Serum Neopterin Is Not Increased in Obese Juveniles
Hindawi Publishing CorporationCopyright Hindawi Publishing Corporation

Harald Mangge
Clinical Institute of Medical and Chemical Laboratory Diagnostics
Medical University of Graz
8036GrazAustria

Florian Freytag
Clinical Institute of Medical and Chemical Laboratory Diagnostics
Medical University of Graz
8036GrazAustria

Gunter Almer
Clinical Institute of Medical and Chemical Laboratory Diagnostics
Medical University of Graz
8036GrazAustria

Daniel Weghuber
Department of Pediatrics
University Hospital of Salzburg
5020SalzburgAustria

Carmen Bauer-Denk
Clinical Institute of Medical and Chemical Laboratory Diagnostics
Medical University of Graz
8036GrazAustria

Dietmar Fuchs
Division of Biological Chemistry
Innsbruck Medical University
6020Biocenter, InnsbruckAustria

Serum Neopterin Is Not Increased in Obese Juveniles

Journal of Obesity
Hindawi Publishing Corporation201110.1155/2011/946795Received 11 June 2010; Accepted 22 December 2010Research Article Correspondence should be addressed to Harald Mangge, firstname.lastname@example.com Academic Editor: A. Halpern
Objective. Cardiovascular disease is associated with inflammation and immune activation, concentrations of immune activation markers like neopterin predict outcome in adults. Methods. Serum neopterin concentrations and early metabolic and preatherosclerotic symptoms were analyzed in 295 obese juveniles and 101 normal weight controls of similar age. Additionally, the influence of a 12 months weight reduction program on neopterin levels was investigated in 31 obese juveniles. Results. Intimamedia thickness of common carotid arteries (IMT) and the concentrations of C-reactive protein (CRP) were increased in the obese juveniles (P < .001). Also triglycerides, oxidized LDL, fasted insulin levels, HOMA-index, leptin, liver transaminases and uric acid were increased compared to the controls. However, serum neopterin was decreased in the obese versus non-obese juveniles (P < .03). The intervention consisting of regular sports, nutritional devices, and a psychologic attendance led after 12 months to an increase of neopterin concentration (P < .05; paired test). Conclusions. Neopterin concentrations in juvenile obesity behaved considerably different from what was demonstrated in adults, levels did not correlate with metabolic and pre-atherosclerotic symptoms found in early phases although early vascular burden and chronic low grade inflammation was indicated by increased IMT and CRP. Neopterin concentrations increased after a 12 months intervention program.

Introduction

Nowadays, the role of inflammation and immune activation in atherogenesis is well established [1], and the activation of macrophages appears to be of critical relevance within this process [1,2]. Cardiovascular disease (CVD) is also associated with increased neopterin concentrations [3][4][5][6][7][8][9][10], and predictive information is provided by neopterin in patients at risk of myocardial infarction [11][12][13][14][15][16]. A recent study in 2312 patients undergoing angiography demonstrated that neopterin is an independent predictor of total and cardiovascular mortality [17]. Likewise, in patients with diabetes, both neopterin and C-reactive protein (CRP) were independent predictors of fatal ischemic heart disease [18].

Neopterin is produced by human monocyte-derived macrophages and dendritic cells preferentially upon stimulation with Th1-type cytokine interferon-γ (IFN-γ) [19,20].

Thus, neopterin concentrations reflect activation of the Th1type immune response [21]. In humans, increased neopterin concentrations are detected in patients with virus infections including human immunodeficiency virus, infections by intracellular bacteria and parasites, autoimmune diseases, malignant tumors, and during rejection episodes in allograft recipients [21][22][23][24].

In foregoing studies, we detected an increased carotid intima-media thickness (IMT) indicating preatherosclerosis paralleled by a subclinical inflammation in obese juveniles aged around 13 years [25]. Additionally, we showed an influence of subcutaneous adipose tissue topography on total adiponectin and adiponectin subfraction serum levels in obese juveniles and provided the first evidence that incipient atherosclerosis yet found in adolescents by an increased IMT is associated with a disturbed oligomerisation from lowmolecular to high-molecular-weight adiponectin [26,27].

In a group of otherwise healthy adults, a positive correlation was observed between neopterin concentrations and body mass index [28]. From the data obtained in adults, it appears that neopterin serum levels are associated with chronic subclinical inflammation and preatherosclerotic symptoms in early phases of obesity in a particular manner. In this study, we analysed serum concentrations of neopterin with carotid IMT, and cardiovascular risk factors in a cohort of 295 obese juveniles and adolescents compared to 101 normal weighted, healthy controls of the same age and gender distribution. Further, we investigated the influence of a weight reduction program performed for 12 months in a subgroup of 40 obese juveniles on neopterin serum concentrations.

Materials and Methods

Patients.

Study participants were from the STYrian Juvenile Obesity Study (STYJOBS), which is designed to investigate early stages of atherosclerosis and metabolic disorders in obese juveniles. STYJOBS is registered at http://clinicaltrials.gov/ (Identifier NCT00482924), where detailed information of the study is available. The inclusion criterium for the obese probands was body mass index (BMI) > 97th percentile if under 18 years of age, BMI > 30 kg/m 2 if over 18 years of age. Exclusion criteria were endocrine diseases (e.g., hypothyroidism), infectious, inflammatory, or any other chronic diseases. Controls were healthy juvenile volunteers recruited from the Department of Pediatric Surgery, where they underwent minor elective surgery (e.g., herniotomia). Fasted blood samples were collected prior to anaesthesia and surgery. All controls had to be normal weight, free of infectious, and endocrine or any other diseases. 295 juveniles (mean age 12.9 ± 3.3 SD years) and 101 normal weight healthy controls of similar age and gender distribution were investigated.

Out of this cohort, 40 juveniles with BMI > 97th percentile (male 17, female 23, mean age 13.6 + 2.3 years (SD)) underwent a 12-month weight reduction program during the year 2006. The intervention schedule encompassed regular sports (2 times per week, one hour, 12 months), nutritional devices (1x/week, 1st three months, 1x/2 weeks, 2nd three months, 1x/4 weeks within the remaining 6 months), and a psychologic attendance. Thirtyone obese juveniles (male 13, female 18) who fully completed this program were included in the analysis of the neopterin kinetics during intervention.

The study was approved by the Ethical Committee of the Medical University of Graz. Blood collection and ultrasonography were performed after written informed consent was given by the probands if aged over 18 years, or by their parents, if they were under 18 years old. At the time of blood collection, the probands were fasting. Venous puncture was performed in a standard procedure (cubital vein approach with butterfly); blood samples were immediately centrifuged at 3500 rpm at ambient temperature and stored at −80 • C until analysis.

Carotid Artery

Ultrasound. The ultrasound protocol involved scanning of the bulbous near the common carotid artery on both sides with a 12-to-5-MHz broad-band linear transducer on an HDI 5000 (ATL, Bothell, Washington, DC, USA). All scans were performed by the same investigator. Longitudinal images directed through the center of the artery were taken at each vessel site. Measurements were made from stored digital images by an experienced reader. The carotid IMT was assessed at the far wall as the distance between the interface of the lumen and intima, and the interface between the media and adventitia. The maximal IMT was recorded at each of the vessel segments and averaged for the left and right sides. The lumen diameter was calculated as the interadventitial diameter minus twice the maximum far wall IMT. All diameters were measured during diastole to avoid image blurring due to systolic arterial wall motion and to minimize the influence of blood pressure [29].

Lipometry.

Measurements of total percentual body fat were performed as described elsewhere by means of a lipometer, a patented optical device (EU Pat.Nr. 0516251) [30].

Laboratory Analysis.

Neopterin concentrations were determined by enzyme-linked immunosorbant assay (ELISA) (BRAHMS Diagnostica, Hennigsdorf, Germany). Total adiponectin, leptin, and resistin were determined by ELISA (Biovendor Laboratory Medicine Inc., Brno, Czech Republic) according to manufacturer's instructions. Intra-and interassay coefficients of variation for all ELISAs in our study were below 10 percent. Cholesterol and triglycerides were measured by means of ECLIA (Electro Chemi Luminiscence Assay) on an Elecsys 2010 analyser (Roche Diagnostics Mannheim, Germany), and lipoproteins were separated by a combined ultracentrifugation-precipitation method (βquantification). Blood lipids inclusive of fatty acids were analysed as outlined elsewhere [31]. Oxidized low-dense lipoprotein (oxLDL) was measured by a commercially available ELISA (Mercodia oxidized LDL Competitive ELISA, 754 50 Uppsala, Sweden). Concentrations of CRP were analysed with a highly sensitive particle-enhanced immunoturbidimetric assay (Tina-quant C-reactive protein latex ultrasensitive assay, Roche diagnostics). Plasma insulin was measured by ELISA (Mercodia, Uppsala, Sweden) and plasma glucose was measured by the glucose hexokinase method on a Hitachi 917 chemical analyser. HOMA-IR (homeostatic model assessment-insulin resistance) was calculated as the product of the fasting plasma insulin value (in μU/ml) and the fasting plasma glucose value (mm/L), divided by 22.5 [27]. Liver transaminases AST/GOT, ALT/GPT, γ-GT, creatinine, and uric acid were measured by routine laboratory methods on a Hitachi 917 chemical analyser.

Statistic

Analysis. Statistical analysis was performed by SPSS version 14. Kolmogorov-Smirnov test was used to examine for normal distribution. Means were compared by a two-tailed unpaired sample t-test or by Mann-Whitney Test, depending on the distribution of the data. A value of P < .05 was considered statistically significant. Least squares regression analysis was performed to test the correlation between variables according to Pearson. Significance of correlation was determined by univariate ANOVA and subsequent multiple comparison analysis. Bonferroni correction was used for multiple comparisons after ANOVA. To exclude a dependence of the resulting correlations from other variables, the correlations were adjusted by partial correlations. Stepwise multiple regression analysis was applied to investigate correlations between neopterin as dependent variable and different metabolic and vascular measures as independent variables.

Results

The clinical and laboratory characteristics of the study probands are summarized in Table 1. Obese juveniles had a significantly increased IMT (P < .001) and elevated CRP, (P < .001, see also Figure 1) indicating early stages of atherosclerosis and chronic low-grade inflammation (Table 1). Oxidized LDL, fasted insulin, HOMAindex, resistin, and systolic-, diastolic-blood pressure were significantly increased, and HDL cholesterol significantly decreased in the obese cohort (Table 1). Compared to the controls, obese juveniles had significantly lower neopterin serum concentrations (Table 1 and Figure 1). Neopterin concentrations did not correlate with percentual body fat or BMI-SDS values in the whole study cohort and were independent from any other variable investigated in this study and listed in Table 1 (data not shown).

After the 12-month weight reduction program consisting of regular sports, nutritional devices, and a psychologic attendance, the total percentual total body fat, oxLDL, and resistin serum concentrations decreased significantly whereas the HDL-cholesterol levels were significantly increased ( Table 2). CRP concentrations increased upon intervention as compared with baseline, but the difference was not significant. Fasted blood glucose, insulin levels, IMT of common carotid arteries, and systolic and diastolic blood pressure did not differ significantly from baseline (details not shown). After the 12-month intervention program, neopterin concentrations increased and the mean value became higher than baseline ( Table 2). This increase of neopterin concentrations did not correlate with the decrease of the BMI-SDS.

Discussion

Our study shows that serum neopterin concentrations are not increased in obese adolescents compared to nonobese controls, rather a trend to subnormal levels was observed. Although neopterin concentrations were lowered only by approximately 10% compared to the nonobese controls, the difference was significant. This result is astonishing compared to results obtained in several studies performed earlier by various research groups in adults: adults with high risk for atherosclerosis presented with slightly but significantly increased serum neopterin concentrations compared to lowrisk persons [11][12][13][14][15][16][17][18]32]. Myocardial infarction (MI) is usually accompanied by a transient increase of neopterin [33]. Although only moderately elevated compared to other clinical conditions like acute virus infections or cancer, neopterin levels showed a predictive potential for future clinical events in symptomatic and stable CVD patients [12,15,17,18,34]. This predictive value of neopterin was found to be independent from other biomarkers such as CRP [16][17][18]32]. The ability to enhance oxidative stress and to induce proinflammatory cytokine cascades [33,35] via induction of nuclear factor κB suggests neopterin  to act as a proinflammatory biomarker in atherogenesis [36]. Neopterin appears to play an important role in the pathogenesis of CVD. However, to our surprise this seems to be true only in adults but not in juvenile obesity. There is already information available from adults that CAD activity more than the size of lesions is important for the elevation of neopterin, but in contrast, despite detectable CAD activity in juvenile obesity, neopterin concentrations tended to even lower levels than in nonobese controls of similar age. Increased neopterin concentrations are characteristic for an activated Th1-type immune response [22]. Accordingly, the decreased or low-neopterin concentrations found in our study cohort of obese adolescents even if rather small in absolute terms might indicate a suppressed Th1type immunity and thus lowered IFN-γ production [37]. In fact, only the neopterin concentrations found in the obese groups were lower than normal, the mean value of neopterin concentrations measured in the nonobese control population (5.3 ± 1.9 nmol/L; Table 1) was almost identical with the one found in larger populations of adult blood donors and healthy controls as 5.3 ± 2.7 nmol/L [21,22,38]. At present, increased neopterin concentrations are well established in patients with several diseases which are known to be associated with immune activation such as virus infections, autoimmune syndromes, and various types of cancer [21,22]. However, subnormal or suppressed neopterin release in clinical disease is rather rare. Only from in vitro studies, it is known that several antioxidant compounds are able to slowdown neopterin production in peripheral blood mononuclear cells. Among them are antioxidant food supplements like preservatives and colorants, but also high dose of antioxidant vitamins C and E suppresses Th1-type cytokines and inducees a Th2-type immunity pattern in vitro [39].

Whereas antioxidant compounds were observed to suppress Th1-type immunity and neopterin production in vitro, physical exercise and sports like long-distance running and hiking were found to induce a transient increase of neopterin concentrations [40,41]. An increase of neopterin concentrations was also observed in a rather small subgroup of our patients who attended an intervention therapy during one year that comprised improved dietary habits and the increase of exercise. This increase of neopterin indicates a proinflammatory response which is induced by exercise and corresponds nicely to the above-mentioned earlier findings in healthy individuals. Thus, the most direct and consistent interpretation would be that physical exercise was responsible for the increase of neopterin concentrations.

One may conclude that low or even subnormal neopterin concentrations may relate to a low incidence of physical activities in obese juveniles. Moreover, in diabetes patients, antioxidants have been recently found to prevent the healthpromoting effects of physical exercise [42]. In conclusion, low frequency of physical exercise and sports as well as antioxidant food supplements could contribute to the low neopterin levels in obese juveniles, and it may contribute to weight gain. However, this is merely speculation and still the increase of body weight and the decrease of neopterin might well develop in parallel and are not necessarily linked directly.

Unfortunately, the findings of the intervention program appear somewhat hampered by the fact that those individuals who were willing to enter this program presented with baseline levels somewhat different from the whole study group, for example, neopterin concentrations were slightly lower and resistin and CRP levels were slightly but not significantly higher. The intervention program increased neopterin concentrations, but the average neopterin level reached was only slightly higher as compared to the whole group without any special sports activities. Still the difference of neopterin concentrations before and after intervention was significant when paired samples of the subgroup were compared. Certainly a larger study will be necessary to adequately address this issue, but it is not easy to get.

According to the findings on neopterin concentrations, obesity in adolescents might be classified as a condition linked with Th2-type immunity. It will be interesting to investigate in future whether the balance between Th1-and Th2-type immunity is essentially involved in the observed neopterin kinetics and in obesity development. It can be speculated that the early low-grade inflammation, as found in our obese juveniles in context with an already increased IMT, may be a more Th2-driven process, whereas the advanced atherosclerosis of adulthood is associated with a stronger Th1-type immune activation. There might be a role of mast cells which are known to be strongly engaged in the allergic response but also have the potential to promote atherosclerosis by releasing proinflammatory cytokines which could become relevant with older age [43].

In conclusion, the finding of low-serum neopterin concentrations in obese juveniles forces one to consider that the pathogenesis of cardiovascular disease development in children and juveniles may differ from adults. Further studies should help to better define age-dependent differences and to define optimized preventive strategies, not only the increase of physical exercise and sports but also avoidance of antioxidant food supplements such as preservatives or extra vitamins might be of benefit.

Abbreviations

BMI:

Body mass index CVD: Cardiovascular disease CRP:

Figure 1 :
1Serum concentrations of neopterin and C-reactive protein in obese and nonobese adolescents. Neopterin concentrations are given as mean values (boxes) and S.E.M. (bars) and because of nonnormal distribution C-reactive protein is given as medians (boxes) and 25-75th percentiles (bars).

Table 1 :
1Characteristics of the study subjects.Variable
Controls (n = 101)
Obese adolescents (n = 295)
Age (Years)
12.9 ± 2.7 (4-18)
12.4 ± 2.9 (4-18)
Body Length (m)
1.56 ± 0.14
1.56 ± 0.14
Body Weight (kg)
47.2 ± 13.4
74.5 ± 24.2  *  *
BMI (kg/m 2 )
18.7 (16.9-20.4)
28.9 (26.2-32.4) +++
BMI-SDS
0.21 (−0.7-0.8)
5.4 (4.0-7.2) +++
Carotid IMT (cm)
0.052 ± 0.009
0.070 ± 0.09  *  *  *
Neopterin (nmol/L)
5.3 ± 1.9
4.9 ± 1.8  *
C-Reactive protein (mg/dl)
0.26 (0.17-0.84)
1.9 (0.9-3.8) +++
HDL-cholesterol (mg/dl)
48.4 ± 12.0
41.9 ± 11.2  *  *  *
Oxidized LDL (mg/dl)
35.1 ± 15.3
44.3 ± 16.9  *  *  *
Insulin (uE/ml)
7.9 (5.1-12.7)
15.4 (8.8-29.1) +++
HOMA-IR
1.7 (1.0-2.7)
3.5 (1.8-6.9) +++
Resistin (ng/ml)
3.7 ± 1.5
4.3 ± 1.6  *  *
SBP (mmHg)
115 ± 4.9
125 ± 15.5  *  *  *
DBP (mmHg)
63.1 ± 9
6 8 ± 8.8  *  *  *

Results are expressed as mean ± S.D. analysed by Student's t-test (  *  P < .05,  *  *  P < .01,  *  *  *  P < .001) or as median (25th-75th percentile) and Mann Whitney
U test ( + P < .05, ++ P < .01, +++ P < .001) depending on the distribution of data.
BMI, body mass index; BMI-SDS, BMI-standard deviation score; HOMA-IR, homeostatic model assessment-insulin resistance; SBP, systolic blood pressure;
DBP, diastolic blood pressure; carotid IMT, carotid intima-media thickness.

Table 2 :
2Neopterin, body fat, HDL cholesterol, resistin, oxLDL, and C-reactive protein concentrations (mean ± S.D.) before and after 12 months of intervention therapy.Start of intervention
End of intervention (12 months)

AcknowledgmentThe authors thank Miss Astrid Haara and Miss Kerstin Hingerl for excellent technical assistance.
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Robertsonian chromosomes and the nuclear architecture of mouse meiotic prophase spermatocytes

Soledad Berríos
Catalina Manieu
Julio López-Fenner
Eliana Ayarza
Jesús Page
Marisel González
Marcia Manterola
Raúl Fernández-Donoso
Robertsonian chromosomes and the nuclear architecture of mouse meiotic prophase spermatocytes
R E S E A R C H A R T I C L E Open Access
Background: The nuclear architecture of meiotic prophase spermatocytes is based on higher-order patterns of spatial associations among chromosomal domains from different bivalents. The meiotic nuclear architecture depends on the chromosome characteristics and consequently is prone to modification by chromosomal rearrangements. In this work, we consider Mus domesticus spermatocytes with diploid chromosome number 2n = 40, all telocentric, and investigate a possible modification of the ancestral nuclear architecture due to the emergence of derived Rb chromosomes, which may be present in the homozygous or heterozygous condition. Results: In the 2n = 40 spermatocyte nuclei random associations mediated by pericentromeric heterochromatin among the 19 telocentric bivalents ocurr at the nuclear periphery. The observed frequency of associations among them, made distinguishable by specific probes and FISH, seems to be the same for pairs that may or may not form Rb chromosomes. In the homozygote Rb 2n = 24 spermatocytes, associations also mediated by pericentromeric heterochromatin occur mainly between the three telocentric or the eight metacentric bivalents themselves. In heterozygote Rb 2n = 32 spermatocytes all heterochromatin is localized at the nuclear periphery, yet associations are mainly observed among the three telocentric bivalents and between the asynaptic axes of the trivalents.Conclusions:The Rb chromosomes pose sharp restrictions for interactions in the 2n = 24 and 2n = 32 spermatocytes, as compared to the ample possibilities for interactions between bivalents in the 2n = 40 spermatocytes. Undoubtedly the emergence of Rb chromosomes changes the ancestral nuclear architecture of 2n = 40 spermatocytes since they establish new types of interactions among chromosomal domains, particularly through centromeric and heterochromatic regions at the nuclear periphery among telocentric and at the nuclear center among Rb metacentric ones.

Background

During the meiotic prophase of spermatocytes, chromosomes form a distinctive arrangement in the nuclear space, governed mainly by the synapses between homologous chromosomes that form bivalents and by the union of their ends to the nuclear envelope [1][2][3]. The bivalent configuration generates a meiotic nuclear architecture dependent on the chromosome characteristics [4][5][6]. Consequently, this architecture is subject to modification by chromosomal rearrangements.

In this work, we investigate how the ancestral nuclear architecture of Mus domesticus spermatocytes with a diploid chromosome number 2n = 40, all of them telocentric, is modified by the emergence of derived Rb metacentric chromosomes, which may be present in the homozygous or heterozygous condition.

Synapsis between homologous chromosomes is a process that culminates with the formation of the synaptonemal complex (SC). The SC is a tripartite proteinaceous scaffold consisting of two lateral elements (homologous chromosome axes) and a medial component that stabilizes the joint between the homologous chromosomes forming the bivalents [7][8][9][10].

Numerous proteins are involved in the SC structure. SYCP3 protein is the main constituent of the axis of each homologous chromosome, and SYCP1 protein is located between the axes of homologous chromosomes, binding them together in synapsis with transverse filaments [11,12]. Both ends of each bivalent SC are attached to the nuclear envelope, so that each SC forms an arc of different extension depending on the length of the synapsed chromosomes. The chromatin, organized in loops, is connected to the lateral elements of the SC, hence the chromatin domains are sequentially ordered along each bivalent SC. Finally, the SC'trajectory determines the place for the chromosomal domains to occupy within the nuclear space. This is not trivial, because interactions or associations among heterologous chromosomal domains depend on the real possibility of establishing contacts between them together with their structural and functional affinities that could favor the consolidation of such interactions.

In this regard, the spermatocytes of Mus domesticus 2n = 40 containing 19 autosomal telocentric bivalents with abundant pericentromeric heterochromatin near their proximal ends naturally favor the associations among them over the nuclear envelope [4]. Comparative analysis of the observed combinations of associated and non-associated bivalents in the spermatocytes, and the predictions of an ad-hoc developed probabilistic model for associations between indistinguishable elements, suggest that these associations could indeed take place randomly [6]. Notice that associations are not a phenomenon unique to meiotic prophase cells. They have also been described in Mus somatic cells with highly complex chromocenters, involving several chromosomes and the nucleoli [13].

Large blocks of satellite DNA (pericentromeric heterochromatin) surround the centromere and extend towards the proximal end of each chromosome, thus favoring the occurrence of Rb translocations in Mus. Double-strand breaks of DNA with nearly identical base components, in addition to physical proximity, facilitates the fusion of different chromosomes [14]. Rb translocations involve double-strand DNA breaking at the centromere level in two telocentric (acrocentric) chromosomes, followed by a repair (fusion) that binds the respective long arms, creating a metacentric Rb chromosome. The short arms (p) of the original telocentric chromosomes, including the proximal telomeres, part of the satellite DNA, and generally one centromere, are all lost. However, this loss of DNA does not significantly alter the total amount of DNA as compared with the standard Mus karyotype [15]. Rb translocation is the most common chromosomal rearrangement in mammals [16] and represents the type of chromosomal change that most effectively contributes to differentiation or speciation of natural populations [17].

In Mus, Rb translocations have resulted in more than 40 different chromosomal races (or subspecies), ranging from 2n = 40 to 2n = 22. These chromosomal races are natural populations characterized by altogether about 100 Rbs chromosomes with different combinations of arms [18], many of which emerged and spread extremely rapidly within populations of the standard karyotype [19].

Metacentric Rb chromosomes can become numerous in the Mus genome, leading to a reduction of ancestral telocentric chromosomes and to an emergence of new mixed karyotypes. Furthermore, crossing between wild homozygotes 2n = 40 and Rb homozygotes produce F1 hybrids in whose genomes the ancestral telocentric chromosomes are reunited with the metacentric derivatives. Trivalents are formed in the meiotic prophase spermatocytes of these mice, in which a metacentric chromosome is synapsed with the long arms of two telocentric chromosomes. Varying degrees of synapses are established among the proximal ends of the involved telocentric chromosomes, despite being heterologous [20].

The presence of Robertsonian (Rb) metacentric chromosomes, which can be multiple in this species, constitutes a valuable opportunity to study how these new chromosomes modify the original, ancestral nuclear architecture built just for telocentric bivalents.

To this end, we studied the meiotic nuclear organization of:

We further compared both families of spermatocytes with the nuclear architecture of the ancestral homozygote 2n = 40.

We found different patterns of nuclear architecture according to chromosome constitution of the spermatocytes. The Rb chromosomes present in 2n = 24 and 2n = 32 spermatocytes drastically restrict the possibilities for interaction between the heterochromatic domains as compared with the wealth of random associations observed in 2n = 40 spermatocytes.

Results

Early prophase and the chromosome nuclear distribution in 2n = 40 and 2n = 24 spermatocytes

In early prophase nuclei, chromosome/bivalents are aggregated in a configuration known as the bouquet, in which the telomeric regions of all chromosomes cluster over a small area of the nuclear envelope. In this meiotic stage, the 2n = 40 spermatocytes showed centromeres aggregated in one or two main groups at the nuclear periphery, while the respective axes or SCs described arcs toward the nuclear space (Figure 1a & a'). In the bouquet configuration of the 2n = 24 spermatocytes, the centromeres appeared in two major clusters: one at the nuclear periphery, grouping the centromeres of the telocentric bivalents, and the other one toward the nucleus center, grouping the centromeres of the metacentric Rb bivalents (Figure 1b & b').

When the early prophase nuclei were treated with DAPI that stains DNA rich in AT sequences, which in Mus is the DNA that underlies the pericentromeric heterochromatin, the 2n = 40 spermatocyte nucleus showed one large DAPI-positive chromocenter whose location matched the regions of aggregated centromere. The 2n = 24 spermatocytes showed at least two DAPIpositive chromocenters, one at the nuclear periphery and the other toward the center of the nucleus. We showed previously that the initial aggregation of centromeres occurring in lepto-zygotene of 2n = 40 spermatocytes will eventually resolve into several smaller aggregates that we can observe in pachytene or more advanced stages of meiotic prophase [6].

Bivalent configuration and associations in 2n = 40 pachytene spermatocytes

In these meiotic nuclei all the telocentric SCs described regular arcs with the centromeres located in one extreme near the nuclear envelope. Figure 2a shows a wellpreserved pachytene nucleus with this chromosomal configuration. The focal plane is over the SC of one of the smallest bivalent where the centromeric domain close to the nuclear limit can be distinguished ( Figure 2a). The abundant pericentromeric heterochromatin in all the bivalents preserving associations among them, produce chromocenters of different sizes, scattered throughout showing the configuration of autosomal chromosomes in the "bouquet" of a 2n = 40 spermatocytes. The chromatin of the sex bivalent is also represented (a). The centromeres (red) aggregate in one main group at the nuclear periphery, while the chromosomal axes (green) describe arches toward the nuclear space. Diagram (b) and nucleus (b') showing the configuration of autosomal chromosomes in the "bouquet" of a 2n = 24 spermatocyte. The chromatin of the sex bivalent is also represented (b). The centromeres appear in two major clusters: one at the nuclear periphery, grouping the centromeres of the telocentric bivalents, and the other toward the nucleus center, grouping the centromeres of the metacentric Rb bivalents. The axes that emerge from the peripheral centromeres are shorter than those linked to the central centromeres. The chromosomal axes (green) and centromeres (red) in nuclei with preserved sphericity from 2n = 40 and 2n = 24 spermatocytes were identified by immunochemistry using, respectively, anti-SYCP3 and anti-CENPA antibodies. Bar = 5 μm. the nuclear envelope ( Figure 1b). Therefore, each chromocenter is corresponds to a cluster of pericentromeric heterochromatin formed by the joint contribution of all those bivalents that remain associated between them throughout the prophase. The size of the chromocenter was proportional to the number of the associated bivalents. In pachytene nuclear spreads the clusters of heterochromatin were well preserved. By immunochemistry, both the axial elements of the SC as well as the pericentromeric heterochromatin, could be identified and, consequently, the number of bivalents participating in each chromocenter could be determined (Figure 2c & d). The associations among chromosomes were studied in 100 microspreads of 2n = 40 pachytene spermatocytes, considering the 19 autosomal bivalents as indistinguishable elements from one another. Each nucleus exhibited different combinations of chromosomal clusters. More than 60% of the pachytene spermatocytes presented groups of 4, 5 or 6 bivalents, as the highest number of associated bivalents (Figure 2c & d). Spermatocytes with no associations among bivalents or forming a cluster involving more than 11 associated bivalents were never observed (See [6]). Besides, we also studied the associative behavior of specific bivalents, identifying by FISH and chromosomal probes two bivalents at a time in 100 spermatocytes each ( Figure 3). The probes were specific for chromosomes 9, 14, 16, and 17, and they were chosen because the chromosome pairs 9/14 and 16/17 form Rb metacentric chromosomes. In approximately 60% of spermatocytes, the two marked bivalents were found to be associated with other bivalents in a number of combinations, but not among themselves (Figure 3a & b). In approximately 30 to 32% of the spermatocytes, the identified bivalents were alone, not associated with each other or with other bivalents. In about 8% to 11% of the studied spermatocytes, the two identified bivalents were associated to each other ( Figure 3c). No differences was observed in the frequency of association between pairs of chromosomes that may form Rb metacentric chromosomes (9/14, 16/17), as compared to those pairs that do not form Rb chromosomes (9/17, 14/16) were observed (See Table 1). A computer simulation of all possible associations between the 19 Figure 2 Nuclear architecture of spermatocytes 2n=40. a Distribution of telocentric bivalents in a 3D pachytene nucleus from 2n = 40 spermatocytes. The synaptonemal complexes (green) and centromeres (red) were identified by immunochemistry using anti-SYCP3 and anti-CENPA antibodies. The 19 autosomal bivalents form regular arcs with the centromeres (red) (and the heterochromatin) in one extreme of each and consequently localized at the nuclear periphery. The focus is over one telocentric bivalent. Bar = 5 μm. b Scheme representing the nuclear architecture of 2n = 40 spermatocytes. The centromeric regions (red) of all telocentric bivalents are located at the nuclear periphery. The bivalentes can be alone or associated in different numbers. The XY chromatin is also represented (blue). c & d. Associations between telocentric bivalents through heterochromatin in 2n = 40 spermatocytes. The SC (green) and the pericentromeric heterochromatin (red) were identified by immunochemistry using, respectively, anti-SYCP3 and anti-H3K9me3 antibodies. XY = sex bivalent. elements, which included only 2 distinguishable bivalents, revealed that the frequency of events in which they can be associated with each other over all conditions was close to 12% (López-Fenner J, Berrios S, Manieu C, Page J, Fernández-Donoso R. Bivalent associations in Mus domesticus 2n = 40 spermatocytes. Are they random? Submitted). If we use this theoretical value as a reference, we can consider that the observed frequencies of association between distinguishable elements could also be random, at least for the four pairs of bivalents here studied.

Bivalent configuration and associations in 2n = 24 pachytene spermatocytes

In these nuclei only three SCs described regular arcs with the centromeres localized in one extreme near the nuclear envelope because only three autosomal bivalents are even telocentrics. The eight metacentric Rb bivalents formed longer SCs than those of the telocentric bivalents and the centromeres were localized in the middle of the metacentric Rb bivalents, far from the nuclear envelope. In a wellpreserved nucleus the focus was over one metacentric Rb bivalent (Figure 4a). In 2n = 24 spermatocytes, associations were also produced through the pericentromeric heterochromatin, but mostly within groups of telocentric or metacentric bivalents (Figure 4b). Nuclear spreads with well-preserved clusters of heterochromatin showed two or more groups of metacentric bivalents associated in different number combinations and the telocentric bivalents mainly associated among themselves (Figure 4c & d). The nuclear frequency per each chromosomal combination is One hundred spermatocyte nuclei for each pair of bivalents were scored. The nuclei were stained with DAPI, and the specific bivalents were identified by FISH and specific DNA probes. shown in Table 2. In 27% of the spermatocytes the 3 telocentric bivalents were all together, in 56% of the nuclei only two bivalents were found associated and one single; and in approximately 17% of the nuclei the 3 telocentric bivalents were not in association, neither among themselves nor with other bivalents. Only 12% of the examined spermatocytes also showed associations between telocentric and metacentric bivalents ( Table 2). In about 19% of the spermatocytes some bivalents were associated with the XY bivalent.

Trivalent configurations in spermatocyte nuclei of heterozygotes 2n = 32

In well-preserved nuclei of heterozygote spermatocytes, each trivalent showed three points of attachment to the nuclear envelope, two of which corresponded to the distal telomeres of the metacentric chromosomes synapsed with the telocentric chromosomes and the third given by the attachment of the proximal telomeres of the telocentric chromosomes. The third point of attachment to the nuclear envelope drags the pericentromeric heterochromatin of the 3 chromosomes involved in the trivalent towards the nuclear periphery. Throughout the meiotic prophase, the distance between the three telomere attachment points over the nuclear envelope increases, so that each trivalent may have different spatial configuration options. These configurations are observed in the spermatocyte shown in Figure 5b and represented in the diagram shown in Figure 5a. In early prophase, most trivalents occupy a small space at the nuclear periphery, due to the close attachment on the nuclear envelope of the three telomeres. This configuration is modified in more advanced spermatocytes, where the distal telomeres appear attached at the opposite pole of the nuclear envelope with respect to the proximal telomere, so that the trivalent occupies almost the complete nuclear diameter. No associations between chromosomal axes were observed in any of the configurations in well-synapsed Figure 4 Nuclear architecture of spermatocytes 2n=24. a Distribution of metacentric and telocentric bivalents in a 3D pachytene nucleus from 2n = 24 spermatocytes. The synaptonemal complexes (green) and centromeres (red) were identified by immunochemistry using anti-SYCP3 and anti-CENPA antibodies. The metacentric bivalents form SCs longer than those of the telocentric bivalents and the centromeres are localized in the middle of the metacentric bivalents unlike the terminal position in the telocentric bivalents. The focus is over one metacentric Rb bivalent. Bar = 5 μm. b. Scheme representing the nuclear architecture of 2n = 24 spermatocytes. The centromeric regions of the three telocentric bivalents (T) are located at the nuclear periphery and the centromeric regions of the metacentric bivalents at the nuclear center. The associations are produced mostly within groups of telocentric or metacentric bivalents. The XY chromatin is also respresented (blue). c & d Associations between metacentric and telocentric bivalents through heterochromatin in 2n = 24 spermatocytes. The SC (green) and the pericentromeric heterochromatin (red) were identified by immunochemistry using anti-SYCP3 and anti-H3K9me3 antibodies. The associations were mainly between the telocentric or the metacentric bivalents. c) Nucleus with 6 associated metacentric bivalents and 2 singles; the 3 telocentric bivalents appear all together. The XY bivalent is indicated. d) Nucleus with three groups of metacentric bivalents: 4 associated, 2 associated and 1 single; the 3 telocentric bivalents appear all together. The XY bivalent is indicated.

trivalents. A different situation was observed on the trivalents in which the short arms of telocentric chromosomes remain asynapsed. These associations involved an endto-end junction between one or both single axes from two or more trivalents (Figure 5c) or between a trivalent single axis and the X chromosome single axis (Figure 5d). In both cases, as revealed by immunocytochemistry, the SYCP1 protein is detected at the end-to-end contact region (Figure 5c & d). The asynapsed trivalents also presented ectopic associations just mediated by heterocromatin from different trivalents or between a trivalent and the XY chromatin (Figure 5e & f ). In these situations the heterochromatin was modified as it can be demonstrated by the presence of the histone protein γH2AX (Figure 5e & f). In 68% of mid pachytene, the single axes of partially-asynapsed trivalents established ectopic associations amongst themselves (15,7%), with the XY bivalent (27,1%) or both (23,6%) ( Table 3). Only about 5% of the mid pachytene with well-synapsed trivalents presented ectopic associations, which were just mediated by heterocromatin from different trivalents or between a trivalent and the XY bivalent (Table 3). In late pachytene or diplotene spermatocytes, which mostly present just one asynapsed trivalent or the 8 trivalents completely synapsed, ectopic associations were rarely observed (not shown).

Discussion

The nuclear architecture of spermatocytes in meiotic prophase is primarily determined by the synaptic organization of the bivalents, bound by their telomeres to the nuclear  100 nuclear microspreadings from 2n = 24 spermatocytes were examined. The SC and the pericentromeric heterochromatin were identified by immunochemistry using, respectively, anti-SYCP3 and anti-H3K9me3 antibodies (as in Figure 4c & d). The first column shows the combination of associated and single metacentric bivalents per nucleus; The 2 nd column the combination of associated and single between 3 telocentric bivalents; the 3th column the spermatocytes that showed associations between metacentric and telocentric bivalents; The 4 th column the frequency of spermatocytes per nuclear combination; and the 5 th column the frequency of spermatocytes per Class, considering as such, all the nuclei sharing the same highest combination of metacentric bivalent. For example the third line of this Table means: In 5 spermatocytes the metacentrics were in a group of 6 and in another of two bivalents. In 3 of these spermatocytes the telocentrics were all together and in 2 they were in a combination 2 together and one alone. 12 spermatocytes were Class 6 or 12 had 6 as the higher number of associated metacentric bivalents.

envelope and describing arc-shaped trajectories through the nuclear space. However, over this basic meiotic organization, the spermatocyte nuclear architecture is also conditioned by the individual characteristics of the chromosomes and the opportunity for interactions between their domains. The homogeneity in the morphology of the chromosomes of Mus domesticus 2n = 40, in addition to the numerous subspecies with Figure 5 Configurations of synapsed trivalents in pachytene nuclei of 2n = 32 spermatocytes (a-b) and ectopic associations between asynapsed trivalents and the XY bivalent (d-f). a-b.Trivalent configurations in a diagram (a) and in a well preserved nucleus (b). The synaptonemal complexes (green) and centromeres (red) were identified by immunochemistry, using, respectively, anti-SYCP3 and anti-CENPA antibodies. The trivalents present 3 points of attachment to the nuclear envelope, two of which correspond to the distal telomeres of the metacentric chromosomes synapsed with the long arms of the telocentric chromosomes (arrowheads), and the third to the attachment of the proximal telomeres of the telocentric chromosomes (arrow). The third point drags the pericentromeric heterochromatin of the 3 chromosomes involved in the trivalent towards the nuclear periphery. c-d. In nuclear spreads the chromosomal axes (red) and central element of the SC (green) were identified by immunochemistry, using, respectively, anti-SYCP3 and anti-SYCP1 antibodies. c) Two asynapsed trivalents are associated through both single axes from the two telocentric short arms (arrow). In both bonding zones, the protein SYCP1 was detected (yellow). The 6-remaning trivalents are totally synapsed. d) The single axis of an asynapsed trivalent is bound to the X chromosome single axis. At the end-to-end contact region, the SYCP1 protein was detected (arrowhead). e-f. γH2AX antibodies intensely labels the XY chromatin (blue), the chromatin surrounding asynapsed trivalent AEs, and is absent in the chromatin of synapsed bivalents. e) One asynapsed trivalent is associated to the XY bivalent only through chromatin where the modified histone γH2AX is detected (arrowhead). The 7-remaning trivalents are totally synapsed, not associated at all and without detection of γH2AX. f) Two asynapsed trivalents (arrows) and one asynapsed trivalent and the XY bivalent (arrowhead), are associated among them only through chromatin where the histone γH2AX is detected.

reduced diploid numbers and carriers of metacentric Rb chromosomes, makes this species a convenient model to evaluate the above proposal. In this sense, the comparative analysis of the nuclear architecture of Mus spermatocytes with different chromosomal constitutions (2n = 40, 2n = 24 and 2n = 32) allowed us to establish which differences in nuclear organization are attributable to Rb chromosomes. Moreover, all telocentric chromosomes of Mus 2n = 40 exhibit abundant pericentromeric heterochromatin that is composed of two distinct repetitive DNA sequences, the minor and major satellites. It has been shown that the major satellites with the heterochromatin protein 1alpha form clusters, creating an association of several bivalents [21]. Previous analyses have allowed us to demonstrate that associations between autosomal bivalents mediated by heterochromatin are very frequent and can be considered as being random [6]. In this work, in which two bivalents were distinguishable each time, we found that the frequency of association between them was close to 10%, coinciding with the expected rate if associations were at random. There were no significant differences in the frequency of association by spermatocyte between chromosome pairs that form Rb chromosomes (9/14 and 16/17) versus those that do not (9/16 and 14/17), nor did we find higher frequencies of association between bivalents 16 and 17 that are nucleolar and whose NOR regions are both near the pericentromeric heterochromatin. Ribosomal gene expression occurs in pachytene spermatocytes; this situation may have contributed to the association of NORs from different bivalents in the production of a common nucleolus, as has been described in somatic and meiotic cells [5,13]. However, as Mus has multiple nucleolar chromosomes, we cannot rule out preferential associations among other nucleolar chromosomes [22]. In any case, frequent random associations among all bivalents would be consistent with the necessary physical proximity among their heterochromatic regions for Rb fusions to occur among any of the 19 bivalents, as has been described for the different subspecies of Mus [18]. On the other hand, the homology of satDNA sequences shared by the mouse telocentric chromosomes [17] might have occurred by means of multiple small exchanges among these large tracts of tandemly repeated DNA [23]. The associations among the autosomal bivalents during meiotic prophase, precisely through their heterochromatic domains, would also provide a scenario in which these frequent recombinational exchanges between non-homologous chromosomes may have occurred. A similar origin has been proposed for the concerted evolution of the ribosomal DNA spacers on non-homologous nucleolar chromosomes [24][25][26].

In many organisms, a meiosis-specific organization of chromosomes called the "bouquet configuration," which is a clustering of telomeres on the inner nuclear envelope, appears to facilitate homologous recognition and alignment by concentrating all chromosomes within a limited region of the nuclear volume [27][28][29][30]. In the bouquet of the 2n = 40 spermatocytes, all of the heterochromatic ends of the telocentric chromosomes cluster together, forming a large chromocenter. Later, the progress of the prophase increases the nuclear volume and the movement of telomeres over the nuclear envelope, and consequently the great original chromocenter breaks down into the smaller chromocenters seen in the pachytene stage [6]. In contrast, during the bouquet of 2n = 24 spermatocytes, it was remarkable, that only two chromocenters were observed, a small one at the nuclear periphery and another with abundant heterochromatin toward the center of the nucleus. Therefore, two different areas for association are characteristic for these nuclei: one among the heterochromatins of telocentric bivalents, and another among the heterochromatins of metacentric bivalents. This organization continues toward the pachytene stage, where most of the spermatocytes show the telocentric bivalents tightly associated among themselves and the metacentric bivalents associated in a looser way forming two or more groups. We suggest that this new nuclear architecture would favor the progressive fusion of the remaining telocentric chromosomes. It is also possible that this narrowing of association opportunities is related to the overall trend toward production of metacentric chromosomes, as observed in the chromosomal evolution Table 3 Number of partially asynapsed trivalents and the type of ectopic asociations in 2n = 32 heterocygote  spermatocytes   Asynapsed trivalents  0  1  2  3  4  5  6  7  8  Total  %   Ectopic associations  none  35  1  3  4  3  2  0  0  0  47  33,6   XY/triv  2  13  8  6  8  3  0  0  0  38  27,1   triv/triv  0  0  5  7  4  4  0  0  0  22  15,7   both  0  0  0  3  9  12  3  2  3  33  23,6   Total nuclei  37  14  16  20  24  21  3  2  3 140 100 140 nuclear microspreadings from 2n = 32 pachytene spermatocytes were examined. The single axes and central element of the SC were identified by immunochemistry, using, respectively, anti-SYCP3 and anti-SYCP1 antibodies (as in Figure 5c & d).

of this species [16,31]. On the other hand, association between the heterochromatic regions of the metacentric bivalents is the scenario in which WARTS may occur. In these Rb rearrangements, described also in the karyotypes of Mus, two Rb metacentric chromosomes exchange their arms due to breakage and reciprocal fusion at the pericentric heterochromatin level of both [32,33]. The described topological nuclear organization allows for the repeated encounter between chromosomal domains that may eventually experience rearrangements among them. However, under this topographic scenario it is also necessary to consider that during meiotic prophase, a programmed induction of DNA double-strand breaks (DSBs) leads to the exchange of genetic material between homologous chromosomes [34]. DSBs preferentially occurs at discrete sites called hotspots whose localization seems to be influenced by both local chromatin and higher-order chromosome structures [35]. It is not known if hotspots are preferentially localized at the heterochromatic domains of the Mus spermatocytes. However, on these meiotic prophase nuclei the full DNA repair machinery would be available, which could account for an exchange between heterologous DNA that may change the structure of the involved chromosomes.

In heterozygote 2n = 32 spermatocytes, another nuclear architecture emerges, which in this case accounts for the meeting of two different chromosome complements, one from 2n = 40 homozygotes and the other from 2n = 24 homozygotes. These hybrids, or similar, may occur naturally in any overlapping area of two subpopulations of Mus, one bearing the ancestral karyotype and other that has diverged by the presence of multiple Rb chromosomes. In Rb heterozygote spermatocytes, recognition and synapsis between the two parental chromosome complements occur, forming eight trivalents, three telocentric bivalents, and a sexual bivalent, similar to what has been previously described for other Rb hybrids of Mus [36]. Each trivalent has three points of attachment to the nuclear envelope, two corresponding to the distal telomeres and the third to the heterologous proximal ends of the telocentric chromosomes. This organization leads to the three centromeres clustering together at the nuclear periphery. Depending on the distance between the distal and proximal telomeres the trivalents may take different configurations at the nuclear space. The ultimate reason why telomeres move away or why the trivalents present different configurations remains unknown. On the other hand, in any of these configurations, the centromeric and heterochromatic domains of the very same eight pairs of telocentric chromosomes are always together in every trivalent and spermatocyte. This repeated convergence of the same pairs of heterochromatic domains should be also decisive in favoring the formation of new metacentric Rb chromosomes.

It has been reported that approximately 80% of the heterozygote spermatocytes in early prophase shows 2 to 6 trivalents with varying degrees of asynapsis between the short arms of telocentric chromosomes [20]. Chromatin surrounding these areas of asynapsis experiences changes in condensation and modifications on the underlying proteins resulting in transcriptional repression. This entire phenomenon is known as MSUC for "Meiotic Silencing of Asynapsed Chromatin" [37]. Some of the asynapsed trivalents were only connected between them or with the XY bivalent through this kind of chromatin. It has been reported that MSUC also delay the progress of meiotic prophase, which gives the opportunity that these chromosomal regions reach the synapsis what apparently it happens in many of the spermatocytes [20]. Despite of this, we observed that in 68% of mid pachytene nuclei, the single axes of partially asynapsed trivalents established ectopic associations amongst themselves (15,7%), with the XY bivalent (27,1%) or both (23,6%). Furthermore, the presence of SCP1 protein between the heterologous axes demonstrates the formation of the SC medial element and thus the possibility of recombination between heterologous chromosomal regions, which can later lead to segregational problems between those chromosomes. Therefore, the configuration of trivalents and the relationships among their unsynaptic axes impact globally the architecture of the heterozygote pachytene nuclei and possibly may have consequences for the survival of the spermatocytes. Thus, ectopic bonds could be quite deleterious to the normal progress of prophase and also to the subsequent segregation of the involved chromosomes. Spermatocytes carrying these ectopic joints may be eliminated by apoptosis in prophase or metaphase I, a phenomenon that has been observed in the Rb heterozygote male germ line [38,39]. All of these situations could explain the reduced fertility found in Rb hybrids [38,[40][41][42].

Clearly, the multiple presence of Rb metacentric chromosomes in 2n = 24 spermatocytes, as in the 2n = 32 heterozygotes, determines a new organization and distribution of chromosomal domains in the meiotic prophase nucleus and consequently changes the possibilities for interchromosomal relationships as compared to those present in the 2n = 40 spermatocytes. The nuclear architecture can achieve a new optimum as have been seen in homozygote spermatocytes 2n = 24. However, in heterozygote nuclei, the areas for meeting and interaction between chromosomal domains, which actually define the nuclear architecture, are unclear and their emerging trials rather seem to produce additional instability.

On the other hand, the significant changes in the spermatocyte nuclear architecture described here allow us to better understand the difficulties that a new Rb chromosomes face in surviving meiosis, and therefore to be inherited by the progeny and propagated into the reproductive community.

Conclusions

The Rb chromosomes pose sharp restrictions for interactions in the 2n = 24 and 2n = 32 spermatocytes, as compared to the ample possibilities for interactions between bivalents in the 2n = 40 spermatocytes.

The emergence of Rb chromosomes changes the ancestral nuclear architecture of 2n = 40 spermatocytes since they establish new types of interactions among chromosomal domains.

The associations are produced through centromeric and heterochromatic regions at the nuclear periphery among telocentric bivalents and at the nuclear center among Rb metacentric ones.

Methods

We analyzed spermatocytes from four male threemonth-old Mus domesticus 2n = 40 CD1 mice with all telocentric chromosomes; spermatocytes from four males of the Milano II 2n = 24 with eight pairs of homozygote Rb metacentric chromosomes; and spermatocytes from four heterozygote Rb mice 2n = 32 with eight single Rb metacentric chromosomes. The heterozygote mice were generated by mating females of the laboratory strain CD1 2n = 40 and males of the Milano II race. Male and female specimens from the original natural populations were donated to our laboratory, by Drs Carlo Redi and Silvia Garagna from the Pavia University, Italy, as part of a collaborative research proyect.

Mice were maintained at 22°C with a light/dark cycle of 12/12 hours and fed ad libitum. Procedures involving the use of the mice were reviewed and approved by the Ethics Committee of the Faculty of Medicine, Universidad de Chile and by the Ethics Committee of the Chilean National Science Foundation FONDECYT-CONICYT.

Spermatocyte squashes with 3-D preserved nuclei

Spermatocyte squashes that preserved the nuclear volume were obtained following the procedure described by Page et al. [43]. Testes were removed and fixed in 2% formaldehyde in PBS containing 0.05% Triton X-100. Pieces of tubules were placed in a drop of fixing solution on a slide. They were gently minced with tweezers, and then a coverslip was added. Exerting pressure on the coverslip squashed the cells. The slides were frozen in liquid nitrogen, and coverslips were then removed.

Spermatocyte nuclear spreads

Spermatocyte spreads were obtained following the procedure described by Peters et al. [44]. Briefly, a testicular cell suspension in 100 mM sucrose was spread onto a slide dipped in 1% paraformaldehyde in distilled water containing 0.15% Triton X-100 then left to dry for two hours in a moist chamber. The slides were subsequently washed with 0.08% Photoflo (Kodak), air-dried, and rehydrated in PBS.

Immunochemical identification of bivalents

The slides were incubated for 45 minutes at 37°C in a moist chamber with the primary antibodies: rabbit anti-SYCP3 1:100 (Abcam, ab15093); mouse anti-CENPA 1:200 (Abcam, ab13939); rabbit anti-H3K9me3 1:200 (Abcam ab8580); or mouse anti-phospho-histone H2AX (Ser139) 1:1000 clone JBW301 (Upstate, 05-636). Then, the slides were incubated for 30 minutes at room temperature with the secondary antibodies: FITC-conjugated goat antimouse IgG (1:50) (Sigma), or Texas red-conjugated goat anti rabbit IgG (1:200) (Jackson). Slides were counterstained with 1 μg/ml DAPI (4,6-diamidino-2-phenylindole). Finally, slides were rinsed in PBS and mounted in Vectashield (Vector).

In situ hybridization

In the microspreads of the 2n = 40 spermatocytes, chromosomes 9, 14, 16, or 17 were identified by in situ hybridization using commercial probes (MetaSystems). The slides were treated for 5 minutes in 1XPBS, dehydrated in a series of 70, 80, 90, and 100% ethanol for 2 minutes each, and air-dried at room temperature. The slides with the samples and the DNA probe were denatured together at 75°C for 2 minutes. Then, the slides were incubated in a humid chamber at 41°C for 16 hours. Next, the coverslip was removed, and the slides were washed in: 0.4X SSC at 72°C for 2 minutes; 2XSSC with 0.05% Tween20 at room temperature for 30 seconds, and twice in 1× PBS for 5 minutes each. Nuclear contrast was performed with DAPI, and coverslips were mounted with Vectashield.

Images

Observations were made using a Nikon (Tokyo, Japan) Optiphot or Olympus BX61 microscope equipped with epifluorescence optics, and the images were photographed on a DS camera control unit DS-L1 Nikon or captured with an Olympus DP70 digital camera. All images were processed using Adobe Photoshop CS5.1 software or the public domain software ImageJ (National Institutes of Health, United States; http://rsb. info.nih.gov/ij).

Figure 1
1Bouquet configuration in early meiotic prophase nuclei of 2n = 40 and 2n = 24 spermatocytes. Diagram (a) and nucleus (a')

The19 autosomal bivalents appear singly or associated in different numerical combinations per spermatocyte. The combinatory presents in the nucleus of figure c) is:

Figure 3
3Associations between specific bivalents in pachytene nuclear squashs from 2n = 40 spermatocytes. a, b, and c: The bivalents 16 (red) and 17 (green) appear identified by FISH and specific probes. a', b' and c': Same nuclei counterstained with DAPI; a", b" and c": Representation of the painted bivalents 16 and 17 and the DAPI heterochromatin of the all bivalents. In the nucleus a", the bivalent 16 through the heterochromatin is associated with 5 bivalents while the bivalent 17 is single; In the nucleus b", the bivalent 16 as the bivalent 17 are independently associated with other 2 bivalents; in the nucleus c", both bivalents 16 and 17 are associated with each other and with two more bivalents through their heterochromatins.

Table 1
1Frequency of 2n = 40 spermatocyte bearers with two given distinguished bivalents associated between themPair of distinguishable
bivalents

Spermatocyte percentage with those
bivalents associated between them

16 and 17
8

9 and 14
10

14 and 16
11

9 and 17
10

Table 2
2Combination of associated and single metacentric/telocentric bivalents per nucleus observed in 2n = 24 spermatocytesCombinations between
8 metacentric bivalents

Combinations between
3 telocentric bivalents

Metacentric/Telocentric
associations

Number of spermatocytes
per nuclear combination

% of spermatocytes
per Class

http://www.biolres.com/content/47/1/16
Competing interestsThe authors declare that they have no competing interests.Authors' contributions
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Robertsonian chromosomes and the nuclear architecture of mouse meiotic prophase spermatocytes. Berríos, Biological Research. 4716Cite this article asCite this article as: Berríos et al.: Robertsonian chromosomes and the nuclear architecture of mouse meiotic prophase spermatocytes. Biological Research 2014 47:16.