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.

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.

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.

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.

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.

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.

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.

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.

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.

Abstract 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 accumulating, 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 72% 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 6 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.

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-like factor (RLF)/insulin-like peptide 3 (INSL3) is secreted from testicular Leydig cells as a monomeric protein comprising three domains B-C-A with full biological activity in boars.

RLF (relaxin-like factor), also known as INSL3 (insulin-like peptide 3), is a novel member of the relaxin/insulin gene family that is expressed in testicular Leydig cells. Despite the implicated role of RLF/INSL3 in testis development, its native conformation remains unknown. In the present paper we demonstrate for the first time that boar testicular RLF/INSL3 is isolated as a monomeric structure with full biological activity. Using a series of chromatography steps, the native RLF/INSL3 was highly purified as a single peak in reverse-phase HPLC. MS/MS (tandem MS) analysis of the trypsinized sample provided 66% sequence coverage and revealed a distinct monomeric structure consisting of the B-, C- and A-domains deduced previously from the RLF/INSL3 cDNA. Moreover, the N-terminal peptide was four amino acid residues longer than predicted previously. MS analysis of the intact molecule and PMF (peptide mass fingerprinting) analysis at 100% sequence coverage confirmed this structure and indicated the existence of three site-specific disulfide bonds. RLF/INSL3 retained full bioactivity in HEK (human embryonic kidney)-293 cells expressing RXFP2 (relaxin/insulin-like family peptide receptor 2), the receptor for RLF/INSL3. Furthermore, RLF/INSL3 was found to be secreted from Leydig cells into testicular venous blood. Collectively, these results indicate that boar RLF/INSL3 is secreted from testicular Leydig cells as a B-C-A monomeric structure with full biological activity.

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.

Identification of SAMT family proteins as substrates of MARCH11 in mouse spermatids.

MARCH11, a RING-finger transmembrane ubiquitin ligase, is predominantly expressed in spermatids and localized to the trans-Golgi network (TGN) and multivesicular bodies (MVBs). Because ubiquitination acts as a sorting signal of cargo proteins, MARCH11 has been postulated to mediate selective protein sorting via the TGN-MVB pathway. However, the physiological substrate of MARCH11 has not been identified. In this study, we have identified and characterized SAMT1, a member of a novel 4-transmembrane protein family, which consists of four members. Samt1 mRNA and its expression product were found to be specific to the testis and were first detected in germ cells 25 days after birth in mice. Immunohistochemical analysis further revealed that SAMT1 was specifically expressed in haploid spermatids during the cap and acrosome phases. Confocal microscopic analysis showed that SAMT1 co-localized with MARCH11 as well as with fucose-containing glycoproteins, another TGN/MVB marker, and LAPM2, a late endosome/lysosome marker. Furthermore, we found that MARCH11 could increase the ubiquitination of SAMT1 and enhance its lysosomal delivery and degradation in an E3 ligase activity-dependent manner. In addition, the C-terminal region of SAMT1 was indispensable for its ubiquitination and proper localization. The other member proteins of the SAMT family also showed similar expression profile, intracellular localization, and biochemical properties, including ubiquitination by MARCH11. These results suggest that SAMT family proteins are physiological substrates of MARCH11 and are delivered to lysosomes through the TGN-MVB pathway by a ubiquitin-dependent sorting system in mouse spermatids.

Relaxin ameliorates salt-sensitive hypertension and renal fibrosis.

BACKGROUND: Although relaxin (RLX) has potent vasodilatory and anti-fibrotic properties, there is no information on its effects on salt-sensitive hypertension. METHODS: We investigated the effects of short-term treatment with RLX on blood pressure (BP) and nitric oxide synthase (NOS) protein in the kidneys of male Dahl salt-sensitive (DS) and Dahl salt-resistant (DR) rats after 1 week consumption of an 8% NaCl diet. We also evaluated the inhibitory effects of each specific NOS inhibitor on BP during 1-week RLX treatment under high-salt diet. Next, we examined the long-term effects of RLX treatment for 6 weeks on renal histology and transforming growth factor-beta1 (TGF-ß1) expression in male DS and DR rats placed on the 8-week high-salt diet. RESULTS: The short-term RLX treatment significantly attenuated the high-salt diet-induced rise in BP in DS rats with increasing neuronal NOS and endothelial NOS protein in kidneys. Selective inhibition of each of the three NOS isoforms significantly blocked the anti-hypertensive effects of RLX in DS rats after 1-week high-salt diet. The long-term treatment of DS rats with RLX for 6 weeks significantly reduced systolic BP, lessened glomerular and tubulointerstitial changes and reduced TGF-ß signaling compared to saline-treated controls. CONCLUSIONS: The results suggested that RLX converted salt sensitivity to salt resistance, at least in part, by up-regulating NOS. RLX is a potentially useful therapeutic agent for salt-sensitive hypertension.

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.

Zona pellucida protein ZP2 is expressed in the oocyte of Japanese quail (Coturnix japonica).

The avian perivitelline layer (PL), a vestment homologous to the zona pellucida (ZP) of mammalian oocytes, is composed of at least three glycoproteins. Our previous studies have demonstrated that the matrix's components, ZP3 and ZPD, are synthesized in ovarian granulosa cells. Another component, ZP1, is synthesized in the liver and is transported to the ovary by blood circulation. In this study, we report the isolation of cDNA encoding quail ZP2 and its expression in the female bird. By RNase protection assay and in situ hybridization, we demonstrate that ZP2 transcripts are restricted to the oocytes of small white follicles (SWF). The expression level of ZP2 decreased dramatically during follicular development, and the highest expression was observed in the SWF. Western blot and immunohistochemical analyses using the specific antibody against ZP2 indicate that the 80 kDa protein is the authentic ZP2, and the immunoreactive ZP2 protein is also present in the oocytes. Moreover, ultrastructural analysis demonstrated that the immunoreactive ZP2 localizes to the zona radiata, the perivitelline space, and the oocyte cytoplasm in the SWF. By means of western blot analysis and immunofluorescence microscopy, we detected a possible interaction of the recombinant ZP2 with ZP3 and that this interaction might lead to the formation of amorphous structure on the cell surface. These results demonstrate for the first time that the avian ZP gene is expressed in the oocyte, and that the ZP2 protein in the oocyte might play a role for the PL formation in the immature follicles of the ovary.

Detection of relaxin mRNA in the corpus luteum, uterus, and uterine cervix in the bitch.

In the pregnant bitch, the placenta is a major source of circulating relaxin, but its local expression in the reproductive organs is not clear. This study demonstrated expression of relaxin mRNA in the corpus luteum, uterus, uterine cervix as well as placenta in the pregnant and nonpregnant bitch by reverse transcriptase-polymerase chain reaction (RT-PCR).

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.

Effects of relaxin and IGF-I on capacitation, acrosome reaction, cholesterol efflux and utilization of labeled and unlabeled glucose in porcine spermatozoa.

Aim: Relaxin and insulin-like growth factor (IGF)-I have pronounced effects on the male and female reproductive tracts. The aim of this study was to investigate the effects of relaxin and IGF-I on the motility, capacitation, acrosome reaction, cholesterol efflux and utilization of glucose in porcine spermatozoa. Methods: Swim-up separated spermatozoa that had been washed twice were incubated at 37°C for 1 or 4 h in modified Tyrode's albumin lactate pyruvate (mTALP) medium supplemented without (control) or with relaxin (20 ng/mL) or IGF-I (20 ng/mL) or both (10 + 10 ng/mL). Results: Progressive motility and the induction rate of capacitation and acrosome reaction were increased (P < 0.05) by relaxin and IGF-I alone or in combination, especially after 4 h of incubation. Relaxin alone or combined with IGF-I enhanced (P < 0.05) the cholesterol efflux after 4 h, whereas IGF-I alone did not show any significant effect on the cholesterol efflux compared with the control at any time point. The utilization rates of labeled and unlabeled glucose increased (P < 0.05) in spermatozoa incubated with relaxin or IGF-I alone or in combination compared with the control. Conclusion: Thus, supplementation of relaxin alone or combined with IGF-I into the medium possibly plays a beneficial role in porcine spermatozoal prefertilization events in vitro. (Reprod Med Biol 2008; 7: 29-36).

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.