Erwana Harscoe ¨t Nathalie Daniel-Carlier

Dominique The

Aure

Eric Pailhoux

Genevie ` Ve Jolivet
email: genevieve.jolivet@jouy.inra.fr

Gonad Differentiation in the Rabbit: Evidence of Species-Specific Features

The rabbit is an attractive species for the study of gonad differentiation because of its 31-day long gestation, the timing of female meiosis around birth and the 15-day delay between gonadal switch and the onset of meiosis in the female. The expression of a series of genes was thus determined by qPCR during foetal life until adulthood, completed by a histological analysis and whenever possible by an immunohistological one. Interesting gene expression profiles were recorded. Firstly, the peak of SRY gene expression that is observed in early differentiated XY gonads in numerous mammals was also seen in the rabbit, but this expression was maintained at a high level until the end of puberty. Secondly, a peak of aromatase gene expression was observed at two-thirds of the gestation in XX gonads as in many other species except in the mouse. Thirdly, the expression of STRA8 and DMC1 genes (which are known to be specifically expressed in germ cells during meiosis) was enhanced in XX gonads around birth but also slightly and significantly in XY gonads at the same time, even though no meiosis occurs in XY gonad at this stage. This was probably a consequence of the synchronous strong NANOS2 gene expression in XY gonad. In conclusion, our data highlighted some rabbit-specific findings with respect to the gonad differentiation process.

Introduction

In mammals, gonad differentiation is initiated early during foetal life. In a first step, genital crests arising from the adjacent mesonephros are colonized by germ cells. Somatic cells and germ cells then differentiate in a coordinated manner: somatic cells differentiate in cells expressing steroids or cells acting as nursing or supporting cells for germ cells. At the same time, germ cells differentiate to acquire the ability to enter meiosis. It is now well established that multiple, complex and reciprocal interactions between all these cell types are mandatory from early foetal life until puberty to ensure the differentiation of fully functional gonads (see [START_REF] Wilhelm | Sex determination and gonadal development in mammals[END_REF] for review).

The molecular mechanisms that underlie these events have already been studied intensively in mouse species, because this species is a model of choice for the study of gene function through gene invalidation (for a review see [START_REF] Brennan | One tissue, two fates: molecular genetic events that underlie testis versus ovary development[END_REF]). A cascade of genes has thus been described as the main actors which regulate specific steps. It is now accepted that early events which occur during foetal life profoundly determine the sequence of further gonad development. However, foetal life is extremely short in mice when compared with other mammalian species (especially livestock species such as cows, goats, sheep and pigs) and humans. Even though the same events punctuate overall differentiation, the different milestones are not reached at the same time and probably do not have the same effect in each species. It is therefore difficult to rely on data obtained in the mouse to further explain the origin of aberrations in gonad development that may be encountered in other species, especially in humans.

Rabbit species occupy a particular position among livestock species. Rabbit gestation is longer than that of the mouse (30 days, rather than 21 days in the mouse), but still is short enough to enable regular and systematic sampling throughout foetal life. Meiosis occurs at birth in females and not during foetal life, as is the case in numerous other mammalian species. Moreover, there is a 15-day delay between the appearance of genital crests and the onset of meiosis around birth in the female, while this interval is only 3 days long in the mouse. The rabbit genome sequence is now available and despite some missing annotations, it is easy to extract gene sequences from it. Finally, the recent possibility to modify the genome through specifically targeted nucleases (such as meganucleases, zinc finger nucleases or Transcription Activator-Like Effector nucleases) has provided an opportunity to investigate gene function in the rabbit, as has recently been published by other groups [START_REF] Flisikowska | Efficient immunoglobulin gene disruption and targeted replacement in rabbit using zinc finger nucleases[END_REF].

Numerous histological features have previously been described in detail by other groups; the principal events are summarized in Figure 1. From a macroscopic point of view, rabbit gonads become evident at 14 dpc. At this stage, the mesonephros and gonads are still connected and interactions between tissues are probable. A regression of the mesonephros was described from 16 dpc to 25 dpc [START_REF] Bernier | Structure et re ´gre ´ssion du me ´sone ´phros du foetus de lapin[END_REF]. It should be noted that at 23 dpc, the gonadal and mesonephric tissues are separated by connective tissue that is supposed to prevent the migration of cells and other substances [START_REF] Hayashi | Sexual dimorphism in the regulation of meiotic process in the rabbit[END_REF].

In both sexes, the first germ cells are detected from the 9 th day post coı ¨tum [START_REF] Chretien | Etude de l'origine, de la migration et de la multiplication des cellules germinales chez l'embryon de lapin[END_REF]. At 16 dpc, most germ cells have already entered the genital crests. In XY and XX gonads, germ cells undergo a series of mitoses, starting from the early stages and lasting until their final differentiation at meiosis [START_REF] Chretien | Etude de l'origine, de la migration et de la multiplication des cellules germinales chez l'embryon de lapin[END_REF][START_REF] Gondos | Germ cell kinetics in the neonatal rabbit testis[END_REF][START_REF] Iczkowski | Morphometric study of the prepubertal rabbit testis: germ cell numbers and seminiferous tubule dimensions[END_REF]. In XX gonads, the first signs of meiosis have been reported around birth; meiosis spans a period of two weeks after birth. Follicle differentiation initiates after meiosis, as it has been described extensively [START_REF] Hutt | Primordial follicle activation and follicular development in the juvenile rabbit ovary[END_REF]. In XY gonads, meiotic activity appears by 50 days post partum (dpp) and becomes abundant by 63 dpp [START_REF] Iczkowski | Morphometric study of the prepubertal rabbit testis: germ cell numbers and seminiferous tubule dimensions[END_REF]. Interestingly, XY germ cells at preleptoten stages have been detected around birth and for two weeks thereafter in the testis, at the same time as meiosis occurs in the ovary, but without any further signs of more advanced meiotic stages [START_REF] Hayashi | Sexual dimorphism in the regulation of meiotic process in the rabbit[END_REF][START_REF] Gondos | Germ cell kinetics in the neonatal rabbit testis[END_REF]. At 10 weeks of age (70 dpp), the blood-testis barrier is definitively complete [START_REF] Sun | Formation of the blood testis barrier in the rabbit[END_REF]. Puberty is achieved at 5-6 months.

The aim of the present study was to report a first insight into gonad differentiation in the rabbit by studying the expression of genes already characterized as being important actors in sex determination and gonad differentiation in mouse species. A series of genes was retained, as presented in Table 1. At the same time, gonads were analysed histologically throughout the developmental period, alongside immunohistological localizations of proteins when this was possible. Our results showed that the rabbit is a highly interesting species for the study of gonad development during foetal life, as it enables the characterization of original and specific gene expression patterns which coincide better with those seen in human and other livestock species than in mouse species.

Materials and Methods

Sample Collection, Sexing and Staging

New-Zealand (NZ 1077 strain) rabbit embryos and foetuses were collected from the UCEA rabbit facility (Unite ´Commune d'Expe ´rimentation Animale, Jouy-en-Josas, France). The onset of pregnancy was established as the time of observed copulation. The day of coitus was termed day 0. A minimum of three animals was sampled for each stage from 14 dpc until adulthood (6 months). The gonads were rapidly dissected after death, snap frozen in liquid nitrogen and kept at 280uC until RNA extraction. At specific stages (20 dpc, 28 dpc, 4 dpp, 14 dpp, 28 dpp and in adults), only one gonad was kept for RNA extraction. The other gonad was treated immediately for histological study. Except in 14 and 16 dpc samples, the gonads were dissected from the mesonephros. Embryo sexing was performed on the basis of anatomical observation [START_REF] Nielsen | Anatomy of fetal rabbit gonads and the sexing of fetal rabbits[END_REF] and confirmed by determining the presence of SRY using PCR with head genomic DNA and specific primers (see Table S1).

All experiments were performed with the approval of the local committee for animal experimentation (COMite ´d'ETHique applique ´a `l'expe ´rimentation Animale (COMETHEA), Jouy-en-Josas, accreditation number 12/028). All researchers working directly with the animals possessed an animal experimentation license delivered by the French veterinary services.

Histology and Immunohistochemistry

Freshly dissected gonads were fixed in 4% paraformaldehyde in phosphate saline buffer (PBS) at 4uC for times ranging between 2 hours and overnight, depending on the size of the tissue. After washing 3 times in PBS, each tissue was transferred successively to 30% ethanol for 1 hour then to 70% ethanol and processed in an automated Shandon Citadel 2000 (ThermoFisher Scientific, Illkirch, France) before being embedded in paraffin wax for histological or immunohistochemical analysis.

For histology studies, all testis or ovary sections were stained with Harris haematoxylin and eosin (H&E) using standard protocols in order to examine basic tissue morphology. For immunohistochemical analysis, 6 mm tissue sections were used. Briefly, tissues sections underwent antigen retrieval by pressurecooking in 0.01 M Citrate buffer, pH 6.0 for 5 minutes. The sections were then incubated for 2 hours in Blocking Reagent (MOM Kit, Vector Laboratories) and incubated overnight at 4uC with primary antibody diluted in MOM kit buffer (see Table S2 for the list of antibodies and dilutions used). The slides were then washed in PBS and incubated with the appropriate secondary antibody (anti-rabbit IgG Fab2 Alexa Fluor 488 or 555, Cell Signalling; anti-mouse IgG Cy3, Millipore) for 45 minutes at room temperature. The slides were then rinsed in PBS and mounted in Vectashield mounting medium containing DAPI (Vector Laboratories). Observations were performed using a Leica DMRB epifluorescence microscope coupled to a DP50 CCD camera (Olympus).

Quantitative RT-PCR

Total RNAs were extracted from each gonad using Trizol reagent (Invitrogen Life Technologies, Cergy-Pontoise, France) and the RNeasy mini kit (Qiagen SA, Courtaboeuf, France) according to the manufacturer's instructions. All samples were treated with DNase I to remove most of the contaminant genomic DNA. Reverse Transcription (RT) was performed on 500 ng-1 mg of total RNA using the High Capacity cDNA Archive kit (Applied Biosystems) and the random primer mix included in the kit.

Quantification was achieved using SYBR Green quantitative PCR (Applied Biosystems) with dilutions of the RT reactions and sets of primers designed by the Primer Express software (Applied Biosystem). Whenever possible, qPCR primers were chosen on separate exons in order to avoid DNA amplification, and all amplicons were 100 base-pairs long. Moreover, for all samples, a RT minus reaction was performed with all RT components except the reverse transcriptase enzyme, and assayed as a complete RT reaction to ensure that no amplification was due to contaminant DNA.

A series of normalizing genes (b-ACTIN, YHWAZ, HPRT, CPR2, H2AFX) was tested on all samples for their stable expression in gonad tissues. The GeNorm program included in Biogazelle QBasePlus software (Biogazelle NV, Ghent, Belgium) was used automatically to select a combination of the most stable genes for the rabbit gonad samples. Finally, three genes were used as references for this study (YHAWZ, H2AFX, CPR2). In order to correct for inter-run fluctuations, a set of samples was chosen as calibrators and was assayed in all compared runs. Care was taken to consider Ct values within the linear amplification zone. Gene expression was considered as significant when Ct values obtained using 2-5 ng of cDNA in each q-PCR reaction were lower than 34, and when one single DNA fragment with the expected size was amplified as template in each q-PCR reaction.

Results

Histological Features Since the Onset of Gonad Development until Early Puberty

In the present study, sampling for the histological study was initiated at 28 dpc in both sexes. At 28 dpc, the rabbit ovary was primarily made up of naked oocytes located in nests in the ovarian cortex (Figure 2Aa). After birth, clear figures of meiosis appeared. Nuclei at preleptoten, leptoten and zygoten stages were observed (1 dpp, Figure 2Ab). At 4 dpp, germ cells were grouped in oocyte nests with zygoten-stage nuclei (Figure 2Ac). As the age of the rabbit increased, more developmentally advanced follicles were detected in the ovary: primordial follicles with squamous granulosa cells at 14 dpp (Figure 2Ad), primary follicles with cuboidal granulosa cells at 18 and 28 dpp (Figures 2Ae and2Af), and secondary follicles with several layers of granulosa cells in 2 month-old rabbit ovaries (Figure 2Ag, 60 dpp). At the same time, advanced figures of meiosis were observed with nuclei at pachyten and diploten stages.

The foetal testis was clearly distinguishable from the foetal ovary at 28 dpc, at which stage seminiferous tubes were individualized (Figure 2Ba). At 4 dpp, Sertoli cells and large round germ cells were recognized (Figure 2Bb). We were not able to identify preleptoten stage germ cells in 4 dpp testes by opposition to what has been previously described by others [START_REF] Gondos | Germ cell kinetics in the neonatal rabbit testis[END_REF] [START_REF] Hayashi | Sexual dimorphism in the regulation of meiotic process in the rabbit[END_REF]. Besides, at 7 and 18 dpp (Figures 2Bc and2Bd), we observed several nuclei of germ cells with preleptoten like figures. At 28 dpp (Figure 2Be), we detected no further figures of meiosis; instead, condensed nuclei were observed in few cells. Four (120 dpp) and six months after Table 1.

SRY

no intron mammalian Y-chromosomal testis-determining gene [START_REF] Kashimada | Sry: the master switch in mammalian sex determination[END_REF]; already characterized in the chinchillarabbit species [START_REF] Dı ´az-Herna ´ndez | Expression profiles of SRY and SOX9 in rabbit gonads: the classical model of mammalian sex differentiation[END_REF].

SOX9 exon 1-2 transcription factor, target of SRY and SF1, induces Sertoli cell differentiation, stimulates the expression of AMH [START_REF] Sekido | Sex determination involves synergistic action of SRY and SF1 on a specific Sox9 enhancer[END_REF]; already characterized in the chinchilla-rabbit species [START_REF] Dı ´az-Herna ´ndez | Expression profiles of SRY and SOX9 in rabbit gonads: the classical model of mammalian sex differentiation[END_REF].

DMRT1 exon 3-4 transcription factor, interacts with Sox9 [START_REF] Matson | The Mammalian Doublesex Homolog DMRT1 Is a Transcriptional Gatekeeper that Controls the Mitosis versus Meiosis Decision in Male Germ Cells[END_REF]; antagonist of Foxl2 [START_REF] Matson | DMRT1 prevents female reprogramming in the postnatal mammalian testis[END_REF]

Analysis of Gene Expression

As shown in Table 1, a series of genes was studied in order to investigate the molecular mechanisms governing the sex determination and organogenesis of gonads in rabbits. Sampling was initiated at 14 dpc. However, at this stage, the genital ridge is still morphologically undifferentiated. Sex determination was thus based on the presence or the absence of the SRY gene, as determined by PCR on genomic DNA.

Moreover, at 14 and 16 dpc, the mesonephros and gonads were not dissociated prior to RNA extraction. Consequently, at these stages, the level of expression of gonad-specific genes was probably underestimated.

An attempt was made to localize proteins using immunohistological analysis when a specific antibody was available. However, because most commercially available antibodies are produced in the rabbit species, this method suffered frequently from a high background level; this, combined with the weak sensitivity of this method, meant that it was impossible to confirm the presence of some of the studied proteins at all developmental stages. The immunohistological analysis was thus performed with the aim to localize proteins at the cellular level whenever possible. However, in the absence of histological localization, we have no indication concerning which cells express certain genes.

Testis differentiation: SRY, DMRT1, SOX9 and AMH. In mammals, SRY (Sex-determining Region on the Y chromosome) is a major part of a pathway that induces male sex determination [START_REF] Koopman | Male Development of Chromosomally Female Mice Transgenic for Sry[END_REF]. It acts as a transcription factor that regulates the activity of a series of target genes (see for a review [START_REF] Kashimada | Sry: the master switch in mammalian sex determination[END_REF] and references therein).

In the chinchilla rabbit, SRY gene expression was barely but significantly detected by in situ hybridization at 13 dpc in the genital ridges of XY embryos, and by quantitative PCR on gonad RNA from 14 dpc [START_REF] Dı ´az-Herna ´ndez | Expression profiles of SRY and SOX9 in rabbit gonads: the classical model of mammalian sex differentiation[END_REF]. During the present study using quantitative PCR, significant expression was found throughout foetal life in XY embryos, from the first stage studied (14 dpc) to 60 dpp (at least 2 months after birth). It further decreased but was still expressed at a significant level in adults (Figure 3A). As anticipated, no significant expression was found in XX gonads.

DMRT1 is a critical regulator of gonad development in mammals, being involved in testis differentiation specifically through preventing female reprogramming in the postnatal testis in the mouse [START_REF] Matson | DMRT1 prevents female reprogramming in the postnatal mammalian testis[END_REF]. In the rabbit, DMRT1 gene expression was detected at significant levels in both XY and XX gonads (Figure 3B). In the latter, expression reached its maximum at the onset of meiosis around birth at 28-30 dpc, which was 6 times higher than the level on 16 dpc; it then dropped down by 14 dpp, to reach an extremely low level in adults. In XY gonads, DMRT1 gene expression rose from 18 dpc until 28-30 dpc. A major peak was observed with the first wave of meiosis at around 49-60 dpp. The level then remained intermediate until adulthood.

SOX9 is a mandatory gene for satisfactory development of the male phenotype, and is one of the firstly stimulated genes of the SRY-induced pathway. SRY-induced SOX9 transcription is a key event in achieving Sertoli cell differentiation. In the chinchilla rabbit, SOX9 gene expression has been reported in both XY and XX gonads from 13 dpc using in situ hybridization and from 14 dpc using quantitative PCR [START_REF] Dı ´az-Herna ´ndez | Expression profiles of SRY and SOX9 in rabbit gonads: the classical model of mammalian sex differentiation[END_REF]. In the present study using quantitative PCR, very low levels were found in the ovary compared to the testis (Figure 3C1). The level of SOX9 gene expression became significantly higher in the testis than in the ovary from 18 dpc, rising gradually five-fold between 18 dpc and 60 dpp, and then falling to reach a basal level similar that seen at 18 dpc. As shown by immunofluorescence labelling, the SOX9 protein was only detected in the nucleus of Sertoli cells (Figure 3C2).

The anti Mu ¨llerian hormone (AMH) is necessary to induce the regression of Mu ¨llerian ducts that otherwise contribute to the female genital tract. The AMH gene is one of the targets of the SOX9 gene. In XY rabbit, AMH gene expression was low at 14 and 16 dpc, had increased by 18 dpc (3-4-fold), and peaked at 2-4 weeks after birth (Figure 3D1). Expression of the AMH gene was at its lowest in adults. In XX rabbits, between 14 dpc and adulthood, a low level was observed without any significant changes. The AMH protein was detected by immunofluorescence labelling in the cytoplasm of Sertoli cells of 20 dpc testis (Figure 3D2).

Ovary

differentiation: FOXL2, RSPO1, WNT4, CYP19A1. In mammals, a series of genes is expressed specifically in the XX gonad when the male sex determining gene SRY is absent. The genes most extensively studied include FOXL2, genes of the Wnt/b-catenin pathway (RSPO1 and WNT4), the CYP19A1 (Aromatase) gene that is required for oestrogen synthesis and the BMP-15 gene involved in folliculogenesis.

FOXL2 is a mandatory gene for female sex determination that maintains the ovarian phenotype through an active process [START_REF] Uhlenhaut | Somatic sex reprogramming of adult ovaries to testes by FOXL2 ablation[END_REF]. In the rabbit, the testis did not significantly express the FOXL2 gene. In females, the first signs of significant FOXL2 gene expression were detected at around 16-18 dpc (Figure 4A). By 20 dpc until adulthood, the level of FOXL2 gene expression rose gradually to reach a maximum that was sustained in adults. FOXL2 was clearly detected by immunohistofluorescence analysis in granulosa cells (Figure 4A2, 14 dpp).

In the mouse, WNT4 and R-spondin-1 (RSPO1) promote ovarian fate and block testis development through induction of the b2catenin pathway [START_REF] Parma | Rspondin1 is essential in sex determination, skin differentiation and malignancy[END_REF][START_REF] Chassot | RSPO1/b-catenin signaling pathway regulates oogonia differentiation and entry into meiosis in the mouse fetal ovary[END_REF][START_REF] Vainio | Female development in mammals is regulated by Wnt-4 signalling[END_REF]. In the rabbit, the two genes are expressed in the gonads of both sexes from the early stages of differentiation (14-16 dpc; Figures 4B andC). Interestingly, from 18 dpc, the expression levels of both genes became higher in ovary than in testis. In females, WNT4 gene expression reached its maximum at 24 dpc and then fell gradually to reach a low level in adults. RSPO1 gene expression peaked later than WNT4 gene expression, at 7 dpp, one week after birth, then decreased to a minimal level in adult gonads. RSPO1 was detected by immunofluorescence in the cytoplasm of germ cells in the cortical zone at 4 dpp (Figure 4B2).

The cytochrome P450 aromatase is responsible for the irreversible conversion of androgens into estrogens. In goat and fish, the CYP19A1 gene encoding the aromatase is a target of the FOXL2 transcription factor that acts on regulatory elements located on the CYP19A1 gene promoter [START_REF] Pannetier | FOXL2 activates P450 aromatase gene transcription: towards a better characterization of the early steps of mammalian ovarian development[END_REF][START_REF] Wang | Foxl2 up-regulates aromatase gene transcription in a female-specific manner by binding to the promoter as well as interacting with ad4 binding protein/ steroidogenic factor 1[END_REF][START_REF] Fleming | Aromatase Is a Direct Target of FOXL2: C134W in Granulosa Cell Tumors via a Single Highly Conserved Binding Site in the Ovarian Specific Promoter[END_REF]. In the rabbit, aromatase activity has been characterized by other groups in vitro Figure 2. Histological features from the onset of gonad development until early puberty. Paraffin sections of ovaries (2A) and testes (2B) were stained as described in Materials and Methods at 28 dpc, 4, 7, 14, 18 and 28 dpp, 2 months (ovary, 60 dpp), 4 (120 dpp) or 6 months post partum (testis, 180 dpp). Images magnified 2.56 (testes) or 36 (ovaries) are presented for each stage. Arrows point some nuclei harbouring characteristic figures: Sertoli cells (S), granulosa cells (G), germ cells (GC), stages of meiosis (PL = preleptoten; L = leptoten; Z = zygoten; P = pachyten; D = diploten), R = round spermatid, E = elongated spermatid, CN = condensed nuclei. The blue star labels proliferating germ cells (2Bc). doi:10.1371/journal.pone.0060451.g002 by measuring the ability of the embryos to convert androgens into oestrogens. Thus two windows of aromatase activity have already been observed: the first in pre-implantation embryos (male and female) at 6-11 dpc [START_REF] Dickmann | Steroidogenesis in rabbit preimplantation embryos[END_REF][START_REF] Milewich | Estrogen Formation by the Ovary of the Rabbit Embryo[END_REF][START_REF] George | Estrogen formation in the early rabbit embryo[END_REF][START_REF] Hoversland | Aromatase activity in the rabbit blastocyst[END_REF] and the second in XX but not XY gonads at 20 dpc [START_REF] Gondos | Granulosa cell differentiation and estrogen synthesis in the fetal rabbit ovary[END_REF]. In the present study, CYP19A1 gene expression was extremely low in testis during foetal and perinatal life (Figure 4D). By contrast, in ovary, it peaked at 20 dpc and then returned gradually to lower values by 28 dpp. Moderate CYP19A1 gene expression levels were observed in adult XX rabbits. Interestingly, very high levels of aromatase gene expression were detected in the testis 4 months after birth (120 dpp) and in older animals (180 dpp and older adults).

The BMP-15 factor is an ovary-specific secreted factor that acts in a paracrine manner. It is known to play a fundamental role in the growth and maturation of follicles from primordial to primary, and subsequent secondary follicles [START_REF] Otsuka | Integral role of GDF-9 and BMP-15 in ovarian function[END_REF]. In the female rabbit, the growth of primordial follicles starts about two weeks after birth, at the end of meiosis. Significant BMP-15 gene expression was found in ovaries from 14 dpp, when primordial follicles were clearly detected (Figure 4E). The highest level was measured at 60 dpp. A lower level was detected in adults. As expected, the BMP-15 gene was not expressed significantly in testes.

Expression of germ cell marker genes. OCT4 is commonly used to delineate the germ stem cell population, since it is characteristic of totipotency. In the rabbit, we observed that the OCT4 gene was significantly expressed at all studied stages and in both sexes (Figure 5A1). The level of expression rose from 14 dpc in the early-differentiated gonad in line with the number of germ cells. In both testes and ovaries, the maximum level of expression was found at around 24-28 dpc, while the number of germ cells was the highest at 26 dpc [START_REF] Chretien | Etude de l'origine, de la migration et de la multiplication des cellules germinales chez l'embryon de lapin[END_REF]. From 28 dpc in ovaries and 1 dpp in testes, OCT4 gene expression declined gradually to stabilize in adults at a level similar to that seen at the early stages. As expected, the OCT4 protein was localized in the nuclei of germ cells (Figure 5 A2 and A3, in ovaries and testes, respectively). Interestingly, despite a significant expression of the OCT4 gene in young rabbits and adults, we never succeeded in immunolabelling any positive cells after birth in either testes or ovaries. This was probably due to the fact that pluripotent germ cells which still express the OCT4 gene after birth are too rare to be visualized.

Previous studies in the mouse had characterized a number of genes whose expression is necessary for germ cell survival and maturation in both sexes [START_REF] Fujiwara | Isolation of a DEAD-family protein gene that encodes a murine homolog of Drosophila vasa and its specific expression in germ cell lineage[END_REF][START_REF] Raz | The function and regulation of vasa-like genes in germ-cell development[END_REF][START_REF] Onohara | Localization of mouse vasa homolog protein in chromatoid body and related nuage structures of mammalian spermatogenic cells during spermatogenesis[END_REF]. One such gene is the Mvh gene (mouse VASA homolog). In the rabbit, VASA gene expression was observed in both sexes in all the samples studied. The lowest expression was found at 14 and 16 dpc (Figure 5 B1). It reached a first peak after birth, at around 4 dpp in the testis and 14 dpp in the ovary. In the testis, the level of expression then fell at around 10-18 dpp, rising subsequently to being the highest in the adult gonad. The VASA protein was detected by immunohistology in germ cells only with a cytoplasmic location (ovary 18 dpp Figure 5B2 and testis 150 dpp, Figure 5B3).

Mechanisms of meiosis induction and the retinoic acid

transduction pathway. In the female rabbit, the onset of meiosis occurs around birth. The first signs of leptoten stages are visible at 26-28 dpc, and later stages are clearly observed from birth. Meiosis spans for two weeks after birth. Finally, ovocytes are blocked at the diploten stage [START_REF] Hayashi | Sexual dimorphism in the regulation of meiotic process in the rabbit[END_REF]. In XY rabbit, several authors have reported figures of germ cells in the preleptoten stage before birth, at the same stage as in the female, without any signs of further true meiosis; however, the first signs of true meiosis were only observed in the male 2-3 months after birth [START_REF] Hayashi | Sexual dimorphism in the regulation of meiotic process in the rabbit[END_REF][START_REF] Gondos | Germ cell kinetics in the neonatal rabbit testis[END_REF][START_REF] Iczkowski | Morphometric study of the prepubertal rabbit testis: germ cell numbers and seminiferous tubule dimensions[END_REF].

In this study, the expression of genes specific to meiotic events was analysed throughout gestation until adulthood. Three genes were studied in particular (Figure 6). The STRA8 gene is expressed under retinoic acid (RA) activation, and is known to be mandatory for the onset of meiosis [START_REF] Anderson | Stra8 and its inducer, retinoic acid, regulate meiotic initiation in both spermatogenesis and oogenesis in mice[END_REF]. DMC1 is a recombinase A related protein necessary for the progression of meiosis [START_REF] Handel | Genetics of mammalian meiosis: regulation, dynamics and impact on fertility[END_REF]. In the mouse, NANOS2 is essential for the survival of primordial germ cells, and it has been suggested that it acts in the testis as a meiotic repressor [START_REF] Suzuki | Nanos2 suppresses meiosis and promotes male germ cell differentiation[END_REF].

As expected, the STRA8 gene was significantly expressed in the ovary from 24-28 dpc, at the onset of meiosis (Figure 6A). Its expression was maintained throughout meiosis, and then fell after the arrest of meiosis on day 14 pp, after which it was no longer significantly expressed. The pattern of expression of the DMC1 gene was roughly similar to that of the STRA8 gene. A significant but extremely low level of NANOS2 gene expression was observed in the ovary from 24 to 14 dpp. At other stages, the NANOS2 gene was not significantly expressed in the ovaries.

In testes, the STRA8 and DMC1 genes were clearly expressed when meiosis started, from 49 dpp until adulthood. Surprisingly, a low but significant expression of the STRA8 and DMC1 genes was observed from 24-28 dpc, and was maintained for approximately 14 days after birth. Thus, at the time of onset of meiosis in the ovary, genes specific to the induction and advancement of meiosis were also expressed significantly in the testis. Interestingly, a peak of NANOS2 gene expression was observed from day 28 dpc, immediately after the peak of STRA8 gene expression. This peak was followed by a decrease (day 10-14 dpp), and then a second peak (day 28 dpp).

Discussion

The present study reports on a first transcriptomic analysis of gonads in the rabbit throughout development, from 14 days postconception to adulthood. Numerous studies have been performed to date in the mouse to investigate the gene pathways involved in sex determination and gonad differentiation mechanisms, and to achieve a temporal profiling in somatic and germ cells RNAs such as that reported recently ( [START_REF] Jameson | Testis development requires the repression of Wnt4 by Fgf signaling[END_REF] and references therein). But despite the huge quantity of data that has accumulated in recent years, backed by functional genomic studies in the mouse, several authors have suggested that the mouse model is not wholly relevant to other mammalian species. As reported by others ( [START_REF] Ross | Profiles of gonadal gene expression in the developing bovine embryo[END_REF][14] and references therein), a Carnegie stage comparison of the major developmental stages in male sexual development across several mammalian species showed that genital ridge emergence, SRY and SOX9 gene expression, and testis cord formation occur at an earlier stage in the mouse than in all other species. Furthermore, in the other mammals studied, such as the rabbit species, these events were reported at comparable developmental stages. Considering the length of gestation, the occurrence of meiosis at around birth in the female, the high number of foetuses in the litter and recent opportunities to investigate gene function in non-mouse species by using enzymes designed to target specific regions of any genome, the rabbit species could be used conveniently to study the gene pathways involved in sex determination and gonad development.

The main features of the present study have been reported in a schematic conclusion figure (Figure 7). Based on our present data, we are able to confirm in the rabbit the relevance of the classic model for mammalian sex determination. Indeed, as it is clearly visible in Figure 7, in the testis, expression of the SRY gene started from 14 dpc just after genital crest individualization, together with that of the DMRT1, SOX9 and AMH genes, while in the ovary, the onset of expression of the FOXL2, RSPO1 and WNT4 genes marked the female commitment of the gonad from 18 dpc. However, it is worth highlighting some specific features of the rabbit, as reported in the present study. The patterns of SRY and Figure 4. Expression of genes involved in ovary differentiation. Expression levels of the FOXL2, WNT4, RSPO1, CYP19A1 and BMP-15 genes were determined in males (black bars) and females (grey bars). Values are given as percentages after normalisation to the highest value (100%). The horizontal scale represents the age of animals in dpc and dpp. ad = adults, 2-year old rabbit. Note that at 120 and 180 dpp, only male samples were collected. A2, B2: immunofluorescence detection of FOXL2 (A2) and RSPO1 (B2) on paraffin sections of gonads. The antibody against FOXL2 specifically stains the nuclei of granulosa cells (female gonad at 18 dpp). The antibody against RSPO1 specifically stains the cytoplasm of germ cells (female gonad at 4 dpp). doi:10.1371/journal.pone.0060451.g004 SOX9 gene expression have already been compared by others in the mouse [START_REF] Hacker | Expression of SRY, the mouse sex determining gene[END_REF], chinchilla-rabbit [START_REF] Dı ´az-Herna ´ndez | Expression profiles of SRY and SOX9 in rabbit gonads: the classical model of mammalian sex differentiation[END_REF], goat [START_REF] Montazer-Torbati | A study of goat SRY protein expression suggests putative new roles for this gene in the developing testis of a species with long-lasting SRY expression[END_REF] and cow [START_REF] Ross | Profiles of gonadal gene expression in the developing bovine embryo[END_REF]. The authors distinguished two kinds of profiles: the mouse profile, with a short peak of SRY gene expression during early fetal life and no expression later; and the other profiles with a low but sustained SRY expression shortly after the early fetal peak until adulthood. Our present study indicates that the rabbit SRY gene expression profile is closer to the chinchilla-rabbit, goat and cow profile. However, in the rabbit, the SRY gene expression is remarkably high for two months after birth, which is not the case in the other species. To date, the physiological importance of this high level is unknown.

The pattern of aromatase gene expression also necessitates some comments. i) In the ovary, our study highlighted a peak of aromatase gene expression at around 18-20 dpc, followed by a gradual decline. Such a peak of aromatase gene expression has previously been reported in the goat at the onset of ovary differentiation [START_REF] Pannetier | FOXL2 activates P450 aromatase gene transcription: towards a better characterization of the early steps of mammalian ovarian development[END_REF]. Likewise, a peak of aromatase activity has been described at this stage in the rabbit [START_REF] Gondos | Granulosa cell differentiation and estrogen synthesis in the fetal rabbit ovary[END_REF], sheep [START_REF] Maule ´on | Very early and transient 17 b-estradiol secretion by fetal ovary. In vitro study[END_REF] and humans [START_REF] George | Conversion of androgen to estrogen by the human fetal ovary[END_REF], but not in the rat or mouse [START_REF] Weniger | Aromatase activity in fetal gonads of mammals[END_REF]. To date, the physiological importance of this early aromatase activity -or of the peak of aromatase gene expression -remains unknown. ii) Unlike what has been described in the goat [START_REF] Pannetier | FOXL2 activates P450 aromatase gene transcription: towards a better characterization of the early steps of mammalian ovarian development[END_REF][START_REF] Pannetier | Telling the story of XX sex reversal in the goat: highlighting the sex-crossroad in domestic mammals[END_REF], our study did not report any increase in aromatase gene expression during follicular growth. In the rabbit ovary, we reported a lower level after meiosis than during foetal life, while in the goat, the principal peak of aromatase gene expression was observed during the growth of follicles, and was 4-5 times higher than that observed during foetal life. However, it should be pointed out that in the rabbit, the level of aromatase gene expression was far from being null throughout follicle growth and differentiation. Consequently, it can be assumed that aromatase probably plays an important role after meiosis in the gonad development of young rabbits, as has already been proposed in other species. iii) The level of aromatase gene expression was extremely high in the rabbit testis late in puberty (120 dpp) and in the adult. Unfortunately, we were not able to localize the protein by immunohistochemistry using commercially available antibodies. Since the level of circulating oestrogens is very low in the male rabbit, it is reasonable to suppose that aromatase gene-expressing cells are enclosed within the seminiferous tubules. Thus the presence of the blood-testis barrier that appears at 70 dpp in the rabbit [START_REF] Sun | Formation of the blood testis barrier in the rabbit[END_REF] could explain why the oestrogens produced are not secreted in peripheral blood. Additional experiments need to be performed to determine the role of oestrogens produced by the testis in the adult rabbit, but this could be involved in the later process of spermatogenesis, as has already been described in several species (see a review in [START_REF] Carreau | Aromatase expression and role of estrogens in male gonad : a review[END_REF]). To conclude on aromatase data, it is of importance to undertake a further study in order to elucidate the role of this enzyme in rabbit gonad differentiation during fetal and adult life. Experiments are in progress in our laboratory through aromatase gene invalidation in this species.

Expression of the BMP-15 gene was studied in order to investigate a gene that has been shown by others to be involved in follicle growth in many other mammalian species [START_REF] Paulini | The role of oocyte-secreted factors GDF9 and BMP15 in follicular development and oogenesis[END_REF][START_REF] Mandon-Pepin | Expression Profiles and Chromosomal Localization of Genes Controlling Meiosis and Follicular Development in the Sheep Ovary[END_REF]. Here, we report a BMP-15 mRNA profile in complete agreement with this hypothesis, since BMP-15 mRNA was detected in the ovaries from 14 dpp when primordial follicles were observed, peaked during follicle growth and diminished thereafter.

As expected from previous studies in the mouse, and as we observed by immunofluorescence, OCT4 and VASA are germ cellspecific genes. OCT4 gene expression varied with the number of undifferentiated germ cells. In ovaries, it was high until 28 dpc, and dropped down around birth, when in the mouse OCT4 gene expression is down regulated long before birth. This apparent discrepancy is due to the fact that the rabbit is a species presenting a delayed XX meiosis, compared to the mouse, with the first signs of meiosis at 28 dpc in the ovaries, just before birth and not as in the mouse during fetal life. It is thus not surprising that the OCT4 gene expression decreased around birth, which coincides with germ cell differentiation at the onset of meiosis. Interestingly, in testes also, the expression of OCT4 fell dramatically around birth. However, meiosis starts two-three months after birth in testes. Thus it can be hypothesised that XY germ cells were committed to a differentiation pathway at around birth and onwards, even in the absence of meiosis.

VASA gene expression, which is characteristic of germ cells in the mouse from early stages [START_REF] Toyooka | Expression and intracellular localization of mouse Vasa-homologue protein during germ cell development[END_REF], was detected by RT-PCR in the earliest studied stages in the rabbit. It increased gradually as OCT4 gene expression declined, in both testes and ovaries. As already shown in the mouse, in the rabbit also this probably reflected the number of germ cells under differentiation. In ovaries, its expression fell after meiosis. As found by other authors, this corresponds to a phase of germ cell degeneration around meiosis. In rabbit testes, a phase of germ cells degeneration has been described a few days after birth [START_REF] Gondos | Germ cell kinetics in the neonatal rabbit testis[END_REF]. This phenomenon was probably responsible for the decrease in VASA gene expression in the testis that we reported around birth. The subsequent rise in VASA gene expression from 28 dpp until adulthood in the testes probably underlines the progress of mitosis in numerous germ cells, which precedes meiosis at the end of puberty.

Of the various genes that characterize meiosis, we chose to study three genes: STRA8, which is characteristic of the RAdependent induction of meiosis; DMC1, which encodes a protein involved in the mechanism of meiosis; and NANOS2, which is supposed to be involved in the repression of meiosis in the testis. In the rabbit ovary, STRA8 and DMC1 genes were expressed significantly around birth and for two weeks after, as meiosis progressed, but not after that. The model for the RA induction of meiosis is thus probably acceptable in the rabbit ovary, as has previously been demonstrated in the mouse.

Interestingly, we observed a low but significant expression of STRA8 and DMC1 genes around birth and during a few days after birth in rabbit testes. Considering that RA induces meiosis in both sexes, this probably indicates that in the testis, an RA-dependent stimulus could induce meiosis in the same way as in ovaries around birth. This concurs with previously reported observations of XY germ cells at the preleptoten stage in rabbit testes at around birth and for two weeks after [START_REF] Hayashi | Sexual dimorphism in the regulation of meiotic process in the rabbit[END_REF][START_REF] Gondos | Germ cell kinetics in the neonatal rabbit testis[END_REF][START_REF] Iczkowski | Morphometric study of the prepubertal rabbit testis: germ cell numbers and seminiferous tubule dimensions[END_REF]. In the present study as well, the presence of preleptoten like figures at 7 and 18 dpp indicates that meiosis was initiated in the testes but aborted thereafter since no more advanced stages of meiosis were further detected. Consequently, it should be of importance to study genes involved in the synthesis or the metabolism of RA in the rabbit gonad to further clear up the role of the RA induced pathway, as it has been elegantly done in the mouse [START_REF] Raverdeau | Retinoic acid induces Sertoli cell paracrine signals for spermatogonia differentiation but cell autonomously drives spermatocyte meiosis[END_REF].

In the mouse, it has been supposed that the NANOS2 gene is responsible for the absence of meiosis in the foetal testis [START_REF] Suzuki | Nanos2 suppresses meiosis and promotes male germ cell differentiation[END_REF]. In the rabbit testis also, the NANOS2 gene may be at the origin of meiosis arrest. Interestingly, the pattern of NANOS2 gene expression displayed two peaks, like the pattern of VASA gene expression. This was not surprising because it has been described in the mouse that both the NANOS2 and VASA genes are expressed by germ cells. This means that the NANOS2 gene was expressed in rabbit XY germ cells in a sustained manner from few days before birth (24-28 dpc) until the first signs of meiosis at the end of the pre-puberty period (60 dpp). Finally, it is important to point out that in the rabbit ovary, the NANOS2 gene was expressed significantly as in the testis from few days around birth and for two weeks afterwards, but at an extremely low level. This could suggest that in the ovary, NANOS2 gene expression is repressed around birth and subsequently, while this is not the case in the testis.

In conclusion, this study has provided some detailed findings regarding gene expression by the rabbit gonads during early foetal life until adulthood, in both sexes. Most of the genes that have already been characterized in other species (essentially the mouse) as mandatory actors in gonad differentiation were studied, showing that in the rabbit also, they probably play fundamental roles. Notably, the length of the foetal period between the formation of genital crests and birth is sufficiently long (15 days) to offer an opportunity to investigate this period in detail. However, further studies now need to be performed in order to take advantage of this uniqueness of the rabbit species and gain insight into some of the gene pathways involved in the mechanisms of gonad differentiation, such as the aromatase gene pathway in the foetal female gonad or the RA-induced pathway involved in the induction and progress of meiosis.

Figure 1 .

 1 Figure 1. Schematic representation of the principal features of gonad differentiation in the rabbit species. doi:10.1371/journal.pone.0060451.g001

  by binding to phosphoserine-containing proteins testis differentiation

Figure 3 .

 3 Figure 3. Expression of genes involved in testis differentiation. Expression levels of the SRY, DMRT1, SOX9 and AMH genes were determined in males (black bars) and females (grey bars) by quantitative RT-PCR, as described in the Materials and Methods. The levels of expression are given as percentages after normalisation to the highest level (100%). Values are means +/2 sem of expression levels determined on gonads from at least 3 animals per stage. The horizontal scale indicates the age of the rabbit in dpc (days post coitum) and dpp (days post partum). ad = adults, 2-year old rabbit. Note that at 120 and 180 dpp, only testes were collected and analysed. C2, D2: immunofluorescence detection of SOX9 (C2) and AMH (D2) on paraffin sections of gonads. The antibody against SOX9 specifically stains the nuclei of Sertoli cells (male gonad at 28 dpp). The white arrows label some of the Sertoli cells. The antibody against AMH stains the cytoplasm of Sertoli cells (gonads at 20 dpc). doi:10.1371/journal.pone.0060451.g003

Figure 5 .

 5 Figure 5. Expression of germinal-specific marker genes. Expression levels of the OCT4 and VASA genes were determined in testes and ovaries. Values are given as percentages after normalisation to the highest value (100%). A2, A3 and B2, B3: immunofluorescence detection of OCT4 and VASA proteins in paraffin sections of gonads. Immunofluorescence pictures are given with each DAPI counterstaining. The antibody against OCT4 stains the nuclei of germ cells in 28 dpc testis (A2) or ovary (A3). Using the antibody against VASA, a cytoplasmic fluorescence is visible in germ cells in 18 dpp ovaries (B2) and 150 dpp testes. Red arrows point to the specifically labelled nuclei. Horizontal bars represent 100 mm (A2 and A3) or 200 mm (B2 and B3). doi:10.1371/journal.pone.0060451.g005

Figure 6 .

 6 Figure 6. Expression of genes involved in meiosis. Expression levels of the STRA8, DMC1 and NANOS2 genes were determined in male and female gonads. Values are given as percentages after normalisation to the highest value (100%). Note that at 120 and 180 dpp, only male samples were collected. doi:10.1371/journal.pone.0060451.g006

Figure 7 .

 7 Figure 7. Schematic representation of gene expression patterns and main histological observations. Continuous lines represent a significant gene expression in ovaries (pink) or testes (blue) extracts. Dotted lines indicate that expression is significant but at a lower level. Thin black lines are drawn when no samples were assayed. doi:10.1371/journal.pone.0060451.g007

PLOS ONE | www.plosone.org

April 2013 | Volume 8 | Issue 4 | e60451

Acknowledgments

We would like to thank Michel Baratte, Nicolas Stadler and other staff working in the rabbit facility (UCEA, INRA, Jouy-en-Josas) for their care of the animals.

This work was supported by the Agence Nationale de la Recherche (ANR-GenAnimal GENIDOV 2010/13). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Supporting Information

Table S1.

(XLS)

Table S2.

(XLS)

Author Contributions


R Rupp

V Clément

A Piacere

C Robert-Granié

E Manfredi

and relationship with production and udder type traits in dairy Alpine and Saanen primiparous goats

Keywords: somatic cell count, genetic parameter, dairy goat Genetic parameters for milk somatic cell score

Goat milk somatic cell counts have been collected for several years in France by the national milk recording organization. Information is used for health management, because repeatedly elevated somatic cell counts are a good indirect predictor of intramammary infection. Genetic parameters were estimated for 67,882 and 49,709 primiparous goats of the dairy Alpine and Saanen breeds, respectively, with complete information for milk somatic cell counts and milk production traits. About 40% of the goats had additional information for 11 udder type traits scored by official classifiers of the breeders' association CAPGENES. Estimates were obtained by REML with an animal model. The studied trait was lactation somatic cell score (LSCS), the weighted mean of somatic cell score (log-transformed SCC) adjusted for lactation stage. Heritability of LSCS was 0.20 and 0.24 in the Alpine and Saanen breeds, respectively. Relationships with milk production and udder type traits were additionally estimated by using multitrait analyses. Heritability estimates in first lactation ranged from 0.30 to 0.35 for lactation milk, fat, and protein yields; from 0.60 to 0.67 for fat and protein contents; and from 0.22 to 0.50 for udder type traits. Genetic correlations of somatic cell score with milk production traits were generally low, ranging from -0.13 to 0.12. Slightly more negative correlations were estimated for fat content: -0.18 and -0.20 in Saanen and Alpine breeds, respectively. Lactation somatic cell score was genetically correlated with udder floor position (r g = -0.24 and -0.19 in the Alpine and Saanen breeds, respectively), and, in Saanen, teat length, teat width, and teat form (r g = 0.29, 0.34 and -0.27, respectively). These results suggest that a reduction in somatic cell count can be achieved by selection while still improving milk production and udder type and teat traits.

INTRODUCTION

Milk SCC has been used widely in dairy ruminants as an indirect predictor of udder health status. Indeed, milk SCC mainly reflects the number of neutrophils that migrate from blood to the mammary gland in response to infection. As in dairy cattle or sheep, an increase in SCC has been reported in goat as a consequence of infection [START_REF] Poutrel | Control of intramammary infections in goats: Impact on somatic cell counts[END_REF][START_REF] Paape | Milk somatic cells and lactation in small ruminants[END_REF]Bergonier and Berthelot, 2003;Bergonier et al., 2003;[START_REF] Luengo | Influence of intramammary infection and non-infection factors on somatic cell counts in dairy goats[END_REF][START_REF] Moroni | Risk factors for intramammary infections and relationship with somaticcell counts in Italian dairy goats[END_REF][START_REF] Koop | Short communication: Repeatability of differential goat bulk milk culture and associations with somatic cell count, total bacterial count, and standard plate count[END_REF]. Because the frequency of clinical mastitis is very low in dairy goats, relationships between SCC and mastitis were established using milk bacteriology examination. [START_REF] Contreras | Physiological threshold of somatic cell count and California Mastitis Test for diagnosis of caprine subclinical mastitis[END_REF], for instance, reported that 500,000 cells/mL was a useful threshold to discriminate between infected and uninfected udder halves. It is difficult, however, to interpret single SCC measures and to define fixed infection thresholds because numerous factors influence the SCC of infected and noninfected animals, such as age and physiological status of the host, infection stage, and pathogen [START_REF] Paape | Milk somatic cells and lactation in small ruminants[END_REF]Bergonier et al., 2003;[START_REF] Luengo | Influence of intramammary infection and non-infection factors on somatic cell counts in dairy goats[END_REF][START_REF] Moroni | Risk factors for intramammary infections and relationship with somaticcell counts in Italian dairy goats[END_REF][START_REF] Koop | Short communication: Repeatability of differential goat bulk milk culture and associations with somatic cell count, total bacterial count, and standard plate count[END_REF]. Distributions of SCC of infected or noninfected animals overlap considerably. Furthermore, the ability of milk SCC to predict intramammary infection is lower in goat than in cattle and sheep [START_REF] Poutrel | Cell content of goat milk: California Mastitis Test, Coulter counter, and Fossomatic for predicting half infection[END_REF]Boettcher et al., 2005). Accordingly, prediction rules would be better based on repeated SCC measures over a lactation, as proposed by De Crémoux and Poutrel (2001). The latter authors suggest using several SCC measures over lactation and 2 thresholds, of 750,000 and 1,750,000 cells/mL, to discriminate between uninfected animals, animals infected with minor pathogens, and animals infected with major pathogens. With these rules, they achieved a sensitivity of 61 to 83% and a specificity of 61 to 80%. Such prediction, although imperfect, can be used efficiently for prophylactic purposes, including culling of persistently infected animals.

Somatic cell count can be recorded at low cost in routine milk recording, whereas direct mastitis diagnosis is more expensive. Recording of SCC has been extensively developed in dairy cattle and in several dairy sheep populations. In those species, evidence is accumulating that SCC is under genetic control [START_REF] Mrode | Genetic and statistical properties of somatic cell count and its suitability as an indirect means of reducing the incidence of mastitis in dairy cattle[END_REF][START_REF] El-Saied | Genetic parameters of lactation cell counts and milk and protein yields in dairy ewes[END_REF][START_REF] Rupp | Genetics of resistance to mastitis in dairy cattle[END_REF]Rupp et al., 2003). Accordingly, many countries [START_REF] Rupp | Selection for mastitis resistance in French dairy sheep[END_REF][START_REF] Miglior | Selection indices in Holstein cattle of various countries[END_REF] have updated their breeding objective to include a linear decrease of milk SCC as a tool to decrease both subclinical and clinical intramammary infections. Results from a divergent selection experiment in dairy sheep suggest that SCC-based selection can efficiently decrease the rate, duration, and severity of intramammary infections with staphylococci [START_REF] Rupp | Response to somatic cell count-based selection for mastitis resistance in a divergent selection experiment in sheep[END_REF]. Considering SCCbased selection in dairy goat for improved udder health, however, requires accurate estimates of genetic parameters for goat milk SCC that are not currently available in peer-reviewed papers.

The objective of this study was to estimate heritability for milk SCC and genetic correlations between SCC and currently selected traits (milk production and udder type) in Alpine and Saanen goats.

MATERIALS AND METHODS

Data

Data originated from Alpine and Saanen goats first kidding between September 1, 1998, andAugust 31, 2003. Only SCC data collected in A-type sampling schemes as part of the official milk recording system were used; that is, milk samples from evening and morning milkings were mixed before chemical analysis. Somatic cell counts were measured by using a Fossomatic cell counter (Foss Electric, Hillerød, Denmark) at interprofessional milk analysis laboratories. The SCC data were stored in regional genetic information centers and were extracted for the present genetic analysis. The SCC information consisted of an average of 6 individual SCC per lactation collected on a monthly basis. Data on SCC were then merged with pedigree and milk production information from the national goat database of the CTIG (Processing Centre of Genetic Information, Jouy en Josas, France).

As natural mating is predominant in goats, edits were applied to maximize connections between flocks. Disconnectedness affects the estimates of variance components, especially when the genetic level is variable among flocks [START_REF] Clément | Simulation analysis to test the influence of model adequacy and data structure on the estimation of genetic parameters for traits with direct and maternal effects[END_REF]. In the goat populations studied, several groups of farms were identified with different genetic levels given by their mating and replacement policies. Sires could be from the national AI scheme or sons of AI bucks or sons of natural mating bucks. Dams could be daughters of AI bucks, granddaughters of AI bucks, or unrelated to AI bucks. Connected flocks by year were defined as flock-by-year combinations with at least one daughter sired by a connecting AI buck. Connecting bucks had to have at least 20 daughters raised in at least 5 different flock-years. Accordingly, 67,882 and 49,709 Alpine and Saanen goats from 1,814 and 1,324 flock-years, respectively, were selected. Alpine and Saanen goats were sired by 2,624 and 2,125 bucks, respectively, 7% of them ensuring connection among flocks via AI.

Of those selected animals, 24,388 Alpine (36%) and 18,741 Saanen (44%) goats were scored by official classifiers of the breeders' association CAPGENES (Mignaloux Beauvoir, France) for 11 udder type traits during first or second lactation: fore udder, udder profile, udder floor position, teat length, teat width, teat form, teat angle, teat placement, teat orientation, rear udder, and rear udder attachment, as was previously detailed by [START_REF] Manfredi | Genetic parameters of type appraisal in Saanen and Alpine goats[END_REF].

Definition of SCC, Milk Production, and Udder Type Traits

Test-day SCC were transformed to SCS by a classical logarithmic transformation [START_REF] Ali | An optimum transformation for somatic cell concentration in milk[END_REF] to achieve normality of distribution. A mean lactation somatic cell score (LSCS) was computed as the weighted arithmetic mean of test-day SCS adjusted for DIM, following the procedure developed for French dairy cattle [START_REF] Boichard | Genetic analysis and genetic evaluation for somatic cell score in French dairy cattle[END_REF]. Briefly, adjustment factors for DIM at each test were estimated using a quadratic function of time for a subset of goats likely to be unaffected by mastitis. Unaffected goats were selected according to diagnosis rules established by De Crémoux and Poutrel (2001); that is, with all test-day SCC (between 1 and 235 DIM), except 1, below 750,000 cells/mL. Adjustment factors should therefore mainly reflect the dilution effect of DIM. They were estimated independently for the 2 breeds. Adjusted test-day SCS were then averaged using the ratio R d /σ d as weighting factor, where d was the lactation stage in months (d = 1 to 8 stages, up to 240 d in milk), R d was the correlation between test-day SCS at stage d and the average of the other SCS measured during first lactation, and σ d was the standard deviation of SCS at stage d. The highest R d /σ d ratios were for SCS collected between 60 and 180 DIM. Those weights and adjustments should accommodate a lack of strict periodicity in the recording design and the variability in the number of records available.

Milk production traits were those considered for genetic evaluation [START_REF] Clément | Genetic evaluation of French goats for dairy and type traits[END_REF]: 250-d lactation for milk, fat, and protein yields (kg) and fat and protein contents (g/kg). Udder type traits were linear scores from 1 to 9, and included measured traits such as teat length and teat width [START_REF] Manfredi | Genetic parameters of type appraisal in Saanen and Alpine goats[END_REF]. They were considered as continuous quantitative traits with a single measure per animal. Characteristics of the data set are summarized in Table 1.

Method

Variance components for LSCS were estimated separately in the Saanen and Alpine breeds with REML applied to multitrait animal models, using the VCE package [START_REF] Neumaier | Restricted maximum likelihood estimation of covariances in sparse linear models[END_REF]. Genetic and environmental correlations of LSCS with production traits and udder type traits were estimated by multivariate REML using 2-trait analyses because of computing requirements. Four generations of ancestors were traced back with the relationship matrix, resulting in a total number of animals of 162,121 and 121,411 for the Alpine and the Saanen goats, respectively.

For LSCS and the 5 milk production traits recorded in first lactation, the model included, as fixed effects, the combination between year of kidding and flock (1,814 and 1,324 levels in Alpine and Saanen breeds, respectively), the combination between age at kidding and year (25 levels), and the combination between kidding month and year (28 levels), and the random effect of animal.

For udder type traits, the model included, as fixed effects, the interaction between flock, year, and parity (1,646 and 1,225 levels in Alpine and Saanen breeds, respectively), the interaction between age at scoring and year (73 levels), the interaction between lactation stage and year (84 levels), and the random effect of animal. The flock-year-parity interaction included the classifier effect as scoring of each flock-year was done by a single classifier.

RESULTS AND DISCUSSION

Descriptive Statistics

Averages of milk somatic score (LSCS) in primiparous goats were 5.1 (±1.4) and 5.3 (±1.2) in the Alpine and Saanen breeds, respectively (Table 1), corresponding to an average SCC of about 1,000,000 cells/mL. Those levels are in the range of SCC values usually reported in dairy goats, as reviewed by Bergonier et al. (2003) in 3 Spanish, Italian, and French dairy goat populations. Lower values are reported in various sheep breeds (Bergonier et al., 2003) and in dairy cattle. For instance, average values of LSCS in French primiparous Holstein [START_REF] Rupp | Genetic parameters for clinical mastitis, somatic cell score, production, udder type traits, and milking ease in first lactation Holsteins[END_REF] and French primiparous Lacaune dairy sheep (Rupp et al., 2003) were 2.6 (1.2) and 3.1 (1.5), respectively.

The prevalence of intramammary infections and the type of pathogens (mainly staphylococci) are rather similar in the 3 dairy species, except for the higher frequency of clinical mastitis in dairy cattle (Bergonier et al., 2003). High SCC levels in goat are probably due, at least to some extent, to the importance of lentiviral mastitis and to noninfectious factors of inflammation such as parity, stage of lactation, or season [START_REF] Paape | Milk somatic cells and lactation in small ruminants[END_REF].

Genetic Parameters for LSCS

Genetic parameters for LSCS are reported in Table 2. Heritability was 0.20 and 0.24 for the Alpine and Saanen breeds, respectively. The higher value for the Saanen breed resulted from a lower environmental variance. To our knowledge, this is the first published report on genetic parameters for SCC in goat. Conversely, in dairy cattle and sheep, estimates of genetic parameters for SCC have been published over the past 2 decades. Heritability of LSCS in goat was slightly higher than the published heritability for cattle, around 0.15, as reviewed earlier [START_REF] Mrode | Genetic and statistical properties of somatic cell count and its suitability as an indirect means of reducing the incidence of mastitis in dairy cattle[END_REF]; [START_REF] Rupp | Genetics of resistance to mastitis in dairy cattle[END_REF] and for dairy sheep, around 0.13 [START_REF] El-Saied | Genetic parameters of lactation cell counts and milk and protein yields in dairy ewes[END_REF][START_REF] Barillet | Genetic analysis for mastitis resistance and milk somatic cell score in French Lacaune dairy sheep[END_REF]Rupp et al., 2003;[START_REF] Serrano | Genetic analysis of somatic cell count and milk traits in Manchega ewes Mean lactation and test-day approaches[END_REF][START_REF] Legarra | Genetic parameters of udder traits, somatic cell score, and milk yield in Latxa sheep[END_REF][START_REF] Riggio | Parameters for milk somatic cell score and relationships with production traits in primiparous dairy sheep[END_REF]. This higher heritability in goats might be the consequence of a higher true genetic variability (genetic variances in Table 2 were higher than corresponding typical values for cows). In addition, the choice of connected data prevented biases, under-or overestimation, of true genetic parameters as shown in simulation studies [START_REF] Clément | Simulation analysis to test the influence of model adequacy and data structure on the estimation of genetic parameters for traits with direct and maternal effects[END_REF]. Environmental variances were lower than those reported in other studies on small ruminants (e.g., [START_REF] Barillet | Genetic analysis for mastitis resistance and milk somatic cell score in French Lacaune dairy sheep[END_REF]Rupp et al., 2003). One explanation is the standardized conditions of the field data used here, evening and morning sampling each month, and the consequent amount of information on each goat, with an average of 6 records per lactation. Including later lactations could have yielded somewhat lower heritabilities than those reported here. In this regard, we plan to expand on the present study to include lactations >1 and random regression models of analysis [START_REF] Reents | Estimation of genetic parameters for test day records of somatic cell score[END_REF]. Moreover, an ongoing experiment in the experimental farm of INRA Bourges (France) is designed to study the relationships between SCS, clinical mastitis, and infectious status for divergent lines selected for extreme LSCS.

Relationships with Production Traits

Heritabilities for milk production traits (Table 3) were close to previous French estimates in the same breeds [START_REF] Bélichon | Genetic parameters of dairy traits in the Alpine and Saanen goat breeds[END_REF]. Genetic correlations between LSCS and milk production traits were generally low (Table 3). In the Alpine breed, genetic correlations with milk yield and protein yield were not significantly different from zero (P > 0.05). Genetic correlations, however, were slightly positive, and therefore unfavorable, in the Saanen breed (0.06 to 0.12). On the contrary, correlations with fat content and with fat yield (only in the Alpine breed) were significantly (P < 0.05) negative and favorable.

Genetic antagonism between milk yield and SCS has been consistently reported in dairy cattle [START_REF] Mrode | Genetic and statistical properties of somatic cell count and its suitability as an indirect means of reducing the incidence of mastitis in dairy cattle[END_REF] and in some sheep breeds (Rupp et al., 2003;[START_REF] Riggio | Parameters for milk somatic cell score and relationships with production traits in primiparous dairy sheep[END_REF], but the antagonism found in goats was limited, especially in the Alpine breed.

Relationships with Udder Type Traits

Genetic parameters for udder type traits and genetic correlations (r g ) with LSCS are summarized in Table 4. Heritabilities for udder type traits were in close agreement with 2 previous reports in French Saanen and Alpine goats [START_REF] Manfredi | Genetic parameters of type appraisal in Saanen and Alpine goats[END_REF][START_REF] Clément | Elaboration of a total merit index combining dairy and udder type traits[END_REF]. In general, genetic associations of LSCS with udder and teat traits were moderate.

Genetic correlations were significantly (P < 0.05) negative for udder floor position in the 2 breeds (r g = -0.24 in Alpine and r g = -0.19 in Saanen), indicating that better attached udders were associated with lower cell counts. Consistently, udder depth has generally shown the most reliable results across species, breeds, and studies, indicating that higher and more tightly attached udders are associated with lower SCC (review by [START_REF] Rupp | Genetics of resistance to mastitis in dairy cattle[END_REF]. Udder depth can therefore be considered as an easy-to-collect predictor of udder health. In addition, higher udder floor position was genetically associated with lower milk production in previous studies [START_REF] Manfredi | Genetic parameters of type appraisal in Saanen and Alpine goats[END_REF][START_REF] Clément | Elaboration of a total merit index combining dairy and udder type traits[END_REF]. Accordingly, a trade-off has been made to select simultaneously for udder floor position and milk production traits [START_REF] Clément | Elaboration of a total merit index combining dairy and udder type traits[END_REF].

Except for udder depth, genetic correlations between cell counts and udder type traits are highly inconsistent in dairy cattle and sheep literature [START_REF] Rupp | Genetics of resistance to mastitis in dairy cattle[END_REF], which complicates comparisons across species.

In the present goat study, genetic correlations between

3633

LSCS and type traits were highest for teat length, teat width, and teat form in the Alpine breed (Table 4), showing that, in this breed, the shortest and tightest teats are associated with the lowest LSCS. Teat length and width are strongly correlated with teat form: schematically, short and tight teats are cylindrical, whereas long and large teats are conic. Accordingly, goats with cylindrical teats have lower LSCS than goats with conic teats. Teat external anatomy might be associated with prevalence of mammary infections. However, further bacteriological status information of the mammary gland will be necessary to confirm this hypothesis. Correlations with teat traits were significantly (P < 0.05) lower in the Saanen breed than in the Alpine breed (Table 4).

CONCLUSIONS

In the present study, genetic parameters for the weighted lactation mean of somatic cell scores (LSCS) in Alpine and Saanen primiparous goats were reported. Estimates of heritability for SCS at the lactation level obtained in this study will allow implementation of genetic evaluation in the short term. Genetic correlations between LSCS and production traits were generally low. The LSCS trait was genetically favorably associated with tightly attached udders and, in the Saanen breed, with some teat characteristics. A reduction in SCS can be achieved by selection, with minor consequences on present genetic gains in milk production and udder type traits.

Table 1 .

 1 Structure and descriptive statistics of the data set (goats kidding betweenSeptember 1, 1998, and August 31, 2003)

	Item

Journal of Dairy Science Vol. 94 No. 7, 2011

Table 2 .

 2 Genetic parameters (± SE) for lactation SCS in primiparous Saanen (n = 49,709) and Alpine (n = 67,882) goats (means of five 2-trait analyses including each 1 of the 5 milk production traits)

	Parameter	Saanen	Alpine
	Heritability	0.24 ± 0.01	0.20 ± 0.01
	Genetic variance	0.30 ± 0.01	0.31 ± 0.01
	Environmental variance	0.93 ± 0.01	1.26 ± 0.01

1 In a 250-d lactation. Journal of Dairy Science Vol. 94 No. 7, 2011 GENETIC PARAMETERS FOR SOMATIC CELL SCORE IN GOAT

Table 4 .

 4 Mean and heritability (h 2 , ± SE) of udder type traits, and genetic correlation (r g , ± SE) with lactation SCS (LSCS) in 24,388 and 18,741 Alpine and Saanen goats

		Mean (SD)	h 2		r g with LSCS
	Udder type trait 1	Alpine	Saanen	Alpine	Saanen	Alpine	Saanen
	Fore udder Rear udder 1 Udder floor position 1 Udder profile 1 Rear udder attachment 1	3.33 (1.10) 5.57 (1.23) 6.34 (1.06) 5.81 (1.33) 4.96 (1.36)	3.49 (1.20) 5.19 (1.16) 6.17 (1.14) 6.21 (1.28) 5.35 (1.49)	0.30 ± 0.014 0.29 ± 0.015 0.34 ± 0.015 0.40 ± 0.016 0.23 ± 0.015	0.25 ± 0.018 0.24 ± 0.017 0.37 ± 0.018 0.28 ± 0.017 0.29 ± 0.016	-0.16 ± 0.034 -0.16 ± 0.034 -0.24 ± 0.032 0.11 ± 0.030 -0.10 ± 0.039	-0.01 ± 0.040 -0.08 ± 0.040 -0.19 ± 0.036 -0.11 ± 0.037 -0.10 ± 0.039
	Teat length, cm	5.52 (1.49)	6.20 (1.73)	0.50 ± 0.014	0.46 ± 0.017	0.29 ± 0.029	0.15 ± 0.030
	Teat width, cm	2.96 (0.93)	3.16 (1.07)	0.41 ± 0.015	0.45 ± 0.016	0.34 ± 0.030	0.11 ± 0.030
	Teat form	4.90 (1.30)	4.92 (1.40)	0.27 ± 0.016	0.26 ± 0.016	-0.27 ± 0.034	-0.07 ± 0.038
	Teat placement	3.61 (0.99)	4.13 (0.92)	0.38 ± 0.016	0.30 ± 0.017	0.15 ± 0.030	-0.00 ± 0.036
	Teat angle	5.02 (0.87)	5.10 (0.80)	0.22 ± 0.015	0.20 ± 0.016	0.04 ± 0.039	-0.08 ± 0.041
	Teat orientation	3.67 (0.89)	4.07 (0.84)	0.35 ± 0.016	0.32 ± 0.016	0.13 ± 0.032	0.06 ± 0.007

Udder type trait currently included in the goat udder type index.

ACKNOWLEDGMENTS

Authors acknowledge Claire Proffit and Christine Bertrand from the CTIG (Processing Centre of Genetic Information, Jouy en Josas, France) for their assistance in providing SCC data.


Barbara Viginier

Christine Dolmazon

Isabelle Lantier

Fre ´de ´ric Lantier

Fabienne Archer

Caroline Leroux

Christophe Terzian
email: christophe.terzian@univ-lyon1.fr

Copy Number Variation and Differential Expression of a Protective Endogenous Retrovirus in Sheep

The Jaagsiekte sheep retrovirus exJSRV and its endogenous counterpart enJSRV co-exist in sheep. exJSRV, a betaretrovirus, is the etiological agent of ovine pulmonary adenocarcinoma, and it has been demonstrated in vitro that an enJSRV Gag variant bearing the R-to-W amino acid change at position 21 was able to block exJSRV budding from the cells, providing a potential protective role for the host. In this work, we developed a fast mutation detection assay based on the oligo ligation assay (OLA) that permits the quantification of the relative proportions of the R21 and W21 Gag variants present in individual genomes and in cDNA obtained from normal and exJSRV-induced lung tumors. We have shown that the W21/R21 ratio is variable within and between breeds. We also describe for the first time that putative protecting enJSRV variants were expressed in alveolar type II cells (AECII), the major target of exJSRV.

Introduction

Endogenous retroviruses (ERVs) represent a class of retroviruses that are inherited through the germline of their hosts like cellular genes. ERVs represent a significant portion of the host genomes (i.e., 8% of the human genome) and are generally inactivated by point mutations and/or indels [START_REF] Jern | Effects of retroviruses on host genome function[END_REF]. However, some of these endogenous retroviruses still encode functional retroviral proteins. Two main hypotheses have been proposed to understand the maintenance of the coding capacities of ERVs: (1) endogenous retroviral proteins are necessary for the replication of ERVs, and (2) endogenous retroviral proteins are beneficial to their hosts. Several important results have shed light on the benefit of ERV proteins to their hosts. One well-illustrated beneficial impact of ERVs concerns the protection of the host from infection by related exogenous retroviruses. Protection can occur at the entry level of exogenous retrovirus through receptor interference resulting from competition for receptor binding between the exogenous and endogenous envelopes [START_REF] Ikeda | Fv-4 resistance gene: a truncated endogenous murine leukemia virus with ecotropic interference properties[END_REF]. Protection can also occur later during the replication cycle of the retrovirus, either before the integration of the retroviral genome into the host nucleus [START_REF] Pryciak | Fv-1 restriction and its effects on murine leukemia virus integration in vivo and in vitro[END_REF] or during the assembly of the retroviral particles [START_REF] Black | TRIM5alpha disrupts the structure of assembled HIV-1 capsid complexes in vitro[END_REF].

The enJSRV/exJSRV retroviruses in sheep represent a powerful model for the study of interactions between an ERV, its exogenous counterpart and their host. The sheep genome contains approximately 30 copies of the endogenous retrovirus enJSRV, which is highly related to the exogenous betaretrovirus exJSRV (90%-98% identity at the amino acid level), the etiological agent of ovine pulmonary adenocarcinoma [START_REF] Palmarini | Jaagsiekte sheep retrovirus is necessary and sufficient to induce a contagious lung cancer in sheep[END_REF][START_REF] Palmarini | Molecular cloning and functional analysis of three type D endogenous retroviruses of sheep reveal a different cell tropism from that of the highly related exogenous jaagsiekte sheep retrovirus[END_REF][START_REF] Carlson | Chromosomal distribution of endogenous Jaagsiekte sheep retrovirus proviral sequences in the sheep genome[END_REF][START_REF] Leroux | Jaagsiekte Sheep Retrovirus (JSRV): from virus to lung cancer in sheep[END_REF].

Among the enJSRVs, it has been shown that the enJS56A1 provirus was able to block the release of infectious JSRV particles when co-expressed in cultured cells, illustrating a novel mechanism of retroviral interference [START_REF] Murcia | The transdominant endogenous retrovirus enJS56A1 associates with and blocks intracellular trafficking of Jaagsiekte sheep retrovirus Gag[END_REF]. The determinant of this blocking process resides at the level of a single residue at position 21 in the Gag precursor protein. The arginine residue at position 21 (R21) of the exJSRV Gag sequence is replaced by a tryptophan (W21) in the enJSR56A1 Gag sequence. This single change confers to enJS56A1 the ability to block the normal trafficking of JSRV particles to the plasma membrane. A complete set of enJSRV proviruses was characterized from a genomic library for the Texel breed, cloned and sequenced, revealing the presence of a second provirus containing non-synonymous mutations inducing the R-.W change; this provirus was named enJSRV20 [START_REF] Arnaud | A paradigm for virus-host coevolution: sequential counter-adaptations between endogenous and exogenous retroviruses[END_REF]. Interestingly, phylogenetic analysis strongly suggested that these two protecting proviruses became fixed in the sheep during its domestication [START_REF] Arnaud | A paradigm for virus-host coevolution: sequential counter-adaptations between endogenous and exogenous retroviruses[END_REF].

Nothing is known concerning the variation of W21 enJSRV between individuals and between breeds. Therefore, we screened 42 sheep genomes and quantified the presence of W21 using an approach based on the oligo ligation assay (OLA), also known as the fast mutation detection assay [START_REF] Landegren | A ligase-mediated gene detection technique[END_REF]. Moreover, one can ask if protection against sheep exJSRV infection by W21 enJSRV, which was shown in vitro, is also relevant in vivo. Hence, previous data have shown that the enJSRVs are predominantly expressed in the placenta, and exJSRV infects and replicates in AECII [START_REF] Leroux | Jaagsiekte Sheep Retrovirus (JSRV): from virus to lung cancer in sheep[END_REF] but it has never been shown if W21 RNA is present or not in these target cells. We then quantified the relative level of the W21 variant RNAs in AECII derived from tumoral and non-tumoral lungs.

Our results indicate that W21 is present at variable frequencies in the genomes of several breeds and is expressed in the lung, the target of JSRV-induced transformation. Furthermore, we observed that tumoral lung samples rarely express the W21 variant, and when expressed, that it is expressed at a low level compared to that the level in normal lung samples.

Materials and Methods

Ethics Statement

All animal experiments have been performed in compliance with our institutional and national guidelines in accordance with the European Community Council Directive 86/609/EEC. The INRA Tours ethics committee approved the experimental protocols.

Sample Collection

Forty-one DNA samples extracted from total blood have been collected from 10 ovine breeds and five inbred lines (IL) from the experimental flock maintained at the large animal facility, INRA, Tours Nouzilly France (Table 1). IL 16 to 63 are inbred lines derived from the Pre-Alpes breed. Fifteen samples of total lung DNA have been prepared from 7 Manech sheep from the French Basque Region that presented with respiratory symptoms and massive tumoral lesions upon gross examination and from 8 sheep of mixed breeds from a local slaughterhouse (Corbas, France) without macroscopic pulmonary lesions. Total DNA has been extracted using the Fast DNA kit as recommended by the manufacturer (Qbiogene, France). The exJSRV statuses of the sheep have been confirmed by histopathological examination to identify tumoral lesions (Anatomy and Pathological Cytology laboratory, Hospices Civils de Lyon, Pr Franc ¸oise Thivolet-Be ´jui) and by PCR detection of the exJSRV genome using exJSRVspecific primers targeting the 3'end of the env gene and the LTR (primer sequences available upon request). From the same 15 sheep, total RNA has been extracted using the standard RNeasy protocol (Qiagen, France) from primary AECII derived from nontumoral (n = 5) or tumoral (n = 7) lung tissues as previously reported [START_REF] Archer | Alveolar type II cells isolated from pulmonary adenocarcinoma: a model for JSRV expression in vitro[END_REF]. The derivation of primary AECII failed for 3 tissues due to the poor conditions of the tissues. For each sample, 500 ng total RNA have been reverse-transcribed into cDNAs using random hexamers and M-MLV RT (Invitrogen) according to the manufacturer's protocol and have been analyzed by PCR to determine their exJSRV status using specific primers.

DNA and cDNA Gag Amplification

The PCRs were performed using DNAs or cDNAs and the primers enJSRV 482 (5'-TCCTCGCCACTACTCTTATT-3') and enJSRV 953 (5'-GGAGGGTCGCTTTTAACC-3') to amplify specifically the Gag region of enJSRV (Fig. 1A); the corresponding exJSRV region is not amplified using these same primers (not shown). The PCR reactions were performed in 50 ml volumes containing Taq polymerase buffer, 1.65 mM MgCl 2 , 200 mM each dNTP, 0.2 mM each primer, 100 to 500 ng of genomic DNA, and 1.25 U of Taq polymerase (Eurobio). The amplification conditions consisted of an initial 5 min denaturation at 95uC followed by 35 cycles of 94uC for 30 sec, 55uC for 30 sec, and 72uC for 1 min and a final extension of 10 min at 72uC. Positive and negative controls were based on PCR reactions performed using the penJS65A1 and pJS21 plasmids, which contain, respectively, enJSRV and exJSRV proviral sequences.

OLA Assay

OLA is a genotypic assay for the detection of known mutations based on the covalent joining of two adjacent oligonucleotides by a DNA ligase when they are hybridized to a DNA target [START_REF] Arnaud | A paradigm for virus-host coevolution: sequential counter-adaptations between endogenous and exogenous retroviruses[END_REF]. For each reaction, 4 ml of purified amplicon, 4 U of thermostable Thermus aquaticus ligase (New England Biolabs, Beverly, MA), 5 nM upstream HEX or FAM-modified oligonucleotides (HEX-GCA-TATGTTATCTGTAATGTTAAAACATT, FAM-AAAAGCA-TATGTTATCTGTAATGTTAAAACATT, or FAM-AAAAG-CATATGTTATCTGTAATGTTAAAACATA), and 5 nM downstream phospho-modified oligonucleotide (Ph-GGGGAAT-TACTGTTTCTAAACCTAAATTAAT) were added together in a 20 ml reaction (Fig. 1); the phospho-modification is necessary for ligation to occur. The ligation assay was performed using an initial 2 min denaturation at 94uC followed by 20 cycles at 94uC for 30 sec and 45uC for 3 min and a final denaturation of 10 min at 98uC. Only probes that matched to the target were ligated, resulting in a 60 nt HEX-labeled oligonucleotide for the W21 coding sequence, and in a 64 nt FAM labeled oligonucleotide for the R21 coding sequences (Fig. 1). Two microliters of a 1/10 dilution of the OLA products was mixed with 7.5 ml of deionized formamide and 0.5 ml of ROX-1000 size marker (PE Biosystems). The mixture was denatured at 95uC for 3 min and rapidly cooled on ice prior to analysis by a DNA sequencer (ADNid, France). The FAM and HEX peak heights values were determined by Gene Scan analysis (Applied Biosystems), and the data were analyzed using STR and 2.3.79 (Veterinary Genetics Laboratory, University of California, Davis, CA). The results have been expressed as the ratio of the HEX/FAM peak areas.

Results and Discussion

Validation of the OLA Assay to Estimate the Ratio of W21 vs. R21 Variants Our first aim was to estimate the number of enJSRV proviruses coding the W21 Gag variant relative to the number of wild-type (R21) enJSRV proviruses. We performed a molecular assay based on the oligo ligation assay (OLA) of 41 genomes sampled from 10 sheep breeds and five inbred strains derived from the Pre-Alpes du Sud breed (Table 1). We developed an assay involving a PCR followed by a quantitative OLA that allowed the determination of the ratio of the copy numbers of the R21 and W21 enJSRV gag variants (Fig. 1). The PCR specifically amplifies a 470 bp region of the enJSRV gag gene encompassing the R21/W21 variant position. This specificity was provided by the reverse primer that targeted a segment within a variable region (VR1), which is highly divergent between exJSRV and enJSRV [START_REF] Hallwirth | Variable regions 1 and 2 (VR1 and VR2) in JSRV gag are not responsible for the endogenous JSRV particle release defect[END_REF]. Four OLA probes were designed: the downstream phospho-oligonucleotide probe was common to the R21 and W21 gag sequences, whereas the three labeled upstream oligonucleotides were specific for the mutations responsible for the R21 and W21 variants (Fig. 1). Ligation only occurred when there was a perfect match between the upstream and downstream oligonucleotides. Labeled ligation products were then identified based on their fluorescent dyes and their sizes: 60 nt for W21 vs. 64 nt for R21 (Fig. 1).

The repeatability of the estimation of the ratio of W21 to R21 variants was tested in two steps. First, the penJS56A1 plasmid [START_REF] Palmarini | Molecular cloning and functional analysis of three type D endogenous retroviruses of sheep reveal a different cell tropism from that of the highly related exogenous jaagsiekte sheep retrovirus[END_REF] containing the W21 variant was diluted to 0%, 20%, 50% and 100% vs. the pJaag101 plasmid containing the R21 variant; for each dilution, a PCR followed by the OLA was then performed twice on two replicates, and two curves (one for each replicate) were obtained by plotting the HEX/FAM peak area ratios vs. the serial dilutions. The two corresponding R-squared values were 0.96 and 0.99, reflecting the very high repeatability of the assay and the good prediction values of the curves (F-statistics and pvalues = 10 26 and 10 28 , respectively). Thus, the standard curve allowed intrapolation of the W21/R21 ratio for test specimens using the measured HEX/FAM ratio. Second, PCRs were performed on genomic and cDNA samples. Ten amplicons were randomly chosen, and OLAs were performed three times on each amplicon. The W21/R21 ratio was estimated using the previously constructed standard curves (Fig. 2), and the high repeatability of the W21/R21 measurement was confirmed by the non-parametric Friedman test (p-value = 0.41), indicating no significant difference between the triplicate measurements from the same sample. The OLA was then performed once for the 41 genomic samples from the breeds and twice for the others samples; in the latter case, the means of the two measurements were used.

W21 Gag Variant is Present in the Genomes of Many Ovine Breeds

Our results indicate that the W21/R21 ratios are significantly different between ovine breeds (Kruskall-Wallis test, p = 0.01). This result suggests that the R21 and/or W21 enJSRV copy number varies significantly from breed to breed (Fig. 3). Interestingly, the W21/R21 ratio varies between individuals from the same inbred line (e.g., IL16) (Fig. 3), suggesting ongoing proviral amplification. We determined by Southern blotting that the enJSRV proviral copy number varies between but also within the IL16, 19, 36, 39 and 63 inbred lines (data not shown). Likewise, it was recently shown that enJS65A1 copy number can vary 5-fold between several individuals from Texel [START_REF] Armezzani | The signal peptide of a recently integrated endogenous sheep betaretrovirus envelope plays a major role in eluding gag-mediated late restriction[END_REF]. This result implies that the two enJS65A1 copies among the 30 enJSRV copies described in a single Texel individual by Arnaud et al. [START_REF] Arnaud | A paradigm for virus-host coevolution: sequential counter-adaptations between endogenous and exogenous retroviruses[END_REF] are not representative of Texel individuals. Thanks to the OLA approach, we are now able to estimate the R21/W21 ratio in a large sample of individuals, and do not restrict our screen to enJS65A1 but consider all enJSRV types coding the W21 Gag mutant.

We then tested the hypothesis that a significant level of W21 Gag could provide a selective advantage for the hosts. Thus, we looked for the presence of W21 gag expression in AECII, the primary target of exJSRV together with Clara cells in the lung.

The W21 Gag Expression Level in Sheep Lung Cells Depends on the Tumoral Status of the Sample

We compared the W21/R21 variant frequencies in DNA extracted from the 15 studied lungs (7 tumoral and 8 non-tumoral tissues) and, when available, i.e., for 12 of the 15 animals, from the DNA from the corresponding in vitro-derived primary AECII (5 tumoral and 7 non-tumoral), established as previously reported [START_REF] Archer | Alveolar type II cells isolated from pulmonary adenocarcinoma: a model for JSRV expression in vitro[END_REF]. All the tumoral alveolar type II cells harbored the JSRV provirus genome, while the non tumoral cell lines were negative [START_REF] Archer | Alveolar type II cells isolated from pulmonary adenocarcinoma: a model for JSRV expression in vitro[END_REF]. We first estimated the W21/R21 ratios in the genomic DNA. The W21/R21 ratio varied from 13 to 48%, and the medians from the tumoral (T) and non-tumoral (NT) genomic samples were not significantly different (Wilcoxon test, p-value = 0.8). However, we observed a significant difference when we compared the medians between genomic DNA and cDNA extracted from tumoral and non-tumoral samples (Fig. 4): the median of W21/ R21 cDNA (RNA) ratio was significantly lower than the median of W21/R21 genomic copies (DNA) in tumoral samples, whereas such a difference did not exist in non-tumoral samples (Fig. 4). This result indicates that the expression of W21 gag was null or lower in tumoral AECII and lung tissues than in non-tumoral samples. Whether the absence of W21 expression favors in vivo the replication of exJSRV could not be assessed in this study.

However, it is known that the site of proviral integration can affect the expression by mechanisms that involve local epigenetic modifications, as described for position effects on the expression of transgenic integrants [START_REF] Wang | Quantitative analysis of lentiviral transgene expression in mice over seven generations[END_REF]. Comparing the W21 proviral insertion sites between infected sheep that develop a JSRV-induced lung adenocarcinoma and infected sheep that do not develop this adenocarcinoma would help us to answer to this question.

Conclusions

In this study using a rapid, discriminative and quantitative molecular technique, we have shown that the W21/R21 ratio varies between individuals from different breeds. Moreover, our results indicate for the first time that W21 and R21 enJSRV are expressed in normal lung tissues and that W21 is not expressed or is expressed at a low level in tumor-derived AECIIs. Our present study did not demonstrate that the absence of W21 gag RNA (and therefore W21 Gag proteins) was the causal factor in the development of tumoral cells, but this study does provide new insights into the putative protective role of some endogenous retroviruses against infection by their exogenous counterparts.

Figure 1 .

 1 Figure 1. A. Canonical structure of the enJSRV provirus. The W/R variant at position 21 is indicated by a star. The 470 bp amplicon is represented by a gray bar. B. Ligation products obtained from the OLA detection assay of W21 and R21 variants. The sequence of the phosphatemodified downstream oligo is indicated in italics. The upstream oligo that detected the R21 to W21 mutation is labeled with HEX, whereas the two FAM-labeled oligos detected the R21 variant. Probes that were mismatched to the target by a single nucleotide at their junctions were not ligated. The ligation products were discriminated by size (60 nt for W21 coding sequence vs. 64 nt for R21 coding sequences) and color (HEX for W21 vs. FAM for R21). The W and R codons are underlined. doi:10.1371/journal.pone.0041965.g001

Figure 2 .Figure 3 .

 23 Figure 2. Boxplot representation of triplicate W21/R21 ratio measurements among randomly selected 10 samples. For each sample, W21/R21 measurement was repeated three times. Each dot represents a single measurement. Numbers correspond to the samples listed in Figure 1. NT1 and NT2 correspond to DNA extracted from two non-tumoral samples. doi:10.1371/journal.pone.0041965.g002

Figure 4 .

 4 Figure 4. Distribution of the W21/R21 ratios between the DNA and RNA extracts from non-tumoral (respectively n = 6 and n = 5) and tumoral samples (respectively n = 7 and n = 7). Two Wilcoxon tests were used to compare the medians of W21/R21 ratios measured in DNA and RNA extracts from non-tumoral (p-value = 0.41, non significant) and tumoral samples (p-value = 0.001, highly significant). doi:10.1371/journal.pone.0041965.g004

Table 1 .

 1 List of breeds analyzed. IL16 to 63 are inbred lines derived from the Pre-Alpes du Sud French breed.

	Breed	No. of individuals
	IL16	4
	IL19	2
	IL36	3
	IL39	4
	IL63	4
	Texel (Tex)	2
	Ile de France (IdF)	2
	Bleu de Maine (BdM)	2
	Rouge de l'Ouest (RdO	2
	Vende ´en (Ven)	3
	Solognot (Sol)	3
	Pol Dorset (Pol)	3
	Suffolk (Suf)	3
	Tho ˆnes et Marthod (Tho)	2
	Romane (Rom)	2
	doi:10.1371/journal.pone.0041965.t001

PLoS ONE | www.plosone.org 1 July 2012 | Volume 7 | Issue 7 | e41965

PLoS ONE | www.plosone.org

July 2012 | Volume 7 | Issue 7 | e41965

Acknowledgments

We are grateful to Dr Hung Fan for the penJSRV65A and pJSR21 plasmids. We also thank Dr Franc ¸ois Guiguen and Dr Jean-Christophe Natorp for the post mortem collection of tissue samples.

This work was supported by Institut National de la Recherche Agronomique, Ecole Pratique des Hautes Etudes, Agence National Recherche ''Genomobile'' and Fondation Innovation en Infectiologie. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Author Contributions

Conceived and designed the experiments: BV CT. Performed the experiments: BV CD. Analyzed the data: BV CT. Contributed reagents/ materials/analysis tools: BV IL FL FA CL. Wrote the paper: BV CL CT.