Abstract
It is well established in both humans and rodents that the state of hyperprolactinaemia leads to reduced reproductive capacity, as a consequence of suppressed luteinising hormone secretion. Return of reproductive function can be achieved by physiological gonadotropin-releasing hormone (GnRH) administration in humans. In rodents, there is no decrease in pituitary GnRH responsiveness in the presence of elevated prolactin. These findings indicate that prolactin, an anterior pituitary hormone capable of direct action in the central nervous system, is affecting hypothalamic GnRH secretion, rather than pituitary gonadotropin secretion. However, as prolactin receptors are only expressed on a small minority of GnRH neurons, a direct suppressive action by prolactin on these neurons is unlikely. Hence, an indirect mechanism utilising neurons afferent to GnRH neurons may be in place. The neuropeptide kisspeptin has recently been discovered to be a key afferent regulator of GnRH secretion. Prolactin receptors are present on the majority of kisspeptin neurons, leading to the hypothesis of this thesis; that prolactin inhibits kisspeptin neurons, providing an indirect pathway through which prolactin alters GnRH output. To investigate this hypothesis, several experiments were carried out. Firstly, double-label immunohistochemistry, staining for pSTAT5 (an intracellular signal transducer of prolactin signalling) and kisspeptin, was performed throughout the AVPV/PeN and arcuate nuclei of the rat hypothalamus. Colocalisation of pSTAT5 nuclear-staining within kisspeptin neurons was evident in ovine prolactin (oPRL)-treated animals, indicating that the prolactin receptors expressed by kisspeptin neurons are functional in vivo. Secondly, Kiss1 mRNA expression in a lactational model of hyperprolactinaemia was analysed by qPCR. There was a significant suppression of Kiss1 mRNA expression in each nucleus during lactation compared to diestrous levels. This was not reversed by prolactin removal (by bromocriptine-treatment), suggesting a suckling-induced suppression not mediated solely by prolactin. A third treatment group, where pups were removed and oPRL was administered, however, suggested the presence of an additional suppressive effect of prolactin in the arcuate nucleus. Finally, in order to investigate the effects of hyperprolactinaemia without the confounding factors of a lactation, a nonlactational model of chronic hyperprolactinaemia was developed. This trial involved ovariectomy with low level oestradiol replacement, and oPRL administration every 8 hours for 48 hours. Serum oPRL concentration, profiled by serial blood sampling through indwelling jugular cannulae, was found to peak 1 -3 h post-injection (approximately 80 ng/ml) and drop to 0 ng/ml by 6 h post-injection. This oPRL-treatment did not suppress LH concentrations compared to vehicle-treated controls, and thus in this regard, the model was unsuccessful. Nevertheless, the hypothalamic tissue obtained was analysed by qPCR to investigate whether Kiss1 mRNA expression was altered by oPRL-treatment. No significant changes were detected in the AVPV/PeN, whilst in the arcuate, there was a significant four-fold increase in Kiss1 mRNA expression in vehicle-treated, ovariectomised rats. This increase was significantly dampened by approximately half, in oPRL-treated ovariectomised rats.
Each of these experiments provide evidence in support of the hypothesis; indicating that prolactin does regulate kisspeptin neurons. This finding could hold important implications for further investigations into the use of kisspeptin as treatment of hyperprolactinaemic infertility, a condition that hinders many patients.