Sperm Guidance in Zebrafish

The core challenge of the fertilization process is one of pathfinding: how do motile sperm cells locate the immotile oocyte? In some species, this process is well studied, e.g. sea urchin and human. Both species employ chemotaxis—the orientation along a three-dimensional gradient of a chemoattractant—to facilitate pathfinding of sperm towards the oocyte. But the molecular implementation differs considerably between the two species. Overall, great variation is found in fertilization processes even between closely related species. Zebrafish is a widely used vertebrate model system, yet very little is known about how their sperm function. In my thesis, I approached the question of pathfinding in zebrafish sperm from several angles. First, I performed extensive bioinformatic studies in jawless, cartilaginous, and bony fishes to elucidate the presence or absence of several components of the signaling cascades involved in pathfinding. I showed a fine- grained pattern of many independent gene losses for the CatSper channel complex and soluble adenylate cyclase (sAC), both of which are considered essential for sperm motility in species as distantly related as sea urchin and human. In particular, both CatSper and sAC are clearly absent in zebrafish. Synteny analysis between the genome of zebrafish and the closely related goldfish, which possesses CatSper, showed chromosomal breakpoints as likely origin of the loss for all four subunits of the CatSper channel. By comparison with related species I could deduce that CatSper was lost at the origin of the family Danionidae, whereas sAC was already lost earlier, at the origin of the order Cypriniformes. The absence of these proteins in zebrafish means that signaling in zebrafish sperm must function in a manner yet unknown. Second, I investigated the proteome of the egg envelope (chorion) using mass spectroscopy to identify potential chemoattractant candidates for haptotaxis (orientation along a two-dimensional gradient of a tethered chemoattractant). Zebrafish chorion possess a micropyle, an opening that allows the sperm access to the oocyte. I found no difference in the proteome between chorion samples that contained the micropyle and those that did not. The main components of the zebrafish chorion are zona pellucida (ZP) proteins. I identified 20 ZP proteins in the proteome and assigned their respective subfamilies. Interestingly, ZPB and ZPC proteins are present, which are required for the acrosome reaction in mammals, although zebrafish possess a micropyle and therefore do not use the acrosome reaction. Third, I established a baseline of motility behavior and internal ion changes of activated but unstimulated zebrafish sperm through dark-field and fluorescence microscopy. Swimming paths vary greatly in curvature, although the majority displays lower curvature. Cytosolic calcium increases during initiation of motility, via release from internal stores. The sperm also acidify, in contrast to sea urchin, human, and mouse, where the sperm alkalizes. Next, I tested oocyte-derived stimulus on the sperm, but found no indication that zebrafish sperm employ chemotaxis. Last, I discovered regularly spaced, dye-accumulating structures in the sperm flagellum. They may correspond to vesicular bodies of unknown function described so far only morphologically. My results form a basis for future studies to fully elucidate signaling in zebrafish sperm

Reconstruction of the birth of a male sex chromosome present in Atlantic herring

The mechanisms underlying sex determination are astonishingly plastic. Particularly the triggers for the molecular machinery, which recalls either the male or female developmental program, are highly variable and have evolved independently and repeatedly. Fish show a huge variety of sex determination systems, including both genetic and environmental triggers. The advent of sex chromosomes is assumed to stabilize genetic sex determination. However, because sex chromosomes are notoriously cluttered with repetitive DNA and pseudogenes, the study of their evolution is hampered. Here we reconstruct the birth of a Y chromosome present in the Atlantic herring. The region is tiny (230 kb) and contains only three intact genes. The candidate male-determining gene BMPR1BBY encodes a truncated form of a BMP1B receptor, which originated by gene duplication and translocation and underwent rapid protein evolution. BMPR1BBY phosphorylates SMADs in the absence of ligand and thus has the potential to induce testis formation. The Y region also contains two genes encoding subunits of the sperm-specific Ca2+ channel CatSper required for male fertility. The herring Y chromosome conforms with a characteristic feature of many sex chromosomes, namely, suppressed recombination between a sex-determining factor and genes that are beneficial for the given sex. However, the herring Y differs from other sex chromosomes in that suppression of recombination is restricted to an similar to 500-kb region harboring the male-specific and sex-associated regions. As a consequence, any degeneration on the herring Y chromosome is restricted to those genes located in the small region affected by suppressed recombination

Selective regain of <it>egfr </it>gene copies in CD44⁺/CD24^-/lowbreast cancer cellular model MDA-MB-468

Abstract Background Increased transcription of oncogenes like the epidermal growth factor receptor (EGFR) is frequently caused by amplification of the whole gene or at least of regulatory sequences. Aim of this study was to pinpoint mechanistic parameters occurring during egfr copy number gains leading to a stable EGFR overexpression and high sensitivity to extracellular signalling. A deeper understanding of those marker events might improve early diagnosis of cancer in suspect lesions, early detection of cancer progression and the prediction of egfr targeted therapies. Methods The basal-like/stemness type breast cancer cell line subpopulation MDA-MB-468 CD44high/CD24-/low, carrying high egfr amplifications, was chosen as a model system in this study. Subclones of the heterogeneous cell line expressing low and high EGF receptor densities were isolated by cell sorting. Genomic profiling was carried out for these by means of SNP array profiling, qPCR and FISH. Cell cycle analysis was performed using the BrdU quenching technique. Results Low and high EGFR expressing MDA-MB-468 CD44+/CD24-/low subpopulations separated by cell sorting showed intermediate and high copy numbers of egfr, respectively. However, during cell culture an increase solely for egfr gene copy numbers in the intermediate subpopulation occurred. This shift was based on the formation of new cells which regained egfr gene copies. By two parametric cell cycle analysis clonal effects mediated through growth advantage of cells bearing higher egfr gene copy numbers could most likely be excluded for being the driving force. Subsequently, the detection of a fragile site distal to the egfr gene, sustaining uncapped telomere-less chromosomal ends, the ladder-like structure of the intrachromosomal egfr amplification and a broader range of egfr copy numbers support the assumption that dynamic chromosomal rearrangements, like breakage-fusion-bridge-cycles other than proliferation drive the gain of egfr copies. Conclusion Progressive genome modulation in the CD44+/CD24-/low subpopulation of the breast cancer cell line MDA-MB-468 leads to different coexisting subclones. In isolated low-copy cells asymmetric chromosomal segregation leads to new cells with regained solely egfr gene copies. Furthermore, egfr regain resulted in enhanced signal transduction of the MAP-kinase and PI3-kinase pathway. We show here for the first time a dynamic copy number regain in basal-like/stemness cell type breast cancer subpopulations which might explain genetic heterogeneity. Moreover, this process might also be involved in adaptive growth factor receptor intracellular signaling which support survival and migration during cancer development and progression.</p

Interlaboratory variability of Ki67 staining in breast cancer

Background Postanalytic issues of Ki67 assessment in breast cancers like counting method standardisation and interrater bias have been subject of various studies, but little is known about analytic variability of Ki67 staining between pathology labs. Our aim was to study interlaboratory variability of Ki67 staining in breast cancer using tissue microarrays (TMAs) and central assessment to minimise preanalytic and postanalytic influences. Methods Thirty European pathology labs stained serial slides of a TMA set of breast cancer tissues with Ki67 according to their routine in-house protocol. The Ki67-labelling index (Ki67-LI) of 70 matched samples was centrally assessed by one observer who counted all cancer cells per sample. We then tested for differences between the labs in Ki67-LI medians by analysing variance on ranks and in proportions of tumours classified as luminal A after dichotomising oestrogen receptor–positive cancers into cancers showing low (<14%, luminal A) and high (≥14%, luminal B HER2 negative) Ki67-LI using Cochran's Q. Results Substantial differences between the 30 labs were indicated for median Ki67-LI (0.65%–33.0%, p < 0.0001) and proportion of cancers classified as luminal A (17%–57%, p < 0.0001). The differences remained significant when labs using the same antibody (MIB-1, SP6, or 30-9) were analysed separately or labs without prior participation in external quality assurance programs were excluded (p < 0.0001, respectively). Conclusion Substantial variability in Ki67 staining of breast cancer tissue was found between 30 routine pathology labs. Clinical use of the Ki67-LI for therapeutic decisions should be considered only fully aware of lab-specific reference values

Interlaboratory variability of Ki67 staining in breast cancer

Background Postanalytic issues of Ki67 assessment in breast cancers like counting method standardisation and interrater bias have been subject of various studies, but little is known about analytic variability of Ki67 staining between pathology labs. Our aim was to study interlaboratory variability of Ki67 staining in breast cancer using tissue microarrays (TMAs) and central assessment to minimise preanalytic and postanalytic influences. Methods Thirty European pathology labs stained serial slides of a TMA set of breast cancer tissues with Ki67 according to their routine in-house protocol. The Ki67-labelling index (Ki67-LI) of 70 matched samples was centrally assessed by one observer who counted all cancer cells per sample. We then tested for differences between the labs in Ki67-LI medians by analysing variance on ranks and in proportions of tumours classified as luminal A after dichotomising oestrogen receptor–positive cancers into cancers showing low (<14%, luminal A) and high (≥14%, luminal B HER2 negative) Ki67-LI using Cochran's Q. Results Substantial differences between the 30 labs were indicated for median Ki67-LI (0.65%–33.0%, p < 0.0001) and proportion of cancers classified as luminal A (17%–57%, p < 0.0001). The differences remained significant when labs using the same antibody (MIB-1, SP6, or 30-9) were analysed separately or labs without prior participation in external quality assurance programs were excluded (p < 0.0001, respectively). Conclusion Substantial variability in Ki67 staining of breast cancer tissue was found between 30 routine pathology labs. Clinical use of the Ki67-LI for therapeutic decisions should be considered only fully aware of lab-specific reference values