Sigrid Hoyer-Fender

Sequence and developmental expression of a mRNA encoding a putative protein of rat sperm outer dense fibers.

We have isolated a cDNA from rat testis homologous to the Drosophila melanogaster gene mst(3)gl-9 by screening a rat testis cDNA library with mst(3)gl-9 and by direct PCR amplification of upstream sequences out of the cDNA library. Homologous genes are also expressed in testes of different mammalian species. In rat testis, two different transcripts are found. Evidences are presented which suggest that these two transcripts are alternative splicing products. As proved by Northern blot analysis of testis RNA prepared from rats of different ages and by in situ hybridization to rat testis tissue sections, the mRNAs are first transcribed in early spermatids. The longest open reading frame of the cDNA encodes a polypeptide of 244 amino acids which contains 16.4% cysteine, 9.9% proline, and 5.7% glycine and closely resembles the sizes and amino acid compositions of two major polypeptides isolated from outer dense fibers of rat spermatozoa. The COOH-terminal end consists mostly of the tripeptide motif Cys-Gly-Pro, the main motif in D. melanogaster mst(3)gl-9. It is suggested that the isolated rat cDNA encodes a polypeptide which is a protein component of the outer dense fibers of spermatozoa.

The Small Heat Shock Protein ODF1/HSPB10 Is Essential for Tight Linkage of Sperm Head to Tail and Male Fertility in Mice

ABSTRACT Sperm motility and hence male fertility strictly depends on proper development of the sperm tail and its tight anchorage to the head. The main protein of sperm tail outer dense fibers, ODF1/HSPB10, belongs to the family of small heat shock proteins that function as molecular chaperones. However, the impact of ODF1 on sperm tail formation and motility and on male fecundity is unknown. We therefore generated mutant mice in which the Odf1 gene was disrupted. Heterozygous mutant male mice are fertile while sperm motility is reduced, but Odf1-deficient male mice are infertile due to the detachment of the sperm head. Although headless tails are somehow motile, transmission electron microscopy revealed disturbed organization of the mitochondrial sheath, as well as of the outer dense fibers. Our results thus suggest that ODF1, besides being involved in the correct arrangement of mitochondrial sheath and outer dense fibers, is essential for rigid junction of sperm head and tail. Loss of function of ODF1, therefore, might account for some of the cases of human infertility with decapitated sperm heads. In addition, since sperm motility is already affected in heterozygous mice, impairment of ODF1 might even account for some cases of reduced fertility in male patients.

Molecular aspects of XY body formation

More than a century ago, a densely stained area inside the nucleus of male meiotic cells was described. It was later shown to harbor the sex chromosomes which undergo transcriptional inactivation in conjunction with heterochromatinisation and synapsis to form the XY body. Formation of the XY body is conserved throughout the mammalian phylogenetic tree and is thought to be essential for successful spermatogenesis. However, its biological role as well as the molecular mechanisms underlying XY body formation are still far from being understood. A lot of effort has already been undertaken to characterize components of the XY body and to investigate their functional implications in sex chromatin heterochromatinisation and meiotic sex chromosome inactivation (MSCI). This review gives an overview of those components and their possible implications in XY body formation and function.

CHD8 interacts with CHD7, a protein which is mutated in CHARGE syndrome

CHARGE syndrome is an autosomal dominant disorder caused in about two-third of cases by mutations in the CHD7 gene. For other genetic diseases e.g. hereditary spastic paraplegia, it was shown that interacting partners are involved in the underlying cause of the disease. These data encouraged us to search for CHD7 binding partners by a yeast two-hybrid library screen and CHD8 was identified as an interacting partner. The result was confirmed by a direct yeast two-hybrid analysis, co-immunoprecipitation studies and by a bimolecular fluorescence complementation assay. To investigate the function of CHD7 missense mutations in the CHD7-CHD8 interacting area on the binding capacity of both proteins, we included three known missense mutations (p.His2096Arg, p.Val2102Ile and p.Gly2108Arg) and one newly identified missense mutation (p.Trp2091Arg) in the CHD7 gene and performed both direct yeast two-hybrid and co-immunoprecipitation studies. In the direct yeast two-hybrid system, the CHD7-CHD8 interaction was disrupted by the missense mutations p.Trp2091Arg, p.His2096Arg and p.Gly2108Arg, whereas in the co-immunoprecipitation studies disruption of the CHD7-CHD8 interaction by the mutations could not be observed. The results lead to the hypothesis that CHD7 and CHD8 proteins are interacting directly and indirectly via additional linker proteins. Disruption of the direct CHD7-CHD8 interaction might change the conformation of a putative large CHD7-CHD8 complex and could be a disease mechanism in CHARGE syndrome.

Mouse Odf2 cDNAs consist of evolutionary conserved as well as highly variable sequences and encode outer dense fiber proteins of the sperm tail.

The outer dense fibers (ODF) of the mammalian sperm tail comprise a unique, specialized, and very prominent structure, consisting of nine fibers surrounding the axoneme. The ODF may play an important but as yet undefined role in sperm morphology, integrity and function. Study of the ODF is hampered by insufficient knowledge of their protein composition and the genetic regulation of their synthesis. We report here on the characterization of cDNAs encoding the Odf2 proteins of outer dense fibers of mouse sperm. We isolated two cDNA clones with variable 5′ regions. Variability in sequence is restricted to specific regions in the N‐terminal part of the encoded proteins, whereas the C‐terminal part is highly conserved in Odf2 proteins both between species and within a species. This variability is confirmed at the protein level. The outer dense fibers could be detected immunologically in total sperm tails allowing a direct comparison of their length in relation to the length of the sperm tail. Odf2 transcripts could be demonstrated in testicular RNA and are restricted to germ cells. The start of transcription is in step 5 spermatids of tubular stage V and the RNA could be detected in the cytoplasm of differentiating spermatids in all subsequent tubular stages. Mol. Reprod. Dev. 51:167–175, 1998.

Centriole maturation and transformation to basal body

Centrioles and basal bodies are fascinating and mysterious organelles. They interconvert and seem to be crucial for a wide range of crucial cellular processes. However, intense research over the last years suggested that centrioles/basal bodies are essential mainly for the generation of cilia. Although a neglected organelle over a long time, interest in the primary cilia was recently rekindled by the notion that they are affected in a number of human diseases. Cilia formation is an intricate process that starts with the transformation of centrioles to basal bodies and their docking to the apical plasma membrane. Disturbance of basal body formation thus might cause ciliopathies. This review focuses on the formation of basal bodies in mammalian cells with an emphasis on basal bodies sprouting a primary cilium.

SPAG4L/SPAG4L-2 are testis-specific SUN domain proteins restricted to the apical nuclear envelope of round spermatids facing the acrosome

SUN domain proteins are integral proteins of the inner nuclear membrane and functions in linkage of the nuclear lamina to the cytoskeleton. Moreover, SUN domain proteins seem to mediate the tethering of the centrosome to the nuclear membrane, and they are involved in telomere attachment to the nuclear envelope in meiotic cells, and in germ cell development in invertebrates. In contrast to the widely expressed SUN domain proteins in mammals, SUN1 and SUN2, which have been analysed in great detail, there is virtually nothing known about testicular SUN domain proteins. Since a hallmark of male germ cell development is the profound remodelling of the nuclear envelope, emphasized, for example, by the reshaping of the nucleus during spermiogenesis, and the biogenesis of its tightly associated acrosome, SUN domain proteins might be engaged in these processes. We have isolated a novel SUN domain protein, SPAG4L-2, that differs from SPAG4L by an N-terminal insertion of 25 amino acids. Spag4l and Spag4l-2 are exclusively expressed in testis at about equimolar amounts, and show elevated transcription during ongoing spermiogenesis coincident with the appearance of round spermatids. Molecular dissection of the protein followed by cytological and biochemical investigations revealed that SPAG4L-2 and SPAG4L are transmembrane proteins that localize to the nuclear envelope. SPAG4L/4L-2 are restricted to the apical nuclear region of round spermatids that face the acrosomic vesicle, and thus are most probably involved in linkage of the acrosomic vesicle to the spermatid nucleus, and in acrosome biogenesis.

Outer dense fibre protein 2 (ODF2) is a self-interacting centrosomal protein with affinity for microtubules

Outer dense fibre protein 2 (ODF2) is a major protein of sperm tail outer dense fibres which are prominent sperm tail-specific cytoskeletal structures. Moreover, ODF2 was also identified as a widespread component of the centrosomal scaffold and was found to associate preferentially with the appendages of the mother centriole [Nakagawa, Y., Yamane, Y., Okanoue, T., Tsukita, S. and Tsukita, S. (2001) Mol. Biol. Cell 12, 1687-1697]. Secondary structure predictions indicated ODF2 as an overall coiled-coil protein with a putative fibre forming capacity. To investigate its potential functions in generating the centrosomal scaffold and in microtubule nucleation we asked whether ODF2 is able to form a fibrillar structure by self-association in vivo and if it interacts with microtubules. By cytological investigation of transfected mammalian cells expressing ODF2-GFP fusion proteins and in vitro coprecipitation assays we could demonstrate that ODF2 is a self-interacting protein that forms a fibrillar structure partially linked to the microtubule network. Microtubule cosedimentation and coprecipitation assays indicated ODF2 as a microtubule-associated protein. However, we could not demonstrate a direct interaction of ODF2 with tubulin, suggesting that binding of endogenous ODF2 to the axonemal as well as to centrosomal microtubules may be mediated by, as yet, unknown proteins.

Reduction of Spermatogenesis but Not Fertility in Creb3l4-Deficient Mice

ABSTRACT Creb3l4 belongs to the CREB/ATF family of transcription factors that are involved in mediating transcription in response to intracellular signaling. This study shows that Creb3l4 is expressed at low levels in all organs and in different stages of embryogenesis but is present at very high levels in the testis, particularly in postmeiotic male germ cells. In contrast to CREB3L4 in the human prostate, of which specific expression was detected, Creb3l4 transcripts in the mouse prostate could be detected only by RT-PCR. To identify the physiological function of Creb3l4, the murine gene was inactivated by replacement with the gene encoding green fluorescent protein. Surprisingly, Creb3l4-deficient mice were born at expected ratios, were healthy, and displayed normal long-term survival rates. Despite a significant reduction in the number of spermatozoa in the epididymis of Creb3l4− / − mice, the breeding of mutant males with wild-type females was productive and the average litter size was not significantly altered in comparison to wild-type littermates. Further analyses revealed that the seminiferous tubules of Creb3l4 − / − mice contained all of the developmental stages, though there was evidence for increased apoptosis of meiotic/postmeiotic germ cells. These results suggest that Creb3l4 plays a role in male germ cell development, but its loss is insufficient to completely compromise the production of spermatozoa.

Disruption of the Pelota Gene Causes Early Embryonic Lethality and Defects in Cell Cycle Progression

ABSTRACT Mutations in either the Drosophila melanogaster pelota or pelo gene or the Saccharomyces cerevisiae homologous gene, DOM34, cause defects of spermatogenesis and oogenesis in Drosophila, and delay of growth and failure of sporulation in yeast. These phenotypes suggest that pelota is required for normal progression of the mitotic and meiotic cell cycle. To determine the role of the pelota in mouse development and progression of cell cycle, we have established a targeted disruption of the mouse Pelo. Heterozygous animals are variable and fertile. Genotyping of the progeny of heterozygous intercrosses shows the absence of Pelo −/− pups and suggests an embryo-lethal phenotype. Histological analyses reveal that the homozygous Pelo deficient embryos fail to develop past day 7.5 of embryogenesis (E7.5). The failure of mitotic active inner cell mass of the Pelo −/− blastocysts to expand in growth after 4 days in culture and the survival of mitotic inactive trophoplast indicate that the lethality of Pelo-null embryos is due to defects in cell proliferation. Analysis of the cellular DNA content reveals the significant increase of aneuploid cells in Pelo −/− embryos at E7.5. Therefore, the percent increase of aneuploid cells at E7.5 may be directly responsible for the arrested development and suggests that Pelo is required for the maintenance of genomic stability.