Recent evidence has linked human phthalate exposure to abnormal reproductive and hormonal effects. Phthalates are plasticizers that confer flexibility and transparency to plastics, but they readily contaminate the body and the environment. In this study, timed pregnant CD1 outbred mice were treated with di-(2-ethylhexyl) phthalate (DEHP) from Embryonic Day 7 (E7) to E14. The subsequent generation (F1) offspring were then bred to produce the F2, F3, and F4 offspring, without any further DEHP treatment. This exposure scheme disrupted testicular germ cell association and decreased sperm count and motility in F1 to F4 offspring. By spermatogonial transplantation techniques, the exposure scheme also disrupted spermatogonial stem cell (SSC) function of F3 offspring. The W/W V recipient testes transplanted with F3 offspring germ cells from the DEHP-treated group had a dramatically lower percentage of donor germ cellderived spermatogenic recovery in seminiferous tubules when compared to the recipient testes transplanted with CD1 control germ cells. Further characterization showed that the major block of donor germ cell-derived spermatogenesis was before the appearance of undifferentiated spermatogonia. Interestingly, the testes transplanted with the F3 offspring germ cells from the DEHP-treated group, when regenerated, replicated testis morphology similar to that observed in the testes from the F1 to F3 offspring of the DEHP-treated group, suggesting that the germ cell disorganization phenotype originates from the stem cells of F3 offspring. In conclusion, embryonic exposure to DEHP was found to disrupt testicular germ cell organization and SSC function in a transgenerational manner.

Primary Sertoli cells isolated from mouse testes survive when transplanted across immunological barriers and protect cotransplanted allogeneic and xenogeneic cells from rejection in rodent models. In contrast, the mouse Sertoli cell line (MSC-1) lacks immunoprotective properties associated with primary Sertoli cells. In this study, enriched primary Sertoli cells or MSC-1 cells were transplanted as allografts into the renal subcapsular area of naive BALB/c mice, and their survival in graft sites was compared. While Sertoli cells were detected within the grafts with 100% graft survival throughout the 20-day study, MSC-1 cells were rejected between 11 and 14 days, with 0% graft survival at 20 days posttransplantation. Nonetheless, the mechanism for primary Sertoli cell survival and immunoprotection remains unresolved. To identify immune factors or functional pathways potentially responsible for immune privilege, gene expression profiles of enriched primary Sertoli cells were compared with those of MSC-1 cells. Microarray analysis identified 2369 genes in enriched primary Sertoli cells that were differentially expressed at ±4-fold or higher levels than in MSC-1 cells. Ontological analyses identified multiple immune pathways, which were used to generate a list of 340 immune-related genes. Three functions were identified in primary Sertoli cells as potentially important for establishing immune privilege: suppression of inflammation by specific cytokines and prostanoid molecules, slowing of leukocyte migration by controlled cell junctions and actin polymerization, and inhibition of complement activation and membrane-associated cell lysis. These results increase our understanding of testicular immune privilege and, in the long-term, could lead to improvements in transplantation success.

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Exposure to di-(2-ethylhexyl) phthalate (DEHP) has been linked to male reproductive abnormalities. Here, we assessed transgenerational actions of DEHP on several behaviors and stress responses. We used 2 doses of DEHP (150- and 200-mg/kg body weight) and a treatment regimen previously shown to produce transgenerational effects on male reproduction. Mice, 3 generations removed from DEHP exposure (F3), were tested for social behavior and anxiety on the elevated plus maze. We collected blood and pituitaries from undisturbed and restrained mice. Body weights, anogenital distances, and reproductive organ weights were collected at killing. In social interaction tests juvenile males from the DEHP lineage (200 mg/kg) displayed more digging and less self-grooming than did controls. Interestingly, 150-mg/kg lineage males, killed in early puberty, had smaller seminal vesicle weights than their controls. However, the 200-mg/kg males (killed on average 10 d later) did not show this effect. Females from a DEHP lineage had lower corticosterone concentrations than controls after restraint stress. We also found sex- and DEHP-specific mRNA expression changes in the pituitary in 2 of the 6 stress-related genes we measured. In particular, Gnas mRNA was elevated by the combination of DEHP lineage and stress. Thus, transgenerational effects of DEHP are noted in male behavior, and in females, DEHP had transgenerational effects on levels of corticosterone. Both of these results may be related to transgenerational modifications in the expression of several pituitary hormones involved in the hypothalamic-pituitary-adrenal axis.