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doi: 10.1242/10.1242/jcs.00111

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Meiotic sex chromosome inactivation in male mice with targeted disruptions of Xist

¹ Division of Developmental Genetics, National Institute for Medical Research, Mill Hill, London NW7 1AA, UK
² Institute of Human Genetics, University of Newcastle Upon Tyne, NE1 7RU, UK
³ INSERM U25, Hôpital Necker, 75743 Paris Cedex 15, France
⁴ Department of Animal Biology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA
⁵ Whitehead Institute for Biomedical Research and Massachusetts Institute of Technology, Cambridge, Massachusetts 02142, USA

View larger version (7K): [in a new window]   Fig. 1. Schematic of the Xist disruptions and primer pairs used in the present study. Closed arrowheads indicate the interval deleted in Xisttrun males and open arrowheads indicate that deleted in Xist1lox males. Asterisks denote primer positions (see Materials and Methods).

View larger version (41K): [in a new window]   Fig. 2. Xist and Tsix expression during spermatogenesis analysed by strand-specific RTPCR using primer pair JT4/MIX20 (product 230 bp). ES cells serve as Xist and Tsix positive controls, and testes from Xisttrun males serve as negative controls. Hprt serves as RTPCR control. (a) In the male mouse, Xist is expressed in the testis and at very low levels in the heart, while Tsix is detected only in the testis. (b) Expression of both Xist and Tsix is detectable from 11.5 dpc. (c) Germ cell-dependency of Xist and Tsix, with germ cells detected by Dazla RTPCR. Wild-type adult testis, 13.5 dpc wild type and We/+ heterozygous testis all express Xist and Tsix, while neither are detectable in adult XOSry and embryonic We/We testes that lack germ cells as evidenced by the absence of Dazla transcripts.

View larger version (119K): [in a new window]   Fig. 3. Analysis of sex body formation and disappearance in wild type (a-c), Xisttrun (a'-c') and Xist1lox (a''-c'') spermatocytes using the sex body marker -H2AX. (-H2AX, green; SYCP3, red; X chromosome, short arrow; Y chromosome, arrowhead). -H2AX labels the sex chromosomes prior to their incorporation into the sex body (a,a',a'') and then associates with the sex body throughout pachytene (b,b',b''). It finally disappears as sex bodies are disassembled, at metaphase I (c,c',c''). Bar, 10 µm.

View larger version (48K): [in a new window]   Fig. 4. Analysis of sex bodies in wild type (a-d), Xisttrun (a'-d') and Xist1lox (a''-d'') late pachytene and diplotene spermatocytes by DAPI staining and immunostaining for XY77 and M31 (DAPI, blue; SYCP3, red; green is XY77 in b,b',b'' and M31 in d,d',d''; sex body, arrowhead). In late pachytene wild type, Xisttrun and Xist1lox spermatocytes the sex body protrudes from the edge of the nucleus (a,a',a'') and stains positively for XY77 (b,b',b''). Later, during diplotene, in wild type, Xisttrun and Xist1lox spermatocytes the sex body becomes more densely staining for DAPI (c,c',c'') and in all three genotypes is M31-positive (d,d',d''). Bar, 5 µm.

View larger version (96K): [in a new window]   Fig. 5. Analysis of macroH2A1.2 localisation in wild type (a,b), Xisttrun (a',b') and Xist1lox (a'',b'') pachytene spermatocytes. (MacroH2A1.2, green; SYCP3, red; X chromosome, short arrow; Y chromosome, arrowhead). MacroH2A1.2 is enriched in the chromatin of the sex chromosomes during early pachytene in all three genotypes. Bar, 10 µm.

View larger version (143K): [in a new window]   Fig. 6. Analysis of MSCI by exclusion of RNA POLII from the sex body in wild type (a-c), Xisttrun (a'-c') and Xist1lox (a''-c'') late pachytene/early diplotene spermatocytes (RNA POLII, green; SYCP3, red; X chromosome, short arrow; Y chromosome, arrowhead). RNA POLII is excluded from the sex body in all three genotypes. Bar, 10 µm.

View larger version (58K): [in a new window]   Fig. 7. Analysis of MSCI by Rbmy-inactivation in wild type (a-f), Xisttrun (a'-f') and Xist1lox (a''-f'') spermatogenic cells (RBMY, red; XMR, green; forming sex body, black arrowhead, mature sex body, white arrowhead). Spermatogonia, identified by their lack of staining for XMR (a,a',a'') abundantly express RBMY (b,b',b''). Late zygotene spermatocytes, identified by their low level nuclear XMR staining with brighter superimposed sex chromosome XMR staining (c,c',c''), express RBMY at lower levels (d,d',d''). Pachytene spermatocytes, identified by their mature XMR-positive sex bodies and absent whole nuclear XMR staining (e,e',e'') contain undetectable levels of RBMY (f,f',f'') in all three genotypes. Bar, 5 µm.

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