Effects of long term exposure to a high dose GnRH agonist deslorelin Thank you for coming to my research proposal. My proposal today is meant to give you all some insights of the research that I have completed, my current research, and potential future research directions. And, also, to and get some good feedback or suggestions or even other possible paths to take or look into for my research. So please don’t hesitate to interrupt me and ask questions throughout my talk.

Outline Introduction Long term treatment/high dose HPG axis overview Gonadotropes/gonadotropins Long term treatment/high dose GnRH agonist research Potential mechanism

HPG-axis overview GnRH is secreted from the hypothalamus binds to GnRH-R on gonadotropes controls production/secretion of LH and FSH The axis is controlled by a negative feedback loop Testosterone is a key regulating factor FSH LH Testosterone -controls production of the gonadotropins LH and FSH which are transported down to the testes to control production of testosterone and spermatogenesis -the axis is controlled by a negative feedback loop in which testosterone and inhibin secreted from the testes feedback at the level of the hypothalamus and pituitary to control GnRH, LH, and FSH production

LH and FSH are heterodimeric glycoproteins Common α-subunit αGSU Unique β-subunits LHβ β-subunit is used to measure FSH and LH levels in the pituitary FSHβ -the gonadotropins LH and FSH are heterodimeric glycoproteins conisisting of a common alpha subunit known as alpha-gonadotropin subunit and unique beta subunits (these are responsible for the interaction with LH and FSH receptors in the testes) -The common alpha subunits combine with the unique beta subunits to form the glycoprotein's FSH and LH -So we use the beta subunit in measuring activity levels to infer specific biological activity of the hormone. The beta subunit is also the rate limiting step for the hormone -Chorionic gonadotropin (comes from the placenta not pituitary) also shares the same common alpha subunit as FSH, LH, and TSH FSH LH (Bousfield et al. 2006)

GnRH secreted in pulses (Belchetz et al. 1978) -Gonadotropins are differentially regulated via GnRH being released in a pulsatile manner ( which is key to reproduction) in which faster-frequency pulses favor LH release and slower-frequency pulses favor FSH release. But, the pulsatility of GnRH doesn’t completely explain gonadotropin the differential secretion of gonadotropins. -Continuous administration of GnRH using a GnRH agonist causes receptor downregulation on rat gonadotropes. Subsequently leading to suppression of LH and FSH hence causing a decrease in testosterone production. Though rendering that gonadotrope in desensitized state to GnRH -Graph doesn’t show evidence of it but after agonist administration there is a subsequent rise in LH and FSH before the downregulation -an important note as that when GPCR are continuously stimulated they become desensitized via phosphorylation and beta arrestin binding of the C-terminal tail. GnRHType 1 receptor is completely different compared to regular GPCRs in that they are resistant to desensitization and internalize slowly due to the lack of the C-terminal tail. The exact mechanism through which the GnRHr-1 is internalized and the route though which agonists induce there desensitizing effect are not entirely understood -graph shows a study in rhesus monkeys Subsequently leads to suppression of LH and FSH Testosterone and spermatogenesis

High Dose Long term treatment -Testosterone levels did not recover in 9% of the patients. (Kaku et al. 2006) High Dose -In the first study male dogs were treated with different doses of deslorelin and they found that the higher the dose the longer the time it takes until these animals completely recover. -In the second study patients with prostate cancer received low dose GnRH agonist (via leuprorelin 3.6 mg/month) treatment for 24-30months (which is one of only a few studies detailing long term exposure to a GnRH agonist) and then monitered hormone level recovery time. -48% of the patients testosterone levels didn’t recover for up to 2 years following cessation of treatment and 9% of patients testosterone levels never returned to the normal range. -Thus showing an interaction between duration of treatment and time until full recovery. And that the longer the treatment the longer the time until full recovery. -Increasing dose extends time to full recovery (Junaidi et al. 2009)

Our experimental hypothesis: -Significantly increasing the dose and/or duration of GnRH agonist exposure will permanently suppress the reproductive axis. -So we used this model and the information and formulated a hypothesis that if we increase the dose of deslorelin, could we permanently shut down the reproductive axis -We were not sure of the effects deslorelin would have at such a high exposure, but we were really not sure how important duration of exposure would be

Low dose (1.1 mg) for 6 weeks Mono-FSHβ Bi-hormonal Mono-LHβ αGSU -The outline of our first study went like this: -We had 5 groups ranging from deslorelin, desl plus a 4 week and 12 week recovery, deslorelin plus a Testosterone implant to see if the effects of desl was soley mediated through androgen levels, and our control animals -here is a cartoon illustration of how the implants were administered. They are simlpy injected in the scapular region. -a few methods: Used adult male rats roughly 6 months old. Used a massive dose of deslorelin at 14.1mg for 6 weeks. At the end of each treatment period I performed cardiac perfusions to fix tissue to allow for immunocytochemistry. Obtained blood via cardiac puncture to assay hormone levels. And then weighed all organs to detail effects GnRH has on other organs outside of the hypothalamic-pituitary axis (extrapituitary sites). (Smith et al. 2012)

Different treatments: -High dose (14.1 mg) for 6 weeks + recovery -High dose for (14.1 mg) 6 months + recovery -The outline of our first study went like this: -We had 5 groups ranging from deslorelin, desl plus a 4 week and 12 week recovery, deslorelin plus a Testosterone implant to see if the effects of desl was soley mediated through androgen levels, and our control animals -here is a cartoon illustration of how the implants were administered. They are simlpy injected in the scapular region. -a few methods: Used adult male rats roughly 6 months old. Used a massive dose of deslorelin at 14.1mg for 6 weeks. At the end of each treatment period I performed cardiac perfusions to fix tissue to allow for immunocytochemistry. Obtained blood via cardiac puncture to assay hormone levels. And then weighed all organs to detail effects GnRH has on other organs outside of the hypothalamic-pituitary axis (extrapituitary sites).

FSHβ DESL Control DESL + 4 week recovery -Used ICC to label for FSHbeta -As you can see the amount of immunoreactive cells was significantly downregulated in the desl group compared to controls. -in the testosterone replaced group there were more ir-cells but was still reduced compared to controls. -and 4 weeks following implant removal we had complete recovery in immunoreactivity when compared to controls.

LHβ DESL Control DESL + 4 week recovery -this is the labeling for LHbeta in which the DESL group had a decrease in ir compared to controls -Testosterone replaced group had an even further decrease in immunoreactivity -and we had complete recovery within 4 weeks following implant removal.

FSH Total T -these are the bar graphs just representing the same thing shown previously. So we were not able to completely suppress the HPG axis. But there were some really cool features that we noticed going away from this experiment -That was the differential effects Testosterone had on FSH and LH. -As you can see in the top graph the testosterone replaced group was still significantly decreased compared to control, but when compared to the desl group using a T-test the testosterone replaced group was significantly increased. -Looking at Lhbeta cell percentage Deslorelin decreased, but the testosterone replaced group was reduced even more compared to controls -Testosterone rescues Deslorelins suppressive effects on FSH while it enhances the suppressive effects on LH -These differences in the testosterone replaced groups also hold true for plasma hormone levels in that we are seeing testosterone has divergent effects on FSH and LH. -Gonadotrope cells may be multipotent; hence changing storage/synthesis patterns of LH and FSH LH

6 week treatment (14.1mg) DESL Control 66% decrease in testes weight -The testes weights are in milligrams per 100 grams body weight -Deslorelin caused decreased testes weights by 66% of control values -Testosterone replacement increased testes weights to were there is no significant differences compared to control values -4 weeks following implant removal testes weights were still significantly suppressed and complete recovery of testes weights didn’t occur until 12 weeks following implant removal. Implying that testes are more downstream in the recovery from deslorelin compared to percentage of immunoreactive cells in the anterior pituitary and plasma hormone levels. -SO…this study was good in that we were able to induce a chemically castrated state, but we were not successful in permanently suppressing the reproductive axis for the long term 66% decrease in testes weight

Different treatments: -High dose (14.1 mg) for 6 weeks + recovery -High dose for (14.1 mg) 6 months + recovery -The outline of our first study went like this: -We had 5 groups ranging from deslorelin, desl plus a 4 week and 12 week recovery, deslorelin plus a Testosterone implant to see if the effects of desl was soley mediated through androgen levels, and our control animals -here is a cartoon illustration of how the implants were administered. They are simlpy injected in the scapular region. -a few methods: Used adult male rats roughly 6 months old. Used a massive dose of deslorelin at 14.1mg for 6 weeks. At the end of each treatment period I performed cardiac perfusions to fix tissue to allow for immunocytochemistry. Obtained blood via cardiac puncture to assay hormone levels. And then weighed all organs to detail effects GnRH has on other organs outside of the hypothalamic-pituitary axis (extrapituitary sites).

FSH Total T -The duration of exposure to deslorelin had different results in that as you expects cell % for both FSH and LH in the deslorelin groups was significantly decreased. -The difference this time compared to the 6 week group is that we did not see any recovery in the cell % of gonadotropes for up to 12 weeks following implant removal. -Implying that we see a possible permanent cytoarchitectual change in the anterior pituitary specific to gonadotropes up to 12 weeks post exposure. -Interestingly as you will see in a few slides that we don’t see a decrease in the amount of gonadotropes per se (by looking at the alpha subunit) but there does seem to be a change in the ability to synthesize the beta subunits of LH and FSH. -If there was a loss in gonadotropes there would subsequently be a drop in alpha subunit as well and we are not seeing this. -So we are seeing that the mechinery of gonadotropes becomes altered under the exposure of a high dose deslorelin being administered for an extended period of time. LH

6 month treatment (14.1mg) 81% decrease in testes weight -we completely ablated the testes with the longer duration to deslorelin by massively decreasing the testes weight by 81% compared to control values -we still had recovery within 12 weeks following implant removal -morphological analysis on the deslorelin testes shows no resemblance of any intact seminiferous tubules nor leydig and sertoli cells. Analysis still needs to be completed on the 12 week recovery group to check for intact tubules -the increase in weight on the 12 week recovery group may be contributed to edema from all of the cells lysing (but no confirmation as of yet). -testes weights have reached an even further decrease, but what is the minimum value can we get and how long would treatment have to be?

Conclusion Dose and duration is key for suppression of the endocrine profile Long term alteration in cellular composition in the pituitary Seems the testes are starting to recover But testosterone levels are still low -Now we are going to switch gears and talk about mechanisms. A lot of GnRH agonist research really focuses on short term exposure, but what is going on at the level of the gonadotrope during long term exposure to deslorelin. So the next few slides focuses on the potential mechanism through which this works. -Now here are a few slides demonstrating some novel potential mechanisms through which GnRH agonists specifically deslorelin exerts its actions.

GnRH agonist mechanisms of action -Activin stimulates FSH secretion and FSH-β mRNA levels (ling et al. 1986, Vale et al. 1986, Weiss et al. 1993). -Follistatin binds activin, reducing FSH secretion and FSH-β mRNA levels (Ying 1988) -Pituitary adenylate cyclase-activating polypeptide (PACAP) stimulates follistatin expression thus suppressing FSHβ mRNA (Tsujii et al. 1994) -PACAP enhances α-subunit transcription (Schomerus et al. 1994) GnRH agonists also increase α-subunit -Remember when I talked earlier about potential mechanisms through which GnRH exerts its actions are not completely understood -here are a few possible explanations of continued FSH production independent of GnRH and could also explain the continued alpha subunit production -activin stimulation of FSH is via in vitro and in vivo -now I am going to try and present a few possible scenarios were both of these two pathways are related and possibly how agonists exert there actions

Signaling network DESL follistatin FSHβ gene expression activin-βB PACAP PACAP GnRH-R follistatin αGSU follistatin follistatin -this brings me to this point of all the intra-pituitary signaling, known as paracrine (a mode of action in which a cell secretes a factor and that factor acts on surrounding cells nearby) and autocrine (a mode of action in which a cell produces a factor and that factor acts back on the same cell that produced it) signaling. -So we have this elaborate network of cells secreting all kinds of autocrine and paracrine factors and that this signaling could explain how FSHbeta subunit is regulated independently of GnRH. -activin B is composed of two identical beta subunits that acts to increase both FSHbeta gene expression and secretion. Activin binds to the type II receptor, and then an activin Type I receptor is recruited into the complex. Once activated the type 1 receptor stimulates a novel family of proteins called Smads. This then stimulates the FSHbeta promoter through Smad3 and is augmented by Smad-4. Activin also stimulates GnRH-receptor synthesis. -Follistatin (autocrine/paracrine factor produced in gonadotropes and folliculostellate cells) is a single-chain polypeptide that is known as an activin binding protein . It binds to activin with extremely high affinity (irreversible) and bioneutralizes activins activity (forming a follistatin-activin complex) hence decreasing FSHbeta gene expression and secretion. This complex then prevents binding of activin to type II activin receptor subtypes. -But what is controlling follistatin expression? -Pituitary adenylate cyclase-activating polypeptide (PACAP) stimulates follistatin gene expression in rat pituitary cell cultures. Though decreasing FSHbeta gene expression. PACAP is also known to stimulate alpha subunit transcription and lengthen the Lhbeta mRNA transcript and presumably prolonging its half-life (Fujii et al. 2002). -Another factor controlling follistatin gene expression is testosterone. In which testosterone suppresses follistatin gene expression hence stimulating FSHbeta mRNA and FSH secretion in cultured rat pituitary cells (Kawakami et al. 2002). Testosterone could also be acting on -Therefore T may increase FSHbeta gene expression indirectly by decreasing FS and increasing activin availiability -PACAP to decrease alpha subunit indirectly via downregulation by testosterone..?? Remember gonadotropes contain an androgen receptor FSHβ gene expression Folliculo-stellate cell Activin-βB Activin-R Gonadotrope

Acknowledgements: Dr. Donal Skinner (Advisor) Cheryl Asa Andrew Taylor Carole Hertz Arik Smith