Revealing the endocrine landscape of INSL3/RXFP2 signaling in hamster

The Insulin-like 3 peptide hormone (INSL3) and its cognate relaxin-family peptide receptor 2 (RXFP2) are predominantly synthesized and expressed in testicular Leydig cells. In mammalian tissues, the INSL3/RXFP2 signaling pathway plays a decisive role in male reproductive physiology [ 1 , 2 ]. During mouse embryogenesis, this pathway has a physiological function in the development of the gubernaculum ligament for the initial transabdominal descent of the testis, a condition common in placental mammals [ 3 – 5 ]. Adult Insl3 − / − mice have shown impaired testicular descent due to gubernaculum developmental abnormalities, which results in abnormal spermatogenesis and infertility [ 6 , 7 ]. Moreover, INSL3 has been reported as a biomarker of Leydig cell functional capacity in adult male mice [ 8 ]. Concurrently, genomic analysis has revealed that human gene mutations in INSL3 and RXFP2 cause testicular torsion and cryptorchid testes, respectively [ 9 , 10 ]; however, genetic analysis in a Greek pediatric cohort suggests that INSL3 gene mutations are not a common cause of testicular maldescent in humans [ 11 ]. Recently, mutation studies in human patients with bilateral cryptorchidism and male infertility reported bi-allelic loss-of-function (LoF) variants in INSL3 and RXFP2, while carriers of the heterozygous variant remain phenotypically unaffected [ 12 ]. Collectively, these studies have indicated a potential role of INSL3 and RXFP2 genes in male pathophysiology; therefore, future assays are crucial to establish a direct cause-effect relationship. However, in female mammals, it has been shown that INSL3 is synthesized in the steroidogenic theca interna cells of antral follicles [ 13 ]. The INSL3 gene is also expressed in the human corpus luteum, trophoblast [ 14 ], mammary glands [ 15 ], and endometrium [ 16 ]; while the RXFP2 gene expression has been identified in the fetal membranes and placenta [ 17 ]. In bovines, RXFP2 mRNA expression has been observed in theca cells, luteal cells, and oocytes [ 18 , 19 ]. Therefore, a biological role has been suggested for the INSL3/RXFP2 signaling pathway in maintaining healthy uterine function. RXFP2 is also intimately involved in female reproductive processes. It is expressed in the ovaries and has been implicated in follicle development, ovulation, and the regulation of the menstrual cycle [ 13 ]. Overall, the INSL3/RXFP2 signaling pathway is associated with reproductive health and may have implications for fertility and reproductive disorders. Ongoing molecular studies on this pathway continue to shed light on their physiological functions. However, further studies are required to fully comprehend its potential therapeutic value in the treatment of various diseases and possible clinical applications. The exact gene expression and potential physiological role of the INSL3/RXFP2 signaling pathway in females remain unclear, mandating more experimental assays to completely understand the signaling mechanisms of INSL3/RXFP2 in various tissues and their biological relevance. Additionally, DNA cloning and differing expression levels of INSL3 between various biological species remain largely unexplored. Thus, the current study aims to ascertain the gene expression profiles of the INSL3/RXFP2 signaling pathway cloned from a rodent model, such as hamsters ( Mesocricetus auratus ). Preceding reports in this biological species, specifically in the Harderian gland (HG), described a sexual dimorphism controlled by the levels of sex-steroid hormones, making it a compelling model to study the molecular mechanism of gene regulation by sex-steroid hormones and their physiological effects. [ 20 ]. There are some relevant reasons for selecting the hamster as a model species for investigating INSL3 and RXFP2 expression. Hamsters have well-defined seasonal reproductive cycles, which allows for the study of how the expressions of genes such as INSL3 and RXFP2 vary at different stages of gonadal development, as well as reproductive tissues regulated by steroid/protein hormones. Therefore, studying their regulation in hamsters may provide information that could be extrapolated to other species, including humans [ 21 , 22 ].

In this study, Sanger sequence analysis revealed that the cloned sequences of INSL3 and RXFP2 from hamsters are 888 bp and 3233 bp in length, respectively. A 281 bp 5′-untranslated region (UTR) and a 229 bp 3′-UTR with a putative polyadenylation consensus signal (AATAAA) were identified in INSL3, while a 175 bp 5′-UTR and an 844 bp 3′-UTR were identified in RXFP2. Additionally, the cDNAs of INSL3 (GenBank accession number PQ 606061 ) and RXFP2 (GenBank accession number PQ 606062 ) contain open reading frames (ORFs) of 375 bp ( Fig 1 ) and 2211 bp ( Fig 2 ) encoding 125 amino acids (molecular weight 13786) with a theoretical pI value of 8.33 and 737 amino acids (molecular weight 83270) with a theoretical pI value of 8.71, respectively. The initiation codon of ATG and stop codon TGA are indicated in bold. The polyadenylation signal AATAAA is underlined. Numbers indicate the amino acid and nucleotide positions. The nucleotides and amino acids are numbered from right to left. The positions of the cDNA and deduced amino acid sequences are indicated by numbers on the right. The start codon was ATG and the stop codon is marked by asterisks (***). Using Clustal Omega multiple sequence alignment and NCBI Protein–BLAST analysis, the amino acid sequences of hamster INSL3 ( Fig 3 ) and RXFP2 ( Fig 4 ) were aligned and characterized structurally from other mammalian species. Consequently, the hamster INSL3 sequence revealed five distinct structural domains ( Fig 3A ), namely signal peptide (residues 1–15), cleavage site (residues 16 and 17), B-chain (residues 18–51), C-chain (residues 54–98), A-chain (residues 100–125), and a disulfide bond region (residues 109–114). The MEGA X program and multiple sequence alignment were used for a maximum likelihood phylogenetic analysis/JTT matrix-based model of the hamster INSL3 amino acid sequence, confirming a 57–81% identity and homology ( Fig 3B ) to the amino acid sequences of B. taurus , Capra hircus , Sus scrofa , Canis lupus familiaris , H. sapiens , Macaca mulatta , M. musculus , and R. norvegicus . The hamster INSL3 demonstrated a high amino acid sequence similarity (80–81%) with rodent species. Robetta and PyMOL 3.0 were used to predict the 3D structural model of hamster INSL3 ( Fig 3C ), illustrating its classification within the insulin-like hormone superfamily. Spatial alignment results revealed that the 3D structure of each functional hamster INSL3 domain was similar to human INSL3, confirming structural conservation. (A) The alignment of INSL3 amino acid sequence from B. taurus ( NP_776790.1 ), C. hircus ( NP_001272508.1 ), S. scrofa ( NP_999135.1 ), C. lupus familiaris ( NP_001002962.1 ), H. sapiens ( NP_005534.2 ), M. mulatta ( NP_001191290.1 ), M. musculus ( NP_038592.3 ), and R. norvegicus ( NP_446132.1 ). Asterisks and boxed yellow indicate conserved residues. Structural domains are indicated by boxed colors. (B) Evolutionary analysis of INSL3 from hamster using the Maximum Likelihood method and JTT matrix-based model in MEGA X. This analysis involved 9 amino acid sequences with percentage similarities ranging from 57.02% to 81.45%. The hamster INSL3 was clustered with the Rodentia group. (C) Detailed structural analyses, including INSL3 3D structure obtained computational modeling, indicated key insights into the peptide signal; B-chain (Ala 16 –Glu 51 ), C-chain (Pro 54 –Gli 98 ), and A-chain (Ser 100 –His 125 ); disulfide bond region; and receptor binding mechanisms (B-chain). (A) Multiple amino acid sequence alignment of RXFP2 of hamster with other representative mammals, H. sapiens ( NP_570718.1 ), R. norvegicus ( NP_001012493.1 ), and M. musculus ( NP_536716.2 ). Identical and similar amino acids are marked with asterisks. The deduced amino acids were predicted to contain conserved domains of the RXFP2, such as a signal peptide (1–19 residues), cleavage site (19–20 residues), low-density lipoprotein receptor class A domain (LDLa, 27–64 residues) which contains six disulphide-bound cysteines (C 28 C 35 C 41 C 48 C 54 C 63 ) and a highly conserved cluster of negatively charged residues, LRR domain, hinge region (359–402 residues), and 7-transmembrane domain. (B) The Maximum Likelihood evolutionary tree of RXFP2 constructed by MEGA X software of representative mammals. The hamster RXFP2 branch was further clustered with the rodentia group. Sequence identities are indicated at right. (C) PyMOL 3D-structural analyses localized the signal peptide in the extracellular N-terminus, LRR domain, hinge region, 7-transmembrane domain in the intracellular C-terminus. 3D-conformational structure showed seven alpha-helices interconnected by extracellular and intracellular loops. Comparative sequence studies and the identification of conserved domains have been conducted on the hamster RXFP2 protein ( Fig 4 ), revealing insights into its structure and evolutionary relationships. The research uncovered six evolutionarily conserved domains ( Fig 4A ) which can be classified as a signal peptide (1–19 residues), a cleavage site (19–20 residues), and a low-density lipoprotein receptor class A domain (LDLa, 27–64 residues). This LDLa contains six disulfide-bound cysteines (C 28 C 35 C 41 C 48 C 54 C 63 ) and a highly conserved cluster of negatively charged residues. Other structural features include a leucine-rich repeat (LRR) domain (LRR1 through LRR10, with residues ranging from 121–358), a hinge region (359–402 residues), and a 7-transmembrane domain (seven TMs with residues ranging from 403–675). Phylogenetic analysis strongly supports a close relationship between hamsters and other mammalian species as well as a sister relationship with rodents. Molecular diversity analysis of the RXFP2 protein showed high degree of similarity with R. norvegicus (91.99%) and M. musculus (92.81%), with a notably lower homology to H. sapiens RXFP2 protein at 82.72% ( Fig 4B ). The 3D structural model of the hamster RXFP2 indicates its classification within the G protein-coupled, 7-transmembrane receptor (GPCR) family ( Fig 4C ). Proteins within the same branch display structural domains with high homology, conserved motifs, and similar 3D structures, indicating the robust conservation of RXFP2 proteins. To understand the endocrine landscape of the INSL3/RXFP2 signaling pathway, we examined the mRNA expression profiles in adult hamster tissues from both sexes, along with the gene regulation in the estrous cycle, from INSL3 ( Fig 5 ) and RXFP2 ( Fig 6 ) genes. Our findings showed significant expression of the INSL3 transcript in the testes, male adrenal glands, and ovaries, whereas lower expression was observed in the female hypothalamus, male HG, female adrenal glands, and male hypothalamus (statistical significance =  p  < 0.0001). Furthermore, no detectable mRNA expressions of INSL3 were observed in tissues such as the lung, liver, duodenum, cerebellum, spleen, pancreas, epididymis, and uterus ( Fig 5A ). Similarly, differential INSL3 expression was observed in the ovary, adrenal glands, hypothalamus, and HG during the estrous cycle, with the analysis revealing that the INSL3 mRNA expression level was relatively higher in the ovary tissue during the proestrus phase ( p  > 0.0001 and p  < 0.001). INSL3 expression was not detected in the lung, spleen, uterus, and pancreas across different phases of the estrous cycle ( Fig 5B ). (A) A panel of tissues was collected and examined in this study, including the lung, liver, duodenum, cerebellum, testis, ovary, adrenals, HG, hypothalamus, spleen, epididymis, uterus, and pancreas. (B) Relative expression profiles were determined during different stages of the estrous cycle such as proestrus P, estrus E, metestrus M, and diestrus D. The INSL3 gene under study was normalized using ACTB as the housekeeping gene. The means with the asterisks are statistically significantly different between males (in blue) and females (in pink). The statistical significance was calculated using a one-way analysis of variance (ANOVA), followed by Sidak´s and Tukey’s test ( p  < 0.05). Data are shown as means ± SD (n = 5). **** indicate p  < 0.0001, *** indicate p  < 0.001. (A) In males (in blue) and females (in pink) hamsters, the relative gene expression was analyzed in lung, liver, duodenum, hypothalamus, epididymis, uterus, cerebellum, testis, ovary, spleen, HG, pancreas, and adrenals. (B) Additionally, differential gene expression analyses were performed at estrous cycle; proestrus P, estrus E, metestrus M, and diestrus D. Values are expressed as mean ± SD of five independent qPCR assays (n = 5). ACTB gene was used as an internal reference to examine the mRNA levels of RXFP2. Asterisks (****, indicate p  < 0.0001) above each vertical bar indicate statistical differences, as determined by ANOVA followed by Sidak´s and Tukey’s tests ( p  < 0.05). To investigate the variations in RXFP2 relative expression profiles in hamster tissues, we performed qPCR reactions using the standard method ( Fig 6 ). The highest relative expression levels were found in the male hypothalamus, uterus, female cerebellum, epididymis, and ovary. In contrast, the least abundant transcripts were detected in the female hypothalamus, male cerebellum, and testis ( p  < 0.0001). Tissue distribution analysis in hamsters showed that RXFP2 mRNA was not detected in the lung, liver, duodenum, spleen, HG, pancreas, and adrenals ( Fig 6A ). Moreover, differential RXFP2 expression was noticed in the uterus, hypothalamus, and ovary during the estrous cycle. This analysis indicated that RXFP2 mRNA expression levels were significantly higher in uterine tissue during the proestrus phase. Specifically, this uterus had twofold and threefold more RXFP2 mRNA than the female hypothalamus and ovary hamster ( p  < 0.0001), respectively. The adrenal had the lowest mRNA levels during the estrous cycle. Meanwhile, transcripts in the lung, spleen, HG, and pancreas during the estrous cycle were not detected ( Fig 6B ).

In summary, the present study enhances our understanding of the endocrine INSL3/RXFP2 signaling and the sites of gene expression involved during the reproductive stages of both male and female hamsters. Nevertheless, the potential gene regulation by sex-steroid hormones in male and female hamsters remains to be molecularly characterized, necessitating further research to fully grasp their role in female reproductive physiology, especially in the uterus. Although our findings support an evolutionarily conserved role for INSL3/RXFP2 structure and expression in reproductive endocrinology, it is yet to be established to what extent this signaling function has been altered in the human uterus and cerebellum, as well as its likely association with uterine clinical disorders.

All experimental assays involving hamsters ( Mesocricetus auratus ) were approved by our institution’s ethical committee (INCMNSZ, BRE-1930-18-19-1). The hamsters used in this study were offspring of a colony maintained at Universidad Autónoma Metropolitana-Xochimilco (UAM-X; México City, México; code number AUT-B-C-0215–016). We used adult hamsters (10 months old; 150–200 g; 6 inches in length), with five males, and five females each in proestrus, estrus, metestrus, and diestrus, who all had access to food and water ad libitum. The environment was regulated for temperature, humidity, and a 12-h light/darkness cycle. The stage of the estrous cycle was confirmed via standard vaginal smear examination. The hamsters were anesthetized with ketamine:xylazine (80 mg/kg:8 mg/kg, intramuscularly). Male and female adult hamsters were sacrificed by decapitation; we procured samples from the testis, ovary, uterus, lung, heart, pancreas, adrenal glands, liver, epididymis, duodenum, spleen, brain, and HG for tissue distribution detection. These tissues were immediately placed on dry ice and then stored in a −70 °C freezer. Total RNA was isolated using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. It was collected in diethyl pyrocarbonate (DEPC)-treated distilled water. RNase-free DNase I was added to the total RNAs to remove genomic DNA. The quantity and quality of total RNA were evaluated by spectrophotometry at an A 260 /A 280 ratio of 1.8 (Beckman DU 650, Fullerton, CA, USA) through duplicate samples for each tissue. Additionally, its integrity (20 µg) was assessed based on the localization of ribosomal RNA (28S:18S rRNA ratio) using denaturing formaldehyde/MOPS/1.5% agarose electrophoresis. The quantity, quality, and integrity of the isolated total RNA were deemed sufficient for subsequent cDNA synthesis. Following the manufacturer’s recommendation, 2 µg of the total RNA was used to synthesize single-stranded cDNA using both a Transcriptor First Strand cDNA Synthesis kit for cloning (Roche Diagnostics, IN, USA) and a Maxima First Strand cDNA Synthesis kit (ThermoScientific, Vilnius, Lithuania) for real-time quantitative PCR (qPCR). The synthesized first-strand cDNAs were stored at −20 °C until further analysis was performed. Full-length cDNA sequences of the INSL3 and RXFP2 genes were isolated from hamster testis by RACE, following the instructions for the SMARTer® RACE 5′/3′ kit (Takara Bio Inc, Mountain View, CA, USA). The 5´ and 3´ end cDNA sequences of INSL3 and RXFP2 were obtained via RACE, using 5 µg of total RNA extracted from hamster testis. Four gene-specific primers (GSPs) based on known sequences ( Mus musculus , Rattus norvegicus , and Homo sapiens ) of conserved regions were designed for RACE (INSL3: RACE 5′ = 5´-ccacagagcttgtcacgcgcctcag-3´, RACE 3′ = 5′-ctcaccggctgcacccagcaagac-3´; RXFP2: RACE 5′ = 5´-gtcaggacgatgacatgaagtag-3´, RACE 3′ = 5´-gggacaatataatgaagccagtgtc-3´). PCR bands were separated by 1.2% agarose gel electrophoresis, and the gel was cut under a transilluminator. The PCR-amplified DNA fragments were recovered according to the instructions of the GeneJET Gel Extraction Kit (Thermo Fisher Scientific, Vilnius, Lithuania). The PCR products obtained by RACE were sequenced, and four specific primers (INSL3: 5´-cgaccttgtgggtgctg-3´, 5´-gggcatgtgaccatccttt-3´; RXFP2: 5´-gggaggcaccagactcta-3´, 5´-gtctcagacgccatcttcc-3´) were designed to amplify the full-length cDNA of INSL3 and RXFP2. The PCR products isolated through RACE were cloned using a TOPO TA cloning kit for sequencing, following the manufacturer’s instructions (Invitrogen Co., Carlsbad, CA, USA). After transforming into competent Escherichia coli DH5α cells, the pCR2.1-TOPO TA Vector-Taq-amplified PCR products were purified as per the instructions given by the E.Z.N.A. Plasmid DNA Mini and Maxi kits (Omega Bio-Tek, Inc., Norcross, Georgia, USA). The positive clones were selected for Sanger sequencing. Full-length cDNA sequences were sequenced bidirectionally (in both sense and antisense directions) using a BigDye Terminator v3.1 Cycle Sequencing kit (Applied Biosystems, Austin, TX, USA). This was carried out with an initial denaturation at 96 °C for 1 min, followed by 35 cycles at 96 °C for 10 s, 50 °C for 5 s, and 60 °C for 4 min (Veriti 96 well Thermal Cycler, Applied Biosystems, Marsiling, Singapore). Reactions were purified using a BigDye XTerminator™ Purification kit (Applied Biosystems, Austin, TX, USA) according to the manufacturer’s instructions. The products from the sequencing reaction were electrophoresed using an ABI-PRISM 310 genetic analyzer (Applied Biosystems, Foster City, CA, USA). The conditions for sequencing included a temperature of 50 °C, injection voltage of 15 kV, injection time of 5–7 s, and a current of 5–8 μA. All the obtained nucleotide sequence data was analyzed using Chromas sequencing software version 2.6.6. The INSL3/RXFP2 amino acid sequences were deduced via the Expert Protein Analysis System. ExPaSy proteomic tools were utilized to predict the isoelectric point and molecular weight of INSL3/RXFP2. The sequence data were compared using the Basic Local Alignment Search Tool and the Clustal Omega program. The DeepLoc-2.1 was used for the prediction of eukaryotic protein subcellular localization. SignalP-2.0 was used to predict signal peptides and their cleavage site. TMHMM – 2.0 was used to identify the prediction of transmembrane helices. The evolutionary history was inferred using the Maximum Likelihood method, the JTT matrix-based model, and the Molecular Evolutionary Genetics Analysis (MEGA) software. Three-dimensional (3D) models of INSL3/RXFP2 were generated with the Robetta software package. The homology model was rendered using PyMOL version 2.3 to observe the 3D structure of INSL3/RXFP2. Gene expression levels of hamster INSL3/RXFP2 were assessed via qPCR from various tissues. The assays were performed using 2 μg total RNA and the Maxima First Strand cDNA Synthesis kit (ThermoScientific, Vilnius, Lithuania), as per the instructions. Briefly, every qPCR reaction (20 μL) included 0.2 μL of 10 µM probe library, 0.2 μL of 20 µM forward primer, 0.2 μL of 20 µM reverse primer (INSL3 = 5′-tgacaagctctgtggccac-3′ and 5′-caagtgcatgcaggagctg-3′; RXFP2 = 5′-acttccagtcaaagttttcagcaaa-3′ and 5′-aaaaatgccttcctggatatgtgtg-3′), 4.0 μL of TaqMan Master LightCycler, and 5.0 μL of cDNA template. INSL3 / RXFP2 gene expressions were detected on a LightCycler 2.0 instrument (Roche Diagnostics, Indianapolis, IN, USA). The β- actin ( ACTB ) gene (5′-agctatgagctgcctgatgg-3′ and 5′-caggaaggaaggctggaaa-3′) was used as the internal reference. Amplification was done with 45 cycles of 95 °C for 10 s and 60 °C for 30 s, and 72 °C for 1 second. Each sample was set up for five independent biological replicates, and the relative expression levels were determined using the 2 −ΔΔCT method. Data are presented as the mean and standard deviation (SD) of five animals per group. GraphPad Prism software (California Corporation, USA) was used to analyze and graph gene expressions. One-way analysis of variance (ANOVA) was used to compare groups under different physiological conditions, taking into account both sex and group. A P -value < 0.05 was considered statistically significant. Additionally, an ANOVA with a post hoc Tukey test was conducted to compare means between different tissues in male and female hamsters. Data were also evaluated with post hoc Sidak tests during the estrous cycle.