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In this work, we report the design and application of a new ratiometric fluorescent probe, which contains different-colored quantum dots (QDs) as dual fluorophores, ultrathin silica shell as spacer, and meso-tetra(4-sulfonatophenyl)porphine dihydrochloride (TSPP) as receptor, for Zn(2+) detection in aqueous solution and living cells. In the architecture of our designed probe, the silica shell plays the key roles in controlling the locations of QDs, TSPP, and Zn(2+), preventing the direct contact between QDs and Zn(2+) but affording fluorescence resonance energy transfer (FRET) from dual-color QDs to TSPP. In the presence of Zn(2+), the analyte-receptor reaction changes the absorption in the range of the Q-band of TSPP and accordingly the efficiencies of two independent FRET processes from the dual-colored QDs to the acceptor, respectively, leading to fluorescence enhancement of green-emission QDs whereas fluorescence quenching of yellow-emission QDs. Benefiting from the well-resolved dual emissions from different-colored QDs and the large range of emission ratios, the probe solution displays continuous color changes from yellow to green, which can be clearly observed by the naked eye. Under physiological conditions, the probe exhibits a stable response for Zn(2+) from 0.3 to 6 μM, with a detection limit of 60 nM in aqueous solutions. With respect to single-emission probes, this ratiometric probe has demonstrated to feature excellent selectivity for Zn(2+) over other physiologically important cations such as Fe(3+) and Cu(2+). It has been preliminarily used for ratiometric imaging of Zn(2+) in living cells with satisfying resolution.

SUMMARYObesity, an increasingly frequent societal disease can also be accompanied by declines in spermatozoa quality and male subfecundity. To determine if there are obesity-associated proteomic changes potentially affecting sperm quality and motility, differential proteomic analysis was performed on spermatozoa from both obesity-associated asthenozoospermia and clinically healthy individuals, using a label-free quantitative LC-MS/MS approach. We resolved 1975 proteins in the human sperm proteome, amongst which, 105 proteins were less abundant, whereas 22 other proteins increased in obesity-associated asthenozoospermia. Functional category analyses indicated that the differentially expressed proteins are mainly related to cytoskeletal regulation, vesicle biogenesis, metabolism, and protein degradation involved in spermiogenesis and sperm motility. Furthermore, declines in endoplasmic reticulum protein 57 (ERp57) and actin-binding-related protein T2 (ACTRT2) expression were verified by immunofluorescence, Western blot, and flow cytometry analyses. It is evident that ERp57 is localized in the acrosome region, neck and principal piece of human spermatozoa, whereas ACTRT2 is localized in the post-acrosomal region and middle piece. Thus, these differences in protein expression in asthenozoospermia may contribute to the underlying sperm quality defects afflicting these individuals. Notably, declines in ERp57 and ACTRT2 expression in obesity-associated asthenozoospermia may play critical roles in reducing sperm motility.

Sperm motility is essential for male reproduction or natural fertilization. The cyclic AMP (cAMP)/cAMP-dependent protein kinase A (PKA) signaling pathway is generally recognized as one of the significant signaling pathways in the regulation of mammalian spermatozoan motility. Since Zn-a2-glycoprotein (ZAG) activity in mammalian adipose tissue is mediated via the b 3 -adrenoreceptor, with upregulation of the cAMP pathway, we hypothesize that ZAG may play the same role in sperm motility regulation, a new factor of regulation of sperm motility. Therefore, the gene encoding human ZAG was cloned and polyclonal antibodies were generated, and then laser scanning confocal microscopy and flow cytometry were employed to identify this protein in human spermatozoa. The results showed that ZAG protein was mostly localized on the pre-equatorial region covering the acrosome, neck, and middle piece of the flagellum of spermatozoa. Furthermore, using computer-assisted sperm analysis, we found that anti-human ZAG antibodies could significantly reduce the motility of human swim-up spermatozoa after 90-or 120-min incubation (P!0.05 and P!0.01 respectively), together with the decreasing of intracellular cAMP and PKA levels. In conclusion, these data suggest that ZAG is present in human spermatozoa and may be involved in the regulation of sperm motility via the cAMP/PKA signaling pathway.