Background-Premenopausal women treated for early stage breast cancer (ESBC) are at risk for chemotherapy-related amenorrhea (CRA). Prospectively-validated, predictive markers of CRA are needed.

We have identified 7-ketocholesterol (7-KC) as an endogenous modulator that inhibits transactivation by the arylhydrocarbon receptor (

Maternal smoking during pregnancy is associated with a variety of adverse neonatal outcomes including altered reproductive performance. Herein we provide molecular evidence for a pathway involved in the elimination of the female germline due to prepregnancy and/or lactational exposure to polycyclic aromatic hydrocarbons (PAHs), environmental toxicants found in cigarette smoke. We show that ovaries of offspring born to mice exposed to PAHs contained only a third of the ovarian follicle pool compared with offspring of unexposed female mice. Activation of the cell death pathway in immature follicles of exposed females was mediated by the aryl hydrocarbon receptor (Ahr), as ovarian reserve was fully rescued by maternal cotreatment with the Ahr antagonist, resveratrol, or by inactivation of the Ahr gene. Furthermore, in response to PAHs, Ahr-mediated activation of the harakiri, BCL2 interacting protein (contains only BH3 domain), was necessary for execution of cell death. This pathway appeared to be conserved between mouse and human, as xenotransplanted human ovarian cortex exposed to PAHs responded by activation of the identical cell death cascade. Our data indicate that maternal exposure to PAHs prior to pregnancy and/or during lactation compromises ovarian reserve of female offspring, raising the concern about the transgenerational impact of maternal smoking on ovarian function in the human.

Improved culture conditions that support the development of human embryos to the blastocyst stage in vitro led to the prospect of blastocyst transfer to increase pregnancy rates. Thus, there is a need for characterization of possible biochemical markers able to predict the implantation potential of human blastocysts. In this study, the expression of three placental markers that are expressed prior to implantation, beta-human chorionic gonadotrophin (HCG), human leukocyte antigen (HLA)-G and pregnancy specific beta-1 glycoprotein (SP-1), was investigated. beta-HCG transcript could be detected as early as the two-cell stage, which is one to two cleavage divisions earlier than previously reported. Both beta-HCG and HLA-G transcripts could be detected in the majority of blastocysts, but their levels were highly variable. No association could be found between the amount of transcript for these genes, total cell number or cell death rate. Interestingly, there was a highly positive correlation between accumulation of beta-HCG and HLA-G transcripts. SP-1 protein concentrations were assessed in the culture medium of blastocysts using enzyme-linked immunosorbent assay. There was a significant positive correlation between SP-1 concentrations and blastocyst cell numbers. Moreover, synthetic oviductal medium enriched with potassium resulted in an SP-1 concentration twice as high as that observed using human tubal fluid medium. These data suggest that SP-1 may be used to select blastocysts with higher cell number, possibly resulting in higher pregnancy rates.

Unlike most cytochrome P450 (CYP) enzymes, murine hepatic CYP2A5 is induced during pathological conditions that result in liver injury including hepatotoxicity mediated by xenobiotics, hepatitis caused by various microbial agents and liver neoplasia. Since CYP2A5 metabolizes various important xenobiotics including nicotine and pro-carcinogens such as nitrosamines and aflatoxin B(1), altered gene expression could affect tobacco addiction, hepatotoxicity and hepatocarcinogenesis. This article synthesizes the current knowledge concerning hepatic expression of Cyp2a5 including the transcriptional and post-transcriptional regulatory mechanisms, pathophysiological conditions associated with enzyme induction such as oxidative and endoplasmic reticulum stress and altered lipid and energy homeostasis as well as the known exogenous and putative endogenous substrates. Knowledge of the stimuli responsible for the unique overexpression of CYP2A5 during liver injury may provide clues to a functional role for this enzyme and the impact of variable CYP2A5 expression on xenobiotic metabolism and toxicity, disease development and the adaptive response to hepatocellular stress.

There is critical demand in contemporary medicine for gene expression markers in all areas of human disease, for early detection of disease, classification, prognosis, and response to therapy. The integrity of circadian gene expression underlies cardiovascular health and disease; however time-of-day profiling in heart disease has never been examined. We hypothesized that a time-of-day chronomic approach using samples collected across 24-h cycles and analyzed by microarrays and bioinformatics advances contemporary approaches, because it includes sleep-time and/or wake-time molecular responses. As proof of concept, we demonstrate the value of this approach in cardiovascular disease using a murine Transverse Aortic Constriction (TAC) model of pressure overload-induced cardiac hypertrophy in mice. First, microarrays and a novel algorithm termed DeltaGene were used to identify time-of-day differences in gene expression in cardiac hypertrophy 8 wks post-TAC. The top 300 candidates were further analyzed using knowledge-based platforms, paring the list to 20 candidates, which were then validated by real-time polymerase chain reaction (RTPCR). Next, we tested whether the time-of-day gene expression profiles could be indicative of disease progression by comparing the 1- vs. 8-wk TAC. Lastly, since protein expression is functionally relevant, we monitored time-of-day cycling for the analogous cardiac proteins. This approach is generally applicable and can lead to new understanding of disease.

The biosynthetic pathway governing inhibin heterodimer (alpha/beta) and activin homodimer (beta/beta) assembly and secretion from ovarian granulosa cells is not fully understood. Here, we examined the role of inhibin subunit glycosylation in the assembly and secretion of mature inhibin A and activin A. Inhibition of subunit glycosylation by tunicamycin treatment of alpha- and beta(A)-expressing CHO cell lines reduced inhibin but not activin secretion. Dimeric inhibin A is preferentially secreted from parental isogenic wild-type (wt) cell lines (alpha(wt)beta(wt)). Mutation of a single glycosylation site at asparagine 268 (alpha(Delta268)beta(wt)) reduces inhibin secretion by 78% and permits beta/beta assembly and secretion. Conversely, gain of a glycosylation (GOG) site in the analogous region of the beta(A)-subunit (alpha(wt)beta(GOG327)) enhances inhibin A secretion. The present study demonstrates that N-linked glycan sites direct heterodimer vs. homodimer assembly, and prevention of glycosylation abrogates inhibin secretion. These data support a definitive role for site-specific N-glycosylation in governing inhibin/activin dimer assembly and secretion.

Elevated activin A levels in serum, cyst fluid, and peritoneal fluid of ovarian cancer patients suggest a role for this peptide hormone in disease development. We hypothesize that activin A plays a role in ovarian tumor biology, and analyzed activin-mediated pro-oncogenic signaling in vitro and the expression of activin signaling pathway molecules in vivo. Activin A regulation of Akt and GSK, and the effects of repressing the activities of these molecules (with pharmacological inhibitors) on cellular proliferation were assessed in the cell line, OVCA429. Activin A activated Akt, which phosphorylated GSK, repressing GSK activity in vitro. Activin A stimulated cellular proliferation and repression of GSK augmented activin-regulated proliferation. To validate in vitro observations, immunostaining of the betaA-subunit of activin A and phospho-GSKalpha/beta (Ser9/21) was performed, and the correlation between immunoreactivity levels of these markers and survival was evaluated in benign serous cystadenoma, borderline tumor, and cystadenocarcinoma microarrays. Analysis of tissue microarrays revealed that betaA expression in epithelia did not correlate with survival or malignancy, but expression was elevated in stromal cells from carcinomas when compared with benign tumors. Phospho-GSKalpha/beta (Ser9/21) staining was more intense in mitotically active carcinoma cells and exhibited a polarized localization in benign neoplasms that was absent in carcinomas. Notably, lower phospho-GSKalpha/beta (Ser9/21) immunoreactivity correlated with better survival for carcinoma patients (P=0.046). Our data are consistent with a model in which activin A may mediate ovarian oncogenesis by activating Akt and repressing GSK to stimulate cellular proliferation.