Comparative Expression Profile of RFRP-3 in Hypothalamus and Testis of Adult Rats

Hypothalamic-pituitary-gonadal axis plays a pivotal role in regulating mammalian reproduction (Harris, 1964). Kisspeptins and RFamide-related peptides (RFRPs) are two crucial neuropeptides that regulate GnRH secretion from the hypothalamus. Kisspeptins stimulate GnRH secretion, whearse RFRP-3 inhibits GnRH secretion in birds and mammals. RFRP-3 regulates reproduction and influences food intake, stress response and sexual behavior (Tsutsui and Ubuka, 2020). RFRPs (RFRP-1 and RFRP-3) are RFamide peptide, which contains arginine-phenylalanine-amide at C-terminal end.

The RFamide-related peptide-3 (RFRP-3), also known as gonadotropin inhibiting hormone (GnIH) in mammals. Different studies have shown that GnIH (RFRP-3) acts directly in the hypothalamic hypophysial system, offers direct actions on the gonads and prevents gonadotropin release (Tsutsui and Ubuka, 2020). RFRP-3 and its receptor have been detected in testicular interstitial cells and germ cells and pseudostratified columnar epithelial cells in the epididymis. GnIH (RFRP-3) and its receptors are also expressed in the gonads and reproductive organs of birds (Bentley et al., 2008; McGuire and Bentley, 2010) and mammals (Zhao et al., 2010). The sites of secretion and expression patterns of GnIH/RFRP-3 and its receptor suggest a role in steroid synthesis, gamete maturation and apoptosis. Recent studies indicate that GnIH/RFRPs can have an autocrine or paracrine effect on a wide range of gonadal functions in birds and mammals.

In mice, the expression of RFRP-3 and its effect on steroidogenesis on GnRH receptor (GnRHR) and LH-receptor (LHR) in the testis has been tested (Anjum et al., 2012; Anjum et al., 2014). The presence of RFRP-3 and its receptors in the Syrian hamster (Zhao et al., 2010) suggested a role in spermatogenesis and androgens synthesis (Zhao et al., 2010). The effects of RFRP-3 depend on gonadotropins in primates and rats (Anjum et al., 2014). The expression of RFRP-3 is affected by photoperiod and reproductive conditions (Zhao et al., 2010).

Several studies were conducted on different avian and mammalian species for molecular characterization of RFRP-3 and its physiological distribution and function; however, there are very few studies on the expression analysis of RFRP-3 gene in the mammalian testis. Therefore, it is the need of the hour to comprehensively analyze the expression level of the RFRP-3 gene in the hypothalamic region of the brain and testis in rats. Present study was designed to localize RFRP-3 secreting cells and comparative expression analysis of RFRP-3 gene in hypothalamus and testis of the adult male rats.

The experiments were performed on adult male Wister rats free from physical and anatomical abnormalities. The animals were maintained under 12/12-h light-dark cycle and constant temperature (24°C) and given ad-libitum access to pelleted food and fresh drinking water. All the experiments were performed following the guidelines of the regional Animal Ethics Committee and the rules for experimental animals of the University. 

Brain (hypothalamus) and testiscular tissue samples were collected from the rats after lightly anesthetized with isoflurane in 10% neutral buffered formalin (NBF) for histological and immunohistochemical studies.

The samples for immunochemistry were fixed in NBF for 48h at room temperature. They were dehydrated through a graded series of ethyl alcohol and frozen before being cut into 5-7 µm sections. For RNA extraction, brain (hypothalamus), pituritary, testis and muscle tissue samples were removed and immersed in RNA-later solution (Thermo Fisher Scientific, Santa Clara, CA, USA) and frozen at -80°C until used.

The sections of the samples of 5µm thickness were prepared with the help of Leica 2125 DM rotary microtome after preparing a stanadred paraffin blocks and fixed for histological studies in 10% NBF solution. The slides were treated with Mayer’s Haematoxylin followed by 2% Eosin and rinsed in 90% ethyl alcohol. Finally, mounted with DPX.

The sections were treated with phosphate buffered saline PBS and 0.3% H2O2 in methanol for quenching endogenous peroxidase. Blocking of the background was done by 10% normal horse serum. Tissue sections were then incubated for 4 hours at 37°C with RFRP-3 antibody (RFRP-3 antibody was kindly given by Kazuyoshi Tsutsui, Professor, Center for Medical Life Science Waseda University, Tokyo, Japan) diluted in PBS. The slides were then incubated with the secondary antibody using the ABC universal staining kit (Wuhan Boster Biological Technology Co., Ltd., China).  The peroxidise activity was assessed using 0.03% 3,3’-diaminobenzidine tetrahydrochloride (Wuhan Boster Biological Technology Co, Ltd., China) in 0.05 M Tris, pH 7.6 and 0.1% H₂O₂ for 5 min. For testing immunoreaction specificity, the primary antiserum was replaced in the control phase with 1% normal horse serum; the pre-adsorption of the RFRP-3 antiserum with the RFRP-3 antigen (10 µg/ml). The slides of both histology and immunohistochemistry were observed under Leica DM 2000 Microscope in different magnifications and localizing RFRP-3-immunoreactive (ir) cells were done.

Tissue samples stored in RNA-later solution were homogenized in a Polytron homogenizer and total RNA was extracted using Tri reagent (Sigma-Aldrich, St. Louis, MO, USA) following the manufacturer’s instruction. After checking the integrity of RNA (1.5% agarose gel) and determining the concentration of RNA (NanoDrop ND-1000 spectrophotometer, Thermo Fisher Scientific, Wilmington, DE, USA) total RNA was treated with RNase-free DNAseI (Fermentas Life Sciences, Canada) to exclude genomic DNA contamination. The absence of contaminant genomic DNA in RNA preparations was tested using RNA as a template in real-time PCR assays (RNA not reverse-transcribed to cDNA). Total RNA from each sample was reverse transcribed into cDNA in a 20 μl reaction mixture using High Capacity RNA-to-cDNA synthesis kit (Applied Biosystems, Foster City, CA, USA) following the manufacturer’s instructions.

The primers for RFRP-3 were designed from the available sequences in NCBI GenBank (Table 1), optimized synthesized and standardized (Fig 1) along with the housekeeping gene (RPS18) for amplification by quantitative real-time polymerase chain reaction (qPCR). The relative expression level of mRNA transcripts of the RFRP-3 gene was measured by qPCR (Roche LC480) using the SYBR Green I (Roche Diagnostics, Switzerland). Relative quantification of a target gene was done by comparing the expression of RPS18 as per Pfaffl (2001) method. The specificity of each primer pair was confirmed using melt curve analysis and samples run on a 2% agarose gel with a 100-bp DNA ladder (Invitrogen) to verify the generation of a single product of the correct size (Fig 1A).





Data were analyzed by one-way analysis of variance (ANOVA) considering significant level at p<0.05. All statistical analyses of the qPCR data used CT values, whereas graph (Fig 4) indicate the fold changes (2-DDCT).

Localization of RFRP-3 secreting cells in the hypothalamus and testis
 
Different nucleus were identified by histological study with HE staining (Fig 2). RFRP-3 labeled cells were mainly identified (by immunochemistry) in the dorsomedial nucleus of the hypothalamus (DMH) and ventromedial nucleus of the hypothalamus (VMH) with some labeled cells along the sides of the third ventricle (3V) in the rostral-caudal extent of the hypothalamus (Fig 2). RFRP-3-ir fibers are also detected along the sides of the and above the dorsal edge of the 3V. A higher density of fibers was found in the caudal extent of the hypothalamus.

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The results of this study confirmed the presence of scattered RFRP-3-ir cell bodies in the VMH, DMH and surroundings the third ventricles in male rats. Similar findings, viz. RFRP3-secreting cells in DMN and fibers of neurons extend to the hippocampus, preoptic area (POA), paraventricular nucleus (PVN), anterior hypothalamus and the lateral hypothalamus’ rostral aspects were recorded in rats by Rizwan et al., (2009); in mouse DMH (Kriegsfeld et al., 2006), surrounding the VMH in the rat (Johnson et al., 2007) and in PVN and DMH in Sheep (Smith et al., 2008). The expression of GnIH mRNA in the neurons of DMH was observed in mice by Ukena and Tsutsui (2001), in hamster (Ubuka et al., 2012) and in DMH and PVN of the sheep (Qi et al., 2009) and in mare (Thorson et al., 2014).

Thompson et al., (1996) reported the projections of DMH axons in the intrahypothalamic, i.e., neuroendocrine and pre-autonomic hypothalamic regions and to some extent in the extrahypothalamic sites, namely, brainstem and the telencephalon. Thompson et al., (2003) explained the DMH, with five other preoptic nuclei, i.e., anteroventral and anterodorsal preoptic, parastrial, median preoptic and anteroventral periventricular, form a complex interconnected network that plays a key role in coordinating neuroendocrine, autonomic and somatic responses to external stimuli, sensory feedback and cognitive/motivational input. Any lesions in DMH prevent the seasonal onset of reproductive quiescence which is mediated by increased gonadal steroid negative feedback in the Syrian hamster (Lewis et al., 2002).

In our study, the RFRP-3 was observed in both Leydig cells and the seminiferous tubules at the level of germ and Sertoli cells of rats (Fig 3). Similar findings were obtained in different species, viz. in avian species (McGuire and Bentley, 2010), pigs (Zheng et al., 2015), rodents (Zhao et al., 2010; Anjum et al., 2012). Therefore, our study revealed the testes as a site of RFRP-3 biosynthesis in rats, according to the earlier work in birds (Bentley et al., 2008) and mammals (Iwasa et al., 2017). It was reported that Leydig cells is the main site of RFRP-3 synthesis and have a local effect on Leydig cells to inhibit secretion of testosterone in birds (McGuire and Bentley, 2010) as well as in mammals (Anjum et al., 2014; Lents et al., 2017) via the repression of LHR, steroidogenic acute regulatory (StAR) protein, cytochrome P450 side-chain cleavage and 3β-hydroxysteroid dehydrogenase expression (Anjum et al., 2014; Zheng et al., 2015) and probably preventing protein kinase A (PKA) activation (Lents et al., 2017).

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Relative expression profile of RFRP-3 gene
 
Melting curve analysis did not yield any non-specific peak from each primer set tested. Additionally, every PCR product generated a prominent band with an expected size in the gel electrophoresis analysis (Fig 1). These indicated that non-specific amplification with the primer sets tested was not detected in the real-time PCR analysis. Present study also indicated that the mRNA of RFRP-3 was significantly highly expressed (p<0.01) in the hypothalamus than the pituitary and testis; pituitary had a higher (p<0.05) expression than the testis and no expression was observed in muscle tissue (Fig 4).

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Our results support previous studies on other species (Legagneux et al., 2009; Tan et al., 2021). Ubuka et al., (2014) observed the expression of GnIH precursor mRNA in the interstitium. Rizwan (2012) observed that RFRP mRNA is mainly expressed at high levels in the hypothalamus and eye and a very low level in the testis. The presence of RFRP and its receptors in the seminiferous tubules and developing spermatids have confirmed in the Syrian hamster and suggested the inhibitory role of RFRP in the local regulation of testicular functions during the regressive phase of the reproductive cycle (Zhao et al., 2010). Expression of RFRP and its receptor (GPR147) has been demonstrated in spermatocytes and suggests a possible role of RFRP in the final maturation of spermatids (Zhao et al., 2010). McGuir and Bentley et al., (2008) confirmed the expression of GnIH and its receptor, GPR147, in Leydig cells of the testis and GnIH reduced the effect of LH on testosterone secretion. Apoptotic cell death in the testis may result from a GnIH-induced decrease in testicular testosterone (Tsutsui et al., 2010). Based on current understanding, it appears that GnIH may have the potential for healing of the disease that requires down-regulation of reproductive function.

This study confirmed the presence of RFRP-3 secreted neurons in the hypothalamus in male rats. Scattered RFRP-3-ir cell bodies were identified to be mainly surrounding the VMH, DMH and third ventricles. RFRP-3-ir cells are clustered in DMH. DMH projects extensively to neuroendocrine and preautonomic hypothalamic regions. Immunohistochemical studies revealed the presence of RFRP-3 peptide in rat testes. This finding is consistent with the results obtained in different species. RFRP -3 was mainly expressed at high levels in the hypothalamus and very low levels in the testes.

The authors are thankful to Kazuyoshi Tsutsui, Professor, Laboratory of Integrative Brain Sciences, Department of Biology, Waseda University, Tokyo, Japan for providing the RFRP-3 antibody for this study.

None.