Central Injection of Substance P Antagonizes the RF Amide-Related Peptide-3 Impacts on Hypothalamic KISS-1 and GnRH Gene Expressions in Male Wistar Rats

Introduction: Gonadotropin-inhibitory hormone (GnIH) and its mammalian orthologue RF amide-related peptide (RFRP) are known to inhibit the secretion of gonadotropins. In addition, substance P (SP), a member of tachykinin’s family, can increase the firing rate of kisspeptin/ neurokinin B/dynorphin (KNDy) neurons and provoke the secretion of gonadotropins. In this experimental study, we investigated the effects of co-administration of RFRP-3 and SP on the expression of KISS-1 and GnRH genes, as gonadotropin regulator genes, in male rats. Methods: Forty-two mature Wistar rats were randomly allocated into 7 groups (n=6 in each group). Animals in each group intracerebroventricularly received either saline+DMSO, SP (1nmol), RFRP-3 (5 nmol), SP (1nmol) + RFRP-3 (5 nmol), SP (1 nmol) + RF9 (RFRP-3 receptor antagonist, 10 nmol), SP (1 nmol) + P234 (kisspeptin receptor antagonist, 1 nmol) + RFRP-3 (5 nmol), or SP (1 nmol) + CP-96,345 (SP receptor antagonist, 5 nmol) + RFRP-3 (5 nmol). Two hours after injections, hypothalamic samples were collected to evaluate the expression of target genes by real-time PCR. Results: Injections in SP and SP + RF9 groups increased the expression of both GnRH and KISS-1 genes (P < 0.05). Injections in RFRP-3 and SP + RFRP-3 + CP-96,345 groups significantly decreased the expression of GnRH and KISS-1 genes (P < 0.05). However, injections of SP + RFRP-3 and SP + RFRP-3 + P234 did not significantly change the expression of GnRH and KISS-


Introduction
Gonadotropin releasing hormone (GnRH) plays a critical role in gametogenesis and steroidogenesis. 1 Gonadotropin-inhibitory hormone (GnIH) is one of the numerous neurohormones and neuromodulator molecules that are involved in the control of reproductive axis.GnIH, which was identified for the first time in quail, is known to inhibit the secretion of gonadotrophins by inhibiting the transcription of common α subunit and specific β subunits of gonadotropins. 2,3he function of GnIH, which is a member of RF amide family, is mediated by 2 G-protein-coupled receptors; GPR 147 and GPR 74. 4 One GnIH molecule and 2 GnIH-related peptides are encoded by the precursor mRNA of GnIH.These molecules possess LPXRF amide (X = leucine or glutamine) at their C terminus which is flanked by glycine and a single basic amino acid, arginine or lysine.Glycine and basic amino acids act as amidation signals and the endoproteolytic sites, respectively. 57][8] Dorsomedial hypothalamic (DMH) region contains GnIH neural cell bodies and the fibers of these neurons are extended to different locations including paraventricular nucleus (PVN), preoptic area (POA), arcuate nucleus (ARC), median eminence (ME), ventral paleostriatum, optic tectum, septal area, and dorsal motor of vagus nucleus. 9n rodents, almost 33% of GnRH neurons and 9%-16% of rostral periventricular kisspeptin neurons express the mRNA of the GnIH receptor. 10In hypothalamic regions of rat, there are 2 populations of kisspeptin neurons: one in the arcuate nucleus (ARC) and the other in the anteroventral periventricular nucleus (AVPV), which are involved in the negative and positive feedbacks of steroids on GnRH, respectively.Both populations as well as GnRH neurons are opposed by RFRP-3 immunoreactive fibers.Therefore, GnIH can suppress the GnRH-stimulated gonadotropin secretion directly or in a kisspeptindependent manner. 11Accordingly, there are findings suggesting that the central administration of RFRP-3 reduces KISS-1 mRNA in the hypothalamus. 12here are accumulating evidence, demonstrating that tachykinins family including substance P (SP), neurokinin A and B exert serious impacts on the reproductive axis. 13he functions of these closely-related peptides are mediated by three G-protein-coupled receptors known as NK1R, NK2R, and NK3R. 14SP is the undecapeptide neuromodulator which is associated with nociception and inflammatory processes in the brain.SP has been reported to have a significant effect on the events leading to the preovulatory surge of luteinizing hormone (LH) and follicle stimulating hormone (FSH) in human and mammals. 13,15The information obtained from light microscopic immunohistochemical investigations in human and rats indicated that SP neurons establish axosomatic and axodendritic inputs to GnRH neurons. 16here is strong evidence demonstrating that SP and specific NK1R agonists can promote the firing rate of kisspeptin neurons in the ARC nucleus that acts as a chief upstream pulse generator for GnRH neurons.Furthermore, the central administration of SP elevated the LH serum levels followed by an increase in the expression level of GnRH mRNA. 17otwithstanding precise interaction of GnIH and SP signaling pathways as opposite inputs in modulating the expression of GnRH gene and its critical upstream regulator, KISS-1 gene expression is unclear.The goal of the present investigation was to determine the impacts of central interaction of SP and RFRP-3 on the expression of GnRH and KISS-1 mRNA.

Materials and Methods
Forty-two mature male Wistar rats (220-250 g body weight) were housed individually in cages under controlled temperature (22±2°C), lighting (12-hour light/ dark cycle), and humidity (approximately 46%), with ad libitum access to food and water all the time.
For intracerebroventricular (ICV) injections, SP (Ana Spec Co, USA), P234 (Phoenix Pharmaceutical Inc, USA), RFRP-3 (Tocris Co, USA), CP96-345 (Tocris Co, USA), and RF9 (Tocris Co, USA) were dissolved in 50% physiologic normal saline and 50% DMSO.Solutions in the final volume of 3 µL were injected by a 27-gauge stainless steel injector that protruded 0.5 mm beyond the cannula and was connected to a Hamilton micro syringe by a polyethylene tube via the third cerebral ventricle at 8:30-9:00 am.[22][23] Tissue Collection Two hours after the injections, all rats were anesthetized and decapitated.Brains were immediately removed and hypothalamus was obtained.The ARC nucleus (arcuate nucleus; containing kisspeptin/neurokinin B/dynorphin [KNDy] neurons) and the POA (preoptic area; containing GnRH neurons) were dissected by micro punctuation method.Tissues were stored at -80°C until further assessment.

Gene Expression Assay
Total mRNAs were extracted from hypothalamic samples by using the total RNA extraction kit (Pars Tous, Iran), and were used for cDNA synthesis by Easy cDNA synthesis kit (Pars Tous, Iran).These procedures were performed according to the manufacturer's instructions.Quantitative real-time PCR with duplicate reactions were performed to evaluate mRNA levels of GnRH and KISS-1 genes using gene-specific primers as shown in Table 1.
The GAPDH gene was considered as a housekeeping gene for normalizing copy number of target genes.Relative expression of GnRH and KISS-1 was determined by SYBER ® Premix Ex Taq™ II (Takara, Japan) using Corbett-RG 6000X (Corbett Research, Australia) as follows: samples were heated for 10 minutes at 95°C, then 40 cycles of denaturation at 95°C for 30 seconds, annealing at 60°C for 30 seconds, and extension at 72°C for 30 seconds.Random samples were chosen and their qualification was determined by the agarose gel electrophoresis (ethidium bromide stained 1% agarose gel).All the amplicons represented a single peak.RT-qPCR data analysis was carried out using comparative cycle-threshold (CT) method and the relative expression of target mRNAs in comparison with the reference levels was calculated by the 2 -ΔΔCT formula. 24atistical Analysis SPSS software (version 25.0) was utilized for data analysis.In order to determine the normal distribution of data, onesample Kolmogorov-Smirnov test was applied.All values were presented as mean ± SEM.The data analysis was performed using one-way analysis of variance (ANOVA) followed by post hoc Tukey test.The significance level was considered as P < 0.05.

GnRH mRNA Expression in POA
The central infusion of RFRP-3 (5 nmol) significantly decreased the level of GnRH mRNA expression compared to that of control group (P < 0.05).Moreover, ICV injection of SP (1 nmol) resulted in a meaningful increase in GnRH mRNA levels (P < 0.05, Figure 1).
The co-administration of SP and RFRP-3 with the mentioned dosages did not alter the expression of GnRH while the co-administration of SP and RF9 led to a considerable and meaningful elevation in the expression of GnRH mRNA (P < 0.05, Figure 2).
In addition, concomitant injection of SP + RFRP-3 + P234 (1 nmol) had no significant influence on the expression of GnRH mRNA compared to the control group.The expression of GnRH mRNA in POA following the injection of SP + CP-96,345 (SP receptor antagonist, 5 nmol) + RFRP-3 was suppressed significantly compared to that in the control group (P < 0.05, Figure 3).

KISS-1 mRNA Expression in ARC Nucleus
The central infusion of RFRP-3 (5 nmol) decreased the level of KISS-1 mRNA in ARC region in comparison with the control group (P < 0.05).Moreover, ICV injection of SP (1 nmol) resulted in a meaningful increase in KISS-1 mRNA levels (P < 0.05, Figure 4).
The co-administration of SP and RFRP-3 with the mentioned dosages did not alter the expression of KISS-1.This is while the co-administration of SP and RF9 led to a significant increase in the expression of KISS-1 mRNA in ARC (P < 0.05, Figure 5).
The expression of KISS-1 mRNA did not significantly change following the concomitant injection of SP + RFRP-3 + P234 (1 nmol) compared to the control group.The expression of KISS-1 mRNA in the ARC nucleus was significantly inhibited following the injection of SP + CP-96,345 (5 nmol) + RFRP-3 compared to the control group (P < 0.05, Figure 6).

Discussion
Based on our results, the injection of RFRP-3 (5 nmol) resulted in decrement in both KISS-1 and GnRH mRNA expression levels.These data are in a good agreement with previous studies demonstrating that RFRP-3 suppressed the expression of GnRH and KISS-1 genes by acting through its G-protein-coupled receptors.In addition, there are findings that demonstrate both GnRH and KISS-1 expressing neurons in both ARC and AVPV nuclei also express RFRP-3 receptors (GPR 147 and GPR 74). 25 In this context, other studies showed that KISS-1 expressing neural populations either in ARC or AVPV nuclei as well as GnRH expressing neurons receive inputs from RFRP-3 neurons that in rats are mainly located in DMH area.However, the proportion of the KISS-1 expressing neurons that receive these inputs varies in a species-

*
dependent manner. 11urthermore, our results showed that injection of SP (1 nmol) resulted in the elevation of both KISS-1 and GnRH mRNA expression; these data are in accordance with the previous findings that suggested SP, by acting through NK1R receptor, increases the activity of KNDy neurons in ARC nucleus which contains kisspeptin neurons and acts as critical upstream pulse generator of GnRH system. 26There are also findings, though unrevealed, that neural clusters in ARC nucleus received inputs from SPimmunoreactive fibers. 27n the present study, we demonstrated that SP antagonized the function of RFRP-3 on the expression of hypothalamic KISS-1 and GnRH genes.Furthermore, our study was the first study that determined the interaction between SP and RFRP3 as 2 opposite inputs on GnRH/kisspeptin system with the co-administration of them resulted in neutralizing the effects of both on the expression of KISS-1 and GnRH genes.
Besides, it was demonstrated that RF9, which is an RFRP-3 receptor antagonist and a kisspeptin agonist, had a stimulatory effect on GnRH system by either stimulation of KISS-1 pathway or inhibition of RFRP-3 signaling. 28s previous studies showed, the administration of RF9 increased the secretion of gonadotropins and promoted the effects of kisspeptin in rats. 28These functions are mediated by GPR 54 which is expressed in GnRH expressing neurons (agonistic effect) or by GPR147 and GPR74 which is expressed in kisspeptin and GnRH expressing neurons (antagonistic effect).
Furthermore, the result of our study for the first time showed that concomitant administration of SP and RF9 enhanced the effects of SP on the expression of GnRH mRNA.Kisspeptin signaling pathway is considered as the main upstream regulator of GnRH system.There are accumulating evidence indicating that the effects of neuromodulators and neuropeptides such as SP and RFRP-3 on GnRH system are mediated by kisspeptin signaling pathway. 29P234 is a potent kisspeptin receptor antagonist which was introduced in 2009.P234 inhibits the impacts of kisspeptin on HPG axis in mice, ewes, and monkeys. 30The results from previous studies indicate that the administration of P234 results in a considerable decrease in LH serum concentration level by acting through G-protein-coupled receptor 54 (GPR54) on GnRH expressing neurons. 31n the present study, we found that the co-administration of SP, RFRP-3, and P234 had no significant effect on the GnRH mRNA expression.In other words, when kisspeptin signaling is blocked by P234, SP exerts its effect independent from kisspeptin signaling pathway and suppresses the function of RFRP-3.Hence other pathways may be involved in SP function on GnRH system.These data correspond with the studies that showed SP immunoreactive neurons projected GnRH neurons and provided axosomatic and axodendritic inputs to them. 16Nevertheless, the presence of NK1R on GnRH expressing neurons has not been reported. 32P96,345 is a selective, high affinity, and non-peptide antagonist of the NK1 receptor.Studies have shown that central infusion of SP antagonist elevates the plasma levels of adrenocorticotropic hormone (ACTH) and corticosterone which are considered as stimulatory factors for the secretion of endogenous RFRP-3. 33This evidence is in accordance with our results that demonstrated suppressive effect of SP on RFRP-3 function was eliminated by the ICV injection of SP receptor antagonist resulting in the elevation of the expression of GnRH and KISS-1 genes.Nonetheless, SP receptor antagonist (CP96,345) did not significantly reinforce the effects of RFRP-3 on the expression of neither GnRH nor KISS-1.
Further studies are needed to examine the interactions between other productions of KNDy neurons such as neurokinin B or dynorphin, which are the modulating factors involved in the activity of HPG axis.

Conclusion
In the present study, the interaction between SP and RFRP-3 on the reproductive axis in male rats was investigated for the first time.Our results indicated that SP antagonized the effects of RFRP-3 on the expression of hypothalamic KISS-1 and GnRH genes.In addition to kisspeptin signaling pathways, other pathways may be involved in the effect of SP on HPG axis.

Figure 2 .
Figure 2. GnRH mRNA Expression in the Rat Preoptic Area 2 Hours After Injection of SP + RFRP-3 and SP + RF9 Compared to the Control Group (n = 6 per group).Data was shown as mean ± SEM (*P < 0.05).

Figure 1 .
Figure 1.GnRH mRNA Expression in the Rat Preoptic Area (POA) 2 Hours After Injection of SP, and RFRP-3 Compared to the Control Group (n = 6 per group).Data was shown as mean ± SEM. (*P < 0.05).

Figure 5 .
Figure 5. Relative Expression of KISS-1 mRNA in the Rat Arcuate Nucleus 2 Hours After Injection of SP + RFRP-3 and SP + RF9 Compared to the Control Group (n = 6 in each group).Data was shown as mean ± SEM (*P < 0.05).

Figure 4 .
Figure 4. KISS-1 mRNA Levels in the Rat Arcuate Nucleus 2 Hours After Injection of SP, and RFRP-3 in Comparison With the Control Group (n = 6 per group).Data was shown as mean ± SEM (*P < 0.05).

Table . 1
Primers Applied for Amplification of Selected Genes in Real Time PCR