Expression of Relaxin Family Peptide Receptors 1 and 3 in the Ovarian Follicle of Japanese Quail.

In our previous studies, we demonstrated that the primary source of relaxin 3 (RLN3) in Japanese quail is ovarian granulosa cells. Although several relaxin family peptide (RXFP) receptors have been sequenced, the intricacies of these receptors in avian species remain insufficiently clarified. Therefore, we assessed the expression of RXFP receptors, RXFP1 and 3, in Japanese quail. Using RT-PCR, we found that both RXFP1 and 3 were ubiquitously expressed. The expression level of RXFP1 is significantly higher in the ovarian theca layer, indicating that it is the primary receptor for RLN3 in the ovary. During follicular development, there was an elevation in thecal RXFP1 expression, but it declined after the luteinizing hormone (LH) surge. We found that the protease activity of the 60 kDa band increased after the LH surge, suggesting the involvement of RLN3 signaling in ovulation. These results suggest a paracrine role of RLN3, involving its binding with RXFP1 in ovarian theca cells. This interaction may elicit biological actions, potentially initiating ovulation after the LH surge.

Identification and characterization of dystrophin-locus-derived testis-specific protein: A testis-specific gene within the intronic region of the rat dystrophin gene.

The mammalian X chromosome exhibits enrichment in genes associated with germ cell development. Previously, we generated a rat model of Becker muscular dystrophy (BMD) characterized by an in-frame mutation in the dystrophin gene, situated on the X chromosome and responsible for encoding a protein crucial for muscle integrity. Male BMD rats are infertile owing to the absence of normal spermatids in the epididymis. Within the seminiferous tubules of BMD rats, elongated spermatids displayed abnormal morphology. To elucidate the cause of infertility, we identified a putative gene containing an open reading frame situated in the intronic region between exons 6 and 7 of the dystrophin gene, specifically deleted in male BMD rats. This identified gene, along with its encoded protein, exhibited specific detection within the testes, exclusively localized in round to elongated spermatids during spermiogenesis. Consequently, we designated the encoded protein as dystrophin-locus-derived testis-specific protein (DTSP). Given the absence of DTSP in the testes of BMD rats, we hypothesized that the loss of DTSP contributes to the infertility observed in male BMD rats.

Expression of Relaxin 3 in the Ovarian Follicle of Japanese Quail.

The relaxin (RLN) gene is expressed in the reproductive tracts, such as the ovary and uterus, of mammalian species. Although RLN expression is detected in the chicken ovary, detailed clarification of the physiological role of RLN has not yet been reported. To address this issue, in the present study we aimed to examine the spatiotemporal expression and hormonal control of RLN in Japanese quail. By performing semi-quantitative and quantitative reverse transcription-polymerase chain reaction analysis, we found that RLN mRNA was mainly expressed in the granulosa and theca layers of the ovary. The expression level in the granulosa layer increased with the stage of follicular development. Results from granulosa layer culture experiments revealed that RLN mRNA expression increased with the addition of estradiol-17ß, whereas the addition of progesterone suppressed RLN transcription. More detailed analysis indicated that RLN expression was highest in the stigma region of the follicle but significantly decreased as the time of the expected luteinizing hormone (LH) surge approached. Together, our findings demonstrated that the granulosa cells in the mature preovulatory follicles constitute the main source of RLN in the Japanese quail. Because RLN expression was highest in the stigma region and the expression dramatically decreased following the LH surge, the results further suggest that RLN may be related to tissue remodeling for the ovulation process in birds.

Relaxin exerts a protective effect during ischemia-reperfusion in the rat model.

BACKGROUND: Testicular torsion, which causes ischemia-reperfusion (IR) injury, is a serious urological emergency that can lead to testicular dysfunction, including infertility, primarily among newborn and pubertal males; thus, effective drugs should be administered during or after ischemia. OBJECTIVES: Using a rat model of testicular IR injury, the present study investigated the protective effects of relaxin (RLN) against oxidative stress, testicular dysfunction, inflammation, histological damage, arrested spermatogenesis, and germ cell apoptosis as well as explored the usefulness of RLN as a potential protective drug for IR injury combined with surgical treatment. MATERIALS AND METHODS: Male Sprague-Dawley rats were subjected to left testicular ischemia for 2 h, followed by 24 h of reperfusion. They were subsequently divided into three groups: sham, IR, and IR + RLN groups. Porcine RLN (500 ng/h) or saline was infused using an implanted osmotic mini-pump 90 min after inducing ischemia. The RLN dose used herein was that which resulted in serum RLN levels comparable to those in mid-pregnant rats based on previous studies. RESULTS: Testicular IR increased germ cell apoptosis and histological damage as well as promoted disorganized and arrested spermatogenesis, accompanied by a significant increase in oxidative stress and inflammation. However, RLN administration ameliorated the adverse consequences associated with IR injury by attenuating oxidative stress and mitigating apoptosis and inflammation. DISCUSSION AND CONCLUSION: The study findings clearly demonstrated that RLN exerts a protective effect against IR-induced testicular injury by attenuating oxidative stress, apoptosis, and inflammation, suggesting that RLN together with surgical treatment is a potentially efficacious approach toward ameliorating testicular dysfunction following testicular torsion.

Monitoring the reactive oxygen species in spermatozoa during liquid storage of boar semen and its correlation with sperm motility, free thiol content and seasonality.

Oxidative stress is an important factor affecting the quality of spermatozoa during liquid storage of boar semen; however, monitoring of reactive oxygen species (ROS) that provides direct insight into the oxidative status is not yet attempted. This study aimed to monitor ROS in boar sperm during liquid semen storage to determine its correlation with sperm motility and free thiol (SH) content, and seasonality. Ejaculate was collected from mature Duroc boars in a commercial farm in autumn and spring, diluted in Mulberry III extender, stored at 15°C, and examined daily for sperm ROS level, SH content and motility. The ROS levels in spermatozoa prepared during autumn and spring were constantly low until days 4 and 5 of storage, respectively, which thereafter progressively increased in association with the loss of sperm motility. The increased sperm ROS level correlated with the higher SH level and lower motility, which was accentuated from day 4 of storage and was higher in September, or early autumn. This study indicates that increased sperm ROS levels during liquid storage results in oxidative damage, causing loss of sperm motility, presumably through decreased sperm viability, suggesting that sperm ROS monitoring effectively evaluates the quality of boar semen.

Evidence for existence of insulin-like factor 3 (INSL3) hormone-receptor system in the ovarian corpus luteum and extra-ovarian reproductive organs during pregnancy in goats.

Insulin-like factor 3 (INSL3), initially described as a male hormone, is expressed in female reproductive organs during the estrous cycle and pregnancy but its function has not yet been established. This study explores the function of INSL3 in pregnant Saanen goats by characterizing the expression dynamics of INSL3 and its receptor, relaxin family peptide receptor 2 (RXFP2) and by demonstrating specific INSL3 binding in reproductive organs, using molecular and immunological approaches and ligand-receptor interaction assays. We demonstrate that the corpus luteum (CL) acts as both a source and target of INSL3 in pregnant goats, while extra-ovarian reproductive organs serve as additional INSL3 targets. The expression of INSL3 and RXFP2 in the CL reached maximum levels in middle pregnancy, followed by a decrease in late pregnancy; in contrast, RXFP2 expression levels in extra-ovarian reproductive organs were higher in the mammary glands but lower in the uterus, cervix and placenta and did not significantly change during pregnancy. The functional RXFP2 enabling INSL3 to bind was identified as an ~ 85 kDa protein in both the CL and mammary glands and localized in large and small luteal cells in the CL and in tubuloalveolar and ductal epithelial cells in the mammary glands. Additionally, INSL3 also bound to multiple cell types expressing RXFP2 in the uterus, cervix and placenta in a hormone-specific and saturable manner. These results provide evidence that an active intra- and extra-ovarian INSL3 hormone-receptor system operates during pregnancy in goats.

Physiology and evolution of the INSL3/RXFP2 hormone/receptor system in higher vertebrates.

Although the insulin-like peptide hormone INSL3 and its cognate receptor RXFP2 (relaxin-family peptide receptor 2) have existed throughout chordate evolution, their physiological diversification appears to be linked closely with mammalian emergence and radiation. In contrast, they have been lost in birds and reptiles. Both hormone and receptor are expressed from autosomal genes which have maintained their synteny across vertebrate evolution. Whereas the INSL3 gene comprises only two exons closely linked to the JAK3 gene, RXFP2 is normally encoded by 18 exons. Both genes, however, are subject to alternative splicing to yield a variety of possibly inactive or antagonistic molecules. In mammals, the INSL3-RXFP2 dyad has maintained a probably primitive association with gametogenesis, seen also in fish, whereby INSL3 promotes the survival, growth and differentiation of male germ cells in the testis and follicle development in the ovary. In addition, however, the INSL3/RXFP2 system has adopted a typical 'neohormone' profile, essential for the promotion of internal fertilisation and viviparity; fetal INSL3 is essential for the first phase of testicular descent into a scrotum, and also appears to be associated with male phenotype, in particular horn and skeletal growth. Circulating INSL3 is produced exclusively by the mature testicular Leydig cells in male mammals and acts as a potent biomarker for testis development during fetal and pubertal development as well as in ageing. As such it can be used also to monitor seasonally breeding animals as well as to investigate environmental or lifestyle conditions affecting development. Nevertheless, most information about INSL3 and RXFP2 comes from a very limited selection of species; it will be especially useful to gain further information from a more diverse range of animals, especially those whose evolution has led them to express unusual reproductive phenotypes.

Efficacy of relaxin for cisplatin-induced testicular dysfunction and epididymal spermatotoxicity.

BACKGROUND: Cisplatin (CP) is an extremely effective anticancer agent widely used to treat various cancer types, however, the potential side effects include testicular dysfunction. This study was to investigate, using a rat model of CP-induced testicular dysfunction, the protective effects of relaxin (RLN) against oxidative stress, testicular function, histological damage, spermatogenesis, germ-cell apoptosis, and sperm output, and to explore the usefulness of RLN as a potential protective drug for use with CP in chemotherapeutic treatments. METHODS: Sprague-Dawley male rats were used, which were divided into three groups: sham control, CP, and CP + RLN. Porcine RLN (500 ng/h) or saline was infused for 5 days using an implanted osmotic mini-pump following intraperitoneal injection of CP (6 mg/kg). RLN dose was chosen based on previous studies showing that it resulted in serum relaxin levels comparable to those in rats at the middle of pregnancy. At 5 days after CP administration, samples were collected and assessment of testicular histopathology, germ-cell apoptosis, oxidative stress, lipid peroxidation, and sperm quality was performed as main measures. RESULTS: The testicular CP model showed reduced testis weight and significantly decreased spermatogenesis scores. Additionally, CP administration induced a 4.6-fold increase in the apoptotic index associated with a significant increase in oxidative stress and upregulation of pro-apoptotic Casp3 and downregulation of anti-apoptotic Bcl2 levels, resulting in a marked reduction in sperm concentration. However, RLN administration caused a significant reduction in CP-mediated damage by attenuating oxidative stress and cell apoptosis. RLN administration efficiently scavenged ROS via the activation of SOD, CAT, and GPx and upregulation of GSH to prevent lipid peroxidation and decreased apoptosis by altering Bcl2 and Casp3 expression, thereby reducing histopathological damage and restoring spermatogenesis. Furthermore, RLN ameliorated attenuated sperm motility in the cauda epididymis resulting from CP treatment. CONCLUSIONS: This study clearly indicates that RLN exerts a protective effect against CP-induced testicular damage through attenuation of oxidative stress and suppression of apoptosis. Our findings suggest RLN as a potentially efficacious drug for use with cisplatin chemotherapy in order to ameliorate CP-induced side effects and testicular injury adversely affecting spermatogenesis, sperm quality, and oxidative-stress parameters.

CONTEXTE: Le cis platine (CP) est un agent anticancéreux extrêmement efficace largement utilisé pour traiter divers types de cancer. Parmi les effets secondaires potentiels associés aux traitements par CP on compte le dysfonctionnement des testicules. Le propos de ce manuscrit est d'étudier, à l'aide d'un modèle rat de dysfonctionnement testiculaire induit par la prise de CP, l'action protectrice de la relaxine (RLN) contre les effets délétères dus au CP lesquels incluent le stress oxydant, la perte de fonction testiculaire, les dommages histologiques au testicule, l'apoptose des cellules germinales et la baisse de la qualité des spermatozoïdes. L'objectif est d'explorer l'utilité de la RLN comme médicament protecteur potentiel à utiliser avec le CP dans les traitements chimiothérapeutiques. MÉTHODES: Des rats mâles Sprague-Dawley ont été utilisés. Trois groupes : contrôle, CP, et CP + RLN ont été comparés. Après une injection intrapéritonéale de CP (6mg/kg), de la RLN porcine (500 ng/h) ou du sérum physiologique a été perfusé pendant 5 jours en utilisant une mini-pompe osmotique implantée. La dose de RLN a été choisie en fonction d'études antérieures qui avaient montré qu'elle entraînait des taux sériques de RLN comparables à ceux de rats en milieu de la gestation. Cinq jours après l'administration de la CP, des échantillons ont été prélevés afin d'évaluer l'histopathologie, l'apoptose des cellules germinales, le stress oxydant, la peroxydation des lipides et les paramètres spermatiques. RÉSULTATS: Le groupe CP a montré une réduction du poids des testicules et une diminution significative des scores de spermatogenèse. De plus, l'administration de CP a entraîné une augmentation de l'apoptose de 4,6 fois associée à une augmentation significative du stress oxydant, de la régulation à la hausse de la Caspase 3 pro-apoptotique et à la baisse de Bcl2 anti-apoptotique conduisant in fine à une réduction marquée de la concentration en spermatozoïdes. La RLN a ainsi significativement corrigée les effets négatifs du CP en atténuant le stress oxydant et l'apoptose. La RLN a permis d'éliminer efficacement les ROS via l'activation de la triade enzymatique anti-oxydante superoxyde dismutase (SOD)/catalase (CAT)/glutathion peroxydase (GPx) et via la régulation à la hausse du GSH prévenant ainsi la lipopéroxydation. La RLN a par ailleurs diminué les atteintes histopathologiques testiculaires préservant la spermatogenèse. En parallèle, la RLN a amélioré la mobilité spermatique des spermatozoïdes prélevés dans la queue de l'épididyme. CONCLUSIONS: Cette étude montre clairement que la RLN exerce un effet protecteur contre les lésions testiculaires par l'atténuation du stress oxydant et la suppression de l'apoptose induite par le CP. Nos résultats suggèrent que la RLN est un médicament potentiellement pertinent à utiliser afin de diminuer les effets secondaires induits par le CP sur la fonction testiculaire et sur les spermatozoïdes lors de la chimiothérapie cancéreuse.

Insulin-like peptide 3 expressed in the silkworm possesses intrinsic disulfide bonds and full biological activity.

Insulin-like peptide 3 (INSL3) is a member of the relaxin/insulin superfamily and is expressed in testicular Leydig cells. Essential for fetal testis descent, INSL3 has been implicated in testicular and sperm function in adult males via interaction with relaxin/insulin-like family peptide receptor 2 (RXFP2). The INSL3 is typically prepared using chemical synthesis or overexpression in Escherichia coli followed by oxidative refolding and proteolysis. Here, we expressed and purified full-length porcine INSL3 (pINSL3) using a silkworm-based Bombyx mori nucleopolyhedrovirus bacmid expression system. Biophysical measurements and proteomic analysis revealed that this recombinant pINSL3 exhibited the correct conformation, with the three critical disulfide bonds observed in native pINSL3, although partial cleavage occurred. In cAMP stimulation assays using RXFP2-expressing HEK293 cells, the recombinant pINSL3 possessed full biological activity. This is the first report concerning the production of fully active pINSL3 without post-expression treatments and provides an efficient production platform for expressing relaxin/insulin superfamily peptides.

Transduction of a Neospora caninum antigen gene into mammalian cells using a modified Bombyx mori nucleopolyhedrovirus for antibody production.

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) can easily enter and transduce foreign genes into mammalian cells, but these functions are difficult for Bombyx mori nucleopolyhedrovirus (BmNPV). In this study, we investigated the induction of antibody production in mice immunized with an engineered BmNPV. The GP64 of BmNPV (BmGP64) was replaced with the GP64 of AcMNPV (AcGP64); this construct, designated BmNPVΔbgp/AcGP64, displays AcGP64 on the surface of BmNPV. The Neospora caninum antigen (NcSRS2) expression cassette, consisting of the cytomegalovirus immediate-early promoter and NcSRS2 from N. caninum, was inserted into BmNPVΔbgp/AcGP64; this construct was designated BmNPVΔbgp/AcGP64/SRS2. For comparison, AcMNPV/SRS2, which contains the same NcSRS2 expression cassette as for BmNPVΔbgp/AcGP64, was also constructed. NcSRS2 was expressed in HEK293T cells when the engineered BmNPVs were transduced at a multiplicity of infection of 150. BmNPVΔbgp/AcGP64/SRS2 induced the production of NcSRS2-specific antibodies in mice, whereas AcMNPV/SRS2 and the control BmNPV did not. These results suggest that BmNPV prepared from silkworm hemolymph induces the production of antigen-specific antibodies in immunized mice and can be used for antibody production and vaccine development.

Functional insulin-like factor 3 (INSL3) hormone-receptor system in the testes and spermatozoa of domestic ruminants and its potential as a predictor of sire fertility.

Insulin-like factor 3 (INSL3) is essential for fetal testis descent, and has been implicated in the testicular and sperm functions in adult males; however, similar functions in domestic ruminants remain largely unknown. This study investigated the functional INSL3 hormone-receptor system in adult ruminant testes and spermatozoa, and explored its potential to diagnose the fertility of sires. Testes and spermatozoa were obtained from fertile bulls, rams and he-goats, whereas subfertile testes and spermatozoa were obtained only from bulls. As expected, INSL3 was visualized in Leydig cells, while we clearly demonstrated that the functional receptor, relaxin family peptide receptor 2 (RXFP2), enabling INSL3 to bind was identified in testicular germ cells and in the sperm equatorial segment of bulls, rams and he-goats. In comparison to fertile bulls, the percentage of INSL3- and RXFP2-expressing cells and their expression levels per cell were significantly reduced in the testes of subfertile bulls. In addition, the population of INSL3-binding spermatozoa was also significantly reduced in the semen of subfertile bulls. These results provide evidence for a functional INSL3 hormone-receptor system operating in ruminant testes and spermatozoa, and its potential to predict subfertility in sires.

Lactic acid is a sperm motility inactivation factor in the sperm storage tubules.

Although successful fertilization depends on timely encounters between sperm and egg, the decoupling of mating and fertilization often confers reproductive advantages to internally fertilizing animals. In several vertebrate groups, postcopulatory sperm viability is prolonged by storage in specialized organs within the female reproductive tract. In birds, ejaculated sperm can be stored in a quiescent state within oviductal sperm storage tubules (SSTs), thereby retaining fertilizability for up to 15 weeks at body temperature (41°C); however, the mechanism by which motile sperm become quiescent within SSTs is unknown. Here, we show that low oxygen and high lactic acid concentrations are established in quail SSTs. Flagellar quiescence was induced by lactic acid in the concentration range found in SSTs through flagellar dynein ATPase inactivation following cytoplasmic acidification (

Expression of insulin-like factor 3 hormone-receptor system in the reproductive organs of male goats.

Relaxin-like factor (RLF), generally known as insulin-like factor 3 (INSL3), is essential for testis descent during fetal development. However, its role in adult males is not fully understood. We investigate the function of INSL3 in male Saanen goats by identifying cell types expressing its receptor, relaxin/insulin-like family peptide receptor (RXFP)2 and by characterizing the developmental expression pattern of INSL3 and RXFP2 and the binding of INSL3 to target cells in the male reproductive system. A highly specific RXFP2 antibody that co-localizes with an anti-FLAG antibody in HEK-293 cells recognizes RXFP2-transcript-expressing cells in the testis. INSL3 and RXFP2 mRNA expression is upregulated in the testis, starting from puberty. INSL3 mRNA and protein expression has been detected in Leydig cells, whereas RXFP2 mRNA and protein localize to Leydig cells, to meiotic and post-meiotic germ cells and to the epithelium and smooth muscle of the cauda epididymis and vas deferens. INSL3 binds to all of these tissues and cell types, with the exception of Leydig cells, in a hormone-specific and saturable manner. These results provide evidence for a functional intra- and extra-testicular INSL3 ligand-receptor system in adult male goats.

Evaluation of recombinant Neospora caninum antigens purified from silkworm larvae for the protection of N. caninum infection in mice.

Three antigens (NcSAG1, NcSRS2 and NcMIC3) from Neospora caninum were expressed using the BmNPV bacmid system in silkworm larvae and purified from the hemolymph. From 20 silkworm larvae, 1.5, 1.2 and 1.4 mg of purified recombinant NcSAG1, NcSRS2 and NcMIC3 were obtained, respectively. When each purified recombinant antigen was immunized with Freund's incomplete adjuvant (FIA) to mice, recombinant NcSAG1 induced a Th2 immune response in immunized mice and produced a SAG1-specific antibody. In the experiment where NcSAG1-immunized mice were challenged with N. caninum, the cerebral N. caninum burden was significantly reduced compared with that of either the FIA- or PBS-immunized mice. Recombinant NcSRS2 or NcMIC3 induced both Th1 and Th2 immune responses, but NcMIC3-immunization did not induce significant production of NcMIC3-specific antibodies. These results suggest that the silkworm can produce recombinant antigens of N. caninum, which can be used as a recombinant vaccine against N. caninum.

The insulin-like factor 3 (INSL3)-receptor (RXFP2) network functions as a germ cell survival/anti-apoptotic factor in boar testes.

Relaxin-like factor, commonly known as insulin-like factor (INSL3), is essential for testis descent during fetal development; however, its function in the adult testis is still being elucidated. The study aimed to identify a relaxin family peptide receptor 2 (RXFP2)-specific antibody suitable for immunological approaches, analyze which testicular germ cell types express RXFP2, and clarify its expression dynamics in the boar testis. In addition, the function of INSL3-RXFP2 signaling on the germ cells was explored by neutralizing INSL3 using long-term active immunization. Samples were collected from Duroc boars, and a commercially available RXFP2-specific antibody directed against the human RXFP2 endodomain was identified by characterizing its specificity in HEK-293 cells expressing mouse RXFP2, and by demonstrating the suitability for analyzing RXFP2 expression in porcine tissues. RXFP2 mRNA and protein were both localized mainly in meiotic and post-meiotic germ cells, but not in Leydig cells. Functional RXFP2, which enables INSL3 to bind, was detected as an ∼85-kDa band, which increased in intensity from the pubertal stage onward. Interestingly, INSL3 immunization significantly reduced testis weight and induced a 4-fold increase in the frequency of apoptotic germ cells, which was associated with the up-regulation of pro-apoptotic caspase-3 (CASP3) and BAX, and the down-regulation of anti-apoptotic XIAP and BCL2, and a substantial reduction in sperm concentration. These results revealed that RXFP2 was expressed in boar meiotic and post-meiotic germ cells, where INSL3 neutralization led to increased germ cell apoptosis and reduced sperm output, suggesting that INSL3 acts as a survival/anti-apoptotic factor in maintaining sperm production.

Bombyx mori nucleopolyhedrovirus displaying neospora caninum antigens as a vaccine candidate against N. caninum infection in mice.

Baculovirus display systems have been utilized for cell-specific gene transfer, regenerative medicine, and as vaccine vectors. In particular, baculovirus particles displaying surface antigens have been used as vaccines against some parasites and viruses. In this study, Bombyx mori nucleopolyhedrovirus (BmNPV) particles displaying Neospora caninum antigens (NcSAG1, NcSRS2, and NcMIC3) purified from the hemolymph or fat body of silkworm larvae were prepared to vaccinate mice against N. caninum. Each antigen was expressed on the surface of BmNPV particles through glycoprotein 64 transmembrane and cytoplasmic domains. Antigen-specific antibody production was induced in mice by immunization with each recombinant BmNPV particle. NcMIC3-displaying BmNPV particles purified from the fat body induced a lower antibody titer than particles purified from the hemolymph. Antigen-specific IgG2a was predominantly produced in mice by immunization with NcSAG1-displaying BmNPV particles compared to IgG1, and induction of IFN-γ was dominant, indicating that antigen-displaying BmNPV particles can elicit a Th1 immune response in mice. Semi-quantitative PCR analysis revealed that immunization with each antigen-displaying BmNPV particle partially protected mice from cerebral N. caninum infection. These results suggest that antigen-displaying BmNPV particles can provide an alternative method of controlling neosporosis in cattle and represent a new generation of N. caninum vaccines.

Protective effects of relaxin against cisplatin-induced nephrotoxicity in rats.

BACKGROUND: Cisplatin (CDDP)-induced acute kidney injury (AKI) involves pro-inflammatory responses, apoptosis of renal tubular epithelial cells and vascular damage. AKI increases the risk of chronic kidney disease. Relaxin (RLX) has anti-apoptotic and anti-fibrosis properties. The aim of this study was to investigate the effects of RLX on CDDP-induced nephrotoxicity. METHODS: We investigated the mitigating effects of RLX based on the etiopathology of AKI induced by CDDP, and also the anti-fibrotic effect of RLX on renal fibrosis after AKI. In the short-term experiments, rats were divided into the control group, CDDP group, and CDDP+RLX group. In the latter group, RLX was infused for 5 or 14 days using an implanted osmotic minipump. CDDP was injected intraperitoneally (6 mg/kg) after RLX or saline infusion. At 5 and 14 days post-CDDP, the kidneys were removed for analysis. The effect of RLX on renal fibrosis after AKI was evaluated at 6 weeks post-CDDP. RESULTS: In short-term experiments, CDDP transiently increased plasma creatinine and blood urea nitrogen with peaks at day 5, and RLX prevented such rises. Semiquantitative analysis of the histological lesions indicated marked structural damage and apoptotic cells in the CDDP group, with the lesions being reduced by RLX treatment. Overexpression of Bax, interleukin-6 and tumor necrosis factor-α observed in the kidneys of the CDDP group was reduced in the CDDP+RLX group. In the long-term experiments, RLX significantly reduced renal fibrosis compared with the CDDP group. CONCLUSIONS: The results suggested that RLX provided protection against CDDP-induced AKI and subsequent fibrosis by reducing apoptosis and inflammation.

Dynamics of insulin-like factor 3 and its receptor expression in boar testes.

Relaxin-like factor (RLF), now mainly known as insulin-like factor 3 (INSL3), is essential for testis descent during fetal development; however, its function in the adult testis is still being elucidated. As a major step toward understanding the as-yet-unknown function of INSL3 in boars, this study aimed to develop a time-resolved fluoroimmunoassay for boar INSL3, characterize the dynamics of INSL3 expression during development, and demonstrate the expression of the INSL3 hormone-receptor system in the testis. All samples were collected from Duroc boars. The sensitivity of the assay system established was 8.2 pg/well (164 pg/ml), and no cross-reactivity with other hormones, such as porcine relaxin, was observed. Circulating INSL3 was shown to increase progressively during development. INSL3 secreted from the Leydig cells was released not only into the blood circulation but also into the interstitial and seminiferous compartments in sufficient concentrations. A testicular fractionation study revealed that its receptor RXFP2 transcripts were expressed mainly in testicular germ cells. In addition, INSL3 bound to the germ cell membranes in a hormone-specific and saturable manner. These results reveal that INSL3 secreted into the interstitial compartment from the Leydig cells is transported into the seminiferous compartments, where its receptor RXFP2 is expressed mainly in the germ cells to which INSL3 binds, suggesting that INSL3 functions as a paracrine factor on seminiferous germ cells.

The active form of goat insulin-like peptide 3 (INSL3) is a single-chain structure comprising three domains B-C-A, constitutively expressed and secreted by testicular Leydig cells.

Relaxin-like factor (RLF), also called insulin-like peptide 3 (INSL3), is a member of the insulin/relaxin gene family and is produced by testicular Leydig cells. While the understanding of its effects is growing, very little is known about the structural and functional properties of native INSL3. Here, we demonstrate that native INSL3 isolated from goat testes is a single-chain structure with full biological activity, and is constitutively expressed and secreted by Leydig cells. Using a series of chromatography steps, native INSL3 was highly purified as a single 12-kDa peak as revealed by SDS-PAGE. MS/MS analysis provided 81% sequence coverage and revealed a distinct single-chain structure consisting of the B-, C-, and A-domains deduced previously from the INSL3 cDNA sequence. Moreover, the N-terminal peptide was six amino acid residues longer than predicted. Native INSL3 exhibited full bioactivity in HEK-293 cells expressing the receptor for INSL3. Immunoelectron microscopy and Western blot analysis revealed that INSL3 was secreted by Leydig cells through the constitutive pathway into blood and body fluids. We conclude, therefore, that goat INSL3 is constitutively secreted from Leydig cells as a B-C-A single-chain structure with full biological activity.

Relaxin protects against renal ischemia-reperfusion injury.

Relaxin, a pregnancy hormone, has antiapoptotic and anti-inflammatory properties. The aim of this study was to determine the effects of relaxin on ischemia-reperfusion (IR)-induced acute kidney injury. Male rats underwent unilateral nephrectomy and contralateral renal IR (45 min of renal pedicle clamping). Rats were divided into three groups: 1) sham group, 2) IR group, and 3) IR-RLX group (rats treated with relaxin before ischemia). In this group, relaxin was infused at 500 ng/h via subcutaneous osmotic minipump for 24 h beginning 2 h before renal ischemia. At 24 h after reperfusion, renal function was assessed and kidneys were removed for analysis. There was no significant difference in blood pressure among the three groups. IR increased plasma levels of creatinine and urea nitrogen, and relaxin provided protection against the increases in these two parameters. Relaxin significantly decreased plasma TNF-α levels and renal TNF receptor 1 mRNA expression, compared with the IR group. Semiquantitative assessment of the histological lesions showed marked structural damage in IR rats compared with the IR-RLX rats. RLX significantly reduced apoptotic cell counts compared with the IR group. Overexpression of caspase-3 observed in the IR kidneys was reduced in the IR-RLX group. The results demonstrated that relaxin provided protection against IR-induced renal injury by reducing apoptosis and inflammation.