Altered microRNA expression patterns during the initiation and promotion stages of neonatal diethylstilbestrol-induced dysplasia/neoplasia in the hamster (Mesocricetus auratus) uterus.

Treatment of Syrian hamsters on the day of birth with the prototypical endocrine disruptor and synthetic estrogen, diethylstilbestrol (DES), leads to 100% occurrence of uterine hyperplasia/dysplasia in adulthood, a large proportion of which progress to neoplasia (endometrial adenocarcinoma). Consistent with our prior gene expression analyses at the mRNA and protein levels, we now report (based on microarray, real-time polymerase chain reaction, and in situ hybridization analyses) that progression of the neonatal DES-induced dysplasia/neoplasia phenomenon in the hamster uterus also includes a spectrum of microRNA expression alterations (at both the whole-organ and cell-specific level) that differ during the initiation (upregulated miR-21, 200a, 200b, 200c, 29a, 29b, 429, 141; downregulated miR-181a) and promotion (downregulated miR-133a) stages of the phenomenon. The biological processes targeted by those differentially expressed miRNAs include pathways in cancer and adherens junction, plus regulation of the cell cycle, apoptosis, and miRNA functions, all of which are consistent with our model system phenotype. These findings underscore the need for continued efforts to identify and assess both the classical genetic and the more recently recognized epigenetic mechanisms that truly drive this and other endocrine disruption phenomena.

Soluble Fas ligand activates the sphingomyelin pathway and induces apoptosis in luteal steroidogenic cells independently of stress-activated p38(MAPK).

Fas ligand (FasL) is implicated as a mediator of luteolysis. However, a gap exists in our understanding of the Fas-mediated signaling mechanisms that are involved in either the loss of progesterone production or the structural regression of the corpus luteum. In the present study we investigated the acute and chronic effects of FasL with respect to activation of cytokine/stress-induced signaling pathways and apoptosis in bovine steroidogenic cells. More specifically, we investigated soluble FasL (sFasL)-activated production of ceramide, a second messenger of the sphingomyelin pathway, and activation of p38(MAPK), a member of the MAPK family. sFasL activated the sphingomyelin pathway, as evidenced by a 2-fold increase (P < 0.05) in the production of ceramide. Pretreatment with imipramine (50 micro M), an inhibitor of acid sphingomyelinase activity, attenuated (75%; P < 0.05) sFasL-induced ceramide production, suggesting that the increase in ceramide was partially the result of acid sphingomyelinase-mediated hydrolysis of sphingomyelin. Treatment of luteal cells with sFasL or a cell-permeable ceramide analog (C8) for 24-48 h resulted in a significant increase (P < 0.05) in apoptosis. Western blot analysis revealed that sFasL had little effect on the activation of p38(MAPK) in primary bovine luteal steroidogenic cells. Furthermore, pretreatment with the p38(MAPK) inhibitor SB203580 failed (P > 0.05) to inhibit sFasL- or C8-induced death. Although sFasL did not alter basal progesterone levels detected in the culture medium, C8 caused a significant increase (P < 0.05) in progesterone concentrations within the medium. Collectively, these data suggest that the role of FasL in luteolysis may be to activate the stress-induced sphingomyelin pathway that, in turn, serves as a mediator of apoptosis.

Altered gene expression patterns during the initiation and promotion stages of neonatally diethylstilbestrol-induced hyperplasia/dysplasia/neoplasia in the hamster uterus.

Neonatal treatment of hamsters with diethylstilbestrol (DES) induces uterine hyperplasia/dysplasia/neoplasia (endometrial adenocarcinoma) in adult animals. We subsequently determined that the neonatal DES exposure event directly and permanently disrupts the developing hamster uterus (initiation stage) so that it responds abnormally when it is stimulated with estrogen in adulthood (promotion stage). To identify candidate molecular elements involved in progression of the disruption/neoplastic process, we performed: (1) immunoblot analyses and (2) microarray profiling (Affymetrix Gene Chip System) on sets of uterine protein and RNA extracts, respectively, and (3) immunohistochemical analysis on uterine sections; all from both initiation stage and promotion stage groups of animals. Here we report that: (1) progression of the neonatal DES-induced hyperplasia/dysplasia/neoplasia phenomenon in the hamster uterus involves a wide spectrum of specific gene expression alterations and (2) the gene products involved and their manner of altered expression differ dramatically during the initiation vs. promotion stages of the phenomenon.

Neonatal diethylstilbestrol exposure disrupts female reproductive tract structure/function via both direct and indirect mechanisms in the hamster.

We assessed neonatal diethylstilbestrol (DES)-induced disruption at various endocrine levels in the hamster. In particular, we used organ transplantation into the hamster cheek pouch to determine whether abnormalities observed in the post-pubertal ovary are due to: (a) a direct (early) mechanism or (b) an indirect (late) mechanism that involves altered development and function of the hypothalamus and/or pituitary. Of the various disruption endpoints and attributes assessed: (1) some were consistent with the direct mechanism (altered uterine and cervical dimensions/organization, ovarian polyovular follicles, vaginal hypospadius, endometrial hyperplasia/dysplasia); (2) some were consistent with the indirect mechanism (ovarian/oviductal salpingitis, cystic ovarian follicles); (3) some were consistent with a combination of the direct and indirect mechanisms (altered endocrine status); and (4) the mechanism(s) for one (lack of corpora lutea) was uncertain. This study also generated some surprising observations regarding vaginal estrous assessments as a means to monitor periodicity of ovarian function in the hamster.