Abstract
In mammals, lactation is accompanied by a period of infertility. This adaptive change is an essential part of a successful reproductive strategy, and enables a mother’s metabolic resources to be directed towards caring for her newborn, rather than supporting another pregnancy. Lactational infertility is characterized by a lactation-induced suppression of kisspeptin immunoreactivity, reduced pulsatile luteinizing hormone (LH) secretion and a lack of ovulation, as well as chronically elevated levels of the anterior pituitary hormone prolactin. Despite elevated prolactin, per se, being a well-recognized cause of infertility, the specific role that prolactin plays in lactational infertility, as distinct from other suckling or metabolic cues, is currently unclear. The aim of this thesis was to determine the contribution of prolactin action in lactational infertility in mice.
Wild-type C57BL/6J mice normally experience prolonged diestrus during lactation, lasting until weaning of pups (around 21 days in mice). However, if the suckling stimulus is removed during the first week following parturition, through premature weaning, then this diestrus persists for a prolonged period (on average 8 days). By blocking prolactin action, through the use of the dopamine receptor agonist bromocriptine, pup separated mice showed a significantly faster return to estrus, indicating that prolactin action plays a key role in prolonging the diestrus in mice following premature weaning.
To investigate whether prolactin action in the brain is necessary to maintain lactational infertility, two mouse models with specific deletions of the prolactin receptor (Prlr), either in forebrain neurons (Prlrlox/lox/Camk2aCre) or kisspeptin neurons (Prlrlox/lox/Kiss1Cre) were used. Estrous cycle, patterns of LH secretion and kisspeptin immunoreactivity were monitored in mice of both genotypes during lactation. Additionally, the activity of ARC kisspeptin neurons in vivo was investigated in Prlrlox/lox/Kiss1Cre mice using GCaMP6 in vivo fibre photometry. While no control animals experienced estrus until after weaning of pups (>20 days of lactation), both Prlrlox/lox/Camk2aCre and Prlrlox/lox/Kiss1Cre mice did not exhibit the normal period of lactational infertility, returning to estrus within 6-10 and 6-17 days of lactation, respectively. In both Prlrlox/lox/Camk2aCre and Prlrlox/lox/Kiss1Cre mice, immunohistochemical analysis of kisspeptin in the rostral periventricular region of the third ventricle (RP3V) and/or arcuate nucleus (ARC) showed a significant attenuation of the normal lactation-induced suppression of kisspeptin immunoreactivity. Pulsatile LH secretion was found to return prior to the observed estrus in Prlrlox/lox/Camk2aCre mice, while in vivo fibre photometry was able to demonstrate that Prlrlox/lox/Kiss1Cre mice had a reactivation of ARC kisspeptin population activity prior to premature estrus in early lactation. These data provide evidence that high levels of prolactin during lactation, acting specifically on kisspeptin neurons in the brain, is necessary for the suppression of fertility during lactation.
To determine whether progesterone may play a role in inhibiting pulsatile LH secretion during lactation, alongside prolactin, progesterone levels were manipulated in Prlrlox/lox/Camk2aCre mice using the progesterone receptor antagonist mifepristone. Mice treated with mifepristone showed no change in pulsatile LH secretion LH, and so progesterone levels were measured in C57BL/6J mice in virgin, pregnant and lactating states to determine whether progesterone was elevated during lactation in mice. Interestingly, progesterone was found to be at low levels during lactation, similar to those of diestrus virgin controls. These data combined indicate that progesterone likely does not play a significant role in suppressing pulsatile LH secretion during lactation in mice.
In vivo fibre photometry was also used to investigate the activity of ARC kisspeptin neurons across different reproductive states. This ARC population of kisspeptin neurons is defined as the GnRH “pulse generator”, responsible for pulsatile secretion of LH. Neuronal activity was monitored in real time in the same mice (Kiss1Cre) across different reproductive states using kisspeptin neuron-selective GCaMP6 fibre photometry. During the virgin (nulligravida) state, periodic events of elevated intracellular calcium (indicative of synchronous activity of the ARC kisspeptin population) precede the release of LH, with approximately one event observed every hour (described in McQuillan et al. (2019)). However, dynamics and frequency of these events changed during pregnancy. A profound suppression of synchronised events was observed by mid pregnancy, however, low amplitude, “normal” frequency events were observed in late pregnancy (day 18). Evaluation of neuronal activity during lactation revealed a complete suppression on day 7 of lactation, which corresponded with an absence of pulsatile LH secretion. This lactation-induced suppression of activity was gradually relieved throughout the second half of lactation, returning to pre-pregnancy levels following weaning. These observations show dynamic variation in the activity of ARC kisspeptin neurons, likely associated with the hormonal changes of these two reproductive states. The independent role of progesterone or other hormones prolactin in mediating these changes is still unclear.
Together the data presented in this thesis provide substantial evidence that prolactin action is necessary for the chronic suppression of kisspeptin activity during early lactation in mice.