Label-free based quantitative proteomics analysis of primary neonatal porcine Leydig cells exposed to the persistent contaminant 3-methylsulfonyl-DDE

Evidence that persistent environmental pollutants may target the male reproductive system is increasing. The male reproductive system is regulated by secretion of testosterone by testicular Leydig cells, and perturbation of Leydig cell function may have ultimate consequences. 3-Methylsulfonyl-DDE (3-MeSO₂-DDE) is a potent adrenal toxicants formed from the persistent insecticide DDT. Although studies have revealed the endocrine disruptive effect of 3-MeSO₂-DDE, the underlying mechanisms at cellular level in steroidogenic Leydig cells remains to be established. The current study addresses the effect of 3-MeSO₂-DDE on viability, hormone production and proteome response of primary neonatal porcine Leydig cells. The AlamarBlue™ assay was used to evaluate cell viability. Solid phase radioimmunoassay was used to measure concentration of hormones produced by both unstimulated and Luteinizing hormone (LH)-stimulated Leydig cells following 48h exposure. Protein samples from Leydig cells exposed to a non-cytotoxic concentration of 3-MeSO₂-DDE (10μM) were subjected to nano-LC-MS/MS and analyzed on a Q Exactive mass spectrometer and quantified using label-free quantitative algorithm. Gene Ontology (GO) and Ingenuity Pathway Analysis (IPA) were carried out for functional annotation and identification of protein interaction networks. 3-MeSO₂-DDE regulated Leydig cell steroidogenesis differentially depending on cell culture condition. Whereas its effect on testosterone secretion at basal condition was stimulatory, the effect on LH-stimulated cells was inhibitory. From triplicate experiments, a total of 6804 proteins were identified in which the abundance of 86 proteins in unstimulated Leydig cells and 145 proteins in LH-stimulated Leydig cells was found to be significantly regulated in response to 3-MeSO₂-DDE exposure. These proteins not only are the first reported in relation to 3-MeSO₂-DDE exposure, but also display small number of proteins shared between culture conditions, suggesting the action of 3-MeSO₂-DDE on several targeted pathways, including mitochondrial dysfunction, oxidative phosphorylation, EIF2-signaling, and glutathione-mediated detoxification. Further identification and characterization of these proteins and pathways may build our understanding to the molecular basis of 3-MeSO₂-DDE induced endocrine disruption in Leydig cells.

Proposed role for COUP-TFII in regulating fetal Leydig cell steroidogenesis, perturbation of which leads to masculinization disorders in rodents

Reproductive disorders that are common/increasing in prevalence in human males may arise because of deficient androgen production/action during a fetal 'masculinization programming window'. We identify a potentially important role for Chicken Ovalbumin Upstream Promoter-Transcription Factor II (COUP-TFII) in Leydig cell (LC) steroidogenesis that may partly explain this. In rats, fetal LC size and intratesticular testosterone (ITT) increased ~3-fold between e15.5-e21.5 which associated with a progressive decrease in the percentage of LC expressing COUP-TFII. Exposure of fetuses to dibutyl phthalate (DBP), which induces masculinization disorders, dose-dependently prevented the age-related decrease in LC COUP-TFII expression and the normal increases in LC size and ITT. We show that nuclear COUP-TFII expression in fetal rat LC relates inversely to LC expression of steroidogenic factor-1 (SF-1)-dependent genes (StAR, Cyp11a1, Cyp17a1) with overlapping binding sites for SF-1 and COUP-TFII in their promoter regions, but does not affect an SF-1 dependent LC gene (3β-HSD) without overlapping sites. We also show that once COUP-TFII expression in LC has switched off, it is re-induced by DBP exposure, coincident with suppression of ITT. Furthermore, other treatments that reduce fetal ITT in rats (dexamethasone, diethylstilbestrol (DES)) also maintain/induce LC nuclear expression of COUP-TFII. In contrast to rats, in mice DBP neither causes persistence of fetal LC COUP-TFII nor reduces ITT, whereas DES-exposure of mice maintains COUP-TFII expression in fetal LC and decreases ITT, as in rats. These findings suggest that lifting of repression by COUP-TFII may be an important mechanism that promotes increased testosterone production by fetal LC to drive masculinization. As we also show an age-related decline in expression of COUP-TFII in human fetal LC, this mechanism may also be functional in humans, and its susceptibility to disruption by environmental chemicals, stress and pregnancy hormones could explain the origin of some human male reproductive disorders.

A switch in the cellular localization of macrophage migration inhibitory factor in the rat testis after ethane dimethane sulfonate treatment.

Macrophage migration inhibitory factor (MIF), one of the first cytokines to be discovered, has recently been localized to the Leydig cells in adult rat testes. In the following study, the response of MIF to Leydig cell ablation by the Leydig cell-specific toxin ethane dimethane sulfonate (EDS) was examined in adult male rats. Testicular MIF mRNA and protein in testicular interstitial fluid measured by ELISA and western blot were only marginally reduced by EDS treatment, in spite of the fact that the Leydig cells were completely destroyed within 7 days. Immunohistochemistry using an affinity-purified anti-mouse MIF antibody localized MIF exclusively to the Leydig cells in control testes. At 7 days post-EDS treatment, there were no MIF immunopositive Leydig cells in the interstitium, although distinct MIF immunostaining was observed in the seminiferous tubules, principally in Sertoli cells and residual cytoplasm, and some spermatogonia. A few peritubular and perivascular cells were also labelled at this time, which possibly represented mesenchymal Leydig cell precursors. At 14 and 21 days, Sertoli cell MIF immunoreactivity was observed in only a few tubule cross-sections, while some peritubular and perivascular mesenchymal cells and the re-populating immature Leydig cells were intensely labeled. At 28 days after EDS-treatment, the MIF immunostaining pattern was identical to that of untreated and control testes. The switch in the compartmentalization of MIF protein at 7 days after EDS-treatment was confirmed by western blot analysis of interstitial tissue and seminiferous tubules separated by mechanical dissection. These data establish that Leydig cell-depleted testes continue to produce MIF, and suggest the existence of a mechanism of compensatory cytokine production involving the Sertoli cells. This represents the first demonstration of a hitherto unsuspected pattern of cellular interaction between the Leydig cells and the seminiferous tubules which is consistent with an essential role for MIF in male testicular function.

Localization of follistatin in the rat testis.

The cellular localization of the activin-binding protein, follistatin, in the rat testis has been a matter of some controversy with different investigators claiming that Sertoli cells, Leydig cells or germ cells are the primary cell types containing this protein. The localization of mRNA encoding follistatin was re-examined using reverse transcription-polymerase chain reaction (RT-PCR) and in situ hybridization as well as the distribution of follistatin by immunohistochemistry. The results demonstrate that mRNA encoding follistatin is located in many germ cells including type B spermatogonia, primary spermatocytes with the exception of the late leptotene and early zygotene stages, and spermatids at steps 1 to 11. It is also found in Sertoli cells and endothelial cells but not in Leydig cells. Immunohistochemistry, using two different antisera to follistatin, showed that this protein was localized to spermatogonia, primary spermatocytes at all stages except the zygotene stage, spermatids at all stages and to endothelial cells and Leydig cells in the intratubular regions. The failure to detect mRNA for follistatin in Leydig cells using RT-PCR and in situ hybridization suggests that the immunohistochemical localization in these cells reflects binding of follistatin produced elsewhere. The widespread localization of follistatin, taken together with its capacity to neutralize the actions of activin, may indicate that follistatin modulates a range of testicular actions of activin, many of which remain unknown.