Metastin 45-54 of human origin (Kp10, h-YNWNSFGLRF-NH₂) was provided by Santa Cruz Biotechnology, Inc., Milan, Italy (sc-221883). For in-vivo experiments, Kp10 was dissolved in saline at a concentration of 1 mg/ml, while N-arachidonylethanolamine (AEA, sc-396321A) and rimonabant [SR141716A, SR, N-(piperidino-1-yl)-5-(4-chlorophenyl)-1-(2,4dichlorophenyl)-4-methyl-pyrazole-3-carboxamide sc-205491A] (all by Santa Cruz Biotechnology, Herculer, California, USA) were dissolved in ethanol at a concentration of 20 mg/ml and 5 mg/ml, respectively. Stock solutions were further diluted in saline just before use as indicated below. A detailed list and the working conditions of the primary and secondary antisera used for Western blot, immunofluorescence (IFL), and immunohistochemistry (IHC) are given in Table 1 .

Early peripubertal Wistar male rats (body weight 250–300 g) [38 post-natal days (pnd), n = 10/experimental group (Harlan Laboratories, Italy)] were housed two per cage with food and water available ad libitum and maintained on a 12-h light/dark cycle. Then, animals were divided into four groups identified using tail tags and treated as follows: group 1 control (saline 1% EtOH, C), group 2 KP (Kp10 0.1 mg/kg bw), group 3 AEA (2 mg/kg bw), and group 4 AEA (2 mg/kg bw) + SR (0.5 mg/kg bw). Treatments were performed two times a week. KP, AEA, and SR doses were chosen based on previously published works [KP (48, 49), AEA (24), and SR (23)]. Drugs were dissolved in a vehicle containing 99% saline and 1% ethanol, and SR was administered through intraperitoneal injection, whereas all the other animal groups received an equal volume of vehicle alone. Then after 30 min, rats were reinjected i.p. with the appropriate drug. After 3 weeks of treatment, rats were sacrificed by urethane (2 g/kg bw, Sigma-Aldrich, Milan, Italy) at 66 pnd (sexually mature). Blood was collected by cardiac puncture and processed for plasma collection. After being sacrificed, the testes and brain were removed; one testis/animal was frozen in liquid nitrogen and stored at −80°C until processing for RNA extraction and expression analysis by quantitative PCR (RT-qPCR) or Western blot; the other testis was fixed in Bouin’s fluids, embedded in paraffin following standard procedures, and then processed for morphological evaluation, IF, IHC, and Harris hematoxylin and eosin (H&E) staining. Brains were divided into two hemispheres that were randomly processed for molecular and morphological evaluations. In detail, one hemisphere of the brain was dissected to collect the mediobasal hypothalamus and immediately frozen in liquid nitrogen and stored at −80°C until processed for Western blot and qPCR; the contralateral hemisphere was fixed in 4% paraformaldehyde overnight and further processed for morphological evaluation, IF, and IHC. For IF and IHC, fixed brains were transferred to 70% ethanol, until they were included in paraffin. Serial coronal sections were sampled as already described (50) from bregma −2.04 mm to −5.04 mm according to the Paxinos and Watson rat brain atlas (51). The research protocol was approved by the Ethical Committee of the University of Salerno and the Italian Ministry of University and Research (authorization no. 66/2020-PR dated 29 January 2020).

At sacrifice, the gonadosomatic index (GSI) was calculated using the formula:

The growth of the animals was evaluated as:

Blood was centrifuged at 800×g for 15 min at 4°C for plasma collection. Plasma was immediately frozen at −80°C until used for the measure of Kiss1 and sex steroid by enzyme-linked immunosorbent assay (ELISA).

The plasma levels of Kiss1 were measured by ELISA kit specifically designed for rat Kiss1 (CEC559Ra, Cloud-Clone Corp., Katy, TX 77494, USA) (50 µl/sample, n = 6 for the SR + AEA group; n = 7 for the control, KP, and AEA groups; intra-assay coefficient of variation<10%, interassay coefficient of variation<12%, detection range: 24.69–2,000 pg/ml, minimal detectable Kiss1 dose 9.22 pg/ml).

Plasma testosterone and estradiol levels, at basal conditions and following treatment, were measured using ELISA (Cat. No. DEV9911 and Cat. No. DEV9999, respectively; Demeditec Diagnostics GmbH, Germany). For the testosterone assay, 10 µl/sample was run in triplicate following the manufacturer’s instructions (n = 7; intra-assay coefficient of variation range, 6.50%–11.07%; interassay coefficient of variation range 9.3–11.3; detection range, 0.1–25 ng/ml; minimal detectable testosterone concentration, 0.066 ng/ml). For estradiol assay, 75 µl/sample was run in triplicate following the manufacturer’s instructions (n = 7; intra-assay coefficient of variation range, 3.0%–6.1%; interassay coefficient of variation range, 4.0%–7.1%; detection range, 5–1,280 pg/ml; minimal detectable estradiol concentration, 2.5 pg/ml).

All plates were read on a Bio-Rad plate reader (Bio-Rad, Milan, Italy), and data were calculated automatically using a 4-parameter logistics (4PL) curve fit (https://www.myassays.com/index.html).

Rat testes (n = 6) and hypothalamus (n = 4) from in-vivo-treated animals were processed for the extraction of total proteins by the RIPA Lysis Buffer System (Santa Cruz Biotechnology sc-24948) supplied with protease inhibitor cocktail (10 µl/ml of RIPA), 200 mM of phenylmethylsulfonyl fluoride (PMSF, 10 µl/ml of RIPA), and 100 mM of sodium orthovanadate (10 µl/ml RIPA) following the manufacturer’s instructions. Cleared protein extracts were collected by centrifugation at 11,000×g for 30 min at 4°C and quantified by Lowry assay (52).

Total proteins (40 µg) were resolved on precast 4%–20% Mini-Protean TxG gels (Bio-Rad Laboratories, CA, USA) and electrotransferred into PVDF filters by the TransBlot Turbo Transfer System (Bio-Rad Laboratories). Membranes were blocked in UltraCruz Blocking solution (sc-516214, Santa Cruz Biotechnology) for 1 h at room temperature (RT) and incubated overnight with primary antiserum (details in Table 1 ). After washing three times for 10 min in T-TBS (0.25% Tween-20 in Tris-buffered saline, TBS, pH 7.6) at RT, membranes were incubated 1 h at RT with the appropriate horseradish peroxidase-conjugated (HRP) secondary antiserum (details in Table 1 ) in UltraCruz Blocking solution and then washed again in T-TBS. Western blotting luminol reagent was used to detect the signals (Santa Cruz Biotechnology sc-2048). To quantify the protein signal, each membrane was stripped at RT for 30 min in stripping buffer (Santa Cruz Biotechnology sc-281698) and then reprobed with the mouse monoclonal anti-α-tubulin (details in Table 1 ). Autoradiographic films were scanned; signals were plotted and quantified with ImageJ. Western blot data are reported as specific protein optic density (OD)/housekeeping OD ± SD (n = 3 at least).

Total RNA was extracted by TRIzol reagent (Thermo Fisher Scientific, Milan, Italy) following the manufacturer’s instructions and treated with DNAseI to eliminate any contamination of genomic DNA (Invitrogen Life Technologies: Carlsbad, California, USA). After quantification and quality evaluation on agarose gel, 2.5 µg of total RNA was reverse-transcribed in a final volume of 20 µl using SuperScript-III RnaseH^– Reverse Transcriptase (Thermo Fisher Scientific), 0.5 µg of oligo dT (18), 0.5 mM of dNTP mix, 5 mM of DTT, 1X first-strand buffer (Thermo Fisher Scientific), and 40 U of RNase Out (Thermo Fisher Scientific) following the manufacturer’s instructions. As a negative control, total RNA not treated with reverse transcriptase was used.

All real-time PCR assays were carried out in duplicate in the Mastercycler CFX-96 (Bio-Rad) using 1:2.5 diluted cDNA, 0.5 µM of each primer, and 10 µl of SYBR Green Master Mix (Bio-Rad) in a final volume of 20 µl. cDNAs were replaced by water in the negative controls. The primer sequences were as follows: Aromatase, Cyp19, forward, 5′-GCTTCTCATCGCAGAGTAT-3′, reverse, 5′-CAAGGGTAAATTCATTGGG-3′ (GenBank accession number: NM_017085); GnRH, forward, 5′-AGGAGCTCTGGAACGTCTGAT-3′, reverse, 5′-AGCGTCAATGTCACACTCGG-3′ (GenBank accession number: NM_012767); 3β-Hydroxysteroid dehydrogenase/Δisomerase (3βHsd), forward 5′-TCTACTGCAGCACAGTTGAC-3′, reverse 5′-ATACCCTTATTTTTGAGGGC-3′ (GenBank accession number: L17138); β-Actin, forward primer 5′-agatgacccagatcatgtttgaga-3′, reverse primer 5′-accagaggcatacagggacaa-3′ (GenBank accession number: NM_031144); or hypoxanthine-guanine phosphoribosyltransferase (HPRT), forward primer 5′-TTGTTGGATATGCCCTTGACT-3′, reverse primer 5′-CCGCTGTCTTTTAGGCTTTG-3′ (GenBank accession number: NM_012583) used as endogenous control.

The 2^−ΔΔCt method was used for the relative quantification of gene expression (53). Assays also included a melting curve analysis for which all samples displayed single peaks for each primer pair. Data were reported as mean fold change ± SEM over the value one arbitrarily assigned to the control group.

For the analysis of miRNA expression, miRNAs were extracted from the testis by the miRNeasy tissue/cells/advanced mini kit and cleared from DNA by the gDNA Eliminator Spin Columns (Qiagen, Milan, Italy). Following the manufacturer’s instructions, reverse transcription was carried out using the miRCURY LNA-RT Kit; qPCR was carried out in miRCURY LNA miRNA PCR Assay for the custom-validated primers for hsa-miR145-5p, has-miR-132-3p, has-let7a-5p, and has-let7b-5p and the housekeeping U6 (reverse transcription kit, master mix, and validated primers all from Qiagen).

Hypothalamus coronal sections were processed for Nissl staining as already described (54). Briefly, after deparaffinization and rinsing in distilled H₂O, sections were incubated for 1 min in thionine. Slides were then dehydrated through serial ethanol solutions (70% to 100%), cleared in xylene, and mounted with Eukitt® Quick-hardening mounting medium. Images were acquired by using a bright-field microscope (Olympus SC180) at ×4 and ×10 magnification. Sections obtained from the left cerebral hemisphere were imaged for bright-field microscopy using the Olympus SC180 microscope. The areas of the lateral ventricle were manually traced and measured using ImageJ, and the measurements (n = 4 animals for each group) were recorded in square millimeters (54).

For paraffin-embedded sections of the testis and hypothalamus, the IF protocol included deparaffination in Clear-Rite 3 (Thermo Scientific, Waltham, MA, USA) rehydration through descending degrees of alcohol up to water and non-enzymatic antigen retrieval in sodium citrate buffer 10 mM, pH 6.0, for 30 min at 95°C. Slides were washed three times with ice-cold PBS for 10 min and permeabilized with 0.1% Triton X-100 in PBS. For Kiss1 and Kiss1R analysis, a treatment with HCl 2 M for 30 min at 37°C was performed. Then, the slides were washed in PBS and blocked with a solution containing 10% of appropriate normal serum in PBS for 1 h at RT. The testis sections were incubated with anti-Kiss1, anti-Kiss1R, anti-CB1, and/or anti-CB2 or stained with 4′,6-diamidine-2′-phenylindole dihydrochloride solution (DAPI, 1.5 µg/ml in distilled H₂O), while the hypothalamus sections were processed using anti-neuronal nuclei (NeuN) and anti-Kiss1. All sections were incubated with primary antisera overnight at 4°C in a humidified chamber. After three washing steps, the slides were incubated with the appropriate fluorescent dye-conjugated secondary antibodies for 1 h at RT. Details of the primary and secondary antisera are given in Table 1 .

Negative controls were performed using all reagents except the primary antibody. Samples were then coverslipped with mounting gel (80% glycerol in PBS).

The samples were analyzed using a confocal laser scanning microscope (Leica SP5, Leica Microsystems, Wetzlar, Germany). Images were acquired in sequential scan mode by using the same acquisition parameters (laser intensities; gain photomultipliers; pinhole aperture; magnification ×20, ×40, or ×63). Figures were assembled using Adobe Photoshop 7 and Adobe Illustrator 10.

Ten-micron-thick brain sections, mounted on poly-L-lysine-coated glass slides, were analyzed by IHC using the anti-Kiss1R antibody (details and working conditions in Table 1 ). The IHC protocol included deparaffination, non-enzymatic antigen retrieval, and permeabilization as reported in the previous section. Then, the slides were immersed in 3.6% hydrogen peroxidase solution for 10 min to inactivate endogenous peroxidases and blocked with a solution containing 10% of normal serum in PBS for 1 h at RT. After rinsing, the samples were incubated overnight at 4°C with anti-Kiss1R antibody. Then, the sections were washed twice in PBS and incubated with the secondary antibody for 1 h at RT, as in Table 1 . Slides were then incubated with streptavidin-horseradish peroxidase (Vector Laboratories) for 1 h at RT. The immunoreaction signal was detected using a substrate chromogen solution containing 3,3-diaminobenzidine tetrahydrochloride (DAB substrate kit, Vector Laboratories). Finally, the sections were coverslipped with the Eukitt® Quick-hardening mounting medium (Sigma-Aldrich). For histological analysis, H&E staining was performed on the testis sections following standard procedures (54). Slides were analyzed using a brightfield microscope (Olympus SC180) at ×10 magnification. The number of the Kiss1R-positive cells within the arcuate nucleus (ARC) was counted in each section for n = 4 per experimental group using ImageJ software, and the mean number of cells/animal was used for further statistical analysis.

Student’s t-test and ANOVA followed by Duncan’s test were carried out to assess the significance of the differences in gene/protein expression, IFL, and IHC signal. Student’s t-test was used to assess the statistically significant differences between the treatment groups and the control group; ANOVA followed by Duncan’s test was used for multigroup comparison. Data were considered significant at p<0.05.

Weight gain is a recognized tool of puberty and SR has a possible anorectic effect (55). Hence, we measured the body weight and the length of the tibia to evaluate the correct growth of the treated animals vs. the control group during the 3-week-long treatments. At sacrifice, no differences in body weight, tibial length, testes weight, GSI, and growth were observed between the control and treated animals ( Table 2 ).

Kisspeptin is the main upstream modulator of GnRH, from puberty onset onward (5); both kisspeptin and estradiol (5, 6, 31, 40), the sex steroid produced by the irreversible aromatization of testosterone by Cyp19 aromatase, were found as positive modulators of the KS. Conversely, the HPG axis is downregulated in CB1-/- mice (16, 17). To test the possible interplay between KS and ECS on the above issues, we evaluated the effects of Kp10 and AEA on Kiss1R, Kiss1, and Cyp19 proteins and GnRH mRNA in the mediobasal hypothalamus of the treated animals by Western blot and qPCR, respectively. The KP administration caused a slight increase of Kiss1R, Kiss1, and Cyp19 proteins and GnRH mRNA ( Figures 1A–E ), but the change was statistically significant only for Kiss1 and Cyp19 proteins and GnRH mRNA. AEA significantly increased Kiss1R protein (p< 0.01), decreased Kiss1 and Cyp19 proteins (p< 0.01), and increased GnRH mRNA, but the trend was not statistically significant (p = 0.065). Pretreatment with SR reverted the AEA-dependent effect on both Kiss1R and Kiss1 proteins but lowered Kiss1R protein below the control group, had no effect on Cyp19, and increased GnRH mRNA (p< 0.05).

Since treatments were carried out in pubertal animals and stopped at sex maturation, the effects of Kp10 and AEA treatments on the expression of miRs notably involved in the hypothalamic control of reproduction at puberty (56) were analyzed. The expression rate of miR-132-3p, let-7a-5p, and let-7b-5p did not change vs. the control group following KP or AEA treatment ( Figures 1F–I ); the expression of miR-145-5p was significantly higher in the AEA-treated group only and SR reverted this AEA-dependent effect ( Figure 1G ).

Based on the Western blot results, we investigated whether the treatments specifically affected Kiss1 and Kiss1R proteins in the hypothalamic ARC, where GnRH is preferentially produced. Hence, we evaluated Kiss1 and Kiss1R in the ARC of treated and untreated rats ( Figures 2A–C ). Both KP and AEA treatments increased the number of Kiss1R-expressing neurons in the ARC compared with controls (p< 0.01 and 0.05, respectively) ( Figure 2B ), and the AEA-mediated effect was attenuated by SR administration. KP administration and the combined AEA/SR treatment increased the number of Kiss1-expressing neurons in the ARC (p< 0.01 and p< 0.05, respectively) ( Figure 2C ).

Interestingly, Kiss1 was present not only within the ARC but also along the ependyma of the lateral ventricle as shown in the representative image of the control in Figure 2C . The KP treatment exacerbated this condition suggesting a role for this protein also in cerebrospinal fluid homeostasis ( Figure 2C ). To further investigate this issue, we analyzed the morphology and size of the lateral ventricle in KP-treated animals. A significant expansion of the ventricle area was observed compared with controls (p< 0.01) ( Figure 3 ).

Changes in the circulating gonadotropins and sex steroids in turn are the main consequences of the overstimulation or inhibition of the HPG axis (1), and the possible effects of KP and AEA administration on the circulating levels of Kiss1 have never been investigated before in rodents. Thus, we measured Kiss1 and sex steroids in the blood to ascertain to what extent treatments affected the physiology of the HPG axis.

The circulating levels of Kiss1, testosterone, and estradiol were measured by ELISA following the in-vivo treatment with KP, AEA, and SR + AEA ( Table 3 ) revealing the lack of any statistically significant change in the measured hormonal levels.

To further confirm the functionality of the HPG axis and the lack of significant effects on steroid biosynthesis, we investigated by Western blot the effects of the treatments on 3βHsd and Cyp19, the two main enzymes involved in testosterone biosynthesis and conversion into estradiol (57). In addition, the expression rate of intratesticular GnRH mRNA, involved in the control of the physiology of both Leydig cells and spermatogenesis (57), was also considered. Results revealed the absence of any statistically significant difference between the control group and the KP-treated group ( Figures 4A–C ). The trend of the corresponding mRNA was toward a decrease in the KP group for both 3βHsd and Cyp19 ( Figure 4D ) but was statistically significant only for Cyp19 mRNA (p< 0.05 vs. the control group). The intratesticular expression of GnRH mRNA was significantly lower in the KP-treated group (p< 0.05).

Due to the possible acceleration of spermatogenesis and tissue damage related to prolonged KP treatments (58, 59), molecular markers of spermatogenesis and testis morphology were investigated. The protein levels of molecular markers of the S phase of the cell cycle (i.e., PCNA) (60), spermatogenesis progression at puberty (i.e., Lin28A) (61), and cell survival (i.e., Sirt1) (62) were analyzed by Western blot ( Figure 5A ). The protein level of PCNA did not differ between the control and the KP-treated group ( Figure 5B ), while both Lin28A and Sirt1 protein levels increased following KP treatment (p< 0.05) ( Figures 5C, D ).

The expression rate of miRNAs notably involved in the control of spermatogenesis at puberty (i.e., miR145-5p, miR-1323p, let7a-5p, let7b-5p) (61) was also analyzed by qPCR following KP and AEA treatments ( Figure 5E ). The expression rate of miR145-5p, miR-1323p, and let7a-5p was significantly lower in AEA-treated animals (p< 0.01 vs. the control group and vs. the KP group). The expression rate of miR-132-3p and let7a-5p was significantly higher in the KP-treated group (p< 0.05 vs. control), and the expression rate of let7b-5p did not change in all treatments. Pretreatment with SR reverted the AEA-dependent effects on miR145-5p, miR-132-3p, and let7a-5p.

Testis morphology in the control and treated animals was comparable ( Figure 5F ).

The possible self-modulatory loop of KP on the intratesticular KS was analyzed by Western blot ( Figure 6A ). The KP treatment significantly increased the protein level of the corresponding receptor ( Figure 6B ) (p< 0.05), without any significant effect on the ligand ( Figure 6C ).

Following the in-vivo treatment with AEA ± SR141716A, Kiss1R and Kiss1 proteins were analyzed by Western blot ( Figure 6D ). The AEA treatment significantly reduced the protein level of Kiss1R ( Figure 6E ) but did not have any significant effect on Kiss1 ( Figure 6F ) although an increasing trend could be observed. Pretreatment with SR141716A fully reversed the AEA-dependent effects on Kiss1R.

In control animals, the intratesticular localization of Kiss1 and Kiss1R was quite overlapping within the germinal epithelium ( Figure 7 ). It marked mostly elongating spermatids close to the lumen and, to a lesser extent, premeiotic stages (i.e., preleptotene spermatocytes) close to the basal compartment within the germinal epithelium. In the interstitial compartment, a few Kiss1 and numerous Kiss1R-positive elements were detectable. Following the KP treatment, more intense staining was observed for both Kiss1 and Kiss1R in the interstitial compartment and the germinal epithelium. In detail, within the germinal epithelium, the immunoreactivity of Kiss1 and Kiss1R still characterized elongating spermatids but was also detectable in spermatocytes and a few spermatogonia. Following the AEA treatment, Kiss1 and Kiss1R localization in the spermatids was limited to a few scattered spots but was still evident in spermatocytes ( Figure 7 ). A stronger staining for Kiss1 and Kiss1R was detectable in the SR + AEA-treated animals in both preleptotene and pachytene spermatocytes.

The possible effects of in-vivo administration of KP on the testicular ECS were evaluated by Western blot focusing on the main cannabinoid receptors, CB1 and CB2, on FAAH and N-acylphosphatidylethanolamine-phospholipase D (NAPE-PLD), the main enzymes responsible for AEA degradation and biosynthesis, respectively. KP treatment significantly increased the protein level of CB1 ( Figure 8A ) (p< 0.05) but did not have any effect on CB2 ( Figure 8B ). Kp10 treatment significantly increased the protein level of FAAH ( Figure 8C ) (p< 0.05), without any statistically significant effect on NAPE-PLD ( Figure 8D ).

Since CB1 and FAAH were responsive to the KP treatment, a possible self-modulatory loop of AEA on its canonical receptor and its hydrolyzing enzyme was analyzed by Western blot following the AEA treatment ( Figure 8E ); a lack of any statistically significant change in CB1 and FAAH proteins was observed ( Figures 8F, G ).

In control animals, CB1 was localized in both somatic (i.e., interstitial Leydig cells and Sertoli cells) and germ cells (i.e., spermatogonia in the basal compartment and round/elongating spermatids in the luminal compartment). CB2 had a more scattered and diffuse localization within the interstitium and the germinal epithelium, particularly in Sertoli cells and germ cells at mitotic and meiotic stages. CB1 and CB2 merge revealed a quite overlapping distribution ( Figure 9 ). Following the KP treatment, CB1 was detectable in the interstitium and along the contour of the seminiferous tubule; a scattered and punctuate signal for CB2 was detectable in the germinal epithelium ( Figure 9 ). In AEA-treated animals, CB1 and CB2 spotted signals were well evident in the interstitial and the germinal compartment, with overlapping signals ( Figure 9 ). Pretreatment with rimonabant reduced CB1 but not CB2 staining in both interstitial and germinal compartments ( Figure 9 ) corroborating the CB1-dependent action of AEA at the testicular level.