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Physiological characterization of PK2 in the control of fertility, and its interaction with kisspeptins

Final Report Summary - PK2-KISS (Physiological characterization of PK2 in the control of fertility, and its interaction with kisspeptins)

Reproduction is a tightly regulated process that enables perpetuation of species. Given its importance, development and function of the reproductive axis is sensitive to modulation by a variety of central and peripheral signals that ultimately dictate the pattern of gonadotropin-releasing hormone (GnRH) release. GnRH is the final central switch responsible for the activation of pituitary secretion of gonadotropins, which are in turn responsible for trophic maturation and function of the gonads. However, the neurons producing GnRH lack the ability to efficiently respond to most of the crucial regulators of reproductive function, such as estradiol, leptin and other metabolic signals, as well as environmental cues. This feature suggests the existence of intermediate afferent pathways, responsible for the fine-tuning of GnRH secretion and, hence, of the reproductive axis.

In recent years, human genetic studies, especially in patients with isolated forms of hypogonadotropic hypogonadism (iHH), have revealed essential regulatory pathways involved in the physiological control of reproductive maturation and function. In late 2003, studies in this front led to the identification of a new indispensable player of the reproductive brain with potent activity as stimulator of GnRH secretion: the Kiss1/GPR54 system. More recently, human genetics also revealed that mutations, affecting the genes encoding the neuropeptide, prokineticin 2 (PK2) and prokineticin receptor 2 (PK2R), were associated to iHH phenotypes in humans and rodents, thus suggesting that the PK2 system is also a key player in the central circuitries governing the migration and/or function of GnRH neurons.

Kisspeptins (products of the Kiss1 gene) are potent elicitors of GnRH secretion through direct action on GnRH neurons. Importantly, Kiss1 neurons reported to possess receptors to the major central and peripheral regulators of the GnRH system. Therefore, they were proposed to act as a funnel and integratory node, conveying critical information for the proper functioning of the gonadotropic axis. In this context, characterisation of the regulatory afferents of the Kiss1 system and their interactions with kisspeptins in the control of GnRH neurons became a priority in the field of Reproductive Physiology; a mandate that remains to date largely ill defined.

Thus, this project was aimed towards the characterisation of -but not limited to- the roles of the PK2 system in the control of key facets of reproductive maturation and function. As specific goals, the present project intended to answer whether PK2:

(i) stimulates gonadotropin secretion;
(ii) interacts with the Kiss1 system; or
(iii) participates in the generation of the pre-ovulatory GnRH/LH surge (and hence ovulation), and/or whether expression of PK2 and/or PK2R genes is regulated by developmental or hormonal factors.

In addition to these conceptual objectives, this Marie Curie initiative was built on the complementary expertise and formative capacities of the outgoing and return groups, which synergised to allow an integral formation of the trainee in different (and related) aspects of neuroendocrinology.

Initial work undertaken towards the specific aims revealed that the stimulatory effects of PK2 on gonadotropin secretion are mild or null, and PK2 (and PK2R) do not seem to co-localise with Kiss1 in the rodent brain, probably reflecting a more relevant role in photoperiodic strains. These initial results casted some doubts on the potential role of PK2 as major afferent for the Kiss1 system, and prompted us to evaluate related regulatory pathways to provide additional physiological information of the systems governing Kiss1/GnRH secretion and reproductive function.

In this context, during the initial phase of the outgoing period, a revealing study by Topaloglu and colleagues demonstrated that, in clear analogy with GPR54 and PK2/PK2R, patients with inactivating mutations in the genes encoding neurokinin B (NKB; TAC3 gene), or its receptor, NK3R (TACR3 gene) display iHH. These findings prompted us to evaluate the physiology of NKB/Kiss1 interactions and have provided conclusive evidence for a novel regulatory circuit, involving NKB and dynorphin as reciprocal regulators of kisspeptin output onto GnRH neurons. This hypothetical model has been constructed on the basis of the experimental work initiated during the outgoing phase and continued during the return phase (mostly in rodent species), and has been confirmed via external collaborations in other relevant (goat) mammalian species. This model would provide a tenable basis for the state of iHH observed in patients with either TAC3/TACR3 or KISS1/GPR54 mutations. Given the suggested relevance of the NKB system in the control of the reproductive axis, we extended our studies to characterise the role of this system in the maturation of the gonadotropic axis and its interaction with the Kiss1 system. In this sense, results from the return phase evidence a prominent action of NKB in the timing of puberty onset. Importantly, these findings also manifest a contribution of the NKB system in the interaction between energy balance and reproductive function. Situations of energy shortage, such as fasting or caloric restriction, deplete Tac2 expression, corresponding with their associated suppression of the reproductive axis, which, importantly, can be restored through exogenous administration of NK3R agonists. Further studies have been initiated during the returning phase to document the postnatal development and regulation of the NKB system as major contributor of kisspeptin / GnRH / LH release.

Altogether, results from this project have enlarged our current understanding of the mechanisms of physiological control of the reproductive axis, this helping. In this line, the collaborative work conducted between the trainee and the host groups has offered a better understanding of some additional neuroendocrine mechanisms involved in the control of brain sexual maturation, puberty and reproductive function. As examples, the work of the trainee has documented the effects of early estrogen exposure on the development of the Kiss1 system and the roles of the neuropeptide, nesfatin-1, in the control of puberty onset. Again, this basic knowledge has not only contributed to provide the trainee with a wider knowledge of key aspects of Neuroendocrinology, but also will help to define new suitable targets for the pharmacological manipulation and, potentially, treatment of different pathological conditions such as advanced puberty, PCOS, prostate cancer, new contraceptive methods, etc. As index of the accomplishments obtained in this front, the trainee has published a total of 18 peer-reviewed papers (involving original articles and invited reviews) on the subjects indicated above which supports the relevance and thoroughness of the studies performed during this Marie Curie fellowship.