Graduate Thesis Or Dissertation
 

Effect of PFAS Exposure on Reproduction : a Comparative Investigation between Kisspeptin-secreting AVPV and Arcuate Nuclei

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  • Ovulation requires preovulatory surges of gonadotropin-releasing hormone (GnRH) from preoptic hypothalamic neurons, initiated by elevated ovarian estradiol (E₂). Rising estradiol activates a subset of sexually dimorphic kisspeptin (Kiss-1) neurons in the female, located in the anteroventral periventricular nuclei (AVPV). Conversely, estradiol negative feedback on GnRH secretion is mediated by a neuroanatomically separate population of Kiss-1 neurons in the arcuate nuclei. Kisspeptin stimulates GnRH expression and secretion in vivo, and the development of this system is critical for the initiation of puberty. To elucidate how phenotypically similar Kiss-1 neuronal populations react differentially to estradiol exposure, we have generated two immortalized Kiss-1 cell lines from kiss1-GFP post-pubertal female mice. These cell models recapitulate in vivo differential responsiveness to estradiol, with KTaV-3 (AVPV-derived) demonstrating ~6-fold increases in kiss1 expression under higher estradiol doses (5pM – 50pM E₂), while kiss1 expression in KTaR-1 cells is suppressed up to 80% under lower E2 concentrations (2pM – 10pM). We probed temporal patterns of kiss1 and core clock gene expression in these lines in response to estradiol, and found distinct antiphasic patterns of bmal1 and per2 in KTaV-3 cells irrespective of estradiol exposure. Treatment of KTaV-3 cells with 25pM E₂, however, elicited distinct patterns of kiss1 expression over time in contrast to vehicle, suggesting differential coupling of intracellular oscillators to kiss1 transcriptional activity in the presence of estradiol. Further, we have found that expression alterations between nuclear receptor ERα and ERβ genes, esr1 and esr2, respectively, fluctuate divergently between these lines. We have implicated that the peaks of kiss1 expression demonstrated by the KTaV-3 lines may be mediated by both classical and non-classical estrogen signaling. In addition, we provide evidence that the negative regulation of kiss1 expression in KTaR-1 cells may be a function of mutual antagonism due to overabundance of contemporaneously expressed esr1 and esr2 genes that is not observed in KTaV-3 lines. Lastly, we explored the impact of an endocrine disrupting class of perfluorinated alkyl substances (PFASs) on these neurons, with preliminary results illustrating kiss1, esr1, and esr2 transcriptional activation and/or repression at relevant doses of perfluorooctanoic acid (PFOA), perfluorooctanesulfonic acid (PFOS), and perfluorohexanoic acid (PFHxA) in the two lines. At extremely low doses of PFOA, typical estrogenic demonstrations of kiss1 expression are presented by KTaV-3 and KTaR-1 neurons. However at the same dose of PFOS the expression modulation of kiss1 gated by estrogen signaling in this hypothalamic populations is flipped. This implicates that a sufficient exposure to these ubiquitous chemicals can have a potent effect on neuronal expression profiles for estrogen sensitive genes that is a complex function of dose, particular PFAS, and tissue type. Ongoing delineation of responsiveness to estradiol in these lines could reveal novel molecular mechanisms underlying differential expression patterns demonstrated in vivo between these neuronal populations. Furthermore, investigating the impact of select PFASs on secretory dynamics of kisspeptin and on the activity ERα and ERβ between these two cell lines could elucidate the consequence of estrogen mimicry during sex-steroid sensitive developmental phases.
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