Cell medium-dependent dynamic modulation of size and structural transformations of binary phospholipid/ω-3 fatty acid liquid crystalline nano-self-assemblies: Implications in interpretation of cell uptake studies.

Lyotropic non-lamellar liquid crystalline (LLC) nanoparticles, with their tunable structural features and capability of loading a wide range of drugs and reporter probes, are emerging as versatile injectable nanopharmaceuticals. Secondary emulsifiers, such as Pluronic block copolymers, are commonly used for colloidal stabilization of LLC nanoparticles, but their inclusion often compromises the biological safety (e.g., poor hemocompatibility and enhanced cytotoxicity) of the formulation. Here, we introduce a library of colloidally stable, structurally tunable, and pH-responsive lamellar and non-lamellar liquid crystalline nanoparticles from binary mixtures of a phospholipid (phosphatidylglycerol) and three types of omega-3 fatty acids (ω-3 PUFAs), prepared in the absence of a secondary emulsifier and organic solvents. We study formulation size distribution, morphological heterogeneity, and the arrangement of their internal self-assembled architectures by nanoparticle tracking analysis, synchrotron small-angle X-ray scattering, and cryo-transmission electron microscopy. The results show the influence of type and concentration of ω-3 PUFAs in nanoparticle structural transitions spanning from a lamellar (Lα) phase to inverse discontinuous (micellar) cubic Fd3m and hexagonal phase (H2) phases, respectively. We further report on cell-culture medium-dependent dynamic fluctuations in nanoparticle size, number and morphology, and simultaneously monitor uptake kinetics in two human cell lines. We discuss the role of these multiparametric biophysical transformations on nanoparticle-cell interaction kinetics and internalization mechanisms. Collectively, our findings contribute to the understanding of fundamental steps that are imperative for improved engineering of LLC nanoparticles with necessary attributes for pharmaceutical development.

Control of bull sperm cell volume during epididymal maturation.

Mature spermatozoa have a mechanism by which they can reduce cellular swelling caused by hypo-osmotic stress. The development of this ability during epididymal maturation in the bull was investigated. Caput and cauda sperm preparations were exposed to various osmotic stresses at 38 degrees C and measurements of cell volume made by electronic cell sizing. (1) Epididymal spermatozoa recovered and incubated in a medium isotonic with caudal epididymal plasma (360 mOsm kg(-1)) showed better viability and better volume regulatory ability than those incubated in a medium isotonic with seminal plasma (300 mOsm kg(-1)) or in seminal plasma itself. (2) Preparations of both caput and cauda spermatozoa, isolated in a medium isotonic with caudal epididymal plasma, contained two volumetric subpopulations, unrelated to the presence or absence of attached cytoplasmic droplets. (3) The cell volume of both subpopulations of caput spermatozoa was always greater than that of the corresponding cauda spermatozoa subpopulations. (4) After exposure to hypotonic challenge, both caput and cauda spermatozoa were able to reduce their relative volumes, demonstrating that both immature and mature cells are able to express regulatory volume decrease under physiological conditions. (5) When spermatozoa were incubated in chloride- or sodium-free media, although two subpopulations remained present, the volume of the caput sperm populations decreased to that of their counterparts in cauda sperm preparations. It is concluded that immature caput spermatozoa are capable of regulating their volume in a similar fashion to mature cauda spermatozoa but are less able to control their isotonic volume, probably due to poorly controlled sodium and chloride ion transport.

Fibronectin type II-module proteins in the bovine genital tract and their putative role in cell volume control during sperm maturation.

The male reproductive tract of ungulates contains two protein families bearing tandemly arranged fibronectin II (Fn2) modules; one (small Fn2 proteins) bears two modules (e.g. BSP-A1/2), the other (long Fn2 proteins) bears four (e.g. epididymal sperm-binding protein 1 (ELSPBP1)). While it is well known that small Fn2 proteins are present in bull semen, nothing is known about long Fn2 proteins. In the present study, the presence of ELSPBP1 proteins in the bull epididymis and their association with maturing spermatozoa were investigated using a specific antibody against canine ELSPBP1. Analysis of western blots showed ELSPBP1 to be present in the caput, corpus and cauda regions of the epididymis. The protein, which bound phosphorylcholine (PC) strongly, appeared to associate with the spermatozoa during maturation because it was absent from caput spermatozoa but present on cauda spermatozoa. Immunocytochemistry of cauda spermatozoa showed the protein to be bound to the post-acrosomal and midpiece regions. ELSPBP1 could not be detected on freshly ejaculated spermatozoa but was revealed after a capacitating treatment. Our previous studies have shown differences between bovine caput and cauda spermatozoa in terms of their ability to control cell volume. Because of the close homology of BSP-A1/2 PC binding regions with Fn2 regions in ELSPBP1, BSP-A1/2 was used as a model to investigate the effect of a PC-binding Fn2 protein on cell volume control. While the protein had no effect on cauda spermatozoa, it caused caput spermatozoa to swell more in response to hypotonic stress, similarly to untreated cauda spermatozoa.

Chromatin-unstable boar spermatozoa have little chance of reaching oocytes in vivo.

In the present study, the prevalence of chromatin instability in the fertilizing-competent sperm population in the porcine oviduct in vivo was examined through qualitative analysis of the chromatin structure status of accessory boar sperm found in in vivo-derived embryos. The binding of chromatin-unstable sperm to oviductal epithelium in vitro was also studied. To examine the sperm chromatin state, a modified fluorescence microscopic sperm chromatin structure assay was used. Among a population of 173 fertile boars, individuals were selected for according to their chromatin status: 25 animals showed more than 5% of chromatin-unstable sperm in their ejaculates, and 7 showed consistently elevated percentages of chromatin-unstable sperm in three successively collected semen samples. A positive correlation was found between incidence of chromatin instability and attached cytoplasmic droplets (r=0.44, P<0.01). Analyses of accessory spermatozoa from in vivo-derived embryos demonstrated that the proportion of chromatin-unstable sperm was significantly (P<0.05) reduced in the population of fertilizing-competent sperm in the oviduct compared with the inseminated sperm. Populations of sperm bound to the oviduct in vitro had significantly (P<0.05) lower percentages of chromatin instability than in the original diluted semen sample. In conclusion, numbers of sperm with unstable chromatin are reduced in the oviductal sperm reservoir, possibly because of associated changes in the plasma membrane that prevent sperm from binding to the oviductal epithelium. We conclude that in vivo the likelihood that sperm with unstable chromatin will reach the egg and fertilize it is low.