne, A., Lahoz, B., Jarrier, P., Bodin, L., Folch, J., Alabart, J. L., Fabre, S., Monniaux, D. ( 2017 Comment citer ce document : Estienne, A., Lahoz, B., Jarrier, P., Bodin, L., Folch, J., Alabart, J. L., Fabre, S., Monniaux, D. ( 2017). BMP15 regulates the inhibin/activin system independently of ovulation rate control in sheep. Reproduction, 153 (4), 395-404. DOI : 10.1530/REP-16-0507 Comment citer ce document : Estienne, A., Lahoz, B., Jarrier, P., Bodin, L., Folch, J., Alabart, J. L., Fabre, S., Monniaux, D. ( 2017). BMP15 regulates the inhibin/activin system independently of ovulation rate control in sheep. Reproduction, 153 (4), 395-404. DOI : 10.1530/REP-16-0507

  BMP4 and Activin A all enhanced FST and, in a lesser extent, INHA expression, but inhibited the expression of TGFBR3. In addition, INHBA expression was specifically enhanced by BMP15 and BMP4, whereas ACVR2A expression was inhibited by Activin A only. The similar effects of BMP4 and BMP15 can be explained by their common activation of the SMAD1/5/8 signaling pathway[START_REF] Liao | Effect of intracellular interactions on the processing and secretion of bone morphogenetic protein-15 (BMP-15) and growth and differentiation factor-9. Implication of the aberrant ovarian phenotype of BMP-15 mutant sheep[END_REF], whereas GDF9 and Activin A are known to signal through SMAD2/3 proteins[START_REF] Mazerbourg | Growth differentiation factor-9 signaling is mediated by the type I receptor, activin receptor-like kinase 5[END_REF]). From our results, no effect of Comment citer ce document : Estienne, A., Lahoz, B., Jarrier, P., Bodin, L., Folch, J., Alabart, J. L., Fabre, S., Monniaux, D. (2017). BMP15 regulates the inhibin/activin system independently of ovulation rate control in sheep. Reproduction, 153 (4), 395-404. DOI : 10.1530/REP-16-0507 14 GDF9 alone was observed, in agreement with the presence of a low activation level of the SMAD2/3 signaling pathway by GDF9 in ovine granulosa cells(Pierre et al. 2016). However, GDF9 was found to enhance and decrease the stimulating effects of BMP15 on the expression of FST and INHBA, respectively. GDF9 and BMP15 are known to form heterodimers[START_REF] Liao | Effect of intracellular interactions on the processing and secretion of bone morphogenetic protein-15 (BMP-15) and growth and differentiation factor-9. Implication of the aberrant ovarian phenotype of BMP-15 mutant sheep[END_REF][START_REF] Mcintosh | The proregion of mouse BMP15 regulates the cooperative interactions of BMP15 and GDF9[END_REF]) and exhibit synergistic interactions by activating both SMAD pathways(Mottershead et al. 2015). These interactions might be involved in the observed regulations by BMP15 and GDF9, when acting together.

FecX

  Gr mutation, when present at the homozygous state in the French Grivette breed, led to a 2.1-fold increase in ovulation rate, in agreement with previous observations[START_REF] Lahoz | Effect of the FecX(R) polymorphism in the bone morphogenetic protein 15 gene on natural or equine chorionic gonadotropin-induced ovulation rate and litter size in Rasa Aragonesa ewes and implications for on-farm application[END_REF][START_REF] Demars | Genome-wide association studies identify two novel BMP15 mutations responsible for an atypical hyperprolificacy phenotype in sheep[END_REF]. From our results, compared to non-carrier ewes, BMP15 mutant ewes exhibited a higher number of large dominant (preovulatory) follicles during the follicular phase of the estrous cycle, associated with a small decrease in their diameter, suggesting an earlier final differentiation of their granulosa cells, as previously proposed for the Inverdale ewes carrying the FecX I mutation in BMP15[START_REF] Mcnatty | Gonadotrophin-responsiveness of granulosa cells from bone morphogenetic protein 15 heterozygous mutant sheep[END_REF]) and more strikingly for the Booroola ewes carrying the FecB B mutation in BMPR1B (Henderson et al.

Fabre

  Estienne A, Pierre A, di Clemente N, Picard JY, Jarrier P, Mansanet C, Monniaux D & Fabre S 2015 Anti-Mullerian hormone regulation by the bone morphogenetic proteins in the sheep ovary: deciphering a direct regulatory pathway. Endocrinology 156 301-13. of ovulation rate in mammals: contribution of sheep genetic models. Reproductive Biology and Endocrinology 4 20. Forage RG, Brown RW, Oliver KJ, Atrache BT, Devine PL, Hudson GC, Goss NH, Bertram KC, Tolstoshev P, Robertson DM, et al. 1987 Immunization against an inhibin subunit produced by recombinant DNA techniques results in increased ovulation rate in sheep. Journal of Endocrinology 114 R1-4. Galloway SM, Gregan SM, Wilson T, McNatty KP, Juengel JL, Ritvos O & Davis GH 2002 Bmp15 mutations and ovarian function. Molecular and Cellular Endocrinology 191 15-8. Comment citer ce document : Estienne, A., Lahoz, B., Jarrier, P., Bodin, L., Folch, J., Alabart, J. L., Fabre, S., Monniaux, D. (2017). BMP15 regulates the inhibin/activin system independently of ovulation rate control in sheep. Reproduction, 153 (4), 395-404. DOI : 10.1530/REP-16-0507 21 Harris RA, Tardif SD, Vinar T, Wildman DE, Rutherford JN, Rogers J, Worley KC & Aagaard KM 2014 Evolutionary genetics and implications of small size and twinning in callitrichine primates. Proceedings of the National Academy of Sciences of the United States of America 111 1467-72. Henderson KM, Franchimont P, Lecomte-Yerna MJ, Hudson N & Ball K 1984 Increase in ovulation rate after active immunization of sheep with inhibin partially purified from bovine follicular fluid. Jornal of Endocrinology 102 305-9. Henderson KM, Kieboom LE, McNatty KP, Lun S & Heath D 1985 Gonadotrophinstimulated cyclic AMP production by granulosa cells from Booroola x Romney ewes with and without a fecundity gene. Journal of Reproduction and Fertility 75 111-20. Hillard MA, Wilkins JF, Cummins LJ, Bindon BM, Tsonis CG, Findlay JK & O'Shea T 1995 Immunological manipulation of ovulation rate for twinning in cattle. Journal of Reproduction and Fertility Suppl 49 351-64. Hoekstra C, Zhao ZZ, Lambalk CB, Willemsen G, Martin NG, Boomsma DI & Montgomery GW 2008 Dizygotic twinning. Human Reproduction Update 14 37-47. Knight PG & Glister C 2006 TGF-beta superfamily members and ovarian follicle development. Reproduction 132 191-206. Knight PG, Satchell L & Glister C 2012 Intra-ovarian roles of activins and inhibins. Molecular and Cellular Endocrinology 359 53-65. Comment citer ce document : Estienne, A., Lahoz, B., Jarrier, P., Bodin, L., Folch, J., Alabart, J. L., Fabre, S., Monniaux, D. (2017). BMP15 regulates the inhibin/activin system independently of ovulation rate control in sheep. Reproduction, 153 (4), 395-404. DOI : 10.1530/REP-16-0507 Comment citer ce document : Estienne, A., Lahoz, B., Jarrier, P., Bodin, L., Folch, J., Alabart, J. L., Fabre, S., Monniaux, D. (2017). BMP15 regulates the inhibin/activin system independently of ovulation rate control in sheep. Reproduction, 153 (4), 395-404. DOI : 10.1530/REP-16-0507 24 15 and growth differentiation factor 9 co-operate to regulate granulosa cell function. Reproduction 129 473-80. McNatty KP, Juengel JL, Reader KL, Lun S, Myllymaa S, Lawrence SB, Western A, Meerasahib MF, Mottershead DG, Groome NP, et al. 2005b Bone morphogenetic protein 15 and growth differentiation factor 9 co-operate to regulate granulosa cell function in ruminants. Reproduction 129 481-7. McNatty KP, Juengel JL, Wilson T, Galloway SM, Davis GH, Hudson NL, Moeller CL, Cranfield M, Reader KL, Laitinen MP, et al. 2003 Oocyte-derived growth factors and ovulation rate in sheep. Reproduction Suppl 61 339-51. Monestier O, Servin B, Auclair S, Bourquard T, Poupon A, Pascal G & Fabre S 2014 Evolutionary origin of bone morphogenetic protein 15 and growth and differentiation factor 9 and differential selective pressure between mono-and polyovulating species. Biology of Reproduction 91 83. Monniaux D, di Clemente N, Touzé JL, Belville C, Rico C, Bontoux M, Picard JY & Fabre S 2008 Intrafollicular steroids and anti-mullerian hormone during normal and cystic ovarian follicular development in the cow. Biology of Reproduction 79 387-96. Montgomery GW, Galloway SM, Davis GH & McNatty KP 2001 Genes controlling ovulation rate in sheep. Reproduction 121 843-52. Comment citer ce document : Estienne, A., Lahoz, B., Jarrier, P., Bodin, L., Folch, J., Alabart, J. L., Fabre, S., Monniaux, D. (2017). BMP15 regulates the inhibin/activin system independently of ovulation rate control in sheep. Reproduction, 153 (4), 395-404. DOI : 10.1530/REP-16-0507 25 Moore RK, Otsuka F & Shimasaki S 2003 Molecular basis of bone morphogenetic protein-15 signaling in granulosa cells. Journal of Biological Chemistry 278 304-10. Moore RK & Shimasaki S 2005 Molecular biology and physiological role of the oocyte factor, BMP-15. Molecular and Cellular Endocrinology 234 67-73. Mottershead DG, Sugimura S, Al-Musawi SL, Li JJ, Richani D, White MA, Martin GA, Trotta AP, Ritter LJ, Shi J, et al. 2015 Cumulin, an oocyte-secreted heterodimer of the transforming growth factor-beta family, is a potent activator of granulosa cells and improves oocyte quality. Journal of Biological Chemistry 290 24007-24020. Otsuka F, Yamamoto S, Erickson GF & Shimasaki S 2001 Bone morphogenetic protein-15 inhibits follicle-stimulating hormone (FSH) action by suppressing FSH receptor expression. Journal of Biological Chemistry 276 11387-92. Otsuka F, Yao Z, Lee T, Yamamoto S, Erickson GF & Shimasaki S 2000 Bone morphogenetic protein-15. Identification of target cells and biological functions. Journal of Biological Chemistry 275 39523-8. Palmer JS, Zhao ZZ, Hoekstra C, Hayward NK, Webb PM, Whiteman DC, Martin NG, Boomsma DI, Duffy DL & Montgomery GW 2006 Novel variants in growth differentiation factor 9 in mothers of dizygotic twins. Journal of Clinical Endocrinology and Metabolism 91 4713-6. Comment citer ce document : Estienne, A., Lahoz, B., Jarrier, P., Bodin, L., Folch, J., Alabart, J. L., Fabre, S., Monniaux, D. (2017). BMP15 regulates the inhibin/activin system independently of ovulation rate control in sheep. Reproduction, 153 (4), 395-404. DOI : 10.1530/REP-16-0507 Persani L, Rossetti R, Di Pasquale E, Cacciatore C & Fabre S 2014 The fundamental role of bone morphogenetic protein 15 in ovarian function and its involvement in female fertility disorders. Human Reproduction Update 20 869-83. S, et al. 2016 The Bone Morphogenetic Protein 15 Up-Regulates the Anti-Mullerian Hormone Receptor Expression in Granulosa Cells. Journal of Clinical Endocrinology and Metabolism 101 2602-11. Tannetta DS, Feist SA, Bleach EC, Groome NP, Evans LW & Knight PG 1998 Effects of active immunization of sheep against an amino terminal peptide of the inhibin alpha C subunit on intrafollicular levels of activin A, inhibin A and follistatin. Journal of Endocrinology 157 157-68. Visser JA & Themmen AP 2005 Anti-Mullerian hormone and folliculogenesis. Molecular and Cellular Endocrinology 234 81-6. Visser JA & Themmen AP 2014 Role of anti-Mullerian hormone and bone morphogenetic proteins in the regulation of FSH sensitivity. Molecular and Cellular Endocrinology 382 460-465. Walton KL, Makanji Y & Harrison CA 2012 New insights into the mechanisms of activin action and inhibition. Molecular and Cellular Endocrinology 359 2-12. Comment citer ce document : Estienne, A., Lahoz, B., Jarrier, P., Bodin, L., Folch, J., Alabart, J. L., Fabre, S., Monniaux, D. (2017). BMP15 regulates the inhibin/activin system independently of ovulation rate control in sheep. Reproduction, 153 (4), 395-404. DOI : 10.1530/REP-16-0507 27 Yan L, Li H & Shi Z 2015 Immunization against inhibin improves in vivo and in vitro embryo production. Animal Reproduction Science 163 1-9.

Figure legends Figure 1 :

 legends1 Figure legends

Figure 2 :

 2 Figure 2: Consequences of the presence of natural loss-of-function mutations in the BMP15

Figure 3 :

 3 Figure 3: Consequences of the presence of natural loss-of-function mutations in the BMP15

Figure 4 :

 4 Figure 4: Consequences of the presence of natural loss-of-function mutations in the BMP15

Figure 5 :

 5 Figure 5: Consequences of the presence of natural loss-of-function mutations in the BMP15

Figure 6 :

 6 Figure 6: Effect of BMP4, BMP15, GDF9 and Activin A on the expression of genes

Figure 1 :Figure 2 :BMP15Figure 3 :Figure 4 :

 1234 Figure 1: Consequences of the presence of natural loss-of-function mutations in the BMP15 gene on the expression of functional gene markers in the granulosa cells of ewes. Results represent CYP19A1 (a marker of fully differentiated granulosa cells, upper panels) and IGFBP5 (a marker of apoptotic granulosa cells, lower panels) expression in small growing (1-3 mm) and large dominant (> or equal to 5 mm) follicles from +/+ (n=6, black bars) and R/+ (n=6, open bars) Rasa Aragonesa ewes (left panels), and +/+ (n=5, black bars) and Gr/Gr (n=5, open bars) Grivette ewes (right panels). All the follicles were recovered on the ovaries of ewes in the follicular phase of a synchronized estrous cycle. CYP19A1 and IGFBP5 mRNA accumulation was studied by reverse transcription and quantitative PCR and represented as mRNA relative level, using RPL19 as an internal reference. In each panel, data were analyzed by one-way ANOVA and different letters indicate significant differences (p < 0.05) between follicular classes and genotypes.

FollicularFigure 5 :

 5 Figure 5: Consequences of the presence of natural loss-of-function mutations in the BMP15 gene on the expression of genes encoding activin/inhibin binding proteins in the granulosa cells of ewes. Results represent FST (encoding follistatin, the specific activin-binding protein, upper panels) and TGFBR3 (encoding betaglycan, the co-receptor of inhibin, lower panels) expression in small growing (1-3 mm) and large dominant (> or equal to 5 mm) follicles from +/+ (n=6, black bars) and R/+ (n=6, open bars) Rasa Aragonesa ewes (left panels), and +/+ (n=5, black bars) and Gr/Gr (n=5, open bars) Grivette ewes (right panels), analyzed as described in Figure 1. In each panel, different letters indicate significant differences (p < 0.05) between follicular classes and genotypes. *: p<0.05, **: p<0.01 vs. the corresponding follicle class in +/+ Rasa Aragonesa ewes.

Follicular

  ., Lahoz, B., Jarrier, P., Bodin, L., Folch, J., Alabart, J. L., Fabre, S., Monniaux, D. (2017). BMP15 regulates the inhibin/activin system independently of ovulation rate control in sheep. Reproduction, 153 (4), 395-404. DOI : 10.1530/REP-16

Figure 6 :

 6 Figure 6: Effect of BMP4, BMP15, GDF9 and Activin A on the expression of genes encoding different members of the inhibin/activin system in ovine granulosa cells. Sheep granulosa cells were cultured in 96well plates for 48 hours with or without 50 ng/ml of the different factors. Each treatment was tested in 12 replicate wells, then cells were pooled according to treatment at the end of culture for mRNA preparation. Messenger RNA accumulation of INHA, INHBA, ACVR1B, ACVR2A, FST and TGFBR3 was studied by reverse transcription and quantitative PCR relatively to RPL19 as internal reference. The results were expressed in percentage of expression relatively to that of cells cultured without ligand (Ctrl, fixed at 100%). Data represent the results of 15 independent experiments performed for each treatment. Comparisons of means between different treatments were made by repeated measures one-way ANOVA with the Greenhouse-Geisser correction of the degrees of freedom, followed by Fisher's LSD test for multiple comparisons. (*): p<0.1, *: p < 0.05, **: p < 0.01, ***: p < 0.001, vs. Ctrl, or between BMP15 and BMP15+GDF9 conditions.

  . In this study, 6 Rasa Aragonesa ewes heterozygous carriers of the FecX R mutation (R/+; ovulation rate = 1.89 ± 0.16), 6 Rasa Aragonesa non-

carrier ewes (+/+; ovulation rate = 1.00), 5 Grivette ewes homozygous carriers of the FecX Gr mutation (Gr/Gr; ovulation rate = 4.88 ± 0.55) and 5 Grivette non-carrier ewes (+/+; ovulation rate = 2.29 ± 0.20) were used after genotyping. All animals were treated during their seasonal period of reproduction with intravaginal progestagen sponges (fluogestone acetate, 20 mg; Intervet, Angers, France) for 14 days to synchronize estrus, before slaughtering of the ewes 36 h after sponge removal, corresponding to the follicular phase of the cycle. Ovaries from all genotypes were recovered in a local slaughterhouse at Zaragoza (Spain) and Toulouse (France), for the Rasa Aragonesa and Grivette breeds, respectively. All procedures were approved by the Agricultural and Scientific Research Government Committees and local ethical committees (Approval numbers C37-175-2 in Nouzilly, C31-429-01 in Toulouse and -CITA 2011-08 in Zaragoza) in accordance with the guidelines for Care and Use of Agricultural Animals in Agricultural Research and Teaching.

  atresia or apoptosis (Supplementary Figure2and Supplementary Table1) in this size class containing the subordinate follicles already engaged in the atresia process.The presence of loss-of-function BMP15 mutations in vivo had little effect on the expression of the components of the inhibin/activin system and the differences found between genotypes were not consistent with the effects of BMP15 observed on ovine granulosa cells in vitro. For instance, the expression of INHA, INHBA and FST was not, or only slightly, affected by the presence of BMP15 mutations in vivo in both breeds, despite the existence of a stimulating effect of BMP15 on the expression of these genes in vitro. From our in vitro results, Activin A was able to enhance the expression of INHA and FST expression and could such participate in balancing the expression level of these components in vivo. However, this hypothesis cannot explain why INHBA was unaffected by the presence of BMP15 mutations

	in vivo.

). BMP15 regulates the inhibin/activin system independently of ovulation rate control in sheep. Reproduction, 153 (4), 395-404. DOI : 10.1530/REP-16-0507 15 markers of

  affecting BMP15 are not yet fully understood. Alternatively, the in vivo expression level of each component of the inhibin/activin system could depend on the specific equilibrium of the different components existing within each breed. This hypothesis is supported by the observation that the granulosa cells of non-mutant Grivette ewes expressed higher levels of INHA, INHBA, ACVR1B and ACVR2A, but lower levels of FST and

Comment citer ce document : Estienne, A., Lahoz, B., Jarrier, P., Bodin, L., Folch, J., Alabart, J. L., Fabre, S., Monniaux, D. (

2017

). BMP15 regulates the inhibin/activin system independently of ovulation rate control in sheep. Reproduction, 153 (4), 395-404. DOI : 10.1530/REP-16-0507 16 molecular mechanisms

Table 1 :

 1 Quantitative PCR primers sequences and their efficiencyComment citer ce document : Estienne, A., Lahoz, B., Jarrier, P., Bodin, L., Folch, J., Alabart, J. L., Fabre, S., Monniaux, D. (2017). BMP15 regulates the inhibin/activin system independently of ovulation rate control in sheep. Reproduction, 153 (4), 395-404. DOI : 10.1530/REP-16-0507 1

	Gene	Accession number	Forward sequence 5'→3'	Reverse sequence 5'→3'	Efficiency
	CYP19A1	NM_001123000.1	TCATCCTGGTCACCCTTCTG	CCAGACGAGACCAGCGACCG	1.95
	IGFBP5	NM_001129733.1	CTACAAGAGAAAGCAGTG	CAGCTTCATCCCATACTT	1.99
	INHA	NM_001308579.1	GACCAAGATGTCTCCCAG	CAGTATGGAACCACAGCT	2.00
	INHBA	NM_001009458.1	GAAGAGGAAAGAAGAGGAA	GTAGTGGTTGATGACTGT	2.08
	ACVR1B	XM_012174234.2	TTGTGTACTGGGAGATTG	ACTAGGTCATAATATGGCAG	1.96
	ACVR2A	NM_001009293.1	GATTAGTCCTGTGGGAAC	GTCTTCAAGAGAGGGATG	2.05
	FST	NM_001257093.1	TGGACCAGACTAATAATG	TATCTTCACAGGACTTTG	1.87
	TGFβR3	XM_012176015.2	CTCTTTCTCCAGTGTGAG	GATCATTGAGGCATCCAG	1.98
	RPL19	XM_004012837	AATGCCAATGCCAACTC	CCCTTTCGCTACCTATACC	1.95

Table 2 :

 2 Follicular populations in the ovaries of Rasa Aragonesa and Grivette ewes with 1

	2	different genotypes
	Breed		Rasa Aragonesa	Grivette
	Genotype	+/+	R/+	+/+	Gr/Gr
	(N° of ewes)	(n=6)	(n=6)	(n=5)	(n=5)
	N° of small follicles (1-3 mm)	43.0 ± 7.4	42.8 ± 6.5	48.2 ± 3.7	56.6 ± 6.8
	N° of large follicles (> or equal to 5 mm)	2.3 ± 0.3	3.7 ± 0.3*	2.2 ± 0.2	3.8 ± 0.9
	Mean diameter of the large follicles (mm) 7.25 ± 0.33 5.88 ± 0.11** 7.06 ± 0.28 5.36 ± 0.09***
	3
	4	*: p<0.05, **: p<0.01; ***: p<0.001, vs. +/+ ewes of the same breed.
	5
	6

Comment citer ce document : Estienne, A., Lahoz, B., Jarrier, P., Bodin, L., Folch, J., Alabart, J. L., Fabre, S., Monniaux, D. (2017). BMP15 regulates the inhibin/activin system independently of ovulation rate control in sheep. Reproduction, 153 (4), 395-404. DOI : 10.1530/REP-16-0507

Acknowledgements

We thank the staff of the slaughterhouse of the CIRE platform at Nouzilly (France) for the recovery of sheep ovaries. We are grateful to the technicians of the INRA experimental unit of Langlade who took care of the Grivette animals in France, and the staff of the experimental animal facilities of CITA de Aragón for taking care and providing the Rasa Aragonesa ewes in Spain.

Funding

This work was supported by grants from France via the "Agence Nationale pour la Recherche" (http://www.agence-nationale-recherche.fr/) (ANR-12-BSV1-0034-02, AMHAROC) and EC (FP7/2007-2013), grant 245140, ''3SR'', Sustainable Solutions for Small Ruminants (http://www.3srbreeding.eu/), WP4 (this publication reflects only the authors' views and the EC is not liable for any use that may be made of the information contained herein). A. Estienne was supported by a French Fellowship from the Région Centre and INRA.

Declaration of interest

The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

Comment citer ce document : Estienne, A., Lahoz, B., Jarrier, P., Bodin, L., Folch, J., Alabart, J. L., Fabre, S., Monniaux, D. ( 2017


Katrine S Hoset
email: katrine.hoset@nord.no

Alexandre Villers

Ralf Wistbacka

Jean-Michel Gaillard

Vesa Selonen

Pulsed food resources, but not forest cover, determines lifetime reproductive success in a forest-dwelling rodent

Keywords: arboreal mammal, fecundity, forest-specialist, habitat availability, longevity, Pteromys volans, Siberian flying squirrel, tree mast

HAL is

4. According to generalised additive models female longevity, fecundity and LRS were mainly determined by variation in cumulative lifetime availability of food from masting alder and birch. Instead, habitat and body mass had smaller role. The path analysis indicated that lifetime food availability had direct effect on longevity and fecundity, and these had equal effect on LRS at both study sites. 5. Our results on LRS shows that the occurrence of tree masting events during a flying squirrel female's lifetime have profoundly larger effect on lifetime reproductive success than the cover of suitable forest habitat. Furthermore, this study emphasises the importance of both fecundity and longevity, and the indirect effects of food availability via those components, as determinants of lifetime fitness of female flying squirrels.

INTRODUCTION

Defining habitat quality for a species can be a complex task [START_REF] Gaillard | Habitat-performance relationships: finding the right metric at a given spatial scale[END_REF], especially if no knowledge on the relationship between individual performance and habitat quality exists. Consequently, interpretations of habitat quality are often based on simple proxies that may be misleading [START_REF] Van Horne | Density as a Misleading Indicator of Habitat Quality[END_REF][START_REF] Gaillard | Habitat-performance relationships: finding the right metric at a given spatial scale[END_REF]. For example, forest cover and area of old growth forests are often used as proxies of habitat quality for arboreal animals.

However, efficient conservation efforts of vulnerable species require not only knowledge on their habitat use and food requirements, but also an understanding of how habitat availability and food availability interact to affect individual variation in fitness [START_REF] Colwell | Arrival times, laying dates, and reproductive success of Snowy Plovers in two habitats in coastal northern California[END_REF]. This is particularly important for species living in environments with pulsed resources, such as forests dominated by masting tree species. The relative contributions of habitat and food availability on fitness may provide evidence for key habitat features needed to safeguard population persistence [START_REF] Delibes | Effects of an Attractive Sink Leading into Maladaptive Habitat Selection[END_REF][START_REF] Robertson | A Framework for Understanding Ecological Traps and an Evaluation of Existing Evidence[END_REF].

A reliable and common estimate of fitness is lifetime reproductive success (LRS) or lifetime breeding success (LBS) which can be estimated from longitudinal data (Clutton-Brock 1988;[START_REF] Newton | Lifetime Reproduction in Birds[END_REF][START_REF] Brommer | Single-Generation Estimates of Individual Fitness as Proxies for Long-Term Genetic Contribution[END_REF]. From initiation of first breeding, LRS and LBS are mainly determined by reproductive longevity (number of breeding seasons) and fecundity (average production per breeding season), with contribution from juvenile survival (Clutton-Brock 1988;[START_REF] Newton | Lifetime Reproduction in Birds[END_REF]. Environmental factors, such as food and habitat quality, can affect LRS through fecundity and longevity in conjunction with individual traits. For example, availability of food resources affect LRS directly (e.g., [START_REF] Millon | Pulsed resources affect the timing of first breeding and lifetime reproductive success of tawny owls[END_REF], but can also be mediated through longevity [START_REF] Wauters | Fitness Consequences of Hoarding Behaviour in the Eurasian Red Squirrel[END_REF] This article is protected by copyright. All rights reserved. [START_REF] Bérubé | Individual Differences, Longevity, and Reproductive Senescence in Bighorn Ewes[END_REF][START_REF] Bieber | Habitat differences affect life history tactics of a pulsed resource consumer, the edible dormouse (Glis glis)[END_REF]. Habitat quality or habitat use are often important determinants of LRS in both birds [START_REF] Herman | Lifetime reproductive success of Snowy Plovers in coastal northern California[END_REF] and mammals [START_REF] Mcloughlin | Lifetime reproductive success and density-dependent, multi-variable resource selection[END_REF][START_REF] Bieber | Habitat differences affect life history tactics of a pulsed resource consumer, the edible dormouse (Glis glis)[END_REF][START_REF] Regan | Sex differences in relationships between habitat use and reproductive performance in Soay sheep (Ovis aries)[END_REF]. For example, female St. Kilda Soay sheep that had high cover of key grass habitat within their home ranges had higher LRS than females with low cover of grass habitat [START_REF] Regan | Sex differences in relationships between habitat use and reproductive performance in Soay sheep (Ovis aries)[END_REF]. Of individual traits, body mass is most commonly related with LRS, longevity or fecundity, and usually heavier females achieve higher fitness (e.g., Clutton-Brock 1988;[START_REF] Newton | Lifetime Reproduction in Birds[END_REF][START_REF] Bérubé | Individual Differences, Longevity, and Reproductive Senescence in Bighorn Ewes[END_REF], but see Gaillard et al. 2000a).

The Siberian flying squirrel (Pteromys volans L., hereafter flying squirrel) is a nocturnal, arboreal forest species which has its westernmost distribution in Finland. Due to the long-lasting declining population trends across Finland [START_REF] Hokkanen | Decline of the flying squirrel Pteromys volans l. populations in Finland[END_REF][START_REF] Hanski | Liito-Oravan Pteromys Volans Suomen Kannan Koon Arviointi[END_REF][START_REF] Selonen | Decline of flying and red squirrels in boreal forests revealed by long-term diet analyses of avian predators[END_REF] and possible role as umbrella species potentially indicating biodiversity values in old growth forests [START_REF] Hurme | Role of the Siberian flying squirrel as an umbrella species for biodiversity in northern boreal forests[END_REF], the species has a high conservation value and is strictly protected under European legislation (Habitats Directive 92/43/EEC). The population declines are probably linked with decreasing availability of suitable habitat due to current forestry management practises [START_REF] Hanski | Liito-Oravan Pteromys Volans Suomen Kannan Koon Arviointi[END_REF]Santangeli et al. 2013b). Understanding how habitat characteristics and food availability contribute to the variation in LRS, lifespan and fecundity of flying squirrels may provide key evidence to the mechanisms involved in the population declines and clues to how conservation actions could be improved. Occurrence of flying squirrels is highly correlated with the presence and density of suitable habitat, but species also seem to cope well with fragmentation [START_REF] Selonen | Young flying squirrels (Pteromys volans) dispersing in fragmented forests[END_REF]Santangeli, Hanski & Mäkelä 2013a).

In this paper, we take advantage of a 22 year-long longitudinal data set on two populations of Siberian flying squirrels, combined with annual land cover data on habitat availability, to determine the relative importance of availability of food and suitable forest This article is protected by copyright. All rights reserved.

habitat on local and landscape scale on female lifetime reproductive success (LRS) of this forest specialist. Further, we evaluate the contribution of average annual reproductive success (fecundity) and breeding longevity on LRS, and which component is most influenced by habitat availability and/or food availability. Based on the known occupancy patterns and habitat preferences [START_REF] Selonen | Space use of the Siberian flying squirrel Pteromys volans in fragmented forest landscapes[END_REF][START_REF] Hurme | Role of the Siberian flying squirrel as an umbrella species for biodiversity in northern boreal forests[END_REF]Santangeli et al. 2013a;Selonen & Wistbacka 2016), we predict that LRS will be higher for females with large cover of suitable forest around their core territories and for females experiencing higher food abundance during their lifetime. Further, we predict that females with higher body mass produce more offspring similar to findings in red squirrels (Sciurus vulgaris) (Wauters & Dhondt 1995). Higher food availability and access to suitable habitat should lead to higher body mass and thus increased longevity or fecundity [START_REF] Regan | Sex differences in relationships between habitat use and reproductive performance in Soay sheep (Ovis aries)[END_REF]. High food availability also has a positive effect on annual reproduction in flying squirrels (Selonen & Wistbacka 2016), and is therefore likely positively related with fecundity. However, as flying squirrels can live up to 7+ years, although most females are short lived (< 2 years, see results), we expect that longevity is more important for LRS than fecundity, following e.g. [START_REF] Bérubé | Individual Differences, Longevity, and Reproductive Senescence in Bighorn Ewes[END_REF].

MATERIAL AND METHODS

Study species

Flying squirrels nests in tree cavities, nest-boxes and dreys in spruce-dominated boreal forests. The mating season starts from mid-March. After the first litter born in April, the females can sometimes have a second litter born in June, especially when spring food availability is high [START_REF] Selonen | Food abundance and weather modify reproduction of two arboreal squirrel species[END_REF]. Litter size at both sites is about 2.3-2.5 young for both first and second litters and about 15% of mothers produce summer This article is protected by copyright. All rights reserved. litters (Selonen & Wistbacka 2016). Weaning age for the species is at about 2 months, but exact estimates are lacking. Breeding dispersal is much shorter than natal dispersal, especially in females (average breeding movement distance: 72 ± 130m, average natal dispersal distance: 1830 ± 1390m, Selonen & Wistbacka 2016). Females are territorial, living in nonoverlapping home ranges (on average 7 ha) indicating that competition for nesting sites occur, while males live in overlapping home ranges (on average 60 ha) encompassing several males and females [START_REF] Selonen | Space use of the Siberian flying squirrel Pteromys volans in fragmented forest landscapes[END_REF][START_REF] Selonen | Mating system and reproductive success in the Siberian flying squirrel[END_REF]. Annual local apparent survival in our populations is about 0.43-0.53 [START_REF] Lampila | Survival and Population Growth Rate of the Threatened Siberian Flying Squirrel (Pteromys volans) in a Fragmented Forest Landscape[END_REF], and although most females disappear from the population after their first breeding season (probably from death, and not breeding dispersal, Selonen & Wistbacka 2017;[START_REF] Brommer | Immigration ensures population survival in the Siberian flying squirrel[END_REF] we have recorded individuals as old as 7+ years. Main predators of flying squirrels are larger owls, such as the Eurasian eagle owl (Bubo bubo), Ural owl (Strix uralensis), goshawk (Accipiter gentilis) and the European pine marten (Martes martes) [START_REF] Selonen | Decline of flying and red squirrels in boreal forests revealed by long-term diet analyses of avian predators[END_REF].

Study sites and CMR data

The study was conducted at two study sites along the coast of West Finland from 1992/1993 to 2014 (Figure 1). One site is located in and around Vaasa city (hereafter: Vaasa region, 63°3'N, 21°41'E). The marking of flying squirrels started in 1992 in Vaasa within an area of 400ha, after 2000 the area was 25 km 2 . Vaasa is covered by spruce forest patches, shoreline forests, clear-cuts, and agricultural fields (for more information see [START_REF] Lampila | Survival and Population Growth Rate of the Threatened Siberian Flying Squirrel (Pteromys volans) in a Fragmented Forest Landscape[END_REF]Selonen, Hanski & Wistbacka 2014). The other site is located about 90 km north-east at Luoto (63°49'N, 22°49'E; Figure 1) and covers an area of 44 km 2 . The main forest types in Luoto are shoreline spruce-dominated mixed forests, clear-cuts, and cultivated Scots pine forests. Capture-mark-recapture (CMR) data has been collected since 1993 from this area. This article is protected by copyright. All rights reserved.

At both sites, nest-boxes for flying squirrels were placed in forest patches of various sizes in sets of 2-4 nest-boxes per site, with an average of 2 nest-boxes per mature spruce forest hectare. Box occupancy percentage by the flying squirrel was low (~25%), that is, in most cases a nest box was empty when checked. Flying squirrels (adults and juveniles) were captured by hand in nest-boxes, sexed, weighed, and marked with ear-tags (Hauptner 73850, Hauptner, Germany). Natural cavities were rare in our study areas (on average 0.1 cavities per hectare based on 742 spruce forest hectares surveyed within our study areas); we are thus confident that a large majority of breeding events occurred in monitored nest-boxes. The nest-boxes were checked during one to two sessions in June and August, depending on presence of breeding females in June, but also infrequently throughout the year.

In this study, we used only data on adult breeding females with their assumed full reproductive activity within the study period. Females were defined as dead or emigrated from the population when they had not been seen for three consecutive breeding seasons and thus analyses were based on females born before 2012 to avoid under-estimating LRS due to truncated longevity. Based on earlier analysis [START_REF] Lampila | Survival and Population Growth Rate of the Threatened Siberian Flying Squirrel (Pteromys volans) in a Fragmented Forest Landscape[END_REF]) recapture probability of females was high in our study areas (around 0.9). Only a few females skipped breeding seasons, 15 females (3% of all females in the dataset) skipped 1 breeding season, 1 female skipped 2 seasons, and 17 individuals (3.6%) were not observed breeding until second year after birth. Many of these females were likely not breeding as radio-tracking data from the study area indicate that non-breeding females use dreys more than nest boxes during summer and are therefore less likely trapped (R. Wistbacka & P.Reunanen, unpublished data). Most females were observed only once per breeding season, and we could not separate potential first and second litters in all cases as we observed juveniles close to weaning age. The fate of juveniles after leaving the nest and their mother (i.e., recruitment to the population) was not This article is protected by copyright. All rights reserved. known for most individuals. Therefore, we defined LRS as the total number of offspring produced per female during the whole lifetime. Longevity is defined as the number of breeding seasons a female was observed in the study site, and fecundity is the average production of young per year (i.e., total number of offspring divided by the number of breeding seasons; annual variation in litter size is analysed in Selonen et al. 2016;Selonen & Wistbacka 2016).

Food availability data

The flying squirrel feeds in deciduous trees that occur within or at the edge of spruce (Picea) forests, and in our study sites birch (Betula spp.), alder (Alnus spp.) and aspen (Populus tremula) are the main forage items [START_REF] Mäkelä | Liito-oravan (Pteromys volans L.) ravintokohteet eri vuodenaikoina ulosteanalyysin perusteella[END_REF]Selonen et al. 2016). These common species occur in all parts of the study area. During winter and early spring, birch and alder catkins are the main food of flying squirrels with birch constituting the main part of the winter diet (80% of used food, based on faecal diet analysis; Mäkelä 1996), although they eat pine buds and other food items when catkins are not available [START_REF] Mäkelä | Liito-oravan (Pteromys volans L.) ravintokohteet eri vuodenaikoina ulosteanalyysin perusteella[END_REF]. However, only alder catkins are stored and are preferred over birch [START_REF] Sulkava | Liito-oravan ravinnosta ja ruokailutavoista Keski-Suomessa [Feeding habits of flying squirrels[END_REF][START_REF] Mäkelä | Liito-oravan (Pteromys volans L.) ravintokohteet eri vuodenaikoina ulosteanalyysin perusteella[END_REF].

A recent study indicates that the availability of catkins before initiation of breeding was important for flying squirrel reproduction in our study area by increasing the likelihood of second litters (Selonen & Wistbacka 2016). However, catkin production varies considerably between years, and forms a highly pulsed resource in the forest [START_REF] Hokkanen | Seed crops and seed crop forecasts for a number of tree species. Forest regeneration in the northern parts of Europe[END_REF][START_REF] Ranta | Masting by Betula-species; applying the resource budget model to north European data sets[END_REF][START_REF] Ranta | Male flowering of birch: Spatial synchronization, year-to-year variation and relation of catkin numbers and airborne pollen counts[END_REF]Selonen et al. 2016). Weather conditions during previous summer determine birch mast; catkin production increase when the previous summer has been warm, but trees seldom manage to produce mast for two successive years [START_REF] Ranta | Masting by Betula-species; applying the resource budget model to north European data sets[END_REF]. After the opening of leaves, in the beginning of May in our study areas, leaves form the main diet of flying squirrels in late spring and summer [START_REF] Mäkelä | Liito-oravan (Pteromys volans L.) ravintokohteet eri vuodenaikoina ulosteanalyysin perusteella[END_REF].

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Following Selonen et al. (2016), we used two estimates of landscape-level food availability in this study. First, we used estimates from an annual birch-catkin survey conducted by the Finnish Forest Research Institute [START_REF] Hokkanen | Seed crops and seed crop forecasts for a number of tree species. Forest regeneration in the northern parts of Europe[END_REF]. These data are collected to describe nation-wide pollen situation in Finland. Birch catkins were counted in winter from seed-crop observation stands. The catkin data originated from 15 permanent research stands in different parts of Finland [START_REF] Hokkanen | Seed crops and seed crop forecasts for a number of tree species. Forest regeneration in the northern parts of Europe[END_REF]. We used indices for western

Finland that are sampled in Vaasa. Catkin production of deciduous trees is spatially correlated at scales of up to several hundred kilometres in Finland [START_REF] Ranta | Male flowering of birch: Spatial synchronization, year-to-year variation and relation of catkin numbers and airborne pollen counts[END_REF]. Thus, although the food availability index is less accurate for Luoto than Vaasa, both study sites are located in coastal areas with very similar weather conditions and the spatial correlation in birch catkin production between two sampling sites at similar distance is around r ≈ 0.7.

Consequently, this index is able to properly describe the yearly variation in catkin production in the area.

There was no catkin count data for alder, but we used aerial pollen estimates that correlate with catkin production as a proxy [START_REF] Ranta | Male flowering of birch: Spatial synchronization, year-to-year variation and relation of catkin numbers and airborne pollen counts[END_REF]Selonen et al. 2016). Pollen data was collected by the aerobiology unit at University of Turku with samples collected from 10 different locations in Finland using EU standard methods and Burkard samplers. The data consisted of accumulated sums of average daily counts of airborne pollen in 1 m 3 of air during spring [START_REF] Ranta | Male flowering of birch: Spatial synchronization, year-to-year variation and relation of catkin numbers and airborne pollen counts[END_REF]. Similarly as for the birch catkin data, we used the estimates for western Finland, sampled in Vaasa for both of our study areas.

Land cover data

Yearly maps of habitat classes for each site from 1997 to 2012 were compiled from the SLICE dataset [START_REF] Mikkola | The Slices-project: National classification of land use, land cover and soil, and the production of databases[END_REF] This article is protected by copyright. All rights reserved.

http://www.maanmittauslaitos.fi/en/opendata). To determine the extent of clear-cutting of forest patches between years, we estimated an index of vegetation cover (the Normal Difference Moisture Index) based on an analysis of Landsat images (http://landsat.usgs.gov/).

This index allows identifying areas with and without vegetation by measuring the variation in the moisture level due to the degree of vegetation cover [START_REF] Jin | Comparison of time series tasseled cap wetness and the normalized difference moisture index in detecting forest disturbances[END_REF], which can thus be used to reveal the moment vegetation has been removed from a plot. We were thus able to produce accurate landscapes (on a 25 x 25m pixels grid) on a yearly basis (see method details in [START_REF] Morosinotto | Competitors and predators alter settlement patterns and reproductive success of an intraguild prey[END_REF].

We defined buffer zones around each nest box at 100m and 500m distances and calculated the total area of each land cover class within each buffer of active nest boxes for each study year. The 100m buffer (3.14 ha) represent the core area available for daily use S1). The cover of each habitat type within the 100m and 500m buffers was averaged across breeding years for each female to obtain a relevant measure of habitat use to compare with LRS for a territorial species. Exchanging

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average cover with maximum cover barely changed the values as flying squirrel females are site tenacious [START_REF] Hanski | Female-biased natal dispersal in the Siberian flying squirrel[END_REF]Selonen & Wistbacka 2017) and moved between breeding attempts only over very short distances (see Study Species description above).

Extensive occupancy analyses of flying squirrels indicate that spruce dominated mixed old growth and mature forests are important habitat for the species (e.g., [START_REF] Reunanen | Habitat requirements of the Siberian flying squirrel in northern Finland: comparing field survey and remote sensing data[END_REF]Santangeli et al. 2013a), and also presence of agricultural fields, likely due to preference for edge habitat in fertile soil, are an important determinant of landscape level occupancy (Santangeli et al. 2013a). Our study areas are characterised by a large degree of mixed forest structure where mature pine forests (less preferred forest type) often are small fragments surrounded by spruce/birch forests (preferred mature forest type), where flying squirrels in our study are known to forage (R. Wistbacka, pers. obs.). Several studies also indicate that young forest is not used for nesting, feeding or moving [START_REF] Reunanen | Habitat requirements of the Siberian flying squirrel in northern Finland: comparing field survey and remote sensing data[END_REF]Santangeli et al. 2013a). Therefore, we decided to use cover of all mature forest (hereafter, suitable forest habitat) in our analyses (our results were robust for different definitions of suitable habitat, e.g. including patch size measured from aerial photographs, see Appendix S1).

Statistical analyses

All statistical calculations were conducted using R version 3.3.0 (R Development Core Team, 2016). Data exploration was applied following Zuur et al. ( 2010). The presence of outliers was investigated using Cleveland dotplots, collinearity was assessed using multi-panel scatterplots, Pearson correlation coefficients and variance inflation factors.

The importance of suitable habitat, agricultural fields and landscape-level food availability measures (cumulative lifetime birch catkin availability = the sum of average birch catkins per tree and cumulative lifetime alder pollen availability = the sum of Alder pollen/m 3 of air during the lifetime of each female) for determining LRS of each individual (n = 500)

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was modelled with Generalised Additive Mixed Models (GAMM) using the gamm4 package [START_REF] Wood | Gamm4: Generalized Additive Mixed Models Using Mgcv and Lme4[END_REF] in R. Cover of suitable habitat and of agricultural fields within each buffer zone were centred according to site-specific means to take into account the difference in the site-specific availability of each habitat class (see Table S2). We used the lifetime sum of food availability (number of birch catkins and pollen density) for each female to capture the variability in annual food availability experienced by each female. Annual food availability ranged from 31-1857 catkins per birch tree and 47-3000 alder pollen per m 3 air.

Although the dataset is not optimal for detecting possible silver spoon effects (sensu Grafen 1988), we also tested the importance of food availability during year of birth of each females since food availability early in life can have important consequences for future reproductive success (Gaillard et al. 2000b;[START_REF] Descamps | Cohort effects in red squirrels: the influence of density, food abundance and temperature on future survival and reproductive success[END_REF]. However, food availability in the year of birth did not have any effects on LRS, longevity or fecundity (see Table S6 in Appendix S4) and was not considered further. In addition to habitat and food variables, we included study site as fixed factor to control for site-specific variation in LRS. Year of birth of females was entered as a random effect to take into account for any cohort effects on LRS. The model was fitted assuming Poisson distributed error terms as LRS represent a count of offspring.

Alternative error-terms, such as Generalized Poisson distribution, are found to potentially describe the distribution of variables such as lifetime reproductive success [START_REF] Kendall | A Stochastic Model for Annual Reproductive Success[END_REF]. However, current implementation of Generalized Poisson distribution in regressions do not allow for fitting models including random effects. Therefore, we compared model estimates from the GAMM-models assuming Poisson distribution with model estimates from a GAM-model fitted with the vgam-function in R-package VGAM (Yee 2017). Model estimates were similar (Table S3 in Appendix S2) and we report results from the GAMM-model.

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the final models included not statistically significant terms. For these models, we manually simplified models until only significant terms were left in the model. For each path, we extracted standardised path coefficients and R 2 -values. The path analysis was conducted for the sites combined and for each site separately. The best model explaining variation in LRS of female flying squirrels included nonlinear terms for the lifetime sum of alder pollen concentration and the lifetime sum of birch catkins (Table 1). LRS increased with increasing alder pollen concentrations and increasing number of birch catkins, but levelled off at intermediate birch catkin availability (Figure 2).

RESULTS

LRS

LRS was, on average, higher in Vaasa than Luoto and showed a positive linear relationship with cover of agricultural fields within the 500 m buffer around the nest site that tended to be steeper in Luoto than in Vaasa (Table 1). The model explained about 52% of the variation in LRS (adjusted R 2 = 0.517).

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Fecundity and longevity

The best model describing variation in average fecundity included a positive linear relationship with the lifetime sum of alder pollen for each female and was higher in Vaasa than in Luoto (Table 1). However, the covariates of interest poorly described fecundity and showed poor explanation power (adjusted R 2 = 0.06).

Longevity was best explained by a non-linear relationship with lifetime sum of birch catkin availability and with lifetime sum of alder pollen availability (Table 1). As for LRS, longevity increased with increasing birch catkin and alder pollen availability. The best model after model selection also included a negative effect of cover of agricultural fields within the 100m buffer (Table 1). The model explained about 55% of the variation in longevity (adjusted R 2 = 0.545).

Path analysis

The path model (based on the smaller dataset including body mass) for Luoto and Vaasa combined (Figure 3) showed that the amount of agricultural fields and suitable forest within the 100m buffer had negative effects on female winter body mass, while suitable forest within the 500m buffer and alder availability had positive effects on winter body mass.

However, body mass had only weak effect on longevity, and no effects on fecundity or LRS (Table S5). Longevity was positively affected by availability of birch catkins and alder pollen, as well as the amount of suitable forest within the 500m buffer. These variables together explained about 43% of the variation in longevity. Fecundity, on the other hand, was explained by a strong positive effect of alder availability and a negative effect of amount of suitable forest within the 100m buffer. However, only 10% of the variability in fecundity was explained. LRS was only directly affected by fecundity and longevity, which explained about 79% of the variation in LRS.

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Ovaskainen 2015). Our study supports the view that the currently inefficient management rules might quite easily be improved with a little bit more planning and preserving larger uncut areas around flying squirrel nest sites. The total area of forest cover needed cannot be evaluated from this study, or the above mentioned studies, and needs further investigation.

Our results indicate a possibly regional difference in fitness response to landscape structure. At Vaasa, landscape scale cover of agricultural fields negatively affected winter body mass according to the path analysis, but not in Luoto (Figure 4). In Luoto, where the cover of fields are lowest, the addition of field edges may act as a resource and predictor of suitable habitat for food where the likelihood of finding birch, aspen and alder are higher. In Vaasa, where the cover of fields within buffers is more extensive, fields may act more as a feature that increases fragmentation, and therefore reduces the long-term body condition of female flying squirrels. The positive effect of fields on flying squirrels was not as clear in our study as the association with occupancy reported previously (Santangeli et al. 2013a).

However, the positive association between field edges and flying squirrel occupation is likely related with food availability (Santangeli et al. 2013a), which we analysed separately from fields, and suggests fields may be an indicator of general food availability on a larger scale.

Interestingly, the effect of body mass, which is correlated with reproductive success in our data (see Appendix S3) and in other arboreal squirrels [START_REF] Wauters | Body Weight, Longevity and Reproductive Success in Red Squirrels (Sciurus vulgaris)[END_REF][START_REF] Wauters | Body Mass at Weaning and Juvenile Recruitment in the Red Squirrel[END_REF]Wauters & Dhondt 1995), had only minor effect in our path analysis. This perhaps indicates that body mass correlation with reproduction is mainly mediated through food availability.

Conclusively, we show that the occurrence of masting events of tree species during a flying squirrel female's lifetime, affecting lifetime availability of food, have profoundly larger effect on lifetime reproductive success than the cover of suitable forest habitat. The importance of mast for LRS may have far reaching consequences for population dynamics

(

  average distance moved per night: 111 ± 33 m,[START_REF] Hanski | Home-Range Size, Movements, and Nest-Site Use in the Siberian Flying Squirrel, Pteromys volans[END_REF], with a size about half of an average annual female home range (ca 7 ha,[START_REF] Selonen | Space use of the Siberian flying squirrel Pteromys volans in fragmented forest landscapes[END_REF]. The 500m buffer covers 78.5 ha and represents the availability of habitats on a larger landscape level, but likely to impact settlement decisions. All GIS analyses were done in R version 3.3.0 (R Development Core Team, 2016).The original land cover classes were combined to represent biological relevant classes for flying squirrels (

Figure 1 .

 1 Figure 1. The map shows the location of the two study sites within Finland. Colours in the

  Table S1 in Supporting Information and Santangeli et al. 2013a),

	including mature forest (all mature and old growth forests), young forest, open habitat (clear-
	cuts and peat bogs), agricultural fields, human impacted habitats (roads and buildings), and
	water (see full description of classes in Table

  . Pulsed food resources, but not forest cover, determines lifetime reproductive success in a forest-dwelling rodent. Journal of Animal

Ecology, 86 (5), 1235-1245. , DOI : 10.1111/1365-2656.12715

Acknowledgements. We thank all of the field workers Pasi Reunanen, Rune Jakobsson, Antero Mäkelä and Markus Sundell, who have assisted during data gathering. Research adhered to all applicable international, national, and/or institutional guidelines for the care and use of animals under several project licences issued by the Finnish Animal Experiment Board (project licence no., 2002-2006: Dnr LSU-2002-L-265(254), 2007-2011: Dnr LSU-2007-L-573(254), 2012-2016: Dnr EPOELY/412/07.01/2012). The study was financially supported by Academy of Finland (grant number 259562 to VS), Oskar Öflunds Stiftelse (to RW), Societas Pro Fauna et Flora Fennica (to RW), Svensk-Österbottniska Samfundet (to RW), and Vuokon luonnonsuojelusäätiö (to RW).

Data accessibility

Data are available from the Dryad Digital Repository: http://dx.doi.org/10.5061/dryad.mr11t [START_REF] Hoset | Data from: Pulsed food resources, but not forest cover, determines lifetime reproductive success in a forestdwelling rodent[END_REF]. Since Siberian flying squirrels are of conservation value, any data that potentially reveals exact locations of breeding individuals are omitted from the data package.

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Comment citer ce document : Hoset, K. S., Villers, A., Wistbacka, R., Selonen (2017). Pulsed food resources, but not forest cover, determines lifetime reproductive success in a forest-dwelling rodent. Journal of Animal Ecology, 86 (5), 1235-1245. , DOI : 10.1111/ 1365-2656.12715 This article is protected by copyright. All rights reserved.

The same model structure as for LRS above was used to test the importance of habitat and food in determining average fecundity and longevity (n = 500). In the model for fecundity, we assumed errors to be Gaussian distributed, while errors were assumed to be

Poisson distributed in the model for longevity. For a smaller subset of females, we included average winter body mass (measured at captures from September until March) of females as an explanatory factor in addition to the above mentioned co-variates (results presented in Appendix S3), as body mass may influence reproductive success in this species [START_REF] Selonen | Mating system and reproductive success in the Siberian flying squirrel[END_REF]. As most females were trapped during the breeding season, only a portion of females had body mass measures not confounded by pregnancy (n = 213).

We determined the optimal model for each model set (LRS, fecundity and longevity, with and without body mass, i.e. 6 model sets) implementing the dredge-function in the Rpackage MuMIn [START_REF] Bartoń | MuMIn: Multi-Model Inference[END_REF]. We first fitted a full model, for which we tested nested models. Full models and model selection tables are available in Appendix S4. In addition, model validation was applied on the optimal model to verify the underlying assumptions.

Specifically, we plotted residuals versus fitted values to assess homogeneity of variance, and the residuals versus each covariate to investigate model misfit.

Finally, we used the smaller dataset including body mass in a piecewise path analysis [START_REF] Shipley | Confirmatory path analysis in a generalized multilevel context[END_REF] to get better understanding on how the different variables interacted and were linked with LRS. Each path in the model was fitted with function gamm4 using the same random structure and error-terms as outlined above to allow for non-linear effects. However, none of the best models obtained included non-linear terms. We followed a hierarchal approach where we tested, 1) whether food and habitat variables explained winter body mass,

2) how food and habitat variables and winter body mass explained fecundity and longevity, and finally 3) whether all of the above mentioned variables explained LRS (see hypothetical paths in Figure S1). At each step, we optimised fit as for previous models. However, many of

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When study areas were analysed separately body mass did not have detectable effects, but was weakly affected by alder in Luoto and negatively by fields in Vaasa (Table S5, Figure 4). Suitable forest within the 500m buffer had a positive effect on longevity in Luoto, but no effects in Vaasa (Table S5, Figure 4). Of the food availability variables, lifetime availability of alder was the main determinant of fecundity and longevity at both study sites, although model fit for fecundity in Vaasa was poor (Figure 4). Instead, birch availability had no detectable effects (Table S5, Figure 4). LRS was equally explained by variation in fecundity and longevity at both sites (Table S5, Figure 4).

DISCUSSION

Lifetime mast availability mainly determined fecundity, longevity and lifetime reproductive success of female Siberian flying squirrels. Cover of suitable habitat did not have strong effects, but at landscape scale (cover within the 500m buffer) suitable habitat had positive effects on body mass, longevity or fecundity, and at local sale (100m buffer) no or negative effects. This discrepancy may reflect the preference for edge habitat at the local scale, whereas at the landscape scale the amount of forests may be important for the settlement process of female flying squirrels.

Only a few long-term studies have assessed the direct link between lifetime reproductive success and habitat, and these studies concentrate on birds (e.g., [START_REF] Newton | Lifetime Reproductive Output of Female Sparrowhawks[END_REF][START_REF] Laaksonen | Lifetime reproduction of a forestdwelling owl increases with age and area of forests[END_REF]) and large ungulates (reviewed in [START_REF] Gaillard | Habitat-performance relationships: finding the right metric at a given spatial scale[END_REF]. Our study supports the view that determining habitat quality requires careful evaluation of individual performance, in particular for species that rely on pulsed resources.

The rare mast events have an obvious importance for lifetime fitness (e.g., Wauters et al. 1995;[START_REF] Bieber | Habitat differences affect life history tactics of a pulsed resource consumer, the edible dormouse (Glis glis)[END_REF][START_REF] Millon | Pulsed resources affect the timing of first breeding and lifetime reproductive success of tawny owls[END_REF], and often the effect of food is mediated through longevity (Wauters & Dhondt 1995;[START_REF] Bérubé | Individual Differences, Longevity, and Reproductive Senescence in Bighorn Ewes[END_REF][START_REF] Bieber | Habitat differences affect life history tactics of a pulsed resource consumer, the edible dormouse (Glis glis)[END_REF]. In our

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case, availability of alder had a strong effect on both fecundity and longevity at both study sites while availability of birch catkins only affected longevity when considering both sites together. This partial difference in response paths and effect strength for birch and alder is logical since birch is the main winter food of flying squirrels possibly affecting winter survival [START_REF] Mäkelä | Liito-oravan (Pteromys volans L.) ravintokohteet eri vuodenaikoina ulosteanalyysin perusteella[END_REF], whereas alder is cached and is important for fuelling production of second litters (Selonen & Wistbacka 2016). Our results are in partial contrast to our initial expectations for the importance of longevity for reproductive success. For example, in North American red squirrels [START_REF] Mcadam | Life histories of female red squirrels and their contributions to population growth and lifetime fitness[END_REF]) longevity of females was the major factor behind population growth rate. In our case, the equal contribution of fecundity and longevity on LRS may be related to the fact that most individuals seem to be short lived, and shortlived individuals breeding in a mast year may still have a high fecundity. However, survival probabilities for flying squirrels may be smaller than for some other tree squirrels [START_REF] Lampila | Survival and Population Growth Rate of the Threatened Siberian Flying Squirrel (Pteromys volans) in a Fragmented Forest Landscape[END_REF], which may explain some of the differences between flying squirrels and North-American tree squirrels.

Interestingly, the cover of suitable forest habitat did not explain variation in LRS directly, with non-conclusive positive effects of cover within 500m radius of nest boxes on longevity and negative effects of cover within 100m radius on fecundity (Table 1, Figure 3, and Figure 4). This is quite surprising for an arboreal forest species (e.g., [START_REF] Reunanen | Habitat requirements of the Siberian flying squirrel in northern Finland: comparing field survey and remote sensing data[END_REF]Santangeli et al. 2013a; see also, Holloway & Smith 2011) that is expected to experience better shelter and higher food availability in mature spruce-dominated forests [START_REF] Mönkkönen | Landscape Characteristics Associated with the Occurrence of the Flying Squirrel Pteromys volans in Old-Growth Forests of Northern Finland[END_REF][START_REF] Reunanen | Managing Boreal Forest Landscapes for Flying Squirrels[END_REF]. Our results were also robust against alternative definitions of suitable habitat. As indicated by the positive effect of large scale habitat cover (500m buffer) relative to local habitat cover (100m buffer), the cover of forests might be more important for the initial acceptance of a site as female flying squirrels settle for breeding. We did not test for habitat selection in this study, and all nesting

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Comment citer ce document : Hoset, K. S., Villers, A., Wistbacka, R., Selonen (2017) Thus, we had decoupled the availability of nesting sites from other habitat characteristics.

These features of the physical habitat, availability of tall trees for gliding and shelter, probably reflect variation in perceived predation risk.

Characteristics of the physical habitat of a species are often highly coupled with food availability, which is evident from the niche-based definition of habitat which includes the resources and environmental factors that define the presence and persistence of a species in a given locality (reviewed in [START_REF] Gaillard | Habitat-performance relationships: finding the right metric at a given spatial scale[END_REF]. In our study however, we were able to compare the relative importance of the physical habitat and food availability separately. The important food sources, alder and other deciduous trees, are known pioneer species that often occur at forest edges, which may explain the affinity of flying squirrels to forest/field edges (Desrochers, Hanski & Selonen 2003) and shoreline forests (R. Wistbacka, unpublished data). These results support the notion that flying squirrels cope well with forest fragmentation [START_REF] Selonen | Young flying squirrels (Pteromys volans) dispersing in fragmented forests[END_REF]Santangeli et al. 2013a), and do not necessarily need large forest areas for breeding. However, it is clear that the area of 0.03-0.07 ha of forest (corresponding to 10-15m radius) that recent Finnish guidelines require to be preserved around breeding or resting flying squirrel locations is not sufficient to ensure a viable and breeding population of flying squirrels (Santangeli et al. 2013b; Jokinen, Mäkeläinen &

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Comment citer ce document : Hoset, K. S., Villers, A., Wistbacka, R., Selonen (2017) This article is protected by copyright. All rights reserved. [START_REF] Ostfeld | Pulsed resources and community dynamics of consumers in terrestrial ecosystems[END_REF], source-sink dynamics [START_REF] Pulliam | Sources, sinks, and population regulation[END_REF], habitat selection [START_REF] Wauters | Demography of alpine red squirrel populations in relation to fluctuations in seed crop size[END_REF], and optimal foraging [START_REF] Boutin | Anticipatory Reproduction and Population Growth in Seed Predators[END_REF]) of the species. Identifying the cause of variation in life history traits linked with lifetime fitness is important for guiding and improving conservation of this species as fitness is tightly coupled with population growth and viability through stochastic events [START_REF] Shaffer | Minimum Population Sizes for Species Conservation[END_REF]. Our study emphasises the importance of both fecundity and longevity, and the indirect effects of food availability via those components, as determinants of lifetime fitness of female flying squirrels.

Authors' contributions. VS conceived ideas for the study; RW collected the data; KH and AV analysed the data; KH and VS led the writing of the manuscript. All authors contributed critically to the drafts and gave final approval for publication.

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Comment citer ce document : Hoset, K. S., Villers, A., Wistbacka, R., Selonen (2017) The following Supporting Information is available for this article online:

Table S1. Habitat classification.

Table S2. Availability of habitat classes within buffer zones.

Table S5. Standardized coefficients and statistics for path analysis

Accepted Article Accepted Article

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Comment citer ce document : Hoset, K. S., Villers, A., Wistbacka, R., Selonen (2017)

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Comment citer ce document : Hoset, K. S., Villers, A., Wistbacka, R., Selonen (2017) This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.