Neli Hevir

Jasna Šinkovec

Dr Tea Lanišnik Rižner

Disturbed expression of phase I and II estrogen-metabolizing enzymes in endometrial cancer: Lower levels of CYP1B1 and increased expression of S-COMT

Keywords: Endometrial cancer, Estrogen-metabolizing enzymes, Cytochrome P450 1B1 (CYP1B1), Catechol-O-methyltransferase

Expression levels of genes encoding phase I and II estrogen-metabolizing enzymes: CYP1A1, CYP1A2, CYP1B1, CYP3A5, CYP3A7, SULT1A1, SULT1E1, SULT2B1, COMT

Endometrial cancer (EC) is the fourth most common cancer in women, and the most prevalent gynaecological malignancy in the developed World [START_REF] Amant | Endometrial cancer[END_REF][START_REF] Parkin | Global cancer statistics[END_REF].

Based on clinicopathological and molecular characteristics, two different pathways have been described for tumourigenesis of sporadic endometrial carcinoma [START_REF] Amant | Endometrial cancer[END_REF][START_REF] Samarnthai | Molecular profiling of endometrial malignancies[END_REF]. The first is type-I, or estrogen-dependent, endometrioid endometrial carcinoma, which is usually low-grade and represents the majority of cases (70% to 80%); this forms in a background of endometrial hyperplasias [START_REF] Nieminen | Molecular analysis of endometrial tumorigenesis: importance of complex hyperplasia regardless of atypia[END_REF]. The second is type II, or non-endometrioid endometrial carcinoma, which accounts for the remaining 10% to 20% of cases, and is believed to follow an estrogen-unrelated pathway. The type II cancer is typically high-grade, most frequently papillary serous, and less frequently clear-cell carcinoma.

Some tumours share pathological and molecular features of both types I and II. Therefore, many endometrial carcinomas are in the gray area between types I and II, and have overlapping clinical, morphological, immunohistochemical and molecular features [START_REF] Liu | Molecular carcinogenesis of endometrial cancer[END_REF]. There is, however, a clear association of excessive exposure to estrogens that is not opposed by progesterone, with the development of hyperplastic processes and the initiation and maintenance of malignant tumour growth in the endometrium [START_REF] Henderson | Hormonal carcinogenesis[END_REF].

Estrogens can induce cell proliferation and stimulate cell division, while they can also cause random errors during DNA replication that can result in a malignant phenotype [START_REF] Zhu | Functional role of estrogen metabolism in target cells: review and perspectives[END_REF][START_REF] Henderson | Hormonal carcinogenesis[END_REF][START_REF] Hecht | Molecular and pathologic aspects of endometrial carcinogenesis[END_REF][START_REF] Bolton | Potential mechanisms of estrogen quinone carcinogenesis[END_REF][START_REF] Lanisnik Rizner | Estrogen metabolism and action in endometriosis[END_REF]. The endogenous estrogens can undergo extensive oxidative metabolism at different positions, which is catalyzed by various cytochrome P450 (CYP) isoforms [START_REF] Zhu | NADPH-dependent metabolism of 17beta-estradiol and estrone to polar and nonpolar metabolites by human tissues and cytochrome P450 isoforms[END_REF] (Figure 1). These produce mostly A-ring metabolites by A c c e p t e d M a n u s c r i p t 4 2-and 4-hydroxylation, such as catecholestrogens (CEs), and to a lesser extent, D-ring metabolites by 16α-hydroxylation [START_REF] Zhu | Functional role of estrogen metabolism in target cells: review and perspectives[END_REF]. The metabolic activation of estrogens to form the CEs 2-hydroxy-estrone/estradiol and 4-hydroxy-estrone/estradiol (2-OH-E1/E2 and 4-OH-E1/E2, respectively) is catalyzed predominantly by CYP1A1/1A2 and CYP1B1, respectively [START_REF] Yager | Estrogen carcinogenesis in breast cancer[END_REF]. These CEs can be oxidized to estrogen ortho-quinones (E1/E2-2,3-Q and E1/E2-3,4-Q, respectively), which can react with DNA, primarily forming unstable N3-adenine or N7-guanine DNA adducts. Greater carcinogenic potency is associated with 4-OH-E1/E2 than 2-OH-E1/E2, the latter of which are borderline carcinogens. Additionally, quinones undergo redox cycling via their reduction to semiquinones and subsequent oxidation back to quinones, forming the reactive oxygen species (ROS) superoxide (O 2 -) and then H 2 O 2 , which in the presence of Fe 2+ forms hydroxyl radicals that can damage cellular proteins and DNA [START_REF] Zhu | Functional role of estrogen metabolism in target cells: review and perspectives[END_REF][START_REF] Lee | Characterization of the oxidative metabolites of 17beta-estradiol and estrone formed by 15 selectively expressed human cytochrome p450 isoforms[END_REF]Cavalieri et al., 2006;[START_REF] Yager | Estrogen carcinogenesis in breast cancer[END_REF][START_REF] Bolton | Potential mechanisms of estrogen quinone carcinogenesis[END_REF][START_REF] Salama | Catecholestrogens induce oxidative stress and malignant transformation in human endometrial glandular cells: protective effect of catechol-O-methyltransferase[END_REF]. In human extra-hepatic tissue, 16α-hydroxylation is mainly catalyzed by CYP3A5 [START_REF] Zhu | NADPH-dependent metabolism of 17beta-estradiol and estrone to polar and nonpolar metabolites by human tissues and cytochrome P450 isoforms[END_REF] and CYP3A7 [START_REF] Lee | Characterization of the oxidative metabolites of 17beta-estradiol and estrone formed by 15 selectively expressed human cytochrome p450 isoforms[END_REF]. Like 4-OH-E2, 16α-OH-E1 and 16α-OH-E2 show specificity for the estrogen receptors ERα and ERβ [START_REF] Zhu | Quantitative structureactivity relationship of various endogenous estrogen metabolites for human estrogen receptor alpha and beta subtypes: Insights into the structural determinants favoring a differential subtype binding[END_REF]. Indeed, increased formation of 16α-OH-estrogens has been suggested to be associated with elevated risk of breast cancer [START_REF] Zhu | Functional role of estrogen metabolism in target cells: review and perspectives[END_REF].

The metabolism of CEs and 16α-OH-estrogens is catalyzed by the phase II metabolic enzymes: catechol-O-methyltransferase (COMT) (Cavalieri et al., 2006;[START_REF] Salama | Catecholestrogens induce oxidative stress and malignant transformation in human endometrial glandular cells: protective effect of catechol-O-methyltransferase[END_REF], sulfotransferases (SULTs) [START_REF] Raftogianis | Chapter 6: Estrogen metabolism by conjugation[END_REF][START_REF] Adjei | Catecholestrogen sulfation: possible role in carcinogenesis[END_REF][START_REF] Lee | Characterization of the oxidative metabolites of 17beta-estradiol and estrone formed by 15 selectively expressed human cytochrome p450 isoforms[END_REF], UDP glucuronosyl transferases (UGTs) [START_REF] Lepine | Specificity and regioselectivity of the conjugation of estradiol, estrone, and their catecholestrogen and methoxyestrogen metabolites by human uridine diphosphoglucuronosyltransferases expressed in endometrium[END_REF], and glutathione Stransferases (GSTs) [START_REF] Lee | Characterization of the oxidative metabolites of 17beta-estradiol and estrone formed by 15 selectively expressed human cytochrome p450 isoforms[END_REF]. Quantitatively, methylation is the most important conjugative pathway for CEs [START_REF] Raftogianis | Chapter 6: Estrogen metabolism by conjugation[END_REF]. COMT has an important role in estrogen-induced cancers because it inactivates CEs by converting them into non-A c c e p t e d M a n u s c r i p t 5 carcinogenic methoxyestrogens (MeO-estrogens). Furthermore, MeO-estrogens, and especially 2-MeO-E1/E2, have potent tumour-suppressing properties in vitro [START_REF] Zhu | Inhibition of catechol O-methyltransferase-catalyzed Omethylation of 2-and 4-hydroxyestradiol by quercetin. Possible role in estradiol-induced tumorigenesis[END_REF][START_REF] Mannisto | Catechol-O-methyltransferase (COMT): biochemistry, molecular biology, pharmacology, and clinical efficacy of the new selective COMT inhibitors[END_REF]. The COMT protein exists in two major forms: soluble (S-COMT) and membrane-bound (MB-COMT) [START_REF] Jeffery | Purification and kinetic mechanism of human brain soluble catechol-O-methyltransferase[END_REF]. S-COMT and MB-COMT are products of the same gene and are regulated by the proximal P1 and distal P2 promoters, respectively [START_REF] Tenhunen | Genomic organization of the human catechol O-methyltransferase gene and its expression from two distinct promoters[END_REF][START_REF] Mannisto | Catechol-O-methyltransferase (COMT): biochemistry, molecular biology, pharmacology, and clinical efficacy of the new selective COMT inhibitors[END_REF]. In most human tissues, the majority of COMT is present as S-COMT, with only a small fraction as MB-COMT [START_REF] Tenhunen | Genomic organization of the human catechol O-methyltransferase gene and its expression from two distinct promoters[END_REF]. Both COMT enzymes catalyze a wide variety of endogenous and exogenous catechol substrates. However, the kinetic properties and in-vitro regioselectivity of these enzymes are different [START_REF] Pihlavisto | Separation methods for catechol O-methyltransferase activity assay: physiological and pathophysiological relevance[END_REF][START_REF] Bai | Biochemical and molecular modeling studies of the O-methylation of various endogenous and exogenous catechol substrates catalyzed by recombinant human soluble and membrane-bound catechol-Omethyltransferases[END_REF].

Estrogens and CEs are also inactivated by sulfation, which is catalyzed by cytosolic SULTs [START_REF] Raftogianis | Chapter 6: Estrogen metabolism by conjugation[END_REF]. Among these, SULT1A1 and SULT1E1 are the key enzymes of estrogen and CE inactivation [START_REF] Hirata | CYP1A1, SULT1A1, and SULT1E1 polymorphisms are risk factors for endometrial cancer susceptibility[END_REF], while SULT2B1 might be important in CE conjugation [START_REF] Adjei | Catecholestrogen sulfation: possible role in carcinogenesis[END_REF]. Estrogens and their metabolites can also be eliminated by specific UGT proteins, forming inactive glucuronides. UGT2B7 is the major UGT expressed in the uterus, and it mainly catalyzes glucuronidation of 4-OH-estrogens. This can block the genotoxic oxidation of 4-OH-CEs to the quinone estrogens, and thus reduce their mutagenic potential [START_REF] Zhu | Functional role of estrogen metabolism in target cells: review and perspectives[END_REF][START_REF] Lepine | Specificity and regioselectivity of the conjugation of estradiol, estrone, and their catecholestrogen and methoxyestrogen metabolites by human uridine diphosphoglucuronosyltransferases expressed in endometrium[END_REF]. Also, conjugation of CEs with GSTs has an important role [START_REF] Lo | Genetic polymorphism and function of glutathione Stransferases in tumor drug resistance[END_REF]. Among the GSTs, the piclass enzyme GSTP1 is the major GST isoform expressed consistently in a wide range of tissues, including endometrium [START_REF] Terrier | An immunohistochemical study of pi class glutathione S-transferase expression in normal human tissue[END_REF][START_REF] Chan | Promoter methylation and differential expression of piclass glutathione S-transferase in endometrial carcinoma[END_REF]. Conjugation transforms estrogens into less active, and more polar and water-soluble metabolites, thus facilitating their excretion in the bile and urine [START_REF] Neves | Biochemical and computational insights into the anti-aromatase activity of natural catechol estrogens[END_REF]. Clearly, the balance between the toxification and de-toxification metabolic pathways determines whether formation of the CE-quinones occurs [START_REF] Joosten | Genotoxicity of hormonal steroids[END_REF]. Carcinogenesis is usually viewed as a step-wise process that begins with genotoxic effects (initiation), followed by enhanced cell proliferation (promotion). It is generally accepted that estrogens promote carcinogenesis by ER-mediated cell proliferation [START_REF] Joosten | Genotoxicity of hormonal steroids[END_REF], which results in an increased risk of genomic mutations during DNA replication. Another mechanism involves newly discovered membrane-associated ERs and appears to regulate extranuclear estrogen signaling pathways [START_REF] Bolton | Potential mechanisms of estrogen quinone carcinogenesis[END_REF]. The third pathway of carcinogenesis involves the oxidative metabolism of estrogens to electrophilic/ redox-active estrogen ortho-quinones, with the concurrent formation of ROS, as a mechanism of tumour initiation by endogenous and exogenous estrogens (Cavalieri and Rogan, 2006;[START_REF] Bolton | Potential mechanisms of estrogen quinone carcinogenesis[END_REF]. Many studies have reported the carcinogenicity of estrogens and CEs through their mitogenic and mutagenic affects with regard to breast cancer. However, there are only a few studies of estrogen oxidative metabolism at the mRNA, protein and enzymeactivity levels in endometrial cancer (Sasaki et al., 2003a;[START_REF] Bochkareva | Co-occurrence of hyperplasia, endometrial carcinoma and uterine myoma: role of sex hormones, their receptors and enzymes of estrogen metabolism[END_REF][START_REF] Chan | Promoter methylation and differential expression of piclass glutathione S-transferase in endometrial carcinoma[END_REF][START_REF] Lepine | Circulating Estrogens in Endometrial Cancer Cases and Their Relationship with Tissular Expression of Key Estrogen Biosynthesis and Metabolic Pathways[END_REF][START_REF] Salama | Catecholestrogens induce oxidative stress and malignant transformation in human endometrial glandular cells: protective effect of catechol-O-methyltransferase[END_REF][START_REF] Singh | Quantifiable mRNA transcripts for tamoxifen-metabolising enzymes in human endometrium[END_REF][START_REF] Saini | Functional significance of cytochrome P450 1B1 in endometrial carcinogenesis[END_REF].

In the present study, we have characterized the local metabolism of estrogens in endometrial cancer and adjacent control endometrium at the mRNA as well as at the protein and cellular levels. We hypothesized that the alteration in estrogen phase I and II metabolism contributes to development of endometrial cancer.

Tissue samples

Specimens of endometrial cancer and adjacent control endometrium were collected from 40 patients undergoing hysterectomies (mean age, 63 ±14 years) (Table 1). An additional nine control endometrium samples were obtained following hysterectomies from patients diagnosed with myoma uteri (mean age, 47 ±5 years) (Table 2). The study was approved by the National Medical Ethics Committee of the Republic of Slovenia. The specimens were immediately placed into RNA Later (Qiagene) RNA stabilization solution, and kept at -20 °C until RNA extraction. Diagnosis of carcinoma was confirmed histologically by an experienced gynaecological pathologist (J.Š.).

RNA isolation

Total RNA was isolated from 38 tissue samples using Tri Reagent (Sigma), according to the manufacturer instructions. The quality of the RNA samples was determined using Agilent 2100 Bioanalyser, and they showed an average RIN of 7.7. Total RNA was reverse transcribed using SuperScript® VILO™ cDNA Synthesis Kits (Invitrogen). One μg of total RNA was converted into cDNA (20 l) according to the manufacturer instructions, and then stored at -20 °C.

Selection of normalization gene candidates

A cohort of 16 reference genes was tested on samples of endometrial cancer and adjacent control tissue (see Table 3). The TaqMan® Gene Signature Array Configuration (384) -Human Endogenous Control Array (Applied Biosystems, Foster City, CA, USA) was used.

The most stable reference genes: PPIA, HPRT1 and POLR2A were selected using both, GeNorm and NormFinder algorithms. The expression of eleven genes of interest and three selected reference genes was examined by real-time TaqMan® PCR assay. Expression levels were determined with the exonspanning hydrolysis probes (FAM or VIC dye labeled) that are commercially available as 'Assay on Demand' (Applied Biosystems, Foster City, CA, USA), with optimised primer and probe concentrations (Table 4). Quantification was accomplished with an Applied Biosystems 7500 real-time RT-PCR machine using TaqMan® Universal PCR Master Mix and universal thermocycling parameters recommended by Applied Biosystems. The RT-PCR samples were run in duplicates using 1 μL cDNA. The reactions were performed in 96-well plates (Applied Biosystems) in the reaction volume of 20 μL. The gene expression normalization factor for each sample was calculated based on the geometric mean of all three selected reference genes [START_REF] Vandesompele | Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes[END_REF]. The Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines were considered in the performance and interpretations of the qPCR reactions [START_REF] Bustin | The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments[END_REF].

Western blotting

Proteins were isolated from samples of cancerous endometrium and the adjacent control endometrium previously used for RNA isolation, following the Tri Reagent instructions.

Protein aliquots of 60 μg were separated by SDS PAGE on 10% gels (NuPAGE® Novex 10% Bis-Tris Gel 1.0 mm, Invitrogen). The proteins were transferred from the gels to PVDF membranes (Millipore) and incubated with 5% non-fat milk for 1 h, to avoid non-specific binding. The membranes were then incubated overnight with the primary antibodies: rabbit anti-CYP1B1 (Abcam, ab33586, 1:20000), and rabbit anti-COMT (Millipore, ab5873, 1:7500) in TTBS with 1% non-fat milk powder. The next day, the secondary antibodies A c c e p t e d M a n u s c r i p t 9 (peroxidase-conjugated goat anti-rabbit IgG + IgM (H + L), Jackson ImmunoResearch Laboratories Inc., USA, 1:10000) were incubated with membranes for 2 h. The RapidStep ECL Reagent (Calbiochem) was used for detection of the bound antibodies, according to manufacturer instructions, using Fujifilm LAS4000 (Fujifilm, Japan). β-Actin (Sigma, A5441, 1:3000), cyclophilin A (Abcam, ab58144, 1:1000) and GAPDH (Sigma, G8795, 1:3000) were tested as normalisation controls. The mean values of the detected signals for cyclophilin A and GAPDH showed the best correlations to the amounts of proteins visualized on the gels after Coomassie Blue staining. Therefore, the protein levels of the investigated genes were normalised to these values. Quantification of Western blots was carried out with Multi-Gauge (Fujifilm software, Fujifilm, Japan).

Immunohistochemistry

Formalin-fixed, paraffin-embedded samples of the cancerous endometrium were de-waxed in xylene and rehydrated. Sections were incubated in H 2 O 2 to block endogenous peroxidase.

After antigen retrieval in sodium citrate buffer in a microwave oven, sections were incubated overnight with mouse anti-COMT (Abnova, H00001312-B01, 1:20) and rabbit anti-CYP1B1 antibodies (Abcam,ab33586,1:200). The specificities of the antibodies were first checked by Western blotting. Control staining was performed by replacing the primary antibodies with serum of the same animal species. The peroxidase-antiperoxidase complex with the substrate 3,3'-diaminobenzidine (DAB Plus Substrate System, Thermo Fisher Scientific Inc.) were used to detect the bound antibodies.

Additionally, endometrial cancer/ adjacent paired tissue microarray (TMA; Pantomics, Inc, EMC241) was used to detect cellular CYP1B1 by immunohistochemical staining. TMA sections were first evaluated by a pathologist (J.Š.), to confirm the diagnoses determined by the manufacturer. Eleven pairs of cancerous and adjacent control sections were confirmed 5). In one pair, both of the sections were diagnosed as tumour sections, and so they were excluded from further evaluation. The TMA were processed as described above for fullsection slides. A quick-score system was used for immunohistochemical scoring: the data represent the products of the percentages of positive cells and the intensities of the stain (scoring interval = 0-300).

Statistical analyses

Statistical calculations and tests were performed using the SPSS Software (SPSS Inc., USA).

The Wilcoxon test was used for paired samples, and the Mann-Whitney test for unrelated samples. All of the statistical tests were two-tailed, and differences in p values of less than 0.05 were considered to be significant.

Results

CYP1B1 codes for the most abundant phase I enzyme in human endometrium, and it is down-regulated in endometrial cancer

We initially studied the expression of five genes coding for estrogen phase I metabolizing enzymes by real-time PCR in 38 samples of cancerous and adjacent control endometrium: CYP1A1, CYP1A2, CYP1B1, CYP3A5 and CYP3A7 (Figure 2). There were significantly lower mRNA levels of CYP1B1 (5.4-fold; p <0.0001; 95% of samples) and CYP3A7 (9.6-fold; p = 0.0006; 79% of samples) in the cancerous tissues. Among all of the genes coding for the phase I enzymes, CYP1B1, the prevalent estrogen 4-hydroxylase, was the most abundant CYP isoform in both control and cancerous tissues. The second most abundant was CYP3A5, which is mainly responsible for 16α-hydroxylation, but which was not aberrantly expressed in the tumors (p = 0.4684). The levels of CYP1A1 were much lower (more than 10 4 -fold) than the A c c e p t e d M a n u s c r i p t 11 levels of CYP1B1, and there was no significant difference in CYP1A1 expression between cancerous and adjacent control endometrium (p = 0.7607). The expression of CYP1A2 was hardly detectable. These more than 10 4 -fold lower levels of CYP1A1 and negligible mRNA levels of CYP1A2 compared to CYP1B1 suggested a marginal role for estrogen 2hydroxylation in both control and cancerous endometrial tissue.

Protein levels of CYP1B1 are also decreased in endometrial cancer

Protein expression levels of CYP1B1 were examined in 15 samples of cancerous and adjacent control endometrium. The results were assessed by Western blotting and showed significantly lower protein levels of CYP1B1 in cancerous tissue (2.9-fold; p = 0.0002). Protein levels of CYP1B1 were decreased in 14 cases (93%) (Figure 3). CYP1B1 expression was also examined at the cellular level, using immunohistochemical staining (Figure 4). CYP1B1 was detected all over the tumour samples, predominantly in epithelial cells, but to a lesser extend also in stromal cells. The average scores using the quick-score system (see Methods) for 18 cancerous specimens was 175 ±35 for epithelial and 103 ±36 for stromal cells. In 14 specimens, normal and simple hyperplastic endometrial glands were also present, and were used as controls. The mean score for the control specimens was 197 ±31. The score was lower in 13 of 14 specimens and a small, and statistically significant, difference was seen for the cancerous gland staining, compared to control glands (p = 0.0143). As not all of the specimens contained control endometrial glands, we introduced an additional 8 control endometrial samples of myoma uteri patients. The mean score for these control specimens was 178 ±22 for epithelial and 160 ±23 for stromal cells. No difference was found between cancerous and control endometrial specimens of myoma patients for the staining of epithelial cells. A difference was, however, seen in stromal cells, where the staining was statistically TMA was also used to evaluate CYP1B1 cellular status (Figure 4). The TMA included 11 endometrial cancer samples paired with their corresponding non-involved tissue from the same patients. The mean score for cancerous and adjacent control sections was 146 ±43 and 162 ±33 for epithelial and 73 ±44 and 88 ±33 for stromal cells, respectively. Control sections showed slightly higher scores than cancer sections; this differences was, however, not statistically significant (p = 0.1763) (Figure 5).

Genes coding for phase II enzymes SULT2B1, UGT2B7 and GSTP1 are over expressed in cancerous tissue

The expression levels of genes coding for phase II enzymes, were also measured in 38 samples of cancerous and adjacent control endometrium: SULT1A1, SULT1E1, SULT2B1, COMT, UGT2B7 and GSTP1 (Figure 2). Significantly higher mRNA levels were seen in cancerous than control tissues for SULT2B1 (10.3-fold; p <0.0001; 84% of samples), UGT2B7 (2.1-fold; p = 0.0342; 63% of samples) and GSTP1 (1.4 fold; p = 0.0001; 75% of samples). COMT and SULT1A1 expression were not altered (p = 0.1315; p = 0.6322; respectively). The levels of SULT1E1 were decreased in 71% of cancerous samples, yet not with any statistical significance (p = 0.1173). The mRNA levels of GSTP1 and COMT were the highest, suggesting that these represent the main detoxification force in endometrial tissue.

S-COMT is the prevalent COMT isoform in cancerous endometrium, and MB-COMT prevails in adjacent control tissue

COMT protein levels were examined in 16 samples of cancerous and adjacent control endometrium by Western blotting (Figure 3). Significantly lower protein levels of MB-COMT A c c e p t e d M a n u s c r i p t 13 (2.8-fold; p = 0.0015) were seen in cancerous tissue. Levels of S-COMT were elevated in the tumours of all 16 samples examined, and this increase was statistically significant (2.1-fold; p = 0.0005). The total COMT levels remained unchanged (p = 1.0000), which correlated well with our qPCR experiments. The comparisons of the cancerous samples and the control samples from the same individuals revealed that in control endometrium the levels of MB-COMT were 2.96-fold higher than the levels of S-COMT, whereas in the tumour the levels of MB-COMT were 2.71-fold lower. This MB-COMT/S-COMT ratio was thus increased 8.00fold in adjacent control compared to cancerous endometrium (p = 0.0006), indicating that in cancerous endometrium, S-COMT is the most abundant COMT isoform, while in contrast, MB-COMT appears to be of greater importance in adjacent control tissue.

There are at present no specific antibodies available for MB-COMT or S-COMT, so we could only examine total COMT at the cellular level (Figure 4). Positive immunohistochemical staining was seen mainly in epithelial cells, while stromal staining was very weak. The mean score for 18 cancerous specimens was 141 ±24 for epithelial and 24 ±17 for stromal cells. Normal and hyperplastic endometrial glands were present in 14 of a total of 18 specimens, and the mean score for these control epithelial cell specimens was 147 ±24. As expected, there were no significant differences in these scores between cancerous and control epithelium.

We also stained 9 control endometrium specimens of myoma uteri patients. The mean score of these control specimens was 158 ±35 for epithelial and 84 ±40 for stromal cells. We saw no significant differences in epithelial cell staining between cancerous and control specimens of these myoma patients. Interestingly, the stromal cells scores for the control myoma samples were significantly higher than for the tumours (p = 0.0003).

A c c e p t e d M a n u s c r i p t 14

The significantly lower CYP1B1/COMT ratio in endometrial cancer suggests decreased synthesis of 4-OH-E1/E2 and increased formation of MeO-estrogens

We compared the CYP1B1/COMT expression ratios to determine the contributions of these enzymes in cancerous and adjacent control tissues. We found a 4.84-fold lower CYP1B1/COMT ratio in tumours when comparing mRNA expression levels (p <0.0001) (not shown), and a 3.50-fold decrease when analyzing protein levels (p = 0.0001) (Figure 3). A decreased ratio between the activating enzyme CYP1B1 and the detoxificating COMT suggests reduced synthesis of 4-OH-E1/E2 accompanied by increased formation of protective MeO-estrogens in the cancerous tissue. Furthermore, we compared CYP1B1 protein levels with levels of the MB-COMT and S-COMT isoforms (Figure 3). The CYP1B1/MB-COMT ratio was surprisingly not altered in cancerous compared to adjacent control tissue (1.08-fold; p = 0.3303, statistically significantly effective pairing), suggesting that decreased CYP1B1 expression in tumours is associated with the same fold-lower MB-COMT expression. On the other hand, the CYP1B1/S-COMT ratio was statistically significantly reduced (11.47-fold; p = 0.0001) in the cancer tissue, showing that here a lower expression of CYP1B1 was accompanied by an increased expression of S-COMT.

Discussion

Estrogens have been shown to contribute to the growth and development of endometrial cancer. The most active estrogen, E2, stimulates cell proliferation and gene expression via the ERs, while by forming oxidative metabolites, it causes DNA damage [START_REF] Zhu | Functional role of estrogen metabolism in target cells: review and perspectives[END_REF][START_REF] Parl | Estrogen exposure, metabolism, and enzyme variants in a model for breast cancer risk prediction[END_REF]. In the present study, we assessed the mRNA expression profiles of the 10 most abundant estrogen phase I and phase II metabolizing enzymes. We also studied the The CYP1A1 and CYP1A2 enzymes are mainly responsible for 2-hydroxylation of estrogens [START_REF] Zhu | NADPH-dependent metabolism of 17beta-estradiol and estrone to polar and nonpolar metabolites by human tissues and cytochrome P450 isoforms[END_REF]. 2-OH-E1/E2 can be oxidized to E1/E2-2,3-Q, which is believed to be less carcinogenic. The moderate expression of CYP1A1 and the hardly detectable CYP1A2 levels seen in our study suggest a marginal role for estrogen 2hydroxylation in both control and cancerous endometrial tissues.

The major CYP isoform in human endometrium is CYP1B1, which mainly catalyzes 4-hydroxylation of estrogens. Previous studies have suggested that high levels of estrogen 4hydroxylase activity in estrogen target tissues are important in estrogen-induced carcinogenesis [START_REF] Zhu | Functional role of estrogen metabolism in target cells: review and perspectives[END_REF]Cavalieri and Rogan, 2006;[START_REF] Bolton | Potential mechanisms of estrogen quinone carcinogenesis[END_REF]. However, our data show significantly decreased levels of CYP1B1 in cancerous endometrium compared to adjacent control tissue. This same expression pattern in endometrial cancer was reported by [START_REF] Singh | Quantifiable mRNA transcripts for tamoxifen-metabolising enzymes in human endometrium[END_REF], and recently by Lepine at al. [START_REF] Singh | Quantifiable mRNA transcripts for tamoxifen-metabolising enzymes in human endometrium[END_REF][START_REF] Lepine | Circulating Estrogens in Endometrial Cancer Cases and Their Relationship with Tissular Expression of Key Estrogen Biosynthesis and Metabolic Pathways[END_REF]. Knowing that mRNA levels do not necessarily correlate with the amount of their respective proteins, we examined protein levels of CYP1B1 by Western blotting and immunohistochemical staining. The 2.9-fold decrease in CYP1B1 protein levels in cancerous tissue indicated reduced 4-hydroxylation in the tumours. The same pattern was seen at the cellular level. In contrast to our results Saini et al. reported up-regulation of CYP1B1 in endometrial cancer based on immunohistochemical staining of 48 cases, but their study was not supported by quantification and statistical analysis [START_REF] Saini | Functional significance of cytochrome P450 1B1 in endometrial carcinogenesis[END_REF]. As others have seen previously, we show that in human endometrium, the levels of the 4hydroxylating enzymes exceed those of the 2-hydroxylating enzymes (Sasaki et al., 2003a;[START_REF] Lepine | Circulating Estrogens in Endometrial Cancer Cases and Their Relationship with Tissular Expression of Key Estrogen Biosynthesis and Metabolic Pathways[END_REF]. Due to reduced expression of CYP1B1 in the tumours, its possible role in precancerous tissue has also to be taken into consideration, especially knowing that our A c c e p t e d M a n u s c r i p t 16 adjacent control endometrium might have already undergone some hyperplastic changes, and endometrial hyperplasia is regarded as a preliminary stage of endometrioid carcinoma type 1.

It has been suggested that 16α-OH-E1 has an important role in mammary carcinogenesis [START_REF] Nebert | Elevated estrogen 16 alpha-hydroxylase activity: is this a genotoxic or nongenotoxic biomarker in human breast cancer risk?[END_REF]. CYP3A7 is the main 16α-hydroxylating enzyme, followed by CYP3A5 [START_REF] Lee | Characterization of the oxidative metabolites of 17beta-estradiol and estrone formed by 15 selectively expressed human cytochrome p450 isoforms[END_REF][START_REF] Zhu | NADPH-dependent metabolism of 17beta-estradiol and estrone to polar and nonpolar metabolites by human tissues and cytochrome P450 isoforms[END_REF]. In the present study, both genes, CYP3A7 and CYP3A5 were expressed at high levels. No differences were seen for CYP3A5; however, CYP3A7 was statistically significantly down-regulated in cancerous tissues, suggesting that, as with 4-hydroxylation, 16α-hydroxylation is also decreased in endometrial cancer.

Thus, two out of three reactions of estrogen oxidative metabolism appear to be deficient in endometrial cancer. Our data imply decreased production of potentially carcinogenic 4-OH-E1/E2 and 16α-OH-E1/E2 in the tumours, accompanied by unchanged, but low, 2-hydroxylation. Oxidation of estrogens is followed by their methylation to the less harmful MeOestrogens. COMT is expressed in various human tissues, but its precise role and the ratio between the S-COMT and MB-COMT isoforms remain unclear. A membrane-spanning extension in the amino-terminus of MB-COMT contributes to the higher binding affinity of its substrates [START_REF] Lotta | Kinetics of human soluble and membrane-bound catechol O-methyltransferase: a revised mechanism and description of the thermolabile variant of the enzyme[END_REF]. And the catalytic efficiency of S-COMT for 4-Omethylation is approximately 50% lower than the efficiency for 2-O-methylation, whereas the efficiency of MB-COMT for both CEs is comparable [START_REF] Bai | Biochemical and molecular modeling studies of the O-methylation of various endogenous and exogenous catechol substrates catalyzed by recombinant human soluble and membrane-bound catechol-Omethyltransferases[END_REF]. We observed no differences in the expression of total COMT at the mRNA and protein levels. In contrast to our results, Lepine at al. reported elevated mRNA levels of COMT in endometrial cancer compared to adjacent control tissue [START_REF] Lepine | Circulating Estrogens in Endometrial Cancer Cases and Their Relationship with Tissular Expression of Key Estrogen Biosynthesis and Metabolic Pathways[END_REF]. This difference in expression profiles may have arisen from different normalization procedures used in the qPCR data quantification. We carefully selected normalization controls among 16 genes examined, while A c c e p t e d M a n u s c r i p t immunohistochemical analyses also confirmed our qPCR data. Additionally, Western blotting revealed modified expression of the COMT isoforms: S-COMT was present in cancerous endometrium, while MB-COMT was the most abundant in the adjacent control endometrium.

To the best of our knowledge, this is the first study showing increased levels of S-COMT in endometrial cancer suggesting lower capacity for 4-O-methylation in tumour. Our data showing decreased MB-COMT expression in endometrial cancer can be explained by the elevated CpG methylation of the MB-COMT promoter that was observed by Sasaki et al. in 78.3% of cancer cases, and was also confirmed by lower mRNA levels for MB-COMT.

However, the same study showed that S-COMT was expressed and unmethylated (Sasaki et al., 2003b); therefore, the mechanisms behind the elevated levels of S-COMT in cancerous endometrium are not clear at the moment, and remain to be further defined.

We found no changes in the CYP1B1/MB-COMT ratio, which demonstrates that in the tumours, the decrease in CYP1B1 expression correlates well with the down-regulation of MB-COMT. Even more so, the fold-levels of the decreases here were almost identical (2.9 and 2.8 for CYP1B1 and COMT, respectively). However, the CYP1B1/S-COMT ratio was reduced, implying that the decreased CYP1B1 levels in the tumours were accompanied by elevated S-COMT expression. These data suggest that the expression of CYP1B1 and MB-COMT are linked at either the transcriptional or translational levels. The role of each COMT isoform in endometrial cancer therefore needs further investigation. Also sulfate conjugation is an important pathway for estrogen biotransformation [START_REF] Adjei | Catecholestrogen sulfation: possible role in carcinogenesis[END_REF][START_REF] Adjei | Human estrogen sulfotransferase (SULT1E1) pharmacogenomics: gene resequencing and functional genomics[END_REF][START_REF] Pasqualini | Recent insight on the control of enzymes involved in estrogen formation and transformation in human breast cancer[END_REF]. SULT1E1, SULT1A1 and SULT2B1 catalyze the sulfation of CEs and E2. In the present study, lower levels of SULT1E1, the most efficient of the SULT enzymes, were observed in 70% of cancer cases, thus suggesting decreased sulfation of CEs and estrogens. However, the A c c e p t e d M a n u s c r i p t 18 significant increase in SUL2B1, the most abundant SULT enzyme in the cancerous endometrium, indicated that lower levels of SULT1E1 may be compensated for by much higher levels of SULT2B1.

Another important conjugative pathway of estrogens, CEs and MeO-E1/E2 is by glucuronidation. Several UGTs catalyze these reactions [START_REF] Guillemette | Metabolic inactivation of estrogens in breast tissue by UDP-glucuronosyltransferase enzymes: an overview[END_REF][START_REF] Lepine | Specificity and regioselectivity of the conjugation of estradiol, estrone, and their catecholestrogen and methoxyestrogen metabolites by human uridine diphosphoglucuronosyltransferases expressed in endometrium[END_REF][START_REF] Lepine | Circulating Estrogens in Endometrial Cancer Cases and Their Relationship with Tissular Expression of Key Estrogen Biosynthesis and Metabolic Pathways[END_REF], but UGT2B7 has the highest catalytic efficiency towards the carcinogenic 4-OH-E1/E2 [START_REF] Cheng | Glucuronidation of catechol estrogens by expressed human UDPglucuronosyltransferases (UGTs) 1A1, 1A3, and 2B7[END_REF][START_REF] Raftogianis | Chapter 6: Estrogen metabolism by conjugation[END_REF][START_REF] Guillemette | Metabolic inactivation of estrogens in breast tissue by UDP-glucuronosyltransferase enzymes: an overview[END_REF][START_REF] Lepine | Specificity and regioselectivity of the conjugation of estradiol, estrone, and their catecholestrogen and methoxyestrogen metabolites by human uridine diphosphoglucuronosyltransferases expressed in endometrium[END_REF]. Significantly increased levels of UGT2B7 were seen in the cancerous endometrium suggesting increased 4-OH-E1/E2 detoxification by glucuronidation. This is also in agreement with recently reported up-regulation of UGT2B7 in endometrial cancer [START_REF] Lepine | Circulating Estrogens in Endometrial Cancer Cases and Their Relationship with Tissular Expression of Key Estrogen Biosynthesis and Metabolic Pathways[END_REF].

GSTs catalyze the conjugation of CEs and CE-Qs, and thus have important roles in cell detoxification [START_REF] Butterworth | Formation of catechol estrogen glutathione conjugates and gamma-glutamyl transpeptidase-dependent nephrotoxicity of 17beta-estradiol in the golden Syrian hamster[END_REF][START_REF] Bolton | Potential mechanisms of estrogen quinone carcinogenesis[END_REF]. We observed statistically significantly elevated levels of GSTP1 in cancerous compared to adjacent control endometrium. GSTP1 was increased in 75% of specimens examined. This increase can be explained by the elevated local E2 levels in endometrial cancer [START_REF] Berstein | Tumor estrogen content and clinico-morphological and endocrine features of endometrial cancer[END_REF][START_REF] Ito | Hormone replacement therapy and cancers: the biological roles of estrogen and progestin in tumorigenesis are different between the endometrium and breast[END_REF], as increased GST activities were observed in female rats treated with E2 [START_REF] Sanchez | Induction of NAD(P)H quinone oxidoreductase and glutathione S-transferase activities in livers of female August-Copenhagen Irish rats treated chronically with estradiol: comparison with the Sprague-Dawley rat[END_REF]. In contrast to our data, Chan et al. reported reduced GSTP1 expression in 25 specimens of endometrial cancer [START_REF] Chan | Promoter methylation and differential expression of piclass glutathione S-transferase in endometrial carcinoma[END_REF]. As GSTP1 is highly polymorphic and this study was performed on an Asian population, this may well be the reason for the observed differences. Thus, among six genes coding for the most abundant conjugative enzymes, three were overexpressed (SULT2B1, UGT2B7 and GSTP1), the levels of another two were unchanged (COMT and SULT1A1), and only one was down-regulated (SULT1E1), although not A c c e p t e d M a n u s c r i p t 19 statistically significantly, in cancerous endometrium. Our data suggest that the overall expression of estrogen phase II metabolising enzymes is increased in these tumours.

Conclusions

Our data show imbalance between the phase I and phase II enzymes in endometrial cancer. In cancerous endometrium we found lower levels of CYP1B1 suggesting decreased production of 4-hydroxycatechols. We also observed, for the first time, higher protein levels of S-COMT and lower levels of MB-COMT in cancerous endometrium. Although CYP1B1 and COMT enzymatic activities in cancerous and adjacent control endometrium still need to be evaluated, our data imply that the adjacent control endometrium may represent a preliminary stage of endometrioid carcinoma, where oxidative metabolism exceeds conjugation, thus favouring the production of the carcinogenic CEs and quinones. M a n u s c r i p t

  multiple overlapping mechanisms of estrogen carcinogenesis.

  A c c e p t e d M a n u s c r i p t 12 significantly weaker in tumour cells, compared to control endometrium of myoma patients (p = 0.0009).

  major phase I and II enzymes, CYP1B1 and COMT, at the protein and cellular levels.

  FIGURE LEGENDS

Figure 2 .

 2 Figure 2. Gene expression of phase I and II estrogen-metabolic enzymes in paired

Figure 3 .

 3 Figure 3. Western blotting for CYP1B1 and COMT.

  graphs (whiskers: 5-95 percentile) demonstrate decreased ratios MB-COMT/S-COMT, CYP1B1/S-COMT and CYP1B1/Tot-COMT and unchanged ratios CYP1B1/MB-COMT in endometrial cancer. D. Representative ECL analysis of cyclophilin A and GAPDH expression used for normalization of CYP1B1 and COMT protein levels.

Figure 4 .

 4 Figure 4. Immunohistochemical staining for CYP1B1 and COMT in cancerous and

  C7 T11 C11 T14 C14 P1 T16 C16 T18 C18 T19 C19 T20 C20 P1 C49 T49 C50 T50 C52 T52 C53 T53 P1 C54 T54 C55 T55 C56 T56 C57

Table 1 . Patient details for the cancer group.

 1

	No. Sample	Age	Pathology	Phase	Stage
		(years)			(TNM)
	1	5	39	G2/G3	secretory	T1bN0M0
	2	7	50	G1		T1bN0M0
	3	8	83	G2/G3		T2aN0M0
	4	10	53	G1	proliferative T1aN0M0
	5	11	60	G1		T1cN0M0
	6	13	64	G2		T1cN0M0
	7	14	73	G2		T1cN0M0
	8	16	69	G1/G2		T1bN0M0
	9	18	79	G1		T1cN0M0
	10	19	74	G1/G2		T1bN0M0
	11	20	76	G2/G3		T1bN0M0
	12	21	53	G2	proliferative T1aN0M0
	13	22	36	G1/G2	NA	T1aN0M0
	14	23	45	G1/G2	early s.	T1aN0M0
	15	24	69	G2/G3		T1cN0M0
	16	25	54	G3		T1aN0M0
	17	26	72	G1/G2		T1bN0M0
	18	30	54	G1		T1aN0M0
	19	31	69	G2/G3		T1bN0M0
	20	32	52	G1	sesecsecretor	T1aN0M0
	21	33	77	G3		T1bN0M0
	22	34	57	G1	y	T1bN0M0
	23	35	61	G2		T1aN0M0
	24	38	78	G2/G3		T1bN0M0
	25	40	71	G1		T1aN0M0
	26	42	81	G1		T1bN0M0
	27	44	73	G1		T1bN0M0
	28	46	50	G2/G3		T1aN0M0
	29	47	27	G1/G2	late s.	T1aN0M0
	30	49	70	G1		T1bN0M0
	31 32	50 51	73 75	G1 G2/G3	s.secretory	T1aN0M0 T1bN0M0
	33	52	75	G2/G3		T1bN0M0
	34	53	50	G2/G3	proliferative T1aN0M0
	35	54	71	G1		T1bN0M0
	36	55	75	G2		T1cN1M1
	37	56	55	G1/G2		T1aN0M0
	38	57	43	G1	late s.	T1aN0M0
	39	39	63	G1		T2aN0M0
	40	58	68	G2/G3	ssssecretory	T1aN0M0
	G1-G3, grading according to histological differentiation
	NA: data not available

Table 2 . Patient details for the further control group.

 2

	Table 1

Table 3 . Reference gene details.

 3

	Gene	Assay ID	Gene Name
	symbol
	18S	Hs99999901_s1	Eukaryotic 18S rRNA
	ACTB	Hs99999903_m1	actin, beta
	B2M	Hs99999907_m1	beta-2-microglobulin
	GAPD	Hs99999905_m1	glyceraldehyde-3-phosphate dehydrogenase
	GUSB	Hs99999908_m1	glucuronidase, beta
	HMBS	Hs00609297_m1	hydroxymethylbilane synthase
	HPRT1	Hs99999909_m1	hypoxanthine phosphoribosyltransferase 1 (Lesch-Nyhan
	IPO8 PGK1	Hs00183533_m1 Hs99999906_m1	importin 8 syndrome) phosphoglycerate kinase 1
	POLR2A	Hs00172187_m1	polymerase (RNA) II (DNA directed) polypeptide A,
	PPIA RPLP0	Hs99999904_m1 Hs99999902_m1	peptidylprolyl isomerase A (cyclophilin A) 220kDa ribosomal protein, large, P0
	TBP	Hs99999910_m1	TATA box binding protein
	TFRC	Hs99999911_m1	transferrin receptor (p90, CD71)
	UBC	Hs00824723_m1	ubiquitin C
	YWHAZ	Hs00237047_m1	tyrosine 3-monooxygenase/tryptophan 5-monooxygenase
			activation protein, zeta polypeptide

Table 4 . Details of genes investigated.

 4

	Table 3

Table 4

 4

Acknowledgements

This work was supported by J3-9448 grants to T.L.R. and a young researcher grant to N.H., both from the Slovenian Research Agency. The authors thank Dr. Chris Berrie for critical reading of the manuscript.


Maria Grazia Izzo
email: mariagrazia.izzo@elettra.trieste.it

Filippo Bencivenga

Alessandro ; Gessini

Alessandro Cunsolo

Claudio Masciovecchio

Sincrotrone Grazia

Trieste Elettra

Filippo; ELETTRA Bencivenga

A viscoelastic analysis of IXS spectra from He/Ne mixtures

Keywords: fluids, disordered systems Keywords (user supplied): binary mixtures, viscoelastic model, Inelastic X-ray Scattering fluid binary mixtures, viscoelastic model, Inelastic X-ray Scattering

teaching and research institutions in France or abroad, or from public or private research centers.

A viscoelastic analysis of IXS spectra from He/Ne mixtures

Introduction

It is very well assessed that, in the hydrodynamic limit, a binary mixture behaves as a one-component fluid, its spectrum being characterized by only one inelastic mode whose propagation speed, c s , results from the weighted average of the adiabatic sound velocities of the separate components. In the 1986 a molecular dynamics simulation [START_REF] Bosse | Fast sound in two-component liquid[END_REF] demonstrated that if the two components have very disparate masses a new faster sound mode can be observed at sufficiently short wavelengths (λ) and high frequencies (ω). Moreover, a second excitation dispersing with a group velocity lower than c s has been observed by light scattering experiments [START_REF] Clouter | Light scattering in gas mixtures: Evidence of fast and slow sound modes[END_REF][START_REF] Wegdam | Observation of fast sound in disparate-mass gas mixtures by light scattering[END_REF] and ascribed to density fluctuations separately propagating through the heavier component only.

The whole body of results seems somehow supportive of the occurrence of a bifurcation of the hydrodynamic excitation, which splits into two separate modes whenever the exchanged wave-vector (Q), overcome some threshold value.

Theoretical studies accounting for such phenomenology have been developed either in the frame of the Revisited Enskog theory (RET) [START_REF] Campa | Observable Fast Kinetic Eigenmode in Binary Noble-Gas Mixtures?[END_REF][START_REF] Campa | Fast sound in binary fluid mixtures[END_REF][START_REF] Huck | Possibility of Double Sound Propagation in Disparate-Mass Gas Mixtures[END_REF] or within the so called "two-temperature" approach [START_REF] Goldman | Equations for Gas Mixtures[END_REF]. Recently, a new apprpoach has been proposed on the Generalized Collective Modes (GCM) description [START_REF] Bryk | Longitudinal optical-like excitations in binary liquid mixtures[END_REF][START_REF] Bryk | Collective dynamics in binary liquids: spectra dependence on mass ratio[END_REF]. Even if all these theories provide a consistent account of the observed phenomenology, they strongly differ in quantitative prediction of transport parameters along the crossover region (from hydrodynamic to sound-decoupled regime) and also suggest different physical interpretations of the observed dispersive behavior.

Experimental evidences on the occurrence of two coexisting collective modes mainly come from inelastic scattering experiments performed either in nobles gas mixtures [START_REF] Clouter | Light scattering in gas mixtures: Evidence of fast and slow sound modes[END_REF][START_REF] Montfrooij | Fast sound in a helium-neon mixture determined by neutron scattering[END_REF][START_REF] Montfrooij | Fast and slow sound in a dense gas mixture of helium and neon[END_REF][START_REF] Sampoli | Dynamic Structure of He-Ne Mixtures by Molecular Dynamics Simulation: From Hydrodynamic to Fast and Slow Sound Modes[END_REF] or in molten alloys, such as Li 4 P b [START_REF] Alvarez | High-Frequency Dynamics in a Molten Binary Alloy[END_REF][START_REF] Bosse | Fast sound in two-component liquids[END_REF][START_REF] Bove | Evidence for the coexistence of two density fluctuation modes in molten Li 30 Bi 70 as probed by neutron scattering[END_REF]. The experimental insight on the dynamics of binary mixture is, however, deeply hampered by the scarce hope of properly resolving the two excitations in the spectra [START_REF] Montfrooij | Fast sound in a helium-neon mixture determined by neutron scattering[END_REF] and also by the rather limited Q-range accessible by most of instruments. Actually these limitations have so far prevented an unambiguous validation of any of the mentioned theoretical predictions [START_REF] Montfrooij | Fast and slow sound in a dense gas mixture of helium and neon[END_REF][START_REF] Sampoli | Dynamic Structure of He-Ne Mixtures by Molecular Dynamics Simulation: From Hydrodynamic to Fast and Slow Sound Modes[END_REF]. These difficulties are even worsened by the lack of a well assessed analytical model providing an accurate approximation of the spectrum of mixtures beyond the hydrodynamic limit. In order to amend to this lack, we test here the soundness of a new line-shape description obtained as a generalization of the memory function formalism to the case of binary mixtures [START_REF] Anento | Viscoelastic model for the dynamic structure factors of binary systems[END_REF]. Interestingly enough, such formalism has been extensively employed for single component (both atomic and molecular) fluids in the mesoscopic range [START_REF] Bencivenga | High-frequency dynamics of liquid and supercritical water[END_REF][START_REF] Monaco | Viscoelastic behavior of water in the terahertz-frequency range: An inelastic x-ray scattering study[END_REF][START_REF] Cunsolo | Microscopic relaxation in supercritical and liquid neon[END_REF], yet its "two-components" extension has never been considered to describe experimental spectra of binary mixture.

Experimental method

The experiment was performed at the ID28 beamline of the European Synchrotron Radiation Facility (Grenoble, France). The spectrometer was operated using the Si [START_REF] Montfrooij | Fast and slow sound in a dense gas mixture of helium and neon[END_REF][START_REF] Montfrooij | Fast and slow sound in a dense gas mixture of helium and neon[END_REF][START_REF] Montfrooij | Fast and slow sound in a dense gas mixture of helium and neon[END_REF] reflection from both monochromator and analyzer, which yields an overall energy resolution bandwidth of about 1.5 meV . The present configuration of ID28 spectrometer allows to simultaneously measure Inelastic X-ray Scattering (IXS) spectra at nine different Q-values mutually separated by a constant offset of about 1.5 nm -1 . The chosen ω-range was ± 40 meV with steps of 0.3 meV. The acquisition time for each ω point was ≈ 100 ÷ 200 sec in order to obtain high statistical quality data. The actual Q-values of IXS spectra were established by varying the angle of the spectrometer arm in between 5 o and 20 o interval. The explored Q-range was 6 -14 and 24 -30 nm -1 . The Q indetermination (∆Q ) was determined by the aperture of the analyzer slits and set to be better than 0.3 nm -1 at the lowest Q's. At the highest Q's a more relaxed ∆Q (< 1 nm -1 ) was instead tolerable and this allowed us to keep the slit wide open thus increasing the count rate and compensating the strong intensity decrement in this dynamic region.

The high purity He 0.8 N e 0.2 sample (purchased from Gas Liquide) was embedded in a high pressure hydrostatic cell made of Inconel 625 with 1 mm thick crystal diamond windows. A 12 mm, sample length combined with a 2.3 mm window diameter , allowed a 30 o angular acceptance, necessary to probe the highest exchanged momenta. The cell was connected to an external high pressure generator through a capillary. The pressure stability was monitored by a pressure gauge and found to be better than ± 5 bar, within the typical acquisition time (12 ÷ 18 hours) of a single set of IXS spectra. A closed-cycle He cryostat and a resistive heater in thermal contact with the sample cell were used to steadily keep the sample at the desired temperature. The actual sample temperature was measured by two thermocouples and electronically controlled by a PID device. The overall temperature stability was better than 0.2 K. All the experimental setup was kept under high vacuum (∼ 10 -8 mbar) in order to avoid thermal gradients and spurious scattering from the air surrounding the cell. The IXS spectra of He 0.8 N e 0.2 were collected at a temperature T = 82 K and P = 320 bar, corresponding to a sample number density of 18.5 nm -3 .

Ancillary measurements of the energy resolutions of each of the nine analyzers have been performed through the acquisition of the nearly elastic scattering of a Plexiglas sample at low temperature (5K) and at the Q of its first diffraction peak. It has been found that both the shape and the width (about 1.4 -1.7 meV ) of the

Philosophical Magazine andalo˙izzo˙2

Philosophical Magazine energy resolution function, R(ω) are quite insensitive to the specific experimental conditions (i.e., to the angle of the spectrometer arm and slit aperture). Empty cell measurements were also performed for comparison. The measured scattering was found more than an order of magnitude weaker than the signal from the sample, even at the higher Q's, thus it has been neglected in the analysis to be discussed. Fig. 1 displays two selected IXS spectra at the indicated Q-values along with the measured shape of R(ω) . The spectral profiles I(Q, ω), were approximated by the following model function:

I(Q, ω) = I(Q)[n(ω, T )S ex (Q, ω) ⊗ R(ω)] + c(Q). (1)
Here the symbol ⊗ represents the numerical convolution operator, I(Q) is an overall intensity factor, c(Q) is a background term taking into account both the electronic background of the detector and the environmental one. The factor n(ω, T ) is a frequency dependent factor accounting for the detailed balance:

n(ω, T ) = ω/k B T 1 -e -ω/kBT , (2)
where k B is the Boltzmann's constant and T the temperature. The "experimental" S ex (Q, ω) is related to the partial dynamic structure factors through weighting coefficients, I αβ , which depend on the specific probe and measurement conditions. In the Faber-Ziman representation [START_REF] Faber | A theory of the electrical properties of liquid metals III. The resistivity of binary alloys[END_REF]:

S ex (Q, ω) = I 11 S 11 (Q, ω) + I 12 S 12 (Q, ω) + I 22 S 22 (Q, ω), (3)
where the apices 1 and 2 refer to the two specie components. By comparing eq.1 with the IXS cross section for binary mixtures, one obtains:

∂ 2 σ ∂Ω∂ω f ∝ α,β=1,2 (x α x β ) 1/2 f α (Q)f β (Q)S αβ (Q, ω), (4)
from which it follows that I αβ = (x α x β ) 1/2 f α (Q)f β (Q), being x α and f α (Q) the concentration and the atomic form factor of particles of specie α = 1, 2. Here S αβ (Q, ω) is the partial dynamic structure factor, defined as

S αβ (Q, ω) = 1 (x α x β ) 1/2 V dr ∞ -∞ dt δn α δn β e i(Q•r-ωt) , (5)
being V the volume occupied by the system and δn α the fluctuations of partial number density of specie α. S αβ (Q, ω) are calculated using a generalization of the viscoelastic model to binary systems as described in the next section.

The data analysis consists of a fitting procedure based on a standard minimization of a χ 2 function, defined as:

χ 2 = N i=1 (I(Q, ω i ) -y i ) 2 σ i , ( 6
)
where N is the number of data points in the IXS spectra, y i are the measured counts at the energy transfer ω i and σ i is their relative standard deviation. A Poisson statistics is assumed, so that σ i = √ y i . The minimization was performed with the software MATLAB, employing two routines: one exploiting the Nelder-Mead Simplex algorithm [START_REF] Lagarias | Convergence Properties of the Nelder-Mead Simplex Method in Low Dimensions[END_REF] and, subsequently, a second one based on a large-scale algorithm, which uses the method of preconditioned conjugate gradients (PCG) [START_REF] Coleman | An Interior Trust Region Approach for Nonlinear Minimization Subject to Bounds[END_REF]. Finally, the errors were estimated through the calculation of the covariance matrix.

Data analysis

For a one-component system the dynamic structure factor S(Q, ω) is given by the expression:

S(Q, ω) = 1 π ( F (Q, -iω)), (7)
where the intermediate scattering function, F (Q, -iω) can be evaluated, in the memory function frame, through the continue fraction expansion, truncated to the third order:

F (Q, -iω) = S(Q)   -iω + M (Q) -iω + N (Q) -iω+K(Q)   -1 . (8)
S(Q), M (Q), N (Q) and K(Q) are the zero, first, second and third order memory function calculated at t = 0, respectively. S(Q) can be identified with the static structure factor, while M (Q), N (Q) and K(Q) can be associated to the zero and infinite sound frequencies, ω 0 (Q) and ω ∞ (Q), and to the compliance relaxation time, τ c (Q), through the following relations:

ω 2 ∞ (Q) = N (Q) + M (Q) ( 9
)
ω 2 0 (Q) = M (Q) = k B T M S(Q) (10)
τ c = K -1 (Q) ω 2 ∞ ω 2 0 , (11)
being M the mass of the particles. By imposing that in the Q → ∞ limit the expected short time behavior is reached, it is possible to link K(Q) and N (Q):

K(Q) -1 = √ π 2 N (Q) , (12)
also known as Lovesey relation [START_REF] Copley | The dynamic properties of monatomic liquids[END_REF].

As the memory function formalism holds for a generic set of dynamical variables, ν, the generalization of the viscoelastic model to binary systems is, in principle, quite straightforward. The physical problem is focused on the choice of the suitable set of dynamical variables describing the dynamics of the system. By choosing ν = (δn 1 (r, t), δn 2 (r, t)) (i.e. exploiting the Faber-Ziman representation) the dynamic structure factor result to be a 2 × 2 matrix, S(Q, ω), whose generic elements are S αβ (Q, ω). S(Q, ω) can be obtained from the intermediate scattering matrix, F(Q, -iω), assuring it to be hermitian, i.e.:

S(Q, ω) = 1 2π [ F+ (Q, -iω) + F(Q, -iω)], (13)
where F+ is the adjoint of F. The matrix F(Q, -iω) is, in the viscoelastic model, given by an expression formally equal to eq.8, although in this case S(Q), M (Q), N (Q) and K(Q) are matrixes expressed in the vectors basis (δn 1 (r, t), 0) and (0, δn 2 (r, t)). In order to reduce the number of free parameters in the fitting routine, the Lovesey relation in Eq.12 is used as generalized to the case of binary systems. Explicit calculations of the involved matrix elements K αβ (Q) as a function of N αβ (Q) can be found in [START_REF] Anento | Viscoelastic model for the dynamic structure factors of binary systems[END_REF]. The matrix M(Q) is, moreover, related to S(Q) trough the squared single components isothermal velocities, Q 2 k B T /M 1(2) [START_REF] Anento | Viscoelastic model for the dynamic structure factors of binary systems[END_REF]. The identities N 21 = N 12 (M 22 /M 11 ) and S 12 = S 21 follow from symmetry requirements.

The parameters determined by the fitting procedure are, therefore:

S 11 (Q), S 12 (Q), S 22 (Q), N 11 (Q), N 12 (Q), N 22 (Q), c (
Q) and I(Q), while T was fixed to the measured value. f 1(2) (Q) were calculated [START_REF]Internetional tables for crystallography[END_REF] and kept fix during the minimization routine.

As clearly emerges from Fig. 1 the agreement between the model function and the raw IXS data is satisfactory for both the reported Q values. As a matter of fact, the χ 2 normalized to the number of degrees of freedom turns out to be rather close to 1. The logarithmic scale emphasizes the agreement also in the high frequency spectral tails.

As will be discussed in the next section, the present data analysis allowed to extrapolate S αβ (Q, ω) from a single IXS spectra of the mixture. Matrix expressions equivalent to Eqs.9-11 can be, moreover, defined so that the zero and infinite frequency as well as the compliance relaxation time matrix remain defined. In the chosen representation (i.e. assuming ν = (δn He (r, t), δn N e (r, t))), the ω 0 , ω ∞ , and τ c values associated with He and N e subsystems can be identified with the diagonal elements of the respective 2 × 2 matrix.

Results and discussion

S(Q, ω) can be also expressed in the basis defined by the vectors (δn(r, t), 0) and (0, δc(r, t)), being δn(r, t) and δc(r, t) the fluctuations of the total density and concentration, respectively. These are related to δn 1 (r, t) and δn 2 (r, t) by linear combinations. Consequently, the partial dynamic structure factors S nn (Q, ω) and S cc (Q, ω) can be obtained by S 11 (Q, ω), S 22 (Q, ω) and S 12 (Q, ω) by means of a change of vectors basis. For each partial dynamic structure factor the respective longitudinal current can be finally obtained as:

J(Q, ω) = ω 2 S(Q, ω). (14)
In the left panel of Fig. 1-(a) the longitudinal partial current J HeHe (Q, ω), J N eN e (Q, ω), J HeN e (Q, ω) (upper frame) are reported as directly extrapolated by the fitting procedure for the IXS spectra at Q = 9.4 nm -1 , while J nn (Q, ω) and J cc (Q, ω) are obtained from the former. A low-frequency peak (ω 1 ∼ = 1.9 meV ) is observed in J HeHe (Q, ω), J N eN e (Q, ω), J nn (Q, ω) and J cc (Q, ω). This feature dominates J N eN e (Q, ω) and J cc (Q, ω). It is located in a frequency range where the cross current, J HeN e (Q, ω) touches negative values, which reveal out-of-phase motion of particles of different specie. The peak centroid is, finally, around D 12 Q 2 ∼ = 1.6 meV , which leads to estimate an inter-diffusion coefficient D 12 consistent to the one derived from Generalized Enskog theory [START_REF] Hirschfelder | Molecular theory of gases and liquids[END_REF]. These trends suggest that this lowfrequency feature can be related to interspecies diffusion dynamics. A further higher frequency peak is observable in J HeHe (Q, ω) and J nn (Q, ω) at ω 2 ∼ = 4.6 meV . The phase velocity associated to such mode is c 2 = ω 2 /Q ∼ = 750 m/s, a value similar to the speed of sound of pure He at the same temperature and equivalent density. Moreover, the peak is located in a frequency range corresponding to positive value of the cross correlation. It can be, thus, related to an acoustic-like collective mode mainly supported by the He particles. This scenario strongly suggests a dynamic decoupling of the two components. Similar behavior is observed also in others He/N e mixtures [START_REF] Sampoli | Dynamic Structure of He-Ne Mixtures by Molecular Dynamics Simulation: From Hydrodynamic to Fast and Slow Sound Modes[END_REF]. The partial static structure factors obtained after the minimization routine are S HeHe (Q) = 1.3 ± 0.5, S HeN e (Q) = -0.23 ± 0.06, S N eN e (Q) = 0.61±0.05. A calculation made by using the hypernetted chain (HNC) closure relations in the Ornstein-Zernike (OZ) equation, solved with the Gillan method extended to binary systems, shows consistency with these experimental results [START_REF] Fiocco | [END_REF]. The zero and infinite sound frequencies are ω HeHe . The first relevant observation concerns the cross current J HeN e (Q, ω). Its amplitude is much smaller than these of J HeHe (Q, ω) and J N eN e (Q, ω). S HeN e (Q) is, as well, nearly vanishing (i.e., S HeN e (Q) = -0.009 ± 0.008). This is consistent with the expectation that the dynamical variables δn 1 (r, t) and δn 2 (r, t) became statistically independent in the high Q limit, where the struck atoms basically act as free particles [START_REF] Izzo | The high frequency dynamics of iodine in the Sachs Teller regime[END_REF]. Single peaks are present in J HeHe (Q, ω) and J N eN e (Q, ω) at ω 1 ∼ = 11.1 meV and ω 2 ∼ = 4.9 meV , respectively. The ω 1(2) values are rather close to (2Q 2 k B T /M 1(2) ) 0.5 , being (2Q 2 k B T /M He ) 0.5 ∼ = 10.6 meV and (2Q 2 k B T /M N e ) 0.5 ∼ = 4.7 meV , which is the expression for J(Q, ω) maxima corresponding to a Gaussian function of variance σ = Q 2 k B T /M 1(2) for S(Q, ω). This suggests that the spectral density is gradually approaching its single particle limit. According to this finding, moreover, the values of the compliance relaxation times (τ HeHe c = 0.21 ± 0.08 psec and τ N eN e c = 0.4 ± 0.1 psec) are consistent, within the errors, with the single components collision times.

In conclusion, IXS spectra from He 0.8 N e 0.2 mixture at T = 82 K and n = 18.5 nm -3 in the mesoscopic region have been analyzed through a generalization to binary systems of the viscoelastic model [START_REF] Anento | Viscoelastic model for the dynamic structure factors of binary systems[END_REF]. So far, this model has been largely used to successfully describe the dynamic structure factor of one-component systems in this regime. We applied it for the first time to analyze experimental data of a twocomponent system. These preliminary results are encouraging, yet clearly worth further tests hopefully with the support of parallel molecular dynamics techniques. Moreover, its natural evolution consists in a full characterization of the parameters describing collective dynamics in a binary mixture as function of the thermodynamic conditions.

We'd like to acknowledge R. Cucini, F. D'Amico and R. Verbeni.

0= 2 . 3 ±

 23 0.5 meV , ω HeHe ∞ = 5.2 ± 0.3 meV and ω N eN e 0 = 1.5 ± 0.7 meV , ω N eN e ∞ = 4.5 ± 0.2 meV , i.e ω HeHe(N eN e) 0 /ω HeHe(N eN e) ∞ ∼ 0.3 ÷ 0.4 as usually found for one-component fluids [17, 19, 27]. The compliance relaxation times associated to HeHe and N eN e correlations have been as well calculated. It is found τ HeHe c = 0.8 ± 0.4 psec and τ N eN e c = 1.5 ± 0.3 psec. These values are, respectively, three and for times bigger than the single specie collision times: τ He coll ∼ = 0.27 psec, τ N e coll ∼ = 0.36 psec, respectively. Fig.1-(b) reports the same profiles as in Fig.1-(a), but for Q = 27.6 nm -1

Figure 1 .

 1 Figure 1. IXS spectrum of He 0.8 N e 0.2 mixture at Q = 9.4 nm -1 (panel (a), left) and Q = 27.6 (panel (b), left). The corresponding best fit curve as obtained by the viscoelastic model for binary systems and the instrumental resolution curve are represented by solid red line and dotted black line, respectively. The longitudinal currents extrapolated by the fitting procedure are reported in the right panels.

Taylor & Francis and I.T. Consultant

http://mc.manuscriptcentral.com/pm-pml Philosophical Magazine & Philosophical Magazine Letters


Tim Schmoll
email: tim.schmoll@uni-bielefeld.de

Oddmund Kleven

Sperm dimensions differ between two coal tit Periparus ater populations

Keywords: Sperm morphology, sperm proportions, sperm competition, population differences, intraspecific variation, extra-pair paternity

The evolutionary forces that shape and maintain variation in sperm traits across species in birds have recently attracted increased attention. Much less is known about the patterns, the causes and the consequences of variation in sperm traits within and, in particular, between populations of the same species. We here analyse intraspecific variation in sperm dimensions within and between two populations of the coal tit Periparus ater, a socially monogamous passerine with a high frequency of extra-pair paternity. Spermatozoa from a Norwegian population had a greater mean total length compared to those from a German population, mainly as a result of longer sperm heads. Sperm head length in the Norwegian population also accounted for a larger percentage of sperm total length suggesting differences in sperm proportions between populations. Furthermore, spermatozoa from the Norwegian population showed lower mean within-male variation in sperm length and there was significant betweenmale variation in sperm total length within each of the two populations. We discuss these results in the light of recent comparative evidence suggesting relationships between sperm length, and variation in sperm length, and the frequency of extra-pair paternity across passerine birds.

Introduction

The staggering diversity in form and function of spermatozoa across taxa and the evolutionary forces that have shaped and do maintain this variation have attracted a lot of attention (reviewed in [START_REF] Birkhead | Sperm biology -an evolutionary perspective[END_REF]. In socially monogamous bird species, extra-pair fertilisations are the rule rather than an exception [START_REF] Griffith | Extra pair paternity in birds: A review of interspecific variation and adaptive function[END_REF] and constitute a potentially powerful source of postcopulatory sexual selection on sperm traits. This idea is supported by recent comparative evidence suggesting relationships between sperm total length as well as sperm swimming speed and extra-pair paternity: Both traits were positively though independentlyrelated to the frequency of extra-pair paternity across passerine bird species (Kleven et al. 2009a; see also [START_REF] Briskie | The evolution of sperm size in birds[END_REF]. Furthermore, within-male variation in sperm dimensions [START_REF] Immler | Increased postcopulatory sexual selection reduces the intramale variation in sperm design[END_REF]; see also [START_REF] Kleven | Intraspecific variation in sperm length is negatively related to sperm competition in passerine birds[END_REF]) and between-male variation in sperm length [START_REF] Calhim | Postcopulatory sexual selection is associated with reduced variation in sperm morphology[END_REF][START_REF] Kleven | Intraspecific variation in sperm length is negatively related to sperm competition in passerine birds[END_REF] were negatively related to the frequency of extra-pair paternity across passerines, indicating that postcopulatory sexual selection may not only favour longer and faster spermatozoa, but also sperm of less variable length with increasing risk of sperm competition. While sexual selection through extra-pair fertilisations can explain substantial variation in sperm traits between species, little is known about the intraspecific variation in sperm morphology, its causes and its consequences in natural bird populations [START_REF] Laskemoen | Intraspecific variation in sperm length in two passerine species, the bluethroat Luscinia svecica and the willow warbler Phylloscopus trochilus[END_REF]. This is true for variation within populations, but in particular for variation between populations of the same species.

Here we analyse intraspecific variation in sperm dimensions in the coal tit Periparus ater, a socially monogamous passerine with a high frequency of extra-pair paternity [START_REF] Lubjuhn | High frequency of extra-pair paternity in the coal tit[END_REF][START_REF] Dietrich | Pair identity -an important factor concerning variation in extra-pair paternity in the coal tit (Parus ater)[END_REF]). We address variation within and between individual males in a Norwegian and a German population of coal tits and compare sperm dimensions, and variation in sperm dimensions, between these populations.

Methods

Study populations and field methods

We studied territorial male coal tits during the breeding season in Norway and Germany (for details see Table 1). A total of 20 sperm samples were obtained non-invasively by gently massaging the cloacal protuberance (Wolfson 1952) of birds that had been mistnetted while singing and/or responding to playback (Norway: ten males and Germany: six males) or that were captured while feeding nestlings ten to 14 days old (Germany only: four males). Sperm samples were fixed and stored in 5% formalin solution until further use. If not already ringed, the birds were banded with uniquely numbered metal rings and released immediately after sampling.

Sperm morphometry

A droplet of the formalin solution was placed on a microslide and air-dried. Sperm were then examined by digital light microscopy using a Leica DM6000 B microscope at ×320 magnification and photographed with a digital camera (Leica DC500). Sperm head, midpiece and tail length (for delimitation of sections see Figure 1 in Kleven et al. 2009b) were measured to a precision of 0.1 μm using Leica IM1000 analysis software (Leica Microsystems, Heerbrugg, Switzerland) and sperm total length was calculated as the sum of these components. Ten randomly selected spermatozoa of each of ten males per population were measured by the same person (O. K.) within a 28 day period in autumn 2009. Sampling effort of a similar magnitude has been shown to give representative estimates for the mean and the variation in sperm dimensions in populations of two other passerine birds [START_REF] Laskemoen | Intraspecific variation in sperm length in two passerine species, the bluethroat Luscinia svecica and the willow warbler Phylloscopus trochilus[END_REF]. Measurement repeatabilities (sensu [START_REF] Lessels | Unrepeatable repeatabilities: A common mistake[END_REF], based on 10 spermatozoa measured blindly twice, were high for all sperm dimensions (sperm total length: r = 0.95, F 9,10 = 36.7, p < 0.001; head length: r = 0.68, F 9,10 = 5.2, p = 0.008; midpiece length: r = 0.97, F 9,10 = 74.3, p < 0.001; tail length: r = 0.96, F 9,10 = 54.0, p < 0.001).

Statistical analysis

We used R 2.9.0 (R Development Core Team 2009) for all computations and linear mixed effects models (LME, R function lme in library nlme, [START_REF] Pinheiro | Nlme: Linear and nonlinear mixed effects models[END_REF] to test for differences between populations in sperm traits. Male identity was included as a random effect to account for the non-independence of measurements obtained from the same individual and to estimate between-male random variation in sperm dimensions. P values in the context of LME analysis refer to the increase in model deviance (compared against a 2 distribution) when a term is removed from the current model and the significance of a random effect was tested by removing it from the minimal adequate model. Sperm head, midpiece and tail length relative to sperm total length was compared between populations by testing for differences in sperm head, midpiece and tail length, respectively, while simultaneously controlling for sperm total length in the LME. The coefficient of variation (CV) for a given sample was calculated as CV = standard deviation (SD) of the sample/mean of the sample *100%. The within-male CVs for sperm total length were compared between populations with a t-test. 95% confidence intervals for between-male CVs were obtained by non-parametric bootstrapping (N = 10000 replicates per population). Intraclass correlation coefficients, or repeatability, for sperm total length were calculated according to [START_REF] Lessels | Unrepeatable repeatabilities: A common mistake[END_REF] and the calculation of standard errors for repeatability estimates followed [START_REF] Becker | Manual of quantitative genetics[END_REF]. All statistical tests were two-tailed and the null hypothesis was rejected at p < 0.05.

Results

Spermatozoa from the Norwegian population were on average 3.2% longer compared to those from the German population (Table 2). This difference resulted mainly from a difference in sperm head length: Spermatozoa from the Norwegian population had on average 7.3% longer heads (Table 2), while no significant differences between populations were detectable in midpiece and tail size (Table 2). Sperm head length in the Norwegian population accounted for a slightly larger percentage of sperm total length compared to the German population (Table 2), while no significant differences in relative length were evident for midpiece and tail length (Table 2). While the between-male coefficient of variation in sperm total length was 2.57 (95% confidence interval: 1.19-3.17) for the Norwegian but only 1.77 (0.95-2.31) for the German population, the mean within-male coefficient of variation was nearly 40% higher in the German population (CV ± SE: Norway: 1.44 ± 0.35; Germany: 2.00 ± 0.53; t = -2.78, df = 18, p = 0.01; see also Figure 1). Repeatability analysis within each of the two populations revealed that 74.4% (± SE: 9.8%; F 9,90 = 30.1, p < 0.001) and 38.9% (± 13.7%; F 9,90 = 7.4, p < 0.001) of the total variation in sperm length was explained by variation between males in the Norwegian and the German population, respectively. We found significant between-male random variation within each of the two populations in sperm total length (Norway: χ 2 = 97.4, df = 1, p < 0.001; Germany: χ 2 = 27.3, df = 1, p < 0.001) as well as in all other sperm dimensions measured (all χ 2 > 9.29, all df = 1, all p < 0.01).

Discussion

We have shown differences in the mean, the proportion and the variation of sperm dimensions between a Norwegian and a German coal tit population. But four out of the ten German samples were obtained from males while feeding nestlings which might confound these findings, for example if sperm traits of individual males (and/or variation in these traits) change during the breeding season. However, none of the traits showing significant betweenpopulation differences differed between the four males sampled while feeding nestlings versus males that were sampled earlier in the season in the German population (LME: all p > 0.1). Furthermore, excluding the four males from analysis does not affect conclusions (LME: all p < 0.02) except for proportion head length (LME: p = 0.06) suggesting a fair degree of robustness for these analyses. Finally, although poorly investigated in birds, sperm size traits seem to be highly repeatable across ejaculates for individual males [START_REF] Birkhead | Ejaculate quality and the success of extra-pair copulations in the zebra finch[END_REF][START_REF] Møller | Impaired swimming behaviour and morphology of sperm from barn swallows Hirundo rustica in chernobyl (vol 650, pg 210[END_REF]. We therefore consider it unlikely that the sampling at partly different stages during the reproductive cycle in the two study populations has affected our results. However, to elucidate potential within-seasonal variation in sperm traits, a longitudinal approach that tracks and compares sperm size traits within individual males is required (see [START_REF] Immler | Pronounced within-individual plasticity in sperm morphometry across social environments[END_REF] for a recent example of individual phenotypic plasticity in sperm size traits). Furthermore, storage duration differed for samples from the two populations (two and a half vs. half a year). If storage duration in formalin affects sperm morphology we would assume that spermatozoa tend to shrink with time. In our case, however, the on average shorter spermatozoa (from the German population) are the ones with the shorter storage duration (see methods). We therefore argue that our results were not confounded by a storage duration effect.

Between-population comparisons in sperm morphology are scarce in general and a recent review compiled a few case studies from insect, mollusc and anuran species, but none from birds [START_REF] Pitnick | Sperm biology -an evolutionary perspective[END_REF]. Three other studies have shown differences in sperm morphology between populations in birds. [START_REF] Møller | Impaired swimming behaviour and morphology of sperm from barn swallows Hirundo rustica in chernobyl (vol 650, pg 210[END_REF] compared sperm morphology and sperm motility of barn swallows Hirundo rustica from the Chernobyl region with two less radioactively contaminated control areas further away. Swallow males breeding near Chernobyl showed a higher proportion of sperm with abnormal morphology and had, on average, shorter tails compared to those breeding in control areas (note that "tail" here refers to the sum of midpiece and tail in our terminology, see [START_REF] Møller | Impaired swimming behaviour and morphology of sperm from barn swallows Hirundo rustica in chernobyl (vol 650, pg 210[END_REF]. Kleven and coworkers (2009b) compared published data on sperm length in the sand martin Riparia riparia [START_REF] Helfenstein | Between-male variation in sperm size, velocity and longevity in sand martins Riparia riparia[END_REF] with data obtained from two other populations sampled by themselves. Apparent differences in mean sperm total length between their samples and the data of [START_REF] Helfenstein | Between-male variation in sperm size, velocity and longevity in sand martins Riparia riparia[END_REF], however, were likely due to methodological problems in the latter study (discussed in Kleven et al. 2009b). Finally, [START_REF] Lüpold | Geographical variation in sperm morphology in the red-winged blackbird (Agelaius phoeniceus)[END_REF] recently reported significant between-population variation in sperm morphology in the red-winged blackbird Agelaius phoeniceus. Thus the present study is among the first to demonstrate differences in sperm dimensions between undisturbed populations of the same bird species.

Recent comparative evidence indicates a positive relationship between sperm length

and extra-pair paternity (Kleven et al. 2009a; see also Lüpold et al. 2009a) and a negative relationship between within-male variation [START_REF] Immler | Increased postcopulatory sexual selection reduces the intramale variation in sperm design[END_REF]; see also [START_REF] Kleven | Intraspecific variation in sperm length is negatively related to sperm competition in passerine birds[END_REF] as well as between-male variation [START_REF] Calhim | Postcopulatory sexual selection is associated with reduced variation in sperm morphology[END_REF][START_REF] Kleven | Intraspecific variation in sperm length is negatively related to sperm competition in passerine birds[END_REF] in sperm length and extra-pair paternity across passerine birds. In our study, the Norwegian population showed longer spermatozoa and lower within-male variation in sperm length than the German population, while between-male variation was statistically indistinguishable (although the Norwegian population shows a lower degree of variation here, too, and the limited sample size may have restricted the power of this analysis). Based on this, we would predict that the frequency of extra-pair paternity in the Norwegian is higher compared to the German population. However, while the frequency of extra-pair paternity in the German study population is quite high (around 28% extra-pair offspring in first and up to nearly 50% extrapair offspring in second broods, [START_REF] Dietrich | Pair identity -an important factor concerning variation in extra-pair paternity in the coal tit (Parus ater)[END_REF], no information is available on the frequency of extra-pair paternity in the Norwegian population. The difference in sperm total length resulted mainly from a difference in sperm head length and sperm heads were proportionally larger in the Norwegian population. The sperm head contains the acrosome and the nucleus and it is not clear why postcopulatory sexual selection should favour longer sperm heads. Rather it may be expected that the midpiece (containing the mitochondria) and/or the tail should be under postcopulatory sexual selection for greater length through extra-pair fertilisations (Lüpold et al. 2009b; see also [START_REF] Laskemoen | Sperm quantity and quality effects on fertilization success in a highly promiscuous passerine, the tree swallow Tachycineta bicolor[END_REF]. Furthermore, interspecific associations between traits as revealed by comparative methods are not necessarily found also on intraspecific levels. Thus the observed differences between the two populations may well not be related at all to (the contemporary degree of) sperm competition in these populations.

For example, a different phylogenetic history (involving genetic drift or different correlated selection pressures) may be responsible for the differential patterns of sperm morphology in these two populations as they are likely to belong to different phylogeographic lineages [START_REF] Martens | Molecular phylogeny of Parus (Periparus), a eurasian radiation of tits (Aves: Passeriformes: Paridae)[END_REF]; see also [START_REF] Lüpold | Geographical variation in sperm morphology in the red-winged blackbird (Agelaius phoeniceus)[END_REF], who report a lack of an association between mating system and sperm morphology across populations in red-winged blackbirds).

We conclude that at present it is unclear whether the detected between-population differences in sperm morphology represent the rule or an exception and whether and how they may reflect adaptations to sperm competition and/or to postcopulatory mechanisms of female choice. Thus an important goal for future research is to test for differences in sperm traits across many different populations in this and in other species and to investigate the relation to the degree of sperm competition and possibly other traits.

With respect to within-population variation, it is surprising that only a single study up to date has related individual variation in sperm traits to individual variation in male fertilisation success in a natural bird population. [START_REF] Laskemoen | Sperm quantity and quality effects on fertilization success in a highly promiscuous passerine, the tree swallow Tachycineta bicolor[END_REF] showed that relative midpiece size was positively correlated to male fertilization success in tree swallows Tachycineta bicolor and suggested that this effect is mediated via increased sperm swimming speed due to higher mitochondrial loading. In the German coal tit study population, no consistent predictors of male extra-pair fertilisation success were detectable (T. Schmoll, W.

Winkel, T. Lubjuhn, unpublished data) with the notable exception of male age (Schmoll et al. 2007). Thus the significant between-male variation in sperm morphology suggests a potential for sperm traits to explain individual differences in (extra-pair) fertilisation success in this and possibly in other natural populations of birds. Establishing that variation in sperm traits relates to variation in extra-pair paternity not only between, but also within species may represent a major step towards understanding sexual selection in birds. We therefore propose analysing variation in key sperm traits and their relationship to extra-pair paternity in a within-species context in the future. Tables Table 1 Details b Effect of population on sperm midpiece length while controlling for sperm total length.

Figure captions

c Effect of population on sperm tail length while controlling for sperm total length. *Significant after sequential Bonferroni-Holm correction.

Fig. 1

 1 Fig. 1 Variation in sperm total length for a) ten males of a Norwegian and b) ten males of a

  of the study sites and coal tit study populations investigated. April 13 th to May 5 th 2009, April 18 th to June 5 th

		Norwegian population	German population
	Locations	Three localities near Oslo	D-48488 Elbergen near Lingen/Ems
	Coordinates	Sørkedalen: 59°58' N, 10°37' E,	52°27' N, 7°15' E
		Sørkedalen: 60°01' N, 10°37' E,
		Skien: 59°20' N, 09°33' E
	Habitat	Lowland mixed coniferous forest	Lowland mixed coniferous forest
	Sampling period	2007,

Table 2

 2 Sperm morphometrics for ten spermatozoa of each of ten males from each of the two coal tit study populations. Descriptive statistics refer to population-wide estimates and significance testing used linear mixed effects models that included male identity as a random effect. Effect of population on sperm head length while controlling for sperm total length.

	Norway	Germany

a

Acknowledgments

We would like to thank Doris Winkel ( † 2007) & Wolfgang Winkel for maintaining and monitoring the German study population over the last decades and the Institute for Avian

Research "Vogelwarte Helgoland", Wilhelmshaven, Germany for logistic support. Frode Fossøy, Lars Erik Johannessen, Terje Laskemoen, Jan T. Lifjeld, Trond Øigarden and Heike Wemhoff-de Groot assisted in the field. We thank Bart Kempenaers and anonymous reviewers for helpful comments on earlier versions of this manuscript. This study complies with the current laws of Norway and Germany. R Development Core Team (2009) R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. ISBN 3-900051-07-0, URL http://www.R-project.org Schmoll T, Mund V, Dietrich-Bischoff V, Winkel W, Lubjuhn T (2007) Male age predicts extrapair and total fertilization success in the socially monogamous coal tit. Behav Ecol 18 (6):1073-1081 Wolfson A (1952) The cloacal protuberance -a means for determining breeding condition in live male passerines. Bird Banding 23:159-165