Sexual dimorphism in prophase I of meiosis in the Northern mole vole ( Ellobius talpinus Pallas , 1770 ) with isomorphic ( XX ) chromosomes in males and females

The synaptonemal complex (SC) surface-spreading technique was used to visualize the process of chromosome synapsis in spermatocytes and oocytes of E. talpinus Pallas, 1770, a species with the XX sex chromosome system in both males and females. We used electron microscopy and immunofl uorescent localization of synaptonemal complex protein (SCP3) and centromeric proteins to analyze the structure and behaviour of synaptonemal complexes in prophase I of meiosis, aiming to reveal signs of meiotic sexual dimorphism in this species. We present evidence of considerable differences in the structure and behaviour of the axial structures of sex bivalents in male and female meiosis, despite the isomorphic Gand C-banding patterns of mitotic sex chromosomes. During meiotic prophase I, the sex bivalent in females behaved as autosomal bivalents, but it was not involved in the formation of the bouquet confi guration or it was the fi rst to leave it. The XX chromosomes of males formed closed sex bivalents. Only short tracts of SC were formed at both ends of the sex bivalent, while large middle segments of the lateral elements remained unpaired. The male sex chromosomes also formed characteristic “sex bodies”. In fact, electron microscopy revealed dense nucleolus-like bodies associated with unpaired parts of the axial elements. These regions of the sex chromosomes were poorly immunostained, because the distribution of SCP3 had a peculiar powder-like pattern, but SCP3 was not associated with the nucleolus-like bodies. We also revealed signs of sexual dimorphism in the dynamics of formation and destruction of autosomal SCs. In males, the total SC length was shorter than in females. The chromosome bouquet confi guration was preserved up to the stage of early pachytene in females. The bouquet confi guration in males was not expressed. At late pachytene, gaps were revealed in the structure of autosomal SCs in spermatocytes immunostained with antibodies to SCP3. The pattern of distribution of these gaps was comparable with the G-banding patterns of mitotic chromosomes.


INTRODUCTION
The key point for evolution and development of sexually dimorphic organisms is the production of genetically dissimilar gametes by meiotic division.The knowledge of sexual dimorphism in meiosis has been Comp. Cytogenet., 2010 4(1) Comparative Cytogenetics considerably extended in recent years due to active interest in the problems of regulation in meiosis and the mechanisms of selection of meiotic cells, which are different in females and males (Forejt, 1984;Tease, Hultén, 2004;Morelli, Cohen, 2005).
Meiosis in males is an entirely postnatal event.Germ cells enter meiosis prior to puberty, and gametogenesis continues in waves throughout adult life recruiting a new population of germ cells to enter meiosis during each wave of spermatogenesis (Handel, Eppig, 1998).
Unlike males, female meiosis starts in the ovary in the foetal period.Prophase I is mostly completed during foetal development, and the oocytes then enter the dictyotene arrest.Later, the oocytes remain quiescent until female sexual maturation, when one or more oocytes are recruited in each oestrous cycle to resume meiosis.Therefore, the meiotic cell cycle in mammals is a process that includes several check points (Dyban, Baranov, 1977;Kurilo, Zelenin, 1985;Morelli, Cohen, 2005).Moreover, sexual dimorphism implies that the processes of recombination, chromosome pairing, synapsis, and desynapsis of homologous chromosomes are subjected to different levels of checkpoint control in males and females (Morelli, Cohen, 2005).
The behaviour of the sex chromosomes during prophase I is evidently different in mammalian males and females with heteromorphic sex chromosomes, even if some peculiar species-specifi c features are acknowledged, whereas the systems with XO and XX sex chromosomes, identical in females and males, are extremely interesting for understanding underlying events in the determination of sexual dimorphism in meiosis.
The genus Ellobius Fischer, 1814 represents a group of rodent species with a remarkable system of sex determination.Indeed, it shows three types of sex chromosomes: the common XX/XY in E. fuscocapillus Blyth, 1843, the XO/ XO in E. lutescens, and the indistinguishable XX/XX in the group of sibling species, E. talpinus, E. tancrei, and E. alaicus.This latter system is unique among mammals, which gives a rare opportunity to investigate sex determination and necessity of X chromosome inactivation in males.

Comp. Cytogenet., 2010 4(1)
Comparative Cytogenetics So far, we have studied male meiosis in three Ellobius species and revealed some species-specifi c traits of it (Kolomiets et al., 1991).As yet no comparative study of male and female meioses has been performed for Ellobius.Furthermore, because of the uncertain systematic status of two sibling species E. talpinus and E. tancrei, previously, we used the species name "talpinus" for E. talpinus sensu stricto and for different chromosomal forms of E. tancrei (Bogdanov et al., 1986).It is worth mentioning that the complicated situation in the taxonomy of Ellobius led to confused species descriptions in various reports including the last edition of the "Mammal species of the world" (Musser, Carleton, 2005).In spite of Topachevsky's opinion (1965), the two species were not divided by taxonomists until additional morphological and chromosomal studies were carried out (Vorontsov, Yakimenko, 1984).
To investigate female meiosis, we have started the study with a chromosomally stable species, the Northern mole vole -E.talpinus.The karyotype of this species was described by Ivanov (1967) as 2n =54, NF=54.The author failed to identify sex chromosomes, but hypothesized the typical, XX/XY, sex chromosome system.In E. talpinus, differences in the male and female chromosomal sets and, especially, in chromosomes defi ned as XX did not emerge after either G-banding or Zoo-FISH with X-chromosome probes of Microtus agrestis Linnaeus, 1761, Mus musculus Linnaeus, 1758, Mesocricetus auratus Waterhouse, 1839, human, and rat (Romanenko et al., 2007).
The study of meiotic features in E. talpinus, as a species with isomorphic XX chromosomes in both sexes, may further contribute to general understanding of the role and signifi cance of sex dimorphism.The main goal of our study is to investigate meiotic behaviour of indistinguishable male and female sex bivalents during prophase I.

MATERIAL AND METHODS
Animals.All E. talpinus specimens (three females and three males) were obtained from a laboratory collection of N. K. Koltzov Institute of Developmental Biology, Russian Academy of Sciences.Testes were obtained from mature (not younger than 1 year) males and ovaries from one-day-old females.
Coating of slides.Slides for a subsequent electron-microscopic study were covered with Falcon fi lm.Slides coated with poly-L-lysine were used for immunostaining.
Preparation of cell suspension.The suspension of oocytes was prepared by our own method.Ovaries were removed and transferred onto a drop of Eagle's medium (without glutamine) on the surface of a slide.The ovaries were overlaid with a cover slip.The cover slip was carefully pressed, under visual control, with a dissecting needle to separate the ovarian cells.The cover slip was lifted at one side, and the suspension of cells was collected and put into a centrifuge tube and then homogenized with an automatic pipette and placed on ice.The suspension of spermatocytes was left in a centrifuge tube and washed by centrifugation with 10 ml of Eagle's medium, 2-3 times for 10 minutes at 1500 rpm.After that the tube with cells was placed on ice.
Cell spreading and fi xation.Spreads were prepared and fi xed using the technique of Navarro et al. (1981) with some modifi cations.
Electron microscopy.The slides were stained with 50% AgNO 3 solution in a humid chamber at 56°C for 3 hours.The slides were washed in four changes of distilled water and air-dried.The stained slides were observed in a light microscope, suitably spread cells were selected, and plastic (Falcon fi lm) circles were Comp. Cytogenet., 2010 4(1) Comparative Cytogenetics preleptotene to diplotene.At preleptotene, when the formation of axial elements just begins, and homologous chromosomes are not coupled yet, foci immunostained with ACA are scattered over the whole nucleus, and only single centromeres are joined in pairs or pulled together (Fig. 1, a).At early zygotene, the telomeres are grouped close to one pole of the nucleus.As E. talpinus has only acrocentric chromosomes, the immunolabeled centromere-telomere chromosome ends are visualized most clearly (Fig. 1, b).The chromosome bouquet confi guration is retained in oocytes at the stage of early pachytene (Fig. 1, с).In the immunocytochemical analysis, oocytes with completely formed SCs were detected at the stage of middle pachytene (Fig. 1, d).
As a rule, one large ACA signal was observed on one SC, but two signals were revealed in single SCs, since the positions of the centromeres on homologous chromosomes did not coincide (Fig 1, c-e).Different lengths of the short arms of acrocentric chromosomes of different SCs are worth noting.
At diplotene, desynapsis of homologous chromosomes begins at the telomeric ends in some SCs and, in other SCs, in the interstitial regions of bivalents (Fig. 1 f).
In electron microscopic analysis, no oocytes had completely formed SCs in all chromosomes in concert.At the pachytene, several chromosomes, in which interstitial regions of homologous chromosomes remained asynaptic, were constantly detected.Nonhomologous synapsis of chromosomes was not observed at all (Fig. 2) in either stage of prophase I of meiosis.
Both immunostaining and electronmicroscopic methods did not allow discrimination between the sex bivalent and autosomes.To identify the XX bivalent in oocytes, we compared the relative lengths of cut out with a diamond tap and transferred onto grids.The slides were examined under JEM 100B electron microscope.
Immunostaining.The slides were washed in PBS.Whole mount SCs were blocked with HB (holding buffer: PBS, 0.3% BSA, 0.005% Triton X-100).The slides were incubated overnight at 4°C with rabbit polyclonal antibodies against the human lateral element protein SCP3 (Abcam, 15093Ab, UK Cambridge, UK) diluted to a concentration of 1:200 in ADB (Antibody Dilution Buffer: PBS, 3% BSA, 0.05% Triton X-100) and with human anti-centromere antibodies, ACA, (Antibody Incorporated, California, USA) diluted to a concentration of 1:200 in ADB.The slides were washed in PBS and incubated with goat anti-rabbit Alexa Fluore 488 conjugated antibodies (1:800, Abcam, Cambridge, UK) and goat anti-human Alexa Fluore 546 conjugated antibodies (1:800) at 37°C for 60 min.The slides were washed with PBS, rinsed briefl y with distilled water, dried and mounted in Vectashield with DAPI (Vector Laboratories).
Measurements of autosomal bivalents, in order to determine their relative lengths and their ranking in each cell, were made with MicroMeasure 3.3 (Colorado, USA) using the STATISTICA 8.0 (StatSoft, Inc., 2008).

RESULTS
Female SCs.On the fi rst day after birth, the females of E. talpinus present oocytes at different stages of prophase I, from
We noticed that at the stage of pachytene the sex bivalent is one of the fi rst to become straight and leave the bouquet confi guration (Fig. 1, с).The total synaptonemal complex complement length at pachytene in oocytes is 155±7 μm.Male SCs.In spermatocytes, the total SC length at pachytene is 119±11 μm.The bouquet confi guration of chromosomes in spermatocytes is not expressed.Beginning from zygotene, the sex bivalent is located in the nucleus periphery; the axial elements are surrounded by a cloud of electron-dense material.The XX chromosomes form a closed bivalent with two short paratelomeric SC fragments.In the central part of the sex bivalent, the thin axial elements of XX chromosomes do not approach each other, but are repulsed from one another.An electrondense nucleolus-like body is formed on one of the axial elements (Fig. 4, a).During the progression of prophase I of meiosis, twisting of the sex chromosome axes occurs.The axial asynaptic regions intersect, short thorns are gradually formed on the axial elements, and a nucleolus-like body is also formed on the axial element of the second X chromosome

DISCUSSION
Autosomes.The present study revealed a number of differences in the structure and behaviour of autosomes in oocytes and spermatocytes of E. talpinus.First, the difference in the average total length of autosomes in females and males is conspicuous.Based on the electron microscopy data, the total SC length at the stage of pachytene is greater in females (155 ± 7 μm) than in males (119 ± 11 μm).These data agree with the data of other authors.For example, the total length of the SC complement at pachytene in human female (519 μm) is about twice the respective length in male (Wallace, Hulten, 1985).Accordingly, approximately 50 crossovers occur in one human spermatocyte, while there are 70 crossovers in one oocyte.In addition, the loop size is shorter in oocytes than in spermatocytes (Tease, Hulten, 2004).For that reason, meiotic chromosomes at the pachytene stage of meiosis I, when crossing-over takes place, should be packed in different ways in human Fig. 2. Spread from an E. talpinus oocyte at early diplotene examined with transmission electron microscopy.Numbers of SCs correspond to numbers of metaphase chromosomes of E. talpinus (Romanenko et. al., 2007).Asynaptic zones of SCs are visible (snowfl ake).Bar = 3 μm.

Comp. Cytogenet., 2010 4(1)
Comparative Cytogenetics oocytes and spermatocytes (Wallace, Hulten, 1985;Tease, Hulten, 2004).This is thought to be determined by a special role of female sex cells, which implies not only transmission of genetic information to the next generation, but also provides a the trophic function of a zygote after fertilization.This function is manifested in a high transcriptional activity of DNA of  meiotic chromosomes, which is associated with their lower level of condensation (Tease et al., 2002(Tease et al., , 2006)).Second, our electron microscopic analysis in oocytes of E. talpinus failed to reveal SCs with completely paired lateral elements, although such oocytes were detected immunocytochemically. On the contrary, full synapsis of autosomes at the stage of pachytene was always observed in spermatocytes.Accordingly, abnormal chromosome synapsis is observed in 30% of oocytes obtained from normal human foetuses.Univalents are formed in 15% of cells, and in 16% of cells chromosomes enter nonhomologous synapsis (Speed, 1986;Speed, Chandley, 1990).Actually, asynapsis and nonhomologous synapsis at the stage of pachytene in female meiosis occur much more frequently than in male meiosis (Ohno et al., 1963).Ohno et al. (1963) advanced a hypothesis, according to which the associations of nonhomologous chromosomes in prophase I of meiosis is the reason why oocytes remain blocked for a long time and thus do not enter metaphase I.It should be emphasized that we used females obtained on the fi rst day after birth, while other authors, as a rule, examined foetal oocytes.We managed to obtain oocytes from the preleptotene stage to the stage of diplotene-dictyotene, including

Comp. Cytogenet., 2010 4(1)
Comparative Cytogenetics Kolomiets, 2007).According to literature data, the formation of a sex body, which gradually moves into the nucleus periphery in prophase I of meiosis, is considered to be characteristic of mammalian males with heteromorphic XY sex chromosomes.In E. tancrei as in E. talpinus, XX chromosomes are isomorphic in the pattern of G-banding in females and males, and a sex body is also formed in representatives of these species (Bogdanov et al., 1986) Moreover, the sex bivalent of E. talpinus is surrounded with an electron-dense cloud, which is a morphological feature of silencing of sex chromosome genes of males (Homolka et al., 2007).In contrast to males, the behaviour and SC structure of the sex bivalent SC in females are identical to those of autosomes.The distribution of SCP3, a major protein of the axial elements of chromosomes, in the asynaptic regions of XX chromosomes is powder-like, whereas in mice SCP3 not only contributes to thickening of the axial elements, but it is also a component of the protein coverlet involved in the process of inactivation of sex chromosomes and their isolation from autosomes (Turner et al., 2000).Condensation of X and Y chromosomes during formation of sex (XY) bodies is one of the cardinal characteristics distinguishing male from female meiosis in mammals.It is considered to be a morphological manifestation of inactivation of meiotic sex chromosomes, or MSCI (Forejt, 1984;Turner et al., 2000;Homolka et al., 2007).
During meiotic prophase I the structure and behaviour of the axial structures of the sex bivalent in oocytes were generally similar to those of autosomal bivalents.During prophase I of the male meiosis, XX chromosomes formed a closed sex bivalent and formed a typical sexual body.Thus, despite the isomorphism showed by G-banding of the XX sex chromosomes in males and females of E. talpinus, we present evidence of a considerable dissimilarity in structure and behaviour of male and female sex and autosomal bivalents at prophase I of meiosis.

Fig. 1
Fig. 1, a-f.Spreads from E. talpinus oocytes immunolabeled with antibodies to the centromere (red) and SCP3 (green).a -preleptotene.Centromeres (red) are scattered over the nucleus.Only short fragments of the axial elements (green) have been formed.b -early zygotene.The formation of the axial elements and bouquet confi guration is visible.c -Early pachytene.All 27 SCs have already been formed.Most SCs are in the bouquet confi guration, except sex bivalent (XX).d -middle pachytene.Two ACA signals lie on distance from each other on four SCs (arrows).Short arms of some chromosomes are not visible (arrow heads).e -early diplotene.Two ACA signals lie on distance from each other on three SCs (arrows).Asynaptic zone of two SCs are visible (snowfl ake).e' -two signals of the ACA on SC lie on distance from each other.Bar=1 μm.f -middle diplotene.Desynapsis of chromosomes begins in the telomeric ends and in the interstitial regions of bivalents.Immunolabeling of SCs with antibodies to SCP3 has the banding pattern.Bar = 3 μm.

Fig. 3 .
Fig. 3.The average relative length of SCs of oocytes (columns without fi ll) and spermatocytes (grey columns) of E. talpinus at pachytene.Numbers on the abscissa correspond to the length of autosomes; the 27 pair is the sex chromosomes (XX).

Fig. 4
Fig. 4, a-c.Dynamics of the sex bivalent structure in E. talpinus spermatocytes in prophase I of meiosis.SCs spread from E. talpinus spermatocytes examined by transmission electron microscopy.The arrowhead indicates thorns on axes.The short arrow indicates the electron-dense nucleolus-like body; the long arrows indicate short paratelomeric SC fragments.Bar = 1 μm.a -late zygotene.A closed sex bivalent with short paratelomeric SC fragments and extensive zone of asynapsis between the axial elements of XX chromosomes.b -pachytene.Two electron-dense nucleolus-like bodies at each of XX chromosomes axes.c -diplotene.The compact sex bivalent has a complicated confi guration and is surrounded by a "cloud" of electron-dense material.

Fig. 5
Fig. 5, a-c.Spreads from E. talpinus spermatocyte immunolabeled with antibodies to the SCP3 (green) and centromere (red; ACA) proteins at different stages of prophase I of meiosis.a -early zygotene.The formation of the axial elements without bouquet confi guration is visible.b -middle pachytene.Autosomal SCs are uniformly stained; and the SCP3 distribution in the asynaptic regions of the sex bivalent (XX) axes has the powder-like pattern.Short arms of some chromosomes are visible (arrow heads).Signals of the ACA on one SC lie on distance from each other (arrow).b' -Short acrocentric SC with two ACA signals, which lie on distance from each other.Bar = 0.5 μm.c -late pachytene.Banding pattern of the SCP3 protein distribution over autosomal SCs and a very weak SCP3 fl uorescence signal on the sex bivalent axial elements.Numbers of SCs correspond to numbers of metaphase chromosomes E. talpinus according to Romanenko et al. (2007).Bar = 3 μm.

Fig. 6 .
Fig. 6.Late pachytene.Comparison of the distributions of gaps in the SC for the second biggest bivalent and G-band for the second biggest mitotic chromosomes(Romanenko et al., 2007).