Covalently functionalized Laponite clay was synthesized through a condensation reaction of the clay's silanol groups with mono- and trifunctional alkoxy silanes. Most of the work focuses on primary-amine-containing modifications because that group offers a wide range of derivitization options. Various 3-aminopropyltrimethoxy silane (APS) treatments yielded 4−14 wt % of organic material bound to the clay. APS-treated clay was further reacted to yield attached methacrylate, benzophenone, and tertiary bromine groups capable of polymerization, photoinitiation, and atom transfer radical polymerization initiation, respectively. Studies using a monoalkoxy analogue of APS, aminopropyldimethylethoxysilane (APES), are consistent with the hypothesis that multifunctional alkoxy silanes can cause the clay sheets to link together, hindering the clay's dispersibility and the efficiency of subsequent surface ion-exchange reactions. The attached amines were further reacted to 40 and 80% conversion in the APS- and APES-treated Laponites. Quantification studies show that there are about 200 siloxane-reactive sites per clay sheet at a concentration of 11 mequiv per 100 g of clay. All products were characterized with thermogravimetric analysis and solid-state 13C NMR to study the organic content and composition. 29Si solid-state NMR was also used to compare the treated clay with untreated sodium Laponite. Changes in the 29Si spectra were consistent with the expected silanol reaction.
Although studies of both humans and animals suggest detrimental effects of psychological (restraint) stress on reproduction, reports concerning the direct effect of psychological (restraint) stress on the oocyte are few and conflicting. In the present study, a restraint system that allows mice free intake of feed and water while restraining their movement was established, and effects of maternal restraint on oocyte competence were examined by observing embryo development in vitro and in vivo. The results indicated that restraint stress applied to both gonadotropin-stimulated and unstimulated females during oocyte growth and maturation increased their plasma cortisol level but impaired ovulation and oocyte developmental potential. Injection of cortisol also decreased oocyte developmental potential in both stimulated and unstimulated mice. However, whereas restraint stress reduced the plasma follicle-stimulating hormone (FSH) level of unstimulated mice, injection of cortisol did not. Because the stimulated mice had received very high doses of FSH and luteinizing hormone from injection with equine chorionic gonadotropin injection, the results suggested that whereas cortisol acts directly on the ovary to damage the oocyte, restraint stress impairs oocyte competence by actions on both the hypothalamic-pituitary-gonadal and the hypothalamic-pituitary-adrenal axes. However, exposing the cumulus-oocyte complexes (COCs) to physiological levels of cortisol did not affect oocyte nuclear and cytoplasmic maturation in vitro. Thus, cortisol might have impaired ovulation and oocyte potential by an indirect effect on ovarian tissues other than the COCs.
Laponite clay was modified with combinations of organic ammonium surfactant and/or covalently bound poly(methyl methacrylate) (PMMA). Two polymer attachment methods were explored, one through reaction of a methacrylate compound with the clay's silanol group followed by in situ free-radical polymerization of methyl methacrylate (MMA), and the other through attachment of an ATRP initiator followed by brush polymerization. The free-radical method yielded clays with ca. 75 wt % of polymer bound though multiple attachment sites to the clay, whereas the ATRP method yielded ca. 68 wt % of bound polymer attached only at the chain end. The PMMA-modified clays were very dispersible in organic solvents and were solvent-blended with commercial PMMA at 1, 3, 5, and 10 wt % concentrations. The resulting nanocomposites were optically transparent and homogeneous. TEM images showed mixed intercalated and exfoliated dispersions. DMA analysis showed an increase in room temperature modulus of 50% at 5 wt % concentration for the clay with no surfactant and PMMA free-radical attachment.
Inhibiting oocyte aging is important not only for healthy reproduction but also for the success of assisted reproduction techniques. Although our previous studies showed that cumulus cells accelerated aging of mouse oocytes, the underlying mechanism is unknown. The objective of this paper was to study the effects of pyruvate and cumulus cells on mouse oocyte aging. Freshly ovulated mouse cumulus-oocyte complexes (COCs) or cumulus-denuded oocytes (DOs) were cultured in Chatot-Ziomek-Bavister (CZB) medium or COC-conditioned CZB medium supplemented with different concentrations of pyruvate before being examined for aging signs and developmental potential. Pyruvate supplementation to CZB medium decreased rates of ethanol-induced activation in both COCs and DOs by maintaining their maturation-promoting factor activities, but more pyruvate was needed for COCs than for DOs. Addition of pyruvate to the COC-conditioned CZB also alleviated aging of DOs. Observations on cortical granules, level of BCL2 proteins, histone acetylation, intracellular concentration of glutathione, and embryo development all confirmed that pyruvate supplementation inhibited aging of mouse oocytes. It is concluded that the aging of mouse oocytes, facilitated by culture in COCs, can be partially prevented by the addition of pyruvate to the culture medium.
The objectives of this study were to investigate the effect of heat stress during in vitro maturation on the developmental potential of mouse oocytes and to determine whether the deleterious effect was on the nuclear or cytoplasmic component. While rates of oocyte nuclear maturation (development to the metaphase II stage) did not differ from 37 to 40 8C, rates for blastocyst formation decreased significantly as maturation temperature increased from 38.5 to 39 8C. Chromosome spindle exchange showed that while blastocyst formation did not differ when spindles matured in vivo or in vitro at 37, 40 or 40.7 8C were transplanted into in vivo matured cytoplasts, no blastocyst formation was observed when in vivo spindles were transferred into the 40 8C cytoplasts. While oocytes reconstructed between 37 8C ooplasts and 37 or 40 8C karyoplasts developed into 4-cell embryos at a similar rate, no oocytes reconstituted between 40 8C ooplasts and 37 8C spindles developed to the 4-cell stage. Immunofluorescence microscopy revealed impaired migration of cortical granules and mitochondria in oocytes matured at 40 8C compared with oocytes matured at 37 8C. A decreased glutathione/GSSG ratio was also observed in oocytes matured at 40 8C. While spindle assembling was normal and no MAD2 was activated in oocytes matured at 37 or 40 8C, spindle assembling was affected and MAD2 was activated in some of the oocytes matured at 40.7 8C. It is concluded that 1) oocyte cytoplasmic maturation is more susceptible to heat stress than nuclear maturation, and 2) cytoplasmic rather than nuclear components determine the pre-implantation developmental capacity of an oocyte.
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