Ovarian follicle dynamics in the rat: regulation and flexibility

Reproduction is the most important factor for the maintenance of a species. The key event in this process Is fertilization: combination of haploid cells from male and female parents, spermatozoon and oocyte, respectively. Before spermatozoa and oocytes are formed and ready for fertilization many processes have taken place. which are very different for spermatozoa and oocytes. One of the most striking differences is the continuous production of newly formed spermatozoa in the fertile male, while the formation of oocytes is already complete before birth. The oocyte is surrounded by supporting cells. In the complex of the oocyte with the surrounding cells, called the follicle, close collaboration exists between the oocyte and the surrounding cells. The main part of this thesis describes the dynamics of follicle development and the hormonal factors influencing this development

Specific dose-dependent effects of ethane 1,2-dimethanesulfonate in rat and mouse Leydig cells and non-steroidogenic cells on programmed cell death

The mechanism by which ethane 1,2-dimethanesulfonate (EDS) selectively kills Leydig cells is poorly understood. To characterize further the cell-specific actions of EDS, we studied biochemical and morphological changes during apoptosis in different Leydig cell and non-steroidogenic cell models.Rat testicular and H540 tumor Leydig cells were killed by 1-2 mM EDS, whereas 20 mM EDS were required for MA-10 cells. This higher concentration of EDS was also necessary for activation of apoptosis in non-steroidogenic Chinese hamster ovary cells, whereas COS-1 monkey kidney cells were resistant. These variable effects of EDS on apoptosis were independent of new protein synthesis and, interestingly, could be delayed by co-incubation with dibutyrl cyclic AMP. Along with cell death, we also observed chromosomal fragmentation and other hallmarks indicative of apoptosis as evidenced by DNA laddering and fluorescent microscopy. Time-lapse photography with a confocal microscope showed that the time of onset, duration and even the sequence of apoptotic events between individual H540 cells was heterogeneous. When the dose of EDS was gradually increased from 2 to 10 mM, the proportion of cells showing normal apoptotic features gradually decreased. Intriguingly, treatment with 10 mM EDS did not result in death for most cells and was marked by an absence of DNA laddering and ultrastructural features of apoptosis and necrosis. However, incubation with 20 mM EDS resulted in necrosis.These results demonstrated that the effects of EDS on cell survival are not specific to Leydig cells, that different cell types have different sensitivities to EDS and that stimulation of the cAMP pathway may mitigate EDS action. The data obtained with H540 cells further revealed that EDS can induce two types of programmed cell death

Three-dimensional microscopic analysis of clinical prostate specimens

__Aims:__ Microscopic evaluation of prostate specimens for both clinical and research purposes is generally performed on 5-μm-thick tissue sections. Because cross-sections give a two-dimensional (2D) representation, little is known about the actual underlying three-dimensional (3D) architectural features of benign prostate tissue and prostate cancer (PCa). The aim of this study was to show that a combination of tissue-clearing protocols and confocal microscopy can successfully be applied to investigate the 3D architecture of human prostate tissue. __Methods and results:__ Optical clearing of intact fresh and formalin-fixed paraffin-embedded (FFPE) clinical prostate specimens allowed us to visualize tissue structures up to a depth of 800 μm, whereas, in uncleared tissue, detection of fluorescence was only possible up to 70 μm. Fluorescent labelling with a general nuclear dye and antibodies against cytokeratin (CK) 5 and CK8-18 resulted in comprehensive 3D imaging of benign peripheral and transition prostate zones, as well as individual PCa growth patterns. After staining, clearing, and imaging, samples could still be processed for 2D (immuno)histochemical staining and DNA analysis, enabling additional molecular and diagnostic characterization of small tissue specimens. __Conclusions:__ In conclusion, the applicability of 3D imaging to archival FFPE and fresh clinical specimens offers unlimited opportunities to stud

Androgen receptor complexes probe DNA for recognition sequences by short random interactions

Owing to the tremendous progress in microscopic imaging of fluorescently labeled proteins in living cells, the insight into the highly dynamic behavior of transcription factors has rapidly increased over the past decade. However, a consistent quantitative scheme of their action is still lacking. Using the androgen receptor (AR) as a model system, we combined three different fluorescence microscopy assays: single-molecule microscopy, photobleaching and correlation spectroscopy, to provide a quantitative model of the action of this transcription factor. This approach enabled us to distinguish two types of AR-DNA binding: very brief interactions, in the order of a few hundred milliseconds, and hormone-induced longer-lasting interactions, with a characteristic binding time of several seconds. In addition, freely mobile ARs were slowed down in the presence of hormone, suggesting the formation of large AR-co-regulator complexes in the nucleoplasm upon hormone activation. Our data suggest a model in which mobile hormone-induced complexes of transcription factors and co-regulators probe DNA by briefly binding at random sites, only forming relatively stable transcription initiation complexes when bound to specific recognition sequences

Repair of exogenous DNA double-strand breaks promotes chromosome synapsis in SPO11-mutant mouse meiocytes, and is altered in the absence of HORMAD1

Repair of SPO11-dependent DNA double-strand breaks (DSBs) via homologous recombination (HR) is essential for stable homologous chromosome pairing and synapsis during meiotic prophase. Here, we induced radiation-induced DSBs to study meiotic recombination and homologous chromosome pairing in mouse meiocytes in the absence of SPO11 activity (Spo11YF/YF model), and in the absence of both SPO11 and HORMAD1 (Spo11/Hormad1 dko). Within 30 min after 5 Gy irradiation of Spo11YF/YF mice, 140–160 DSB repair foci were detected, which specifically localized to the synaptonemal complex axes. Repair of radiation-induced DSBs was incomplete in Spo11YF/YF compared to Spo11+/YF meiocytes. Still, repair of exogenous DSBs promoted partial recovery of chromosome pairing and synapsis in Spo11YF/YF meiocytes. This indicates that at least part of the exogenous DSBs can be processed in an interhomolog recombination repair pathway. Interestingly, in a seperate experiment, using 3 Gy of irradiation, we observed that Spo11/Hormad1 dko spermatocytes contained fewer remaining DSB repair foci at 48 h after irradiation compared to irradiated Spo11 knockout spermatocytes. Together, these results show that recruitment of exogenous DSBs to the synaptonemal complex, in conjunction with repair of exogenous DSBs via the homologous chromosome, contributes to homology recognition. In addition, the data suggest a role for HORMAD1 in DNA repair pathway choice in mouse meiocytes

Live cell analyses of synaptonemal complex dynamics and chromosome movements in cultured mouse testis tubules and embryonic ovaries

During mammalian meiotic prophase, homologous chromosomes connect through the formation of the synaptonemal complex (SC). SYCP3 is a component of the lateral elements of the SC. We have generated transgenic mice expressing N- or C-terminal fluorescent-tagged SYCP3 (mCherry-SYCP3 (CSYCP) and SYCP3-mCherry (SYCPC)) to study SC dynamics and chromosome movements in vivo. Neither transgene rescued meiotic aberrations in Sycp3 knockouts, but CSYCP could form short axial element-like structures in the absence of endogenous SYCP3. On the wild-type background, both fusion proteins localized to the axes of the SC together with endogenous SYCP3, albeit with delayed initiation (from pachytene) in spermatocytes. Around 40% of CSYCP and SYCPC that accumulated on the SC was rapidly exchanging with other tagged proteins, as analyzed by fluorescent recovery after photobleaching (FRAP) assay. We used the CSYCP transgenic mice for further live cell analyses and observed synchronized bouquet configurations in living cysts of two or three zygotene oocyte nuclei expressing CSYCP, which presented cycles of telomere clustering and dissolution. Rapid chromosome movements were observed in both zygotene oocytes and pachytene spermatocytes, but rotational movements of the nucleus were more clear in oocytes. In diplotene spermatocytes, desynapsis was found to proceed in a discontinuous manner, whereby even brief chromosome re-association events were observed. Thus, this live imaging approach can be used to follow changes in the dynamic behavior of the nucleus and chromatin, in normal mice and different infertile mouse models

Acanthamoeba castellanii interferes with adequate chlorine disinfection of multidrug-resistant Pseudomonas aeruginosa

Verona-Integron-encoded-Metallo-b-lactamase-positive Pseudomonas aeruginosa (VIM-PA) is a cause of hard-to-treat nosocomial infections, and can colonize hospital water networks alongside Acanthamoeba. We developed an in-vitro disinfection model to examine whether Acanthamoeba castellanii can harbour VIM-PA intracellularly, allowing VIM-PA to evade being killed by currently used hospital disinfectants. We observed that A. castellanii presence resulted in significantly increased survival of VIM-PA after exposure to chlorine for 30 s or for 2 min. This undesirable effect was not observed after disinfection by 70% alcohol or 24% acetic acid. Confocal microscopy confirmed the presence of VIM-PA within A. castellanii pseudocysts. Our data indicate that A. castellanii contributes to persistent VIM-PA colonization of water systems after chlorine treatment

Comparison of high-and low-let radiation-induced dna double-strand break processing in living cells

High-linear-energy-transfer (LET) radiation is more lethal than similar doses of low-LET radiation types, probably a result of the condensed energy deposition pattern of high-LET radiation. Here, we compare high-LET α-particle to low-LET X-ray irradiation and monitor double-strand break (DSB) processing. Live-cell microscopy was used to monitor DNA double-strand breaks (DSBs), marked by p53-binding protein 1 (53BP1). In addition, the accumulation of the endogenous 53BP1 and replication protein A (RPA) DSB processing proteins was analyzed by immunofluorescence. In contrast to α-particle-induced 53BP1 foci, X-ray-induced foci were resolved quickly and more dynamically as they showed an increase in 53BP1 protein accumulation and size. In addition, the number of individual 53BP1 and RPA foci was higher after X-ray irradiation, while focus intensity was higher after α-particle irradiation. Interestingly, 53BP1 foci induced by α-particles contained multiple RPA foci, suggesting multiple individual resection events, which was not observed after X-ray irradiation. We conclude that high-LET α-particles cause closely interspaced DSBs leading to high local concentrations of repair proteins. Our results point toward a change in DNA damage processing toward DNA end-resection and homologous recombination, possibly due to the depletion of soluble protein in the nucleoplasm. The combination of closely interspaced DSBs and perturbed DNA damage processing could be an explanation for the increased relative biological effectiveness (RBE) of high-LET α-particles compared to X-ray irradiation

Three-dimensional analysis reveals two major architectural subgroups of prostate cancer growth patterns

The Gleason score is one of the most important parameters for therapeutic decision-making in prostate cancer patients. Gleason growth patterns are defined by their histological features on 4- to 5-µm cross sections, and little is known about their threedimensional architecture. Our objective was to characterize the three-dimensional architecture of prostate cancer growth patterns. Intact tissue punches (n = 46) of representative Gleason growth patterns from radical prostatectomy specimens were fluorescently stained with antibodies targeting Keratin 8/18 and Keratin 5 for the detection of luminal and basal epithelial cells, respectively. Punches were optically cleared in benzyl alcohol–benzyl benzoate and imaged using a confocal laser scanning microscope up to a depth of 500 µm. Gleason pattern 3, poorly formed pattern 4, and cords pattern 5 all formed a continuum of interconnecting tubules in which the diameter of the structures and the lumen size decreased with higher grades. In fused pattern 4, the interconnections between the tubules were markedly closer together. In these patterns, all tumor cells were in direct contact with the surrounding stroma. In contrast, cribriform Gleason pattern 4 and solid pattern 5 demonstrated a three-dimensional continuum of contiguous tumor cells, in which the vast majority of cells had no contact with the surrounding stroma. Transitions between cribriform pattern 4 and solid pattern 5 were seen. There was a decrease in the number and size of intercellular lumens from cribriform to solid growth pattern. Glomeruloid pattern 4 formed an intermediate structure consisting of a tubular network with intraluminal epithelial protrusions close to the tubule splitting points. In conclusion, three-dimensional microscopy revealed two major architectural subgroups of prostate cancer growth patterns: (1) a tubular interconnecting network including Gleason pattern 3, poorly formed and fused Gleason pattern 4, and cords Gleason pattern 5, and (2) serpentine contiguous epithelial proliferations including cribriform Gleason pattern 4 and solid Gleason pattern 5