Here we focus on the XY₁Y₂ condition in male common shrew Sorex araneus Linnaeus, 1758, applying electron microscopy and immunocytochemistry for a comprehensive analysis of structure, synapsis and behaviour of the sex trivalent in pachytene spermatocytes. The pachytene sex trivalent consists of three distinct parts: short and long synaptic SC fragments (between the X and Y₁ and between the X and Y₂, respectively) and a long asynaptic region of the X in-between. Chromatin inactivation was revealed in the XY₁ synaptic region, the asynaptic region of the X and a very small asynaptic part of the Y₂. This inactive part of the sex trivalent, that we named the ‘head’, forms a typical sex body and is located at the periphery of the meiotic nucleus at mid pachytene. The second part or ‘tail’, a long region of synapsis between the X and Y₂ chromosomes, is directed from the periphery into the nucleus. Based on the distribution patterns of four proteins involved in chromatin inactivation, we propose a model of meiotic silencing in shrew sex chromosomes. Thus, we conclude that pachytene sex chromosomes are structurally and functionally two different chromatin domains with specific nuclear topology: the peripheral inactivated ‘true’ sex chromosome regions (part of the X and the Y₁) and more centrally located transcriptionally active autosomal segments (part of the X and the Y₂).

Sex body, MSCI, synaptonemal complex, γH2AFX, ATR, SUMO-1, ubiH2A, Sorex araneus

At first meiotic prophase, the male sex chromosomes in mammals form a specific heterochromatic nuclear domain (Solari 1974; Handel 2004). The structure and behaviour of the sex bivalent changes from zygotene to late diplotene. In the majority of mammal species the processes of pairing and synapsis of the X and Y chromosomes at zygotene occurs later than the same processes in autosomes. At early and mid pachytene the sex bivalent is usually located in the centre of the meiotic nucleus. At mid pachytene the sex chromosomes become shorter due to condensation and homologous regions of the X and Y are completely paired (Burgoyne 1982). Recombination nodules appear only in the short pseudoautosomal region (PAR) of the sex bivalent. In many mammals irregular thickenings may occur at asynaptic sites of axial elements of the sex bivalent. After that the sex bivalent gradually moves from the centre of the nucleus to its periphery and forms a so-called XY or sex body (Solari 1974).

The chromatin of the sex chromosomes transforms into an inactive condition and this chromatin remodelling process is known as meiotic sex chromosome inactivation (MSCI) (McKee and Handel 1993; Turner et al. 2000). MSCI is the process whereby unsynapsed regions of the sex chromosomes undergo transcriptional silencing (Lifschytz and Lindsley 1972; Handel and Hunt 1992; Turner et al. 2002, 2007); this is a case of MSUC (meiotic silencing of unsynapsed chromatin) (Schimenti 2005). The asynaptic chromatin undergoes inactivation by incorporation and modification of specific proteins (Burgoyne et al. 2009). First, BRCA1 (breast cancer 1) accumulates in non-synaptic areas of the sex chromosomes, which starts the process of phosphokinase ATR (ataxia telangiectasia- and RAD3-related) recruitment and then there is ATR-dependent phosphorylation of the γH2AFX (phosphorylated (Ser139) histone 2 A.X) histone (Turner et al. 2004). At early pachytene, ubiH2A (ubiquitinated histone H2A), SUMO-1 (small ubiquitin-related modifier-1) and other proteins are incorporated into the asynaptic chromatin of the sex chromosomes (Baarends et al. 2005). Such modification of chromatin decreases its transcriptional activity as confirmed using Cot-1 RNA FISH and RNA polymerase type II immunolocalisation (Turner et al. 2005; Baarends et al. 2005). Thus, the chromatin of the sex body is inactive.

MSCI has been well studied for the normal male sex chromosome system in mammals (XY), but there are few data on this process for multiple sex chromosome systems.

Translocation between the X and an autosome results in the formation of multiple sex chromosomes (XY₁Y₂; where the X is a product of a translocation between the ‘true’ X and an autosome, Y₁ is the ‘true’ Y and Y₂ is the autosome). The XY₁Y₂ condition has been demonstrated in insects (Jacobs 2003), fish (Centofante et al. 2006; de Oliveira et al. 2008) and, in particular, among mammals – including marsupials: greater bilby Macrotis lagotis (Sharp 1982), and placentals: Indian muntjac Muntiacus muntjak (Artiodactyla, Fronicke and Schertan 1997), red brocket deer Mazama americana (Artiodactyla, Aquino et al. 2013), big fruit-eating bat Artibeus lituratus (Chiroptera, Solari and Pigozzi 1994), short-tailed fruit bat Carollia perspicillata (Chiroptera, Noronha et al. 2009), delicate mouse Salinomys delicates (Rodentia, Lanzone et al. 2011), Sahel gerbil Taterillus arenarius and Senegal gerbil Taterillus pygargus (Rodentia, Ratomponirina et al. 1986; Volobouev and Granjon 1996) and others (see reviews by Fredga 1970; Sharman 1991; and Yoshida and Kitano 2012). An XY₁Y₂ sex chromosome system also characterises species of shrews (small insectivores) belonging to the Sorex araneus group (Eulipotyphla; Hausser et al. 1985), including the Eurasian common shrew Sorex araneus Linnaeus, 1758 which is a model system for evolutionary cytogenetics with numerous Robertsonian autosomal variants as well as the XY₁Y₂ condition (Searle and Wójcik 1998).

The XY₁Y₂ condition in the common shrew arises from a tandem fusion between an autosome and the true X chromosome (Sharman 1956, 1991; Fredga 1970; Searle et al. 1991) (Fig. 1a). Although the observation of a meiotic sex trivalent was part of the discovery of the XY₁Y₂ condition in the common shrew it was not until the work of Pack et al. (1993) that chromosome pairing in the XY₁Y₂ at meiotic prophase I was first examined. We supplemented those early observations with the discovery that the γH2AFX histone is associated with the true sex chromosome regions of the pachytene sex trivalent (Matveevsky et al. 2012).

Figure 1.

a G-banded sex chromosomes in the male common shrew (left) and ideogram with chromosome arms labelled according to the alphabetic nomenclature of Searle et al. (1991)b Schematic diagram of the shrew pachytene sex trivalent, based on Pack et al. (1993) and our data c Electron micrograph of a shrew sex trivalent, XY₁Y₂ at late pachytene. The true X region and the Y₁ are surrounded by electron-dense material. Scale bar: 5µm. d Diagram of the XY₁Y₂ configuration as represented in Fig. 1c.

In this paper we analyse the distribution of four transcription silencing proteins (ATR, γH2AFX, SUMO-1, ubiH2A) on the sex trivalent XY₁Y₂ at prophase I in common shrew spermatocytes and assess how these participate in MSCI.

Shrews. A total of five adult males of the common shrew were collected from a locality in the vicinity of the Moscow-Neroosa chromosomal hybrid zone (near Ozyory town, Moscow Region) in April 2014, at the beginning of the breeding season. All animals were karyotyped using the method of Pavlova et al. (2008), with modifications. The trypsin-Giemsa staining technique of Král and Radjabli (1974) was used for identification of chromosome arms by G-bands, following the standard nomenclature for the S. araneus karyotype, which uses letters of the alphabet for chromosome arms (Searle et al. 1991).

All karyotypes were characterised by the set of invariant autosomal metacentrics af, bc, jl and tu as well as the XY₁Y₂ sex chromosomes system. Race-specific autosomes differed between individuals, two males had gm, hi, kr, no and pq metacentrics which mark the karyotype of the Moscow race. Other males had go, hi, kr, mn and pq metacentrics which characterise the Neroosa race. All shrews had the same diploid number of chromosomes (2n=21). Spermatocyte spreads were obtained from all males. All necessary national and institutional guidelines for the care and use of animals were followed.

A total of 331 cells were analysed of which 14 were prepared for electron microscopy and 317 for fluorescence microscopy. All the latter were labelled with SYCP3 (synaptonemal complex protein 3) and CREST and a proportion of cells were labelled with other antibodies (γH2AFX: 90; SUMO-1: 59; ubiH2A: 52; ATR: 32; MLH1: 74; SYCP1: 28; RNA Pol II: 10).

Meiotic spread preparations. Synaptonemal complex (SC) preparations were made and fixed using a previously described technique (Kolomiets et al. 2010). AgNO₃-stained slides were screened under a light microscope to select suitably spread cells. Once selected, plastic (Falcon film) circles were cut out with a diamond tip and transferred onto grids and examined in a JEM 100B electron microscope.

Antibodies, immuncytochemistry and multistep immunostaining procedure. Poly-L-lysine-coated slides were used for immunostaining. The slides were placed in phosphate buffer saline (PBS) and incubated overnight at 4°C with the primary antibodies diluted in antibody dilution buffer (3% bovine serum albumin - BSA, 0.05% Triton X-100 in PBS): mouse anti-MLH1 (1:50–1:100, Abcam, Cambridge, UK), rabbit polyclonal anti-SYCP1 (1:500, Abcam, Cambridge, UK), rabbit polyclonal anti-SYCP3 (1:500–1:1000, Abcam, Cambridge, UK), mouse monoclonal anti-ATR (1:200, Abcam, Cambridge, UK), human anticentromere antibody CREST (Calcinosis Raynaud’s phenomenon, Esophageal dysmotility, Sclerodactyly, and Telangiectasia) (1:500, Fitzgerald Industries International, Acton, MA, USA), mouse monoclonal anti-SUMO-1 (1:250, Zymed Laboratories, South San Francisco, CA, USA), mouse monoclonal anti-ubiquityl histone H2A (1:400, Millipore, Billerica, MA, USA), and mouse anti-phospho-histone H2AX (also known as γH2AFX) (1:1000, Abcam, Cambridge, UK).

After washing, we used the following corresponding secondary antibodies diluted in PBS: FITC-conjugated bovine anti-rabbit IgG (1:1000, Santa Cruz Biotechnology, Santa Cruz, CA, USA), goat anti-rabbit Alexa Fluor 488 (1:500, Invitrogen Corporation, Carlsbad, CA, USA), FITC-conjugated horse anti-mouse IgG (1:500, Vector Laboratories, Burlingame, CA, USA), Rodamin-conjugated chicken anti-rabbit IgG (1:400, Santa Cruz Biotechnology, Santa Cruz, CA, USA), goat anti-human Alexa Fluor 546 (1:500, Invitrogen Corporation, Carlsbad, CA, USA), goat anti-mouse Alexa Fluor 546 (1:200, 1:1000, Invitrogen Corporation, Carlsbad, CA, USA).

Immunostaining was carried out sequentially in 3 steps: 1. SYCP3/CREST (or SYCP1/MLH1); 2. ATR (or SUMO-1 or ubiH2A); 3. γH2AFX. After an each step slides were washed in PBS (6–7 times for 7–10 min) and mounted with Vectashield mounting medium containing 4,6-diamino-2-phenylIndol (DAPI) (Vector Laboratories, Burlingame, CA, USA). Slides were examined using an Axioimager D1 microscope (Carl Zeiss, Jena, Germany) equipped with an Axiocam HRm CCD camera. Images were processed using Adobe Photoshop CS3 Extended.

It should be noted that after photobleaching, bound antibodies of the first round still remain attached to the cellular structures. The more antibodies attached to the structures of interest the higher the probability that epitopes of further rounds of immunolocalisation become inaccessible. To ensure that these processes have not impacted our results, we performed control experiments for all antibodies.

Controls. We always conducted parallel control experiments on different slides when immunostaining was performed with a single antibody to a MSCI specific protein (double immunostaining). Our colleague Dr TM Grishaeva has conducted a bioinformatics analysis of the proteins studied. The pairwise sequence alignment of human and mouse proteins, which was performed by the COBALT program (NCBI), demonstrated high conservation of the H2AX, ubiH2A, SUMO-1, ATR and Polo II proteins. Comparison of the proteins did not reveal any problematic similarity between them. The pairwise sequence alignment of ATR and H2AX showed no amino acid sequence similarity. SUMO-1 and H2AX appeared to have 14 coincidences of amino acids, which should not affect the cross-reaction. ubiH2A and H2AX have a high level of similarity except a short sequence in the carboxyl terminus. Nevertheless, an analysis of the fluorescence intensity profile suggests a close, but not identical, picture of distribution for ubiH2A and H2AX (Matveevsky et al. 2016).

Image analysis. Intensity Correlation Analysis (ICA) was carried out according to Reitan et al. (2012). Scatter plots, Pearson’s coefficients (p_r) and overlap correlation coefficients (r) were obtained using a plug-in ICA (Li et al. 2004) of ImageJ 1.45 (Rasband 1997–2016). p_r helps to evaluate the degree of correlation between the different intensities and is ranked from -1 (negative correlation) to +1 (positive correlation) (French et al. 2008). In analysing scatter plots, overlaying green and red signal resulted in a yellow signal. The more yellow in the scatter plot, the higher the level of overlap. The width of the yellow signal distribution in scatter plots corresponded to the degree of co-localisation of the fluorescence signals being compared: the wider the distribution of the signal, the higher the level of overlap of the two channels.

To evaluate the degree of co-localisation of some proteins, we have developed Fluorescent-Intensity Profiles (FIPs) using the ImageJ plug-in RGB profiler (created by Christophe Laummonerie, Jerome Mutterer, Institute de Biologie Moleculaire des Plantes, Strasbourg, France) and following Barak et al. (2010) and Fargue et al. (2013).

Statistical analysis. All of the data are shown as the mean values ± SD. Student’s t-test was performed to determine significant differences in the data. All statistical analyses were conducted using GraphPad Prism Version 5.0 (GraphPad Software, CA, USA).

A pronounced difference in the structure, behaviour and MSCI of the two parts of the shrew sex trivalent has been revealed on the basis of detailed analysis of the organisation and behaviour of XY₁Y₂ at prophase I of meiosis. The ‘head’ part of the trivalent that moves to the periphery of the pachytene nuclei involves the true sex chromosome regions and includes synapsis between the X and Y₁ chromosomes. The ‘tail’ part involves the region of synapsis between the translocated X and Y₂ chromosomes. The structure and behaviour of the ‘head’ part (true X region and the Y₁) including specific MSCI shows patterns which are typical for a male sex bivalent of mammals. At the same time, the ‘tail’ part (the translocated region of the X and the Y₂) is located among other autosomes and does not differ from them morphologically excluding the fact that this part is attached to the ‘head’ part of the sex trivalent. These dual properties of the ‘head’ and ‘tail’ parts of the XY₁Y₂ trivalent in shrew spermatocytes are a notable feature of this system.

It is also noteworthy in this study that we have determined for the first time specific features of MSCI related to the discontinuous distribution of ATR along the SC at the site of synapsis between X and Y₁ and the distribution limits of SUMO-1 which occurs in the same part of the SC.

We are grateful to G.N. Davidovich and A.G. Bogdanov of the Electron Microscopy Laboratory of Biological Faculty of Moscow State University for the technical assistance and to the reviewers for their helpful comments. We thank the Common Use Center of the Vavilov Institute of General Genetics of the Russian Academy of Sciences for the possibility to use some microscopic equipment. This work was partially supported by research grants of the Russian Foundation for Basic Research № 15-29-02649 (to SM), 16-04-01447 (to OL), 15-04-04759 (to SP) and the President Grant for Russian Distinguished Young Scientists MK-4496.2015.4 (to SP).