Abstract
The reproductive or Hypothalamic-Pituitary-Gonadal (HPG) axis is tightly regulated by its own internal negative feedback system, as well as by external factors such as the body’s stress responses. One key regulatory factor is the energy status of the body. Several lines of evidence present the reproductive system as metabolically gated, as sufficient energy stores are required for organism go through puberty and be fertile.
Several hormones are used by the body to signal energy status. Leptin is a major metabolic hormone released from adipose tissue. It travels into the hypothalamus to signal satiety, but also serves as a permissive signal for the HPG axis. Leptin deficiency or leptin receptor knockout results in an obese phenotype and infertility. As the GnRH neurons which drive the HPG axis do not express leptin receptors, several intermediary neurons have been identified as communicating leptin’s fertility actions. A neuronal population recently suggested to fulfil this role is the Pituitary Adenylate Cyclase Activating Polypeptide or PACAP neurons. PACAP is a neurotrophic factor expressed across many different tissues which serves a wide number of functions including as a satiety signalling peptide and regulating fertility. A recent study found the PACAP neurons of the ventral premammillary nucleus (PMV) region synapse with the kisspeptin neurons (which stimulate reproductive activity), and when PACAP is knocked out from the PMV, it causes significant delays in puberty onset and impaired female fertility. As these neurons also express leptin receptors, perhaps these neurons act as conduits for leptin’s actions on the reproductive axis.
The aim of the experiment was to investigate the role of PACAP in leptin’s regulation of fertility, with body weight and adiposity measured as secondary endpoints. We used the Cre-lox system to create two genetic mouse models to manipulate leptin receptor expression. To test whether PACAP is necessary for leptin’s metabolic and reproductive actions, leptin receptors were knockout out selectively from PACAP neurons. These animals had no changes in body weight compared to controls, but females showed a moderate but significant ~2-day delay in puberty onset. To test whether PACAP is sufficient for leptin’s metabolic and reproductive actions, PACAP-Cre animals were crossed with animals containing a loxP-flanked stop sequence within the leptin receptor. In Cre-expressing neurons the stop condon was excised, allowing the leptin receptor to be expressed. Thus, only PACAP neurons had functional leptin receptors. These animals exhibited slightly reduced body weights compared to total leptin receptor knockout animals, but puberty and fertility were not restored. An additional experiment was performed to map hypothalamic PACAP neuron populations from birth to adulthood. Using PACAP-Cre mice crossed to animals with floxed GFP, several regions such as the SCN showed increased PACAP expression at later development stages. These results show PACAP neurons are necessary for normal functional fertility and timing, but they are not sufficient for mediating leptin’s fertility role. PACAP expression may also be linked to development.