Human chorionic gonadotropin (hCG) is a placental hormone produced during pregnancy which regulates in part the development of fetal reproductive tract. hCG is thought to mediate effects of prenatal phthalates on the fetus. We have shown previously that phthalate-hCG and hCG-neonatal genitalia associations are both sex-specific. The aim was to directly measure, within the relevant critical window in the first trimester, modification of hCG regulation by fetal sex and by phthalate levels. Placental tissue samples (N=96, 45% male) were donated by women undergoing elective pregnancy termination at 2 clinics between gestational weeks 7 - 14. Tissues were dissected, snap frozen and stored at -80 C. mRNAs were measured by qPCR: PPARG, CGA, CGB and 2 housekeeping genes, RN18S and UBP1. PPARG encodes a transcription factor that regulates expression of hCG (encoded by CGA and CGB). Quantitation was done by relative standard curve. Sex was determined by measuring 2 Y-linked genes by PCR. All data were normalized, transformed, and analyzed using linear regression. Placental phthalate levels in the same sample set are being analyzed currently. There was no difference in the means of mRNAs between male and female fetuses. We measured a strong interaction of PPARG and fetal sex in the correlation of PPARG and CGA/CGB (p-interaction<0.005). In male placentas, one log10 unit in PPARG was associated with a 2.53 (95% CI 1.80, 3.26) log10 increase in CGA and CGB levels. In female placentas, the same association was null (β= 0.37 log10 units, 95% CI -0.32, 1.05). Associations with placental phthalate levels are forthcoming. These data offer novel insight into the basis for commonly reported sex-specific associations between prenatal phthalates and child health outcomes. hCG offers promise as a molecular biomarker that can conceptually and quantitatively connect sex-specific effects of prenatal exposures on placental mechanisms, and on fetal outcomes.

Expression of c-Fos, or other immediate early gene products, by individual neurons can be used as a marker of cell activation, making staining of these proteins an extremely useful technique for functional anatomical mapping of neuroendocrine systems. Because these proteins are located in the nucleus, identification of the phenotype of the activated neuron using substances located within the cytoplasm can be accomplished with standard double-labeling immunocytochemical techniques. Although it is clear that neurons have the capacity to express a number of immediate early gene products, what remains to be established is whether there is a different pattern of expression following various stimuli. In our studies, we focus primarily on expression of one immediate early gene product, the c-Fos protein. We also include some experiments using expression of other members of the Fos family and Jun proteins as markers for neuronal activation. Our studies describe uses of c-Fos expression in both parvocellular and magnocellular hypothalamic systems to address the following issues: (a) identification of neuroendocrine cells activated by specific treatments and conditions, (b) ascertainment of functional differences in subpopulations activated by specific stimuli, (c) evaluation of neuronal activity in complex areas containing multiple neuroendocrine systems, (d) identification of other brain areas activated in conjunction with neuroendocrine systems following specific stimuli, (e) analysis of connectivity of activated neuroendocrine systems with other parts of the brain, and (f) identification of stimuli that decrease neuronal activity. The neuroendocrine systems studied include those that secrete arginine vasopressin (AVP), oxytocin (OT), corticotropin-releasing hormone (CRH), luteinizing hormone-releasing hormone (LHRH), and dopamine (DA). The use of c-Fos expression has permitted functional neuroanatomical mapping of these systems in response to specific stimuli such as cholecystokinin (CCK), hyperosmolality, and volume depletion, or during various physiological states such as the proestrous ovulatory luteinizing hormone (LH) surge and lactation. Although the use of c-Fos as a marker of neuronal activation will continue to be an extremely powerful technique, future studies will also be directed at relating immediate early gene expression to changes in neuroendocrine gene expression. To this end, we have shown that both c-Fos and c-Jun are expressed in neuroendocrine neurons in response to a number of stimuli, setting the stage for potential regulatory drive to genes containing AP-1 binding sites.

Immediate early genes are rapidly and transiently expressed within neurons following stimulation. While it is likely immediate early gene products alter gene expression in neurons, the products of the Immediate early genes also serve as excellent markers for Identifying activated neurons. The immunocytochemical localization of immediate early gene products enables the analysis of changes In activation of individual neurons in the brain in response to gonadal steroids or other stimuli. The technique presented in this article outlines the strategies for using immediate early gene proteins in the fos family as markers for neuronal activity and focuses principally on the examination of one chemically identified neuron population, the luteinizing hormone-releasing hormone system. Included are comparisons of the staining of brain tissue for c-fos and related antigens following use of different tissue fixatives. Because some of the c-fos antibodies also recognize other proteins in the fos family, we have presented data comparing staining patterns obtained with a number of available antisera. We also describe some of the approaches we use to analyze c-fos expression.