The role of stem/progenitor cells (hereafter referred to as stem cells) in the development of the embryonic pituitary is well established (Perez Millan et al., 2024; Alatzoglou et al., 2020). In 2005, initial evidence was also provided that the adult pituitary contains a putative stem cell population (Chen et al., 2005). A series of studies supporting this hypothesis followed (Fauquier et al., 2008; Gleiberman et al., 2008; Garcia-Lavandeira et al., 2009; Chen et al., 2009). Both in vitro and in vivo studies have shown that SRY-box transcription factor 2 (Sox2) expressing cells function as pituitary stem cells during the embryonic and postnatal periods (Fauquier et al., 2008; Andoniadou et al., 2013; Rizzoti et al., 2013). Morphological and transcriptomic heterogeneity of postnatal Sox2-positive cells has been previously suggested for the rodent pituitary (Yoshida et al., 2016a). Sox2-expressing cells line the marginal zone (MZ), called marginal cell layer (MCL), but also appear in the parenchyma of the anterior lobe (Gremeaux et al., 2012; Chen et al., 2013). We have also reported that Sox2 gene and protein are expressed in the rat pituicytes of the posterior pituitary, in addition to MCL and parenchyma (Fletcher et al., 2023).

Here we studied the heterogeneity of Sox2-expressing cells in mouse pituitary tissue and their roles in postnatal gonadotrophs differentiation. Gonadotrophs are specialized for the synthesis and release of follicle-stimulating hormone (FSH) and luteinizing hormone (LH), which are essential for sexual development and reproduction (McArdle and Roberson, 2015). These hormones are heterodimers consisting of a common alpha chain and hormone-specific beta chains (FSHB and LHB), encoded by the Cga, Fshb, and Lhb, respectively (Cahoreau et al., 2015). Gonadotrophs are also defined by the expression of gonadotropin-releasing hormone (GnRH) receptor gene (Gnrhr) (Hapgood et al., 2005; Janjic et al., 2017). This receptor is activated by the decapeptide GnRH, which is synthetized by a subpopulation of hypothalamic neurons and released into the hypothalamic-pituitary portal system (Herbison, 2018). The activated receptor signals through Gq/11-phospholipase C pathway, which leads to the breakdown of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) to inositol-1,4,5-trisphosphate (InsP3) and diacylglycerol (Naor and Huhtaniemi, 2013). This signaling pathway ultimately controls the expression of Fshb, Lhb, and Gnrhr, as well as the synthesis and release of gonadotropins determined by the pattern of endogenous GnRH release or its external application (Constantin et al., 2022).

Like other pituitary endocrine cell types, gonadotrophs initially appear in the embryonic pituitary, with early expression of a common alpha chain and late expression of FSHB and LHB chains (Zhu et al., 2007; Wen et al., 2010). Postnatally, there is also a progressive growth of gonadotroph population from birth to puberty (Bjelobaba et al., 2015), with cells spreading dorsally throughout the gland (Mollard et al., 2012). The role of Sox2-expressing cells in postnatal gonadotroph differentiation has recently been established (Sheridan et al., 2025). However, the contribution of marginal and/or parenchymal cells expressing Sox2 has not been documented and transition of stem cells into gonadotrophs has not been visualized. The signaling pathways that control postnatal gonadotrophs differentiation have also not been characterized.

If postnatal stem cell proliferation leads to differentiation of hormone-producing cells, including gonadotrophs, we hypothesized that cells in the stem-to-endocrine transition and newly differentiated endocrine cells should be in the vicinity of Sox2-positive cells in the marginal niche and/or parenchymal niche. We visualized gonadotrophs by the specific expression of the fluorescent protein tdTomato via GnRH receptor-driven Cre/Lox recombination of the Rosa-tdTomato locus. We also examined the potential role of plasma membrane phosphoinositides in postnatal gonadotroph differentiation using transgenic mice with a gonadotroph-specific phosphatidylinositol 4-kinase alpha (PI4KA) knockout (GSKO), as previously described (Constantin et al., 2023).