So, our first speaker is Alessandra Mancini from Brigham and Women's and Harvard Medical School. Hi, good morning everyone, and thank you to the organizing committee for inviting us to speak today and present this very interesting study. So the reproductive axis is active during three times, during fetal development, at infancy, and then at puberty. Puberty is triggered by an increase in pulsatile release of GnRH, which is a decapeptide hormone released by GnRH neurons that are localized in the hypothalamus. GnRH stimulates LH and FSH release from the anterior pituitary, which in turn stimulates sex steroid production from the gonads. But now, in the hypothalamus, there is also another population of neurons that is called candy neurons. They take their name by the co-expression of cispeptin, neurokinin B, and dynorphin A. Neurokinin B, which is an excitatory peptide, stimulates the release of cispeptin on GnRH neurons, and that's how GnRH gets triggered to be released. Now, these factors are all extremely important for a correct onset and timing of puberty, and we know that mutations in the genes that encodes for these factors are associated with conditions of delayed puberty, early puberty, as well as absent puberty. But the period preceding puberty is also extremely important, so we know that during childhood, the reproductive axis is completely silenced, and there are factors, obviously, mediating this. One of the most important factors that play an important role in this is MKRN3. Our group, Dr. Kaiser Research Group, found that mutations in MKRN3 cause central precocious puberty, or CPP. MKRN3 is an intron-less gene, is imprinted, and is maternally silenced, so mutations are inherited through the father, as you can see by this pedigree here. It was found that MKRN3 is highly expressed in the arcuate nucleus within the hypothalamus of both female and male mice, and very remarkably, it was found that its expression follows a temporal gradient, in a way that it's highly expressed during early postnatal stages, and then it declines with very low or undetectable levels prior to puberty. Within the arcuate nucleus, MKRN3 is co-expressed with cispeptin, and it was also found that MKRN3 has an inhibitory effect on cispeptin promoter, as well as TAC3 promoter, and TAC3 is the gene that encodes for Neurokinin B. So MKRN3 encodes for a protein that is an E3 ubiquitin ligase, with, and as such, with a role in ubiquitination. It was actually found that MKRN3 auto-ubiquitinates itself, as you can see by the wild-type protein here, and with the typical poly-smear of ubiquitination. But then, interestingly, the mutations that were found in the patients with CPP were unable to have this auto-ubiquitination property, and especially those mutants that are within a particular region, a particular domain called ring finger domain. So overall, MKRN3 inhibits pubertal onset, and to date, we know that MKRN3 mutations are the most common genetic cause of CPP, with a prevalence of up to 46% of familial cases, and 10% of sporadic cases. So the main objective of these studies to identify the exact molecular mechanisms of MKRN3 that are mediating this inhibitory break on pubertal onset. So in our laboratory, it was found that Neurokinin B protein levels were significantly increased in the arcuate nucleus of MKRN3 knockout mice when compared to the control wild-type animals. And similarly, by using a different and opposite model, it was found that Neurokinin B protein levels were significantly decreased in the arcuate nucleus when MKRN3 was overexpressed. So we were fascinated by this relationship between these two factors, and so we took, we dig further, and we used an in vitro model by overexpressing MKRN3 and Neurokinin B in HEK293 cells, and we also confirmed, and we also observed that overexpression of MKRN3 caused Neurokinin B levels to decrease significantly. But not only, we actually saw that the subcellular localization, subcellular distribution, was severely affected when MKRN3 was expressed. As you can see here, MKB alone versus MKB when we overexpressed MKRN3. Next, our lab performed an MKRN3 interactome that identified interacting proteins mainly involved in RNA metabolism and binding. And in particular, we selected two factors, PABPC4 and IGF2BP1, for main reasons being PABC4 is one of the most significant factors that was identified, and IGF2BP1 was also among the most significant, and also as you can see, there are multiple IGF2BPs present in this interactome. So, we performed a co-immunoprecipitation, and we saw that indeed MKRN3 interacts with IGF2BP1 as well as with PABPC4. And then we thought, well, IGF2BP1 and PABC4, these are both RNA-binding proteins, so how about treating these immunocomplexes with RNAase A? And actually, quite remarkably, we saw that upon treatment with RNAase A, we lost completely the interaction between MKRN3 and IGF2BP1, indicating that their interaction is RNA-mediated. Whereas when we were treating MKRN3 and PABPC4 immunocomplex, their interaction is maintained, suggesting that their interaction is not mediated by RNA. So, in order to find out which is the RNA that is mediating this interaction, we performed an RNA co-IP by transline transfecting either MKRN3 or IGF2BP1, and then pulling down MKRN3 or IGF2BP1, and then we amplified the RNA that was associated with the immunocomplexes, and we found that TAC3 was highly enriched in both. So, this suggested that MKRN3 and IGF2BP1 bind to TAC3 mRNA, and then lastly, considering MKRN3 role in ubiquitination, we performed an ubiquitination assay that showed that, as you can see on the bottom here, NKB is normally seen as a very low molecular weight, and then as we co-transfect with MKRN3, NKB gets almost completely degraded, but then once we also add MG132, which is a proteasome inhibitor, we have not only a restoration of NKB expression, but also this molecular shift that is suggesting that there could be some ubiquitin addition, molecule addition to NKB, and also these higher ubiquitin polysmear. So, these suggest that MKRN3 actually targets NKB for degradation. So, to summarize, MKRN3, we found that MKRN3 interacts with IGF2BP1 and PABPC4, that MKRN3 and IGF2BP1 interaction is actually RNA-mediated, whereas MKRN3 and PABPC4 interaction is not RNA-mediated, and that these mRNA is TAC3 from our RNA-CoIP studies, and also we found that MKRN3 targets NKB for ubiquitination and proteasomal degradation. So, lastly, our proposed model suggests that MKRN3 is part of an RNA-processing complex that regulates TAC3 mRNA metabolism and NKB for degradation, ultimately influencing the onset of puberty. I would like to finish my talk by thanking my mentor, Dr. Rusla Kaiser, the Kaiser Lab, the funding bodies, and thank you all for your attention. Thank you. Questions? Dan Bernard, McGill, that was really great. You showed the effect with an AAV, I think, of overexpressing MKRN3 on the neurokinin B expression. Have you taken that further and looked at the effects on fertility? So, to answer that question, I invite you to actually follow the talk that Dr. Roberts will take tomorrow morning, and you'll hear more about it, but, yeah. Okay, and then, sorry, with your interactome study, was that, how did you do that? Was that BioID, was it a CoIP, and in what cellular context did you do that experiment? So, we performed it in HEK293 cells and SHSY5Y, which is a neuroblastoma, human neuroblastoma cell lines, and they've used multiple conditions by transiently or stably transfecting MKRN3, which was tagged with a tag, a JTAG, and so that was pulled down, and then mass spec was used for identification of targets, and then a software was used called Compass that was basically identifying the most significant interacting factors, basically. Okay, and, oh, sorry, maybe one quick last one. Sure. Because when you use a heterologous system, I think we always wonder about whether you would see the same proteins in a homologous system. So, just in the case of IGF2BP1, is that expressed in candy neurons? That's a good question, and that's actually one of our next things to do, because if you read in the literature, IGF2BP1 is highly, highly expressed during development in embryogenesis, and actually, to my knowledge from the literature, it's just reported to be expressed in cancer cell lines in adults, but actually we found that it's expressed in the hypothalamus, so definitely we would like to narrow down where in the hypothalamus it's expressed. Great, thanks. Hello, well, fantastic talk. Just a very brief question, maybe it will be disclosed tomorrow in the same presentation, but have you looked at the interaction with KISS1, and also the over-expression and knockdown that you have done? Is it causing also changes, not only in type 3 or neokininB, but also in KISS1 and KISSpeptin? Yes, so, for example, we have looked, well, my colleagues have looked in animal models for KISSpeptin, and we do have some differences, especially at the pro-syn levels. Regarding the data that I've shown, specifically the RNA CoIP, we actually are in the process of performing RNA co-immunoprecipitation targeting KISSpeptin, so to see whether KISSpeptin is also mediating these pro-syn, pro-syn interactions. Thank you very much, Alison. Thank you all, thank you. Thank you. Our next speaker is Caitlin McIntyre from King's College, London. Thank you for the introduction, and I'd like to thank the organizing committee for giving me the opportunity to present my data. So the GnRH pulse generator is a neural construct located in the arcuate nucleus of the hypothalamus. Pulsatile release, we now know this neural construct is a network of KISSpeptin neurons located in the arcuate named Candi as they co-express neurokinin B and dynorphin A. A KISSpeptin release at the level of the median eminence triggers pulses of GnRH, which will then regulate LH and FSH release to control gonadal function. This talk will focus on the amygdala, a key site, the amygdala and its role in stress and tissue suppression of pulsatile LH secretion. The amygdala is a key site for emotional processing through an anxiety. The amygdala is located here in your temporal lobe. It's a complex structure, but we focused on the posterodorsal sub-nucleus of the medial amygdala, abbreviated MEPD. Recently, we have shown that KISSpeptin signaling in the MEPD is actually an upstream regulator of the GnRH pulse generator. The MEPD is also involved in mediating the stress-induced suppression of LH pulse frequency. It's activated during psychological stress, and if we lesion the MEPD, we block stress-induced suppression of LH pulses. The MEPD also contains the stress neuropeptide uacortin-3, which is also involved in mediating stress-induced suppression of the GnRH pulse generator. Now, the MEPD is a density GABAergic nucleus and has a lot of GABA projections of the hypothalamus. So, our first aim was to address the question, does GABA signaling in the MEPD mediate stress-induced suppression of the GnRH pulse generator? To achieve this, we determined whether chemogenetic inhibition of MEPD GABA neurons can block psychological stress-induced suppression of LH pulses. So, this slide shows our methodology. We take over-rectomized VGAT cream mice and bilaterally inject an adenovirus containing an inhibitory DREADS construct into the MEPD. We can visualize our GABA neurons as they express TD tomato and can confirm co-expression with DREADS by the orange-yellow fluorescence. And we can then use the MEPD to We wait three weeks to ensure adequate bio-expression and get our mice used to handling, and then we begin a blood-sampling protocol to determine LH pulse utility. We cut the very tip of the tail, and then we wait one hour to allow our mice to obituate. And then we begin collecting blood samples every five minutes for one hour to determine baseline LH pulse frequency. Mice are then exposed to inhibitors and the inhibitors are then exposed to inhibitors. Mice are then exposed to either predator odor exposure or restraint stress for one hour while blood sampling continues. 15 minutes before stress exposure, mice receive either CNO to inhibit MEPD GABA neurons or saline as a control. So in A, you can clearly see that TMT, a synthetic extract from fox urine, clearly decreases LH pulse frequency. However, when we administer CNO to inhibit MEPD GABA neurons, we completely block this effect. And this data is summarized in D, here and here. And then as an additional control, we injected mice with a control viral construct that does not contain the inhibitory dreads. And CNO failed to block the TMT-induced suppression of LH pulses. And this data is summarized here. So we repeated these experiments, but this time we used restraint as our psychological stress. And as you can see in A, there's a profound suppression of LH pulses in response to restraint. However, when we chemogenetically silence MEPD GABA neurons, we completely block this effect. And this data is summarized here for CNO and here for saline. And then again, mice expressing a control virus, CNO failed to affect LH pulse frequency, and this data is also summarized in D. So we have shown that GABA signaling in the MEPD is a key mediator of psychological stress-induced suppression of the junior H-pulse generator. But what is the underlying neural circuitry? So we have seen recently that increased kiss-peptin signaling in the MEPD increases LH pulse frequency. And similarly, decreased kiss-peptin signaling decreases LH pulse frequency. But how can we explain this? So we propose that kiss-peptin neurons stimulate GABA interneurons, and these, in turn, inhibit a GABA projection to the junior H-pulse generator. And the resulting decrease in inhibitory GABA tone will increase junior H-pulse generator frequency. So the MEPD is of palatial embryological origin. So a GABA-GABA disinhibitory system is not too outrageous. Now, our DREADS approach would not be able to differentiate between these GABA interneurons and the GABA projection neurons. However, using optogenetics, we can selectively target MEPD GABA terminals in the arcuate. So our second aim was to determine, is there a functional GABA projection from the MEPD to the junior H-pulse generator? So we selectively stimulated MEPD GABA terminals in the arcuate and measured the effect on LH pulse frequency. So again, we take VGAT-CRE mice, but this time we unilaterally inject a adenovirus with a channel adoption into the MEPD. And then position an optic fiber cannula into the arcuate. And this would allow for the selective stimulation of MEPD GABA terminals in the arc. And we can visualize channel adoption expressing fibers from the, in the arcuate as indicated by the arrows. And again, we repeat our bleeding protocol to determine LH pulse satility. So following one hour sampling to determine baseline LH pulse frequency, we continue blood sampling, but expose mice to optogenetic stimulation at different frequencies. So this schematic shows the optogenetic stimulation of GABA terminals in the arc from the MEPD. And in the absence of optogenetic stimulation, there's no effect on LH pulse frequency, and this is shown in A. The same is true for stimulation at two hertz and at five hertz. However, at 10 hertz, we see a clear decrease in LH pulse frequency. And at 20 hertz, a complete suppression in most animals. And this data is summarized in F. So in conclusion, we've shown that MEPD GABA signaling mediates stress-induced suppression of the general H pulse generator. We propose that psychological stress is acting to decrease caspeptin signaling in the MEPD, and the reduced GABA interneuron activity would leave a GABA projection to the generative pulse generator unopposed, and the resulting increase in inhibitory GABA tone would suppress generative pulse generator frequency. However, further work is needed to confirm this neural circuitry. I'd like to thank my supervisor, Kevin, and my teammates and the funding bodies. Thank you. was really beautiful. I might have missed this in your introduction, but how is stress inhibiting cispeptin expression in the MEPD? So that's all like a theory at the minute, that stress might be acting to decrease cispeptin neurons just because we've shown that cispeptin signaling in the MEPD would increase LH pulse frequency and decreased signaling also decreases it. So we're still looking at how stress might be working in the MEPD to suppress LH pulse frequency. We've shown that like you were caught in signaling in the MEPD plays a role in this process. So it might be the case that you were caught in signaling onto cispeptin neurons is like decreasing cispeptin activity. Okay so you don't think it's a direct effect and so you wouldn't affect that you wouldn't expect stress to be also affecting cispeptin in the arcuate? I mean it could be acting because I mean CORT could act on cispeptin neurons in the arcuate as well and stuff and obviously there's probably other mechanisms by which stress might be interacting with the arcuate candy neurons, but this just is one pathway that we propose that psychological stress may be acting on arcuate cispeptin neurons. Okay thank you so much. Thank you. Caitlin I was wondering have you tried a panel of different types of stressors? I'm thinking of things that Tracy Bale and other labs have done if you've seen any different effects with that. Yeah so we mostly focus on psychological stress because the MEPD is like activated by both TMT and restraint. It would be interesting to see if like more physical immunological stressors would also affect it, but yeah that'd be really interesting to look at. Yeah. Thank you very much. Our next speaker is Patrick Chappelle from Oregon State University. This is down here, okay perfect. All right well thanks to this society for having me out. It was actually my undergrad students did this project and I was hoping she could come out but she could not, so I'm doing it in her stead. This is good. I'm not gonna have to explain a lot of this to the folks here because you've already heard about it, although what I am gonna do is pull away from the GnRH pulse generator and go more over to the surge generating type using an in vitro model that we've made. So the basic question that I wanted to ask was actually a question asked to me a while ago after we made these in vitro models from KISS-GFP mice of arcuate and AVPV cells, cuspeptin cells, and that is, you know, are the GnRH receptors present on these cells? I said, oh I'll look, that's a great question, and check it out and let you know. Just a little background on these cells we published a while ago. We made KTAR and KTAV and the R is arcuate and the AV is AVPV, so you don't get confused, and both lines express KIS1, KTARs have slightly higher, and the KTARs seem to express the candy bits as well. What's really interesting and what we enjoy about these lines is that they actually seem to be pretty sensitive to timing and dosage of estradiol. In fact, we found that at sort of pharmacological doses of estradiol we don't see much response, but in the picomolar range we see plenty response commensurate with what's expected in vivo, so really nice stimulation of AVPV, cuspeptin expression in the presence of elevated estrogen, and then a decrement in the arcuate. And we've seen, we started to break this down, so you know, if you increase the dose of estradiol up to a point you see a KIS increase in the KTAVs, progesterone actually brings it, inhibits it, whereas progesterone seems to be actually pretty good, just like serum-free in the arcuate ones, and then in the KTAVs the ER alpha seems to be dynamically regulated by estrogen, but constitutive in the arcuate line. So I'm gonna really focus on mostly this line, and basically the question was asked, do these cells have GnRH receptors, because that would be kind of interesting, and I looked and said, just using cDNAs of, you know, cells and growth media. I even, you know, left some in some steroids for a while and said, nope, I don't see it. And then thinking a little bit ahead, I thought, well since they're pretty sensitive, maybe what we could do is actually try to change the estrogen, change the dose to something that is seen more physiologically in estrous cycle conditions. And so what we were able to see, oh sorry Mark, I just hit you in the head with the laser, was that actually bringing up and going in 48 hours from a low 10 picomolar up to a high picomolar, we began to see induction of this, of GnRH receptor. We kept seeing this, these are all representative gels. Interestingly enough, when you give GnRH halfway through this, in this period, you actually begin to see a repression of this effect, suggesting that GnRH actually brings it back down. And interestingly, although I said I'm not going to talk about it a lot, in the arcuate cells we actually saw the reverse, which we see with cispeptin as well, that decreasing estrogen levels seem to induce that receptor. I'm not exactly sure, I'm not going to talk a lot about what's going on there. But that brought up the question of, is this sort of 48-hour high to low, or low to high estradiol, is this the real timing of this requirement of timing? And so we wanted to actually check it out and try different concentrations in different timing. And what we'd also decided to do is, the previous experiments had not used a synchrome, a serum synchronization, which is, that or dexamethasone is really important for cultured and mortalized cells, right, which can drift out of phase being a culture for a while. And so it sort of acts, I guess, the in vivo correlate would be some kind of rapid peptidergic signal. And so with the serum shock, that actually changed things quite a bit, showing that it actually, within the serum-free conditions, at 4 hours and 24 hours, you could actually see a rise in GnRH receptor. But in the presence of estradiol, at the lower doses and at the higher doses, which is what you would expect to see in the KTABs, that's when you really see GnRH expression increase in the presence of steroids. And then by 24 hours, that's gone again. So the timing seems to be relatively tight. Interestingly, and like I said, I'm not going to go into great detail, we've sort of got the mirror image in the other cells, whereas serum-free conditions seem to increase it within that same timeline, with a return at 24 hours. And this is interesting, because actually in our previous paper, we showed that 12 hours is sort of the sweet spot, particularly for the KTAB neurons, to increase kispeptin release in our parafusion paradigm, in that same timeline. And so, like I said, serum synchronization is obviously an in-vitro technique. What would potentially be the analog here to this that would be occurring in vivo? We don't know, but we used AVP as a likely signal, right, because we know that the receptors are present on these cells, even in vivo. Estrogen actually increases it nicely in these cells, and in the presence of estradiol, 12 hours is actually the sweet spot for this. And we even gave AVP, and at lower doses, you actually see an induction. So earlier than the peak levels, AVP can actually induce generation suppression. And then to finish this off, we sort of kept delving deeper, exposing these cells to sort of longer-term, like let's just give them a whole estro cycle. I know it seems weird in an incubator, but we thought we'd try it. So giving sort of, you know, proestrus through diestrus 2, in this case, levels of estradiol, although there's a serum-free in here, which they don't really typically see. And what we can see is, giving this, that actually sort of the proestrus and diestrus 2, particularly, cases or levels of estradiol, meaning they've seen that—wow, sorry—they've seen that progression over the last 96 hours, seem to maximally induce a gene or H-receptor. Then we step back, added progesterone, also trying to mimic estro cycle conditions, and said, all right, well, let's first of all just leave them in there for 96 hours and see what they do with that. And at the end of that, they all kind of constitutively expressed gene or H-receptor under those conditions, and real-time bears it out as not much different. Now, really making as much of an effort as we can to bring in progesterone dynamically over 96 hours, along with estradiol, trying to really make the different mouse cycles, although 24 hours limited. Now, this shifted it over to more sort of the estrus phase, interestingly. But then, since we're seeing this 12-hour sweet spot, we just decided, look, let's give them these conditions for 12 hours only instead of 96 hours. And that's where we really see a nice induction of gene or H-receptor under the pro-estrus, so the lowest progesterone, highest estrogen conditions. What's cispeptin doing during all this? In some cases, it seems to track really well with gene or H-receptor expression, particularly with just estradiol. It actually seems to not like cispeptin expression that wasn't there at all, just sitting in these disparate conditions for 96 hours. And then, it tracks really well what we see with the, you know, our highest level of mimicry. And then, in the 12-hour, that's where you see the highest expression of cispeptin under those conditions. And we looked also under those 12-hour conditions, did a Western blot, and actually saw at the appropriate size the best expression of gene or H-receptor under the pro-estrus and not the diastereose one conditions. And this did seem to increase, actually, throughout when we added ATP. So, still looking at that. A lot more to do there. Took a quick look, this is still preliminary, as to then what might be occurring intracellularly, sort of at the gene or H-promoter level. Got some vectors from another lab and transfected, co-transfected with FOS and GENE vectors and co-transfection as well under different estrogen conditions, sort of low and high and none. And we sort of see a differential pattern where FOS and GENE, present in the elevated estradiol, seems to be associated with an increase in gene or H-receptor expression, although pretty lame. And then, together, it seems to work better at the low. But this is without serum shock. When we added that, everything changed a bit. FOS is better in the absence of estradiol, although not much. And then, in the presence of the elevated estradiol, kind of like what we saw up here, that the highest levels of estrogen, with FOS and GENE co-transfected, actually brought it back down. And then, one other place we wanted to look, it seemed a little odd, but I thought I'd check, is that there are two transcription factors, DLX3 and MSX1, that have been associated, particularly developmentally, with gene or H-receptor expression in pituitary gonadotropes. And we found it there, and surprisingly, DLX3 was relatively dynamic when it came to changes over time and with estradiol condition. Seems to be best with this lower estradiol condition. MSX1 really doesn't change much at all. And MSX1 is supposed to be somewhat inhibitory to gene or H-receptor expression, but it doesn't seem to be linked here. In all of our conditions, DLX3 wasn't really around for those two. Kind of came up a little bit in the more detailed estrous cycle conditions. And then, seemed to be though, present in the diestrous 2-like conditions, found after 12 hours, sort of preceding gene or H-receptor expression, which was kind of interesting. MSX1, not really any change there. Maybe a decrement later on. So, where does this leave us? The implications are, neurons in the AVPV might possess some sort of capacity to respond to gene or H, but only under a very tight temporal and steroid window. These might increase then, because we saw a lot of these changes occurring sort of prior to the proestrous. A lot of them occurring in this diestrous 2 conditions might allow for an increase in gene or H-receptor expression to be present during the actual point where you'd want to see this positive feedback, which is sort of a classical positive feedback and not the feed-forward that we typically think of. And then, you know, these also might express the gene or H-receptor, and I don't know how that would necessarily fit in right now. And the caveats are obvious, right? This is an in vitro system. It's very different from in vivo. These are immortalized cells, which are, you know, can be very different. They're homogenous, right? There's no other inputs. There's nothing within a brain slice. Another caveat is the levels of expression that we saw here, and I forgot to put that slide on there. The gene or H-receptor expression is there, but it's definitely much lower than what you would see in pituitary gonadotropes, so it's not incredibly high level. And then, of course, the evidence for intrahypothalamic gene or H-secretion. I think there has been some in previous in vivo work, but I don't know that in this context whether they're reaching the cells in the ABPV or not through the ventricle or any other way. So this still needs to be investigated, but this is just a nice way of looking at potential mechanisms, and I wanted to shout out to Noah, who was the one that did this, and her colleague Blake, and then please come tomorrow and check out these three authors, also undergraduates, for the poster session in the afternoon. Thanks. Sorry I went over time. Dan, you know you want to. I know, but I also know it annoys people that I do it all the time. We love it. Nice to see you, by the way, Pat. Jen was saying how old you've gotten. Thanks. I'm joking. Wise. I'm joking. So a couple questions. So you talked about DLX. I'm just wondering, did you look at SF1 expression in those cells, too? Not yet. We plan to, but we have not looked at that. And then, you know, there are these Grick mice, which are a CRE knocked into the GNR intraceptor locus. If you cross them to a reporter strain, we actually don't see GFP in the KISS. And that was, and I think you did look in multiple steroid conditions, right? Yeah. Like I said, it's relatively low, and so, I mean, I don't know that, I'm not saying that you didn't catch it, but yeah, we only see it under this. And so, it could be that because they're by themselves and not in the brain, right? So that, yeah, it is maybe not incredibly physiological, only under very certain conditions. I don't know. And then, I don't know if anyone in the audience, I'm sure there's some of you that are doing single cell analyses. Maybe you can tell us whether you've seen any, because you have to know where with GNR intraceptor expression. I mean, I would love to hear that. Thanks. Pat, you said there was a major role for ER-alpha. Have you looked for ER-beta? We did. In ER-beta, the differences there are the arcuate lines seem to have more ER-alpha in general, even though it's constitutively expressed in different steroid conditions. The AV line has more ER-beta relative. And we've tried, we used, this is in previous work, we used DPN and PPT, and we're able to get some responses. But, yeah, they're both there. And then your upregulation of FOS and JUN by estradiol, what do you think those downstream effects are? What are they doing? Don't know yet. No speculation? Come on, help me out here. Well, I mean, so really, we're trying to just figure out how, if it's possible, which as Dan suggested, it might not be, for GNR-H receptor to be there, what could be the way that they get there? So we're just really looking at the input, right? And so there are multiple signals they use, the AP1, and so I, you know, you can conjecture for a while, but we're not there yet. Thank you very much. Thanks, Pat. Our next speaker is Bruno Azevedo from the University of San Paolo. Hi. Good morning, everyone. First, I would like to thank you, the organizing committee, for the opportunity to bring my work in this session today. And I have nothing to disclose. So, as an introduction, I would like to briefly explain the importance of pituitary gland. Pituitary gland is the master gland of the endocrine system, responsible for regulating multiple physiological processes, including stress response, body growth, reproduction, and metabolism. Much of the control came from five cell types of the anterior pituitary gland, including corticotropes, somatotropes, lactotropes, thyrotropes, and gonadotropes. In part to emphasize the nanorecursation of... I'm sorry, what is the point? Okay, nanorecursation of signaling molecules and transcription factors are required to obtain these five differentiated cell lines just mentioned. In a long story short, you can see on the presentation, the main transcription factors related to the development and differentiation of pituitary cell lineage. And I would like to highlight one of these factors, PROP-1 gene. PROP-1 gene is initially described as an extremely necessary factor to activate a PO-1F1 gene that leads to differentiation of somatotropes, lactotropes, and thyrotropes. And important to mention that when random mutations in PROP-1 gene occur, we see a lack of difference between pituitary hormones in the organism. And one of the first hormone deficient animals to be reported back in 1961 by Shenbaum and Goen was the Ames dwarf mice. These mice have a spontaneous recessive point mutation in the PROP-1 gene and have a lack of multiple hormones such as GH, TSH, prolactin, and reduced levels of gonadotropin and circulating IGF-1. From the standpoint of phenotype characteristics, these animals are small with a low body weight and size when compared to the white type. And clinically, these animals present a hypothyroidism and were initially classified as infertile. Throughout the years, these animals models have been extremely analyzed, especially in age studies. And however, recent works have shown that sexual maturity and fertility of male Ames dwarf mice depend on some factors such as genetic background, mice's feeding, and life's conditions. And according to the current literature, male Ames dwarf mice can spontaneously be fertile or started through hormone treatment. Either way, the molecular mechanism behind it remains barely studied. So the present study aims to characterize sexual maturation and fertility restoration in isogenic strain under or not hormone replacement, given our environment and genetic factors that could influence the reproductive axis. So to evaluate sexual maturity and fertility, the animals are divided in four groups with five animals at each, dividing treated dwarf mice and treated dwarf mice, Y-type mice, and heterozygous mice. And to access additional aspects, such as anthropometric, morphological, and molecular parameters, we divide the animals in three groups with five animals each. The agent-lab turoxin treatment start at third days after birth, and we follow a protocol already established in the literature. And these animals were euthanized at nine days after birth. At the sacrifice day, body length and weight were performed, and also stethoscope weight. Spare were collected for the epididymals for spermogram, and PT tear were collected for gene expression through real-time PCR. And tests are measured and prepared for histological evaluation. So let's move on the results, and then we'll be done. From the standpoint of sexual maturity, we can see that all treated animals, I try again. What's this point? Okay. We can see that all treatment animals reach sexual maturity, but with a significant delay when compared to the Y-type animals. This could be explained by the fact the treatment start at third days old period. And only one animal out of the five animals without treatment reach sexual maturity. And regarding the weight, we have not observed statistical difference in any of the groups. Supports the fact that apparently, sexual maturity is more and more related to corporal age than a chronological age. Okay, so. No, it's this. So the treatment have a positive impact on the anthropometric parameters as well. All groups present in a statistical difference in both weight and national length when compared to the Y-type group. And the testicular, sorry, yes, I'm sorry. I in the wrong slide. So this in the testicular weight of the animals under treatment was similar to the Y-type group. And statistically different from the AMES untreated group. In addition, the treated group had its fertility restored with no significant difference in the number of animals per offspring compared to the Y-type. While the untreated group was confirmed infertile. So now, recording the histological data of the seminiferous tubules, such as the germinal, epithelial thickness, and the luminal area, there is no statistical difference between the treated group and the Y-type. However, when this is converting the total area of the tubule there is a difference between the three groups. In this image we can see the test's histological data where in the first row we have animal tests from the Y-type group, the second animal test from the treated group, and the third row represents animal tests from the untreated group. And based on that we can conclude that the treatment was effective in relation to the development of spermatogenesis. Where both Y-type and treated group had a complete spermatogenesis and present an organized epithelium. While for the dwarf animal without treatment it was observed in the spermatosis stage an interruption of spermatogenesis. So in this slide looking to four different sparse parameters such as number of spermatozoids, motility, concentration, and spermatozoid survival rate, we can see no difference between Y-type and treated group in contrast with the untreated group that presents all parameters decrease. And finally moving to molecular parameters, GH and prolactin expression pattern present a statistical difference among the three groups. However, this is observed in the treated group due to negative feedback that the treatment with human recombinant GH generates. Since this human GH in mice binds on GH and prolactin receptors. The TSH present only one statistical difference between the treated group and the Y-type group. And probably this statistical difference in the treated group could be also explained by the negative feedback that lyotiroxine treatment generates on TSH transcriptor levels in the pituitary gland. And FSH expression pattern present a statistical difference between the Y-type group and the untreated group. And LA present a significant increase in specialist values in the group that received the treatment when compared to the Y-type group. And GATA2, that is important factor for both tyrotrophic and gonadotrophic lineage, only present a statistical difference between Y-type and untreated groups. So in conclusion, AIMS mutant mice under treatment with GH and lyotiroxine replacement should reach sexual maturation and restore fertility. And treatment appears to play a key role in reproduction parameters acting peripherally. And the mechanism behind this phenomenon will be explored using RNA-seq in the future. So thank you all and I'm glad to help if you all have any question. Thank you. Any questions? While people are thinking about it, I was wondering if you had any effects on the age of maturation? And also, what about if you looked at behavior, if there are any changes in behavior, in reproductive behavior? Oh my, sorry, I don't understand your question. Sorry, I was just wondering if you saw any effects, it's sort of an off question, but if you saw any effects on behavior, on the reproductive behavior of these mice? No, we didn't saw any behavior difference in this group. Sorry for this. No, no, no, that's good. Dan? So, looking at the histology, it looked like even in your treated group that the Leydig cells were hypoplastic. I'm just wondering, did you measure intratesticular testosterone? No, we didn't measure, but we want to see that, we want to measure that in the future, but we didn't measure now. Thank you. Thank you very much. And our last speaker of the session is Edouard Mills from Imperial College of London. Thank you, Chair, and thank you to the organizing committee for allowing us to share data from the Dillo Lab at Imperial College London, revealing that kisspeptin increases penile tumescence and sexual brain processing in men with hypoactive sexual desire disorder. So, hypoactive sexual desire disorder, or HSDD, is a common condition characterized by a persistent lack of sexual desire with severe distress in someone who is otherwise entirely healthy, eugenadal, and previously had normal sexual function. It affects up to 8% of men with major effects on quality of life, interpersonal relationships, and fertility. However, despite this high clinical burden, there's currently no licensed pharmacotherapies nor therapies in late-stage development for men with HSDD. So, a therapeutic target could be the neuropeptide kisspeptin, which we know sits at the apex of the reproductive axis to regulate downstream reproductive hormone release, as I'm sure we'll hear in a talk by Dr. Ali Abara this afternoon. However, outside of its established reproductive roles, kisspeptin and its receptor are extensively distributed throughout the limbic brain, including in humans, as shown here by work from Muir and colleagues. So, this therefore provides a neuroanatomical framework for kisspeptin's emerging importance in sexual behavior, with data from the eminent Professor Kevin O'Byrne's lab at King's College London that kisspeptin signaling in the male rodent amygdala stimulates both erections and sexual motivation, and evidence from Professor Julie Bakker that kisspeptin signaling in the hypothalamus of female mice regulates the key reproductive behavior of lumbar lordosis. Now, turning to humans, we've shown previously in healthy men that kisspeptin administration increases both brain responses to sexual images with associated reductions in sexual aversion, and increases brain responses in attraction pathways. So, this therefore led us to hypothesize that kisspeptin administration in men with hypoactive sexual desire disorder would enhance penile tumescence and sexual brain processing in response to sexual stimuli. So, therefore, to address this, we recruited 32 heterosexual men with low sexual desire caused by hypoactive sexual desire disorder who were eugenadal and otherwise entirely healthy and not taking any regular medications. So, here is the protocol. 32 men with hypoactive sexual desire disorder attended for two study visits each, during which they were infused for 75 minutes with kisspeptin or placebo on their first study visit, and then the other infusion on their second study visit, with the order of the infusions randomized and, of course, the participants blinded to the infusion identity. Blood was collected throughout for hormone levels, and participants completed psychometric questionnaires at baseline, and then again towards the end of the infusion period, with functional MRI scanning carried out during the infusions, with the participants performing two tasks. First was a 12-minute short videos task, during which the men were shown 20-second blocks of erotic videos, which for the purpose of today's presentation are pixelated, so please use your imagination, and so these alternated with exercise videos as a non-sexual control. This was then followed by a continuous 8-minute erotic video, during which penile tumescence and subjects of arousal were recorded continuously. So here are the results, starting with the hormonal data. So here's a graph of circulating kisspeptin levels against time, and you can appreciate that kisspeptin administration, represented by the red line, resulted in a robust increase in circulating kisspeptin levels, which reached steady state from 30 to 75 minutes during the fMRI and psychometric questionnaire period. However, testosterone did not increase in the 75-minute study period, which is consistent with the literature that downstream testosterone will increase after 90 minutes in humans following kisspeptin administration. In addition, circulating levels of cortisol did not increase, thereby excluding changes in testosterone or cortisol as contributing to our results. So turning to the behavioral results, we observed that kisspeptin administration caused a significant increase in happiness about sex compared to placebo, as well as flushing, which is a sensitive physiological marker of sexual arousal in men, with these increases likely to be explained by the changes in brain activity observed during the fMRI tasks, which I'll now present. So starting with the brain responses to the short erotic videos, here are cuts through the brain constructed using the collected data from all 32 participants. So areas in yellow and red represent areas of significantly enhanced brain activity, and areas in green and blue represent areas of significantly decreased brain activity during kisspeptin administration compared to placebo on viewing the short erotic videos. And you can appreciate that kisspeptin administration significantly enhanced brain activity in the middle frontal gyrus and anterior cingulate, and significantly decreased activity in the parahippocampus. So next, we took our brain data and we correlated it with our psychometric data. So the posterior cingulate is an important structure in the sexual processing network, and we observed that men with higher baseline sexual desire distress scores had greater posterior enhancement by kisspeptin on viewing the short erotic videos. We also observed that globus pallidus, which is an important reward structure, that greater globus pallidus enhancement by kisspeptin on viewing the short erotic videos predicted how sexually naughty the participants felt at that time. So turning to the long video task, here's a graph of penile tumescence over the eight-minute task, and you can see that kisspeptin administration represented by the red line resulted in a robust increase in penile tumescence. In fact, kisspeptin's pro-erectile effect was most marked by the end of the eight-minute task, where kisspeptin increased penile tumescence by 56% compared to placebo. So next, using this penile tumescence data as a regressor to identify the brain regions where activity is related to penile tumescence, we observed that kisspeptin administration significantly increased activity in both the visual cortex and the fusiform gyrus compared to placebo, which are two frequently activated brain regions in response to sexual stimuli. So then finally, using the subjective arousal data as a regressor to identify the brain regions where activity is related to sexual arousal, we observed that kisspeptin administration significantly decreased activity in the frontal pole, precuneus, and posterior cingulate compared to placebo, which is highly relevant as all three of these areas are involved in self-control and self-judgment and tend to be hyper-activated in HSC. So the putamen is a further reward structure, and we observed that men with lower baseline satisfaction with sex had greater putamen enhancement by kisspeptin administration on viewing the long erotic video compared to placebo. We also observed that in addition, the greater putamen enhancement by kisspeptin on viewing the long erotic video predicted how erect and how horny the participants felt. So in summary, we provide the first clinical evidence that kisspeptin administration in a patient group of men with low sexual desire robustly increases penile tumescence by up to 56%, as well as behavioral measures of sexual desire and arousal by modulating activity in key structures of the sexual processing network. Our data therefore suggests that kisspeptin-based therapies may provide a new therapeutic option for men with low sexual desire. And with that, I'd like to close by thanking my supervisors, Dr. Alexander Korminos and Professor Walter Dillay for their fantastic mentorship over the last five years. The members of the Dillow Lab, our collaborators in Vicro London and St. Mary's Hospital, our funders at the NIHR and MRC, and of course, the men who took part in the study. Thank you very much for your attention. Questions? I'll start Edward. Thank you for the talk. So you mentioned that there were some areas that are increased activity during this hypo, hyperactive sexual desire disorder, and that those areas were decreased activity by kisspeptin. I'm just wondering if you could talk a little bit about that. Yeah, so we looked at a range of regions of interest that we know express kisspeptin receptors in humans. So, for example, the amygdala, the insula, and a range of other limbic areas, and they weren't affected by, or they weren't significantly affected by kisspeptin. And so we looked at a range of regions of interest that we know express kisspeptin receptors in humans. And so we looked at a range of regions of interest that we know express kisspeptin receptors in humans. So, for example, the amygdala, the insula, and a range of other limbic areas, and they weren't affected by, or they weren't significantly affected by kisspeptin. And we know from the range of neuro studies that have been done so far that those regions are not potentially involved so much in men with hyperactive sexual desire disorder. So the regions that we saw were modulated were areas that we know typically are either hyperactivated or hypoactivated in this disorder. All right. Manuel. Very nice talking. Very interesting data. There is some ongoing debate on whether this kisspeptin 54 that you are administering can, I mean, how much of it enters into the brain or the way around. Do you think this is direct action of kisspeptin in the sites that you see deeper or hypoactivated? Or is it actions on a specific zone that are more accessible and then projections go to these areas? Yeah, that's a great question. Thank you. So we know that different kisspeptin isoforms have different degrees of blood-brain barrier penetrance. We know that 54, which we use, is able to access the dendritic terminals of generated neurons outside of the blood-brain barrier. But we know from previous work that we've done that 54 can also cross the blood-brain barrier. So we suggest that it's probably a direct action of kisspeptin on its receptor. We also know that some of the brain areas which kisspeptin modulated in this study are not known to express kisspeptin receptors in humans. But whether kisspeptin is interplaying with another downstream hormone, whether it be GABA, nitric oxide, and that's what's bringing about these behavioral effects, we don't know at the moment. But it's probably an action on its receptor, but also these downstream pathways. Thanks. I had a related question about the route of administration and the effect of this dose of kisspeptin. So you showed that there was no effect on testosterone. But with the same administration, would you see increases in LH? Yeah. So the pattern of LH that we see mirrors the pattern of kisspeptin. So were you surprised that you didn't see an increase in testosterone production? I mean, it's usually pretty acute that LH would stimulate an acute increase in testosterone. So we know that with this administration protocol at one nanomole per kilogram per hour that we see a testosterone rise after 90 minutes, which is why we designed our protocol to finish at 75 minutes. That way we could exclude that confounding effect. It's possible that if we'd continued the actual intravenous infusion that you'd see that increase beyond 90 minutes, but the study had already stopped at that point. Okay. And then if I understood the design of the study, so this was counterbalanced, so each individual would get both treatments. Is that correct? Correct. Yeah. So there were 16 men who had kisspeptin first and placebo second, and then the other 16 had the other order. The images that they saw, were those the same on both times? Yeah. So what we did was we designed a protocol such that most men attended about a month later. The minimum time period was a week, so that avoided necessarily a complete habituation. What we wanted to do was to have the same stimuli, that way we could directly compare the two visits, rather than having stimulus material that may have different arousability. Oh, Kelly, go ahead. Very interesting talk. I'm curious if subsequent trials would enhance activity of these brain regions, and whether you have any evidence that activity is elevated in subsequent trials. So this was an acute study that we've done. Obviously, the next step would be to do more chronic studies, but also to see what the effects are of kisspeptin using different administration protocols that would be more realistic and feasible at home, for example. But we'd envisage that we would hope to see the same sorts of results in further subsequent chronic studies. Thank you. Thank you. Thank you very much, Edward. So please join me in thanking all the speakers.

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