The molecular mechanisms underlying normal and pathological spermatogenesis remain poorly understood. We compared protein concentrations in different germ cell types to identify those proteins specifically or preferentially expressed at each stage of rat spermatogenesis. Crude cytosolic protein extracts and reversed-phase HPLC prefractionated cytosolic extracts from spermatogonia, pachytene spermatocytes, and early spermatids were subjected to two-dimensional difference gel electrophoresis (2-D DIGE). By comparing gels and carrying out statistical analyses, we were able to identify 1274 protein spots with relative abundances differing significantly between the three cell types. We found that 265 of these spots displaying highly differential expression (ratio > or = 2.5 between two cell types), identified by mass fingerprinting, corresponded to 123 nonredundant proteins. The proteins clustered into three clades, corresponding to mitotic, meiotic, and post-meiotic cell types. The differentially expressed proteins identified by 2-D DIGE were confirmed and validated by western blotting and immunohistochemistry, in the few cases in which antibodies were available. 2-D DIGE appears a relevant proteomics approach for studying rat germ cell differentiation, allowing the establishment of the precise expression profiles for a relatively large number of proteins during normal spermatogenesis.

Combinations of therapeutic agents could synergistically enhance the response of lung cancer cells. Co-delivery systems capable of transporting chemotherapeutics with different physicochemical properties and with the simultaneous release of drugs remain elusive. Here, we assess the ability of nanoparticles of 30-nm diameter obtained from the self-assembly of hyaluronan-based copolymer targeting CD44 receptors to encapsulate both gefitinib and vorinostat for effective combinational lung cancer treatment. Drug loading was performed by nanoprecipitation. Drug release experiments showed a slow release of both drugs after 5 days. Using two- and three-dimensional lung adenocarcinoma cell cultures, we observed that the nanoparticles were mostly found at the periphery of the CD44-expressing spheroids. These drug-loaded nanoparticles were as cytotoxic as free drugs in the two- and three-dimensional systems and toxicity was due to apoptosis induction. In mouse models, intravenous injection of hyaluronan-based nanoparticles showed a selective delivery to subcutaneous CD44-overexpressing tumors, despite a significant liver capture. In addition, the systemic toxicity of the free drugs was reduced by their co-delivery using the nanoparticles. Finally, intrapulmonary administration of drug-loaded nanoparticles, to avoid a possible hepatic toxicity due to their accumulation in the liver, showed a stronger inhibition of orthotopic lung tumor growth compared to free drugs. In conclusion, hyaluronan-based nanoparticles provide active targeting partially mediated by CD44, less-toxic drug release and improved antitumor efficiency.