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    • GT cells were treated in two ways

    2021-12-02

    GT1-7 bud were treated in two ways; specifically, they were either dosed or transfected with VP, reflecting the extracellular and intracellular actions of VP, respectively. A dose of 200 pg/ml VP in culture supernatants was effective in stimulating GnRH in the supernatant and cell extracts; this concentration was close to the concentration of VP in the rat serum of the SO group. After preliminarily investigating the effects of VP on the transcription of GnRH and some relevant transcription factors, including Oct-1 and Otx2, which have been shown to bind to the regulatory regions of the GnRH gene and boost GnRH neuron pulse generation [29], the experiments demonstrate that extracellular administration of VP is more effective in stimulating GnRH and relevant transcription factors than transfecting the cells with VP. We surmise that the presence of paracrine-generated or projected VP in close proximity to GnRH neurons may promote the synthesis and secretion of GnRH. The evidence also suggests that the stimulation effects of VP mostly occur through extracellular stimulation, but it is not clear whether VP can act through presynaptic release, although there is a possibility that VP could enter GnRH neurons, considering the intracytoplasmic-positive PLA signal in neurons of the Arc. A series of cell experiments were conducted to study the optimum concentration of extracellular VP promoting GnRH synthesis. Significant dose-effect correlations were detected between supernatant GnRH and VP administration and between VP and intracellular Ca2+, indicating that the effective concentration range of extracellular VP is 25–200 pg/ml. The time-effect correlation indicates the promoting effects of VP on GnRH neurons achieving their apex levels at 6 h post-treatment, lasting approximately 3 h. The phenomenon indicates that the stimulation of VP on GnRH secretion is not immediate; rather, it is durative. Most GnRH neurons generate [Ca2+]i transients throughout development, and calcium influx has been indicated to act as vital facilitators of GnRH secretion. As for the results showing the effects of 200 pg/ml extracellular VP on promoting GT1-7 proliferation and viability, as well as intracellular Ca2+, stimulation on intracellular Ca2+ may be one of the key mechanisms of extracellular VP on GnRH secretion. VP in concentrations as low as 25 pg/ml could increase cell viability, and VP in concentrations as low as 50 pg/ml could increase cell proliferation. While only VP concentrations higher than 50 pg/ml promoted GnRH transcription and promoter activity, and higher than 200 pg/ml VP showed effective on stimulating intracellular Ca2+. These results indicate the promotion on cell viability which is one index of energy mechanisms may be one of the early processes of the effects of VP on GT1-7 cells, and the promotion on intracellular Ca2+ may be partially responsible for the stimulatory effects of 200 pg/ml VP. In addition, the in vitro experiments showed VP in concentrations higher than 400 pg/ml decreased the cell proliferation, and VP in concentrations higher than 2000 pg/ml decreased the intracellular ATP. While VP in concentrations higher than 7000 pg/ml increased Ca2+, which may indicate some cytotoxicity of high concentration of extracellular VP on GT1-7 cells. As for the sensitive changes of intracellular ATP and Ca2+ in accordance with concentration of VP, we speculate that extracellular VP upregulates the synthesis of GnRH through activating energy metabolism and increasing intracellular Ca2+, and further experiments will be conducted to investigate the definite effects and mechanisms of extracellular VP on [Ca2+]i transients and energy metabolism. It is worth noting that VP concentrations > 1000 pg/ml decreased GnRH transcription and secretion without decreasing the expression levels of relevant transcription factors, suggesting that the upregulation of Oct-1 and Otx2 may facilitate the activation of GT1-7 cells but is not crucial for the GnRH stimulatory effects of extracellular VP. In summary, the effects of VP on the synthesis activity of GT1-7 cells based on histomorphological clues of direct correlations were reported for the first time in this study, but the concrete underlying mechanisms have not yet been revealed. These mechanisms will be the research focus of future studies.