Adolescents experiencing gender dysphoria are at risk for depression, anxiety, suicide, substance use disorders, and poor self-esteem (Garg et al., 2024; Shain and Committee On, 2016). Early physical signs of puberty, such as breast budding (Marshall and Tanner, 1969) and testicular/phallic enlargement (Marshall and Tanner, 1970), can elicit extreme clinical distress in adolescents who are questioning their gender identity (Martinerie et al., 2016). However, gender-affirming interventions can improve mental health and quality of life (de Vries et al., 2011; Gomez-Gil et al., 2012). The recommended standard of care for adolescents with gender dysphoria is to temporarily delay further pubertal progression with gonadotropin-releasing hormone (GnRH) agonists (Coleman et al., 2022). An advantage of GnRH agonists is that effects are thought to be reversible, thereby safely providing patients with time to consider their gender expression without the distressing development of incongruent secondary sex characteristics (Cohen-Kettenis and van Goozen, 1998; Giordano, 2008). If young adults transition, GnRH agonists can be continued for long-term suppression of endogenous gonadal hormone secretion once gender-affirming hormones are prescribed (Gava et al., 2020). Unfortunately, little is known about the effects of delaying puberty by suppressing the hypothalamic-pituitary-gonadal (HPG) axis with protracted GnRH agonist treatment on the development of the brain.

Only a few preclinical studies have tested the effects GnRH agonists in animals to model the suppression of puberty in adolescents with gender dysphoria or disorders such as precocious puberty. For instance, in male rats, daily leuprolide acetate (LEU) treatment during the periadolescent period delayed puberty, as measured by preputial separation (PPS) as well as the development of copulatory behavior and sexual motivation after treatment ended (Guarraci et al., 2021). Similarly, male mice exposed to LEU for 6 weeks in late adolescence spent less time investigating a female stimulus, but more time investigating a male stimulus compared to saline controls (Anacker et al., 2021). These results suggest that LEU delays not only puberty but also the maturation of sexual behavior and sexual motivation in male rodents. In female mice (Anacker et al., 2021) and rats (Guarraci et al., 2023), daily periadolescent LEU delayed puberty as measured by vaginal opening (VO) and disrupted estrous cyclicity, such that females failed to enter estrus during treatment. However, sociosexual behaviors were not affected by LEU when tested after treatment ended (Guarraci et al., 2022). Together, these findings indicate a differential effect of LEU on male vs. female behavior, as well as a differential effect within females on VO vs. estrous cyclicity.

Hypothalamic GnRH secretion into the median eminence is critical for the onset of puberty in mammals. At puberty in rodents, GnRH neurons increase in sensitivity to kisspeptin from the arcuate nucleus (ARC) and the anteroventral periventricular nucleus (AVPV) of the hypothalamus (Han et al., 2005; Kallo et al., 2012). During pubertal development, the number of kisspeptin-expressing neurons increases in the ARC of female and male rodents (Bentsen et al., 2010). It is kisspeptin from the ARC that regulates the pulsatile release of GnRH, which in turn triggers luteinizing hormone (LH) and follicle-stimulating hormone (FSH) release from the anterior pituitary starting at puberty and continuing into adulthood. Kisspeptin neurons in the ARC are under negative feedback from gonadal hormones to regulate pulsatile release of GnRH and LH/FSH in both sexes for steroidogenesis and folliculogenesis/spermatogenesis. By contrast, kisspeptin neurons in the AVPV of females are under positive feedback from estradiol to regulate preovulatory surge release of GnRH. Due to perinatal defeminization in males and prepubertal neurogenesis in females, there are fewer kisspeptin neurons in male AVPV and thus no GnRH surge in adult males (Sisk, 2016; Yamada et al., 2024).

The present study was designed to investigate the effects of daily LEU treatment on expression of key genes in the hypothalamus and the pituitary gland, and serum gonadal hormones, of female and male rats. We focused on the POA and MBH, areas of the brain involved in pubertal onset, estrous cyclicity, and sexual behavior and motivation.