Supplementary Material for: Expression of Kisspeptin in Gonadotrope Precursors in the Mouse Pituitary during Embryonic and Postnatal Development and in Adulthood

<p><b><i>Background:</i></b> Kisspeptins are important regulators of the development and function of the hypothalamic-pituitary-gonadal axis. However, the importance of kisspeptin at the pituitary level is unclear. <b><i>Methods:</i></b> We examined the expression profile of kisspeptin in the mouse pituitary during development and in adulthood using RT-PCR, quantitative PCR and immunohistochemistry.<b><i> Results:</i></b> Kiss1 mRNA was detected in both embryonic and postnatal pituitaries. Kisspeptin-immunoreactive (<sup>+</sup>) cells were detected from embryonic day (E) 13.5 throughout adulthood, being localized to the rostroventral portion in the anterior pituitary (AP) in embryos, and also to the dorsocaudal AP postnatally. A large proportion of kisspeptin<sup>+</sup> cells were double-labeled with gonadotrope markers including Foxl2, SF-1, and LHβ, and the percentage of LHβ<sup>+</sup> cells in kisspeptin<sup>+</sup> cells increased during development. No kisspeptin<sup>+</sup> cells were positive for the proliferating cell marker MCM7 (minichromosome maintenance protein 7), but a few kisspeptin<sup>+</sup> cells co-expressed the stem/progenitor cell marker Sox2. Kisspeptin expression was similar between sexes and between agonadal SF-1 knockout embryos and wild-type littermates. Kiss1 mRNA levels were not significantly different between sexes or during early postnatal development, but levels in females increased when puberty began and were significantly higher than in males at postpubertal ages. <b><i>Conclusions:</i></b> These results suggest that kisspeptin is expressed in gonadotrope precursors during gonadotrope differentiation, and that kisspeptin expression begins soon after the initiation of αGSU production and is extinguished soon after the initiation of LH production. Furthermore, pituitary kisspeptin expression may be regulated in a gonad-independent manner during development, but may be associated with gonadotrope function in adulthood.</p>