Effects of di-n-butyl phthalate on male rat reproduction following pubertal exposure

Di-n-butyl phthalate （DBP） is an endocrine-disrupting chemical that has the potential to affect male reproduction. However, the reproductive effects of low-dose DBP are still not well known, especially at the molecular level. In the present study, pubertal male Sprague-Dawley rats were orally administered DBP at a wide range of doses （0.1, 1.0, 10, 100 and 500 mg kg^-1 day^-1） for 30 days. The selected end points included reproductive organ weights, testicular histopathology and serum hormonal levels. Additionally, proteomic analysis was performed to identify proteins that are differentially expressed as a result of exposure to DBP at low doses （0.1, 1.0 and 10 mg kg^-1 day^-1）. Toxic effects were observed in the high-dose groups, including anomalous development of testes and epididymides, severe atrophy of seminiferous tubules, loss of spermatogenesis and abnormal levels of serum hormones. Treatment with low doses of DBP seemed to exert a ＇stimulative effect＇ on the serum hormones. Proteomics analysis of rat testes showed 20 differentially expressed proteins. Among these proteins, alterations in the expression of HnRNPA2/B1, vimentin and superoxide dismutase 1 （SOD1） were further confirmed by Western blot and immunohistochemistry. Taken together, we conclude that high doses of DBP led to testicular toxicity, and low doses of DBP led to changes in the expression of proteins involved in spermatogenesis as well as changes in the number and function of Sertoli and Leydig cells, although no obvious morphological changes appeared. The identification of these differentially expressed proteins provides important information about the mechanisms underlying the effects of DBP on male rat reproduction.

The role of ezrin-associated protein network in human sperm capacitation

Membrane modifications in sperm cells represent a key step in sperm capacitation; however, the molecular basis of these modifications is not fully understood. Ezrin is the best-studied member of the ezrin/radixin/merlin family. As a cross-linker between the cortical cytoskeleton and plasma membrane proteins, ezrin contributes to remodeling of the membrane surface structure. Furthermore, activated ezrin and the Rho dissociation inhibitor, RhoGDI, promote the formation of cortical cytoskeleton-polymerized actin through Rho activation. Thus, ezrin, actin, RhoGDI, Rho and plasma membrane proteins form a complicated network in vivo, which contributes to the assembly of the structure of the membrane surface. Previously, we showed that ezrin and RhoGDI1 are expressed in human testes. Thus, we sought to determine whether the ezrin-RhoGDIl-actin-membrane protein network has a role in human sperm capacitation. Our results by Western blot indicate that ezrin is activated by phosphorylation of the threonine567 residue during capacitation. Co-immunoprecipitation studies revealed that, during sperm capacitation, the interaction between ezrin and RhoGDI1 increases, and phosphostaining of two dimensional electrophoresis gels showed that RhoGDI 1 is phosphorylated, suggesting that RhoGDI 1 dissociates from RhoA and leads to actin polymerization on the sperm head. We speculate that activated ezrin interacts with polymerized actin and the glycosylated membrane protein cd44 after capacitation. Blocking sperm capacitation using ezrin- or actin-specific monoclonal antibodies decreases their acrosome reaction （AR） rate, but has no effect on the AR alone. Taken together, our results show that a network consisting of ezrin, RhoGDI1, RhoA, F-actin and membrane proteins functions to influence the modifications that occur on the membrane of the sperm head during human sperm capacitation.