Potential etiology of PFS/PSSD/PRSD : a novel target RFRP3

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Serguei_Novitchok
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Potential etiology of PFS/PSSD/PRSD : a novel target RFRP3

Unread post by Serguei_Novitchok »

Here I present a potential mechanism of PFS/PSSD/PRSD involving a neuropeptide RFRP3(in humans) (I will abbreviate RFRP). I also present a study protocol to demonstrate this theory.


INTRODUCTION
Here we present two studies of RFRP3 that will help us establish a potential theory about the possible mechanism of these syndromes. Based on these two studies, we will establish a potential etiology for this syndrome and provide an experimental protocol to demonstrate it.



STUDY 1: Hypothalamic RFRP3 knockdown prevents chronic stress-induced infertility and embryonic resorption (http://dx.doi.org/10.7554/eLife.04316.001)

Study Summary:
Female rats exposed to a stressor for a period of 18-days followed by a 4-day recovery period without a stressor were compared to a control group without a stressor. The mating frequency of the stressor-exposed rats was compared to that of a control group. As expected, the stressor-exposed group had a statistically significant decrease in mating frequency compared to the control group.
The level of RFRP mRNA and RFPR receptors (indicator of gene expression) in the hypothalamus and the level of corticosterone (indicator of stress) in venous blood were assessed during both the stress and recovery periods of the brain. As expected, the stress hormone (corticosterone) increased during the stress period and returned to normal during the recovery period. RFRP mRNA and RFRP receptor levels increased compared to control during the stress period and, interestingly, remained elevated even after recovery compared to control.
The study authors hypothesized that this persistent increase in RFRP and RFRP receptor gene expression plays a causal role in lingering sexual behavior. To prove this hypothesis, they used a viral vector containing an shDNA plasmid. The shDNA plasmid was composed of a shRNAmir gene and a promoter region, called TetOFF, controlling the initiation of shRNAmir transcription. The presence of doxicylcine prevented the TetOFF promoter region from initiating transcription. Therefore, transcription of shDNA was prevented by the presence of doxicycline. In other words, doxicycline acts as a switch for shDNAmir transcription. The shDNA is transcribed into shRNA, which will be processed by a protein called Dicer and will yield a small interfering RNA (siRNA). These siRNAs will interfere with the mRNA of the targeted gene (here RFRP) and prevent its translation. This is a well known technique to silence a target gene in vivo using the RNA interference machinery. This process is briefly described here: https://en.wikipedia.org/wiki/Short_hai ... ivirus.svg.
In the presence of doxicycline (the RFRP gene is expressed), rats exposed to stress have a reduced mating rate. Without doxicycline (the RFRP gene is not expressed), the stress-exposed rats had a normal mating rate. This is well summarized in figure 4 of the study. From these observations, we can assume that high RFRP gene expression plays a causal role in the persistence of abnormal sexual behavior after stress exposure.

Why is this study interesting?
In terms of PFS/PSSD/PRSD, this study is of great interest because it demonstrates the role of a protein in an induced persistent sexual dysfunction. But it is of even greater interest in light of another study (see Study 2 below).



STUDY 2: Citalopram (antidepressant) administration causes sexual dysfunction in male mice via RF-amide-related peptide in the dorsomedial hypothalamus (doi:10.1016/j.neuropharm.2010.03.018)

Summary of study:
Mice chronically treated with citalopram (5 mg/kg/BW) for 4 weeks were compared with a control group. As expected, altered sexual behavior of male mice was observed compared to the control group. Cell number, fiber number, and mRNA expression of GnRH, kisspeptin, RFRP, and RFRP receptor were assessed in the following brain regions: POA, AVPV, ARC, and DMH.
The study found no relevant results (cell number, mRNA) for Kisspeptin or GnRH compared to control. However, they did find some interesting results regarding RFRP: increased neurons in DMH, increased RFRP-immunoreactive fiber density in POA, increased RFRP receptor mRNA levels in POA (by "increased," I mean statistically significant; p<0.05). However, no statistically significant change was observed in RFRP mRNA levels in the POA, an increase was observed but did not reach statistical significance (control: 0.63 +/- 0.25 citalopram: 0.79 +/- 0.26; p=unknown).
Based on the change observed following chronic citalopram treatment, we can speculate that RFRP plays a role in the observed sexual dysfunction during SSRI intake.

Why is this study interesting?
This study highlights the potential role of RFRP and its receptor in SSRI-induced sexual dysfunction. This is in agreement with the previous study showing that modulated expression of the RFRP receptor gene causes altered sexual behavior in female rats. Interestingly, the part of the POA brain that showed increased RFRP receptor gene expression is known to be involved in sexual behavior. In fact, lesions in these areas are linked to loss or abnormality of sexual function: Impairment of mating behavior in male rats following lesions in the preoptic-anterior hypothalamic continuum (https://doi.org/10.1016/0006-8993(67)90076-5).
Given that RFRP plays a causal role in the persistent stress-induced sexual dysfunction observed in female rats and given the increase in RFRP receptor gene expression following chronic SSRI treatment, we can assume that SSRI-induced sexual dysfunction involves RFRP.



BUT HOW DO WE PROVE SUCH A HYPOTHESIS?
Here is the interesting part. We can easily propose a study to demonstrate whether RFRP plays a causal role in SSRI-induced sexual dysfunction. The first study we presented uses a viral vector to inject a DNA plasmid into the host genome. The DNA plasmid acts as a gene expression switch for the RFRP gene. The same viral vector and DNA plasmid can be used to demonstrate whether or not RFRP plays a causal role in SSRI-induced sexual dysfunction. Simply replace the stress sessions with chronic SSRI treatment (no recovery period i.e test the hypothesis while mices are on the drug). If, by deactivating the expression of the RFRP gene, sexual dysfunction is alleviated, we can infer that RFRP plays a causal role.
Further studies can be conducted to strengthen this theory. Gene expression of RFRP and RFRP receptor can be assessed in the hypothalamus or POA of mice on 5-alpha reductase or isotretinoin. If the expression of RFRP and RFRP receptor genes is increased in the evaluated regions compared to a control group, this would confirm the involvement of RFRP as a common potential cause of PFS/PSSD/PRSD.
Furthermore, if this theory proves to be true, it is very likely that the same mechanism is involved in patient with sexual dysfunction when taking SSRIs, finasteride, or isotretinoin.



CAN A CASE CONTROL STUDY BE PERFORMED TO DEMONSTRATE THIS IN HUMANS?
Obviously, the mRNA level of RFRP3 in the hypothalamus can only be assessed by a biopsy of the hypothalamus. This requires the subject to be anesthetized and undergo surgery. Such a biopsy can also induce lesions that can have devastating consequences. Therefore, evaluation in the hypothalamus cannot be performed for obvious ethical reasons. However, RFPR3 and its receptor are present in several tissues including the testis: Immunolocalization of RFamide-related peptide 3 in a desert rodent Gerbillus tarabuli during seminiferous epithelium cycle (doi: https://doi.org/10.1016/j.tice.2018.09.005). According to this study, RFPR3 also plays a role in spermatogenesis which appears to be impaired in PFS/PSSD/PRSD. Therefore, biopsy in the peripheral zone can potentially help us explore the role of RFRP in PFS/PSSD/PRSD sufferers.



CONCLUSION
We have presented here, a potential mechanism of PFS/PSSD/PRSD involving RFRP3 and its receptor. A study protocol has been presented to prove this causal mechanism.
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anacleta
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Re: Potential etiology of PFS/PSSD/PRSD : a novel target RFRP3

Unread post by anacleta »

thank you for your research
fellow1
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Re: Potential etiology of PFS/PSSD/PRSD : a novel target RFRP3

Unread post by fellow1 »

Well if only RF9 (RFRP-3 selective antagonist) would have some safety data/studies... It can be easily sorted from some lab.

Kisspeptin and RF9 prevent paroxetine-induced changes in some parameters of seminal vesicle fluid in the male rats
https://pubmed.ncbi.nlm.nih.gov/32052480/

RF9 Acts as a KISS1R Agonist In Vivo and In Vitro
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4655216/

And some more:

https://pubmed.ncbi.nlm.nih.gov/26418326/
https://pubmed.ncbi.nlm.nih.gov/31203909/
Last edited by fellow1 on Mon Aug 29, 2022 2:20 pm, edited 6 times in total.
Brain food
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Re: Potential etiology of PFS/PSSD/PRSD : a novel target RFRP3

Unread post by Brain food »

There might be a relation between RFRP-3 and Kisspeptin. They’re both members of the arginine-phenylalanine-amide (RFamide) family. See below:

Kisspeptin Neurones do not Directly Signal to RFRP-3 Neurones but RFRP-3 may Directly Modulate a Subset of Hypothalamic Kisspeptin Cells in Mice
M. C. Poling*, J.

https://escholarship.org/content/qt3351 ... 4deaff.pdf
Serguei_Novitchok
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Re: Potential etiology of PFS/PSSD/PRSD : a novel target RFRP3

Unread post by Serguei_Novitchok »

fellow1 wrote: Mon Aug 29, 2022 1:47 pm Well if only RF9 (RFRP-3 selective antagonist) would have some safety data/studies... It can be easily sorted from some lab.

Kisspeptin and RF9 prevent paroxetine-induced changes in some parameters of seminal vesicle fluid in the male rats
https://pubmed.ncbi.nlm.nih.gov/32052480/

RF9 Acts as a KISS1R Agonist In Vivo and In Vitro
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4655216/

And some more:

https://pubmed.ncbi.nlm.nih.gov/26418326/
https://pubmed.ncbi.nlm.nih.gov/31203909/
The studies you have mentionned specify conflicting results. The first study (Kisspeptin and RF9 prevent paroxetine-induced changes in some parameters of seminal vesicle fluid in the male rats) consider RF9 as a RFRP-3 receptor ANTAGONIST while the second study (RF9 Acts as a KISS1R Agonist In Vivo and In Vitro) state RF9 act like a KISS1R AGONIST. It seems like the second study is right. The fact Kiss1r−/− mice have a "blunted gonadotropin response" to RF9 administration clearly suggest RF9 act through a pathway involving Kisspeptin. Assesement of their binding activity with RFRP or Kisspeptin receptor seems to settle that RF9 act like a kiss1R agonist and not an antagonist of RFRP receptor. It seems like a lot of research has been skewed by assuming RF9 is an antagonist of RFRP.

The study showing Kisspeptin and RF9 prevent paroxetine induced changes has to be read in light of this mistake. Therefore it provides no information about RFRP as they falsely consider RF9 to be a RFRP receptor antagonist.

It is unfortunate such a mistake has been made. Unfortunately a lot of research have been done assuming RF9 is a RFRP receptor antagonist. You have to take this mistake into account when you read a study.
mhugh
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Re: Potential etiology of PFS/PSSD/PRSD : a novel target RFRP3

Unread post by mhugh »

Is there any updates on this ?
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