4. DISCUSSION
4.1 Brief overview of results
The present work discusses the role of Lissencephaly-1 protein in male germ cell differentiation. The following results were obtained:
1 Characterisation of the gene trap line L39GT/GT:
● H&E staining of testis sections revealed an extensive germ cell degeneration in tubuli seminiferi of 46 d and 2 months old homozygous gene trap males with a markedly reduced number of late meiotic and postmeiotic germ cells. Heterozygous males exhibit full spermatogenesis.
● Expression analysis of Lis1 transcripts by qRT-PCR could show a significant reduction of Lis1 “Ex2a/3” transcript in homozygous gene trap mice of all analysed ages (from 10 d to 2 months). The Lis1 “Ex2/3” transcript was reduced in homozygous males from d 25 on, but the reduction was not as prominent as the reduction of the Lis1
“Ex2a/3” transcript.
● Northern blot analysis of spermatocyte- and spermatid-specific markers in mutant mice showed similar expression levels for all genotypes in 35 d and 60 d old mice.
● Western blot analysis of 4 months old mice revealed a reduction of LIS1 protein in testes of L39GT/GT males in comparison to heterozygous and wild type males, while the amount of LIS1 protein in brain did not differ. Immunohistochemical analysis revealed that the expression of LIS1 protein is restricted to the cytoplasm of all testicular cells, with an intense staining in meiotically dividing spermatocytes and elongating spermatids.
● Presence of a fusion transcript of Lis1 with the gene trap vector was confirmed.
Spermatogonial cells and some early primary spermatocytes were positive for LacZ staining.
● Caudae epididymes of L39GT/GT mice contained a low number of morphologically normal looking spermatozoa.
● Two integration sites of the gene trap vector in intron 2 of Lis1 gene could be shown by a “Genome Walk” approach. The existence of both integration sites in the ES cell clone “2A53” was confirmed.
● The gene trap mutation was analysed on different genetic backgrounds, namely CD-1, FVB, C57BL and 129/Sv. All genetic backgrounds exhibit the same phenotype as the original gene trap line L39/NMRI. All homozygous males were infertile, with caudae epidiymes containing a low number of morphologically normal looking spermatozoa.
H&E staining of testis sections showed an early onset of the degeneration of germ cells, with a defect of spermatogenesis visible in 25 d old L39/C57BLGT/GT mice.
2 Colocalisation of LIS1 with putative interaction partners
● Four putative new interaction partners of LIS1 were identified by a yeast two-hybrid assay (performed by B. Jung, TU Braunschweig), namely Nude-like protein (Nudel), BRCA1-associated protein (BRAP), Ran binding protein 9 and Lim-only protein ACT (ACT).
● An interaction of LIS1 with Nude-like protein, BRCA1-associated protein and Lim-only protein ACT could be confirmed by Bimolecular Fluorescence Complementation (BiFC) assay.
3 Analysis of germ cell specific regulation
● Luciferase activity in different cell lines (NIH 3T3, NS20Y and GC-1 spg) transfected with pGL3-Basic vector containing a putative Lis1 promoter region (1846bp of Lis1 5`flanking region) was not increased in comparison to controls.
● Luciferase activity in differentiated and undifferentiated SSC/129/Sv cells transfected with pGL3-Promoter vectors containing putative Lis1 enhancer regions was not increased in comparison to controls.
● Generation of a transgenic construct containing putative Lis1 enhancer sequences was not finished during course of this work.
4 Genetic rescue of the infertile mice L39GT/GT
● The transgenic line TNP2-Lis1 (Lispi) was generated that overexpresses Lis1 in postmeiotic germ cells under control of the Transition Nuclear Protein 2 (TNP2) promoter. Expression of the transgene was confirmed by Northern blotting analysis.
Transgenic animals are fertile and testis sections of transgenic males displayed no abnormalities in comparison with sections of wild type mice.
● Double transgenic L39GT/GT/LispiTpos males were generated to rescue the infertility phenotype of the gene trap line. Analysed males remained infertile and histological
analysis of testis sections revealed no differences between L39GT/GT mice and rescued males. Electron microscopy confirmed the degeneration of germ cells in testes of
“rescued” mice.
● The transgenic line PGK2-Lis1-c-myc Tag was generated that overexpresses Lis1 from pachytene spermatocytes on under control of the Phosphoglycerate Kinase-2 (PGK2) promoter. Three transgenic lines were analysed. Testis specific expression of the transgenic fusion transcript was proven by RT-PCR, Northern blotting and Western blotting. Immunohistochemical analysis could confirm the expression of the transgene in pachytene spermatocytes and following stages. All transgenic animals were fertile and testis sections of heterozygous and homozygous transgenic males displayed no abnormalities in comparison with wild type sections.
● Two lines of double transgenic L39GT/GT/PGK2-Lis1-c-myc TagTpos males were generated to rescue the infertility phenotype of L39GT/GT. Transgenic expression was confirmed by Western blotting and immunohistochemical analysis. Males remained infertile and number of sperms in caudae epididymes was as low as in L39GT/GT males.
Detailed comparison of defects in spermatogenesis of L39GT/GT and “rescued” males revealed no significant differences.
● The transgenic line hEF-1α-Lis1-c-myc Tag was generated that overexpresses Lis1 in premeiotic germ cells under control of the human Elongation Factor 1α (hEF-1α) promoter. Five transgenic lines were analysed. Testis specific expression of the transgenic fusion transcript was proven by RT-PCR, Northern blotting and Western blotting. Immunohistochemical analysis confirmed restricted expression of the transgene in premeiotic spermatogonial cells. All transgenic animals were fertile and testis sections of heterozygous and homozygous transgenic males displayed no abnormalities in comparison with wild type sections.
● Two lines of double transgenic L39GT/GT/hEF-1α-Lis1-c-myc TagTpos mice were generated to rescue the infertility phenotype of gene trap males. Transgenic expression was confirmed by Western blotting. Males remained infertile and number of sperms in caudae epididymes was not increased in comparison to L39GT/GT males. Testis sections revealed the same severe germ cell degeneration as observed in the gene trap line.