Functional Architecture of the Meiotic Cell Nucleus

Project leader: Ricardo Benavente

Staff: Manfred Alsheimer, Wolfgang Schütz (since 04/01), Rupert Öllinger (since 03/02), Andrea Baier (since 02/03), Elisabeth von Glasenapp (until 07/00)

Objectives: To understand the mechanisms of chromosome pairing during mammalian meiosis.

Approach: Meiosis is essential for sexual reproduction in that it enables the generation of haploid cells. It consists of two successive cellular divisions that take place without intervening DNA replication. During the extended first meiotic prophase homologous chromosomes pair, synapse and recombine. These processes are essential to ensure correct chromosome segregation during the reductional first meiotic division. During the subsequent second division, sister chromatids are separated leading to the formation of haploid gametes. As a result of the recombination process between the homologous chromosomes and their independent assortment during meiosis I, germ cells are genetically different from the original mother cell (spermatogonia or oogonia). The high biological relevance of meiosis is beyond question: it provides the basis for sexual reproduction and represents the largest source of genetic variability. Synaptonemal complexes (SCs) are meiosis-specific karyoskeletal structures, which play a key role during synaptic pairing of homologous chromosomes, chiasmata formation and chromosome segregation. The inability to assemble SCs has dramatic consequences for mammalian gametogenesis. It causes infertility in the male and promotes aneuploidy in the female followed by embryo death, as recently shown in knockout mice lacking SCP3 (a major structural protein of SCs). It is still unclear as to how SCs assemble and how they become attached to meiotic chromosomes. Furthermore, the molecular mechanisms that enable homologous chromosomes to find each other during meiosis are not known. Circumstantial evidence indicates that the nuclear envelope (NE) plays an essential role during this process since the chromosomes interact via their telomeres dynamically with the NE. Therefore, we are investigating the properties and interactions of structural proteins that are involved in the assembly of SCs and NE of mammalian meiotic cells.

Progress: We have characterized a meiosis-specific structural protein of the mammalian NE (the A-type lamin C2) as the first protein, which is enriched at the attachment sites of meiotic chromosomes. Recently, we were able to show that in Lmna^-/- mice lacking A-type lamins, spermatogenesis is disrupted and that spermatocytes show severe defects in synaptic pairing of chromosomes. These results provided first evidence that A-type lamins are important determinants of male fertility.

The attachment of meiotic chromosomes at the nuclear envelope involve terminal morphological specializations of the SC called attachment plates (Fig. 1). The lateral elements (LEs) of SCs are characteristically thicker at the level of the attachment plates. We found that morphogenesis of chromosome ends is altered in mice lacking SCP3, i.e. the major protein component of LEs. The electron-dense end of the LE with its prominent conical thickening was missing in the knockout mice. Nevertheless, chromosomes still attach their telomeres to the inner nuclear membrane via a disc-shaped electron-dense plate that contains the telomere repeats. These results provide strong evidence that LEs are dispensable for telomere attachment to the NE and align with previous evidence, especially in yeast, that telomere repeats are imperative for linking meiotic chromosome ends to the nuclear envelope. Another interesting observation was that SCP3^-/- spermatocytes lack LEs, demonstrating that SCP3 is a major determinant of LE assembly.

Significance: Meiotic pairing of homologus chromosomes via SCs is essential for the correct segregation. As shown in the mouse, the assembly of Les, as well as the interaction of chromosomes with the NE, are critical steps.

Future projects: A better understanding of SC assembly and dynamics will provide a significant progress in our knowledge on the mechanisms of meiosis. To this end, we propose: 1) to investigate the in vivo and in vitro polymerization properties of the structural proteins of SCs and NE of mammalian meiotic cells; 2) to identify proteins interacting with them and to characterize the protein domains involved in these interactions; 3) to investigate by immunocytochemistry and electron microscopy knockout mice showing defective assembly of SCs; and finally 4) to explore the possibility to investigate SC assembly under culture conditions by using a recently developed mammalian spermatogonial cell line.

Collaborations: G. Krohne, Biocenter, Uni Würzburg; M. Schmid, Biocenter, Uni Würzburg; H. Scherthan, MPI für Molekulare Genetik, Berlin; C. Stewart, National Cancer Institute, Frederick; C. Höög, Karolinska Institute, Stockholm; J. Rufas, Autonomous University of Madrid; S. Ortega/M. Blasco, National Cancer Research Center, Madrid; I. Prieto/J.L. Barbero, National Center of Biotechnology, Madrid; G. Vázquez-Nin, National University of Mexico, Mexico City.

Publications:

• Alsheimer, M., E. von Glasenapp, R. Hock & R. Benavente: Architecture of the nuclear periphery of rat pachytene spermatocytes: Distribution of nuclear envelope proteins in relation to synaptonemal complex attachment sites. Mol. Biol. Cell 10:1235-1245(1999). 

• Alsheimer, M., E. von Glasenapp, M. Schnölzer, H. Heid & R. Benavente: Meiotic lamin C2: The unique amino-terminal hexapeptide GNAEGR is essential for nuclear envelope association. Proc. Natl. Acad. Sci. USA 97:13120-13125(2000).

• Alsheimer, M., B. Liebe, L. Sewell, C.L. Stewart, H. Scherthan & R. Benavente: Disruption of spermatogenesis in mice lacking A-type lamins. J. Cell Sci. 117:1173-1178(2004).

• Benavente, R. (editor): Molecular Aspects of Mouse Spermatogenesis. Karger Publishers, ISBN 3-8055-7746-X, Basel (2003).

• Benavente, R., M. Alsheimer & E. von Glasenapp: The nuclear envelope at the attachment sites of mammalian meiotic chromosomes. Chromosomes Today 14:119-126(2004).

• Drabent, B., R. Benavente & S. Hoyer-Fender: Histone H1t is not replaced by H1.1 or H1.2 in pachytene spermatocytes or spermatids of H1t-deficient mice. Cytogenet. Genome Res. 103:307-313(2003).

• Echeverría, O.M., R. Benavente, R. Ortíz & G.H. Vázquez-Nin: Ultrastructural and immunocytochemical analysis of the XY body in rat and guinea pig. Eur. J. Histochem. 47:45-54(2003).

• Franco, S., M. Alsheimer, E. Herrera, R. Benavente & M.A. Blasco: Mammalian meiotic telomeres: Composition and ultrastructure in telomerase-deficient mice. Eur. J. Cell Biol. 81:335-340(2002).

• Geisinger, A., A. Dos Santos, R. Benavente & R. Wettstein: Identification and characterization of Srsp1, a rat gene differentially expressed during spermatogenesis and coding for a serine stretch-containing protein. Cytogenet. Genome Res. 98:249-254(2002).

• Lang, C., M. Paulin-Levasseur, A. Gajewski, M. Alsheimer, R. Benavente & G. Krohne: Molecular characterization and developmentally regulated expression of Xenopus lamina-associated polypeptide 2 (XLAP2). J. Cell Sci. 112:749-759(1999).

• Liebe, B., M. Alsheimer, C. Höög, R. Benavente & H. Scherthan: Telomere attachment, meiotic chromosome condensation, pairing and bouquet stage duration are modified in spermatocytes lacking axial elements. Mol. Biol. Cell 15:827-837(2004).

• Öllinger, R., M. Alsheimer & R. Benavente: SCP1 is a major determinant of mammalian synaptonemal complex assembly. (2004, submitted).

• Parra, M.T., A. Viera, R. Gómez, J. Page, R. Benavente, J.L. Santos, J.S. Rufas & J.A. Suja: Involvement of the cohesin Rad21 and SCP3 in monopolar attachment of sister kinetochores during mouse meiosis I. J. Cell Sci. 117:1121-1234(2004).

• Schmid, M., R. Benavente, M. Guttenbach & C. Steinlein (editors): Abstracts - 14th International Chromosome Conference. Chromosome Res. 9 (suppl. 1):1-160(2001).

• Turner, J.M.A., S.K. Mahadevaiahl, R. Benavente, H.H. Offenberg, C. Heyting & P.S. Burgoyne: Analysis of male meiotic 'sex body' proteins during XY female meiosis provides new insights into their functions. Chromosoma 109:426-432(2000).

• von Glasenapp, E. & R. Benavente: Fate of meiotic lamin C2 in rat spermatocytes cultured in the presence of okadaic acid. Chromosoma 109:117-122(2000).

• Wilson K.L., R. Benavente, B. Burke, R. Craigie, R. Foisner, K. Furukawa, L. Gerace, R.D. Goldman, Y. Gruenbaum, C. Harris, C.J. Hutchison, G. Krohne, G.E. Morris, H. Otto, A.J. Simon & H.J. Worman: Problems with LAP nomenclature. Nature Cell Biol. 3:E90(2001).

Current external funding: DFG-SPP 1050 “Kernarchitektur”, Graduiertenkolleg 639 “Tumorinstabilität”, Graduiertenkolleg 1048 “Organogenesis”, Unibund (until 03/03), VW-Stiftung (until 11/01)