Periodic Reporting for period 1 - Tac1-Ovulation (Addressing the Roles of Tachykinins in the Control of Ovulation: Focus on the Substance-P/Tachykinin Receptor Type 1 (Tac1/Tacr1) System)
Reporting period: 2020-05-01 to 2022-04-30
Infertility/subfertility is a growing challenge in reproductive medicine. Notably, infertility affects one in seven couples. The most common cause of in/subfertility in women is ovulatory dysfunction; anovulation being associated to conditions as polycystic ovary syndrome, hypothalamic amenorrhea and premature ovarian insufficiency. While hypothalamic GnRH neurons are the major output pathway for the brain control of ovulation, upstream Kiss1 neurons, particularly in the rostral hypothalamic area in rodents, have been suggested to be crucial for the timed activation of GnRH neurons and generation of the preovulatory surge of gonadotropins that drives ovulation. However, the major regulators of this Kiss1/GnRH pathway remain ill defined. Substance P (SP, encoded by Tac1), a member of the tachykinin (TAC) family that acts via the receptor, NK1R (encoded by Tacr1), has been shown to centrally regulate gonadotropin release, and, according to our preliminary data, might modulate the pre-ovulatory surge in mice. Yet, the pathophysiological relevance of SP/NK1R signaling in ovulatory control needs to be defined. The objective of this project was to deepen our understanding of the central mechanisms that regulate ovulation, the pre-ovulatory LH surge and LH secretory patterns, by characterizing the roles and mechanisms of action of the SP/NK1R signaling system, with a particular focus on their actions in Kiss1 and GnRH neurons. Implementation of this project has allowed us to expand our understanding of the mechanisms controlling ovulation and female fertility, helping to define novel strategies for reproductive control in the future.
The main activities conducted since initiation of this project can be summarized as follows:
First, we evaluated the physiological relevance of Substance P (SP)/NK1R signaling in Kiss1 neurons during the pre-ovulatory LH surge. To this, we used small interfering RNA (SiRNA), a method to silence, suppress or reduce the expression of certain genes of our interest by genetic engineering techniques. In our case, we knocked down Tacr1 (i.e. the gene encoding NK1R, the receptor of SP) in Kiss1 neurons using SiRNA pools against Tacr1 or nontargeting controls. Our studies suggest that SP could modulate the pre-ovulatory LH surge acting through Kiss1 neurons.
Second, we generated a reporter mouse model, introducing a reporter gene, such as YFP or tdTomato, into the locus of our target gene (Tac1, encoding SP) in order to monitor the expression of that gene. We crossed Tac1-cre mice with mice harboring a reporter, namely YFPm or tdTomato, flanked by loxP sequences, to generate Tac1cre::YFP or Tac1cre::tdTomato mouse models, in which Tac1+ neurons are labelled by the corresponding markers. Then, we identifies the Tac1 neuronal population(s) activated during the LH surge. For this study, we collected brain tissue from female mice between 7:10-7:30 PM after the LH surge induction and used immunohistochemistry to identify Tac1-expressing cells that become activated during the surge (i.e. co-express c-Fos).
Third, we identified the cellular targets of the Tac1 neuronal populations activated during the LH surge. To this end, Tac1-cre mice were stereotaxically injected with an AAV viral vector in the areas previously identified. The viral vector used allowed us to label cell bodies containing Cre-recombinase and their projections with a fluorescent protein.
Fourth, we studied the physiological relevance of Tac1 signaling in the control of LH pulsatility, the pre-ovulatory LH surge and ovulation, using chemogenetic activation or inhibition of Tac1 neurons. To this end, we injected Tac1cre::FP mice, bilaterally, in the specific nuclei activated during the LH surge with excitatory or inhibitory DREADD that depolarizes or hyperpolarizes infected neurons after treatment with the synthetic ligand, clozapine-N-oxide (CNO). We evaluated the LH pulsatility and the magnitude of pre-ovulatory LH surge after the activation or inhibition of Tac1 neurons.
Finally, it is worth to mention that exploitation and dissemination of the results have taken place as planned in the original proposal. All results derived from this project were shared with the scientific community through multidisciplinary seminars and weekly meetings organized by the host group (Tena-Sempere's lab). We maintain also our original plan to disseminate our results via international scientific conferences (e.g. Endocrine Society Annual Meeting and International Congress of Neuroendocrinology), although these have been partially delayed due to the COVID-19 pandemic and will take place in the coming months or year. In addition, the fellow has actively participated in different outreach activities, including 2 European Researcher's Night Programs (Cordoba, 2020 and 2021).
First, we evaluated the physiological relevance of Substance P (SP)/NK1R signaling in Kiss1 neurons during the pre-ovulatory LH surge. To this, we used small interfering RNA (SiRNA), a method to silence, suppress or reduce the expression of certain genes of our interest by genetic engineering techniques. In our case, we knocked down Tacr1 (i.e. the gene encoding NK1R, the receptor of SP) in Kiss1 neurons using SiRNA pools against Tacr1 or nontargeting controls. Our studies suggest that SP could modulate the pre-ovulatory LH surge acting through Kiss1 neurons.
Second, we generated a reporter mouse model, introducing a reporter gene, such as YFP or tdTomato, into the locus of our target gene (Tac1, encoding SP) in order to monitor the expression of that gene. We crossed Tac1-cre mice with mice harboring a reporter, namely YFPm or tdTomato, flanked by loxP sequences, to generate Tac1cre::YFP or Tac1cre::tdTomato mouse models, in which Tac1+ neurons are labelled by the corresponding markers. Then, we identifies the Tac1 neuronal population(s) activated during the LH surge. For this study, we collected brain tissue from female mice between 7:10-7:30 PM after the LH surge induction and used immunohistochemistry to identify Tac1-expressing cells that become activated during the surge (i.e. co-express c-Fos).
Third, we identified the cellular targets of the Tac1 neuronal populations activated during the LH surge. To this end, Tac1-cre mice were stereotaxically injected with an AAV viral vector in the areas previously identified. The viral vector used allowed us to label cell bodies containing Cre-recombinase and their projections with a fluorescent protein.
Fourth, we studied the physiological relevance of Tac1 signaling in the control of LH pulsatility, the pre-ovulatory LH surge and ovulation, using chemogenetic activation or inhibition of Tac1 neurons. To this end, we injected Tac1cre::FP mice, bilaterally, in the specific nuclei activated during the LH surge with excitatory or inhibitory DREADD that depolarizes or hyperpolarizes infected neurons after treatment with the synthetic ligand, clozapine-N-oxide (CNO). We evaluated the LH pulsatility and the magnitude of pre-ovulatory LH surge after the activation or inhibition of Tac1 neurons.
Finally, it is worth to mention that exploitation and dissemination of the results have taken place as planned in the original proposal. All results derived from this project were shared with the scientific community through multidisciplinary seminars and weekly meetings organized by the host group (Tena-Sempere's lab). We maintain also our original plan to disseminate our results via international scientific conferences (e.g. Endocrine Society Annual Meeting and International Congress of Neuroendocrinology), although these have been partially delayed due to the COVID-19 pandemic and will take place in the coming months or year. In addition, the fellow has actively participated in different outreach activities, including 2 European Researcher's Night Programs (Cordoba, 2020 and 2021).
The research work developed by the fellow during the implementation of this project has allowed (i) to gain new insights into the neuroendocrine mechanisms that govern reproductive function, particularly those involving Tac1/SP signaling; and (ii) to identify novel pharmacological targets for a variety of clinical applications, from assisted reproductive techniques to contraception, reproductive hormone replacement and endocrine-dependent cancers, as well as disorders of body weight and metabolism, especially those associated with gonadal dysfunction, e.g. polycystic ovarian syndrome and hypogonadism-induced insulin resistance (frequently linked to obesity). Furthermore, the fellow has become acquainted with new procedures/techniques, including (among others) complementary techniques for functional manipulation of neuronal activity in vivo, via chemo- and optogenetics. Also, during the implementation of this project, the fellow improved her skill in the design of projects and procedures with experimental animals. In terms of wider implications, our project may help to define new strategies for the dissection of complex neuronal/cellular circuits in vivo, as well as to identify novel targets for appropriate management of reproductive and metabolic disorders, of increasing prevalence (and concern) worldwide.