As the main microtubule-organizing center in animal cells, the centrosome has a fundamental role in cell function. Surrounding the centrioles, the Pericentriolar material (PCM) provides a dynamic platform for nucleating microtubules. While the PCM’s importance is established, its amorphous electron-dense nature has made it refractory to structural investigation. By using SIM and STORM sub-diffraction resolution microscopies to visualize proteins critical for centrosome maturation, we demonstrate that the PCM is organized into two major structural domains: a layer juxtaposed to the centriole wall, and proteins extending further away from the centriole organized in a matrix. Analysis of Pericentrin-like protein (Plp) reveals that its C-terminus is positioned at the centriole wall, it radiates outward into the matrix and is organized in clusters having quasi-nine-fold symmetry. By RNAi we show that Plp fibrils are required for interphase recruitment and proper mitotic assembly of the PCM matrix.

The aim of cytochemical techniques is to localize specific biochemical components in particular tissue and cell rumpartments However, since preparation of tissues for strucrural Indeed, washing of the tissue before fixation, the nature of the fixative solution, the chemical basis of the resins, and the physical conditions ofembedding can all introduce changes or cacodylate buffered 1% glutaraldehyde or freshly prepared 4% paraformaldehyde and embedded in various resins. Post-fixation with phosphate-buffered 1% osmium tetroxide was performed before embedding, where indicated. Fixation by vascular perfusion was carried out through the left ventricle at a pressure of 150 cm H30, first with the phosphate buffer for 5 mm and then with the fixative solution for 10 mm. The tissue fragments were further fixed by immersion for a total fixation rime of 2 hr. Embedding in Epoxy Resins. Embedding in Epon 812 (Luft, 1961), Spurr (Spurn, 1969) or Alnadite (Mollenhauer, 1964) was performed according to standard techniques after dehydration of the tissues with ethanol and propylene oxide. Polymerization was performed at 60'C. Embedding in Methacrylate Resins Glyco/Methaciy/ate (GMA). Embedding in glycol methacrylate was performed according to the technique described by Leduc and Bernhard (1967).

Exosomes and microvesicles are extracellular vesicles released from cells and can contain lipids, miRNAs and proteins that affect cells at distant sites. Recently, microvesicles containing miRNA have been implicated in uterine microenvironment of pigs, a species with unique epitheliochorial (non-invasive) placentation. Here we report a novel role of conceptus-derived exosomes/microvesicles (hereafter referred to as extracellular vesicles; EVs) in embryo-endometrial cross-talk. We also demonstrate the stimulatory effects of EVs (PTr2-Exo) derived from porcine trophectoderm-cells on various biological processes including the proliferation of maternal endothelial cells (PAOEC), potentially promoting angiogenesis. Transmission immuno-electron microscopy confirmed the presence of EVs in tissue biopsies, PTr2-Exo and PAOEC-derived EVs (PAOEC-Exo). RT-PCR detected 14 select miRNAs in CD63 positive EVs in which miR-126-5P, miR-296-5P, miR-16, and miR-17-5P were the most abundant angiogenic miRNAs. Proteomic analysis revealed EV proteins that play a role in angiogenesis. In-vitro experiments, using two representative cell lines of maternal-fetal interface, demonstrated bidirectional EVs shuttling between PTr2 and PAOEC cells. Importantly, these studies support the idea that PTr2-Exo and PAOEC-Exo containing select miRNAs and proteins can be successfully delivered to recipient cells and that they may have a biological role in conceptus-endometrial cross-talk crucial for the pregnancy success.

The nature of cholesterol-binding proteins acting upon human spermatozoa during in vitro capacitation was determined by measuring the efflux of [3H]cholesterol and of [3H]cholesteryl sulfate from labeled spermatozoa. Efflux of [3H]sterols was stimulated when the labeled gametes were incubated in Ham's F-10 medium supplemented with female serum or follicular fluid. Upon centrifugation of capacitated spermatozoa and application of the supernatant to density-gradient ultracentrifugation for lipoprotein analysis, both [3H]cholesterol and [3H]cholesteryl sulfate were found to be carried by very-low-density lipoproteins (VLDL), low-density lipoproteins (LDL), high-density lipoproteins (HDL), as well as the albumin fraction (d greater than 1.21) in serum. When the capacitation medium was supplemented with follicular fluid, the [3H]sterols were bound to HDL's and to the albumin fraction; when the latter fraction was analysed by molecular sieve chromatography, 60-70% of the radioactivity eluted in fractions with a mean molecular weight corresponding to that of human serum albumin. Sperm cholesterol efflux was also stimulated when serum or follicular fluid was added to a simplified medium (50 mM Tris-HCl, 0.56% NaCl, pH 7.8); efflux of [3H]cholesterol from labeled gametes progressed in a time-dependent manner, but was low in the absence of serum components. The [3H]cholesterol/cholesterol ratios were higher in the albumin and HDL fractions, indicating some degree of specificity of these sterol acceptors. It was observed that follicular fluid albumin has a [3H]sterol binding capacity that is 2-3-fold higher than that of serum albumin. Commercial human serum albumin also promoted sperm cholesterol efflux. These results provide new information concerning those components of follicular fluid which may play a role in human sperm capacitation and provide further support for the concept that loss of cholesterol from the sperm plasma membrane is an important component of the capacitation process.