The initiation of puberty is caused by the activation of the hypothalamic–pituitary–gonadal axis (HPGA). HPGA activation increases the frequency and amplitude of gonadotropin-releasing hormone (GnRH) pulse secretion; in turn, GnRH acts on GnRH receptors on anterior pituitary gland cells that secrete follicle-stimulating hormone (FSH) and luteinizing hormone (LH) and promotes the secretion of LH and FSH from the pituitary. The latter two act on the gonads to stimulate the secretion of estradiol (E2) from the ovaries in females and testosterone (T) from the testes in males, which promotes the rapid development of reproductive organs and sexual characteristics and the onset of puberty [1]. Puberty is regulated by a complex interaction network among genes, neurotransmitters, and glial neurons in the hypothalamus [2]. For instance, GnRH secretion is regulated by kisspeptin and its receptor KISS1R, a G-protein-coupled receptor, and by neurokinin B and dynorphin (and their respective receptors) [3]. Kisspeptin, encoded by kiss-1 and produced in the hypothalamus, stimulates the reproductive axis and plays a key role in reproductive function [4,5]. Rfrp-3, a mammalian homolog of avian gonadotropin-inhibiting hormone (GnIH), inhibits the reproductive axis [6].

Additionally, lncRNAs are also involved in the regulation of reproductive activities such as ovarian function, embryonic development, and sperm maturation [7,8]. Abnormal expression of lncRNA XIST, which mediates X-chromosome inactivation leads to delayed puberty and ovarian failure in girls [9]. LncRNA GnRH-E1 in the enhancer region of the Gnrh gene regulates the maturation of GnRH neurons [10], and GnRH is a key component of the initiation of puberty. A previous study showed that maternally expressed gene3 (Meg3) is involved in regulating central precocity in female rats [11].

MEG3 is located on human chromosome 14q32.3 within the DLK1-MEG3 locus [12]. It is a maternal imprinting gene that encodes a non-coding RNA of approximately 1.6 kb [13]. Previous studies indicate that MEG3 noncoding RNA is located in the brain, adrenal glands, placenta and ovary and is also highly expressed in the pituitary [12,14]. MEG3 mRNA is widely distributed in the anterior pituitary in humans, and the expression of MEG3 mRNA and FSH-β protein are co-located in human pituitary gonadotropin-secreting cells [14], suggesting the possibility that MEG3 may affect the secretion of FSH and LH. A previous study reported that in a rat model with central precocious puberty (CPP) treated with orexin, the symptoms of precocious puberty were significantly improved (delayed onset of puberty) and Kiss1 and Meg3 expression levels returned to normal, suggesting that Meg3 is involved in regulating CPP in female rats. In vitro experiments found that kisspeptin mRNA and protein levels were increased after transfection of mouse hippocampal neurons with pcDNA-MEG3 [11]. Because Kiss1 promotes the release of GnRH [[15], [16], [17]] and is known as the “gatekeeper” of the initiation of puberty [18], these findings suggest that Meg3 may play an important role in the initiation of puberty by regulating kisspeptin. In addition, Wnt/β-catenin is known to be critical in gonadal development and oogenesis [19]. Low levels of β-catenin have been found in the normal mammary gland of female mice during puberty [20]. Some Wnts, such as Wnt-2, Wnt-4, and Wnt-5b have been shown to be regulated by ovarian hormone levels [20], with the expression of Wnt-4 being induced by E2 through the action of ERα in rat pituitary as well as in cultured mammary epithelial cells [21,22]. Gao et al. found that androgen-mediated signaling loss in female mice resulted in significantly accelerated mammary gland development during puberty as well as disruption of the Wnt/β-catenin signaling pathway [23]. In transgenic mice in which Wnt3 was eliminated from Sertoli cells, on reaching puberty the animals showed low fertility and oligozoospermia [24]. However, Meg3 downregulation leads to the activation of the Wnt/β-catenin signaling pathway, which promotes nerve growth in the rat brain and reduces nerve damage [25]. Studies have found that a disregulated Wnt/β-catenin signaling pathway delays the onset of puberty in female mice and reduces GnRH neurons. Therefore, we speculate that Meg3 may regulate puberty through the Wnt/β-catenin pathway.

However, Meg3 expression in the reproductive axis at different developmental stages and its influence on puberty-related genes and reproductive hormone levels remain unclear. Therefore, this study investigated (1) the expression patterns of Meg3 in the HPGA of female rats at different developmental stages; (2) the effects of Meg3 knockdown on the expression of puberty-related genes and Wnt and β-catenin proteins in hypothalamic cells; and (3) the time of puberty onset, levels of gonadal hormones and expression of puberty-related genes, as well as ovarian morphology in female rats.